Regeneration 1&2

Regeneration of complex structures after injury requires dramatic changes in cellular

behavior. Regenerating tissues initiate a program that includes diverse processes such
as wound healing, cell death, dedifferentiation, and stem (or progenitor) cell
proliferation; furthermore, newly regenerated tissues must integrate polarity and
positional identity cues with preexisting body structures. Gene knockdown approaches
and transgenesis-based lineage and functional analyses have been instrumental in
deciphering various aspects of regenerative processes in diverse animal models for
studying regeneration.
Range of vertebrate regenerative tissues
"Regenerative strategies include the rearrangement of pre-existing tissue, the use of
adult somatic stem cells and the dedifferentiation and/or transdifferentiation of cells, and
more than one mode can operate in different tissues of the same animal. All these
strategies result in the re-establishment of appropriate tissue polarity, structure and
form." During the developmental process genes are activated that serve to modify the
properties of cell as they differentiate into different tissues. Development and
regeneration involves the coordination and organization of populations cells into a
blastema, which is "a mound of stem cells from which regeneration begins."
Dedifferentiation of cells means that they lose their tissue-specific characteristics as
tissues remodel during the regeneration process. Transdifferentiation of cells is when
they lose their tissue-specific characteristics during the regeneration process, and then
re-differentiate to a different kind of cell. Compensatory growth is a type of regenerative
growth that can take place in a number of human organs after the organs are either
damaged, removed, or cease to function. Additionally, increased functional demand can
also stimulate this growth in tissues and organs. The growth can be a result of increased
cell size (compensatory hypertrophy) or an increase in cell division (compensatory
hyperplasia) or both. For instance, if one kidney is surgically removed, the cells of other
kidney divide at an increased rate. Eventually, the remaining kidney can grow until its
mass approaches the combined mass of two kidneys. A large number of growth factors
and hormones are involved with compensatory growth, but the exact mechanism is not
fully understood and probably varies between different organs.[1] Nevertheless,
angiogenic growth factors which control the growth of blood vessels are particularly
important because blood flow significantly determines the maximum growth of an organ.
- Compensatory liver hyperplasia The liver undergoes cellular division after acute
injury, resulting in new cells that restore liver function back to baseline. Approximately
75% of the liver can be acutely damaged or resected with seemingly full regeneration
through hepatocyte division, i.e., hyperplasia. This is what makes living-donor liver
transplants possible.
-Dedifferentiation is a cellular process often seen in more basal life forms such as worms
and amphibians in which a partially or terminally differentiated cell reverts to an earlier
developmental stage, usually as part of a regenerative process. Dedifferentiation also
occurs in plants. Cells in cell culture can lose properties they originally had, such as
protein expression, or change shape. This process is also termed dedifferentiation. A
small molecule dubbed reversine, a purine analog, has been discovered that has proven
to induce dedifferentiation in myotubes. These dedifferentiated cells could then
redifferentiate into osteoblasts and adipocytes
Regenerative biology model organisms
In members of the Salamandroidea (Salamander) superfamily such as the newts, limb
transection stimulates large-scale repair that perfectly replaces all the tissues in the limb
including muscle, bone, nerve, dermis and skin. After the initial sealing of the wound by
migrating epidermis, the injured tissue produces a zone of proliferating, apparently
undifferentiated mesenchymal cells, called the blastema. Blastema formation takes from
5 to 14 days depending on the species and age of animal. These blastema cells along
with the overlying epidermis reactivate developmental programs to replace the missing
portion of the limb.

Mammals: The mechanism for regeneration in Murphy Roths Large (MRL) mice has
been found, and is related to the deactivation of the p21 gene. At least two species of
African Spiny Mice, Acomys kempi and Acomys percivali, are capable of completely
regenerating the autotomically released or otherwise damaged tissue. These species can
regrow hair follicles, skin, sweat glands, fur and cartilage. Adult mammals have limited
regenerative capacity compared to most vertebrate embryos/larvae, adult salamanders
and fish. But the regeneration therapy approach of Robert O. Becker, using electrical
stimulation, has shown promising results for rats and mammals in general. The MRL
mouse is a strain of mouse that exhibits remarkable regenerative abilities for a mammal.
Study of the regenerative process in these animals is aimed at discovering how to
duplicate them in humans. By comparing the differential gene expression of scarless
healing MRL mice and a poorly-healing C57BL/6 mouse strain, 36 genes have been
identified that are good candidates for studying how the healing process differs in MRL
mice and other mice. The regenerative ability of MRL mice does not, however, protect
them against myocardial infarction; heart regeneration in adult mammals
(neocardiogenesis) is limited, because heart muscle cells are nearly all terminally
differentiated. MRL mice show the same amount of cardiac injury and scar formation as
normal mice after a heart attack. However, recent studies provide evidence that this
may not always be the case, and that MRL mice can regenerate after heart damage.
Urodele amphibians
Salamanders - Including newts and axolotl
A model RB system: the axolotl limb
The feature of the salamander that attracts most attention is its healing ability: the
axolotl does not heal by scarring and is capable of the regeneration of entire lost
appendages in a period of months, and, in certain cases, more vital structures. In some
cases, axolotls have been known to repair a damaged limb, as well as regenerating an
additional one, ending up with an extra appendage that makes them attractive to pet
owners as a novelty. These animals have mastered the ability to repair and replace most
of their tissues following damage or amputation even well into adulthood. In fact it
seems that the ability of these organisms to regenerate perfectly is not affected by their
age. They are also easy to breed and rear in the lab. Their care is simplified by the fact
that axolotls do not typically undergo metamorphosis and thus have a completely
aquatic life cycle. Axolotl eggs are larger than frog eggs and they are clear, so one can
easily watch embryogenesis happen without need for a powerful microscope. Also, the
large size makes it possible to perform surgeries on embryos to investigate questions
about development, such as mechanisms that control cell fate and differentiation. The
large collection of axolotls in the Ambystoma Genetic Stock Center ensures that
researchers do not have to rely on salamanders collected from natural populations,
which are genetically variable and susceptible to extinction by over-harvesting and
pathogen transmission by collectors.
Outline of regeneration following axolotl limb amputation
1) Form wound epithelium (Microarray analysis Campbell LJ et al., 2011) some people
screen genes expressed in this seal the wound quickly and if you remove that ability
dont get robust regeneration. 2) Reorganise ECM: upregulated MMPs - matrix
metalloproteinase cleave ECM to allow change in cell shape and move around. 3)
Dedifferentiate cells within a few mm of the amputation. 4) Dedifferentiated cells
migrate and proliferate under the epithelium -start to generate cells of new growing
limb. 5) Blastema forms - group of mesenchymal cells proliferating under wound
epithelium. 6) Blastema cells start to redifferentiate to new limb (inc. epidermis, dermis,
muscle, nerve, blood vessels and skeletal elements).
+ Salamanders have the remarkable ability to regenerate complex structures such as
limbs, tails, retina, and spinal cord, along with some sections of the heart and brain,
during any stage of their life cycle. Salamanders are also able to perform scar-free repair
of deep tissue wounds after injury. Although the cellular mediators and immunological
signaling necessary for regeneration in the salamander have not been described, recent

reports suggest that inflammation may influence the initiation and completion of wound
healing and regeneration, as the cytokine microenvironment directly influences the time
course of leukocyte infiltration, cellular proliferation, angiogenesis, and collagen
remodeling of damaged tissues. In mammals, fibrotic scarring is a major impediment to
tissue regeneration, where cellular infiltration and immune signalling play a key role.
Macrophages are an important source of both inflammatory and anti-inflammatory
signals, arriving in mammalian wounds 4896 h after injury, where they clear dead cells,
release pro-inflammatory cytokines and subsequently produce factors that dampen
inflammation and stimulate angiogenesis, fibroblast migration, and replication. In mice,
macrophage depletion or disruption of macrophage transcriptional regulation after
skeletal muscle injury results in incomplete muscle repair and induction of fibrotic
scarring.
+ Macrophage-depleted axolotl limb stumps also failed to fully activate expression of
TGF- 1, a pleiotropic growth factor and key regulator of embryonic development and
mammalian wound healing, as well as its target genes Runx2 and fibronectin. Inhibition
of TGF-1 signaling blocks successful limb regeneration in the salamander, and
fibronectin forms part of the provisional matrix during scar-free repair that may
contribute to a regeneration permissive environment. The stunted activation of MMP9
and MMP3 on macrophage ablation is consistent with an essential role for matrix
remodeling in successful limb regeneration and implicates macrophages as a key
regulator of matrix degrading enzymes. Other matrix components such as collagen were
markedly altered in the absence of macrophages, Leading to a fibrous cap. The
regenerative Blastema features mainly Collagen type III (thin fibers), with a distinctive
lack of collagen IV, whereas collagen I (thick fibers) is normally down-regulated.
Disruption of collagen production in macrophage-depleted limb stumps is presumably
due to the activation of myofibroblasts, which are mainly absent in normal axolotl limb
and scar-free skin regeneration,contributes to scarring in mammals,and represents a
major difference between fibrotic and scar-free repair in the mouse.
The Blastema: Key to the axolotl regenerative response
i.e. after injury, axolotl forms blastema, mammals form scar tissue typically instead. A
blastema is a mass of undifferentiated cells capable of growth and regeneration into
organs or body parts. Historically, blastemas were thought to be composed of
undifferentiated pluripotent cells, but recent research indicates that in some organisms
blastemas may retain memory of tissue origin. Blastemas are typically found in the early
stages of an organism's development such as in embryos, and in the regeneration of
tissues, organs and bone. Some amphibians and certain species of fish can produce
blastemas as adults. For example, salamanders can regenerate many organs after their
amputation, including their limbs, tail, retina and intestine. Most animals, however,
cannot produce blastemas.
The source of cells for regeneration: The mature tissue source that gives rise to the
regeneration blastema was controversial in early limb regeneration studies. Thorntons
work on limb regeneration in larval axolotl (Ambystoma punctatum) and newt (Triturus
viridescens) revealed trans- formation of muscle, cartilage, and other inner tissues of the
limb into mesenchymal-like cells. An alternative hypothesis at this time was that the
dedi- fferentiating epidermis is a major contributor to the arising blastema. This was
contradicted by with autoradiographic studies using tritiated thy- midine. By tracing
labeled cells, Hay and Fischman could provide the first direct evidence, that blastema
cells in regenerating limbs of the newt indeed originate from the dedifferentiating
internal tissues and not from the apical limb epidermis. To date, a contribution of reserve
stem cells to the blastema has never been ruled out, but there was no direct evidence
for such a mechanism. Very recently, Morrison et al. identified a Pax7-positive satellite
cell population, separated by a basement membrane adjacent to newt myofibers. These
Pax7-positive cells were quiescent in an uninjured limb but became mitotic after
amputation. Further- more, Pax7-positive cells were found in the early blastema,
suggesting that these satellite cells contribute to the regenerating limb. Nevertheless,
dedifferentiation of cells at the wound site appears to be the crucial cellular response to

injury that initiates blastema formation and here, we will focus only on the process of
dedifferentiation. A key question is how does injury initiate the regeneration response
and what are the molecular events occurring inside and outside the cells? Although
experiments following labeled cells or grafts in animals after injury have provided insight
about tissue behavior at the amputation plane, very little is yet known about the process
of regeneration at the molecular and cellular levels, and no factor responsible for the
initiation of dedifferentiation has been identified so far.
Regeneration of skeletal muscle: Studies of regenerating limbs or tails have
revealed enormous tissue reorganization near the wound site. These histological
changes affect all tissues in the regenerating stump; however, the most experimentally
accessible example is the multinucleated skeletal muscle cell. Therefore, muscle has
become the most intensively studied cell type for understanding regeneration at the
cellular and molecular levels. The first proposal that dedifferentiation of multi- nucleated
muscle fibers at the wound site might contribute to the developing blastema came from
Thornton and was confirmed by Elizabeth Hays elegant electron microscopy
observations already 40 years ago. In longitudinal sections of a regenerating axolotl
limb, Hay interpreted changes of the nuclear shape in myofibers, from a normally
elongated to an enlarged and rounded nucleus, as the beginning of DNA synthesis (Fig.
3). In addition, myofibrils disappeared and the syncytial fibers broke up into individual
cells during dedifferentiation (9). However, this conclusion was derived from static
pictures and one could argue as well that the reverse process was occurring, that of cells
fusing to form a muscle fiber.
+ Direct experimental evidence for the dedifferentiation theory of muscle cells was
provided by implantation of labeled myotubes into a regenerating newt limb. Cultured
newt limb myotubes were selectively microinjected with the lineage tracer rhodaminedextran and introduced into regenerating limbs. One week after implantation,
accumulation of rhodamine-dextran-labeled mononucleated cells was seen, strongly
suggesting that the labeled myotubes had fragmented. Furthermore, these
mononucleated cells were found to proliferate, as they were double- labeled with the
cytoplasmic lineage tracer and 3H-Thymidine that had been incorporated into the
nucleus. In contrast, dedifferentiation of newt myotubes, left in cell culture, was never
observed, suggesting a specific environment for reversal of the differentiated state in
the regenerating limb. In later work, these experiments were more elegantly repeated
using retrovirus-labeled implanted myotubes.
+ Although the experiments by Lo and Kumar et al. strongly supported the reversal of
differentiation during the course of regeneration, it remained unclear whether
endogenous muscle fibers close to a wound can undergo dedifferentiation and contribute
to the mass of proliferating blastema cells. This was demonstrated by labeling of single
muscle fibers in axolotl tail. Specifically, a single fiber was labeled by pressure injection
of rhodamine-dextran, followed by distal amputation of the tail close to the labeled fiber.
Dedifferentiation of mature muscle fibers occurred between 3 and 5 days after an
amputation by the synchronous fragmentation of the multinucle- ate muscle fiber into
mononucleated cells followed by rapid proliferation of these cells. Remarkably, in
addition to amputation or severe tissue damage, a direct clipping of the muscle fiber was
required to initiate this process. Based on these experiments, it was calculated that
about 17% of the blastema cell mass derives from muscle dedifferentiation alone. In
parallel, similar observations were made using isolated myofibers from axolotl limb and
dissociated in culture. When striated myofibers were labeled with a cell tracker dye and
then implanted back into the environment of a limb blastema, many examples of labeled
mononucleated cells were observed in the regenerating limb 24 days later. Aside from
fragmentation of multinucleated cells, the second characteristic of dedifferentiating
muscle cells is the reentry from a postmitotic state into a cell cycle accompanied by DNA
replication. Studies with tritiated thymidine both in vivo and with implanted myotubes
indicated that DNA synthesis occurs in the multinucleated myotube before
fragmentation. Using injection of tritiated thymidine into regenerating limbs, followed by

fixation of these tissues, Hay and Fishman found, in sections of the regenerating tissue,
clear evidence for thymidine incorporation in polynucleated muscle fibers as early as the
fourth day after amputation. However, between 10 and 20 days, incorporation was seen
more commonly in rounded nuclei of mononucleated muscle fragments derived from the
syncytial muscle fiber. Similar results were obtained by implanting retrovirus-labeled
cultured myotubes into the environment of a regenerating limb using incorporation of
bromodeoxyuridine (BrdU) as a marker for DNA synthesis. After injection of BrdU into
regenerates 9 days after implantation followed 24 h later by fixation and analysis, all the
nuclei in several retrovirally labeled myotubes were found to be BrdU-positive, indicating
S-phase reentry of these myotubes. Neither of these studies addressed the issue of
whether cell cycle reentry and budding are independent from each other or if both linked
in one pathway. The first support for autonomous mecha- nisms came from implantation
of cultured newt myotubes, where S-phase reentry was irreversibly blocked. When
myotubes, inhibited from progressing through the cell cycle by X-irradiation or
transfection with the cell cycle inhibitor p16INK4, were implanted into the regenerating
newt blastema, fragmentation still occurred.
New insights into blastema biology first results: Kragl M. et al., (Nature, 2009)
labe either embryonic precursors or older in Axolotl limb with stably integrated GFP
gene. Follow what happens to the cells in the blastema after amputation. Overall they
examined fate of 1000s GFP+ cells in many animals. Helped shift our view of the
Blastema containing homogenous pluripotent (or at least multipotent) stem cells but
rather a mixture of different mesenchymal progenitor cells. Experiment: GFP positive
group of cells from another animal and put into animal you amputate limb of. Do
amputation and blastema forms and can see what happened to GFP positive
transplanted cells. Finding found that epidermis didnt contribute to the Blastema.
Dermis can contribute to many cell types but not muscle muscle cells did not come
from cartilage transplant. Could work out which regions could give rise to which cell
types after amputation. Cartilage cells contributed to a lot of the Blastema but didnt
make muscle cells. Pax7 marker for muscles cells. Previous dogma: blastema =
homogenous group of multipotent stem cells. Kragl et al showed blastema = mixture of
different mesenchymal progenitor cells covered by an epidermis (muscle, Schwann cells,
epidermis, dermis/skeleton) i.e. the cells had not completely dedifferentiated. This could
be useful in regenerative medicine. Use to think multipotent stem cells and now study
realize they arent multipotent just progenitors of certain lineages. Cells arent
completely dedifferentiated. For human tissue may not have to go back to multipotent
less risk of tumerogenesis and may be easy as dont have to reprogram entirely.
To thine own self be true: Cellular memory during regeneration
+ In addition to identifying the cellular sources of regeneration in different systems,
researchers have been examining whether blastemal cells are pluripotent, multipotent,
or have more limited potential. Recent experiments using transgene-based lineage
tracing suggest that vertebrate blastema cells can remember the tissue from which
they are derived, and that their fates are largely restricted to forming similar tissues in
the regenerate. For example, transgenesis in axolotl and Xenopus has been combined
with embryonic grafting to specifically label various tissues, including muscle, Schwann
cells, spinal cord, and dermis; this tissue-specific labeling enables the contribution of
various tissues to be analyzed during regeneration. These experiments revealed that
regenerated muscle tissue is derived solely from muscles present in the limb before
amputation. However, in the axolotl, interconversion of dermis and cartilage was
observed. Both of these tissues are derivatives of lateral plate mesoderm, suggesting
that these tissues may dedifferentiate to produce progenitors restricted to lateral plate
fates or that the dermis may contain an uncommitted stem cell population. Lineage
restriction has also been observed during fin regeneration in zebrafish. Tu and Johnson
(2011) have used transposon-based clonal analysis to examine the potency of various
cell lineages during development, growth, and regenerate. This method stably labels one
to a few cells in the developing fin bud, enabling the progeny of single fin bud cells to be
monitored. Examination of hundreds of animals indicated that fin bud cells are greatly

restricted in developmental potential, only generating one to a few cell types in the adult
fine. As in axolotl and Xenopus, zebrafish caudal fin cells remain lineage committed
during regeneration, and do not contribute to lineages other than that from which they
are derived. Similar results were obtained in studies of bone regeneration in the
zebrafish fin. Osteoblasts near the amputation site down-regulate osteoblast
differentiation markers, lose their differentiated morphology, proliferate, and give rise to
new bone in the regenerate. These results reveal that regeneration in these contexts
does not require dedifferentiation to a pluripotent state, and that the cells that make up
the blastema are lineage restricted. These lineage-restricted progenitors even occupy
distinct spatial domains in the blastema, indicating that the initial sorting and positioning
of these cells may be crucial for producing a properly patterned appendage.
+ Although the lineage restrictions observed during limb regeneration are similar to
those observed during limb development, there are dramatic differences between
development and regeneration. In addition to nerve dependence (see next section) and
differences in scale of the structures being formed, limb regeneration differs significantly
from limb development in that some components of the proximo-distal axis of the limb
are retained after amputation. To prevent duplication or deletion of limb structures, cells
at the site of amputation must be able to determine their position along the proximodistal axis and use this information to regenerate only the structures that have been lost.
Interestingly, when distal (wrist) blastemas are transplanted onto proximal (shoulder)
blastemas, the distal blastema cells contribute only to distal structures, suggesting that
they retain memory of their proximo-distal origin. Additionally, when proximal and distal
blastemas are co-cultured, the proximal blastema encapsulates the distal blastema,
suggesting that the adhesive properties of cells differ along the proximo-distal axis.
Lineage tracing and examination of proximal (e.g., nuclear localized MEIS) and distal
limb markers (e.g., hoxA13 expression) indicate that although some cells (such as
cartilage) retain positional identity, other cells (such as Schwann cells) do not. Because
of the important role positional identity plays in limb regeneration, it is critical to
understand the sources that provide positional values along the limb axes.
+ Based on its differential expression along the proximo-distal axis, da Silva et al. (2002)
identified a cell surface protein, referred to as Prod1, that is expressed at high levels
proximally and low levels distally. Treatment with anti-Prod1 antibodies blocked the
encapsulation of distal blastema by proximal blastema tissues, suggesting that Prod1
plays a role in cellcell interactions that mediate identity along the proximo-distal axis.
Furthermore, when distal blastema cells overexpressing Prod1 were transplanted into
proximal blastemas, they contributed to proximal rather than distal structures. The
mechanisms by which differences in Prod1 expression along the proximo-distal axis
translate into faithful regeneration of the limb are not completely clear; however, Prod1
has been found to interact with a secreted protein called newt Anterior Gradient,
providing a link between proximo-distal patterning and the role of innervation in
regeneration.
Summary of this result in paper: Cartilage cells do have proximal-distal positional
identity (cf embryonic limb development debate) - Schwann cells do not and probably
spread along the limb - Tissue origin of the blastema cell must be considered when
studying positional identity. Can alter proximo-distal identity: - RAR receptor inducible by
T3 in distal blastema cells. If transplanted to proximal stump + T3, become proximal.
Distal blastema cells + T3 - Distal blastema cells electroporated with PROD1 or MEIS
become proximal.
Distal transformation i.e. blastema autonomously makes limb structures distal to its
site of origin. Connective tissue derived blastema cells drive this not muscle. The
connective tissue cells can drive muscle development (involves Wnt/b-catenin). During
salamander limb regeneration, only the structures distal to the amputation plane are
regenerated, a property known as the rule of distal transformation. Multiple cell types
are involved in limb regeneration; therefore, determining which cell types participate in
distal transformation is important for understanding how the proximo-distal outcome of
regeneration is achieved. We show that connective tissue-derived blastema cells obey

the rule of distal transformation. They also have nuclear MEIS, which can act as an upper
arm identity regulator, only upon upper arm amputation. By contrast, myogenic cells do
not obey the rule of distal transformation and display nuclear MEIS upon amputation at
any proximo-distal level. These results indicate that connective tissue cells, but not
myogenic cells, are involved in establishing the proximo-distal outcome of regeneration
and are likely to guide muscle patterning. Moreover, we show that, similarly to limb
development, muscle patterning in regeneration is influenced by -catenin signalling. It
was shown through intercalation assays that myogenic cells break the rule of distal
transformation, whereas connective tissue cells obey it. These results suggest that,
similarly to development, the connective tissue cells guide muscle patterning during
regeneration. Therefore, it will be important in the future to study connective tissue cells
in order to understand how the proximo-distal outcome of regeneration is established.
Also, similarly to development, activation of the -catenin pathway influences muscle
patterning, indicating that molecules involved in muscle patterning are likely to be
conserved between regeneration and development. In addition to TCF4/-catenin, TBX4
and TBX5 non-autonomously influence limb muscle patterning during mouse limb
development.
Profiling the genes necessary for blastema formation - Compared genes
expressed in deep wounds (50% forelimb) to those expressed during limb regeneration
until redifferentiation started - so compared lateral wounds to regenerative healing Wound-healing diverges from regeneration at 24 hours - saw at 120hrs start a limb
development program in amputations with 528 hrs like a limb bud i.e. wound-heal, form
blastema, redevelop. First 24 hours is quite key again here. Switch from healing the
wound to deciding you will regenerate a limb in first 24 hours. 120 hours later saw
classical developmental genes on. This program switched off a little bit until 528 hours
later. Gene functions identified in this screen - Cellular stress often starting at 24hrs and
sustained -Cellular stress genes classic responders to free radicals tress were switched
on just cause its been amputated only switched on at 24 hours kept on for a while
dont know why this is yet? Suggest that free radical signalling might be important in
blastema or hydrogen peroxide is important during regeneration? Chromatin modulators
e.g. some known in ES and HSC cells (convert the differentiated cells into regeneration
blastema cells?). Other kind of genes identified were chromatin modulators gone from
adult tissue differentiated to dedifferentiated so going to have chromatin remodelers.
May regulate dedifferentiation?
In situ hybridization database of blastema-associated genes: The regeneration
blastema emerges from the mature tissue in the first days post amputation, and consists
of two major compartments, the mesenchymal blastema that will give rise to skeleton,
connective tissue, muscle, blood vessels and peripheral nerve, and the epithelium that
plays an important signaling role during regeneration. To verify the regeneration-specific
expression of the transcripts identified by this analysis, and to determine the spatial
domains of their expression, we performed in situ hybridization of anti-sense RNA probes
on sections of regenerating and laterally wounded samples at different time points after
injury. Knapp et al (2013) performed in situ hybridization for both, transcripts with a clear
human ortholog, as well as un-annotated transcripts. We obtained interpretable
expression patterns from 46 out of 53 transcripts examined. 40 out of 46 transcripts
displayed re- generation-specific expression, validating our screening approach. They
could classify the gene expression patterns into two broad categories: those that are
expressed in the wound epithelium that directly covers the regeneration blastema, and
those genes expressed in the mesenchymal blastema. The expression patterns in the
epidermis revealed the complex structure of the epidermis, with molecularly distinct
layers. For example Gp2, Cnfn, axAg, Muc2, Umod, Psca, Dnase1l3 were all expressed
from early time points in the outer layer of the wound epidermis. In contrast, Dsg4,
Krt17, Fcgbp were expressed in all layers of the wound epidermis. Two genes were
expressed in the innermost layer of epidermis: Wnt5a and DK18. Finally, DK45 and DK64
expression appeared to be localized to a limited area, usually the posterior wound
epidermis. DK45, DK64 and DK35 represent late onset genes whose expression becomes

visible at 5 days after amputation (DK45 and 64) or at 7 days (DK35). A number of early
wound epidermis genes also showed up-regulation in the blastema mesenchyme
typically at later time points (12 transcripts such as Pcsa, Dnase1l2, Wnt5a). The
mesenchymally expressed group of genes included: Bambi, Gad1, Hmgb2, Hnrnpc,
Hnrnpl, Tecta, Wnt5b, DK23, DK40. Robust expression in the mesenchymal blastema was
most easily observable in later time points, such as 7 and 12 days. In most cases, the
expression was uniformly distributed in the blastema. However, intriguingly, DK40
marked a subset of cells in the mature tissue that were interspersed with muscle fibers.
Whether these cells represent satellite cells or connective tissue, and whether the
positive cells represent blastema cell precursors should be a topic of further
investigation.
Implications for regenerative medicine?
- Lack of complete dedifferentiation. Having positional memory: the cells know where
the injury has occurred and can give rise to the complete correct structure not just
regenerate a piece of tissue. For example, the frog Xenopus can normally regenerate its
limbs at early developmental stages but loses the ability during metamorphosis. This
behavior provides a potential gain-of-function model for measures that can enhance limb
regeneration. Lin et al. (2013) showed that frog limbs can be caused to form multidigit
regenerates after receiving transplants of larval limb progenitor cells. It is necessary to
activate Wnt/-catenin signaling in the cells and to add Sonic hedgehog, FGF10, and
thymosin 4. These factors promote survival and growth of the grafted cells and also
provide pattern information. The eventual regenerates are not composed solely of donor
tissue; the host cells also make a substantial contribution despite their lack of
regeneration competence. Cells from adult frog legs or from regenerating tadpole tails
do not promote limb regeneration, demonstrating the necessity for limb progenitor cells.
These findings have obvious implications for the development of a technology to
promote limb regeneration in mammals.
- It is not understood why some animals are able to regenerate and others apparently
are not; but even from our present limited perspective, there appear to be a number of
differences between mammals and urodeles that prevent or limit regeneration, rather
than any single defect or aberrant pathway. For example, mouse myotubes are
refractory to the action of the thrombin pathway that leads to S phase reentry in newt
myotubes, although mouse nuclei do respond in a mouse/newt heterokaryon. In another
case, the Hox gene C6 is turned off after limb development in the mouse, but its
expression persists into the adult newt forelimb and limb blastemal cells. One aspect of
adult wound healing in mammals that has been discussed in relation to the curtailment
of regeneration is the occurrence of fibrosis, and also of immune and inflammatory
responses. These are all potential targets for genetic and other manipulations. Existing
variation in mouse strains and transgenics encompasses marked ability in tissue
regeneration. For example, the MRL strain has the ability to heal punch wounds in the
pinna of the ear, whereas transgenic mice expressing elevated levels of the muscle
insulin-like growth factor1 isoform show enhanced recruitment of bone marrow cells
and augmented repair mechanisms after injury. Nevertheless, it would be surprising if
such approaches, even in combination, were to confer regeneration on a structure such
as the limb.
Limb regeneration: limb skeletal muscle cell cycle re-entry
Multinucleated fibres fragment into mononucleated progenitors. Experimental evidence
inc: Cultured newt limb myotubes microinjected with lineage tracer rhodamine-dextran
and injected into regenerating limbs: One week later, labelled mononucleated cells seen.
These were double labelled with 3H-Thymidine (also + retroviral labelling) cf. no
dedifferentiation of newt myotubes in vitro Lack niche signals? Post mitotic nucleated
muscle fibers to breaking them up to single mono-nucleate muscle cells. Fragment into
progenitor cells if you label the cells with rhodamine (lipophilic dye) and put into
regenerative limbs, or through retroviruses. Go through fused muscle fibers to

mononucleate cells, though you cant achieve this in vitro some signals in regenerative
limb that we are unaware of at the moment. How you get differentiated cells back into
the cell cycle this has implications for cell cycle control and cancer research.
What are the molecules involved? Cell line tool - A1 cells (myotube cell line) from
cultures of normal newt limb - A1 myotubes +FGF, IGF, PDGF, EGF stayed out of cell
cycle - In higher serum A1 cells re-entered the cell cycle (mammalian myotube cells
dont) - Dose response from 5-20% serum. A1 tubes like myotubes stay out of cell
cycle even if you put in mitogens can put FGF etc and still cant push cells into cell
cycle. For mysterious factor that allows re-entry into cell cycle for differentiated cells.
What do myotubes need to regenerate? Soluble factors in serum / More divide in
low-density culture: a loss of cellcell contact may be necessary for cell cycle re-entry /
In vivo myofibers require direct clipping (just cut off end of fibre) for dedifferentiation
regeneration. Muscle dedifferentiation occurs by the synchronous fragmentation of the
multinucleate muscle fiber into mononucleate cells followed by rapid cell proliferation
and the extension of cell processes. Echeverri et al. (2013) found that direct clipping of
the muscle fiber and severe tissue damage around the fiber are both required to initiate
dedifferentiation. Our observations also make it possible to estimate for the first time
how many of the blastema cells arise specifically from muscle dedifferentiation.
Calculations based on our data suggest that up to 29% of nondermal-derived cells in the
blastema come from dedifferentiation of mature muscle fibers. Overall, these results
show that endogenous multinucleate muscle fibers can dedifferentiate into
mononucleate cells and contribute significantly to the blastema.
The Role of Rb in Cell Cycle Re-entry: The retinoblastoma protein is a negative
regulator of entry into S-phase in all cells. In proliferating cells such as fibroblasts,
growth factor signalling induces the phosphorylation of Rb at the G1 to S transition. This
phosphorylation inactivates Rb and thereby allows progression into the cell cycle. The
kinases involved in phosphorylating Rb are the cyclin-dependent-kinases that control the
timing of cell cycle transitions. In particular, the cyclinD/cdk4 heterodimer is a serumresponsive kinase that phosphorylates Rb and mediates the growth factor stimulation of
cell proliferation. The cyclinE/cdk2 kinase also phosphorylates Rb and promotes the G1-S
transition. During muscle differentiation the regulation of Rb protein phosphorylation by
growth factors is shut down. In wild-type mouse myotubes that are stably withdrawn
from the cell cycle the Rb protein is no longer phosphorylated in response to serum.
Many growth factor receptors are down regulated upon myogenic differentiation, making
the muscle cell blind to those extracellular stimuli. However, during differentiation
muscle cells become refractory to growth factor stimulation well before down-regulation
of receptors indicating the action of an internal inhibitor of cell cycle re-entry. For
example, in the early stages of differentiation mouse myotubes can still induce the early
response genes when challenged with serum but other components of the cell cycle reentry machinery remain repressed and the cells remain out of the cell cycle. This internal
inhibition to cell cycle re-entry is attributable largely to cyclin-dependent-kinase
inhibitors (CKIs).
Does Rb Have a Role in Newt Myotube Cell Cycle Re-entry? First, it should be
noted that newt myotubes are normally as firmly differentiated as wild-type mouse
myotubes. For example in Rb-/- or p27 -/-;p57 -/- mice muscle tissue forms but
widespread BrdU incorporation in muscle fibers and other pathology is observed. In
contrast, newt muscle is stably withdrawn from the cell cycle in the uninjured animal
indicating that the differentiation machinery is completely intact. Exposure of newt
myotubes to injury (in vivo) or serum (in vitro) results in cell cycle re-entry in a highly
controlled manner. Tanaka et al showed that newt myotubes unlike mouse myotubes can
phosphorylate Rb in response to serum. The functional importance of Rb phosphorylation
in newt myotube cell cycle re-entry was demonstrated by expression of an
unphosphorylatable form of Rb in the newt myotubes that dominantly inhibited S- phase
re-entry. These results indicated that somehow in the newt myotubes cyclin- dependentkinases became active in response to serum. This latter conclusion was confirmed by

showing that the forced expression of the human CKI p16 INK4a in the newt myotubes
efficiently blocked the cell cycle response to serum. The specificity of p16 for CDK4/6
indicates that serum-induced activation of the CDK4/cyclinD kinase is important for cell
cycle re-entry in newt myotubes.
Regulators of the Rb Pathway Play an Important Role in Newt Myotube Cell
Cycle Re-entry
It is not yet known how the cyclinD/cdk4 pathway is activated in the newt myotubes
since none of the components of the pathway have been isolated or studied. Two general
classes of models can be proposed to explain the different response to serum of newt
and mouse myotubes. In the first model the newt myotubes may be lacking a factor
present in mouse myotubes that makes cell cycle arrest permanent. An example of the
first class would be if one of the several CKIs that is not necessary for cell cycle
withdrawal but is required for permanence of cell cycle arrest is not expressed in the
newt myotubes. p18INK4C which in mouse myotubes is upregulated in the late stages of
differentiation well after cell cycle withdrawal has already taken place could be such a
molecule. There is no data yet from genomic deletion in mouse to test this hypothesis. It
is interesting, however, that mice lacking p16INK4A, a close relative of p18 that is
expressed in complementary tissues, develop normally but are prone to tumors.
Furthermore fibroblasts derived from p16-/- mice do not undergo cellular senescence
another case of permanent of cell cycle arrest despite accumulation of another CKI,
p21, over time. Intriguingly, primary newt myoblasts and blastema cells are immortal
and do not display cellular senescence supporting the possibility of differences in
regulation of INK4 family members in the newt. The cloning of the newt CKIs has not yet
been achieved most likely because of high sequence divergence. Data from the sole
Xenopus p28 XIC gene, which displays hybrid features of both p27 and p21, indicates
that the CKIs have evolved rapidly. These observations suggest that the cyclinD/CDK4
pathway, in particular the CKIs, may be a fulcrum for evolutionary changes that
modulate the features of cell cycle arrest. In the second class of models the newt and
mouse cells may differentiate along identical pathways but newt cells upon serum
stimulation may be able to activate a signalling pathway that dominantly stimulates
cell cycle re-entry from the differentiated state. Mammalian myotubes may lack this
pathway. This dominant pathway might take several forms. For example, many of the
CKIs are known to be targets of controlled protein degradation and this pathway might
be activated in the newt cells but not in mouse cells. Another possibility is the induction
of CDK4 expression, which is not induced by serum in mouse myotubes at high enough
levels to overcome the inhibitory CKIs. The difference in ability between newt and mouse
myotubes to activate a dominant pathway that overcomes the cell cycle block in
differentiated cells could be due either to a limitation in an intracellular component, or
the mouse myotubes may merely be lacking the receptor for the serum factor that
stimulates the newt myotubes.
Is this so in newt myotubes? Experiments: - injected myotubes with plasmids encoding
CDK 4 or 6 inhibitor: no DNA synthesis - Injected Rb mutant D34 Rb (cant be
phosphorylated): inhibited DNA synthesis
Summary diagram of newt muscle regeneration (1) In vivo muscle fibers retract in
response to injury. Around the fiber, a blood clot is formed and the wound heals over.
The retracted muscle fiber then re-elongates. (2) In vitro an extracellular factor found in
both serum and newt limb blastema extract is capable of pulling the cells out of G0 and
allowing them to progress to S-phase, where they become arrested in a 4N state. The
G1S transition is mediated via the phosphorylation of the Rb protein in newt myotubes.
(3) Ectopic expression of Msx-1 or the presence of newt blastema extract causes
mammalian myotubes to down regulate markers of terminally differentiated muscle.
Newt myotubes upregulate Nrad in the nucleus of muscle fibers at the plane of
amputation. Steps 2 and 3 are likely to occur concomitantly. In vivo, axolotl tail muscle
fibers fragment and form mononucleate cells in response to clipping the end of a muscle
fiber in combination with a signal released in response to severe tissue damage. In vitro

a newt blastema extract can stimulate a response in both newt and mammalian
myotubes, causing 9% of the myotubes in culture to fragment and form mononucleate
cells. The factors responsible for inducing the cells to fragment, divide and eventually redifferentiate are still unknown
p53s role in regenerating limbs
Recently another factor P53 is important for controlling re-entry of differentiated cell.
The activity of p53 initially decreases and then returns to baseline. Its downregulation is required for formation of the blastema, and its up-regulation is
necessary for the redifferentiation phase{Since MAPK p38 activates p53,
MAPK p38 levels may need to have similar trends as p53 levels during
chondrogenic differentiation}. Importantly, we show that a decrease in the
level of p53 activity is critical for cell cycle reentry of postmitotic,
differentiated cells, whereas an increase is required for muscle
differentiation. In addition, we have uncovered a potential mechanism for the
regulation of p53 during limb regeneration, based on its competitive inhibition by
Np73. The regulation of p53 activity is a pivotal mechanism that controls the plasticity
of the differentiated state during regeneration.
- Studies on myogenesis provide direct evidence of the role of p53 in cell differentiation
in urodeles. We found that both protein and activity levels of p53 increase during
myogenesis, consistent with previous reports on the differentiation of mammalian
muscle in culture. Although stabilization of p53 activity during the induction of
myogenesis increases the overall efficiency of the process, inhibition of p53 prevents
myotube formation, demonstrating that p53 is essential during muscle differentiation in
salamander cells. These findings help explain the effects, identified in our study, of both
inhibition and stabilization of p53 during late stages of regeneration. Our observations
are supported by previous reports that demonstrate that inhibition or depletion of p53
abrogates myogenesis. It is notable that during myotube formation the inhibition of p53
does not prevent cell cycle withdrawal but does preclude subsequent stages of
differentiation. This finding indicates that p53 acts on the differentiation program
independent of its functions in cell cycle regulation.
Purification of a serum factor that triggers cell cycle reentry in newt A1
myotubes
A major goal in the laboratory has been to molecularly identify the thrombin-activated
serum factor through classical biochemical approaches using the in vitro newt myotube
cell cycle reentry assay. In an initial characterization of the activity in bovine serum,
neither delipidation nor dialysis against a membrane with a molecular weight cut off
between 6000 and 8000 Da abolished the activity. Gel filtration on Superose-12
suggested that the native molecular weight of the factor in serum is 150 000 300 000
Da. Furthermore, the activity appeared rather robust, as it was resistant to denaturation
by sodium dodecyl-sulfate (SDS). The activity found in serum is always referred to as cell
cycle reentry factor or S-phase reentry factor (SPRF).
The Clotting Cascade Activates a Serum Factor That Stimulates S-Phase Reentry
Serum is the soluble component of blood formed after clotting. The cell cycle stimulatory
activity in serum has several interesting properties that indicate that it is regulated by
clotting. Here, we will refer to the factor in serum that stimulates newt myotubes as Sphase re-entry factor (SPRF). Low serum conditions (1% serum) normally do not elicit
the S-phase response in the newt myotubes while high serum concentrations (10%) do.
However, addition of thrombin to low serum media induced S-phase re-entry in the newt
myotubes. One possibility was that thrombin was SPRFit directly stimulated a cell
surface receptor for thrombin on the newt myotubes to induce re-entry. The existence of
a G-protein coupled receptor for thrombin made this possibility particulary appealing.
However, it was shown that thrombin is not SPRF itself but rather, thrombin cleaves a
component in the low serum media that results in the generation of SPRF activity. This
was demonstrated by pre-treating low serum media with thrombin for 24 hours and

subsequently inhibiting all thrombin protease activity before addition of the preparation
to cells. Such preparations contained SPRF activity whereas preparations where thrombin
and inhibitor were added simultaneously did not contain SPRF activity. The involvement
of thrombin in SPRF biogenesis uncovers the role of blood clotting in triggering
dedifferentiation. Clotting is a highly localized process that is initiated by tissue injury.
Thrombins main role in blood is the terminal protease in the clotting cascade that results
in the cleavage of fibrinogen to fibrin. After cleavage fibrin self-associates to form an
insoluble fibrous network which forms the structural basis for the clot. Though it cleaves
fibrin at relatively specific protein sequences, thrombin also has multiple other
substrates in serum. Fibrin, or its degradation products do not have SPRF activity and so
far, none of the other known, direct substrates of thrombin such as ApoE or
thrombospondin has proven to be SPRF. It is possible that SPRF represents an unknown
substrate of thrombin. Alternatively thrombin does not cleave SPRF directly but rather an
upstream regulator of SPRF.
Purification of S-Phase Re-entry Factor: The identity of the SPRF activity that is
downstream of thrombin proteolysis and acts directly on newt myotubes is critical to
understanding cell cycle re-entry from the differentiated state. The direct SPRF activity
was found to be significantly enriched in commercially available crude thrombin
preparations. These preparations have proved critical to the further characterization of
the SPRF activity. The activity present in total fetal calf serum and in the crude thrombin
preparation has an apparent molecular weight of 200,000 Daltons. Fractionation of crude
thrombin on strong cation exchange resins separates thrombin, an indirect activator of
S-phase re-entry, from SPRF which directly acts on newt myotubes. Direct (SPRF) versus
indirect (e.g., thrombin) activities can be distinguished by the requirement of low serum
in the assay. Thrombin, which generates SPRF from serum always requires the presence
of low levels of serum in the media as a substrate. SPRF present in crude thrombin can
act in the complete absence of serum in the media. Clearly it will be important to
identify the polypeptide sequence of SPRF in order to understand how it stimulates cell
cycle re-entry in newt myotubes but not mouse myotubes. Fractionation of crude
thrombin on cation exchange followed by anion exchange, and finally affinity to heparin
sulfate has been used to purify SPRF 2000-fold over serum. This purification scheme
yields a protein preparation of 10 g/ml with 15-20 bands visible by silver staining. Given
a molecular weight of 200 kD, if SPRF were to represent one of the visible bands, it
would be active at a concentration of 1-5 nM. Interestingly, this represents a potency
similar to those of the anti-angiogenic factors endostatin and angiostatin. These two
molecules represent good paradigms for SPRF, as they are cleavage products of serum
and extracellular matrix proteins that have bioactive properties different from the parent
molecule.
Thrombin promotes cell cycle reentry during dedifferentiation
We have been pursuing the identity of the serum factor that initiates cell cycle reentry in
newt myotubes, as it could provide the first molecular insight for understanding
dedifferentiation. Serum represents the soluble fraction of blood after coagulation, which
involves a proteolytic cascade terminating with thrombin cleaving fibrinogen to form the
fibrin clot. Considering the primary role of thrombin in blood clotting, we examined the
relationship between thrombin proteolytic activity and the myotube cell cycle-inducing
activity. Indeed, thrombin was already known to function not only in fibrinogen cleavage,
but also in activating platelets and fibroblasts by cleaving a specific G-protein-coupled
receptor. To determine if thrombin could directly induce myotube cell cycle reentry, pure
thrombin was assayed on myotubes in differentiation media containing low levels of
serum (1.5%) and in serum-free media (1% bovine serum albumin). Strikingly, only
myotubes in 1.5% FBS reentered the cell cycle, indicating that thrombin-mediated
reentry requires subthreshold concentrations of serum. This result suggested two
possibilities: either thrombin acts directly through cleavage of a thrombin receptor but
additionally requires a second serum-derived growth factor, or thrombin acts indirectly
by cleaving molecules in serum that then, in turn, act on myotubes. To distinguish
between these possibilities, we deter- mined if the cells needed to be exposed to active

thrombin, or whether preincubation of the 1.5% serum with thrombin was sufficient to
induce a cellular response. A medium containing 1.5% serum was incubated with pure
thrombin. After 24 h of incubation with thrombin, the sample was then treated with DPhe-Pro-Arg-chloromethylketone (PPACK) to completely inhibit thrombin protease activity
before addition to cells. As a control, thrombin was inhibited with PPACK before
incubation with 1.5% serum. In these experiments, the incubation of serum with
thrombin prior to addition to cells was sufficient to induce cell cycle reentry. This
indicates that throm- bin proteolysis generates a downstream factor that induces cell
cycle reentry in the newt myotubes. A similar result was obtained when serum was
incubated with the serum protease plasmin using, in this case, a2-antiplasmin for
inhibition. Other proteases that were tested in similar assays were found to be negative,
including trypsin, Factor Xa, Protein Ca, Factor IX, and Factor XII.
This characterization of thrombin activity on the newt myotubes allowed us to integrate
the in vivo observations that thrombin is present and important for initiating
regeneration, with a mechanistic insight that it induces cell cycle reentry in
differentiated cells. Indeed, the exposure of newt iris pigmented epithelial cells to the
thrombin-activated serum factor in culture also induces cell cycle reentry into these
cells, again linking the in vivo observations with
in vivo evidence that selective activation of thrombin is a critical event for
limb and lens regeneration in vertebrates: In vivo evidence that thrombin
activation is involved in initiating dedifferentiation came from ocalizing active thrombin
in histological sections of regenerating tissue. This was performed by overlaying sections
with membranes infused with a fluorogenic thrombin substrate. Thrombin proteolytic
activity was observed exclusively in the blastema at the tip of the 8-day regenerating
limb. By this time point, the cells of the tissue close to the amputation site had
dedifferentiated and contributed to the forming blastema. Moreover, this signal was
completely inhibited by inclusion of PPACK, an irreversible inhibitor of thrombin.
Studies of lens regeneration have provided further compelling in vivo evidence for the
role of thrombin in initiating regenerative events. In adult newts, lens regeneration only
takes place at the pupillary margin of the dorsal iris, where pigmented epithelial cells
reenter the cell cycle and transdifferentiate into the lens. However, in cell culture,
pigmented epithelial cells both from dorsal and ventral iris possess the capacity to
transdifferentiate, suggesting a missing stimulus in the ventral part of the iris after lens
removal. Recently, Imokawa and Brockes demonstrated the transient and selective
activation of thrombin at the dorsal margin in the newt eye after lentectomy is
absolutely required for the activation of pigmented epithelial cells to undergo these
regen- erative events. Membrane overlay assays showed that after lentectomy, active
thrombin is selectively localized to the dorsal pupillary margin. Furthermore, injection of
the irreversible thrombin inhibitor, PPACK, into the eye strongly delayed lens
regeneration. These results indicate that the selective activation of thrombin is a
fundamental signal linking tissue injury to the initiation of regeneration in vertebrates.
The role of thrombin in this aspect of dedifferentiation of cells provides an interesting
link between injury, the initiation of blood coagulation, and the local activation of factors
for regeneration of missing tissue in urodeles. It is also in agreement with experimental
evidence, that injury alone elicits dedifferentiation and cell cycle reentry of cells.
FIG. 4. Schematic diagrams of the S-phase reentry model and summary of cellular
dedifferentiation effects observed in vitro on newt and mouse myotubes. (A) Schematic
diagram of the activation of S-phase reentry by newt A1 myotubes in the context of the
wound healing responses that leads to regeneration: Amputation of the limb triggers
multiple responses such as inflammation, remodeling of the extracellular matrix, and cell
migration. A major aspect of wound healing is activation of the conversion of
prothrombin to thrombin. In mammals, this induces the conversion of fibrinogen to fibrin
polymers and formation of a clot. In newt, thrombin activation, in addition, leads to cell
cycle reentry from the differentiated state. This involves the conversion of a latent
activity (SPRF to SPRFa) within blood (or serum), which can selectively stimulate newt A1
myotubes, but not their mono- nucleate precursors to undergo S-phase. Although the

SPRFa activity was found in all animal sera tested so far and hence is likely to be a
general product of thrombin activation, mouse C2C12 myotubes were refractory to this
activity.
The Drug Myoseverin Induces Mouse Myotubes to Form Mononucleate Cells:
These results suggest that intact nuclei bud off from multinucleate syncytia. As with cell
cycle re-entry, work on mouse myotubes may provide a clue to the mechanisms of
myotube fission. Myoseverin, a derivative of a purine-based synthetic chemical library,
caused multinucleated mouse C2C12 myotubes to resolve into mononucleate, myosinpositive cells. The similarity to the newt myotube phenotype was striking. Cells from
myoseverin-treated cultures incorporated BrdU and formed a higher number of colony
forming units compared to non-treated cultures, providing evidence that the C2C12 cells
may become proliferative after fission although some ambiguity remains concerning this
last issue since the myotube cultures probably contained significant numbers of unfused
mononucleate cells. Myoseverin was shown to bind microtubules and it was proposed
that disruption of the microtubule network caused the severing phenotype. The apparent
ability of another microtubule drug, taxol, to induce myotube fission supports this view.
Screening of DNA chips with RNA from myoseverin-treated cells also revealed the
upregulation of many genes involved in wound-healing and cell cycle regulation. It is not
yet clear to what extent this transcriptional response is also required for the severing
phenotype. It is yet unknown if myoseverin is activating a pathway for mononucleate cell
formation that is normally used in the newt cells. The hypothesis that myoseverin works
via microtubule disruption would predict that the microtubule cytoskeleton in
dedifferentiating newt myotubes would be altered during the process and that blocking
changes in microtubules would inhibit newt myotube fission. Myoseverin acts within 24
hours in C2C12 myotubes and fission of endogenous salamander myotubes takes 3-5
days. The timing is compatible with myoseverin activating a similar pathway to the
endogenous one in newt cells. It will of course be fascinating to determine if myoseverintreatment causes the formation of mononucleate C2C12 cells that have an equivalent
proliferative and lineage potential as dedifferentiated newt cells. The results from newts
suggesting that the cell cycle re-entry and fission are likely independent events casts
doubt on whether the myoseverin-treated C2C12 cells will have true proliferative
potential. However, the possibility that the process of myotube fission promotes the
ability to re-enter the cell cycle has not been ruled out.
Msx1 Expression in Mouse Myotubes Induces Mononucleate Cell Formation and
Proliferation: Recently important molecular insight into the dedifferentiation process
was gained through ectopic expression of the msx1 homeobox gene in C2C12 myotubes.
Msx1 is a homeobox containing gene that when ectopically expressed can prevent
myoblast differentiation. During embryogenesis msx1 is expressed at the growing end of
the limb where the proliferating, undifferentiated cells reside while it is upregulated
during regeneration in newts and fish. The expression of msx1 also correlates with the
ability of post-natal mice to regenerate fingertips. Odelberg et al expressed an inducible
form of msx1 in C2C12 myotubes that had been placed in growth factor rich media and
followed the behavior of these myotubes over time.104 9% of the myotubes fragmented
into smaller myotubes or budded mononucleate cells. In 5% of cases myotubes
generated proliferating mononucleate cells. During the dedifferentiation process
myotubes lost expression first of myogenin and MRF4, then p21CIP1 and myoD. This
loss-of-expression profile is the reverse sequence to the differentiation process. Clones
derived from mononucleate cells that had budded from msx1- expressing myotubes
showed a multipotency that is not observed in normal C2C12 myoblasts. When put into
the appropriate inducing media, msx1-derived mononucleate cells were capable of
forming chondrogenic, osteogenic, or adipogenic cells. This plasticity was also observed
in C2C12 myoblasts that had transiently expressed the msx1 gene. Therefore the
multipotency and the budding process are in some sense separable. These results
indicate that under the correct conditions mammalian myotubes are capable of
dedifferentiating like the newt myotubes. The msx1-induced dedifferentiation required
serum-containing growth media. It will be interesting to determine if the factor in serum

required for msx1-dependent dedifferentiation is SPRF. Although in the C2C12 cells,

msx1 expression was forced, in the newt, an extracellular signal produced by amputation
presumably elicits msx1 expression and dedifferentiation during regeneration. The
identity of the signal that induces msx1 expression in newts and fish and whether this
signal can induce msx1 expression in mammalian myotubes will be an important future
aspect of the problem. In fish, FGF-signalling expression modulates msx expression
during fin regeneration. It is unlikely that FGFs are sufficient to initiate msx1 expression
in myotubes since FGFs are unable to induce cell cycle re-entry in newt myotubes. It will
furthermore be interesting to know if msx1 can induce dedifferentiation of other
mammalian differentiated cell types or if it is limited to myogenic cells.
More on Urodele limb regeneration: does it recapitulate embryonic
development?
+ Then, the underlying stump cells receive signals, initiated by the activation of
thrombin, which induce changes in the severed tissues of muscle, cartilage, bone,
tendons, various types of fibroblasts, and Schwann cells by the process of
dedifferentiation. During this phase, differentiated cells with a highly specialized
morphological and functional state, e.g., muscle, lose their phenotypic identity and
apparently return to a progenitor state. In cartilage tissue, nuclei change shape and size
as the cartilage matrix disappears, freeing the cells. The syncytial muscle fibers of the
amputated stump change into mononucleated mesenchymal cells by dedifferentiation in
Ambystoma larvae as described with light microscopic studies by Thornton. Later, Hay
reaffirmed, with meticulous electron microscopic sequences, that muscle tissue
dedifferentiated in regenerating Ambystoma limbs. The myofibrils first break into pieces
(a fragmentation process later described as cellularization) when the individual
myofilaments, Z bands, and basement membranes are lost. The smooth endoplasmic
reticulum within the cells disappears, while the nuclei synthesize DNA. This process
transforms the highly differentiated tissues into cell types with the attributes of the
mesenchymal cells from which cartilage and muscle originated in the embryo. At the
same time, resident muscle stem cells are prompted to form progenitor cells within their
own lineage. As soon as dedifferentiation takes place, cell proliferation follows and
mitotic cells can be found in all of the tissues near the amputation surface. The
formation of a basal lamina at the wound surface in the late bud stage is essential for
continued development. Once formed, it appears to stabilize the phenotype of adjacent
cells. The epithelium becomes epidermis and the adjacent mesenchyme becomes
dermis. During this period, the wound epidermal cover thickens, changing roughly from
three cell layers to 12 or more cell layers to form an apical epidermal cap (AEC).
+ The AEC is believed to emit molecular signals that stimulate and maintain early stages
of regeneration. Just beneath the AEC, accumulated mesenchymal cells re-enter the cell
cycle, proliferate, and aggregate to form a bud of mitotically active cells called the
blastema. Using H3 thymidine labeling to trace cell proliferation and migration, Hay and
Fischman reported that the blastema cells are derived from dedifferentiating stump
tissue. The undifferentiated mesenchymal cells in the proliferating blastema cells
synthesize RNA and DNA along with heavy protein synthesis, indicating re-entry into the
cell cycle. DNA synthesis begins 45 days after amputation in tissues interpreted as
dedifferentiated muscle, periosteum, cartilage, nerve sheaths, and connective tissues.
The presence of nerves is necessary for the formation of a blastema throughout the
early stages of regeneration. This is one of two important differences between
embryonic limb formation that does not require nerves for early development; the
second is that the embryonic limb bud is vascularized early. Early on, it was found that
denervation of limbs would inhibit limb regeneration if performed any time up until 9
days after amputation, when nerve dependency ceases. The nerves were postulated to
release a trophic factor and the presence of nerves was proposed to be a prerequisite for
the process of dedifferentiation to take place. Extensive investigations by Singer
established that the number or quantity, not the type of nerves, was a controlling factor.
Later work by Mescher and Tassava indicated that denervation does not prevent wound

healing, histolysis, and dedifferentiation , but failure in the proliferation of mesenchyme

cells in the blastema at the amputation surface is the controlling event.
+ Once a blastema forms, the proliferating mesenchymal cells are directly dependent
again on stimuli from the nerves. The neurons release a glial growth factor along with a
fibroblast growth factor 2 (FGF2) that initiates cell proliferation. Bead implants with FGF2
into early blastemas of denervated axolotl limbs stimulates regeneration into the digit
stages. A third factor is a neurotransmitter, substance P, found in newt ganglia, the AEC,
and mesenchyme cells that is mitogenic for blastema cells in vitro.
+ Another factor that affects blastema cell proliferation is transferrin, a protein that
transports iron and is a cofactor for many enzymes. Transferrin can replace the action of
nerve extracts in the stimulation of blastema cell proliferation in vitro. It is released from
nerves in regenerating axolotl limbs and maintains blastema cells in vivo. Mescher
proposes that the release of growth factors, including transferrin, from regenerating
axons is important for cell proliferation during the avascular phase of blastema growth
before such plasma-borne factors are supplied during regeneration. He suggests that
transferrin affects the rate of cellular activity. When nerve extracts are
immunoabsorpted by antiserum to transferrin, it removes the stimulatory effect on
blastema cells. The inhibition can be reversed by the addition of pure transferrin.
+ Other influences controlling blastema cell growth and later dedifferentiation of the
organized limb tissues are hormones, along with several growth factors from the AEC
(FGF1, 2, 8, 10). The AEC begins to secrete extracellular matrix proteins, such as laminin
and collagen. Hyaluronate synthesis is also stimulated by growing axons. An extensive
series of markers for many of these proteins has been identified, along with the
identification of homeobox transcription factors, Dlx-3 and Msx-2, and mitogenic signals
of the fibroblast growth family. The next phase involves morphogenesis of the limb bud,
accompanied by differentiation of the progenitor cells. Redifferentiation of the blastema
follows a proximal-distal axis, a sequence that is also found in the limb bud of the
developing embryo. The identity of this axis is controlled by a local gene-controlled
protein, Prod 1,that is linked to the blastema cell surface. Prod 1 exists as a gradient
along the blastema and establishes the positional (proximal-distal) identity of the
blastema. The discovery of a ligand for Prod 1, a newt protein (nAG), showed that it
plays a key role as a growth factor for blastemal cells and the proximal-distal identity of
the blastema. After amputation, nAG appears in the Schwann cells of the regenerating
nerves and later in gland cells of the epidermis. The expression of nAG is blocked in the
denervated blastema. If the DNA for nAG is introduced by electroporation into the
blastema cells of a denervated amputated limb, the expression of nAG returns and the
blastema is able to resume the formation of a regenerated limb.
Prod1 gives limb (and blastema) proximodistal identity
How do blastemal cells know their position on a limb? Prod 1, a gene that is
regulated by PD location and RA. Prod 1 encodes a small protein that is linked to the cell
surface by a glycosylphosphatidylinositol (GPI) glycolipid anchor. It is apparently the
newt ortholog of mammalian CD59, as evidenced by the prediction of secondary
structure. The difference in expression at mRNA and protein levels is shown for midhumerus and mid-radius blastemas, as well as for the gradient of expression in the
normal limb. The CD59 protein in mammals is associated with the inhibition of the
terminal phase of complement activation, and it is also able to mediate activation of
intracellular nonreceptor tyrosine kinases. When proximal and distal blastemas are
confronted in culture, the proximal member reproducibly engulfs the distal, and
engulfment is selectively blocked by two antibodies against the protein Prod 1.
Compelling evidence for its relevance to PD identity has come from electroporating a
Prod 1 expression vector into distal cells of the limb blastema of the larval axolotl.
Whereas labeled cells in control blastemas maintain their distal location and give rise to
tissues in the regenerated hand, labeled cells in the contralateral blastema receiving the
Prod 1 vector relocate and contribute to the upper arm. Taken together, the evidence
suggests that Prod 1 is a cue for local cell identity that is expressed in the normal limb
and persists in blastemal cells. Questions remain as to which extracellular and surface

ligands may interact with it and how it mediates cell interactions based on differences in
expression between neighbors. We have suggested that neighbors may titrate the
relative expression of Prod 1 by homophilic adhesion between cells, leaving spare Prod 1
molecules on the proximal cell to interact with ligand. This mechanism may dictate the
extent of growth, movement, and adhesion during patterning and hence define the
morphogenetic autonomy of the blastema.
+ Nerves are required in regeneration and are thought to play a major role,
possibly even initiating the entire process. In a study published in 2007, Kumar et
al. searched for molecules that interact with Prod1 and identified a candidate ligand,
dubbed the newt Anterior Gradient or nAG protein, that is secreted by nerves. To do
this, they used a yeast two-hybrid screening assay, a technique commonly used to check
for protein-protein interactions. In this technique, two factors required for transcription
initiation are separately attached to bait and prey proteins of interest. Successful
transcription indicates that the two proteins have interacted. When denervated
(separated from the nerve), the regenerative epithelium (RE) does not contain nAG,
showing that it is supplied to the RE by nerve axons. Specifically, nAG is secreted by
Schwann cells, the principle type of glial cell (support neuron). After identifying nAG as a
Prod1 ligand, Kumar et al. used nAG-specific antibodies to investigate when and where
nAG is expressed in the process of regeneration. They found that nAG is expressed after
amputation in the nerve and, later, in the RE of the regenerating structure. Perhaps the
most interesting question raised by this research is not why adult urodeles
can regenerate, but why other adult vertebrates cannot. One direction for
research could involve the introduction of molecular signals like Prod1 and nAG to nonurodele embryonic tissues. With Prod1 proteins expressed since the beginning of
development, perhaps it would be possible for fully differentiated adult non-urodele cells
to retain the memory of their positional identity. If Prod1 and nAG could not be expressed
for some reason in other vertebrates, a search for orthologs of nAG could help
researchers design a pathway similar to the Prod1-nAG system.
Two more examples of Urodele regeneration
1) Regeneration of newt lens: Regeneration of the lens in adult newts was observed
first by Collucci in 1891 and independently by Wolff in 1895, after whom the process is
often called Wolffian regeneration. A few species of fish and premetamorphic frogs can
also regenerate the lens. In the fish and newts the regenerating lens is derived from the
pigment epithelial cells (PECs) of the dorsal iris, whereas in the frog it is derived from the
cornea.
The process of lens regeneration - PECs re-enter the cell cycle: It is conceivable that this
is a necessary step. PECs must proliferate to create the lens vesicle and the subsequent
lens. However, studies in the past have clearly indicated that the ventral iris PECs reenter the cell cycle as well, eventhough, seemingly at a lower rate. These previous
studies have shown that proliferation ensues at about 4 days post-lentectomy and that
at that time both dorsal and ventral PECs show comparable levels of cell proliferation.
Following that period, proliferation rates are higher in dorsal PECs, but obviously this
must correlate with the process of lens regeneration, which has been initiated from the
dorsal iris. In relation to this, expression of a hyperphosphorylated form of
Retinoblastoma (Rb) has been found in the PECs of both dorsal and ventral iris. Rb is a
paramount player in cell cycle regulation and when hyperphosphorylated is inactive and
leads to dissociation from E2F, which then allows re-entering of the cell cycle. However,
treatment with a CDK inhibitor (which is involved in Rb regulation and inhibits
proliferation) does not completely inhibit transdifferentiation. Also, when ventral cells are
cultured for about two weeks and then implanted as aggregates in the eye cavity they
can never transdifferentiate to lens, eventhough they undergo considerable proliferation.
In different studies it has been demonstrated that thrombin activation is predominant in
the dorsal iris and that its inhibition results in loss of proliferating cells. Nevertheless,
PECs from both dorsal and ventral iris are responsive to thrombin when stimulated in
vitro. This might imply that while proliferation is necessary to prepare cells for the

process of regeneration it might not be sufficient for the subsequent transdifferentiation,

and, thus, events other than the ones involved in cell cycle re-entering are decisive for
transdifferentiation and lens regeneration.
Transdifferentiation: The histological events leading to dedifferentiation have been well
studied at the microscopic level. Soon after lentectomy the pigment epithelial cells
dedifferentiate by shedding their pigment. Macrophages recruited to the site mediate
such a process. Four days post-lentectomy (a time frame that coincides with the onset of
cell proliferation) the iris is decondensing and the PECs begin to elongate and at about 810 days these cells become columnar and depigmented. The first depigmented cells are
obvious at the tip of the dorsal iris. At about 10 days a lens vesicle has been formed, but
no crystallin expression is yet apparent. Many of the genes that are known to be
expressed and involved in lens development, such as pax-6, FGFs, FGF receptors and
prox-1 are expressed in the early lens vesicle. We can confidently say that the genetic
program, which is involved in lens development, is recruited during lens regeneration as
well. This makes sense since the same tissue is produced in both processes. However,
caution should be exercised in equating these two events especially as far as the
induction signals are concerned because of the lack of adequate information at the
moment. After the vesicle is formed the events of lens differentiation are initiated. At the
posterior edge of the vesicle elongation of the cells ensues with concomitant induction of
crystallin synthesis. The elongated cells differentiate to lens fibers and the anterior part
of the vesicle becomes the lens epithelium, which continues to contribute by
proliferation and differentiation to the growing of the lens. Once lens differentiation has
started the process is remarkably similar to lens development (as far as the sequential
appearance of the different crystallins is concerned). Indeed, studies using antibodies to
crystallins or cDNA probes have conclusively shown that there is a parallel of their
synthesis during developing and regenerating lens. The examined crystallins were A,
B1 and . These crystallins appeared all at the same time at the ventral portion of the
lens vesicle (Sato stage IV; nearly 10 days post- lentectomy), with -crystallin (in
contrast with the other two) being specific for the lens fibers and not the lens epithelial
cells. As in normal lens development, when lens epithelial cells differentiate to lens
fibers, they stop dividing. At the same time events of apoptosis are also observed, but at
a minimal rate.
- Regeneration is localised to the dorsal iris and recent work has provided evidence for
intrinsic differences between dorsal and ventral cells in relation to responsiveness and
signalling, as well as for early and preferential activation on the dorsal pupillary margin
- In earlier work the protease thrombin, a critical mediator of hemostasis and other
aspects of the injury response, was found to be selectively activated at the dorsal
margin after lentectomy. If thrombin activity was blocked, this inhibited cell cycle reentry and lens regeneration. In most contexts of tissue injury, prothrombin is converted
to thrombin in association with extracellular complexes of clotting factors nucleated by
the transmembrane protein tissue factor (TF).
- An important contribution of recent work has been the demonstration that dorsal and
ventral iris PECs differ in respect of both BMP and Wnt signalling. The ventral iris is
subject to signalling from the BMP pathway that inhibits transdifferentiation to lens,
while the dorsal iris is apparently subject to Wnt signalling that stimulates
transdifferentiation and expression of lens genes.
2) Newt heart regeneration - Can functionally replace 3050% heart ventricle after
removal - Tritiated thymidine + E.M. shows differentiated cardiomyocytes contributing to
regenerating ventricle - Doesn't involve full dedifferentiation - Dissociated
cardiomyocytes from adult newt ventricle re-enter S phase in culture, about 33% divide
and may divide again (Rb phosphorylation involved). Mammalian hearts dont do this.
The key to this ability to regenerate are the heart muscle cells themselves. When a
newts heart sustains damage, its cells can lose their characteristic properties; they can
dedifferentiate. Braun et al (2006) were able to show that proteins typical of heart
muscle cells - the heavy myosin chain and various troponins - were dramatically downregulated in this process. At the same time, the cells embark on massive cell division to

build up new heart muscle. It takes around two weeks for the heart function to be
restored in the newt. The data shows that at this point the expression of the musclespecific proteins is again normal, i.e. the cells have differentiated again, and have
regained their characteristic properties. The researchers isolated the heart muscle cells
and cultured them. In most of the cells, Braun and his colleagues were able to
demonstrate the existence of a protein called Phospho-H3. This protein is a marker for
the G2 phase of the cell cycle and indicates that the newt heart regenerates without the
involvement of stem cells. It also seems that the heart regeneration does not create
typical wound healing tissue, called a blastema. The heart only has a relatively small
number of different cell types. This could be a reason why the regeneration of heart
tissue does not require a blastema. The researchers in Bad Nauheim found no indication
that stem cells were involved in repairing newt hearts. The process of regenerating lost
extremities is different. Unlike in the process with the heart, newts develop a blastema in
this case. Blastema cells have certain characteristics in common with stem cells, such as
the development into different cell types. The cell biologists in Bad Nauheim injected
isolated heart muscle cells into a newts leg that was regrowing after amputation. In this
environment, the cells began to de-differentiate, as they did in the heart. However, this
did not happen when they were injected into an undamaged extremity. Again, the
researchers registered the very rapid loss of heart muscle-specific proteins. It is possible
that the signal for the de-differentiation comes from the area where the wound is healing
and the cells communicate with each other. These signals could be transmitted via
certain enzymes, for example. An enzyme of this nature - focal adhesion kinase -, which
plays a part in the transmission of signals in the cells, is phosphorylated in the
transplanted cells and is thus active. The Max Planck researchers in Bad Nauheim hope
that better understanding of the molecular issues involved in regeneration in the newt
will open up new possibilities for the repairing human patients damaged hearts.
Salamander brains - Abundant production of new neurons in the adult mammalian
brain is limited to the dentate gyrus of the hippocampus and the subventricular zone of
the lateral ventricles in the forebrain. Neurogenesis may be evoked in quiescent regions,
but the number of persisting new neurons that are generated remains low and
consequently the functional recovery of the animals limited. By contrast, the adult brain
in non-mammalian vertebrates of certain fish and salamander species repairs damage
via processes fuelled by neurogenesis (Zupanc, 2009). Previous studies have revealed
several proliferation hotspots in the adult zebrafish brain from which neurons are
continuously derived. From these and other observations it was hypothesised that the
broad distribution of homeostatic neurogenesis in the brain is an underlying component
of the extensive regenerative ability in these animals. This link, however, needs further
testing, as it would have important implications for the possibility of engaging nongerminal zones for functional neuronal replacement in species where it naturally does
not occur.
- Cells that give rise to new neurons in mammals are of glial character in terms of
morphology and gene expression. Data indicated that glial cells are neural stem cells
also in non-mammalian vertebrates but the glial origin of brain neurons has however not
been directly demonstrated. A recent report suggested that stem cells in the adult nonmammalian brain have neuroepithelial rather than glial features. Here, we have
addressed whether the presence of constitutively active neurogenic niches is a
prerequisite for extensive neuronal regeneration, and revisited the identity of cells that
produce new neurons. We studied an aquatic salamander, the red spotted newt, which
has the widest regenerative repertoire among vertebrates. Adult newts regenerate
among other structures limbs, cardiac muscle, ocular tissues and tails. Central nervous
system (CNS) regeneration in newts has mostly been studied after spinal cord
transection, tail amputation, or by removing a piece of brain tissue.
- Recently, we developed a chemical ablation model in newts by intraventricular injection
of 6-hydroxydopamine (6-OHDA. 6-OHDA acts as a selective neurotoxin and is used to
model aspects of Parkinson's disease in many species by the elimination of midbrain

dopamine (DA) neurons. Similar to other species, midbrain DA neurons in newts express
the evolutionarily conserved markers, tyrosine hydroxylase (TH) and Nurr1. Newts
respond uniquely to the loss of midbrain DA neurons by full regeneration within 4 weeks.
Regeneration of DA neurons depends on cellular proliferation and is characterised by the
gradual birth of new neurons leading to complete histological and locomotor
performance recovery.
- In contrast to mammals, some non-mammalian vertebrate species regenerate neurons
more efficiently in the entire brain. Careful mapping of the Zebrafish showed that this
capacity correlated with the sustained and widespread production of neurons in the adult
brain (Grandel et al.,2006), which suggested a correlation between homeostatic and
injury-responsive cell replacement. (Zupanc, 2009) showed that the adult brain is
capable of maintaining as well as awakening the efficient neurogenic and regenerative
potential of quiescent niches. Neurogenesis is normally limited to the forebrain in newts,
but neuronal loss leads to activation of neuronal stem cells in otherwise quiescent areas.
Thus, the newt is akin to mammals in terms of the extent of normal homeostatic cell turn
over, but distinct in terms of its injury response, which is manifested in complete
regeneration.
Mammalian brains
Much less division and regeneration in postnatal mammalian brain. We have much less
regeneration in our brains increasingly a problem when more people having dementia
and Alzheimers disease.
We just dont regenerate our neurons seems we resist this.
Why cant most of the mammalian brain regenerate? The discovery that adult neural
stem cells in the mammalian brain continue to generate neurons throughout life has
shed new light on the possibility to repair degenerated neurons by endogenous sources.
The similarity of adult neural stem cells to astroglial cells prompts the question whether
glial cells reacting to injury also may have a broader potential. Astrocytes mediate the
wound healing reaction of the central nervous system (CNS) upon injury and reexpress
molecules [vimentin, nestin, tenascin-C, and glia-fibrillary acidic protein (GFAP) that are
absent in mature astrocytes but present during development in radial glial cells and
adult neural stem cells. Whereas during development adult neural stem cells and radial
glia generate neurons, reactive astrocytes do not, despite their partial dedifferentiation
response. However, immature astrocytes can generate neurons in vitro upon stimulation
with intrinsic or extrinsic factors. This neurogenic potential is not restricted to astrocytes,
because NG2-positive glial precursors also can generate neurons upon exposure to
certain culture conditions or transplantation into neurogenic zones in vivo but fail to
regenerate neurons upon injury in vivo. Thus, endogenous precursor and glial cell
populations have the potential to generate neurons but fail to do so upon injury in vivo.
Here, we set out to elucidate the molecular cues involved in the glial fate restriction after
injury. We examined transcription factors, such as Pax6, Ngn2, Mash1, Gsh2, Olig1, and
Olig2, that regulate neuron-glia fate decisions during development and are present in
precursors in the subependymal zone (SEZ) of the adult mouse forebrain. Our results
identify Olig2 as a key factor in reaction of glial cells to brain injury and show that, by
interfering with Olig2 function, some degree of endogenous neurogenesis can be
evoked. Thus, these results show proof of principle evidence that neurogenesis can occur
in the mammalian neocortex, even in severe injury conditions, such as acute stab-wound
lesion.
Mammalian regeneration we could learn from?
Deer antler regeneration Full regeneration of deer antlers, a bona fide epimorphic
process in mammals, is in defiance of the general rule of nature. Revealing the
mechanism underlying this unique exception would place us in a better position to
promote organ regeneration in humans. Antler regeneration takes place in yearly cycles
from its pedicle, a permanent protuberance on the frontal bone. Both growing antlers
and pedicles consist of internal (cartilage and bone) and external components (skin,
blood vessels, and nerves). Recent studies have demonstrated that the regeneration of

both internal and external components relies on the presence of pedicle periosteum (PP).
PP cells express key embryonic stem cell markers (Oct4, Nanog, and SOX2) and are
multipotent, so are termed antler stem cells. Now it is clear that proliferation and
differentiation of PP cells directly forms internal antler components; however, how PP
initiates and maintains the regeneration of external antler components is thus far not
known. Based on the direct as well as indirect evidence that is presented in this review, I
put forward the following hypothesis to address this issue. The full regenerative ability of
external antler tissue components is achieved through PP-derived chemical induction
and PP-derived mechanical stimulation: the former triggers the regeneration of these
external components, whereas the latter drives their rapid elongation. Eventual
identification of the putative PP-derived chemical factors would open up a new avenue
for devising effective therapies for lesions involving each of these tissue components, be
they traumatic, degenerative, or linked to developmental (genetic) anomalies.
African spiny mouse (Acomys) skin At least two species of African spiny mice, Acomys
kempi and Acomys percivali, are capable of autotomic release of skin, e.g. upon being
captured by a predator. They are the first mammals known to do so. They can
completely regenerate the autotomically released or otherwise damaged skin tissue regrowing hair follicles, skin, sweat glands, fur and cartilage with little or no scarring. It is
believed that the corresponding regeneration genes could also function in humans.
Seifert and his colleagues found that compared to Mus musculus, the common
laboratory mouse model, spiny mice skin is less elastic, probably because of the large
hair follicles that take up about 12.5 percent more space than M. musculus hair follicles.
Such large follicles reduce the space available for elastic connective tissue, making spiny
mouse skin easier to tear. When Seifert compared the regrowth of tissue in spiny mice to
the lab specimens, he found that not only did epithelial cells migrate to the wound faster
in spiny mice, but they did not scar. In M. musculus, collagen fibers arranged into dense,
organized bundles that give rise to healed, but scarred, wounds. In contrast, the
regenerating wounds of the spiny mouse had a looser collagen organization
characteristic of undamaged tissue. Spiny mice also regrew their hairs, seemingly as a
result of the same signals used in developing hair follicles; M. musculus mice do not
regrow hair. Furthermore, modifying the hair follicle to elastic tissue ratio is an entirely
novel way of losing a chunk of tissuenot seen in other animals, she noted, pointing
out that for mice, which are nabbed by predators grasping their necks, losing tissue in
this way may provide similar survival benefit as a lizard losing its tail. As it happens, this
isnt the spiny mouses only regenerative trick. Another well-studied regeneration model
is rabbits ability regenerate ear punches. It turns out that spiny mice can do the same.
Seifert and his colleagues found that the mice can regenerate all tissue except muscle,
an ability that appears to stem from a specialized conglomerate of de-differentiated
cells, called the blastema, that initiates limb regeneration in other species. In the future,
Seifert plans to explore how the pathways regulating regeneration in the spiny mouse
are modulated to produce tissue regrowth instead of scarring. It may be possible, he
said, to modulate the immune response, in conjunction with other strategies, to push
healing away from scar production towards a regenerative pathway.
Mammalian Postnatal fingertip regeneration
Stem cells at the base of finger and toenails act as coordinating centers to orchestrate
communication between the nail, bone, and nerve tissues necessary to promote mouse
fingertip regeneration after amputation. Researchers found that nail stem cells use a
signaling pathway important for embryonic limb development to help nerves and new
nail and bone cells coordinate signaling during tissue regeneration, providing insight that
may enable future stem cell therapies. These cells, unlike tissues farther from the tip of
mouse digits, expressed proteins in the Wnt signaling pathway, already known to be
important in nail and limb growth during embryonic development. Conditionally knocking
out -catenina transcription factor downstream of Wntin adult mice prevented nail
differentiation. It turned out that nails only regenerated if some nail stem cells remained
after the rodents digit tips were snipped off, suggesting that nail stem cells serve as a

signaling center to coordinate the regeneration response of both bone and nail tissue,
said Ito. Knocking out -catenin prevented regeneration, suggesting the Wnt pathway
was critical to the nail stem cells role. Loss of -catenin in nail stem cells also prevented
nerves from growing toward the wound sitea process known to be critical to successful
amphibian limb regeneration. The nerves in turn prompted the mices nail epithelia to
secrete fibroblast growth factor (FGF), a protein that prompted the differentiation and
proliferation of bone-forming cells during digit regeneration. Without signals from the
nail stem cells, the interactions between nail and bone tissues failed to occur. However,
when the researchers amputated mouse digits beyond the stem cell sitebut activated
-catenin signalingthe nails still could not regenerate even though bone-forming cells
were induced, suggesting that the nail stem cells secrete other factors that spur the
regeneration process
Mechanisms: Regenerating fingertip genes
Digit regeneration in vitro - Han et al. (2003) used techniques for organ culture to
determine whether the regeneration of stage 11 digits can occur in vitro. Stage 11
autopods were isolated from E14.5 fetuses, and the tips of 2-3 central digits were
amputated and cultured for up to 4 days. After 2 days of culture a clear regeneration
response is evident based on the reformation of the digit blastema, and after 3-4 days of
culture the digit blastema elongates distally. Based on external anatomy, the in vitro
regeneration response is comparable to the in vivo response. The cultured autopods
appear morphologically different than in vivo limbs, primarily because the re-fusion of
digits, which occurs late in limb development (Maconnachie, 1979), does not occur in
cultured autopods. In addition to the anatomical response, we characterized the
expression of genes associated with digit regeneration in vivo. In 4-day-cultured
regenerates, we observed robust expression of Msx1 and Bmp4 in mesenchymal cells
surrounding the terminal phalanx, and Msx2 expression restricted largely to the apical
epidermis. Expression of Ihh in 4-day-cultured regenerates is associated with
skeletogenesis of the terminal phalanx, whereas Hoxc13 expression is associated with
the apical epidermis, and is spatially more variable than in in vivo regenerates. In 2-daycultured regenerates, an apical blastema of mesenchymal cells is present and Msx1 is
expressed strongly throughout the regenerating digit tip. Msx2 is expressed strongly in
the basal layer of the apical epidermis and is expressed weakly in the most distal
mesenchyme. Like Msx1, Bmp4 is expressed in the distal mesenchyme. They
investigated the expression of the differentiation marker genes Ihh and Hoxc13,
associated with the terminal phalanx and the forming nailbed. Overall, they observed an
anatomical regeneration response in more than 90% of cultured wild-type digits, and we
found perfect correlation between a regeneration response and the expression of Msx1,
Msx2 and Bmp4.
Regeneration studies on the mature mammalian digit tip have focused almost
exclusively on the role of the nail organ and the distal growth of the terminal phalanx.
This association is derived from both clinical studies in humans and experimental studies
on rodents. During the normal development of the nail wnt/beta catenin signalling gives
rise to the differentiation of the nails stem cells to produce the components of the digit.
The same pathway is used during regeneration. The signalling induces nerves to come to
the site of amputation which provide the fgf-2 signalling allowing bone cells to
differentiate (osteoblasts). Without the wnt signalling nervesa rent recruited and the
signalling axis between nail/bone and nerves is lost preventing regeneration to occur.
Amputating the digit to a point where NSCs are no longer present prevents regeneration
form occurring. It should be noted MSx1 and 2 are upregulated transiently during
regenerative but not in the blastema and that BMP4 cells are present in the blastema.
Experiments showed the blastema is a collection of lineage specific mesenchymal
progenitors rather than multiopotent stem cells. Similar to results found by Kragl M et al
for the blastema used to generate axolotl limb regeneration. By studying both
developing and mature digits, we found that regenerative potential is associated with
the expression domain of Msx1, but that Msx1 expression in mature digits is restricted to
loose connective tissue fibroblasts subjacent to the nail, and is not found in the nail

organ itself. The demonstration that Msx1 mutant digits display a regeneration defect
indicates that Msx1-expressing cells play a crucial role in the response. These findings
suggest that digit tip regeneration may not be dependent on the nail organ, but rather
on the connective tissue cells underlying the nail organ. Because this population of cells
is closely associated with the nail organ it would be difficult to distinguish between a nail
organ effect and the influence of the underlying connective tissues. The importance of
this cell population in mature digit regeneration is also supported by histological
observations noting a streaming of fibroblastic cells toward the regenerating digit tip,
and by the observation that a regeneration response itself need not include the nail
plate.
Why do humans suppress regeneration?
No adaptive advantage / Regeneration takes energy and time than esp. large and
complex structures
Warm blooded mammals are susceptible to bacterial infection and water loss. Urodeles
(e.g. axolotl and newt) are the only vertebrates that can perfectly regenerate different
body parts throughout their life. Anurans (e.g. Xenopus) are also used as a model to
study regeneration, but they can only regenerate perfectly before metamorphosis. Their
regenerative ability starts to decrease from developmental stage 51 until total loss at
stage 58. By over-expressing -catenin after stage 51, it is possible to maintain a high
regenerative capacity between stages 51 and 57 but regeneration still stops at stage 58.
Mammals are very limited in terms of epimorphic regeneration. Based on the work of
Illingworth in humans and Muneoka in mice, regeneration in mammals is limited to digit
tips. During the first trimester of gestation, mammals are able to perfectly regenerate
their digit tips, but this capacity seems to drop with age. Some rare cases have been
reported where adults were able to regenerate digit tips; unfortunately these cases are
rare and poorly documented. In green are proteins or signalling pathways known to be
essential or implicated in regeneration as discussed in the text. A decreased level in
Delta and a predominant presence of hypophosphorylated Rb (pRb) may explain in part
the loss in regenerative capacity observed in mammals.
What limits regenerative ability during ageing - understanding how aging changes
cells, tissues, and physiological systems is key to identifying mechanisms that limit
regenerative ability. During both development and regeneration, relatively
undifferentiated cells become specified to form organs that then undergo tremendous
growth, but the overall process differs between the two. Regeneration is activated in
response to injury, depends upon tissue-specific progenitor cells, and occurs under
physiological conditions and within an extracellular environment that differs from the
embryonic state. Embryonic cells have great potential for cellular reprogramming, and
cellular reprogramming through epigenetic modifications and changes in transcription
are associated with regenerative responses. During development, cells exhibit changes
in transcription that limit signaling pathways associated with cellular plasticity. For
example, cells differentiate and lose the ability to enter the cell cycle, both of which
must be reversed for limb regeneration to occur in salamanders. The maintenance of
plastic cellular states and cell-cycle re-entry are likely associated with the actions of
tumor suppressor proteins like retinoblastoma protein (RB), the levels and activation
states of which are known to vary during regeneration in salamanders and across
developmental stages in mammals. Taken together, studies suggest that the
abundances of key regulatory molecules are permissive for cell cycle re-entry from a
quiescent state in young life stages, but restrictive as an organism ages, and this limits
regenerative ability. Future studies that quantify levels of such molecules in young and
old salamanders may shed new light on progenitor cell activation, regenerative ability,
and potentially, diseases of aging such as cancer.
What makes axolotls different than humans? There are many questions that arise
as to why axolotls (urodeles in general) can regenerate perfectly and mammals cant.
We can find evidence in the literature that will point to the ability of urodeles to
dedifferentiate their cells at the site of amputation) or that they display an attenuated
immune response following injury. Both of these examples are based on extensive

research and are important aspects of regeneration in these animals, but other data
from humans and mice seem to indicate that these observations may not be enough to
explain the differences in regenerative capacity between urodeles and mammals. One of
the best example is the work of Illingworth which has demonstrated that humans can
regenerate their digit tips if the amputation occurred distal or at least partly distal to the
nail organ. Her results are supported by the work of Muneoka on mouse digit tip
regeneration where similar results were obtained. In addition, work by the group of
Keating has shown that cellular dedifferentiation can be induced in mammalian cells. It
also seems that nude mice which have a deficient immune system do not resist better to
the induction of fibrosis than their wild type littermates. However, this is not a universal
observation for mice with deficient immune systems. In fact, the macrophageless PU1
immunodeficient mice heal their wounds better than wild type littermates with nearly
scarless phenotype similar to that observed during embryonic wound healing. This latter
observation is in agreement with a reduced immune response being an integral part of
the regeneration process. It would indeed be quite interesting to test how well these PU1
immunodeficient mice would do in terms of digit regeneration compared to wild type
littermates. One aspect of limb regeneration that has always been fascinating is the fact
that there is never any residual scar between the stump (limb, tail or jaw) and the
regenerated part. Also, urodeles can regenerate components of their central nervous
system (e.g. spinal cord, fore brain, midbrain dopaminergic neurons, retinotectal
projection) without the appearance of any glial scar (S Roy data not published. In
mammals the appearance of a glial scar in the central nervous system prevents the
growing axons from reaching their targets whenever there is an injury. Therefore, it
seems that one important difference between urodeles and mammals, that may
potentially be crucial for allowing tissues to regenerate as adults, is the formation or not
of scar tissue. Interestingly, if the stump of an amputated axolotl limb is covered by a full
thickness skin flap or a suture is applied to close the wound to mimic the presence of
scar tissue, regeneration is blocked. Again, the work done by Illingworth showed exactly
the same thing for human digit tips where the use of sutures to close the wound
prevented regeneration. One situation for which mammals resemble urodeles is in the
ability of the developing foetuses to achieve scarless wound healing during the first
trimester of gestation. It is tempting to speculate that this is due to the fact that the
foetus is in amniotic fluid a situation with some similarities to axolotls, which live in
water. The caveat is that fishes, frogs and even the sea cucumber, which can regenerate
an important portion of its viscera, all live under water and cannot heal their wounds
perfectly. Another argument that is often heard pertains to the fact that axolotls retain
some neotenic characteristics such as a caudal fin and external gills, indicating a failure
to metamorphose. Interestingly, humans are often considered neotenic amongst
primates and yet they cannot regenerate or heal without scars. Finally, the newt which
metamorphoses can still regenerate perfectly in its terrestrial form. Therefore, the idea
that axolotls can regenerate because they are neotenic does not explain why humans
cannot regenerate and why the newt can. Another recent study has demonstrated that
mammalian myotubes maintain some level of responsiveness to serum stimulation
which is reflected by an upregulation of the immediate early genes jun and fos but still
cannot re-enter the S-phase. Unidentified factors downstream of the aforementioned
immediate early genes might explain this inability of mammalian myotubes to re-enter
the S-phase. Mechanosensory cochlear hair cells, which are part of the central nervous
system, can be regenerated in amphibians but not in mammals. A recent study has
shown that conditional knockout mice for Rb in hair cells are able to regenerate
functional hair cells. Also, Rb was found to be mostly in its hyperphosphorylated state
during rat liver regeneration emphasizing the idea that the inactivation of Rb may be a
key process for regeneration. A better understanding of how urodele cells mediate the
inhibition of Rb compared to their mammalian counterpart following serum stimulation
could yield important knowledge on cellular machinery that may be harvested in order to
promote tissue repair and replacement.
Is there a relationship between aging and regeneration? This is an age old

question that is still without an answer. It is obviously very tempting to say yes to this
question, especially if we consider regeneration in Xenopus which is limited to premetamorphic tadpoles. In addition, muscle regeneration in mammals has been shown to
decrease with age up to the point of failing. Carlson has recently shown that part of this
loss in the ability of older animals to repair muscle is linked to the transforming growth
factor beta signalling via Smad3 which interferes with Notch. Finally, the report from
Illingworth where it seems that younger people regenerate more efficiently also supports
the idea that age affects the regeneration potential although there is some evidence
that this may not be universal as digit regeneration has been reported in a 29 year old
man. In rodents, digit tip regeneration is sustained throughout adulthood as long as the
amputation occurs within the nail organ, which provides a potential source of stem cells.
Neufeld has shown that although adult mice do regenerate a bony growth, which
resembles a normal digit tip, following amputation, the growth is not of the same quality
as that observed in foetal or newly born mice implicating a possible link with age and
regeneration capacity. The champions of regeneration (i.e. urodeles), however, do not
seem to lose their capacity to regenerate with age. It is a well known fact that growth
hormone levels diminish with age in mammals and it has also been shown that growth
hormone supplementation improves wound healing in elderly men. So what about the
role of growth hormone on limb regeneration? The first experiments to report on limb
regeneration in hypophysectomised newts seem to indicate that the pituitary gland was
essential for regeneration and that supplementation with prolactin and thyroxin was
sufficient to rescue the phenotype. However, this was later shown to be incorrect as it
was demonstrated that if you started with very well fed newts that were
hypophysectomised just before amputation you got a normal regenerate. These
experiments support the idea that growth hormone is not required for limb regeneration
but rather for animal growth and perhaps feeding.
The Role of Dedifferentiation in Regeneration
Limb regeneration is a complex process because many cell types contribute to the
blastema. The quantitative contribution of dedifferentiated muscle to the blastema is not
yet known because experiments have depended on the implantation of exogenous
myotubes as indicators of dedifferentiation. Furthermore, muscle dedifferentiation has
yet to be specifically inhibited to test the dependence of regeneration on this process.
Experiments examining the contribution of dermis to the regenerating limb blastema
indirectly sup- port the role of muscle. Local X-irradiation of the limb inhibits
regeneration by inhibiting cell division. A cuff of unirradiated skin including epidermis
and dermis was trans- planted onto an irradiated limb resulting in limbs where the skin
was unirradiated but internal tissues including muscle were irradiated. When such limbs
were amputated through the grafted skin and allowed to regenerate, the skin, bone, and
ligaments were regenerated with perfect skeletal patterning. The regenerated bone and
ligaments derived from the grafted skin. Muscle, however, was largely missing. This
result implies that muscle tissue makes a necessary contribution to the blastema,
although other interpretations are possible. As with other tissue grafting experiments,
these observations also did not provide insight into whether the contribution of muscle
would occur through dedifferentiation or activation of a resident progenitor cell in
muscle. An experimental resolution to these issues would be to transplant purified
myotubes into the X-irradiated limbs with skin grafts to determine if implantation of
myotubes rescues the lack of muscle.
The importance of dedifferentiation to regeneration is best seen in simpler structures
other than the limb. In salamanders, a number of the eye tissues can be regenerated.
For example removal of the lens of the eye induces its regeneration from the dorsal
pigmented iris epithelium to which the lens was originally attached. In this case a single
cell type, the retinal pigment epithelial cell (RPE) clearly gives rise to the cells of the
regenerating lens through dedifferentiation and transdifferentiation. This RPE-to-lens
transition has been rigorously demonstrated in clonal cell culture of embryonic chick RPE
and occurs in two distinct steps where RPE cells lose their differentiated features and

begin to proliferate before the second step of transdifferentiating into lens. + Reversals
of muscle differentiation are also known to occur during regeneration in other,
evolutionarily distinct organisms. The invertebrate hydrozoan jellyfish (Podocoryne
carnea) is capable of undergoing extensive regeneration that involves
transdifferentiation of skeletal muscle into other cell types. This transition has been
analysed in cell culture. When a homogeneous sheet of mononucleated striated body
muscle cells was treated with the proteases pronase or collagenase, digesting the
basement membrane, the muscle cells underwent DNA synthesis, proliferation and
transdifferentiation giving rise to both smooth muscle and RF-amide positive
neurosecretory cells. Interestingly the transition from skeletal to smooth muscle did not
require cell cycle progression, while transdifferentiation to the neurendocrine lineage did.
+ Analysis of the transdifferentiation event in jellyfish indicates that changes in the
muscle cells interaction with the extracellular matrix induce the destabilization of the
differentiated phenotype. Cells that maintained a rigid attachment to the ECM remained
stably differentiated while those that were loosened and did not maintain a spread or
stretched state underwent cell cycle re-entry and transdifferentiation.
Transdifferentiation was observed when pieces of striated muscle that had not been
treated with protease called mechanically isolated pieceswere plated onto a new
piece of extracellular matrix. When the muscle cells were plated onto ECM that was
firmly adherent to glass those muscle cells straddling the junction between the new and
old ECM underwent DNA synthesis and transdifferentiation into neurons. These cells had
apparently lost contact with both ECMs, and did not have a spread morphology. In
contrast, those cells that had moved beyond the junction and had adopted a spread or
stretched state on the new ECM did not undergo proliferation and transdifferentiation. To
test the role of cell tension in triggering transdifferentiation, pieces of mechanically
isolated striated muscle cells were combined with floating pieces of ECM where cells
would migrate onto the new ECM, but they could not adopt a spread morphology and
generate tension across their cell diameters. In this case cells throughout the explant
underwent DNA synthesis and transdifferentiation into neurosecretory cells. In the
jellyfish, the ability to analyse transdifferentiation of a homogeneous sheet of muscle
cells reveals the important role that the reversal of differentiation plays in providing
progenitor cells for regeneration. In the future it will be fascinating to determine whether
the signalling processes controlling dedifferentiation in invertebrate muscle cells have
significant parallels with dedifferentiation of newt muscle.
Muscle Progenitor Cells During Repair: Muscle Dedifferentiation Versus
Satellite Cells
In vertebrates such as mammals and birds, adult skeletal muscle contains a dormant
population of mononucleate progenitor cells called satellite cells that lie between the
differentiated muscle cell and the basal lamina. In growing animals these cells contribute
to muscle tissue growth by proliferating and then fusing with resident fibers. In adults,
injury stimulates the satellite cells to divide and to later fuse into fibers. In essence
satellite cells represent a muscle-specific stem cell. Recent evidence also indicates that
cells of endothelial origin can also act as muscle progenitor cells. + Are muscle satellite
cells or other myogenic progenitors also activated during newt limb regeneration? Adult
newt muscle apparently does not have classically defined satellite cells. However
whether other interstitial mononucleate cells that reside within the mature muscle are
stimulated to produce myogenic cells for regeneration is still an open question. Schrag
and Cameron argued that the outgrowth in culture of mononucleate, myogenic cells from
urodele limb muscle explants represented the migration and proliferation of a
mononucleate precursor population residing between muscle fibers because the authors
could not see evidence of dedifferentiating muscle fibers in their preparations. In these
experiments, however, the origin of the dividing cells in culture was not determined so it
could not be ruled out that muscle fibers had actually dedifferentiated, losing their welldefined morphology and making them difficult to identify. + It is clear however that in
salamanders muscle dedifferentiation occurs in tissues where myogenic progenitor cells
are still present. Hay s histological work on limb regeneration8 and our own recent work

tracing endogenous muscle cell fate during tail regeneration indicate that muscle
dedifferentiation occurs during regeneration in larval salamanders where rapid overall
body growth is still occurring, and where mononucleate muscle pre- cursors must still be
actively contributing to growing muscle. This means that in some parts of the tail close
to the site of injury multinucleate muscle cells are dedifferentiating to form the
regeneration blastema while in the non-injured tissue myoblasts are fusing into growing
fibers. This poses the interesting question why dedifferentiation is invoked at a stage
where myoblast proliferation is clearly still occuring. Recent work described in the last
section of this Chapter indicates that the progenitors produced from dedifferentiation are
more multipotent than myoblasts. This suggests that dedifferentiation may be required
during regeneration to produce flexible cell types that can be influenced by extracellular
signals to form many tissue types.

Heart and Lungs

Cardiac Cell types - The four-chambered heart consists of different cell types. All these
cell types contribute to structural, biochemical, mechanical and electrical properties of
the functional heart. Atrial and ventricular cardiomyocytes form the muscular walls of

the heart (that is, the myocardium). More than 50% of the cells of the heart are cardiac
fibroblasts. Endothelial cells form the endocardium, the interior lining of blood vessels
and cardiac valves. Smooth muscle cells contribute to the coronary arteries and inflow
and outflow vasculature. The epicardium gives rise to the precursors of the coronary
vasculature and cardiac fibroblasts. Pacemaker cells and Purkinje fibres in the
conduction system are specialized cardiomyocytes that generate and conduct electrical
impulses. The sinoatrial node (SAN), which is composed of a group of pacemaker cells,
resides in the right atrium generating impulses to initiate heart contraction. The
atrioventricular node (AVN) is located between the atria and ventricles, and it conducts
an electrical impulse from the atria to the ventricles.
Cardiac regeneration - During embryogenesis, the mammalian heart grows through
the proliferation of cardiomyocytes. After birth, cardiomyocytes undergo another round
of DNA synthesis without cytokinesis, which renders most of them binucleated. At this
point, cell division ceases and postnatal heart growth is achieved primarily through the
hypertrophy (enlargement) of cardiomyocytes. Although the adult human heart clearly
lacks sufficient regenerative potential to replace the billions of cardiomyocytes lost
following myocardial infarction, there is evidence that cardiomyocytes can slowly selfrenew, fostering ongoing debate regarding the extent of cardiomyocyte proliferation and
turnover in the uninjured adult mammalian heart.
Why is cardiac regeneration needed? - Despite current treatment regimens, heart
failure remains the leading cause of morbidity and mortality in the developed world due
to the limited capacity of adult mammalian ventricular cardiomyocytes to divide and
replace ventricular myocardium lost from ischaemia-induced infarct. Hence there is great
interest to identify potential cellular sources and strategies to generate new ventricular
myocardium. Past studies have shown that fish and amphibians and early postnatal
mammalian ventricular cardiomyocytes can proliferate to help regenerate injured
ventricles; however, recent studies have suggested that additional endogenous cellular
sources may contribute to this overall ventricular regeneration. Zhang et al (2013)
developed, in the zebrafish (Danio rerio), a combination of fluorescent reporter
transgenes, genetic fate-mapping strategies and a ventricle-specific genetic ablation
system to discover that differentiated atrial cardiomyocytes can transdifferentiate into
ventricular cardiomyocytes to contribute to zebrafish cardiac ventricular regeneration.
Using in vivo time-lapse and confocal imaging, we monitored the dynamic cellular events
during atrial-to-ventricular cardiomyocyte transdifferentiation to define intermediate
cardiac reprogramming stages. They observed that Notch signalling becomes activated
in the atrial endocardium following ventricular ablation, and discovered that inhibiting
Notch signalling blocked the atrial-to-ventricular transdifferentiation and cardiac
regeneration. Overall, these studies not only provide evidence for the plasticity of
cardiac lineages during myocardial injury, but also more importantly reveal an abundant
new potential cardiac resident cellular source for cardiac ventricular regeneration.
Triggers: 1) Inflammation some might be good but overproduction of cytokines can
lead to damaging inflammatory responses. 2) Fibroblast accumulation. 3) ECM
production and scarring (fibrosis) - Scar may cause severe contractile dysfunction Current drugs/treatments not ideal they do not replace lost cardiac muscle - (+ organ
supply, prevent/treat congenital heart deformations)
Naturally occurring cardiac regeneration: Adult Zebrafish & newts - Damage heart - de
novo cardiogenesis. Rodents & humans -fibrogenesis - (excessive connective tissue) and little cardiomyocyte regeneration
Embryonic and early postnatal mammalian hearts can regenerate: E14 mouse
heart explanted, damaged and cultured serum-free. Quickly regenerates, no
inflammation or scarring. Why? - immature immunoinflammatory response? - Not much
immune response during early development? - limited fibroblasts?
The neonatal mouse heart retains regenerative potential for ~7 days after birth. When
the ventricle apex of the heart is amputated in 1 day-old mouse pups (P1), the heart can
undergo full regeneration without scar formation, similar to adult zebrafish. Lineage
tracing of cardiomyocytes demonstrated that newly formed cardiomyocytes are derived

from the proliferation of pre-existing cardiomyocytes rather than from a stem cell
population. Surgical resection of the ventricle apex 7 days postnatally (P7; that is when
most cardiomyocytes have withdrawn from the cell cycle) results in a loss of the
regenerative capability. The heart also fully regenerates in a model of neonatal
myocardial infarction in which the left anterior descending (LAD) coronary artery is
ligated on day 1 after brith. However, this regenerative capability is lost when LAD
coronary artery ligation is performed at P7 or later. In the absence of regeneration after
removal of the ventricle apex or LAD coronary artery ligation at P7, extensive cardiac
fibrosis ensues, highlighting the reciprocal relationship between regeneration and
fibrosis in the heart. The similarity in cardiac regeneration capability between zebrafish
and neonatal mammals suggests that this process is mainly driven by the
dedifferentiation and proliferation of existing cardiomyocytes, and not by cardiac
progenitor cells. In the future, it will be of interest to identify factors that can promote
cardiomyocyte proliferation to extend the neonatal regenerative response to injury.
Why cant adult mammalian hearts regenerate? The difference in the capacity for
cardiac regeneration between adult fish hearts and mammalian hearts could be due to
the distinct heart anatomies and cardiomyocytes characteristics in these organisms.
Mammalian hearts are four-chambered with double-circulation that allows the chambers
to generate high pressure, whereas fish hearts are two-chambered with single circulation
that pumps blood with a relatively lower force. In addition, most cardiomyocytes in the
zebrafish heart are mononucleated throughout life and retain significant proliferative
capacity. By contrast, ~90% of cardiomyocytes in mice become binucleated shortly after
birth as a consequence of DNA replication without cell division. The lower pressure load
and mononucleated cardiomyocytes of neonatal mice resemble those of adult zebrafish.
Mammalian cardiomyocytes resist mitosis after injury (cf. newts): 1) Withdraw
from cell cycle late gestation (cell cycle regulators and miRNAs downregulated) Withdrawing from cell cycle and inability to go back into cell cycle can get cells in to G1
but need to get past restriction point G1-S although tightly down regulated in
mammals and human systems significant barrier to regeneration.
Studies investigating mammalian cardiomyocyte mitosis after injury can be found as
early as the 1970s (Rumyantsev, 1974), although more definitive evidence for the
potential of embryonic and neonatal mammalian myocardium to regenerate has recently
emerged. Using an elegant mouse model to effectively damage 50% of the developing
cardiac tissue by inactivating the gene encoding holocytochrome c synthase, Cox and
colleagues demonstrated that lost myocardium is replaced by healthy tissue during fetal
development, resulting in only 10% of the cardiac volume occupied by diseased tissue at
birth. Furthermore, Sadek and colleagues showed that the 1-day-old neonatal mouse
heart is capable of regeneration after resection of approximately 15% of the ventricle at
the apex. This neonatal mouse heart regeneration appears to occur as a result of
dedifferentiation followed by proliferation of preexisting cardiomyocytes. However, the
ability to regenerate myocardium is rapidly lost by 7 days after birth; instead, the heart
develops fibrotic scars similar to the response observed following myocardial injury in
adult mice and humans. These experiments raise the critical question of what prevents
mouse heart regener- ation after the first days of life, and point to this first week of life
as a crucial period for understanding inherent regenerative mecha- nisms in mammals.
2) Cardiomyocytes contain highly organized sarcomeres that are required for
synchronous contraction. When cardiomyocytes undergo dedifferentiation, the
sarcomere structure collapses and is displaced to the cell periphery. The mechanisms
responsible for sarcomere collapse and reformation are unclear, but sarcomere
disassembly is probably essential for DNA synthesis and cell division to occur. Adult
human cardiomyocytes are primarily binucleated with highly stable sarcomeres that
probably prevent cytokinesis. By contrast, neonatal mammalian cardiomyocytes and
zebrafish cardiomyocytes contain less elaborate sarcomeres, which probably accounts
for their ability to proliferate. 3) Initial studies concluded that cells from the epicardium
(the outer layer of the heart) were activated in response to injury and gave rise to
cardiomyocytes and other cell types involved in heart regeneration, such as vascular

smooth muscle cells. By contrast, subsequent lineage tracing studies, using the
tamoxifen- inducible CreloxP recombination system to label cardiomyocytes with a
regulatable GFP transgene that is controlled by the cmlc2 (cardiac-specific cardiac
myosin light chain 2) promoter after birth, followed by amputation at adulthood, showed
that newly formed cardiomyocytes were GFP positive. This supports the notion that new
myocardium is generated through dedifferentiation and proliferation of pre-existing
cardiomyocytes rather than from a progenitor stem cell population.
Turnover of adult mammalian cardiomyocytes: The weight of evidence supporting
adult human cardiomyocyte renewal is based on an approach that took advantage of
radiocarbon (14C) that was released for a short time during atomic bomb testing (from
1955 to 1963) and incorporated into human DNA32. Using the integration of 14C to
establish the age of cardiomyocytes in humans, cardiomyocytes were found to turn over
at a rate of ~1% of the entire population per year in a person at the age of 25 years, and
this rate declines to ~0.45% at the age of 75 years. This suggests that nearly 50% of
cardiomyocytes are replenished during a normal lifespan. In an independent analysis of
tissue samples from patients with cancer between 19 and 104 years of age who had
received an infusion of iododeoxyuri-dine, ~22% of cardiomyocytes were found to renew
annually. This suggests that the myocyte compartment of the adult human heart is fully
replaced up to 15 times between the ages of 20 and 100 years. The different
cardiomyocyte renewal rates detected in the two studies are difficult to reconcile, but
both studies indicate that cardiomyocytes in the adult human heart turn over at a
measureable rate, suggesting that regenerative strategies could potentially enhance this
process.
Induce mammalian cardiomyocyte division
- Cardiomyocyte death is a common end-point in many forms of cardiovascular disease.
It is generally agreed that cardiomyocytes in the adult mammalian heart exhibit some
capacity to re-enter the cell cycle. In addition, recent studies suggested that adultderived stem cells might contribute to cardiomyocyte renewal in injured hearts.
However, it is very clear that these intrinsic processes are of insufficient magnitude to
restore cardiac function after myocardial infarction. A number of approaches have been
explored to increase cardiomyocyte number in injured hearts, with the hope that this
would promote functional recovery. These include direct transplantation of
cardiomyocytes or myogenic stem cells, treatment with cytokines to mobilize
endogenous stem cells and cardiomyocyte cell cycle activation.
- It was shown that cardiomyocytes de-differentiation and re-entry back into the cell
cycle was possible. Rb usually acts a protein for the checkpoint of the cell cycle its
phosphorylation allows E2Fs to drive the cell cycle progression. Using transgenic mice
expressing CDK2 under the cardiac alpha myosin heavy chain promoter could drive cell
cycle re-entry of cells and in a myocardial infarction model could allow repair and regrowth of myocytes to replace heart function other than generating fibrosis and scar
tissue reducing heart function.
Need to induce dedifferentiation first? There is a hunt for drugs, which can induce the
reassembly/disassembly of sarcomeres to allow for re-entry into cell cycle, but also have
to deal with the binucleate nature of the cardiomyocytes fibres in humans. Interestingly
chronic atrial fibrillation and the border zone of infarction show de-differentiated cells
could identify the cause and sue to a therapeutic way. When learning to de-differentiate
cells it may be best to induce them back to a progenitor state rather than pluripotent
due to a risk of cancer. Chemical-induced pluripotency has recently been discovered as a
cheap method using small chemical molecules to induce pluripotency could provide a
potential basis to identify reversion back to progenitor states and be useful for
therapeutic cardiac regeneration.
Stem cell based cardiac therapy
1) Exogenous: Exogenous stem cells can be used in treatment, such as injecting,
skeletal myoblast progenitors, HSCs, and mesenchymal stem cells and circulating
endothelial progenitors. However exogenous injection only provides transient benefits

(cells only stay there shortly) through paracrine action (not transdifferentiation of
exogenous cells into cardiac myocytes) but can also have deleterious side-effects such
as with myoblast cells get calcification and bone formation in heart, which prevents
proper function and conduction and thus a decline in health. MSCs are useful as can
interact with different tissue and have migratory abilities, which provides a great
foundation for stem cell therapies in the future.
2. Endogenous heart stem cells
Endogenous cardiac stem cells have been identified such as 3 main types in the heart
c-kit, sca-1 and atpase binding cassette transporter. Injected c-kit cells into infarcted
model could regenerate myocardium and myocytes. Despite these findings, the ability
of c-kit+ cells to activate and support spontaneous regeneration in the adult heart
remains highly controversial.
- All studies are based on the transplantation of very large numbers of c-kit+ cells into
the injured heart, so a cell lineage approach is required to address this issue
conclusively.
- Two other major concerns about these cells are their precise localization and number.
Using genetically engineered mice expressing GFP under the c-kit promoter, Fransioli et
al. (2008) demonstrated that c-kit+ cardiac cells decline markedly in the first two
postnatal weeks and almost disappear after ten weeks. Have to use exogenous cells
then.
- Other progenitors have been identified. For example following lineages from embryo to
post-natal in rats and humans identified ISL-1 surface marker for cardiac progenitor
gives rise to left/right atria and ventricle pretty much. Interestingly this progenitor can
be found in adults suggesting its induction could be useful in inducing regeneration
progenitors may also be more useful as finite division and can be more easily controlled
and can be easily manipulated into cardiac myocytes for heart function, unlike
pluripotent/multipotent stem cells. Other people looked at development of heart from
mesoderm and tried to identify lineages associated with cardiac development then look
at these markers in adult tissue to identify progenitors present. Found more in the
epicardium capable of giving rise to smooth muscle, cardiac fibroblasts and endothelial
cells. It is possible they are derived from ISl-1 cells therefore making the epicardium a
heterogeneous population of progenitors that can be influenced to regenerate the heart
under damage. One paper showed exactly that these epicardium cells can be utilised to
induce regeneration in the heart.
- A significant bottleneck in regenerative cardiac medicine is the identification of viable
stem/progenitor cells to allow de novo cardiomyocytes generation to restore heart
function after injury. It could replace transplantation and avoid limited graft survival,
restricted tissue homing and host rejection. One paper identified that using thymosin
Beta4 they could induce a marker of activated epicardial progenitors to be expressed
(wilt tumor 1 (WT1)) lead to mobilisation of population and transdifferentiation into
myocytes which integrate with resident muscle significant step in regenerative
medicine.
Cardiac stem cell niche: role for bvs?
- A subpopulation of epicardial cells delaminates from the subendocardial space and
migrates to the underlying myocardium using the vascular network as a guide. These
progenitors may also be able to generate the intramyocardial clusters of c-kit+/Sca-1+
stem and progenitor cells that persist into adulthood, but alternative sources of these
cells cannot be excluded. In the developing heart, there may be a cell network that, for
regeneration purposes, connects the epicardium to the myocardium via the coronary
vessels. Further investigations are needed to establish whether such a network persists
in postnatal life.
- Another consideration with respect to the assessment of efficiency of in vivo
reprogramming is that there is substantial heterogeneity among fibroblast populations
within the heart that could display differing potential for cardiac reprogramming. In
addition, blood vessel-associated cells such as mesoangioblasts, which function as a
stem cell population that can differentiate into multiple mesodermal cell types, may also

show selectivity for adopting a cardiac fate in response to reprogramming factors. In this
regard, although lineage tracing demonstrates that fibroblasts are reprogrammed into
cardiomyocytes in vivo, other cell types can also be reprogrammed to adopt a cardiac
fate, and therefore the reprogramming efficiency, solely based on fibroblast conversion,
is likely to be underestimated.
Transdifferentiation (Direct reprogramming of non-myocytes to
cardiomyocytes)
The discovery that expression of MyoD in fibroblasts stimulated their transdifferentiation
into skeletal muscle established the paradigm of cellular reprogramming. This paradigm
has been expanded in recent years in the wake of Takahashi and Yamanakas startling
discovery that fibroblasts can be transcriptionally reprogrammed to pluripotency.
Subsequently, conversion of one differentiated cell type to another without proceeding
through a pluripotent intermediate, known as direct reprogramming, was reported for
several cell types, including cardiomyocytes. Initial results demonstrated that a
combination of three transcription factors, namely GATA4, myocyte-specific enhancer
factor 2C (MEF2C) and TBX5 (collectively referred to as GMT factors), could
reprogramme both mice cardiac and dermal fibroblasts into induced cardiac-like
myocytes (iCLMs) in vitro, with an efficiency of ~57%, depending on the cardiac marker
assayed. A minor fraction of these cells displayed spontaneous contractions after 45
weeks in culture. The efficiency of this approach is variable, and some laboratories have
difficulty reproducing these results. The variability is most likely due to the need for a
precise stoichiometry of reprogramming factors to activate the cardiac gene programme,
the heterogeneity of fibroblasts used in the different studies, the differences in the cell
culture conditions used and other unknown variables. The inclusion of an additional
bHLH transcription factor, HAND2, (in a cocktail referred to as GHMT) increased the
reprogramming efficiency to ~20%. The newly formed iCLMs generated by these
methods seem to arise from direct conversion of fibroblasts, bypassing an intermediate
stem cell state. Moreover, these cells are phenotypically stable following the withdrawal
of exogenous cardiogenic factors. Fibroblasts reprogrammed by GMT or GHMT factors
suppress the expression of fibroblast- specific genes and concomitantly activate a broad
array of genes characteristic of cardiomyocytes. Both atrial and ventricular cardiac
genes are expressed in reprogrammed fibroblasts, suggesting a heterogeneous response
to these factors. A small fraction of reprogrammed cells also displays spontaneous
contractions on tissue culture plates, accompanied by electrical activity and Ca2+
transients. This suggests that they are functional and similar, yet not identical, to
neonatal ventricular cardiomyocytes.
- GATA4 and TBX5 together with BAF60C (BRG1- associated factor 60C), a cardiacrestricted subunit of the SWI/SNF-like BAF chromatin remodelling complex, were reported
to be sufficient to generate beating cardiomyocytes when ectopically overexpressed in
the non-cardiogenic posterior mesoderm and the extra-embryonic mesoderm of the
mouse amnion. BAF60C did not display cardiogenic activity in fibroblasts or enhance the
activity of GMT factors in this setting, suggesting that its activity may require a
permissive cell type or other permissive cardiogenic signals in the embryo. The precise
mechanism responsible for direct cardiac reprogramming remains to be fully defined.
Various combinations of the GHMT factors have been shown to directly activate genes
encoding cardiac structural proteins and to auto- and cross-regulate their own
expression. Presumably, when these factors achieve threshold levels of expression, they
can switch on endogenous cardiac genes that gradually establish a stable cardiac
phenotype.
Induced Cardiomyocytes Exhibit Spontaneous Ca2+ Flux, Electrical Activity and beating
To determine if iCMs possessed the functional properties characteristic of
cardiomyocytes, we analyzed intracellular Ca2+ flux in iCMs after 24 weeks of culture.
Cardiac fibroblast derived iCMs showed spontaneous Ca2+ oscillations with varying
frequency of electrical activity tracings similar to the potential observed in neonatal
cardiomyocytes. Intracellular electrical recording of iCMs displayed action potentials that
resembled those of adult mouse ventricular cardiomyocytes.

Reprogramming into non-myocyte heart cells: In addition to cardiomyocytes,

various other specialized cell types within the heart are essential for cardiac function,
including certain cells of the cardiac conduction system and endothelial and smooth
muscle cells that give rise to the vascular system:
Reprogramming cardiomyocytes into conduction system cells: The cardiac
conduction system coordinates electrical impulses through the heart that mediate
cardiac rhythm. The heartbeat is initiated by an impulse within the sinoatrial node (SAN),
which passes through the atrium to the atrioventricular node, where impulse
propagation is delayed to allow the atria to contract. Electrical impulses then progress
through the ventricular conduction system, which comprises the His bundle, right and
left bundle branches and Purkinje fibres, to coordinate contraction of the heart from the
apex to the base. Abnormalities in the cardiac conduction system cause disturbances in
cardiac rhythm and account for a high fraction of human morbidity and mortality. Current
treatment for such disorders involves the implantation of costly electronic pacemakers.
In recent studies, in vivo reprogramming has been extended to the generation of cardiac
conduction system cells. Forced expression of the TBX18 transcription factor in rodent
ventricular cardiomyocytes, in vitro and in vivo, has been reported to confer the
functional and genetic properties of pacemaker cells from the SAN without passage of
cells through a pluripotent state. Remarkably, the cells generated by this approach
display characteristics of bone fide pacemaker cells such as Ca2+ cycling (the release
and re-uptake of intracellular Ca2+ to control muscle contraction and relaxation) and
automaticity (the ability of cells to depolarize spontaneously). They also retain their
pacemaker phenotype even after exogenous TBX18 has been removed, indicating their
stable conversion. Activation of Notch signalling in newborn rodent cardiomyocytes, in
vitro and in vivo, has been shown to confer a Purkinje-like phenotype81. These initial
findings point towards exciting opportunities to generate biological pacemakers in vivo.
Looking to the future: Deciphering the signalling pathways and regulatory networks
of cardiac development has led to therapeutic strategies to trigger cardiac regeneration
and repair. Although progress made in promoting cardiomyocyte proliferation and in
direct reprogramming of non- myocytes into cardiomyocytes and other cell types of the
heart has offered new perspectives in the quest to enhance repair and regeneration of
the injured adult heart, many challenges remain to be addressed. Efficient cardiac repair
will probably also necessitate the delivery of reprogramming factors specifically to
cardiac fibroblasts, and not to other fibroblast populations throughout the body, and
molecules that promote cardiomyocyte proliferation will need to be delivered selectively
to cardiomyocytes, and not to fibroblasts that develop into fibrotic scar tissue.. Currently,
reprogrammed iCLMs and dedifferentiated dividing cardiomyocytes are immature and
phenotypically heterogeneous, which may contribute to arrhythmogenesis. Therefore, it
is crucial to promote complete cardiomyocyte differentiation after proliferation and to
reprogramme cardiac fibroblasts into homogeneous, mature cardiomyocytes that can
functionally integrate with existing cardiomyocytes in the adult heart to maintain
contractility and conductivity. Another concern is the safety issue associated with the
viral-based delivery of factors to the injured heart. As integration of retroviruses into the
genome can cause cancer, developing a strategy to enable the delivery of transcription
factors without viral vectors would be important. Such methods also need to be noninvasive and yet efficient in promoting cardiomyocyte proliferation and the
reprogramming of non-myocytes into cardiomyocytes and possible reprogramming of
other cardiac cell types. Cardiogenic small molecules and synthetic oligonucleotides will
be attractive alternatives to virus-mediated approaches in the future. Although
discoveries in mouse models have provided important insights into human biology, they
may not be directly applicable to human disease therapies. Preclinical trials in large
animals are necessary to evaluate safety and efficacy issues.
CARDIOMYOCYTE CELL CYCLE REENTRY: Fetal cardiomyocytes actively proliferate,
whereas adult cardiomyocytes largely do not. One attractive strategy to replenish
cardiomyocytes lost in heart disease is to induce mature cardiomyocytes to reenter the
cell cycle. In rodents, cardiomyocytes exit the cell cycle in the first postnatal week, and

this correlates with the loss of functional cardiac regenerative capacity. In humans, the
timing of cardiomyocyte cell cycle withdrawal is uncertain, but initial studies suggest
that human cardiomyocytes continue to cycle beyond the immediate neonatal period
and well into childhood (9). It will be interesting to determine whether there is indeed a
protracted period of proliferative competence in infant human cardiomyocytes, and if so,
how this relates to the regenerative capacity of the human heart and the current timing
and strategies for repair of congenital heart disease
- Existing cardiomyocytes proliferate to replenish lost cardiomyocytes. Cardiomyocyte
refreshment through stimulation of endogenous pathways may allow newly formed
cardiomyocytes to integrate and recruit their own vasculature. In mature rodent
cardiomyocytes, cell cycle genes such as cyclin A, cyclin B, and CDC2 are downregulated, whereas cyclin-dependent kinase inhibitors are actively expressed. These cell
cycle regulators are under transcriptional and epigenetic control in cardiomyocytes. For
instance, the cell cycle inhibitors Ink4a/b are repressed by polycomb repressive complex
2 (PRC2), and inactivation of the PRC2 catalytic subunit EZH2 causes inappropriate fetal
up-regulation of Ink4a/b, reduction of cardiomyocyte proliferation, and myocardial
hypoplasia. Rb/p130-dependent heterochromatin formation at E2F-regulated cell cycle
genes also stimulated cardiomyocyte cell cycle withdrawal, and inactivation of Rb and
p130 led to reduced heterochromatin formation, cell cycle gene derepression, and adult
cardiomyocyte cell cycle reentry.
- Forced expression of individual cell cycle regulators has been evaluated as a means to
stimulate rodent cardiomyocyte cell cycle reentry. In some cases, this strategy
successfully stimulated cardiomyocyte proliferation but caused extensive, lethal cardiac
pathology. Cardiomyocyte-specific, transgenic expression of cyclin D2 was more
promising because it was not deleterious and successfully stimulated adult
cardiomyocyte DNA synthesis. Initial infarct size after MI was no different between
control and cyclin D2 transgenics, and infarct size progressively decreased over time,
consistent with effective regeneration. These data show that overexpression of some cell
cycle regulators may have salutary effects in heart disease.
- Coordinated activation of promitogenic gene programs may have greater success than
overexpression of individual cell cycle regulators. This goal might be achieved through
the redeployment of developmental regulatory circuits. The YAP transcriptional
coactivator is a major target of the Hippo signaling pathway, a highly conserved pathway
that governs cell proliferation and organ size. Mutations in the Hippo/YAP pathway that
enhance YAP transcriptional activity stimulated fetal cardiomyocyte proliferation and
caused profound cardiac overgrowth. In the adult heart, YAP-activating mutations
likewise promoted proliferation of mature cardiomyocytes, albeit less robustly than in the
fetal heart, and improved myocardial recovery after MI.
- MicroRNAs (miRNAs), which, like transcription factors, also coordinately regulate
multiple genes, may also offer a means to coordinately activate a mitogenic program in
adult cardiomyocytes. MiRNAs are attractive as therapeutic targets, because small RNAs
or their antagonists can be efficiently delivered as synthetic small molecules or
expressed in the heart using adeno-associated virus. A screen of miRNAs that enhance
neonatal cardiomyocyte proliferation identified miR-590 and miR-199a, and subsequent
testing showed that these stimulate adult cardiomyocyte proliferation and enhance
myocardial recovery after MI in mice. Gain- and loss-of-function approaches also showed
that the miR-17-92 cluster promotes cardiomyocyte proliferation. On the other hand, the
miR-15 family suppresses cardiomyocyte proliferation, and its inhibition by antagomirs
(sequence-specific miRNA inhibitors) stimulated cardiomyocyte proliferation and
improved outcome in a murine MI model.
- Overall, stimulating cardiomyocyte cell cycle reentry is an attractive strategy for
cardiac regeneration because it appears to be the major endogenous mechanism for
regeneration in both lower vertebrates and mammals. Likely this is because it leverages
endogenous, developmental mechanisms for functional heart growth. These innate
developmental programs generate new, autologous cardiomyocytes and ensure their
mechanical, electrical, and vascular integration into the myocardium.

Lung Damage and Disease

Lung diseases are some of the most common medical conditions worldwide. Tens of
millions of people suffer from lung disease in the U.S. Smoking, infections, and genetics
are responsible for most lung diseases. The lungs are part of a complex apparatus,
expanding and relaxing thousands of times daily to bring in oxygen and expel carbon
dioxide. Lung disease can result from problems in any part of this system. The airways
eventually branch into tiny tubes (bronchioles) that dead-end into clusters of air sacs
called alveoli. These air sacs make up most of the lung tissue. Lung diseases affecting
the alveoli include Pneumonia and Tuberculosis.
The normal response to lung injury: 1) Macrophages and neutrophils become
activated. 2) When you get infiltration of the immune system - secrete growth factors,
cytokines, interleukins and matrix but dont want too much or can lead to inflammatory
damage. 3) Nearby epithelial cells and fibroblasts secrete ECM and /or divide. These cells
secrete MMPs, reorganise cytoskeleton and migrate to close the wound. - Fibrosis can
occur Leads to scarring and causes the lung to become inefficient as scar tissue
reduces gaseous exchange. Injury response stops: cells stop dividing, immune cells go
and macrophages return to resting. Selective apoptosis allows local tissue remodelling
i.e. cell-based regeneration has to occur amongst all this. Its ok to work with 1 cell type
in a dish but regenerating a lung need to work with fibrosis and modulation of the
immune response as well.
Some factors involved in lung epithelium damage repair
EGFs: stimulates mitosis, migration, spreading (EGFR upregulated in asthmatics) - GF
acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell
surface and stimulating the intrinsic protein-tyrosine kinase activity of the receptor (see
the second diagram). The tyrosine kinase activity, in turn, initiates a signal
transduction cascade that results in a variety of biochemicalchanges within the cell - a
rise in intracellular calcium levels, increased glycolysisand protein synthesis, and
increases in the expression of certain genes including the gene for EGFR - that ultimately
lead to DNA synthesis and cell proliferation
HGF stimulates proliferation, migration - Hepatocyte growth factor regulates cell growth,
cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after
binding to the proto-oncogenic c-Met receptor. Hepatocyte growth factor is secreted
by mesenchymal cellsand acts as a multi-functional cytokine on cells of mainly epithelial
origin. Its ability to stimulate mitogenesis, cell motility, and matrix invasion gives it a
central role in angiogenesis, tumorogenesis, and tissue regeneration
KGF (keratinocyte growth factor) can aid repair - The Keratinocyte Growth
Factor (KGF), also known as FGF7, is a growth factor present in the epithelializationphase of wound healing. In this phase, keratinocytes are covering the wound, forming
the epithelium. KGF is a small signaling molecule that binds to fibroblast growth factor
receptor 2b (FGFR2b). For signalling to occur, a dimer is required between two FGF:FGFR
complexes that is linked together by a molecule of heparin
- Chemokine receptor and ligands e.g. CCR3 stimulates repair, increased in asthmatics
- Interleukins e.g. IL-2 via ERKs Stimulated migration, reduce apoptosis
- Eicosanoids inc. COX-1, COX-2, PGE2. Inhibition of COX blocks wound closure; PGE2
enhances wound closure Hormone molecule involved in signalling include
prostaglands/leukotrienes. They have various roles in inflammation, fever, regulation of
blood pressure, blood clotting, immune system modulation, control of reproductive
processes and tissue growth, and regulation of the sleep/wake cycle.
Lung stem cells
Endogenous lung stem cells: The lung is a complex organ containing many distinct
epithelial cell types that are distributed in several different regional microenvironments
along the pulmonary tract. Other considerations specific to the lung include a low
constitutive epithelial turnover rate. As such, lung injury models specific to particular

regions of lung epithelium, for example, sulfur dioxide, ozone, or nitrogen dioxide
inhalation (trachea, large airways), naphthalene administration (nonciliated club cells
[formerly known as Clara cells] in the bronchiolar epithelium), or bleomycin (alveolar
epithelium) have been used in mice and other adult animal models to identify
stem/progenitor cells by inducing cellular proliferation and repopulation of the lung
epithelium. For example, lineage tracing studies in mice have suggested that basal cells
can give rise to club cells and ciliated cells in the proximal airways during homeostasis
as well as after sulfur dioxide injury in mice.
Cell signaling pathways including b-catenin, Notch, and tissue factor appear to regulate
function and fate of the basal epithelial cells and other putative epithelial progenitor
populations. Similar conclusions have been derived using human proximal airway basal
epithelial cells in ex vivo or in vitro culture systems.
However, the situation is complex and there may be sub- populations of basal epithelial
cells that have more restricted lineages or specific roles. In distal mouse lung airways,
differentiated club epithelial cells, although exhibiting a low steady- state proliferative
index, can both self-renew and also function to replenish ciliated cells in both the trachea
as well as in the distal airways during normal homeostasis and also during injury.
- Alveolar epithelial repair and regeneration remain centered on the type 2 (ATII) alveolar
epithelial cell and the long held concept that ATII cells are precursors for type 1 alveolar
epithelial (ATI) cells. However, recent data suggest that several populations of distal
airway epithelial and other progenitor cells in adult mice, including BASCs and CK51/
p631 cells can differentiate into ATII and ATII cells in vitro and conceivably might
contribute to repair of damaged alveoli in in vivo models.
- There is also interest in the possible roles of endogenous or alveolar airway stem or
progenitor cells as lung cancer stem or tumor-initiating cells. Furthermore, MSCs, EPCs,
and fibrocytes may contribute to development of primary and metastatic lung carcinoma
and other malignancies in mouse models, in part, by providing a supportive stroma for
the cancers and/or by participating in tumor vascularization . In contrast, MSCs and EPCs
have been demon- strated to home to areas of tumor development and EPCs and MSCs,
engineered to express antitumor substances, including the tumor necrosis factor-related
apoptosis inducing ligand or IFNb, have been used to suppress tumor growth in mouse
tumor models of primary lung cancers, metastatic lung cancers, and of other cancers
metastatic to the lung. As such, a growing yet cautious investigation of MSCs and
perhaps other cell types in treatment of lung cancers is anticipated to occur over the
next several years.
Generating lung progenitor cells from ES cells: Early findings from several
laboratories demonstrated that both mouse and human ESCs could be induced in culture
to express surfactant proteins and lamellar bodies and even form pseudoglandular
structures suggestive of type 2 alveolar epithelial (ATII) cell phenotype. Other early
studies suggested development of cells with phenotypic markers of airway epithelial
cells following culture of the ESCs under air-liquid interface conditions. However, these
studies were limited by focus on generally one or two immunophenotypic markers, for
example, expression of surfactant protein, and it has never been clear that the derived
cells acquired appropriate functions of airway or alveolar cells. More recent protocols
incorporating more sophisticated understanding and application of cell signaling
pathways guiding embryologic lung development and development of definitive
endoderm, as well as newly developed lineage tracing tools such as Nkx2.1-GFP
expressing mice, have yielded more robust in vitro derivation of cells with phenotypic
characteristics of airway cells and of both type 2 (ATII) and type 1 (ATI) alveolar epithelial
cells from murine and human ESCs as well as from iPS cells, including those derived from
iPS cells obtained from patients with cystic fibrosis (CF). These derived cells can
repopulate decellularized whole lung scaffolds but other functional properties have yet
to be elucidated. The generation of disease-specific human ESC cells from patients with
CF and of iPS cell lines from patients with both genetic and acquired lung diseases
including CF, alpha 1 antitrypsin deficiency, sickle cell, and scleroderma provides further
opportunity to use iPS for study of lung diseases. Upon induction, they can express a

broad repertoire of markers indicative of lung and thyroid lineages and can recellularize
a 3D lung tissue scaffold.
Generating lung progenitors from human iPSCs
- Fortunately, a solution to the political and ethical hurdles that have limited ES cell
research is on the horizon. The remarkable discovery of reprogramming technology, by
Takahashi and Yamanaka in 2006, revealed that four transcription factors (Oct4, Klf4,
Sox2, and c-Myc) could reset the epigenetic state of somatic cells, such as skin
fibroblasts, into an embryonic- like stage virtually indistinguishable from ES cells.
Reproduction and refinement of this discovery have now established reprogramming as
an accepted technique to engineer induced pluripotent stem (iPS) cells from dermal
fibroblasts or peripheral blood cells, such as those obtained from simple skin punch
biopsies, or banked blood. Like ES cells, the broad differentiation repertoire of iPS cells
suggests their potential to form any desired somatic cell type, including lung epithelium .
In contrast to ES cells, iPS cells are genetically identical to the individual from whom they
are derived, raising the prospect of using iPS cells for autologous cellbased therapies
without risk of rejection.
- To establish clinically relevant pluripotent stem cell platforms for lung disease research,
some investigators have developed new reprogramming technologies able to derive
clinical grade iPS cells from human skin or blood and have successfully applied these
technologies to generate banks of lung disease specific iPS cell lines from patients
with a variety of end-stage lung diseases. Many groups are now focused on using
patient-specific iPS cell lines (and ES cells) to model lung diseases in vitro, to screen
drugs and gene therapy approaches, and to derive de novo replacement lung epithelia
and endothelial cells that may one day be transplanted back into the patients from
whom they have been derived . The lessons learned from the failed attempts to deliver
bone marrow derived cells to the injured lung epithelium have taught us that delivery of
iPS cellderived lung cells (or any other lung cell) in vivo will not be easy or
straightforward. - -- Still, precedent has already been set in rat models of Parkinsons
disease and mouse models of sickle cell disease, for example, where iPS-derived
replacement neurons and hematopoietic stem cells, respectively, can result in clinical
improvement when technology for the effective differentiation and transplantation of
these cells is carefully developed. In almost any field of research in which cells
reminiscent of the early embryo are employed, such as iPS cells or ES cells, recapitulating the milestones of early development of that tissue lineage has proven to be
the most effective and most efficient way to derive desired differentiated lineages in
vitro. Un- fortunately, little is known about the earliest stages of embryonic lung
development, a deficit that severely limits progress in deriving de novo lung epithelia
from pluripotent stem cells in vitro and hampers our ability to properly understand and
modulate repair after lung injury.
*****Deriving lung progenitors from patient-specific pluripotent cells is a key step in
producing differentiated lung epithelium for disease modeling and transplantation. By
mimicking the signaling events that occur during mouse lung development, we
generated murine lung progenitors in a series of discrete steps. Definitive endoderm
derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm,
then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic
lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP,
FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+
progenitor cells formed respiratory epithelium (tracheospheres) when transplanted
subcutaneously into mice. We then adapted this strategy to produce disease-specific
lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs),
creating a platform for dissecting human lung disease. These disease-specific human
lung progenitors formed respiratory epithelium when subcutaneously engrafted into
immunodeficient mice.
**Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the CFTR (cystic
fibrosis transmembrane conductance regulator) gene, which regulates chloride and

water transport across all epithelia and affects multiple organs, including the lungs. Here
we report an in vitro directed differentiation protocol for generating functional CFTRexpressing airway epithelia from human embryonic stem cells. Carefully timed treatment
by exogenous growth factors that mimic endoderm developmental pathways in vivo
followed by air-liquid interface culture results in maturation of patches of tight junction
coupled differentiated airway epithelial cells that demonstrate active CFTR transport
function. As a proof of concept, treatment of CF patient induced pluripotent stem cell
derived epithelial cells with a small-molecule compound to correct for the common CF
processing mutation resulted in enhanced plasma membrane localization of mature
CFTR protein. Our study provides a method for generating patient-specific airway
epithelial cells for disease modeling and in vitro drug testing.
Recapitulate lung development? - Human lung epithelial cell line + vascular
endothelial cells in 3D culture (matrigel) - The endothelial cells stimulate epithelial
branching. Background - Lungs develop from the fetal digestive tract where epithelium
invades the vascular rich stroma in a process called branching morphogenesis. In
organogenesis, endothelial cells have been shown to be important for morphogenesis
and the maintenance of organ structure. The aim of this study was to recapitulate
human lung morphogenesis in vitro by establishing a three dimensional (3D) co-culture
model where lung epithelial cells were cultured in endothelial-rich stroma. Methods - We
used a human bronchial epithelial cell line (VA10) recently developed in our laboratory.
This cell line cell line maintains a predominant basal cell phenotype, expressing p63 and
other basal markers such as cytokeratin-5 and -14. Here, we cultured VA10 with human
umbilical vein endothelial cells (HUVECs), to mimic the close interaction between these
cell types during lung development. Morphogenesis and differentiation was monitored by
phase contrast microscopy, immunostainings and confocal imaging. Results - We found
that in co-culture with endothelial cells, the VA10 cells generated bronchioalveolar like
structures, suggesting that lung epithelial branching is facilitated by the presence of
endothelial cells. The VA10 derived epithelial structures display various complex patterns
of branching and show partial alveolar type-II differentiation with pro-Surfactant-C
expression. The epithelial origin of the branching VA10 colonies was confirmed by
immunostaining. These bronchioalveolar-like structures were polarized with respect to
integrin expression at the cell-matrix interface. The endothelial-induced branching was
mediated by soluble factors. Furthermore, fibroblast growth factor receptor-2 (FGFR-2)
and sprouty-2 were expressed at the growing tips of the branching structures and the
branching was inhibited by the FGFR-small molecule inhibitor SU5402.
1) Circulating stem/progenitor cells can be recruited after lung damage
e.g. Adult bone marrow-derived progenitors including MSCs home to and extravasate
into sites of lung injury. Integrate and differentiate but at very low rate. MSCs of bone
marrow, adipose, placental tissue, and other origins are being widely investigated for
their immunomodulatory effects in a wide range of inflammatory and immune diseases MSCs relatively easily transduced and expanded (need to avoid senescence), retain
growth and multi-lineage potential - MSCS do not elicit an immune response (low HLAI,
low HLAII, low co-stimulatory molecules, and change cytokine secretion by immune
cells).
Mesenchymal stem cells (MSC) are a population of tissue-resident adult
progenitor cells that were origi- nally identified in bone marrow, but have now
been identified in many organs including the lung. Although their precise role
in organ function remains incom- pletely defined, mounting evidence suggests
that they are an important component of the parenchymal pro- genitor cell
niche and orchestrate organ homeostasis and repair following injury.
MSCs can be important immune modulators - Clinical trials e.g. healthy donor MSCs to
treat COPD preliminary data show reduced systemic inflammation but no significant
improvement in pulmonary function.
- The mechanisms by which MSCs are acting in the different lung disease models are not
fully understood and are likely to be different reflecting the different inflammatory and
immune environments in each disease. Following systemic administration, a number of

studies have demonstrated that MSCs initially localize in lung and that lung injury results
in increased localization and/or retention of marrow-derived cells in lung. Retention in
the lung may also trigger the cells to have functional effects. For example, embolization
of systemically administered MSCs in lung was felt to result in secretion of an antiinflammatory protein, TSG-6. In contrast, a growing number of reports suggest that
administration of conditioned media obtained from MSCs may mimic many of the
ameliorating effects resulting from MSC administration in different lung injury models.
- MSCs can also exert effects on lung inflammation and injury through primary
interactions with the immune system rather than through direct actions in lung. For
example, a growing body of evidence suggests that MSCs ameliorate allergic airways
inflammation in mice by increasing T- regulatory cells or by promoting a Th1 phenotype
in vivo in antigen-specific CD4 T-cells and in circulating antigen-specific immunoglobulins
as a means of abrogating Th2-mediated lung injury. As such, MSCs appear to be capable
of a spectrum of effects in different lung injuries and also in critical illnesses such as
sepsis. This is a critically important point as clinical use of MSCs must be tailored toward
the specific disease process.
- Other relevant factors about optimal cell preparations, storage and vehicle buffers,
dosing, and route of administration (systemic vs. direct airway) are poorly understood.
Furthermore, cell-based immunomodulation of lung diseases may not be restricted to
MSCs as a populations of bone marrow-derived mononuclear cells, human amniotic fluid
cells, and human amnion epithelial cells have each been recently described to decrease
lung injury in several immune-competent mouse models. These are ripe areas for further
study.
Optimising MSC-based Therapies - A robust preclinical literature supports use of
EPCs in pulmonary hypertension and MSCs in acute lung injury or inflammatory critical
illnesses and in more chronic inflammatory and immune mediated conditions such as
asthma, bronchiolitis obliterans, BPD, and others. Nonetheless, as preclinical lung
disease models do not necessarily fully mimic human disease pathogeneses or predict
clinical behaviors, clinical investigations of cell-based therapies for lung diseases have
been relatively slow to develop. A recent multicenter, double-blind, placebo-controlled
Phase II trial of systemic administration of a bone marrow- derived MSC preparation in
patients with moderate-severe COPD in the U.S. demonstrated safety with no acute
infusional toxicity and no attributable mortality or serious adverse events over a
subsequent 2-year follow-up period. Although the study was not powered to assess
efficacy, a significant early decrease in the systemic inflammatory marker C-reactive
protein (CRP) occurred in a subpopulation of MSC-treated patients with elevated CRP
levels at study onset. This trial provides a firm basis for safety of MSC use, including
multiple infusions, in patients with chronic lung diseases and also provides a potential
mechanistic clue of in vivo MSC effects. However, chronic persistent lung diseases with
low level or smoldering inflammation, such as COPD, or diseases in which currently
available preclinical data suggest that MSCs may worsen the disease process, such as
IPF, may not be the best therapeutic targets for MSC intervention at present. More acute
inflammatory lung or systemic diseases such as adult respiratory distress syndrome
(ARDS) or sepsis/septic shock, or chronic immune-mediated lung diseases such as
severe asthma, may be better targets. To this end, clinical trials of MSCs for ARDS and
for septic shock are currently in development in the U.S. and in Canada, respectively.
HUMAN STUDIES OF MSC THERAPY IN LUNG DISEASE - The multifaceted activity of
MSC has translated into a large body of clinical trial activity outside the lung, most
notably in the treatment of steroid refractory graft versus host disease following
allogeneic bone marrow transplant, but also in other immune- mediated diseases like
Crohns disease, multiple sclerosis, lupus and in the renal transplant setting. The tissue
repair capability of MSC is being investigated in clinical trials for cardiac repair, bone
disorders (osteogenesis imperfecta), bone fracture and following meniscectomy.
However, there has only been one large clinical trial of MSC therapy for lung disease,
which is as yet unpublished. The safety and efficacy of intravenous ex vivo cultured
adult human MSC for subjects with moderate to severe chronic obstructive pulmonary

disease was tested in an industry- sponsored phase II, double-blind, placebo-controlled

trial which has just been published. Human adult MSC were derived from normal healthy
adult volunteer bone marrow donors. A total of 62 patients, with a diagnosis of moderate
(n = 23) or severe (n = 39) chronic obstructive pulmonary disease were enrolled. All
patients completed the planned course of four infusions without any evidence of
infusional toxicity. Adverse event rates were comparable for patients receiving MSC and
placebo, but the pulmonary function efficacy end-point was not met.
- Our group has initiated two human phase I trials of MSC therapy for lung disease. In the
first study (NCT01175655), the primary objective is to establish the safety of infusions of
MSC from related or unrelated HLA-identical or HLA-mismatched donors in the
management of BOS after lung transplantation. The secondary objectives are to
document changes in lung function, 6-min walk distance and survival following MSC
infusion. Patients (n = 10) with single, bilateral or heart-lung allografts and deteriorating
chronic allograft dysfunction manifesting as either BOS grades 2 & 3, or grade 193 with
an additional risk factor for subsequent death, receive open-label treatment with 2X106
MSC/kg bodyweight intravenously twice weekly for 2 weeks. Thus far, six patients have
been treated with no evidence of toxicity. The second study (NCT01385644) is an openlabel, non- randomized, dose-escalation evaluation of the safety and feasibility of
intravenous placental MSC treatment for subjects diagnosed with idiopathic pulmonary
fibrosis. This study has recently closed to recruitment. MSC have been delivered
intravenously to eight patients with moderate (forced vital capacity > 50% of predicted
and diffusing capacity for carbon monoxide > 25% of predicted) idiopathic pulmonary
fibrosis. Four received 1X106 cells/kg and the next four received 2X106 cells/kg. The
primary end- point was again safety, with particular interest in the potential for acute
infusional toxicity and in the medium term, increased fibrosis. There has been no
evidence of acute infusional toxicity, but follow up is ongoing to assess the medium-term
safety of MSC treatment for this indication.
2) The role of mechanical forces during organ (inc. lung) regeneration
- In lung tissue engineering, bioreactor design that allows the application of physiological
forces in vitro to support lung cell survival and differentiation is proving to be important
for whole lung regeneration. By studying the in vitro growth of native, explanted lungs,
our group has demonstrated the importance of ventilatory movements for retaining the
epithelial differentiation state of AT1, AT2, and Clara cells. Furthermore, ventilatory
movements alone were sufficient to enable maintenance of cell differentiation state and
cell survival of native lungs in vitro for 1 week. In engineered lungs, perfusion of medium
through the vasculature increased endothelial adherence and survival. Additionally,
ventilation was found to impact the epithelial differentiation state, specifically the
flattening of cultured alveolar epithelium to a phenotype more consistent with AT1
epithelium. However, much more work will be required to identify the proper balance
and exact characteristics of perfusion and ventilation necessary for in vitro whole lung
regeneration.
- Externally applied mechanical forces are transmitted to cells via the ECM, primarily via
integrin-mediated interactions, where integrins essentially function as force transducers.
Mechanical forces are known to play key roles in cell function: cyclic mechanical strain
enhances replication of fetal rat epithelium; and short periods of cyclic mechanical
stretch (in contrast to long periods of cyclic stretch and static stretch) enhance adult AT2
surfactant secretion. Furthermore, mechanical transduction is matrix dependent: straininduced SPC mRNA expression by fetal AT2 is higher on laminin-1 and collagen I than on
vitronectin, fibronectin, and elastin. This strain-induced SPC expression is mediated via
integrins 3, 6, and 1 and is transduced intracellularly via a transcellular cytokeratin
network. For an in-depth review of mechanical force transduction in epithelium, the
reader is referred to recent comprehensive reviews.
- Exposure of lung epithelium to an air interface has been shown to be beneficial for
maintaining and promoting epithelial differentiation. When airway epithelial cells are
cultured in vitro, ciliogenesis is induced at an air interface and is reduced in the absence
of an air interface. Furthermore, an air-liquid interface is required for maintenance of AT2

differentiation state, and encourages surfactant secretion by AT2 cells. Besides matrix
effects, the air-liquid interface is important in maintaining AT2 differentiation in many in
vitro systems.
In vitro work on mechanical forces and cell biology
To facilitate tissue maturation, Ott et al (2009) designed a bioreactor system that
subjects acellular lung scaffolds to mechanical stretch similar to that imposed by normal
fetal circulation and breathing movements, necessary components of fetal lung growth
and development. Within 5 d in isolated organ culture, seeded cells engrafted on
acellular lung scaffolds, formed endothelium and formed rudimentary respiratory and
alveolar epithelium. Most importantly, recellularization and isolated organ culture led to
reestablishment of an intact alveolar-capllary barrier of physiologic thickness,
regenerating the morphologic basis of gas exchange. Number and size of alveoli as
functional units did not differ from those of native lung. In vitro ventilation and blood
perfusion confirmed physiologic ventilatory mechanics and gas exchange capacity.
Orthotopic transplantation of regenerated lungs requires physiologic size, shape, airway
and vascular structures, and mechanical properties that allow ventilation by means of
the recipients respiratory muscles. After cell seeding of acellular lung scaffolds and
tissue maturation in vitro, we successfully transplanted regenerated left lung constructs
after experimental left pneumonectomy. Regenerated lungs were perfused through the
recipients pulmonary artery and vein and were ventilated through the recipients
trachea. Rats could be extubated after transplantation and breathed room air without
the support of a ventilator. The observed development of pulmonary secretions and
interstitial edema may be related to lack of lymphatic drainage, capillary leak of
immature vessels, and ventilation trauma.
Mechanical force effects: Both repair mechanisms following injury and normal
epithelial turnover occur within an environment and upon a substrate that is undergoing
cyclic mechanical deformation. Furthermore, the role of interactions between mechanical
and biochemical signals in the lung is not well understood. This is particularly important
in light of the potential for alterations in repair in diseases that involve changes in lung
mechanics such as IPF, asthma, and COPD. In addition, patients with ALI placed on
mechanical ventilators may experience elevated levels of mechanical distention that
may contribute to injury or affect repair mechanisms. To investigate the latter possibility
in vitro, we used cell lines (Calu-3, 1HAEo-) and primary cultures of human bronchial
epithelial cells to demonstrate that both cyclic stretch and cyclic compression
significantly inhibited wound closure by inhibiting cell spreading and cell migration.
Inhibition was dependent on the magnitude of mechanical strain, and, interestingly, the
extent of inhibition was dependent on the duration of strain during each cycle and not
specifically the frequency of stretch. In contrast to the inhibitory effects on spreading
and migration, mechanical stretch and compression were shown to stimulate BrdU
incorporation in wounded bronchial epithelial cells in culture. It was also found that
treatment with KGF abrogated the inhibitory effects of mechanical strain on wound
repair by promoting spreading and migration. More recently, it was demonstrated that
mechanical stretch of wounded 16HBE14o- cells caused loss of focal adhesion kinase
(FAK) phosphorylation, dissociation of JIP-3 (JNK-interacting protein 3) from FAK, and
decreased activation of JNK. Also, ATII cells isolated from rats following 2 h of high tidal
volume mechanical ventilation (25 ml/kg with 50% oxygen) exhibited decreased
phosphorylation of FAK and paxillin, increased RhoA activity, and decreased cell
adhesion. Wound repair of cultured rat ATII cells was inhibited by adenoviral expression
of a kinase- inactive from of FAK (FRNK), whereas repair was stimulated by
overexpression of wild-type FAK. Also, cyclic stretch of rat ATII cells caused decreased
wound repair through mechanisms involving decreased Tiam1-mediated Rac1 activity.
Although it is well-recognized that there are spatial variations in the localization and
activity of Rho GTPases, focal adhesion, and other signaling molecules during
coordinated cell migration, the relationship between these dynamic processes and the
local mechanical properties of the cells are less well understood. In addition to inhibitory
effects on early processes of epithelial restitution, mechanical forces may also negatively

impact epithelial repair in other ways. Several groups have demonstrated that
mechanical stretch of cells in culture or high tidal volume in vivo causes ATII cell injury
and necrosis. However, the interplay between mechanical and biochemical signaling
during repair processes in vivo has not been extensively studied.
Lung on a chip
- Lung on a chip describes a biomimetic microsystem that reconstitutes the critical
functional alveolar-capillary interface of the human lung. This bioinspired microdevice
reproduces complex integrated organ-level responses to bacteria and inflammatory
cytokines introduced into the alveolar space. In nanotoxicology studies, this lung mimic
revealed that cyclic mechanical strain accentuates toxic and inflammatory responses of
the lung to silica nanoparticles. Mechanical strain also enhances epithelial and
endothelial uptake of nanoparticulates and stimulates their transport into the underlying
microvascular channel. Similar effects of physiological breathing on nanoparticle
absorption are observed in whole mouse lung. Mechanically active organ-on-a-chip
microdevices that reconstitute tissue-tissue interfaces critical to organ function may
therefore expand the capabilities of cell culture models and provide low-cost alternatives
to animal and clinical studies for drug screening and toxicology applications.
- Preclinical drug development studies currently rely on costly and time-consuming
animal testing because existing cell culture models fail to recapitulate complex, organlevel disease processes in humans. There is proof of principle for using a biomimetic
microdevice that reconstitutes organ-level lung functions to create a human disease
model-on-a-chip that mimics pulmonary edema. The microfluidic device, which
reconstitutes the alveolar-capillary interface of the human lung, consists of channels
lined by closely apposed layers of human pulmonary epithelial and endothelial cells that
experience air and fluid flow, as well as cyclic mechanical strain to mimic normal
breathing motions. This device was used to reproduce drug toxicityinduced pulmonary
edema observed in human cancer patients treated with interleukin-2 (IL-2) at similar
doses and over the same time frame. Studies using this on-chip disease model revealed
that mechanical forces associated with physiological breathing motions play a crucial
role in the development of increased vascular leakage that leads to pulmonary edema,
and that circulating immune cells are not required for the development of this disease.
Next, the authors checked if the IL-2-induced pulmonary edema-on-a-chip model is
applicable to testing pharmacological agents. Using the microchip, they first tested
angiopoietin-1 (Ang-1), a known endothelial junction stabilizer (Peters, 1998). Consistent
with the previous reports, Ang-1 co-administration with IL-2 completely restored IL-2induced loss of integrity in vascular-epithelial barrier function. Additionally, Huh et al.
demonstrated that a new transient receptor potential vanilloid 4 (TRPV4) ion channel
inhibitor, GSK2193874 (GlaxoSmithKline) completely blocked IL-2-induced increase in
vascular-epithelial permeability (Thodeti et al., 2009). They also confirmed the
physiological relevance of these in vitro results by duplicating IL-2-induced pulmonary
edema using an ex vivo mouse ventilation-perfusion model. However, there are
limitations to Huh et al.'s microdevice: (1) The lung epithelial cells used in the system
are cancer cells not primary cells, which raises the question that the system might not
closely recapitulate the real pulmonary function; (2) The system lacks interstitial
fibroblasts and alveolar macrophages, cells involved in maintenance of lung
homeostasis; (3) The microchip-based experiment, which requires well-equipped
laboratory and an experienced technician, would not be easily reproduced.
Extracellular matrix (ECM) and regeneration
The extracellular matrix (ECM) provides tissue structure, modulates fluid dynamics, and
regulates cell behavior. The lung extracellular matrix is composed principally of collagen
and elastin, which are interwoven with fibronectin fibrils and proteoglycans, and allow
plastic deformation of the lung parenchyma. In addition, other native lung ECM
components include laminins, heparan sulfate proteoglycans, entactin, hyaluronate,
chondroitin sulfate, and glycosaminoglycans. In the lung, collagen subtypes I, III, IV, and
V are predominant. Types I and III are the principal structural collagens, while type IV is a
key basement membrane component. Elastin is an important ECM component in tissues

that require reversible distension, and elastin allows for the intrinsic recoil property of
lung tissue.
Fibronectin is a dimeric cell-adhesive glycoprotein that is important for the adherence of
a variety of pulmonary cell types to the extracellular matrix and interacts with cells to
impact their morphology, motility, and differentiation. Fibronectin is important in
vascular and airway development and during wound healing, providing a cell adhesion
and guidance system. Fibronectin is deposited at airway-matrix interfaces during wound
repair and stimulates epithelial migration. Fibronectin, fibrin, and vitronectin are
components of the provisional matrix secreted initially after lung injury, which is
important in epithelial growth and repair.
- Proteoglycans are proteins that are found on cell surfaces, within intracellular vesicles,
and incorporated into the ECM. Proteoglycans can impact a protein's structure and
function, and also help control macromolecular and cellular movement across the basal
lamina. Laminins assist cell binding through integrins and other receptors. There are at
least eleven different types of these large molecules, and they interact with cells in
complex fashions, allowing laminins to affect cell shape and permeability.
- A wide range of matrix molecules are known to mediate epithelial attachment and
survival, depending on cell type, including collagens I and IV, laminin, fibronectin, and
vitronectin. In the case of AT2 cells, adherence is strongest to fibronectin, and weaker to
laminin, vitronectin, or collagens I, III, or IV. Laminin-rich matrices favor AT2
differentiation, while fibronectin-rich matrices accelerate dedifferentiation54. The ECM
also significantly impacts epithelial differentiation. For example, adult-derived AT2 cells
maintain an AT2 phenotype on collagen and laminin-5 substrates, but differentiate
towards AT1 cells when cultured on collagen or laminin-5 alone. It is likely that local
matrix cues within acellular lung matrix direct not only cell adhesion, but also
differentiation of seeded cell types.
- The ECM also impacts epithelial migration via specific integrin-mediated interactions
between cells and the surrounding ECM. For example, epidermal growth factor facilitates
wound repair via integrin 1 binding on epitopes present in collagen type IV, laminin-1,
and laminin-2 substrates. Additionally, binding of integrin subunits 2, 3, and 6 enables
migration of airway epithelium on collagen type IV substrates. Integrin mediators of
adult AT2 cell adherence to fibronectin are primarily 1 and v3, while fetal-derived AT2
adherence to laminin-1 is primarily mediated via integrins 6 and 1.
- The ECM significantly impacts epithelial differentiation, including stem cell
differentiation. Lin et al. reported that laminin-332 and Matrigel coatings led to increased
SPC expression by ESC-derived AT2 cells. Adult-derived AT2 cells maintained an AT2
phenotype on collagen and laminin-5 substrates, but differentiated towards AT1 cells
when cultured on collagen or laminin-5 alone. The interplay between the matrix
substrates cues, and those provided by specific growth factors and by exposure to airliquid interface, together regulate epithelial differentiation and survival. Therefore, the
ECM is an important part of the milieu that controls epithelial survival, differentiation,
and function of pulmonary epithelium. The evidence that the ECM impacts the growth of
engineered lung tissue is accumulating. Studies have shown the importance of natural
matrices in enabling alveolar development in vitro, as well as the beneficial effects of a
native, decellularized matrix on whole engineered lung growth. However, the specific
components of lung matrix that are responsible for lung tissue growth in engineered
systems are not yet clear. This will become one of the most important focus areas for
lung regeneration in the near term.
Laryngeal tissue engineering
Although considerable progress has been made in regenerative medicine in recent
years, replacement of functional, reinnervated tissues using tissue-engineered
approaches remains a major challenge for generating active movement, as required for
replacing partial or entire larynges that require mobile vocal cords, or an esophagus that
is capable of normal peristalsis. Thus, although replacement of airways, blood vessels, or
urogenital tissue using stem cell-based techniques has been achieved, engineering of

entire larynges and vocal cords has only been partially explored.
- Autologous tissue-engineered solutions have the major advantage of not requiring
immunosuppression, but clinical applications are presently limited to static organs and
tissues, such as skin, or those which can function through passive movement alone, such
as trachea, heart valves, blood vessels, bladder, and urethra. The field, however,
continues to expand, and tissue bioengineering has already provided, or is close to
delivering, functional human organ replacements elsewhere. The ability to produce
innervated and revascularized tissues would hugely extend the possible clinical
applications of regenerative medicine and has been discussed elsewhere.
- It is hoped that in time, the experience gained in tissue engineering tracheas can be
applied to more complex airways, namely tissue engineering of entire larynges and vocal
cords. Indeed, progress is starting to be made in this field. The key requirement for
successful laryngeal transplantation is restoration of normal vocal cord movements
necessary for normal breathing and speech. The larynx has one of the most
sophisticated actions of any muscular-based organ in the human body, as may be
appreciated by listening to high-performance singing. This complexity is reflected by the
comparatively high density of motor axons in the recurrent laryngeal nerve, as
compared with the small sizes of the intrinsic laryngeal muscles that they innervate.
Restoration of near-normal mobility of the arytenoids, and hence of the vocal cords, is
essential. Reinnervation, however, remains a significant challenge. Recent research in
this area, however, suggests that this may be overcome by the simultaneous use of
nerve grafts, neurotrophic factors, and/or electrical (laryngeal) pacing.
- Full-scale phase I/II clinical trials are now required and are currently underway in the UK
(RegenVOX study) to fully assess the stem cell-based tissue-engineered airway's efficacy
and safety. In the case of this trial, decellularized laryngotracheal implants will be
seeded with autologous-derived stem cells and implanted into 10 patients with severe
acquired laryngotracheal stenosis (Myer Cotton Grade 3 or 4) due to traumatic,
inflammatory, iatrogenic, or idiopathic causes who have exhausted conventional
therapies. Patients will be followed up for at least 2 years. Longer follow-up (i.e., 2 years
or longer) is necessary to assess the long-term retention of the graft's biomechanical
properties, to determine the fate of the seeded stem cells and their contribution toward
the regeneration seen, and to monitor for signs of restenosis, rejection, or the presence
of anti-donor antibodies.
***Paolo Macchiarini and colleagues (Dec 13, p 2023)1 present a well developed,
streamlined process by which a tracheal graft could be regenerated in a bioreactor, and
applied to treat a patient with a large airway defect. They developed a nature-derived
tracheal matrix from donor tracheal tissues. By the 25th cycle of decellularisation, the
matrices were fully removed of antigens. Macchiarini and colleagues then used this
tubular matrix as a scaffold on which to engineer native tracheal tissues by seeding
autologous epithelial cells and mesenchymal stem cells. However, Macchiarini and
colleagues do not indicate whether this tracheal matrix would eventually be replaced by
newly formed cartilage tissues. Such a decellularised matrix would degrade completely
in vivo, and would lose its original supporting role, which would lead to the collapse of
the airway. We suggest detailed follow-up of morphological changes to the patients
trachea.
- Furthermore, Macchiarini and colleagues seeded cells with a density of 10106/mL
onto the surface of the scaffold. For cartilage tissue engineering, the preferred cell
density is 50107/mL, because of chondrocytes limited proliferate and migrating ability
in vivo. In this study, how many cells could penetrate into the pores of the scaffold, and
could the above cell density provide sufficient cells for further tissue formation? Perhaps
such data could be presented in in-vitro specimens.
- Although revascularisation has been noticed in the inner surface of the graft, we think
it will be difficult for the seeded epithelial cells to survive and form mucosal tissue at this
site, because of the length of time full revascularisation will take and of the very limited
nutritional perfusion from surrounding tissues. We suggest that Macchiarini and
colleagues collect the patients sputum postoperatively and analyse the cells in it so that

we can ascertain whether the seeded epithelial cells have been chipped out and
discharged.
- The main drawback of the proposed reconstruction is the lack of an intrinsic blood
supply. Avascular tissue is known for its unpredictable healing when used for airway
reconstruction. In Macchiarini and colleagues procedure, reconstruction was done with a
tracheal transplant colonised by the recipients epithelial cells and chondrogenic
mesenchymal cells. Histological analysis of the full thickness of the cartilaginous and
membranous tracheal construct at the time of implantation would have allowed for a
comparison of the transplant histology with that of the normal tracheal wall. Moreover,
although the volume- rendered CT images and virtual bronchoscopic images provide
superb pictures of a stenotic and a patent main bronchus, they provide no clear
information about the reconstructive value of the tissue-engineered tracheal transplant.
Have now sped up trachea production: Baiguera et al (2010) performed the first
clinical successful transplantation of a fully tissue engineered trachea. Despite the
clinically positive outcome, the graft production took almost 3 months, a not feasible
period of time for patients with the need of an urgent transplantation. They improved
decellularization process and herein and described the obtainment of human tracheal
bioactive supports. Histological and molecular biology analysis demonstrated that all
cellular components and nuclear material were removed and quantitative PCR confirmed
it. SEM analysis revealed that the decellularized matrices retained the hierarchical
structures of native trachea, and biomechanical tests showed that decellularization
approach did not led to any influence on tracheal morphological and mechanical
properties. Moreover immunohistological staining showed the preservation of angiogenic
factors and angiogenic assays demonstrated that acellular human tracheal scaffolds
exert an in vitro chemo-active action and induce strong in vivo angiogenic response
(CAM analysis). They are now able to obtained, in a short and clinically useful time
(approximately 3 weeks), a bioengineered trachea that is structurally and mechanically
similar to native trachea, which exert chemotactive and pro-angiogenic properties and
which could be successfully used for clinical tissue engineered airway clinical
replacements.
Quality control of tissue for grafting: Baiguera et al (2010) work suggests that 25
cycles of the DEM generates a bioengineered human tracheal matrix that is structurally
and mechanically similar to native trachea and that contains angiogenic factors which
exert chemotactive and pro-angiogenic properties. Moreover, additional positive aspect
of tracheal matrices obtained with DEM has to be highlighted. The time taken to
complete the decellularization process can have a significant clinical (and commercial)
impact reducing production, and hence, patient costs. In their approach, 25
decellularization cycles correspond to more or less 30 days of processing; however, the
length of the single steps can be arranged (without altering the final number of DEM
cycles) in order to reduce the clinic time necessary to obtain complete trachea
decellularization (17 days). This is the first study which completely describes and
characterizes the obtainment of human tracheal bioactive supports which provide a
natural environment for cellular growth and differentiation and which will allow clinically
functioning, fully in vivo tissue engineered airway replacements in the very near future.
Immunogenicity of Implanted Scaffolds: A critical assumption for clinical use of
decellularized lung scaffolds is that they will be relatively non-immunogenic and
minimize any detrimental host response following implantation. However, ECM and other
proteins remaining in decellularized scaffolds can provoke immune responses.
Interestingly, some of these may be beneficial as growing literature suggests that
decellularized scaffolds can polarize macrophages to anti- inflammatory M2 phenotype
with subsequent permissive effects on implanted scaffolds. With respect to lung,
proteomic assessments utilizing mass spectrometry and/ or western blotting
demonstrate that a wide range of residual proteins, including intracellular, nuclear,
cytoskeletal, and others can remain in the lungs, despite apparent effective decellularization. Whether these residual proteins provoke immune responses is currently
the focus of intense inquiry. Theoretically, despite conservation of ECM proteins, any

denuded basement membrane may provoke an immune response, in part to mobilize

the necessary cells to cover the damaged membrane. Whether cells inoculated into
decellularized scaffolds will secrete ECM and other proteins and remodel the scaffold
accordingly is also the subject of intense current investigation.
The grafts are pro-angiogenic: - In the study by Haag et al (2011), the obtainment
and characterization of decellularized rat tracheal grafts are described. The detergentenzymatic method, already used to develop bioengineered pig and human trachea
scaffolds, has been applied to rat tracheae in order to obtain airway grafts suitable to be
used to improve our knowledge on the process of tissue-engineered airway
transplantation and regeneration. The results demonstrated that, after detergentenzymatic cycles, almost complete decellularized tracheae, retaining the hierarchical
and mechanical properties of the native tissues with strong in vivo angiogenic
characteristics, could be obtained. Moreover, to improve the mechanical properties of
decellularized tracheae, genipin is here considered as a naturally derived cross-linking
agent. The results demonstrated that the treatment increased mechanical properties, in
term of secant modulus, without neither altering the pro-angiogenic properties of
decellularized airway matrices or eliciting an in vivo inflammatory response.
- Macroscopic observations of CAM treated with decellularized and genipin-treated
(0.25%, 0.5% and 1%) tracheal matrices showed that all samples resulted surrounded by
allantoic vessels that developed radially toward the implant in a spoke-wheel pattern.
New vessels were sometimes arranged in loops around the samples, suggesting that rat
tracheal decellularized and genipin-treated matrices could positively affect the growth
and the organization of the network of CAM vessels. The effect of tracheal matrices on
direct blood vessel growth was quantified as the total number of blood converging
vessels. All the evaluated samples induced a significant (p < 0.05) increased compared
to that induced by PBS (used as negative control), and a comparable effect to the one
induced by VEGF (used as positive control). In particular, the increase induced by
tracheal matrices treated with genipin 1% resulted significantly (p < 0.05) different from
the one induced by all the other samples, suggesting a stronger pro-angiogenic effect.
The CAM assay is quick, technically simple, and inexpensive. However, a major drawback
is that it is labor intensive due to the large number of eggs that are required to obtain
consistent results. The investigator should note that this is a non-mammalian system
which should be taken into consideration when interpreting results.
Next step: Because adult lung tissue has limited regeneration capacity, lung
transplantation is the primary therapy for severely damaged lungs. To explore whether
lung tissue can be regenerated in vitro, Peterson et al (2010) treated lungs from adult
rats using a procedure that removes cellular components but leaves behind a scaffold of
extracellular matrix that retains the hierarchical branching structures of airways and
vasculature. We then used a bioreactor to culture pulmonary epithelium and vascular
endothelium on the acellular lung matrix. The seeded epithelium displayed remarkable
hierarchical organization within the matrix, and the seeded endothelial cells efficiently
repopulated the vascular compartment. In vitro, the mechanical characteristics of the
engineered lungs were similar to those of native lung tissue, and when implanted into
rats in vivo for short time intervals (45 to 120 minutes) the engineered lungs
participated in gas exchange. Although representing only an initial step toward the
ultimate goal of generating fully functional lungs in vitro, these results suggest that
repopulation of lung matrix is a viable strategy for lung regeneration.
Whole lung regeneration: Recent work has demonstrated progress toward the goal of
developing functional engineered lungs. In two recent studies employing similar
techniques, investigators used decellularized lung scaffolds as the basis for functional
lung tissue that can participate in gas exchange. The lungs of adult rats were first
treated with detergent solutions to remove all cellular material, leaving behind a scaffold
of extracellular matrix, principally composed of collagen and elastin. These scaffolds
were then reseeded with endothelial and epithelial cells through the vasculature and
airways, respectively, and cultured ex vivo to allow the cells to repopulate the scaffold.
Two independent groups were able to demonstrate that the resultant engineered tissues

could be transplanted into rats and could efficiently exchange gas during time periods of
up to 6 hours.
- Lungs can be decellularized in a number of ways, and the optimal method for removing
cells while retaining salient matrix properties has not yet been identified. In work from
our laboratory, we utilized a combination of hypertonic salt solutions, zwitterionic
detergents, and alkalinity, while other groups have used other detergents including: the
ionic detergent sodium dodecyl sulfate (SDS); the ionic detergent sodium deoxycholate;
and the nonionic detergent triton X. An impressive difference between decellularization
methodologies is the time required for treatment, which ranges from approximately 2 3
hours to 5 weeks. Complete characterization of these varying methods and identification
of optimal methodologies will require continued in-depth analysis of the resultant
decellularized matrices. In particular, retention of the fine ultrastructure of the lung
matrix, including intact capillary basement membranes and alveolar septae, will be an
important index of decellularization success. In addition, any decellularization method
must create a matrix that supports both epithelial and vascular endothelial adhesion and
differentiation. Beyond examining the micro-architecture of the lung matrix, we must
develop tools to allow us to characterize the proteins and glycoproteins present at each
anatomical location in the lung, so that we can have benchmarks for assessing adequacy
of matrix preservation after decellularization.
- Cell sources utilized thus far in whole lung regeneration have been multiple. Our group
used neonatal rat epithelial cells (post-natal day 7) and adult rat endothelial cells, while
Ott and colleagues used a combination of rat fetal epithelium (day 18) and human adult
endothelium. Meanwhile, Price and co-workers employed fetal mouse cells and Cortiella's
group used mouse embryonic stem cells. In all cases, AT2 cells were most abundant,
while most groups also noted Clara cells. Ciliated epithelium, however, was less
commonly observed in repopulated lung matrices: our studies found only sparse ciliated
epithelia in large airways when lungs were cultured under air ventilation, while Cortiella
found isolated regions of ciliated epithelium in the trachea. Better repopulation of large
airway epithelium may be aided by culture with mesenchymal stem cells, which have
been shown to support the differentiation ability of human bronchial epithelial cells by
paracrine mechanisms. Additional cell types found in some reports of whole engineered
lungs included basal cells and sparse BASC. Since differentiated human pulmonary
epithelium is difficult to expand in vitro, it is likely that future studies in human lung
regeneration will focus on epithelial cells sourced from progenitor populations, such as
embryonic stem cells or induced pluripotent stem cells.
Acellular matrix scaffolds: An alternative approach is to utilize whole lungs in which
all cells and cellular materials are removed leaving an intact 3-dimensional scaffold
comprised of innate extracellular matrix (ECM) proteins in a bio-mimetically similar 3dimensional architecture. This approach, termed decellularization, preserves native
airway and vascular structure and provides an acellular matrix for cell seeding and
functional recellularization. This approach also provides a novel culture system to study
cell-matrix interactions and environmental factors such as mechanical stretch on lung
cell growth and development. This technique was originally described many years ago,
one classic example is by Lwebuga- Mukasa and colleagues in 1986 in which a
decellularized rat lung was utilized to study the effect of the basement membrane on
growth of type II alveolar epithelial (AEII) cells.
Seeding the bioscaffold: - Mixed populations of neonatal rat lung epithelial cells into
the airway - The cells stuck to alveoli, small and medium airways - Rapidly proliferated
and rarely died (much better than cell culture plastic) -Microvascular lung endothelial
cells injected into the pulmonary artery adhered throughout the scaffold vasculature They took source of rat lung epithelial cells and put onto 3D lung scaffold could do this
with more sophisticated sub types of cells.
Seeding decellularized lung scaffolds with whole lung fetal or post-natal cell suspensions
has the potential advantage of providing a model of spontaneous self-assembly and
facilitating natural cell-cell interactions that could potentially improve organotypic

growth. When fetal rat (E17) whole lung suspensions were delivered intratracheally to
decellularized rat lungs and cultured in a bioreactor, cells adherent in the alveolar
compartment expressed markers of ATII (CK18 and pro-surfactant C (proSPC)); markers
of type I alveolar epithelial (ATI), endothelial, mesenchymal, or club cells (formerly
known as Clara cells) were absent31. E17 lung cells are enriched for ATII cells so it is
possible that this observation relates to the difficulty to induce ATII cells to convert to ATI
cells under these conditions. In contrast, seeding with rat fetal lung cells (E1720)
resulted in multilineage engraftment again, mainly in the alveolar compartment, with
expression of markers of ATII cells such as pro-surfactant A (proSPA), proSPC, thyroid
transcription factor (TTF-1/Nkx2.1), ATI (T1), and fibroblasts (vimentin); concurrent
inoculation with human umbilical vein endothelial cells (HUVEC) by way of the
pulmonary artery showed retention along the entire vasculature and close apposition of
endothelial and alveolar epithelial cells suggestive of perfusion of distal lung33. Larger
airways were only sparsely engrafted in this model. Neonatal (P7) whole lung cell
suspensions exhibited similar multilineage engraftment including evidence of distal
airway repopulation with club cells (club cell secretory protein (CCSP+,CC10)) and basal
cells (CK14+), as well as alveolar engraftment of ATII cells (proSPC+) and ATI cells
(Aqp5+); endothelium engrafted the pulmonary vasculature and formed tight junctions.
While it is difficult to ascertain the precise geospatial distribution of cells, it is clear that
fetal or post-natal lung homogenates can repopulate scaffolds with cells exhibiting a
wide range of phenotypes with preferential distribution to the alveoli and distal airways.
Seeding acellular rat scaffolds with mouse ESCs resulted in both greater survival
compared to cells seeded onto non-lung matrices (Matrigel, gelfoam, or collagen) and
also apparent differentiation toward multiple lineages that exhibited region-specific
distribution, including club cells (CC10+), ATII cells (CK18+, proSPC+), endothelial cells
(CD31+), and mesenchymal cells (PDGFR+). Thus it is theoretically possible to observe
differentiation of the most primitive of stem cells along the lines of development simply
by seeding them on acellular scaffolds. However, the efficiency of this system was not
explicitly evaluated. Seeding of decellularized mouse lungs with definitive endoderm
derived from ESCs and subsequent culture of lung slices resulted in spontaneous
differentiation to elongated type I alveolar epithelium expressing T1 (podoplanin) that
distributed along the alveolar septae; in contrast, inoculation with parent ESCs produced
hypercellular sheets of disorganized cells lining both the alveoli and some ciliated cells
along airways. Similarly, mouse ESCs differentiated to Nkx2.1+, proSPC+ ATII-like cells
directly seeded onto mouse acellular scaffolds that were implanted subcutaneously
distributed to airways (FoxJ1pos) and alveolar regions (proSPC+ or PDGFR+ cells) and
maintained phenotype expression for 14 days. In parallel, host-derived endothelial cells
infiltrated the scaffolds suggesting that functional vascularization might occur. These
studies also demonstrated that Matrigel as a vehicle for seeding increased the frequency
of proSPC, TTF-1, PDGFR, and FoxJ1 positive cells after 14 days, implying that
biomimetic basement membranes may preserve heterogeneity in populations of lung
endoderm derived from ESC or iPS cells, particularly in the airway epithelial fractions.
However, no data is as yet available examining the behavior of iPS cells at any stage of
differentiation in decellularized lung scaffolds.
Used a biomimetic bioreactor: Decellularized lung is mounted in bioreactor - Culture
medium is perfused through at physiological pressures - Air pumped in and out of
reactor to allow lung to inhale and exhale , The injected cells were cultured on the
scaffold for 8 days, Perfusion and the breathing movements helped the cells adhere
and survive. Exposure to air is beneficial for maintaining and promoting epithelial
differentiation, so a transition may have to be made from liquid to air ventilation in vitro.
Because of the leaky nature of acellular and newly seeded extracellular matrix
scaffolds, quantitative models or visual information about the actual stretch
experienced by the cells within the scaffold will be of use as well.
Transplanted the engineered lung

- No visible air leaks - Rapidly perfused with host blood - Gas exchange occurred (short
period)
Methods of DecellularizationCreation of organ scaffolds requires removal of the
native cell population while minimizing alterations to the dimensions and mechanical
characteristics of the organ, the structural support for the airway, vascular and
lymphatic networks, and to the composition of the native matrix including important cell
binding ligands. Common methods for decellularization of lung tissue pieces include
sonication, sieving, and extraction of thin pieces of lung tissue and digestion with acetic
acid followed by sonication. While useful techniques for developing in vitro systems to
study lung biology, these methods did not preserve the 3 dimensional architecture of the
lung. Recently, several techniques have emerged for decellularizing whole lungs which
retain the 3 dimensional architecture as well as key extracellular matrix proteins.
Human tests of this technique needs improvements
- Alveolar barrier function must be improved to prevent blood leakage - production of
surfactant should be increased - differentiated columnar ciliated epithelium - the
efficiency of vascular endothelial coverage must be very high to prevent exposure of
collagen-containing basement membrane to the circulation, with consequent thrombosis,
because some clotting was noted - Need a suitable, autologous source of pulmonary
epithelial cells
Discussion from paper- To date, cell therapy and tissue engineering have been
applied less extensively to lung than to other tissues and organs. Many efforts in lung
regeneration have involved synthetic scaffolds or simple in vitro culture systems. Such
systems can regenerate certain microscopic features of alveolar architecture but have
not yet produced tissue that can participate in gas exchange. Use of the decellularization
paradigm for respiratory tissue was described in 2008, when Macchiarini and colleagues
implanted a reseeded tracheal matrix into a patient with severe bronchomalacia.
Peterson et al (2010) have demonstrated the feasibility of producing an engineered lung
that displays much of the microarchitecture of native lung and that can effect gas
exchange for short periods of time when implanted into rats. Although these results are
encouraging, multiple issues remain to be addressed before long-term engineered lung
function can be realized. For example, alveolar barrier function must be improved so as
to prevent any leakage of blood components into the airways. This can be accomplished
through iterative improvement in the decellularization procedure in order to minimize
alveolar septal damage. Production of surfactant should be increased, and differentiated
columnar ciliated epithelium should be enhanced by more prolonged air breathing in
culture. In addition, the efficiency of vascular endothelial coverage must be very high
throughout the engineered lung vasculature. This is to prevent exposure of collagencontaining basement membrane to the circulation, with consequent thrombosis, because
some clotting was noted at explant. Lastly, a decellularization strategy for lung
regeneration will only become clinically useful when a suitable, autologous source of
pulmonary epithelium can be identified, such as a resident lung stem cell or induced
pluripotent stem cell.
Rapid progress
- Uygun et al., 2010 Nature Medicine - Same approach taken with liver - Orthotopic liver
transplantation is the only available treatment for severe liver failure, but it is currently
limited by organ shortage. One technical challenge that has thus far limited the
development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we
demonstrate a novel approach to generate transplantable liver grafts using
decellularized liver matrix. The decellularization process preserves the structural and
functional characteristics of the native microvascular network, allowing efficient
recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for
in vitro culture. The recellularized graft supports liver-specific function including albumin
secretion, urea synthesis and cytochrome P450 expression at comparable levels to
normal liver in vitro. The recellularized liver grafts can be transplanted into rats,
supporting hepatocyte survival and function with minimal ischemic damage. These

results provide a proof of principle for the generation of a transplantable liver graft as a
potential treatment for liver disease.
Combine stem cell derived cells and bioscaffold - You can combine bioscaffold with
genetically modified stem cells can do procedure with iPSCs and combine with scaffold
to generate a lung mimic with loads of different cell types. Could engineer to not have
CF as well. Two populations of Nkx2-1(+) progenitors in the developing foregut
endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is
known about these cells because they are difficult to isolate in a pure form. Longmire et
al (2012) demonstrate here the purification and directed differentiation of primordial
lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Following
BMP/TGF inhibition, subsequent combinatorial induction of BMP and FGF signaling
promotes initial lineage specification of endodermal Nkx2-1+ lung and thyroid
progenitors. These primordial progenitors can be sorted to purity and thereafter
expanded and differentiated in culture. In contrast, prolonged exposure to BMP/TGF
inhibitors results in highly efficient derivation in culture of Nkx2-1+ neuroectodermal
progeny that do not express any endodermal, lung, or thyroid markers, revealing precise
control in culture over the germ layer lineage specification of cells expressing an
otherwise nonspecific transcriptional regulator. Upon induction, they can express a broad
repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D
lung tissue scaffold. Thus, we have derived a pure population of progenitors able to
recapitulate the developmental milestones of lung/thyroid development.
Moving closer to the lung clinic?
- Currently, patients with end-stage lung disease are limited to lung transplantation as
their only treatment option. Unfortunately, the lungs available for transplantation are
few. Moreover, transplant recipients require life-long immune suppression to tolerate the
transplanted lung. A promising alternative therapeutic strategy is decellularization of
whole lungs, which permits the isolation of an intact scaffold comprised of innate
extracellular matrix (ECM) that can theoretically be recellularized with autologous stem
or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly
relevant preclinical model with which to assess the feasibility of recellularized lung
scaffolds for human lung transplantation. Our laboratory has successfully accomplished
lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an
NHP model.
- Decellularization of normal adult rhesus macaque lungs as well as the biology of the
resulting acellular matrix have been extensively characterized. Acellular NHP matrices
retained the anatomical and ultrastructural properties of native lungs with minimal effect
on the content, organization, and appearance of ECM components, including collagen
types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to
decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue
extracts due to the removal of cells and cellular proteins by decellularization. Cellular
DNA was effectively removed after decellularization (92% reduction), and the
remaining nuclear material was found to be highly disorganized, very-low-molecularweight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells
(MSC) attach to the decellularized lung matrix and can be maintained within this
environment in vitro, suggesting that these cells may be promising candidates and
useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which
MSC were seeded showed that the cells attached to the decellularized matrix, elongated,
and proliferated in culture. Future investigations will focus on optimizing the
recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue.
Bringing this technology to eventual human clinical application will provide patients with
an alternative therapeutic strategy as well as significantly reduce the demand for
transplantable organs and patient wait-list time.
Hippo signalling impedes adult heart regeneration - Heallen et al. (2013)
investigated Hippo signaling in adult cardiomyocyte renewal and regeneration. They
found that unstressed Hippo-deficient adult mouse cardiomyocytes re-enter the cell

cycle and undergo cytokinesis. Moreover, Hippo deficiency enhances cardiomyocyte

regeneration with functional recovery after adult myocardial infarction as well as after
postnatal day eight (P8) cardiac apex resection and P8 myocardial infarction. In
damaged hearts, Hippo mutant cardiomyocytes also have elevated proliferation. Our
findings reveal that Hippo signaling is an endogenous repressor of adult cardiomyocyte
renewal and regeneration. Targeting the Hippo pathway in human disease might be
beneficial for the treatment of heart disease.
- The Hippo signalling pathway, which was discovered in Drosophila melanogaster, is
evolutionarily conserved and has a key role in regulating cell proliferation and organ
size. This pathway comprises a series of adaptors and kinases that promote the
phosphorylation of the transcriptional co-activator Yes-associated protein (YAP), thereby
preventing its nuclear entry. Specifically, the mammalian STE20-like protein kinase 1
(MST1) and MST2 kinases interact with the cofactor Salvador (SAV); this enables them to
phosphorylate and activate a complex consisting of large tumour suppressor homologue
1 (LATS1) or LATS2 and MOB (Mps one binder), which phosphorylates and inactivates
YAP. Inactivation of YAP has a role in maintaining organ size during normal development,
and dysregulation of this inhibition has been reported to cause cancer. Genetic loss of
function of Sav in the mouse heart results in perinatal lethality owing to extreme
enlargement of the heart, which is due to expansion of the trabecular and subcompact
ventricular myocardial layers. Ablation of the SAV-interacting kinases MST1 and MST2, or
of the downstream kinase LATS2, causes a similar myocardial phenotype in embryonic
mouse hearts. Enlarged hearts resulting from alterations in the Hippo signalling pathway
are not attributable to cardiomyocyte hypertrophy but are due to increased
cardiomyocyte proliferation.
Strategies for the Use of MSCs in Lung Tissue Engineering: To date, several kinds
of stromal cells, such as bone marrow and adipose-derived mesenchymal stem cells,
have been introduced to decellularized lung scaffolds. Decellularized lungs that are
inoculated with MSCs result in cellular deposition mainly in the parenchyma, and to a
lesser extent in the small and larger airways. Where the MSCs attach and bind is largely
deter- mined by the ECM components of the decellularized scaffold, which in turn is
highly defined by the method utilized to de- cellularize the scaffold. Bonvillain et al.
found that the murine MSC initially adhere to areas rich in collagen I and laminin as well
as fibronectin-rich areas, while in a macaque model they showed substantial preference
for laminin above all other matrix molecules. Studies by Daly et al. revealed substantial
homing of inoculated MSCs to collagen I, collagen IV, and laminin-rich areas, but very
little binding of MSCs to fibronectin. There is mounting evidence that the ECM contributes to the functional behavior of MSCs. Cells with different known integrin
expression have different binding characteristics on the decellularized matrix. Overall,
determination of cell-matrix interactions will be key in under- standing the regional
specification of MSCs on a decellularized lung scaffold. Although MSCs bind to and
proliferate on decellularized scaffolds, it is unclear what phenotypes they assume under
dif- ferent culture conditions. Over a 28-day culture of MSCs on decellularized mouse
lungs in small airway growth medium and basal medium, only transient expression of
TTF1 was detected, and no other epithelial markers. Other groups reported that rhesus
marrow-derived MSCs as well as adipose MSCs in the decellularized matrix did not have
epithelial marker expression after seven days of culture. Overall, successful employment
of interstitial cells for lung tissue engineering may require a combination of fibroblasts
and MSCs. Based on the ratio of cell types in an intact lung, a 1:1 or 2:1 ratio of
interstitial cells to epithelium would be most appro- priate. A combination of MSCs with
epithelium, followed by supplementation with differentiated fibroblasts, would provide
epithelial support while minimizing the likelihood of the epithe- lium being overrun by
MSCs. MSCs are less proliferative, less migratory, and produce less ECM than fibroblasts.
As a source of MSCs, bone marrow and adipose-derived MSCs are most accessible. Each
of these cell types has been used clinically for other applications; these populations are
generally regarded as safe. Bone marrow MSCs have even demonstrated the ability to

support the lung epithelium during acute injury by mitochondrial transfer. However, the
ability of these cells to perform appropriately in the context of lung tissue engineering
has not been clearly established. Efforts in the near term will center on exploring bone
marrow and adipose-derived MSCs for tissue engineering. Long-term, lung-resident MSCs
will be the most appropri- ate MSC source. Human lung-MSCs instilled into mouse lungs
localize to the alveolar compartment, where they secrete keratinocyte growth factor, a
key growth factor for lung epithelium. They also show higher initial retention and greater
longevity in the lung, as compared to fibroblasts [144]. One caveat is that there may be
a variety of lung-MSC subpopulations. One recent report of MSCs derived from the
mature lung, for example, in- dicates that CD166- lung-MSCs support the formation of
lung epithelium stem cell colonies in vitro, while CD166+ lung- MSCs do not. Like
epithelial cells used for lung tissue engineering, the MSC population that is used may
ultimately have to be a heterogeneous one. Regardless, interstitial cells, including both
MSCs and fibroblasts, will be an indispensable component of a successfully engineered
lung.

Bioscaffolds and Tissue Engineering

Bioscaffolds
Cells are often implanted or 'seeded' into an artificial structure capable of supporting
three-dimensional tissue formation. Bioscaffolds in therapy need to be tolerated by the
patient as it is no use stimulating a huge immune response as it would create more
problems and inserting a bioscaffold is of importance as it can create another entry route
for further infections. The bioscaffold has to interact with surrounding tissue as well
especially difficult with bone (and skin) bone has to resist lots of forces. But bioscaffold
can damage surrounding tissue. Design bioscaffold so it disappears after a few weeks
designed to degrade make sure it doesnt degrade into toxic compounds.
- To achieve the goal of tissue reconstruction, scaffolds must meet some specific
requirements. A high porosity and an adequate pore size are necessary to facilitate cell
seeding and diffusion throughout the whole structure of both cells and nutrients.
Biodegradability is often an essential factor since scaffolds should preferably be
absorbed by the surrounding tissues without the necessity of a surgical removal. The
rate at which degradation occurs has to coincide as much as possible with the rate of
tissue formation: this means that while cells are fabricating their own natural matrix
structure around themselves, the scaffold is able to provide structural integrity within
the body and eventually it will break down leaving the neotissue, newly formed tissue
which will take over the mechanical load. Injectability is also important for clinical uses.
- With new technologies, an alternative to the classical way of forming the scaffold has
become possible, namely, socalled organ printing, which allows the production of more
complex scaffolds. In the area of Tissue Engineering, 3D printers combined with image
processing software, allow scaffolds to be produced from small fragments. It is possible
to direct the assembly of these fragments to form a more complex scaffold, with
complete control of the internal structure, including a vascular tree. This latter feature is
critical and tends to be the limiting factor in the production of various TE prototypes.
Another important source of scaffolds are native tissues and organs, which have been
decellularized, by physical and chemical means. Decellularization enables all the
extracellular matrix of a tissue to be recovered, removing the original cells and replacing
them by cultured autologous cells. With this technique scaffolds have been obtained
from the intestinal mucosa, dermis, tendons and adipose tissue. Decellularization
techniques, first described some years ago, have come back into vogue. One of the
advantages of this approach is that the native vascular tree can be used to deliver
chemical agents to achieve organ decellularization. With this strategy, it has been
possible to obtain organs such as the heart, liver and even lungs16 with their internal

structure intact, including the blood vessels. These scaffolds may be the basis for the
future development of complex organs using TE techniques.
Scaffold requirements: Numerous scaffolds produced from a variety of biomaterials
and manufactured using a plethora of fabrication techniques have been used in the field
in attempts to regenerate different tissues and organs in the body. Regardless of the
tissue type, a number of key considerations are important when designing or
determining the suitability of a scaffold for use in tissue engineering:
(i) Biocompatibility: The very first criterion of any scaffold for tissue engineering is that
it must be biocompatible; cells must adhere, function normally, and migrate onto the
surface and eventually through the scaffold and begin to proliferate before laying down
new matrix. After implantation, the scaffold or tissue engineered construct must elicit a
negligible immune reaction in order to prevent it causing such a severe inflammatory
response that it might reduce healing or cause rejection by the body.
(ii) Biodegradability: The objective of tissue engineering is to allow the body's own
cells, over time, to eventually replace the implanted scaffold or tissue engineered
construct. Scaffolds and constructs, are not intended as permanent implants. The
scaffold must therefore be biodegradable so as to allow cells to produce their own
extracellular matrix. The by-products of this degradation should also be non-toxic and
able to exit the body without interference with other organs. In order to allow
degradation to occur in tandem with tissue formation, an inflammatory response
combined with controlled infusion of cells such as macrophages is required. Now that
tissue engineering strategies are entering clinical practice more routinely, the field of
immunology is a playing a role of increasing prominence in the research area.
(iii) Mechanical properties: Ideally, the scaffold should have mechanical properties
consistent with the anatomical site into which it is to be implanted and, from a practical
perspective, it must be strong enough to allow surgical handling during implantation.
While this is important in all tissues, it provides some challenges for cardiovascular and
orthopedic applications specifically. Producing scaffolds with adequate mechanical
properties is one of the great challenges in attempting to engineer bone or cartilage. For
these tissues, the implanted scaffold must have sufficient mechanical integrity to
function from the time of implantation to the completion of the remodeling process 8. A
further challenge is that healing rates vary with age; for example, in young individuals,
fractures normally heal to the point of weight-bearing in about six weeks, with complete
mechanical integrity not returning until approximately one year after fracture, but in the
elderly the rate of repair slows down. This too must be taken into account when
designing scaffolds for orthopedic applications. However, as the field has evolved, it
could be argued that too much focus has been placed on trying to develop scaffolds with
mechanical properties similar to bone and cartilage. Many materials have been produced
with good mechanical properties but to the detriment of retaining a high porosity and
many materials, which have demonstrated potential in vitro have failed when implanted
in vivo due to insufficient capacity for vascularization. It is clear that a balance between
mechanical properties and porous architecture sufficient to allow cell infiltration and
vascularization is key to the success of any scaffold.
(iV) Scaffold architecture: The architecture of scaffolds used for tissue engineering is
of critical importance. Scaffolds should have an interconnected pore structure and high
porosity to ensure cellular penetration and adequate diffusion of nutrients to cells within
the construct and to the extra-cellular matrix formed by these cells. Furthermore, a
porous interconnected structure is required to allow diffusion of waste products out of
the scaffold, and the products of scaffold degradation should be able to exit the body
without interference with other organs and surrounding tissues. The issue of core
degradation, arising from lack of vascularization and waste removal from the centre of
tissue engineered constructs, is of major concern in the field of tissue engineering.
Another key component is the mean pore size of the scaffold. Cells primarily interact
with scaffolds via chemical groups (ligands) on the material surface. Scaffolds
synthesized from natural extracellular materials (e.g. collagen) naturally possess these
ligands in the form of Arg-Gly-Asp (RGD) binding sequences, whereas scaffolds made

from synthetic materials may require deliberate incorporation of these ligands through,
for example, protein adsorption. The ligand density is influenced by the specific surface
area, i.e. the available surface within a pore to which cells can adhere. This depends on
the mean pore size in the scaffold. The pores thus need to be large enough to allow cells
to migrate into the structure, where they eventually become bound to the ligands within
the scaffold, but small enough to establish a sufficiently high specific surface, leading to
a minimal ligand density to allow efficient binding of a critical number of cells to the
scaffold. Therefore, for any scaffold, a critical range of pore sizes exists, which may vary
depending on the cell type used and tissue being engineered. The final criterion for
scaffolds in tissue engineering, and the one which all of the criteria listed above are
dependent upon, is the choice of biomaterial from which the scaffold should be
fabricated.
Example of a common bioscaffold: collagen: As a major ECM protein of the dermal
layer of the skin, collagen is the most widely studied and clinically utilized natural
scaffold available for TES substitutes. The advantages include good biocompatibility,
proper porous structure, as well as low immunogenicity. However, the poor mechanical
strength and rapid biodegradation rate of natural collagen scaffolds limiting the graft
instability are the critical disadvantages that hamper its applications. Therefore, to
control the degradation, numerous works have focused on the mechanical properties of
collagen, such as chemical and biophysical cross-linking techniques or a structural
modification method like dense film. For example, addition of matrix protein tropoelastin
to type I collagen enhanced the proliferation and migration rates of dermal fibroblasts in
vitro. Cross-linking collagen with chitosan after electrospinning resulted in a good
potential for keratinocyte migration and wound re-epithelialization. Addition of fibroblast
growth factor 2 (FGF-2) or vascular endothelial growth factor (VEGF) to heparin crosslinked collagen scaffold increased its angiogenic potential. Although effective
modification and improvement have been made, other discouraging problems in
collagen-based polymers for skin tissue engineering still exist, including the high cost of
purification process, potential viral and prior contamination, and variability in the
physicochemical properties depending on source and processing. Plaiting collagen Collagen-based biomaterials are a viable option for tendon reconstruction and repair.
However, the weak mechanical strength of collagen constructs is a major limitation.
Kishore et al (2012) have previously reported a novel methodology to form highly
oriented electrochemically aligned collagen (ELAC) threads with mechanical properties
converging on those of the natural tendon. In this study, they assessed the in vivo
response of rabbit patellar tendon (PT) to braided ELAC bioscaffolds. Rabbit PTs were
incised longitudinally and the ELAC bioscaffold was inlaid in one limb along the length of
the tendon. The contralateral limb served as the sham-operated control. Rabbits were
euthanized at 4 or 8 months postoperatively. High-resolution radiographs revealed the
absence of ectopic bone formation around the bioscaffolds. Four months postimplantation, the histological sections showed that the ELAC bioscaffold underwent
limited degradation and was associated with a low-grade granulomatous inflammation.
Additionally, quantitative histology revealed that the cross-sectional areas of PTs with the
ELAC bioscaffold were 29% larger compared with the controls. Furthermore, ELACtreated PTs were significantly stiffer compared with the controls. The volume fraction of
the tendon fascicle increased in the ELAC-treated PT compared with the controls. By 8
months, the ELAC bioscaffold was mostly absorbed and the enlargement in the area of
tendons with implants subsided along with the resolution of the granulomatous
inflammation. They conclude that ELAC is biocompatible and biodegradable and has the
potential to be used as a biomaterial for tendon tissue engineering applications.
Bioscaffolds dont have to be passive e.g. cell signalling by Integrins - Might be
enough to have strong woven fibrous mesh put into wound site but might also want it to
be less passive, might want it so signal? Could engineer it to have cross links to initiate
cell signalling. Collagen can have integrins bound to it can have downstream effects in
cell drive protein synthesis? Or cell suicide? Cell proliferation and survival, division and
migration?

Case study: collagen scaffolds for bone tissue engineering - Collagen is the most
common protein in the body and provides strength and structural stability to tissues in
the body including skin, blood vessels, tendon, cartilage and bone. Along with
hydroxyapatite, collagen is one of the two major components of bone. It makes up 89 %
of the organic matrix and 32 % of the volumetric composition of bone. As such, it has
significant potential for culturing cells to produce bone. In our laboratory, we typically
combine collagen (Type I) with gycosaminoglycan, a polysaccharide found in many
tissues in the body, and, using a controlled freeze drying process, produce a highly
porous collagen-GAG (CG) scaffold. The scaffolds we have developed are variants of the
very first scaffold developed for tissue engineering applications. These were developed
by Prof. Ioannis Yannas at MIT and received FDA approval to regenerate skin in burns
patients. The development of the scaffold by Yannas, and subsequent clinical approval,
was one the key steps in the embryogenesis of the field of tissue engineering and led to
the formation of Integra Life Sciences who still sell the CG dermal graft and are now one
of the leading companies in regenerative medicine worldwide.
- In common with all natural polymers, one major problem with using collagen as the
main constituent of a scaffold for orthopedic tissue engineering is that it has relatively
poor mechanical properties. However, we have demonstrated that the compressive and
tensile mechanical properties of collagen and CG scaffolds can be improved through
physical and chemical cross-linking methods. We have also identified the optimal
composition for osteogenesis and optimal pore structure to facilitate bone tissue
formation. Taken together, these studies have led to the development of a CG scaffold
with an optimized composition, cross-linking density and pore size for bone regeneration
and we have demonstrated the ability of these scaffolds to heal bone defects in vivo in
minimally loaded calvarial defects. However, while these CG scaffolds have shown
immense promise for bone repair in minimally weight-bearing regions of the body; in
order to facilitate repair of regions where the scaffolds are subjected to higher levels of
loading, we have strengthened these collagen-based scaffolds by introducing a ceramic
phase and have thus developed a series of highly porous biomimetic collagenhydroxyapatite (CHA) scaffolds based on the two primary constituents of bone.
- These scaffolds not only possess significantly increased mechanical properties
compared to a CG scaffold while retaining the highly porous and interconnected pore
structure, but also show improved permeability which benefits cell infiltration and
subsequent vascularization. A comparative in vivo analysis between the CHA and CG
scaffolds has demonstrated enhanced healing in the former scaffold. The reasons for this
are two-fold: (i) the enhanced mechanical properties and permeability provided by the
CHA scaffold allowed improved cellular infiltration and vascularization and (ii) the
presence of calcium phosphate produced an osteoinductive response whereby its
chemical composition enhanced the osteogenic potential of the host cells resulting in
increased bone formation. The osteoinductive potential of calcium phosphates has been
noted previously. The presence of the HA phase in these biomimetic scaffolds thus also
imparts a bioinstructive facet to the materials. We also compared healing in cell-free
scaffolds and scaffolds which had been cultured with mesenchymal stem cells (MSCs) for
4 weeks prior to implantation, i.e. an in vitro tissue engineered approach. Interestingly,
we found reduced healing in the tissue engineered constructs which was caused, as
revealed by immunological analysis, by excess matrix deposited by MSCs during in vitro
culture, which adversely affected healing by acting as a barrier to macrophage-led
remodeling when implanted in vivo. In other words, the study demonstrated that in
addition to tissue engineered constructs failing after implantation because the scaffolds
are insufficiently porous, they may also fail if the in vitro engineered tissue which has
been formed on the scaffolds has been over-engineered leading to core degradation
following implantation. This is consistent with a number of studies, which have
demonstrated how a major barrier to clinical success in the field of tissue engineering is
this issue of core degradation.
- Further developments aimed at differentiating mesenchymal stem cells directly in
collagen-based biomaterial, to permanently solve osteochondral defects on a long-term

basis, are currently under investigation. Optimization of pore size and distribution is also
a concern considering the effect of these parameters on cell adhesion, proliferation and
migration. Decellularization of complex structures like meniscus has also shown
promising results in order to produce an optimal replacement scaffold for specific
osteochondral defects.
Skin and cornea - Skin and cornea share a similar tissue structure: dermis and stroma
both being connective tissues; epidermis and cornea being stratified epithelia. Collagenbased wound dressings have been applied for decades for burn coverage applications
and ulcer treatment. Highly sophisticated and innovative tissue-engineered skin models
have been developed with melanocytes, a capillary- like network, dendritic cells, sensory
innervation, adipose tissue, and tissue reproducing psoriatic or sclerotic phenotypes. A
living allogenic reconstructed skin (Apligraf), made of a collagen gel-populated
fibroblasts overlayed by an epidermis, is commercialized for ulcer treatment as a
temporary dressing. Skin, dermal substitutes and dressing such as Integra (acellular
collagen-GAG scaffold), AllodermTM (human dermis), AmniographTM (amniotic
membrane) and OasisTM (porcine SIS) are currently available for medical applications.
Mesenchymal stem cell delivery to the wound bed in collagen-based biomaterial is a
growing topic in wound healing. The combination of collagenous biomaterials and stem
cells could also be a valuable strategy to treat corneal defects. In the last decade,
collagen scaffolds have been intensively studied for the delivery of limbal epithelial stem
cells to damaged cornea. Advances in collagen-based corneal scaffolds also include the
utilization of recombinant human collagen, the secretion of collagen by the fibroblasts
themselves (self-assembled fibroblasts sheets) and surface modification to reduce
extensive endothelialization.
Tissue engineering is a field in which our knowledge in the life sciences and medical
fields is applied using engineering principles, in order to design therapeutic treatments
that positively affect tissue function. While tissue engineering encompasses three
approaches: Cells and cell substitutes, factors for tissue induction and the seeding of
cells onto matrices, the most common tissue engineering approach is the third one, to
place cells onto a biomaterial matrix. This combination is designed to grow tissue in
vitro, prior to implantation within the subject. The design of the scaffold prior to
exposure to cells is of vital importance. The scaffold must present a surface that
promotes cell attachment, growth and differentiation, while providing a porous network
for tissue growth. The material chosen is of great importance when designing a scaffold.
It must degrade at a rate matching that of the new tissue formation must be
biocompatible and the products of its degradation must be biocompatible. Once
implanted, the scaffold must have the mechanical properties necessary to temporarily
offer structural support until the new tissue has formed. In addition to consisting of an
effective biomaterial, the scaffold must also possess key morphological characteristics. It
must be highly porous and offer a suitable path for nutrient transmission and tissue
ingrowth. To achieve these requirements, tissue-engineering scaffolds are often designed
to mimic the structure of the naturally occurring extracellular matrix (ECM).
Examples of materials used in Bioscaffolds
Many different materials (natural and synthetic, biodegradable and permanent) have
been investigated. Most of these materials have been known in the medical field before
the advent of tissue engineering as a research topic, being already employed as
bioresorbable sutures. Examples of these materials are collagen and some polyesters.
New biomaterials have been engineered to have ideal properties and functional
customization: injectability, synthetic manufacture, biocompatibility, nonimmunogenicity, transparency, nano-scale fibers, low concentration, resorption rates,
etc. PuraMatrix, originating from the MIT labs of Zhang, Rich, Grodzinsky and Langer is
one of these new biomimetic scaffold families which has now been commercialized and
is impacting clinical tissue engineering. A commonly used synthetic material is PLA polylactic acid. This is a polyester which degrades within the human body to form lactic
acid, a naturally occurring chemical which is easily removed from the body. Similar

materials are polyglycolic acid (PGA) and polycaprolactone (PCL): their degradation
mechanism is similar to that of PLA, but they exhibit respectively a faster and a slower
rate of degradation compared to PLA. Scaffolds may also be constructed from natural
materials: in particular different derivatives of the extracellular matrix have been studied
to evaluate their ability to support cell growth. Proteic materials, such as collagen or
fibrin, and polysaccharidic materials, like chitosan or glycosaminoglycans (GAGs), have
all proved suitable in terms of cell compatibility, but some issues with potential
immunogenicity still remains. Among GAGs hyaluronic acid, possibly in combination with
cross linking agents (e.g. glutaraldehyde, water soluble carbodiimide, etc), is one of the
possible choices as scaffold material. Functionalized groups of scaffolds may be useful in
the delivery of small molecules (drugs) to specific tissues. Another form of scaffold under
investigation is decellularised tissue extracts whereby the remaining cellular
remnants/extracellular matrices act as the scaffold.
e.g. tissue engineering Bioengineered skin
- The skin is the largest organ of the body in vertebrates and is composed of the
epidermis and dermis with a complex nerve and blood supply. A third layer the
hypodermis, is composed mainly of fat and a layer of loose connective tissue. These
three layers play an important role in protecting the body from any mechanical damage
such as wounding. The epidermis is thin and highly cellular, composed mainly of
keratinocytes and in the lower epidermal layers, melanocytes, for pigmentation. It does,
however, have sufficient thickness to provide vital barrier function. Mammalian
epidermis and its appendages (hair, nail, sweat and sebaceous glands) maintain
homeostasis by constant recycling of the basal cell layer. The dermis situated directly
below the epidermis, constitutes the bulk of the skin and is composed of collagen with
some elastin and glycosaminoglycans (GAG's). Fibroblasts are the major cell type
present in the dermis, and are capable of producing remodelling enzymes such as
proteases and collagenases, which play an important role in the wound healing process.
The dermis also plays a major role in the biomechanics of the skin, and this will be
discussed in a later section of this review. The final layer, the hypodermis, contains
adipose tissue that is well vascularised and contributes to both the thermoregulatory
and mechanical properties of the skin.
- The stability of this complex organ is threatened by damage caused by trauma or injury
and millenary evolutionary development has led to the development of rapid wound
repair mechanisms in mammals as opposed to a more regenerative outcome. Rational
design of a smart skin replacement for grafting, must therefore account for the cellular
mechanisms used during wound repair. According to MacNeil, three factors are key to
developing tissue-engineered materials: - patient safety - clinical efficacy and convenience of use. These three factors, however, encompass a larger series of
functional parameters that must be addressed which include adequate attachment of
the tissue-engineered construct to the wound bed, supported by neovascularisation and
graft acceptance by the host immune system. Scarring at the margins of a skin graft is
also problematic clinically, physically, mechanically, psychologically and from an
aesthetic and cosmetic viewpoint.
Skin: several stem cell populations
The epidermis comprises the interfollicular epidermis, hair follicles, sebaceous glands
and sweat glands.
Interfollicular epidermis: The interfollicular epidermis generates a protective barrier
against dehydration and external aggressions. Hair follicles are scattered in the
interfollicular epidermis and give rise to the hair shafts. The permanent portion of the
hair follicle consists of the infundibulum, the junctional zone, isthmus and bulge. The
schematic shows the hair follicle in resting phase (telogen), during which the dermal
papilla lies right below the secondary hair germ. Attached to the side of the hair follicle is
the sebaceous gland, which contributes to the generation of the isolating barrier of the
epidermis with its secretions. Interestingly, the higher expression of 1 integrin by
quiescent mouse interfollicular stem cells has also been observed for human epidermal

stem cells, with quiescent and proliferative pools being distinguished by several
markers. For instance, quiescent human interfollicular stem cells not only express higher
levels of 6 and 1 integrins but also lower levels of the transferrin receptor CD71.
Quiescence of human interfollicular stem cells also relies on the activity of Delta 1, MCSP
(melanoma-associated chondroitin sulphate proteoglycan), LRIG1 (Leu-rich repeats and
immunoglobulin-like domains 1)34, the polycomb protein.
- However, the location of human quiescent epidermal stem cells within the basal layer
is still under debate, and three different hypotheses have been proposed. One of these
suggests that cells with the highest expression of 6 integrin and keratin 15 located at
the bottom of the rete ridges (structures on the basal layer of the human interfollicular
epidermis) are relatively quiescent and display a higher clonogenic potential than the
more proliferative and keratin 15 dimmer upper-rete ridge basal cells. However, there is
also strong evidence indicating that the brightest 1 integrin, MCSP and LRIG1+
quiescent cells clustered at the top of the rete ridges harbour the highest stemness of all
basal cells. Interestingly, the basal layer of the epidermis of infants shows homogeneous
expression of keratin 15 throughout, whereas in the adult human epidermis basal cells at
the tip of the ridge express higher levels of this protein than those at the bottom.
Considering that the studies regarding 6 integrin and keratin 15 used adult
keratinocytes, whereas those characterizing 1 integrin+ and LRIG1+ cells were carried
out in keratinocytes obtained from young donors, this apparent controversy might simply
reflect the different age of the epidermis used.
-- Hair follicles contain several pools of spatially distributed stem cells that are defined
by unique molecular signatures and differentially contribute to hair follicle cycling. The
vertical axis of the hair follicle determines different proliferative and regenerative
capabilities. Apart from that, attachment to the basal lamina also dictates the
proliferative behaviour of bulge stem cells. The more proliferative cells in the lower
portion of the bulge are in close contact with the secondary hair germ and express LGR5
(Leu-rich repeat-containing G protein-coupled receptor 5) and the Hedgehog
transcription factor GLI1. A separate population at the lower bulge cells expresses high
levels of SOX9, keratin 15 and CD34. At the upper fringe of the bulge is a population that
is rich in expression of GLI1 and might act as a reserve population, with roles in wound
repair. Above this is a population of LGR6+ and MTS24+ stem cells that predominantly
contributes to the maintenance of the junctional zone and sebaceous glands. An MTS24+
stem cell population that does not express LGR6 has also been identified. Further up
resides a quiescent population of stem cells that expresses LRIG1 (Leu-rich repeats and
immunoglobulin-like domains 1) and contributes to follicular, sebaceous and
interfollicular lineages. BLIMP1+ (B lymphocyte-induced maturation protein 1) cells have
also been identified and act as sebaceous gland progenitors.
Sweat glands. Sweat glands are not part of the pilosebaceous unit comprising hair
follicles and sebaceous glands, but they regulate the temperature of the skin and whole
body by secreting an aqueous solution. Sweat buds emerge during late embryonic
development from keratin 14+ progenitors and share some developmental traits with
other glands such as those of the mammary tissue. However, in adulthood they are
distinguished from these in that they do not require branching but instead a coiling
process that generates the ductal and glandular parts of the gland. In addition, they
undergo little renewal during adult life compared with other glandular structures that
show extensive morphogenesis. In any case, sweat glands do undergo self-renewal, but
until recently nothing was known about the identity of their progenitors in adulthood.
Sweat glands contain ductal, myoepithelial and luminal progenitors. Similar to the
mammary gland, each of these progenitors is unipotent. Although ductal and epidermal
progenitors can help to repair the gland when wounded, the myoepithelial and luminal
glandular components cannot. Interestingly, however, when removed from their natural
environment, both ductal and myoepithelial progenitors, but not luminal progenitors, can
become multipotent in a manner dependent on the microenvironment. For instance,
when transplanted into the shoulder fat or mammary fat pads of non-lactating
recipients, both types of progenitors reconstituted functional sweat glands, but

intriguingly, when transplanted into the mammary fat pad of lactating mice, 20%
displayed features of mammary glands. However, when transplanted into the dorsal skin
they only generated a stratified epidermis.
Microenvironmental influence on hair follicles
Signals emanating from structures adjacent to the bulge (that is, the basal membrane)
or from specialized cell types (that is, the dermal papilla, the keratin 6+ bulge layer or
mature and progenitor adipocytes) control the behaviour of bulge stem cells and
consequently affect hair follicle cycling. The dermal papilla maintains bulge stem cells
and secondary hair germ cells quiescent during telogen by producing bone
morphogenetic protein 4 (BMP4) and triggers their proliferation during anagen through
BMP inhibitors (BMPihh), fibroblast growth factor 7 (FGF7) and FGF10. Adipocytes
maintain bulge stem cell quiescent through BMP2 secretion, whereas adipocyte
progenitors promote their proliferation through platelet-derived growth factor- (PDGF)
secretion. Keratin 6+ cells promote bulge stem cell quiescence through the secretion of
BMP6 and FGF18. Hair follicle stem cell lineage determination is also dictated by
microenvironmental cues, such as nerve-derived sonic hedgehog (SHH) signalling. The
crosstalk is mutual, as signals generated by hair follicle stem cells affect other cell types,
including muscle cells and melanocyte stem cells. Positional cues for the correct
attachment of the arrector pili muscle (APM) to the bulge depend on nephronectin
(NPNT) deposition by bulge stem cells. The survival (transforming growth factor-
(TGF)), quiescence, proliferation (WNT and endothelin 2 (END2)) or differentiation (KIT)
of melanocyte stem cells relys on signals from hair follicle stem cells and dermal papilla
fibroblasts, which ensure their coordinated behaviour with the rest of the hair follicle
cells during each stage of the hair cycle.

Understanding skin wound healing

The loss of skin can occur for many reasons, including genetic disorders (bullous
conditions), acute trauma, chronic wounds or even surgical interventions. Superficial
partial-thickness wounds affect the epidermis and superficial parts of the dermis, with
epidermal blistering and severe pain accompanying this type of injury, especially in the
case of thermal trauma. Such wounds heal by epithelialization from the margins of the
wound, where basal keratinocytes change into a proliferating migratory cell type and
cover the damaged area. Cells migrate either from the wound edge, hair follicle or from
sweat gland remnants that lie in the deeper dermis, which has been preserved in this
depth of injury. Each hair follicle and sweat gland is lined with epithelial cells capable of
contributing to epithelial regeneration across the wounded surface. In addition, the hair
follicles of human skin contain a reserve of stem cells, located in the bulge region of the
follicle, which are capable of self-renewal. Full-thickness injuries are characterized by
the complete destruction of epithelial-regenerative elements. This type of injury heals by
contraction, with epithelialization from only the edge of the wound, leading to cosmetic
and functional defects. All full-thickness skin wounds which are more than 1 cm in
diameter require skin grafting as they cannot epithelialize on their own and may lead to
extensive scarring, resulting in limitations in joint mobility and severe cosmetic
deformities. Currently, the clinical gold standard in full-thickness injuries treatment
is split-thickness autologous skin grafting. Epidermis with a superficial part of the dermis
is harvested with a dermatome from an undamaged skin donor site and applied to the
full-thickness wound. Being applied to the wound, capillaries of the split skin graft (SSG)
form anastamoses or plug in into the existing capillary network to provide nutrients for
graft survival; this is referred to as graft take.
Skin healing

1) Inflammation 2) Synthesis of new connective tissue 3) Epithelial wound closure 4)

Scarring/remodelling 5) Immune response, cell proliferation, migration, differentiation
and cell death 6) ECM remodelling
- In some instances, such as skin, partial regeneration may be induced by the use of
biodegradable (collagen-glycoaminoglycan) scaffolds. These scaffolds are structurally
analogous to extracellular matrix (ECM) found in normal/un-injured dermis.[58]
Interestingly, fundamental conditions required for tissue regeneration often oppose
conditions that favor efficient wound repair, including inhibition of (1) platelet activation,
(2) inflammatory response, and (3) wound contraction.[1] In addition to providing support
for fibroblast and endothelial cell attachment, biodegradable scaffolds inhibit wound
contraction, thereby allowing the healing process to proceed towards a moreregenerative/less-scarring pathway. Pharmaceutical agents have been investigated
which may be able to turn off myofibroblast differentiation
Scarring
Simultaneously with angiogenesis, fibroblasts begin accumulating in the wound site.
Fibroblasts begin entering the wound site two to five days after wounding as the
inflammatory phase is ending, and their numbers peak at one to two weeks postwounding. By the end of the first week, fibroblasts are the main cells in the wound.
Fibroplasia ends two to four weeks after wounding. In the first two or three days after
injury, fibroblasts mainly migrate and proliferate, while later, they are the main cells that
lay down the collagen matrix in the wound site. Origins of these fibroblasts are thought
to be from the adjacent uninjured cutaneous tissue (although new evidence suggests
that some are derived from blood-borne, circulating adult stem cells/precursors). Initially
fibroblasts utilize the fibrin cross-linking fibers (well-formed by the end of the
inflammatory phase) to migrate across the wound, subsequently adhering to fibronectin.
Fibroblasts then deposit ground substance into the wound bed, and later collagen, which
they can adhere to for migration.
- Granulation tissue functions as rudimentary tissue, and begins to appear in the wound
already during the inflammatory phase, two to five days post wounding, and continues
growing until the wound bed is covered. Granulation tissue consists of new blood
vessels, fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and the
components of a new, provisional extracellular matrix (ECM). The provisional ECM is
different in composition from the ECM in normal tissue and its components originate
from fibroblasts. Such components include fibronectin, collagen, glycosaminoglycans,
elastin, glycoproteins and proteoglycans. Its main components are fibronectin and
hyaluronan, which create a very hydrated matrix and facilitate cell migration. Later this
provisional matrix is replaced with an ECM that more closely resembles that found in
non-injured tissue.
- Growth factors (PDGF, TGF-) and fibronectin encourage proliferation, migration to the
wound bed, and production of ECM molecules by fibroblasts. Fibroblasts also secrete
growth factors that attract epithelial cells to the wound site. Hypoxia also contributes to
fibroblast proliferation and excretion of growth factors, though too little oxygen will
inhibit their growth and deposition of ECM components, and can lead to excessive,
fibrotic scarring.
Roles of connective tissue fibroblasts in wound healing
Investigating the signals necessary for wound healing
- During the wound healing process, it starts of with a dominant immune response. The
connective tissue is repaired exclusively through the action of fibroblasts, which migrate
into the wound site where they synthesize and remodel a new extracellular matrix
(ECM). This immune response will drive granulation tissue formation and the scar
remodelling process typically after the epithelial wound closure has occurred. Typically
scarring will not occur in embryonic stages of development because they have reduced
inflammation, express an array of TGFbeta isotypes and have reduced fibrin clot
formation and platelet degranulation (produces growth factors and chemoattractants)
compared to adult wound repair. Therefore understanding or inducing an embryonic
state of repair or on a scaffold would be beneficial for skin regeneration within adults.

The granulation tissue that forms consists of blood vessels, fibroblasts/ other
mesenchymal cells and the production of the new ECM connective tissue after wound
injury.
- The ability of cells to migrate, attach to and remodel ECM is mediated by specialized
cell surface structures termed focal adhesions (FAs), which mediate adhesion between
the ECM and the actin cytoskeleton through integrin cell surface receptors. The
fibroblasts that generate the adhesive and biomechanical forces required for wound
closure are termed myofibroblasts, because they express highly contractile proteins
including -smooth muscle actin (-SMA). This depends on a finely tuned balance
between a proinflammatory or a transforming growth factor (TGF)-beta-dominated
environment. As the phenotype of fibroblasts from different tissues or body sites
becomes better defined, we may understand their individual contribution in wound
healing in more detail and possibly explain different clinical outcomes. Although
myofibroblasts normally disappear from the healed wound, the persistence of the
myofibroblast appears to be a major contributor to the phenotype of excessive scarring
and fibrotic disorders. Controlling myofibroblast action is therefore essential to control
normal tissue repair and to develop novel, effective and rational anti-fibrotic therapies.
Integrins are believed to be crucial for tissue repair, but their tissue-specific role in this
process is poorly understood. Liu et al. (2010) showed that mice containing a fibroblastspecific deletion of integrin 1 exhibit delayed cutaneous wound closure and less
granulation tissue formation, including reduced production of new ECM and reduced
expression of -smooth muscle actin (-SMA). Integrin-1-deficient fibroblasts showed
reduced expression of type I collagen and connective tissue growth factor, and failed to
differentiate into myofibroblasts as a result of reduced -SMA stress fiber formation. Loss
of integrin 1 in adult fibroblasts reduced their ability to adhere to, to spread on and to
contract ECM. Within stressed collagen matrices, integrin-1-deficient fibroblasts showed
reduced activation of latent TGF. Addition of active TGF alleviated the phenotype of
integrin-1-deficient mice. Thus integrin 1 is essential for normal wound healing, where
it acts, at least in part, through a TGF-dependent mechanism in vivo. Restoring TGF
beta to the K/K cells prevented this delayed wound closure response. Understanding the
signalling events that occur between the different cell types involve in the wound
response can help elucidate the necessary cells and signals required to be produced on a
bioscaffold that can further aid in developing regenerative abilities for excessive skin
damage.
2) Bioengineer skin replacement
Dermo-epidermal or composite skin substitutes aim to mimic the histological structure of
normal skin where both epidermal and dermal layers are present. This similarity also
provides some functional resemblance to the normal skin. These are not only the most
advanced and sophisticated products, when compared with epidermal and dermal
substitutes, but also the most expensive tissue-engineered biological constructs for
tissue repair (Jones et al. 2002). Most of these products are based on allogeneic skin
cells, incorporated into a dermal scaffold. This approach allows the production of large
quantities of uniform batches of the product, with a relative off-the-shelf availability.
There are reports of host immunogenic tolerance to allogeneic fibroblasts (Coulomb et
al. 1998) and their survival in the host up to three weeks. Long-term preservation of
allogeneic fibroblasts and their proliferation up to two months in the host without signs
of immune rejection have also been reported. However, porcine studies could not
confirm allogeneic fibroblast survival beyond a 7-day time point, nor could some clinical
studies when allogeneic fibroblasts were transplanted onto burn wounds.
Allogeneic keratinocytes provide effective pain relief and accelerate wound healing, but
they do not survive longer than a few weeks when applied to the wound because they
are rejected by the host. It is possible that the expression of the human leucocyte
antigen (HLA) is different in fibroblasts and keratinocytes, hence allogeneic fibroblasts
are less prone to tissue rejection initiated by the antigen complex. The inability to induce
T-cell proliferation by fibroblasts through the cytokine production when HLA class II

molecules are involved may indirectly support this observation (Ohyama et al. 2002). In
vivo models to investigate acute graft-versus-host disease to study the immunologic
tolerance to allogeneic fibroblasts in the host have been suggested. Therefore, in order
to produce permanent dermo-epidermal skin substitutes, it appears that either
allogeneic or autologous fibroblasts can be used but only autologous keratinocytes can
be used to achieve permanent closure of the skin defect.
Safe: non-toxic, non-immunogenic, no excessive inflammation, not transfer disease
Aims: Have similar physical & mechanical properties to skin. Provide pain relief, prevent
fluid and heat loss and protect wound from infection.
Needs to be: - Biodegradable, repairable, able to support reconstruction of normal skin
- Ideally: cost-effective, readily available, user-friendly, long shelf life.
Future aims: Innervated, quickly vascularised - try get nerve innovation of healed
wounds or implanted tissue and to quickly get blood circulation established otherwise it
cant support cell growth
Cross-disciplinary - Highly sensitive flexible pressure sensors with microstructured
rubber dielectric Layers (Mannsfeld SC et al., Nature Materials, 2010). Touch also
remains stubbornly difficult to mimic. The difficulty is that touch emulation necessitates
the development of high spatial-resolution, pressure-sensitive artificial skins capable of
discriminating between local stimuli on a textured surface. - Boland et al. (2010)
addressed this problem in two different ways. Both employ an active matrix array of
transducers using flexible materials. Flexibility is desirable because it enables the
fabrication of transducer arrays that can conform to curved surfaces, which is essential if
these engineered materials are to serve as artificial skins for prosthetic devices or in
applications where a high degree of spatial resolution is required. Where the groups
differ is in their approach to the transduction mechanism and the types of substrate
used. Ali Javey and co-workers used Ge/Si-nanowire-array field-effect transistors (FETs)
laminated on a flexible polyimide substrate with a pressure-sensitive rubber layer that
acts as a tunable resistor in series with the nanowire FET. On the other hand, Zhenan
Bao and collaborators microstructured polydimethylsiloxane (PDMS) films to produce
pressure-sensitive capacitor arrays that are integrated into the gate dielectrics of an
organic FET array. Both report pressure sensors with response times of less than 100 ms
and a dynamic range of 0.520 kPa or better, and each represents a significant advance
in the state of the art. By tailoring the microstructured PDMS film, it is possible to
achieve static load sensitivities as low as 3 Pa, and ultrafast millisecond response
times4. In the approach of Bao and colleagues4, array integration involves laminating
the PDMS film onto a non-flexible silicon substrate, so the overall conformability is lost.
However, these authors also reported a proof-of-concept flexible capacitive matrix-type
pressure sensor. In contrast, the nanowire-on-polyimide approach ensures flexibility, and
Javey and collaborators have demonstrated repeated bending to a 2.5 mm radius
without appreciable degradation in performance.
- There remain, however, significant opportunities for further innovation. The millimetrescale separation between pressure-sensitive pixels in the present active matrix arrays
must be decreased to better mimic the tactile sensing properties of human skin, or
calibrated to account for off-centre loading of the transducing elements. That said, the
prototype artificial skins reported have successfully emulated the pressure sensitivity of
human skin, and have done so in a manner that allows for macroscale integration of
sensor arrays. One can readily imagine the incorporation of sensors that enable the
detection of local temperature, the presence of light or electromagnetic fields, or even
humidity levels. The addition of piezoelectric nanowire arrays may even help emulate
the enhanced sensitivity provided by human hair follicles.
ALLOGENIC
Apligraf consists of viable allogeneic neonatal fibroblasts, grown in a bovine type I
collagen gel matrix, combined with viable allogeneic neonatal keratinocytes, forming a
confluent superficial layer of the construct, thus mimicking the normal structure of
human skin. Although this product does not cause immunological rejection, allogeneic
cells of the construct do not survive after one to two months in vivo. Hence, Apligraf,

which was marketed initially as an organotypic skin substitute, can only be considered
as a temporary bioactive dressing. It is known to deliver ECM components to the wound
bed, as well as cytokines and growth factors, such as interferons and , PDGF,
interleukins 1, 6 and 8. Nevertheless, reports of Apligraf use in burns treatment are
available, and some authors consider it to be an alternative to traditional skin grafting in
partial-thickness burns. The product cannot be used, however, to deliver a definitive
wound closure in full-thickness injuries because of the temporary nature of the grafted
allogeneic cells, and therefore it needs co-grafting with an autologous epithelial source.
In the study undertaken by Waymack and colleagues, Apligraf was combined with
autologous meshed SSG, and better cosmetic and functional outcomes were reported
when compared with the conventional meshed SSG treatment. The material is licensed
only for the treatment of venous leg and diabetic foot ulcers and no results of large
clinical trials in burns treatment have yet been reported. Drawbacks include a product
shelf life of 5 days, it requires delicate handling and possesses the risk of disease
transfer from its allogeneic constituents. Despite these complications it is reported to be
the most clinically successful product in its category giving a 25 per cent improvement in
ulcer treatment when compared with conventional treatments (Clark et al. 2007). Taking
into account its high cost of $28 per cm2, very short shelf life, safety considerations and
the temporary nature of cover it is unclear whether this product will find widespread use
in burns practice and skin reconstruction in large wounds.
OrCell: This tissue-engineered skin construct includes cultured allogeneic fibroblasts
and keratinocytes obtained from the same neonatal foreskin. Fibroblasts are seeded into
a bovine type I collagen sponge, which has a non-porous collagen-gel coating, on top of
which keratinocytes are added to form a confluent layer. The product was licensed in
2001 to treat donor sites in burns and recessive dystrophic epidermolysis bullosa. This
bilayered product is reported to produce an array of cytokines and growth factors such
as fibroblast growth factor-1, keratinocyte growth factor-1, platelet-derived growth
factor, vascular endothelial growth factor and transforming growth factor-, which are all
favourable for host cell migration and wound healing, thus conditioning the wound bed
for further treatment with skin grafts. This artificial skin substitute product showed
reduced scarring, and a shorter healing time was also reported when compared with the
acellular bioactive wound dressing Biobrane (Still et al. 2003). Being composed of
allogeneic cells, the product performs a temporary role, resorbs in 714 days (similar to
Apligraf) and no cellular DNA from the product can be found in the wound 1421 days
post-application.
AUTOLOGOUS
As it became possible to cultivate human keratinocytes serially in vitro and to rapidly
expand the number of patient keratinocytes ex vivo, this technology was rapidly
transferred into clinical applications where it contributed to improve patients' survival
rates. To initiate a culture of autologous cells, a skin biopsy of 25 cm2 is usually taken
along with initial wound debridement upon the patient's arrival at the clinic. The
epidermis is separated from the dermis and single keratinocytes are released from the
sheet by exposure to enzymes. These keratinocytes are plated into tissue culture vessels
where single cells start to divide to form colonies in the presence of mitotically
inactivated mouse fibroblasts and culture medium containing foetal calf serum, and
necessary supplements. It is possible to expand keratinocytes in xenogeneic-free
conditions where murine fibroblasts and bovine serum are avoided (Notara et al. 2007)
but the proliferative lifespan of cells cultured under these conditions is noticeably
reduced. Single colonies of keratinocytes merge together and form stratified epithelial
layers which can be enzymatically detached from the culture flasks, mounted onto
backing supports (such as paraffin gauze) to maintain basalapical orientation and the
sheet then applied to the wound.
Laserskin (Vivoderm) was designed and manufactured by Fidia Advanced Biopolymers
(Italy) with rights to manufacture and distribute this product granted to ConvaTec, a
division of Bristol-Myers Squibb Company under the Vivoderm trade name. The product
consists of autologous keratinocytes cultured on a hyaluronic acid membrane, which is

laser-microperforated. This allows the keratinocyte migration from a support material

down to the wound bed. Hyaluronic acid is a naturally synthesized polymer of the human
skin ECM which is reported to promote fibroblast and keratinocyte migration and
proliferation. It is also reported to participate in scarless foetal wound healing (Price et
al. 2007). Preliminary studies with Laserskin have shown the promising potential of the
product giving good graft take rate, biocompatibility as well as low infection rates in a
pre-clinical animal model and small clinical trials.
Autologous Adipose Stem Cells Use for Skin Regeneration and Treatment in
Humans
There is growing evidence that adipose stem cells contribute to the restoration of tissue
vascularization, and have a potentially large therapeutic effect in the field of
regenerative medicine. The purpose of this study was to assess the clinical effectiveness
of lipoaspirate transplantation on the treatment of skin lesions in humans. This study
began with surgical procedures in 2009 followed by a follow-up plan for 4 months.
Twenty clients underwent therapy for skin lesions and follow-up. Adipose derived stem
cells can promote human dermal fibroblast, proliferation, and re-epithelialization of
cutaneous wounds, rejuvenation of the aging skin and related skin lesions. These stem
cells replenish dying cells, and have the capacity to regenerate new tissues. Adverse
events including pain, swelling and allergy were minimal. All participants expressed their
satisfaction of the results. This surgical procedure is a low-invasive therapeutic approach
that can resolve the problem of depressed skin, skin lesions, and wrinkles. Adopting this
procedure decreases the cost of skin care, and improves clients long-term outcome.
Furthermore, it facilitates cell-mediated skin repair and regeneration.
3) Making artificial matrices
Novel potential skin biomaterials/scaffolds include:
Hyalomatrix PA, Hyalograft 3D - Both products are based on hyaluronic acid
derivates. Hyaluronic acid is one of the main polysaccharide components of dermal ECM
and promotes migration and proliferation of skin fibroblasts and keratinocytes.
Hyalomatrix PA has a temporary silicone layer which acts like an epidermis, while the
dermal component of the construct incorporates into the wound so preparing it for the
subsequent skin grafting. Hyalograft 3D has no pseudo-epidermal layer but the product's
effects are strengthened by the cultured autologous fibroblasts that provide the healing
wound with growth factors and cytokines. It also lays down ECM components
conditioning the wound for split skin grafting. This material is reported to improve in
vitro epithelial organization and dermalepidermal junction maturation in organotypic
skin bioconstructs. Clinically, Hyalograft 3D is primarily used for feet ulcer treatment in
combination with Laserskin autologous epidermal bioconstructs. Such combinations of
hyaluronic acid-based products incorporating autologous fibroblasts and keratinocytes
did not show any improvements in plantar diabetic feet ulcer treatment when compared
with standard treatment. Contrarily, successful treatment of severe scleroderma
cutaneous ulcers employing this technique has been reported. Similar material
combinations for deep burns treatment have also revealed advantages of Hyalograft 3D
grafting, which enhanced keratinocyte take, and reduced hypertrophy and wound
contracture rates when compared with exclusive application of keratinocyte cultures.
Hyalograft 3D also contributes towards rapid basement membrane formation (Stark et
al. 2004). Hyalomatrix PA has been investigated with favourable outcomes in a porcine
preclinical model of full-thickness wounds (Myers et al. 2007), and it has also been used
clinically for the treatment of deep partial-thickness burns. The material served as a
temporary dressing to stimulate wound regeneration after dermabrasion and was
reported to be a good and feasible approach for such wound treatments. It is also
reported to give favourable outcomes in deep paediatric burns treatment. Both products
are also appealing from the safety point of viewthey do not contain any animal or
allogeneic human-derived components.
Biomechanical properties of skin substitutes
- In terms of the effects of such cross-linking, Powell & Boyce (2006) have cross-linked
collagenglycosaminoglycan matrices with increasing amounts of the water-soluble

carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The stiffness of the

matrix increases to a point where brittleness renders the matrix mechanically unsuitable
for tissue replacement, but when tested for the growth of human fibroblasts and
keratinocytes, cytotoxicity was observed at higher EDC concentrations, an optimum
between increased mechanical stability and reduced cytotoxicity existing at 5 mM EDC.
The introduction of the glutamyl-lysine cross-links into the collagen matrix modified its
properties such that wound contraction in an in vivo model was significantly reduced.
This approach to mechanical modification also avoids potential cytotoxicity contributed
by chemical cross-linkers.
- The mechanical behaviour of these collagen matrices, but measured on the nanoscale,
has been investigated by Chaudhry et al. (2009). Using a nanoindentation approach,
they were able to discriminate between surface and bulk properties, and reported values
for the storage modulus of 0.71 GPa, and for the loss modulus of 0.40 GPa.
Constantinides et al. (2008) report a value for the elastic modulus of porcine skin of 222
kPa also using a nanoindentation approach, but this was derived from a creep
compliance approach. Nevertheless, this does suggest at least some discrepancy
between collagen matrix and skin in terms of mechanical properties at the nanometre
scale. This is important as cell attachment is likely to be influenced more at dimensions
similar to those of focal adhesions than bulk dimensions, and cells are known to respond
to the mechanical stiffness of their substrate. A direct comparison of ultimate tensile
strength, stressstrain behaviour, stress relaxation and creep parameters between
native excised human skin and four tissue engineering scaffolds was attempted by
Zhang et al. (2007). Unfortunately, the scaffolds used are not well characterized in the
report, but are two cross-linked dermal matrices from human and pig, and two non-crosslinked matrices from pig and goat. The parameters for the non-cross-linked matrices
approximated those of the natural excised skin, while the gluteraldehyde-fixed matrices
gave results significantly higher than the natural skin.
- In an effort to improve the mechanical properties of skin substitutes at the bulk level,
several workers have attempted to introduce synthetic polymers into natural polymer
matrices such as collagen. Interestingly, Powell & Boyce (2009) have added increasing
amounts of PCL to an electrospun collagen scaffold, and have shown that increasing PCL
ratios increase the stiffness of the material, making it easier to handle for the surgeon.
However, after growth of human keratinocytes and fibroblasts into the scaffolds, the
mechanical properties were either not significantly different, or even worse than those of
the collagen alone. This suggests that investigation of the mechanical properties of
tissue-engineering matrices both before and during the invasion of cellular components
is critical in the design of artificial skin.
Future perspectives
Another approach where both true skin regeneration and avoidance of scarring may be
successfully achieved is the use of either embryonic or adult stem cells. While embryonic
stem cell research is delayed by ethical and political debate, the number of
investigations in the use of adult stem cells is constantly growing, including skin
research. It is well established that the hair follicle outer root sheath contains pluripotent
epithelial stem cells capable of self-renewal. Although many in vitro and in vivo assays
exist to label, isolate and analyse skin stem cells, it is still impossible to identify a stem
cell within an intact human skin tissue without ambiguity. Nevertheless, pluripotent
epithelial stem cells are isolated from hair follicles and grown into epithelial sheets to be
used clinically for epithelial restoration. Although positive results regarding wound
epithelialization were reported, no restoration of hair follicles or sweat glands was seen;
such stem cells exhibited stable self-renewal potential, but their plasticity was limited.
Varying microenvironmental stimuli may affect stem cell plasticity, and result in
acquiring stem cell functions by more differentiated cells and limiting stem capacity by
pushing stem cells into a more differentiated state. Hence, unsuitable biochemical and
mechanical conditions in a wound may limit plasticity and proliferative activity of
implanted stem cells. Multipotent adult stem cells isolated from rodent dermis have been
shown to transdifferentiate into cells of several embryonic lineages such as adipocytes,

smooth muscle cells, glial and neuron cells retaining their regenerative capacity. Studies
have shown that these cells can be subcultivated as distinct diverse cell types for at
least 1 year without the loss of regenerative capacity. Novel therapeutical approaches in
wound healing could well be established if such precursors can be identified within a
human dermis and isolated. Another study on murine skin stem cells of hair follicle
bulges, isolated for in vitro culture, revealed their ability to retain stem cell capacity and
plasticity by producing multiple cell types like keratinocytes, hair follicles and
functionally active sebaceous glands, when engrafted in vivo. Successful replication of
these experiments in humans would hold out promise to produce fully functional true
skin equivalents.
Microenvironmental reprogramming by three-dimensional culture enables
dermal papilla cells to induce de novo human hair-follicle growth
- Growth of de novo hair follicles in adult skin occurs by a process known as hair
neogenesis. One way of initiating neogenesis is to place dermal papillae isolated from
the hair follicle in contact with an overlying epidermis where they reprogram the
epidermis to adopt a follicular fate. This approach, however, has not been successful
using cultured human dermal papilla cells in human skin because the cells lose their
ability to induce hair growth after expansion in vitro. In this paper, we demonstrate that
by manipulating cell culture conditions to establish three-dimensional papilla spheroids,
we restore dermal papilla inductivity. We also use several systems biology approaches to
gain a comprehensive understanding of the molecular mechanisms that underlie this
regenerative process.
- De novo organ regeneration has been observed in several lower organisms, as well as
rodents; however, demonstrating these regenerative properties in human cells and
tissues has been challenging. In the hair follicle, rodent hair follicle-derived dermal cells
can interact with local epithelia and induce de novo hair follicles in a variety of hairless
recipient skin sites. However, multiple attempts to recapitulate this process in humans
using human dermal papilla cells in human skin have failed, suggesting that human
dermal papilla cells lose key inductive properties upon culture. Here, we performed
global gene expression analysis of human dermal papilla cells in culture and discovered
very rapid and profound molecular signature changes linking their transition from a 3D
to a 2D environment with early loss of their hair-inducing capacity. We demonstrate that
the intact dermal papilla transcriptional signature can be partially restored by growth of
papilla cells in 3D spheroid cultures. This signature change translates to a partial
restoration of inductive capability, and we show that human dermal papilla cells, when
grown as spheroids, are capable of inducing de novo hair follicles in human skin.
Summary of Bioengineering skins
The bioengineering of skin presents several challenges that may be relevant to the
fabrication of solid organs and other complex tissues. A common multilayered design
consists of a highly cellular keratinocyte layer overlying a fibroblast-incorporated dermal
matrix to mimic the epidermis and dermis, respectively. The dermal matrix can be
derived from natural sources (eg, decellularized human or pig skin), created from natural
proteins (eg, collagens, fibronectin, or chitosan), or engineered from synthetic molecules
(eg, glycolic acid or polycaprolactone). The use of decellularized scaffolds has also been
extended to heart, liver, and lung engineering, providing a 3-dimensional prepatterned
scaffold onto which delivered cells can organize and mature.
- Skin engineering has traditionally relied on multilayered construction of epithelial
sheets overlying a dermal-type matrix. Advances in control-release systems,
nanotopography, biomechanics, materials science, and stem cell biology will enable
researchers to design increasingly sophisticated engineered skin grafts with the potential
to treat acute or chronic wounds. A multidisciplinary approach will be needed to
integrate these novel technologies and implement effective skin regeneration strategies.
- Stem cells have also proved highly promising for regenerating skin based on tissueengineering strategies. For example, the clinical use of cultured epithelial autografts
(sheets of patient-derived keratinocytes fabricated ex vivo) for massive burn injuries is
based on the ability of progenitor cells to expand keratinocyte populations. Skin grafts

are the gold standard for severe burn injuries, and their restorative abilities may also
rely on stem cellmediated processes. Moreover, hair follicle stem cells have been shown
to regulate wound repair and may play a critical role in regenerating functional skin. The
concept of integrating different populations of stem cells to create complex tissue
structures may prove more applicable than using individual stem cell populations in
isolation.
- Engineered skin constructs have also benefitted from advances in nanotechnology and
biomechanics.87 Nanofabrication techniques allow researchers to design complex
scaffolds that mimic microenvironment domains that facilitate skin regeneration.
Topographical modifications to biomaterial surfaces can regulate cell behavior and
potentially guide stem cell differentiation. The mechanical properties of engineered
matrices also play an important role in how incorporated cells behave. These factors are
especially critical for the engineering of skin, a flexible, pliable, and resilient structure
that has viscoelastic properties similar to biomaterial hydrogels. These mechanical
properties have been characterized on a microscopic scale and may influence future
designs of engineered skin grafts.
- Cytokines play an instrumental role in facilitating cellular communication within and
across different skin compartments. These signaling pathways are centrally involved in
skin development, homeostasis, and disease, but the ability of bioengineers to
recapitulate these biochemical networks remains limited.99 Progress has been made in
developing biomaterial substrates that contain various growth factors and that can be
activated to release stored contents under controlled conditions, hence the term
controlled-release or control-release systems. These delivery systems can be regulated
by factors such as temperature, pH, time, and solubility, providing an important means
of re-creating the complex biochemical milieu during tissue regeneration.89 Advances in
microfluidics technology (interconnected microchannel networks capable of precise
delivery of biomolecules) may enable researchers to reproduce morphogenic gradients
in temporospatial scales never before achieved.100 Ultimately, successful skin
engineering will rely on characterizing and recapitulating the optimal cellular, matrix,
biochemical, and biophysical cues that drive tissue regeneration.
The need for tissue-engineered skin substitutes
Patients with 50 per cent TBSA full-thickness wounds have only 50 per cent of
undamaged skin left which could be used for split-thickness skin harvesting. Donor sites
would add to the total wound size resulting in a wound area covering 100 per cent of the
body. An impaired epidermal barrier combined with reduced immunity of heavily burned
patients can result in bacterial sepsis which is the main complication in deep extensive
burns (Stoilova et al. 2007). Donor sites also heal with some scarring and may be very
painful; hence an additional analgesic pharmacological load is required. Moreover,
depending on the thickness of the dermis, only three to four split-thickness skin harvests
are possible from the same site and re-cropping is delayed by the time necessary for reepithelialization (Atiyeh & Costagliola 2007).
- In the case of a more extensive injury, donor sites are extremely limited and in such
cases, meshing techniques can be used where grafted skin is uniformly perforated and
stretched to cover greater areas of the wound. Although this method allows greater area
coverage and reduces mortality rates, the cosmetic and functional outcomes of such a
treatment are inferior when compared with the standard SSG application. This is because
of the lack of dermis in the interstices of the stretched meshed skin graft, and slow
epithelialization from graft margins across interstices, resulting in a greater graft
contraction, delayed healing, scar tissue formation and pronounced crocodile skin
appearance of the scar. In near-total full-thickness skin injuries even meshing techniques
are no help owing to the unavailability of donor sites. In such cases wounds are covered
with temporary dressings or cadaver skin to form a mechanical barrier in order to
prevent fluid loss and microbial contamination. Only delayed serial autologous split skin
grafting can be used to heal injured skin in these cases (Papini 2004). Such wounds are
left unhealed for a long time over the course of treatment while awaiting epithelial
regeneration, and are prone to severe complications which can result in death.

- Alternative life-saving approaches in the treatment of extensive full-thickness wounds,

where donor sites for SSG harvesting are not available, include the use of cultured
autologous keratinocytes and/or bioengineered skin substitutes. Significant progress has
been made recently in the development and clinical use of these products Off-the-shelf
availability or the possibility of producing, in a relatively short period of time, sufficient
quantities of epithelium capable of permanent wound closure sometimes make these
approaches the treatments available in extensive deep injuries.
- All tissue-engineered skin substitute bioconstructs need to comply with three major
requirements. They must be safe for the patient, be clinically effective and be
convenient in handling and application. Properties of the ideal skin substitute for in vivo
use have been described elsewhere and recently reviewed by MacNeil (2007). In
general, such biomaterials must not be toxic, immunogenic or cause excessive
inflammation, and should also have no or low level of transmissible disease risk. The
biomaterial for skin reconstruction should be biodegradable, repairable and able to
support the reconstruction of normal tissue, with similar physical and mechanical
properties to the skin it replaces. It should provide pain relief, prevent fluid and heat loss
from the wound surface and protect the wound from infection. It is also of great
advantage if the skin substitute bioconstruct is cost-effective, readily available, userfriendly and possesses a long shelf life.
Biomaterials: In the first Consensus Conference of the European Society for
Biomaterials (ESB) in 1976, a biomaterial was defined as a nonviable material used in a
medical device, intended to interact with biological systems. Biomaterials have moved
from merely interacting with the body to influencing biological processes toward the goal
of tissue regeneration. Typically, three individual groups of biomaterials, ceramics,
synthetic polymers and natural polymers, are used in the fabrication of scaffolds for
tissue engineering. Each of these individual biomaterial groups has specific advantages
and, needless to say, disadvantages so the use of composite scaffolds comprised of
different phases is becoming increasingly common. Although not generally used for soft
tissue regeneration, there has been widespread use of ceramic scaffolds, such as
hydroxyapatite (HA) and tri-calcium phosphate (TCP), for bone regeneration applications.
Ceramic scaffolds are typically characterized by high mechanical stiffness (Young's
modulus), very low elasticity, and a hard brittle surface. From a bone perspective, they
exhibit excellent biocompatibility due to their chemical and structural similarity to the
mineral phase of native bone. The interactions of osteogenic cells with ceramics are
important for bone regeneration as ceramics are known to enhance osteoblast
differentiation and proliferation. Various ceramics have been used in dental and
orthopedic surgery to fill bone defects and to coat metallic implant surfaces to improve
implant integration with the host bone. However, their clinical applications for tissue
engineering has been limited because of their brittleness, difficulty of shaping for
implantation, and because new bone formed in a porous HA network cannot sustain the
mechanical loading needed for remodeling19. In addition, although HA is a primary
constituent of bone and might seem ideal as a bone graft substitute, problems also exist
in that it is difficult to control its degradation rate.
- Numerous synthetic polymers have been used in the attempt to produce scaffolds
including polystyrene, poly-l-lactic acid (PLLA), polyglycolic acid (PGA) and poly-dl-lacticco-glycolic acid (PLGA). While these materials have shown much success as they can be
fabricated with a tailored architecture, and their degradation characteristics controlled
by varying the polymer itself or the composition of the individual polymer, they have
drawbacks including the risk of rejection due to reduced bioactivity. In addition, concerns
exist about the degradation process of PLLA and PGA as they degrade by hydrolysis,
producing carbon dioxide and therefore lowering the local pH which can result in cell and
tissue necrosis25. The third commonly used approach is the use of biological materials as
scaffold biomaterials. Biological materials such as collagen, various proteoglycans,
alginate-based substrates and chitosan have all been used in the production of scaffolds
for tissue engineering. Unlike synthetic polymer-based scaffolds, natural polymers are
biologically active and typically promote excellent cell adhesion and growth.

Furthermore, they are also biodegradable and so allow host cells, over time, to produce
their own extracellular matrix and replace the degraded scaffold. However, fabricating
scaffolds from biological materials with homogeneous and reproducible structures
presents a challenge. In addition, the scaffolds generally have poor mechanical
properties, which limits their use in, for example, load-bearing orthopedic applications.
- The problems described above that exist with using scaffolds fabricated from a single
phase biomaterial have resulted in considerable research being devoted to the
development of composite scaffolds comprising a number of phases. For example, a
number of groups have attempted to introduce ceramics into polymer-based scaffolds
while others have combined synthetic polymers with natural polymers in order to
enhance their biological capacity. While composite scaffolds such as these have shown
some promise, each consists of at least one phase which is not found naturally in the
body and they all have associated problems with biocompatibility, biodegradability or
both. A more typical approach is the use of collagen-based scaffolds, either alone or with
an additional phase incorporated to enhance biological and/or mechanical properties.
This is the approach used in our laboratory particularly for bone regeneration but
increasingly for other applications such as cartilage and cardiovascular repair.
Example of bioengineered tissue 2: Bone
- Bone tissue engineering has the potential to reach millions annually through the repair
of bone defects. Therefore, researchers in bone tissue engineering are working to
develop alternatives to allogenic and autologous bone grafts in order to address the
growing needs of the population, and the much of the research is scaffold. A scaffold can
be used to guide bone regeneration and repair defects or be combined with cell and/or
biologics, which are added to further enhance bone regeneration. Tadic et al. studied on
calcium phosphate phase that is equivalent in composition and crystallinity to the
mineral phase of bone which was prepared by a continuous precipitation method. The
powder was compacted by cold isostatic pressing into desired shapes with high
compressive strength in the range of 20 50MPa. It is concluded that such implant
materials can be prepared with a fine-tuned biodegradability in combination with a high
mechanical strength. The high mechanical strength of the objects also permits further
mechanical shaping procedures like drilling or cutting.
- A successful tissue engineering method for bone replacement would imitate natural
bone graft by providing the essential elements for new bone formation using synthetic
scaffolds, osteogenic cell populations, and bone induction factors. Thomson et al.
evaluated the suitability of various formulations of poly (DL-lactic-co-glycolic acid)
(PLGA) foams to provide a tissue conducting scaffold in an bovine model for bone flap
fabrication. Three formulations were used of different copolymer ratio and molecular
weight. Porous wafers of PLGA were stacked into a closed rectangular chambers with one
side open. Some chambers also contained autologous morcellized bone graft (MBG). The
chambers were inserted with the open face adjacent to the cambium layer of the
periosteum in rib beds of seven sheep and harvested after 8 weeks in vivo. Gross and
histologic examination of the resulting tissue specimens demonstrated molded units of
vascularized tissue generally conforming to the shape of the chambers and firmly
attached to the periosteum. Polymer degradation appeared to occur by varying degrees
based on polymer formulation. New bone formation was observed only in areas
containing MBG. A PLGA foam scaffold is an efficient conductor of new tissue growth but
not osteoinductive, it contributes to the shape of molded tissue, and biocompatible when
used in this model.
- Surgeons and other researchers have long sought a synthetic material capable of
accelerating the bone healing process, integrating with the surrounding tissue, and later
allowing or encouraging tissue remodeling such that the material resembles or is
replaced by native bone. Historically, a variety of alloplastic materials have been
investigated toward this end, including celluloid, aluminum, gold, vitallium, tantalum,
stainless steel, titanium, methylmethacrylate resins, polyethylene, silicone elastomers,

and hydroxyapatite ceramics. When compared with autologous bone grafts, the current
material gold standard for these applications, alloplastic materials are deficient in a
number of areas. For example, a non-degradable alloplastic material may not respond to
mechanical stress in the same manner as the surrounding host bone, resulting in
structural failure of the implant under load or pathologic changes in the surrounding
bone, as seen in stress shielding. Biologically inactive materials may facilitate
inflammatory scarring, neoproliferative reactions in the neighboring tissues, and may
serve as a nidus for bacteria, resulting in infectious complications. Bioactive implants
such as demineralized bone matrix obtained from allogeneic (cadaver) or xenogeneic
sources have shown promise as reconstructive materials because of their high
osteoinductivity and propensity for remodelling, although drawbacks include the
theoretical risk of disease transmission as well as cost and availability. The benchmark
for comparison of new bone grafting materials continues to be autogenous bone as a
result of its potential for growth and remodeling, as well as the ability to osseointegrate
and resist infection.
The tissue engineering paradigm typically incorporates three components for tissue
regenerationa degradable support or scaffold material, bioactive factors such as growth
factors or other pharmaceuticals, and cells. The clinical strategies outlined in the
previous section generally do not include components of this paradigm, with the
exception of autogenous bone and demineralized bone from other sources, which may
contain a number of bioactive factors (Reddi, 1998). Recently, a number of new products
for bone regeneration have entered into widespread clinical use that incorporate key
elements of the tissue engineering paradigm. One such product, Infuse (Medtronic,
USA) incorporates a bioactive factor, bone morphogenetic protein-2 (BMP-2) into a
degradable, acellular collagen sponge and is indicated for clinical use in a number of
applications including spinal fusion, traumatic tibial fractures, and certain oral-facial
applications. The clinical success of this material (Govender et al., 2002) illustrates the
potential for tissue engineering-based therapies in the clinic.
Bioactive glass - Several variations of glass beads called Bioglass (US Biomaterials,
Alachua, FL, USA) are currently being developed, and one formulation (PerioGlas) has
been approved in the USA for periodontal use. The beads are composed of silica (45%),
calcium oxide (24.5%), disodium oxide (24.5%), and pyrophosphate (6%). When
implanted, they bind to collagen, growth factors, and fibrin to form a porous matrix to
allow infiltration of osteogenic cells. Recently, a novel nanocomposite hydrogel made of
collagen and mesoporous bioactive glass nanoparticles with surface amination has been
developed. The addition of bioglass into the collagen hydrogel significantly increases the
bioactivity of the scaffold and improves its mechanical properties; this novel strategy
would therefore be suitable for bone tissue engineering applications. Moreover, the
bioactive glass foam produced by solgel is an osteoinductive material with a network of
interconnected macropores necessary for cell colonization. It has been shown that
bioactive glass can differentiate human adipose-derived stem cells into osteoblasts, in
vitro. Moreover, Gu et al. used scaffolds composed of a mixture of two different bioactive
glasses (silicate 1393 and borate 13-93B3) to evaluate their response to osteogenic
MLO-A5 cells in vitro and their capacity to regenerate bone in rat calvarial defects in
vivo. The scaffolds can guide bone regeneration and have a controllable degradation
rate. A combination of glass and HA has also been used in bone regeneration. Fredericks
et al. determined the performance characteristics of a novel silicate-substituted HA bone
graft substitute (BGS), SiCa-P EP (Baxter Healthcare/ApaTech, Elstree, UK), in a standalone mode, a stand-alone with bone marrow aspirate (BMA) mode, and an extender
mode with iliac crest autograft (ICBG) in a rabbit posterolateral spine fusion model. The
SiCa-P EP utilized as a stand-alone, as a stand-alone with BMA, and as an autograft
(ICBG) extender produced results that were clinically and radiographically similar to
ICBG.
Calcium phosphate and its derivatives - Different types of mono-, bi-, and tricalcium
phosphate bioceramics and molecules have been extensively used in bone tissue

engineering researches and developments 2) Calcium phosphate ceramics, specifically

-tricalcium phosphate (-TCP) and synthetic HA, have recently been used in composites
and in fibrous composites formed using the electrospinning technique for bone tissue
engineering applications. Calcium phosphate ceramics are sought because they can be
bone bioactive, so that apatite forms on their surface, facilitates bonding to bone tissue,
and is osteoconductive. In a recent study, the bioactivity of electrospun composites
containing calcium phosphates and their corresponding osteogenic activity was
investigated. Electrospun composites consisting of (20/80) HA/TCP nanoceramics and
poly(-caprolactone) were fabricated, and the results demonstrated that after seeding
the hybrid scaffold with human MSCs, the cells not only showed greater osteogenic
differentiation but also proliferated and produced more bony matrix in vitro. 3) Piccinini
et al. implanted a new mineralogical formulation, HA/tetracalcium phosphate (TTCP), as
a biomaterial for bone regeneration in the maxillary sinus of rats. After 17 weeks from
implantation, HA/TTCP synthetic bone graft performed very well as osteoconductive
material: bone graft contact was found very high, and bone volume and vital bone
showed an ideal bone density for implant placement. Farahpour et al. radiographically
evaluated the effects of biphasic calcium phosphate scaffold with 5%, 10%, and 20% of
porosity on cortical bone repair in rabbits. They showed that TCP+HA scaffold is an
osteoconductive and osteoinductive biomaterial. TCP+HA scaffold can increase the
amount of newly formed bone and more rapid regeneration of bone defects. Velasquez
et al. reported the in vitro and in vivo behavior of -tricalcium phosphate (-TCP) and
also -TCP with either 1.5 or 3.0 wt.% of dicalcium silicate (C2 S). The in vivo behavior of
the ceramics matched the in vitro results, independent of the C2 S content in -TCP. A
fully mineralized new bone growing in direct contact with the implants was found under
the in vivo conditions. The bioactivity and biocompatibility of the implants increased with
the C2 S content in -TCP. The C2 S-doped ceramics also favored a phase transformation
of -TCP into CHA, which is important for full implant integration during the natural bone
healing processes.
Bone components
***There are several types of cells constituting the typical bone: 1) Osteoblasts are
mononucleate bone-forming cells that descend from osteoprogenitor cells. They are
located on the surface of osteoid seams and make a protein mixture known as osteoid,
which mineralizes to become bone. The osteiod seam is a narrow region of newly formed
organic matrix, not yet mineralized, located on the surface of a bone. Osteoid is
primarily composed of Type I collagen. Osteoblasts also manufacture hormones, such as
prostaglandins, to act on the bone itself. They robustly produce alkaline phosphatase, an
enzyme that has a role in the mineralisation of bone, as well as many matrix proteins.
Osteoblasts are the immature bone cells, and eventually become entrapped in the bone
matrix to become osteocytes- the mature bone cellscBone lining cells are essentially
inactive osteoblasts. They cover all of the available bone surface and function as a
barrier for certain ions. 2) Osteocytes originate from osteoblasts that have migrated into
and become trapped and surrounded by bone matrix that they themselves produce. The
spaces they occupy are known as lacunae. Osteocytes have many processes that reach
out to meet osteoblasts and other osteocytes probably for the purposes of
communication. Their functions include, to varying degrees: formation of bone; matrix
maintenance; and calcium homeostasis. They have also been shown to act as mechanosensory receptors regulating the bone's response to stress and mechanical load. They
are mature bone cells. 3) Osteoclasts are the cells responsible for bone resorption, thus
they break down bone. New bone is then formed by the osteoblasts (remodeling of bone
to reduce its volume). Osteoclasts are large, multinucleated cells located on bone
surfaces in what are called Howship's lacunae or resorption pits. These lacunae, or
resorption pits, are left behind after the breakdown of the bone surface. Because the
osteoclasts are derived from a monocyte stem-cell lineage, they are equipped with
phagocytic-like mechanisms similar to circulating macrophages. Osteoclasts mature
and/or migrate to discrete bone surfaces. Upon arrival, active enzymes, such as tartrate

resistant acid phosphatase, are secreted against the mineral substrate

***Bone regeneration by autogenous osteoblast or osteoblast progenitor transplantation
is one of the most promising new techniques being developed because it would
eliminate problems of donor scarcity, immune rejection, and pathogen transfer.
Osteoblasts and osteoblast progenitors obtained from patient bone marrow can be
expanded in culture and seeded onto an appropriate degradable scaffold, which will
slowly degrade as cells grow and secrete new bone in vivo. In addition to bone marrow
progenitors, other cell sources have recently been identified as promising precursors
capable of differentiation into osteoblast-like cells. Kern et al. 2006 compared
mesenchymal stem cells isolated for bone marrow aspirates, adipose tissue, and
umbilical cord blood and found that while umbilical cord blood-derived progenitor cells
were the most difficult to isolate and were incapable of adipogenic differentiation, all
three cell sources were promising for bone regeneration. The transcription profiles of
differentiating bone marrow and adipose derived progenitor cells have been shown to be
very similar, although bone marrow mesenchymal stem cells more efficiently
differentiate into fully mature osteoblasts. Additional cells with multipotent or stem-like
potential have been isolated from various tissues including dental tissues and dermal
fibroblasts, and the selection of the most appropriate cell source likely depends on the
intended clinical application and any present comorbidity.
A second cell-based approach: normal osteogenesis
Stem cells: The combination of stem cells with scaffolds as a polytherapy is a new
option. Collagen and demineralized bone powder have been used to produce a novel
scaffolds for bone tissue engineering. Human periosteum-derived cells (PD cells) were
cultured on this scaffold: the hybrid scaffolds exhibited greater osteoinductive potential
than collagen scaffolds. The PD cells with hybrid scaffolds possessed higher ALP activity,
calcium deposition, and superior behavior (e.g., attachment, differentiation, and
proliferation) than those with collagen scaffolds.
2) The feasibility of applying calcined bovine bone (CBB) coated with allograft bone
marrow mesenchymal stem cell (BMSC)-sheet as a 3D scaffold material in bone healing
has been investigated recently. The new scaffold material was implanted into
osteoporosis rat cranial bone defects and critical size bone defects (8-mm diameter).
The CBB-BMSC-sheet combination had a stronger potential in osteogenic differentiation
and mineralized formation both in vitro and in vivo than CBB-BMSC combination. Threedimensional reconstruction of micro-CT, H&E staining, and bone strength results showed
that the area and volume of the newly formed bone in CBB-BMSC-sheet group was
significantly higher than that of the CBB-BMSC group after 4 to 12 weeks. 3) Adiposederived stem cells (ASCs) with multilineage differentiation capacities have been
demonstrated as an alternative cell candidate for in vitro and in vivo bone regeneration.
This suggests that they may be a potential candidate to repair the bone defects.
Yang et al. attempted to demonstrate the use of new biomimetic constructions of
undifferentiated rabbit ASCs with fully interconnected porous -TCP scaffolds
encapsulated by collagen I hydrogel in the regeneration of a critical-sized defect of
rabbit radii. Critical-sized defects in the left radii of rabbits were prepared and inserted
with rASCs/collagen I/-TCP scaffold composites or collagen I/-TCP scaffold composites.
Twelve weeks after implantation, the defects were almost completely repaired as
confirmed by the presence of the cortical bone and medullary cavity. In addition, a
greater number of ASCs in the scaffold enhanced osteogenesis in critical-sized defects.
Pourebrahim et al. [144] compared bone regeneration of tissue-engineered bone from
ASCs and autogenous bone graft in a canine maxillary alveolar cleft model. The
undifferentiated cells were incubated with a HA/-TCP scaffold in specific osteogenic
medium for 21 days. Four mongrel dogs were prepared by removal of two of the three
incisors bilaterally and a 15-mm defect in bone was created from the crest to the nasal
floor. After healing, repair was followed by a tissue-engineered bone graft from ADCs on
one side and cortico-cancellous tibial autograft on the other side. Bone regeneration was
evaluated by histomorphometry on days 15 and 60 after implantation. The bone

formation on the autograft sides was higher than on the stem cell sides at 15 and
60 days, and 45% and 96% versus 5% and 70%, respectively. 4) Pang et al. observed
the therapeutic effects of hybrid RP scaffolds comprising PLGA, -TCP, collagen I, and
apatite (PLGA/-TCP-collagen I/apatite) on segmental bone defects in conjunction with
combined bone marrow MSCs. The bone defect remained unconnected in the original RP
scaffolds (PLGA/-TCP) during the study. In hybrid RP scaffold group, woven bone united
the radial defect at 12 weeks and consecutively remodeled into lamellar bone at
24 weeks post-operation and finally matured into cortical bone with normal marrow
cavity after another 12 weeks. No bone formation but connective tissue was detected in
the RP scaffold at the same time.
5) Xuan et al. produced polycaprolactone/HA tissue scaffolds with individualized
grooves to repair the sternal defect. The defects were surgically produced in a
sternocostal joint of Beagle dogs. The animals were allocated to one of the three groups
(n=6): no treatment group, PCL/HA group, and PCL/HA/BMSCs group. The application of
the PCL/HA scaffolds with specific grooves resulted in satisfactory repair of the sternal
defect. Furthermore, the BMSCs-seeded scaffolds enhanced the amount of bone
ingrowth and seemed to be more promising. Calcium phosphate nanoparticles such as
HA/fluorapatite (FA), with chitosan gel filled with unrestricted somatic stem cells (USSCs),
have been used for healing calvarial bone in a rat model [147]. The combination of
scaffold especially with USSC could be considered a useful method for bone
regeneration. In another study, autogenic and allogenic bone-marrow-derived MSCs were
compared for repair of bone gap defect in rabbits. The defects were filled with HA alone,
HA with autogeneic BM-MSCs, and HA with allogenic BM-MSCs. Histologically, increased
osteogenesis, early and better reorganization of cancellous bone, and more bone
marrow formation were discernible in treatment groups as compared to the control
group. In vitro culture expanded allogenic and autogenic BM-MSCs induced similar but
faster and better healing as compared to control.
Creating in vivo bioreactors
In vivo tissue engineering of musculoskeletal tissues
A key benefit is that the in vivo approach to generating neo-tissue limits excessive
manipulation of cells in vitro and utilizes the body's own regenerative capacity for the regrowth and development of tissues, creating a more physiological niche for new tissue
formation. We achieved this effectively at the simplest level by injection of an alginatebased hydrogel between the cambium layer of the periosteum and the underlying bone
to create an artificial in vivo bioreactor space. Progenitor cells from the cambium rapidly
proliferated to fill the in vivo bioreactor space and generated histologically normal,
mechanically competent bone tissue. However, as with in vitro tissue engineering
strategies, in vivo tissue engineering may require a combination of bioactive scaffolds,
local administration of growth factors, and seeding of autologous cells. Furthermore, in
vivo tissue engineering may also rely on recruitment of progenitor/stem cells and
selection of an appropriate anatomical site.
Bioactive scaffolds and growth factors as regulators of tissue formation in vivo
Scaffold materials can be engineered to control cell behavior based on their
physicochemical properties, or the incorporation of biological recognition elements such
as cell adhesive peptides or growth factor proteins. Immobilized peptides or growth
factors can enhance stem cell homing capacity as well as having significant implications
for immune responses in vivo. For example, immobilization of stromal derived factor 1-
(SDF 1-) has been shown to attract both primitive and CXCR4-expressing cells from the
circulation. Growth factor release from implanted scaffolds can not only control local
cellular responses, but also promote neo-vascularization and attract mesenchymal stem
cells from surrounding tissues. Local sustained release of Vascular Endothelial Growth
Factor (VEGF) and its inhibitors has been shown to create spatially restricted zones of
angiogenesis. Physico-chemical properties of scaffold materials can be tailored to
regulate the local microenvironment in vivo. Modifications in the chemical structure and
crystallinity of calcium-phosphate biomaterials have been shown to affect protein

adsorption, release of ionic species, and ability to promote ectopic and orthotopic bone
growth. Other ceramics such as bioactive glasses can be tailored to release ionic
species with potent biological effects, such as strontium, cobalt and calcium. Sustained
release of strontium is able to promote osteoblast proliferation and differentiation
(leading to bone formation), while inhibiting osteoclast activity (hence bone resorption)
Aim: smart matrix + cells
i.e. 3D scaffold with signalling cues that induce cellular differentiation and correct ECM
deposition
Controlled spatial and conformational display of immobilised bone
morphogenetic protein-2 and osteopontin signalling motifs regulates
osteoblast adhesion and differentiation in vitro
It has long been recognised that cell regulatory molecules, such as growth factors and
cytokines, exert powerful influences on the behaviour of eukaryotic cells at the interface
of tissues. Indeed the immobilized activity of these signalling mediators in combination
with the extracellular matrix (ECM) underpins many fundamental biological processes
including embryo-, morpho- and tumorogenesis and wound healing. Numerous studies
have established the principle that tethered ligands regulate cell behavior quite
distinctly from their freely diffusible counterpart. For example, extracellular matrix
proteins like fibronectin promote adhesion [2] and migration [3] on a surface but when
added exogenously to cells can have adhesion-independent effects, for example
activating intracellular signalling cascades [4]. Furthermore, soluble growth factors, like
fibroblast growth factor (FGF)-2, vascular endothelial growth factor (VEGF) and
interleukin (IL)-1 exhibit elevated activity when presented within the context of matrix
such as fibrin [5-7]. Whilst these and many other studies have provided fundamental
insight into the underlying biology, the experimental approaches have meant that
immobilised ligands are often presented in orientations and conformations that are not
well controlled, at densities that are poorly characterised and under conditions in which
the durability and stability of the ligand is undefined.
- Mitchell (2010) designed and fabricated protein scaffolds that can display protein
ligands on self-assembled monolayers (SAM) in a controlled and reproducible way. This
work uses a TolAIII-fusion expression system to provide the engineered protein at high
yields and solubility. A cysteine residue engineered into the C-terminal end of the protein
ensures durable chemisorption onto gold surfaces whilst a 12 amino acid stretch termed
the switch-tag (ST) can switch from a water soluble coil to a hydrophobic helix that coassembles with the alkane thiols of the SAM. Therefore formation of a SAM containing
both the scaffold protein and an amphiphile produces a surface where the orientation of
the protein is controlled and non-specific interactions are low. These surfaces provide the
opportunity to address how immobilised ligands can both provide insights into cell
function and contribute to tissue engineering.
They demonstrated the ability of this system to influence the long term function of cells
in vitro via the incorporation of two distinct ligand motifs. The first is derived from the
extracellular matrix protein, osteopontin (OPN), and was identified as supporting cell
adhesion through the 91 integrin and subsequently shown to promote adhesion and
migration of endothelial cells leading to enhanced angiogenesis. The second is the
knuckle epitope from bone morphogenetic protein (BMP)-2, a member of the
transforming growth factor (TGF) superfamily, which has been widely shown to support
commitment of cells to the osteoblastic lineage, their differentiation and functional
ability to make bone.
- Here we show that Tol-OPN-ST dose-dependently supported the adhesion and spreading
through vinculin adhesion sites of primary rat osteoblasts. Biological activity of the BMP
motif within the scaffold protein was verified by adding the recombinant protein to cells
transfected with a Sma- and MAD-related protein (SMAD) responsive-reporter construct
and demonstrating SMAD1 activation. Tol-BMP2-ST reproduced BMP signalling and
produced bone-like nodules on patterned surfaces.

This technology effectively immobilises bioactive protein motifs on a surface for analysis
of cell behaviour in vitro and may provide the basis for future tissue engineering
approaches.
Discussion: To design and produce an efficient bone graft, the researchers and
orthopedic surgeons should have sufficient knowledge of the characteristics of grafts
such as osteogenesis, osteoinductivity, and osteoconductivity, and their other
advantages and disadvantages. Autografts are the gold standard for bone regeneration.
Among the available strategies to improve fracture healing and enhance the outcome of
incorporation of the grafts, tissue engineering is a suitable option. An ideal tissueengineered product should have characteristics similar to those of autografts without
their limitations. The bone scaffolds should additionally be highly porous and have pores
of suitable sizes at all locations of the scaffold to provide an optimal environment for
new bone matrix and bone regeneration. Furthermore, growth factors such as basic
fibroblastic growth factor, which may affect cell functions, proliferation, or differentiation;
healing promotive agents such as hPRP; and also Tarantula cubensis extract can be
included in the scaffolds to enhance the healing performance of the injured connective
tissues. Agents such as glycosaminoglycans, including hyaluronic acid, chondroitin, and
dermatan sulfate have modulatory roles in bone and fracture healing and can improve
the quality of the tissue-engineered scaffolds. More recently, zoledronate, simvastatin,
and alendronate have been shown to have promising effects on bone healing and
regeneration. The vascularity of the scaffold is critical because if not present, the
scaffold will undergo ischemia and the cells will die. Therefore, application of growth
factors such as VEGF, PDGF, and FGF can be useful to stimulate angiogenesis in the
scaffolds and the grafts. A combination of stem cells with scaffolds and healing
promotive factors, especially the growth factors, could be one possible strategy
providing all the necessary characteristics for bone repair and regeneration.
Scaffolds for tissue engineering: state of the art and future directions
The challenge of tissue engineering is to mimic what happens in nature. Attempts are
being made to engineer in vitro practically every tissue and organ in the body. Work is
proceeding in creating tissue-engineered liver, nerve, kidney, intestine, pancreas and
even heart muscle and valves. In the area of connective tissues, work has been ongoing
worldwide for many years in the engineering of tendon, ligament, bone and cartilage. To
date the highest rates of success have been achieved in the areas of skin 11, bladder 53,
airway 54 and bone, where tissue-engineered constructs have been used successfully in
patients. In addition, autologous chondrocyte implantation (ACI) and matrix-induced
autologous chondrocyte implantation (MACI) are showing some success for cartilage
repair. This latter approach involves culturing harvested autologous chondrocytes on a
collagen-based membrane for a number of weeks in Genzyme's GMP-certified
laboratories prior to the construct being shipped back to the patient's hospital, followed
by implantation at the injured site. This product is currently approved for use in Europe
and Australasia.
- While major breakthroughs have taken place and economic activity within the tissue
engineering sector has grown exponentially, with increasing numbers of products
entering the market place and into clinical trials, and with sales of regenerative
biomaterials already exceeding US$240 million per annum57, significant research is
required in a number of specific areas in the field58. As described above, lack of
vascularity in scaffolds and tissue engineered constructs is a major challenge, and
improving vascularization strategies is considered one of the areas requiring the most
extensive research in the field of tissue engineering. One way to improve vascularization
might to be engineer microvasculature by cells in the scaffolds prior to implantation and
a large body of work in this area is ongoing by a number of groups including ourselves.
This is a complex approach and from a clinical perspective would involve initially
harvesting cells from a patient/donor, then engineering a nascent microvasculature

followed by engineering the desired matrix/tissue around this microvasculature; all of

which would have to be achieved prior to implantation into a patient. Many clinicians
question the efficacy of in vitro tissue engineering due to the requirement for at least
two procedures and the delay in treatment while the construct is being cultured in vitro.
From a commercial perspective, this approach also poses problems due to the prolonged
regulatory process required before such a tissue engineered construct can be approved
for clinical use. However, in tissues such as cartilage, which do not have the ability to
regenerate themselves when damaged, long term in vitro tissue engineering may be the
only solution to prevent the requirement of an eventual joint arthroplasty.
- Other tissues, such as bone for example, have an intrinsic ability to repair, remodel and
regenerate. The task in the field of tissue engineering is therefore to try and harness this
innate regenerative capacity. One way to do so might be to engineer the scaffold in such
a way that the scaffold itself provides regenerative signals to the cells which might
negate the requirement for prolonged in vitro culture prior to implantation. Therefore,
much ongoing research is devoted to developing more sophisticated biomimetic
biomaterials with added levels of complexity to incorporate multi-functionality and to
encourage the biomaterials to have, for example, bioinstructive and stimuli-responsive
properties (see review 67). Cells derive a vast wealth of information from their
environment. The native extra-cellular matrix (ECM) surrounded, and produced, by cells
is instructive, providing a dynamic and spatially heterogeneous constellation of
microstructural, compositional and mechanical cues that can influence cell behavior.
Harnessing the mechanosensitive capacity of cells, in particular, provides immense
opportunities. The mechanical properties of tissues, biomaterials, cells, and biomolecules
have profound biological consequences in terms of implant bioactivity versus failure,
transmission of mechanical stimuli, and for a wide range of processes at the tissue, cell
and subcellular levels. Key roles in molecular signaling pathways are played by cell
adhesion complexes and the cellular cytoskeleton, whose contractile forces are
transmitted through transcellular structures. Therefore, the mechanical properties of the
substrate to which the cells are attached are critical to the regulation of cellular
mechanotransduction and subsequent cellular behavior. This has important implications
for development, differentiation, disease, and regeneration. It is now clear, for example,
that substrate stiffness can regulate both the behavior of mature cells and the
differentiation pathway of stem cells (see review 70). For example, when MSCs were
grown on firm gels that mimic the elasticity of muscle, differentiation down a myogenic
(muscle-forming) lineage was observed, whereas when MSCs were grown on rigid gels
that mimic pre-calcified bone the cells differentiated down an osteogenic pathway 71.
Similarly, with neural stem cells, neuron differentiation is favored on soft scaffolds that
mimic normal brain tissue, whereas differentiation into neuron-supporting glial cells is
promoted on harder matrices 72. In addition, cardiomyocytes (heart muscle cells) have
been shown to beat best on a substrate with heart-like elasticity, whereas on harder
substrates, that mechanically mimic a post-infarct fibrotic scar, cells overstrain
themselves and stop beating 73. Therefore, increasing research is now being directed at
utilizing the mechanosensitive capacity of cells to develop scaffolds and biomaterials
with specific mechanical properties that can be used to direct the behavior of the cells
with which they interact.
- In addition to biomechanical signals, cellular behavior is strongly influenced by
biological and biochemical signals from the extracellular matrix. Therefore, the use of
scaffolds as delivery systems for growth factors, adhesion peptides and cytokines is
receiving considerable attention in the field. The incorporation of, for example,
angiogenic growth factors in scaffolds might also improve their vascular potential.
Similarly, methods of providing nerve supply to newly formed tissues also need to be
considered80. Another area of critical importance is controlling, and understanding, the
host immune response and preventing infection following implantation. To this end, the
incorporation of drugs (i.e. inflammatory inhibitors and/or antibiotics) into scaffolds has
been proposed as a method to reduce the possibility of infection after surgery81. Finally,
the use of scaffolds as delivery systems for therapeutic genes is undergoing

considerable investigation82, 83, 84, 85, 86 and 87. Gene therapy approaches (viral and
non-viral) which utilize DNA encoding for therapeutic genes potentially provide a more
stable and effective approach to allow sustained and controlled release of therapeutic
factors. Gene therapy can thus be a valuable tool to avoid the limitations of the local
delivery of growth factors, including the short half-life, large dose requirement, high cost,
need for repeated applications, and poor distribution88. The idea of a gene delivery
vector contained within a scaffold, although not new, is a recent development in the field
of regenerative medicine and the system has been coined as a gene activated matrix
(GAM) by Bonadio and co-authors89 who developed the very first system. Despite the
prolonged regulatory process required to enter the clinical arena, such a GAM might offer
advantages by increasing the rate of DNA delivery into the cells which are in contact
with the gene-eluting scaffold and provide spatial and temporal control of the desired
gene. These areas of new and expanding research demonstrate just how
multidisciplinary the field of tissue engineering has become and while the challenges are
vast, the opportunities for improving human health in a whole variety of areas are
immense. Undoubtedly exciting times lie ahead in this field, which is only now beginning
to define itself as more technologies enter the clinical and commercial arenas.