Elite Masters Programme in

Advanced Materials and Processes

Literature Review
Summer Semester 2014

Strategies and materials for blood vessel regeneration

This literature review is in partial fulfilment of the M4 Module portfolio exam
for the focal subject Biomaterials and Bioprocessing
Students Name

Lakhe, Kasturi

Matriculation Number

21845280

Lecture Subject

Biomaterials for Tissue Engineering

Professor/Lecturer

Prof. Dr.-Ing. habil. Aldo R. Boccaccini

Number of words

2342

Abstract
With increase in cardiovascular diseases, there is a great need for blood vessel grafts for the
replacement of damaged arteries to restore the blood supply to the tissues. The use of Autografts is
common for cardiac tissue replacement; however the lack of availability of donor tissue, calls for the
requirement of biomaterial that can regulate the vascularised tissue replacement or reconstruction.
There are various strategies used by the researchers to fulfil the requirements such that the neo-tissue
formed mimics the native tissue in both structure and function and maintains long term patency. This
review aims to briefly address a few such approaches and materials for blood vessel regeneration or
replacement.

I hereby declare that this literature review is my own work, that I have only made use of the cited
documents and that this review has not been previously submitted as academic coursework elsewhere.
Erlangen, 30/09/14
City, Date

Signature

Elite Masters Programme in

Advanced Materials and Processes
Literature Review
Summer Semester 2014

Strategies and materials for blood vessel regeneration

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Lakhe, Kasturi
21845280

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City, Date

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Note to grader: Please fill in and sign this form and return by mail or electronically to Dagmar Senft
(MAP Office or dagmar.senft@ww.uni-erlangen.de) by 31 October 2014

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1. Introduction
Due to limited regeneration potential of most Human tissues, the recovery of tissue after damage
usually leads to impaired functionality. Replacement therapy is the most frequently applied method for
treatment of cardiovascular tissue damages. In particular for blood vessels, the use of Autografts( e.g.
coronary artery bypass grafts with autologous vein) and artificial prosthesis(synthetic vascular grafts)
is common. However due to limitations like shortage of donor tissue; immune rejection, pathogen
transfer and limited durability are seen. Furthermore, they do not allow full regeneration and
functional recovery of the blood vessels 1. The development of autologous tissue engineered grafts
shows potential of improvement over artificial prosthesis.
Tissue Engineering utilizes three basic components (Fig:1) (i) the cells, (ii) scaffolds for organisation
of extracellular matrix for neo-tissue formation, and (iii) humoral and mechanical signals. 1

Figure 1: Basic concept of Tissue Engineering

Early strategies, also known as Top-Down strategies of blood vessel Tissue Engineering are based
on seeding of cells of porous biodegradable scaffolds or embedding the cells in hydrogels to support
the formation of tubular tissue engineered grafts. However, foreign body reaction and inflammation
due to bacterial colonization can be observed for such approaches. The Bottom-Up approach tends
to be scaffold free and thus offers potential of less inflammation and toxicity due to scaffold
degradation. In this approach, individual sections of tissue are generated and gradually assembled to
get the complete Tissue Engineered construct that replicates the native tissue. 2

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This review briefly discusses a few approaches and the biomaterials proposed to be used for the
regeneration of blood vessels.

2. The Blood Vessel

There are three types of blood vessels in the vascular system, the arteries, the capillaries and the veins.
The walls of blood vessels have concentric layers of distinct cell and protein composition. The layer
on the luminal side is the tunica intima, composed of Endothelial cells (EC). The middle layer, tunica
media, consists of circumferentially arranged alternating layers of smooth muscle cells and elastin
sheets. The tunica adventitia is the outermost layer composed of collagen fibers. It is responsible for
the reinforcement and protection of blood vessel and its attachment to the surrounding. Elastin in the
vessel wall prevents vascular dialation that can occur due to creep of collagen due to high blood
pressure. In muscular arteries (e.g. in heart as coronary artery), the blood pressure is typically around
10-16Kpa; 80-120mmHg. The inner diameter of these vessels can be from 0.3mm to 1cm depending
on the location and wall thickness of about 1mm. The wall shear stress ranges 0.75-2.25Pa during a
cardiac cycle. Strain in circumferential wall ranges 10-15%. The longitudinal strains are higher
because of pumping of heart. The burst pressure for human coronary artery is about
2000mmHg(266Kpa).3
An EC lining and the smooth muscle cell (SMC) layer, both are important for the full functionality of
blood vessels. Furthermore the Extracellular matrix influences the architecture and robustness of
vessels. Thus, over past decade, there have been five approaches of manufacturing Tissue Engineered
vascular grafts, (i) EC seeded synthetic grafts, (ii) collagen based blood vessel analogs, (iii)
biodegradable synthetic polymer based constructs, (iv) self assembly blood vessels, (v) decellularised
tissue grafts.4

3. Strategies
3.1Scaffolds for Blood vessel Tissue Engineering
Cell growth and differentiation in a culture requires a structured environment for cell attachment.
Thus, the use of scaffolds forms the basis of classical tissue-engineering. The cells can be grown on
biodegradable synthetic scaffolds or biological scaffolds which are ultimately replaced by the
extracellular matrix secreted by the cells. 1Scaffolds for Blood vessel Tissue Engineering
Xu C.Y. et al,6 developed biodegradable nanofibrous scaffold from aligned poly(l-lactid-co-ecaprolactone) [P(LLA-CL)] (75:25) copolymer by electrospinning. The three dimensional mesh had
fibers of diameters ranging 200-800nm and thickness of 500nm. Aligned topography of fibers was
seen (Fig 2), mimicking the circumferential orientation of native arterial cells. The smooth muscle

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cells attached and expressed a spindle-like phenotype along the aligned nanofibers. The SMC proteins
inside the cells also organised parallel to the nanofibers.

Figure 2: SEM micrograph of aligned P(LLA-CL) nanofibrous scaffold

Vaz C.M. et al.5 also used electrospinning to produce a bi-layered tubular scaffold. The outer layer of
the scaffold composed of stiff and oriented PLA and the inner fibrous layer composed of randomly
oriented pliable PCL. The fiber diameter ranges 0.4-6m. The pore size upto 15m was observed. The
strong and elastic anisotropic scaffolds showed maximum stress 4.3MPa and elastic behaviour
maintained upto 10% strain and may provide the compliance required by the blood vessels. Further
mouse fibroblast showed attachment to the fibers and proliferation through the aligned fibrous
network, thus proving the capability to support the attachment spread and growth of cells.
Biodegradable synthetic polymer scaffolds, like P(LLA-CL) and PLA, show premature loss of
mechanical strength as the cells fail to form ECM before the degradation of the synthetic polymers. 7
Zhang et al.7 thus, used electrospun silk fibroin scaffolds. Silk has unique mechanical properties like
excellent biocompatibility, processasibility and controlled degradability. The fiber diameter showed an
average of 377nm. The scaffold supported the growth and expansion of human aeortic endothelial
cells and human coronary artery smooth muscle cells. Prior to cell culture these small diameter silk
fibroin electrospun scaffolds displayed a burst strength of 81177.2 mmHg, sufficient to withstand
arterial pressure.8 The tensile properties were also similar to the native vessels. The ultimate tensile
strength of 2.420.48 MPa, and linear modulus of 2.45047 MPa were observed. 8
The advantage of using electrospining for the fabrication of nanofibers is its high controllability. Thus
the nanofibers have a defined architecture replication in-vivo. The shortcomings of this technology are
limited substrate choice, high expenses, difficulty in achieving nanometer scale and limitations in
making 3D constructs.6
Poly(-caprolactone) is also a favoured biomaterials for making scaffolds for blood vessel tissue
engineering. Ekaputra et al 9 prepared a 3D hybrid mesh of poly(-caprolactone)-collagen blend
3

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(PCL/collagen) with hyaluronic acid (HA). Due to limitations of electrospining technique for
preparation of 3D constructs, electrodeposition was used to prepared the 3D scaffolds. The hybrid
scaffold allowed controlled release of angeogenic factors VEGF 165 and PDGF-BB over a period of 5
weeks in vitro. The fiber diameter were in range 1.670.27m. Fengyi Du et al 10 developed a 3D
gradient hiparinized nanofibrous scaffold that will aid EC and the lumen of blood vessel to prevent
thrombosis. The scaffolds composed of vertical graded chitosan/ poly(-caprolactone) nanofibrous
vessels fabricated with chitosan and PCL by sequential quantity graded co-electrospining. The natural
blood vessel environment was mimicked by hiparinization and immobilization of VEGF. Human
Umbilical vein endothelial cells and SMCs were seeded on top and bottom of the surface of scaffold.
The quantity of chitosan PCL nanofibers increased gradually from tunica adventitia to lumen surfaces
in the gradient wall of tissue engineered vessel.

3.2Scaffold-free tissue Engineering

1. Scaffolds have been subjected to prolific research and development for last three decades and
in general show a lot of advantages. But there are some general as well as some requirement
specific challenges in the use of scaffolds for tissue engineering. Scaffold choice, toxicity od
degradation products, host inflammatory responses, mechanical mismatch with surrounding
tissue are some of such challenges that may affect the long term behaviour of engineered
constructs.11

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Auger et al12 used self assembly approach of tissue engineering in which SMCs and fibroblasts were
cultured in a medium composed of serum and absorbic acid(50g/ml). Under these conditions the
cells produce their own Extracellular matrix. The SMC sheets were rolled over dehydrated
fibroblast tissue layer. Fibroblast sheets were rolled on it after a week of maturation. After further
8 weeks maturation the ECs were seeded on the inner membrane to form endothelium. The
matured blood vessels contained all three histological layers from human vessels. The tissue
engineered blood vessels showed burst strength of 2500mmHg.
Norotte et al.11 produced a fully biological self assembly through a rapid prototyping bioprinting
method to make scaffold-free small diameter vessels. Different vascular cell types like SMCs and
fibroblasts were aggregated in discrete units. Extruded agarose rods were used as building blocks
of moulding template. In Figure 3 Agarose rods and multicellular spheroids were deposited layer
by layer.

Figure 3: Design template for tubular structures. (AE) Deposition scheme for the smallest diameter
tube that can be built of agarose rods (pink) and multicellular spheroids (orange)of the same
diameter. (FH) More complex tubular structures. (I) Scheme for a branching structure. 11

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(a)

(b)
Figure 4: Bioprinting tubular structures with cellular cylinders. (a) The bioprinter outfitted with two vertically
moving print heads (b) The printed construct. E. Engineered pig SMC tubes of distinct diameters resulted after 3
days of post-printed fusion (left: 2.5 mm OD; right: 1.5 mm OD). 11

After the assembly, multicellular cylinder fused within 2-4 days into final tubular structure. The
supporting agarose rods were then removed. The method gives flexibility of variation in parameters
such as size, thickness of wall, patterning of cells, single and double layered tubes ranging from 0.92.5mm outer diameter. The technology relies on multicellular three dimensional spheroids or cylinders
as building blocks made of self adhering cell-types. Using spheroids for long-vascular tubes is
laborious and time-consuming, thus cylindrical building blocks are preferred for long tubes. The
method is scalable and reliable.
The limitation for this bioprinting prototype is lack of resolution. The micropipettes used for extrusion
of building blocks were relatively large in diameter (300-500m) and the smallest tube diameter has
900m OD. This leads to nutrients and oxygen limitation and as a result sparsely distributed apoptotic
cells were observed throughout the vascular wall. The authors propose the use of smaller diameter
pipettes for extrusion (e.g.100m diameter building blocks would lead to 300m tubes of 100m
thickness). Authors suggest that thinner wall would lead to better viability.
Other limitation is the removal of agarose rods from the mature tubes. Authors propose the use of
other moulding gels, which are for example thermoreversible or photosensitive, would eliminate the
limitation
S. Rayatpisheh et al2 propose the combination of top-down and bottom-up approaches of tissue
engineering to overcome the limitations of both the strategies and produce a tubular construct of
circumferentially aligned SMCs showing higher expression of contractile genes and enough
mechanical strength. The SMCs were cultured on micropatterned and N-isopropylacrylamide-grafted
polydimethylsiloxane. A small portion of this was covered by aligned electrospun scaffolding. This
resulted in single sheet of unidirectional cells. Upon cooling, the cell sheet detaches from the scaffold

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and could be collected on a mandrel to generate the tubular constructs with contractile gene
expression. (Figure 5).

Figure 5: Schematic of scaffold-assisted cell sheet engineering of vascular media: (A) Assembly of cell culture
surface which combines aligned and electrospun PCL scaffolding with thermoresponsive and micropatterned
PDMS, (B) alignment of cells during 4 days of culture, and (C) rolling nanofiber scaffold and aligned cell sheet
over the mandrel to generate tubular constructs. (For the size of 6 well plates the mandrel diameter is 3 mm.) 2

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3.3Synthetic Grafts
Tissue engineered grafts require long fabrication times generally due to the requirement of cells from
different sources to produce a robust, load bearing ECM. Thus tissue engineered constructs have
yet to gain wide-spread acceptance as an alternative to autologous artery or vein 13.
V. A. Kumar et al.13 propose design strategy (Fig 6) for the fabrication of tubular grafts composed of
collagen fiber network and elastin-like protein polymers to mimic native tissue structure and
function.

Figure 5 : Schematic of fabrication scheme for vascular grafts. (A) Collagen gels are neutralized in a
buffer at 4 _C for 24 h in a rectangular mold (10 x 7x 0.4 cm), yielding 4 mm thick gels. (B) Gels
are dried to thin ~40lm mats and placed on 0.45 m filter paper (*) atop a fritted filter ($). (C)
Collagen mat on filter paper is placed on a glass slide at 4 C for 30 min. (D) Green plastic shims
are placed around the collagen mat and elastin, blue, is poured atop, with a mapping glass slide
placed to spread the elastin, and heated to 25 C. (E) Composite matrices are rolled on 1.3 mm or
4.0 mm mandrels. (F) Rolled matrices are cooled and reheated to allow elastin bonding of collagen
layers.13

Dense fiber collagen network exhibited ultimate tensile strength of 0.710.06MPa, strain to failure
37.12.2% and Youngs modulus 2.090.42 MPa. Compliance and burst pressure exceeded
2.70.3%/100mmHg and 830131 mmHg respectively. Rat aortic interposition model confirmed
limited early inflammatory response.

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Figure 6: Mechanical characterization of composite vascular grafts

Synthetic grafts made of PET(Decron) and expanded PTFE are options for large diameter vascular
grafts, but mismatch in compliance of these polymers to the native tissue results in inflammatory
responses and intimal hyperplasia that lead to re-occlusion in small diameter vessels. Developing
vascular grafts for replacement of small diameter vessel, in the range 2-5mm is a clinical
challenge.14
S. Liu et al.14 prepared a bilayered vascular graft(Fig 8) based on silk fibroin (SF) composed of an
inner silk-fiber reinforced tube containing Heparin, and highly porous external SF layer using
predefined methods like moulding, electrospinning, gel-spinning, coating with the aim of
providing suitable mechanical properties and blood compatibility. They proposed that addition of
degummed silk fibers to regenerated silk solution results in flexible silk films, even in dry state.
Silk fiber scaffolds with nanofibrous structure were coated on these tubes to afford a favourable
growth environment for cells. The measured compliance, mechanical strength, burst pressure, and
suture retention strength were comparable to that of sephanous vein (3.42%/mmHg). In vitro
studies showed good cytocompatibility and hemocompatibility.

Figure 8: Representative morphological assessment of the grafts. (A) Macroscopic appearance of

inner tubes. (B) Macroscopic appearance of the tubular bilayered vascular grafts 14

4. Conclusion
Lot of work has been done in the field of blood vessel regeneration for vascular
systems. Various approaches propose a better healing mechanism for the

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impaired tissue. These can broadly be classified as scaffold based tissue
engineered grafts, scaffold free tissue engineered grafts and synthetic grafts.
However each strategy has some downfalls and the selection of a specific
approach seems application specific. A combination of these approaches are also
proposed to combat the limitations. Selection of right material and right approach
is important, as the constructs should not only look like blood vessels but should
fulfil their purpose in conjugation with native tissue inside the living system.

References
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Cleary, Animesh Rathore, Corey Fein, Spencer Church, Christopher Breuer; Advanced Drug
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(2) Shahrzad Rayatpisheh, Daniel E. Heath, Amir Shakouri, Pim-On Rujitanaroj, Sing Yian
Chewa, Mary B. Chan-Park; Biomaterials 35 (2014) 2713-2719
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