Accepted Manuscript

Title: Chitosan based Biocomposite Scaffolds for Bone

Tissue Engineering

Author: S. Saravanan R.S. Leena N. Selvamurugan

PII: S0141-8130(16)30115-5
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.01.112
Reference: BIOMAC 5802

To appear in: International Journal of Biological Macromolecules

Received date: 11-11-2015

Revised date: 27-1-2016
Accepted date: 29-1-2016

Please cite this article as: S.Saravanan, R.S.Leena, N.Selvamurugan, Chitosan based
Biocomposite Scaffolds for Bone Tissue Engineering, International Journal of
Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.01.112

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 Chitosan based Biocomposite Scaffolds for Bone Tissue Engineering

S. Saravanan, R. S. Leena and N. Selvamurugan*

Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur,

Tamil Nadu, India.


To whom correspondence should be made:
Nagarajan Selvamurugan, Ph. D.
Professor
Department of Biotechnology
School of Bioengineering
SRM University
Kattankulathur 603 203.Tamil Nadu.
India.
Cell: 91-9940632335
Email: selvamurugan.n@ktr.srmuniv.ac.in
selvamn2@yahoo.com

Abstract
The clinical demand for scaffolds and the diversity of available polymers provide
freedom in the fabrication of scaffolds to achieve successful progress in bone tissue
engineering (BTE). Chitosan (CS) has drawn much of the attention in recent years for its
use as graft material either as alone or in a combination with other materials in BTE. The
scaffolds should possess a number of properties like porosity, biocompatibility, water
retention, protein adsorption, mechanical strength, biomineralization and biodegradability
suited for BTE applications. In this review, CS and its properties, and the role of CS along
with other polymeric and ceramic materials as scaffolds for bone tissue repair applications
are highlighted.

1
Abbreviations

3D : Three dimensional

Alg : Alginate

BG : Bioglass ceramic

n-BGCs: Nano bioactive glass ceramics

BSA : Bovine serum albumin

BTE : Bone Tissue Engineering

CAD : Computer aided design data

CaP : Calcium phosphate

CMC : Carboxymethyl cellulose

Col : Collagen

CS : Chitosan

ECM : Extracellular matrix

EDAC : Ethyl-3[3-dimethylaminopropyl] carbodiimide hydrochloride

GAGs : Glycosaminoglycans

Gn : Gelatin

HAp : Hydroxyapatite

mWS: Mesoporous wollastonite

NaCl : Sodium chloride

NAG : N-acetyl-D-glucosamine

nBGC : Nano Bioglass ceramic

2
NHS : N-hydroxysuccnimide

NPs : Nanoparticles

nSiO2 : Nano silicon dioxide

nZrO2 : Nano zirconium oxide

nβ-TCP: Beta tricalcium phosphate nano particles

PCL : Polycaprolactone

PEG : Poly(ethylene oxide)

PLLA : Poly (L-lactic acid)

SF : Silk fibroin

SiO2 : Silicon dioxide

TCP : Tricalcium phosphate

Ti : Titania

TPP : Sodium tripolyphosphate

ZrO2 : Zirconium oxide

β-TCP : Beta-tricalcium phosphate

Keywords: Chitosan, scaffold, protein adsorption, biodegradability, bone tissue

engineering

3
Contents
1. Introduction--------------------------------------------------------------------------------------
2. Bone and its components-----------------------------------------------------------------------
3. Bone tissue engineering------------------------------------------------------------------------
3.1 Chitosan---------------------------------------------------------------------------------
3.2 Physico-chemical properties -------------------------------------------------------------
3.3 Biological properties
4. Chitosan scaffold fabrication methods-----------------------------------------------------
5. Chitosan scaffold properties------------------------------------------------------------------
5.1 Porosity--------------------------------------------------------------------------------------
5.2 Water retention-----------------------------------------------------------------------------
5.3 Protein adsorption--------------------------------------------------------------------------
5.4 Biomineralization---------------------------------------------------------------------------
5.5 Biodegradation------------------------------------------------------------------------------
5.6 Mechanical properties--------------------------------------------------------
6. Conclusions---------------------------------------------------------------------------------------

1. Introduction
Bone is a dynamic tissue and it undergoes continuous remodeling during the
lifetime of an individual. It involves in locomotion with load bearing role and protects
delicate vital organs of the body [1]. Large or critical sized bone defects occurring due to
tumor resections, non-union fractures and correction of birth defects often require major
surgical intervention in correction of those defects despite high regenerative potential.
Bone grafts are widely utilized clinically to augment critical sized bone defects and
promote bone regeneration. Autografting is the gold standard method for treating bone loss
[2] and the major problems associated with this are the availability of donor tissue and
donor site morbidity [3-16]. Over the few decades, the steeping demand for bone grafts has
been found to be rising with human population. The necessity in the use of biomaterials
with improved properties has always been an alternative to the traditional use of
autogenous bone grafts.

4
 Bone tissue engineering (BTE) encompasses the principles of bone biology and
engineering disciplines to augment bone loss through the use of temporary matrices called
as scaffolds. Scaffolds are fabricated by materials belonging with various classes including
polymers, ceramics. Chitosan (CS), a natural polymer has been widely used as one of the
scaffolding materials, and its use alone or along with other polymers or ceramics as
scaffolds in bone tissue repair applications are reviewed in the following sections.
2. Bone and its components
Bone is a highly functionalized connective tissue forming the skeletal framework of
human body with its involvement in various physiological functions [17]. The anatomical
representation of bone and its components are shown in Fig. 1. Collagen and calcium
phosphate (CaP) apatite crystals are the chief constituents of bone. Apatite crystals are
observed as embodiments within the collagen matrix. The density based classification
compartmentalizes bone into cortical, a dense tissue and cancellous or trabecular, highly
porous. Bone marrow is homed in the interior of cancellous bone. Bone tissue undergoes
continuous remodeling through the action of its cellular components osteoblasts- bone
forming cells, osteoclasts- bone resorbing cells and osteocytes-senile osteoblasts (Fig. 1). It
possesses various units from lamella to individual collagen fibrils arranged hierarchically
and sized under nano dimensions. Bone mineral hydroxyapatite (HAp) is architectured
down to few hundred nanometers. Bone cells work in harmony to maintain the structural
integrity and help in regeneration of diseased bone tissue. However, under non unions and
critical sized defects, healing of bone through normal remodeling process remains
unreachable and hence, bone grafting is required to facilitate bridging of those defects.
Figure 1
3. Bone tissue engineering
Critical sized bone defects are addressed by the use of cells and biomaterials, and
with a combination of cells, biomaterials and bioactive molecules (Fig. 2). Scaffolding
materials exerting the following properties such as osteogenicity, osteoconductivity,
biocompatibility and biodegradability are often selected for BTE [18, 19]. The scaffolds
must be highly porous with interconnectivity providing space for cellular infiltration,
nutrient transfer, waste disposal, neovascularization and space for new bone tissue
ingrowth. Pores ranging between 200-600 μm is often found be best suited for BTE

5
applications [20]. Surface roughness and wettability of the scaffolding materials is essential
for improving biomaterial-cell interactions. Suitable mechanical properties possessed by
the scaffolds decide their site of application including whether in load bearing or non-load
bearing sites [21, 22]. The degradation properties are strongly dependent on the scaffolding
materials, and tailoring of the scaffold degradation in vivo can be obtained through
adjusting materials properties [23].
Figure 2
Ceramics such as calcium phosphate (biphasic) (CaP), tricalcium phosphate (TCP)
and HAp are widely employed in scaffold fabrication due to their structural similarity with
the mineral components of human bone. Zirconium oxide (ZrO2), silicon dioxide (SiO2),
bioactive glass ceramics (BGCs), titania (Ti) are few other ceramics employed in BTE [24-
30]. Polymers include both natural and synthetic, and synthetic polymers such as poly(L-
lactic acid) (PLLA), Polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA) have
been studied for their use as scaffolding materials [31-33]. Natural polymer based
composite materials are gaining an increased attention for their use in BTE. Chitosan,
alginate (Alg), collagen (Col), gelatin (Gn), silk fibroin (SF) and glycosaminoglycans
(GAGs) are widely used natural polymers [34-43].

3.1 Chitosan
Chitosan, the deacetylated form of chitin is the structural component found in the
exoskeleton of crustaceans like shrimps, crabs and lobsters. It is a natural polymer, with a
linear structure consisting of β(1-4) glycosidic bonds linked D-glucoasmine residues with a
variable number of randomly located N-acetyl-D-glucosamine (NAG) groups [44].
Chitosan has the properties of bioactive, biodegradable, anti-bacterial and biocompatible,
and it possesses hydrophilic surface, which is absent in many synthetic polymers. A role
for CS in enhanced cell adhesion, proliferation, and osteoblast differentiation and
mineralization has been reported [45-48]. Chitosan can be engineered to form 3D scaffolds
with varied pore structures and composites with a number of materials including ceramics
and polymers, for its application in BTE [24-30]. Knowing the physico-chemical and
biological properties of CS would promote its usage as one of the base materials in
designing and fabrication of scaffolds to obtain the enhanced effect in bone tissue repair.

6
3.2 Physico-chemical properties
When the number of NAG is greater than 50%, then it is said to be chitin and when
the number of N-glucosamine is greater than 50%, it is said to be CS. The different forms
of CS are varied based on their degree of acetylation (ranging from 50-95%) and their
molecular weights (ranging from 300 to 1000 kDa) [49]. Also, the degree of acetylation
and the molecular weights influence the physicochemical properties of CS such as
crystallinity, solubility and degradation [50]. Thus, 0% deacetylated chitin and 100%
deacetylated CS are highly crystalline in nature, and other intermediate degrees of
deacetylated CS are semi-crystalline in nature. The solubility of CS is based on the amount
of free amino and N-acetyl groups, which enable it to be soluble in both organic and
inorganic acids (pKa 6.5), and insoluble in neutral and basic solutions [51]. Chitosan is said
to be cationic in nature which enables CS to form complexes with anionic macromolecules
such as glycosaminoglycans (GAGs) that modulate the activity of cytokines and growth
factors, aiding in its applications in BTE.
The primary amines and the secondary hydroxyl groups seen on CS facilitate the
addition of side groups, peptides or amino acids that aid in functionalization and
optimization of CS for BTE [52]. Lysozyme is the enzyme that degrades CS in vitro and in
vivo, by hydrolyzing the glucosamine-glucosamine, glucosamine–NAG and NAG–NAG
bonds. The resulting CS oligosaccharides on hydrolysis are then fused into GAG or
excreted [53, 54]. The degree of degradation and molecular weight of CS are inversely
related to the degree of deacetylation. If the degree of deacetylation is higher, the
degradation rate of it is lower, due to high polymer crystallinity and when the molecular
weight of CS is higher, the degree of degradation is said to occur at a lower rate [55]. Thus,
the fabrication of CS scaffolds for BTE can be done by altering the degree of deacetylation
according to the required bone ingrowth.
3.3 Biological properties
Chitosan has been widely used in accelerating the wound healing capacity and
antimicrobial activities due to its cationic nature [53]. Chitosan by activating and
modulating the inflammatory cells facilitates the growth of granular tissue, which makes it
as a potent wound healing accelerator [56]. It acts as an important component in wound
dressing; as it binds to the anionic red blood cells, promoting clotting [54]. Chitosan

7
possesses antimicrobial properties by associating to the anions in the bacterial cell walls
and thus suppresses the biosynthesis of cell wall, which eventually kills the bacteria [57]. A
supporting role for CS in cell proliferation, osteoblast differentiation and mineralization has
been documented [45-48].
4. Chitosan scaffold fabrication methods
Bone tissue engineering involves the basic idea of using a 3D biodegradable
polymeric scaffold to promote bone tissue growth and remodeling. The physical properties
of CS have made it possible to be shaped into various structures, membranes, sponges,
fibers and porous scaffolds for BTE. Chitosan based scaffolds are prepared through various
fabrication methods and few of the methods are listed in Fig. 3. Amongst the methods, the
most common methodology for producing CS scaffolds is by lyophilization [24-30].
Freezing the CS solution results in formation of ice crystals via phase separation and on
sublimation, the original space occupied by the crystals is emptied and leading to the
formation of pores. This technique requires high precision control over temperature. The
disadvantages associated with it are collapsing of pores structure if the temperature is not
controlled. There is possibility of formation of larger pores and the choice of solvent is
limited. The use of solvent-exchange/phase-separation which is based on the gelation of a
solution using alkaline solution below its gelation point can be used. Through these
techniques, 3D scaffold with various geometries can be obtained but the major
disadvantage is the lack of proper mechanical strength. The technique of salt leaching
involves the incorporation of porogens such as sodium chloride (NaCl) of desired sized into
CS matrix and successive leaching of the salt particles using water results in formation of
porous CS scaffolds [58, 59].
Electrospinning allows the production of fibrous scaffolds. It utilizes electric field
created between the nozzle tip of the polymeric reservoir and ground collector. Applied
electric field causes the elongation of the polymer drop and leads to the formation of long
fibers. This versatile technique allows the production of both nano and micro fibers [60-
62]. Electrospinning of CS is quite difficult and the stability inside aqueous environment is
a major concern and often requires crosslinking with agents like poly(ethylene oxide)
(PEG). Blending with other polymers such as SF, Col, PCL can significantly reduce the
degradation rate of CS in electrospun membranes under aqueous conditions. Majority of

8
the scaffold fabrication methods has been widely utilized for generating CS based scaffolds
with no control over the scaffold architecture. Rapid prototyping utilizes the computer-
aided design data (CAD) to produce 3D scaffolds with precise control over its architecture
[63]. Highly reproducible geometries with enhanced mechanical properties are possible
with this technique. Chitosan based scaffolds using melt-based routes were also
synthesized and investigated by various research groups. Irrespective of the fabrication
technologies, the application of CS scaffolds in most of the load bearing sites is often
doubted. Blending CS with various natural, synthetic polymers or ceramic particles have
been found to possess enhanced bioactivity and mechanical properties compared to native
CS scaffolds.
Figure 3
5. Chitosan scaffold properties
Biomaterials offer substantial advantages compared to the conventional metallic
implants in terms of cell adhesion, spreading, proliferation, differentiation and other
cellular processes. The success of BTE scaffolds depends on its physico-chemical and
biological properties of the materials. The scaffolds should satisfy various properties suited
for BTE applications. They are (i) porosity, (ii) biocompatibility, (iii) water retention, (iv)
protein adsorption, (v) biomineralization, (vi) biodegradability and (vii) mechanical
properties. These parameters can be tuned by the addition of other polymers and ceramic to
CS for the improvement of scaffolds’ properties and applications in BTE.
5.1. Porosity
Porosity provides support for cell infiltration, adhesion, secretion of ECM
components and bone tissue ingrowth [64-66]. Cell attachment to scaffold depends on pore
dimensions. If pores are too small, it results in limited cell permeability and capsule
formation around the edges of the scaffold. Conversely, if pores are too large, it results in
limited specific area and reduces the ligand density available for the cell to bind. Cells can
easily recognize subtle changes in the ECM and that may affect cellular behavior [67, 68].
This indirectly influences cellular activity through integrin-ligand interaction between cells
and biomaterials. Hence, it is crucial in maintaining optimal pore size for cell migration
and attachment.

9
 Studies reported that pores greater than 20-100 µm favor cell infiltration, and
beyond 100 µm neovascularization is greatly improved [69]. Various reports highlighted
that pores greater than 300 µm promote direct osteogenesis and pores below 300 µm
encourage endochondral ossification [70, 71]. Despite various reports on pore size and
cellular interaction, the relationship connecting pores and osteoblast activity is not fully
understood due to the conflicting reports. Pores of the CS scaffolds depend on various
parameters like polymer concentration, crosslinkers, freezing temperature and amount of
micro or nanoparticles (NPs) addition. Chitosan scaffold containing HAp and beta-
tricalcium phosphate (β-TCP) was fabricated by freeze drying and processed at both -80°C
and -20°C. The scaffold processed at -80°C exhibited elongated pores with irregularity in
shape; whereas the scaffold processed at -20°C showed more irregularities in pores and
highly layered pores with collapsed morphologies. Pore distributions appeared to be less
homogenous when processed at -20°C. Upon crosslinking with sodium tripolyphosphate
(TPP), CS caused the changes in microstructural properties in the scaffold porosity [72].
Crosslinkers such as TPP, ethyl-3[3-dimethylaminopropyl] carbodiimide hydrochloride
(EDAC), genipin are also employed to improve the pores morphology and porous
interconnectivity in the scaffolds [73-78].
Addition of nanoparticles (NPs) to CS and its composite matrix may also have
effect on the porous architecture of the scaffolds. The addition of NPs can decrease the
pore size or may have no effect on the pore dimensions in the scaffolds. Addition of HAp
particles to CS-SF scaffolds significantly reduced the porosity from 92.4% to 89.7% [79].
nZrO2 on its incorporation into CS-SF scaffold decreased the pore sizes from 100 to 300
µm to 50 to 150 µm. In contrast, pores with interconnectivity were obtained on addition of
Zr [80]. Sufficient interconnectivity of pores is crucial for nutrient and oxygen transport,
metabolic waste removal and neovascularization. Addition of HAp to CS-Alg composite
scaffold was assessed and above 30% addition, the pores were collapsed and agglomerated.
In situ synthesis of HAp with CS solution and subsequent freeze gelation resulted in round
shaped porous structure. However, on increasing the content of inorganic phase into CS
matrix resulted in more non-defined pores. Higher amount of inorganic phase disrupts the
organized structures of CS microstructure and eliminates pore boundaries [81].

10
 Pore volume is one of the important features that determines the success of the
implanted scaffolds owing to its role in supporting cells harboring. Bioglass particles-CS
scaffolds formed a new hybrid biocomposite for releasing gentamicin sulfate, an
antimicrobial agent. With minimal addition of BG particles to CS matrix, fine pores with
large mean diameter were observed. On addition equal amounts of BG particles to CS
matrix, pore diameter was found to be decreased; whereas there was no change in the
morphology of pores but with less homogeneous surfaces. Addition of twice the amount of
BG particles to CS matrix resulted in wider pores and decreased pore volume [82].
Chitosan scaffolds possessed interconnected open pores with pore sizes from 300 to 400
µm and it reduced to 250 - 300 µm upon blending with Gn, and addition of nSiO2 particles
further decreased the pore size to 200 - 250 µm [83]. Chicken feather derived keratin NPs
incorporation into CS scaffolds had no change in the pore dimensions [84]. Inclusion of
mesoporous wollastonite (mWs) particles synthesized from rice straw ash into CS-
Carboxymethyl cellulose (CMC) matrix had no effect on pores morphology [30].
5.2. Water retention ability
Water retention ability refers to the ability of tissue engineering scaffolds to retain
water and also known as swelling ability. In vivo implantation of scaffolds results in the
adsorption of water from surrounding tissues and results in increased pore size which avails
cellular infiltration deep into the internal structures of the scaffolds [85]. Increased swelling
leads to the loosening of the implant and retraction away from the site of implantation.
Scaffolds with reduced swelling ability decrease cellular interaction. The swelling of
polymers depends on the ionizable groups in the structures and the surrounding medium.
Chitosan chains swell through protonation of amine/imine groups and lead to the
mechanical relaxation of coiled chains [86]. Amine groups of CS chains are the key player
of determining the swelling ability. Due to cationic nature of CS, it would have
electrostatic interaction with anionic polymers, and most of the amine groups in CS would
involve in polymeric complexation, leading to decreased swelling. Chitosan-Gn scaffolds
were fabricated and its swelling ratio was significantly reduced through the addition of n-
BGC particles [87]. Besides complexation of CS to Gn, the free hydrophilic groups of Gn
are vulnerable and decide the swelling upon exposure to fluids. On addition of nBGC
particles, these hydrophilic groups were engaged in the interaction. This caused reduction

11
in the number of free hydrophilic groups resulting in reduced swelling ability of the
scaffolds. To CS matrix, inclusion of nSiO2 and nZrO2 particles significantly reduced the
swelling behavior of matrix [24]. Cross-linking the reactive groups of CS with chemical
crosslinkers enhances the stability of the polymeric scaffolds. EDAC triggers carboxylic
groups and crosslinks with free primary amine groups in the polymers. TPP interacts with
CS amine groups [88]. Chitosan/nβ-TCP scaffolds were crosslinked with genipin, and they
were found to be stable by exhibiting a reduced swelling ratio [89]. Hence, it is possible to
tailor the swelling rate of CS based scaffolds through addition of other materials at micro or
nano scale and crosslinkers.
5.3. Protein adsorption
Rapid adsorption of proteins onto the tissue engineering scaffolds is required for
subsequent cellular interaction with biomaterials when introduced in a physiological
system [90-94]. The adsorbed proteins could influence cell adhesion, spreading and other
cellular events. The biological responses mediated by adsorbed proteins are based on their
amino acids content. It has been reported that fibronectin derived peptide REDV facilitates
endothelial cells adhesion but not fibroblasts or muscle cells [94]. These adsorbed proteins
regulate cellular functions via integrin mediated signaling pathways resulting in cell
spreading, proliferation and differentiation [95-98]. Primary integrin binding promotes the
clustering of additional adhesive proteins that initiate local cellular cytoskeletal remodeling
and cell arrangement [99]. Studies depicted that the presence of surface functional groups
(oxygen, nitrogen, carbonyl and hydroxyl) in the biomaterials influence protein adsorption
[100, 101]. In general, the factors influencing protein adsorption on the surface of
implanted biomaterials are (i) surface chemistry-functional groups, (ii) wettabillity and (iii)
surface topography. Fibronectin is secreted by osteoblasts, and it is one of the earliest cell-
binding proteins which functions as a link between the cells and the implants [102-109].
The amino and carboxyl groups of CS are the key players in protein adsorption. These
functional groups on CS impart hydrophilicity and interact with the functional groups of
proteins via electrostatic forces, van der waals force and induce protein adsorption onto the
surface of CS scaffolds. Chitosan blended with its electrostatically opposite polymers
generates increased mechanical strength and controlled swelling but the number of reactive
free functional groups may be diminished hence, the protein adsorption is hindered. In

12
order to enhance protein adsorption onto the scaffolds, NPs can be included. Nanoparticles
generate more surface area and focal adhesion points for the cells. Addition of nSiO2 to
CS/Alg matrix significantly increased the amounts of proteins adsorbed on the matrix [25].
Similarly, addition of nZrO2 increased protein adsorption onto CS based biocomposite
scaffolds [24]. It is most likely that addition of biomaterials at nano scale to the scaffolds
could promote protein adsorption on their surfaces [84, 110-112].
Diopside particles containing extensive silanol (-Si-OH) groups also increased
adsorption of proteins when complexed with CS, indicating the importance of silanol
groups in protein adsorption [86]. Protein adsorption onto the scaffolds is essential for
successive cell attachment, spreading and proliferation. Cell proliferation was found to be
potentially improved on CS upon its modification with nano-structured carbon [113]. Even
though bovine serum albumin (BSA) whose adsorption does not influence any
physiological changes, it is likely to improve cell proliferation and to enhance the stretched
pseudopodia in promotion of biological function. The actin micro filaments are vital in
facilitating movement of cells along the substratum/biomaterial [114]. Thus, pre adsorption
of proteins onto the scaffolds is essential for cellular interaction, and modification/addition
of NPs into CS matrix would improve its characteristics for protein adsorption and
regulation of cell behavior.
5.4. Biomineralization
Biomineralization refers to the deposition of ions and minerals on the surfaces of
biomaterials on exposure to body fluids. The deposition of CaP on the implants enhances
the activity of osteoblasts [115]. Since human contains 65-70% inorganic crystals mostly
with HAp, it is a key regulator in determining the bonding of scaffolds to bone [123].
Excellent integration of the scaffolding materials with bone can be achieved if a bone like
apatite is formed on the surface of the scaffolds in situ [124-126]. Hence, the
biomineralization potential of the scaffolding materials is tested on their ability to form
CaP crystals when exposed to simulated body fluid (SBF). Chitosan can act as a template
for biomineralization due to the presence of –NH2 and –C=O functional groups. The CS
based biocomposite scaffolds were found to have enhanced biomineralization potential
compared to CS alone. Addition of nZrO2 to CS/nSiO2 matrix improved biomineralization
as earlier at 7d [24]. Inclusion of nBGC particles to CS/Gn scaffolds enhanced the number

13
of nucleation sites, resulting in efficient apatite formation [87]. Thus, incorporation of NPs
tends to increase surface area thereby providing more nucleation sites for apatite formation.
Increasing the number of reactive groups in the scaffolds would generally enhance
the formation of bone like apatite with a Ca/P ratio of 1.6 close to human bone. The anionic
groups such as COO-, OH- and –NH2 can act as nucleation sites for the formation of
crystalline HAp particles [119]. Chitosan scaffolds exhibited only marginal
biomineralization; whereas addition of diopside particles enhanced apatite deposition [86].
It was suggested that the involvement of silanol groups in diopside particles could have
been contributed to increased apatite formation. Further, the release of calcium from
diopside particles also enhanced apatite formation [86]. Similarly, an increase in
biomineralization potential of CS/Alg scaffolds was achieved upon addition of nSiO2
particles [25, 120]. As mentioned earlier, the biomineralized layer is essential for direct
bonding of the scaffolds to host bone tissue and this would indirectly determine the success
of the implanted scaffolds.
5.5. Biodegradation
Biodegradation refers to the chemical process of gradual breakdown of the
implanted biomaterials in a biological system [121]. It is initiated on exposure of the
scaffolds to tissue fluids containing various enzymes and other active substances, whose
action tightly is regulated under physiological conditions. The implanted graft materials
should undergo degradation over time and should match with the formation of new bone.
Biodegradation is an important criterion to be considered prior to the design of any BTE
scaffolds for long-term success [122, 123]. There are few biomaterials which are degraded
in a non-regulated way on their exposure to water or serum [124]. The biodegradation
involves the scission of chemical bonds between the monomeric units of biopolymers,
between two polymers or between the polymer and ceramic/NPs added into the system.
Erosion is another phenomenon through which degradation occurs. Water soluble polymers
based scaffolds follow erosion and the scaffolds are degraded inside the biological system
through the absorption of water. The resulting degraded products should be non-toxic and
should not prove any immune response. They should be small so as to dissolve in body
fluids, excreted or incorporated into metabolic pathways [125-127].

14
 Chitosan is mainly degraded by the action of lysosyme [128, 129]. Higher degree of
deacetylation of CS leads to faster degradation by lysosyme [55]. The NAGs of CS are
hydrolyzed by the action of lysosyme and the degraded products are amino sugars which
are incorporated into GAG and glycoprotein metabolic pathways and excreted. The
magnitude of tissue response to the implanted biodegradable material is dependent upon
the site of implantation [130]. The faster degradation rates of CS scaffolds often limit their
long term persistence in vivo. Addition of other polymers, NPs to CS matrix may have
effect in controlling degradation kinetics. Diopside incorporation into CS scaffolds had no
effect on the percentage of degradation even after 72 h [86]. It has been reported that the
rate of degradation of CS/nHAp scaffolds was higher but in the presence of nano silver
(nAg), the degradation was significantly lowered. Due to the Ag ion complexes with the
amino and hydroxyl groups of CS, there might be only few free reactive functional groups
resulting in decreased hydration upon fluid exposure and thereby the decreased rate of
degradation [34]. Addition of nBGC particles to CS/Gn scaffolds significantly reduced
their degradation and that could be due to neutralization of the acidic degraded products of
CS because the persistence of acidic condition would cause dissolution of CS [87]. The
degradation rate can be controlled with the availability of free functional groups,
complexation with electrostatically opposite polymers and inclusion of crosslinkers such as
TPP, glutaraldehyde and EDAC [128]. The rate of degradation was found to be reduced in
glutaraldehyde crosslinked CS/Gn biocomposite scaffolds compared to uncrosslinked
scaffolds of the same composite [131]. Over all, the tailored degradation of CS based
biocomposite scaffolds is often required to tune with the rate of bone formation.
5.6. Mechanical properties
The bone tissue engineering scaffolds should possess appropriate mechanical
properties for their application in load bearing areas. The mechanical properties of the
scaffolds should match the host bone for proper load transfer to the adjacent tissues. The
mechanical properties of natural bone vary depending upon the type such as cortical or
cancellous bone. The cortical bone possesses young’s modulus and compressive strength of
15-20 GPa and 100-200 MPa while, the trabecular bone possesses between a range of 0.1-2
GPa and 2-20 MPa, respectively [132]. Studies reported the presence of a strong
relationship between scaffold stiffness and cell behavior. It influences the differentiation of

15
mesenchymal stem cells to specific cell types [133-136]. Chitosan has low to moderate
mechanical properties which limits its use in load bearing applications [137]. However,
addition of various polymers or NPs increased the mechanical properties [25, 137]. Such
reinforcement with opposite charged polymers or other addition changes the degradation
rate, scaffold chemistry and improves the mechanical properties. Addition of Gn and β-
TCP increased the compressive strength by approximately 70% [89]. Another report stated
that addition of CNT to CS/BG scaffolds increased the compressive strength up to 5.95±0.5
MPa [138]. As described earlier, the addition of negatively charged polymer improves the
mechanical strength, in accordance to that, addition of Gn, a negatively charged polymer
forms polyelectrolyte complex due to ionic interaction between these two molecules and
improves the mechanical strength [139].
In addition to improving the mechanical properties, inclusion of other polymers,
NPs or ceramics also determines the stiffness of the CS scaffolds. Stiffness of such
implanted scaffolds limits its use in load bearing sites. Mechanical tests indicated that
addition of ceramics (β-TCP) affects the stiffness of materials. Addition of TCP at 50%
concentration showed young modulus values about 0.5 MPa; when TCP added at 15%
concentration to CS sponges exhibited about 0.02 MPa. Thus, the compression strength of
the foams was found to be higher with increased TCP concentrations [140]. The possible
reason behind such increase in compressive strength could due to the decreased porosity
upon the addition of TCP particles. Scaffolds on implantation in vivo would absorb fluids
and the mechanical strength would vary on dry and wet conditions. Chitosan-Gn
membranes upon testing for tensile properties in dry condition showed significant increase
in break stress on increasing Gn content. Conversely, the increased Gn content decreased
break strain under wet conditions. In dry condition, increased stiffness was observed with
more Gn addition but in wet conditions, the presence of Gn decreased the stiffness of CS-
Gn membranes [111]. Although many reports stated that the mechanical properties of CS
based scaffolds can be tuned, no clear reasons were concluded for identifying an optimal
addition to match the properties with host bone. Hence, such additions have to be
considered and tuned for matching the mechanical properties of CS based scaffolds for
BTE.

16
 A literature analysis for the availability CS based biocomposite scaffolds with
enhanced properties suitable for BTE is shown in Table 1. Additionally, the in vivo studies
evaluating the CS based bone scaffolds are mentioned in Table 2.
Table 1
Table 2
6. Conclusions
Bone tissue engineering is regarded as an effective alternative approach to the
conventional grafting strategies. Amongst the polymers, the use of CS based biocomposite
scaffolds for promoting new bone tissue growth has been intensively researched. Although
many CS scaffolds are fabricated, there remain serious drawbacks in developing an
effective scaffold with optimal mechanical strength, neovascularization and proper
reproducibility. The inclusion of bioactive materials/molecules and newly developed
technologies in scaffold preparation, characterization and biological studies would further
promote the potential use of CS and its biocomposite scaffolds in bone tissue repair
applications.

Acknowledgement
This work was supported by the Council for Scientific and Industrial Research,
India (No. 37(1574)/12/EMR-II; No. 60(0110)/13/EMR-II to N.S).

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FigureLegends
Figure 1. Hierarchical structure of bone indicates the presence of various elements (osteon,
lamella, fibre bundles) and occurrence of nanostructured hydroxyapatite minerals
embedded on collagen fibrils. Osteoprogenitors, osteoblasts, osteoclasts and osteocytes are
the major cellular constituents of bone.

Figure 2. Bone tissue engineering involves the use of cells and biomaterials, or a
combination of cells loaded onto biodegradable scaffolds to treat critical sized bone
defects. Mesenchymal stem cells isolated from the patient donor are differentiated into
osteoblasts in vitro prior to its loading into scaffolds. Cell free construct is also used along
with bioactive molecules and nanoparticles for enhancing bone formation.

Figure 3. Schematic representation of the commonly used fabrication methods for

producing chitosan based scaffolds - freeze drying, freeze gelation, salt leaching,
electrospinning and 3D printing.

36
37
38
39
Table 1. Addition of polymers, ceramic particles and others to chitosan matrix for the
improvement of their physical, chemical and biological properties required for bone tissue
engineering applications.

Chitosan + Chitosan + Properties observed References

Polymers Ceramic or
others
Alginate Nano silicon  Improved protein adsorption, 25
dioxide controlled swelling
 Improved apatite deposition
Carboxymethyl Mesoporous  Reduced swelling
cellulose wollastonite  Decreased susceptibility to 30
lysosome
 Increased biomineralization
 Up regulated pre-mir-15b
- Hydoxyapatite  Enhanced tensile properties
and β-TCP  Controlled water retention and 72
maintained structural stability
Silk fibroin Hydroxyapatite  Reduced the porosity
 Supported growth of SaoS-2 cells 79
and increased viability
 Enhanced ALP activity
Silk fibroin Nano zirconium  Increased water uptake ability and 80
oxide compressive strength by
controlling the porosity
-  Controlled pore morphology upon
Bioglass varying the amounts of bioglass 82
 Better release kinetics of
gentamicin sulfate

Gelatin Nano silicon  Increased the swelling ability 83

dioxide  Decreased degradation rate
 Improved protein adsorption and
biomineralization
 Enhanced cell attachment and
ALP activity
- Keratin  Increased protein adsorption and
nanoparticles improved biodegradation 84
 Improved compressive strength
Gelatin β-TCP by 70% 89
 Increased the swelling and
biomineralization
- Bioglass and  Increased mechanical properties 138
carbon nanotube and compressive strength
 Promoted attachment and
proliferation of MG-63 cells
-  TCP addition decreased the 140

40
 β-TCP degradation of scaffolds
- Simvastatin  Reduced swelling, increased 141
loaded PLGA compressive modulus
mircoparticles  Enhanced cell proliferation of
hFOB and improved
differentiation
Alginate Hydroxyapatite  Controlled and uniform porosity 142
 Increased compressive strength
and elastic modulus
 Promoted differentiation and
mineralization of MC3T3-E1 cells
Collagen VEGF loaded  Promoted vascularisation in vivo 143
PLGA-PEG in rats
microspheres and
hASCs
- Nano bioactive  Increased compressive modulus 144
glass
- PLGA  Increased mechanical strength
nanoparticles and  Reduced swelling behaviour 145
bioactive glass
 Improved compression strength
Fucoidan β-TCP  Enhanced apatite deposition 146
 Increased osteocalcin secretion by
hMSCs
Poly(propylene -  Enhanced hydrophilicity
carbonate) favouring fibroblast attachment 147
and proliferation
 26% increased in young’s
modulus
Mimosa -  Improved cell viability,
tenuiflora cortex proliferation and differentiation 148
and ALP expression in rat
cavarial cells

Polu-3- Hydroxyapatite  High ultimate tensile strength

hydroxybutyrate- upon varying HA addition. 149
co-3-  Higher ALP activity in human
hydroxyvalerate fetal osteoblasts
(PHBV)  Chelated apatite crystals and
increased biomineralization
 Genipin crosslinked decreased
Polyvinyl 45S5 Bioglass degradation 150
pyrroidone  Supported cell growth by
controlling the dissolution of
bioglass
 Improved cell attachment with
extended cytoplasmic processes
with polygonal morphology
Alginate In situ  Highly interconnected porosity 151

41
 synthesised with thicker pore walls
hydroxyapatite
Gelatin HA,  Decreased degradation rate 152
montmorillonite  Increased apatite deposition and
swelling property
- Multiwalled  Promoted water uptake ability 153
carbon nanotube  Controlled the porosity
 Enhanced cell proliferation,
protein content and ALP activity
 Mineralization was higher
Chondroitin Hydroxyapatite  Increased cell proliferation, ALP 154
sulphate and activity and collagen-1 expression
amylopectin in MG-63 cells
Chitin Nano zirconium  Controlled swelling and 155
oxide degradation
 Enhanced osteogenesis
Alginate and -  Controlled swelling 156
polypyrrole  Improved cell viability and cell
attachment.
 Biomineralization in SBF and
under culture conditions were
enhanced
Silk fibroin -  Decreased degradation rate and 157
possessed antibacterial activity
 Supported adhesion and growth of
fibroblasts
Poly(vinyl Bioglass particles  Controlled porosity, swelling and 158
alcohol), degradation
collagen  High compressive modulus
 Increased ALP activity
 Controlled release of protein
(BSA)
Gelatin Calcium  Increased material degradation 159
phosphate  Improved cell attachment
- RGD  Increased pore size and 160
mechanical properties
 Promoted cell attachment and
proliferation
 Increased calcium deposition
-- HA and iron  Promoted bone healing upon 161
nanoparticles magnetic stimulation
β-1,3-glucan HA  Favoured cell adhesion, spreading 162
and proliferation and up regulated
the ALP, OC, Col-I levels
Polycaprolactone -  Improved bioactivity, protein 163
adsorption and adhesion of
osteoblasts
Poly(l-lactic  Tuned porosities 164
acid)

42
 PLGA  Supported MSC attachment and 165
nanocapsules spreading
loaded with  Sequentially delivered growth
BMP-2 and factors
PHBV loaded  Enhanced differentiation and
with BMP-7 increased ALP activity
poly(lactic acid-  Controlled porosity 166
glycolic acid)  Matched the compressive
microspheres modulus and strength of
trabecular bone.
 Increased ALP activity
 ALP, OPN, BSP, and OCN levels
were up regulated
Collagen HA  Exhibited higher cell proliferation 167
alginate and mineralization
Alginate Chondroitin 4-  Increased compressive modulus 168
sulfate and enhanced apatite formation
 Promoted cell spreading,
proliferation and osteogenic
differentiation
- Bioactive glass  Exhibited shape memory 169
nanoparticles properties
 Induced apatite formation

43
Table 2: In vivo studies on chitosan based biocomposite scaffolds

Chitosan + Polymers Bioactive Animal model Defect Site References

or other ceramic molecules
nanoparticles

Diopside - Rat Thigh 86

muscle

Alginate and - Severe combined Calvaria 151

hydroxyapatite immuno-deficient
mice

Alginate BMP-2 Rat Calvaria 170

Hydroxyapatite - Rat Tibia 171

Alginate and PLA-H VEGF Rat Femur 172

Nanohydroxyapatite, BMP-2 Rabbit Femoral 173

collagen, PLLA condyle

Nanohydroxyapatite - Rabbit Femoral 174

condyle

Collagen rhBMP-2 Rabbit Radius 175

Collagen, PLGA rhBMP-2 Dog Mandible 176

Collagen - Miniature pigs Femur 177

- - Rat Muscle 178

packets

44