Accepted Manuscript

Title: Chitosan based nanofibers in bone tissue engineering

Author: K. Balagangadharan S. Dhivya N. Selvamurugan

PII: S0141-8130(16)32362-5
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.12.046
Reference: BIOMAC 6861

To appear in: International Journal of Biological Macromolecules

Received date: 11-11-2016

Revised date: 30-11-2016
Accepted date: 16-12-2016

Please cite this article as: K.Balagangadharan, S.Dhivya, N.Selvamurugan, Chitosan

based nanofibers in bone tissue engineering, International Journal of Biological
Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.12.046

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Chitosan based nanofibers in bone tissue engineering

K. Balagangadharan, S. Dhivya and N. Selvamurugan*

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

Tamil Nadu, India.

To whom correspondence should be made:

N. 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

Bone tissue engineering involves biomaterials, cells and regulatory factors to make
biosynthetic bone grafts with efficient mineralization for regeneration of fractured or
damaged bones. Out of all the techniques available for scaffold preparation, electrospinning
is given priority as it can fabricate nanostructures. Also, electrospun nanofibers possess
unique properties such as the high surface area to volume ratio, porosity, stability,
permeability and morphological similarity to that of extra cellular matrix. Chitosan (CS) has a
significant edge over other materials and as a graft material, CS can be used alone or in
combination with other materials in the form of nanofibers to provide the structural and
biochemical cues for acceleration of bone regeneration. Hence, this review was aimed to
provide a detailed study available on CS and its composites prepared as nanofibers, and their
associated properties found suitable for bone tissue engineering.

Keywords: Chitosan, Nanofibers, Bone tissue engineering

Abbreviations

AA : Acetic Acid
Alg : Alginate
BSA : Bovine Serum Albumin
Col : Collagen
CS : Chitosan
DCM : Dichloromethane
DMAc : Dimethylacetamide
DMSO : Dimethyl sulfoxide
ECM : Extracellular matrix
GAGs : Glycosaminoglycons
GBR : Guided bone regeneration
HAp : Hydroxyapatite
HFIP : Hexafluoro-2-isopropanol
HFP : Hexafluoropropene
MSNs : Mesoporous silica nanoparticles
NCF : Nano carbon fiber
OCN : Osteocalcin
PCL :Polycaprolactone
PEO : Polyethylene oxide
PET : Polyethylene terephthalate
PHBV : Poly-3-hydroxybutyrate-co-3-hydroxyvalerate
PLGA : Poly(lactic-co-glycolic acid)
PLLA : Poly(L-lactic acid)
PPC : Poly(propylene carbonate)
PVA : Polyvinyl alcohol
TFA : Trifluoroacetic acid

Contents
1. Introduction--------------------------------------------------------------------------
2. Bone biology------------------------------------------------------------------------
3. Bone tissue engineering------------------------------------------------------------
3.1 Chitosan-------------------------------------------------------------------
3.2 Physico-chemical properties -------------------------------------------
3.3 Biological properties-----------------------------------------------------
4. Fabrication methods----------------------------------------------------------------
5. Electrospinning
6. Chitosan based nanofibers---------------------------------------------------------
6.1 Mechanical Strength-----------------------------------------------------
6.2 Biocompatibility----------------------------------------------------------
6.3 Degradation---------------------------------------------------------------
6.4 Cell adhesion and proliferation-----------------------------------------
7. Comparison of chitosan with other polysaccharides
8. Applications of chitosan in human medicine
9. Conclusions-------------------------------------------------------------------------
1. Introduction

Bone is an essential supportive structure of the body, characterized by its rigidity,

hardness, and regeneration ability. It serves to guard necessary organs, produces blood cells,
acts as mineral reservoir for calcium and maintains acid–base balance [1]. Bone injuries due
to fractures, joint arthroplasties, and dental defects are some clinical challenges that hinder
the natural bone healing pattern where autogenous and allogenous bone grafts fail to
circumvent the issues. Bone graft (natural/synthetic) acts as a substitute in stimulation and
augmentation of the body’s regenerative capabilities [2]. Recent scientific progress in
biomaterials based therapeutics has created significant advances with the development of
engineered tissues. Ceramics, natural and synthetic polymers are used in scaffold preparation
[3]. Chitosan (CS) is a natural polymer which is generally used alone or in combination with
different polymers or ceramics as scaffolds in bone tissue engineering (BTE) [4-9]. Scaffolds
are prepared by several techniques such as fiber bonding, melt molding, solvent casting, gas
foaming and phase separation [10]. Electrospinning is one of the techniques to prepare
scaffolds ranging from nanoscale to microscale fibers, and the nanostructures prepared by
this technique resemble the native components of the extracellular matrix (ECM) [11].

2. Bone biology

Bone is a highly vascularized tissue, consists of 60% mineral, 30% matrix and 10%
water [12]. Bone tissue can be categorized in two discipline forms: (a) cortical bone, which
provides mechanical and protective function, and (b) trabecular bone, which is more
metabolically active and facilitates movement of the joints and limbs [13-15]. In the process
of ossification, the formation of new bone by cells called osteoblasts occur. These osteoblasts
and bone matrix are the two essential elements involved in the formation of bone. Two
important processes involved in bone formation are (a) Intramembranous ossification: It is
the process through which the primitive connective tissue is directly involved (mesenchyme)
in the formation of bones (skull, clavicle, and mandible); (b) Endochondral ossification: It is
a process in which mesenchymal cells are initially differentiated into cartilage and further
replaced by bone cells (femur, tibia, humerus, and radius).

The constitution of the bone structure is as follows: (a) Inorganic (69%) component
containing hydroxyapatite (HAp) (99%), which is a crystalline complex of calcium phosphate
responsible for the hardness and rigidity of bone; (b) Organic (22%), consists of collagen
(Col) (90%) and non-Col structural proteins which include proteoglycans, sialoproteins and
glycoproteins. Growth factors and cytokines are the functional components of bone [16-18].
There are four types of active cells embedded in mineralized bone matrix namely,
osteoblasts, osteoclasts, osteocytes and bone lining cells, and these cells dynamically regulate
bone remodeling including bone formation and bone resorption [19].

3. Bone Tissue Engineering

Bone tissue engineering (BTE) can be described as a blend of different scientific

fields of interest. It offers prospective alternatives to autografts and allografts by making
effective use of synthetic grafts to assist tissue regeneration [20-22]. BTE focuses on
designing 3D scaffolds which have the ability to mimic the ECM, provide mechanical
assistance and help in the formation of new bone. Scaffolds must possess osteo-conductive,
osteo-inductive and osteogenic properties to promote the adhesion, survival, and migration of
osteogenic cells. Also, they are fabricated in such a way to provide physical and biochemical
factors which induce osteoblastic lineage [23-25]. Scaffolds with appropriate morphological
structure facilitate cell adhesion, proliferation and differentiation. The site of action of the
scaffolds is determined by its mechanical property. It is also essential that the scaffolds
degrade after the formation of natural tissues thereby mediating a completely natural tissue
replacement [26].
Natural polymers due to their ideal properties such as biocompatibility and
biodegradability have a greater option to use as scaffolds. Moreover, the optimization of
polymer concentrations and conditions result in control of porosity, charge, and mechanical
strength. Bioactivity is another highly rated property which can be controlled by the
introduction of additional chemicals, proteins, peptides, and cells. CS, Col/gelatin, silk,
alginate (Alg), hyaluronic acid, and peptides are some of the commonly used natural
polymers in the field of BTE. The study of these natural polymers as 3D scaffolds for bone
regeneration and experimentation of various optimization conditions are carried out to
improve their osteogenic capacities [27, 28]. Natural polymers when blended with other
materials as composite improves the physical and or biological properties of the scaffold [29-
35]. In general, BTE utilizes three components containing biomaterials, cells and regulatory
signals for cell lineage-specific commitment towards bone regeneration (Fig. 1).
Figure 1
3.1 Chitosan

Chitin is one of the most abundant natural resources found on the earth. The common
sources of chitin are the skeletal materials of crustaceans and insects and the cell walls of
bacteria and fungi. Chitosan is a linear polysaccharide composed of glucosamine and N-
acetyl glucosamine units linked by β(1-4) glycosidic bonds. It is the deacetylated form of
chitin. The degrees of deacetylation and molecular weights determine the purity level of the
CS. CS has the excellent properties such as biodegradability, anti-bacterial activity and bio-
compatibility [36-39]. The physico-chemical and biological properties of CS play a
significant role in design and fabrication of scaffolds used in BTE. There are a number of
reports available indicating the role played by CS for the enhancement of cell adhesion,
proliferation, osteoblast differentiation and mineralization [5-7, 29-37, 40].

3.2 Physico-chemical properties of chitosan

Chitosan is a heteropolysaccharide that consists of reactive hydroxyl groups (at C(2),
C(3) and C(6) positions), amino group and a linear polyamine. These groups are essential for
CS to undergo modification such as graft copolymerization. This, in turn, is used to produce
various useful scaffolds for tissue engineering applications. The 100% deacetylated CS is
highly crystalline in nature whereas CS with less than 100% degree of deacetylation is semi-
crystalline in nature. CS is soluble in both organic and inorganic acids (pKa 6.5); whereas it
is insoluble in neutral and basic solutions. The amount of free amino and N-acetyl groups in
CS determines its solubility [41].

3.3 Biological properties of chitosan

The presence of protonable amino groups and breakable glycosidic bonds in CS has
advantages of its utilization in biomedical applications. The protonable amino groups of CS
bind with the negatively charged mucin (cell membrane) resulting in mucoadhesion [42]. The
presence of positive charges on CS backbone is responsible for the haemostatic activity.
Protons released in the inflammatory area facilitate the protonation of the amino groups of D-
glucosamine residues resulting in an analgesic effect. CS contains breakable glycosidic
bonds, and there are proteases available which can degrade CS by breaking these bonds in
vivo [43]. As of now, eight human chitinases have been discovered, out of which three of
them possess enzymatic activity on CS. The degree of deacetylation determines the rate of
CS degradation. Non-toxic oligosaccharides of variable length are formed due to
biodegradation of CS. These oligosaccharides can be incorporated in metabolic pathways or
excreted. CS was approved by the Food and Drug Administration (FDA) for its use in wound
dressings due to its biocompatibility [44-49].

4. Fabrication methods
Scaffolds can serve as the template for cell adhesion, growth and function. They can
also act as the delivery vehicle or matrix to facilitate the migration, binding, or transport of
cells or bioactive molecules that are used for tissue regeneration. It is essential that the
scaffolds should get degraded after the formation of tissue. Based on the nature of the
polymer and its intended applications, the following methods such as fiber bonding, solvent
casting, lyophilization, phase separation have been developed to create highly porous
scaffolds.
• Fiber bonding: Primary polymer fibers are bound at their cross points using a
secondary polymer. Scaffolds prepared using fiber bonding have large surface area,
which is suitable for cell attachment and provides sufficient space for the regeneration
of ECM.
 Solvent casting: A polymer/salt/organic solvent mixture is cast which is followed by
solvent evaporation and dissolution of the salt particles in an aqueous medium.
Solvent casting is very simple, easy and inexpensive. There are two routes for a
scaffold to be prepared using solvent casting. In the first method, the mold is dipped
into the polymeric solution, and the solution is drawn off after a particular period.
This results in the formation of the polymeric membrane. In the other method, the
polymeric solution is immersed into a mold and sufficient time is provided for the
solvent to evaporate which results in the formation of a polymeric membrane layer
which then adheres to the mold.
• Lyophilization: A polymer solution at desired concentration is formed by the
dissolution of the polymer and is allowed to freeze, resulting in formation of the ice
crystals. Then, sublimation occurs which is the basic principle behind lyophilization,
and during this process, the original space occupied by the crystals is emptied and
further leads to pore formation and inter connectivity.
• Phase separation: Thermal energy is used as the latent solvent which initiates phase
separation. The polymer solution is dragged below the freezing point of the solvent
and dried as such. The resulting porous structure can be modified by adjusting the
thermodynamic and kinetic parameters.
 Due to the presence of several components at nano scale in ECM, scaffolds with
nanofibrous structure would mimic the ECM aiding the beneficial effects of bone
regeneration. These biomimetic scaffolds consist of biodegradable polymer nanofibers can be
fabricated by several methods, and electrospinning is also one of the techniques used in the
preparation of nanofibers. Electrospinning technique is considered to be the best alternative
method providing excellent spatial control over polymer architecture for achieving precise
control of pore size, geometry and interconnectivity [50-54].

5. Electrospinning

Electrospinning is a process of converting viscoelastic solution into nanofibers using a

high electrostatic force [55]. Polymeric solution is loaded into the syringe at a slow flow rate
by a syringe pump that has to be electrospun. As a high DC voltage is applied, the solution is
charged and causes a repulsive force within it. When a large amount of electric field is
applied, the liquid droplet from the tip forms a cone shape known as Taylor cone. A narrow
jet of liquid is originated from Taylor cone when applied voltage is sufficient to overcome the
surface forces and finally this narrow jet moves toward the collector. Opposite polarity or
neutral (grounded) charged electrode is placed nearby that attracts and collects the fibers.
While the liquid jet travels towards the collector, the solvent evaporates from fiber jet, and a
solid fiber is deposited on the collector (Fig. 2). The end product is the formation of
continuous nanofibers, and these nanofibers are used in many different applications including
bone tissue regeneration.
Figure 2

There are four main adjustable parameters to determine the morphology of

electrospun structure such as 1) flow rate of the polymer solution through the syringe, 2)
concentration of the polymer solution, 3) voltage applied to the needle, and 4) the working
distance between the needle and the collecting plate. In BTE, scaffolds should be orientated
into uniform nanofibers without beads. Because the uniformity of nanofibers is essential for
the enhancement of cells growth, communication, and differentiation [56-60]. Due to high
surface area to volume in nanofibers, the nanofibrous structures lead to enhanced cell
adhesion and proliferation. The porosity of nanofibers supports oxygen permeability resulting
in the creation of micro environment for bone repair. Most importantly, these nanofibers are
associated with the properties of mimicking the ECM, biocompatibility and controlled
biodegradability, and they are capable of promoting cell adhesion, proliferation and
differentiation (Fig. 3). Reports are indicating the preparation of the nanofibers containing CS
and other polymers at various conditions using electrospinning technique. These nanofibers
were found to have enhanced properties which are essential to support BTE (Table 1).
Figure 3
Table 1

6. Chitosan based nanofibers

Even though CS has been widely used in BTE, its utilization alone or with other
materials as nanofibers using electrospinning is not yet greatly explored. Based on the
literature available, in the following sections, the advantages of CS based nanofibers
regarding their properties such as mechanical strength, biocompatibility, degradation, cell
adhesion and proliferation for BTE are discussed.

6.1 Mechanical strength

Based on the types of natural bone, their mechanical properties are varied. Young’s
modulus and compressive strength of cortical bone are 15-20 GPa and 100-200 MPa whereas,
the trabecular bone ranges between 0.1-2 GPa and 2-20 MPa, respectively. Hence, designing
the scaffolds for bone regeneration and fracture healing, the mechanical properties of the
scaffolds should be considered [73]. There is a strong relationship between scaffold stiffness
and cell behavior, and it was shown that such kind of relationship influenced the
differentiation of mesenchymal stem cells [74]. In load bearing applications, CS cannot be
used as such due to its low mechanical strength. But its mechanical properties can be
increased by including other polymers. The mechanical properties of the scaffolds can be
improved in the presence of oppositely charged polymers [75].
Electrospun fibers have been shown to withstand forces originated during bone
regeneration [76]. The mechanical strength of the nanofibrous scaffolds provides an initial
support to the cells. Increasing the fiber diameter and decreasing the porosity of PCL/CS
nanofibers significantly improved their mechanical properties in both ambient and
physiological conditions [77]. Large amounts of hydrophilic CS were introduced into PLGA
solution, and the PLGA/CS hybrid nanofiberous membranes were synthesized by dual-power
electrospinning. A number of factors like fiber structure, properties of the constituent
polymers, and their interactions determine the mechanical properties of electorspun
nanofibrous membranes. Interaction of CS with PLGA at points of fiber intersection due to
cross-linking appeared to have a positive effect that provides frictional entanglements
between the fibers [78]. The altered compositions of PLGA/CS polymers enhanced their
structural and mechanical properties [79]. The strong ionic interactions between alginate–
CS–HA composite has also been reported to show their enhanced mechanical strength. The
mechanical properties of CS–starch composite was enhanced by the addition of nano-HAp
(nHAp) and very small amounts of nano carbon fiber (NCF). The bonding formed between
the inorganic bio-ceramic nHAp and the organic biopolymers promoted the compressive
modulus and strength of CS/starch nanofibers. The addition of 10% nHAp increased their
compressive strength and compressive modulus from 16.42 MPa to 71.84 MPa and 525.16
MPa to 1326.5 MPa, respectively. Consequently, an amino silane treatment introduced amine
groups to the surface of nHAp particles which in turn strengthened the interfacial interactions
in starch/CS/NCF [80]. Mesoporous silica nanoparticles (MSNs) belong to the category of
bioactive inorganic ceramic materials that structurally mimic a honey comb-like pattern with
prominent features like controllable pore sizes, high surface areas, high pore volumes. MSNs
are increasingly used as advanced reinforcement agents with polymers to improve the surface
properties and mechanical strength. MSNs incorporated CS based nanofibers yielded the
mechanical interlocking, and the high surface area inherent to MSNs could lead to an
efficient stress transfer mechanism, thus increasing the strength of MSN/polymer composites
[71].
6.2 Biocompatibility
Cell–material interactions play an important function which is regulated by the
surface properties of the material such as chemistry, roughness, and surface energy [81]. One
of the basic requirements of the qualified material is biocompatibility. Biocompatibility
testing is a standardized biological test for biomaterials, and it attempts to find an effective
and safe testing protocol determining the suitability of material for its downstream
biomedical applications. The biocompatible material can be able to interact with host cells
without producing any an adverse effect. The material must possess non-toxic, non-
carcinogenic, non-allergenic, and non-immunogenic properties. In general, the material does
not elicit toxicity or any chronic adverse effects [82]. The biocompatibility of the material
can be tested under in vitro and in vivo conditions. In an in vitro test, the cultured cells that
react with the experimental material will allow us to directly determine the material toxicity
properties. Under in vivo conditions, material implanted in the subcutaneous or intramuscular
region of rats and rabbits can be allowed for a particular period of observation and can be
assessed for tissue response [83]. CS scaffolds showed biocompatibility when it was
implanted in a mammalian model. Early accumulation of neutrophils in the implantation area
reduced the inflammatory response. The granulation of tissue formation with angiogenesis
referred as the healing response was also found, and thus CS has a high degree of
biocompatibility in vivo [81].
Chitosan nanofibers have been shown to not altering the morphologies of osteoblasts
cells when they were seeded on CS nanofibers [84, 85]. Strong intermolecular interactions
between PET and CS enhanced their antibacterial and biocompatibility properties compared
to the PET fibers alone [86]. The topography of nano-scaled structures improved by PLLA
and CS hybrid polymer increased their biocompatibility level [87]. Honey with PVA-CS
nanofibers has been shown to have their effective biocompatibility [88]. Accounting to
improve the bioactivity of CS/PCL nanofiber system, Vaidya et al., 2015 have reported a two
step process comprising of (i) a coaxial electrospinning followed by (ii) peptide grafting. In
the coaxial electrospinning of CS/PEO-PCL, CS/PEO was the shell and PCL served as the
core. The resulting nanofibers possessed superior mechanical properties regarding young’s
modulus and tensile strength. The surface of this core-shell nanofibers upon functionalization
by RGD peptide grafting resulted in enhanced cell attachment and metabolic activity [89].
Poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), a polyhydroxyalkanoate
derived naturally from bacteria is a biodegradable, biocompatible and cost-effective polymer
with superior mechanical properties. Also, the degradation products of PHBV are less toxic
due to which recent investigations focus on using PHBV based polymers for tissue
engineering applications. To further improve the biocompatibility of these polyesters, CS
being a natural polymer with high biocompatibility was blended with PHBV in an
electrospinning method. Such nanofibers were developed by Zhang et al., 2014 where HAp
was additionally composited to increase the bioactivity of nanofibers. The prepared PHBV-
CS/HAp fibers showed improved osteo-integrative properties regarding cell attachment and
integrin responses for increased expression of osteocalcin (OCN) [90].
Surface modification of biomaterials is a method adopted to improve their biological
performance by immobilizing biochemical cues like growth factors, drugs, proteins/ peptides.
Some common limitations in the incorporation of bioactive molecules via co-electrospinning
include non-homogeneous distribution, insufficient loading efficiency, instability/loss of
activity due to harsh experimental conditions and compromised mechanical strength. Cheng
et al., 2014 used an alternative method of surface modification to immobilize Col and bovine
serum albumin (BSA) by covalent conjugation. CS is often preferred as a polymer of choice
for bio-conjugation mainly due to the availability of amine functionality in its structure that
could easily be conjugated with other small bioactive molecules. Previous attempts to
incorporate Col with CS based nanofibers had failures due to loss of mechanical integrity due
to rapid degradation. The current study has employed, EDC/NHS chemistry as a fitting
strategy that could maintain the bioactivity and structural property of the protein Col.
Covalent coupling was found to result in maximum immobilization efficiency and uniform
distribution of proteins (BSA and Col) when compared to a physical adsorption process. The
resulting PCL/CS nanofibers conjugated with collagen and BSA. The cells attached on these
functionalized matrices showed well organized F-actin filaments and had the polygonal shape
with larger surface area. The expression of osteogenic markers namely ALP, osteocalcin and
osteopontin was found to be upregulated in rBMScs at 14 d and 21 d of growth on these Col,
BSA functionalized PCL-CS nanofibers compared to their unfunctionalized counterpart [91].

6.3 Degradation
The degradation of polymer-based materials is based on a surface erosion mechanism
when catalytic molecules or substances such as enzymes and alkalis are present in the
degradation media or environment. In this mechanism, ions act only on the surface of
materials rather than diffusing into the material. This results in the erosion of the material
from the surface whereas the core part remains as such. On the other hand, bulk erosion
mechanism is responsible for the degradation of most of the biodegradable polymers. This is
due to the absence of catalytic molecules. The thickness of the material determines the
conversion of bulk erosion to surface erosion mechanism. The erosion mechanism was found
to be due to the hydrolytic degradation, which was shown to be dependent on the structure of
the materials [92]. Biodegradation of polymeric biomaterials involves the breaking down of
hydrolytically or enzymatically sensitive bonds present in the polymers. Non-toxicity and
avoiding of immunogenicity are crucial properties which should be essentially possessed by
the degradation products. The products thus obtained must be smaller in sizes as they have to
be dissolved in the body fluids. They also play a vital role in transportation and excretion [93-
95]. The rate of degradation of scaffolds is an important parameter to be considered as it
generates space for the tissue and thereby assists in matrix deposition. This is highly essential
for qualitative and quantitative regeneration of bone. The N-acetyl glucosamine (NAG) group
in the CS chain is degraded by lysozyme which is a primary degradation enzyme [96]. The
CS and Col interaction may cause steric hindrance to specific cleavage sites of lysozyme. The
degradation rate of the scaffolds must be tailored appropriately with the growth rate of the
new tissue. The enzymatic mixture with lysozyme and lipase had a strong positive effect on
the scaffolds degradation [97]. PCL is elastic with hydrophobic nature while CS is brittle and
hydrophilic. The hydrophobic PCL interacted with hydrophilic CS and formed hybrid
nanofibers in the aqueous medium [98].
The controlled degradation and biocompatible nature of CS favour its use as barrier
membranes for guided bone regeneration (GBR). GBR is a surgical procedure which employs
resorbable/non-resorbable barrier membranes for space maintenance at the defective site
(mainly in periodontal bone defects) that could promote the recruitment and growth of
osteogenic cells and block the migration of competing soft tissue cells from overlying
mucosa [99]. Such membranes are also meritorious in protecting the exposed wound against
mechanical disruption, saliva-borne contamination. The use of both resorbable and non-
resorbable membranes as GBR materials has been reported. Non-resorbable membranes
initiate the unfavorable immune response by the host, and a surgery may be required for
subsequent removal of them. On the other hand, CS and Col based natural resorbable
polymers are superior over non-resorbable synthetic grafts as they possess features like (i)
elimination of the need for a second surgery for membrane removal, (ii) cost-effective, (iii)
decreasing patient morbidity. Electrospun CS based barrier membranes due to their tunable
degradability and anti-infective properties established their candidature as potential GBR
barrier membranes. The CS/genipin crosslinked nanofiber mats have been reported as
effective barrier membranes with increased tensile strength and prolonged degradation
capacity compared to uncrosslinked CS and Col. Further, these membranes supported the
growth and proliferation of human osteoblastic cells (SaOS-2) [100].
Although CS has diverse biological features that could facilitate their use as organic
polymer-template for tissues, one major drawback encountered in electrospinning CS
solutions is that highly viscous CS solution results in chain entanglement. In general,
nanocomposite fibers containing organic-inorganic materials aim at utilizing the advantage of
organic material in providing flexibility, moldability while the inorganic material confers heat
stability, high strength and chemical resistance. Toskas et al., 2013 developed the core-shell
nanocomposite fibers containing CS/PEO as the organic component with silica as the
inorganic component. PEO, a source of neutral moieties was able to reduce the CS solution
viscosity which in turn alleviated the chain entanglement issues resulting in finely
electrospun fibers. The resulting electrospun core-shell type nanofibers possessed silica at the
shell surface encapsulating the organic component. The addition of PEO minimized the hyper
reactivity of silicates by establishing a two-phase dispersion that prevented the formation of
silicate beads during electrospinning. The study highlighted the following interesting
observations: (i) Silicate ions rendered silanol groups that effectively initiated nucleation of
HAp deposits upon exposure to SBF (ii) Silica precursor sourced from sol-gel method was
found to provide a large surface area for biomineralization (iii) A strong hydrogen bonding
was formed between the silicate and CS/PEO system while a covalent bonding was formed
between epoxy group of silanol groups at the shell surface and the amine groups of CS at the
organic core which in turn was capable of preventing the decomposition of the core
components [101].
In a study by Cui et al., 2013, CS grafted PDLLA nanofibers were developed by a
two-step process, and these nanofibers enhanced biomineralizing ability in SBF due to the
incorporation of bioactive amino groups on their surfaces. Also, the grafted amino groups
promoted the proliferation of mouse pre-osteoblastic cells (MC3T3-E1) and increased their
ALP activity [102]. A major limitation in using CS as a standalone polymer for tissue
engineering applications is its faster degradation. To overcome the higher degradation ability
of Col, Yu et al., 2013 developed CS/Alg mat coated with Col and HAp to form a
polyelectrolyte layer aiming to lower the solubilisation of Col at the implanted site [103].
They successfully employed electrospinning method to develop a CS/Alg fibrous mat with a
core–sheath 3D structure resulting in fibrous mats with high a specific surface area. The
presence of CS in the fibers rendered positive charge distribution on the surface of fibers.
Further, they attempted to coat Col and HAp on the surface of the fibrous mat by a physical
adsorption technique resulting in a composite that could act as a template for controlled
degradation of Col over a long period. The prepared CS/Alg/Col-HAp reinforced nanofibrous
mats facilitated rapid cell attachment, spreading, proliferation and mineralization of
osteoblast cells. This might be a novel approach to stabilizing the bioactivity of Col over a
longer period which is required in BTE applications [104]. The morphological aspects and
diameter of fibers are the factors to be considered while examining the degradation of
electrospun fibers. Honey has several properties favourable for tissue engineering
applications namely antibacterial activity, wound healing ability and ability to increase
surface to volume ratio etc. Recently, Sarhan et al., 2016 developed a nanocomposite fiber
system comprising of CS/PVA/honey (HPCS) crosslinked by glutaraldehyde. The resulting
cross-linked HPCS nanofibers exhibited decreased degradation compared to the
uncrosslinked system [105].
6.4 Cell adhesion and proliferation
Scaffolds should promote cell adhesion which is the initial step required for cell
proliferation and differentiation. Nanofibrous structures have been shown to facilitate the
attachment of human osteoblasts and chondrocytes. They also helped in maintaining
characteristic cell morphology and viability. The nanofibers composed of polymers were
found to be conductive for cellular attachment. The CS/PEO fibers prepared by
electrospinning promoted the attachment of cells, without causing any hindrance to their
morphology and structure. CS nanofibers showed higher surface area and better porosities
which are essential for the improvement of cell activity and cell maturation, and these
nanofibers also stimulated adhesion of osteoblasts [106, 107]. The synthetic PCL and natural
CS were used to form a core-shell electrospun structure with micro- and nano-sized fibers
having inter fiber pores. These PCL/CS core-shell structures then resembled the native ECM.
This interconnected open pores of the nanofibers increased the diffusion rate of the nutrients
[108]. The porosity and pore morphology of PVA/CS mats enhanced the migration of the
cells and blood vessels formation. They also promoted the nutrients and waste products
between the cells. The higher surface amine group of CS with lower water contents of the
PVA influenced the fiber diameter. This fiber diameter attributed to the enhanced cell
adhesion, proliferation, and migration [109].
Cell viability on CS/HAp nanofibers was found to be distinctly higher than that on CS
film, CS nanofibers, or CS/HAp film. The high surface area of CS/HAp nanofibers
contributed to this property [110]. Bioactivity of the material can be viewed by its protein
adsorption. Adsorption of proteins such as fibronectin and vitronectin from serum can be
achieved using the HAp/CS nanofibers, and these proteins facilitated better binding with
integrins. The subsequent signaling pathways then got activated resulting in the promotion of
cell signalling and proliferation [111]. Nanometric topologies of the fibrous or microporous
structure of electrospun fibers are important, and they are recognized by the cells. The
decrease in the fiber diameter decreased the porosity, but increased its fiber density.
Electrospun PLGA/CS/PVA membranes were able to provide a suitable nanotopography
aiding favourable cell-cell, cell-matrix interactions that promoted attachment and
proliferation of fibroblast cells [112]. The effect of the addition of nHAp and nBGC in
PCL/CS nanofibers for periodontal bone tissue engineering applications was studied. While
the incorporation of both nHAp and nBGC in PCL/CS nanofibers increased their protein
adsorption. The ALP activity of human periodontal ligament fibroblasts and MG-63 cultured
on PCL-CS fibers also increased [113]. A hybrid containing PCL and PVA/CS facilitated the
attachment of osteoblasts [114]. The CS–Alg nanofibers enhanced cell adhesion properties
indicating their potential towards bone tissue engineering applications [115]. The biological
properties associated with nanofibers containing CS and others, and the in vivo applications
of these nanofibers in BTE are summarized in Table 2 and Table 3, respectively.
Table 2
Table 3
7. Comparison of chitosan with other polysaccharides
Other polysaccharides such as cellulose, its derivatives and Alg have been
investigated polysaccharides in bone tissue engineering applications. However, cellulose
based biopolymers are associated with poor degradation properties limiting their use for
biomedical applications. Alg is a naturally available polysaccharide comprising of L-
guluronic acid and b-D mannuronic acid established for its use in tissue engineering
application. Pristine Alg has very low cyto-compatibility and hence it is often composited
with other natural/synthetic polymers or chemically modified with functionally bioactive
peptides to promote cell adhesion [124]. Alternatively, CS complexed Alg scaffolds showed
improved cell attachment behavior due to the enhanced adsorption of serum proteins
mediated by the cationic nature of CS [125, 126].
Fucoidan, ulvan and acemannan are found to be associated with notable osteogenic
properties. Fucoidan, a sulfated polysaccharide derived from brown algae promoted FGF-2
activity in stem cells and as a result, there was increased expression of osteoblast marker
genes. The presence of heparin binding domain in the structure of fucoidan resulted in higher
binding affinity to growth factors [127, 128]. Despite the osteogenic property of fucoidan, the
highly hydrophilic nature of this polysaccharide makes it difficult to be incorporated as a
blend in composite systems to support bone tissue regeneration. Upon addition of fucoidan to
CS, Alg, PCL and bioceramics, the physically stable composite scaffolds could be developed
with enhanced osteogenic property [128-130]. Algal polysaccharide ulvan has captured
attention due to its bioactivity and non-cytotoxic behavior. The unusual complex gelling
behavior of ulvan and its undesirable salvation properties in water are the major limitations
associated with ulvan in its pristine form like most polysaccharides. Toskas et al., 2012
assessed the cyto-compatibility and the fiber forming ability of a ulvan-CS polyelectrolyte
complex in 7F2 osteoblast cells [132]. Barros et al., 2013 reported novel bone cement
comprising of modified ulvan-CS composite [131]. Dash et al., 2014 identified the osteogenic
capacity of a photo-crosslinked ulvan scaffold [133]. Further, in vitro and in vivo
investigations on ulvan based composites are needed to validate its use in bone tissue
engineering applications.
Acemannan, a plant polysaccharide derived from the leaves of aloe vera showed the
bone forming properties [134, 135]. The extracts of aloe vera plant stimulated the levels of
VEGF, BMP-2, alkaline phosphatase activity, bone sialoprotein, osteopontin expression, and
mineralization in bone marrow stromal cells (BMSCs) and primary dental pulp cells. Aloe gel
possesses several bioactive compounds namely glycoproteins, polysaccharides, antioxidants,
growth factors, vitamins and others, and it confers proliferation and differentiation of cells
[134, 135]. PCL/aloe vera/silk fibroin nanofibrous scaffold enhanced the proliferation and
differentiation of adipose-derived stem cells. An aloe vera based biocomposite scaffold
containing mesoporous HAp, polyurethane with antimicrobial properties was reported [136].

8. Applications of chitosan in human medicine

Chitosan as a natural polymer with high biocompatibility, osteoconductivity and

versatile biological properties markedly certifies its use as scaffolding material for tissue
engineering. Although the discussions of the current review had focused on the application of
CS based biocomposite nanofibers in bone tissue engineering, it is crucial to discuss other
biomedical applications of CS as well. CS has the ability to bind to red blood cells and thus
possesses exceptional immunomodulatory properties enabling its usage as a hemostatic agent
for bandages and wound dressings [137-139]. Any wound dressing material is expected to
prevent infection so as to eliminate the need for oral or site specific delivery of antibiotics.
Being inherently antimicrobial, CS based wound dressings combat against wound associated
infections and prevents dehydration at the site of injury. The addition of metallic
nanoparticles like copper, silver, zinc and zirconium with antimicrobial properties [140]
could further improvise the anti-infective properties of CS based wound dressings [140, 141].
Demineralization of enamel is a serious dental problem where entry of acids from the
surrounding oral circulation leaches out the minerals from the tooth enamel. Effective
mechanical barriers termed as guided membranes are often required to prevent the
demineralization processes. Natural polymers such as CS, Col have advantageous over
synthetic polymers as guide barrier membranes for treating periodontitis; whereas the poor
mechanical stability and the high cost of Col make CS as the preferred material of choice
[142]. A most significant wide spread biomedical application of CS is in the fields of drug
delivery and gene therapy. In DNA transfection, the cationic nature of CS is beneficial in
protecting the negatively charged DNA molecules thereby preventing its immediate
degradation upon exposure to physiological environments [143]. Also, CS nanofibers are
widely used as efficient drug delivery vehicles for the controlled and sustained release of
drugs [144].

9. Conclusions
Bone tissue engineering is considered as an efficient alternative approach to the
typical graft methods. Amongst other available fabrication techniques for making scaffolds in
BTE, electrospinning is a relatively simple, unique widely used for the fabrication of fibers
with a high porosity and high surface area. Blending with other polymers/bioactive materials
in CS matrices resulted in functionally improved materials regarding physico-chemical and
biological properties, and hence, CS based nanofibers have wide relevance for their use in the
field of bone tissue engineering.

Acknowledgements
This work was supported by the SRM University and the Council of Scientific and
Industrial Research, India (60 (0110)/13/EMR-II to N.S.).
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Figure legends:

Figure 1. Three components namely biomaterials, cells and regulatory factors are essential in
bone tissue engineering.

Figure 2. A schematic diagram of the electrospinning setup.

Figure 3. The properties associated with nanofibers, and interaction of cells with nanofibers
for bone tissue engineering.
 Table 1. Preparation of Chitosan based nanofibers by electrospinning and their significances
in bone tissue engineering.

Distance
-Natural Ratio Solvent Voltage between Flow rate Significances References
(kv) syringe
-Synthetic and
polymers collector
(cm)

Chitosan 50/50 w% Acetic acid 25-28 11 0.4 and 1.0 Promoted cell attachment 61
(Polyethylene mL/h and proliferation, and
oxide)/Silica increased apatite
formation
N-methylene 1:1 (v%) Deionized water 18 8 5 µl/min Enhanced cell attachment 62
phosphonic and proliferation of MG-
chitosan/PVA 63 cells, increased mRNA
expressions of ALP and
Col-1, non-immunogenic,
and accelerated bone
healing in rat tibial defect
Chitosan/PCL 0.8:8 1,1,1,3,3,3- 15 - 10 µl/min Antibacterial activity 63
/Rifampicin (w%) hexafluoro-2- against S. epidermidis and
1:1 (v%) propanol (HFIP) prevented biofilm
formation and upregulated
the gene expression levels
of ALP and Col-1
Chitosan/PVA 7%:7% Glacial AA &DDW 20 10 0.5 ml/hr Decreased tensile strength 64
30:70 (70/30) and supported fibroblast
cells attachment and
proliferation

Ag-NPs- 0.2%: 0.1% (v/v) 25 20 3 ml/hr Enhanced stability 65

chitosan/ 10% AA: Water
Gelatin (w/w)
Chitosan 20:80 HFP/TFA (v/v, 16 10 0.8 ml/hr Facilitation of nanometer 66
/Collagen (w/w) 90/10) scale architecture of
extracellular matrix
Chitosan/ 15%: DMSO/DMAc 14 11 1 ml/hr Possessed antibacterial 67
PAN 15% (50/50, v/v) activity and increased
(Wt %) surface area and water
insolubility
Chitosan/Silk 10% TFA/DCM (7:3 18 12 0.5 ml/hr Regulated ALP activity, 68
Fibroin ( 1:1) w/w) increased in osteogenic
(w/w) gene expression and
enhanced mineral
deposition
Chitosan/ 20%:5% HFIP/ AA (9:1) 20 17 0.5 ml/hr Enhanced adhesion, 69
polyamide- (Wt %) proliferation and
6,6 maturation of osteoblasts
Chitosan/ 3% DCM : DMSO 20 20 0.01ml/s Promoted cell growth and 78
Poly lactic 70/30 increased in cell adhesion
acid
Chitosan/PH 9.5:85.5:5 Trifluoroacetic 13 10-15 1 ml/h Increased cell attachment, 70
BV/HAp (w:w:w) acid:dichloromethan proliferation and ALP
e activity, increased
hydrophilicity, tensile
strength, and promoted
HAp
Chitosan/Mes 20%/10% Acetic acid:dimethyl 16 15 1 ml/h Increased mechanical 71
oporou silica (w%) sulphoxide strength, cytocompatible
nanoparticles to osteoblasts, and
increased ALP activity,
Chitosan/ 2:3 (w%) Acetic 10 30 0.3ml/h Increased tensile strength 73
PVA/ Fe3O4 acid:deionized water and supported cell
adhesion and proliferation
of MG-63 cells
Table 2. Properties of chitosan based nanofibers

Chitosan+ Natural
/Synthetic Mechanical Biocompatibility Cell adhesion References
polymers Strength and Degradation
/ Others Proliferation
PCL + + - ± 77
PLGA + + - - 78
Alginate–HAP + + - - 80
PET - + + - 86
PLLA - + - - 87
Honey /PVA - + - - 88
PCL core-shell - + + - 108
PVA - + + - 109
HA - + + - 110
PCL -PVA + + + - 114
Alginate - + + - 115
Collagen + + - + 97
Honey/PVA - + - + 98
Table 3. In vivo studies on chitosan based nanofiber scaffolds

Chitosan +
Polymers Bioactive Animal model Defect Site References
or others molecules

Hydroxyapatite - Rat Calvarial 116

PEO Nanohydroxyapatite Rat Femur 117

Collagen Minocycline Rat Calvarial 118
Collagen - Rabbit Calvarial 119
Alginate- β-TCP - Rat Osteochondral 120
- Genipin Rat Calvarial 121
iPSCs+collagen Hydroxyapatite mouse Cranium 122
Hydroxyapatite-genipin mouse Calvarial 123