A Review of Chitosan and Its Derivatives in Bone Tissue Engineering

Carbohydrate Polymers 151 (2016) 172188
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Review
A review of chitosan and its derivatives in bone tissue engineering

R. LogithKumar 1 , A. KeshavNarayan 1 , S. Dhivya 1 , A. Chawla 1 , S. Saravanan,
N. Selvamurugan
Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu, India
a r t i c l e
i n f o
Article history:
Received 4 March 2016
Received in revised form 24 April 2016
Accepted 15 May 2016
Available online 18 May 2016
Keywords:
Biomaterials
Chitosan
Stem cells
Osteoblasts
Bone tissue engineering
a b s t r a c t
Critical-sized bone defects treated with biomaterials offer an efcient alternative to traditional methods involving surgical reconstruction, allografts, and metal implants. Chitosan, a natural biopolymer is
widely studied for bone regeneration applications owing to its tunable chemical and biological properties.
However, the potential of chitosan to repair bone defects is limited due to its water insolubility, faster
in vivo depolymerization, hemo-incompatibility, and weak antimicrobial property. Functionalization of
chitosan structure through various chemical modications provides a solution to these limitations. In
this review, current trends of using chitosan as a composite with other polymers and ceramics, and its
modications such as quaternization, carboxyalkylation, hydroxylation, phosphorylation, sulfation and
copolymerization in bone tissue engineering are elaborated.
2016 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Quaternized chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
2.1.
N,N,N-trimethyl chitosan (TMC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
2.2.
N-(2-hydroxyl) propyl-3-trimethylammonium chitosan chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
2.3.
Other quaternized chitosans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Carboxyalkyl chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
3.1.
Carboxymethyl chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
3.2.
Carboxymethyl chitosan derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Abbreviations: ACP, amorphous calcium phosphate; ALP, alkaline phosphatase; BBC, bioactive bone cement; bFGF, basic broblast growth factor; BTE, bone tissue engineering; BHA, butylated hydroxy anisole; BMP-2, bone morphogenetic protein-2; CDH, calcium-decit hydroxyapatite; CHPTAC, (N-(3-chloro-2-hydroxypropyl) trimethyl
ammonium chloride); CS, chitosan; CMC, carboxymethyl chitosan; COL-I, collagen-I; CPC, calcium phosphate cement; CT, computed tomography; DAH, 1,6-diaminohexane;
DCPA, dicalcium phosphate anhydrous; DCPD, dicalcium phosphate dihydrate; DD, degree of dimethylation; DDD, degenerative disc disease; DMC, dimethyl chitosan;
DQ, degree of quaternization; ECM, extracellular matrix; EDAX, energy dispersive X-ray analysis; GAG, glycosaminoglycans; GEN, genipin; GLU, glutaraldehyde; GP, glycerophosphate; GTMAC, glycidyl trimethyl ammonium chloride; HACC, hydroxypropyltrimethylammonium chloride chitosan; HBC, hydroxybutyl chitosan; HDPs, human
dermal progenitor cells; HEC, hydroxyethylchitosan; HPLCs, human periodontal ligament cells; HTCC, N-(2-hydroxyl) propyl-3-trimethylammonium chitosan chloride;
HPC, hydroxypropyl chitosan; HPLCs, human periodontal ligament cells; HTEC, N-(2-hydroxyl) propyl-3-triethyl ammonium chitosan chloride; HyA, hyaluronic acid;
LBL, layer by layer; MMA, methyl methacrylate; MMT, montmorillonite; MMA, methyl methacrylate; MPCM, monocalcium phosphate monohydrate; MRSA, methicillinresistant Staphylococcus aureus; MSCs, mesenchymal stem cells; MTX, methotrexate; nHAp, nano hydroxyapatite; NBHPDCS, N-(5-bromic-2-hydroxyl-phenyl)-N, N-dimethyl
chitosan; NHNPDCS, N-(2-hydroxyl-5-nitro-phenyl)-N, N-dimethyl chitosan; NHPDCS, N-(2-hydroxyl-phenyl)-N, N-dimethyl chitosan; NMPC, N-methylene phosphonic chitosan; NHS-QPS, (4-(2,5-Dioxo-pyrrolidin-1-yloxycarbonyl)-benzyl)-triphenyl-phosphonium bromide; N, N-DCMCS, N, N-dicarboxymethyl chitosan; NP, nanoparticle; OBs,
osteoblasts; OREC, organic rectorite; PAA, polyacrylic acid; PAMPS, poly(2-acrylamido-2-methylpropanesulfonic acid); P-COS, phosphorylated chito-oligosaccharides; PCPC-1,
monocalcium phosphate monohydrate; PCTS, sodium-phosphorylated chitosan; PEC, polyelectrolyte complex; PEG, polyethylene glycol; PHEMA, polymethylmethacrylateco-polyhydroxyethylmethacrylate; PIA, polysaccharide intracellular adhesin; PMMA, poly methyl methacrylate; PVA, poly vinyl alcohol; PVP, poly vinyl pyrrolidone; TBDMS,
di-tert-butyl dimethylsilyl; TCP, tricalcium phosphate; TMC, N, N, N trimethyl chitosan; TMCMC, tri methyl carboxymethyl chitosan; TNF, tumor necrosis factor; 26SCS, 2-N,
6-O-sulfated chitosan; CT, micro computed tomography.
Corresponding author at: Department of Biotechnology School of Bioengineering SRM University, Kattankulathur 603 203, Tamil Nadu, India.
E-mail addresses: selvamn2@yahoo.com, selvamurugan.n@ktr.srmuniv.ac.in (N. Selvamurugan).
1
These authors equally contributed.
http://dx.doi.org/10.1016/j.carbpol.2016.05.049
0144-8617/ 2016 Elsevier Ltd. All rights reserved.
R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172188
4.
5.
6.
7.
8.
173
Hydroxyalkyl chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Phosphorylated chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Sulfated chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Copolymer of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
1. Introduction
A worldwide increase in the occurrence of pathological fractures
coupled with risk factors during surgical interventions underlines the importance of bone tissue engineering (BTE). Cell-free
constructs and cell-loaded constructs with various chemical and
biological cues act as a template to support bone regeneration
process. They can serve as an alternative treatment method to
conventional grafting procedures. Polymers that can recapitulate natural bone extracellular matrix (ECM) architecture with
the necessary biochemical and load-bearing properties have been
analyzed for in vivo bone regeneration applications (Swetha
et al., 2010; Saranya, Moorthi, Saravanan, Devi, & Selvamurugan,
2011).
Chitosan (CS) is a deacetylated form of chitin procured
mainly from the exoskeleton of crustaceans. It is a linear
polymer composed of randomly distributed units namely: (1 4)2-acetamido-2-deoxy--d-glucan (N-acetyl-d-glucosamine; NAG)
and (1 4)-2-amino-2-deoxy--d-glucan (d-glucosamine) linked
by (1 4) linkages (Jayakumar, Nagahama, Furuike, & Tamura,
2008; Jayakumar, Prabaharan, Nair, Tamura, & 2010b) as shown
in Fig. 1. The degree of deacetylation represents the molar ratio
of the d-glucosamine units to the sum of both NAG and dglucosamine units (Croisier and Jerome, 2013). Depolymerization
of CS can occur by enzymes like glucosaminidases, lipases, and
lysozyme. Chitosan possesses structural resemblance with glycosaminoglycans (GAG), one of the components of ECM that
interacts with collagen bers playing an important role in cellcell adhesion. Chitosan, upon depolymerisation yields bioactive
chito-oligosaccharides with superior anti-microbial properties, and
its monomeric products (glucosamine) metabolized or excreted
from the body. Therefore, CS is biodegradable and has excellent
biocompatibility with almost all the tissues of the body. Chitosan has displayed signicant osteoconductivity, but minimal
osteoinductive property. It induces proliferation of osteoblast cells,
mesenchymal cells and induces in vivo neovascularization (CostaPinto, Reis, & Neves, 2011; Kim et al., 2008; Saravanan, Sameera,
Moorthi, & Selvamurugan, 2013). For orthopedic applications, CS
in various geometries like sponges, bers, lms and other complex
structures are prepared. Thus, CS satises most of the properties supporting its candidature for tissue engineering applications
(Jiang, James, Kumbar, & Laurencin, 2014; Niranjan et al., 2013;

Saranya, Saravanan, Moorthi, Ramyakrishna, & Selvamurugan,
2011).
To enhance the properties of CS for BTE applications, other
polymers and inorganic materials added to CS, and used as 3D
lyophilized scaffolds, hydrogels/lm, and electrospun mats. Chitosan hydrogel membranes with nano-hydroxyapatite (nHAp)
signicantly increased the crystallinity of the composite and
showed excellent biocompatibility with MG-63 cells (Madhumathi
et al., 2009). The addition of nHAp and nano-bioactive glass ceramics (nBGC) to CS/gelatin signicantly improved cell attachment and
proliferation of MG-63 osteoblast-like cells (Peter et al., 2010a,
2010b). The inclusion of nanoscale silicon dioxide and zirconia particles into CS matrix signicantly reduced the degradation rate of
pristine CS scaffolds and enhanced protein adsorption property
(Pattnaik et al., 2011). Protein adsorption and biomineralization are
essential factors that determine the bioactivity of material used in
BTE. Incorporation of carbohydrate anionic moiety like chondroitin
4-sulfate into CS matric increased apatite deposition which facilitated the spreading of bone marrow stromal cells and signicantly
enhanced the compressive modulus (Park et al., 2013). The addition of diopside particles (CaMgSi2 O6 ) to CS matrix up regulated
the expression of osteoblast differentiation marker genes namely
alkaline phosphatase (ALP) and type I collagen (COL-I) and exhibited biocompatibility in vivo in a rat model (Kumar et al., 2014).
The inclusion of bioceramic materials to polymers may enhance
their mechanical property. There was an increase in compressive strength by 33.07% and enhancement in the proliferation of
mouse preosteoblastic cells (MC3T3-E1) upon addition of nHAp to
CS (Zhang, Myers, Wallace, Brandt, & Choong, 2014).
Chitosan as one of the biopolymers in bio-composite scaffolds
has been widely used for BTE applications. The blending of CS with
an anionic polysaccharide alginate stabilized the system by electrostatic interaction of them (Venkatesan, Bhatnagar, Manivasagan,
Kang, & Kim, 2015). Incorporation of nano-silicon dioxide particles to CS-alginate composite displayed a signicant increase in
protein adsorption and apatite deposition, and they were nontoxic to osteoblasts (Sowjanya et al., 2013). Also, incorporation
of nHAp to CS/alginate matrix enhanced the mechanical property of the scaffold as well as stimulated the differentiation of
mouse pre-osteoblastic cells (MC3T3-E1) cells to osteocytes to
Fig. 1. Chemical Structures of chitin and chitosan. Reprinted with permission from Younes and Rinaudo (2015). Chitin and chitosan preparation from marine sources.
Structure, properties and applications. Marine drugs, 13(3), 11331174. 2015, MDPI.
174
Cs/alginate scaffold (Kim et al., 2015). Cold atmospheric plasma

treated CS/HAp scaffolds displayed surface roughness and wettability, which selectively enhanced the protein adsorption of
bronectin and vitronectin. This modication resulted in greater
mesenchymal stem cells (MSCs) inltration into the scaffolds with
increased collagen deposition and mineralization by the end of 3
weeks (Wang, Cheng et al., 2013; Wang, Xie et al., 2013). The inclusion of chicken feather keratin nanoparticles with CS signicantly
improved the protein adsorption, and they were biocompatible
with human osteoblastic cells (Saravanan et al., 2013). Doping and
addition of antibacterial metal ions such as nanophase copper,
zinc and silver to CS polymer signicantly improved the antimicrobial property (Niranjan et al., 2013; Saravanan et al., 2011;
Tripathi et al., 2012). Chitosan/hyaluronic acid scaffolds upon addition of calcium phosphate cement exhibited a signicant increase
in ALP activity with no signicant change in the rate of osteoblastic cell proliferation (Hesaraki and Nezafati, 2014). The presence
of fucoidan in CS/alginate scaffold stimulated the proliferation of
MG-63 osteoblast-like cells with signicant enhancement in ALP
activity and apatite deposition over the scaffolds (Venkatesan,
Bhatnagar, & Kim, 2014). The addition of nHAp to CS/gelatin matrix
not only increased the mechanical property of the scaffolds but also
stimulated the proliferation and differentiation of induced pluripotent stem cells of gingival broblasts to osteocytes (Isikli, Hasirci, &
Hasirci, 2012; Ji et al., 2015, 2016). Fucoidan in TCP-CS scaffolds
increased the compressibility, apatite deposition, and osteocalcin
release, which favor osteogenic differentiation of human mesenchymal stromal cells in vitro (Puvaneswary et al., 2015).
Chitosan and its bio-composite scaffolds have been widely
tested under in vivo conditions. Chitosan/gelatin scaffolds promoted osteoblast proliferation in vivo, their degradation occurred
completely in 12 weeks, and thus, these scaffolds have the potential for bone healing process (Ma et al., 2014). The encapsulation
of MC3T3-E1 osteoblast-like cells in -tricalcium phosphateCS-alginate microcapsules and their injection in the dorsal
subcutaneous area of mice showed collagen deposition and neovascularization at the site of injection. Also, the characteristic
osteoid-like structures were seen at the end of 8 weeks (He, Dong,
Chen, & Lin, 2013; Qiao et al., 2013). An osteoinductive bone graft
prepared by combining calcium phosphate with CS and its implantation in the rats calvarial defects resulted in a signicantly higher
matrix deposition rate as well as osteon formation. Surprisingly,
implantation with the scaffolds replaced the entire calvarial defect
with host bone tissue. An injectable thermosensitive hydrogel containing CS along with Zn, nHAp and -glycerolphosphate (-GP)
showed its potential towards bone formation in vivo as evidenced
by increased apatite and collagen deposition in the defective bone
area (Dhivya, Saravanan, Sastry & Selvamurugan, 2015).
Despite the numerous applications of CS as a stand-alone polymer or as a composite with other polymers and ceramic particles,
its use is limited. Firstly, CS solubility in aqueous solutions is pH
dependent. Since CS has a pKa of 6.5 and its semi-crystalline nature
favors strong intra/inter molecular hydrogen bonding, the solubility of CS at neutral pH is limited. Secondly, CS is quickly and
easily degraded in vivo due to the presence of (1 4) glycosidic linkages, which are hydrolytically cleaved by the abundance
of lysozyme (approximately, 1315 mg/L) present in the body (Lim
et al., 2008). Hence, the study focused on CS with its side chain
modications to address their solubility and controlled biodegradation (Feng and Xia, 2011; Lafeur et al., 2013). Additionally, the
polycationic nature of CS can induce thrombosis, red blood cell
aggregation and hemolysis making it unsuitable for tissue engineering applications. Therefore, many studies focused on altering
the surface charge on the CS chain to enhance its hemocompatibility (Balan and Verestiuc, 2014; Yalinca, Yilmaz, Taneri, Bullici,
& Tuzmen, 2013). Scaffolds with a suitable antimicrobial prop-
erty were required to prevent the implant-associated infections

(Hedrick, Adams, & Sawyer, 2006; Qiu, Zhang, An, & Wen, 2007).
However, CS has little antimicrobial property at neutral pH due to
the protonation of amino groups that occurs only in acidic medium.
Finally, CS has low bioactivity due to the lack of charged and reactive
groups in its structure, which otherwise are necessary to facilitate protein adsorption. Thus, immobilizing scaffolds with proteins
like bronectin was essential for increasing the bioactivity of CS
based scaffolds (Custodio, Alves, Reis, & Mano, 2010). Table 1 summarizes several modications at C2 or C5 positions of CS by an
introduction of various reactive groups or by copolymerization
with other polymers of interest or by grafting with biological and
synthetic macromolecules, which render appropriate bone regenerative properties.
2. Quaternized chitosan
The modication of CS side chain yields to a variety of derivatives
for biomedical applications. Chitosan was modied into N,N,Ntrimethyl chitosan (TMC) by reaction with methyl iodide catalyzed
by sodium hydroxide at 60 C into N-methyl-2-pyrrolidinone. In a
second step, the substitution of iodide ions by chloride ion by an ion
exchange process yielded TMC (Fig. 2). In another example, N-(2hydroxyl) propyl-3-trimethylammonium chitosan chloride (HTCC)
was prepared by dissolving CS in NaOH/CHPTAC aqueous solution by a pressure-equalizing dropping funnel (Fan et al., 2015).
Other modied CSs were such as N-(2-hydroxyl) propyl-3-triethyl
ammonium chitosan chloride (HTEC) and N-(2-hydroxyl-phenyl)N,N-dimethyl chitosan (NHPDCS) (Benediktsdottir, Gudjonsson,
Baldursson, & Masson, 20114; de Britto, Celi Goy, Campana Filho,
& Assis, 2011; Guo et al., 2007; Yang et al., 2015). Chitosan exerts
antimicrobial activity by disrupting the negatively charged outer
membrane of the microbes, and the modications of CS structure
would further enhance its antimicrobial activity that is one of the
features required in BTE.
2.1. N,N,N-trimethyl chitosan (TMC)
Positive charge rich trimethyl chitosan (TMC) displayed
enhanced antibacterial activity compared to CS against both E. coli
and S. aureus (Jia and Xu, 2001). The ratio of DD (degree of
dimethylation) to DQ (degree of quaternization) seems to control
mucoadhesive, cytotoxic and other physicochemical properties of
TMC (Haas, Ravi Kumar, Borchard, Bakowsk, & Lehr, 2005). The
anionic groups of lipopolysaccharide in Gram-negative and lipoteichoic acids in Gram-positive bacteria act as a molecular linkage for
quaternized and trimethylated amino groups in TMC (Raafat, Von
Bargen, Haas, & Sahl, 2008). The electrostatic interaction between
these groups can disrupt the function and integrity of the bacterial membrane. The antibacterial property of TMC increased with
increase in pH of the medium (Xu, Xin, Li, Huang, & Zhou, 2010). It is
important to note that chitosan chemistry inuences the shielding
effect of surface ion pairs resulting in either partial O-methylation
or free of O-methylation. Removal of these surface ion pairs by
dialysis showed an increase in the mobility of alkyl chains, conferring an active interaction at the surface of the bacterial envelope
resulting in accelerated the antibacterial activity. In addition, TMC
without O-methylation reduced the cytotoxicity and increased its
solubility at physiological pH. Di-tert-butyl dimethylsilyl (TBDMS)
is a commonly used protectant of OH group during methylation,
which is highly stable under the reaction conditions while the
removal is also a facile process (Benediktsdottir et al., 2011; Martins
et al., 2015).
In addition to the antibacterial property of TMC, TMC could also
used as a polycation in developing polyelectrolyte complexes by
175
Table 1
Chemically modied derivatives of Chitosan and the improved properties offered by their reactive groups.
Derivatives
Subtypes
Quaternized Chitosan
N,N,N-Trimethyl chitosan (TMC)
High absorption efciency (Britto & Assis, 2007)

Mucoadhesion (Pardeshi and Belgamwar, 2016; Raafat,
Von Bargen, Haas, & Sahl, 2008)
N-(2-hydroxyl)
propyl-3-trimethylammonium
chitosan chloride (HTCC or HACC)
Better moisture retention and absorption compared to

chitosan (Huang et al., 2014)
Inhibited the expression of intracellular adhesion genes
which can mediate biolm formation on implants (Peng
et al., 2011)
High transfection efciency when compared to Chitosan
(Xiao et al., 2012)
N-(2-hydroxyl-substituted
phenyl)-N,N-dimethyl chitosan
(NXRPDCS)
Strong antifungal activities against many resistant

strains (Guo et al., 2007)
N-Carboxy-methyl Chitosan (N-CMC)
High metal chelation ability-bound strongly to Ca2+ ions

and promoted apatite formation (Mourya and Inamdar,
2008; Mourya et al., 2010)
O-Carboxymethyl Chitosan (O-CMC)
Promoted adhesion and proliferation in human

broblast cells (Wongpanit et al., 2005)
N,O-Carboxymethyl chitosan
(N,O-CMC)
High water retentivity and excellent gel forming

capacity (Jayakumar, Prabaharan, Nair, & Tamura, 2010)
Exhibited negligible immunogenicity (Shalumon et al.,
2009).
Hydroxyethyl chitosan
High moisture absorption rate and highly hydrophilic
Hydroxypropyl chitosan
99% inhibition against most of the bacteria (Peng, Han,

Liu, & Xu, 2005)
Hydroxybutyl chitosan
Rapid gelation kinetics and displayed good in vivo

stability (Dang et al., 2006)
N-Acetyl chitosan
Due to hydrophobic pockets it caused lesser lysozyme

absorption and higher bronectin adsorption
(Tangpasuthadol, Pongchaisirikul, & Hoven, 2003)
Lower swelling index maintains its mechanical strength
(Le Tien, Lacroix, Ispas-Szabo, & Mateescu, 2003)
Carboxyalkyl Chitosan
Hydroxyalkyl
Chitosan
N-Acyl Chitosan
Reactive group
Important properties
176
Table 1 (Continued)
Derivatives
Thiolated Chitosan
Subtypes
Reactive group
N-Carboxyacyl chitosan
Formed stable hydrogels owing to its amphoteric nature

(Hirano & Moriyasu, 2004)
Chitosan cysteine conjugate
Rapid formation of hydrogel promotes cell attachment

and inltration (Bernkop-Schnurch, Hornof, & Zoidl,
2004; Riva et al., 2011)
Gel interacted with BMP-2 causing no change in its
swelling behavior (Kurniawan Fudholi, & Susidarti,
2012)
Chitosan2-iminothiolane conjugate
(or Chitosan-4-Thio-butylamidine)
Highly porous structure for cell inltration (Vijapur

Sreenivas, Patil, Vijapur, & Patwari, 2012)
High solubility over wide range of pH (Masuko et al.,
2005)
Chitosan-Thioglycolic acid
Excellent in situ gelling capacity: Formed gels rapidly at

body temperature (Sakloetsakun, Hombach, &
Bernkop-Schnurch, 2009)
Attributing to its negative charge, low interfacial energy

and high binding capacity to Ca2 +, and it acted as a
strong nucleator for high biomineralization (Xu, Neoh,
Lin, & Kishen, 2011)
Enhanced adipose derived stem cell growth in vitro (Yeh
and Lin, 2012)
Phosphorylated Chitosan
Sulfated Chitosan
2-N, 6-O-sulfated chitosan (26SCS)
Structural analogy to heparin, made it highly

hemo-compatible (Shen et al., 2011)
Semi-synthetic resins
of Chitosan
Copolymer of chitosan with methyl

methacrylate
Good mechanical and compressive strength (>10 MPa)

(Endogan et al., 2014)
Uniformly arranged nanoporous structure which caused
sustained time-release of drugs and proteins (Shen et al.,
2011)
Copolymer of Chitosan with

Acrylamide
Thermo-responsive gels with 3-D porous architecture,

good interconnectivity, elasticity and displayed
appreciable osteoconductivity (Liao, Chen, & Chen, 2011)
Glycol Chitosan
High water solubility at wide range of pH (Chung et al.,

2003)
Faster biodegradability for early bone healing
application (Park, Cho, Chung, Kwon, & Jeong, 2003)
Glucosamine Chitosan
Better water solubility with enhanced antimicrobial

properties (Chen & Zhao, 2012)
Sugar derivatives of
Chitosan
177
Table 1 (Continued)
Derivatives
Subtypes
Specic Ligand conjugated Chitosan
Arginylglycylaspartic acid
(RGD),TyrIleGlySer- Arg(YIGSR),
le-Lys-Val-Ala-Val(IKVAV),
A99a(ALRGDN) and others
Reactive group
Acceleration of integrin mediated osteoblast
attachment, proliferation and differentiation (Tsai, Chen,
& Liu, 2013; Wang et al., 2014)
Formed viscoelastic injectable hydrogels with

biostability and high metal adsorption capacity (Wang,
2006)
Glycol Chitosan
Glutaraldehyde Chitosan
found biocompatible and biostable complex which

induced vascularization and tissue formation (Ma et al.,
2003; Wu, Black, Santacana Laftte, & Patrick, 2007)
Chitosan-ascorbate conjugate
Excellent antioxidant property with high scavenging

and chelation abilities (Tian Tian, Wang, & Mo, 2009)
Cyanoethyl Chitosan
Enhanced bactericidal nature
Imidazole Chitosan
Inhibited Ca2 + release from bone (Di Martino, Sittinger,

& Risbud, 2005)
Slightly osteoinductive (Muzzarelli et al., 1994)
Fig. 2. Quaternization of chitosan. Chitosan was modied into N,N,N-trimethyl chitosan. Reprinted with permission from, Mourya and Inamdar (2008). Chitosan-modications
and applications: opportunities galore. Reactive and Functional polymers, 68(6), 10131051. Elsevier, 2008.
layer by layer (LBL) assembley, a surface modication strategy benecial over crosslinking, in preventing the effects of altered charge
density, reactivity, and degradability of the material. Almodovar
and Kipper (2011) developed an LBL assembley using TMC as a
polycation, heparin as a polyanion. This PEC-LBL assembley showed
to promote FGF-2 adsorption serving as suitable matrices for the
delivery of growth factors for cell and tissue engineering. A yet
another study on the coating of cortical bone with TMC-heparin
polyelectrolyte multilayers showed that they can serve as periosteum mimics, deliver osteoprogenitor cells, and improve bone
allograft healing (Romero et al., 2015). Thus, while the application
of TMC much explored regarding its antibacterial properties, efforts
are needed in considering the polycationic nature of TMC to extend
further its applications in tissue engineering as vehicles for growth
factor delivery.
O-carboxymethyl-N,N,N-trimethyl-chitosan (CMTMC) scaffolds
had no negative inuence when treated with human dermal
progenitor (HDP) cells (Patrulea et al., 2015). Further, by immobilization of 10 nmol cm2 and ve nmol cm2 RGDC peptide on the
surface of 1,6-diaminohexane (DAH)-CMTMC, there was the successful attachment of HDP cells as well as the promotion of cell proliferation compared to DAH-CMTMC scaffolds without the RGDC
peptide (Patrulea et al., 2016). Novel Electrospun silver nanoparticles (Ag Nps) loaded nanobrous mats containing TMC, polyacrylic
acid (PAA) or poly(2-acrylamido-2-methylpropanesulfonic acid)
(PAMPS) showed superior antibacterial activity against E. coli and
S. aureus than the membranes without TMC and Ag NPs (Kalinov
et al., 2015). Polyalanine treatment to quaternized CS improved
its biocompatibility and antibacterial activities (Zhao, Li, Guo, &
Ma, 2015). The antibacterial nature and the wound healing properties of TMC bers, prepared by the methylation of CS bers
using iodomethane under alkaline conditions has been recently
assessed by Zhou et al. (2016). The resulting TMC bers with varying degrees of quaternization indicated their higher antibacterial
178
Fig. 3. Effects of CS and TMC bers in rat would model in vivo over 12-day treatment period. Micrographs displaying the wounds at day 0, 3, 6, 9, 12. The modied CS bers
(TMC) had enhanced wound healing property. Reprinted with permission from, Zhou et al. (2016). Biomaterials based on N, N, N-Trimethyl chitosan bers in wound dressing
applications. International journal of biological macromolecules. Elsevier, 2016.
activity against E. coli and S. aureus compared to CS bers. The TMC

bers also exhibited enhanced swelling properties attributed to the
positive surplus charges in its chain. These modied CS bers tested
in vivo in rat full-thickness excision models displayed reduced
wound maceration, wound re-epithelialization, eschar development over 12 days compared to the unmodied CS bers (Fig. 3).
2.2. N-(2-hydroxyl) propyl-3-trimethylammonium chitosan

chloride
N-(2-hydroxyl) propyl-3-trimethylammonium chitosan chloride (HTCC) showed stronger antibacterial activity under alkaline
condition (Qin et al., 2004). It had enhanced antibacterial potency
compared to CS against Methicillin-resistant Staphylococcus aureus
and Staphylococcus epidermidis Porphyromonas gingivalis (P. gingivalis ATCC33277), Prevotella intermedia (P. intermedia ATCC 25611),
Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans
Y4) and Streptococcus mutans (S. mutans Ingbritt C) (Martinez
et al., 2010; Peng et al., 2010). In addition to antibacterial activity,
the role of HTCC in tissue regeneration was studied. Hydroxypropyltrimethylammonium chloride chitosan accelerated the
proliferation of human periodontal ligament cells (HPDLCs) even at
the lower concentration as compared to pristine CS (Qiu Xia et al.,
2010). The CS-HTCC/GP aqueous solution injected intramuscularly
into the rumps of spraguedawley rats formed a gel-like plug at
the injection site and showed a good amount of collagen deposition at the end of 9th week. Chitosan-HTCC/glycerophosphate (GP)
thermosensitive hydrogel loaded with basic broblast growth factor (CS-HTCC/GPbFGF) effectively enhanced the new periodontal
support tissues in dogs. The regenerative tissues seemed complete to ll the fraction areas, and a signicant amount of new
bone formation observed (Croisier and Jerome, 2013). Quaternized
CS derivatives like O-(2-hydroxyl) propyl-3-trimethyl ammonium
chitosan chloride (O-HTCC) and HTEC have also displayed superior
antioxidant properties exhibiting dose-dependent reducing power
and lipid peroxidation inhibition effect (Cui, Tang, & Yin, 2012;
Wan, A., Xu, Sun, & Li, 2013). The addition of CMC to HTCC improved
its mechanical property (Hu, Wang, & Wang 2016). Their results
indicated the formation of strong intermolecular hydrogen bond
between OH group of HTCC and CMC, which lead to the increased
rigidity and enhanced the tensile strength and elongation at break
values.
2.3. Other quaternized chitosans

The quaternized CS (QCS) displayed a signicant role in inhibition of the expression of icaA gene (that is involved in secretion
of polysaccharide intracellular adhesin) in bacteria on titanium
implants in a biolm prevention assay (Peng et al., 2011). Use
of antibiotics such as gentamicin loaded in PMMA (Poly (methyl
methacrylate)) bone cement was standard practice for preventing
infections in joint arthroplasty and osteomyelitis. This practice had
not only given rise to antibiotic resistant bacteria like methicillinresistant Staphylococcus aureus (MRSA) but also shown to reduce
proliferation of hMSCs and the viability of osteoblast cells (Parker,
Clegg, & Taylor, 2012). However, hydroxypropyl trimethylammonium chloride chitosan (HACC) with 26% degree of substitution
loaded in PMMA inhibited biolm formation (Tan, Guo, Yang, Xu,
& Tang, 2012). It also resulted in better apatite deposition, cell
attachment and differentiation of hMSCs towards osteoblast lineage. Expression levels of COL-I and ALP were signicantly higher
compared to both gentamicin loaded PPMA and CSPMMA (Tan
et al., 2012b). The quaternized CS derivatives with increasing
degree of substitution (DS) showed antifungal potency in vitro
on the mycelial growth of Aspergillus avus. Increasing the DS of
CS exhibited an almost six-fold increase in the antifungal activity at only one-fourth concentration of the deacetylated chitosan
polymer. Quaternized CSsincluding N-(2-hydroxyl-phenyl)-N,
N-dimethyl CS (NHPDCS), N-(5-chloro-2-hydroxyl-phenyl)-N, Ndimethyl CS (NCHPDCS), N-(2-hydroxyl-5-nitro-phenyl)-N, Ndimethyl CS (NHNPDCS) and N-(5-bromic-2-hydroxyl-phenyl)N,N-dimethyl CS (NBHPDCS), N,N,N-(diethylcinnamyl) CS (QC1)
and N,N,N-(diethyl-p-dimethylaminobenzyl) CS (QC3) showed better antifungal activities compared to the unmodied CS (Badawy
and Rabea, 2014; Guo et al., 2007).
An injectable in situ forming an electroactive hydrogel, containing QCS grafted polyaniline with oxidized dextran (o-dex) as crosslinker showed excellent antimicrobial properties in vitro and in vivo
(Zhao, Li, Guo, & Ma, 2015). The design and synthesis of a series of
novel quaternized derivatives of CS, dihydroxy quaternary ammonium salts (QAS) containing long chain alkyl bromides reported.
The results indicated that the increase in the alkyl chain length
increased the antibacterial activity (Wang et al., 2016). Additionally, a reactive antibacterial compound (4-(2,5-dioxo-pyrrolidin1-yloxycarbonyl)-benzyl)-triphenyl-phosphonium bromide (NHSQPS) showed effective antimicrobial activity towards E. coli and S.
aureus compared to CS and also the compound was solubilized in

water between a wide range of pH 312 (Zhu et al., 2016).
Layer by layer self-assembling polymers into composite lms
is a technique to improve the antibacterial efciency of implant
materials. Organic rectorite (OREC) is a layered silicate material
well known for their superior mechanical, anti-infective properties owing to their larger interlayer distance. Self-assembled
QCMC/OREC/alginate nanobrous mats layered onto cellulose
nanobers displayed enhanced antibacterial activity against E. coli
and S. aureus compared to cellulose lms (Jiang et al., 2015). As cellulose based composites are mechanically strong matrices, the LBL
assembly of QCMC/OREC/Alg onto cellulose mats could act as cytocompatible, mechanically strong antiinfective materials for tissue
engineering applications. Recently, a novel method of modication
to QCS was adopted by reacting CS with (3-chloro-2-hydroxypropyl
trimethyl ammonium chloride) in the presence of alkali or urea
resulting in the highest degree of substitution (You et al., 2016). The
prepared QCS was used to develop a hydrogel by in situ polymerization with PAA. There existed a strong electrostatic interaction
between the quaternary ammonium group of CS and the carboxyl
group of PAA resulting in enhanced toughness, self-recovery/shape
memory properties with tunable mechanical properties. An electrostatic LBL lm assembly comprising of heparin (HP), CS and
its derivatives namely DMC, TMC was aimed to develop an efcient anti-inammatory coating to medical devices and implants
(Follmann et al., 2016). Because inammatory responses are universal host defense mechanisms against foreign agents, which on
the other hand hinders the success of many medical implants.
3. Carboxyalkyl chitosan
Amongst other derivatives of CS, carboxyalkyl CS was widely
studied due to its ease of synthesis, a high degree of hydrophilicity and numerous potential applications (Riva et al., 2011).
Carboxyalkylation of CS yields carboxyethyl, carboxybutyl, carboxymethyl CS and other derivatives. The carboxy ( COOH) groups
impart anionic charges on the CS chain, making it amphoteric with
a wide range of biomedical applications. Carboxyalkyl CSs were
water soluble at a wide range of pH, biodegradable, biocompatible,
and were non-toxic to human tissues. Grafting succinic acid to carboxyalkyl CS improved water solubility and transfection efciency
(Ahmed and Ikram, 2015).
3.1. Carboxymethyl chitosan
The most commonly used CS derivative was carboxymethyl
chitosan (CMC). CMC is amphiprotic ether derived from CS. The
alkylation of CS with monohalocarboxylic acid at different reaction
conditions conferred the selectivity form of N- or O-carboxymethyl
derivatives (Fei Liu, Lin Guan, Zhi Yang, Li, & De Yao, 2001).
Carboxymethylation introduced carboxymethyl ( COOCH3 ) group
in the primary or secondary hydroxyl groups and the amino
groups bonded to the glucopyranose unit (Riva et al., 2011).
Amphoteric nature, enhanced aqueous solubility, biocompatibility,
biodegradability and minimal immunogenicity of CMC supported
its candidature in BTE applications (Ibrahim, El-Zairy, & Mosaad,
2015; Shalumon et al., 2009). By reductive alkylation and direct
alkylation, carboxymethylation of CS prepared. However, the former one was not preferred as it utilized expensive chemicals,
produces toxic by-products like NaCN and HCN, and would be difcult to scale-up to industrial level (Mourya, Inamdar, & Tiwari,
2010; Shalumon et al., 2009). The degree of substitution of CS was
directly proportional to the concentration of NaOH used in the
direct alkylation process, but it was independent of the degree of
deacetylation of CS. The optimum concentration of NaOH was found
179
to be%; higher concentrations could lead to the depolymerization

of the CS chains (Chen, Tian, & Du, 2004).
There are reports available showing the potential role played
by CMC along with other polymers and biomaterials in BTE. Carboxymethyl chitosan/HAp scaffolds showed better bone formation
than HAp alone with negligible cytotoxicity on MC3T3-E1 cells
and high blood adsorbing capacity (Tokura and Tamura, 2001).
The layering of CMC on titanium implants prevented implantassociated infection and subsequent implant failure. This strategy
highly reduced S. aureus and S. epidermidis adhesion on the implants
(Shi, Neoh, Kang, Poh, & Wang, 2009). Carboxymethyl chitosan/PVA
(poly vinyl alcohol) electrospun scaffold supported hMSC attachment and proliferation with no apparent cytotoxicity (Shalumon
et al., 2009). Injectable gels of CMCgelatinnHAp stimulated proliferation of osteoblasts and showed no evidence of inammation
at the site of implantation (Mishra et al., 2011). Carboxymethyl
chitosan/gelatin/-TCP composites fabricated via ultrasonic radiation mediated crosslinking were porous in structure with optimum
mechanical strength. These scaffolds showed biocompatible and
promoted bone regeneration when implanted in the mandibular
region of beagle dogs. Furthermore, the micro-CT analysis clearly
indicated new bone formation with a volumetric density (BV/TV%)
of 13.3% (Zhou et al., 2012). MTT assay showed that CMC (800 mg/L)
with a combination of platelet-derived growth factor-BB (10 g/L)
could promote proliferation as well as differentiation of HPDLCs (Ji
et al., 2010).
3.2. Carboxymethyl chitosan derivatives
Derivatives of CMC can be amino substituted (N-CMC), hydroxyl
substituted (O-CMC) or both amino and hydroxyl substituted (N, OCMC). Fig. 4 represents a schematic representation of the chemical
synthesis of these derivatives. These derivatives were cytocompatible, biodegradable and exerted superior antibacterial properties.
Further, N-CMC supported chondrocyte adhesion and possesses
metal ion chelating ability (Mourya et al., 2010). O-CMC stimulated
broblast proliferation (Fei Liu et al., 2001) while N, O-CMC had
unique gel forming capacity with excellent water retentive properties (Jayakumar, Nagahama et al., 2010; Jayakumar, Selvamurugan,
2010; Jayakumar, Prabaharan et al., 2010). N-CMC possessed selective metal adsorption capacity and it readily bound to Ca2+ ions and
promoted biomineralization at the site of implantation (Kyzas and
Bikiaris, 2015). O-CMC-BMP-2 modied titanium surfaces proved
the great extent of biomineralization of both osteoblast cells and
hMSCs along with enhancement of osseointegration and longevity
of the titanium implants (Shi, Neoh, Kang, Poh, & Wang, 2009).
N, O-CMC/n--TCP (tricalcium phosphate) composites in a mass
proportion of 1:1 highly promoted apatite formation on its surface when subjected to varied chemical conditions of simulated
body uids (Liu et al., 2013). Lee and Kamarul (2014) prepared a
copolymer scaffold comprising of PVA/N-O-CMC/PEG, physically
cross-linked by 50 KGy e-beam radiation. The strong crosslinking
mediated by the e-beam radiation enhanced the physical properties
of the scaffold such as porosity, pore size, and conferred superior
swelling properties. The scaffolds also showed increased chondrocyte proliferation on its surface with high expression of GAG and
collagen type II (Lee and Kamarul, 2014).
O-Carboxymethyl chitosan-based injectable hydrogel containing alginate (Alg), nano brin particles displayed higher resistance
to mechanical compression. The Alg/O-CMC/nano brin (AOF) composite promoted the differentiation of adipose derived stem cells
to adipocytes for adipose tissue engineering (Jaikumar et al., 2015).
PVA/CMC/montmorillonite (MMT) based hydrogel nanocomposite system displayed enhanced antimicrobial activity better than
the standard drug penicillin G (Sabaa, Abdallah, Mohamed, &
Mohamed, 2015). Li et al. (2015) developed a CMC/modied starch
180
Fig. 4. Carboxyalkylation of Chitosan. Chitosan was modied into N-carboxymethyl chitosan, O-carboxymethyl chitosan and N, O-carboxymethyl chitosan. Reprinted with
permission from, Mourya and Inamdar (2008). Chitosan-modications and applications: opportunities galore. Reactive and Functional polymers, 68(6), 10131051. Elsevier,
2008.
based in situ injectable hydrogel with a superior mechanical property. Oxidized cholesterol starch as a non-cytotoxic crosslinker
used in the hydrogel synthesis mediated stable covalent bond formation, by the condensation of aldehyde groups of oxidized starch
with the primary amine group of CMC. Besides being an antibacterial material, upon optimizing the crosslinker concentration, the
structural and mechanical properties of the hydrogel could be
tailor made to specic applications. Hao, Chen, Yu, Liu, and Sun
(2016) recently developed the CMC-multiwalled carbon nanotube
based strong antibacterial composite upon coordination of metal
ions Cu and Zn. They displayed enhanced antimicrobial activity
against S. aureus, E. coli and V. anguillarum. In another application,
CMC grafted polyacrylic acid served as superabsorbent polymers
in the case of serious hemorrhages, and these polymers promoted

the concentration of coagulation factors, RBCs and platelets. The
enhanced roughness, porous surface structure, high water retention ability and availability of positively charged NH3 + cations of
the polymers promoted the adhesion of negatively charged RBCs
onto the surface of the wound thereby facilitated hemostasis (Chen,
Huang et al., 2016; Chen, Zhang et al., 2016).
N, O-CMC nanoparticles displayed better antibacterial property
against S. aureus than both O-CMC and CS nanoparticles, and the
inhibition of bacterial growth seemed to increase with an increase
in the concentration of these nanoparticles (Anitha et al., 2009).
N, O-CMCzinc compound exerted a better antimicrobial activity than CSzinc complex against S. aureus (Patale and Patravale,
2011). Carboxymethyl chitosan displayed an inhibition efciency

of >90% against bacterial adhesion. It prevented biolm formation
at the efciency of 63.1% of Gram-positive bacteria and 70.6% of
Gram-negative bacteria after 1 of biolm initiation. The proposed
mechanism behind this phenomenon is the neutralization of the
bacterial surface charge leading to occulation of the bacterial cell
population (Tan, Han, Ma, & Yu, 2011). Kaya et al. (2014) demonstrated the antimicrobial and antioxidant activities possessed by
O-CMC. Water soluble N, O-CMC developed by Patrulea, Applegate,
Ostafe, Jordan, and Borchard (2015) showed its non-cytotoxic and
non-immunogenic nature, which are the features also required in
BTE.
4. Hydroxyalkyl chitosan
Hydroxyalkyl chitosans can be obtained by the substitution
reaction of CS with epoxides at either amino or hydroxyl groups
giving N-hydroxyalkyl or O-hydroxyalkyl CS derivatives, respectively (Fig. 5). The ratio of O/N-substitution was determined by
the choice of catalyst (NaOH or HCl) used in the reaction and the
reaction temperature (Donges, Reichel, & Kessler, 2000; Mourya
and Inamdar, 2008). For tissue engineering applications, only a few
derivatives of hydroxyalkyl CS such as hydroxybutyl, hydroxyethyl
and hydroxypropyl chitosan have been explored. Hydroxypropyl
chitosan (HPC) at a concentration of, grafted with maleic acid,
owing to its capability to form coordination bonds displayed better
inhibitory effects up to 99.9% against both S. aureus and E.coli within
30 min of contact time (Peng, Han, Liu, & Xu, 2005). Hydroxybutyl
chitosan (HBC) provided an extra advantage of a rapid gelation
kinetics and better in vivo stability over that of collagen and therefore, established itself as a suitable polymer (Dang et al., 2006).
Hydroxybutyl chitosan was shown to be biocompatible and was
minimally toxic to ve-wt% when exposed to hMSCs and intervertebral disk cells as a potential candidate to treat degenerative disc
disease (DDD). Its thermosensitive properties underlined its probable use to form gels at body temperature in as quick as 30. The
cell viability in the presence of HBC was similar to that in the presence of collagen in a 2-weeks treatment period. It promoted the
proliferation of L929 mouse broblast cells with minimal toxicity suggesting its application in wound healing (Wei et al., 2009).
Cellulose/O-hydroxyethyl chitosan bers with an HEC concentration of 6.2% displayed reduction of E. coli growth and had moisture
absorption rate of 13.55% (Xu, Xin et al., 2010; Xu, Zhuang et al.,
2010).
Clay nanocomposites emerged to be superior materials in
improving the mechanical properties of polymeric materials
with strength, modulus, and dimensional stability. A modied
HTCC-nanoclay (montmorillonite) composite showed antibacterial
activity, cytocompatibility, and cell growth (Aliabadi, Dastjerdi, &
Kabiri, 2013). The inclusion of nanoclay to HTCC resulted in strongly
intercalated network structure, which allowed the bacteria to effectively entrapped, and damaged them. However, further studies are
warranted to investigate and support the applications of hydroxyalkyl CS in BTE.
5. Phosphorylated chitosan
The synthesis of phosphorylated CS (P-CS) involves the simultaneous reaction of the phosphorous acid and formaldehyde to CS
in the acidic aqueous acidic resulting in the formation of watersoluble N-mono- and di-phosphonicmethylene chitosan (Fig. 6).
Phosphorylated CS displays better ionic conductivity and swelling
index. Although it decreased crystallinity, its tensile strength
remained similar to CS. Phosphorylated CS exhibited considerably rough surface morphology unlike CS (Jayakumar, Nagahama,
181
Furuike, & Tamura, 2008; Jayakumar, Selvamurugan, Nair, Tokura,

& Tamura, 2008). Phosphorous pentoxide (P2 O5 ) reaction with CS
yielded water-soluble phosphorylated CS with a high degree of substitution (Tachaboonyakiat, Netswasdi, Srakaew, & Opaprakasit,
2010). Phosphorylated CS blend alginate lms showed induction
of apatite layer after immersing it in simulated body uid (SBF)
for 21 days (Jayakumar et al., 2009). The solubility of CS based
hydrogels above pH 6 reduced its potential for tissue engineering applications. To overcome this limitation, the phosphorylated
derivative of CS, N-methylene phosphonic chitosan (NMPC) was
synthesized by cross-linking of NMPC with either glutaraldehyde
(NMPC-GLU) or genipin (NMPC-GEN). While NMPC-GEN hydrogel was better among the two, both the hydrogels showed higher
elastic modulus, better compressive strengths and increased the
proliferation of cells.
A biodegradable P-CS/monetite (dicalcium phosphate
anhydrous-DCPA) composite showed enhanced mechanical
performance compared to DCPA or unphosphorylated CS-DCPA
(Boroujeni, Zhou, Luchini, & Bhaduri, 2014). The addition of 5 w%
P-CS powders to DCPA decreased the setting time as well as
increased the youngs modulus, compressive strength by two-fold
(Fig. 7a,b). However, it was observed that further increase in
w% of P-CS from 5% to 10% reversed the increasing mechanical
properties. The composite showed cytocompatibility, supported
the proliferation of MC3T3-E1 cells, and hence suggested as ideal
bone cement for load bearing applications.
Calcium phosphate cement has been used as llers at the site
of injury for its remarkable osteoconductive and osseointegrative
properties (Winge, Reikeras, & Rokkum, 2011), yet they lack the
mechanical strength required for bone tissue implants. The binding afnity of calcium to reactive functional groups followed the
order: phosphorylate > carboxylate > amino > hydroxyl groups. The
addition of P-CS up to 2 wt% to monocalcium phosphate monohydrate (PCPC-1) and calcium oxide cement, and up to 10 wt%
to dicalcium phosphate dihydrate and calcium hydroxide cement
(PCPC-2) increased their compressive strength and youngs modulus. The leaching experiment showed that P-CS did not leach out
for up to two months when implanted in vivo. Hence, P-CS could
bind tightly to inorganic phase during the setting process of CPC
(calcium phosphate cement), thus, can be effective as bone ller in
clinical applications.
Enzymatic degradation of CS formed chito-oligosaccharides
(Aam et al., 2010) and phosphorylated chito-oligosaccharides (PCOS) up to 100 g/mL concentration showed no cytotoxicity to
MG-63 osteoblast-like cells. Also, low molecular weight P-COS
seemed to promote cell proliferation. ALP activity was signicantly
increased in the presence of P-COS as compared to CS (Venkatesan,
Pangestuti, Qian, Ryu, & Kim, 2010). To achieve the mechanical
strength, a P-CS/CS/n-HAp composite was prepared by the coprecipitation method and it displayed a compressive strength of
70.25 Mpa. The compressive strength of the composite remained
64% of the original (45.07 Mpa) after soaking in simulated body
uid (SBF) for . Hence, it displayed controlled biodegradation and
excellent bioactivity for BTE purposes (Li, Huang, Wang, Ma, & Xie,
2011). The incorporation of P-CS powders at ve-wt% to the widely
used bone cement DCPA reduced its setting time and doubled its
compressive strength. Both DCPA and DCPAP-CS increased cell
proliferation (MC3T3-E1). The results showed that both the materials were as cytocompatible as HAp. The cell morphology of attached
osteoblast cells in 7 days culture testied the biocompatibility of
DCPAP-CS with up to 5-wt% of P-CS (Boroujeni et al., 2014).
The lack of negatively charged functionalities on the surface of
CS limits the osteoblast cell attachment. Hence, its modications
with plasma induced phosphonic groups signicantly improved
osteoblast attachment, viability, and proliferation (Lopez-Perez, Da
Silva, Sousa, Pashkuleva, & Reis, 2010). It has been reported that P-
182
Fig. 5. Hydroxyalkylation of chitosan. Reprinted with permission from, Mourya and Inamdar (2008). Chitosan-modications and applications: opportunities galore. Reactive
and Functional polymers, 68(6), 10131051. Elsevier, 2008.
CS was completely blood compatible, had lesser platelet reactivity

unlike CS, and enhanced adipose-derived stem cell growth in vitro
(Yeh and Lin, 2012). Metals like Zn have been widely used for their
antimicrobial properties in complex with biomaterials (Thian et al.,
2013). However, metals can cause signicant cytotoxicity if used
above a threshold concentration. Sodium-phosphorylated chitosan
(PCTS) and ZnO showed their application in periodontal dressings.
The PCTS did not change the crystalline nature of ZnO and reduced
its cytotoxicity by forming a complex with its protonated hydroxyl
groups. The composite was biocompatible with primary human
gingival broblast cells (Srakaew, Ruangsri, Suthin, Thunyakitpisal,
& Tachaboonyakiat, 2011). Phosphorylated CS signicantly promoted proliferation of both primary human osteoblasts (OBs) and
the OB like stromal cell (the component of the giant cell tumor
of bone cells; up to 1000-g ml1 concentration) only after seven
days of treatment. A study on the osteogenic effect of the P-CS suggested that it regulates the level of osteoclastogenic factors, and
receptor activator of nuclear factor kappa B ligand (NFKBL) and
osteoprotegerin (OPG) expression (Tang et al., 2011). Partially demineralized dentine sections modied by covalent immobilization of
P-CS showed better deposition of calcium phosphate on the dentine
surface (Xu, Neoh, Lin, & Kishen, 2011). All these properties ensure
P-CSs candidature as a promising biomaterial for BTE applications.
6. Sulfated chitosan
Chitosan chain can be sulfated using sulphuric acid or sulphonic
acid salts (Rajasree and Rahate, 2013; Zhang, Zhang, Ma, Yang, &
Nie, 2015). The sulfated CS played a major in bone metabolism
and enhanced bone morphogenetic protein-2 (BMP-2) activity in
bone (Takada et al., 2003). The half-life of BMP-2 in culture was
prolonged with heparin, a sulfated polysaccharide (Hosseinkhani,
Khademhosseini, & Kobayashi, 2007; Zhao et al., 2006). The addition of sulfated CS to calcium-decit hydroxyapatite (CDH) loaded
with BMP-2 increased in release prole of BMP-2 as compared

to the composite without sulfated CS. The initial burst following gradual release was essential for the growth of new bone
tissue (Zhao et al., 2011). Sulfated CS stimulated proliferation of
both primary human osteoblasts and the OB like stromal cell
component of the giant cell tumor (GCT) of bone cells at a concentration of 100 g ml1 . However, it inhibited cell proliferation
above 1000 g ml1 ; this effect was more pronounced in primary
human GCT stromal cells as compared to osteoblast cells (Tang et al.,
2011).
Neovascularization at the site of a bone defect is necessary for
the development of a new brous tissue. Modied heparin displayed vascular tissue formation as reported previously (Janairo
et al., 2012; Wang, Cheng et al., 2013; Wang, Xie et al., 2013). Sulfated N-carboxymethyl CS inhibited thrombin and factor Xa similar
to antithrombin. It also caused no hemolysis when human blood
samples treated with it (Zhou et al., 2013). In another study, 2N, 6-O-sulfated chitosan (26SCS) based nanoparticle (S-NP) was
successfully developed and loaded with BMP-2 (BMP-2/S-NP) and
then fabricated with gelatin BMP-2/S-NP (BMP-2/S-NP/G) to repair
critical-sized bone defect of rabbit radius. The gelatin BMP-2/S-NP
treatment signicantly increased both peripheral and new vessel
formation. Therefore, vascularisation contributed by BMP-2/S-NP
in the critical defect site and controlled release of BMP-2 led to
increased bone augmentation (Cao et al., 2014). The photopolymerizable hydrogel incorporated with rhBMP-2 and 2-N, 6-O-sulfated
CS nanoparticles displayed excellent cytocompatibility, cell attachment and cell in the growth of human MSCs. In vitro results
indicated higher ALP activity and mineralization in the presence of
2-N, 6-O-sulfated CS nanoparticles. Bone formation was conrmed
in rat thigh defect and rabbit radius defect models (Cao et al., 2014;
Peschel, Zhang, Fischer, & Groth, 2012). These results indicated that
sulfated CS can have potential towards BTE purposes.
183
Fig. 6. Synthesis of Phosphorylated Chitosans. Reprinted with permission from, Mourya and Inamdar (2008). Chitosan-modications and applications: opportunities galore.
Reactive and Functional polymers, 68(6), 10131051. Elsevier, 2008.
Fig. 7. Effect of p-CS loaded DCPA cements on the mechanical properties. a) Compressive strength of DCPA, DCPAp-chitosan (5 wt%), DCPAp-chitosan (10 wt%) and
DCPAuntreated chitosan (5 wt%). b) Youngs modulus of DCPA, DCPAp-chitosan (5 wt%), DCPAp-chitosan (10 wt%), and DCPAuntreated chitosan (5 wt%). Reprinted with
permission from Boroujeni et al. (2014). Development of monetite/phosphorylated chitosan composite bone cement.Journal of Biomedical Materials Research Part B: Applied
Biomaterials,102(2), 260266. 2013 Wiley Periodicals, Inc.
7. Copolymer of chitosan
The grafted copolymers improve the physicochemical properties of synthetic or natural polymers. Poly(methyl methacrylate)
(PMMA) is one of the most commonly used bone cement owing

to its ability to form a strong mechanical bond with the implant.
This acrylic resin is currently the widely used material for the construction of removable denture and is the basis of dental composite
184
Table 2
Superior properties of chitosan derivatives/chemically modied chitosan based composites favouring bone tissue engineering.
Derivatives/Modications
of Chitosan
Bioactive
Cells treated
molecules/Crosslinkers/proteins
Properties
References
Carboxymethyl
chitosan
Ulvan
Immortalized mouse lung

broblasts cell line (L929)
Barros et al., 2013
Carboxymethyl
Chitosan
genipin carbodiimide local

recombinant human
BMP-2
Zein Silica
Human osteoblastic cell

line (SaOS-2)
Increased Ca and P absorption in

the bulk, enchanced mechanical
performance.
Cytocompatible, promoted cell
attachment and proliferation.
Reves, Bumgardne, &

Haggard (2013)
hMSCs
Enhanced antibacterial activity.
Zhou et al. (2014)
BMP-2 photopolymerisable
hydrogels
hMSCs
Sustained growth factor delivery

and enhanced bioactivity.
Cao et al. (2014)
Chitosan Hydroxyapatite
Good compressive strength.
alkaline phosphatase (ALP)

polydopamine Ti
polyethylene glycol
dimethacrylate (PEGDA)
N,N-dimethylacrylamide
(DMMA)
Hydroxyapatite
hMSCs
Enhanced cellular ALP activity and

calcium deposition by osteoblasts.
Improved mechanical behaviour,
thermal stability. Promoted cell
attachment and proliferation.
Li, Huang, Wang, Ma & Xie

(2011)
Zheng, Neoh & Kang (2016)
hydroxypropyltrimethyl
ammonium chloride
chitosan(HACC)
2-N, 6-O-sulfated
chitosan
nanoparticles
4-phosphorylated
chitosan
carboxymethyl
chitosan
Methacryloyloxy ethyl
carboxyethyl
chitosan
chitosanpolylactic
acid
chitosan-graftpolycaprolactone
(CHS-g-PCL)
N-methylene
phosphonic chitosan
(NMPC)
SW1353
Greatly inuenced nucleation and

growth of crystalline HA.
Enhanced cell mineralization and
ALP activity
Silicatein
SaOS-2
Genipin
Human osteoblastic cell

line (MG-63)
Thiolated chitosan
beta-glycerophosphate
hydroxyapatite
hMSCs
Glycol chitosan (G-CS)
Hyaluronic acid/nHAp
MC-3T3-E1
HACC
Alginate oyster shell

powder
MC-3T3-E1, MG-63 cells
lling materials (Nayak, 2010). It is better than other materials

regarding aesthetics, easy manipulation, low cost, and its ability to
distribute the implant load at the site of implantation. However,
PMMA does not adhere so well to the surrounding bone tissue,
and it is not suitable for the high load bearing sites (Dorozhkin,
2011). The addition of hydroxypropyltrimethylammonium chloride chitosan (HACC) to PMMA showed signicantly higher apatite
deposition, ALP activity and promoted attachment and proliferation of hMSCs. These cells on PMMA-HACC displayed signicantly
higher expression of COL-1, osteopontin (OPN) and osteocalcin
(OC) by the end of 21 days as compared to PMMA-CS, PMMAgentamicin, and PMMA (Tan, Guo et al., 2012). In another study,
the addition of PMMA-co-PHEMA (polymethylmethacrylateco-polyhydroxyethylmethacrylate) to CS/HAp blend showed a
signicant increase in Youngs modulus and stiffness, and enhanced
water absorption capacity of the scaffolds (Bhowmick, Banerjee,
Kumar, & Kundu, 2013). PMMA-HACC promoted a stronger bond
with the surrounding bone, as compared to others in rabbit femoral
condyle (Tan, Ao, Ma, & Tang, 2013). The CS-PMMA composite
increased its compressive strength and promoted new osteoid formation compared to PMMA alone (Endogan et al., 2014). The effects
of using CMC as an additive to methotrexate (MTX) loaded PMMA
bone cement signicantly increased the bending modulus, bending strength and compressive strength of the PMMA composite
(Liu et al., 2015). In comparison to the MTX-PMMA group, CMCMTX-PMMA improved the osseointegration with the host tissue
proving to be an ideal composite over MTX-PMMA as an antitumour bone cement. CS-g-PVCL is a grafted copolymer where CS
grafted with PVCL polymers of varying chain lengths through ami-
Better cell adhesion, cell-cell

interaction, proliferation. Increased
ALP activity and calcium
deposition.
Porous structure with a uniform
distribution of nHAp with
appropriate degradation rate and
low cytotoxicity.
Faster enzymatic degradation of
the scaffold within 4 weeks.
Increased compressive strength,
protein absorption &
biomineralization.
Ma et al. (2010)
Cai et al. (2009)

Wiens, Elkhooly,
Schroder,Mohamed and
Muller (2014)
Datta, Dhara and
Chatterjee (2012)
Liu et al. (2014)
Huang et al., 2016 Huang,

Zhang, Wu and Xu (2016)
Chen, Huang et al. (2016);
Chen, Zhang et al. (2016)
dation reaction by activation of the terminal carboxylic group. The

copolymers were water-soluble at low temperature and were thermosensitive. The thermal behavior was dependent on the grafted
chain length. For future drug delivery purposes, BTE and regenerative medicine, these thermosensitive characteristics can be useful
(Fernandez-Quiroz et al., 2015)
The nanocomposites of CS-graft-(methyl methacrylate) (CS-gMMA) containing Ag nanoparticles used to avoid the implantassociated infections, and the result showed excellent antimicrobial properties (up to 9398%) with increased mechanical strength.
Even though PMMA loaded with antibiotics were used in dermal
llers, and joint arthroplasties, the release of antibiotics was quick
and hence the possibility of altering the mechanical and fatigue
strength of PMMA (Zhang, Myers et al., 2014). The quaternized
CS (HACC) loaded with PMMA in the cavity displayed signicantly
better antibacterial property against the resistant bacteria as compared to PMMA, PMMA-gentamicin or PMMA-CS (Tan, Ao, Ma, Lin,
& Tang, 2014). The treatment of CS with carbon disulde under
alkaline condition formed dithiocarbamate CS. Dithiocarbamate CS
with a metal ion complex showed a prolonged antibacterial activity (Kim et al., 2014). To understand better about CS derivatives
and their properties, we listed the various modications of chitosan
along with bioactive molecules employed in bone tissue engineering (Table 2).
8. Conclusion
This review summarized CS composites and chemically
modied CS for their application in BTE. Modied-CS based scaf-
folds/hydrogels displayed superior physical, chemical, mechanical

and biological properties unlike its counterpart serving to be excellent vehicles for accelerating bone regeneration. Also, this review
consolidated that the empirical renement of CS has candidly
thrown open new avenues for the treatment of bone defects. Thus,
an effort took here provided insights into the past and current
trends of using modied CS polymer. Integration and processing of
the differential properties offered by various modied CS composites would be further benecial for treating bone and bone related
defects.
Acknowledgements
We thank Anbuselvan Thambidurai and Pallavi Chatterjee for
their technical help in the manuscript preparation. We also thank
the Council of Scientic and Industrial Research (CSIR), India (Grant
No. 60(0110)/13/EMR-II to N.S.) and the SRM University for nancial support.
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A review of chitosan and its derivatives in bone tissue engineering

R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172188

Hydroxyalkyl chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

(Jiang, James, Kumbar, & Laurencin, 2014; Niranjan et al., 2013;

R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172188

Cs/alginate scaffold (Kim et al., 2015). Cold atmospheric plasma

erty were required to prevent the implant-associated infections

R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172188

N,N,N-Trimethyl chitosan (TMC)

High absorption efciency (Britto & Assis, 2007)

Better moisture retention and absorption compared to

Strong antifungal activities against many resistant

N-Carboxy-methyl Chitosan (N-CMC)

High metal chelation ability-bound strongly to Ca2+ ions

O-Carboxymethyl Chitosan (O-CMC)

Promoted adhesion and proliferation in human

High water retentivity and excellent gel forming

High moisture absorption rate and highly hydrophilic

99% inhibition against most of the bacteria (Peng, Han,

Rapid gelation kinetics and displayed good in vivo

Due to hydrophobic pockets it caused lesser lysozyme

R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172188

Formed stable hydrogels owing to its amphoteric nature

Chitosan cysteine conjugate

Rapid formation of hydrogel promotes cell attachment

Highly porous structure for cell inltration (Vijapur

Excellent in situ gelling capacity: Formed gels rapidly at

Attributing to its negative charge, low interfacial energy

2-N, 6-O-sulfated chitosan (26SCS)

Structural analogy to heparin, made it highly

Copolymer of chitosan with methyl

Good mechanical and compressive strength (>10 MPa)

Copolymer of Chitosan with

Thermo-responsive gels with 3-D porous architecture,

High water solubility at wide range of pH (Chung et al.,

Better water solubility with enhanced antimicrobial

R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172188

Specic Ligand conjugated Chitosan

Formed viscoelastic injectable hydrogels with

found biocompatible and biostable complex which

Excellent antioxidant property with high scavenging

Enhanced bactericidal nature

Inhibited Ca2 + release from bone (Di Martino, Sittinger,

R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172188

activity against E. coli and S. aureus compared to CS bers. The TMC

2.2. N-(2-hydroxyl) propyl-3-trimethylammonium chitosan

2.3. Other quaternized chitosans

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aureus compared to CS and also the compound was solubilized in

to be%; higher concentrations could lead to the depolymerization

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in the case of serious hemorrhages, and these polymers promoted

R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172188

2011). Carboxymethyl chitosan displayed an inhibition efciency

Furuike, & Tamura, 2008; Jayakumar, Selvamurugan, Nair, Tokura,

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CS was completely blood compatible, had lesser platelet reactivity

with BMP-2 increased in release prole of BMP-2 as compared

R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172188

(PMMA) is one of the most commonly used bone cement owing

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Immortalized mouse lung