a review of chitosan and its derivatives in bone tissue engineering

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Carbohydrate Polymers 151 (2016) 172–188

Contents lists available at ScienceDirect

Carbohydrate Polymers

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review of chitosan and its derivatives in bone tissue engineering

. LogithKumar 1, A. KeshavNarayan 1, S. Dhivya 1, A. Chawla 1, S. Saravanan,. Selvamurugan ∗

epartment of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu, India

r t i c l e i n f o

rticle history:eceived 4 March 2016eceived in revised form 24 April 2016ccepted 15 May 2016vailable online 18 May 2016

a b s t r a c t

Critical-sized bone defects treated with biomaterials offer an efficient alternative to traditional meth-ods involving surgical reconstruction, allografts, and metal implants. Chitosan, a natural biopolymer iswidely 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, fasterin vivo depolymerization, hemo-incompatibility, and weak antimicrobial property. Functionalization of

eywords:iomaterialshitosantem cellssteoblasts

chitosan structure through various chemical modifications provides a solution to these limitations. Inthis review, current trends of using chitosan as a composite with other polymers and ceramics, and itsmodifications such as quaternization, carboxyalkylation, hydroxylation, phosphorylation, sulfation andcopolymerization in bone tissue engineering are elaborated.

© 2016 Elsevier Ltd. All rights reserved.
one tissue engineering
ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1732. Quaternized chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

2.1. N,N,N-trimethyl chitosan (TMC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1742.2. N-(2-hydroxyl) propyl-3-trimethylammonium chitosan chloride. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1782.3. Other quaternized chitosans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

3. Carboxyalkyl chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1. Carboxymethyl chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2. Carboxymethyl chitosan derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: ACP, amorphous calcium phosphate; ALP, alkaline phosphatase; BBC, beering; BHA, butylated hydroxy anisole; BMP-2, bone morphogenetic protein-2; CDH, cmmonium chloride); CS, chitosan; CMC, carboxymethyl chitosan; COL-I, collagen-I; CPC,CPA, dicalcium phosphate anhydrous; DCPD, dicalcium phosphate dihydrate; DD, degQ, degree of quaternization; ECM, extracellular matrix; EDAX, energy dispersive X-rayrophosphate; GTMAC, glycidyl trimethyl ammonium chloride; HACC, hydroxypropyltrermal progenitor cells; HEC, hydroxyethylchitosan; HPLCs, human periodontal ligamePC, hydroxypropyl chitosan; HPLCs, human periodontal ligament cells; HTEC, N-(2-hBL, layer by layer; MMA, methyl methacrylate; MMT, montmorillonite; MMA, methyl

esistant Staphylococcus aureus; MSCs, mesenchymal stem cells; MTX, methotrexate; nHAphitosan; NHNPDCS, N-(2-hydroxyl-5-nitro-phenyl)-N, N-dimethyl chitosan; NHPDCS, N-osan; NHS-QPS, (4-(2,5-Dioxo-pyrrolidin-1-yloxycarbonyl)-benzyl)-triphenyl-phosphonsteoblasts; OREC, organic rectorite; PAA, polyacrylic acid; PAMPS, poly(2-acrylamido-2-monocalcium phosphate monohydrate; PCTS, sodium-phosphorylated chitosan; PEC, pol

o-polyhydroxyethylmethacrylate; PIA, polysaccharide intracellular adhesin; PMMA, polyi-tert-butyl dimethylsilyl; TCP, tricalcium phosphate; TMC, N, N, N trimethyl chitosan; T-O-sulfated chitosan; �CT, micro computed tomography.∗ Corresponding author at: Department of Biotechnology School of Bioengineering SRM

E-mail addresses: [email protected], [email protected] (N. Selva1 These authors equally contributed.

ttp://dx.doi.org/10.1016/j.carbpol.2016.05.049144-8617/© 2016 Elsevier Ltd. All rights reserved.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

ioactive bone cement; bFGF, basic fibroblast growth factor; BTE, bone tissue engi-alcium-deficit hydroxyapatite; CHPTAC, (N-(3-chloro-2-hydroxypropyl) trimethyl

calcium phosphate cement; CT, computed tomography; DAH, 1,6-diaminohexane;ree of dimethylation; DDD, degenerative disc disease; DMC, dimethyl chitosan;

analysis; GAG, glycosaminoglycans; GEN, genipin; GLU, glutaraldehyde; GP, glyc-imethylammonium chloride chitosan; HBC, hydroxybutyl chitosan; HDPs, humannt cells; HTCC, N-(2-hydroxyl) propyl-3-trimethylammonium chitosan chloride;ydroxyl) propyl-3-triethyl ammonium chitosan chloride; HyA, hyaluronic acid;

methacrylate; MPCM, monocalcium phosphate monohydrate; MRSA, methicillin-, nano hydroxyapatite; NBHPDCS, N-(5-bromic-2-hydroxyl-phenyl)-N, N-dimethyl

(2-hydroxyl-phenyl)-N, N-dimethyl chitosan; NMPC, N-methylene phosphonic chi-ium bromide; N, N-DCMCS, N, N-dicarboxymethyl chitosan; NP, nanoparticle; OBs,ethylpropanesulfonic acid); P-COS, phosphorylated chito-oligosaccharides; PCPC-1,yelectrolyte complex; PEG, polyethylene glycol; PHEMA, polymethylmethacrylate-

methyl methacrylate; PVA, poly vinyl alcohol; PVP, poly vinyl pyrrolidone; TBDMS,MCMC, tri methyl carboxymethyl chitosan; TNF, tumor necrosis factor; 26SCS, 2-N,

University, Kattankulathur 603 203, Tamil Nadu, India.murugan).

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R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172–188 173

4. Hydroxyalkyl chitosan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1815. Phosphorylated chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1816. Sulfated chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1827. Copolymer of chitosan. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1838. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185. . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

A worldwide increase in the occurrence of pathological fracturesoupled with risk factors during surgical interventions under-ines the importance of bone tissue engineering (BTE). Cell-freeonstructs and cell-loaded constructs with various chemical andiological cues act as a template to support bone regenerationrocess. They can serve as an alternative treatment method toonventional grafting procedures. Polymers that can recapitu-ate natural bone extracellular matrix (ECM) architecture withhe necessary biochemical and load-bearing properties have beennalyzed for in vivo bone regeneration applications (Swethat al., 2010; Saranya, Moorthi, Saravanan, Devi, & Selvamurugan,011).

Chitosan (CS) is a deacetylated form of chitin procuredainly from the exoskeleton of crustaceans. It is a linear

olymer composed of randomly distributed units namely: (1 → 4)--acetamido-2-deoxy-�-d-glucan (N-acetyl-d-glucosamine; NAG)nd (1 → 4)-2-amino-2-deoxy-�-d-glucan (d-glucosamine) linkedy � (1 → 4) linkages (Jayakumar, Nagahama, Furuike, & Tamura,008; Jayakumar, Prabaharan, Nair, Tamura, & 2010b) as shown

n Fig. 1. The degree of deacetylation represents the molar ratiof the d-glucosamine units to the sum of both NAG and d-lucosamine units (Croisier and Jerome, 2013). Depolymerizationf CS can occur by enzymes like glucosaminidases, lipases, and

ysozyme. Chitosan possesses structural resemblance with gly-osaminoglycans (GAG), one of the components of ECM thatnteracts with collagen fibers playing an important role in cell-ell adhesion. Chitosan, upon depolymerisation yields bioactivehito-oligosaccharides with superior anti-microbial properties, andts monomeric products (glucosamine) metabolized or excretedrom the body. Therefore, CS is biodegradable and has excellentiocompatibility with almost all the tissues of the body. Chi-osan has displayed significant osteoconductivity, but minimalsteoinductive property. It induces proliferation of osteoblast cells,esenchymal cells and induces in vivo neovascularization (Costa-

into, Reis, & Neves, 2011; Kim et al., 2008; Saravanan, Sameera,
oorthi, & Selvamurugan, 2013). For orthopedic applications, CS
n various geometries like sponges, fibers, films and other complextructures are prepared. Thus, CS satisfies most of the proper-ies supporting its candidature for tissue engineering applications

ig. 1. Chemical Structures of chitin and chitosan. Reprinted with permission from Yotructure, properties and applications. Marine drugs, 13(3), 1133–1174. ©2015, MDPI.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

(Jiang, James, Kumbar, & Laurencin, 2014; Niranjan et al., 2013;Saranya, Saravanan, Moorthi, Ramyakrishna, & Selvamurugan,2011).

To enhance the properties of CS for BTE applications, otherpolymers and inorganic materials added to CS, and used as 3Dlyophilized scaffolds, hydrogels/film, and electrospun mats. Chi-tosan hydrogel membranes with nano-hydroxyapatite (nHAp)significantly increased the crystallinity of the composite andshowed excellent biocompatibility with MG-63 cells (Madhumathiet al., 2009). The addition of nHAp and nano-bioactive glass ceram-ics (nBGC) to CS/gelatin significantly improved cell attachment andproliferation of MG-63 osteoblast-like cells (Peter et al., 2010a,2010b). The inclusion of nanoscale silicon dioxide and zirconia par-ticles into CS matrix significantly reduced the degradation rate ofpristine CS scaffolds and enhanced protein adsorption property(Pattnaik et al., 2011). Protein adsorption and biomineralization areessential factors that determine the bioactivity of material used inBTE. Incorporation of carbohydrate anionic moiety like chondroitin4-sulfate into CS matric increased apatite deposition which facili-tated the spreading of bone marrow stromal cells and significantlyenhanced the compressive modulus (Park et al., 2013). The addi-tion of diopside particles (CaMgSi2O6) to CS matrix up regulatedthe expression of osteoblast differentiation marker genes namelyalkaline phosphatase (ALP) and type I collagen (COL-I) and exhib-ited biocompatibility in vivo in a rat model (Kumar et al., 2014).The inclusion of bioceramic materials to polymers may enhancetheir mechanical property. There was an increase in compres-sive strength by 33.07% and enhancement in the proliferation ofmouse preosteoblastic cells (MC3T3-E1) upon addition of nHAp toCS (Zhang, Myers, Wallace, Brandt, & Choong, 2014).

Chitosan as one of the biopolymers in bio-composite scaffoldshas been widely used for BTE applications. The blending of CS withan anionic polysaccharide alginate stabilized the system by electro-static interaction of them (Venkatesan, Bhatnagar, Manivasagan,Kang, & Kim, 2015). Incorporation of nano-silicon dioxide parti-cles to CS-alginate composite displayed a significant increase inprotein adsorption and apatite deposition, and they were non-
toxic to osteoblasts (Sowjanya et al., 2013). Also, incorporationof nHAp to CS/alginate matrix enhanced the mechanical prop-erty of the scaffold as well as stimulated the differentiation ofmouse pre-osteoblastic cells (MC3T3-E1) cells to osteocytes to
unes and Rinaudo (2015). Chitin and chitosan preparation from marine sources.

1 drate

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74 R. LogithKumar et al. / Carbohy

s/alginate scaffold (Kim et al., 2015). Cold atmospheric plasmareated CS/HAp scaffolds displayed surface roughness and wet-ability, which selectively enhanced the protein adsorption ofbronectin and vitronectin. This modification resulted in greateresenchymal stem cells (MSCs) infiltration into the scaffolds with

ncreased collagen deposition and mineralization by the end of 3eeks (Wang, Cheng et al., 2013; Wang, Xie et al., 2013). The inclu-

ion of chicken feather keratin nanoparticles with CS significantlymproved the protein adsorption, and they were biocompatible

ith human osteoblastic cells (Saravanan et al., 2013). Doping andddition of antibacterial metal ions such as nanophase copper,inc and silver to CS polymer significantly improved the antimi-robial property (Niranjan et al., 2013; Saravanan et al., 2011;ripathi et al., 2012). Chitosan/hyaluronic acid scaffolds upon addi-ion of calcium phosphate cement exhibited a significant increasen ALP activity with no significant change in the rate of osteoblas-ic cell proliferation (Hesaraki and Nezafati, 2014). The presencef fucoidan in CS/alginate scaffold stimulated the proliferation ofG-63 osteoblast-like cells with significant enhancement in ALP

ctivity and apatite deposition over the scaffolds (Venkatesan,hatnagar, & Kim, 2014). The addition of nHAp to CS/gelatin matrixot only increased the mechanical property of the scaffolds but alsotimulated the proliferation and differentiation of induced pluripo-ent stem cells of gingival fibroblasts to osteocytes (Isikli, Hasirci, &asirci, 2012; Ji et al., 2015, 2016). Fucoidan in �TCP-CS scaffolds

ncreased the compressibility, apatite deposition, and osteocalcinelease, which favor osteogenic differentiation of human mes-nchymal stromal cells in vitro (Puvaneswary et al., 2015).

Chitosan and its bio-composite scaffolds have been widelyested under in vivo conditions. Chitosan/gelatin scaffolds pro-

oted osteoblast proliferation in vivo, their degradation occurredompletely in 12 weeks, and thus, these scaffolds have the poten-ial for bone healing process (Ma et al., 2014). The encapsulationf MC3T3-E1 osteoblast-like cells in �-tricalcium phosphate-S-alginate microcapsules and their injection in the dorsalubcutaneous area of mice showed collagen deposition and neo-ascularization at the site of injection. Also, the characteristicsteoid-like structures were seen at the end of 8 weeks (He, Dong,hen, & Lin, 2013; Qiao et al., 2013). An osteoinductive bone graftrepared by combining calcium phosphate with CS and it’s implan-ation in the rat’s calvarial defects resulted in a significantly higher

atrix deposition rate as well as osteon formation. Surprisingly,mplantation with the scaffolds replaced the entire calvarial defect

ith host bone tissue. An injectable thermosensitive hydrogel con-aining CS along with Zn, nHAp and �-glycerolphosphate (�-GP)howed its potential towards bone formation in vivo as evidencedy increased apatite and collagen deposition in the defective bonerea (Dhivya, Saravanan, Sastry & Selvamurugan, 2015).

Despite the numerous applications of CS as a stand-alone poly-er or as a composite with other polymers and ceramic particles,

ts use is limited. Firstly, CS solubility in aqueous solutions is pHependent. Since CS has a pKa of 6.5 and its semi-crystalline nature

avors strong intra/inter molecular hydrogen bonding, the solu-ility of CS at neutral pH is limited. Secondly, CS is quickly andasily degraded in vivo due to the presence of � (1 → 4) glyco-idic linkages, which are hydrolytically cleaved by the abundancef lysozyme (approximately, 13–15 mg/L) present in the body (Limt al., 2008). Hence, the study focused on CS with its side chainodifications to address their solubility and controlled biodegra-

ation (Feng and Xia, 2011; Laffleur et al., 2013). Additionally, theolycationic nature of CS can induce thrombosis, red blood cellggregation and hemolysis making it unsuitable for tissue engi-
eering applications. Therefore, many studies focused on alteringhe surface charge on the CS chain to enhance its hemocompati-ility (Balan and Verestiuc, 2014; Yalinca, Yilmaz, Taneri, Bullici,
Tuzmen, 2013). Scaffolds with a suitable antimicrobial prop-

Polymers 151 (2016) 172–188

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 tothe protonation of amino groups that occurs only in acidic medium.Finally, CS has low bioactivity due to the lack of charged and reactivegroups in its structure, which otherwise are necessary to facili-tate protein adsorption. Thus, immobilizing scaffolds with proteinslike fibronectin was essential for increasing the bioactivity of CSbased scaffolds (Custodio, Alves, Reis, & Mano, 2010). Table 1 sum-marizes several modifications at C2 or C5 positions of CS by anintroduction of various reactive groups or by copolymerizationwith other polymers of interest or by grafting with biological andsynthetic macromolecules, which render appropriate bone regen-erative properties.

2. Quaternized chitosan

The modification of CS side chain yields to a variety of derivativesfor biomedical applications. Chitosan was modified into N,N,N-trimethyl chitosan (TMC) by reaction with methyl iodide catalyzedby sodium hydroxide at 60◦ C into N-methyl-2-pyrrolidinone. In asecond step, the substitution of iodide ions by chloride ion by an ionexchange process yielded TMC (Fig. 2). In another example, N-(2-hydroxyl) propyl-3-trimethylammonium chitosan chloride (HTCC)was prepared by dissolving CS in NaOH/CHPTAC aqueous solu-tion by a pressure-equalizing dropping funnel (Fan et al., 2015).Other modified CSs were such as N-(2-hydroxyl) propyl-3-triethylammonium 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 exertsantimicrobial activity by disrupting the negatively charged outermembrane of the microbes, and the modifications of CS structurewould further enhance its antimicrobial activity that is one of thefeatures required in BTE.

2.1. N,N,N-trimethyl chitosan (TMC)

Positive charge rich trimethyl chitosan (TMC) displayedenhanced antibacterial activity compared to CS against both E. coliand S. aureus (Jia and Xu, 2001). The ratio of DD (degree ofdimethylation) to DQ (degree of quaternization) seems to controlmucoadhesive, cytotoxic and other physicochemical properties ofTMC (Haas, Ravi Kumar, Borchard, Bakowsk, & Lehr, 2005). Theanionic groups of lipopolysaccharide in Gram-negative and lipotei-choic acids in Gram-positive bacteria act as a molecular linkage forquaternized and trimethylated amino groups in TMC (Raafat, VonBargen, Haas, & Sahl, 2008). The electrostatic interaction betweenthese groups can disrupt the function and integrity of the bacte-rial membrane. The antibacterial property of TMC increased withincrease in pH of the medium (Xu, Xin, Li, Huang, & Zhou, 2010). It isimportant to note that chitosan chemistry influences the shieldingeffect of surface ion pairs resulting in either partial O-methylationor free of O-methylation. Removal of these surface ion pairs bydialysis showed an increase in the mobility of alkyl chains, con-ferring an active interaction at the surface of the bacterial enveloperesulting in accelerated the antibacterial activity. In addition, TMCwithout O-methylation reduced the cytotoxicity and increased itssolubility 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; Martinset al., 2015).
In addition to the antibacterial property of TMC, TMC could alsoused as a polycation in developing polyelectrolyte complexes by


Table 1Chemically modified derivatives of Chitosan and the improved properties offered by their reactive groups.

Derivatives Sub−types Reactive group Important properties

Quaternized Chitosan N,N,N-Trimethyl chitosan (TMC) • High absorption efficiency (Britto & Assis, 2007)• Mucoadhesion (Pardeshi and Belgamwar, 2016; Raafat,

Von Bargen, Haas, & Sahl, 2008)

N-(2-hydroxyl)propyl-3-trimethylammoniumchitosan chloride (HTCC or HACC)

• Better moisture retention and absorption compared tochitosan (Huang et al., 2014)

• Inhibited the expression of intracellular adhesion geneswhich can mediate biofilm formation on implants (Penget al., 2011)

• High transfection efficiency when compared to Chitosan(Xiao et al., 2012)

N-(2-hydroxyl-substitutedphenyl)-N,N-dimethyl chitosan(NXRPDCS)

• Strong antifungal activities against many resistantstrains (Guo et al., 2007)

Carboxyalkyl Chitosan N-Carboxy-methyl Chitosan (N-CMC) • High metal chelation ability-bound strongly to Ca2+ ionsand promoted apatite formation (Mourya and Inamdar,2008; Mourya et al., 2010)

O-Carboxymethyl Chitosan (O-CMC) • Promoted adhesion and proliferation in humanfibroblast cells (Wongpanit et al., 2005)

N,O-Carboxymethyl chitosan(N,O-CMC)

• High water retentivity and excellent gel formingcapacity (Jayakumar, Prabaharan, Nair, & Tamura, 2010)

• Exhibited negligible immunogenicity (Shalumon et al.,2009).

HydroxyalkylChitosan

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 vivostability (Dang et al., 2006)

N-Acyl Chitosan N-Acetyl chitosan • Due to hydrophobic pockets it caused lesser lysozymeabsorption and higher fibronectin adsorption

• (Tangpasuthadol, Pongchaisirikul, & Hoven, 2003)• Lower swelling index maintains its mechanical strength

(Le Tien, Lacroix, Ispas-Szabo, & Mateescu, 2003)

176 R. LogithKumar et al. / Carbohydrate Polymers 151 (2016) 172–188

Table 1 (Continued)


N-Carboxyacyl chitosan • Formed stable hydrogels owing to its amphoteric nature(Hirano & Moriyasu, 2004)

Thiolated Chitosan Chitosan cysteine conjugate • Rapid formation of hydrogel promotes cell attachmentand infiltration (Bernkop-Schnurch, Hornof, & Zoidl,2004; Riva et al., 2011)

• Gel interacted with BMP-2 causing no change in itsswelling behavior (Kurniawan Fudholi, & Susidarti,2012)

Chitosan–2-iminothiolane conjugate(or Chitosan-4-Thio-butylamidine)

• Highly porous structure for cell infiltration (VijapurSreenivas, 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 atbody temperature (Sakloetsakun, Hombach, &Bernkop-Schnurch, 2009)

Phosphorylated Chitosan • Attributing to its negative charge, low interfacial energyand high binding capacity to Ca2+, and it acted as astrong nucleator for high biomineralization (Xu, Neoh,Lin, & Kishen, 2011)

• Enhanced adipose derived stem cell growth in vitro (Yehand Lin, 2012)

Sulfated Chitosan 2-N, 6-O-sulfated chitosan (26SCS) • Structural analogy to heparin, made it highlyhemo-compatible (Shen et al., 2011)

Semi-synthetic resinsof Chitosan

Copolymer of chitosan with methylmethacrylate

• Good mechanical and compressive strength (>10 MPa)(Endogan et al., 2014)

• Uniformly arranged nanoporous structure which causedsustained time-release of drugs and proteins (Shen et al.,2011)

Copolymer of Chitosan withAcrylamide

• Thermo-responsive gels with 3-D porous architecture,good interconnectivity, elasticity and displayedappreciable osteoconductivity (Liao, Chen, & Chen, 2011)

Sugar derivatives ofChitosan

Glycol Chitosan • High water solubility at wide range of pH (Chung et al.,2003)

• Faster biodegradability for early bone healingapplication (Park, Cho, Chung, Kwon, & Jeong, 2003)

Glucosamine Chitosan • Better water solubility with enhanced antimicrobialproperties (Chen & Zhao, 2012)


Table 1 (Continued)


Specific Ligand conjugated Chitosan Arginylglycylaspartic acid(RGD),Tyr–Ile–Gly–Ser- Arg(YIGSR),le-Lys-Val-Ala-Val(IKVAV),A99a(ALRGDN) and others

• Acceleration of integrin mediated osteoblastattachment, proliferation and differentiation (Tsai, Chen,& Liu, 2013; Wang et al., 2014)

Glycol Chitosan • Formed viscoelastic injectable hydrogels withbiostability and high metal adsorption capacity (Wang,2006)

Glutaraldehyde Chitosan – • found biocompatible and biostable complex whichinduced vascularization and tissue formation (Ma et al.,2003; Wu, Black, Santacana Laffitte, & Patrick, 2007)

Chitosan-ascorbate conjugate – • Excellent antioxidant property with high scavengingand 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)

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ig. 2. Quaternization of chitosan. Chitosan was modified into N,N,N-trimethyl chitosnd applications: opportunities galore. Reactive and Functional polymers, 68(6), 10

ayer by layer (LBL) assembley, a surface modification strategy ben-ficial over crosslinking, in preventing the effects of altered chargeensity, reactivity, and degradability of the material. Almodovarnd Kipper (2011) developed an LBL assembley using TMC as aolycation, heparin as a polyanion. This PEC-LBL assembley showedo promote FGF-2 adsorption serving as suitable matrices for theelivery of growth factors for cell and tissue engineering. A yetnother study on the coating of cortical bone with TMC-heparinolyelectrolyte multilayers showed that they can serve as perios-eum mimics, deliver osteoprogenitor cells, and improve bonellograft healing (Romero et al., 2015). Thus, while the applicationf TMC much explored regarding its antibacterial properties, effortsre needed in considering the polycationic nature of TMC to extend
urther its applications in tissue engineering as vehicles for growthactor delivery.
O-carboxymethyl-N,N,N-trimethyl-chitosan (CMTMC) scaffoldsad no negative influence when treated with human dermal

printed with permission from, Mourya and Inamdar (2008). Chitosan-modifications51. ©Elsevier, 2008.

progenitor (HDP) cells (Patrulea et al., 2015). Further, by immobi-lization of 10 nmol cm−2 and five nmol cm−2 RGDC peptide on thesurface of 1,6-diaminohexane (DAH)-CMTMC, there was the suc-cessful attachment of HDP cells as well as the promotion of cell pro-liferation compared to DAH-CMTMC scaffolds without the RGDCpeptide (Patrulea et al., 2016). Novel Electrospun silver nanoparti-cles (Ag Nps) loaded nanofibrous mats containing TMC, polyacrylicacid (PAA) or poly(2-acrylamido-2-methylpropanesulfonic acid)(PAMPS) showed superior antibacterial activity against E. coli andS. aureus than the membranes without TMC and Ag NPs (Kalinovet al., 2015). Polyalanine treatment to quaternized CS improvedits biocompatibility and antibacterial activities (Zhao, Li, Guo, &Ma, 2015). The antibacterial nature and the wound healing prop-
erties of TMC fibers, prepared by the methylation of CS fibersusing iodomethane under alkaline conditions has been recentlyassessed by Zhou et al. (2016). The resulting TMC fibers with vary-ing degrees of quaternization indicated their higher antibacterial


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ctivity against E. coli and S. aureus compared to CS fibers. The TMCbers also exhibited enhanced swelling properties attributed to theositive surplus charges in its chain. These modified CS fibers tested

n vivo in rat full-thickness excision models displayed reducedound maceration, wound re-epithelialization, eschar develop-ent over 12 days compared to the unmodified CS fibers (Fig. 3).

.2. N-(2-hydroxyl) propyl-3-trimethylammonium chitosanhloride

N-(2-hydroxyl) propyl-3-trimethylammonium chitosan chlo-ide (HTCC) showed stronger antibacterial activity under alkalineondition (Qin et al., 2004). It had enhanced antibacterial potencyompared to CS against Methicillin-resistant Staphylococcus aureusnd Staphylococcus epidermidis Porphyromonas gingivalis (P. gingi-alis ATCC33277), Prevotella intermedia (P. intermedia ATCC 25611),ctinobacillus actinomycetemcomitans (A. actinomycetemcomitans4) and Streptococcus mutans (S. mutans Ingbritt C) (Martinezt al., 2010; Peng et al., 2010). In addition to antibacterial activity,he role of HTCC in tissue regeneration was studied. Hydrox-propyltrimethylammonium chloride chitosan accelerated theroliferation of human periodontal ligament cells (HPDLCs) even athe lower concentration as compared to pristine CS (Qiu Xia et al.,010). The CS-HTCC/GP aqueous solution injected intramuscularly

nto the rumps of sprague–dawley rats formed a gel-like plug athe injection site and showed a good amount of collagen deposi-ion at the end of 9th week. Chitosan-HTCC/glycerophosphate (GP)hermosensitive hydrogel loaded with basic fibroblast growth fac-or (CS-HTCC/GPbFGF) effectively enhanced the new periodontalupport tissues in dogs. The regenerative tissues seemed com-lete to fill the fraction areas, and a significant amount of newone formation observed (Croisier and Jerome, 2013). QuaternizedS derivatives like O-(2-hydroxyl) propyl-3-trimethyl ammoniumhitosan chloride (O-HTCC) and HTEC have also displayed superiorntioxidant properties exhibiting dose-dependent reducing powernd lipid peroxidation inhibition effect (Cui, Tang, & Yin, 2012;

an, A., Xu, Sun, & Li, 2013). The addition of CMC to HTCC improvedts mechanical property (Hu, Wang, & Wang 2016). Their results
ndicated the formation of strong intermolecular hydrogen bondetween OH group of HTCC and CMC, which lead to the increasedigidity and enhanced the tensile strength and elongation at breakalues.
iod. Micrographs displaying the wounds at day 0, 3, 6, 9, 12. The modified CS fibers. (2016). Biomaterials based on N, N, N-Trimethyl chitosan fibers in wound dressing

2.3. Other quaternized chitosans

The quaternized CS (QCS) displayed a significant role in inhibi-tion of the expression of icaA gene (that is involved in secretionof polysaccharide intracellular adhesin) in bacteria on titaniumimplants in a biofilm prevention assay (Peng et al., 2011). Useof antibiotics such as gentamicin loaded in PMMA (Poly (methylmethacrylate)) bone cement was standard practice for preventinginfections in joint arthroplasty and osteomyelitis. This practice hadnot only given rise to antibiotic resistant bacteria like methicillin-resistant Staphylococcus aureus (MRSA) but also shown to reduceproliferation of hMSCs and the viability of osteoblast cells (Parker,Clegg, & Taylor, 2012). However, hydroxypropyl trimethylammo-nium chloride chitosan (HACC) with 26% degree of substitutionloaded in PMMA inhibited biofilm formation (Tan, Guo, Yang, Xu,& Tang, 2012). It also resulted in better apatite deposition, cellattachment and differentiation of hMSCs towards osteoblast lin-eage. Expression levels of COL-I and ALP were significantly highercompared to both gentamicin loaded PPMA and CS–PMMA (Tanet al., 2012b). The quaternized CS derivatives with increasingdegree of substitution (DS) showed antifungal potency in vitroon the mycelial growth of Aspergillus flavus. Increasing the DS ofCS exhibited an almost six-fold increase in the antifungal activ-ity at only one-fourth concentration of the deacetylated chitosanpolymer. Quaternized CSs—including N-(2-hydroxyl-phenyl)-N,N-dimethyl CS (NHPDCS), N-(5-chloro-2-hydroxyl-phenyl)-N, N-dimethyl CS (NCHPDCS), N-(2-hydroxyl-5-nitro-phenyl)-N, N-dimethyl 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 bet-ter antifungal activities compared to the unmodified CS (Badawyand Rabea, 2014; Guo et al., 2007).

An injectable in situ forming an electroactive hydrogel, contain-ing QCS grafted polyaniline with oxidized dextran (o-dex) as cross-linker showed excellent antimicrobial properties in vitro and in vivo(Zhao, Li, Guo, & Ma, 2015). The design and synthesis of a series ofnovel quaternized derivatives of CS, dihydroxy quaternary ammo-nium 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). Addition-ally, a reactive antibacterial compound (4-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)-benzyl)-triphenyl-phosphonium bromide (NHS-QPS) showed effective antimicrobial activity towards E. coli and S.

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ureus compared to CS and also the compound was solubilized inater between a wide range of pH 3–12 (Zhu et al., 2016).

Layer by layer self-assembling polymers into composite filmss a technique to improve the antibacterial efficiency of implant

aterials. Organic rectorite (OREC) is a layered silicate materialell known for their superior mechanical, anti-infective prop-

rties owing to their larger interlayer distance. Self-assembledCMC/OREC/alginate nanofibrous mats layered onto celluloseanofibers displayed enhanced antibacterial activity against E. colind S. aureus compared to cellulose films (Jiang et al., 2015). As cel-ulose based composites are mechanically strong matrices, the LBLssembly of QCMC/OREC/Alg onto cellulose mats could act as cyto-ompatible, mechanically strong antiinfective materials for tissuengineering applications. Recently, a novel method of modificationo QCS was adopted by reacting CS with (3-chloro-2-hydroxypropylrimethyl ammonium chloride) in the presence of alkali or ureaesulting in the highest degree of substitution (You et al., 2016). Therepared QCS was used to develop a hydrogel by in situ polymer-

zation with PAA. There existed a strong electrostatic interactionetween the quaternary ammonium group of CS and the carboxylroup of PAA resulting in enhanced toughness, self-recovery/shapeemory properties with tunable mechanical properties. An elec-

rostatic LBL film assembly comprising of heparin (HP), CS andts derivatives namely DMC, TMC was aimed to develop an effi-ient anti-inflammatory coating to medical devices and implantsFollmann et al., 2016). Because inflammatory responses are uni-ersal host defense mechanisms against foreign agents, which onhe other hand hinders the success of many medical implants.

. Carboxyalkyl chitosan

Amongst other derivatives of CS, carboxyalkyl CS was widelytudied due to its ease of synthesis, a high degree of hydrophilic-ty and numerous potential applications (Riva et al., 2011).arboxyalkylation of CS yields carboxyethyl, carboxybutyl, car-oxymethyl CS and other derivatives. The carboxy ( COOH) groups

mpart anionic charges on the CS chain, making it amphoteric with wide range of biomedical applications. Carboxyalkyl CSs wereater soluble at a wide range of pH, biodegradable, biocompatible,

nd were non-toxic to human tissues. Grafting succinic acid to car-oxyalkyl CS improved water solubility and transfection efficiencyAhmed and Ikram, 2015).

.1. Carboxymethyl chitosan

The most commonly used CS derivative was carboxymethylhitosan (CMC). CMC is amphiprotic ether derived from CS. Thelkylation of CS with monohalocarboxylic acid at different reactiononditions conferred the selectivity form of N- or O-carboxymethylerivatives (Fei Liu, Lin Guan, Zhi Yang, Li, & De Yao, 2001).arboxymethylation introduced carboxymethyl ( COOCH3) group

n the primary or secondary hydroxyl groups and the aminoroups bonded to the glucopyranose unit (Riva et al., 2011).mphoteric nature, enhanced aqueous solubility, biocompatibility,iodegradability and minimal immunogenicity of CMC supported

ts candidature in BTE applications (Ibrahim, El-Zairy, & Mosaad,015; Shalumon et al., 2009). By reductive alkylation and directlkylation, carboxymethylation of CS prepared. However, the for-er one was not preferred as it utilized expensive chemicals,

roduces toxic by-products like NaCN and HCN, and would be dif-cult to scale-up to industrial level (Mourya, Inamdar, & Tiwari,
010; Shalumon et al., 2009). The degree of substitution of CS wasirectly proportional to the concentration of NaOH used in theirect alkylation process, but it was independent of the degree ofeacetylation of CS. The optimum concentration of NaOH was found
Polymers 151 (2016) 172–188 179

to be%; higher concentrations could lead to the depolymerizationof the CS chains (Chen, Tian, & Du, 2004).

There are reports available showing the potential role playedby CMC along with other polymers and biomaterials in BTE. Car-boxymethyl chitosan/HAp scaffolds showed better bone formationthan HAp alone with negligible cytotoxicity on MC3T3-E1 cellsand high blood adsorbing capacity (Tokura and Tamura, 2001).The layering of CMC on titanium implants prevented implant-associated infection and subsequent implant failure. This strategyhighly 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 attach-ment and proliferation with no apparent cytotoxicity (Shalumonet al., 2009). Injectable gels of CMC–gelatin–nHAp stimulated pro-liferation of osteoblasts and showed no evidence of inflammationat the site of implantation (Mishra et al., 2011). Carboxymethylchitosan/gelatin/�-TCP composites fabricated via ultrasonic radia-tion mediated crosslinking were porous in structure with optimummechanical strength. These scaffolds showed biocompatible andpromoted bone regeneration when implanted in the mandibularregion of beagle dogs. Furthermore, the micro-CT analysis clearlyindicated 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 (Jiet al., 2010).

3.2. Carboxymethyl chitosan derivatives

Derivatives of CMC can be amino substituted (N-CMC), hydroxylsubstituted (O-CMC) or both amino and hydroxyl substituted (N, O-CMC). Fig. 4 represents a schematic representation of the chemicalsynthesis of these derivatives. These derivatives were cytocompat-ible, biodegradable and exerted superior antibacterial properties.Further, N-CMC supported chondrocyte adhesion and possessesmetal ion chelating ability (Mourya et al., 2010). O-CMC stimulatedfibroblast proliferation (Fei Liu et al., 2001) while N, O-CMC hadunique gel forming capacity with excellent water retentive proper-ties (Jayakumar, Nagahama et al., 2010; Jayakumar, Selvamurugan,2010; Jayakumar, Prabaharan et al., 2010). N-CMC possessed selec-tive metal adsorption capacity and it readily bound to Ca2+ ions andpromoted biomineralization at the site of implantation (Kyzas andBikiaris, 2015). O-CMC-BMP-2 modified titanium surfaces provedthe great extent of biomineralization of both osteoblast cells andhMSCs along with enhancement of osseointegration and longevityof the titanium implants (Shi, Neoh, Kang, Poh, & Wang, 2009).N, O-CMC/n-�-TCP (tricalcium phosphate) composites in a massproportion of 1:1 highly promoted apatite formation on its sur-face when subjected to varied chemical conditions of simulatedbody fluids (Liu et al., 2013). Lee and Kamarul (2014) prepared acopolymer scaffold comprising of PVA/N-O-CMC/PEG, physicallycross-linked by 50 KGy e-beam radiation. The strong crosslinkingmediated by the e-beam radiation enhanced the physical propertiesof the scaffold such as porosity, pore size, and conferred superiorswelling properties. The scaffolds also showed increased chondro-cyte proliferation on its surface with high expression of GAG andcollagen type II (Lee and Kamarul, 2014).

O-Carboxymethyl chitosan-based injectable hydrogel contain-ing alginate (Alg), nano fibrin particles displayed higher resistanceto mechanical compression. The Alg/O-CMC/nano fibrin (AOF) com-posite promoted the differentiation of adipose derived stem cellsto adipocytes for adipose tissue engineering (Jaikumar et al., 2015).
PVA/CMC/montmorillonite (MMT) based hydrogel nanocompos-ite system displayed enhanced antimicrobial activity better thanthe standard drug penicillin G (Sabaa, Abdallah, Mohamed, &Mohamed, 2015). Li et al. (2015) developed a CMC/modified starch


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ased in situ injectable hydrogel with a superior mechanical prop-rty. Oxidized cholesterol starch as a non-cytotoxic crosslinkersed in the hydrogel synthesis mediated stable covalent bond for-ation, by the condensation of aldehyde groups of oxidized starchith the primary amine group of CMC. Besides being an antibacte-

ial material, upon optimizing the crosslinker concentration, thetructural and mechanical properties of the hydrogel could beailor made to specific applications. Hao, Chen, Yu, Liu, and Sun2016) recently developed the CMC-multiwalled carbon nanotubeased strong antibacterial composite upon coordination of metal

ons Cu and Zn. They displayed enhanced antimicrobial activitygainst S. aureus, E. coli and V. anguillarum. In another application,MC grafted polyacrylic acid served as superabsorbent polymers

osan, O-carboxymethyl chitosan and N, O-carboxymethyl chitosan. Reprinted withpportunities galore. Reactive and Functional polymers, 68(6), 1013–1051. ©Elsevier,

in the case of serious hemorrhages, and these polymers promotedthe concentration of coagulation factors, RBCs and platelets. Theenhanced roughness, porous surface structure, high water reten-tion ability and availability of positively charged NH3

+ cations ofthe polymers promoted the adhesion of negatively charged RBCsonto 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 propertyagainst S. aureus than both O-CMC and CS nanoparticles, and theinhibition of bacterial growth seemed to increase with an increase
in the concentration of these nanoparticles (Anitha et al., 2009).N, O-CMC–zinc compound exerted a better antimicrobial activ-ity than CS–zinc complex against S. aureus (Patale and Patravale,

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011). Carboxymethyl chitosan displayed an inhibition efficiencyf >90% against bacterial adhesion. It prevented biofilm formationt the efficiency of 63.1% of Gram-positive bacteria and 70.6% ofram-negative bacteria after 1 of biofilm initiation. The proposedechanism behind this phenomenon is the neutralization of the

acterial surface charge leading to flocculation of the bacterial cellopulation (Tan, Han, Ma, & Yu, 2011). Kaya et al. (2014) demon-trated the antimicrobial and antioxidant activities possessed by-CMC. Water soluble N, O-CMC developed by Patrulea, Applegate,stafe, Jordan, and Borchard (2015) showed its non-cytotoxic andon-immunogenic nature, which are the features also required inTE.

. Hydroxyalkyl chitosan

Hydroxyalkyl chitosans can be obtained by the substitutioneaction of CS with epoxides at either amino or hydroxyl groupsiving N-hydroxyalkyl or O-hydroxyalkyl CS derivatives, respec-ively (Fig. 5). The ratio of O/N-substitution was determined byhe choice of catalyst (NaOH or HCl) used in the reaction and theeaction temperature (Donges, Reichel, & Kessler, 2000; Mouryand Inamdar, 2008). For tissue engineering applications, only a fewerivatives of hydroxyalkyl CS such as hydroxybutyl, hydroxyethylnd hydroxypropyl chitosan have been explored. Hydroxypropylhitosan (HPC) at a concentration of, grafted with maleic acid,wing to its capability to form coordination bonds displayed better

nhibitory effects up to 99.9% against both S. aureus and E.coli within0 min of contact time (Peng, Han, Liu, & Xu, 2005). Hydroxybutylhitosan (HBC) provided an extra advantage of a rapid gelationinetics and better in vivo stability over that of collagen and there-ore, established itself as a suitable polymer (Dang et al., 2006).ydroxybutyl chitosan was shown to be biocompatible and wasinimally toxic to five-wt% when exposed to hMSCs and interver-

ebral disk cells as a potential candidate to treat degenerative discisease (DDD). Its thermosensitive properties underlined its prob-ble use to form gels at body temperature in as quick as 30. Theell viability in the presence of HBC was similar to that in the pres-nce of collagen in a 2-week’s treatment period. It promoted theroliferation of L929 mouse fibroblast cells with minimal toxic-

ty suggesting its application in wound healing (Wei et al., 2009).ellulose/O-hydroxyethyl chitosan fibers with an HEC concentra-ion of 6.2% displayed reduction of E. coli growth and had moisturebsorption rate of 13.55% (Xu, Xin et al., 2010; Xu, Zhuang et al.,010).

Clay nanocomposites emerged to be superior materials inmproving the mechanical properties of polymeric materials

ith strength, modulus, and dimensional stability. A modifiedTCC-nanoclay (montmorillonite) composite showed antibacterialctivity, cytocompatibility, and cell growth (Aliabadi, Dastjerdi, &abiri, 2013). The inclusion of nanoclay to HTCC resulted in strongly

ntercalated network structure, which allowed the bacteria to effec-ively entrapped, and damaged them. However, further studies arearranted to investigate and support the applications of hydrox-

alkyl CS in BTE.

. Phosphorylated chitosan

The synthesis of phosphorylated CS (P-CS) involves the simul-aneous reaction of the phosphorous acid and formaldehyde to CSn the acidic aqueous acidic resulting in the formation of water-oluble N-mono- and di-phosphonicmethylene chitosan (Fig. 6).
hosphorylated CS displays better ionic conductivity and swelling
ndex. Although it decreased crystallinity, its tensile strengthemained similar to CS. Phosphorylated CS exhibited consider-bly rough surface morphology unlike CS (Jayakumar, Nagahama,

Polymers 151 (2016) 172–188 181

Furuike, & Tamura, 2008; Jayakumar, Selvamurugan, Nair, Tokura,& Tamura, 2008). Phosphorous pentoxide (P2O5) reaction with CSyielded water-soluble phosphorylated CS with a high degree of sub-stitution (Tachaboonyakiat, Netswasdi, Srakaew, & Opaprakasit,2010). Phosphorylated CS blend alginate films showed inductionof apatite layer after immersing it in simulated body fluid (SBF)for 21 days (Jayakumar et al., 2009). The solubility of CS basedhydrogels above pH 6 reduced its potential for tissue engineer-ing applications. To overcome this limitation, the phosphorylatedderivative of CS, N-methylene phosphonic chitosan (NMPC) wassynthesized by cross-linking of NMPC with either glutaraldehyde(NMPC-GLU) or genipin (NMPC-GEN). While NMPC-GEN hydro-gel was better among the two, both the hydrogels showed higherelastic modulus, better compressive strengths and increased theproliferation of cells.

A biodegradable P-CS/monetite (dicalcium phosphateanhydrous-DCPA) composite showed enhanced mechanicalperformance 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 asincreased the young’s modulus, compressive strength by two-fold(Fig. 7a,b). However, it was observed that further increase inw% of P-CS from 5% to 10% reversed the increasing mechanicalproperties. The composite showed cytocompatibility, supportedthe proliferation of MC3T3-E1 cells, and hence suggested as idealbone cement for load bearing applications.

Calcium phosphate cement has been used as fillers at the siteof injury for its remarkable osteoconductive and osseointegrativeproperties (Winge, Reikeras, & Rokkum, 2011), yet they lack themechanical strength required for bone tissue implants. The bind-ing affinity of calcium to reactive functional groups followed theorder: phosphorylate > carboxylate > amino > hydroxyl groups. Theaddition of P-CS up to 2 wt% to monocalcium phosphate mono-hydrate (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 young’s mod-ulus. The leaching experiment showed that P-CS did not leach outfor up to two months when implanted in vivo. Hence, P-CS couldbind tightly to inorganic phase during the setting process of CPC(calcium phosphate cement), thus, can be effective as bone filler inclinical applications.

Enzymatic degradation of CS formed chito-oligosaccharides(Aam et al., 2010) and phosphorylated chito-oligosaccharides (P-COS) up to 100 �g/mL concentration showed no cytotoxicity toMG-63 osteoblast-like cells. Also, low molecular weight P-COSseemed to promote cell proliferation. ALP activity was significantlyincreased in the presence of P-COS as compared to CS (Venkatesan,Pangestuti, Qian, Ryu, & Kim, 2010). To achieve the mechanicalstrength, a P-CS/CS/n-HAp composite was prepared by the co-precipitation method and it displayed a compressive strength of70.25 Mpa. The compressive strength of the composite remained64% of the original (45.07 Mpa) after soaking in simulated bodyfluid (SBF) for . Hence, it displayed controlled biodegradation andexcellent bioactivity for BTE purposes (Li, Huang, Wang, Ma, & Xie,2011). The incorporation of P-CS powders at five-wt% to the widelyused bone cement DCPA reduced its setting time and doubled itscompressive strength. Both DCPA and DCPA–P-CS increased cellproliferation (MC3T3-E1). The results showed that both the materi-als were as cytocompatible as HAp. The cell morphology of attachedosteoblast cells in 7 days culture testified the biocompatibility ofDCPA–P-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 modificationswith plasma induced phosphonic groups significantly improvedosteoblast attachment, viability, and proliferation (Lopez-Perez, DaSilva, Sousa, Pashkuleva, & Reis, 2010). It has been reported that P-


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S was completely blood compatible, had lesser platelet reactivitynlike CS, and enhanced adipose-derived stem cell growth in vitroYeh and Lin, 2012). Metals like Zn have been widely used for theirntimicrobial properties in complex with biomaterials (Thian et al.,013). However, metals can cause significant cytotoxicity if usedbove a threshold concentration. Sodium-phosphorylated chitosanPCTS) and ZnO showed their application in periodontal dressings.he PCTS did not change the crystalline nature of ZnO and reduced

ts cytotoxicity by forming a complex with its protonated hydroxylroups. The composite was biocompatible with primary humaningival fibroblast cells (Srakaew, Ruangsri, Suthin, Thunyakitpisal,

Tachaboonyakiat, 2011). Phosphorylated CS significantly pro-oted proliferation of both primary human osteoblasts (OBs) and

he OB like stromal cell (the component of the giant cell tumorf bone cells; up to 1000-�g ml−1 concentration) only after sevenays of treatment. A study on the osteogenic effect of the P-CS sug-ested that it regulates the level of osteoclastogenic factors, andeceptor activator of nuclear factor kappa B ligand (NFKBL) andsteoprotegerin (OPG) expression (Tang et al., 2011). Partially dem-

neralized dentine sections modified by covalent immobilization of-CS showed better deposition of calcium phosphate on the dentineurface (Xu, Neoh, Lin, & Kishen, 2011). All these properties ensure-CS’s candidature as a promising biomaterial for BTE applications.

. Sulfated chitosan

Chitosan chain can be sulfated using sulphuric acid or sulphoniccid salts (Rajasree and Rahate, 2013; Zhang, Zhang, Ma, Yang, &ie, 2015). The sulfated CS played a major in bone metabolismnd enhanced bone morphogenetic protein-2 (BMP-2) activity in
one (Takada et al., 2003). The half-life of BMP-2 in culture wasrolonged with heparin, a sulfated polysaccharide (Hosseinkhani,hademhosseini, & Kobayashi, 2007; Zhao et al., 2006). The addi-
ion of sulfated CS to calcium-deficit hydroxyapatite (CDH) loaded

dar (2008). Chitosan-modifications and applications: opportunities galore. Reactive

with BMP-2 increased in release profile of BMP-2 as comparedto the composite without sulfated CS. The initial burst follow-ing gradual release was essential for the growth of new bonetissue (Zhao et al., 2011). Sulfated CS stimulated proliferation ofboth primary human osteoblasts and the OB like stromal cellcomponent of the giant cell tumor (GCT) of bone cells at a con-centration of 100 �g ml−1. However, it inhibited cell proliferationabove 1000 �g ml−1; this effect was more pronounced in primaryhuman GCT stromal cells as compared to osteoblast cells (Tang et al.,2011).

Neovascularization at the site of a bone defect is necessary forthe development of a new fibrous tissue. Modified heparin dis-played vascular tissue formation as reported previously (Janairoet al., 2012; Wang, Cheng et al., 2013; Wang, Xie et al., 2013). Sul-fated N-carboxymethyl CS inhibited thrombin and factor Xa similarto antithrombin. It also caused no hemolysis when human bloodsamples treated with it (Zhou et al., 2013). In another study, 2-N, 6-O-sulfated chitosan (26SCS) based nanoparticle (S-NP) wassuccessfully developed and loaded with BMP-2 (BMP-2/S-NP) andthen fabricated with gelatin BMP-2/S-NP (BMP-2/S-NP/G) to repaircritical-sized bone defect of rabbit radius. The gelatin BMP-2/S-NPtreatment significantly increased both peripheral and new vesselformation. Therefore, vascularisation contributed by BMP-2/S-NPin the critical defect site and controlled release of BMP-2 led toincreased bone augmentation (Cao et al., 2014). The photopolymer-izable hydrogel incorporated with rhBMP-2 and 2-N, 6-O-sulfatedCS nanoparticles displayed excellent cytocompatibility, cell attach-ment and cell in the growth of human MSCs. In vitro resultsindicated higher ALP activity and mineralization in the presence of2-N, 6-O-sulfated CS nanoparticles. Bone formation was confirmedin rat thigh defect and rabbit radius defect models (Cao et al., 2014;
Peschel, Zhang, Fischer, & Groth, 2012). These results indicated thatsulfated CS can have potential towards BTE purposes.


Fig. 6. Synthesis of Phosphorylated Chitosans. Reprinted with permission from, Mourya and Inamdar (2008). Chitosan-modifications and applications: opportunities galore.Reactive and Functional polymers, 68(6), 1013–1051. ©Elsevier, 2008.

F presD wt%)p hitosaB

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ig. 7. Effect of p-CS loaded DCPA cements on the mechanical properties. a) ComCPA–untreated chitosan (5 wt%). b) Young’s modulus of DCPA, DCPA–p-chitosan (5ermission from Boroujeni et al. (2014). Development of monetite/phosphorylated ciomaterials,102(2), 260–266. © 2013 Wiley Periodicals, Inc.

. Copolymer of chitosan

The grafted copolymers improve the physicochemical proper-ies of synthetic or natural polymers. Poly(methyl methacrylate)

sive strength of DCPA, DCPA–p-chitosan (5 wt%), DCPA–p-chitosan (10 wt%) and, DCPA–p-chitosan (10 wt%), and DCPA–untreated chitosan (5 wt%). Reprinted withn composite bone cement.Journal of Biomedical Materials Research Part B: Applied

(PMMA) is one of the most commonly used bone cement owingto its ability to form a strong mechanical bond with the implant.This acrylic resin is currently the widely used material for the con-struction of removable denture and is the basis of dental composite


Table 2Superior properties of chitosan derivatives/chemically modified chitosan based composites favouring bone tissue engineering.

Derivatives/Modificationsof Chitosan

Bioactivemolecules/Crosslinkers/proteins

Cells treated Properties References

Carboxymethylchitosan

Ulvan Immortalized mouse lungfibroblasts cell line (L929)

Increased Ca and P absorption inthe bulk, enchanced mechanicalperformance.

Barros et al., 2013

CarboxymethylChitosan

genipin carbodiimide localrecombinant humanBMP-2

Human osteoblastic cellline (SaOS-2)

Cytocompatible, promoted cellattachment and proliferation.

Reves, Bumgardne, &Haggard (2013)

hydroxypropyltrimethylammonium chloridechitosan(HACC)

Zein Silica hMSCs Enhanced antibacterial activity. Zhou et al. (2014)

2-N, 6-O-sulfatedchitosannanoparticles

BMP-2 photopolymerisablehydrogels

hMSCs Sustained growth factor deliveryand enhanced bioactivity.

Cao et al. (2014)

4-phosphorylatedchitosan

Chitosan Hydroxyapatite – Good compressive strength. Li, Huang, Wang, Ma & Xie(2011)

carboxymethylchitosan

alkaline phosphatase (ALP)polydopamine Ti

hMSCs Enhanced cellular ALP activity andcalcium deposition by osteoblasts.

Zheng, Neoh & Kang (2016)

Methacryloyloxy ethylcarboxyethylchitosan

polyethylene glycoldimethacrylate (PEGDA)N,N-dimethylacrylamide(DMMA)

SW1353 Improved mechanical behaviour,thermal stability. Promoted cellattachment and proliferation.

Ma et al. (2010)

chitosan–polylacticacid

Hydroxyapatite - Greatly influenced nucleation andgrowth of crystalline HA.

Cai et al. (2009)

chitosan-graft-polycaprolactone(CHS-g-PCL)

Silicatein SaOS-2 Enhanced cell mineralization andALP activity

Wiens, Elkhooly,Schroder,Mohamed andMuller (2014)

N-methylenephosphonic chitosan(NMPC)

Genipin Human osteoblastic cellline (MG-63)

Better cell adhesion, cell-cellinteraction, proliferation. IncreasedALP activity and calciumdeposition.

Datta, Dhara andChatterjee (2012)

Thiolated chitosan beta-glycerophosphatehydroxyapatite

hMSCs Porous structure with a uniformdistribution of nHAp withappropriate degradation rate andlow cytotoxicity.

Liu et al. (2014)

Glycol chitosan (G-CS) Hyaluronic acid/nHAp MC-3T3-E1 Faster enzymatic degradation ofthe scaffold within 4 weeks.

Huang et al., 2016 Huang,Zhang, Wu and Xu (2016)

63 cel

firdPa2rdth(gtcswKwcimobi(Mptg

HACC Alginate oyster shellpowder

MC-3T3-E1, MG-

lling materials (Nayak, 2010). It is better than other materialsegarding aesthetics, easy manipulation, low cost, and its ability toistribute the implant load at the site of implantation. However,MMA does not adhere so well to the surrounding bone tissue,nd it is not suitable for the high load bearing sites (Dorozhkin,011). The addition of hydroxypropyltrimethylammonium chlo-ide chitosan (HACC) to PMMA showed significantly higher apatiteeposition, ALP activity and promoted attachment and prolifera-ion of hMSCs. These cells on PMMA-HACC displayed significantlyigher expression of COL-1, osteopontin (OPN) and osteocalcinOC) by the end of 21 days as compared to PMMA-CS, PMMA-entamicin, and PMMA (Tan, Guo et al., 2012). In another study,he addition of PMMA-co-PHEMA (polymethylmethacrylate-o-polyhydroxyethylmethacrylate) to CS/HAp blend showed aignificant increase in Young’s modulus and stiffness, and enhancedater absorption capacity of the scaffolds (Bhowmick, Banerjee,umar, & Kundu, 2013). PMMA-HACC promoted a stronger bondith the surrounding bone, as compared to others in rabbit femoral

ondyle (Tan, Ao, Ma, & Tang, 2013). The CS-PMMA compositencreased its compressive strength and promoted new osteoid for-

ation compared to PMMA alone (Endogan et al., 2014). The effectsf using CMC as an additive to methotrexate (MTX) loaded PMMAone cement significantly increased the bending modulus, bend-

ng strength and compressive strength of the PMMA compositeLiu et al., 2015). In comparison to the MTX-PMMA group, CMC-

TX-PMMA improved the osseointegration with the host tissueroving to be an ideal composite over MTX-PMMA as an anti-umour bone cement. CS-g-PVCL is a grafted copolymer where CSrafted with PVCL polymers of varying chain lengths through ami-

ls Increased compressive strength,protein absorption &biomineralization.

Chen, Huang et al. (2016);Chen, Zhang et al. (2016)

dation reaction by activation of the terminal carboxylic group. Thecopolymers were water-soluble at low temperature and were ther-mosensitive. The thermal behavior was dependent on the graftedchain length. For future drug delivery purposes, BTE and regenera-tive medicine, these thermosensitive characteristics can be useful(Fernandez-Quiroz et al., 2015)

The nanocomposites of CS-graft-(methyl methacrylate) (CS-g-MMA) containing Ag nanoparticles used to avoid the implant-associated infections, and the result showed excellent antimicro-bial properties (up to 93–98%) with increased mechanical strength.Even though PMMA loaded with antibiotics were used in dermalfillers, and joint arthroplasties, the release of antibiotics was quickand hence the possibility of altering the mechanical and fatiguestrength of PMMA (Zhang, Myers et al., 2014). The quaternizedCS (HACC) loaded with PMMA in the cavity displayed significantlybetter antibacterial property against the resistant bacteria as com-pared to PMMA, PMMA-gentamicin or PMMA-CS (Tan, Ao, Ma, Lin,& Tang, 2014). The treatment of CS with carbon disulfide underalkaline condition formed dithiocarbamate CS. Dithiocarbamate CSwith a metal ion complex showed a prolonged antibacterial activ-ity (Kim et al., 2014). To understand better about CS derivativesand their properties, we listed the various modifications of chitosanalong with bioactive molecules employed in bone tissue engineer-ing (Table 2).

8. Conclusion

This review summarized CS composites and chemicallymodified CS for their application in BTE. Modified-CS based scaf-

drate

falctattid

A

ttNc

R

A

A

A

A

A

B

B

B

B

B

B

B

C

C

C

C

C


olds/hydrogels displayed superior physical, chemical, mechanicalnd biological properties unlike its counterpart serving to be excel-ent vehicles for accelerating bone regeneration. Also, this reviewonsolidated that the empirical refinement of CS has candidlyhrown open new avenues for the treatment of bone defects. Thus,n effort took here provided insights into the past and currentrends of using modified CS polymer. Integration and processing ofhe differential properties offered by various modified CS compos-tes would be further beneficial for treating bone and bone relatedefects.

cknowledgements

We thank Anbuselvan Thambidurai and Pallavi Chatterjee forheir technical help in the manuscript preparation. We also thankhe Council of Scientific and Industrial Research (CSIR), India (Granto. 60(0110)/13/EMR-II to N.S.) and the SRM University for finan-

ial support.

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a review of chitosan and its derivatives in bone tissue engineering

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