International Journal of Biological Macromolecules 93 (2016) 1354–1365

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Chitosan based biocomposite scaffolds for bone tissue engineering S. Saravanan, R.S. Leena, 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 11 November 2015 Received in revised form 27 January 2016 Accepted 29 January 2016 Available online 1 February 2016 Keywords: Chitosan Scaffold Protein adsorption Biodegradability Bone tissue engineering

a b s t r a c t The clinical demand for scaffolds and the diversity of available polymers provide freedom in the fabrication of scaffolds to achieve successful progress in bone tissue engineering (BTE). Chitosan (CS) has drawn much of the attention in recent years for its use as graft material either as alone or in a combination with other materials in BTE. The scaffolds should possess a number of properties like porosity, biocompatibility, water retention, protein adsorption, mechanical strength, biomineralization and biodegradability suited for BTE applications. In this review, CS and its properties, and the role of CS along with other polymeric and ceramic materials as scaffolds for bone tissue repair applications are highlighted. © 2016 Elsevier B.V. All rights reserved.

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

the few decades, the steeping demand for bone grafts has been found to be rising with human population. The necessity in the use of biomaterials with improved properties has always been an alternative to the traditional use of autogenous bone grafts. Bone tissue engineering (BTE) encompasses the principles of bone biology and engineering disciplines to augment bone loss through the use of temporary matrices called as scaffolds. Scaffolds are fabricated by materials belonging with various classes including polymers, ceramics. Chitosan (CS), a natural polymer has been widely used as one of the scaffolding materials, and its use alone or along with other polymers or ceramics as scaffolds in bone tissue repair applications are reviewed in the following sections.

2. Bone and its components Abbreviations: 3D, three dimensional; Alg, alginate; BG, bioglass ceramic; nBGCs, nano bioactive glass ceramics; BSA, bovine serum albumin; BTE, bone tissue engineering; CAD, computer aided design data; CaP, calcium phosphate; CMC, carboxymethyl cellulose; Col, collagen; CS, chitosan; ECM, extracellular matrix; EDAC, ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride; GAGs, glycosaminoglycans; Gn, gelatin; Hap, hydroxyapatite; mWS, mesoporous wollastonite; NaCl, sodium chloride; NAG, N-acetyl-d-glucosamine; nBGC, nano bioglass ceramic; NHS, N-hydroxysuccnimide; NPs, nanoparticles; nSiO2 , nano silicon dioxide; nZrO2 , nano zirconium oxide; ␤-TCP, beta tricalcium phosphate nano particles; PCL, polycaprolactone; PEG, poly(ethylene oxide); PLGA, poly (lactic-co-glycolic acid); PLLA, poly(l-lactic acid); SF, silk fibroin; SiO2 , silicon dioxide; TCP, tricalcium phosphate; Ti, titania; TPP, sodium tripolyphosphate; ZrO2 , zirconium oxide; ␤-TCP, betatricalcium phosphate. ∗ Corresponding author at: Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, 603 203 Tamil Nadu, India. E-mail addresses: [email protected], [email protected] (N. Selvamurugan) http://dx.doi.org/10.1016/j.ijbiomac.2016.01.112 0141-8130/© 2016 Elsevier B.V. All rights reserved.

Bone is a highly functionalized connective tissue forming the skeletal framework of human body with its involvement in various physiological functions [17]. The anatomical representation of bone and its components are shown in Fig. 1. Collagen and calcium phosphate (CaP) apatite crystals are the chief constituents of bone. Apatite crystals are observed as embodiments within the collagen matrix. The density based classification compartmentalizes bone into cortical, a dense tissue and cancellous or trabecular, highly porous. Bone marrow is homed in the interior of cancellous bone. Bone tissue undergoes continuous remodeling through the action of its cellular components osteoblasts-bone forming cells, osteoclasts-bone resorbing cells and osteocytessenile osteoblasts (Fig. 1). It possesses various units from lamella to individual collagen fibrils arranged hierarchically and sized under

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

nano dimensions. Bone mineral hydroxyapatite (HAp) is architectured down to few hundred nanometers. Bone cells work in harmony to maintain the structural integrity and help in regeneration of diseased bone tissue. However, under non unions and critical sized defects, healing of bone through normal remodeling process remains unreachable and hence, bone grafting is required to facilitate bridging of those defects. 3. Bone tissue engineering Critical sized bone defects are addressed by the use of cells and biomaterials, and with a combination of cells, biomaterials and bioactive molecules (Fig. 2). Scaffolding materials exerting the following properties such as osteogenicity, osteoconductivity, biocompatibility and biodegradability are often selected for BTE [18,19]. The scaffolds must be highly porous with interconnectivity providing space for cellular infiltration, nutrient transfer, waste disposal, neovascularization and space for new bone tissue ingrowth. Pores ranging between 200–600 ␮m is often found be best suited for BTE applications [20]. Surface roughness and wettability of the scaffolding materials is essential for improving biomaterialcell interactions. Suitable mechanical properties possessed by the scaffolds decide their site of application including whether in load bearing or non-load bearing sites [21,22]. The degradation properties are strongly dependent on the scaffolding materials, and tailoring of the scaffold degradation in vivo can be obtained through adjusting materials properties [23]. Ceramics such as calcium phosphate (biphasic) (CaP), tricalcium phosphate (TCP) and HAp are widely employed in scaffold fabrication due to their structural similarity with the mineral components of human bone. Zirconium oxide (ZrO2 ), silicon dioxide (SiO2 ), bioactive glass ceramics (BGCs), titania (Ti) are few other ceramics employed in BTE [24–30]. Polymers include both natural and synthetic, and synthetic polymers such as poly(l-lactic acid) (PLLA), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA) have been studied for their use as scaffolding materials [31–33]. Natural polymer based composite materials are gaining an increased attention for their use in BTE. Chitosan, alginate (Alg), collagen (Col), gelatin (Gn), silk fibroin (SF) and glycosaminoglycans (GAGs) are widely used natural polymers [34–43].

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

3.2. Physico-chemical properties When the number of NAG is greater than 50%, then it is said to be chitin and when the number of N-glucosamine is greater than 50%, it is said to be CS. The different forms of CS are varied based on their degree of acetylation (ranging from 50 to 95%) and their molecular weights (ranging from 300 to 1000 kDa) [49]. Also, the degree of acetylation and the molecular weights influence the physicochemical properties of CS such as crystallinity, solubility and degradation [50]. Thus, 0% deacetylated chitin and 100% deacetylated CS are highly crystalline in nature, and other intermediate degrees of deacetylated CS are semi-crystalline in nature. The solubility of CS is based on the amount of free amino and N-acetyl groups, which enable it to be soluble in both organic and inorganic acids (pKa 6.5), and insoluble in neutral and basic solutions [51]. Chitosan is said to be cationic in nature which enables CS to form complexes with anionic macromolecules such as glycosaminoglycans (GAGs) that modulate the activity of cytokines and growth factors, aiding in its applications in BTE.

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

The primary amines and the secondary hydroxyl groups seen on CS facilitate the addition of side groups, peptides or amino acids that aid in functionalization and optimization of CS for BTE [52]. Lysozyme is the enzyme that degrades CS in vitro and in vivo, by hydrolyzing the glucosamine-glucosamine, glucosamine—NAG and NAG—NAG bonds. The resulting CS oligosaccharides on hydrolysis are then fused into GAG or excreted [53,54]. The degree of degradation and molecular weight of CS are inversely related to the degree of deacetylation. If the degree of deacetylation is higher, the degradation rate of it is lower, due to high polymer crystallinity and when the molecular weight of CS is higher, the degree of degradation is said to occur at a lower rate [55]. Thus, the fabrication of CS scaffolds for BTE can be done by altering the degree of deacetylation according to the required bone ingrowth. 3.3. Biological properties Chitosan has been widely used in accelerating the wound healing capacity and antimicrobial activities due to its cationic nature [53]. Chitosan by activating and modulating the inflammatory cells facilitates the growth of granular tissue, which makes it as a potent wound healing accelerator [56]. It acts as an important component in wound dressing; as it binds to the anionic red blood cells, promoting clotting [54]. Chitosan possesses antimicrobial properties by associating to the anions in the bacterial cell walls and thus suppresses the biosynthesis of cell wall, which eventually kills the bacteria [57]. A supporting role for CS in cell proliferation, osteoblast differentiation and mineralization has been documented [45–48]. 4. Chitosan scaffold fabrication methods Bone tissue engineering involves the basic idea of using a 3D biodegradable polymeric scaffold to promote bone tissue growth and remodeling. The physical properties of CS have made it possible to be shaped into various structures, membranes, sponges, fibers and porous scaffolds for BTE. Chitosan based scaffolds are prepared through various fabrication methods and few of the methods are

listed in Fig. 3. Among the methods, the most common methodology for producing CS scaffolds is by lyophilization [24–30]. Freezing the CS solution results in formation of ice crystals via phase separation and on sublimation, the original space occupied by the crystals is emptied and leading to the formation of pores. This technique requires high precision control over temperature. The disadvantages associated with it are collapsing of pores structure if the temperature is not controlled. There is possibility of formation of larger pores and the choice of solvent is limited. The use of solvent-exchange/phase-separation which is based on the gelation of a solution using alkaline solution below its gelation point can be used. Through these techniques, 3D scaffold with various geometries can be obtained but the major disadvantage is the lack of proper mechanical strength. The technique of salt leaching involves the incorporation of porogens such as sodium chloride (NaCl) of desired sized into CS matrix and successive leaching of the salt particles using water results in formation of porous CS scaffolds [58,59]. Electrospinning allows the production of fibrous scaffolds. It utilizes electric field created between the nozzle tip of the polymeric reservoir and ground collector. Applied electric field causes the elongation of the polymer drop and leads to the formation of long fibers. This versatile technique allows the production of both nano and micro fibers [60–62]. Electrospinning of CS is quite difficult and the stability inside aqueous environment is a major concern and often requires crosslinking with agents like poly(ethylene oxide) (PEG). Blending with other polymers such as SF, Col, PCL can significantly reduce the degradation rate of CS in electrospun membranes under aqueous conditions. Majority of the scaffold fabrication methods has been widely utilized for generating CS based scaffolds with no control over the scaffold architecture. Rapid prototyping utilizes the computer-aided design data (CAD) to produce 3D scaffolds with precise control over its architecture [63]. Highly reproducible geometries with enhanced mechanical properties are possible with this technique. Chitosan based scaffolds using meltbased routes were also synthesized and investigated by various research groups. Irrespective of the fabrication technologies, the application of CS scaffolds in most of the load bearing sites is often

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Fig. 3. Schematic representation of the commonly used fabrication methods for producing chitosan based scaffolds—freeze drying, freeze gelation, salt leaching, electrospinning and 3D printing.

doubted. Blending CS with various natural, synthetic polymers or ceramic particles have been found to possess enhanced bioactivity and mechanical properties compared to native CS scaffolds. 5. Chitosan scaffold properties Biomaterials offer substantial advantages compared to the conventional metallic implants in terms of cell adhesion, spreading, proliferation, differentiation and other cellular processes. The success of BTE scaffolds depends on its physico-chemical and biological properties of the materials. The scaffolds should satisfy various properties suited for BTE applications. They are (i) porosity, (ii) biocompatibility, (iii) water retention, (iv) protein adsorption, (v) biomineralization, (vi) biodegradability and (vii) mechanical properties. These parameters can be tuned by the addition of other polymers and ceramic to CS for the improvement of scaffolds’ properties and applications in BTE. 5.1. Porosity Porosity provides support for cell infiltration, adhesion, secretion of ECM components and bone tissue in growth [64–66]. Cell attachment to scaffold depends on pore dimensions. If pores are too small, it results in limited cell permeability and capsule formation around the edges of the scaffold. Conversely, if pores are too large, it results in limited specific area and reduces the ligand density available for the cell to bind. Cells can easily recognize subtle changes in the ECM and that may affect cellular behavior [67,68]. This indirectly influences cellular activity through integrin-ligand interaction between cells and biomaterials. Hence, it is crucial in maintaining optimal pore size for cell migration and attachment. Studies reported that pores greater than 20–100 ␮m favor cell infiltration, and beyond 100 ␮m neovascularization is greatly improved [69]. Various reports highlighted that pores greater than 300 ␮m promote direct osteogenesis and pores below 300 ␮m encourage endochondral ossification [70,71]. Despite various reports on pore size and cellular interaction, the rela-

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

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ing cells harboring. Bioglass particles-CS scaffolds formed a new hybrid biocomposite for releasing gentamicin sulfate, an antimicrobial agent. With minimal addition of BG particles to CS matrix, fine pores with large mean diameter were observed. On addition equal amounts of BG particles to CS matrix, pore diameter was found to be decreased; whereas there was no change in the morphology of pores but with less homogeneous surfaces. Addition of twice the amount of BG particles to CS matrix resulted in wider pores and decreased pore volume [82]. Chitosan scaffolds possessed interconnected open pores with pore sizes from 300 to 400 ␮m and it reduced to 250–300 ␮m upon blending with Gn, and addition of nSiO2 particles further decreased the pore size to 200–250 ␮m [83]. Chicken feather derived keratin NPs incorporation into CS scaffolds had no change in the pore dimensions [84]. Inclusion of mesoporous wollastonite (mWs) particles synthesized from rice straw ash into CS-carboxymethyl cellulose (CMC) matrix had no effect on pores morphology [30]. 5.2. Water retention ability Water retention ability refers to the ability of tissue engineering scaffolds to retain water and also known as swelling ability. In vivo implantation of scaffolds results in the adsorption of water from surrounding tissues and results in increased pore size which avails cellular infiltration deep into the internal structures of the scaffolds [85]. Increased swelling leads to the loosening of the implant and retraction away from the site of implantation. Scaffolds with reduced swelling ability decrease cellular interaction. The swelling of polymers depends on the ionizable groups in the structures and the surrounding medium. Chitosan chains swell through protonation of amine/imine groups and lead to the mechanical relaxation of coiled chains [86]. Amine groups of CS chains are the key player of determining the swelling ability. Due to cationic nature of CS, it would have electrostatic interaction with anionic polymers, and most of the amine groups in CS would involve in polymeric complexation, leading to decreased swelling. Chitosan-Gn scaffolds were fabricated and its swelling ratio was significantly reduced through the addition of n-BGC particles [87]. Besides complexation of CS to Gn, the free hydrophilic groups of Gn are vulnerable and decide the swelling upon exposure to fluids. On addition of nBGC particles, these hydrophilic groups were engaged in the interaction. This caused reduction in the number of free hydrophilic groups resulting in reduced swelling ability of the scaffolds. To CS matrix, inclusion of nSiO2 and nZrO2 particles significantly reduced the swelling behavior of matrix [24]. Cross-linking the reactive groups of CS with chemical crosslinkers enhances the stability of the polymeric scaffolds. EDAC triggers carboxylic groups and crosslinks with free primary amine groups in the polymers. TPP interacts with CS amine groups [88]. Chitosan/n␤-TCP scaffolds were crosslinked with genipin, and they were found to be stable by exhibiting a reduced swelling ratio [89]. Hence, it is possible to tailor the swelling rate of CS based scaffolds through addition of other materials at micro or nano scale and crosslinkers. 5.3. Protein adsorption Rapid adsorption of proteins onto the tissue engineering scaffolds is required for subsequent cellular interaction with biomaterials when introduced in a physiological system [90–94]. The adsorbed proteins could influence cell adhesion, spreading and other cellular events. The biological responses mediated by adsorbed proteins are based on their amino acids content. It has been reported that fibronectin derived peptide REDV facilitates endothelial cells adhesion but not fibroblasts or muscle cells [94]. These adsorbed proteins regulate cellular functions via integrin mediated signaling pathways resulting in cell spreading,

proliferation and differentiation [95–98]. Primary integrin binding promotes the clustering of additional adhesive proteins that initiate local cellular cytoskeletal remodeling and cell arrangement [99]. Studies depicted that the presence of surface functional groups (oxygen, nitrogen, carbonyl and hydroxyl) in the biomaterials influence protein adsorption [100,101]. In general, the factors influencing protein adsorption on the surface of implanted biomaterials are (i) surface chemistry-functional groups, (ii) wettabillity and (iii) surface topography. Fibronectin is secreted by osteoblasts, and it is one of the earliest cell-binding proteins which functions as a link between the cells and the implants [102–109]. The amino and carboxyl groups of CS are the key players in protein adsorption. These functional groups on CS impart hydrophilicity and interact with the functional groups of proteins via electrostatic forces, van der waals force and induce protein adsorption onto the surface of CS scaffolds. Chitosan blended with its electrostatically opposite polymers generates increased mechanical strength and controlled swelling but the number of reactive free functional groups may be diminished hence, the protein adsorption is hindered. In order to enhance protein adsorption onto the scaffolds, NPs can be included. Nanoparticles generate more surface area and focal adhesion points for the cells. Addition of nSiO2 to CS/Alg matrix significantly increased the amounts of proteins adsorbed on the matrix [25]. Similarly, addition of nZrO2 increased protein adsorption onto CS based biocomposite scaffolds [24]. It is most likely that addition of biomaterials at nano scale to the scaffolds could promote protein adsorption on their surfaces [84,110–112]. Diopside particles containing extensive silanol ( Si OH) groups also increased adsorption of proteins when complexed with CS, indicating the importance of silanol groups in protein adsorption [86]. Protein adsorption onto the scaffolds is essential for successive cell attachment, spreading and proliferation. Cell proliferation was found to be potentially improved on CS upon its modification with nano-structured carbon [113]. Even though bovine serum albumin (BSA) whose adsorption does not influence any physiological changes, it is likely to improve cell proliferation and to enhance the stretched pseudopodia in promotion of biological function. The actin micro filaments are vital in facilitating movement of cells along the substratum/biomaterial [114]. Thus, pre adsorption of proteins onto the scaffolds is essential for cellular interaction, and modification/addition of NPs into CS matrix would improve its characteristics for protein adsorption and regulation of cell behavior.

5.4. Biomineralization Biomineralization refers to the deposition of ions and minerals on the surfaces of biomaterials on exposure to body fluids. The deposition of CaP on the implants enhances the activity of osteoblasts [115]. Since human contains 65–70% inorganic crystals mostly with HAp, it is a key regulator in determining the bonding of scaffolds to bone [116]. Excellent integration of the scaffolding materials with bone can be achieved if a bone like apatite is formed on the surface of the scaffolds in situ [116–118]. Hence, the biomineralization potential of the scaffolding materials is tested on their ability to form CaP crystals when exposed to simulated body fluid (SBF). Chitosan can act as a template for biomineralization due to the presence of NH2 and C O functional groups. The CS based biocomposite scaffolds were found to have enhanced biomineralization potential compared to CS alone. Addition of nZrO2 to CS/nSiO2 matrix improved biomineralization as earlier at 7d [24]. Inclusion of nBGC particles to CS/Gn scaffolds enhanced the number of nucleation sites, resulting in efficient apatite formation [87]. Thus, incorporation of NPs tends to increase surface area thereby providing more nucleation sites for apatite formation.

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

Chitosan + ceramic or others

Properties observed

References

Alginate

Nano silicon dioxide

• Improved protein adsorption, controlled swelling • Improved apatite deposition

[25]

Carboxymethyl cellulose

Mesoporous wollastonite

• • • •

[30]

−

Hydoxyapatite and ␤-TCP

• Enhanced tensile properties • Controlled water retention and maintained structural stability

[72]

Silk fibroin

Hydroxyapatite

• Reduced the porosity • Supported growth of SaoS-2 cells and increased viability • Enhanced ALP activity

[79]

Silk fibroin

Nano zirconium oxide

• Increased water uptake ability and compressive strength by controlling the porosity

[80]

−

Bioglass

• Controlled pore morphology upon varying the amounts of bioglass • Better release kinetics of gentamicin sulfate

[82]

Gelatin

Nano silicon dioxide

• • • •

[83]

−

Keratin nanoparticles ␤-TCP

• Increased protein adsorption and improved biodegradation

[84]

• Improved compressive strength by 70% • Increased the swelling and biomineralization

[89]

Gelatin

Reduced swelling Decreased susceptibility to lysosome Increased biomineralization Up regulated pre-mir-15b

Increased the swelling ability Decreased degradation rate Improved protein adsorption and biomineralization Enhanced cell attachment and ALP activity

−

Bioglass and carbon nanotube

• Increased mechanical properties and compressive strength • Promoted attachment and proliferation of MG-63 cells

[138]

−

␤-TCP

• TCP addition decreased the degradation of scaffolds

[140]

−

Simvastatin loaded PLGA mircoparticles Hydroxyapatite

• Reduced swelling, increased compressive modulus • Enhanced cell proliferation of hFOB and improved differentiation

[141]

• Controlled and uniform porosity • Increased compressive strength and elastic modulus • Promoted differentiation and mineralization of MC3T3-E1 cells

[142]

• Promoted vascularisation in vivo in rats

[143]

• Increased compressive modulus

[144]

• Increased mechanical strength • Reduced swelling behaviour

[145]

Alginate

Collagen

− −

VEGF loaded PLGA-PEG microspheres and hASCs Nano bioactive glass PLGA nanoparticles and bioactive glass

Fucoidan

␤-TCP

• Improved compression strength • Enhanced apatite deposition • Increased osteocalcin secretion by hMSCs

[146]

Poly(propylene carbonate)

−

• Enhanced hydrophilicity favouring fibroblast attachment and proliferation • 26% increased in Young’s modulus

[147]

Mimosa tenuiflora cortex

−

• Improved cell viability, proliferation and differentiation and ALP expression in rat cavarial cells

[148]

Poly-3-hydroxybutyrate-co-3hydroxyvalerate (PHBV)

Hydroxyapatite

• High ultimate tensile strength upon varying HA addition • Higher ALP activity in human fetal osteoblasts • Chelated apatite crystals and increased biomineralization

[149]

Polyvinyl pyrroidone

45S5 bioglass

• Genipin crosslinked decreased degradation • Supported cell growth by controlling the dissolution of bioglass • Improved cell attachment with extended cytoplasmic processes with polygonal morphology

[150]

Alginate

In situ synthesised hydroxyapatite

• Highly interconnected porosity with thicker pore walls

[151]

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Table 1 (Continued) Chitosan + polymers

Chitosan + ceramic or others

Properties observed

References

Gelatin

HA, montmorillonite

• Decreased degradation rate • Increased apatite deposition and swelling property

[152]

−

Multiwalled carbon nanotube

• • • •

[153]

Chondroitin sulphate and amylopectin

Hydroxyapatite

• Increased cell proliferation, ALP activity and collagen-1 expression in MG-63 cells

[154]

Chitin

Nano zirconium oxide

• Controlled swelling and degradation • Enhanced osteogenesis

[155]

Alginate and polypyrrole

−

• Controlled swelling • Improved cell viability and cell attachment • Biomineralization in SBF and under culture conditions were enhanced

[156]

Silk fibroin

−

• Decreased degradation rate and possessed antibacterial activity • Supported adhesion and growth of fibroblasts

[157]

Poly(vinyl alcohol), collagen

Bioglass particles

• • • •

[158]

Gelatin

Calcium phosphate

• Increased material degradation • Improved cell attachment

[159]

−

RGD

• Increased pore size and mechanical properties • Promoted cell attachment and proliferation • Increased calcium deposition

[160]

–

• Promoted bone healing upon magnetic stimulation

[161]

␤-1,3-glucan

HA and iron nanoparticles HA

• Favoured cell adhesion, spreading and proliferation and up regulated the ALP, OC, Col-I levels

[162]

Polycaprolactone

−

• Improved bioactivity, protein adsorption and adhesion of osteoblasts

[163]

• Tuned porosities

[164]

PLGA nanocapsules loaded with BMP-2 and PHBV loaded with BMP-7 poly(lactic acid–glycolic acid) microspheres

• Supported MSC attachment and spreading • Sequentially delivered growth factors • Enhanced differentiation and increased ALP activity

[165]

• • • •

[166]

Collagen alginate

HA

• Exhibited higher cell proliferation and mineralization

[167]

Alginate

Chondroitin 4-sulfate

• Increased compressive modulus and enhanced apatite formation • Promoted cell spreading, proliferation and osteogenic differentiation

[168]

−

Bioactive glass nanoparticles

• Exhibited shape memory properties • Induced apatite formation

[169]

Poly(l-lactic acid)

Promoted water uptake ability Controlled the porosity Enhanced cell proliferation, protein content and ALP activity Mineralization was higher

Controlled porosity, swelling and degradation High compressive modulus Increased ALP activity Controlled release of protein (BSA)

Controlled porosity Matched the compressive modulus and strength of trabecular bone. Increased ALP activity ALP, OPN, BSP, and OCN levels were up regulated

Increasing the number of reactive groups in the scaffolds would generally enhance the formation of bone like apatite with a Ca/P ratio of 1.6 close to human bone. The anionic groups such as COO , OH and NH2 can act as nucleation sites for the formation of crystalline HAp particles [119]. Chitosan scaffolds exhibited only marginal biomineralization; whereas addition of diopside particles enhanced apatite deposition [86]. It was suggested that the involvement of silanol groups in diopside particles could have been contributed to increased apatite formation. Further, the release of calcium from diopside particles also enhanced apatite formation [86]. Similarly, an increase in biomineralization potential of CS/Alg

scaffolds was achieved upon addition of nSiO2 particles [25,120]. As mentioned earlier, the biomineralized layer is essential for direct bonding of the scaffolds to host bone tissue and this would indirectly determine the success of the implanted scaffolds. 5.5. Biodegradation Biodegradation refers to the chemical process of gradual breakdown of the implanted biomaterials in a biological system [121]. It is initiated on exposure of the scaffolds to tissue fluids containing various enzymes and other active substances, whose action tightly

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Table 2 In vivo studies on chitosan based biocomposite scaffolds. Chitosan + polymers or other ceramic nanoparticles

Bioactive molecules

Animal model

Defect site

References

Diopside Alginate and hydroxyapatite Alginate Hydroxyapatite Alginate and PLA-H Nanohydroxyapatite, collagen, PLLA Nanohydroxyapatite Collagen Collagen, PLGA Collagen −

− − BMP-2 − VEGF BMP-2 − rhBMP-2 rhBMP-2 − −

Rat Severe combined immuno-deficient mice Rat Rat Rat Rabbit Rabbit Rabbit Dog Miniature pigs Rat

Thigh muscle Calvaria Calvaria Tibia Femur Femoral condyle Femoral condyle Radius Mandible Femur Muscle packets

[86] [151] [170] [171] [172] [173] [174] [175] [176] [177] [178]

is regulated under physiological conditions. The implanted graft materials should undergo degradation over time and should match with the formation of new bone. Biodegradation is an important criterion to be considered prior to the design of any BTE scaffolds for long-term success [122,123]. There are few biomaterials which are degraded in a non-regulated way on their exposure to water or serum [124]. The biodegradation involves the scission of chemical bonds between the monomeric units of biopolymers, between two polymers or between the polymer and ceramic/NPs added into the system. Erosion is another phenomenon through which degradation occurs. Water soluble polymers based scaffolds follow erosion and the scaffolds are degraded inside the biological system through the absorption of water. The resulting degraded products should be non-toxic and should not prove any immune response. They should be small so as to dissolve in body fluids, excreted or incorporated into metabolic pathways [125–127]. Chitosan is mainly degraded by the action of lysosyme [128,129]. Higher degree of deacetylation of CS leads to faster degradation by lysosyme [55]. The NAGs of CS are hydrolyzed by the action of lysosyme and the degraded products are amino sugars which are incorporated into GAG and glycoprotein metabolic pathways and excreted. The magnitude of tissue response to the implanted biodegradable material is dependent upon the site of implantation [130]. The faster degradation rates of CS scaffolds often limit their long term persistence in vivo. Addition of other polymers, NPs to CS matrix may have effect in controlling degradation kinetics. Diopside incorporation into CS scaffolds had no effect on the percentage of degradation even after 72 h [86]. It has been reported that the rate of degradation of CS/nHAp scaffolds was higher but in the presence of nano silver (nAg), the degradation was significantly lowered. Due to the Ag ion complexes with the amino and hydroxyl groups of CS, there might be only few free reactive functional groups resulting in decreased hydration upon fluid exposure and thereby the decreased rate of degradation [34]. Addition of nBGC particles to CS/Gn scaffolds significantly reduced their degradation and that could be due to neutralization of the acidic degraded products of CS because the persistence of acidic condition would cause dissolution of CS [87]. The degradation rate can be controlled with the availability of free functional groups, complexation with electrostatically opposite polymers and inclusion of crosslinkers such as TPP, glutaraldehyde and EDAC [128]. The rate of degradation was found to be reduced in glutaraldehyde crosslinked CS/Gn biocomposite scaffolds compared to uncrosslinked scaffolds of the same composite [131]. Over all, the tailored degradation of CS based biocomposite scaffolds is often required to tune with the rate of bone formation.

5.6. Mechanical properties The bone tissue engineering scaffolds should possess appropriate mechanical properties for their application in load bearing

areas. The mechanical properties of the scaffolds should match the host bone for proper load transfer to the adjacent tissues. The mechanical properties of natural bone vary depending upon the type such as cortical or cancellous bone. The cortical bone possesses Young’s modulus and compressive strength of 15–20 GPa and 100–200 MPa while, the trabecular bone possesses between a range of 0.1–2 GPa and 2–20 MPa, respectively [132]. Studies reported the presence of a strong relationship between scaffold stiffness and cell behavior. It influences the differentiation of mesenchymal stem cells to specific cell types [133–136]. Chitosan has low to moderate mechanical properties which limits its use in load bearing applications [137]. However, addition of various polymers or NPs increased the mechanical properties [25,137]. Such reinforcement with opposite charged polymers or other addition changes the degradation rate, scaffold chemistry and improves the mechanical properties. Addition of Gn and ␤-TCP increased the compressive strength by approximately 70% [89]. Another report stated that addition of CNT to CS/BG scaffolds increased the compressive strength up to 5.95 ± 0.5 MPa [138]. As described earlier, the addition of negatively charged polymer improves the mechanical strength, in accordance to that, addition of Gn, a negatively charged polymer forms polyelectrolyte complex due to ionic interaction between these two molecules and improves the mechanical strength [139]. In addition to improving the mechanical properties, inclusion of other polymers, NPs or ceramics also determines the stiffness of the CS scaffolds. Stiffness of such implanted scaffolds limits its use in load bearing sites. Mechanical tests indicated that addition of ceramics (␤-TCP) affects the stiffness of materials. Addition of TCP at 50% concentration showed young modulus values about 0.5 MPa; when TCP added at 15% concentration to CS sponges exhibited about 0.02 MPa. Thus, the compression strength of the foams was found to be higher with increased TCP concentrations [140]. The possible reason behind such increase in compressive strength could due to the decreased porosity upon the addition of TCP particles. Scaffolds on implantation in vivo would absorb fluids and the mechanical strength would vary on dry and wet conditions. Chitosan-Gn membranes upon testing for tensile properties in dry condition showed significant increase in break stress on increasing Gn content. Conversely, the increased Gn content decreased break strain under wet conditions. In dry condition, increased stiffness was observed with more Gn addition but in wet conditions, the presence of Gn decreased the stiffness of CS-Gn membranes [111]. Although many reports stated that the mechanical properties of CS based scaffolds can be tuned, no clear reasons were concluded for identifying an optimal addition to match the properties with host bone. Hence, such additions have to be considered and tuned for matching the mechanical properties of CS based scaffolds for BTE. A literature analysis for the availability CS based biocomposite scaffolds with enhanced properties suitable for BTE is shown in

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

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Acknowledgement [29]

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

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