Chitin (Figure 1) is the second most abundant natural polymer after cellulose. CTS is produced from chitin, which is a natural polysaccharide found in crab, shrimp, lobster, coral, jellyfish, butterfly, ladybug, mushroom and fungi. However, marine crustacean shells are widely used as primary sources for the production of CTS [8,9]. Crab and shrimp are important marine species of great commercial importance in the tropical and subtropical waters of the Pacific, Atlantic and Indian oceans. The waste from crab and shrimp processing has recently become a serious issue in coastal areas. Selective isolation of bioactive material from these wastes is the simplest way to decrease the pollution. It not only reduces the environmental pollution because of the disposal of this under utilized by products of crabs and shrimps, but also increases the potential applications of CTS. Moreover, the chemical hydrolysis and enzymatic methods, widely used for the isolation for CTS from marine crustaceans shell, are quite inexpensive.

In chemical hydrolysis method, four main steps are involved in order to produce CTS from marine crustacean shell as depicted in Figure 2. They are (i) demineralization; (ii) deproteinization; (iii) discoloration and (iv) deacetylation. To produce 1 kg of 70% deacetylated CTS from shrimp shells, 6.3 kg of HCl and 1.8 kg of NaOH are required [8,9].

Chitosan can be isolated directly from the cell walls of certain fungi, but commercially available CTS are usually prepared from chitin. CTS can be produced via traditional chemical method, however, problems exist related to poor quality and environmental chemical pollution [10]. The enzymatic method could provide an alternative to the current chemical production method. The degree of deacetylation and molecular weight of the CTS depends on the source and preparation method (molecular weight ranges from 300 to over 1,000 kDa, degree of deacetylation from 30% to 95%). CTS can be degraded by enzyme and form various small molecular weight fragments. Chitosanase has been used for the preparation of small molecular weight CTS with various methods such as batch reactor, column reactor and ultrafiltration membrane reactor [11]. The CTS has been extracted with various enzymes like lysozyme, snailase, neutral protease and novel chitin deacetylase from Scopulariopsis brevicaulis. The average molecular weight of CTS obtained using the enzymatic method (267.97 kDa) has been reported to be far greater than that obtained by chemical method (84.04 kDa) [10,12].

CTS is a copolymer consisting of β-(1→4)-2-acetamido-d-glucose and β-(1→4)-2-amino-d-glucose unit linkages (Figure 3) [13,14]. Over the past two decades, CTS has been developed considerably in biomedical applications due to its high biocompatibility, biodegradability, porous structure, suitability for cell ingrowth, osteoconduction and intrinsic antibacterial nature [15], CTS offers a wide range of applications, including cartilage tissue engineering [16], wound healing [17] and orthopedic applications [15]. Degradable polymeric implants eliminate the need for a second surgical operation and can prevent some of the problems associated with stress shielding during post-healing, and can also be used simultaneously to deliver therapeutic drugs to treat infections or growth factors to accelerate new bone growth [18]. There is a growing interest in exploiting the field of bone tissue engineering for composite preparation. This has created a wide range of application in the preparation of artificial organs.

CTS can be easily modified into various forms like films, fibers, beads, sponges, and more complex shapes for orthopedic treatment [15]. The cationic nature of CTS is responsible for attracting various negative charged proteoglycans. Porous materials have a highly significant role in the bone implantation process. Porous CTS structures can be formed by freezing and lyophilizing CTS acetic acid solutions in suitable molds [19,20]. CTS have been combined with a variety of materials such as HAp, alginate, hyaluronic acid, calcium phosphate, poly (methyl methacrylate), poly-l-lactic acid and growth factors for potential application in orthopedics.

Numerous methods have been used to prepare CTS/HAp composites, especially for nanocomposite preparation, which play an excellent role in the extra-space arrangement in the matrix and have the best biomedical properties and biomaterial applications [22]. CTS/HAp nanocomposite with a homogeneous microstructure has been prepared using in situ synthesis [33], precipitation method [34], in situ co-precipitation synthesis with an electrospinning process [21,23,30,35,36], simple in situ hybridization [18], solvent casting and evaporation method [24], in situ chemical method [25,37], freezing and lyophilization [26,27], combined sintering and freeze-drying technique [28], self-assembly of static electricity [38], simple mixing and heating method [29], biomimetic method [39–41], low temperature wet chemical method [42], thermally induced phase separation technique [43], dual membrane diffusion system [44], electrochemical deposition [45], electrochemistry assisted deposition [46,47], electrophoretic deposition [48,49], natural HAp mixed with CTS [50], double diffusion technique [51] and wet spinning method [52].

The mechanical properties of the CTS/HAp composites play a significant role in bone tissue engineering. The intermolecular hydrogen bond and chelate interaction between the CTS and HAp contribute to good mechanical properties. There is a possible interaction between the NH₂ group and primary and secondary –OH group of CTS with Ca²⁺ (metal coordination interaction) of HAp. The possible interaction between CTS and HAp is discussed in a later section. This interaction might be responsible for the higher mechanical strength of the composite scaffolds as compared to CTS and HAp alone. Compressive strength has been a widely used parameter to find out the mechanical strength of porous scaffolds. Li et al. compared the compressive strengths of different composite ratio of CTS/HAp and found the maximum compressive strength to be 119.86 MPa (30:70 CTS/HAp ratio). Increasing the HAp ratio leads to an increase in the compressive strength [53]. The incorporation of CTS into HAp matrix via blending methods would result in the decrease of mechanical properties of composite material due to the weaker interfacial bonding between CTS and HAp matrix and as a result, the compressive strength reduces to 47.8 MPa [54]. The molecular weights of CTS also contributes significantly to the mechanical properties. In general, the high molecular weight CTS scaffolds have higher compression modulus than medium molecular weight CTS. The compression moduli of high molecular CTS and CTS/nHAp (1%) nanocomposite scaffold were found to be 6.0 ± 0.3 kPa and 9.2 ± 0.2 kPa, respectively [27]. The mechanical properties of CTS composite scaffold also depend on the temperature and it increases with an increase in the temperature. A possible rationale to this may be that with an increase in the temperature, the interfacial bonding between CTS and HAp increases [30]. Water content of the scaffold may also have a major role to play in the mechanical strength. In a study, it was found that the nHAp/CTS/carboxymethyl cellulose (40/30/30% w/v) has highest mechanical property of 40 MPa in dry and 12 MPa in wet state [38].

CTS have been widely used in orthopedic treatment since it was shown to promote osteoblastic cell growth. When CTS is incorporated with HAp, it remarkably increases the osteoblastic cell growth on the scaffolds [15,21,24,25,27]. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method has been frequently used to determine the cytotoxicity of CTS composite materials. All the scaffolds’ results showed that the CTS/HAp composite had no cytotoxicity with positive cell attachment and proliferation growth of the osteoblast cells [15,23–25,50,51]. The composite scaffolds possess a pore size of 100–200 μm, providing a spatial arrangement of cells (10–30 μm) and thus cells are able to migrate towards the composite [55]. It was observed that when the osteoblast cells were cultured in the medium of phosphorylated CTS/HAp, the cell morphology changed within 30 min of seeding and later became triangular at 24 h, polygonal at 48 h, and finally, aggregated to be indistinct at 5 days [56]. Apart from HAp, other calcium phosphate minerals also provide adhesion and cell proliferation when combined with CTS. The cell proliferation and adhesion has been found with osteoblast mouse cells MC3T3-E1 and L929 cells in CTS/calcium phosphate specimens [28,57].

The alkaline phosphatase activity (ALP) is considered to be an important marker of the differentiation of osteoblast cells at a relatively early bone forming stage and has been widely used to evaluate ALP for scaffold materials. The CTS/HAp composite scaffolds possess higher ALP activity compared to the CTS scaffold whereas the highest ALP activity has been achieved in the composite containing 30–40% of HAp with good cell proliferation; however, cell proliferation decreases with an increase in the HAp concentration [21,51,58]. The modified CTS and its composites are found to have good cell proliferation and higher ALP activity then compared to non modified CTS. CTS glutamate and HAp containing cultured osteoblasts were found to be promising biomaterials for repair bone defects in vivo [56,59].

Ca²⁺ ions appear on the terminated surface of HAp crystals, which have coordination number of seven and are strictly held in the structure (Figure 4). Therefore, there is a possibility to form coordination bonds between the -NH₂ of CTS and Ca²⁺ of HAp [24,60].

One of the major problems in the preparation of CTS with CNT materials is the difficulty to disperse the CNTs well in the polymer matrix. Several methods have been used for this purpose such as controlled surface deposition and cross linking process [66], simple solution evaporation method [63], thermally induced phase separation followed by freeze drying [67], electrodeposition [68], spray layer by layer technique [69], pH and electrical actuation [70] and wet spinning method [71].

Wang et al. reported that CNT is homogeneously dispersed throughout the CTS matrix. When 0.8% of CNT was introduced in the CTS matrix, the mechanical properties, including the tensile modulus and strength of the nanocomposite were greatly improved by about 93% and 99% [63]. A small addition of CNTs significantly improves the tensile properties of CTS matrix, and the mechanical properties increase with the increase of CNTs’ loading (0.4 wt% of CNTs filler) whereas the tensile modulus and strength of the nanocomposite increases dramatically by about 78% and 94%. Due to the aggregation of CNTs within the CTS matrix at higher concentrations, with further increases of the loading level of CNTs, the tensile modulus only increases slightly, while the tensile strength remains stable. For instance, with 2.0 wt% of MWNTs filler, the tensile modulus of the CTS matrix is 2.15 GPa, only 0.07 GPa higher than that of 0.8 wt% of MWNTs. The tensile test results indicate the mechanical properties of the CTS/CNTs [63].

In another study, the tenacity of neat CTS fiber was recorded as 96 MPa, whereas that of CTS reinforced with purified but non-functionalized single-walled CNTs (p-SWCNTs) increased from 132 MPa for 0.01 wt% SWCNT addition to 180 MPa for 0.4 wt% SWCNT addition. Even at very low SWCNT concentration (0.01 wt%), a significant increase in tenacity was observed. On the other hand, on incorporation of functionalized CNT (f-CNT), the tenacity increased from 136 MPa for 0.01 wt% SWCNT addition to 226 MPa for 0.4 wt% addition. This continuous improvement in tenacity (even at higher SWCNT concentrations) may be ascribed to better dispersion as well as interaction of functionalized nanotubes in CTS [70]. This might be because of the strong chemical interaction as functionalized CNT’s COOH group interacts with amine group of CTS matrix.

CNTs are like an inert matrix material in which cells can proliferate in easily. Zanello et al. suggested that CNTs can be used as an alternative material for the treatment of bone pathologies with the potential for the regrowth of normal bone. Osteoblast-like cells were grown on electrically neutral CNTs and they were also found to produce mineralized bone [72]. The impurities in commercial SWCNTs, such as metal catalysts and amorphous carbon particles, are reported to be toxic to cells and could induce intracellular reactive oxygen species. However, the addition of CTS with amine functional groups should decrease the cytotoxicity. Compared to p-SWCNTs, f-SWCNT grafted with CTS provides better cell proliferation and cell attachment. An explanation to this may be that the cellular membranes are negatively charged, and thus cells can more easily attach and grow on more positively charged surfaces [73].

Wörle-Knirsch et al. suggested that the MTT method is not reliable for finding out the cytotoxity of CNT based materials, as the MTT formazan crystals formed in the MTT reaction are lumped. As a consequence, false results may show a strong cytotoxic effect in the MTT assay after 24 h and show that roughly 50% of the cells die. But other methods, such as WST-1, lactate dehydrogenase, fluorescence-activated cell sorting assisted mitochondrial membrane potential determination and annexin-V/PI staining revealed no cytotoxicity [74]. Very little work has been done with CTS/CNT composite materials for bone tissue engineering. CNT can mimic the strength of natural bone in composite or scaffold materials. Research strategies still need to be developed in the field of CTS/CNT composite material preparation and in vitro as well as in vivo activity of CNT based polymeric materials.

The raw CNTs are mainly hydrophobic and poorly miscible with water. The acid treated CNTs contain many defects and hydrophilic groups, such as -OH and -COOH, which are very helpful for improving the solubility of CNTs in water. CTS, a hydrophilic biopolymer, possesses three kinds of functional groups viz. amino, primary, and secondary hydroxyl groups in a glucosamine unit, and the functionalized CNTs contain carboxylic and hydroxyl groups. There is great possibility that strong hydrogen bonds may form between chitosan and the CNTs (Figure 5). The compatibility and strong interaction between MWCNTs fillers and the matrix greatly enhances the dispersion as well as the interfacial adhesion, thus significantly increasing the mechanical properties of the matrix [63]. The covalent functionalisation of CTS and CNT improves the interaction between CTS with CNT, reduces the damage of CNT and increases its mechanical properties.