**9. Chitin, chitosan and their derivatives**

**Figure 4.** Model of pathogenesis of heparin-induced thrombocytopenia (HIT). Heparin binds with Platelet factor 4 (PF4), which exposes neoepitopes on PF4 and leads to antibody production (1). Heparin-PF4-IgG immune complexes form (2), and IgG in multimolecular complex triggers platelet activation via binding to Fc receptors (3). Activated pla‐ telet releases additional PF4 (4a) and prothrombotic platelet microparticles (4b). Thrombotic risk is further promoted by binding of PF4 to heparin-like molecules on endothelial cells (EC), contributing to immune system–mediated endo‐

thelial damage (5) [112].

122 Advances in Biomaterials Science and Biomedical Applications

Chitosan is a naturally occurring linear polysaccharide, consisting of glucosamine and Nacetyl-glucosamine, normally made of deacetylated chitin which is the structural poly‐ mer found in the shells of crabs and shrimp (lobster, squid, some yeast and mould), by N-deacetylation using strong alkali [120]. More than 40 years have lapsed since this bio‐ material had aroused the interest of the scientific community around the world for its potential biomedical applications [121]. Until now chitosan possess a number of commer‐ cial and biomedical applications in wound dressing, drug delivery and tissue engineer‐ ing. For example, chitosan based scaffold biomaterials have demonstrated versatile properties to promote the epithelial and soft tissue regeneration in the body [122,123]. Chitosan patches in various sizes that have been cleared by the FDA are a topical hemo‐ stat for moderating severe bleeding. Nevertheless, an obvious disadvantage of this im‐ plantable, absorbable biomaterial is that chitosan initiates serious host inflammation reactions (Figure 5) [13,124]. Additionally, chitosan is bioadhesive and has the ability to transiently open tight junctions in the nasal epithelia, thereby permitting drugs to dif‐ fuse through this barrier. Advantages of this nasal route of administration include: a higher permeability of the nasal mucosa than in the gastrointestinal tract; a low degree of pre-systemic metabolism; and a high level of patient compliance, compared to injecta‐ ble systems [125].

It is very interesting that when the number of N-acetyl-glucosamine units in a chitin/ chitosan mixture is higher than 50%, the biopolymer is termed chitin. 50% deacetylated chitosan has a less inflammation reaction than the others when they are implanted *in vivo* [126]. Cross-linking of chitosan membrane using genipin and some other chemical agents can increase the membrane's ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio [127]. In the author's own group, an ammonia treat‐ ed chitosan sponge was implanted subcutaneously in rats for 8 weeks (Figure 5). One week after implantation, the chitosan sponges were entirely retained and wrapped with a layer of purulent cells. The purulent cells had infiltrated the outside chitosan sponges (Figure 5A). Two weeks after implantation, the encapsulated purulent layer was en‐ larged at the periphery of chitosan sponges. More acute inflammatory cells had infiltrat‐ ed the chitosan sponges and there was no sign of biodegradation of the chitosan sponges (Figure 5B). Four weeks after implantation, the chitosan sponges still main‐ tained their porous structure. A much thicker purulent layer and more acute inflamma‐ tory cells were found around or in the chitosan sponges (Figure 5C). Six weeks after implantation, most of the chitosan still maintained their scaffold integrity with numerous interspersed purulent cells. Some purulent cells even formed large channels throughout the chitosan sponges (Figure 5D). Eight weeks after implantation, purulent cell infiltra‐ tions had further increased in the chitosan sponges. Some collapsed matrix structures were detected at the outer margins of the implants and more channel structures were found between the remnants of chitosan lamellae (Figure 5E).

**Figure 5.** Light-microscope evaluation of the tissue response to chitosan sponges with HE staining: (A) 1 week after implantation; (B) 2 weeks after implantation; (C) 4 weeks after implantation; (D) 6 weeks after implantation; (E) 8 weeks after implantation. The scale bar indicates a distance of 50 μm in (A), (C), and (D), and a 25 μm in (B) and (E) [13].

Also in this author's own group, a series of bone repair materials were fabricated by adding three chitosan derivatives, such as phosphorylated chitin (P-chitin), phosphorylated chito‐ san (P-chitosan), and disodium (1→4)-2-deoxy-2-sulfoamino-β-D-glucopyranuronan (S-chi‐ tosan) into two kinds of biodegradable calcium phosphate cements (CPCs). All the chitosan derivatives can greatly improve the mechanical properties and reduce the biodegradation rates of the CPCs. At least six totally different tissue responses were detected when the im‐ plants were examined in tibial and radial defects of rabbits. Large bone defect (9 mm in length for radii and 3 mm in depth and diameter for tibias) repair in rabbits with the P-chi‐ tosan incorporated CPCs exhibits excellent tissue compatibilities with no any adverse or negative effects, such as fibrous encapsulation, osteolysis, hyperplasia, and inflammation, no matter the concentrations of P-chitosan is high or low (Figure 6) [128,129]. Tissue re‐ sponses to P-chitin are highly sensitive (Figure 7) [130,131]. Three different bone formation types in the resorption lacuna of the P-chitin incorporated CPCs due to the P-chitin concen‐ trations were found during the 22 weeks implantation. The first is that with low P-chitin content trabeculae formed directly from the implant (Figure 7A). The second is that with middle P-chitin content cartilages formed from the outside of fibers before they turned into trabeculae (Figure 7B, 7C). The third is that with high P-chitin content callous formed from the outside of fibers before they turned into trabeculae (Figure 7D, 7E). P-chitin content has a negative relationship with the biodegradation rate of the cements. However, the degrada‐ tion rates are compatible with the ingrowth of trabeculae. A mild foreign-body reaction in the high P-chitin content sample during the first three time spans did not impair its place‐ ment by a newly formed bone. The generally properties of these biomaterials have met the main requirements for bone repair (Figure 7) [130,131]. Different from the above mentioned bone repair types, tissue responses to water-soluble S-chitosan, prepared from chitin by suc‐ cessive N-deacetylation, specific carboxylation at C-6 and sulfonation, was rather obtuse. No inflammation or other negative response was found in the S-chitosan containing samples (S-CPCs). After 4 weeks implantation, newly formed trabeculae contacted with the implant di‐ rectly in the lower S-chitosan sample, while a thin layer of fibers formed between the newly formed bone and the implant in the higher S-chitosan samples [132,133]. These results indi‐ cate that the concentrations and functional groups in a linear polysaccharide play a key role in determining the ultimate biocompatibilities of an implantable biomaterial. In addition, as a derivative of chitin, chitosan initiates blood coagulation while S-chitosan inhibits blood co‐ agulation when they are used as hemo-contact biomaterials.

Recently, chitosan and its derivatives have been widely used in skin wound, burn and dis‐ ease treatments. For instance, a chitosan-gelatin-hyaluronic acid scaffold was found flexible with good mechanical properties when it was used as artificial skin substitutes [134]. A bac‐ terial cellulose synthesized by Acetobacter xylinum and modified by chitosan was found to be optimal in providing wound dresses with a moist environment for wound healing [135]. When an artificial chitosan skin regenerating template was implanted subcutaneously it showed a similar inflammatory pattern as Integra, a two-layer skin regeneration system, constructed of a matrix of crosslinked fibers [136,137].

**Figure 5.** Light-microscope evaluation of the tissue response to chitosan sponges with HE staining: (A) 1 week after implantation; (B) 2 weeks after implantation; (C) 4 weeks after implantation; (D) 6 weeks after implantation; (E) 8 weeks after implantation. The scale bar indicates a distance of 50 μm in (A), (C), and (D), and a 25 μm in (B) and (E)

Also in this author's own group, a series of bone repair materials were fabricated by adding three chitosan derivatives, such as phosphorylated chitin (P-chitin), phosphorylated chito‐ san (P-chitosan), and disodium (1→4)-2-deoxy-2-sulfoamino-β-D-glucopyranuronan (S-chi‐ tosan) into two kinds of biodegradable calcium phosphate cements (CPCs). All the chitosan derivatives can greatly improve the mechanical properties and reduce the biodegradation rates of the CPCs. At least six totally different tissue responses were detected when the im‐ plants were examined in tibial and radial defects of rabbits. Large bone defect (9 mm in

[13].

124 Advances in Biomaterials Science and Biomedical Applications

With the combination with other natural polymers, such as collagen, gelatin, hyaluronan, fi‐ brin, the strong host inflammation reactions of chitosan can be reduced to a certain degree. It was found that a bioactive glass-chitosan composite containing 17% (wt%) chitosan pro‐ duced by a freeze-drying process and implanted in the femoral condyl of an ovariectomised rat can promote a highly significant bioactive and osteoinductive property [138-140]. The ul‐ timate biocompatibility of a chitosan compound depends largely on the ratio of the different components. Host tissues, such as smooth muscle and hepatic tissue have a similar response to the chitosan containing collagen/chitosan mixtures [141]. A collagen/chitosan matrix crosslinked by agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in a N-hydroxysucci‐ nimide and 2-morpholinoethane sulfonic acid buffer system has exhibited improved blood and cell compatibilities than the pure chitosan samples [142,143].

**Figure 6.** Tissue responses to the P-chitosan incorporated CPC specimen at different time points with MT staining. (A) 1 week after implantation in the high P-chitosan content (0.12 g/mL) sample with very little hematoma. (B) 4 weeks after implantation in the high P-chitosan content (0.12 g/mL) sample newly formed woven bone clearly appeared with tightly bonding between the implant and host bone. No macrophage was found around the implant. The im‐ plant was directly changed into new trabeculae after degradation. (C) 12 weeks after implantation newly formed long bone in the low P-chitosan content (0.02 g/mL) sample. (D) 12 weeks after implantation newly formed long bone in the middle P-chitosan content (0.07 g/mL) sample. (E) 12 weeks after implantation newly formed long bone in the high P-chitosan content (0.12 g/mL) sample. Trabeculae formed after the implant was gobbled up (infiltrated) by body fluid. Clear evidence of remodeling around the implant surface was displayed. (F) 22 weeks after implantation the newly formed dense trabeculae in the high P-chitosan content (0.12 g/mL) sample [129].

crosslinked by agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in a N-hydroxysucci‐ nimide and 2-morpholinoethane sulfonic acid buffer system has exhibited improved blood

**Figure 6.** Tissue responses to the P-chitosan incorporated CPC specimen at different time points with MT staining. (A) 1 week after implantation in the high P-chitosan content (0.12 g/mL) sample with very little hematoma. (B) 4 weeks after implantation in the high P-chitosan content (0.12 g/mL) sample newly formed woven bone clearly appeared with tightly bonding between the implant and host bone. No macrophage was found around the implant. The im‐ plant was directly changed into new trabeculae after degradation. (C) 12 weeks after implantation newly formed long bone in the low P-chitosan content (0.02 g/mL) sample. (D) 12 weeks after implantation newly formed long bone in the middle P-chitosan content (0.07 g/mL) sample. (E) 12 weeks after implantation newly formed long bone in the high P-chitosan content (0.12 g/mL) sample. Trabeculae formed after the implant was gobbled up (infiltrated) by body fluid. Clear evidence of remodeling around the implant surface was displayed. (F) 22 weeks after implantation

the newly formed dense trabeculae in the high P-chitosan content (0.12 g/mL) sample [129].

and cell compatibilities than the pure chitosan samples [142,143].

126 Advances in Biomaterials Science and Biomedical Applications

**Figure 7.** Tissue responses to the P-chitin incorporated CPC specimen 4 weeks after implantation. (A) P-chitin: 0.02 g/mL with MT staining. Magnification ×100. (B) P-chitin: 0.08 g/mL with MT staining. Magnification ×40. (C) A magni‐ fication of (B) with MT staining. Magnification ×400. (D) P-chitin: 0.14 g/mL with MT staining. Magnification ×40. (E) A magnification of (D) with HE staining. Magnification ×400 [131].

Current advances in some drug delivery systems make it possible to improve the therapeu‐ tic efficacy and minimized the side effects associated with toxicity of the drug. Chitosan has shown promise in the development of non-parenteral delivery systems for challenging drugs. For example, a 5-Fluorouracil (5-FU) loaded scaffold composed of chitosan fibers were prepared by a modified wet spinning technique [144]. Thermosensitive hydrogel com‐ posed of chitosan and glycerophosphate is proposed to be the potential candidate of *in situ* gel-forming implant for long-term drug delivery [145]. However, unpredictable body re‐ sponses to the chitosan systems as stated above can complicate their applications to some degree. The composite chitosan-collagen-soybean phosphatidylcholine film impregnated with MMC-PLA-nanoparticles for treatment of hepatocellular carcinoma in mice has exhib‐ ited some special characteristics compared with pure chitosan delivery systems. In vivo, the growth of the tumors were inhibited considerably and dose-dependently by the MMC-film (P<0.05) with no any signs of vice reactions, such as inflammation, infection, and fibrous en‐ capsulation after 20d of implantation [16,146,147]. Thus a careful balance between the im‐ mune reaction and drug effectiveness is needed when a chitosan pertaining template is used for biomedical applications.
