Kazuo Azuma

Chitin, Chitosan, and Its Derivatives for Wound Healing: Old and New Materials

Chitin (β-(1-4)-poly-N-acetyl-d-glucosamine) is widely distributed in nature and is the second most abundant polysaccharide after cellulose. It is often converted to its more deacetylated derivative, chitosan. Previously, many reports have indicated the accelerating effects of chitin, chitosan, and its derivatives on wound healing. More recently, chemically modified or nano-fibrous chitin and chitosan have been developed, and their effects on wound healing have been evaluated. In this review, the studies on the wound-healing effects of chitin, chitosan, and its derivatives are summarized. Moreover, the development of adhesive-based chitin and chitosan are also described. The evidence indicates that chitin, chitosan, and its derivatives are beneficial for the wound healing process. More recently, it is also indicate that some nano-based materials from chitin and chitosan are beneficial than chitin and chitosan for wound healing. Clinical applications of nano-based chitin and chitosan are also expected.

Anticancer and Anti-Inflammatory Properties of Chitin and Chitosan Oligosaccharides

Previous reports indicate that N-acetyl-d-glucosamine oligomers (chitin oligosaccharide; NACOS) and d-glucosamine oligomers (chitosan oligosaccharide; COS) have various biological activities, especially against cancer and inflammation. In this review, we have summarized the findings of previous investigations that have focused on anticancer or anti-inflammatory properties of NACOS and COS. Moreover, we have introduced recent evaluation of NACOS and COS as functional foods against cancer and inflammatory disease.

Effects of oral administration of fucoidan extracted from Cladosiphon okamuranus on tumor growth and survival time in a tumor-bearing mouse model

We evaluated the anti-tumor activities of the oral administration of fucoidan extracted from Cladosiphon okamuranus using a tumor (colon 26)-bearing mouse model. The materials used included low-molecular-weight fucoidan (LMWF: 6.5–40 kDa), intermediate-molecular-weight fucoidan (IMWF: 110–138 kDa) and high-molecular-weight fucoidan (HMWF: 300–330 kDa). The IMWF group showed significantly suppressed tumor growth. The LMWF and HMWF groups showed significantly increased survival times compared with that observed in the control group (mice fed a fucoidan-free diet). The median survival times in the control, LMWF, IMWF and HMWF groups were 23, 46, 40 and 43 days, respectively. It was also found that oral administration of fucoidan increased the population of natural killer cells in the spleen. Furthermore, from the results of the experiment using Myd-88 knockout mice, it was found that these effects are related to gut immunity. These results suggest that fucoidan is a candidate anti-tumor functional food.

α-Chitin nanofibrils improve inflammatory and fibrosis responses in inflammatory bowel disease mice model

We evaluated the anti-inflammatory and anti-fibrosis effects of α-chitin nanofibrils in a mouse model of dextran sulfate sodium (DSS)-induced acute ulcerative colitis (UC). α-Chitin nanofibrils decreased positive areas of nuclear factor-κB staining in the colon tissue (7.2±0.5%/fields in the α-chitin nanofibrils group vs. 10.7±0.9%/fields in the control group; p<0.05). α-Chitin nanofibrils also decreased serum monocyte chemotactic protein-1 concentration in DSS-induced acute UC (24.1±7.8 pg/ml in the α-chitin nanofibrils group vs. 53.5±3.1 pg/ml in the control group; p<0.05). Moreover, α-chitin nanofibrils suppressed the increased positive areas of Massons trichrome staining in colon tissue (6.8±0.6%/fields in the α-chitin nanofibrils group vs. 10.1±0.7%/fields in the control group; p<0.05). On the other hand, α-chitin powder suspension did not show these effects in DSS-induced acute UC mice model. Our results indicated that α-chitin nanofibrils have the anti-inflammatory effect via suppressing NF-κB activation and the anti-fibrosis effects in DSS-induced acute UC mice model.

Evaluation of the effects of chitin nanofibrils on skin function using skin models.

Chitins are highly crystalline structures that are predominantly found in crustacean shells. Alpha-chitin is composed of microfibers, which are made up of nanofibrils that are 2-5 nm in diameter and 30 nm in length and embedded in a protein matrix. Crystalline nanofibrils can also be prepared by acid treatment. We verified the effect of chitin nanofibrils (NF) and nanocrystals (NC) on skin using a three-dimensional skin culture model and Franz cells. The application of NF and NC to skin improved the epithelial granular layer and increased granular density. Furthermore, NF and NC application to the skin resulted in a lower production of TGF-β compared to that of the control group. NF and NC might have protective effects to skin. Therefore, their potential use as components of skin-protective formulations merits consideration.

Biological adhesive based on carboxymethyl chitin derivatives and chitin nanofibers

Novel biological adhesives made from chitin derivatives were prepared and evaluated for their adhesive properties and biocompatibility. Chitin derivatives with acrylic groups, such as 2-hydroxy-3-methacryloyloxypropylated carboxymethyl chitin (HMA-CM-chitin), were synthesized and cured by the addition of an aqueous hydrogen peroxide solution as a radical initiator. The adhesive strength of HMA-CM-chitin increased when it was blended with chitin nanofibers (CNFs) or surface-deacetylated chitin nanofibers (S-DACNFs). HMA-CM-chitin/CNFs or HMA-CM-chitin/S-DACNFs have almost equal adhesive strength compared to that of a commercial cyanoacrylate adhesive. Moreover, quick adhesion and induction of inflammatory cells migration were observed in HMA-CM-chitin/CNF and HMA-CM-chitin/S-DACNF. These findings indicate that the composites prepared in this study are promising materials as new biological adhesives.

Favorable effects of superficially deacetylated chitin nanofibrils on the wound healing process.

Previous reports indicate that the beneficial effect of chitin nanofibrils (CNFs), and chitosan nanofibrils (CSNFs) for wound healing. In this study, the wound healing effects of superficially deacetylated chitin nanofibrils (SDACNFs) were evaluated using an experimental model. In the experiments using circular excision wound model, SDACNFs induced re-epithelium and proliferation of the fibroblasts and collagen tissue. In the chitin, CNFs, and CSNFs, on the other hand, the e-epithelium and proliferation of the fibroblasts and collagen tissue were not induced perfectly compared with the SDACNFs group. In particular, re-epithelization was observed on day 4 in the only SDACNF group. Moreover, SDACNFs did not induce severe systemic inflammation in the linear incision wound model. The data indicated that SDACNFs effectively induced the proliferation and re-modeling phases compared with chitin, CNFs, and CSNFs in the wound. These data indicate that SDACNFs can be beneficial as a new biomaterial for wound healing.

Anti-inflammatory effects of orally administered glucosamine oligomer in an experimental model of inflammatory bowel disease.

Anti-inflammatory effects of oral administration of the glucosamine oligomers (chito-oligosaccharides: COS) were evaluated in an experimental model of inflammatory bowel disease (IBD). Oral administration of COS improved shortening of colon length and tissue injury (as assessed by histology) in mice. Oral administration of COS inhibited inflammation in the colonic mucosa by suppression of myeloperoxidase activation in inflammatory cells, as well as activation of nuclear factor-kappa B, cyclooxygenase-2, and inducible nitric oxide synthase. Oral administration of COS also reduced serum levels of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-6). Moreover, it prolonged survival time in mice. These data suggest that COS have anti-inflammatory effects in an experimental model of IBD, and could be new functional foods for IBD patients.

Biomineralization of calcium phosphate crystals on chitin nanofiber hydrogel for bone regeneration material.

We previously reported a chitin nanofiber hydrogel from squid pen β-chitin by a simple NaOH treatment. In the present study, a calcium phosphate/chitin nanofiber hydrogel was prepared for bone tissue engineering. Calcium phosphate was mineralized on the hydrogel by incubation in a solution of diammonium hydrogen phosphate solution followed by calcium nitrate tetrahydrate. X-ray diffractometry and Fourier transform infrared spectroscopy showed the formation of calcium phosphate crystals. The morphology of the calcium phosphate crystals changed depending on the calcification time. After mineralization, the mechanical properties of the hydrogel improved due to the reinforcement effect of calcium phosphate crystal. In an animal experiment, calcium phosphate/chitin nanofiber hydrogel accelerated mineralization in subcutaneous tissues. Morphological osteoblasts were observed.

Anti-tumor properties of orally administered glucosamine and N-acetyl-D-glucosamine oligomers in a mouse model.

The current study evaluated the anti-tumor activities of N-acetyl-d-glucosamine oligomer (NACOS) and glucosamine oligomer (COS) after their oral administration in a tumor (colon 26)-bearing mouse model. Compared to the control group, NACOS and COS groups showed significantly suppressed tumor growth, and apparent, marked apoptosis in tumor tissues. Furthermore, serum interleukin-12p70 and interferon-γ levels significantly increased in the NACOS and COS groups compared to the corresponding levels in the control group. Collectively, the results indicate the oral administration of NACOS and COS could enhance innate immunity. Results of experiments in Myd-88 knockout mice revealed that the apparent effects were related to both Myd-88-dependent and Myd-88-independent pathways. The data indicated that oral administration of NACOS and COS produced anti-tumor effects through the induction of apoptosis and stimulation of the immune system, which suggests that NACOS and COS are candidate anti-tumor functional foods.