Yoshiharu Okamoto

Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro

The effects of chitin and its derivatives on the proliferation of fibroblasts and on the production of cytokines were examined in vitro. Chitin and its derivatives showed almost no acceleratory effect on the proliferation of cultured fibroblasts. On the contrary, high-concentration 500 micrograms ml-1) D-glucosamine cultures supplemented with or without a 10% fetal calf serum (FCS) supplementation showed a significant (P < 0.05) reduction in the rate of proliferation of L929 fibroblast cells relative to control. High-concentration chitosan cultures supplemented with 10% FCS showed a significant (P < 0.05) reduction in the rate of L929 fibroblast proliferation. However, the inhibition of cell proliferation by high concentrations of chitosan did not show in cultures without FCS. Interleukin-8 (IL-8) was induced in the supernatants of rat primary cultured dermal fibroblasts stimulated with chitin and its derivatives. Chitin and its derivatives did not stimulate the production of IL-6 by mouse dermal primary cultured fibroblasts. IL-1 alpha, IL-1 beta and tumour necrosis factor-alpha were not detected in the fibroblast supernatants. These observations support the notion that cell proliferation is accelerated indirectly by chitin and its derivatives when these materials are used in vivo. In vivo findings of a angiogenesis and migration of neutrophils may be due to persistent release of IL-8 from fibroblasts.

Effects of chitin and chitosan on blood coagulation

Abstract The effects of chitin and chitosan on blood coagulation and platelet aggregation using canine blood were evaluated. Whole blood was mixed with chitin and chitosan suspensions (0.0001–1.0 mg/ml), and then the blood coagulation time (BCT) was measured using the modified Ree-White method. Chitin and chitosan reduced BCT in a dose-dependent manner. Platelet rich plasma (PRP) was mixed with chitin and chitosan suspensions, and then platelet aggregation (PA) was measured using a Dual aggregometer. The PA level induced by chitin was the strongest in all samples including chitosan, cellulose, and latex. When the washed platelet was used, the PA level induced by chitin was similar to that of chitosan, while the rate of coagulation was lower than that of PRP. Chitin and chitosan enhanced the release of the platelet derived growth factor-AB (PDGF-AB) and the transforming growth factor-β1(TGF-β1) from the platelets, particularly, more with chitosan

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.

Effects of chitin/chitosan and their oligomers/monomers on migrations of fibroblasts and vascular endothelium

Effects of chitin/chitosan and their oligomers/monomers on migrations of fibroblasts (3T6) and vascular endothelial cells (human umbilical vascular endothelial cell: HUVEC) were evaluated in vitro. In direct migratory assay using the blind well chamber method, migratory activity of 3T6 was seen to be reduced by chitin, chitosan and the chitosan monomer (GlcN). Migratory activity of HUVECs was enhanced by chitin, chitosan and the chitin monomer (GlcNAc), and was reduced by chitosan oligomers and GlcN. Supernatant of 3T6 preincubated with chitin or chitosan reduced migratory activity of 3T6 cells. Supernatant of HUVECs preincubated with chitosan also reduced migratory activity of HUVECs, but supernatant preincubated with chitin had no effect on them. In a proliferation (MTT reduction) assay, none of the samples affected proliferation of either type of cell.

Chitin and chitosan stimulate canine polymorphonuclear cells to release leukotriene B4 and prostaglandin E2

The effects of chitin and chitosan on the release of arachidonic acid products were investigated in this study. Supernatants of canine polymorphonuclear cell (PMN) suspensions incubated with chitin and chitosan contained a leukotriene B4 (LTB4) concentration high enough to induce canine PMN migration in vitro. The supernatants also contained the same concentration of prostaglandin E2 (PGE2) as that normally found in the peripheral blood of dogs. Intraperitoneal administration of chitosan to dogs induced peritoneal exudative fluid (PEF), but chitin did not. The PEF contained numerous PMNs and macrophages. The supernatant of PEF contained both heat-stable and heat-labile chemotactic factors for canine PMNs. It also contained enough LTB4 to attract the canine PMNs in vitro.

Analgesic effects of chitin and chitosan

The analgesic effects of chitin and chitosan on inflammatory pain were evaluated using the acetic-acid-induced writhing test in mice. When chitin and chitosan suspensions were mixed with the 0.5% acetic acid solution (chitin-AC and chitosan-AC, respectively) and administered intraperitoneally in mice, both agents induced a dose-dependent decrease in the number of the abnormal behaviors (writhing) due to pain, including extension of the hind legs, abdominal rigidity, and abdominal torsion. This effect was greater in the animals administered the chitosan-AC than in those administered the chitin-AC. In vitro study indicated that addition of the chitin or chitosan suspension increased the pH of the AC, and that this effect was greater in the chitosan than the chitin. Furthermore, the level of bradykinin in the peritoneal lavage fluid in the animals administered the chitin-AC was lower than in the animals administered the chitosan-AC. In vitro study showed that the chitin particles absorbed bradykinin more extensively than the chitosan particles. These results suggest that the main analgesic effect of chitosan is the absorption of proton ions released in the inflammatory site, while that of chitin is the absorption of bradykinin.

Effects of chitin and chitosan particles on BALB/c mice by oral and parenteral administration

Chitin and chitosan were administered orally and parenterally into mice and their toxicity was investigated. When 5 mg of chitin were injected intraperitoneally every 2 weeks over a 12-week period, the mice were apparently normal, but histologically, many macrophages with hyperplasia were observed in the mesenterium and foreign-body giant-cell-type polykaryocytes were observed in the spleen. The polykaryocytes were also observed in the spleen of the mice injected subcutaneously with 5 mg of chitin, but no other changes were observed. When 5 mg of chitosan were injected intraperitoneally, the body weights of the mice decreased significantly and inactivity was observed in the fifth week. Histologically, many macrophages with hyperplasia were observed in the mesenterium. Subcutaneous injection of 5 mg of chitosan did not evoke the general and cellular abnormalities. Oral administration of 5% chitosan via a casein diet caused mouse body weights to decrease and also decreased the number of Bifidobacterium and Lactobacillus in normal flora of the intestinal tract. These results indicate that special care should be taken in the clinical use of chitin and chitosan over a long time period.

Chitin and chitosan activate complement via the alternative pathway

The effect of chitosan on the serum C3 concentration was investigated in dogs and mice after subcutaneous administration. Chitosan (10 mg/kg) induced an increase of the C3 level in dogs, but not in mice. To attain the same C3 level in mice as in dogs, the dose of chitosan had to be increased five-fold. Chitin and chitosan activated complement components C3 and C5, but not C4. The intensity of complement activation was greater with chitosan than with chitin. Chitin and chitosan both activated complement via the alternative pathway.

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.

Influence of physico-chemical properties of chitin and chitosan on complement activation

Abstract To recognize the complement activation by chitin and chitosan, various physico-chemical aspects were studied. Complement activation was determined by change of plasma C3 concentration using single radial immunodiffusion method. Results were as follows: in samples with homogeneous acetylation (oDAC), C3 concentration was decreased with the increase in the degree of acetylation of oDAC. In oDACs 50 and 42 (water soluble materials), however, C3 was not decreased. In samples with heterogeneous acetylation or deacetylation samples (eDAC), all samples showed C3 activation even in eDAC 53 (solid material) and have almost same activity on complement activation. C3 activation was not seen in low molecular weight materials including d -glucosamine. The important factors inducing complement activation by chitosan-based mucopolysaccharide should be solids. The samples with heterogeneous acetylation (eDACs) had a stable ability on complement activation than with homogeneous acetylation (oDACs).