Katsuyuki Miyatake

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

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.

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.

Enhanced healing of cartilaginous injuries by N-acetyl-D-glucosamine and glucuronic acid

Abstract We investigated the restorative effect of orally administered glucose, N -acetyl- d -glucosamine (GlcNAc) and glucuronic acid (GlcUA) on the experimentally produced cartilaginous injuries in rabbits. A total of three holes in the left stifle joint, including one in the medial trochlear ridge, and two in the trochlear sulcus (proximal and distal) of articular cartilage were made surgically using a drill. For the control group, only tap water was administered daily and for the glucose, GlcNAc, GlcUA groups, a water based solution (1 g/head/day) of glucose, GlcNAc, glucuronolactone was administered daily, respectively. We observed the clinical symptoms daily and the condition of the injured part was observed macroscopically and histologically at 3 weeks after the operation. There was no difference in body weight or general conditions among each group. With respect to medial trochlear injury, 1/3 holes were not cured in the control, but all were cured in the glucose, GlcNAc and GlcUA groups, respectively. With respect to the proximal hole, 4/6 in the control group, 3/3 in the glucose and 2/3 in the GlcNAc were not cured. However, 2/3 in the GlcUA were cured. There was significant difference ( p p On histological examination, the injured parts were covered by fibrous connective tissues in the control and the glucose, whereas in the GlcNAc and GlcUA groups, the massive proliferation of matured cartilaginous tissues was observed, and the regenerated cartilaginous tissues were surrounded by the proliferation of chondroblast cells. In the regenerated tissue, matured cartilage substrate was also observed. Safranin O and Alcian blue stains marked a more significantly dense in the GlcNAc and GlcUA group than in the control ( p

Collagen typing of granulation tissue induced by chitin and chitosan

Granulation tissue induced by chitin- or chitosan-coated polyester nonwoven fabric (chitin-NWF, chitosan-NWF) and uncoated NWF (control) implanted in the feline abdominal wall was evaluated by macroscopic and microscopic observation. Tissue growth around the implant was most marked with chitosan-NWF and least with chitin-NWF. The inflammatory reaction was much stronger with chitosan-NWF. In the tissue surrounding the implant, many giant cells and prominent angiogenesis were induced by chitin-NWF compared to the other NWFs. Types I, III and IV collagen were synthesized at higher levels in the chitin-NWF implants, especially type IV collagen, compared with the other implants. At 14 days after implantation, the levels of types III and IV collagen were also greater in the tissue surrounding the chitin-NWF. The pattern of synthesis of each type of collagen induced by chitosan-NWF in the surrounding tissue closely resembled that of the control.

Chitosan-inducing hemorrhagic pneumonia in dogs

Abstract Various amounts of chitosan (10–200 mg/kg) were administered subcutaneously to dogs. Anorexia and mortality were observed in dogs given doses above 50 mg/ kg, and above 150 mg/kg, respectively. In hematologic findings leukocytosis and increasing of serum LDH2 and LDH3 isoenzymes were characteristic. From the findings of autopsy, severe hemorrhagic pneumonia was observed in all dead dogs. Chitosan causes lethal pneumonia to dogs.

Chitin induces type IV collagen and elastic fiber in implanted non-woven fabric of polyester

The non-woven fabric of polyester (control) and the composite material of the non-woven fabric of polyester and chitin (Chitipack P) were implanted to bovine flexor tendon. After 3 weeks implantation, type IV collagen and elastic fibers were significantly increased and type I collagen was decreased in Chitipack P in comparison with control. The breaking strength was about twice as high in Chitipack P than in control. The polykaryocytes in the control were more difficult to digest for the collagens. Angiogenesis in the implanted non-woven fabric and in the neighboring resected tendons was much stronger in Chitipack P. Chitin induced type IV collagen and elastic fibers in the prostheses.

Anti-inflammatory effect of chemically modified chitin

Abstract Anti-inflammatory effects of the three types of chitin derivatives namely phosphated chitin (P-chitin), phosphated–sulfated chitin (PS-chitin), and sulfated chitin (S-chitin) were investigated using a canine model of chitosan-induced pneumonia. After simultaneous administration of chitosan with or without each chitin derivative (chitosan alone: n=6, chitosan and P-chitin: n=6, chitosan and PS-chitin: n=1, and chitosan and S-chitin: n=3), hematological examination and X-ray image processing were performed for up to 24 h. Then the lungs were recovered and were evaluated by softex imaging after inflation and fixation. The hematological findings showed that PS-chitin and S-chitin did not prevent the decrease in white blood cell (WBC) count as seen in dogs administered chitosan, while P-chitin prevented such decrease in WBC count. The surface of the inflated and fixed lung specimens was hemorrhagic in the PS- and S-chitin groups as well as in the chitosan group, while the lung looked like normal in the P-chitin group. The pulmonary blood vessels of the chitosan group showed severe change while the P-chitin group showed no changes with softex findings. Furthermore, the pattern of histogram density obtained with image processing of thoracic X-ray in P-chitin group did not change among pre and post administration while chitosan group showed rightward movement and significant changes on parameters. The cause of which is attribured to an attenuation of X-ray permeability by angiectasis of the lung.

Phosphated chitin (P-chitin) exerts protective effects by restoring the deformability of polymorphonuclear neutrophil (PMN) cells

In continuation of our on-going investigation on P-chitin, we screened 11 more formulations, out of which only two of them were found effective in vivo. It was not known at that moment, why all the formulations were not effective in vivo. Based on the results of the in vivo screening experiment, in vitro tests were carried out to determine the influence of two effective and non-effective samples on migrating ability of polymorphonuclear neutrophil (PMN) cells. PMN cells were made to loose migrating ability to mimic the in vivo condition of PMN sequestration as it occurs during the acute lung injury, by pre-incubating with chitosan activated serum (CAS). To our surprise, such CAS treated PMN cells with reduced ability to migrate, when subjected to P-chitin treatment were found to have restored the migrating ability. Interestingly, only those samples which were effective in vivo enhanced the deformability or the migrating ability of PMN cells, whereas non-effective samples hindered it. How effective P-chitin restores PMN cells deformability is under investigation.