Protective Effect of Chitin Urocanate Nanofibers against Ultraviolet Radiation.

Urocanic acid is a major ultraviolet (UV)-absorbing chromophore. Chitins are highly crystalline structures that are found predominantly in crustacean shells. Alpha-chitin consists of microfibers that contain nanofibrils embedded in a protein matrix. Acid hydrolysis is a common method used to prepare chitin nanofibrils (NFs). We typically obtain NFs by hydrolyzing chitin with acetic acid. However, in the present study, we used urocanic acid to prepare urocanic acid chitin NFs (UNFs) and examined its protective effect against UVB radiation. Hos: HR-1 mice coated with UNFs were UVB irradiated (302 nm, 150 mJ/cm²), and these mice showed markedly lower UVB radiation-induced cutaneous erythema than the control. Additionally, sunburn cells were rarely detected in the epidermis of UNFs-coated mice after UVB irradiation. Although the difference was not as significant as UNFs, the number of sunburn cells in mice treated with acetic acid chitin nanofibrils (ANFs) tended to be lower than in control mice. These results demonstrate that ANFs have a protective effect against UVB and suggest that the anti-inflammatory and antioxidant effects of NFs influence the protective effect of ANFs against UVB radiation. The combination of NFs with other substances that possess UV-protective effects, such as urocanic acid, may provide an enhanced protective effect against UVB radiation.

Metabolomic Analysis of Blood Plasma after Oral Administration of N-acetyl-d-Glucosamine in Dogs.

N-acetyl-d-glucosamine (GlcNAc) is a monosaccharide that polymerizes linearly through (1,4)-ß-linkages. GlcNAc is the monomeric unit of the polymer chitin. GlcNAc is a basic component of hyaluronic acid and keratin sulfate found on the cell surface. The aim of this study was to examine amino acid metabolism after oral GlcNAc administration in dogs. Results showed that plasma levels of ectoine were significantly higher after oral administration of GlcNAc than prior to administration (p < 0.001). To our knowledge, there have been no reports of increased ectoine concentrations in the plasma. The mechanism by which GlcNAc administration leads to increased ectoine plasma concentration remains unclear; future studies are required to clarify this mechanism.

Metabolomic analyses of blood plasma after oral administration of D-glucosamine hydrochloride to dogs.

D-Glucosamine hydrochloride (GlcN∙HCl) is an endogenous amino monosaccharide synthesized from glucose that is useful in the treatment of joint diseases in both humans and animals. The aim of this study was to examine amino acid metabolism in dogs after oral administration of GlcN∙HCl. Accelerated fumarate respiration and elevated plasma levels of lactic acid and alanine were observed after administration. These results suggest that oral administration of GlcN∙HCl induces anaerobic respiration and starvation in cells, and we hypothesize that these conditions promote cartilage regeneration. Further studies are required to evaluate the expression of transforming growth factor-beta (TGF-ß).

Effect of fucoidan extracted from mozuku on experimental cartilaginous tissue injury.

We investigated the effect of fucoidan, a sulfated polysaccharide, on acceleration of healing of experimental cartilage injury in a rabbit model. An injured cartilage model was surgically created by introduction of three holes, one in the articular cartilage of the medial trochlea and two in the trochlear sulcus of the distal femur. Rabbits in three experimental groups (F groups) were orally administered fucoidan of seven different molecular weights (8, 50, 146, 239, 330, 400, or 1000 kD) for 3 weeks by screening. Control (C group) rabbits were provided water ad libitum. After the experimental period, macroscopic examination showed that the degree of filling in the fucoidan group was higher than that in the C group. Histologically, the holes were filled by collagen fiber and fibroblasts in the C group, and by chondroblasts and fibroblasts in the F groups. Image analysis of Alcian blue- and safranin O-stained F-group specimens showed increased production of glycosaminoglycans (GAGs) and proteoglycans (PGs), respectively. Some injured holes were well repaired both macroscopically and microscopically and were filled with cartilage tissues; cartilage matrices such as PGs and GAGs were produced in groups F 50, F 146, and F 239. Thus, fucoidan administration enhanced morphologically healing of cartilage injury.

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.

Effects of oral glucosamine hydrochloride administration on plasma free amino acid concentrations in dogs.

We examined the effects of oral glucosamine hydrochloride (GlcN), N-acetyl-D-glucosamine (GlcNAc) and d-glucose (Glc) administration on plasma total free amino acid (PFAA) concentrations in dogs. The PFAA concentrations increased in the control group and the GlcNAc group at one hour after feeding, and each amino acid concentration increased. On the other hand, in the GlcN group and the Glc group PFAA concentrations decreased at one hour after feeding. A significant decrease in amino acid concentration was observed for glutamate, glycine and alanine. Our results suggest the existence of differences in PFAA dynamics after oral administration of GlcN and GlcNAc in dogs.

Anatomical variations of the extrahepatic ducts in dogs: knowledge for surgical procedures.

Variations of the hepatic duct terminal were examined in 50 cadavers of mixed-breed dogs. The hepatic duct was formed by 4 major tributaries in 70% of the dogs and by 3 tributaries in 30% of the dogs. The order of hepatic duct termination, proximal to the gallbladder, was as follows: the duct from the right medial lobe, then the quadrate, then the common duct from the right lateral and caudate lobes and finally the duct from the left lobes in most of the dogs (90%). In only 10% of the dogs, the duct from the quadrate lobe terminated proximal to the duct from the right medial lobe. The length of the cystic duct was more than 5 mm in most dogs (88%). This study showed anatomical variations of the extrahepatic biliary tree and their preoperational imaging. These results may be useful for shortening the operative period and provide basic information for the application of laparoscopic cholecystectomies.

Mydriasis associated with local dysfunction of parasympathetic nerves in two dogs.

In clinical practice, photophobia resulting from persistent mydriasis may be associated with dysfunction of ocular parasympathetic nerves or primary iris lesions. We encountered a 5-year-old Miniature Dachshund and a 7-year-old Shih Tzu with mydriasis, abnormal pupillary light reflexes, and photophobia. Except for sustained mydriasis and photophobia, no abnormalities were detected on general physical examination or ocular examination of either dog. We performed pharmacological examinations using 0.1% and 2% pilocarpine to evaluate and diagnose parasympathetic denervation of the affected pupillary sphincter muscles. On the basis of the results, we diagnosed a pupillary abnormality due to parasympathetic dysfunction and not to overt primary iris lesions. The test revealed that neuroanatomic localization of the lesion was postciliary ganglionic in the first dog.

Preparation of stable chitosan-carboxymethyl dextran nanoparticles.

Chitosan-carboxymethyl dextran nanoparticles (CHI-CMD NPs) were prepared by the formation of polyelectrolyte complex via the interaction of the positive charged amino group of chitosan and the negative charged carboxyl group of carboxymethyl dextran. Biocompatible organic reagents such as lactic acid, phosphate-buffered saline, and carbonate-bicarbonate buffer were used to make CHI-CMD NPs. The pH value of narrowly distributive CHI-CMD NP suspension was close to the physiological value of 6.8, which made it possible for it to be directly applied in an in vitro test without further additional operations. In the presence of sugars (30 mM), the colloidal stability of CHI-CMD NPs could be extended to 5 to 7 days when the particle size could be maintained within a nanometer scale, while the addition of xylose or lactose was found to most effectively extend the colloidal stability of CHI-CMD NPs to 10 days. Scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction analysis revealed that the structural integrity of CHI-CMD NPs was not destroyed after high steam pressure sterilization (121 degrees C, 30 min), which confirmed the autoclavable property of CHI-CMD NPs.

Retraction: Azuma, K. et al. Chitin, Chitosan, and Its Derivatives for Wound Healing: Old and New Materials. J. Funct. Biomater. 2015, 6, 104⁻142.

The Journal of Functional Biomaterials Editorial Office have been made aware that some parts of the article [1] are duplicated from other publications[...].

Chronic myocardial infarction due to arteriosclerosis of coronary arteries followed by acute thromboembolism of caudal abdominal aorta in a cat.

A 10-year-old cat with the paresis of hind limbs was initially diagnosed as a hypertrophic cardiomyopathy followed by acute thromboembolism of caudal abdominal aorta from the findings of the medical examinations. However, this case was proved to be an chronic myocardial infarction due to arteriosclerosis of coronary arteries by the pathologic diagnosis. In the left ventricular, the hypertrophy and the narrowing were slight, and a coagulative infarction was seen obviously. The intramural coronary arteriosclerosis showed thickening of the wall due to medial hyperplasia by fibrosis, and arterial stenosis. Myocardial infarction and arteriosclerosis are scarcely any reports of these lesions in cats. This case is valuable for an extremely rare case of myocardial infarction in the cat.

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.

Chitin, chitosan, and its derivatives for wound healing: old and new materials.

Chitin (ß-(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.

Clinical systemic lupeol administration for canine oral malignant melanoma.

Canine oral malignant melanoma (COMM) is the most aggressive malignant tumor in dogs. Lupeol is a triterpene extracted from various fruits and vegetables that reportedly inhibits melanoma cell proliferation in vitro and in vivo. In this study, the efficacy of subcutaneous lupeol for spontaneous COMM was evaluated. A total of 11 dogs (3, 5 and 3 dogs diagnosed with clinical stage I, II and III melanoma, respectively) were evaluated. Subcutaneous lupeol (10 mg/kg) was administered postoperatively at various time points to treat these 11 COMM cases. Of the 11 subjects, 7 exhibited no local recurrence 180 days postoperatively and no severe adverse effects were observed in any of the cases. Furthermore, no distant metastasis was observed during the experimental period. Therefore, systemic lupeol may prevent local tumor progression and distant metastasis and may be a novel adjuvant treatment for the treatment of COMM.

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.

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.

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.

Effects of chitin and chitosan on collagen synthesis in wound healing.

Collagen synthesis was evaluated by measuring prolyl hydroxylase (PHL) activity induced within rat granulation tissue by a polyester non-woven fabric (NWF, 1 x 1 cm, 0.6 mm in thickness) impregnated with a chitin or chitosan suspension ranging in concentration from 0.1 to 10 mg/ml. In addition, PHL activity induced in rat granulation tissue by a NWF impregnated with a phosphate buffer solution was examined as a control. The PHL activity in each group remained low until 4 days post-implantation (Day 4). However, in the 10 mg chitin group, the PHL activity increased rapidly without scatter of the data at Day 7 and remained at a plateau until Day 14. In other groups, PHL activity increased linearly until Day 14. The data varied widely at Day 7. Compared to chitosan, chitin at the higher concentration was found to induce stable collagen synthesis in the early wound healing process.

Tissue retention and adverse reaction after intravenous injection of hematoporphyrin derivatives in dogs.

We evaluated changes in hematology and chemical profile, and the tissue retention of hematoporphyrin derivative (HpD) following the intravenous injection in dogs. HpD at concentrations of 1, 5, 10, and 15 mg/kg was intravenously injected to 16 dogs (n=4 each) and complete blood count (CBC) and blood chemistry were performed on days 1, 3, 5, and 7 after the injection. To examine tissue retention, HpD (5 mg/kg) was administered to 15 dogs and 3 dogs were euthanized on days 1, 2, 7, 14, and 28 after the injection, respectively, to collect the skin, muscle, small intestine, spleen, kidney and liver as tissue samples. There were no significant changes in CBC and blood chemical profile except for an increase in LDH concentrations in dogs given 10 and 15 mg/kg of HpD at day 3. The levels of HpD retention in the tissues were ranked in the following order: liver > kidney > spleen > intestine > muscle > skin.