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[...].

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.

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.

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.

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.

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.

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.

Anti-inflammatory effects of ozonated water in an experimental mouse model.

Previous studies have suggested that ozonated water is safe and possesses antibacterial effects for treatment of experimental peritonitis rats. In this study, we evaluated the anti-inflammatory effects of ozonated water that was intraperitoneally injected into an experimental inflammatory mouse model. The concentrations of dissolved ozone decreased constantly and lineally, while the half-life of dissolved ozone was 36.8±2.7 min (27°C). The 10-ppm ozonated water was injected intraperitoneally into mice with lipopolysaccharide (LPS)-induced acute inflammation. The results showed that the intraperitoneal injection of ozonated water decreased the levels of tumor necrosis factor-α (TNF-α) and increased the activity of superoxide dismutase (SOD). The results suggest that ozonated water has anti-inflammatory properties and is a potential therapeutic option for acute inflammation.

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.

Effects of photodynamic hyperthermal therapy with indocyanine green on tumor growth in a colon 26 tumor-bearing mouse model.

The present study used indocyanine green (ICG) and a broadband light source apparatus [photodynamic hyperthermal therapy (PHT) group] in order to treat a colon 26 tumor-bearing mouse model. The other groups were administered either ICG alone (ICG group), light alone (light group) or no treatment (control group). Following the treatment, tumor growth was measured. Nine days after the treatment, the tumors were resected and histological and immunohistological examinations were performed. In the PHT group, the growth rates of the tumor tissues were significantly decreased compared with those observed in the other groups (P<0.05). The proportion of necrotic areas in the PHT and light groups were increased significantly compared with those observed in the ICG and control groups. However, there were no significant differences between the PHT and light groups. The proportion of Ki-67 in the PHT and light groups was less than that observed in the ICG and control groups. The number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the PHT group was significantly increased compared with that observed in the other groups. These data indicate that PHT is effective in vivo and in vitro.

Preparation and biomedical applications of chitin and chitosan nanofibers.

Chitin (ß-(1-4)-poly-N-acetyl-D-glucosamine) is widely distributed in nature and is the second most abundant polysaccharide after cellulose. Chitin occurs in nature as ordered macrofibrils. It is the major structural component in the exoskeleton of crab and shrimp shells and the cell wall of fungi and yeast. As chitin is not readily dissolved in common solvents, it is often converted to its more deacetylated derivative, chitosan. Chitin, chitosan, and its derivatives are widely used in tissue engineering, wound healing, and as functional foods. Recently, easy methods for the preparation of chitin and chitosan nanofibers have been developed, and studies on biomedical applications of chitin and chitosan nanofibers are ongoing. Chitin and chitosan nanofibers are considered to have great potential for various biomedical applications, because they have several useful properties such as high specific surface area and high porosity. This review summarizes methods for the preparation of chitin and chitosan nanofibers. Further, biomedical applications of chitin and chitosan nanofibers in (i) tissue engineering, (ii) wound dressing, (iii) cosmetic and skin health, (iv) stem cell technology, (v) anti-cancer treatments and drug delivery, (vi) anti-inflammatory treatments, and (vii) obesity treatment are summarized. Many studies indicate that chitin and chitosan nanofibers are suitable materials for various biomedical applications.

Photodynamic hyperthermal chemotherapy with indocyanine green: a novel cancer therapy for 16 cases of malignant soft tissue sarcoma.

Sixteen cases of malignant soft tissue sarcoma (STS; 10 canines and six felines) were treated with a novel triple therapy that combined photodynamic therapy, hyperthermia using indocyanine green with a broadband light source, and local chemotherapy after surgical tumor resection. This triple therapy was called photodynamic hyperthermal chemotherapy (PHCT). In all cases, the surgical margin was insufficient. In one feline case, PHCT was performed without surgical resection. PHCT was performed over an interval of 1 to 2 weeks and was repeated three to 21 times. No severe side effects, including severe skin burns, necrosis, or skin suture rupture, were observed in any of the animals. No disease recurrence was observed in seven out of 10 (70.0%) dogs and three out of six (50.0%) cats over the follow-up periods ranging from 238 to 1901 days. These results suggest that PHCT decreases the risk of STS recurrence. PHCT should therefore be considered an adjuvant therapy for treating companion animals with STS in veterinary medicine.

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-ß 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.

Systemic and local injections of lupeol inhibit tumor growth in a melanoma-bearing mouse model.

Melanoma is the most aggressive type of skin cancer and it is procured from activated or genetically altered epidermal melanocytes. In the present study, the tumor-suppressive effects of systemic and local injections of lupeol, a triterpene extracted from Indian lettuce (Lactuca indica), in a melanoma-bearing mouse model were evaluated. Mice were injected once with lupeol or olive oil (solvent control) subcutaneously into the skin of the back or into the tumor tissue. Seven days after the injection, the tumor growth rates were calculated and the tumor tissues were collected. Immunohistochemical staining for Ki-67 and proliferating cell nuclear antigen (PCNA) were performed. The tumor growth rates in the lupeol-injected group were significantly decreased compared to those observed in the non-treated (NT) and solvent control groups. Lupeol also significantly decreased the areas positively stained for Ki-67 and PCNA in the tumor tissues compared to those in the NT and solvent control groups. The results of the present study demonstrated that systemic and local injections of lupeol suppress tumor growth and induce cell cycle arrest in a melanoma-bearing mouse model. These data suggest that lupeol may be effective as a novel therapeutic option for melanoma patients.

Correlation of plasma amino acid concentrations and chondroprotective effects of glucosamine and fish collagen peptide on the development of osteoarthritis.

We investigated the correlation of plasma amino acid concentrations and the effects of glucosamine and fish collagen peptide (FCP) on osteoarthritis (OA). OA was induced according to the rabbit anterior cruciate ligament transection (ACLT) model. After surgery, the rabbits were orally administered FCP (F group), glucosamine (G group) or both (FG group) for 4 weeks. The control group (C group) was not administered anything. Blood was collected before surgery (pre-ACLT) and before euthanasia (post-ACLT). Changes in the alanine, threonine and methionine concentrations were significant between pre- and post-ACLT. The correlation between the histological assessment and arginine concentration post-ACLT was significant. These findings indicate that measurement of plasma amino acids is useful for evaluation of the efficacy of intervention for OA.

Evaluation of the chondroprotective effects of glucosamine and fish collagen peptide on a rabbit ACLT model using serum biomarkers.

The aim of this study was to investigate the correlations of severity of osteoarthritis (OA) and serum biomarkers including keratan sulfate (KS), hyaluronic acid (HA) and chondroitin sulfate (CS) 846 epitope. We also investigated the effect of glucosamine and fish collagen peptide (FCP) on OA. OA was induced in 12 rabbits (12 weeks of age) by anterior cruciate ligament transection (ACLT). After the surgery, the rabbits were orally administered FCP (F group), glucosamine (G group) or FCP and glucosamine (FG group) for 4 weeks. The control group was provided water ad libitum (C group). Blood was collected before surgery (pre-ACLT) and before euthanasia (post-ACLT) for serum marker measurement. Biomarker levels were measured by using commercial kits. We evaluated OA severity both macroscopically and histologically. Macroscopic evaluation showed mildly eroded condylar surfaces in the C group. Histological findings were significantly different from the FG and other groups. There were no significant differences between each group at post-ACLT in terms of serum KS, HA and CS 846. Histological assessment and serum biomarker measurements performed at post-ACLT showed a significant correlation between HA concentration and OA severity. Variations in the CS 846 concentration at pre-ACLT and post-ACLT were significantly correlated with OA severity. Administration of glucosamine and FCP had chondroprotective effects in the ACLT model. Serum biomarker concentrations were significantly correlated with cartilage injury. Serum biomarker measurement would be useful for monitoring articular cartilage damage in the clinical setting.

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.

Plasma free amino acid profiles of canine mammary gland tumors.

The purpose of this study was to elucidate the relationship between plasma free amino acid (PFAA) levels and the clinical stages of mammary gland tumors (MGT) in dogs. PFAA levels in canines with malignant mammary tumors were decreased compared to those of healthy animals. The levels of aspartate and ornithine, in the dogs with tumor metastasis were significantly decreased when compared to those of dogs that did not have metastases. Results of this study indicate that PFAA levels could be a risk factor or biomarker for canine MGT metastasis.