Tsuyoshi Kadosawa

Accelerating effects of chitosan for healing at early phase of experimental open wound in dogs

Chitosan is a polymeric beta(1 --> 4) glucosamine (2-amino-2-deoxy-D-glucose) and N-acetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucose) which has been reported as a wound healing accelerator. In order to evaluate the efficacy of chitosan as an accelerator of wound healing, experimental open skin wounds were made on the dorsal side in three normal beagles. Cottonfiber-type chitosan (degree of acetylation = 18%) was applied for 15 days, and the process of wound healing was evaluated histologically and immunohistochemically. On day 3 postwounding, the chitosan-treated wounds showed histologically severe infiltration of polymorphonuclear (PMN) cells and an increase in effusion compared with that in the control. Granulation was more pronounced by the chitosan treatment on day 9 and 15 postwounding. Immunohistochemical typing of collagen I, III and IV showed increase of the production of type III collagen in the chitosan group. The appearance of mitotic cells occurred numerously in the control on postwounding day 3, and in the chitosan group on postwounding day 6. These results suggest chitosan to be having a function in the acceleration of infiltration of PMN cells at the early stage of wound healing, followed by the production of collagen by fibroblasts.

Evaluation effects of chitosan for the extracellular matrix production by fibroblasts and the growth factors production by macrophages

Chitosan is reported as an accelerator of wound healing. Histological findings of previous reports indicate that chitosan accelerates the reformation of connective tissue, however the details of the mechanism are not clear. In this study, firstly L929 mouse fibroblasts were cultured with chitosan and the production of extracellular matrix (ECM) was evaluated in vitro. Type I and III collagens and fibronectin were secreted by L929 with or without chitosan; however there was no significant difference in the amount of ECM between the control and the chitosan groups. Secondly, macrophages were stimulated with chitosan, and then transforming growth factor-beta 1 (TGF-beta1) and platelet-derived growth factor (PDGF) messenger ribonucleic acid (mRNA) expressions and production of their proteins were assayed in vitro. As a result, chitosan promoted the production of TGF-beta1 and PDGF. These results indicate that chitosan does not directly accelerate ECM production by fibroblast and the ECM production may increase by the growth factors.

Chitosan accelerates the production of osteopontin from polymorphonuclear leukocytes.

Chitosan is a copolymer of beta(1 --> 4) glucosamine and N-acetyl-D-glucosamine, which accelerates the infiltration of polymorphonuclear leukocytes (PMN) in the early phase of wound healing. In the granulation tissue treated with chitosan in canine experimental wound, osteopontin (OPN) was strongly positive in PMN immunohistochemically. OPN is a glycosylated phosphoprotein and promotes the attachment or spread of a variety of cell types. In addition, OPN may play a role in granulomatous inflammation. Production of OPN in PMN was therefore investigated in vitro using human PMN in this study. PMN stimulated with granulocyte-colony stimulating factor (G-CSF) and chitosan accumulated OPN mRNA, and released OPN into their culture supernatants. These findings suggest that OPN is synthesized by migrating PMN which plays the novel role of regulating the evolution of wound healing with chitosan treatment at the early phase of healing.

Occurrence and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus pseudintermedius in an Academic Veterinary Hospital

ABSTRACT Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.

Artificial intervertebral disc replacement using bioactive three-dimensional fabric: design, development, and preliminary animal study.

Study Design. A new artificial intervertebral disc was developed, and its intrinsic biomechanical properties, bioactivity, and the effectiveness as a total disc replacement were evaluated in vitro and in vivo. Objectives. To introduce a new artificial intervertebral disc and to evaluate the in vitro mechanical properties, fusion capacity to bone, and segmental biomechanics in the total intervertebral disc replacement using a sheep lumbar spine. Summary of Background Data. The loss of biologic fusion at the bone–implant interface and prosthetic failures have been reported in previous artificial discs. There have been no clinically applicable discs with detailed experimental testing of in vivo mechanics and interface fusion capacity. Methods. The artificial intervertebral disc consists of a triaxial three-dimensional fabric (3-DF) woven with an ultra-high molecular weight polyethylene fiber, and spray-coated bioactive ceramics on the disc surface. The arrangement of weave properties was designed to produce mechanical behavior nearly equivalent to the natural intervertebral disc. Total intervertebral disc replacement at L2–L3 and L4–L5 was performed using 3-DF disc with or without internal fixation in a sheep lumbar spine model. The segmental biomechanics and interface histology were evaluated after surgery at 4 and 6 months. Results. The tensile-compressive and torsional properties of prototype 3-DF were nearly equivalent to those of human lumbar disc. The lumbar segments replaced with 3-DF disc alone showed a significant decrease of flexion–extension range of motion to 28% of control values as well as partial bony fusion at 6 months. However, the use of temporary fixation provided a nearly physiologic mobility of the spinal segment after implant removal as well as excellent bone–disc fusion at 6 months. Conclusion. An artificial intervertebral disc using a three-dimensional fabric demonstrated excellent in vitro and in vivo performance in both biomechanics and interface histology. There is a potential for future clinical application.

Endothelial cell responses to chitin and its derivatives.

The effects of chitin and its derivatives on the proliferation of human umbilical vein endothelial cells (HUVECs) and on the production of cytokines were examined in vitro. Chitin and its derivatives had no effect on the proliferation of cultured HUVECs. N-Sulfonated 70% deacetylated chitin (S-DAC70) stimulated the production of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha from HUVECs. Compared to S-DAC70, the other materials tested in the present study showed less effect in the stimulation of IL-8 and TNF-alpha production and had no effect in the stimulation of IL-1beta and IL-6 production. These results indicated that S-DAC70 affects HUVECs function but not proliferation.

Flow Cytometric Analysis of Peripheral Blood and Tumor-Infiltrating Regulatory T Cells in Dogs with Oral Malignant Melanoma:

It is well known that tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) from patients with advanced-stage cancer have a poor immune response. Regulatory T cells (Tregs), characterized by the expression of a cluster of differentiation 4 and intracellular FoxP3 markers, can inhibit antitumor immunoresponse. In the present study, the prevalence of Tregs in peripheral blood and tumor tissue from dogs with oral malignant melanoma was evaluated by triple-color flow cytometry. The percentage of Tregs in the peripheral blood of the dogs with malignancy was significantly increased compared with healthy control dogs, and the percentage of Tregs within tumors was significantly increased compared with Tregs in peripheral blood of dogs with oral malignant melanoma. This finding suggests that the presence of tumor cells induced either local proliferation or selective migration of Tregs to tumor-infiltrated sites. A better understanding of the underlying mechanisms of Treg regulation in patients with cancer may lead to an effective anticancer immunotherapy against canine malignant melanoma and possibly other tumors.

Biomechanical and Morphologic Evaluation of a Three-Dimensional Fabric Sheep Artificial Intervertebral Disc : In Vitro and In Vivo Analysis

Study Design. We have developed a new artificial intervertebral disc consisting of triaxial three-dimensional fabric for the sheep lumbar spine. To clarify the characteristics of the new implant, a series of biomechanical tests and morphologic evaluations were conducted. Objectives. To investigate the static, viscoelastic, and fatigue properties of the three-dimensional fabric disc in comparison with natural sheep disc and to evaluate their biomechanical and morphologic alteration in vivo. Summary of Background Data. In its human dimensions the three-dimensional fabric disc revealed mechanical properties similar to a natural human disc. Methods. The disc-body units from sheep spine and the sheep three-dimensional fabric discs underwent tensile-compressive (200 N), torsional (5 Nm), and creep-recovery tests (30 minutes–30 minutes, 200 N). After fatigue loading (2 million, compressive 200 N) the biomechanical changes and the debris were investigated. For in vivo evaluation after placing in the sheep psoas muscles for 6 months, the surface of the three-dimensional fabric disc was evaluated using macroscopy and scanning electron microscopy, followed by previous biomechanical tests. Results. The behavior of the sheep three-dimensional fabric disc was similar to that of natural sheep disc in tensile-compressive and creep-recovery tests. In torsional testing the behavior of natural sheep disc was more rigid than that of the sheep three-dimensional fabric disc. After fatigue loading there was no biomechanical change and no debris detected. Six months after surgery no morphologic deterioration was observed nor were there changes in biomechanical parameters. Conclusions. The sheep three-dimensional fabric disc exhibited biomechanical and morphologic biostability, appropriate viscoelasticity, and excellent fatigue properties. The three-dimensional fabric disc has a potential for clinical application of human intervertebral disc replacement.

Relationship of Disease Progression and Plasma Histamine Concentrations in 11 Dogs with Mast Cell Tumors

Plasma histamine concentrations (PHCs) were measured serially over 9 months or until death in 11 dogs with mast cell tumors (MCTs). Eight dogs had grossly visible disease and the other 3 dogs had microscopic disease. Initial PHCs in the dogs with gross disease were significantly higher than PHCs in healthy dogs (median, 0.73 ng/mL and 0.19 ng/mL respectively; P < .009), whereas initial PHCs in dogs with microscopic disease showed no difference from controls. Seven dogs subsequently had progressive increases in PHC, and developed hyperhistaminemia (median, 14.0 ng/mL; range, 5.11-30.1 ng/nL). These 7 dogs died from MCTs, and 1 had general weakness with rapid lysis of a large tumor burden after radiation therapy. PHCs of the other 4 dogs were less than 1 ng/mL during the study. These 4 dogs were still alive with adequate control of the tumor at the conclusion of the study. Four of the 11 dogs initially had gastrointestinal (G1) signs, which abated soon after administration of histamine-2 (H-2) blockers. No significant difference was found between PHCs in dogs with GI signs and those without GI signs (median, 0.86 ng/mL and 0.35 ng/mL. respectively). Thereafter, 7 dogs had serious GI complications for which H-2 blocker therapy was ineffective. PHCs in these 7 dogs were extremely high (median, 12.2 ng/mL; range, 3.42-30.1 ng/nL). Results of this study demonsrated that PHC was one factor related to disease progression, and indicated that marked hyperhistaminemia was associated with the GI signs refractory to H-2 blocker therapy in dogs with MCTs.

Bone ingrowth fixation of artificial intervertebral disc consisting of bioceramic-coated three-dimensional fabric.

Study Design. The bone-bonding characteristic of the new artificial intervertebral disc consisting of bioceramic-coated three-dimensional fabric was evaluated mechanically and histologically in an in vivo sheep model. Objectives. To investigate the mechanical properties and the histologic appearance of the interface between the three-dimensional fabric disc and the vertebral body, and to evaluate these alterations in vivo under a spinal segmentally mobile condition. Summary of Background Data. Bone ingrowth to the bioceramic-coated three-dimensional fabric surface had been demonstrated already under a stable environment in preliminary animal studies. Methods. For this study, 20 sheep underwent two-level lumbar intervertebral disc replacement with three-dimensional fabric discs (Group I) or bioceramic spacers as a comparative material (Group II). All operative segments were stabilized temporarily with spinal instrumentation for the initial ingrown phase. Four animals each were killed at 4, 6, 15, and 24 months in Group I and at 6 months in Group II, and the operative segments were subjected to either a detachment test or histologic evaluation. Results. The interfacial tensile strength at 6 months was significantly higher in Group I than in Group II. No significant decrease in tensile strength was detected until 24 months after surgery in Group I. Histologically, bone ingrowth to the three-dimensional fabric surface was observed 4 months after surgery, and no aseptic loosening occurred until 24 months after surgery. Conclusions. The findings show that the three-dimensional fabric disc was firmly fixed to the vertebral body by bone ingrowth, and that this biologic fixation was preserved even under the spinal segmentally mobile condition.