Toru Fujinaga

Topical formulations and wound healing applications of chitosan

Chitosan is being used as a wound-healing accelerator in veterinary medicine. To our knowledge, chitosan enhances the functions of inflammatory cells such as polymorphonuclear leukocytes (PMN) (phagocytosis, production of osteopontin and leukotriene B4), macrophages (phagocytosis, production of interleukin (IL)-1, transforming growth factor beta 1 and platelet derived growth factor), and fibroblasts (production of IL-8). As a result, chitosan promotes granulation and organization, therefore chitosan is beneficial for the large open wounds of animals. However, there are some reported complications of chitosan application. Firstly, chitosan causes lethal pneumonia in dogs which are given a high dose of chitosan. In spite of application of chitosan to various species, this finding is observed only in dogs. Secondly, intratumor injection of chitosan on mice bearing tumor increases the rate of metastasis and tumor growth. Therefore, it is important to consider these effects of chitosan, prior to drug delivery.

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.

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.

Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells

The bone marrow harbors a population of mesenchymal stem cells (MSCs) that possess the potential to differentiate into bone, cartilage, and fat, and along other tissue pathways. To date, MSCs from various species have been studied. Despite the bovine experimental model being widely used in experiments in vivo and in vitro, only a limited amount of information regarding bovine MSCs is available. The aim of this study was to isolate and induce the multilineage mesenchymal differentiation of bovine MSCs, thereby initiating further research on these cells. Bovine MSCs were isolated from eight calves, and osteogenic, chondrogenic, and adipogenic differentiation was induced by using a combination of previously reported protocols for other species. The level of differentiation was evaluated by histological examination and by analyzing the expression of tissue-specific genes by a quantitative “real time” reverse transcription/polymerase chain reaction technique. Following osteoinduction, the isolated fibroblast-like cells transformed into cuboidal cells and formed alkaline-phosphatase-positive colonies; during differentiation, these colonies transformed into mineralized nodules. In addition, osteogenesis was followed by osteocalcin and collagen type I mRNA expression. Chondrogenesis was confirmed by the demonstration of collagen type II, aggrecan, and sox9 mRNA expression in the cells stimulated by transforming growth factor β1 in monolayer culture. After being cultured in an adipogenesis-inducing medium, the MSCs responded by the accumulation of lipid vacuoles and the expression of adipocyte-specific genes. We have therefore demonstrated that cells harvested from bovine bone marrow are capable of in vitro extensive multiplication and multilineage differentiation, making them a relevant and invaluable model in the field of stem cell research.

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.

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.

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.

PHYSIOLOGICAL PRODUCTION OF SINGLET MOLECULAR OXYGEN IN THE MYELOPEROXIDASE-H2O2-CHLORIDE SYSTEM

The putative role of singlet oxygen (1O2) in the respiratory burst of neutrophils has remained elusive due to the lack of reliable means to study its quantitative production. To measure 1O2 directly from biological or chemical reactions in the near infrared region, we have developed a highly sensitive detection system which employs two InGaAs/InP pin photodiodes incorporated with a dual charge integrating amplifier circuit. Using this detection system, we detected light emission derived from a myeloperoxidase (MPO)‐mediated reaction in physiological conditions: pH 7.4, 1–30 nM MPO, 10–100 μM H2O2 and 100–130 mM Cl− in place of Br− without the use of deuterium oxide. The MPO‐H2O2‐Cl− system exhibited a single emission peak at 1.27 μm with a spectral distribution identical to that of delta singlet oxygen. Our results suggest physiological production of 1O2 in the MPO‐H2O2‐Cl− system at an intravacuolar neutral pH. The MPO‐mediated generation of 1O2, which may have an important role in host defense mechanisms, is discussed in connection with previous results.

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.