Extracorporeal high-pressure therapy (EHPT) for malignant melanoma consisting of simultaneous tumor eradication and autologous dermal substitute preparation.

Surgical resection of skin tumors leads to large defects in surrounding normal tissues, which should be reconstructed thereafter using the patient's own tissues taken from the other site. Our challenge is to solve this problem in dermal malignant melanoma (MM) by a novel process, named extracorporeal high pressure therapy (EHPT), in which the tissue containing tumor is resected and pressurized, and the treated tissue is re-transplant back to the same position as a tumor-free autologous dermal substitute. The key points are complete tumor death and preservation of native extra cellular matrix (ECM) by the hydrostatic pressure. We found that high hydrostatic pressure at 200 MPa for 10 min at room temperature is completely cytocidal against MM cells in suspension form, in monolayer form, and even in the solid tumor form. MM tumor-bearing nude mice were established by injected human MM cells intradermally and treated by EHTP. The denaturation of the dermal extra cellular matrices was so mild that the pressurized skin was well engrafted as tumor free autologous dermal tissues, resulting in the complete eradication of the MM without any unnecessary skin reconstruction surgery. This very simple and short pressing treatment was proved to make the tumor tissue to the transplantable and tumor-free autologous dermal substitute, which can be applicable to the other temporally resectable tissues.

Peripheral nerve regeneration and electrophysiological recovery with CIP-treated allogeneic acellular nerves.

Acellular nerve grafts are a desirable alternative to autografts, both because the source of acellular nerves is potentially unlimited and because they have the same matrix structure as natural nerves, which would facilitate axon growth from the defective nerve stump. Although some acellular nerves have been developed, most of them were studied in isogenic transplantation models and evaluated only by histological observation. In the present study, novel allogeneic acellular nerves prepared using the cold isostatic pressuring (CIP) method were developed and assessed as a potential substitute for autografts. The host immune response to acellular nerves and fresh nerves was analyzed using Lewis rats as donors and SD rats as recipients, which is the allogeneic transplantation model, by subcutaneous implantation for one month. In addition, sciatic nerve transplantation into a 10-mm nerve gap was carried out using the same model, and the axonal growth in acellular nerve transplantation was evaluated histologically and electrophysiologically, and compared with that of axons in the autograft transplant area. The subcutaneously implanted acellular nerves contained more macrophages and less vasculature than the allogeneic fresh nerves. In spite of these results of the subcutaneous implantation, Schwann cell infiltration in the graft transplanted into the sciatic nerve gap was observed after the short-term transplantation. The myogenic potential, which was measured as an index of electrophysiological function in acellular nerve transplantation, was also recovered in the long-term transplantation. Our results indicate that the acellular nerves developed herein have the potential to support nerve regeneration and might be useful as an alternative to autografts.

Novel adhesion prevention membrane based on a bioresorbable copoly(ester-ether) comprised of poly-L-lactide and Pluronic: in vitro and in vivo evaluations.

Block copolymers consisting of poly(L-lactide) (PLLA) and poly(oxyethylene-co-oxypropylene), with various compositions, were synthesized and characterized in vitro and in vivo for their application as postoperative adhesion prevention membranes. It was found that the flexibility and degradability of the cast films of the block copolymers grew with increasing Pluronic F68 [PN; poly(oxyethylene-co-oxypropylene] composition. The receding contact angle of the copolymer films against water became lower than that of the PLLA film, because the surface was predominantly covered with more hydrophilic PN segments in a wet state. This surface property significantly affects the cell attachment property of the copolymer films, and the fibroblasts cultured on the films exhibit a spheroid-like morphology. The copolymer films subcutaneously implanted in the back of rats induced milder tissue responses compared with PLLA homopolymers, because of the increased surface hydrophilicity in the former. In vivo evaluation using a uterus horn model in rats revealed that the performance of these copolymer films as an adhesion-prevention membrane is comparable to that of a conventionally utilized membrane of oxidized regenerated cellulose. These results indicate that the copolymer films are biocompatible materials with controllable mechanical properties and biodegradability as adhesion-prevention membranes.

Cross-linking of amniotic membranes.

Human amniotic membrane was cross-linked with chemical and radiation methods to investigate the effect of cross-linking on its physicochemical and biodegradation properties. Radiation cross-linking was performed with gamma-ray and electron beam while chemical cross-linking was with glutaraldehyde (GA). Both gamma-ray and electron beam irradiation decreased the tensile strength and elongation at break of the amniotic membrane with an increase in the irradiation dose, whereas GA cross-linking had no effect on the tensile properties. This is probably due to the scission of collagen chains through irradiation. No significant change was observed on the water content of cross-linked amniotic membranes for any of the crosslinking methods and in marked contrast with cross-linking of a gelatin membrane. A permeation study revealed that protein permeation through the amniotic membrane was not influenced by the GA concentration at cross-linking. These findings are ascribed to the structure characteristic of the amniotic membrane. The membrane is composed of a fibrous mesh structure from an assemblage of collagen fibers. It is possible that cross-linking takes place in the interior of the fiber assembly without impairing the mesh structure, resulting in no change of the water content and protein permeability. In vitro degradation of cross-linked amniotic membranes revealed that radiation cross-linking appeared to be much less effective than GA cross-linking in retarding the degradation, probably because of low cross-linking densities. GA-cross-linked amniotic membranes were degraded more slowly as the GA concentration at cross-linking increased. When the GA-cross-linked amniotic membrane was subcutaneously implanted in the rat, the tissue response was mild, similar to that of the non-cross-linked native membrane.

Effect of basic fibroblast growth factor on cartilage regeneration in chondrocyte-seeded collagen sponge scaffold.

A chondrocyte-collagen composite was prepared in an attempt to regenerate cartilage by its subcutaneous implantation in nude mouse. When the composite was impregnated with basic fibroblast growth factor (bFGF) prior to implantation, regeneration of the cartilage tissue was remarkably accelerated. Histological staining of the implanted composites with Safranin O-fast green revealed that the cells incorporated in the composites exhibited their phenotype and formed a new matured cartilage. A thin layer of fibrous capsule was observed surrounding the implanted composite and the inflammatory response of the host to the implant was mild. Specific proteoglycans were accumulated in the composite even 1 week after implantation. At 2 weeks after implantation, the chondrocytes regenerated the cartilage tissue, although still immature, but at 4 weeks almost all of the chondrocytes transferred to the mature stage. Conversely, such mature cartilage tissue was not noticed up to 4 weeks after implantation if the collagen scaffold was not impregnated with bFGF. Moreover, the mature area was limited to only a small fraction of the implanted composite, unless bFGF was incorporated in it.

Protein permeation through poly(vinyl alcohol) hydrogel membranes.

Hydrogel membranes were prepared by radiation and chemical cross-linking of poly(vinyl alcohol) (PVA) in aqueous solutions. Effects of PVA concentration, PVA molecular weight and radiation dose, as well as concentration of cross-linking agent, in the case of the chemical cross-linking procedure, on the permeation of insulin, albumin and immunoglobulin (IgG) through the membranes were investigated. Glucose permeation was also studied. The cross-linking density affected the size of the macromolecular mesh of the hydrogel network and thus the water content of the membrane responsible for the diffusion of the solutes. The diffusion coefficient linearly increased for all the solutes with increasing water content in the PVA hydrogels, indicating that diffusion occurs primarily through the water hydrating the polymer network. The permeability study showed that the water content as well as the mesh size had an influence on the diffusion of low molecular weight glucose and insulin. Although the diffusion of higher molecular weight solutes, such as albumin and IgG, was not so much affected by the mesh size of elaborated PVA hydrogel membranes, the diffusion of these proteins was very low.

Insulin release from a bioartificial pancreas using a mesh reinforced polyvinyl alcohol hydrogel tube. An in vitro study.

Islet transplantation with a bioartificial pancreas is a potential alternative to whole pancreas transplantation. The authors constructed a bioartificial pancreas using mesh reinforced polyvinyl alcohol hydrogel tubes (MRPT), in an attempt to clarify the in vitro responsiveness to glucose of islets seeded in the MRPT. When the MRPT were perfused in a small chamber with buffer containing 3.3 mmol or 16.7 mmol glucose, insulin release from the MRPT began to increase at 9 +/- 3 min, reaching a plateau at approximately 40 min after the glucose concentration in the perfusate increased from 3.3 to 16.7 mmol. When MRPT seeded with islets were subjected to static incubation in buffer containing 3.3 mmol or 16.7 mmol glucose, insulin release from the MRPT remained elevated for 3 hr of high glucose stimulation, the amount of secreted insulin depending upon the number of islets seeded. Although pre incubation of semipermeable membranes in culture medium containing fetal bovine serum prior to seeding with islets has recently been reported to improve insulin release, the authors found that such pre treatment of the MRPT did not have a beneficial effect. Their in vitro findings in this study suggest that the bioartificial pancreas using MRPT could be a promising therapeutic approach to human diabetes mellitus.

Effects of nicardipine on tube formation of bovine vascular endothelial cells in vitro.

BACKGROUND AND PURPOSE: The purpose of this study was to assess the effect of nicardipine, a Ca2+ channel blocker, on angiogenesis in vitro. METHODS: Bovine carotid artery endothelial cells were cultured between type I collagen gel layers with 10(-9) to 10(-5) M nicardipine. The morphological changes were monitored by phase-contrast microscopy and photographed. The total length of tubular structures was measured with an image analyzer system. Endothelial proliferation and migration assays were also performed with the same doses of nicardipine. RESULTS: Cultured endothelial cells form tubular structures between collagen gel layers. Tube formation of endothelial cells was suppressed by culture with 10(-9) to 10(-5) M nicardipine in a dose-dependent manner. Migration of endothelial cells was also suppressed by the same doses of nicardipine. However, proliferation of endothelial cells was not enhanced. CONCLUSIONS: Nicardipine acts as an inhibitor of angiogenesis in vitro by inhibiting the migration of endothelial cells. This result suggests that nicardipine may have therapeutic potential in angiogenic disorders such as tumor growth, atherogenesis, and diabetic retinopathy.

Rapid morphological changes in bovine brain microvascular endothelial cells on extracellular matrices.

The morphological changes in endothelial cells derived from bovine brain microvasculature, carotid artery, and aorta during growth on extracellular matrices were compared. All cells formed tubular structures on a basement membrane. Ultrastructural studies showed that the tubular structures had lumens surrounded by many endothelial cells. On type I collagen gel, brain microvascular endothelial cells still formed tubular structures, but the other two cell types formed confluent monolayers. However, when a second layer of collagen gel was laid over these cells, tubular structures developed within 2-3 days. Brain microvascular endothelial cells form tubular structures more readily than endothelial cells derived from large vessels on both basement membrane and type I collagen gel.

[Rapid morphogenesis of brain microvascular endothelial cells in culture system].

Endothelial cells of bovine brain microvascular vessels (BBECs), carotid artery (BCECs) and aorta (BAECs) were cultured on type I collagen and Matrigel. BBECs make tubular structures and BCECs and BAECs grow and make confluent monolayer on type I collagen gel. But BCECs and BAECs make tubular structures when second layer was overlaid. BBECs, BCECs and BAECs make tubular structures on Matrigel. These morphological changes were not affected by basic fibroblast growth factor. These results suggest that BBECs have more potent angiogenic ability than BCECs and BAECs.

[The effect of nicardipine on angiogenesis in vitro].

We studied the effect of nicardipine, a calcium channel blocker, on the morphological change of endothelial cells in vitro. Cultured endothelial cells derived from bovine carotid artery make tubular structures between collagen gel layers. Tube formation of endothelial cells was suppressed by culture with 10(-9)-10(-5) M of nicardipine in a dose dependent manner. Migration of endothelial cells was also suppressed by the same dose of nicardipine. However, proliferation of endothelial cells was not enhanced. These findings suggest that nicardipine acts as an inhibitor of angiogenesis in vitro by inhibiting the migration of endothelial cells.

Experimental hybrid islet transplantation: application of polyvinyl alcohol membrane for entrapment of islets.

In this study, we first examined in vitro a polyvinyl alcohol membrane to be used to contain hybrid islet cells, and second we tested a bioartificial pancreas with entrapment of pancreatic islets in polyvinyl alcohol membrane in rats with experimentally induced diabetes. The permeability of the polyvinyl alcohol membrane to different substances was studied in a two-cell chamber system. Glucose, insulin, and nutrients passed through the membrane easily, whereas the passage of immunoglobulin G was completely prevented, indicating that this membrane could be effective in protecting the bioartificial pancreas from immunorejection. Approximately 2,000 islets collected from three Sprague-Dawley rats were enclosed in a mesh-reinforced polyvinyl alcohol tube and transplanted into the peritoneal cavity of six Wistar rats with streptozotocin-induced diabetes. Their nonfasting serum glucose levels were significantly decreased for at least 12 days. Six diabetic rats receiving intraperitoneal transplantation of free islets without the tube showed a slight but significant decrease in nonfasting serum glucose levels for only 3 days. One diabetic rat with transplantation of the bioartificial pancreas had a significant and sustained decrease in nonfasting glucose levels from pretransplanted levels of 440-500 mg/dl to a mean value of 162 +/- 13 mg/dl for over 3 months without immunosuppression. The bioartificial pancreas was then removed, and glucose levels gradually increased to over 500 mg/dl. The results of the present study suggest that a bioartificial pancreas with entrapment of islets in a polyvinyl alcohol membrane could be a promising therapeutic approach to diabetes mellitus.