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Some ceramics show bone-bonding ability, i.e. bioactivity. Apatite formation on ceramics is an essential condition to bring about direct bonding to living bone when implanted into bony defects. A controlled surface reaction of the ceramic is an important factor governing the bioactivity and biodegradation of the implanted ceramic. Among bioactive ceramics, glass-ceramic A-W containing apatite and wollastonite shows high bioactivity, as well as high mechanical strength. In this study, glass-ceramics containing zinc oxide were prepared by modification of the composition of the glass-ceramic A-W. Zinc oxide was selected to control the reactivity of the glass-ceramics since zinc is a trace element that shows stimulatory effects on bone formation. Glass-ceramics were prepared by heat treatment of glasses with the general composition: xZnOx(57.0-x)CaOx35.4SiO(2)x7.2P(2)O(5)x0.4CaF(2) (where x=0-14.2mol.%). Addition of ZnO increased the chemical durability of the glass-ceramics, resulting in a decrease in the rate of apatite formation in a simulated body fluid. On the other hand, the release of zinc from the glass-ceramics increased with increasing ZnO content. Addition of ZnO may provide bioactive CaO-SiO(2)-P(2)O(5)-CaF(2) glass-ceramics with the capacity for appropriate biodegradation, as well as enhancement of bone formation.

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Bone cement consisting of polymethylmethacrylate (PMMA) powder and methylmethacrylate (MMA) liquid is clinically used for fixation of implants such as artificial hip joints. However, it does not show bone-bonding ability, i.e., bioactivity. The lack of bioactivity would be one of factors which cause loosening between the cement and the implant. The present authors recently showed the potential of bioactive PMMA-based bone cement through modification with gamma-methacryloxypropyltrimethoxysilane (MPS) and calcium acetate. In this study, the effects of the kinds of PMMA powder on setting time, apatite formation and compressive strength were investigated in a simulated body fluid (Kokubo solution). The cement modified with calcium acetate calcined at 220 degrees C could set within 15 min when the PMMA powder had an average molecular weight of 100,000 or less. The addition of calcium acetate calcined at 120 degrees C in the PMMA powder required a much longer period for setting. The modified cements formed an apatite layer after soaking in the Kokubo solution within 1 day for cement starting from PMMA powder with a molecular weight of 100,000 or less. Compressive strengths of the modified cements were more than 70 MPa for cements starting from 100,000 and 56,000 in molecular weight. After soaking in Kokubo solution for 7 days, the modified cement consisting of PMMA powder of 100,000 in molecular weight showed a smaller decrease in compressive strength than that consisting of 56,000 in molecular weight. These results indicate that bioactive PMMA cement can be produced with appropriate setting time and mechanical strength when PMMA powders with a suitable molecular weight are used. Such a type of design of bioactive PMMA bone cement leads to a novel development of bioactive material for bone substitutes.

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Covalent immobilization of heparin has been developed to reduce the amount of heparin administered systematically during long-term dialysis. Recently, it was doubted partially because of the complexion during immobilization process. In this study, we investigated a novel method for specific immobilization of heparin on polysulfone (PSF) via free electron laser (FEL) irradiation. Laser wavelengths of 6.18 or 6.31 microm, the typical absorption bands of carboxyl groups of heparin and aromatic rings in PSF, respectively, were chosen to irradiate the thin heparin membrane formed on PSF surfaces. The amount of heparin immobilized on PSF was measured by the toluidine blue method. The binding of heparin on PSF was analyzed by X-ray photoelectron spectroscopy (XPS). The immobilization of heparin resulted in a hydrophilic surface on which decreased platelet adhesion was observed. The efficiency differences, depending on laser wavelengths, were discussed from the point of view of structural and environmental differences of light-absorbing groups.

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The construction of organs by tissue engineering and regenerative engineering, using an artificial extracellular matrix, is an innovative method that is expected to replace artificial organs and organ transplantation. We have produced an artificial extracellular matrix of alginate and demonstrated that the matrix stimulated the regeneration of skin, nerve, and bone. In this report, the new matrix, which consists of heparin and alginate covalently crosslinked with ethylenediamine, was produced to stabilize and control the release of growth factors. Heparin content of the new matrix was confirmed by toluidine blue absorption, elementary analysis, and Fourier transform infrared spectrum. In vitro experiments showed that the new matrix significantly suppressed the initial burst of basic fibroblast growth factor, which is a representative member of heparin-binding growth factors, and released biologically active basic fibroblast growth factor for 1 month under physiological conditions. Obvious cellular infiltration and angiogenesis were shown to occur in the new matrix which was implanted subcutaneously in the dorsal area of rat with 1 microg of basic fibroblast growth factor for 2 weeks. This new matrix may be useful for not only the construction of transplantable blood vessels of small diameter, but also the induction of angiogenesis in regenerated skin constructed by tissue engineering.

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A novel material for nerve regeneration, alginate, was employed in both tubulation and nontubulation repair of a long peripheral nerve defect injury. Twelve cats underwent severing of the right sciatic nerve to generate a 50-mm gap, which was treated by tubulation repair (n = 6) or nontubulation repair (n = 6). In the tubulation group, a nerve conduit consisting of polyglycolic acid mesh tube filled with alginate sponge was implanted into the gap and the tube was sutured to both nerve stumps. In the nontubulation group, the nerve defect was repaired by a simple interpolation of two pieces of alginate sponge without any suture. The animals in both groups exhibited similar recovery of locomotor function. Three months postoperatively, successful axonal elongation and reinnervation in both the afferent and efferent systems were detected by electrophysiological examinations. Intracellular electrical activity was also recorded, which is directly indicative of continuity of the regenerated nerve and restoration of the spinal reflex circuit. Eight months after operation, many regenerated myelinated axons with fascicular organization by perineurial cells were observed within the gap, peroneal and tibial branches were found in both groups, while no alginate residue was found within the regenerated nerves. In morphometric analysis of the axon density and diameter, there were no significant differences between the two groups. These results suggest that alginate is a potent material for promoting peripheral nerve regeneration. It can also be concluded that the nontubulation method is a possible repair approach for peripheral nerve defect injury.

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Freeze-dried alginate sponge crosslinked with covalent bonds was developed in our laboratory and has been demonstrated to enhance peripheral nerve regeneration. In this study, we examined spinal cord repair using alginate sponge in infant rats. On postnatal day 8-12, the spinal cord was transversely resected at Th7-Th8 to produce a 2-mm gap. The gap was filled with alginate sponge in the alginate group. For the control group, the gap was left empty. In the alginate group, the recovery of evoked electromyogram and sensory-evoked potentials 6 weeks after surgery indicated that elongation of axons could establish electrophysiologically functional projections through the gap. A histological study revealed that myelinated and unmyelinated axons, surrounded by a perineurial-like structure, had elongated across the gap. An immunohistochemical examination revealed that elongation of astrocytic processes and/or migration of astrocytes into the alginate sponge was induced, whereas astrocyte gliosis was reduced at the interface between the implanted alginate and the host spinal cord, compared with the control group. However, a horseradish peroxidase tracing study revealed ascending and descending fibers had also elongated into the gap and reentered the other stump of the transected spinal cord beyond the gap. These results suggest that alginate might provide a permissive microenvironment for elongation of spinal cord axons.

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Poly (methyl methacylate) (PMMA) bone cement is one of the popular bone-repairing materials for fixation of artificial hip joints. Significant problems on the PMMA bone cement are caused by loosening at the interface between bone and the cement, since the cement does not show bone-bonding, i.e. bioactivity. Development of PMMA bone cement capable of bone-bonding has been therefore long desired. The prerequisite for an artificial material to show bone-bonding is the formation of a biologically active bone-like apatite layer on its surface when implanted in the body. The same type of apatite formation can be observed on bioactive materials even in a simulated body fluid (Kokubo solution) with ion concentrations nearly equal to those of human blood plasma. Fundamental researches for bioactive glasses and glass-ceramics revealed that the apatite deposition is initiated by release of Ca2+ ions from the material into the body fluid, and by catalytic effect of Si-OH groups formed on the surface of the material. These findings lead an idea that novel bioactive cement can be designed by incorporation of Si-OH groups and Ca2+ ion into PMMA bone cement. In the present study, PMMA bone cement is modified with 20 mass % of various kinds of alkoxysilanes and calcium salts, and its apatite-forming ability was evaluated in Kokubo solution. The apatite formation was observed on the surface of the modified cements containing 20 mass % of CaCl2, irrespective of the kind of the examined alkoxysilane. On the other hand, the apatite formation was observed on the cement containing CaCl2, Ca(CH3COO)2 or Ca(OH)2, but not on the cement containing CaCO3 or beta-Ca3(PO4)2, even when the cement contains 3-methacryloxypropyltrimethoxysilane (MPS). The results indicate that modification with alkoxysilane and calcium salts showing high water-solubility is effective for providing PMMA bone cement with bioactivity.

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The irradiation effects of oxygen on polysulfone have been investigated at energies of 20 keV, 150 keV and 2 MeV. The strong improvement of endothelial cell adhesion and proliferation is found on ion irradiated polysulfone at 20 keV. Such improvement is declined with increasing ion energy. The changes of surface color and free energy are strongly dependent on ion energy and dose. The formation of amorphous carbon phase is demonstrated by Raman spectroscopy and its degree is correspondent to the color changes observed. The formations of hydroxyl and carboxyl groups are confirmed by the attenuated total reflectance (ATR) FTIR spectroscopy. The depletions of heteroatoms are conjectured by detail analysis of X-ray photoelectron spectroscopy (XPS). Since no single one of these changes can be related directly to the improved adhesion and proliferation of endothelial cells on irradiated surface, we argue that the distribution of functional groups is crucial in promoting the adhesion of endothelial cells. Although the distribution cannot directly be detected at present, the irradiation effects were related to the results of TRIM simulation. The surface changes can be controlled by adjusting the size energy and dose of irradiating ion for the optimum morphology to cell adhesion.

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A new process for surface modification of polymers with multi-source cluster deposition apparatus has been reported in our previous work. The apparatus simultaneously supplies reactant of ammonium sulfamate and activator of energetic Ar(+) ion. In this work chemical changes are analyzed on the basis of XPS spectra and the relations of contact angle and platelet adhesion with chemical changes are discussed. Polymer film, setting on a turning holder, was irradiated by Ar(+) ions during bombardment with ammonium sulfamate clusters. The Ar(+) ion source served for activation of polymer surface and a cluster ion source supplied ammonium sulfamate molecules to react with activated surface. After thorough washing with deionized sterile water, the modified surfaces were evaluated in terms of contact angle of water, elemental composition and binding state on XPS and platelet adhesion with platelet rich plasma (PRP). The modification of polysulfone decreased the contact angle of water on surfaces from 82.6 down to 34.5 degrees. The adhesion number of platelets were decreased to one-tenth of the original surface. Ammonium, amine, sulfate and thiophene combinations were formed on the modified surfaces. The primary studies showed successful modification of polysulfone with ammonium sulfamate by assistance of Ar(+) ion irradiation. The polar groups like N-sulfate were formed on surfaces and contribute to the decrease of surface contact angle and adhesion number of platelets. Since the same process can also be applied to other polymeric materials with various substrates, combining with the features of no solvent and no topographic changes, this method might be developed in a promising way for modification of polymers.

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Bone morphogenetic proteins (BMP) are unique molecules with a specific biological activity for inducing ectopic bone formation when implanted with a suitable carrier matrix. However, incorporation of BMP into the carrier has disadvantages, including early burst release and protein degradation in biological environments. Therefore, we considered that the next greatest challenge in achieving successful clinical use was the development of a carrier system for site-specific delivery of the morphogenetic signal of BMP. In this study, a novel BMP-2-derived oligopeptide, NSVNSKIPKACCVPTELSAI, was coupled covalently to alginate. Then NSVNSKIPKACCVPTELSAI-linked alginate hydrogel composites were implanted into the calf muscle of rats and harvested 3 or 8 weeks after surgery. Ectopic bone formation was observed in alginate hydrogel linked with BMP-2-derived peptide. It is suggested that alginate hydrogel linked with an oligopeptide derived from BMP-2 might provide an alternative system for topical delivery of the morphogenetic signal of BMP-2.

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Many materials have been used for artificial tubular prostheses to assist peripheral nerve gap reconstruction. However, the clinical use of these devices has been restricted because a microsurgical procedure requires specialized techniques and expensive equipment, such as operating microscope systems. Therefore the authors developed a new gluing method, without sutures, that uses freeze-dried alginate gel. A 7-mm gap in the sciatic nerve of rats was bridged with freeze-dried alginate gel. Regeneration was evaluated by electrophysiologic testing and histologic study. Eighteen weeks after surgery, functional reinnervation of motor and sensory nerves had occurred, as demonstrated by recovery of compound muscle action potentials (CMAP), compound nerve action potentials (CNAP), and somatosensory-evoked potentials (SEP). Histologically, many regenerated nerve fasciculi, including myelinated and unmyelinated fibers, were observed and the implanted alginate gel had disappeared. In conclusion, a gluing technique using alginate gel is a potential alternative to the conventional nerve autograft technique. Advantages include simple application and rapid repair. Freeze-dried alginate gel is a promising material for artificial nerve guides for peripheral nerves and also could be used for repair of disrupted pathways in central nervous tissue that is amorphous and cannot be sutured.

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We have recently reported that freeze-dried alginate gel, which was developed in our laboratory, enhanced peripheral nerve regeneration. The purpose of this study was to examine whether alginate gel is capable of promoting nerve regeneration in the severed spinal cord of adult mammals. Using Wistar rats at 30 days of age (P30), the T9-T10 spinal cord was totally resected and alginate gel was implanted across the gap. Forty-five days after surgery myelinated and unmyelinated axons regenerated throughout the gap with remaining alginate gel. The elongated axons established electrophysiologically functional projections across the gap. In conclusion, freeze-dried alginate gel could be a promising material as an artificial nerve guide for repair of injured central nervous system.

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Alginate dressings are currently used in the management of epidermal and dermal wounds, and provide a moist environment that leads to rapid granulation and reepithelialization. However, a cytotoxic effect on proliferation of fibroblasts and residual material with inflammation in healing wounds have been reported recently. We have developed a new alginate dressing (AGA-100), which does not have an inhibitory effect on proliferation of fibroblasts. The purpose of this study was to evaluate the new alginate dressing with respect to wound healing in full- and partial-thickness pig wounds and with respect to biodegradation following implantation into rabbit muscle. Kaltostat and Sorbsan, both well-established commercial dressings, were used as control. The closure rate of full-thickness wounds treated with AGA-100 was significantly higher on day 15 compared with that with Kaltostat and Sorbsan. Reepithelialization rate of partial-thickness wounds treated with Sorbsan was statistically significantly lower on day 3 than those with the other two dressings. As to dressing debris remained in the healing wound, a large amount of foreign debris was noted in all the full-thickness wounds treated with Kaltostat or Sorbsan, while only about one-third of wounds treated with AGA-100 showed a little dressing debris. AGA-100 implanted into the muscle of rabbits was bioresorbed completely within 3 months. Therefore, dressing residue in AGA-100-treated full-thickness wounds might be fully absorbed in a few months. In conclusion, it is shown that our newly developed AGA-100 possesses superior properties compared with typical alginate dressings.

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The aim of this study was to construct a novel drug delivery system suitable for controlled release of antibiotics. There is a need for devices that release antibiotics only during microbial infection, because prophylactic or prolonged use of antibiotics leads to serious problems, such as renal and liver toxicity and the emergence of drug-resistant bacteria (e.g., meticillin-resistant Staphylococcusaureus). We found previously that Staphylococcus aureus-infected wound fluid showed high thrombin-like activity; therefore, in this study we designed an antibiotic release system triggered by thrombin activity. We synthesized an insoluble polymer-drug conjugate in which gentamicin was bound to poly(vinyl alcohol) hydrogel through a newly developed thrombin-sensitive peptide linker. The conjugate released gentamicin when it was incubated with Staphylococcus aureus-infected wound fluid, with thrombin and leucine aminopeptidase, or with human plasma and Ca2+, whereas no biologically active gentamicin was released when the conjugate was incubated with noninfected wound fluid, with leucine aminopeptidase alone, with thrombin alone, or with plasma. Furthermore, the conjugate reduced the bacterial number in an animal model of Staphylococcus aureus infection. These results demonstrated that the conjugate has sufficient specificity and excellent potential as a stimulus-responsive, controlled drug release system.

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We have developed a novel artificial nerve guide composed of biodegradable freeze-dried alginate gel covered by polyglycolic acid mesh, and evaluated its effect on peripheral nerve regeneration, using a 50-mm gap cat sciatic nerve model. Functional reinnervation of motor and sensory nerves occurred 13 weeks after implantation, as demonstrated by recovery of compound muscle action potential (CMAP) and somatosensory evoked potential (SEP). For histologic evaluation, samples of tissue were harvested from the grafted material segment 7 months after operation. Many newly developed nerve fasciculi were found, and the implanted nerve guidance material had completely disappeared with little inflammation. These results indicate that freeze-dried alginate gel allows the nerve to regenerate across longer gaps than described in previous literature.

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An ideal drug delivery system (DDS) releases an appropriate drug at specific locations and times. We tried to create a new antibiotic delivery system that releases gentamicin only when wounds are infected by Pseudomonas aeruginosa (P.A.). Exudate from the dorsal pouch of rats infected with P.A. showed significantly higher hydrolytic activity-thrombin-like activity-toward Boc-Val-Pro-Arg-MCA than exudate from noninfected wounds. We therefore constructed a device for controlled release of an antimicrobial drug triggered by thrombin-like activity. Briefly, gentamicin was bound to a polyvinyl alcohol derivative (PVA) hydrogel through a newly developed peptide linker cleavable by the proteinase, PVA-(linker)-gentamicin. In vitro experiments showed that proteinases from wounds infected with P.A. cleaved the linker and gentamicin was released while the exudate from noninfected wounds had no hydrolytic activity toward the linker. This device shows potential as an occlusive dressing with an effective antibiotic delivery system for treating infected wounds.

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We have previously reported an elevation of thrombin-like activity in infected wound exudates. Therefore, using this enzymatic activity as a biological signal, a system which can release an antimicrobial drug at infected wounds was investigated. In this paper, we report thrombin-sensitive peptide linkers, the key component of this system. Starting from amino acid sequences of the cleavage site in fibrinogen, which is the substrate of thrombin, we synthesized some thrombin-sensitive peptide linkers. We constructed devices in which the thrombin-sensitive peptide linker interconnected between polyvinyl alcohol hydrogel and gentamicin. The device was able to release gentamicin in response to thrombin.

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Calcium alginate dressings have beneficial effects on wound healing by providing a moist wound environment. However, cytotoxicity and the nonbiodegradable nature of calcium alginate dressings induce unresolved chronic foreign-body reaction. In this study, a novel freeze-dried alginate gel dressing (AGA-100) low in calcium ions was evaluated for cytotoxicity to L929 cells in vitro and in full-thickness pig wounds in vivo. Cytotoxicity testing on L929 cells showed the cytocompatibility of AGA-100 extracts, while extracts from Kaltostat, a well-established alginate dressing, induced cytopathic effects. In an in vivo study using pigskin, AGA-100, Kaltostat, and gauze were applied on 1-in-diameter circular full-thickness wounds on the back of pigs and the time course of wound closure was evaluated. Kaltostat and gauze dressings were used as controls. For histologic evaluation, wound tissue was harvested on day 18. AGA-100-treated wounds showed rapid wound closure compared to control wounds on day 15. Foreign-body reaction was marked in Kaltostat- and gauze-treated wounds, and differed significantly from AGA-100-treated wounds. Based on these data, AGA-100 could reduce the cytotoxicity to fibroblasts and foreign-body reaction that have been observed with currently available calcium alginate dressings; it was also found to be useful as an alginate dressing.

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The aim of this study was to develop a new wound dressing with controlled release of antibiotics only in the presence of infection. In the first experiment using an infected dorsal pouch of rats, exudate containing proteinases from pouches contaminated with Staphylococcus aureus or Pseudomonas aeruginosa showed significantly higher hydrolytic activity toward Boc-Val-Pro-Arg-MCA than that from noninfected wounds. The authors then developed a new type of wound dressing (AGX), a drug delivery system in which gentamicin is bound to polyvinylalcohol hydrogel through an enzymatically degradable peptide linker containing a -(D)-Phe-Pro-Arg-sequence. To investigate in vitro effectiveness, AGX was incubated with exudate from S. aureus infected or P. aeruginosa infected wounds. Gentamicin was selectively released from AGX in the presence of the exudate from S. aureus infected or P. aeruginosa infected wounds, but was not released in the presence of exudate from noninfected wounds. Next, AGX or the polyvinylalcohol hydrogel that served as control was incubated with S. aureus in the presence of human plasma. After 24 hours, S. aureus concentration was markedly lower in the case with AGX than in that with polyvinylalcohol hydrogel. These results indicate that proteinases from wounds infected with S. aureus or P. aeruginosa cleaved the linker and gentamicin was released.

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