Papers by Author: Yasuhiko Tabata

Abstract: Gelatin, a hydrophilic protein derived from collagen, has been widely used in drug delivery system because of its biocompatibility and biodegradability. However, its high water solubility limited its interaction with hydrophilic drugs. The current works propose a method to improve surface activity of gelatin by modifying it into an amphiphilic molecule via conjugating with cholesterol. High contents of cholesterol were conjugated to amino groups (-NH2) of gelatin using N,N’-Disuccinimidyl carbonate. Cholesterol contents were varied from 100% by mole of free -NH2 group in gelatin. The reduction of free -NH2 groups on gelatin determined by 2,4,6-trinitrobenzenesulfonic acid (TNBS) decreased with the increases of cholesterol used in the conjugation. The percentage of reduction of -NH2 content was 74.63 by mole respectively. Hydrophilicity/hydrophobicity changes were evaluated from water and ethylene glycol contact angles. The conjugated-gelatin were aggregated to form micelles at a critical micelle concentration (CMC) of 8 mg/ml in DI water (pH 5), determined by surface tension testing. Average size of the micelles were in range of 459.05±54.59 nm. The micelles were tested for curcumin entrapment for a cancer research.

Abstract: Currently, most commercialized peripheral nerve regenerative products are constructed from biodegradable polymers into hollow conduits. To speed up the regeneration rate, we proposed a development of a biocompatible protein-filled conduit for anastomosis amputated peripheral nerve with growth factor controlled release function. Glutaraldehyde-crosslinked protein sponges were tested for their abilities to controlled release of nerve growth factor (NGF) in vitro in our previous experiments. Type B gelatin sponges were able to limit diffusions of NGF due to electrostatic interactions between them. The rate of growth factor releases would be depended on degradation of the crosslinked gelatin. A nerve conduit model was produced using perfluoro alkoxy (PFA) tubes filled with gelatin which had been crosslinked using X-ray from Argon plasma treatment. This method of crosslinking provided 21.22±3.03 % degree of crosslinking. Hollow nerve conduits fabricated from poly(l-lactide-co-caprolactone) (PLCL) had a thicknesses and an inner diameters of 0.31±0.03 mm and 1.63±0.07 mm respectively. Average pore sizes of the inner surfaces and outer surfaces were 9.70±3.44 µm and 1.24±0.77 µm respectively. PLCL film supported growth of L929 mouse fibroblasts. For continuing works, we are testing the protein-filled conduits for peripheral nerve regeneration in animals.

Abstract: The aim of this study is to investigate in vivo local BMP-2 PK and bone induction in two bioceramics blocks (HAp, β-TCP), based on different composition and surface structures. We estimated the in vivo release profile of 125I–labeled BMP-2 and bone induction of hard tissues histologically. β-TCP is more effective for both BMP-2 retention and bone induction, compared to HAp, in the ectopic model.

Abstract: Bone microstructure and its functions are maintained by the activity of bone cells such as osteoclast for bone resorption and osteoblast for bone formation. In this study, we examined the role of osteoclast on the formation of the preferential orientation of biological apatite (BAp) as a bone quality parameter using OPG-KO and op/op mouse models in which the expression of osteoclast increases for osteoporosis and decreases for osteopetrosis. The orientation degree of the BAp c-axis was analyzed by a microbeam X-ray diffraction system. We found more decrease in the preferential alignment of the BAp c-axis along the longitudinal direction of bone in the femoral bones of both OPG-KO and op/op mice at 12 weeks compared with normal control mice. We concluded that changes in the amount and activity of osteoclast affect BAp alignment, resulting in the degradation of bone microstructure in osteoporosis and osteopetrosis.

Abstract: A new therapeutic trial based on the self-healing potential of cells to naturally induce tissue regeneration, has been recently noted. To realize this regenerative medical therapy, it is highly required to efficiently combine cells with their local environment which basically allows cells to survive and biologically function in vivo through the essential interaction. Tissue engineering is a biomedical technology or methodology to create the local environment which promotes the proliferation and differentiation of cells to induce tissue regeneration. There are some cases where tissue regeneration can be induced only by supplying a cell scaffold of biomaterials. Drug delivery system (DDS) with biomaterials enhanced the in vivo biological activities of un-stable growth factor and gene for cell-induced tissue regeneration. The controlled release technology enabled growth factors to achieve the regeneration of various tissues experimentally and clinically. The DDS technology also augmented the biological functions of plasmid DNA and small interference RNA. The cells genetically engineered by the DDS gene system showed an enhanced therapeutic efficacy in cell-based tissue regeneration (cell-gene hybrid therapy). By making use of DDS technology, it is possible to suppress the deterioration and proceeding of chronic fibrotic diseases based on the self-healing potential inherently equipped in the living body. This paper emphasizes significance of biomaterials in tissue engineering for regenerative medical therapy.

Abstract: Bone mechanical function is given as a result of the material and structural parameters of bone tissue. We previously reported that the material parameter of regenerated bone can be evaluated dominantly using two indices of the density and the preferred orientation degree of biological apatite (BAp). In addition, bone morphology remarkably changes during bone regeneration, which may lead to a dynamic change in the mechanical function of whole bone. In this study, therefore, material and structural parameters of regenerated bone are analyzed separately. A 5-mm-long defect was introduced in rabbit ulna and spontaneously regenerated, and then a three-point bending test was conducted at the regenerated portion. The important parameter which dominantly controls the whole bone mechanical function shifts from a structural to material parameter during bone regeneration. Moreover, it was statistically demonstrated that the increase in the material parameter is strongly determined by recovery of the orientation degree of the BAp c-axis.

Abstract: Since preferential orientation of c-axis of biological apatite (BAp) crystallites depends strongly on the shape of hard tissue, closely relating to the in vivo stress distribution, it is a useful parameter to judge the bone quality. In this study, preferential alignment of BAp crystallites in original and regenerated hard tissues were analyzed by the micro-beam X-ray diffractometer (μ-XRD) with a beam spot of 50 or 100 μm in diameter. Regenerating processes of bone defects introduced artificially in the rabbit ulna or skull were healed by inserting a biodegradable gelatin hydrogel incorporating basic fibroblast growth factor-2 (FGF-2). Recovery of BAp orientation alignment depends strongly on the regenerated portion and period, which is insufficient to recover the original level, while bone mineral density (BMD) is almost improved to the original level. This means that BMD recovers prior to improvement of the BAp orientation and the related mechanical function in the regenerated tissues. Thus, reloading on the regenerated portion caused by BMD restoration is suggested to accelerate to produce the appropriate BAp preferential alignment due to the remodeling process. The BAp orientation was finally concluded to be one of the most important indices to check the regenerative degree and process in the regenerated bone under the tissue engineering technique.

Abstract: Hydroxyapatite (HAp) has been used as a biomaterial for hard tissues. Critical characteristics of biomaterials will include surface geometry, hydrophobicity and hydrophilicity, crystallinity, biodegradation rates, and release pharmacokinetics (PK) of incorporated molecules such as BMP-2. Optimizing BMP-2 for clinical application may be dependent on localized sustained release from biomaterials. We forcused on in vivo local BMP-2 PK and bone induction in two ceramics systems, based on different surface structures. The functionally graded apatites (fg-HAp) was designed by the step-wise calcinations and partial dissolution-precipitation methods. We estimated the in vivo release profile of 125I-labeled BMP-2 from fg-HAp and the dose response of bone induction by BMP-2 in the back subcutis histologically. Bulk-HAp (b-HAp) by only the step-wise calcinations was prepared as a control. The amount of BMP-2 remaining in the fg-HAp at 1 day after implantation was 83.8%, while that was 34.6% in the b-HAp. Moreover, ectopic bone formation were found surely in the fg-HAp/BMP-2 (0.5μg) system at 3 weeks, not in the b-HAp/BMP-2 system. By using fg-HAp, it is likely that an extremely low dose of BMP-2 is enough to enhance bone induction if BMP-2 is appropriately delivered to the site of action.

Abstract: Absorption and formation of hard tissues are repeated in vivo by the activity of osteoclast and osteoblast, respectively. The preferential alignment of BAp or collagen fibril is thought to be closely related to the activity of the bone cells. In this study, changes in bone mineral density (BMD) and preferential alignment of biological apatite (BAp) were examined focusing on the role of osteoblast using the model of osteopetrotic (op/op) mice in which osteoblast activity was normal but the expression of osteoclast was reduced. Osteopetrotic (op/op) mice and their normal littermates aged 5, 12 and 24 weeks were analyzed. The BMD and BAp texture of femoral diaphysis were measured using peripheral quantitative computed tomography (pQCT) and a microbeam X-ray diffractometer system with a 50 µmφ diameter beam spot respectively. The decrease in osteoclast expression induced both the reduction of the skeletal system and calcification of the medullary cavity, which are typical features of osteopetrosis. As a result, the shape, BMD and preferential BAp alignment of the femur in the op/op mice were remarkably different from those in the control group. At the center of the femoral diaphysis, BMD in the cortical area showed no significant difference between the two groups, but preferential alignment of the BAp c-axis in the op/op mice group had a lower value than that in the control group. This suggests that the decrease in the number of osteoclasts suppresses normal remodeling, resulting in a decrease in bone quality, especially the preferential alignment of the BAp c-axis.