Papers by Author: Shinji Kamakura

Abstract: The present study was designed to investigate the mechanism of in vivo conversion from synthetic octacalcium phosphate (OCP) into hydroxyapatite (HA) at ultrastructural level, where the implanted OCP is enhancing bone regeneration in mouse calvarial bone defect. OCP granules were implanted into the subperiosteal area of the calvaria of 7-week-old BALB/c mice for 3 weeks. Transmission electron microscopy of undecalcified frontal sections, obtained from the acrylic resin-embedded skull specimens showed that the bone crystals in newly formed bone directly bonded to the OCP particles implanted. The morphological characteristic of original plate-like OCP particles was remained unchanged even after the implantation, whereas a number of de novo nano-particles were also directly formed onto the plate-like OCP particles. Some of OCP particles were linked with other OCP particles through these nano-particles. The results suggest that the OCP-apatite conversion, involving the enhanced bone regeneration, advances via topotaxial conversion without changing the original OCP morphology, accompanied by solution-mediated de novo nano-apatite formation, in the vicinity of the implanted OCP particles.

Abstract: Bone regeneration by calcium phosphates has been known to be intricately dependent on material properties or implanted milieu of host animals, such as site and species. Critical sized calvarial defects of mouse were recently used as the model for investigating bone regeneration ability and the mechanisms. The purpose of the present study is to investigate whether the critical sized mouse calvarial defects can be utilized to examine bone regeneration with synthetic octacalcium phosphate (OCP). OCP , prepared by wet synthesis methods, was sieved 0.3 ~ 0.5 mm in diameter and used for the animal experiment. At 14 days after surgery, histological examination showed that implantation of OCP grafted defects significantly enhanced bone formation compared with the control defect. OCP tended to convert to hydroxyapatite with time. The tartrate-resistant acid phosphatase (TRAP) positive osteoclastic cells were observed around the OCP particles. The results suggest that the mouse critical sized calvarial bone defects are useful model to investigate the bone formation by the OCP implantation.

Abstract: Our previous studies showed that synthetic octacalcium phosphate (OCP) enhances bone regeneration more than hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP). Also, a synthetic bone substitute constructed of synthetic octacalcium phosphate (OCP) and porcine atelocollagen sponge (OCP/Col) showed stable bone regeneration. The present study was designed to investigate the difference of bone regeneration by OCP/Col and other calcium phosphate-collagen composites. OCP/Col, β-TCP$-collagen% composite (β-TCP/Col), or HA$-collagen% composite (HA/Col) sponge was prepared from pepsin-digested atelocollagen isolated from the porcine dermis and OCP, β-TCP, or HA granules, respectively. A standardized critical-sized defect was made in the rat calvarium, and various calcium phosphate-collagen composites were implanted into the defect. The rats were fixed at four weeks after implantation and radiographic and histological examinations were performed by undecalcified cross sections of implants. Radiographic examination showed that uniform radiopaque masses were observed in the created defects treated with OCP/Col, whereas granulous and foggy radiopacity was observed in β-TCP/Col and HA/Col. Histological examination showed that newly formed bone was observed in the reticulum of OCP/Col and around the implanted OCP. The regenerated bone by β-TCP/Col or HA/Col seemed to be less than that by OCP/Col and would not to be nucleated by the granules of β-TCP or HA. The present study indicated that bone regeneration by OCP/Col was different from those of β-TCP/Col and HA/Col. Application of OCP/Col would be expected for clinical use in the future.

Abstract: Our previous studies suggested that synthetic octacalcium phosphate (OCP) enhances bone regeneration more than hydroxyapatite (HA). However, the molecular mechanisms to induce osteogenic phenotype in osteoblast by OCP have not been identified. OCP tended to convert into an apatite structure in vivo and in vitro, and its process was accompanied by calcium consumption from the surrounding solution and the release of phosphate ions into the solution at a physiological condition. The present study was designed to investigate whether the dissolution of ionic products of OCP affects on proliferation and differentiation of mouse bone marrow stromal ST-2 cells in vitro. The number of cells treated with OCP-conditioned medium was slightly decreased in comparison to that of control at day 7. On the other hand, the level of alkaline phosphatase activity increased in OCP-conditioned medium. These results demonstrated that OCP is capable of inducing osteoblastic cell differentiation in ST-2 cells.

Abstract: The present study was designed to investigate whether porous titanium (Ti) having Young’s modulus similar to bone has osteoconductive characteristics in rat critical-sized calvarial bone defect. The effect of coating by octacalcium phosphate (OCP) was also examined. OCP is known as a precursor of initial mineral crystals of biological apatite in bones and teeth. Ti powder was prepared by plasma rotating electrode process in an Ar atmosphere. Then, porous Ti disks, 8 mm in diameter with 1 mm thick, were obtained using the particles ranging from 300 to 500 +m, by sintering at 1573 K without applied pressure. The disks had about 35 vol% in porosity and about 10 GPa in Young’s modulus which corresponds to that of human cortical bone. Newly formed bone was observed so as to fill the pore up at 12 weeks, confirming the ability to conduct the ingrowths of the bone tissue. Although in vitro study showed that proliferation of mouse bone marrow stromal ST-2 cells was inhibited on the dishes coated by OCP rather than the control dish, OCP coating on porous Ti seemed to stimulate the bone formation in vivo. Taken together, it seems likely that porous Ti having Young’s modulus similar to bone shows osteoconductive characteristics to conduct bone ingrowths. OCP could be a potential coating agent to assist bone regeneration on porous Ti.

Abstract: Our previous study showed that synthetic octacalcium phosphate (OCP) enhanced bone regeneration more than hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP). Recently, we have engineered a composite of synthetic OCP and collagen (OCP/Collagen), which improved the handling performance and synergistically enhanced bone regeneration up to eight weeks after implantation. The present study investigated whether the regenerated bone by OCP/Collagen could be stable for long period. OCP/Collagen sponge was prepared from pepsin-digested atelocollagen isolated from the porcine dermis and OCP granules. A standardized critical-sized defect was made in the rat calvarium, and an OCP/Collagen was implanted into the defect. Five rats were fixed at twenty-four weeks after implantation and examined radiographically and histologically. Radiographic examination showed that radiopaque figure was occupied throughout the defect, whereas OCP/Collagen itself was no radiopacity before implantation. Histological examination showed that newly formed bone was observed throughout the defect in OCP/Collagen. The implanted OCP/Collagen tended to be resorbed and was replaced by newly formed bone. The regenerated bone was stable and matured. The present study indicated that bone regeneration by the implantation of OCP/Collagen was stable for long-term periods. Application of OCP/Collagen without both cell transplantation and exogenous osteogenic cytokines would result in cost-effective bone regenerative therapy in the future.

Abstract: It has been shown that fluoride ions enhance OCP hydrolysis into Ca-deficient apatite and that fluoridation in hydroxyapatite (HA) affects osteoblast activity. The present study was designed to investigate whether fluoridated Ca-deficient apatite (F-HA) formed via OCP enhances bone regeneration. F-HA was obtained through hydrolysis of the OCP in a solution containing 2 ppm fluoride at 37 °C and pH 7.4. A standardized critical-sized defect was made in the rat calvarium, and granules of F-HA were implanted into the defect. Five rats from each group were fixed through four to twelve weeks after implantation. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) confirmed that F-HA corresponded well to apatite structure. In week four, new bone matrix was formed around F-HA. In week twelve of F-HA group, newly formed bone matrix was more abundant, whereas the implanted F-HA was unresorbed and still remained. A statistical analysis in week twelve showed that the newly formed bone in the defect with F-HA was higher than that with untreated group. The fact that new bone was directly formed on F-HA implant suggests F-HA formed via OCP could be used as a bone substitute material.