Shinji Kamakura

Implantation of Octacalcium Phosphate (OCP) in Rat Skull Defects Enhances Bone Repair

Synthetic octacalcium phosphate (OCP) enhances bone formation if implanted into the subperiosteal region of murine bone. Such implanted OCP may be resorbed and replaced by bone with time. We hypothesized that OCP could be used as an effective bone substitute. To test this hypothesis, we designed the present study to investigate if bone repair in a rat skull defect is enhanced by the implantation of OCP. Rats were divided into two groups: OCPtreated animals and untreated controls. Six rats from each group were fixed at 4, 12, and 24 weeks after implantation. A full-thickness standardized trephine defect was made in the parietal bone, and synthetic OCP was implanted into the defect. After being examined radiographically, the specimens were decalcified and processed for histology. OCP implantation significantly promoted bone repair compared with the controls. A statistical analysis showed an increase in the area of radiopacity within the skull defect between week 4 and week 12. Histologically, bone was formed on the implanted OCP and along the defect margin at week 4. At week 12, the implanted OCP was surrounded by newly formed bone. At week 24, the defect was almost completely filled with bone. In the control, bone formation was observed only along the defect margin. The present results demonstrate that OCP could be used as an effective bone substitute.

Bone Regeneration by Synthetic Octacalcium Phosphate and its Role in Biological Mineralization

Octacalcium phosphate (Ca8H2(PO4)6 * 5H2O; OCP) has been advocated to be a precursor of biological apatite crystals in bone and tooth. Recent studies, using physical techniques, showed that OCP is present as a transient phase during biological apatite formation in human dentin, porcine enamel and murine bone. However, there is still a controversy regarding the chemical nature of the first mineral formed in the biominerals. A number of studies have demonstrated that synthetic OCP shows bone regenerative and biodegradable characteristics, rather than other calcium phosphate bone substitute materials, such as hydroxyapatite (Ca10(PO4)6(OH)2; HA) ceramic. It seems likely that synthetic OCP may be an alternative to autogenous bone graft. It is known that OCP contains alternative layers of water molecules and an apatite structure, and that the transition of OCP to HA is likely to be spontaneous and irreversible. The conversion process induces modification of local environment adjacent to OCP surface, including the changes in adsorption of serum proteins and concentration of calcium and inorganic phosphate ions. This article reviews the possible application to bone regeneration by synthetic OCP and the mechanism to enhance bone regeneration in relation to biological mineralization in bone and tooth.

The Effect of Microstructure of Octacalcium Phosphate on the Bone Regenerative Property

The present study was designed to investigate whether the microstructure of synthetic octacalcium phosphate (OCP) affects its intrinsic bone regenerative properties as a scaffold and its conversion process into hydroxyapatite (HA). Our previous studies indicated that an agregate of OCP crystals, consisting of randomly oriented plate-like crystals, are capable of enhancing both osteoblastic cell differentiation in vitro and bone regeneration. While the transformation of OCP into HA has been considered in relation to the stimulatory capacity of OCP in bone regeneration, little is known about the effect of the microstructure of OCP granules on these capabilities. Two types of OCP granules, with identical diameters (300-500 microm) but composed of crystals with distinct crystal dimensions (4.0 and 26.6 microm length), were prepared (hereafter referred to as fine OCP granules [F-OCP] and coarse OCP granules [C-OCP], respectively). The intergranule distances and the porosity, including the intergranule spaces, were 108.5 microm and 93.7% for F-OCP, and 67.5 microm and 95.7% for C-OCP, as estimated by mercury intrusion. The OCP granules were implanted in mouse critical-sized calvarial defects for up to 14 days. Histological examination demonstrated that osteoblastic cells aligned on the surface of F-OCP at day 7 and formed new bone around the granules up to day 14. On the other hand, cells around C-OCP were sparse at day 7, and resulted in only slight bone formation around the granules at day 14. X-ray diffraction showed that both OCP granules tended to be converted to an apatite structure with similar conversion velocity by the implantation. Adhesion of mouse bone marrow stromal ST-2 cells was markedly inhibited on C-OCP compared to F-OCP in vitro. These results suggested that the microstructure consisting of plate-like crystals of OCP controls cell adhesion on the crystal surfaces and their resultant bone regenerative properties as well as the physicochemical effect associated with the transitory nature of OCP previously reported.

Synthetic Octacalcium Phosphate Augments Bone Regeneration Correlated with Its Content in Collagen Scaffold

Previous studies have shown that synthetic octacalcium phosphate (OCP) facilitates in vitro osteoblastic cell differentiation in an OCP dose-dependent manner and that a complex of OCP and collagen (OCP/collagen) enhances critical-sized rat calvaria defects more than OCP alone. The present study was designed to investigate whether the bone regenerative properties of OCP/collagen are augmented in an OCP dose-dependent manner, thereby establishing a suitable composition of this composite as a bone substitute material. OCP/collagens with a wide range of mixing ratios from 23:77 to 83:17, including the previously examined composition (77:23), were prepared by blending granules of OCP with atelocollagen and molded into a disk as an implant. A critical-sized defect was made in rat calvaria, and each disk was implanted into the defect for 4 or 12 weeks and then examined radiographically, histologically, and histomorphometrically. Mouse bone marrow-derived stromal ST-2 cells were cultured in dishes pre-coated with OCP/collagen or OCP alone with different OCP contents to determine the capacity of cell attachment and proliferation up to 14 days. Histological and radiographic examinations showed that newly formed bone was observed in relation to OCP granules within the collagen matrix. Histomorphometric analysis confirmed that increasing the amount of OCP in collagen matrices resulted in progressive enhancement of bone regeneration and that the ratio 83:17 generated the maximum repair level of approximately 64% of the defect at 12 weeks. OCP/collagen promoted the proliferation and attachment of ST-2 cells more than OCP alone regardless of OCP content. Fourier transform infrared spectroscopy analysis of the coatings after the incubation indicated that OCP tended to convert to apatite regardless of the presence of collagen. The present study demonstrated that the osteoconductive characteristics of OCP/collagen can be displayed in an OCP dose-dependent manner. The results suggest that collagen promotes the proliferation and attachment of host osteoblastic cells on OCP/collagen composite implants.

Expression of major bone extracellular matrix proteins during embryonic osteogenesis in rat mandibles.

It is not known how bone proteins appear in the matrix before and after calcification during embryonic osteogenesis. The present study was designed to investigate expressions of the five major bone extracellular matrix proteins – i.e. type I collagen, osteonectin, osteopontin, bone sialoprotein and osteocalcin – during osteogenesis in rat embryonic mandibles immunohistochemically, and their involvement in calcification demonstrated by von Kossa staining. Wistar rat embryos 14 to 18 days post coitum were used. Osteogenesis was not seen in 14-day rat embryonic mandibles. Type I collagen was localized in the uncalcifed bone matrix in 15-day mandibles, where no other bone proteins showed immunoreactivity. Osteonectin, osteopontin, bone sialoprotein and osteocalcin appeared almost simultaneously in the calcified bone matrix of 16-day mandibles and accumulated continuously in 18-day mandibles. The present study suggested that type I collagen constitutes the basic framework of the bone matrix upon which the noncollagenous proteins are oriented to lead to calcification, whereas the noncollagenous proteins are deposited simultaneously by osteoblasts and are involved in calcification cooperatively.

Initiation of alveolar ridge augmentation in the rat mandible by subperiosteal implantation of octacalcium phosphate

The study was designed to investigate the process of bone formation caused by implantation of octacalcium phosphate as well as stability of the bone formed at the alveolar ridge. Synthetic octacalcium phosphate was implanted into a subperiosteal pocket in the rat mandible. Bone formation at the alveolar ridge was examined radiographically and histologically between 1 and 48 week(s) after implantation. Radiopacity of the octacalcium phosphate implant became obvious in week 2. Osteogenesis was initiated from the bone surface near the implantation site and multinucleated giant cells appeared on the implanted octacalcium phosphate in week 1. More apposition of new bone was observed on the implanted octacalcium phosphate in week 2 or later. Some implants were directly enclosed by newly formed bone and no cellular component was seen between the implant and the bone matrix. Many octacalcium phosphate implants were enclosed by bone, whereas the augmented ridge was not seen radiographically in week 24 or later. If its persistence can be improved, octacalcium phosphate could be used to augment atrophic alveolar ridges.

The effect of an octacalcium phosphate co-precipitated gelatin composite on the repair of critical-sized rat calvarial defects

This study was designed to investigate the extent to which an octacalcium phosphate/gelatin (OCP/Gel) composite can repair rat calvarial critical-sized defects (CSD). OCP crystals were grown with various concentrations of gelatin molecules and the OCP/Gel composites were characterized by chemical analysis, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), selected area electron diffraction (SAED) and mercury intrusion porosimetry. The OCP/Gel composite disks received vacuum dehydrothermal treatment, were implanted in Wistar rat calvarial CSD for 4, 8 and 16 weeks, and then subjected to radiologic, histologic, histomorphometric and histochemical assessment. The attachment of mouse bone marrow stromal ST-2 cells on the disks of the OCP/Gel composites was also examined after 1 day of incubation. OCP/Gel composites containing 24 wt.%, 31 wt.% and 40 wt.% of OCP and with approximate pore sizes of 10-500 μm were obtained. Plate-like crystals were observed closely associated with the Gel matrices. TEM, XRD, FTIR and SAED confirmed that the plate-like crystals were identical to those of the OCP phase, but contained a small amount of sphere-like amorphous material adjacent to the OCP crystals. The OCP (40 wt.%)/Gel composite repaired 71% of the CSD in conjunction with material degradation by osteoclastic cells, which reduced the percentage of the remaining implant to less than 3% within 16 weeks. Of the seeded ST-2 cells, 60-70% were able to migrate and attach to the OCP/Gel composites after 1 day of incubation, regardless of the OCP content. These results indicate that an OCP/Gel composite can repair rat calvarial CSD very efficiently and has favorable biodegradation characteristics. Therefore, it is hypothesized that host osteoblastic cells can easily migrate into an OCP/Gel composite.

Implantation of octacalcium phosphate combined with transforming growth factor-β1 enhances bone repair as well as resorption of the implant in rat skull defects

In our previous study, we reported that synthetic octacalcium phosphate (OCP) enhances bone repair if implanted in rat skull defects. We hypothesized that OCP can be used as an effective carrier for transforming growth factor-beta1 (TGF-beta1) to promote bone repair. We designed the present study to investigate histomorphometrically whether combination with recombinant human TGF-beta1 could promote bone repair caused by OCP per se (Control/OCP). A full-thickness standardized trephine defect was made in the rat parietal bone and OCP combined with recombinant human TGF-beta1 (TGF-beta1/OCP) or Control/OCP was implanted into the defect. Four rats from each group were fixed at 2, 4, and 8 weeks after implantation. Histomorphometrical analysis of the percentage of newly formed bone (n-Bone %) and remaining implants (r-Imp %) in the defect was performed. The statistical analysis showed the n-Bone % of TGF-beta1/OCP was significantly higher than that of the Control/OCP in week 4, whereas the r-Imp % of TGF-beta1/OCP was significantly lower than that of the Control/OCP. The present study demonstrated that OCP can be used as an effective carrier for TGF-beta1 and their combination enhances bone repair as well as resorption of the carrier OCP in the early stage of bone formation.

Intraoral reconstruction with innervated forearm flap: A comparison of sensibility and reinnervation in innervated versus noninnervated forearm flap

OBJECTIVE To evaluate the cutaneous sensibility and sensory reinnervation in patients who underwent intraoral reconstruction with an innervated or noninnervated forearm flap. STUDY DESIGN Results of the use of innervated forearm flaps in oral reconstruction was compared with the use of noninnervated flaps. The evaluation of sensibility and reinnervation comprised clinical sensibility tests and immunohistochemical investigation of postoperative biopsy specimens against S-100 and neurofilament. RESULTS The innervated flaps (4 patients) provided earlier and qualitatively better recovery of sensation than the noninnervated flaps (9 patients). Immunohistochemical investigation revealed the existence of a larger number of regularly arranged sensory nerve fibers in the cutaneous tissue of the innervated flaps than in the noninnervated flaps. Examination with an electron microscope found the structure of these nerve fibers to be well preserved in the innervated flaps, whereas nerve fibers in the noninnervated flaps were degenerative. CONCLUSION These findings suggest (1) that the innervated flaps are superior to the noninnervated flaps not only for the repair of defects but also for the restoration of function and (2) that the innervated flaps contribute to the improvement of the quality of life for patients.

Implanted octacalcium phosphate (OCP) stimulates osteogenesis by osteoblastic cells and/or committed osteoprogenitors in rat calvarial periosteum.

Our previous studies demonstrated that the octacalcium phosphate (OCP) causes new appositional bone formation on the OCP when implanted into the subperiosteal region of murine calvaria. The OCP may stimulate the cell population committed to the osteoblastic differentiation in the periosteum and have them express the phenotype. The present study was designed to investigate which periosteal cell population is involved in bone formation on the OCP with applying the OCP implants on top of and underneath the periosteum. The periosteum of the rat parietal bones was flapped and the OCP was implanted on top of or underneath the periosteum, in which the implantation sites were defined using the membrane filter. The histology was examined to see if new appositional bone formation occurs on the OCP implant under each condition. New bone was deposited on the OCP on the bone surface separated from the periosteum by the filter, whereas no bone was formed either under the periosteum separated from the bone surface by the filter or on the periosteum. The present study suggests that the OCP acts on osteoblasts, bone lining cells and/or their closely committed progenitors on the bone surface to express the phenotype and deposit new bone on the OCP implant. Anat Rec 256:1–6, 1999.