Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP--apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage.

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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.