Dose-Dependent Osteogenic Effect of Octacalcium Phosphate on Mouse Bone Marrow Stromal Cells

Abstract

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.

Stimulatory Capacity of an Octacalcium Phosphate/Gelatin Composite on Bone Regeneration in a Rabbit Tibia Defect Model

“…Octacalcium phosphate (OCP) crystals, having specific material characteristics, have been recognized to display a stimulatory capacity on osteoblast differentiation from stromal cells 1,2 and osteoclast formation from the precursor cells in co-culturing with osteoblasts 3 in vitro. In fact, OCP and OCP-based materials with natural polymers, such as OCP/collagen composite, enhance bone regeneration coupled with their own biodegradation in various experimentally created bone defects.…”

Transitory Nature of Octacalcium Phosphate in Physiological Milieu and Its Osteoconductive Property

“…27 OCP also enhanced the expressions of the mRNA of collagen type Ia (Col-1) and alkaline phosphatase (ALP) in ST-2 cells in a dose-dependent manner. 16 However, HA did not show such dose-dependent effect 16. As explained above, an increase in the amount of OCP in the culture medium accelerates OCP-HA conversion through dissolution processes. 7,16 A recent study confirmed that a subtle change in the stoichiometry of OCP, caused by partial hydrolysis, markedly affects its osteoconductive and biodegradable characteristics in rat tibia bone marrow.…”

“…16 However, HA did not show such dose-dependent effect 16. As explained above, an increase in the amount of OCP in the culture medium accelerates OCP-HA conversion through dissolution processes. 7,16 A recent study confirmed that a subtle change in the stoichiometry of OCP, caused by partial hydrolysis, markedly affects its osteoconductive and biodegradable characteristics in rat tibia bone marrow. 28 The results suggest that OCP promotes osteoblastic cell differentiation induced by the process of OCP-HA conversion if implanted in bony sites where osteoblasts are present, resulting in the enhancement of bone formation.…”

“…The concentration of calcium and inorganic phosphate ions in the culture medium was determined quantitatively using commercially available calcium and phosphate analysis kits, respectively. 16 An α-minimal essential medium (αMEM) was added to each well of a 48-well tissue culture plate with or without the OCP or HA coatings. After the plates were incubated for 3 days at 37°C in a 5% CO 2 environment, the supernatants were collected for calcium and phosphate quantitative analyses.…”

“…16 Osterix is known as a transcription factor in osteoblastic cells that indicates its differentiation into mature osteoblasts. 27 OCP also enhanced the expressions of the mRNA of collagen type Ia (Col-1) and alkaline phosphatase (ALP) in ST-2 cells in a dose-dependent manner.…”