To elucidate the biochemical mechanism of osteogenesis, the effect of matrix geometry upon the osteogenesis induced by bone morphogenetic protein (BMP) was studied. A series of five porous hydroxyapatites with different pore sizes, 106-212, 212-300, 300-400, 400-500, and 500-600 microns, was prepared. A block (approximately 5 x 5 x 1 mm, 40.0 mg) of each hydroxyapatite ceramics was combined with 4 micrograms of recombinant human BMP-2 and implanted subcutaneously into the back skin of rat. Osteoinductive ability of each implant was estimated by quantifying osteocalcin content and alkaline phosphatase activity in the implant up to 4 wk after implantation. In the ceramics of 106-212 microns, the highest alkaline phosphatase activity was found 2 wk after implantation, and the highest osteocalcin content 4 wk after implantation, consistent with the results observed with particulate porous hydroxyapatite [Kuboki, Y. et al. (1995) Connect. Tissue Res. 32: 219-226]. Comparison of the alkaline phosphatase activities at 2 wk and the osteocalcin contents at 4 wk after implantation revealed that the highest amount of bone was produced in the ceramics implants with pore size of 300-400 microns. In the ceramics with smaller or larger pore sizes, the amount of bone formation decreased as the pore size deviated from 300-400 microns. The results indicated that the optimal pore size for attachment, differentiation and growth of osteoblasts and vascularization is approximately 300-400 microns. This study using chemically identical but geometrically different cell substrata is the first demonstration that a matrix with a certain geometrical size is most favorable for cell differentiation.

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Bone morphogenetic protein (BMP) is known to require a suitable carrier to induce ectopic bone formation in vivo. Hydroxyapatite ceramics have been reported to be effective in some forms but ineffective in others as a carrier of BMP-induced bone formation. In this study we compare three geometrically different forms of hydroxyapatite to examine their functions as carriers of BMP-induced bone formation. A fraction containing all the active BMPs (BMP cocktail) was partially purified from a 4M guanidine extract from bovine bone by a three-step chromatographic procedure. The BMP cocktail was combined with each of three forms of hydroxyapatite--solid particles (SPHAP), porous particles (PPHAP), and coral-replicated porous tablets (coral-HAP)--and implanted subcutaneously into rats. Both the PPHAP and coral-HAP systems induced osteogenesis 2 weeks after implantation, as evidenced by morphological and biochemical observations. Details of the osteogenetic process were followed by double-fluorescence labeling in the coral-HAP system to confirm bone formation on the surface of hydroxyapatite. However, there was no evidence of osteogenesis or chondrogenesis in the SPHAP system. The results indicate that the geometry of the interconnected porous structure in PPHAP and coral-HAP create spaces for vasculature that lead to osteogenesis while the smooth structure and close contact of particles in SPHAP inhibit vascular formation and proliferation of mesenchymal cells, preventing bone and cartilage formation. It was concluded that the geometrical structure in hydroxyapatite ceramics that induces vasculature is crucial as a carrier for BMP-induced bone formation.

Bone marrow cells are multipotent cells. When bone marrow cells were cultured with type I collagen matrix gels, they showed high alkaline phosphatase activity, collagen synthesis, and formed mineralized tissues. Furthermore, cells expressed osteocalcin and bone sialoprotein genes, which are osteoblast-specific genes. These findings indicate that type I collagen matrix gels induce osteoblastic differentiation of bone marrow cells. Type I collagen interacts with the alpha 2 beta 1 integrin receptor on the cell membrane and mediates extracellular signals into cells. DGEA peptide is a cell-binding domain of type I collagen molecule. When collagen-integrin interaction was interrupted by the addition of Asp-Gly-Glu-Ala (DGEA) peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. Furthermore, anti-alpha 2 integrin antibody, which interacts with alpha subunit of integrin and blocks the binding of integrin with collagen, suppressed the expression of osteoblastic phenotypes. These findings imply that collagen-alpha 2 beta 1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.

Bone marrow contains multipotent cells that differentiate into fibroblasts, adipocytes, and osteoblasts. Recently we found that type I collagen matrix induced the osteoblastic differentiation of bone marrow cells. Three weeks after cells were cultured with type I collagen, they formed mineralized tissues. In this study, we investigated the expression of osteoblast-related genes (alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and cbfa-1) during the osteoblastic differentiation. The expression of alkaline phosphatase and osteopontin genes increased time-dependently during the osteoblastic differentiation. Osteocalcin and bone sialoprotein genes were expressed in cells that formed mineralized tissues, and both were expressed only after cells reached the mineralized tissue-formation stage. On the other hand, the cbfa-1 gene was expressed from the early differentiation stage. The Asp-Gly-Glu-Ala (DGEA) amino acid domain of type I collagen interacts with the alpha2beta1 integrin receptor on the cell membrane and mediates extracellular signals into cells. When the collagen-integrin interaction was interrupted by the addition of DGEA peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. These findings imply that the collagen-alpha2beta1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.

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