Stimulatory effect of hydrothermally synthesized biodegradable hydroxyapatite granules on osteogenesis and direct association with osteoclasts

Calcium-deficient hydroxyapatite (HA) granules with a unique spherical shape were prepared using an applied hydrothermal method. Spherical stoichiometric HA granules were also prepared by normal sintering and both granules were used for implantation into rat tibiae to compare the biological responses to each implant. Twelve and 24 weeks after implantation, the volume of calcium-deficient HA granules was significantly less than that of stoichiometric HA granules, and the biodegradability of calcium-deficient HA granules was confirmed. The larger number of osteoclasts, larger osteoblast surface and larger bone volume in the implanted area of calcium-deficient HA than those of stoichiometric HA suggested that osteoclastic resorption of calcium-deficient HA affected osteogenesis in that area. To analyze the direct contribution of osteoclasts to osteogenesis, C2C12 multipotent myoblastic cells, which have the potential to differentiate into osteoblasts in the presence of bone morphogenetic protein 2, were cultured with supernatants of osteoclasts cultured on calcium-deficient HA, stoichiometric HA, P-tricalcium phosphate disks or plastic dishes, or bone marrow macrophages cultured on plastic dishes. Supernatants of osteoclasts but not bone marrow macrophages stimulated the expression of Runx2 and osteocalcin in C2C12 cells in concert with bone morphogenetic protein 2. The expression of alkaline phosphatase was stimulated with supernatants of osteoclasts cultured on ceramic disks. These results suggested that osteoclasts produced certain soluble factors which stimulated osteoblastic differentiation and they were thought to be associated with the induction of a larger osteoblast surface and bone volume in the animals implanted with calcium-deficient HA granules. (c) 2009 Elsevier Ltd. All rights reserved.