Application of low-pressure system to sustain in vivo bone formation in osteoblast/porous hydroxyapatite composite

Recently, synthetic porous hydroxyapatite (HA) has attracted a great deal of attention as a bone graft substitute in the field of tissue engineering. Combining with bone marrow-derived osteoblasts (BMO), bone formation has been confirmed in vivo and in vitro. However, it is still necessary to obtain more bone formation within porous HA for clinical usage, and, thus, new methods need to be developed. We hypothesized that low pressure during subculture would cause more osteoblastic cells to migrate into the pores of porous HA blocks, resulting in more bone tissue formation in vivo. In the present study, we examined six experimental groups with different pressures from 760 to 10 mm Hg applied to porous HA blocks loaded by bone marrow-derived osteoblasts. For in vivo testing, the 2-week subcultured HA/BMO composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested at 2, 4 or 8 weeks after implantation. Then, they were prepared for biochemical analysis of alkaline phosphatase (ALP) activity, bone osteocalcin (OCN) content and histological analysis. ALP activity and OCN content in the 100-mm Hg pressure group were highest among the different groups 4 and 8 weeks after implantation (P<0.001). Light microscopy revealed mature bone formation in HA/BMO composite at 4 weeks after implantation. In the scanning electron microscopy (SEM) study, mineralized collagenous extracellular matrix as well as active osteoblasts was observed in HA/BMO composite at 2 weeks after implantation. We concluded that the application of low-pressure system to subculture of bone cells to porous HA blocks is beneficial to increase bone tissue formation in vivo.