Viable bone formation in porous hydroxyapatite: marrow cell-derived in vitro bone on the surface of ceramics

With the aim of in vitro production of bone fragments more closely resembling autogenous bone, rat cultured bone marrow cells were combined with porous hydroxyapatite (HA) discs and cultured in the presence of dexamethasone (Dex). Bone marrow cells were collected from the femoral diaphyses of a 7-week-old male Wistar rat, and primary culture was performed for six days. Then, cell suspensions were prepared by trypsin treatment, and combined with the porous HA discs. After a 2-hour incubation, the composites were additionally cultured for up to 4 weeks (subculture) in the presence of Dex. In a control group, the subculture was performed without Dex. After 1, 2, 3 and 4 weeks of subculturing, the HA discs were removed, and alkaline phosphatase (ALP) activity, bone Gla protein (BGP), and DNA were quantitated. A portion of the disc was prepared for scanning electron microscopy (SEM), from which bone formation was evaluated morphologically. ALP activity peaked at 2-3 weeks and decreased at 4 weeks. BGP levels began to increase at 3 weeks. In the SEM study, mineralized collagenous extracellular matrix was noted at 3 and 4 weeks. In the control group, neither significant ALP activity nor increased BGP was detected. These biochemical and morphological results suggest that with the culture technique, active bone formation in the pore regions of HA can be fabricated in vitro. It is anticipated that when composites are subcultured in this way they will function as a bone graft with properties similar to those of autogenous bone.