Bone regeneration with osteogenic matrix cell sheet and tricalcium phosphate: An experimental study in sheep

AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate (TCP) on osteogenesis. METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium (MEM) containing ascorbic acid phosphate (AscP) and dexamethasone (Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and beta-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase (ALP) activity and osteocalcin (OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs were implanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk. RESULTS In the in vitro part, the mean ALP activity was 0.39 +/- 0.03 mg/well in the negative control group, 0.67 +/- 0.04 mg/well in the sheet group, and 0.65 +/- 0.07 mg/well in the positive control group. The mean OC levels were 1.46 +/- 0.33 ng/well in the negative control group, 3.92 +/- 0.16 ng/well in the sheet group, and 4.4 +/- 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group (P < 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group (P > 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. CONCLUSION This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.