The effects of varying degrees of allograft decalcification on cultured porcine osteoclast cells

Background: Demineralized freeze-dried bone allograft (DFDBA) is widely used in periodontal therapy as a scaffold for new bone formation in periodontal defects. It is demineralized, theoretically, to expose osteoinductive or osteoconductive bone matrix proteins that should facilitate osteogenesis. The degree of DFDBA demineralization varies between tissue banks and may affect clinical regeneration. A 2% residual calcium level in DFDBA has been shown to result in the highest alkaline phosphatase activity levels in cultured human periosteal cells and is optimally osteoinductive or osteoconductive for new bone formation. The purpose of this study was to evaluate the effect of 4 different residual calcium levels in commercially available DFDBA samples on porcine osteoclast activity as measured by resorption on calcium phosphate-coated disks. Methods: Bone marrow was harvested from the femurs of 3-week-old farm pigs and cultured for 3 weeks. Hematopoietic stem cells were allowed to differentiate into mature active polykaryons displaying genuine osteoclast characteristics. The osteoclast cells displayed a dense actin band inside the margins of the cytoplasm under light microscopy. Culture media was decanted and collagenase added to free the attached cells. Equal cell samples were pipetted onto calcium phosphate-coated disks in 24-well plates. DFDBA samples with 1.44%, 2.41%, and 5.29% residual calcium; FDBA (30% residual calcium); and control cultures without allograft samples were prepared and all samples incubated for 1 week. Cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), Oregon Green 488-phalloidin, a stain for cytoskeletal proteins, and counterstained with propidium iodide. Specimens were examined by light and fluorescence microscopy using epi-illumination. Calcium phosphate disks were then rinsed in 5% sodium hypochlorite to remove adherent osteoclasts, and substrate surface changes were measured by white light interferometry and image analysis. Results: A higher yield of TRAP-positive cells was produced without DFDBA; however, resorptive activity appears to be significantly increased in the presence of 2.41% residual calcium as compared to all other experimental groups (P <0.0065). Conclusion: In this in vitro model, porcine osteoclasts show significantly more resorptive activity as measured on calcium phosphate-coated disks in the presence of 2.41% residual calcium in DFDBA than in other DFDBA residual calcium levels.