Design of two natural deproteinized bovine bone scaffolds and evaluation of the effect of initial cell seeding density on repairing bone defects.

Engineering functional bone using combinations of cells, scaffolds, and bioactive factors is a promising strategy for the bone-tissue regeneration, while challenge remains. Chemical methods deproteinizing natural bovine cancellous bone to remove immunogenic are poorly understood, and the cell seeding density to promote bone formation still needs to be clarified. In this study, 8.0 × 8.0 × 2.0 mm bovine cancellous bones were either treated with H2 O2 for 8 hr or pepsin for 24 hr and then inoculated with MC3T3-E1 osteoblasts with two cell densities (1 × 106 cells/ml or 4 × 106 cells/ml)separately. We compared the appearance of the bones treated by the two chemical deproteinizing methods, as well as the proliferation ability of the inoculating cell density at 1 × 106 cells/ml. Moreover, scanning electron microscopy was done to analyze the growth of cells on the surface of the material, and an alkaline phosphatase assay was performed to assess osteogenic differentiation. We showed that both treated bones treatments are biocompatible, but bones treated with H2 O2 were more conducive to osteoblast differentiation and ALP secretion, especially when seeded at the higher cell density at 4 × 106 cells/ml. We concluded that chemical deproteinized bovine cancellous bones met the basic bone graft material requirements. Cell seeding density is an important factor to promote the material's osteogenic ability, with H2 O2 -deproteinized bones exhibiting enhanced osteoblast differentiation.