Osteoblast response to nanocrystalline calcium hydroxyapatite depends on carbonate content

Normal bone mineral is a carbonated-apatite, but there are limited data on the effect of mineral containing carbonate on cell response. We characterized surface chemical compositions of three experimental carbonated hydroxyapatite (CO3(2-) HA) substrates and investigated their effect on osteoblast differentiation. Carbonate was incorporated into the hydroxyapatite powders while phosphate and hydroxyl groups were shown to be reduced by analyzing the chemical composition of the substrate surfaces. CO3(2-) HA powders with increasing carbonate concentrations designated as C1 (3.88%), C2 (4.85%), and C3 (5.82%) were molded, pressed, and fired into 14 mm discs. We observed that calcium phosphate ratios increased monotonically with increasing carbonate content, whereas differentiation of MG63 cells decreased. CO3(2-) HA surfaces also affected factor production. Addition of carbonate caused a 70% reduction in osteoprotegerin (OPG) compared to cultures on pure HA, but the effect of carbonate was not dose-dependent. Low carbonate content reduced VEGF-A by 80%, but higher levels of carbonate reversed this effect in a concentration dependent manner, with the C3 VEFG-A levels approximately twice that of C1 levels. These observations collectively indicate that bone cells are sensitive to carbonate content in bone mineral and the effects of carbonate substitution vary with the outcome being measured. Overall, this study provides a preliminary understanding of how carbonate substitution within hydroxyapatite modulates cellular behavior.