Analysis of osteoblast mineral deposits on orthopaedic/dental implant metals

Neonatal rat calvarial osteoblasts were cultured on Ti-6Al-4V, Co-Cr-Mo alloy, 316L stainless steel and polystyrene (reference substrate) in the presence of ascorbic acid and 10 mimage β-glycerophosphate for 16, 17, 18, 19, 20, 21, 24 and 28 d. Scanning electron microscopy examination revealed that osteoblasts cultured on these orthopaedic/dental implant metals synthesized and deposited an extracellular matrix containing collagenous and non-collagenous components, as well as mineral nodules of various morphologies. Energy dispersive spectrometry revealed that the mineral deposits consisted of three distinct chemical compositions: calcium phosphate, calcium-sulphur-phosphorus, and calcium only. Backscattered electron imaging demonstrated that both the calcium phosphate and calcium-only deposits were electron dense, while the calcium-sulphur-phosphorus deposits were electron translucent. X-ray diffraction analysis indicated that the bulk of the osteoblast mineral deposits was amorphous hydroxyapatite; in addition, electron diffraction analysis revealed small regions of crystalline hydroxyapatite.