A METHOD FOR TRANSMISSION ELECTRON-MICROSCOPY INVESTIGATION OF THE OSTEOBLAST/HYDROXYAPATITE INTERFACE

Neonatal rat calvaria osteoblasts were cultured on hydroxyapatite (as received or relatively rough surface and mechanically polished to a 0.3-mu m finish) and on glass (reference material) in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 50 mu g/mL ascorbic acid, and 10 mM beta-glycerophosphate under standard, sterile, cell-culture conditions for 1,3,7,14, and 21 days. At the end of the prescribed time periods, the cells were fixed and embedded in resin before removing the material substrate's by exposure to acid solutions. Transmission electron microscopic examination of stained, ultrathin sections of the biological structures revealed osteoblast monolayers at 1 day of culture but multilayered cell structures at later time periods (14 and 21 days). The osteoblasts exhibited continuous contact and intimate apposition on polished hydroxyapatite and on glass; in contrast, osteoblasts on as received or rough hydroxyapatite made contact with discrete high points, spanned low regions of the material surface, and did not conform Co all substrate contours. An electron dense layer (composed of mucopolysaccharides and proteins) was observed on all substrates tested after 7 days of culture. Collagen fibrils were seen interspersed among the osteoblasts as early as 3 days of culture; at later culture times, (i.e., 21 days) mineralized loci were observed in the extracellular matrix. (C) 1994 John Wiley and Sons, Inc.