The bone-titanium interface in vitro.
Commercially pure 5-mm-diameter titanium (cpTi) discs received droplet inoculations of cells derived from rat bone marrow and were maintained in supplemented culture medium for 2-3 weeks. The cells and extracellular matrix (ECM) were processed for observation by light (LM), scanning (SEM), and transmission electron (TEM) microscopy. The latter was achieved by freeze-fracturing the solid metal from the resin-embedded tissue using a method which preserved the interface. Surface staining of whole discs revealed cells separated from the metal substratum by areas of ECM which stained positively using von Kossa's method to identify mineralization. At SEM, the ECM comprised dense interwoven collagen fiber networks which were partially obscured by globular masses (GMs). Individual GMs were associated with collagen fibers, especially at fiber intersections. EDAX line scan analysis confirmed the presence of Ca and P in these areas which were assumed to be spheritic foci of calcification since the Ca and P peaks diminished in areas which demonstrated only collagen fibers or the underlying cpTi. TEM examination confirmed the presence of globular mineralization and also revealed the presence of an interfacial zone between the metal substratum and the mineralized ECM elaborated by osteoblasts during the culture period. The interfacial zone comprised two layers, a bonding zone containing few collagen fragments and a ruthenium red positive layer containing more densely packed collagen fibers. We believe that this is the first report of both the formation of bonelike tissue on solid titanium substrata in vitro and demonstration of an interface which bears close morphological similarities to that known to develop in vivo.