An {\it in vitro\/} model for mineralization of human osteoblast-like cells on implant materials

An in vitro mineralizing cell-implant system was developed to study osteoblast attachment, secretion of extracellular matrix proteins (ECM) and mineralization. Human osteoblast-like cells (Saos-2) were plated on Tivanium (Tiv, Ti-6Al-4V) and Zimaloy (Zim, CoCrMo) disks prepared similar to clinical implants. Glass disks served as controls. Although cell attachment was highest on glass in the first 24 hours, calcium content in long term cultures were significantly higher on Tiv, less on Zim and least on glass. Addition of $\beta$-glycerophosphate ($\beta$-GP) increased mineralization, with a higher calcium content on Zim than on Tiv. Phalloidin/calcein immunofluorescence microscopy demonstrated nodules composed of multilayered osteoblasts, overlying calcified matrix. On Tiv, nodules were connected in a trabecular-like pattern. On Zim, calcification was diffusely dispersed in a dystrophic manner rather than in nodules alone. Northern blots for alkaline phosphatase, bone sialoprotein, osteocalcin and $\alpha1$(I) procollagen mRNAs showed differential responses on different substrates. The results indicated that cells on Tiv sustained the production of ECM message levels and mineralized matrix for 6 weeks, while cells on Zim or glass showed a peak in ECM mRNA levels which decreased before 6 weeks. Primary human osteoblasts, obtained from surgical samples, mineralized on the implant materials in a similar fashion, although calcium content and the number of nodules were significantly lower than Saos-2 cultures. Transforming growth factor-$\beta1$ (TGF-$\beta1)$ had a biphasic effect on calcification on Tiv with maximal mineralization at 0.2 ng/ml. Continuous presence of 0.2 ng/ml TGF-$\beta$ significantly enhanced mineralization compared to a single treatment. In conclusion, early cell responses to implants may not necessarily predict how well the cell cultures mineralize at later time points. Although the amount and pattern of calcification as well as the expression of ECM-mRNAs differed on each implant material, mineralization on Tiv appeared less dystrophic, and sustained throughout the culture period compared to Zim. This mineralization process by human osteoblast-like Saos-2 cells was comparable to that of primary human osteoblasts and was enhanced by TGF-$\beta,$ depending on the concentration and duration of growth factor. This cell-implant system is able to increase our understanding of the cellular and molecular events leading to osseointegration.