As a means of improving the biological properties of materials for use as bone substitutes, functionally graded carbonate apatite containing Mg, FGMgCO3Ap, was synthesized at 60 degrees C and pH 7.4 using a gradient magnesium supply system. X-ray diffraction analysis of FGMgCO3Ap showed a poorly crystallized apatitic pattern, similar to that of human bone. ESCA analysis clearly showed the negative gradient distribution in Mg1s intensity (atomic concentration) of magnesium from the crystal surface toward the inner core. When the FGMgCO3Ap crystals were mixed with collagen, the resulting FGMgCO3Ap-collagen composite, irradiated with UV light for 4 h, retained their features in the saline solution. After washing away the nonadhesive cells, a cell adhesion assay showed that the optical density of the FGMgCO3Ap-collagen composite was higher than that of the CO3Ap-collagen composite. SEM observation showed that the osteoblast-like cells adhered well to the surface of the FGMgCO3Ap-collagen composite. Staining with hematoxylin-eosin and alizarin red confirmed the existence of a great many more cells and a thicker extracellular matrix layer on the FGMgCO3Ap-collagen composite than on the CO3Ap-collagen composite. This result demonstrated the acceleration effect of magnesium ions on osteoblast adhesion on the FGMgCO3Ap-collagen composite.
Inorganic polyphosphate (poly(P)) has recently been found to play an important role in bone formation. In this study, we found that tartrate-resistant acid phosphatase (TRAP), which is abundantly expressed in osteoclasts, has polyphosphatase activity that degrades poly(P) and yields Pi as well as shorter poly(P) chains. Since the TRAP protein that coprecipitated with anti-TRAP monoclonal antibodies exhibited both polyphosphatase and the original phosphatase activity, poly(P) degradation activity is dependent on TRAP and not on other contaminating enzymes. The ferrous chelator α, α’-bipyridyl, which inhibits the TRAP-mediated production of reactive oxygen species (ROS), had no effect on such poly(P) degradation, suggesting that the degradation is not dependent on ROS. In addition, shorter chain length poly(P) molecules were better substrates than longer chains for TRAP, and poly(P) inhibited the phosphatase activity of TRAP depending on its chain length. The IC50 of poly(P) against the original phosphatase activity of TRAP was 9.8 µM with an average chain length more than 300 phosphate residues, whereas the IC50 of poly(P) with a shorter average chain length of 15 phosphate residues was 8.3 mM. Finally, the pit formation activity of cultured rat osteoclasts differentiated by RANKL and M-CSF were markedly inhibited by poly(P), while no obvious decrease in cell number or differentiation efficiency was observed for poly(P). In particular, the inhibition of pit formation by long chain poly(P) with 300 phosphate residues was stronger than that of shorter chain poly(P). Thus, poly(P) may play an important regulatory role in osteoclastic bone resorption by inhibiting TRAP activity, which is dependent on its chain length.
These results suggest that use of slow degradation-type bFGF-gelatin hydrogel complex may accelerate bone regeneration around fenestrated implants at an early stage of bone regeneration.
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