Calcium and phosphate supplementation promotes bone cell mineralization: implications for hydroxyapatite (HA)-enhanced bone formation.

Organic phosphate, in particular beta-glycerophosphate (beta-GP), has been used to induce mineralization in cell culture systems. It serves as a source of inorganic phosphate when hydrolyzed by alkaline phosphatase. This study examined the effect of supplemental calcium and phosphate as well as the influence of various metabolic inhibitors on mineralization in a rat osteoblast-like cell-culture system. Mineralization was induced by supplementation of 1.8 mM of Ca(+2) and 5 mM of beta-GP or Pi. Mineral deposits associated with in vitro mineralization were revealed under SEM and TEM. Levamisole (10-100 microM) inhibited alkaline phosphatase activity and effectively reduced mineral formation. Actinomycin (500 ng/mL) and cycloheximide (50 microg/mL) also reduced mineral depositions by blocking RNA synthesis and protein synthesis, respectively. Levamisole and beta-GP did not appear to influence DNA synthesis. Spontaneous precipitation of calcium phosphate mineral was not detected in the culture medium with calcium and phosphate supplements in the absence of cell culture. The findings suggest that an elevated concentration of calcium and phosphate is crucial for in vitro mineralization. Furthermore, the mineralization process is associated with biologic events rather than with a spontaneous precipitation of calcium phosphate mineral. In view of the degradation potential of hydroxyapatite (HA)-coated implants, these results may be a viable indication that HA enhances bone formation through a similar mechanism.

In vitro attachment of osteoblast-like cells to osteoceramic materials.

OBJECTIVE: The objective of this work was to examine osteoblast-like cell attachment and morphology in vitro to osteoceramic materials with three different surface morphologies. METHODS: Osteoceramic composite disks were fabricated from tricalcium phosphate and magnesium-aluminate spinel (MgAl2O4) in a 50 vol% ratio. The disks were prepared with three different surface morphologies, including as-fired (irregular), etched (rough), or polished through 1 mm diamond paste (smooth). Osteoblast-like cell cultures were plated onto the prepared disks for 2 h, and the number of attached cells was determined. ANOVA and Student Newman-Kuels tests were used to test for significant differences in cell attachment (p < 0.05). SEM was used to visually evaluate the nature of the cellular adaptation on the osteoceramic surfaces. RESULTS: Some additional surface roughening resulted from the interaction between the osteoceramic disks and the biological culture media during the attachment assay. A statistically larger number of cells was found to be attached to the etched osteoceramic surfaces compared to the as-fired and polished osteoceramic surfaces or the tissue culture plastic control. Cellular adaptation was extensive on all three osteoceramic surfaces at 2 h. SIGNIFICANCE: These results are consistent with previous in vivo work and continue to support the hypothesis that osteoceramic materials have potential for implants and bone substitute materials.

In vitro mineralization and implant calcium phosphate-hydroxyapatite crystallinity.

Biological dissolution of implant calcium phosphate coatings release local concentrations of divalent ions, which may influence mineralization. The objective of this study was to determine the effects of calcium phosphate release from coated commercially pure titanium discs using a bone-like cell culture bioassay. Sandblasted discs were prepared with or without hydroxyapatite crystallinities (50, 75, and 90 percent). Samples of each coating were randomly assigned and either preincubated for 24 hours with media or not before the addition of cells (2200/ mm2). Cultures were grown for 72 hours in culture medium containing 0.5 microCi/mL45 Ca. After rinsing, the remaining calcium phosphate surface was dissolved and counted. Three independent trials were performed. Results indicated proliferation was not altered as a function of crystallinity (P > 0.05) among any of the groups. However, a significant (P < 0.01) inverse relationship was found for biologically mediated mineralization as a function of calcium phosphate crystallinity. Low crystalline surfaces (nominally 50 percent) had the highest level of mineralization, with 75 percent crystalline surfaces being intermediate and 90 percent crystalline samples having the lowest amount of relative mineral formation. Mineralization only occurred on sandblasted commercially pure titanium upon supplementation of the growth medium with an organophosphate (beta-glycerophosphate), although this was less than on culture plastic. The results suggest calcium phosphate dissolution, as a function of implant coating crystallinity, can alter biological mineralization and may be one means in which enhanced mineral formation occurs around calcium phosphate-coated dental implants.

Bond strength and surface characterization of a Ni-Cr-Be alloy.

OBJECTIVES: Since resin composite cements adhere directly to base metal surfaces, the composition and integrity of the surface oxide layer is considered critical for adequate bonding. The aims of this study were to determine the tensile bond strength, and assess predominant surface oxides and oxide layer depth on the surface of a Ni-Cr-Be alloy after subjecting the alloy to various surface treatments. METHODS: Cylinders of Ni-Cr-Be (Rexillium III, Jeneric/Pentron) were cast and the following test groups were investigated: Group 0: no furnace oxidation, no air abrasion; Group 1: air fired, starting temperature 650 degrees C, ending temperature 1000 degrees C (1 min hold); Group 2: same firing sequence under vacuum; Group 3: same air firing sequence followed by air abrasion with 50 micrometers Al2O3; Group 4: vacuum fired, air abraded with 50 micrometers Al2O3; Group 5: air fired, air abraded with 280 micrometers Al2O3; Group 6: vacuum fired, air abraded with 280 micrometers Al2O3; Group 7: no furnace oxidation, air abraded with 50 micrometers Al2O3; and Group 8: no furnace oxidation, air abraded with 280 micrometers Al2O3. The oxide composition of three cylinders per group was analyzed with x-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES). Twenty cylinders from each group were bonded together with Panavia 21 (Kuraray), stored in water for 30 d, and tensile tested. Data were analyzed using one-way ANOVA and Duncan's Multiple-Range test (p=0.05). RESULTS: Furnace oxidation brought Be to the surface and increased the oxide thickness. Air abrasion removed the oxide layer and resulted in surfaces containing significant amounts of Al2O3. Bond strengths for Group 0 were significantly lower than bond strengths for Groups 1, 3, 4, 5, 6, 8, but not significantly different from bond strengths for Groups 2 and 7 (p<0.05). SIGNIFICANCE: Results of this study suggest differences in oxide composition and thickness due to various surface treatments. Furnace oxidation did not significantly affect bond strength and may not be required as a separate step for metal preparation if a resin adhesive is used.