Titanium implant surfaces with rough microtopographies exhibit increased pullout strength in vivo suggesting increased bone-to-implant contact. This is supported by in vitro studies showing that as surface microroughness increases, osteoblast proliferation decreases whereas differentiation increases. Differentiation is further enhanced on microrough surfaces by factors stimulating osteogenesis including 1alpha,25(OH)2D3. Levels of PGE2 and TGF-beta1 are increased in cultures grown on rough microtopographies; this surface effect is enhanced synergistically by 1alpha,25(OH)2D3-treatment. PGE2 and TGF-beta1 regulate osteoclasts as well as osteoblasts, suggesting that surface microtopography may modulate release of other factors from osteoblasts that regulate osteoclasts. To test this hypothesis, we examined the effects of substrate microarchitecture on production of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL), which have been identified as a key regulatory system of bone remodeling. We also examined the production of 1alpha,25(OH)2D3, which regulates osteoblast differentiation and osteoclastogenesis. MG63 osteoblast-like cells were grown on either tissue culture plastic or titanium disks of different surface microtopographies: PT (Ra <0.2 microm), SLA (Ra = 4 microm), and TPS (Ra = 5 microm). At confluence, cultures were treated for 24 h with 0, 10(-8) M or 10(-7) M 1alpha,25(OH)2D3. RANKL and OPG were determined at the transcriptional level by RT-PCR and real time PCR and soluble RANKL, OPG and 1alpha,25(OH)2D3 in the conditioned media were measured using immunoassay kits. Cell number was reduced on SLA and TPS surfaces and 1alpha,25(OH)2D3 caused further decreases. OPG mRNA levels increased on rougher surfaces and 1alpha,25(OH)2D3 treatment caused a further synergistic increase. While the cells expressed RANKL mRNA, levels were low and independent of surface microtopography. OPG protein was greater when cells were grown on SLA and TPS. 1alpha,25(OH)2D3 increased OPG by 50% on the smooth Ti surface but on SLA, 10(-8) M 1alpha,25(OH)2D3 caused a 100% increase and 10(-7) M 1alpha,25(OH)2D3 increased OPG by 200%. On TPS 10(-7) M 1alpha,25(OH)2D3 increased OPG 350%. Soluble RANKL was not detected in the conditioned media of any of the cultures. 1alpha,25(OH)2D3 was produced endogenously and levels were positively correlated with surface roughness. Thus, on surfaces with rough microtopographies, osteoblasts secrete factors that enhance osteoblast differentiation while decreasing osteoclast formation and activity.
