Local factor production by MG63 osteoblast-like cells in response to surface roughness and 1,25-(OH)₂D₃ is mediated via protein kinase C- and protein kinase A-dependent pathways

Titanium (Ti) surface roughness affects bone formation in vivo and osteoblast attachment, proliferation and differentiation in vitro. MG63 cells exhibit decreased proliferation and increased differentiation when cultured on rough Ti surfaces (R_a >2μm) and response to 1,25-(OH)₂D₃ is enhanced, resulting in synergistic increases in TGF-β1 and PGE₂. To examine the hypothesis that surface roughness and 1,25-(OH)₂D₃ exert their effects on local factor production through independent, but convergent, signaling pathways, MG63 cells were cultured on tissue culture plastic or on smooth (PT, R_a=0.60μm) and rough (SLA, R_a=3.97μm; TPS, R_a=5.21μm) Ti disks. At confluence (5 days), cultures were treated for 24h with 10^-8M 1α,25-(OH)₂D₃ and active and latent TGF-β1 in the conditioned media measured by ELISA. Cell layers were digested with plasmin and released TGF-β1 was also measured. 1,25-(OH)₂D₃ regulated the distribution of TGF-β1 between the media and the matrix in a surface-dependent manner; the effect was greatest in the matrix of cells cultured on SLA and TPS. Inhibition of PKA with H8 for the last 24h of culture increased PGE₂ on SLA and TPS, but when present throughout the entire culture period H8 caused an increase in PGE₂ on all surfaces. 1,25-(OH)₂D₃ reduced the effect of H8 an PGE₂ production in cultures treated for 24h. H8 had no effect on TGF-β1 in the media by itself but caused a complete inhibition of the 1,25-(OH)₂D₃ dependent increase. Inhibition of PKC with chelerythrine increased PGE₂ in a surface-dependent manner and 1,25-(OH)₂D₃ reduced the effect of the PKC inhibitor. Chelerythrine also increased TGF-β1 but the effect was not surface dependent; however, 1,25-(OH)₂D₃ reduced the effects of chelerythrine with the greatest effects on the smooth surface. Thus, the distribution of TGF-β1 between the media and the matrix is regulated by 1,25-(OH)₂D₃ in a surface-dependent manner. Surface roughness exerts its effects on TGF-β1 production via PKC but not PKA. The effect of 1,25-(OH)₂D₃ on TGF-β1 production is not via PKC. PKA is involved in the surface-dependent regulation of PGE₂ but not in the regulation of PGE₂ by 1,25-(OH)₂D₃ on rough surfaces. Regulation of PKC affects PGE₂ production but it is not involved in the surface roughness-dependent response to 1,25-(OH)₂D₃. These results suggest two independent but interconnected pathways are involved.