Maturation state determines the response of osteogenic cells to surface roughness and 1,25-dihydroxyvitamin D-3

In this study we assessed whether osteogenic cells respond in a differentia l manner to changes in surface roughness depending on their maturation stat e. Previous studies using MG63 osteoblast-like cells, hypothesized to be at a relatively immature maturation state, showed that proliferation was inhi bited and differentiation (osteocalcin production) was stimulated by cultur e on titanium (Ti) surfaces of increasing roughness. This effect was furthe r enhanced by 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3]. In the present st udy, we examined the response of three additional cell lines at three diffe rent maturation states: fetal rat calvarial (FRC) cells (a mixture of multi potent mesenchymal cells, osteoprogenitor cells, and early committed osteob lasts), OCT-1 cells (well-differentiated secretory osteoblast-like cells is olated from calvaria), and MLO-Y4 cells (osteocyte-like cells). Both OCT-I and MLO-Y4 cells were derived from transgenic mice transformed with the SV4 0 large T-antigen driven by the osteocalcin promoter. Cells mere cultured o n Ti disks with three different average surface roughnesses (R-a): PT, 0.5 mu m; SLA, 4.1 mu m; and TPS, 4.9 mu m. When cultures reached confluence on plastic, vehicle or 10(-7) M or 10(-8) M 1,25(OH)(2)D-3 was added for 24 h to ah of the cultures. At harvest, cell number, alkaline phosphatase-speci fic activity, and production of osteocalcin, transforming growth factor bet a 1 (TGF-beta 1) and prostaglandin E-2 (PGE(2)) were measured. Cell behavio r was sensitive to surface roughness and depended on the maturation state o f the cell line. Fetal rat calvarial (FRC) cell number and alkaline phospha tase-specific activity were decreased, whereas production of osteocalcin, T GF-beta 1, and PGE(2) were increased with increasing surface roughness. Add ition of 1,25(OH)(2)D-3 to the cultures further augmented the effect of rou ghness for all parameters in a dose-dependent manner; only TGF-beta 1 produ ction on plastic and PT was unaffected by 1,25(OH)(2)D-3. OCT-1 cell number and alkaline phosphatase (SLA > TPS) were decreased and production of PGE( 2), osteocalcin, and TGF-beta 1 were increased on SLA and TPS. Response to 1,25(OH)(2)D-3 varied with the parameter being measured. Addition of the ho rmone to the cultures had no effect on cell number or TGF-beta 1 production on any surface, while alkaline phosphatase was stimulated on SLA and TPS; osteocalcin production was increased on all Ti surfaces but not on plastic; and PGE(2) was decreased on plastic and PT, but unaffected on SLA and TPS. In MLO-Y4 cultures, cell number was decreased on SLA and TPS; alkaline pho sphatase was unaffected by increasing surface roughness; and production of osteocalcin, TGF-beta 1, and PGE(2) were increased on SLA and TPS. Although 1,25(OH)(2)D-3 had no effect on cell number, aIkaline phosphatase, or prod uction of TGF-beta 1 or PGE(2) on any surface, the production of osteocalci n was stimulated by 1,25(OH)(2)D-3 on SLA and TPS. These results indicate t hat surface roughness promotes osteogenic differentiation of less mature ce lls, enhancing their responsiveness to 1,25(OH)(2)D-3. As cells become more mature, they exhibit a reduced sensitivity to their substrate but even the terminally differentiated osteocyte is affected by changes in surface roug hness.