The effect of surface roughness on osteoblast proliferation, differentiation, and protein synthesis was examined. Human osteoblast-like cells (MG63) were cultured on titanium (Ti) disks that had been prepared by one of five different treatment regimens. All disks were pretreated with hydrofluroic acid-nitric acid and washed (PT). PT disks were also: washed, and then electropolished (EP); fine sandblasted, etched with HCl and H2SO4, and washed (FA); coarse sandblasted, etched with HCl and H2SO4, and washed (CA); or Ti plasma-sprayed (TPS). Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by brightfield and darkfield microscopy, cold field emission scanning electron microscopy, and laser confocal microscopy, while chemical composition was mapped using energy dispersion X-ray analysis and elemental distribution determined using Auger electron spectroscopy. The effect of surface roughness on the cells was evaluated by measuring cell number, [3H]thymidine incorporation into DNA, alkaline phosphatase specific activity, [3H]uridine incorporation into RNA, [3H]proline incorporation into collagenase digestible protein (CDP) and noncollagenase-digestible protein (NCP), and [35S]sulfate incorporation into proteoglycan. Based on surface analysis, the five different Ti surfaces were ranked in order of smoothest to roughest: EP, PT, FA, CA, and TPS. A TiO2 layer was found on all surfaces that ranged in thickness from 100 A in the smoothest group to 300 A in the roughest. When compared to confluent cultures of cells on plastic, the number of cells was reduced on the TPS surfaces and increased on the EP surfaces, while the number of cells on the other surfaces was equivalent to plastic. [3H]Thymidine incorporation was inversely related to surface roughness. Alkaline phosphatase specific activity in isolated cells was found to decrease with increasing surface roughness, except for those cells cultured on CA. In contrast, enzyme activity in the cell layer was only decreased in cultures grown on FA- and TPS-treated surfaces. A direct correlation between surface roughness and RNA and CDP production was found. Surface roughness had no apparent effect on NCP production. Proteoglycan synthesis by the cells was inhibited on all the surfaces studied, with the largest inhibition observed in the CA and EP groups. These results demonstrate that surface roughness alters osteoblast proliferation, differentiation, and matrix production in vitro. The results also suggest that implant surface roughness may play a role in determining phenotypic expression of cells in vivo.

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Titanium (Ti) surface roughness affects proliferation, differentiation, and matrix production of MG-63 osteoblast-like cells. Cytokines and growth factors produced in the milieu surrounding an implant may also be influenced by its surface, thereby modulating the healing process. This study examined the effect of surface roughness on the production of two factors known to have potent effects on bone, prostaglandin E2 (PGE2) and transforming growth factor beta 1 (TGF-beta 1). MG-63 cells were cultured on Ti disks of varying roughness. The surfaces were ranked from smoothest to roughest: electropolished (EP), pretreated with hydrofluoric acid-nitric acid (PT), fine sand-blasted, etched with HCl and H2SO4, and washed (EA), coarse sand-blasted, etched with HCl and H2SO4, and washed (CA), and Ti plasma-sprayed (TPS). Cells were cultured in 24-well polystyrene (plastic) dishes as controls and to determine when confluence was achieved. Media were collected and cell number determined 24 h postconfluence. PGE2 and TGF-beta 1 levels in the conditioned media were determined using commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. There was an inverse relationship between cell number and Ti surface roughness. Total PGE2 content in the media of cultures grown on the three roughest surfaces (FA, CA, and TPS) was significantly increased 1.5-4.0 times over that found in media of cultures grown on plastic or smooth surfaces. When PGE2 production was expressed per cell number, CA and TPS cultures exhibited six- to eightfold increases compared to cultures on plastic and smooth surfaces. There was a direct relationship between TGF-beta 1 production and surface roughness, both in terms of total TGF-beta 1 per culture and when normalized for cell number. TGF-beta 1 production on rough surfaces (CA and TPS) was three to five times higher than on plastic. These studies indicate that substrate surface roughness affects cytokine and growth factor production by MG-63 cells, suggesting that surface roughness may modulate the activity of cells interacting with an implant, and thereby affect tissue healing and implant success.

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Surface roughness has been shown to affect differentiation and local factor production of MG63 osteoblast-like cells. This study examined whether surface roughness alters cellular response to circulating hormones such as 1 alpha,25-(OH)2D3. Unalloyed titanium (Ti) disks were pretreated with HF/HNO3 (PT) and then were machined and acid-etched (MA). Ti disks also were sandblasted (SB), sandblasted and acid etched (CA), or plasma sprayed with Ti particles (PS). The surfaces, from smoothest to roughest, were: PT, MA, CA, SB, and PS. MG63 cells were cultured to confluence on standard tissue culture polystyrene (plastic) or the Ti surfaces and then treated for 24 h with either 10(-8) M or 10(-7) M 1 alpha,25-(OH)2D3 or vehicle (control). Cellular response was measured by assaying cell number, cell layer alkaline phosphatase specific-activity, and the production of osteocalcin, latent (L) TGF beta, and PGE2. Alkaline phosphatase activity was affected by surface roughness; as the surface became rougher, the cells showed a significant increase in alkaline phosphatase activity. Addition of 1 alpha,25-(OH)2D3 to the cultures caused a dose-dependent stimulation of alkaline phosphatase activity that was synergistic with the effect caused by surface roughness alone. 1 alpha,25-(OH)2D3 also caused a synergistic increase in osteocalcin production as well as local factor (LTGF beta and PGE2) production on the rougher CA, SB, and PS surfaces, but it had no effect on the production on smooth surfaces. The inhibitory effect of surface roughness on cell number was not affected by 1 alpha,25-(OH)2D3 except on the SB surface. 1 alpha,25-(OH)2D3 decreased cell number, increased alkaline phosphatase activity and osteocalcin production, and had no effect on LTGF beta or PGE2 production by MG63 cells grown on tissue culture polystyrene. These data suggest that bone cell response to systemic hormones is modified by surface roughness and that surface roughness increases the responsiveness of MG63 cells to 1 alpha,25-(OH)2D3. They also suggest that the endocrine system is actively involved in normal bone healing around implants.