The results of this study show that high surface energy is an important variable but by itself it is insufficient to cause marked increases in osteoblast responses to Ti substrates with low surface roughness. In contrast, when substrates with complex micron scale and submicron scale roughness are fabricated to retain the high surface energy of uncontaminated TiO2, the cells exhibit synergistic enhancement of their response to the surface topography alone. This includes reduced cell number together with increased differentiation and release of factors into the media that stimulate osteogenesis and reduce osteoclastic activity.
Scanning electron microscopic analysis and immunofluorescence of cell morphology identified significant differences in cell shape as a function of both surface chemistry and surface topography, confirming our previous work and the work of others [32
]. Immunofluorescence images of MG63 cells over the first 24 hours after seeding demonstrated that osteoblasts on smooth and low energy surfaces spread faster than those on rough and higher energy surfaces. Scanning electron micrographs showed that differences in cell shape were retained even after cell density increased. MG63 cells on PT surfaces, like those on plastic, in general were flat and had two or three major extensions. On A and modA, the cells were elongated and formed spindle like shape; on SLA and modSLA, the cells were polygonal in shape with many thin filopodia to attach to the surfaces.
The observation that osteoblasts grown on smooth Ti surface had fewer cytoplasmic extensions than cells grown on micron and submicron structured surfaces, is consistent with previous findings [8
]. Although both A and SLA surfaces had similar 1–2µm pits produced by acid etching, the MG63 cells grown on SLA extended many more filopodia to attach themselves to the substrates. This suggests that the cells not only sense direct focal contacts with the substrate, but they also respond to the broader waviness created by sand blasting. Others have reported comparable effects of surface topography on epithelial cells [35
], suggesting that this is a general property of cell interactions with surface microarchitecture.
Our results confirmed our previous findings that submicron scale roughness, while contributing to the overall response of the cells, is not a major determinant of cell behavior in the absence of the larger craters [20
]. In the present study, with the exception of OPG production, growth on A did not alter osteoblast number, differentiation or local factor levels to any great extent in comparison with growth on the PT surface. However, in combination with micron scale roughness, typified by SLA, there were significant increases in local factor levels to produce an osteogenic environment [20
]. This phenomenon may be due to interference between surface topography and surface energy. The potential effect of submicron scale roughness is counteracted by lower surface energy. Other researchers have observed that osteoblasts grown on nano-textured surfaces express higher osteopontin and bone sialoprotein [36
]. Full characterization of surface energy along with surface roughness is necessary to understand cell/substrate interactions.
Our results show a strong synergistic effect between micron scale roughness and surface energy. When MG63 cells were grown on A and modA substrates, the increase in surface energy had no effect on alkaline phosphatase activity or latent TGF-β1 levels, and only a small increase in osteocalcin or PGE2. In contrast, when the surface presented a complicated micron scale roughness, increased surface energy greatly decreased cell number and enhanced cell differentiation by more than 100%. Both modA and modSLA were produced using the same acid etching procedure to ensure the same surface chemistry. Therefore the differences observed in the cell response were only dependent on micron scale roughness. We did not include modified PT substrates to examine the effect of high surface energy on a relatively smooth surface because PT surfaces were mechanically machined and chemically degreased, resulting in a final modified PT surface chemistry that would be different from modA or modSLA.
It is likely that the differential response of MG63 cells to surface roughness and topography reflects differences in integrin mediated signaling. Cells respond to biomaterial surfaces through interaction between plasma membrane receptor integrins and adsorbed extracellular matrix proteins including fibronectin [37
]. Protein adsorption is highly influenced by surface chemistry, hydrophilicity and topography; small proteins tend to adsorb on hydrophobic surfaces, but large proteins are less affected by surface wettability [38
]. In addition, proteins adsorbed onto hydrophobic surfaces are more sensitive to unfolding and denaturing processes due to electrostatic forces between the surface and cells. Thus, fibronectin fragments adsorb faster on hydrophobic -CH3
surfaces, but lack cell adhesion activity [39
]. Protein adsorption also depends on the scale of surface roughness. Nano scale surface texture seems have little to no effect on protein adsorption and cell proliferation [40
]. However, microrough surfaces adsorb more fibronectin and the protein orientation is different from that on machined smooth Ti, which further alters integrin adhesion. In addition, the profile of integrin expression in osteoblasts is sensitive to surface roughness [9
]. These findings indicate cell behavior is not determined by a single surface feature, but is in response to combinations of different surface properties as a whole.
Previous studies show that osteoblasts grown on microrough surfaces produce an osteogenic environment to promote osteoblast differentiation by paracrine and autocrine pathways [1
]. Our results suggest that the increase in the rate and extent of peri-implant bone formation seen with modSLA implants in vivo [27
] reflects enhanced osteogenesis as well. Osteocalcin levels were increased whereas alkaline phosphatase specific activity was decreased, indicative of mature secretory osteoblasts [41
, which is necessary for osteoblastic differentiation [1
], was also increased. Interestingly, levels of OPG, a local factor produced by osteoblasts that reduces osteoblast-dependent activation of osteoclasts [42
], were higher as well. These results indicate that the increase of OPG on modSLA surfaces plays an important role in controlling osteoclast differentiation in bone remodeling cycle, and contributes to early upregulation of bone to implant contact [27
TGF-β1 also reduces osteoclast activity [43
] and increases osteoblast differentiation [44
]. We previously showed that cells grown on SLA deposit more TGF-β1 in their extracellular matrix than do cells grown on smooth Ti [45
], and cells grown on modSLA release increased levels of latent TGF-β1 into their media than do cells grown on SLA. In the present study, we also found higher levels of latent growth factor in the conditioned media of cells grown on SLA and this was further increased when cells were cultured on modSLA. This suggests that cells on the more reactive surface are producing a larger reservoir of growth factors on high energy surfaces that can be used downstream to control osteoclast formation and activity.