1α,25(OH)₂D₃ Regulation of integrin expression is substrate dependent

Abstract

Osteoblasts are attachment-dependent cells that interact with their surface through integrin-mediated mechanisms. Their differentiation is regulated by 1,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D(3)] and is affected by substrate chemistry and microtopography, suggesting that 1alpha,25(OH)(2)D(3) may regulate integrin expression in a surface-specific manner. To test this hypothesis, osteoblast-like human MG63 cells were grown on tissue culture plastic and on grit-blasted and acid-etched titanium disks with a complex microtopography to induce osteoblast differentiation. Expression of alpha(2), alpha(5), alpha(v), beta(1), and beta(3) integrins were quantified by real-time polymerase chain reaction (PCR) as a function of time in culture and treatment with 1alpha,25(OH)(2)D(3). Results were correlated with expression of osteocalcin, a marker of a differentiated osteoblast. Osteocalcin mRNA increased with time and 1alpha,25(OH)(2)D(3) treatment and these changes were greater in cultures on the titanium disks. Integrin expression varied with time in culture and this was also surface dependent. At each time point, beta(1) and alpha(2) mRNAs were greater on titanium than on plastic, whereas alpha(5) expression was reduced and alpha(v),beta(3) expression was unaffected. 1alpha,25(OH)(2)D(3) increased beta(1) mRNA on both surfaces at all time points, but it increased alpha(2) expression only in 8-d cultures. 1alpha,25(OH)(2)D(3) caused reduced alpha(5) expression only in cultures grown on plastic for 8 d, and had no effect on either alpha(v) or beta(3) expression regardless of surface. These results show that integrin expression in human osteoblast-like cells is differentially modulated by 1alpha,25(OH)(2)D(3) in a time-dependent manner that is sensitive to the surface on which the cells are grown.

Integrin α2β1 plays a critical role in osteoblast response to micron-scale surface structure and surface energy of titanium substrates

“…We previously noted that MG63 cells exhibit increased expression of ␣2 and ␤1 but not ␣5 when cultured on Ti compared with TCPS (43). Here, we report that microstructure can modulate ␣2 and ␤1 expression and that surface energy also plays a regulatory role.…”

“…In contrast, antibodies to ␣2␤1 reduced osteoblast differentiation, suggesting a role for ␣2. Moreover, ␤1 and ␣2 mRNAs were increased when osteoblasts were grown on Ti substrates rather than TCPS, whereas ␣5 expression was unaffected (43). 1␣,25(OH) 2 D 3 further increased ␣2 and ␤1 mRNAs in cells cultured on Ti, but had no effect on ␣5.…”

“…However, integrin expression is substratesensitive (40,41); thus assumptions about cell behavior based on TCPS may not be relevant for cells on implant materials (37,42). Osteoblasts express primarily ␣5␤1 when grown on TCPS, but they shift to expression of ␣2␤1 when grown on Ti and Ti-6Al-4V (43,44). The consequences of this shift to the cell are not well understood, nor is it known whether integrin expression is sensitive to surface morphology or surface energy.…”

Beta-1 integrins mediate substrate dependent effects of 1α,25(OH)2D3 on osteoblasts

“…Because integrins signal through some of the same pathways as 1α, 25(OH) 2 D 3 , cross talk is possible. We found that mRNA levels for integrin subunits beta-1 (β 1 ), alpha-2 and alpha-5, but not for alpha-v or beta-3, were affected by substrate roughness and that the effects were specific to each subunit and varied with time in culture [14]. 1α,25 (OH) 2 D 3 modulated levels of β 1 and the effects were greater on the rougher surface.…”

Requirement for both micron- and submicron scale structure for synergistic responses of osteoblasts to substrate surface energy and topography

“…Studies examining the contribution of surface micro-roughness show that differences in surface topography, including roughness, affect fibronectin and albumin adsorption in vitro [5], and growth of fibroblasts and osteoblast-like cells in culture [6]. Sandblasted Ti supports stronger osteoblast adhesion [7,8], related at least in part to altered integrin expression, higher focal contact density and reorganized cytoskeleton of cells on the rough surface [9]. Cells cultured on microrough Ti surfaces also exhibit decreased proliferation and increased differentiation [8,10].…”