Wound contraction is mediated by myofibroblasts, specialized fibroblasts that appear in large numbers as the wound matures and when resistance to contractile forces increases. We considered that the regulation of myofibroblast differentiation by wound-healing cytokines may be dependent on the resistance of the connective tissue matrix to deformation. We examined transforming growth factor-beta1 (TGF-beta1) induction of the putative fibroblast contractile marker, alpha-smooth muscle actin (alpha-SMA), and the regulation of this process by the compliance of collagen substrates. Cells were cultured in three different types of collagen gels with wide variations of mechanical compliance as assessed by deformation testing. The resistance to collagen gel deformation determined the levels of intracellular tension as shown by staining for actin stress fibers. For cells plated on thin films of collagen-coated plastic (ie, minimal compliance and maximal intracellular tension), TGF-beta1 (10 ng/ml; 6 days) increased alpha-SMA protein content by ninefold as detected by Western blots but did not affect beta-actin content. Western blots of cells in anchored collagen gels (moderate compliance and tension) also showed a TGF-beta1-induced increase of alpha-SMA content, but the effect was greatly reduced compared with collagen-coated plastic (<3-fold increase). In floating collagen gels (high compliance and low tension), there were only minimal differences of alpha-SMA protein. Northern analyses for alpha-SMA and beta-actin indicated that TGF-beta1 selectively increased mRNA for alpha-SMA similar to the reported protein levels. In pulse-chase experiments, [35S]methionine-labeled intracellular alpha-SMA decayed most rapidly in floating gels, less rapidly in anchored gels, and not at all in collagen plates after TGF-beta1 treatment. TGF-beta1 increased alpha2 and beta1 integrin content by 50% in cells on collagen plates, but the increase was less marked on anchored gels and was undetectable in floating gels. When intracellular tension on collagen substrates was reduced by preincubating cells with blocking antibodies to the alpha2 and beta1 integrin subunits, TGF-beta1 failed to increase alpha-SMA protein content in all three types of collagen matrices. These data indicate that TGF-beta1-induced increases of alpha-SMA content are dependent on the resistance of the substrate to deformation and that the generation of intracellular tension is a central determinant of contractile cytoskeletal gene expression.

Only through such recognition can appropriate definitive diagnostic testing be conducted, and appropriate therapeutic intervention for the oral condition and the systemic condition be considered.

Enamel matrix proteins (EMP) induce periodontal regeneration and accelerate dermal wound healing, but the cellular mechanisms of these processes are unclear. We investigated the binding of EMP to the wound matrix proteins fibronectin, laminin-1, collagen type I, and collagen type IV and analyzed the interaction of epithelial cells and periodontal ligament fibroblasts (PDLF) with EMP and composite matrices of EMP + fibronectin or EMP + collagen. The adhesion of PDLF to EMP was concentration- and integrin-dependent and did not require de novo protein synthesis. EMP supported PDLF migration. In contrast, keratinocytes did not adhere to EMP if their protein synthesis was blocked. EMP showed concentration-dependent binding of fibronectin, peaking at 100 microg ml(-1) (before the precipitation point) of EMP. Type I collagen binding to EMP peaked at a low (1 microg ml(-1)) and narrow concentration range. Neither laminin-1 nor type IV collagen bound to EMP. Collagen and fibronectin, bound to EMP, showed significantly reduced (> 50%) binding of both epithelial cells and PDLF compared with the equivalent concentration of these proteins alone. PDLF, but not epithelial cell, adhesion was rescued by increasing the EMP concentration. These findings show that EMP binds to wound extracellular matrix proteins and regulates their adhesive properties. Such interactions may favor fibroblast adhesion over epithelial cells, potentially promoting connective tissue regeneration.

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