Association of fibroblast orientation around titanium in vitro with expression of a muscle actin

The objective of this study was to investigate the association of cell orientation around a biomaterial with expression of a contractile actin isoform. Selected cytokines and a fungal metabolite known to alter the cytoskeleton were used to modulate the fibroblast orientation around titanium in vitro and the synthesis of a specific muscle actin in order to reveal an association between these processes. A novel culture system using a fibronectin-coated silicone surface was employed to evaluate the orientation of human gingival fibroblasts around titanium discs. Round glass cover slips, 25 mm in diameter, were coated with polydimethylsiloxane. During the heat-induced polymerization process, two commercially pure titanium discs, 5 mm in diameter, were placed on the silicone at a distance of approximately 0.5 mm apart. The rubbery consistency of the silicone stabilized the metal discs on the cover slip and eliminated the risk of developing a lip at the edge of the titanium sample. The cover slip was then heated to complete polymerization of the silicone and subsequently coated with fibronectin. One hundred thousand human gingival fibroblasts were plated onto each glass cover slip containing the titanium discs. The cells were treated with one of the following prior to seeding on the cover slips: transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor-BB (PDGF-BB), interferon-gamma (IFN-gamma) for cytochalasin-D. Untreated cells served as controls. The orientation of the cells at the surface of the titanium discs was evaluated microscopically and the cell content of alpha-smooth muscle actin (SMA) was determined by Western blot analysis and immunohistochemistry. A notable finding was the high correlation between the percentage of cells oriented perpendicular to the titanium surface and SMA synthesis. TGF-beta1, IFN-gamma and cytochalasin-D increased synthesis of SMA while PDGF-BB decreased it. The findings support the proposition that SMA-enabled cell contraction may play a role in the orientation of cells to a biomaterial surface.