THE SEQUENCE OF ALIGNMENT OF MICROTUBULES, FOCAL CONTACTS AND ACTIN-FILAMENTS IN FIBROBLASTS SPREADING ON SMOOTH AND GROOVED TITANIUM SUBSTRATA

Contact guidance refers to the reactions of cells with the topography of their substratum. Current hypotheses on the mechanism of contact gu idance focus on the dynamic behaviour of the cytoskeletal components, but most observations have been made on cells that have already become oriented with topographic features of the substratum. The purpose of this study was to examine the sequence in which microtubules, focal co ntacts and microfilament bundles become aligned to the substratum topo graphy as fibroblasts spread on grooved substrata. Human gingival fibr oblasts were trypsinized and seeded onto grooved titanium surfaces pro duced by micromachining, as well as onto control smooth surfaces. Afte r observation and photography of the spreading cells at times up to 6 hours, the cells were fixed and exposed to one or more of the followin g antibodies or fluorescent stains: phallacidin to stain actin filamen ts, monoclonal anti-tubulin, monoclonal anti-vinculin, anti-mouse IgG labelled with Texas-Red or FITC, and/or an aldehyde-reactive stain to identify the cell outline. The cells were photographed and cell area, shape and orientation were calculated. Cells were also examined with c onfocal microscopy to obtain optical sections so that cell height as w ell as the precise locations of the cytoskeletal components with respe ct to the vertical dimension of the grooved substrata could be determi ned.Microtubules were the first element to become oriented parallel to the direction of the grooves and were first aligned at the bottom of the grooves. This alignment of microtubules was evident as early as 20 minutes after plating and preceded the orientation of the cell as a w hole. Aligned actin microfilament bundles were not observed until 40-6 0 minutes and were observed first at the wall-ridge edges. At early ti mes, focal contacts were distributed radially, but only after 3 hours did the majority of cells demonstrate aligned focal contacts. If the f irst cytoskeletal component to become aligned is the prime determinant of cell orientation, then these data suggest that microtubules in hum an gingival fibroblasts may determine cell orientation on grooved tita nium surfaces. By analogy with microtubule behaviour in other systems, we suggest that microtubule orientation on grooved substrata may occu r as a result of the substratum establishing shear-free planes.