Effects of multigrooved surfaces on fibroblast behavior

Microgrooves have been investigated as substrates for the control of cell alignment. However, they are relatively too narrow and shallow for controlling the orientation of extracellular matrices (ECM) such as collagen. Multigrooves, a combination of microgrooves and macrogrooves, are expected to be able to control the orientation of both cells and ECM. This study investigated a method for fabricating multigrooves and evaluated fibroblast behavior on these novel surfaces. Multigrooved patterns were fabricated on a gold-alloy metal die, in which 90-degree V-shaped microgrooves with a 2-microm pitch were cut on trapezoidal macrogrooves. The macrogrooves had a 50- microm ridge width, a 50-microm wall width, a 50-microm bottom width, and a 25-microm depth. The grooves were made by an ultraprecision micromachine using a single crystal diamond. This metal die served as a template for making surface replicas from polystyrene. Microgrooved and smooth polystyrene replicas also were prepared as comparative substrates. Mouse fibroblast L929 cells were cultured in each type of replica substrate for 7 to 21 days. After these periods, the cells were fixed with 2.5% glutaraldehyde, treated with conventional methods, and, finally, observed by SEM. Confocal laser scanning microscopy was performed to investigate ECM formation. The multigrooved metal die exhibited the desired sharp configuration without defects. The dimensional values of the multigrooves on the polystyrene replicas were almost the same as the designed values. The fibroblasts on the multigrooved and microgrooved substrates were aligned parallel to the surface grooves after 7 days of incubation. In contrast to the microgrooved and flat surfaces, a dense extracellular matrix was produced along the multigrooves after 21 days of incubation. These results suggest that multigrooves can control the orientation of ECM as well as cells and thus enhance the production of ECM.