SUBSTRATE TOPOGRAPHY DESIGN AND FABRICATION FOR OSTEOBLAST AND DENTAL PULP STEM CELLS STUDIES

Research for cell guidance based tissue engineering has rapidly grown due to the increasing interest in tissue engineering and reconstructive medicine applications, such as cranial reconstruction. Many research teams have begun the process of identifying what factors influence cell behavior (including cell growth, proliferation, alignment and spreading). Published studies have pointed to the influence of the cell medial pH, substrate stiffness, chemistry and topography. Conclusive results are often hard to identify since researchers often vary many of these factors simultaneously and it is hard to decouple individual factor influence. These reports also points out that the cellular response can be cell type dependent. This work aims to identify the influence of substrate topography on cell response by designing a variety of substrate micropatterns with identical roughness, uniform stiffness and chemistry. These studies were designed primarily to give insight into dental stem cell response and features used were selected for their similarity to naturally occurring dental tissue. Using photolithography techniques developed for the semiconductor industries, Au micropatterned arrays with four feature shapes (lines, dots, holes and hexagons) were fabricated. The forty-eight unique micropatterns were produced with a range of feature heights (100, 500 and 1000 nm), widths (5, 10, 25 and 50 μm) and shapes (lines, dots, holes and hexagons). Subsequent processing (an additional 5 nm Au coating and 3 nm of 11-amino-1-undecanoth hydroxide) provided uniform roughness (RMS is 2 nm to 9 nm) and surface chemistry. Micropatterns were characterized for uniformity, feature width and heights and surface roughness using atomic force microscopy, profilometry and optical microscopy. To study cell response, two types of cells were utilized- mice 7F2 osteoblasts and porcine dental pulp cells. The mice 7F2 osteoblast cells were plated as a control, since there are already published studies characterizing this cell\u27s type response to microarrays of holes and lines. No published studies have been done to characterize the response of dental pulp stem cells. Each cell type was plated and characterized for cell density, alignment and spreading over three days. Initial results of the osteoblast cells confirmed earlier findings that the cells aligned on the anisotropic patterns (lines) and spread on the isotropic patterns (dots and holes). The dental pulp cells did not show any cell alignment or cell proliferation (as indicated by cell density) with the isotropic or anisotropic micropatterns. Significance of the osteoblast and dental pulp cell normalized densities were analyzed with statistical software (SAS using procedure PROC GLM). This analysis showed that there were no significant effects in terms of geometry. However, it did indicate that there was significant variation between each repetition of cell plating and when repetition is taken into account, the feature height significantly influenced the cell density increase over three days. Characterization of the micropatterns after the cell plating showed that the micropatterns could be used for multiple runs without significant degradation when interlayers were used between the Au and Si substrates. However, handling techniques could produce scratches in the micropatterns and residual stresses could cause buckling