Microfabricated Grooved Substrates Influence Cell–Cell Communication and Osteoblast Differentiation In Vitro

Abstract

Implant topography influences osteointegration, but the architectural parameters that dictate cell behavior and the molecular mechanisms remain unclear. Polycarbonate grooved substrates 10, 15, or 30 μm wide and 7 μm deep with an inter-groove distance of 2 μm were contrasted for their ability to influence osteoblasts in vitro. Osteoblasts grew well on the substrates and became alkaline phosphatase–positive under osteogenic conditions, although extensive mineralization of bone-like nodules was only observed on flat surfaces. Osteoblasts were aligned according to the grooves, and on 10-μm grooves had a significantly smaller area than on other surfaces. Vinculin staining revealed that there were significantly smaller and fewer focal adhesions per cell on the grooves than on the flat surfaces. Localization of the gap junction protein, connexin-43, showed lower expression in cells on the 10-μm grooves with lower frequency of large (>3 μm²) gap junction complexes. Cell–cell communication was assessed using fluorescence recovery after photobleaching, demonstrating that cells on the 10-μm grooves exhibited significantly slower dye uptake after bleaching. Cells in the layers above the grooves also had slower dye recovery times. Maintaining appropriate cell–cell communication structures is pivotal in the process of osteoblast differentiation, and the design of orthopedic biomaterials should ensure that interacting cells can maintain these critical interactions.