Modulation of Osteoblast Signaling and Differentiation by Novel Substratum Topographies

Objectives: Substratum topography has proven to be a powerful inducer of bone formation, possibly via altered adhesion formation. Focal adhesions are one of the main sites of cellular phosphorylation, where extracellular regulated kinase 1/2 (ERK 1/2), c-src, and focal adhesion kinase (FAK) are activated. The transcription factors, STAT3 and Runx2, have been associated with skeletogenesis. The aim of this study was to investigate the activation and regulation of these intracellular signaling molecules by novel substratum topographies in osteoblasts cultured on osteoinductive micro-fabricated topographies.

Methods: Osteogenic cells from newborn-rat calvaria were isolated and cultured as previously described [Hamilton et al, 2006]. Substrata were produced in the laboratories of the Center for Advanced Technology in Microelectronics, at University of British Columbia. Epoxy-resin replicas were cast of Gap-Cornered-Boxes (GCB) (34 x 34 to 65 x 65µm in width, 4-10µm in depth), grooves (5-60µ deep, 30-90µm pitch)), pits (130nm in depth, varying pitch) and smooth controls. Cells were cultured from 24 hours to 6 weeks. For western blot analysis, protein lysates were extracted and separated on 12% SDS-page gels, transferred to PVDF membranes, stained with monoclonal antibodies, and visualized using enhanced chemiluminescence. Immunocytochemical staining was performed for osteocalcin, ERK1/2, STAT3, and Runx2.

Results: Immunocytochemistry revealed alterations in focal and fibrillar adhesion formation on GCB and nanopits. FAK phosphorylation increased both on GCBs and nanopits, localizing to focal, but not fibrillar adhesions. Osteocalcin expression increased on GCBs and microgrooves, but not nanopits, and osteocalcin expression correlated with nuclear translocation of ERK 1/2, STAT3 and Runx2.

Conclusions: Topographies that are osteoinductive alter focal and fibrillar adhesion formation in RCOs. We suggest that altered adhesion patterns and associated signaling is a pre-requisite for osteoblast differentiation on GCBs and microgrooves. Furthermore, we propose that nanotopographies could provide important information on control of cell signaling through modulation of FAs.