Influence of nanotopography on MC-3T3 cell adhesion and filopodia formation

摘要

Introduction: The interaction of cells with the surface of biomaterials determines the response of the host tissue and the ultimate success of an implant. Surface topography at all size scales is one of the key factors that influence the functional activity of cells in contact with biomaterials. Our group has exploited simple chemical treatments to create unique nanoporous titanium surfaces that favor cell adhesion and formation of filopodia. However, the influence of these filopodia on the adhesion strength remains unknown. Previous biomechanical studies by AFM faced some problems caused by irregularities on the titanium. In this study, we have tested the effect of chemical treatment time on generating a more planar nanoporous surface that will be amenable to the characterization of the composition, structure and adhesion of cell filopodia. Our ultimate aim is to better understand the biomechanical properties of filopodia and their contribution to adhesion strength and cell signaling. Materials and Methods: Commercially pure titanium discs (cp-Ti) were polished to a mirror finish. The polished discs were treated for various times ranging from 1 to 4 hours with a mixture of H_2SO_4 / H_2O_2 50/50 at room temperature to generate a nanoporous surface. Mouse catvaria-derived osteogenic cells (MC-3T3) were cultured in MEM-α and plated on polished (control) and nanoporous cp-Ti discs at a cell density of 10 000 cells/well. The cells were grown for periods up to 6h, 1 day and 3 days. Surface topography and cell morphology were examined using SEM and AFM. The actin network was visualised by florescence microscopy using rhodamin-phalloidin. Results and Discussion: SEM and AFM characterization showed that a 1.5 h treatment was adequate to produce a nanoporous surface with an overall planarity. SEM revealed that the cells developed more filopodia on this surface as compared with the control surface, confirming previous results with longer treatment times. The filopodia displayed lateral protrusions known as nanopodia that can contribute to the adhesive interaction of the filopodia with the surface. Figure 1. SEM micrograph of MC-3T3 grown for 6h on nanoporous titanium surface. Noteworthy, the reticulated nature of some cell projections on the nanoporous surface. Fluorescence microscopy also showed that the resulting nanoporous surface affects the organization of the actin network. Simple adjustment of the treatment time was therefore sufficient to achieve a mono planar nanoporous surface that allows cellular changes suggestive of increased osteogenic activity. Conclusions: We have shown that it is possible to create a planar nanoporous titanium surface that will be amenable to measuring the filopodia adhesion force. Such measurements will help to elucidate how nanoscale cell biomechanics induces and modulates cellular signalling so that surface topography can be used as an alternative to bioactive molecules.