Abstract
Aim
To control the growth and function of osteoblasts on Titanium alloy surfaces produced by electrochemical patterning.
Methods
Samples of Ti6Al4V were prepared with three different finishes; no surface preparation following machining, polishing on a grinding wheel with sequential grit papers up to 4000 to achieve a mirror finish and treatment in a flat electrochemical cell with a 3M sulphuric acid in methanol using 9V supplied over 60 seconds to produce a surface with defined nano/microscale roughness. Glass coverslips were used as control surfaces. Surfaces were seeded with primary rat calvarial osteoblasts and incubated in Dulbecco's Modified Eagle Medium with 10% (v/v) sera for 24 hours before fixing and performing immunofluorescence staining with anti-vinculin antibody. Photomicrographs of the surfaces were analysed with Image J and analySIS FIVE programs. Results for cell number, cell area, focal adhesion area and polarity (lack of roundness) were analysed (using the Mann Whitney test) for ANOVA using SPSS.
Results
Cells adhered to all surfaces with the most cells on the polished surface and the fewest on the glass and 9V60s surfaces. There were significant differences in cell number only between the polished surface and the glass control (p=0.026) and the 9V60s surface (p=0.006). Cells grown on the glass control surfaces exhibited the largest areas (mean = 840micron2) whilst those on the machined surface were the smallest (mean = 601micron2). A significant difference in cell area was seen between the machined and polished surfaces (p=0.025). The area of the focal adhesions was significantly different between the cells on 9V60s surface and the glass control (p=0.004), machined (p=0.003) and polished surfaces (p=0.006). Significant differences in polarity were seen between the cells on machined surface and the glass control (p=0.004), polished (p=0.004) and 9V60s surfaces (p=0.004).
Discussion
Differences in cell numbers on glass and two of the Ti surfaces may be explained by the smooth nature of the glass coverslips in comparison to the nanoscale topography on the polished and 9V60s treated surfaces. Cell area was noted to be different between the machined and smoother polished surface. This may be explained by the grooves present on the machined surfaces preventing normal cell spreading by the process of contact guidance. There was a marked difference in polarity between the most polarised cells on the machined surface and the more rounded cells on the smoother surfaces, again consistent with the behaviour of contact guidance, with cells growing in the direction of the surface grooves. Focal adhesions present on the 9V60s treated surface were very small in comparison to those on other surfaces. Several features of implant surfaces may affect osteoblast growth, including surface roughness, chemical composition, surface charge and surface energy. These features influence the adsorption of proteins onto the surfaces, in turn influencing the growth and behaviour of the adherent cell population.
Conclusion
Mechanical and electrochemical treatment of titanium alloy can significantly affect the growth and behaviour of osteoblasts grown on the surface. This has potential applications in arthroplasty and fracture fixation.
