THE INFLUENCE OF TITANIUM ALLOY SURFACE TOPOGRAPHY ON KERATINOCYTE GROWTH AND EXPRESSION OF ADHESION COMPLEX MOLECULES

Abstract

Introduction: Transcutaneous Amputation Prosthesis (ITAP) is an alternative for transfemoral amputees to conventional stump-socket prostheses which have many problems. These include: poor fit, stump pressure sores, pain, infections and unnatural gait. ITAP aims to overcome these by being osseointegrated into the femoral medulla with a pin protruding through the skin to which the external prosthesis attaches. Thus, the forces normally encountered by the stump soft tissues are now transferred directly to the skeleton. However, the transcutaneous pin produces a route for infection from the external to internal environment. Therefore, a key feature to the success of the ITAP is to produce a biological seal at the transcutaneous interface. Epithelial cells have been shown to attach to dental transcutaneous titanium devices via hemidesmosomes (HD).2 Focal contacts (FC) are also important in cell adhesion and to the underlying substratum.3 We grew human keratinocytes on different titanium surfaces to assess their morphology, ability to proliferate and produce HD and FC. Hypothesis: Surface topography influences keratinocytes morphology and proliferative capacity and expression of HD and FC.

Materials and Methods: 4 titanium alloy (Ti6Al4V) surface topographies were used (10mm x 4mm discs): polished, machine finished, sandblasted and hydrofluoric acid etched (HF) and a control – plastic thermanox. Surface roughness profiling of titanium discs were measured (Mitutoyo Surftest SV-400). HaCaT keratinocytes were grown on disc surfaces in wells of culture medium at +37oC, 5% CO2 and analysed at 1, 2, 3 and 4 days. Cells were processed to visualise HD with fluorescence microscopy using antibodies to the 6-integrin and plec-tin. Anti-vinculin antibodies were used to visualise FC. Fluorescein isothiocyanate (FITC) secondary antibodies enabled counting of structures (all product: Sigma-Aldrich, UK). Alamar blue (Serotec, UK) measured cell proliferation and SEM (surface morphology, cell area) and TEM were also performed. Cells grown on polished, machined and thermanox discs supported a regular, confluent layer with many cytoplasmic processes and dividing cells. HF and sandblasted discs grew an irregularly layer with fewer cytoplasmic processes and fewer dividing cells (not quantified). Day 3 TEM revealed HD, FC and desmosomes; cells on polished and thermanox were more closely packed and in layers.

Conclusion: Keratinocytes are significantly influenced by titanium surface topography. Smooth polished titanium alloy may be the ideal surface for a transcutaneous pin in the ITAP. Further experiments into isolating favourable biological components needed to encourage keratinocytes to attach onto titanium should be carried out.

Results: No significant difference shown in cell proliferation between titanium discs but cells on thermanox grew significantly more (p< 0.05). FC and HD numbers increased on all surfaces (days 1–3); a negative correlation between surface roughness and HD and FC numbers observed (lower Ra values = more HD and FC expressed).