Summary. Osseointegrated Amputation Prostheses can be functionalised by both biological augmentation and structural augmentation. These augmentation techniques may aid the formation of a stable skin-implant interface. Introduction. Current clinical options are limited in restoring function to amputees, and are associated with contact dermatitis and infection at the stump-socket interface. Osseointegrated Amputation Prosthesis attempts to solve issues at the stump-socket interface by directly transferring axial load to the prosthesis, via a skin-penetrating abutment. However, development is needed to achieve a seal at the skin-implant interface to limit infection. Fibronectin, an Extracellular Matrix protein, binds to integrins during wound healing, with the RGD tripeptide being part of the recognition sequence for its integrin binding domain. In vitro work has found silanization of RGD to polished titanium discs up regulates fibroblast attachment compared to polished control. Electron Beam Melting can produce porous titanium alloy implants, which may encourage tissue attachment. This study aims to test whether a combination of biological RGD coatings and porous metal manufacturing techniques can encourage the formation of a seal at the skin-implant interface. Materials and Methods. We developed four different augmented transcutaneous devices: Porous, Porous RGD coated, drilled and drilled RGD coated. These were implanted in tibial transcutaneous ovine model, n=6, for a period of 6 months. Following explantation we performed hard grade resin histology to assess soft tissue attachment at the transcutaneous interface. Results. Histological analysis revealed no statistical difference in epithelial downgrowth and epidermal attachment values between the four augmented devices. There were significant increases (p<0.05) in the number of blood vessels and the number of cells in the Porous RGD devices compared with both drilled implant devices. Both Porous and Porous RGD implant groups observed significant increase (p<0.05) in soft tissue infiltration compared with both Drilled implant devices. Discussion. The use of porous structures and RGD coatings increases tissue ingrowth and revascularisation in ITAP devices despite having no effect on epithelial downgrowth and epidermal attachment in a long-term ovine model. There were no detrimental effects in the transcutaneous interface formation observed. These augmentation techniques may prove beneficial in preclinical and clinical developments of transcutaneous osseointegrated devices

Intraosseous Transcutaneous Amputation Prosthesis (ITAP) is a new generation of limb replacements that can provide to amputees, an alternative solution to the main problems caused by the most common used external prosthesis such as pressure sores, infections and unnatural gait. ITAP is designed as one pylon osteointegrated into the bone and protruding through the skin, allowing both the mechanical forces to be directly transferred to the skeleton and the external skin being free from frictions and infections. The skin attachment to the implant is fundamental for the success of the ITAP, as it prevents the implant to move and consequently fail. In this study we wanted to test if cell viability and attachment was improved using TiO2 nanotubes. Human keratinocytes and human dermal fibroblasts were seeded for three days on TiO2 nanotubes with different sizes (18–30nm, 40–60nm and 60–110nm), compared with controls (smooth titanium) and tested for viability and attachment. A Mann-Whitney U test was used to compare groups where p values < 0.05 were considered significant. The results showed that the viability and cell attachment for keratinocytes were significantly higher after three days on controls comparing with all nanotubes (p=0.02), while attachment was higher on bigger nanotubes and controls. Cell viability for fibroblasts was significantly higher on nanotubes between 40 and 110nm comparing with smaller size and controls (p=0.03), while investigation of cell attachment is ongoing. From these early results, we can say that TiO2 nanotubes can improve the soft tissue attachment on ITAP. Further in-vitro and ex-vivo experiments on cell attachment will be carried out

To try and aid the formation of a soft tissue seal to promote dermal and epidermal attachment to Intraosseous Transcutaneous Amputation Prostheses we compared the effect of titanium surfaces functionalised with fibronectin (fn) or YRGD peptide sequences on human dermal cell (HDF) attachment. We hypothesise that YRGD and fn coatings will significantly increase HDF attachment to titanium alloy substrates. Titanium alloy 10mm discs were polished and acted as control substrates, functionalised surfaces had YRGD or fn adsorbed or silanised onto the polished surface. HDFs were seeded at 10,000/disc and cultured for 1, 4, 24 and 96 hours, fixed and fluorescent immnolocalisation for vinculin was performed. Individual vinculin markers were counted and density calculated as a measure of cell attachment. All assays were performed in triplicate and data were analysed in SPSS 19.0 and results were considered significant at the 0.05 level. Results showed an up-regulation of Focal adhesion density (FA) against controls at all time-points (excluding ad-fn at 4 hours, p=0.057), p values < 0.05, the use of functionalised titanium surfaces may lead to long-term clinical success of ITAP. We have shown a significant positive effect on cell attachment when a synthetic peptide sequence is used. Using synthetic peptide sequence may also be more beneficial from a regulatory stand-point compared with using isolated proteins

Infection is the primary failure modality for transcutaneous implants because the skin breach provides a route for pathogens to enter the body. Intraosseous transcutaneous amputation prostheses (ITAP) are being developed to overcome this problem by creating a seal at the skin-implant interface to prevent bacterial invasion. Oral gingival epithelial cell adhesion creates an infection free seal around dental implants; however this has yet to be demonstrated outside the oral environment. All epithelial cells attach via hemidesmosomes (HD) and focal adhesions (FA) and their expression is an indicator of adhesion efficiency. The aim of this study was to compare epidermal keratinocyte with oral gingival epithelial cell adhesion on titanium alloy in vitro to determine whether these two cell types differ in their speed and strength of adhesion. It was hypothesised that oral gingival epithelial cells attach to titanium alloy earlier than epidermal keratinocytes; with greater expression of hemidesmosomes and focal adhesions.

Human oral gingival epithelial cell (HGEP) and primary human epidermal keratinocyte (HPEK) adhesion to titanium alloy, was assessed at 4, 24, 48 and 72 hrs. Adhesion was measured by the number of FAs per unit cell area and expression of HDs using a semi-quantitative scale.

At 4 and 24hrs, there was a significant increase in vinculin marker expression per unit cell area of 4.3 and 4.7 times in HGEP compared with HPEK (p=0.000). At 48 and 72hrs there were no significant differences.

HD expression was significantly greater in HGEP at 4 and 24hrs (p=0.002) compared with HPEK. Up-regulation of HD expression in HPEK lagged that of HGEP until 48hrs, after which no significant differences were observed.

This study has demonstrated that oral gingival cells up-regulate both focal adhesion and hemidesmosome expression at earlier time points compared with epidermal keratinocytes. Expression of hemidesmosomes lags that of focal adhesions, suggesting that focal adhesion formation is a prerequisite for hemidesmosome assembly. We postulate that early attachment of oral gingival epithelial cells to dental implant biomaterials may be responsible for the formation of an infection-free seal.