Introduction: Normal limb use in amputees with made to measure external prostheses can be impaired by problems at the stump – socket interface. The development of an Intraosseous Transcutaneous Amputation Prosthesis (ITAP) would overcome the problems by protecting the soft tissues, whilst redistributing high stresses to bone. ITAP creates a breach in the skins protective barrier to infection, hence requires a sufficient soft tissue – implant seal to prevent implant failure. Deer antlers are natural analogues of ITAP, and successfully overcome the problems associated with skin penetrating implants such as infection, marsupilisation and avulsion. In this study, an ITAP device has been developed, with a successful soft tissue – implant interface, based on deer antler morphology. It is hypothesised that sub-epithelial dermal fibroblastic, but not epithelial layer adhesion, is directly responsible for the degree of downgrowth observed around ITAP.

Methods: Eleven pairs of deer antler were used to histologically evaluate the interface between the antler and pedicle, and the soft tissue seal around the antler-pedicle structure. The findings were used to develop a titanium alloy (Ti6Al4V) ITAP device in a goat model. Three to five transcutaneous pins were implanted into the medial aspect of the right tibia of skeletally mature female goats. Four implant designs were tested, Machine Finished Straight (MFS), Hydroxyapatite (HA) Coated MFS, Machine Finished Flanged (MFF) and HA Coated MFF. The 70μ thick HA coating was applied to the implant region abutting the sub-epithelium. The implants remained in situ for four weeks after which the histology of the resulting interfaces were analysed qualitatively and quantitatively for degrees of epithelial downgrowth (marsupilisation) and epithelial/sub-epithelial layer attachment to the implant surface.

Results: The histology of the deer antler showed there to be an extremely small area of epithelial attachment, with negligible downgrowth, arrested by soft tissue adhesion to the underlying pedicle surface. There was a significant increase in pore size and frequency in the pedicle structure (abutting the soft tissues), compared to the antler proper. The MFS ITAP implants were associated with significantly greater downgrowth and reduced epithelial and sub-epithelial layer attachment compared to all other implant designs. The HA coating, and porous flange structure significantly reduced downgrowth and increased sub-epithelial layer attachment. Regression correlation showed that there is a significant negative correlation between the extent of downgrowth and the degree of sub-epithelial dermal fibroblastic layer attachment observed around ITAP implants (All p values < 0.05).

Discussion: Deer antlers successfully overcome the potential problems for ITAP. By artificially recreating some of the aspects of the antler, including layering of porous and bioactive surfaces for tissue adhesion, we have successfully developed an ITAP implant that minimises downgrowth and actively encourages epithelial and sub-epithelial soft tissue adhesion.

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).

Introduction: Functional outcome following proximal tibial replacement can be impaired by extensor mechanism inefficiency. Current methods used to re-attach the patellar tendon result in varied levels of extensor function. Successful attachment of the patellar tendon requires initial mechanical stability and long-term biological fixation. We have employed a prosthesis, to model patellar tendon re-attachment, to test the hypothesis that biological augmentation of an implant which can provide sufficient mechanical integrity will allow a tendon-implant interface to develop that is similar in function and morphology to a normal tendon-bone interface.

Methods: The right patellar tendon in 24 Skeletally mature Friesland ewes was transfixed between the interlocking spikes of a hydroxyapatite-coated, customized tendon clamp to simulate patellar tendon re-attachment to a proximal tibial replacement. In 12 animals (Autograft group) the clamp attachment was augmented with autologous cancellous bone and marrow graft harvested from the ipsilateral iliac crest at the time of surgery, whilst the remaining animals (HA group) served as un-supplemented controls. Functional outcome was assessed using force plate measurements and two-dimensional optical kinematic gait analysis. Animals were euthanised at 6 and 12 weeks. The specimens were harvested, processed for histology and examined using light microscopy.

Results: The clamp device provided sufficient mechanical fixation of the patellar tendon to allow immediate weight bearing. Gait analysis showed that the range of movement of the stifle (knee) joint was not compromised by the surgical intervention at 6 or 12 weeks post-operation. An extensor lag observed at 6 weeks in both the Autograft and HA group was seen to fully recover by 12 weeks post-operation. There was a significant increase in functional weight bearing through the operated limb of the Autograft group animals between 6 and 12 weeks, which was not observed in the HA group. The tendon-implant interface in the HA group animals showed a fibrous tissue encapsulation of the HA coated surface, with collagen fibrils running parallel to the implant surface. In the Autograft group at 6 weeks post-operation a soft tissue – bone – HA interface had developed, similar in morphology to that of an indirect-type enthesis. Perpendicular orientated Sharpey’s-like fibres were observed spanning the region between the tendon and the HA coated implant and the bone graft material was seen to be undergoing active remodelling. By 12 weeks post-operation the interface was layered with regions of fibrocartilage clearly visible, more closely resembling the morphology of a direct-type enthesis.

Discussion: The clamp device provided sufficient mechanical fixation of the patellar tendon to allow immediate use the operated limb. The incorporation of a bio-active implant coating and biological augmentation encouraged a neo-enthesis to develop with near normal functional properties, and morphology similar to that of a normal patellar tendon-bone direct-type enthesis.