Intraosseous transcutaneous amputation prosthesis: an in vitro investigation of the effect of fibroblast pre-seeding on keratinocyte attachment to titanium alloy

Transcutaneous implants have been successfully employed in dentistry since the 1960’s; however subsequent attempts to translate this into extra-oral solutions for amputees have been beset with complications, with infection being the primary failure modality. 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 and provide secure attachment of artificial limbs for amputees. In normal skin, dermal fibroblasts and epidermal keratinocytes are known to effect one another’s differentiation and cellular activities, for example; production and constitution of a basment membrane. Cells do not adhere directly to biomaterial substrate, but attach to the surface via a layer of ECM extruded from cells. In the case of keratinocytes this layer is principally laminin-5 (Ln5). It is possible that pre-seeding ITAP biomaterials with fibroblasts could up-regulate Ln5 expression and result in improved adherence of keratinocytes, and this could improve the infection-resistance of the skin-ITAP seal in vivo. We hypothesise that adhesion of keratinocytes, measured by the number of focal adhesions per unit cell area, and the order and intensity of Ln5 production will be significantly greater on titanium alloy substrates pre-seeded with fibroblasts compared with controls.