Intraosseous transcutaneous amputation prostheses versus dental implants: a comparison between keratinocyte and gingival epithelial cell adhesion in vitro.

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. Oral gingival epithelial cell attachment creates an infection-free seal around dental implants. However, this has yet to be achieved consistently outside of the oral environment. Epithelial cells attach to metal substrates by means of hemidesmosomes and focal adhesions. Their density per unit cell is an indicator of attachment strength. We postulate that gingival epithelial cells express more hemidesmosomes and focal adhesions at earlier time points, compared with epidermal keratinocytes, and this increased speed and strength of attachment may be the reason why an infection-free seal is often achieved around dental implants but less frequently around ITAP. The aim of this study was to compare epidermal keratinocyte with oral gingival cell attachment on titanium alloy in vitro, to determine whether these two cell types differ in their speed and strength of attachment. We aimed to test the hypothesis that gingival cells up-regulate focal adhesion and hemidesmosome formation at earlier time points compared with extra-oral keratinocytes. To test this hypothesis we cultured epidermal keratinocytes and oral gingival cells on titanium alloy substrates and assessed cell attachment by focal adhesions and hemidesmosome expression at 4, 24, 48 and 72 hours. Formation and expression of hemidesmosomes temporally lagged behind that of focal adhesions in both cell types. Gingival derived cells up-regulated focal adhesion and hemidesmosome expression at earlier time points compared with epidermal keratinocytes. Hemidesmosome expression in oral gingival cells was 3 times greater compared with epidermal keratinocytes at 4 hours. Our findings indicate that earlier attachment may be key to the success of the dental implant transcutaneous interface.

The development of fibronectin-functionalised hydroxyapatite coatings to improve dermal fibroblast attachment in vitro.

The success of long-term transcutaneous implants depends on dermal attachment to prevent downgrowth of the epithelium and infection. Hydroxyapatite (HA) coatings and fibronectin (Fn) have independently been shown to regulate fibroblast activity and improve attachment. In an attempt to enhance this phenomenon we adsorbed Fn onto HA-coated substrates. Our study was designed to test the hypothesis that adsorption of Fn onto HA produces a surface that will increase the attachment of dermal fibroblasts better than HA alone or titanium alloy controls. Iodinated Fn was used to investigate the durability of the protein coating and a bioassay using human dermal fibroblasts was performed to assess the effects of the coating on cell attachment. Cell attachment data were compared with those for HA alone and titanium alloy controls at one, four and 24 hours. Protein attachment peaked within one hour of incubation and the maximum binding efficiency was achieved with an initial droplet of 1000 ng. We showed that after 24 hours one-fifth of the initial Fn coating remained on the substrates, and this resulted in a significant, three-, four-, and sevenfold increase in dermal fibroblast attachment strength compared to uncoated controls at one, four and 24 hours, respectively.

Tendon re-attachment to metal prostheses in an in vivo animal model using demineralised bone matrix.

We used demineralised bone matrix (DBM) to augment re-attachment of tendon to a metal prosthesis in an in vivo ovine model of reconstruction of the extensor mechanism at the knee. We hypothesised that augmentation of the tendon-implant interface with DBM would enhance the functional and histological outcomes as compared with previously reported control reconstructions without DBM. Function was assessed at six and 12 weeks postoperatively, and histological examination was undertaken at 12 weeks. A significant increase of 23.5% was observed in functional weight-bearing at six weeks in the DBM-augmented group compared with non-augmented controls (p = 0.004). By 12 weeks augmentation with DBM resulted in regeneration of a more direct-type enthesis, with regions of fibrocartilage, mineralised fibrocartilage and bone. In the controls the interface was predominantly indirect, with the tendon attached to the bone graft-hydroxyapatite base plate by perforating collagen fibres.

A comparison of augmentation techniques for reconstruction of the extensor mechanism following proximal tibial replacement in an experimental animal model.

We used an in vivo model to assess the use of an autogenous cancellous bone block and marrow graft for augmenting tendon reattachment to metallic implants. We hypothesised that augmentation of the tendon-implant interface with a bone block would enable retention of the graft on the implant surface, enhance biological integration, and result in more consistent functional outcomes compared with previously reported morcellised graft augmentation techniques. A significant improvement in functional weight-bearing was observed between six and 12 weeks. The significant increase in ground reaction force through the operated limb between six and 12 weeks was greater than that reported previously with morcellised graft augmented reconstructions. Histological appearance and collagen fibre orientation with bone block augmentation more closely resembled that of an intact enthesis compared with the morcellised grafting technique. Bone block augmentation of tendon-implant interfaces results in more reliable functional and histological outcomes, with a return to pre-operative levels of weight-bearing by 24 weeks.

Sealing the skin barrier around transcutaneous implants: in vitro study of keratinocyte proliferation and adhesion in response to surface modifications of titanium alloy.

Conventional amputation prostheses rely on the attachment of the socket to the stump, which may lead to soft-tissue complications. Intraosseous transcutaneous amputation prostheses (ITAPs) allow direct loading of the skeleton, but their success is limited by infection resulting from breaching of the skin at the interface with the implant. Keratinocytes provide the skin's primary barrier function, while hemidesmosomes mediate their attachment to natural ITAP analogues. Keratinocytes must attach directly to the surface of the implant. We have assessed the proliferation, morphology and attachment of keratinocytes to four titaniumalloy surfaces in order to determine the optimal topography in vitro. We used immunolocalisation of adhesion complex components, scanning electron microscopy and transmission electron microscopy to assess cell parameters. We have shown that the proliferation, morphology and attachment of keratinocytes are affected by the surface topography of the biomaterials used to support their growth. Smoother surfaces improved adhesion. We postulate that a smooth topography at the point of epithelium-ITAP contact could increase attachment in vivo, producing an effective barrier of infection.

Fibronectin silanized titanium alloy: a bioinductive and durable coating to enhance fibroblast attachment in vitro.

Long term success of percutaneous implants is dependent on soft tissue attachment to prevent infection and epithelial downgrowth, which leads to failure of the implant. Fibronectin coatings are known to enhance fibroblast attachment in vitro, but are subject to desorption from serum protein competition in vivo. This paper quantifies the binding of fibronectin to titanium alloy by silanization and the durability of this attachment when soaked in protein-rich fluid compared with adsorbed fibronectin. The biological activity of fibronectin bound to silanized titanium alloy was confirmed by analyzing cell area, morphology, immunolocalization of focal contacts, and metabolism of dermal fibroblasts. This was compared with both adsorbed fibronectin and uncoated titanium alloy. Silanized titanium alloy bound over twice the amount of fibronectin compared to untreated titanium alloy. On soaking in fetal calf serum there was no significant loss of fibronectin (p = 0.589) from the silanized surface but a significant 44% loss (p = 0.002) from untreated surfaces. Fibroblasts on silanized fibronectin had significantly larger cell areas and more vinculin focal contact markers when compared to untreated surfaces (p < 0.005). The results confirm the durability of silanized fibronectin from protein competition and bioactive effect on fibroblasts.

The novel use of resorbable Vicryl mesh for in vivo tendon reconstruction to a metal prosthesis.

We examined the mechanical properties of Vicryl (polyglactin 910) mesh in vitro and assessed its use in vivo as a novel biomaterial to attach tendon to a hydroxyapatite-coated metal implant, the interface of which was augmented with autogenous bone and marrow graft. This was compared with tendon re-attachment using a compressive clamp device in an identical animal model. Two- and four-ply sleeves of Vicryl mesh tested to failure under tension reached 5.13% and 28.35% of the normal ovine patellar tendon, respectively. Four-ply sleeves supported gait in an ovine model with 67.05% weight-bearing through the operated limb at 12 weeks, without evidence of mechanical failure. Mesh fibres were visible at six weeks but had been completely resorbed by 12 weeks, with no evidence of chronic inflammation. The tendon-implant neoenthesis was predominantly an indirect type, with tendon attached to the bone-hydroxyapatite surface by perforating collagen fibres.

Nature's answer to breaching the skin barrier: an innovative development for amputees.

The human body has evolved to maintain homeostasis through the covering of skin and mucous membranes, which separate the internal environment from the harsh and variable external milieu. Few structures naturally penetrate these coverings, and teeth are the only exception in human beings. Attempts to breach these barriers, to develop skin- penetrating, bone-anchored amputation prostheses, can lead to opportunist invasion by microorganisms and subsequent infection, which can jeopardize the life of the individual. There are only a few fascinating examples where the integument of other species is interrupted without such dire consequences, and the deer antler is one such case. Deer antlers are cephalic bony appendages arising from the frontal bones of the skull of the males of most deer species, and are true transient skin-penetrating structures. Antlers are subject to extreme loading during the rutting season and yet the skin-bone barrier remains intact. Here we show how deer antlers can be used as natural analogues for the successful development of specialized orthopaedic amputation prosthetics. We have used quantitative and qualitative findings from a study of the morphology of deer antlers to develop a device that mimics their structure, which creates a tight seal between the implant and the host tissues, for use in amputation prosthetics.

Assessment of reproducibility and accuracy in templating hybrid total hip arthroplasty using digital radiographs.

In 20 patients undergoing hybrid total hip arthroplasty, the reproducibility and accuracy of templating using digital radiographs were assessed. Digital images were manipulated using either a ten-pence coin as a marker to scale for magnification, or two digital-line methods using computer software. On-screen images were templated with standard acetate templates and compared with templating performed on hard-copy digital prints. The digital-line methods were the least reliable and accuracy of sizing compared with the inserted prostheses varied between -1.6% and +10.2%. The hard-copy radiographs showed better reproducibility than the ten-pence coin method, but were less accurate with 3.7% undersizing. The ten-pence coin method was the most accurate, with no significant differences for offset or acetabulum, and undersizing of only 0.9%. On-screen templating of digital radiographs with standard acetate templates is accurate and reproducible if a radiopaque marker such as a ten-pence coin is included when the original radiograph is taken.

Extensor mechanism reconstruction after proximal tibial replacement.

We developed an in vivo model of the attachment of a patellar tendon to a metal implant to simulate the reconstruction of an extensor mechanism after replacement of the proximal tibia. In 24 ewes, the patellar tendon was attached to a hydroxyapatite (HA)-coated titanium prosthesis. In 12, the interface was augmented with autograft containing cancellous bone and marrow. In the remaining ewes, the interface was not grafted. Kinematic gait analysis showed nearly normal function of the joint by 12 weeks. Force-plate assessment showed a significant increase in functional weight-bearing in the grafted animals (p = 0.043). The tendon-implant interface showed that without graft, encapsulation of fibrous tissue occurred. With autograft, a developing tendon-bone-HA-implant interface was observed at six weeks and by 12 weeks a layered tendon-fibrocartilage-bone interface was seen which was similar to a direct-type enthesis. With stable mechanical fixation, an appropriate bioactive surface and biological augmentation the development of a functional tendon-implant interface can be achieved.

A histomorphological study of tendon reconstruction to a hydroxyapatite-coated implant: regeneration of a neo-enthesis in vivo.

The attachment of tendons and ligaments to massive endoprostheses remains a clinical challenge due to the difficulty in achieving a soft tissue implant interface with a mechanical strength sufficient to transmit the forces necessary for locomotion. We have used an in vivo animal model to study patellar tendon attachment to an implant surface. The interface generated when the patellar tendon was attached to a hydroxyapatite (HA) coated implant was examined using light microscopy and a quantitative histomorphological analysis was performed. In the Autograft Group, the interface was augmented with autogenous cancellous bone and marrow graft, and at six weeks an indirect-like insertion was observed. At twelve weeks, the interface was observed to be a layered neo-enthesis, whose morphology was similar to a normal direct tendon insertion. In the HA Group, the tendon-implant interface was not augmented, and the implant was enveloped by a dense collagenous fibrous tissue. This study shows that a tendon-implant neo-enthesis can develop in situ by employing a suitable implant surface in association with biological augmentation.