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.

Background

Re-attachment of tendon to bone is challenging with surgical repair failing in up to 90% of cases. Poor biological healing is common and characterised by the formation of weak scar tissue. Previous work has demonstrated that decellularised allogenic demineralised bone matrix (DBM) regenerates a physiologic enthesis. Xenografts offer a more cost-effective option but concerns over their immunogenicity have been raised. We hypothesised that augmentation of a healing tendon-bone interface with DBM incorporated with autologous mesenchymal stem cells (MSCs) would result in improved function, and restoration of the native enthesis, with no difference between xenogenic and allogenic scaffolds.

Aims

The Intraosseous Transcutaneous Amputation Prosthesis (ITAP) may improve quality of life for amputees by avoiding soft-tissue complications associated with socket prostheses and by improving sensory feedback and function. It relies on the formation of a seal between the soft tissues and the implant and currently has a flange with drilled holes to promote dermal attachment. Despite this, infection remains a significant risk. This study explored alternative strategies to enhance soft-tissue integration.

Summary

Our results prove that Demineralised Cortical Bone (DCB) can be used as biological tendon graft substitute, combined with correct surgical technique and the use of suture bone anchor early mobilisation can be achieved.

Summary

Our study shows that a tendon rupture can be successfully augmented with Demineralised Cortical Bone (DCB) giving initial appropriate mechanical strength suitable for in vivo use providing the biological reactions to the graft are favourable.

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

Bone-anchored devices have been used as skin-crossing conduits to record neuromuscular signals in sedated animals. Long-term recordings from cognisant subjects must be assessed. Hypothesis A bone-anchored device is suitable as a conduit for epimysial EMG (Electromyogram) recordings and is reliable in the long-term.

Repair of tendon injuries aims to restore length, mechanical strength and function. We hypothesise that Demineralised Cortical Bone (DCB) present in biological tendon environment will result in remodelling of the DCB into ligament tissue. A cadaveric study was carried out to optimize the technique. The distal 1cm of the patellar tendon was excised and DCB was used to bridge the defect. 4 models were examined, Model-1: one anchor, Model-2: 2 anchors, Model-3: 2 anchors with double looped off-loading thread, Model-4: 2 anchors with 3 threads off-loading loop. 6 mature sheep undergone surgical resection of the distal 1cm of the right patellar tendon. Repair was done using DCB with 2 anchors. Immediate mobilisation was allowed, animals were sacrificed at 12 weeks. Force plate assessments were done at weeks 3, 6, 9 and 12. Radiographs were taken and pQCT scan was done prior to histological analysis. In the cadaveric study, the median failure force for the 4 models; 250N, 290N, 767N and 934N respectively. In the animal study, none of the specimens showed evidence of ossification of the DCB. One animal failed to show satisfactory progress, X-rays showed patella alta, on specimen retrieval there was no damage to the DCB and sutures and no evidence of anchor pullout. Functional weight bearing was 79% at week12. Histological analysis proved remodelling of the collagen leading to ligamentisation of the DCB. Results prove that DCB can be used as biological tendon substitute, combined with the use of suture bone anchor early mobilisation can be achieved.

Treatment of tendon and ligament injuries remains challenging; the aim is to find a biocompatible substance with mechanical and structural properties that replicate those of normal tendon and ligament. We examined the mechanical properties of Demineralised Cortical Bone (DCB) after gamma irradiation (GI) and freeze drying (FD). We also used different techniques for repairing bone-tendon-bone with DCB in order to measure the mechanical performance of the construct. DCB specimens were allocated into 4 groups; FD, GI, combination of both or none. The maximum tensile forces and stresses were measured. 4 cadaveric models of repair of 1cm patellar tendon defect using DCB were designed; model-1 using one bone anchor, Model-2 using 2 bone anchors, Model-3 off-loading by continuous thread looped twice through bony tunnels, Model-4 off-loading with 3 hand braided threads. Force to failure and mode were recorded for each sample. FD groups results were statistically higher (p=<0.05) compared to non-FD groups, while there was no statistical difference between GI and non-GI groups. The median failure force for model-1: 250N, model-2: 290N, model-3: 767N and model-4: 934N. There was no statistical significance between model-1 and model-2 (p=0.249), however statistical significance was found between other models (p=<0.006). GI has no significant effect on mechanical strength of the CDB while FD may have positive effect on its mechanical strength. Our study shows that a tendon rupture can be successfully augmented with CDB giving initial appropriate mechanical strength suitable for in vivo use providing the biological reactions to the graft are favourable.

Introduction

Tendon injuries remain challenging, secondary healing and prolonged immobilisation result in suboptimal outcome. Previous study by our group showed that demineralised bone matrix (DBM) can result in faster healing of a tendon enthesis. The aim of this study is to test different ways augmenting tendon with DBM to enhance tendon repair and regeneration.

Introduction

Demineralised Bone Matrix (DBM) is widely used in Orthopaedics and dentistry as a bone graft substitute and may be used to augment bone formation in load bearing applications.

In this study we examine the effect of gamma irradiation and freeze drying on the tensile strength of Demineralised Cortical Bone (DCB).

Introduction

Intraosseous transcutaneous amputation prostheses (ITAP) provide an alternative means of attaching artificial limbs for amputees. Conventional stump-socket devices are associated with soft tissue complications including; pressure sores and tissue necrosis. ITAP resolves these problems by attaching the exo-prosthesis transcutaneously to the skeleton. The aim of this study is to increase the attachment of dermal fibroblasts to titanium alloy in vitro. Fibronectin (Fn) and laminin 332 (Ln) enhance early cell growth and adhesion. We hypothesize that silanized dual coatings of fibronectin and laminin (SiFnLn) will be more durable when compared with adsorbed dual coating (AdFnLn), and will enhance early fibroblast growth and adhesion compared to single coatings.

Introduction

Following amputation, residual stumps used to attach the external prostheses can be associated with sores, infection and skin necrosis. These problems could be overcome by off loading the soft tissues. Intraosseous transcutaneous amputation prostheses (ITAP) attach external implants directly to residual bone reducing these complications. However, a tight seal at the skin implant interface is crucial in preventing epithelial down-growth and infection. Fibronectin (Fn) and laminin 332 (Ln), enhance early cell growth and adhesion of keratinocytes. Silanization to titanium alloy (Ti) allows these proteins to bond to the metal directly. We hypothesize that silanized dual coatings of fibronectin and laminin (SiFnLn) will be more durable than absorbed proteins and that keratinocyte adhesion will be increased compared with Ti controls and single silanized 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.

Introduction: Hydrogenated (acetylene:C2H2) and silanized (tetra methyl silane:TMS) diamond-like-carbon coatings (DLC) are applied to titanium alloy to reduce surface energy, cell adhesion and hydrophilicity. The incorporation of silicon into DLC reduces its surface energy. It was hypothesized that surfaces that have high surface energy and high hydrophilicity favoured the adhesion and maturation of fibroblasts when compared with C2H2 and TMS coated substrates in vitro. This would help in achieving a seal at the prosthesis – soft tissue interface, thereby helping in reducing infection.

Methods: and Materials: Fibroblasts were cultured on 10 mm diameter titanium alloy, C2H2 and TMS coated titanium alloy discs for 4 hours and 24 hours (2500 cells per disc). Cell area, adhesion plaque numbers, number of plaques per unit area (plaque density) and the total area of adhesion plaques per cell were analysed. The results were compared between experimental groups and controls at 4 and 24 hours. In order to measure the strength of adhesion of cells fibroblasts were cultured on discs (30 mm diameter)[machine finished and polished(Ra = 0.031)](density-300,000 cells per disc) for 4 and 24 hours with similar coatings and exposed to radial shear by flow (100 mls/min) of culture media over their surface. These discs were then stained and analysed using Photoshop (ver.5.5) and SPSS (ver.16). Mann-Whitney tests were used to calculate significance (p< 0.05).

Results: At 4 and 24 hours, the number of adhesion plaques was significantly greater on control and C2H2 compared with TMS. At 4 hours, cell area on control discs was significantly greater than C2H2 and TMS. At 24 hours, cell area on control and C2H2 was significantly greater than TMS. Between 4 and 24 hours, the number of adhesion plaques increased significantly on all the surfaces. Cell area increased significantly on C2H2 and TMS between 4 and 24 hours. At 4 hours, shear stress needed to dislodge the cells was highest for polished C2H2 and least for titanium unpolished surface. Cells on polished surfaces in corresponding groups required higher shear stress to remove the cells than cells on unpolished surfaces. At 24 hours, cells on polished C2H2 required significantly higher shear stresses to detach them than cells on unpolished C2H2 and TMS (polished and unpolished). Cells on unpolished Ti required higher stress to dislodge than cells on unpolished TMS. From 4 to 24 hours, a significant increase in shear stress to remove the cells was required on all unpolished surfaces and polished C2H2. A significant correlation was seen between adhesion plaque density at 4 hours and shear stress.

Discussion: This work supports the hypothesis that surfaces with high surface energy and high hydrophilicity lead to increased cell attachment and cell area. It also shows the correlation between adhesion plaque density and the shear stress needed to dislodge fibroblasts from bioactive surfaces.

Background: Osseointegrated amputation prostheses avoid soft tissue complications associated with traditional socket prostheses. Forces are transmitted directly to the skeleton resulting in improved function. However, approximately 50% of transcutaneous implants become infected due to the lack of a successful skin-implant seal. Intraosseous Transcutaneous Amputation Prostheses (ITAP) are designed to integrate with the skin preventing epithelial downgrowth and infection.

Fibronectin adsorption enhances fibroblast adhesion in vitro; however, in vivo, fibronectin becomes desorbed from the implant surface. Covalent attachment of fibronectin by silanisation has been shown to be durable in vitro. The silanisation process for fibronectin includes a stage of passivation with sulphuric acid which alters surface characteristics.

Aims: The aim of this study was to determine if in vitro fibroblast adhesion to silanised fibronectin (SiFn) titanium alloy could be improved by omitting or reducing the length of time of passivation. The study also assessed the effects of SiFn on dermal attachment in vivo comparing the results with adsorbed fibronectin substrates and with uncoated controls.

Methods: Scanning electron microscopy, Ra profilometry and contact angle measurement (n=6) were used for topographical characterization of surfaces. Anti-vinculin antibodies were used to immunolocalize fibroblast adhesion sites after 24 hours. The morphology of fibroblasts on each surface was evaluated using scanning electron microscopy. Subcutaneous plates were implanted onto the tibiae of an ovine model (n=3) in order to evaluate the performance of the modified SiFn surface in vivo. Hydroxyapatite (HA) and adsorption of fibronectin to HA (HAFn) were also tested because HA coatings are currently applied to the dermal section of ITAP in clinical trials. After four weeks, a histological assessment of the percentage of soft-tissue attachment and cell alignment relative to the implant was performed.

Results: Passivation produced rougher, more hydrophobic surfaces with numerous microcracks and was associated with poorer fibroblast adhesion and spreading than un-passivated controls in vitro.

SiFn with passivation resulted in poorer cell adhesion than SiFn without passivation. Reducing the time period for passivation did not reduce the detrimental effects of passivation In vivo, HAFn and SiFn resulted in higher median values for soft-tissue attachment than simple adsorption of fibronectin; however, the differences were not statistically significant. Cell alignment was significantly different for HAFn and SiFn compared with controls (p< 0.05), with cells on the fibro-nectin treated surfaces orientated more perpendicular to the implant surface.

Conclusion: Omission of passivation improves fibro-blast adhesion to SiFn surfaces in vitro. Coating with fibronectin either by silanisation onto titanium alloy or by adsorption onto HA surfaces affected the orientation of cells in vivo, implying that tissue attachment was enhanced. A time course may be of value to determine if fibronectin coatings are lost over time in vivo.

Introduction: Due to uneven distribution of stress between the stump and the socket in amputees pain, infection and necrosis of soft tissue can be problematic (Dudek, Marks, & Marshall 2006)Implants have been developed that allow the external prostheses to attach directly to the skeleton by a percutaneous section by osseointegration that reduces the stresses on the soft tissue alleviating the problems associated with a socket (Lai et al. 1998). It has been postulated that surface coatings can enhance soft tissue attachment and increase the in growth of fibroblastic dermal tissues enhancing the seal at the skin implant interface and reducing infection (Pendegrass et al. 2006). Hydrogenated (acetylene: C2H2) and silanized (tetra methyl silane: TMS) diamond-like-carbon coating (DLC) can be applied to titanium(Ti) alloy to reduce surface energy and hydrophilicity. It was hypothesized that biomaterial surfaces having high surface energy and high hydrophilicity eg, Ti alloy enhance the adhesion and maturation of human dermal fibroblasts when compared with C2H2 and TMS coated substrates in vitro.

Methods: Fibroblasts were cultured on 10 mm diameter Ti alloy, C2H2 and TMS coated Ti alloy discs for 4 hours and 24 hours (2500 cells per disc). Cell area and attachment were analysed using Image Analysis and quantification of immunolocalised vinculin containing adhesion plaques respectively. The number of plaques per cell and cell area were compared between experimental groups and controls at 4 and 24 hours. The change in cell area and number of adhesion plaques between 4 and 24 hours were compared for each substrate type. SPSS version 10 was used for the statistical analysis.

Results: At 4 and 24 hours, the number of adhesion plaques was significantly greater on control and C2H2 compared with TMS (p< 0.001). No significant difference was observed between control and C2H2 discs (p> 0.05). At 4 hours, cell area was significantly greater in control compared to both C2H2 and TMS (p< 0.001). At 4 hours, the cell area in TMS was significantly greater than C2H2 (p< .001). At 24 hours, the cell area on control and C2H2 was significantly greater than TMS(p< 0.001). However, there was no significant difference between cell area on control and C2H2 (p> 0.05). From 4 to 24 hours, the number of adhesion plaques increased significantly on all the surfaces (p< 0.001). Cell area increased significantly on C2H2 and TMS between 4 and 24 hours. No significant increase in the cell area was observed on control substrates

Discussion: This supports the hypothesis that surfaces with high surface energy and high hydrophilicity lead to increased cell attachment and cell area. Thus, it can be concluded that the hydrophilic surfaces with higher surface energies favour the adhesion of dermal fibroblasts.

Introduction: Cell adhesion to titanium alloy implants is important in osseointegration [1,2] and attachment of the soft tissues to skin penetrating implants e.g. external fixator pins and Intraosseous Transcutaneous Amputation Prostheses [3,4]. Cell adhesion can be assessed using cell area data and immunolocalisation of focal contact proteins e.g. vinculin; however no method of assessing biophysical attachment is performed routinely. Cell adhesion can be enhanced with adhesion proteins including fibronectin (Fn)[5]. We have previously shown that covalently binding Fn to titanium also increases cell adhesion, and produces a more robust protein coating [6]. However the strength of adhesion of cells to this coating has not been measured. Our hypothesis was that biophysical cell adhesion measured using novel radial flow apparatus would correlate with cell area and focal contact data and that covalently bound fibronectin substrates would increase cell adhesion compared with adsorbed and uncoated controls.

Method: Dermal fibroblasts were cultured for 1, 4, and 24 hours on 30mm and 10mm diameter polished titanium alloy discs (n = 6). Cells on 30mm discs were calcein stained and subjected to shear stress in a submerged, media filled, custom-made radial flow apparatus at 37¬C at 1.66ml/s for 15s. Cells were fixed in 10% formal saline and photographs were taken using a tangential light source. Fluorescent microscopy was performed at 2mm intervals along two perpendicular diameters. Using image analysis, the central cell free zone was measured and radial distance and shear stress calculated. Cells on 10mm discs were fixed, permeablised and vinculin stained (mouse vinculin antibody (1:200) 2hrs; FITC mouse antibody (1:100)1hr). Images were analyzed with a Zeiss microscope linked to image analysis software and the number of focal contacts were counted per cell area. The medians of the radial flow data were compared with data for cell area and focal contact production at the same time points using Spearman¡s regression correlation. This method was subsequently used to compare cell adhesion at one hour with adsorbed and covalently bound Fn substrates (10¥ìg/disc).

Results/Discussion: The shear strength of cells increased between 4 and 24hrs (p=0.002) on polished untreated control substrates. Attachment values (dynes/cm2) were 84.90 (73.98–97.19), 96.30 (91.66–100.89), and 136.69 (134.68–140.30) for 1, 4 and 24 hours respectively. At 1hr, covalently bound Fn (509.90 dynes/cm2 (490.55–528.49) significantly increased cell adhesion compared with adsorbed Fn(434.45 dynes/cm2(385.25–465.62)) and control substrates(p=0.002). There was significant correlation between shear stress and focal contacts/cell (1.00(p< 0.01)) and focal contacts/cell area (0.900(p=0.037)), but not cell area (0.600(p=0.285)).

Conclusion: Radial flow measurement is a useful direct method to quantify cell adhesion to orthopaedic implants and correlates well with other methods of measurement. Covalently bound Fn significantly increases biophysical cell attachment compared with adsorbed and uncoated controls.

Introduction: Intraosseous Transcutaneous Amputation Prostheses (ITAP) could overcome the problems associated with conventional stump-socket prostheses for amputees (pressure sores, pain, infections and unnatural gait), by attaching the external prosthesis directly to the skeleton via a skin penetrating abutment. Despite this, the skin breach introduces a potential route for infection. For success, a biological seal at the skin-ITAP interface is essential.

The protein Laminin-5 (L-5) is a ‘biological glue’, which is integral to epitheial cell adhesion. Covalently bonding L-5 to the ITAP titanium alloy (Ti6Al4V), may enhance the strength of the skin-ITAP interface.

Silanisation, a chemical technique that covalently bonds proteins to metals, could be used to bond L-5 to Ti6Al4V. We have assessed the characteristics L-5 silanised Ti6Al4V as a potential substrate for ITAP.

Method: To determine the maximum quantity of L-5 that could be silanised to Ti6Al4V, and its relative stability when soaked in foetal calf serum (FCS) over time; polished Ti6Al4V discs were silanised by immersing in aminopropyltriethoxysilane followed by glutaraldehyde. Radiolabelled rat laminin-5-I125 was then added. Discs were immersed in FCS for 4 days (37 C) and analysed at 24 hour intervals in a liquid scintillation counter. Un-silanised discs were used as controls.

Results: L-5 was successfully covalently bound to Ti6Al4V. 10ng, 100ng, 250ng and 500ng droplets yielded significantly more silanised L-5 (p< 0.05), but no difference was observed between 750ng and 1000ng. Percentage L-5 covalently bound ranged from 33% and 65%.

A small decrease in bound L-5 occurred after 24 hours of FCS soaking (p< 0.05), but subsequent to this no significant reduction was observed for 4 days (p< 0.05). Controls showed a significantly larger reduction after 24 hours (p< 0.05).

Conclusion: Covalently bonding L-5 to Ti6Al4V by silanisation can be achieved with predictable results. Large enough quantities can be immobilised to influ-ence cellular function. L-5 silanised to Ti6Al4V remains stable in vitro over time and is not removed. Following the study of cellular interactions with silanised L-5, a stable skin seal may be achieved at the transcutaneous portion of the ITAP.

Introduction: Templating of radiographs is part of pre-operative planning in Total Hip Replacement (THR). Digital radiograph technology allows the manipulation of images, altering magnification and therefore affecting accuracy and reproducibility in templating. We have performed a study to investigate templating for hybrid total hip arthroplasty comparing digital hard copies with three computer methods to scale for magnification, in order to assess whether on-screen images can be templated directly with existing acetate templates.

Methods: 20 patients undergoing hybrid THR had pre-operative radiographs taken with a 10 pence coin attached to the skin overlying their greater trochanter. On-screen computer images were manipulated using either the 10p coin as a marker to scale for magnification, or two digital line methods using computer software against external ruler scales. Templating were performed for acetabular size, femoral offset, stem offset and stem size by three grades of observer, and the on-screen images were compared with hard copy digital prints. Intraclass Correlation (ICC) analyses were performed to assess intra-observer and inter-observer variability for the four methods. Comparisons were also made between templated results and the sizes of the inserted prostheses.

Results: All methods showed good reproducibility with all ICC values for intra-observer variability greater than 0.7. Inter-observer variability was less consistent, and the two digital line methods were the least reliable, with accuracy of sizing compared with the inserted prostheses varying between −1.6% to +10.2%. The hard copy radiographs showed better reproducibility than the 10p method, but less accuracy with 3.7% under-sizing. The 10p method was most accurate, with no significant differences for offset or acetabulum compared with the inserted prostheses, and templated under-sizing of only 0.9%.

Discussion: On-screen templating of digital radiographs with standard acetate templates is accurate and reproducible if a radio-opaque marker such as a 10p coin is included when taking the original radiograph.