Introduction. Infection of endoprostheses is a serious complication in orthopedic surgery. As silver is known for its antibactierial effects, silver-coated endoprostheses have gained increased attention to decrease infection rates. However, cytotoxic effects of silver on bone cells have not been investigated in detail. We aimed to investigate whether silver nano-/microparticles and ionic silver exert cytotoxic effects on osteoblasts and osteoclasts in vitro and to correlate potential effects with the antibacterial effect on Staph. epidermidis. Methods. Murine osteoclasts (OC) and murine osteoblasts (OB) were treated with silver particles (avg. sizes: 50nm, 3μm, 30μm, 8μg/ml–500μg/ml) and Ag+NO3- (0.5μg/ml–500μg/ml). Silver treatment started on day 3 to prevent interference with cell adhesion. XTT assays were performed to assess cell viability. Tartrate resistant acidic phosphatase (TRAP) activity and alkaline phosphatase (ALP) activity served as measures for OC and OB differentiation, respectively. The release of silver ions from silver particles was quantified with atomic emission spectometry (AES). Titanium particles (avg. sizes: 50nm and 30μm) were used as controls to investigate whether potential silver effects were particle- or ion-mediated. The antimicrobial activity of silver ions and particles was tested with Staph. epidermidis agar inhibition assays. Results. Ionic silver had the strongest impact on cell differentiation and viability of OC and OB (OC differentiation: mean IC50 = 5 μg/ml, OC viability: mean IC50 = 14 μg/ml, OB differentiation: mean IC50 = 1 μg/ml, OB viability: mean IC50 = 1 μg/ml). Silver nanoparticles decreased cell differentiation and viability in a dose dependent manner (OC differentiation: mean IC50 = 5μg/ml, OC viability: mean IC50 = 14μg/ml, OB differentiation: mean IC50 = 1μg/ml, OB viability: mean IC50 = 1μg/ml). Silver microparticles as well as titanium nano- and microparticles had no effect on cell differentiation and viability. AES showed a size and dose dependent release of silver ions from silver nano- and microparticles. Agar inhibition assays showed a dose correlation of the antibacterial effect of silver with the cytotoxic effects on OB and OC. Conclusion. Silver nanoparticles and silver ions exert dose-dependent cytotoxic effects on OB and OC in vitro resulting in a severe alteration of cell differentiation and viability. The effect of silver on OB and OC seems to be mediated primarily by silver ions and correlates with the substance's antibacterial effects. The cytotoxicity of silver nanoparticles is mediated primarily by the size-dependent liberation of silver ions. Disturbance of OB and OC survival may have deleterious effects on the osseointegration of orthopedic implants. Further in vivo studies are needed to investigate the osseointegration of silver coated implants prior to their widespread clinical application

INTRODUCTION. Analysis of retrieved ceramic components have shown areas of localized ‘stripe wear’, which have been attributed to joint laxity and/or impingement resulting in subluxation of the head, causing wear on the edge of the cup. Studies have been conducted into the effects of mild subluxation, however few in vitro tests have looked at severe subluxation. The aim of this study was to develop a more clinically relevant subluxation protocol. MATERIALS & METHODS. Seven (Subluxation n=4; standard test n=3) of 36mm Biolox Forte (R3, Smith & Nephew) ceramic devices were tested for 0.5m cycles (mc). Two of the subluxed joints were further tested to 1 Mc. The devices were subjected to subluxation under standard testing conditions. The flex/ext was 30° and 15° respectively, with internal/external rotation of ±10°. The force was Paul type stance phase loading with a maximum load of 3 kN, and a standard ISO swing phase load of 0.3 kN at 1 Hz. The test was conducted on a ProSim hip joint wear simulator (SimSol, UK). The simulator is equipped with a novel mechanism to achieve translation of the head, to achieve subluxation. During the ISO swing phase load of 0.3kN, a controlled lateral force required for the translation of the head is applied by a cam mechanism, head retraction then occurs during heel strike. The lubricant used was new born calf serum diluted with de-ionised water to achieve average protein concentration of 20 g/l, with 0.2 wt % concentration NaN3, and changed every 250k cycles. Measurements have been taken at 0.5 & 1 mc stages. RESULTS. Linear wear measurements conducted on the subluxed joints resulted in stripe wear similar to that reported in vivo. Average length, width and depth dimensions were 25.34±1.96 mm, 8±1.60 mm and 16.95±3.87 μm (± 95% CL) respectively. Linear wear at 0.5 Mc for standard joints, were undistinguishable from the original profile. Gravimetrically, weight loss was undetectable for joints tested under standard conditions. The volume loss of the joints under subluxation was 1.9± 0.7 mm3 at 0.5 mc. Two joints tested to 1mc generated an average volume loss of 3.1±2.3 mm3. The stripe wear length, width and depth at 1 Mc were 25.30±3.33mm, 8±3.92mm and 35±17.07 μm respectiveley. DISCUSSION. The current study presents test results of a hip joint simulator with a novel subluxation mechanism to simulate severe and clinically relevant hip joint. Past techniques have had to reduce the swing phase load to achieve stripe wear patches of varying size and depth. The subluxed joints produced significantly higher volumetric wear than the standard joints. Dimensional measurements in terms of length, width and depth of wear patches of subluxed joints generated similar results to that which have been observed following retrieval analysis. Tests that can simulate different types of activity in hip joint simulators will help to improve the design and understanding of implant behaviour in vivo

INTRODUCTION. Ceramic-on-metal hip replacements (COM, where the head is a Biolox Delta ceramic and liner is Co Cr alloy), have demonstrated reduced wear under standard conditions in vitro compared to metal-on-metal (MOM) [1]. Early clinical results are also encouraging [2]. Recently concerns have been raised regarding the poor clinical performance of MOM hip resurfacings [3], particularly when cups are steeply inclined. Laboratory hip simulator testing has been used to replicate edge loading, also demonstrating elevated wear [4]. Therefore, a range of conditions to replicate sub-optimal use clinically to better predict in vivo performance should be used. The aim of this study was to compare the wear rates of MOM and COM under adverse edge loading conditions in an in vitro hip simulator test. METHODS. Ceramic-on-metal (n=3) and metal-on-metal (n=3) 36mm hip prostheses (supplied by DePuy International Ltd, UK) were tested in the Leeds Physiological Anatomical Hip Joint Simulator. Liners were mounted to provide a clinical angle of 45o, and stems positioned anatomically. A simplified gait cycle and microseparation was applied as previously described [5] for two million cycles in 25% new born calf serum. Gravimetric analysis was completed every million cycles and wear volumes calculated. RESULTS. The overall mean volumetric wear rate of COM bearings was 0.36 ± 0.55mm3 per million cycles, this was significantly less than the MOM bearing wear (1.32 ± 0.91mm3 per million cycles). For both COM and MOM bearings wear under these edge loading conditions was significantly greater if compared to previously reported wear under standard conditions [1]. DISCUSSION. The reduced wear of COM has been attributed to the differential hardness decreasing adhesive wear and reduced corrosive wear [6]. Wear under the harsh edge-loading conditions in this study is also reported to be significantly less in COM bearings compared to MOM. In MOM bearings in edge contact conditions, the wear zone becomes starved of lubrication, this elevates wear and increases damage at the edge of the cup. In COM bearings the harder head does not become damaged when there is lubricant starvation and hence wear does not accelerate in the same way. In conclusion, COM bearings show reduced wear compared to MOM bearings under standard and adverse conditions and there is some early evidence to support this finding clinically. ACKNOWLEDGEMENTS. Supported by DePuy International Ltd. SW is supported by a Royal Academy of Engineering/EPSRC (UK) fellowship

Introduction. Recently, some case reports have been published, in which nonunions were successfully healed with parathyroid hormone 1–34 (PTH) administration. Previously, we demonstrated that the intervening tissue at the nonunion site contains multilineage mesenchymal progenitor cells and plays an important role during the healing process of nonunion. We investigated the effect of PTH on osteogenic differentiation of human nonunion tissue-derived cells (NCs) in vitro. Hypothesis. We hypothesized that PTH directly promoted osteogenic differentiation of NCs. Materials & Methods. NCs were isolated from 4 patients, and cultured. The cells were divided into two groups: (1) PTH (−) group: cells cultured in osteogenic medium (OM), (2) PTH (+) group: cells cultured in OM with PTH. Osteogenic differentiation potential was analyzed. Results. Real-time PCR analysis showed that gene expression levels of Runx2, ALP, OC and PTHR1 in PTH (+) group were lower than PTH (−) group at day 14. In both groups, there was no significant difference in ALP activity at days 8 and 14, and in the intensity of Alizarin red S staining at day 20. Discussion. Treatment of PTH did not lead to increase osteogenic differentiation of NCs. Nonunion healing by PTH administration may be caused by other mechanisms such as mobilization and recruitment of osteoprogenitor cells

Introduction. Parathyroid hormone 1–34 (PTH) has been reported to accelerate fracture healing. Previously, we demonstrated human fracture hematoma contained osteo-/chondro-progenitor cells. To date, there has been no study investigating the effect of PTH on fracture hematoma-derived cells (HCs) in vitro. Hypothesis. We hypothesized PTH treatment affected osteogenesis and chondrogenesis of HCs. Materials & Methods. HCs were divided into 3 groups: control (growth medium), PTH (−) (osteogenic or chondrogenic medium (OM or CM)), and PTH (+) group (OM or CM with PTH). Cell proliferation was assessed by MTS assay. Osteogenesis was assessed by alkaline phosphatase (ALP) activity, real-time PCR, and Alizarin red S staining. Chondrogenesis was assessed by real-time PCR and Safranin-O staining. Results. There was no significant difference in proliferation among 3 groups. ALP activity and expression levels of ALP and Runx2 in PTH (+) group were comparable with PTH (−) group. HCs in PTH (−) and PTH (+) group were strongly stained with Alizarin red S staining. The expression levels of collagen-II and -X in PTH (+) group were significantly lower than PTH (−) group. Pellets in PTH (+) group were slightly stained with Safranin-O staining. Discussion & Conclusion. Our results revealed that PTH treatment did not affect osteogenesis and inhibited chondrogenesis of HCs. PTH treatment after fracture may positively affect other cells such as periosteum-derived cells and circulating stem cells

Introduction

Low-intensity pulsed ultrasound (LIPUS) has been reported to enhance healing of fracture and nonunion. Bone morphogenetic protein-7 (BMP-7) has also been reported to promote bone formation. Recently, we demonstrated progenitor cells with osteogenic/chondrogenic differentiation potential existed in human fracture hematoma and nonunion tissue.

As arthroplasty demand grows worldwide, the need for a novel cost-effective treatment option for articular cartilage (AC) defects tailored to individual patients has never been greater. 3D bioprinting can deposit patient cells and other biomaterials in user-defined patterns to build tissue constructs from the “bottom-up,” potentially offering a new treatment for AC defects. Novel composite bioinks were created by mixing different ratios of methacrylated alginate (AlgMA) with methacrylated gelatin (GelMA) and collagen. Chondrocytes and mesenchymal stem cells (MSCs) were then encapsulated in the bioinks and 3D bioprinted using a custom-built extrusion bioprinter. UV and double-ionic (BaCl2 and CaCl2) crosslinking was deployed following bioprinting to strengthen bioink stability in culture. Chondrocyte and MSC spheroids were also bioprinted to accelerate cell growth and development of ECM in bioprinted constructs. Excellent viability of chondrocytes and MSCs was seen following bioprinting (>95%) and maintained in culture, with accelerated cell growth seen with inclusion of cell spheroids in bioinks (p<0.05). Bioprinted 10mm diameter constructs maintained shape in culture over 28 days, whilst construct degradation rates and mechanical properties were improved with addition of AlgMA (p<0.05). Composite bioinks were also injected into in vitro osteochondral defects and crosslinked in situ, with maintained cell viability and repair of osteochondral defects seen over a 14-day period. In conclusion, we developed novel composite bioinks that can be triple-crosslinked, facilitating successful chondrocyte and MSC growth in 3D bioprinted scaffolds and in vitro repair of an osteochondral defect model. This offers hope for a new approach to treating AC defects

Objectives. Deep bone and joint infections (DBJI) are directly intertwined with health, demographic change towards an elderly population, and wellbeing. The elderly human population is more prone to acquire infections, and the consequences such as pain, reduced quality of life, morbidity, absence from work and premature retirement due to disability place significant burdens on already strained healthcare systems and societal budgets. DBJIs are less responsive to systemic antibiotics because of poor vascular perfusion in necrotic bone, large bone defects and persistent biofilm-based infection. Emerging bacterial resistance poses a major threat and new innovative treatment modalities are urgently needed to curb its current trajectory. Materials and Methods. We present a new biphasic ceramic bone substitute consisting of hydroxyapatite and calcium sulphate for local antibiotic delivery in combination with bone regeneration. Gentamicin release was measured in four setups: 1) in vitro elution in Ringer’s solution; 2) local elution in patients treated for trochanteric hip fractures or uncemented hip revisions; 3) local elution in patients treated with a bone tumour resection; and 4) local elution in patients treated surgically for chronic corticomedullary osteomyelitis. Results. The release pattern in vitro was comparable with the obtained release in the patient studies. No recurrence was detected in the osteomyelitis group at latest follow-up (minimum 1.5 years). Conclusions. This new biphasic bone substitute containing antibiotics provides safe prevention of bone infections in a range of clinical situations. The in vitro test method predicts the in vivo performance and makes it a reliable tool in the development of future antibiotic-eluting bone-regenerating materials. Cite this article: M. Stravinskas, P. Horstmann, J. Ferguson, W. Hettwer, M. Nilsson, S. Tarasevicius, M. M. Petersen, M. A. McNally, L. Lidgren. Pharmacokinetics of gentamicin eluted from a regenerating bone graft substitute: In vitro and clinical release studies. Bone Joint Res 2016;5:427–435. DOI: 10.1302/2046-3758.59.BJR-2016-0108.R1

Tranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce peri-operative bleeding. Increasingly, topical administration as an intra-articular injection or peri-operative wash is being administered at concentrations between 10–100mg/ml. This study investigated effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations. Tendon, synovium and cartilage obtained from routine orthopaedic surgeries were used ex vivo or cultured for in vitro studies using various concentrations of TXA. They were stained with 5-chloromethylfluorescein diacetate and propidium iodide and imaged using confocal microscopy to identify the proportion of live and dead cells. The in vitro effect of TXA on primary cultured tenocytes, synovial like fibroblast (FLS) cells and chondrocytes was investigated using cell viability assays (MTT), fluorescent microscopy and multi-protein apoptotic arrays for cell death. There was significant (p<0.01) increase in cell death in all tissue treated with 100mg/ml TXA, ex vivo. MTT assays revealed significant (p<0.05) decrease in cell viability following treatment with 50 or 100mg/ml of TXA within 4 hours of all cell types cultured in vitro. Additionally, there was significant (p<0.05) increase in cell apoptosis detected by fluorescent microscopy within 1 hour of exposure to TXA. Furthermore, multi-protein apoptotic arrays detected increased apoptotic proteins within 1 hour of TXA treatment in tenocytes and FLS cells. Our study provides evidence of TXA cytotoxicity to human peri-articular tissues ex vivo and in vitro at concentrations and durations of treatment routinely used in clinical environments. Clinicians should therefore show caution when considering use of topical TXA administration

Aims

To determine whether platelet-rich plasma (PRP) injection improves outcomes two years after acute Achilles tendon rupture.

Aims

Our objective was to conduct a systematic review and meta-analysis, to establish whether differences arise in clinical outcomes between autologous and synthetic bone grafts in the operative management of tibial plateau fractures.

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. The need for a novel, cost effective treatment option for osteochondral defects has therefore never been greater. As an emerging technology, three-dimensional (3D) bioprinting has the capacity to deposit cells, extracellular matrices and other biological materials in user-defined patterns to build complex tissue constructs from the “bottom up”. Through use of extrusion bioprinting and fused deposition modelling (FDM) 3D printing, porous 3D scaffolds were successfully created in this study from hydrogels and synthetic polymers. Mesenchymal stem cells (MSCs) seeded onto polycaprolactone scaffolds with defined pore sizes and porosity maintained viability over a 7-day period, with addition of alginate hydrogel and scaffold surface treatment with NaOH increasing cell adhesion and viability. MSC-laden alginate constructs produced via extrusion bioprinting also maintained structural integrity and cell viability over 7 days in vitro culture. Growth within osteogenic media resulted in successful osteogenic differentiation of MSCs within scaffolds compared to controls (p<0.001). MSC spheroids were also successfully created and bioprinted within a novel, supramolecular hydrogel with tunable stiffness. In conclusion, 3D constructs capable of supporting osteogenic differentiation of MSCs were biofabricated via FDM and extrusion bioprinting. Future work will look to increase osteochondral construct size and complexity, whilst maintaining cell viability

Aims. Demineralised bone matrix (DBM) is rarely used for the local delivery of prophylactic antibiotics. Our aim, in this study, was to show that a graft with a bioactive glass and DBM combination, which is currently available for clinical use, can be loaded with tobramycin and release levels of antibiotic greater than the minimum inhibitory concentration for Staphylococcus aureus without interfering with the bone healing properties of the graft, thus protecting the graft and surrounding tissues from infection. Materials and Methods. Antibiotic was loaded into a graft and subsequently evaluated for drug elution kinetics and the inhibition of bacterial growth. A rat femoral condylar plug model was used to determine the effect of the graft, loaded with antibiotic, on bone healing. Results. We found that tobramycin loaded into a graft composed of bioglass and DBM eluted antibiotic above the minimum inhibitory concentration for three days in vitro. It was also found that the antibiotic loaded into the graft produced no adverse effects on the bone healing properties of the DBM at a lower level of antibiotic. Conclusion. This antibiotic-loaded bone void filler may represent a promising option for the delivery of local antibiotics in orthopaedic surgery. Cite this article: Bone Joint J 2016;98-B:1126–31

Background. Skeletal stem cells (SSCs) have been used for the treatment of osteonecrosis of the femoral head to prevent subsequent collapse. In isolation SSCs do not provide structural support but an innovative case series in Southampton, UK, has used SSCs in combination with impaction bone grafting (IBG) to improve both the biological and mechanical environment and to regenerate new bone at the necrotic site. Aims. Analysis of retrieved tissue-engineered bone as part of ongoing follow-up of this translational case series. Methods. With Proof-of-Concept established in vitro and in vivo, the use of a living bone composite of SSCs and allograft has been translated to four patients (five hips) for treatment of osteonecrosis of their femoral heads. Parallel in vitro culture of the implanted cell-graft construct was performed. Patient follow-up was by serial clinical and radiological examination. In one patient collapse occurred in both hips due to more advanced disease than was originally appreciated. This necessitated bilateral hip arthroplasty, but allowed retrieval of the femoral heads. These were analyzed for Type 1 Collagen production, bone morphology, bone density and mechanical strength by micro computed tomography (CT), histology (A/S stain, Collagen Type 1 immunostain, biorefringence) and mechanical testing. Representative sections of cortical, trabecular and tissue engineered bone were excised from the femoral heads using a diamond-tipped saw-blade and tested to failure by axial compression. Results. Parallel in vitro analysis demonstrated sustained cell growth and viability on the allograft. Three patients currently remain asymptomatic at up to three year follow-up. Histological analysis of the two retrieved femoral heads demonstrated, critically, Type 1 collagen production in the regenerated tissue as well as mature trabecular architecture, indicative of de novo tissue engineered bone. The trabecular morphology of regenerated bone was evident on CT, and this had a bone density of 1400 Grey scale units, (compared to 1200 for natural trabecular bone and 1800 for cortical bone). On axial compressive testing the regenerated bone on the left showed a 24.8% increase in compressive strength compared to ipsilateral normal trabecular bone, and a 22.9% increase on the left. Conclusions. Retrieval analysis data has demonstrated the translational potential of a living bone composite, while ongoing clinical follow-up shows this to be an effective new treatment for osteonecrosis of the femoral head. Regeneration of the necrotic bone may prevent subsequent collapse, thereby delaying, or possibly avoiding, the need for hip arthroplasty in early stage osteonecrosis. Evaluation of this tissue engineering construct has confirmed the potential for clinical treatment of bone defects using SSC based strategies

Nanoscale topography increases the bioactivity of a material and stimulates specific responses (third generation biomaterial properties) at the molecular level upon first generation (bioinert) or second generation (bioresorbable or bioactive) biomaterials. We developed a technique (based upon the effects of nanoscale topography) that facilitated the in vitro expansion of bone graft for subsequent implantation and investigated the optimal conditions for growing new mineralised bone in vitro. Two topographies (nanopits and nanoislands) were embossed into the bioresorbable polymer Polycaprolactone (PCL). Three dimensional cell culture was performed using double-sided embossing of substrates, seeding of both sides, and vertical positioning of substrates. The effect of Hydroxyapatite, and chemical stimulation were also examined. Human bone marrow was harvested from hip arthroplasty patients, the mesenchymal stem cells culture expanded and used for cellular analysis of substrate bioactivity. The cell line specificity and osteogenic behaviour was demonstrated through immunohistochemistry, confirmed by real-time PCR and quantitative PCR. Mineralisation was demonstrated using alizarin red staining. Results showed that the osteoinduction was optimally conferred by the presence of nanotopography, and also by the incorporation of hydroxyapatite (HA) into the PCL. The nanopit topography and HA were both superior to the use of BMP2 in the production of mineralised bone tissue. The protocol from shim production to bone marrow harvesting and vertical cell culture on nanoembossed HaPCL has been shown to be reproducible and potentially applicable to economical larger scale production

Clinical evidence that patients with type 2 diabetes mellitus (T2DM) have increased risk of fractures is reported. Furthermore, thiazolidinediones, used to treat T2DM increases the risk of secondary osteoporosis & subsequent fractures. The osteogenic potency of metformin is reported in vitro, few studies have investigated the effects of metformin on bone mass and fracture healing in vivo. We aimed to investigate the effects of metformin on fracture healing in vivo. Method. 20 female Wistar rats aged 3 months were randomly divided in two groups, one group receiving saline, the other group receiving metformin administered orally via the drinking water at a concentration of 2mg/ml. After 4 weeks of metformin treatment, a mid-diaphyseal, open External fixation fracture was performed. Rats were sacrified 4 weeks later. Right contralateral tibia and left osteotomised femora were excised, bone architecture analysed by micro-CT in the right tibia. Results. No significant differences were noted between the two groups. Fracture callus volume and mineral content after 4 weeks were similar in metformin and saline groups. Discussion Our results indicate that while metformin has no adverse effects on bone, it does not promote bone mass, as suggested by in vitro studies. This confirms clinical data which have not shown direct links between metformin and decreased fracture risk

In a randomized study of 60 patients allergic reactions are evaluated in three joint prosthesis groups, a resurfacing arthroplasty (ReCap), a non-cemented, large metal-on-metal head (Bimetric Magnum) and a non-cemented, alumina ceramic-on-ceramic bearing in a titanium shell (Bimetric C2a). The inclusion criteria were osteoarthritis, ASA I–II, MRI-scan without caput necrosis, DXA-scan without osteoporosis. The exclusion criteria were short neck (<2cm.), large cysts (>1cm.), medical treatment affecting the bone metabolism, severe deformity of the femoral head, impaired kidney function and inability to co-operate. Blood samples were drawn prior to and 6 weeks, 6 months, 1 year, and 3 years after surgery; two tubes from which plasma was prepared, and two tubes for serum. From the last included 20 patients in each group was also taken blood one and three years after surgery for an in vitro lymphocyte assay for scoring of possible hypersensitivity to prosthesis metals. The isolated lymphocytes were subjected to measurement of proliferation and expression of CD69 by flow cytometry and measurement of the Migration Inhibitory Factor (MIF) by ELISA. Plasma concentrations of the cytokines IL-1, IL-4, IL-6, IL-8, IL-12p70, IL-15, interferon-and osteoprotegerin were determined by multiplex-immunoassay. Serum concentrations of chromium and cobalt were determined by graphite furnace atomic absorption spectrometry. The serum concentrations of chromium and cobalt were lowest in patients with the C2a implant and highest with Magnum, some of these differences were significant at 6 weeks, 6 months, and 1 year after surgery. No patient had a very high serum metal concentration. The values of the variables measured in the in vitro lymphocyte assay mainly changed in the expected direction depending on the concentration of the same metal in the serum sample drawn at the same time, but no significant correlation was seen. One patient had uncertain symptoms of metal hypersensitivity and relatively high serum metal concentrations 3 years after arthroplasty with a Magnum prosthesis and was assessed extraordinarily, and elicited the marginally highest MIF responses in the lymphocyte assay. A strong correlation was found between the plasma concentrations of most cytokines, but the cytokine concentrations were not correlated to contemporary metal concentrations

Engineered bone tissue to recreate the continuity of damaged skeletal segments is one of the field of interest of tissue engineering. Trabecular titanium has very good mechanical properties and high in vitro and in vivo biocompatibility: it can be used in biomedical applications to promote osteointegration demonstrating that it can be successfully used for regenerative medicine in orthopaedic surgery (1). Purpose of this investigation was to evaluate the behavior of adipose tissue derived stem cells (hASCs) cultured on scaffolds of Trabecular TitaniumTM (Lima-Lto) (TT). hASCs are considered to be multipotent mesenchymal stem cells that are easily induced to differentiate into functional osteoblasts both in vitro and in vivo (2). The hASCs were obtained from the subcutaneous adipose tissue of healthy donors during total hip replacement procedures after digestion with collagenase. They were seeded on monolayer and on the TT scaffolds, and incubated at 37 degrees C in 5% CO2 with osteogenic medium or control medium. The expression of bone-related genes using RT-PCR, time course of alkaline phosphatase activity and morphological investigation with Scanning Electron Microscopy (SEM) were performed to evaluate the osteogenic differentiation of hASCs. Alkaline phosphatase activity, marker of the differentiation toward the osteogenic pattern, was significantly higher in hASCs grown with osteogenic medium than in cells grown with control medium, both in monolayer and TT scaffolds; moreover, also alkaline phosphatase of hASCs grown on TT scaffolds in the presence of control medium increased with time, differently from that of cells grown on monolayer. The osteogenic differentiated hASCs expressed the bone-related genes type I collagen, osteocalcin, Runx-2 and alkaline phosphatase. SEM observations showed that hASCs differentiated toward osteoblast-like cells: they produced a big amount of extracellular matrix that covered the surface of the porous scaffolds with bridges between the pore walls. These data suggest that hASCs are able to adhere to TT scaffolds, to acquire an osteoblastic phenotype and to produce abundant extracellular matrix, with but also without osteogenic medium. We can therefore conclude that this material carries osteinductive properties being responsible of ostegenic differentiation; consequently, this scaffold/cells construct is effective to regenerate damaged tissue and to restore the function of bone tissue

Introduction. ACL reconstruction using hamstring tendons has gained general acceptance. However, it has been recommended to seek a tight fit of the tendon in the bone canal in order to provide circumferential contact and healing of the graft, and to prevent secondary tunnel widening. Recent findings show, that the graft dynamically adapts to pressure in the canal resulting in a potentially loose graft-bone contact. It was the goal of this study to understand the viscoelastic behaviour of hamstring grafts under pressure and to develop a new method for tendon pre-conditioning to reduce the graft volume before implantation, in order to reduce the necessary bone canal diameter to accommodate the same graft. Material and Methods. Flexor digitorum tendons of calf and extensor digitorum tendons of adult sheep were identified to be suitable as ACL grafts substitutes for human hamstring tendons in vitro. The effect of different compression forces on dimensions and weight of the grafts were determined. Further, different strain rates (1mm/min vs 10mm/min), compression methods (steady compression vs. creep) and different compression durations(1, 5, 10min) were tested to identify the most effective combination to reduce graft size by preserving its macroscopic structure. Results. The effect of compression on volume reduction (25% of initial volume) reached a plateau at 6000N. Both, steady compression and creeping were able to reduce dimensions of the graft, however, creeping was more effective. There was no difference in effect with different durations for compression (p>0.05) in both methods. With a strain rate of 1mm/min no macroscopic destruction was documented, however with 10mm/min some parts were ruptured. During all pressure tests, considerable amounts of liquid were pressed out from the tendons, and if the graft was submersed in saline solution overnight, the volume reduction was mostly reversible. Conclusion. Compression reduces the dimensions of the ACL graft reversibly, to the greatest part by squeezing out of interstitial water. It is reasonable to assume that this effect also occurs if tendons are under constant pressure in the body, such as at the bend where entering a bone tunnel or under the pressure of interference screws. This in vitro experiment suggests that preconditioning of a 8mm hamstring graft is achieved best by creeping compression with 6kN at a strain rate of 1mm/min. By using this technique, indeed a canal of approximately 10–15% less diameter (i.e. 7 instead of 8mm) may be drilled for the same tendon, resulting in a tight fit of the graft in the bone

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. Methods. The kinetics of dual single and dual protein coating attachment onto titanium alloy was quantified on silanized 10mm diameter discs using radiolabelled Fn (125I-Fn) and Ln (125I-Ln). Sixty discs were polished, sterilized and silanized. Coating durability was assessed when soaked in fetal calf serum (FCS) for 0, 1, 24, 48 and 72hrs. Data was compared to un-silanized Ti discs with the same coatings. Five thousand human dermal fibroblasts were seeded on discs (n = 6) of Ti polished alone (Pol), Ti with adsorbed fibronectin (AdFn), Ti with adsorbed laminin (AdLn), Ti adsorbed dual coating (AdFnLn), Ti silanized (Si), Ti silanized with fibronectin (SiFn), Ti silanized with laminin (SiLn), Ti silanized with a dual coating (SiFnLn) for 24hrs. In order to measure cell adhesion fibroblasts were fixed, vinculin stained using mouse vinculin antibody and alexa fluor. Axiovision Image Analysis software was used to measure cell area, vinculin focal adhesion markers per cell and per unit cell area. Data was analysed in SPSS and significance was assumed at the 0.05 level. Results. Silanized dual coatings bonded to Ti alloy in significantly larger quantities compared with adsorbed coatings at all time points (all p values < 0.05). Fibroblasts cultured on dual coatings were significantly larger, produced more vinculin markers per cell, and per unit cell area compared with single coatings. Cells on SiFnLn were larger with more numerous vinculin markers per cell, and per unit cell area compared with AdFnLn (p<0.05). Conclusion. This study has demonstrated that covalently bonding both fibronectin and laminin to Ti alloy provides a durable, dual coating that enhances early fibroblast growth and attachment compared with either protein coating alone in vitro. Our study showed that there is non-competitive binding of laminin on Ti surfaces in the presence of fibronectin. Dual coatings may be applied to the skin-penetrating region of transcutaneous devices to improve the skin seal and this may have positive implications for the development of ITAP