Introduction. Ilizarov fixators are reliant on tensioned fine wires for stability. The tension in the wires is generated using specific tensioning devices. Loss of wire tension over time may lead of loss a stability and complications. A series of in vitro experiments were undertaken to explore wire tensioner accuracy, the impact of fixation bolt torque and initial tension on loss of tension in ilizarov constructs under static and dynamic loads. Materials & Methods. Medical grade materials were applied to a synthetic bone analogue using surgical instruments in all experiments. Bolt torque was fixed at 6, 10 or 14 Nm using a torque limiting wrench. Wire tension was assessed using a strain measurement bridge. Wires were tensioned to 90, 110 and 130kg as measured by a commercial dynamometric tensioner. Static and dynamic testing was undertaken using an instron testing machine. Cyclical loads from 50–750N were applied for 5000 cycles. Results. Actual wire tension was approximately 15% less than indicated by the tensioner device. Using fixation bolt torques of 10Nm and 14Nm achieved final wire tensions of around 60% and 80% of that applied at 90 and 130kg of applied tension. Static load testing demonstrated self stiffening to similar levels in all pre-tensions. Dynamic testing demonstrated significant loss of tension, most of which occured in the first 3 cycles, inversely proportional to the tension initially applied. Conclusions. These experiments provides insight into the effect of initially applied wire tension on Illizarov mechanical performance. It is important surgeons understand how the different ways that these devices are applied affects mechanical performance. Further research examining what factors affect performance across different manufacturers equipment would therefore be relevant, alongside the development of novel fixation methods to reduce wire slippage and the further development of equipment for clinical use

Despite the increasing availability of bone grafting materials, the regeneration of large bone defects remains a challenge. Especially infection prevention while fostering regeneration is a crucial issue. Therefore, loading of grafting material with antibiotics for direct delivery to the site of need is desired. This study evaluates the concept of local delivery using in vitro and in vivo investigations. We aim at verifying safety and reliability of a perioperative enrichment procedure of demineralized bone matrix (DBM) with gentamicin. DBM (DBMputty, DIZG, Germany) was mixed with antibiotic using a syringe with an integrated mixing propeller (Medmix Systems, Switzerland). Gentamicin, as powder or solution, was mixed with DBM at different concentrations (25 −100 mg/g DBM), release and cytotoxicity was analyzed. For in vivo analysis, sterile drill hole defects (diameter: 6 mm, depth: 15 mm) were created in diaphyseal and metaphyseal bones of sheep (Pobloth et al. 2016). Defects (6 – 8 per group and time point) were filled with DBM or DBM enriched with gentamicin (50 mg/g DBM) or left untreated. After three and nine weeks, defect regeneration was analyzed by µCT and histology. The release experiments revealed a burst release of gentamicin from DBM independent of the used amount, the sampling strategy, or the formulation (powder or solution). Gentamicin was almost completely released after three days in all set-ups. Eluates showed an antimicrobial activity against S. aureus over at least three days. Eluates had no negative effect on viability and alkaline phosphatase activity of osteoblast-like cells (partially published Bormann et al. 2014). µCT and histology of the drill hole defects revealed a reduced bone formation with gentamicin loaded DBM. After nine weeks significantly less mineralized tissue was detectable in metaphyseal defects of the gentamicin group. Histological evaluation revealed new bone formation starting at the edges of the drill holes and growing into the center over time. The amount of DBM decreased over time due to the active removal by osteoclasts while osteoblasts formed new bone. Using this mixing procedure, loading of DBM was fast, reliable and possible during surgical setting. In vitro experiments revealed a burst and almost complete release after three days, antimicrobial activity and good biocompatibility of the eluates. Gentamicin/DBM concentration was in the range of clinically used antibiotic-loaded-cement for prophylaxis and treatment in joint replacement (Jiranek et al. 2006). The delayed healing seen in vivo was unexpected due to the good biocompatibility found in vitro. A reduced healing was also seen in spinal fusion where DBM was mixed with vancomycin (Shields et al. 2017), whereas DBM with gentamicin or DBM/bioactive glass with tobramycin had no negative effect on osteoinductivity or femur defect healing, respectively (Lewis et al. 2010, Shields et al. 2016). In conclusion, loading of DBM with gentamicin showed a proper antibiotic delivery over several days, covering the critical phase shortly after surgery. Due to the faster and complete release of the antibiotic compared to antibiotic loaded cement, the amount of antibiotic should be much lower in the DBM compared to cement

Aim. The efficacy of various irrigation solutions in removing microbial contamination of a surgical wound and reducing the rate of subsequent surgical site infection (SSI), has been demonstrated extensively. However, it is not known if irrigation solutions have any activity against established biofilm. This issue is pertinent as successful management of patients with periprosthetic joint infection (PJI) includes the ability to remove biofilm established on the surface of implants and necrotic tissues. The purpose of this study was to evaluate the efficacy of various irrigation solutions in eradicating established biofilm, as opposed to planktonic bacteria, in a validated in vitro model. Method. Established biofilms of Staphylococcus aureus and Escherichia coli were exposed to different irrigation solutions that included Polymyxin 500,000U/L plus bacitracin 50,000U/L, Vancomycin 1g/L, Gentamicin 80mg/L, Normal saline 0.9%, off-the-shelf Betadine 0.3%, Chlorhexidine 0.05%, Benzalkonium 1.3g/L, Sodium hypochlorite 0.125%, and Povidone-iodine 0.5%. Each experiment was conducted in a 96-well microtiter plate with a peg lid and standardized per the MBEC assay manufacturer's protocol. Following 2 minutes of solution exposure to the irrigation solution, residual biofilms were recovered by sonication. Outcome measures for antibiofilm efficacy were residual colony forming units (CFU) and optical density (690nm). Experiments were conducted in 24 replicates and the observations recorded by two blinded observers. Statistical analysis involved t-tests with Bonferonni adjustment. Results. Povidone-iodine 0.5%, Betadine 0.3%, Benzalkonium 1.3g/L, and Sodium hypochlorite 0.125% were significantly more efficacious against S.aureus biofilm versus all other solutions (p<0.001). Against E.coli biofilm, Povidone-iodine-0.5%, Benzalkonium-1.3g/L and Sodium hypochlorite-0.125% were also most effective compared to other irrigation solutions (p<0.001). Polymyxin-bacitracin, Gentamicin, Vancomycin, and Saline solutions had minimal activity against both E.coli and S.aureus biofilms (p<0.001). Similar trends were observed using both experimental endpoints (CFU and Turbidity) and both investigators (interrater reliability; r=0.99). Conclusion. This in vitro study observed that topical antibiotic solutions do not have any activity against established biofilms. Irrigations solutions containing adequate amount of povidone-iodine, betadine, sodium hypochlorite, and benzalkonium appear to have activity against established biofilm by gram positive and gram negative organisms. The use of these irrigation solutions may need to be considered in patients with established PJI

Aim. Quadrupled hamstring anterior cruciate ligament plasties (4xHp) have been described as having a higher risk of infection than bone patellar tendon bone plasties (BPTBp). There are 2 theories that might explain this phenomenon. One is the presence of sutures in a 4xHp that could act as a foreign body, The other is the more complex preparation of a 4xHp that might lead to higher contamination rates during the process. The objective of the present study was to evaluate the formation of biofilm in these plasties and to compare it between a 4xHp and a BPTBp. The hypothesis was that the presence of sutures in 4xHp would increase the amount of biofilm present in them in comparison to BPTBp. Method. A descriptive in vitro study was conducted. One 4xHp and one BPTBp were prepared. They were subsequently divided into 8 fragments. Three of them were reserved for negative control, and the rest were contaminated with a strain of S. Epidermidis (ATCC 35984) 10–5. Finally, a quantitative analysis was carried out by means of microcalorimetry and sonication with plating. Additionally, a qualitative analysis was carried out by means of electron microscopy. Results. In isothermal microcalorimetry, both contaminated plasties showed the same growth dynamics with a population peak (200uW) at 8h. No significant differences were found between the bacterial growth profiles of 4xHp and BPTBp. The product of sonication was plated and the number of colony forming units per milliliter (CFU/ml) was counted at 24 hours. No significant differences were detected between the 4×Hp (mean +/− sem = 3,5×107 +/− 3450000) and the BPTBp (4,6 ×107 +/− 1,455e+7). With a p value of 0.6667, there were no differences of significance (Mann-Whitney test). In the samples analyzed with electron microscopy, no specific biofilm growth pattern was identified upon comparing BPTBp with 4xHp. Conclusions. There were no significant differences at either the quantitative or qualitative level when comparing bacterial growth in BPTBp and 4xHp. Therefore, the presence of sutures in 4xHp cannot be established as a predisposing factor to higher infection rates. These findings may be justified in the sense that the plasties themselves already behave like foreign bodies. Therefore, the presence of sutures does not increase the possibility of biofilm forming on their surface

The inflammatory cascade associated with prosthetic implant wear debris, in addition to diseases such as rheumatoid arthritis and periodontitis, it is shown to drastically influence bone turnover in the local environment. Ultimately, this leads to enhanced osteoclastic resorption and the suppression of bone formation by osteoblasts causing implant failure, joint failure, and tooth loosening in the respective conditions if untreated. Regulation of this pathogenic bone metabolism can enhance bone integrity and the treatment bone loss. The current study used novel compounds that target a group of enzymes involved with the epigenetic regulation of gene expression and protein function, histone deacetylases (HDAC), to reduce the catabolism and improve the anabolism of bone material in vitro. Human osteoclasts were differentiated from peripheral blood monocytes and cultured over a 17 day period. In separate experiments, human osteoblasts were differentiated from human mesenchymal stem cells isolated from bone chips collected during bone marrow donations, and cultured over 21 days. In these assays, cells were exposed to the key inflammatory cytokine involved with the cascade of the abovementioned conditions, tumour necrosis factor-α (TNFα), to represent an inflammatory environment in vitro. Cells were then treated with HDAC inhibitors (HDACi) that target the individual isoforms previously shown to be altered in pathological bone loss conditions, HDAC-1, −2, −5 and −7. Analysis of bone turnover through dentine resorptive measurements and bone mineral deposition analyses were used to quantify the activity of bone cells. Immunohistochemistry of tartrate resistant acid phosphatase (TRAP), WST-assay and automated cell counting was used to assess cell formation, viability and proliferation rates. Real-time quantitative PCR was conducted to identify alterations in the expression of anti- and pro-inflammatory chemokines and cytokines, osteoclastic and osteoblastic factors, in addition to multiplex assays for the quantification of cytokine/chemokine release in cell supernatant in response to HDACi treatments in the presence or absence of TNFα. TNFα stimulated robust production of pro-inflammatory cytokines and chemokines by PBMCs (IL-1β, TNFα, MCP1 and MIP-1α) both at the mRNA and protein level (p < 0 .05). HDACi that target the isoforms HDAC-1 and −2 in combination significantly suppressed the expression or production of these inflammatory factors with greater efficacy than targeting these HDAC isoforms individually. Suppression of HDAC-5 and −7 had no effect on the inflammatory cascade induced by TNFα in monocytes. During osteoclastic differentiation, TNFα stimulated the size and number of active cells, increasing the bone destruction observed on dentine slices (p < 0 .05). Targeting HDAC-1 and −2 significantly reduced bone resorption through modulation of the expression of RANKL signalling factors (NFATc1, TRAF6, CatK, TRAP, and CTR) and fusion factors (DC-STAMP and β3-integerin). Conversely, the anabolic activity of osteoblasts was preserved with HDACi targeting HDAC-5 and −7, significantly increasing their mineralising capacity in the presence of TNFαthrough enhanced RUNX2, OCN and Coll-1a expression. These results identify the therapeutic potential of HDACi through epigenetic regulation of cell activity, critical to the processes of inflammatory bone destruction

Aim. Implant-associated infection usually require prolonged treatment or even removal of the implant. Local application of antibiotics is used commonly in orthopaedic and trauma surgery, as it allows reaching higher concentration in the affected compartment, while at the same time reducing systematic side effects. Ceftriaxone release from calcium sulphate has a particularly interesting, near-constant release profile in vitro, making it an interesting drug for clinical application. Purpose of the present study was to investigate the potential cytotoxicity of different ceftriaxone concentrations and their influence on osteogenic differentiation of human pre-osteoblasts. Method. Human pre-osteoblasts were cultured up to 28 days in different ceftriaxone concentrations, ranging between 0 mg/L and 50’000 mg/L. Cytotoxicity was determined quantitatively by measuring lactate dehydrogenase release, metabolic activity, and cell proliferation. Gene expression analysis of bone-specific markers as well as mineralization and protein expression of collagen-I (Col-I) were investigated to assess osteogenic differentiation. Results. Cytotoxic effects on human pre-osteoblasts could be shown above 15’000 mg/L after 1 and 2 days, whereas subtoxic effects could be observed at concentrations at 500 mg/L after 10 days. Cell proliferation showed no clear alteration up to 1000 mg/L, though a notable decline at 1500 mg/L could be seen after 10 days. Gene and protein expression of Col-I showed a concentration-dependent decrease at day 10 and 14, but also mineralization levels of human pre-osteoblasts presented a similar trend at day 28. Interestingly, the degree of mineralization was already impaired at concentrations above 250 mg/L. Conclusions. These findings provided extensive insights into the influence of different ceftriaxone concentrations on viability, proliferation, gene, and protein expression but also mineralization of human bone pre-osteoblasts. While short-term cytotoxicity is observed only at very high concentrations, metabolism may be impaired at much lower concentrations when exposure is prolonged. Release of ceftriaxone expected from calcium sulphate however remains below thresholds of impaired bone mineralization, even after 4 weeks of exposure. This study demonstrates the importance of properly selecting and monitoring antibiotic concentrations during clinical application

Aim. In cases of prosthetic joint infections the sensitivity of bacterial cultivation of tissue samples is not 100%. In fact, the reported sensitivity based on standardized criteria and rigorous tissue sampling technique probably differs between 86 to 89%. It has been claimed that sonication of explanted prostheses with subsequent culturing of sonication fluid can increase the sensitivity of the test compared to culturing of tissue samples. To what degree bacteria embedded in biofilm is dislodged during the sonication process has to our knowledge not been fully elucidated. We studied the effect of sonication as a method to dislodge biofilm embedded Staphylococcus epidermidis in vitro. Method. 46 steel plates were colonized with biofilm forming S. epidermidis ATCC 35984 in TSB with 1% glucose aerobically at 37°C for 24 hours. Plates were cleansed for non-adherent bacteria before microscopy. Biofilm embedded bacteria were stained with LIVE/DEAD ™ BacLight ™ Bacterial Viability Kit for microscopy and visualized under vital conditions using EVOS™ FL Auto 2 Imaging System (epifluorescence) and an inverse confocal laser scanning microscope LSM510 (CLSM). All steel plates were subjected to epifluorescence microscopy before and after sonication. CLSM and SEM were used to confirm the presence of biofilm embedded bacteria after sonication. Pictures from epifluorescence microscopy were processed for image analysis with help of a macro application (Fiji) and the data was expressed as biofilm coverage rate (BCR). The sonication was performed using a BactoSonic® Bandolin sonicator and the applied effect in each glass test tube (40 kHz, 800W) was measured with a Bruel og Kjær 8103 hydrophone. The amount of bacteria in the sonication fluid was quantified by counting the number of colony forming units (CFU). Three steel plates acted as negative controls. Results. The BCR was highly variable on the plates after sonication. The biofilm was eradicated from the majority of the plates but a considerable number of plates still had biofilm attached to the surface in a highly variable manner. The amount of bacteria in the sonication fluid correlated poorly with BCR on corresponding plates. Conclusions. Our conclusion is that the ability of sonication to dislodge biofilm embedded S. epidermidis in vitro is not as effective as current opinion might suggest. After sonication biofilm still adhere to a significant number of plates in a highly varying manner. This prompts the need to investigate the effect of sonication on biofilm embedded bacteria formed in vivo

INTRODUCTION. Shoulder joint prostheses have become the most commonly replaced after knee and hip artificial implants. Reverse shoulder arthroplasty (RSA) is the treatment option for patients with severe osteoarthritis, rotator cuff arthropathy or a massive rotator cuff tear with pseudoparalysis. Though successful, the long-term survival of such implants are limited by wear of the materials in contact [1, 2]. The aim of this study was to investigate RSA wear in vitro using a clinically relevant activities of daily living (ADLs). MATERIALS AND METHODS. Four new JRI Orthopaedics Reverse Shoulder 42 mm diameter VAIOS with cobalt-chromium (CoCr) glenospheres and ultra-high molecular weight polyethylene (UHMWPE) humeral components were tested. A five million cycles wear test was undertaken using the unique Newcastle Shoulder Wear Simulator with dilute bovine serum as a lubricant. “Mug to mouth” was performed as the ADL to the test prostheses in intervals of 100 cycles, following by 5 seconds of high load (450N) with no motion simulating an ADL such as “lifting an object”. This combined load cycle was then repeated. A fifth reverse shoulder prosthesis was subject to dynamic loading only in a soak control station. Wear was assessed gravimetrically and roughness (Sa) of the articulating surfaces was measured with a non-contacting profilometer. RESULTS AND DISCUSSION. The mean wear rate and standard deviation of the UHMWPE components was 11.4±3.7 mm. 3. /million cycles, while the CoCr components showed minimal wear over the test duration of 0.01± 0.02 mm. 3. /million cycles (Fig.1). Wear rates are comparable with Kohut et al. (14.1 mm. 3. /million cycles) [3] and Smith et al. (14.3±1.6 mm. 3. /million cycles) [4]. The CoCr glenospheres roughness was unchanged, from 32±8 nm Sa to 28±8 nm Sa over the 5 million cycles of the test (p=0.017), while the UHMWPE humeral components became smoother, from 692±123 nm Sa to 42±29 nm Sa (Fig.2), showing a statically significant change (p<0.001). CONCLUSION. The present study is the first wear test of artificial shoulder joints using a Motion-Load-Motion configuration as a clinically relevant physiological pattern of motion and load. Results are comparable with those found in the literature for in vitro testing of reverse shoulder replacements. For any figures or tables, please contact the authors directly

Aim. To evaluate bacterial adhesion and biofilm formation to metallic cerclage wire versus polymer cerclage system (SuperCable®). Methods. Experimental in vitro study to evaluate quantitative bacterial adherence to different cerclage wire materials. Two types of cerclage wires were compared: a metallic versus a polymer based wire (SuperCable®). A two-centimeter cerclage wire piece of each material was included in 2 mL of tryptic soy broth (TSB) culture media, inoculated with 10 microliters of a 0.5 McFarland of a Staphylococcus epidermidis strain and cultivated at 37°C during 2h for adhesion and 48h for biofilm formation. After this time, the cerclages were washed using a 1% phosphate buffered saline (PBS) and sonicated in new culture medium. After sonication, dilutions of each culture were spread in TSB agar and incubated 37°C during 24h. The number of colonies were counted and the cfu/cm2 was calculated. Results. There were no differences in the number of colonies counted at 2 hours. At 48 hours, the polymer cerclage system showed a clinically and statistically reduction of 95.2% in the biofilm formation of S. epidermidis. The highest bacterial counts were observed in metallic cerclages after 48h. Conclusion. In in vitro conditions, the polymer cerclage system may offer decreased biofilm formation compared with metallic cerclage wires. However, there are many other factors in in vivo conditions that could play a role in bacterial adhesion to cerclage wires. Further research is needed in order to recommend the use of polymer cerclage systems for septic revision surgery

INTRODUCTION. Squeaking after total hip replacement has been reported in up to 10% of patients. Some authors proposed that sound emissions from squeaking hips result from resonance of one or other or both of the metal parts and not the bearing surfaces. There is no reported in vitro study about the squeaking frequencies under lubricated regime. The goal of the study was to reproduce the squeaking in vitro under lubricated conditions, and to compare the in vitro frequencies to in vivo frequencies determined in a group of squeaking patients. The frequencies may help determining the responsible part of the noise. METHODS. Four patients, who underwent THR with a Ceramic-on-Ceramic THR (Trident(r), Stryker(r)) presented a squeaking noise. The noise was recorded and analysed with acoustic software (FMaster(r)). In-vitro 3 alumina ceramic (Biolox Forte Ceramtec(r)) 32 mm diameter (Ceramconcept(r)) components were tested using a PROSIM(r) hip friction simulator. The cup was positioned with a 75° abduction angle in order to achieve edge loading conditions. The backing and the cup liner were cut with a diamond saw, in order to avoid neck-head impingement and dislocation in case of high cup abduction angles (Figure1). The head was articulated ± 10° at 1 Hz with a load of 2.5kN for a duration of 300 cycles. The motion was along the edge. Tests were conducted under lubricated conditions with 25% bovine serum without and with the addition of a 3. rd. body alumina ceramic particle (200 μm thickness and 2 mm length). Before hand, engineering blue was used in order to analyze the contact area and to determine whether edge loading was achieved. RESULTS. Edge loading was obtained. In-vitro, no squeaking occurred under edge loading conditions. However, with the addition of an alumina ceramic 3. rd. body particle in the contact region squeaking was obtained at the beginning of the tests and stopped after ∼20 seconds (dominant frequency 2.6 kHz). In-vivo, recordings had a dominant frequency ranging between 2.2 and 2.4 kHz. DISCUSSION. For the first time, squeaking was reproduced in vitro under lubricated conditions. In-vitro noises followed edge loading and 3. rd. body particles and despite, the severe conditions, squeaking was intermittent and difficult to reproduce. However, squeaking is probably more difficult to reproduce because the cup was cut and the head was fixed in the simulator, preventing vibration to occur. Squeaking noises of a similar frequency were recorded in-vitro and in-vivo. The lower frequency of squeaking recorded in-vivo, demonstrates a potential damping effect of the soft tissues. Therefore, the squeaking in the patients was probably related to the bearing surfaces and modified lubrication conditions that may be due to edge loading. Varnum et al reported recently (3) that all the revised squeaking patients had a neck-cup impingement with metal 3. rd. body particles. These metallic wear particles may generate squeaking as shown in vitro. However, a larger cohort of squeaking patients is needed to confirm these results

Pre-clinical validation of implantable devices, including prostheses, generally aims at demonstrating that a new device offers some advantage compared to existing ones, while not introducing additional hazards. This process involves the assessment of a number of possible failure scenarios and claimed benefits, in order to obtain certification of the device (e.g. FDA or CE-mark), and to support its marketing strategy. While until the 90ies in vitro tests were regarded as the golden standard, nowadays the trend is to rely more and more on numerical models (chiefly Finite Element models, FE). The truth is that neither numerical models nor in vitro tests are self-sufficient. FE models require the support of in vitro tests for a number of reasons. First of all, to construct reliable FE models a number of input parameters are required (e.g. material properties, friction coefficients) that can only be measured experimentally. Furthermore, FE models, like any model, can only address the scenarios they are intended for, and cannot predict something that is totally unexpected: for this reason, some preliminary indication is mandatory from in vitro tests. Finally, FE models cannot be assumed true until this is proven by validation against in vitro measurements. At the same time, in vitroexperiments have several limitations that make them unsuitable in a number of cases, for which FE models are better suited. First of all, experiments need optimization, which can be performed efficiently using FE models. Secondly, experiments typically inspect the outer surface of the in vitro specimen. Finally, in vitro experiments are ineffective in exploring multiple similar conditions (sensitivity analysis). A possible paradigm for pre-clinical validation can be summarized as follows (Fig. 1): . 1). Preliminary in vitro experiments should be performed on implants with a prototype of the prosthesis to understand which failure scenarios should be expected. 2). Potential hazards must be identified. For each hazard, the probability of occurrence and the risk must be identified using either a top-down Fault Tree Analysis (FTA), or a bottom-up Failure Mode and Effect Analysis (FMEA). 3). To assess the risk of occurrence of each mode of failure, the most appropriate approach must be chosen (either experimental, or numerical). For instance, in vitro experiments are necessary to: . Preliminarily assess the intended implant performance, and explore possible failure modes. Measure the actual material properties and interface conditions. Perform tests on specimens that include a real bone, the typical uncertainty related to implantation (interface condition, press-fit), etc. Conversely, numerical models are advantageous to:. Estimate biomechanical quantities (e.g. state of stress/strain) in regions that are not accessible experimentally. Explore the effect of design factors (material, surface finish, geometric features, etc), surgical factors (e.g. implant malpositioning) on the outcome. . Predict the post-operative evolution of the implant over time, including progressive failure, tissue adaptation, etc. Therefore, in vitro experiments and numerical models should be designed concurrently, to enable maximal synergy. The aim of this paper is to illustrate a framework where numerical models and in vitro tests synergistically complement each other (Fig. 2)

Introduction. Osteoporosis is a common skeletal disorder characterised by a reduced bone mass and a progressive micro-architectural deterioration in bone tissue leading to bone fragility and susceptibility to fracture. With a progressively aging population, osteoporosis is becoming an increasingly important public health issue. The Wnt/β-catenin pathway is a major signalling cascade in bone biology, playing a key role in regulating bone development and remodelling, with aberrations in signalling resulting in disturbances in bone mass. Objectives. To assess the effects of silencing the expression of the Wnt antagonist Dickkopf-1 (Dkk1) on the bone profile of primary human osteoblasts exposed in vitro to 10-8M dexamethasone. Methods. Primary human osteoblasts (HOBs) were cultured in vitro and exposed to 10-8M dexamethasone over a time course of 4hr, 12hr and 24hr. Dkk1 expression was silenced using small interfering RNA (siRNA). Quantitative RT-PCR was performed to confirm gene knockdown. Control and Dex-treated phObs (silenced & non-silenced) were compared with respect to bone turnover. Markers of bone turnover analyzed included alkaline phosphatase activity, calcium deposition and osteocalcin expression as determined by pNPP assay, quantitative alizarine red staining and ELISA respectively. Results. Dkk1 expression in HOBs was increased in response to dexamethasone exposure with an associated reduction in alkaline phosphatase activity, calcium deposition and osteocalcin expression. Silencing of Dkk1 expression, as confirmed by quantitative RT-PCR, was associated with a rescue effect in dexamethasone-induced bone loss in vitro. Conclusions. Dkk1 is an antagonist of Wnt/β-catenin signalling and plays a key role in regulating bone development and remodelling. Silencing the expression of Dkk1 in primary human osteoblasts has been shown to rescue the effects of dexamethasone-induced bone loss in vitro. The pharmacological targeting of the Wnt/β-catenin signalling pathway offers an exciting opportunity for the development of novel anabolic bone agents to treat osteoporosis and disorders of bone mass

The role of anconeus in elbow stability has been a long-standing debate. Anatomical and electromyographic studies have suggested a potential role as a stabilizer. However, to our knowledge, no clinical or biomechanical studies have investigated its role in improving the stability of a lateral collateral ligament (LCL) deficient elbow. Seven cadaveric upper extremities were mounted in an elbow motion simulator in the varus position. An LCL injured model was created by sectioning of the common extensor origin, and the LCL. The anconeus tendon and its aponeurosis were sutured in a Krackow fashion and tensioned to 10N and 20N through a transosseous tunnel at its origin. Varus-valgus angles and ulnohumeral rotations were recorded using an electromagnetic tracking system during simulated active elbow flexion with the forearm pronated and supinated. During active motion, the injured model resulted in a significant increase in varus angulation (5.3°±2.9°, P=.0001 pronation, 3.5°±3.4°, P=.001 supination) and external rotation (ER) (8.6°±5.8°, P=.001 pronation, 7.1°±6.1°, P=.003 supination) of the ulnohumeral articulation compared to the control state (varus angle −2.8°±3.4° pronation, −3.3°±3.2° supination, ER angle 2.1°±5.6° pronation, 1.6°±5.8° supination). Tensioning of the anconeus significantly decreased the varus angulation (−1.2°±4.5°, P=.006 for 10N in pronation, −3.9°±4°, P=.0001 for 20N in pronation, −4.3°±4°, P=.0001 for 10N in supination, −5.3°±4.2°, P=.0001 for 20N in supination) and ER angle (2.6°±4.5°, P=.008 for 10N in pronation, 0.3°±5°, P=.0001 for 20N in pronation, 0.1°±5.3°, P=.0001 for 10N in supination, −0.8°±5.3°, P=.0001 for 20N in supination) of the injured elbow. Comparing anconeus tensioning to the control state, there was no significant difference in varus-valgus angulation except with anconeus tensioning to 20N with the forearm in supination which resulted in less varus angulation (P=1 for 10N in pronation, P=.267 for 20N in pronation, P=.604 for 10N in supination, P=.030 for 20N in supination). Although there were statistically significant differences in ulnohumeral rotation between anconeus tensioning and the control state (except with anconeus tensioning to 10N with the forearm in pronation which was not significantly different), anconeus tensioning resulted in decreased external rotation angle compared to the control state (P=1 for 10N in pronation, P=.020 for 20N in pronation, P=.033 for 10N in supination, P=.001 for 20N in supination). In the highly unstable varus elbow orientation, anconeus tensioning restores the in vitro stability of an LCL deficient elbow during simulated active motion with the forearm in both pronation and supination. Interestingly, there was a significant difference in varus-valgus angulation between 20N anconeus tensioning with the forearm supinated and the control state, with less varus angulation for the anconeus tensioning which suggests that loads less than 20N is sufficient to restore varus stability during active motion with the forearm supinated. Similarly, the significant difference observed in ulnohumeral rotation between anconeus tensioning and the control state suggests that lesser degrees of anconeus tensioning would be sufficient to restore the posterolateral instability of an LCL deficient elbow. These results may have several clinical implications such as a potential role for anconeus strengthening in managing symptomatic lateral elbow instability

Purpose of the study. In a recent study, O'Leary et al. [2005] reported their observations on the patterns of Charité disc prosthesis motion under physiologic loads. The purpose of this study was to investigate whether the motion patterns observed in the in vitro model are replicated in clinical practice. Methods. 55 patients with implanted SB Charité 111 artificial lumbar discs were subjected to flexion extension x-rays. Two consultant spinal surgeons and a neuro-radiological consultant were asked to classify the pattern of motion in the clinical subjects based on the patterns observed in the in vitro model. The results were recorded independently then collated. Following this first round of observations an algorithm was devised and the method of measurement was standardised. Summary of findings: There was modest correlation amongst the three observers in distinguishing motion from nonmotion (Kappa 5.6). There was less agreement on what type of motion was present. On both counts using the algorithm there was no correlation. The clinical study based on patients' flexion-extension radiographs identified the following patterns of prosthesis motion:. angular motion between both the upper and lower endplates and core, with visual evidence of core motion;. angular motion predominantly between the upper endplate and core, with little visual evidence of core motion;. lift-off of upper prosthesis endplate from core or of core from lower endplate;. core entrapment and deformation; and. no motion. There are difficulties associated with the interprtation of these using only flexion extension views. Conclusions. In devising artificial discs the requirement would be to replicate spinal motion as closely as possible. O'Leary et al have found non-physiological motions in in vitro models. These have been found in this clinical study but interobserver correlations were far from conclusive. Demonstrating non physiological patterns may have important implications for outcome

Aim. Ceftobiprole, a broad-spectrum cephalosporin, could be used for post-operative treatment of bone implant-associated infections. The aim of the study is to evaluate the in vitro susceptibility of bacteria isolated from bone implant-associated infections to ceftobiprole. Method. We conducted an in vitro, retrospective and comparative study between July 2013 to April 2017 including patients with bone implant-associated infections (prosthesis joint infection (PJI) and osteosynthesis material (OM)). To evaluate MIC distribution of ceftobiprole against Gram positive and Gram negative strains and to compare activity of ceftobiprole to vancomycin for Gram positive and ceftriaxone or ceftazidime for Gram negative strains, we tested all strains (stored in Cryobank storage system) for minimal inhibitory concentrations (MIC) determination by E-test bandelet for ceftobiprole and comparator antibiotics. Results. We collected 63 Gram negative strains (57 Enterobateriaceae and 6 Pseudomonas aeruginosa), isolated from 25 patients with OM and 38 patients with PJI (23 hips and 15 knees), and 100 Gram positive strains (85 Staphylococcus sp, 8 E. faecalis, 7 Propionibacterium sp.) isolated from 38 patients with OM and 62 patients with PJI (33 hips, 28 knees, 1 shoulder). A total of 61.4% of Enterobacteriaceae were susceptible both with ceftobiprole and ceftriaxone, 100% of P. aeruginosa were susceptible with ceftazidime and 83,3% with ceftobiptrole and finally 100% of Gram positive were susceptible both with ceftobiprole and vancomycin (susceptibility interpretation was based on EUCAST breakpoints). Conclusions. Our results suggest that ceftobiprole has a good in vitro activity against strains isolated from bone implant-associated infections. It could be an effective alternative to vancomycin and ceftriaxone or ceftazidime in post-operative treatment but pharmacokinetics and pharmacodynamics studies must be performed in bone tissue

Aim

To evaluate the efficiency of pulse lavage combined with electrical fields to remove biofilm from a metallic surface.

Joint hemiarthroplasty replaces one side of a synovial joint and is a viable alternative to total joint arthroplasty when one side of the joint remains healthy. Most hemiarthroplasty implants used in current clinical practice are made from stiff materials such as cobalt chrome or ceramic. The substitution of one side of a soft cartilage-on-cartilage articulation with a rigid implant often leads to damage of the opposing articular cartilage due to the resulting reductions in contact area and increases in cartilage stress. The improvement of post-operative hemiarthroplasty articular contact mechanics is of importance in advancing the performance and longevity of hemiarthroplasty. The purpose of the present study was to investigate the effect of hemiarthroplasty surface compliance on early in-vitro cartilage wear and joint contact mechanics. Cartilage wear tests were conducted using a six-station pin-on-plate apparatus. Pins were manufactured to have a hemispherical radius of curvature of 4.7 mm using either Bionate (DSM Biomedical) having varying compliances (80A [E=20MPa], 55D [E=35MPa], 75D [E=222MPa], n=6 for each), or ceramic (E=310GPa, n=5). Cartilage plugs were cored from fresh unfrozen bovine knee joints using a 20 mm hole saw and mounted in lubricant-containing chambers, with alpha calf serum diluted with phosphate buffer solution to a protein concentration of 17 g/L. The pins were loaded to 30N and given a stroke length of 10 mm for a total of 50,000 cycles at 1.2 Hz. Volumetric cartilage wear was assessed by comparing three-dimensional cartilage scans before and during wear testing. A two-way ANOVA was used for statistical analysis. To assess hemiarthroplasty joint contact mechanics, 3D finite element modelling (ABAQUS v6.12) was used to replicate the wear testing conditions. Cartilage was modeled using neo-Hookean hyper-elastic material properties. Contact area and peak contact stress were estimated. The more compliant Bionate 80A and 55D pins produced significantly less volumetric cartilage wear compared with the less compliant Bionate 75D and ceramic pins (p 0.05). In terms of joint contact mechanics, the more compliant materials (Bionate 80A and 55D) had significantly lower maximum contact stress levels compared to the less compliant Bionate 75D and ceramic pins (p < 0 .05). The results of this study show a relationship between hemiarthroplasty implant surface compliance and early in vitro cartilage wear, where the more compliant surfaces produced significantly lower amounts of cartilage wear. The results of the joint contact mechanics analysis showed that the more compliant hemiarthroplasty materials produced lower maximum cartilage contact stresses than the less compliant materials, likely related to the differences in wear observed. More compliant hemiarthroplasty surfaces may have the potential to improve post-operative cartilage contact mechanics by increasing the implant-cartilage contact area while reducing peak contact stress at the implant-cartilage interface, however, such materials must be resistant to surface fatigue and longer-term cartilage wear/damage must be assessed

Introduction. First-generation annealed HXLPE has been clinically successful at reducing both clinical wear rates and the incidence of osteolysis in total hip arthroplasty. However, studies have observed oxidative and mechanical degradation occurring in annealed HXLPE. Thus, it is unclear whether the favorable clinical performance of 1st generation HXLPE is due to the preservation of bearing surface tribological properties or, at least partially, to the reduction in patient activity. The purpose of this study was to evaluate the in vitro wear performance (assessed using multidirectional pin-on-disk (POD) testing) of 1. st. -generation annealed HXLPE with respect to in vivo duration, clinical wear rates, oxidation, and mechanical properties. Materials and Methods. 103 1. st. -generation annealed HXLPE liners were collected at revision surgery. 39 annealed HXLPE liners were selected based on their implantation time and assigned to three equally sized cohorts (n=13 per group); short-term (1.4–2.7y), intermediate term (5.2–8.0y) and long-term (8.3–12.5y). From each retrieved liner, two 9-mm cores were obtained (one from the superior region and one from the inferior region). Sixteen cores were fabricated from unimplanted HXLPE liners that were removed from their packaging and six pins from unirradiated GUR 1050 resin served as positive controls. Multidirectional POD wear testing was conducted against wrought CoCr disks in a physiologically relevant lubricant (20 g/L protein concentration) using a 100-station SuperPOD (Phoenix Tribology, UK). Each pin had its own chamber with 15mL lubricant maintained at 37±1°C. An elliptical wear pattern with a static contact stress of 2.0 MPa was employed. Testing was carried out to 1.75 million cycles at 1.0 Hz and wear was assessed gravimetrically. POD wear rates were calculated using a linear regression of volumetric losses. In vivo penetration was measured directly using a calibrated micrometer. Oxidation was assessed on thin films obtained from superior and inferior regions of the liners (ASTM 2102). Mechanical properties were assessed using the small punch test (ASTM 2183). Results. In vitro wear rates from the SuperPOD were positively correlated with implantation time (Rho=0.27; p=0.015) and average oxidation (Rho=0.36; p=0.004) at the bearing surface of the retrieved HXLPE liners. All retrieved HXLPE cohorts had lower in vitro wear rates than uncrosslinked positive control (p≤0.03) and higher wear rates than the never-implanted HXLPE cohort (p <0.001). POD wear rates were negatively correlated with small punch ultimate load (p<0.01). However, the in vitro wear rates were not correlated with clinical penetration rates (p=0.71). Discussion. This study investigated the effects of in vivo degradation on 1. st. -generation annealed HXLPE liners. The data in this study suggest that the tribological properties degrade due to in vivo oxidation as the liner is exposed to the in vivo environment. The clinical implications of these findings, however, are not clear as the clinical penetration rates were not correlated with the in vitro POD wear rates. This may be partially due, to decreasing patient activity as they age. These findings will be useful for comparison for evaluating the in vitro wear properties of other HXLPEs, including 2nd generation HXLPE. Acknowledgements. This study was supported by the NIH(NIAMS) R01AR47904

Introduction. The use of antibiotic-loaded polymethylmethacrylate bone-cement spacers during two-stage exchange procedures is the standard in the treatment of patients with delayed prosthetic joint infection. The real antimicrobial activity of these spacers is unclear because the adherence of bacteria to cement might result in clinical recurrence of infection. The purpose of the study is to evaluate the in vitro formation of Pseudomonas Aeruginosa (PA) and Staphylococcus spp. biofilm on antibiotic-loaded bone cement. Materials and methods. Cement disks (diameter = 6 mm) impregnated with gentamicin and colistin were submerged in bacterial suspensions of Methicillin-resistant Staphylococcus Aureus(MRSA), Staphylococcus epidermidis (SE), and PA. Negative controls (specimen disks without antibiotic) were similarly prepared. Biofilm formation was visualized by confocal scanning laser microscopy (CSLM), after staining the discs with the live/dead BacLight viability stain containing SYTO 9 dye and propidium iodide. Images from five randomly selected areas were acquired for each disc. Sequential optical sections of 2 µm were collected in sequence along the z-axis over the complete thickness of the sample. The resulting stacks of images were analyzed, quantified and rendered into three-dimensional (3D). The biofilm thickness on antibiotic bone cement compared with the controls was automatically evaluated. Results. CSLM showed living bacteria and bacterial biofilm on the surface of all cement disks, either antibiotic-loaded or controls. Mean biofilm thickness on the controls was 29.6 µm for MRSA, 32.3 µm for SE, and 59.7 µm for PA. The 3D rendering showed decrease in the biofilm thickness for all bacterial strains on gentamicin- and colistin-impragnated cement disks as compared with the controls. The incorporation of gentamicin into cement resulted in a 54%, 74%, and 45% reduction in the bacterial biofilm thickness for MRSA, PA and SE, respectively. The use of colistin leaded to a 51 % reduction in the PA biofilm thickness. Conclusion. The bacterial viability and biofilm formation are reduced by adding antibiotics to bone cement but antibiotic-loaded bone cement does not completely inhibit the formation of an infectious biofilm in vitro

Staphylococcus aureus is the most frequently isolated organism in periprosthetic joint infections. The mechanism by which synovial fluid (SF) kills bacteria has not yet been elucidated, and a better understanding of its antibacterial characteristics is needed. We sought to analyze the antimicrobial properties of exogenous copper in human SF against S. aureus.

SF samples were collected from patients undergoing total elective knee or hip arthroplasty. Different S. aureus strains previously found to be sensitive and resistant, UAMS-1 and USA300 WT, respectively, were used. We performed in-vitro growth and viability assays to determine the capability of S. aureus to survive in SF with the addition of 10µM of copper. We determined the minimum bactericidal concentration of copper (MBC-Cu) and evaluated the sensitivity to killing, comparing WT and CopAZB-deficient USA300 strains.

UAMS-1 evidenced a greater sensitivity to SF when compared to USA300 WT, at 12 (p=0.001) and 24 hours (p=0.027). UAMS-1 significantly died at 24 hours (p=0.017), and USA300 WT survived at 24 hours. UAMS-1 was more susceptible to the addition of copper at 4 (p=0.001), 12 (p=0.005) and 24-hours (p=0.006). We confirmed a high sensitivity to killing with the addition of exogenous copper on both strains at 4 (p=0.011), 12 (p=0.011), and 24 hours (p=0.011). Both WT and CopAZB-deficient USA300 strains significantly died in SF, evidencing a MBC-Cu of 50µM against USA300 WT (p=0.011).

SF has antimicrobial properties against S. aureus, and UAMS-1 was more sensitive than USA300 WT. The addition of 10µM of copper was highly toxic for both strains, confirming its bactericidal effect. We evidenced CopAZB-proteins involvement in copper effluxion by demonstrating the high sensitivity of the mutant strain to lower copper concentrations. Thus, we propose CopAZB-proteins as potential targets and the use of exogenous copper as possible treatment alternatives against S. aureus.