Aim. Staphylococcus epidermidis (S. epidermidis) is one of the main pathogens responsible for bone and joint infections especially those involving prosthetic materials (PJI). Although less virulent than S. aureus, S. epidermidis is involved in chronic infections notably due to its ability to form biofilm. Moreover, it is frequently multiresistant to antibiotics. In this context, the development of additional or alternative antibacterial therapies targeting the biofilm is a priority. Method. The aim of this study was to evaluate in vitro the activity of phage lysin exebacase (CF-301) against biofilms formed by 19 S. epidermidis clinical strains responsible for PJI. We determined the remaining viable bacteria inside the biofilm (counting after serial dilution and plating) and the biomass (bacteria and extracellular matrix, using crystal violet staining) after 24h of exposition to exebacase at different concentrations, alone (0.05; 0.5; 5; 50 and 150 mg/L) or in combination (5, 50 and 150 mg/L) with antibiotics commonly used to treat multi-resistant S. epidermidis PJI (rifampin (1 mg/L), vancomycin (10mg/L) and daptomycin (10mg/L)). In this study, synergy was defined as a significantly higher effect of the association in comparison to the sum of the effect of each molecule. Results. Exebacase showed a dose-dependent reduction of biomass, ranging from 11 % at 0.5 mg/L to 66 % at 150 mg/L. Exebacase showed a significant bactericidal activity at 50 and 150 mg/l, with a mean decrease of the inoculum of 0.94 and 1.7 log, respectively. In addition, synergistic effects were observed in association with i) rifampin (1 mg/L) showing a mean decrease up to 84% of the biomass and 3.5 log CFU at 150 mg/L of exebacase, ii) vancomycin (10 mg/L) showing a mean decrease up to 81% of the biomass and 2.82 log CFU at 150 mg/L of exebacase, iii) and daptomycin (10 mg/L) showing a mean decrease up to 85% of the biomass and 3.1 log CFU at 150 mg/L of exebacase. Conclusions. Exebacase showed, in vitro, synergistic activity with antibiotics against S. epidermidis biofilms. It is a promising adjuvant therapy to rifampin, vancomycin and daptomycin in the context of PJI. Further studies are needed, in vitro to understand the mechanism of action on S. epidermidis biofilm and the heterogeneity of strain behaviour and in vivo to confirm the present data

A common step to revision surgery for infected total knee replacement (TKR) is a thorough debridement. Whilst surgical and mechanical debridement are established as the gold standard, we investigate a novel adjuvant chemical debridement using an Acetic Acid (AA) soak that seeks to create a hostile environment for organisms, further degradation of biofilm and death of the bacteria. We report the first orthopaedic in vivo series using AA soak as an intra-operative chemical debridement agent for treating infected TKR's. We also investigate the in vitro efficacy of AA against bacteria isolated from infected TKR's. A prospective single surgeon consecutive series of patients with infected TKR were treated according to a standard debridement protocol. Patients in the series received sequential debridement of surgical, mechanical and finally chemical debridement with a 10 minute 3% AA soak. In parallel, we isolated, cultured and identified bacteria from infected TKR's and assessed the in vitro efficacy of AA. Susceptibility testing was performed with AA solutions of different concentrations as well as with a control of a gentamicin sulphate disc. The effect of AA on the pH of tryptone soya was also monitored in an attempt to understand its potential mechanism of action. Physiological responses during the AA soak were unremarkable. Intraoperatively, there were no tachycardic or arrythmic responses, any increase in respiratory rate or changes in blood pressure. This was also the case when the tourniquet was released. In addition, during the post-operative period no increase in analgesic requirements or wound complications was noted. Wound and soft tissue healing was excellent and there have not been any early recurrent infections at mean of 18 months follow up. In vitro, zones of inhibition were formed on less than 40% of the organisms, demonstrating that AA was not directly bactericidal against the majority of the clinical isolates. However, when cultured in a bacterial suspension, AA completely inhibited the growth of the isolates at concentrations as low as 0.19%v/v. This study has shown that the use of 3% AA soak, as part of a debridement protocol, is safe. Whilst the exact mechanism of action of acetic acid is yet to be determined, we have demonstrated that concentrations as low as 0.19%v/v in solution in vitro is sufficient to completely inhibit bacterial growth from infected TKR's

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, with cumulative treatment costs for all prosthetic joint infections estimated to be ∼ $1 billion per annum (UK and North America). Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics. Low intensity pulsed ultrasound is already licensed for clinical use in fracture management and thus could be translated quickly into a clinical treatment. Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged with gentamicin +/− low-intensity ultrasound (1.5MHz, 30mW/cm2, pulse duration 200µs/1KHz) for 180 minutes and 20 minutes, respectively. The primary outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. Assessment of cellular metabolism was conducted using a liquid-chromatography-mass spectrometry, as well as a triphenyltetrazolium chloride assay coupled with spectrophotometry. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was reduced to 64mg/L. Metabolic activity of biofilm-associated S. aureus was increased by 25% following ultrasound therapy (p < 0 .0001), with identification of key biosynthetic pathways activated by non-lethal dispersal. Low intensity pulsed ultrasound was associated with a four-fold reduction in the effective biofilm eradication concentration of gentamicin, bringing the MBEC of gentamicin to within clinically achievable concentrations. The mechanism of action was due to partial disruption of the extracellular matrix which led to an increase of nutrient availability and oxygen tension within the biofilm. This metabolic stimulus was responsible for the reversal of gentamicin tolerance in the biofilm-associated S. aureus

Bone regeneration includes a well-orchestrated series of biological events of bone induction and conduction. Among them, the Wnt/β-catenin signaling pathway is critical for bone regeneration. Being involved in several developmental processes, Wnt/β-catenin signaling must be safely targeted. There are currently only few specific therapeutic agents which are FDA-approved and already entered clinical trials. A published work has shown that Tideglusib, a selective and irreversible small molecule non-ATP-competitive glycogen synthase kinase 3-β(GSK-3β) inhibitor currently in trial for Alzheimer's patients, can promote tooth growth and repair cavities. [1]Despite some differences, they are some similarities between bone and tooth formation and we hypothesise that this new drug could represent a new avenue to stimulate bone healing. In this work, we locally delivered Tideglusib (GSK3β inhibitor) in the repair of femoral cortical window defects and investigated bone regeneration. A biodegradable FDA-approved collagen sponge was soaked in GSK-3βinhibitor solution or vehicle only (DMSO) and was implanted in 1 × 2 mm unicortical defects created in femora of 35 adult wild-type male mice. Bone defect repair on control and experimental (GSK-3βinhibitor) groups was assessed after 1 week (n=22), 2 weeks (n=24) and 4 weeks (n=24) with microCT and histological analysis foralkaline phosphatase (ALP, osteoblast activity), tartrate resistant acid phosphatase (TRAP, osteoclasts), and immunohistochemistry to confirm the activation of the Wnt/β-catenin pathway. Our results showed that Tideglusib significantly enhanced cortical bone bridging (20.6 ±2.3) when compared with the control (12.7 ±1.9, p=0.001). Activity of GSK-3β was effectively downregulated at day 7 and 14 resulting in a higher accumulation of active β-catenin at day 14 in experimental group (2.5±0.3) compared to the control (1.1±0.2, p=0.03). Furthermore, the onset of ALP activity appears earlier in the experimental group (day 14, 1.79±0.28), a level of activity never reached at any end-point by the control defects. At 4 weeks treatment, we observed a significant drop in ALP in the experimental group (0.47±0.05) compared to the control (1.01±0.19, p=0.02) and a decrease in osteoclast (experimental=1.32±0.36, control=2.23±0.67, p=0.04). Local downregulation of GSK-3β by tideglusib during bone defect repair resulted in significant increase in amount of new bone formation. The early upregulation of osteoblast activity is one explanation of bone healing augmentation. This is likely the effect of upregulation of β-catenin following pharmaceutical inhibition of GSK-3β since β-catenin activation is known to positively regulate osteoblasts, once committed to the osteoblast lineage. As a GSK-3β inhibitor, Tideglusib demonstrates a different mechanism of action compared with other GSK-3β antagonists as treatment was started immediately upon injury and did not interfere with precursor cells recruitment and commitment. This indicates that tideglusib could be used at the fracture site during the initial intraoperative internal fixation without the need for further surgery. This safe and FDA-approved drug could be used in prevention of non-union in patients presenting with high risk for fracture-healing complications

Aim. Biofilm-related infections represent a recurrent problem in the orthopaedic setting. In recent years, great interest was directed towards the identification of novel molecules capable to interfere with pathogens adhesion and biofilm formation on implant surfaces. In this study, two stable forms of α-tocopherol, the hydrophobic acetate ester and the water-soluble phosphate ester, were tested in vitro as coating for titanium prostheses. Method. Antimicrobial activity against microorganisms responsible of prosthetic and joints infections was assessed by broth microdilution method. In addition, α-tocopherol esters were evaluated for both their ability to hamper bacterial adhesion and biofilm formation on sandblasted titanium surfaces. Results. Only α-tocopheryl phosphate displayed antimicrobial activity against the tested strains. Both esters were able to significantly interfere with bacterial adhesion and to prevent biofilm formation, especially by Staphylococcus aureus and Staphylococcus epidermidis. The activity of α-tocopheryl phosphate was greater than that of α-tocopheryl acetate. Alterations at membrane levels have been reported in literature1 and may be likely responsible for the interference on bacterial adhesion and biofilm formation shown by α-tocopherol esters. Conclusions. Although further studies are needed to better investigate the mechanisms of action and the spectrum of activity of α-tocopherol esters, these characteristics, together with the positive effect on wound healing and immune response, make these molecules promising candidate for coating in order to prevent implant-associated infections

Computer assisted orthopaedic surgery (CAOS) is an emerging and expanding filed. There are some old classification systems that are too comprehensive to cover all new CAOS tools and hybrid devises that are currently present and others that are expected to appear in the near future. Based on our experience and on the literature review, we grouped CAOS devises on the basis of their functionality and clinical use into 6 categories, which are then sub-grouped on technical basis. In future, new devices can be added under new categories or subcategories. This grouping scheme is meant to provide a simple guide on orthopaedic systems rather than a comprehensive classification for all computer assisted systems in surgical practice. For example, the number and diversity of tasks of surgical robots is enormous, up to 159 surgical robots with different mechanisms and functions reported in the literature. These can be classified according to their tasks, mechanism of actions, degree of freedom and level of activity but for the purpose of simplicity we subcategorised the orthopaedic robots to only industrial, hand-held and bone-mounted. Table 1 shows the classification system with the 6 categories and other subcategories

Daptomycin has a unique mechanism of action against Gram-positive bacteria. Daptomycin is only bactericidal in the presence of calcium ions. [1]. Kanellakopoulou et al [2] investigated elution of daptomycin from calcium sulfate. The results indicated above MIC elution concentrations out to 28 days. Experience reports that the ability for calcium sulfate to set hard when combined with daptomycin can be problematic.[3] This study aimed to investigate the combination of daptomycin with a synthetic recrystallised form of calcium sulfate and investigate zone of inhibition (ZOI) testing against susceptible organisms. 6mm hemispherical beads, were prepared using a commercially available calcium sulfate hemihydrate powder (CSH) – CaSO4 ·1/2H2O. [4] In order to combine daptomycin [5] with the CSH and enable it to set hard, 7mls of saline solution was added to 20g CSH powder and mixed for 80 seconds to initiate the setting reaction. Then 1g of daptomycin powder was added and mixed for a further 30 seconds. The resultant paste was applied to a bead mat and allowed to set. Tryptone soya agar plates were seeded with 0.2ml of a 10e6 – 10e8 cfu/ml suspension of the relevant organism. The plates were incubated at 33 °C ± 2 °C for 30 minutes. The plates were then removed from the incubator and the beads placed on the surface. The plates were then incubated at 33 °C ± 2 °C for 24 hours before examination for the absence of growth as seen by a clear zone around the test sample. Triplicate samples were tested against Staphylococcus epidermidis, Staphylococcus aureus, MRSA, VRE Enterococcus faecium and Propionibacterium acnes. Repeat tests were carried out for beads that had been stored at 37 °C for 21 days to simulate in-vivo conditions. Setting times for the CSH/daptomycin beads were approximately 20 minutes. ZOIs indicating efficacy were seen for all samples both ‘fresh’ and ‘incubated’ with MRSA and Propionibacterium acnes having the largest ZOIs at 31–33mm. A mixing protocol was established to enable set beads to be formed with daptomycin loaded calcium sulfate. As assessed by ZOI testing, the eluted antibiotic maintained efficacy against susceptible pathogens. Results obtained in-vitro may not be indicative of in-vivo performance

Background. Shockwave therapy has been shown to induce osteoneogenesis in animal models. The mechanism of action is unclear, but experimental evidence suggests micro-fracture formation and increased blood flow as the most likely explanation. Several reports from Europe have suggested good results from the treatment of delayed fracture union with shock-waves. We present the results of a randomised double-blind placebo-controlled pilot study. Method. Fourteen patients with clinically and radiologically confirmed delayed union of long-bones consented to enter the trial. The treatment group had a single application of 3000 high-energy shockwaves using the Stortz SLK unit with image intensifier control. The control group had the exactly the same treatment but with an ‘air-gap’ interposition to create a placebo-shockwave. Each patient was followed-up with serial radiographs as well as visual analogue pain scores and EuroQol assessments. All of the patients were reviewed for a minimum of three years post treatment. Results. There was no difference between the groups in terms of time to fracture union (p=0.781 log-rank test). Nor was there any indication of a treatment effect on any of the secondary outcome measures. Conclusion. We have been unable to recreate the previously reported favourable results of shockwave therapy in the treatment of delayed fracture union. On the basis of this study we have withdrawn a proposal for a multi-centre RCT

Ten percent of fractures end in delayed or non-union. NSAIDs have been linked to an inhibitory action on fracture repair for three decades yet the mechanism of action remains to be elucidated. Cancer research has identified that NSAIDs impede cell proliferation by inhibiting angiogenesis. It is proposed that a similar mechanism occurs in the induction of NSAID induced non-union. We have investigated this hypothesis in a randomised placebo control trial of the NSAID rofecoxib using a murine femoral fracture. All animals had an open femoral fracture treated using an external fixator. Outcomes measures included x-ray, histology and biomechanical testing, with laser Doppler used to assess blood flow across the fracture gap. Radiology showed similar healing patterns in both groups; however, at the later stages (day 32) the NSAID group had significantly poorer healing. Histological analysis showed that controls healed quicker (days 24 and 32), with more callus (day 8) and less fibrous tissue (Day 32). Biomechanical testing showed controls were stronger at day 32. Both groups exhibited a similar pattern of blood flow; however NSAIDs exhibited a lower median flow from day 4 onwards (significant at days 4, 16 and 24). Positive correlations were demonstrated between both histological and radiographic assessments of healing, with increasing blood flow. NSAID animals exhibited lower flows and poorer healing by all outcomes. Regression analysis demonstrates, however, that the negative effect of NSAIDs on fracture repair is independent of its inhibitory action on blood flow. COX-2 inhibitors are marketed as having cleaner side effect profiles and are widely used in trauma patients. Following development of a novel method of analysing functional vascularity across a fracture gap, we have demonstrated that the COX-2 inhibitor rofecoxib has a significant negative effect on blood flow at the fracture gap alongside inhibiting fracture repair

Implant-associated infection is a major source of morbidity in orthopaedic surgery. There has been extensive research into the development of materials that prevent biofilm formation, and hence, reduce the risk of infection. Silver nanoparticle technology is receiving much interest in the field of orthopaedics for its antimicrobial properties, and the results of studies to date are encouraging. Antimicrobial effects have been seen when silver nanoparticles are used in trauma implants, tumour prostheses, bone cement, and also when combined with hydroxyapatite coatings. Although there are promising results with in vitro and in vivo studies, the number of clinical studies remains small. Future studies will be required to explore further the possible side effects associated with silver nanoparticles, to ensure their use in an effective and biocompatible manner. Here we present a review of the current literature relating to the production of nanosilver for medical use, and its orthopaedic applications.

Cite this article: Bone Joint J 2015; 97-B:582–9.