Protocols for processing of tissue from arthroplasty infections vary and might affect the recovery of bacteria. We compared homogenization, bead beating and enzymatic disruption for recovery of live bacteria from tissue samples. Suspensions of Staphylococcus aureus and Escherichia coli were prepared as controls. Three samples were taken from each and the first was bead beaten, the second homogenized, and Proteinase K was added for 10 and 30 minutes to the third sample before culturing. In addition, artificially inoculated pork tissue and known infected human tissue samples were processed by either homogenization or bead beating prior to cultures and results were compared. Number of cycles of bead beating and homogenization and duration of Proteinase K treatment had significant effects. Bead beating for 2 and 4 cycles reduced the yield of S.aureus to 52% and 20% of control, and E.coli to 33% and 8%. Homogenization for 2 and 4 cycles reduced S.aureus to 86% and 65% of control, and E.coli to 90% and 87%. Proteinase K for 10 minutes and 30 minutes reduced the yield of S.aureus to 75% and 33% of control, and E.coli to 91% and 49% respectively. Inoculated Pork tissue showed a reduction in S.aureus recovery of 90% for bead beating compared to homogenization, and 80% in the case of E.coli. Bead beating of infected human tissue samples reduced the yield by 58% compared to homogenization. Bead-beating is a common recommended method of processing tissue from arthroplasty cases. However, even though it produces a homogeneous sample, it does so at the cost of significant loss of viable bacteria. Homogenization and 10 minutes of Proteinase K incubation are almost equivalent, but the homogenizer is preferred being more controllable and cheaper. This should help to define guidelines for diagnosing infections using tissue samples.
A subgroup of patients that undergo TKR surgery have evidence of neuropathic pain and central sensitization that may predispose to severe postoperative pain. This study assesses the correlation of MRI detected bone marrow lesions (BMLs) and synovitis with markers of neuropathic pain and central sensitization in patients undergoing TKR surgery and healthy volunteers. 31 patients awaiting TKR and 5 healthy volunteers were recruited. Each subject underwent a 3-T knee MRI scan that was graded for BMLs (0–45) and synovitis (0–3) using subsets of the MRI Osteoarthritis Knee Score (MOAKS). All subjects were asked to complete the PainDetect questionnaire to identify nociceptive pain (< 13), unclear pain (13–18) and neuropathic pain (>18). Correlation between BMLs and PainDetect score was the primary outcome measure. Secondary outcomes included the correlation of synovitis to PainDetect and temporal summation (TS) a measure of central sensitization to the PainDetect score. TS was determined using a monofilament to evoke pain. Pilot histological analysis of the prevalence of osteoclasts (TRAP+) within BMLs versus normal subchondral bone was performed, implying a role in BML pathology. Increasing BML MOAKS score correlated with neuropathic pain (painDetect), r BMLs and synovitis are more prevalent in neuropathic pain and central sensitization in knee OA. Higher osteoclast prevalence was seen within BMLs which may help explain the association with BMLs and pain in OA.
Painful OA is linked to CNS changes in pain processing. Temporal summation of pain (TSP) is a measure of one such CNS change, central sensitization. TSP is defined using a series (≥0.33Hz) of painful stimuli and is a predictor of postoperative pain, experienced by 20% of patients after total knee replacement (TKR) surgery. This study has developed a protocol to use functional MRI to assess CNS changes in OA pain processing. This pilot includes 3 participants with chronic knee OA pain awaiting TKR (62 ± 4.4) and 5 healthy volunteers (50 ± 13.6). 3-Tesla BOLD fMRI brain scans were recorded during short series of one second painful stimuli, applied using an automated inflatable cuff to the calf muscle of the leg with the affected knee or left side in healthy volunteers. The pain intensity at onset and during the 10 painful stimuli were recorded using a numerical rating scale. The pattern of brain activation was averaged across noxious stimuli, and the differential activation compared the 1st vs. 10th (last) stimulus. Bone marrow lesions (BMLs), synovitis and effusion size were scored from 3-Tesla knee MRI's using MOAKS scoring. TSP was raised in OA patients compared to control group (p=0.023). TSP brain activity in the chronic OA patients displayed higher signal within the subgenual anterior cingulate (sgACC) compared to healthy volunteers. Knee MRI identified OA patient's exhibited higher BML scores (p=0.038) and more knee effusion (p=0.018), but the lack of synovitis did not differ from control group (p=0.107). Enhanced TSP in chronic knee OA pain may be linked with augmented responses in emotional circuitry. BMLs and effusion size appear to contribute more with pain than synovitis. These results may help understand sensitization to improve outcomes for patients with knee OA undergoing TKR surgery.
Bioresorbable materials offer the potential of developing fracture fixation plates with similar properties to bone thereby minimising the “stress shielding” associated with metal plates and obviating the need for implant removal. Phosphate glass fibre reinforced (PGF)-polylactic acid (PLA) composites are bioresorbable and have demonstrated sufficient retention of mechanical properties to enable load bearing applications. To determine the potential in vivo “stress shielding” effects of a novel PGF reinforced PLA composite plate in an animal model.Background
Aim
The type, duration and intensity of exercise required to induce mechanical hypoalgesia is poorly defined. We are interested in identifying the exercise parameters required to induce raised pressure pain thresholds. This pilot study investigates the effect of indoor rowing on pressure pain threshold (PPT) in high performance rowers. Our ultimate aim is to investigate the potential of utilising exercise in the treatment of chronic pain and specifically in relation to the management of knee osteoarthritis. 20 high performance rowers (13M:7F; Mean Age 20.8 years; SD 1.74) were recruited from the University of Nottingham and Nottingham Boat Club high performance rowing teams under a research protocol approved by the University of Nottingham Ethics Committee. PPT measurements were made in triplicate using an algometer (SOMEDIC, Sweden) at the medial knee joint line, anterior tibia and sternum, pre- and post-exercise. Anthropomorphic and rowing ergometer power output data were also recorded. There was significant increase in PPT values at all sites following exercise (Medial joint line: 127.6Nm-2, 26%, p=0.001; Tibia: 110.8Nm-2, 24.7%, p<0.001; Sternum: 48.9Nm-2, 11.7%, p=0.005 – Wilcoxon Signed Rank) statistical power was 97.1%, 100% and 88.1%, respectively. PPT was greater at baseline at the medial joint line compared to other sites, reaching highly significant relative to the sternum (p<0.001). We determined that ten minutes of high intensity indoor rowing induced hypoalgesia in high performance rowers. Further research is required to investigate the detailed interplay between exercise and hypoalgesia, including its duration post exercise, to identify suitability for use in pain management strategies.
Charcot neuropathic osteoarthropathy is a rare, destructive process affecting the bones and joints of feet in patients with diabetic peripheral neuropathy. The aetiology of Charcot remains unknown, although it has been suggested that it is triggered by the occurrence of inflammation in the foot of a susceptible individual, and that the inflammation results in increased osteoclastic activity. The increased bone turnover in acute Charcot is associated with increased concentrations of pro-inflammatory cytokines, related signalling peptides and bone turnover markers.Background
Hypothesis
Injectable scaffolds which also deliver cells and bioactive molecules to augment bone healing overcome many of the limitations associated with current bone graft substitutes. The aim of this study was to develop and test a novel injectable scaffold that self-assembles isothermically in situ to form a biodegradable porous osteoconductive material, and to assess the viability of human mesenchymal stem cells (hMSC) seeded onto the scaffold. Rheological assessment was performed on three different molecular weights (Mw) of poly(lactic-co-glycolic acid) (PLGA) (26kDa, 53kDa and 92kDa) combined with differing ratios of polyethylene glycol (PEG) to control the temperature required for scaffold self-assembly. The strength (MPa) and stiffness (Young's Modulus) patterns of the scaffolds were assessed in compression. The cell viability, proliferation and distribution patterns of hMSCs seeded within the scaffold were assessed through various assays (Alamar Blue), confocal microscopy and micro-CT. The hMSC differentiation in osteogenic media was characterised by the identification of specific bone formation markers (e.g. alkaline phosphatase).Background/Study Aim
Methods
Both the RANK/RANKL system and the endocannabinoid system have roles in bone remodelling. Activation of CB1 receptors on sympathetic nerve terminals in trabecular bone modulates bone remodelling by attenuating adrenergic inhibition over bone formation. CB2 receptors are involved in the local control of bone cell differentiation and function. Osteoblastic CB2 receptor activation negatively regulates RANKL mRNA expression indicating an interaction between the two systems and that efficient bone remodelling requires a balance between these two systems. The aim of the study was to establish the presence of the different components of the endocannabinoid system and the RANK/RANKL signalling pathway in human bone and osteoclast culture. Levels of endocannabinoids (AEA, 2-AG) and their related compounds (OEA, PEA) in human trabecular bone, obtained from patients undergoing elective orthopaedic surgery, were measured using Liquid Chromatography Mass Spectrometry (LC-MS-MS). mRNA for the endocannabinoid synthetic and catabolic enzymes (NAPE-PLD, DAGLa, FAAH, MAGL), cannabinoid-activated receptors (CB1, CB2, PPARs, TRPV1), and RANK, RANKL and NFkB were determined using Taqman Real-Time PCR. Osteoclasts were differentiated from U-937 cells (Human leukaemic monocyte lymphoma cell line), following the sequential treatment using TPA (0.1μg/ml) followed by either TNF-a (3ng/ml) or calcitriol (10−8M), cultured for up to 30 days. Osteoclasts were identified by positive staining with tartrate resistant acid phosphatase (TRAP), multinucleation and the ability to form resorption pits on calcium phosphate coated discs. Taqman Real-Time PCR was performed to detect the expression of the osteoc! last marker genes TRAP and cathepsin K, together with genes of the endocannabinoid and RANK/RANKL signalling pathways.Introduction
Methods
Deep infection rates of 1 - 2% following primary hip and knee arthroplasty are mainly due to endogenous contamination of the surgical site from bacteria within the patient's own skin. However surgical skin preparation removes only bacteria from the surface of the skin, leaving viable bacteria in the deeper layers of the skin within hair follicles and sweat and sebaceous glands. The aim of our study was to test the hypothesis that surface skin swabs taken after skin preparation with alcoholic povidone iodine would not grow bacteria, whereas full thickness biopsies taken from the line of surgical incision would grow bacteria. Under LREC approval, informed consent was obtained from 22 patients undergoing primary hip (n=9) or knee (n=13) arthroplasty. All patients received intravenous antibiotic prophylaxis at the time of induction of anaesthesia. After surgical skin preparation with alcoholic povidone iodine, a surface skin swab and full thickness skin biopsy, using an 8mm x 4 mm elliptical punch, were taken. The swab culture was incubated aerobically and anaerobically at 37°C. The skin biopsy was cut aseptically in half. One half was crushed using artery forceps, placed in 5mL anaerobe basal broth and incubated anaerobically at 37°C. The other half of the skin biopsy was frozen in isopentane and gram – stained after sectioning.Background
Methods
Open fractures occur with an annual incidence of 11.5 per 100,000 (6900 pa in UK). Infection rates, even with intravenous broad-spectrum antibiotics, remain as high as 22%. For this reason necessary bone grafting is usually delayed until soft-tissue cover of the bone injury is achieved. A biodegradable bone graft that released sustained high concentrations of antibiotics and encouraged osteogenesis, that could be implanted safely on the day of injury would reduce infection rates and avoid reoperation and secondary grafting. The non –union rate (approx 350 pa in UK) should also be reduced. Such a graft, consisting of a PLA/PGA co –polymer and containing antibiotics, is under development and here we report assessment of spectrum and duration of antimicrobial activity and effect of addition of antibiotics on mechanical properties. Varying concentrations of gentamicin, colistin, clindamycin and trimethoprim, singly and in combination, were added to the copolymer and test pieces were made. These were then tested using an established method (SPTT) which determines degree and duration of antimicrobial activity as well as risk of emerging resistance. Test bacteria were Staphylococcus epidermidis, Staphylococcus aureus, MRSA and Escherichia coli. Mechanical properties (compressive strength and porosity) were determined using established methods.Introduction
Methods
To determine the reliability, reproducibility, variability and validity of the Osteoarthritis Research Society International (OARSI) Osteoarthritis Cartilage Histopathology (OACH) system and Mankin Histopathology – Histochemical Grading System (HHGS) when applied to the characterisation of the osteoarthritic human knee. Kellgren-Lawrence and Line Drawing Atlas (LDA) radiology scores clinically graded the knees of ten patients undergoing total knee arthroplasty due to osteoarthritis. The tibial plateaux were scored using the Modified Collins (MC) and Société Française d'Arthroscopie (SFA). Three observers, twice scored, using both the OACH and HHGS systems across a single complete medial and lateral tibial plateau transect taken to include the region with the most severe OA lesion. Intra and inter-observer reliability, reproducibility, variability and validity were quantified, and the correlation between the two histopathology scoring systems was calculated.Objective
Method
