Background. Establishing the diagnosis in a child presenting with an atraumatic limp can be challenging. There is particular difficulty distinguishing septic arthritis (SA) from transient synovitis (TS) and consequently clinical prediction algorithms have been devised to differentiate the conditions using the presence of fever, raised erythrocyte sedimentation rate (ESR), raised white cell count (WCC) and inability to weight bear. Within Europe measurement of the ESR has largely been replaced with assessment of C-reactive protein (CRP) as an acute phase protein. We have evaluated the utility of including CRP in a clinical prediction algorithm to distinguish TS from SA. Method. All children with a presentation of ‘atraumatic limp’ and a proven effusion on hip ultrasound between 2004 and 2009 were included. Patient demographics, details of the clinical presentation and laboratory investigations were documented to identify a response to each of four variables (Weight bearing status, WCC >12,000 cells/m3, CRP >20mg/L and Temperature >38.5 degrees C. The definition of SA was based upon microscopy and culture of the joint fluid collected at arthrotomy. Results. 311 hips were included within the study. Of these 282 were considered to have transient synovitis. 29 patients met criteria to be classified as SA based upon laboratory assessment of the synovial fluid. The introduction of CRP eliminated the need for a four variable model as the use of two variables (CRP and weight bearing status) had similar efficacy. An algorithm that indicated a diagnosis of SA in individuals who could not weight-bear and who had a CRP >20mg/L correctly classified SA in 94.8% individuals, with a sensitivity of 75.9%, specificity of 96.8%, positive predictive value of 71.0%, and negative predictive value of 97.5%. CRP was a significant independent predictor of septic arthritis. Conclusions. CRP was a strong independent risk factor of septic arthritis, and its inclusion within a regression model simplifies the diagnostic algorithm, such that a two-variable model correctly classified 95% individuals with SA. Nevertheless, this and similar algorithms are generally more reliable in excluding SA, than confirming SA, and therefore a clinician's acumen remains important in identifying SA in those individuals with a single abnormal variable

The reinfusion of perioperative cell salvage is one method employed to reduce exposure to donor blood. Data on the safety of this process, however, are scant. Notably, the effect of intraoperative, washed cell salvage reinfusion on prothrombotic markers has not been demonstrated. The risk of postoperative venous thromboembolism following major orthopaedic operations is not insignificant. The study objective was to assess the effect of cell salvage reinfusion on coagulation and platelet activation. Twenty-one patients undergoing elective primary hip operations were recruited. Nine patients received washed cell salvage intraoperatively, and were compared with 12 patients undergoing similar surgery that did not. Two patients in the cell salvage group also received postoperative, unwashed cell salvage. Blood samples were collected pre-operatively, immediately post-operatively, and one day post-operatively for assays of platelet activation markers, P-selectin expression and fibrinogen binding by flow cytometry in diluted whole blood; coagulation activation marker, thrombin-antithrombin complex (TAT); D-dimer by ELISA, thrombin generation by chromogenic assay, and full blood count. Samples of cell salvage material were also analysed for prothrombotic markers. There were no significant differences between the groups preoperatively. Postoperatively haemoglobin levels did not differ significantly between the cell salvage group and controls. Postoperative TAT and D-dimer were significantly higher in the cell salvage group compared with controls (p<0.05). One day postoperatively, there were significantly higher platelet P-selectin expression (p=0.006) and platelet fibrinogen binding (p=0.004) in the cell salvage group compared with controls. The white cell count (WCC) was also significantly higher (p=0.04). In the intraoperative washed cell salvage material, and in postoperative cell salvage, the platelet count was low, but significant proportions of platelets were activated, and levels of D-dimer were elevated compared with venous blood. The postoperative salvage material also contained high levels of TAT. The results from this pilot study show the induction of a prothrombotic state following reinfusion of intraoperative, washed cell salvage in recipients undergoing primary elective hip operations. An inflammatory response to reinfusion is also indicated by the raised WCC. Further investigation into the safety of cell salvage is indicated

Fatty marrow and bone loss are prominent pathologic features of osteoporosis. DNA hypermethylation shifts mesenchymal stem cells towards adipocytes impairing bone formation. Brown adipocytes produce growth factors advantageous to osteogenesis, whereas white adipocytes secrete pro-inflammatory cytokines deleterious to bone homeostasis. We assess DNA methylation inhibitor action to brown and white adipocyte formation in marrow fat of osteoporotic skeletons. Osteoporotic skeletons in mice were induced by glucocorticoid, ovariectomy or ageing. Marrow adipose volume and bone structure were quantified using OsO4 contrast-μCT imaging. Brown and white adipocytes were probed using immunostaining, RT-PCR and primary bone-marrow mesenchymal stem cell cultures. Abundant marrow fat and spare trabecular bone existed in osteoporotic skeletons. Osteoporosis increased expressions of general adipogenic markers PPARγ2 and FABP4 and white adipocyte markers TCF21 and HOXc9, whereas expressions of brown adipocyte markers PGC-1α and UCP-1 and osteogenic markers Runx2 and osteocalcin were significantly decreased. Number of UCP-1 immunostaining-positive brown adipocytes also reduced in osteoporotic bone. In vitro, DNA methylation inhibitor 5'-aza-deoxycystidine significantly increased brown adipocyte formation and osteogenic differentiation and mitigated dexamethasone-induced white adipocyte formation in mesenchymal stem cells. 5'-aza-deoxycystidine control of brown adipogenesis and white fat formation appeared to be regulated by increasing Wnt3a/β-catenin and reducing Dkk1. Disintegrated brown adipocyte and white fat cell differentiation contribute to osteoporosis pathogenesis. Maintaining DNA hypomethylation promotes Wnt signalling and brown adipocyte differentiation facilitating osteogenic differentiation. This study shed a new light to the contribution of brown adipocytic cells to bone metabolism during osteoporosis

Summary Statement. Preoperative bone-marrow-derived cell mobilization by G-CSF is a safe orthopaedic procedure and allows circulation in the blood of high numbers of CD34+ve cells, promoting osseointegration of a bone substitute. Introduction. Granulocyte-colony-stimulating-factor(G-CSF) has been used to improve repair processes in different clinical settings for its role in bone-marrow stem cell(CD34+ and CD34-) mobilization. Recent literature suggests that G-CSF may also play a role in skeletal-tissue repair processes. Aim of the study was to verify the feasibility and safety of preoperative bone-marrow cell (BMC) mobilization by G-CSF in orthopaedic patients and to evaluate G-CSF efficacy in accelerating bone regeneration following opening-wedge high tibial valgus osteotomy(HTVO) for genu varum. Patients/Methods. 24 patients were enrolled in a prospective phase II trial. The osteotomy gap was filled by a hydroxyapatite-tricalciumphosphate bone substitute(HATriC). Patients were randomised to receive (GROUP A) or not receive (GROUP B) preoperatively a daily dose of 10µg/kg of G-CSF for three consecutive days, with an additional dose 4 hours before surgery. BMC-mobilization was monitored by white blood cell (WBC)-count, flow-cytometry analysis of circulating CD34+cells and Colony-forming cell assays. Patients were evaluated by: Lysholm and SF-36 scores preoperatively and at 1, 2, 3, 6, and 12 months after surgery;. X-ray evaluation preoperatively and at 1, 2, 3, 6, and 12 months after surgery, in order to compare the percentage of osseointegration of the bone-graft junction using the semi-quantitative score of Dallari[1]. CT-scan of the host bone-substitute interface at 2 months, in order to estimate the quality of the newly formed bone at the bone-graft junction by a quantitative measure of bone density (by Hounsfield unit) at the proximal and distal bone-graft junctions. Results. All patients completed the treatment program without major side effects; G-CSF was well tolerated. BMC-mobilization occurred in all Group A patients, with median peak values of 110/µL (range 29–256) of circulating CD34+ve cells. Circulating clonogenic progenitors paralleled CD34+ve cell levels. A significant improvement in the SF-36-Role-Physical scale and in the Lysholm score was recorded at follow-up in Group A compared to Group B(p<0.05). At the X-ray-evaluation, there was a significant increase in osseointegration at the bone-graft junction in Group A at 1, 2, 3 and 6 months post-surgery compared to Group B(p<0.05). CT-scans of the grafted area at 2 months post-surgery showed no significant difference in the quality of the newly formed bone between the two Groups. Discussion/Conclusions. These results suggest that G-CSF can be safely administered preoperatively in subjects undergoing HTVO. In addition, the clinical, radiographic and CT monitoring indicate that preoperative G-CSF administration promotes bone graft substitute osseointegration. Enhanced osseointegration might be the result of the direct activity of G-CSF on the host bone or a cellular effect mediated by bone marrow-derived progenitors mobilised by G-CSF, or by a combination of all these factors. This study is a proof-of-principle that preoperative G-CSF might be an alternative treatment option to enhance bone regeneration in the field of bone marrow stem cell therapy and reconstructive orthopaedic surgery

Objectives

Platelet-rich fibrin matrix (PRFM) has been proved to enhance tenocyte proliferation but has mixed results when used during rotator cuff repair. The optimal PRFM preparation protocol should be determined before clinical application. To screen the best PRFM to each individual’s tenocytes effectively, small-diameter culture wells should be used to increase variables. The gelling effect of PRFM will occur when small-diameter culture wells are used. A co-culture device should be designed to avoid this effect.

Objectives

Platelet-rich plasma (PRP) is being used increasingly often in the clinical setting to treat tendon-related pathologies. Yet the optimal PRP preparations to promote tendon healing in different patient populations are poorly defined. Here, we sought to determine whether increasing the concentration of platelet-derived proteins within a derivative of PRP, platelet lysate (PL), enhances tenocyte proliferation and migration in vitro, and whether the mitogenic properties of PL change with donor age.

Objectives

This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocols.

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model.

A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 × 10⁷ AdIGF-1 modified chondrocytes and the contralateral joint received 2 × 10⁷ naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated.

Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months.

Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model.

The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.

Post-natal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells migrate, differentiate and incorporate into the nacent endothelium and thereby contribute to physiological and pathological neurovascularisation, has stimulated much interest. Its contribution to neovascularisation of tumours, wound healing and revascularisation associated with ischaemia of skeletal and cardiac muscles is well established. We evaluated the responses of endothelial precursor cells in bone marrow to musculoskeletal trauma in mice.

Bone marrow from six C57 Black 6 mice subjected to a standardised, closed fracture of the femur, was analysed for the combined expression of cell-surface markers stem cell antigen 1 (sca-1⁺) and stem cell factor receptor, CD117 (c-kit⁺) in order to identify the endothelial precursor cell population. Immunomagnetically-enriched sca-1⁺ mononuclear cell (MNC^sca-1+) populations were then cultured and examined for functional vascular endothelial differentiation. Bone marrow MNC^{sca-1+,c-kit+} counts increased almost twofold within 48 hours of the event, compared with baseline levels, before decreasing by 72 hours.

Sca-1⁺ mononuclear cell populations in culture from samples of bone marrow at 48 hours bound together Ulex Europus-1, and incorporated fluorescent 1,1′-dioctadecyl- 3,3,3,’3′-tetramethylindocarbocyanine perchlorate-labelled acetylated low-density lipoprotein intracellularily, both characteristics of mature endothelium.

Our findings suggest that a systemic provascular response of bone marrow is initiated by musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of neovascularisation and the healing of fractures.

We produced large full-thickness articular cartilage defects in 33 rabbits in order to evaluate the effect of joint distraction and autologous culture-expanded bone-marrow-derived mesenchymal cell transplantation (ACBMT) at 12 weeks. After fixing the knee on a hinged external fixator, we resected the entire surface of the tibial plateau. We studied three groups: 1) with and without joint distraction; 2) with joint distraction and collagen gel, and 3) with joint distraction and ACBMT and collagen gel.

The histological scores were significantly higher in the groups with ACBMT collagen gel (p < 0.05). The area of regenerated soft tissue was smaller in the group allowed to bear weight (p < 0.05). These findings suggest that the repair of large defects of cartilage can be enhanced by joint distraction, collagen gel and ACBMT.