Lumbar diseases have become a major problem affecting human health worldwide. Conservative treatment of lumbar diseases is difficult to achieve ideal results, and surgical treatment of trauma, complications, it is imperative to develop a new treatment method. This study aims to explore the regulatory mechanism of cartilage endplate ossification caused by abnormal stress, and design intervention targets for this mechanism, so as to provide theoretical reference for the prevention and treatment of lumbar degeneration. In vivo, we constructed spinal instability model in mice. In vitro, we used a mechanical tensile machine to simulate the abnormal stress conditions of the endplate cartilage cells. Through the high-throughput sequencing, we found the enrichment of Hippo signaling pathway. As YAP is a key protein in the Hippo signaling pathway, we then created cartilaginous YAP elimination mice (Col2::YAPfl/fl). The lumbar spine model was constructed again in these mice for H&E, SOFG and immunofluorescence staining. In vitro lentivirus was used to knock out YAP, immunofluorescence staining, WB and qPCR were performed. Finally, we conducted therapeutic experiments by using YAP agonist and AAV5 carrying YAP plasmids. We collected 8w samples from C57/BL6 mice after modeling. We found ossification of the endplate in mice similar to human disc degeneration. High-throughput sequencing of stretched cells demonstrated high enrichment of the Hippo signaling pathway. By immunofluorescence staining, it was confirmed that Col-II decreased and Col-X gradually increased in the endplate cartilage of mice. This was also confirmed at 7 days after an in vitro stretch of 5% and 12%. Meanwhile, we found that cartilaginous YAP elimination mice developed very severe endplate degeneration. However, the endplate was well protected by intraperitoneal injection of YAP agonist or AAV5-YAP endplate injection, and the results in vitro were consistent with that. In the process of cartilaginous ossification, abnormal stress regulates Col10a1 to promote cartilage endplate ossification through Hippo signaling pathway mediated YAP, and we expect to find potential drug targets for treatment through this mechanism

Despite extensive research aimed at improving surgical outcomes of enthesis injuries, re-tears remain a common problem, as the repairs often lead to fibrovascular scar as opposed to a zonal enthesis. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage [1]. During development, the hedgehog signaling pathway promotes the formation and maturation of fibrocartilage within the zonal tendon-to-bone enthesis [1-4]. However, whether this pathway has a similar role in adult zonal tendon-to-bone repair is not known. Therefore, we developed a murine anterior cruciate ligament (ACL) reconstruction model [5] to better understand the zonal tendon-to-bone repair process and perturb key developmental regulators to determine the extent to which they can promote successful repair in the adult. In doing so, we activated the hedgehog signaling pathway both genetically using transgenic mice and pharmacologically via agonist injections. We demonstrated that both treatments improved the formation of zonal attachments and tunnel integration strength [6]. These improved outcomes were due in part to hedgehog signaling's positive role in proliferation of the bone marrow stromal cell (bMSC) progenitor pool and subsequent fibrocartilage production of bMSC progeny cells that form the attachments. These results suggest that, similar to growth and development, hedgehog signaling promotes the production and maturation of fibrocartilage during tendon-to-bone integration in adults. Lastly, we developed localized drug delivery systems to further improve the treatment of these debilitating injuries in future translational studies. Acknowledgements: This work was supported by NIH R01AR076381, R21AR078429, R00AR067283, F31AR079840, T32AR007132, and P30AR069619, in addition to the McCabe Fund Pilot Award at the University of Pennsylvania

Ankle fractures are among the most common types of fractures. If surgery is not performed within 12 to 24 hours, ankle swelling is likely to develop and delay the operative fixation. This leads to patients staying longer in the ward waiting and increased hospital occupancy. This prolonged stay has significant financial implication as well as it is frustrating for both patients and health care professionals. The aim was to formulate a pathway for the ankle fracture patients coming to the emergency department, outpatients and planned for operative intervention. To identify whether pre-operative hospital admissions of stable ankle fracture patients are reduced with the implementation of the pathway. We formulated an ankle fracture fixation pathway, which was approved for use in December 2020. A retrospective analysis of 6 months hospital admissions of ankle fracture patients in the period between January to June 2020. The duration from admission to the actual surgery was collected to review if some admissions could have been avoided and patients brought directly on the surgery day. A total of 23 patients were included. Mean age was 60.5 years and SD was 17years. 94% of patients were females. 10 patients were appropriately discharged.7 Patients were appropriately admitted. 6 Patients were unnecessarily admitted. These 6 patients were admitted on presentation to ED. Retrospective analysis of this audit showed that this cohort of patients met the safe discharge criteria and could have been discharged. Duration of unnecessary stay ranged from 1 to 11 days (21 days in total). Total saving could have been £6300. Standards were met in 74% of cases. Preoperative hospital admission could be reduced with the proposed pathway. It is a valuable tool to be used and should be implemented to reduce unnecessary hospital admissions

Total hip replacement (THR) is indicated for patients with osteoarthritis where conservative treatment has failed. Metal alloys used in THR implants such as cobalt-chromium (CoCr) have been known to cause pro-inflammatory reactions in patients, therefore leading to the need for costly revision surgery. This study therefore aimed to investigate the role of TLR4 in the activation of a human osteoblast model in response to CoCr particles in vitro. Human osteoblasts (MG-63 cell line) were seeded at a density of 100,000 cells and treated with 0.5, 5, 50mm3 CoCr particles per cell for 24-hours. Trypan blue and the XTT Cell Proliferation Kit II were then used in conjunction with the cells to assess CoCr-induced cytotoxicity. Cells were pre-treated with a commercially available TLR4-specific small molecule inhibitor (CLI-095) for 6 hours. Untreated cells were used as a negative control and lipopolysaccharide (LPS) was used as a positive control. Following treatment the cell supernatant was collected and used for enzyme-linked immunosorbant assay (ELISA) to measure the secretion of interleukin-8 (IL-8), CXCL10, and interleukin-6 (IL-6). Trypan blue and XTT analysis showed that there was no significant changes to cell viability or proliferation at any dose used of CoCr after 24 hours. There was a significant increase in protein secretion of IL-8 (p<0.001), CXCL10 (p<0.001), and IL-6 (p<0.001) in the cells which received the highest dosage of CoCr. This pro-inflammatory secretory response was ameliorated by TLR4 blockade (p<0.001). CoCr particles are not cytotoxic to osteoblasts but they do induce pro-inflammatory changes as characterised by increased secretion of chemokines IL-8, CXCL10, and IL-6. These responses occur via a TLR4-mediated pathway and upon inhibition they can be effectively ameliorated. This is particularly important as TLR4 could be a potential target for pharmacological intervention used in patients experiencing immunological reactions to metal implant debris

Aim. Osteoarthritis (OA) is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control the expression of genes and are likely to regulate the OA transcriptome. We performed integrative genomic analyses to define methylation-gene expression relationships in osteoarthritic cartilage. Patients and Methods. Genome-wide DNA methylation profiling of articular cartilage from five patients with OA of the knee and five healthy controls was conducted using the Illumina Infinium HumanMethylation450 BeadChip (Illumina, San Diego, California). Other independent genome-wide mRNA expression profiles of articular cartilage from three patients with OA and three healthy controls were obtained from the Gene Expression Omnibus (GEO) database. Integrative pathway enrichment analysis of DNA methylation and mRNA expression profiles was performed using integrated analysis of cross-platform microarray and pathway software. Gene ontology (GO) analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Results. We identified 1265 differentially methylated genes, of which 145 are associated with significant changes in gene expression, such as DLX5, NCOR2 and AXIN2 (all p-values of both DNA methylation and mRNA expression < 0.05). Pathway enrichment analysis identified 26 OA-associated pathways, such as mitogen-activated protein kinase (MAPK) signalling pathway (p = 6.25 × 10-4), phosphatidylinositol (PI) signalling system (p = 4.38 × 10-3), hypoxia-inducible factor 1 (HIF-1) signalling pathway (p = 8.63 × 10-3 pantothenate and coenzyme A (CoA) biosynthesis (p = 0.017), ErbB signalling pathway (p = 0.024), inositol phosphate (IP) metabolism (p = 0.025), and calcium signalling pathway (p = 0.032). Conclusion. We identified a group of genes and biological pathwayswhich were significantly different in both DNA methylation and mRNA expression profiles between patients with OA and controls. These results may provide new clues for clarifying the mechanisms involved in the development of OA. Cite this article: A. He, Y. Ning, Y. Wen, Y. Cai, K. Xu, Y. Cai, J. Han, L. Liu, Y. Du, X. Liang, P. Li, Q. Fan, J. Hao, X. Wang, X. Guo, T. Ma, F. Zhang. Use of integrative epigenetic and mRNA expression analyses to identify significantly changed genes and functional pathways in osteoarthritic cartilage. Bone Joint Res 2018;7:343–350. DOI: 10.1302/2046-3758.75.BJR-2017-0284.R1

Introduction. The two-dimensional (2D) monolayer culture paradigm has limited translational potential to physiological systems; chondrocytes and tenocytes in monolayer lose expression of hallmarks of differentiated status (dedifferentiation). Qualitative assessment of three-dimensional (3D) cultures in musculoskeletal biology relative to native tissues has been limited. An understanding of prevailing gene regulatory networks is required to define whether 3D culture systems faithfully restitute the native tissue phenotype (redifferentiation). Using a systems biology approach to explore the gene networks associated with de- and re-differentiation may define targetable regulators associated with phenotypic plasticity of adult musculoskeletal cells. Materials and Methods. Global transcriptomic and proteomic profiling of matrix-depleted chondrocytes and tenocytes from the rat was performed for each of three conditions (native tissue, monolayer at passage three, or tissue-appropriate 3D cultures). Differential analysis of mRNA and protein abundance, gene ontology annotation, pathway topology impact analysis, and derivation of common mechanistic networks was undertaken to define consensus expression profiles, signalling pathways, and upstream regulators for de- and re-differentiation in each cell type. Results. Principal component analysis demonstrated a convergence of gene expression profiles in monolayer, including the expression of musculoskeletal progenitor markers scleraxis (Scx) and Mohawk (Mkx). Three-dimensional culture systems failed to demonstrate parity with native tissue and incited the expression of Il-6 and Ptgs2 (COX2). The CCN-family member Ctgf (CCN2), and the marker of skeletal differentiation Grem1 (gremlin 1), were consistently differentially abundant in de- and re-differentiation at both the mRNA and protein level. Pathway topology impact analysis defined PI-3K/Akt as the common signalling pathway in de- and re-differentiation. Discussion. Historically, the terms de- and re-differentiation have been used with no mechanistic definition. Additionally, there is no standardised phenotype for 3D cultures to benchmark novel progress in bioengineering. Consensus upstream regulators yielded a unified mechanistic network for chondrocyte and tenocyte phenotypes in three conditions. The PI-3K/Akt signalling pathway has been implicated in a range of physiological activities including dedifferentiation, proliferation, matrix synthesis, and cell survival. Pathway analysis suggests that the PI-3K/Akt signalling pathway may contribute to the de- and re-differentiation phenotypes for both chondrocytes and tenocytes and represents a rational target for further network-level analysis

Pulsed electromagnetic fields (PEMFs) have been considered a potential treatment modality for fracture healing. As bone fracture healing and osseointegration share the same biological events, the application of PEMF stimulation to facilitate the osseointegration process of orthopedic implants has been suggested. However, the mechanism of their action remains unclear. Mammalian target of rapamycin (mTOR) signaling may affect osteoblast proliferation and differentiation. This study aimed to assess the osteogenic differentiation of mesenchymal stem cells (MSCs) under PEMF stimulation and the potential involvement of mTOR signaling pathway in this process. PEMFs were generated by a novel miniaturized electromagnetic device (MED). Potential changes in the expression of mTOR pathway components, including receptors, ligands and nuclear target genes, and their correlation with osteogenic markers and transcription factors were analyzed. PEMF exposure increased cell proliferation, adhesion and osteogenic commitment of MSCs. Osteogenic-related genes were over-expressed following PEMF treatment. Our results confirm that PEMFs contribute to activation of the mTOR pathway via upregulation of the proteins AKT, MAPP kinase, and RRAGA, suggesting that activation of the mTOR pathway is required for PEMF-stimulated osteogenic differentiation. In summary, the findings of the present study revealed that MED-generated PEMFs stimulate osteogenic differentiation and the maturation of the adipose tissue-derived MSCs via activation of the mTOR pathways. Even though further research is required to determine an optimal stimulation timing and flux density both in-vitro and in-vivo, this study results may serve a source for an adjuvant therapy to improve orthopedic implant stability, longevity and enhance fracture healing

Bone formation proceeds through two distinct processes. One involves the deposition of bone by osteoblasts (intramembranous ossification) and another through the remodeling of an intermediate cartilaginous matrix formed by chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs) aggregates – a process called endochondral ossification (EO). EO is responsible for formation of long bones during development and most prevalent during facture repair upon callus formation. In adult bone injuries MSCs from periosteum form bone via EO whereas MSCs from bone marrow are primarily differentiate to osteoblast in vivo. We hypothesized that the unique biophysical and biochemical properties of bone mineral phase has a role in programming MSCs. Using a biomimetic bone like apatite (BBHAp) as surrogate for bone mineral phase, we studied the chondrogenic differentiation of human marrow derived MSCs and observed that the BBHAp dictates MSCs fate and strictly dictates the pathway of bone formation in vivo. Through exhaustive dissection of the signaling pathways at play, a prominent role of PTH1R in modulating the effects imposed by the BBHAp has been unraveled. These fundamental insights gained in how bone microenvironment might alter fate of MSCs has important implications for bone repair and regeneration therapies

The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established an ex vivo 3D collagen gel-based system and we studied the remodeling capacity of two different TSPC lines from young, Y-TSPC and aged/degenerative, A-TSPC donors. Here, we specifically focused on investigating the involvement of integrin receptors in the remodeling process. Integrins are transmembrane receptors consisting of alpha (a) and beta (b) subunits, which form cell-to-matrix bonds, activate various pathways and thereby control cell proliferation, differentiation and survival. Y- and A-TSPC were derived from human Achilles tendons and are fully described in Kohler et al. 2013. RT-PCR was used to assess the expression of collagen-binding integrins in the TSPC cultivated in collagen gels. Next, a1 and a11 integrins were silenced by stable lentiviral delivery of target-specific shRNA in the Y-TSPC. Control (con-shRNA), integrin (a1-shRNA) and integrin a11 (a11-shRNA) virus-containing supernatant was given for 24h and then cells were selected with 50 microg./ml zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by quantitative PCR and western blotting. Last, functional tests were carried out by time-lapse recording gel contraction of four cell groups (Y-TSPC+con, Y-TSPC+a1KD, Y-TSPC+a11KD, and A-TSPC). Among the screened integrins we found that integrin a1 and a11 were significantly downregulated in A-TSPC with 3.8 and 5.6 folds, correspondingly. Therefore, to mimic the A-TSPC we carried out a gene KD of a1 and a11 in Y-TSPC. PCR and western blot clearly validated the efficient KD. Analyses of collagen contraction, revealed that Y-TSPC+a11KD significantly reduced collagen contractability comparable to A-TSPC. This indicated the indispensable role of this integrin in the signaling pathway of collagen matrix remodeling. In respect to integrin a1, we found that this receptor did not affect the contraction rate of Y-TSPC, which was similar to Y-TSPC+con. To our knowledge we have now identified for the first time the critical role of a11 integrin receptor in tendon collagen remodeling, and a follow up analysis of its exact downstream cascade is on the way. Future efforts in deciphering how tendon matrix makeover is regulated can lead to innovation in preventive strategies for tendon degeneration

Background/Aims. Bisphosphonates play an important role in the treatment of catabolic bone diseases such as osteoporosis. In addition to their anti-resorptive activity exerted by their proapoptotic effect on osteoclasts, recent data suggest that nitrogen-containing bisphosphonates (N-BP) may also promote osteogenic differentiation by an unknown mechanism. Similar bone-anabolic effects have been attributed to cholesterol-lowering statins, which represent another class of mevalonate pathway inhibitors besides N-BP, suggesting a common mode of action. In vascular endothelial cells statins were recently shown to activate the Mek5/Erk5 mitogen-activated protein kinase cascade, which plays an important role in cellular differentiation, apoptosis or inflammatory processes. Here we evaluated whether N-BPs may also target the Mek5/Erk5 pathway and analysed the consequence of Erk5 activation on bone-relevant gene expression, calcification and osteoblast differentiation. Methods and Results. We show that N-BP dose-dependently activate Erk5 in primary human endothelial cells and osteoblasts. The mechanism likely involves farnesyldiphosphate synthase (FDPS) inhibition and subsequent inactivation of the small GTPase Cdc42 since siRNA-mediated knockdown of both genes could reproduce N-BP-induced ERK5 activation. ERK5 activation resulted in regulation of several bone-relevant genes and was required for calcification and osteoblastic differentiation of mesenchymal stems cells as evident by the lack of alkaline phosphatase induction and alizarin-red staining observed upon Erk5 knockdown or upon differentiation initiation in presence of a pharmacological Erk5 inhibitor. Conclusion. Our data provide first evidence that N-BP activate the Mek5/Erk5 cascade and reveal an essential role of Erk5 in the regulation of bone homeostasis by influencing bone mineralization and osteoblast differentiation

The current study aims to find the role of Enhance Recovery Pathway (ERP) as a multidisciplinary approach aimed to expedite rapid recovery, reduce LOS, and minimize morbidity associated with Non Fusion Anterior Scoliosis Correction (NFASC) surgery. A retrospective analysis of 35 AIS patients who underwent NFASC with Lenke 1 and Lenke 5 curves with a minimum of 1 year of follow-up was done. Patient demographics, surgical details, postoperative analgesia, mobilization, length of stay (LOS), patient satisfaction survey score with respect to information and care, and 90 days complications were collected. The cohort included 34 females and 1 male with a mean age of 15.2 years at the time of surgery. There were 16 Lenke 1 and 19 Lenke 5 in the study. Mean preoperative major thoracic and thoracolumbar/lumbar Cobb's angle were 52˚±7.6˚ and 51˚±4.5˚ respectively. Average blood loss and surgical time were 102 ±6.4 ml and 168 ± 10.2 mins respectively. Average time to commencing solid food was 6.5±1.5 hrs. Average time to mobilization following surgery was 15.5± 4.3 hrs. The average duration to the stopping of the epidural was 42.5±3.5 hrs. The average dose of opioid consumption intraoperatively was 600.5±100.5 mcg of fentanyl i.v. and 12.5±4.5 mg morphine i.v. Postoperatively opioids were administered via an epidural catheter at a dose of 2 mg of morphine every 24 hours up to 2 days and an infusion of 2mcg/hr of fentanyl along with 0.12-0.15% ropivacaine. The average duration to transition to oral analgesia was 55.5±8.5 hrs .20 patients had urinary catheter and the average time to removal of the catheter was 17.5±1.4 hrs. 25 patients had a chest tube and the average time to remove of chest tube was 25.5±3.2 hrs. The average length of hospital stay was 3.1±0.5 days. No patient had postoperative ileus or requirement of blood transfusion or any other complications. No correlation was found between LOS and initial cobb angle. The application of ERP in AIS patients undergoing NFASC results in reduced LOS and indirectly the cost, reduced post-operative opioid use, and overall improve patient satisfaction score

Dupuytren Disease (DD), the most common connective tissue disease in man, presents as a benign fibromatosis of the hands and fingers resulting in the formation of nodules and cords and often leading to flexion contractures in association with keloids or Peyronie disease. Surgical resection of the fibrotic nodules, and more recently intra-lesional collagenase injection are the main therapeutic options for these patients. While the exact cause of DD is still unknown, linkage and Genome Wide Association Studies (GWAS) showed molecular heterogeneity with at least 10 different susceptibility loci 6 of which are close to genes encoding proteins in the Wnt-signaling pathway. We aim to identify the molecular basis of Dupuytren Disease (DD). Twenty patients with Dupuytren disease (including 3 patients with autosomal dominant inheritance, 1 with keloids and congenital torticollis, 2 with Peronie disease), were included in this study. Chromosome Microarray Analysis (CMA), Whole Exome Sequencing (WES) of gDNA and proteomic analysis by LC-Tandem Mass Spectrometry (LC-MSMS) studies were performed. Expression and Network analysis of LCMSMS results was performed using Principal Component Analysis (PCA), ANOVA and Ingenuity Pathway Analysis (IPA). No pathogenic copy number variants (CNVs) were found in CMA (n = 3). WES showed potentially pathogenic variants in POSTN, WNT11, MMP1 and COL3A1. PCA showed three differentially expressed clusters and network-IPA identified ACTB, BAX, COL3A1, FBN1, FN1, MMP1 as potential biomarkers. Comprehensive multi-OMIC analysis of gDNA and tissue proteins in patients with DD identified several connective tissue biomarkers potentially important in the pathogenesis of DD

Introduction. 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. Methods. 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. −8. M), 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. Results. AEA (5.1±0.7pmol/g), 2-AG (527.0±78.6 pmol/g), PEA (122.2±5.1pmol/g) and OEA (122.8±4.3pmol/g) were present in human trabecular bone. All components of the endocannabinoid system and RANK/RANKL pathways were present at the mRNA level in human trabecular bone. TRAP positive, multinucleated, calcium phosphate resorbing osteoclasts were observed from day 8 to day 23 of culture. mRNA expression of the osteoclast specific markers TRAP and cathepsin K and components of the endocannabinoid and the RANK/RANKL systems (with the exception of CB1) were up-regulated with osteoclast maturation with highest levels of expression on day 14. Conclusion. The detection of both synthetic and catabolic enzymes of the endocannabinoids in human trabecular bone and osteoclast culture indicates local skeletal production and regulation of endocannabinoids. The co-expression of all components of the endocannabinoid and the RANK/RANKL systems in human trabecular bone and osteoclast culture suggest possible interactions between the 2 systems in maintaining balanced bone remodelling, which may impact upon bone resorption seen in many bone diseases

Background: The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established a 3D collagen gel system and studied the remodeling capacity of two different TSPC lines: young, Y-TSPC and aged/degenerative, A-TSPC. We specifically investigated the involvement of integrin receptors in the remodeling process. Methods: Y- and A-TSPC were derived from human Achilles tendon. RT-PCR was used to assess the expression of collagen-binding integrins. Integrins a1 and a11 were silenced by lentiviral delivery of shRNA in the Y-TSPC. Control-shRNA, a1-shRNA and a11-shRNA virus was given for 24h and then cells were selected with zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by quantitative PCR and western blotting. Last, time-lapse recording of gel contraction of Y-TSPC+con, Y-TSPC+a1KD, Y-TSPC+a11KD, and A-TSPC were performed. Results: Integrin a1 and a11 were significantly downregulated in A-TSPC. Therefore, to mimic the A-TSPC we carried out a1 and a11 KD in Y-TSPC. PCR and western blot validated very efficient KD. Analyses of collagen contraction revealed that Y-TSPC+a11KD had significant reduction in collagen contractibility comparable to A-TSPC phenotype. Regarding integrin a1, we found that this receptor had no effect on the contraction rate of TSPC. Thus, to our knowledge we have now identified for the first time a novel role of a11 integrin in tendon matrix remodeling, and a follow up analyses of the exact downstream cascade are on the way

CAR (CARSKNKDC) is a systemically administered wound-homing peptide that specifically recognizes angiogenic blood vessels and extravasates into sites of injury. CAR peptide requires heparan sulfate proteoglycans (HSPGs) for its cell penetrating activity. Syndecan-4 (SDC4) is a HSPG and binding to it triggers the wound re-epithelialization process. We have discovered that CAR peptide has the inherent ability to promote wound healing; wounds close and re-epithelialize significantly faster in CAR treated mice than in control groups (PBS and mutant peptide, i.e. mCAR injections). To delineate the molecular mechanism by which CAR accelerates wound healing, we focused on the requirement of HSPG binding for CAR peptide function. We demonstrate that CAR peptide endocytosis and its stimulation of keratinocyte cell migration are both dependent on SDC4. Finally, we show that the systemic administration of CAR peptide stimulates wound re-epithelialization only in WT mice, but not in SDC4 knockout (KO) mice. As SDC4 has very restricted expression in skin wounds, we propose that CAR peptide activates SDC4 function to promote re-epithelialization. CAR peptide may provide an entirely new way of enhancing wound healing, and perhaps tissue regeneration in general. This therapeutic approach is systemic, yet target organ- and cell- specific, and dependent on the naturally occurring SDC4 dependent migratory pathway that is crucial for tissue regeneration

Background. A dedicated referral pathway for patients with bony metastases was introduced at Guy's and St Thomas’ Hospitals (GSTT) in 2009. The aim was to facilitate prompt, consultant-led decision-making and intervention for patients at risk of pathological fracture of long bones. Methods. We performed a clinical audit and service evaluation of the referral pathway through retrospective review of referrals over 3.5 years. Results. 75 patients referred from 7 different specialties, (34:41 male:female), mean age 64. 16 different types of primary cancer identified, the most common being breast (22/75). Location of metastasis was most commonly the femur (59/75). 24 patients underwent surgery, with femoral nail the most common procedure (13/24). Patients in the surgery group were younger, with higher Mirel's score, less visceral metastases and survived longer after surgery than patients treated non-operatively. Median referral-clinic time was 10 days and referral-surgery time was 14 days. Conclusions. Our data demonstrate the importance of this service and the growing demand. We have updated trust guidelines and improved our referral process through GSTT's Electronic Patient Record system. We have presented this data at local level to improve awareness and intend to reduce referral-clinical review time and have recently established a multidisciplinary meeting to improve patient outcome. Level of Evidence. 3

Introduction. Modulation of signaling pathways, which involves tendon development, regeneration, or homeostasis, is one of the potential modalities to facilitate proper regeneration of the injured tendon. Authors have previously reported that activation of Wnt/beta-catenin signaling suppressed the expression of tenogenic genes (i.e. Scleraxis (Scx), Mohawk (Mkx), Tenomodulin (Tnmd)) in rat primary tendon-derived cells (TDCs) and SCX-transduced human mesenchymal stem cells (hMSC-Scx cells), as a tendon progenitor cell line (kindly provided Dr. Docheva). The roles of TGF-beta signaling in tenogenesis have been elucidated. The purpose of the study was to evaluate the effect of TGF-beta signaling on tenogenic genes and relationship between both two signalings in rat TDCs and hMSC-Scx cells. Materials and Methods. Cell cultures. TDCs were harvested from the Achilles tendons of 6 week-old SD rats and the 3rd passage TDCs were used. To evaluate the effect of TGF-beta signaling, TGF-beta 1 protein and SD208 (TGF-beta receptor inhibitor) were utilized. To assess the effect of Wnt/beta-catenin signaling on TGF-beta signaling, BIO (Wnt/b-catenin activator) was utilized. Quantative RT-PCR. TDCs and hMSC-Scx cells were cultured with TGF-beta1 or SD208. Total RNA was isolated from the cells and cDNA was synthesized. Expression of Axin2, as a target gene for Wnt/beta-catenin signaling, as well as tenogenic genes was quantified and normalized by Gapdh. Western blotting. Each cells were cultured with BIO. Whole cell lysates were used for immunoblotting with antibodies against beta-actin and phosphorylated Smad2/Smad3 (p-Smad2/3), which indicates activation of TGF-beta signaling. Band intensity was quantified and normalized by beta-actin. Statistical analysis. Two groups were compared by unpaired Student”s t-test. Multiple groups were analyzed by one-way analysis of variance (ANOVA). (P <0.05). Results. Scx expression was increased by TGF-beta1 and decreased by SD208 in a dose-dependent manner in TDCs. However, TGF-beta1 and SD208 showed no significant effect on Mkx and Tnmd expression in TDCs and hMSC-Scx cells. BIO treatment decreased p-Smad2/3 proteins, while TGF-beta1 and SD208 had no effect on Axin2 expression in both cells. Discussion. Activation of TGF-b signaling induced Scx expression, independent of Wnt/beta-catenin signaling. Activation of Wnt/beta-catenin signaling suppressed p-Smad2/3 amounts for TGF-beta signaling and further Scx expressions. Taken together, activation of Wnt/beta-catenin signaling antagonizes Scx expression induced by TGF-beta signaling. Identification of compounds, which control Wnt/beta-catenin and/or TGF-beta signaling, may lead to developments of novel therapeutic options to facilitate regeneration of injured tendons

In patients with developmental dysplasia of the hip (DDH) chronic joint dislocation induces remodeling of the soft tissue with contractures, muscle atrophy, especially of the hip abductors muscles, leading to severe motor dysfunction, pain and disability (1). The aim pf the present work is to explore if a correct positioning of the prosthetic implants through 3D skeletal modeling surgical planning technologies and an adequate customized rehabilitation can be beneficial for patients with DDH in improving functional performance. The project included two branches: a methodology branch of software development for the muscular efficiency calculation, which was inserted in the Hip-Op surgical planning system (2), developed at IOR to allow surgical planning for patients with complex hip joint impairment; and a clinical branch which involved the use of the developed software as part of a clinical multicentric randomized trial. 50 patients with DDH were randomized in two groups: a simple surgical planning group and an advanced surgical planning with muscular study group. The latter followed a customized rehabilitation program for the strenghtening of hip abductor muscles. All patients were assessed before surgery (T0) and at 3 (T1) and 6 months (T2) postoperatively using clinical outcome (WOMAC, HHS, ROM, MMT, SF12, 10mt WT) and instrumental measures (Dynamometric MT). Pre- and post-operative musculoskeletal parameters obtained by the software (i.e., leg length discrepancy, hip abductor muscle lengths and lever arms) using Hip-Op during the surgical planning were considered. One Way ANOVA for ROM measurement showed a significant improvement at T2 in patients included in experimental group, as well as WOMAC, HHS and SF12 score. The Dynamometric MT score showed significant differences between at T2 (p<0.009). Spearman's rank correlation coefficients showed a significant correlation between both pre- and post-operative abductors lever arm (mm) and hip abductor muscle strength at T2 (ρ = −0.55 pre-op and ρ = −0.51 post-op, p p<0.012 and p<0.02 respectively) and between the operated pre-postoperative leg length variation (mm) and the hip abductor muscle strength (ρ = −0.55, p p<0.013). Results so far obtained showed an improvement of functional outcomes in patients undergoing hip replacement surgery who followed therapeutic diagnostic pathway sincluding a preoperative planning including the assessment of the abductiors lever arm and a dedicated rehabilitation program for the strenghtening of abductios. Particularly interesting is the inverse relationship between the strength of the hip abductor muscles and the variation of the postoperative abductor lever arm

Summary. Corticosteroids (CS) are commonly administered by intra-articular injection to control the symptoms of osteoarthritis; however, CSs also suppress articular chondrocyte matrix synthesis. Both triamcinolone and methylprednisolone acetate significantly suppressed BMPs −2 and −7, and TGF-b1 expression, suggesting a mechanism by which CSs suppress articular chondrocyte matrix synthesis and cartilage homeostasis. Introduction. Osteoarthritis (OA) is a common and debilitating disease that affects approximately 30% of the US population and is also a major clinical problem in companion animals. There are many drugs available to manage the symptoms of OA. Of these, intra-articular corticosteroid (CS) administration is a common and very effective anti-arthritic therapy, and is frequently administered to equine athletes. CSs exert their potent anti-inflammatory effects by blocking phospholipase A and reducing inflammatory mediator production; however, CSs also suppress matrix-biosynthetic activity of articular chondrocytes. This activity, along with ther increased joint use that symptomatic relief allows, has been linked to ‘steroid arthropathy’; a progression of arthritis driven by compromised chondrocyte homeostatic capacity. Several lines of experimental and clinical evidence emphasise the importance of TGF-b and BMP autocrine/paracrine activity in maintaining the homeostatic status of articular chondrocytes (reviewed in Oshin and Stewart 2007). This study was carried out to address the following objectives: 1) To assess the effects of CS on expression of chondro-protective TGF-β and BMP ligands in equine articular chondrocytes, and 2) To determine if exogenous BMP ligand administration can mitigate the suppressive effects of CSs on articular chondrocyte synthesis of collagen type II (Coll II) and glycosaminoglycans (sGAG). Methods. Articular cartilage was collected from clinically normal joints of adult horses, euthanased for reasons other than musculoskeletal disease. Articular chondrocytes were isolated by collagenase digestion and cultured as aggregates in serum-free medium under non-adherent conditions (Stewart et al 2000). Triamcinolone acetate (TA) or methylprednisolone acetate (MPA) was added to the articular chondrocyte cultures at 10. −10. M, 10. −7. M, and 10. −5. M; comparable to in vivo exposure concentrations. Effects on Coll II, aggrecan/sGAG, BMP and TGF-b ligand expression were assessed by QPCR, Coll II ELISAs and DMMB assays. In a separate series of experiments, exogenous BMP-2 was administered to chondrocyte cultures exposed to CS supplementation, to determine whether BMP can prevent or mitigate CS-mediated suppression of matrix synthesis. Results. BMP-2 and BMP-7 mRNA levels were significantly down-regulated by both CS treatments. In contrast, expression of BMPs-4 and 6 was not affected at any of the CS doses tested. TGF-b1 mRNA levels were significantly suppressed by both CSs at all doses tested. Somewhat surprisingly, TGF-b2 expression was increased by CS administration, though not significantly, while TGF-b3 expression was not affected. Exogenous BMP-2 administration (1–100 ng/ml) increased Coll II expression in the control groups but did not prevent the suppression of Coll II or sGAG synthesis in CS-treated chondrocytes. Discussion/Conclusions. Both TA and MPA down-regulated BMP-2, BMP-7 and TGF-b1 mRNA expression in articular chondrocytes. These CS-mediated effects appear to be gene-specific, since BMPs-4 and 6, and TGF-bs 2 and 3 were not similarly affected. Although exogenous BMP-2 administration increased Coll II production under control conditions, this did not effectively prevent CS-mediated suppressive effects on cartilage matrix synthesis. This suggests that other elements of the articular chondrocyte BMP and/or TGF-b signaling pathways are also affected by CS administration

Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms.

Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2^-/-). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR.

Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over hypertrophic chondrocytes to bone-forming osteoblasts was accelerated. Collagen production was increased in mutant fracture callus. Serum levels of BMP-2 were increased in healing KO mice. Isolated integrin deficient osteoblast presented an earlier expression and production of active BMP-2 during the differentiation, which led to earlier mineralisation. Biomechanical testing showed no differences between wild-type and mutant bones.

Knockout of integrin α2β1 leads to a beneficial outcome for fracture repair. Callus maturation is accelerated, leading to faster recovery, accompanied by an increased generation of extra-cellular matrix material. Biomechanical properties are not diminished by this accelerated healing. The underlying mechanism is driven by an earlier availability of BMP-2, one main effectors for bone development. Local inhibition of integrin α2β1 is therefore a promising target to accelerate fracture repair, especially in patients with retarded healing.