Osteoarthritis (OA) is a major global disease with increasing prevalence. It is one of the most significant causes of disability worldwide and represents a major burden in terms of healthcare delivery and impact on the quality of life of patients. It is a cause of severe chronic pain and has given rise to alarming levels of opioid use and addiction. Despite this prevalence, there are no disease-modifying treatments which delay or reverse the degrative changes within joints which are characteristics of the disease. All treatments are symptom-modifying with the exception of joint arthroplasty, which is currently the most common surgical procedure carried out in US hospitals. Several pharmaceutical and biological interventions have been tested in recent years, including metalloproteinase inhibitors, chondrogenic agents such as Kartogenin, IL-1 antagonists and monoclonal antibodies. So far, none of these has provided an effective disease-modifying treatment. Cellular therapies have a great deal of promise because of their anti-inflammatory and regenerative effects. Mesenchymal stromal cells (MSCs) have been widely studied as a treatment for OA in preclinical and clinical assessments with generally positive results. As the clinical testing of these cells proceeds serious questions emerge relating to the quality and consistency of the therapeutic product and the need for better standardisation with regard to, for example, the tissue source and expansion conditions. Of equal importance is the need for deeper insight into the therapeutic mechanism, specifically the activity and phenotype of cells transplanted to the OA environment, their fate and interaction with local cells.

Objectives. The period of post-operative treatment before surgical wounds are completely closed remains a key window, during which one can apply new technologies that can minimise complications. One such technology is the use of negative pressure wound therapy to manage and accelerate healing of the closed incisional wound (incisional NPWT). . Methods. We undertook a literature review of this emerging indication to identify evidence within orthopaedic surgery and other surgical disciplines. Literature that supports our current understanding of the mechanisms of action was also reviewed in detail. . Results. A total of 33 publications were identified, including nine clinical study reports from orthopaedic surgery; four from cardiothoracic surgery and 12 from studies in abdominal, plastic and vascular disciplines. Most papers (26 of 33) had been published within the past three years. Thus far two randomised controlled trials – one in orthopaedic and one in cardiothoracic surgery – show evidence of reduced incidence of wound healing complications after between three and five days of post-operative NPWT of two- and four-fold, respectively. Investigations show that reduction in haematoma and seroma, accelerated wound healing and increased clearance of oedema are significant mechanisms of action. . Conclusions. There is a rapidly emerging literature on the effect of NPWT on the closed incision. Initiated and confirmed first with a randomised controlled trial in orthopaedic trauma surgery, studies in abdominal, plastic and vascular surgery with high rates of complications have been reported recently. The evidence from single-use NPWT devices is accumulating. There are no large randomised studies yet in reconstructive joint replacement. Cite this article: Bone Joint Res 2013;2:276–84

Monomeric C reactive protein (mCRP) presents important proinflammatory effects in endothelial cells, leukocytes, or chondrocytes. However, CRP in its pentameric form exhibits weak anti-inflammatory activity. It is used as a biomarker to follow severity and progression in infectious or inflammatory diseases, such as intervertebral disc degeneration (IVDD). This work assesses for the first time the mCRP effects in human intervertebral disc cells, trying to verify the pathophysiological relevance and mechanism of action of mCRP in the etiology and progression of IVD degeneration. We demonstrated that mCRP induces the expression of multiple proinflammatory and catabolic factors, like nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and lipocalin 2 (LCN2), in human annulus fibrosus (AF) and nucleus pulposus (NP) cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signaling of mCRP. Our results indicate that the effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells. This is the first article that demonstrates the localization of mCRP in intravertebral disc cells of the AF and NP and that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells

Extensive bone defects, caused by severe trauma or resection of large bone tumors, are difficult to treat. Regenerative medicine, including stem cell transplantation, may provide a novel solution for these intractable problems and improve the quality of life in affected patients. Adipose-derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine due to their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. However, the osteogenic potential of ASCs is lower than that of bone marrow-derived stromal/stem cells. To address this disadvantage, our group has employed various methods to enhance osteogenic differentiation of ASCs, including factors such as bone morphogenetic protein or Vitamin D, coculture with bone marrow stem cells, VEGF transfection, and gene transfer of Runx-2 and osterix. Recently, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA-seq gene analysis, we discovered that GSTT1 was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation while GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species and increased GSH/GSSG ratios were also detected in GSTT1- transfected ASCs. GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones

Despite osteoarthritis (OA) representing a large burden for healthcare systems, there remains no effective intervention capable of regenerating the damaged cartilage in OA. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which are a candidate for musculoskeletal cell therapy. However, their precise mechanism of action remains poorly understood. The effects of an intra-articular injection of human bone-marrow derived MSCs into a knee osteochondral injury model were investigated in C57Bl/6 mice. The cell therapy was retrieved at different time points and single cell RNA sequencing was performed to elucidate the transcriptomic changes relevant to driving tissue repair. Mass cytometry was also used to study changes in the mouse immune cell populations during repair. Histological assessment reveals that MSC treatment is associated with improved tissue repair in C57Bl/6 mice. Single cell analysis of retrieved human MSCs showed spatial and temporal transcriptional heterogeneity between the repair tissue (in the epiphysis) and synovial tissue. A transcriptomic map has emerged of some of the distinct genes and pathways enriched in human MSCs isolated from different tissues following osteochondral injury. Several MSC subpopulations have been identified, including proliferative and reparative subpopulations at both 7 days and 28 days after injury. Supported by the mass cytometry results, the immunomodulatory role of MSCs was further emphasised, as MSC therapy was associated with the induction of increased numbers of regulatory T cells correlating with enhanced repair in the mouse knee. The transcriptomes of a retrieved MSC therapy were studied for the first time. An important barrier to the translation of MSC therapies is a lack of understanding of their heterogeneity, and the consequent lack of precision in its use. MSC subpopulations with different functional roles may be implicated in the different phases of tissue repair and this work offers further insights into repair process

Introduction. Homogenous and consistent preparations of mesenchymal stem cells (MSCs) can be acquired by selecting them for integrin α10β1 (integrin a10-MSCs). Safety and efficacy of intra-articular injection of allogeneic integrin a10-MSCs were shown in two post-traumatic osteoarthritis horse studies. The current study investigated immunomodulatory capacities of human integrin a10-MSCs in vitro and their cell fait after intra-articular injection in rabbits. Method. The concentration of produced immunomodulatory factors was measured after licensing integrin a10-MSCs with pro-inflammatory cytokines. Suppression of T-cell proliferation was determined in co-cultures with carboxyfluorescein N-succinimidyl ester (CFSE) labelled human peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/CD28 and measuring the CFSE intensity of CD4+ cells. Macrophage polarization was assessed in co-cultures with differentiated THP-1 cells stimulated with lipopolysaccharide and analysing the M2 macrophage cell surface markers CD163 and CD206. In vivo homing and regeneration were investigated by injecting superparamagnetic iron oxide nanoparticles conjugated with Rhodamine B-labeled human integrin a10-MSCs in rabbits with experimental osteochondral defects. MSC distribution in the joint was followed by MRI and fluorescence microscopy. Result. The production of the immunomodulatory factors indoleamine 2,3-dioxygenase and prostaglandin E2 was increased after inflammatory licensing integrin a10-MSCs. Co-cultures with integrin a10-MSCs suppressed T-cell proliferation and increased the frequency of M2 macrophages. In vivo injected integrin a10-MSCs homed to osteochondral defects and were detected in the repair tissue of the defects up to 10 days after injection, colocalized with aggrecan and type II collagen. Conclusion. This study showed that human integrin a10-MSCs have immunomodulatory capacities and in vivo can home to the site of osteochondral damage and directly participate in cartilage regeneration. This suggests that human integrin α10β1-selected MSCs may be a promising therapy for osteoarthritis with dual mechanisms of action consisting of immunomodulation and homing to damage followed by early engraftment and differentiation into chondrocyte-like cells that deposit hyaline cartilage matrix molecules

Chronic low back pain (cLBP) is a complex, multifaceted disorder where biological, psychological, and social factors affect its onset and trajectory. Consequently, cLBP encompasses many different disease variants, with multiple patient-specific mechanisms. The goal of NIH Back Pain Consortium (BACPAC) Research Program is to develop understanding of cLBP mechanisms and to develop algorithms that optimally match specific treatments to individual patients. To accomplish this, one research activity of BACPAC is to develop theoretical models for chronic low back pain based on the current state of knowledge in the scientific community, and to interrogate the relationships implied by the theoretical models using data generated by or available to BACPAC. The models consider biopsychosocial perspectives, and encompass both peripheral (i.e. low back) and central (i.e. spinal and supra-spinal) factors as well as proposed mechanisms of action of cLBP treatments. However, absent explanations, models/algorithms may fall short of regulatory requirements and clinician expectations, and ultimately may not be embraced by physicians and patients. To address this, BACPAC is developing a clinical utility roadmap (CUR) to clarify how models will be used in practice for selecting optimal treatments, monitoring response to treatment, and reducing health care utilization. This presentation will review the goals of BACPAC and how theoretical models and CUR are being used to support computational knowledge networks to integrate data from deeply phenotyped cLBP patients

Introduction and Objective. Hyaluronic acid (HA) is an effective option for the treatment of osteoarthritis (OA) patients due to several properties such as normalization of the mechanical and rheological properties of the synovial fluid and amelioration of OA symptoms and joints function by promoting cartilage nutrition. Since OA progression is also significantly related to oxidative stress and reactive oxygen species (ROS), sodium succinate (SS) is envisioned as a promising compound for cartilage treatment by providing antioxidant defense able to normalize intracellular metabolism and tissue respiration via mitochondrial mechanism of action. The scope of this study was to investigate on an in vitro inflammatory model the efficacy of Diart. ®. product, a combination of HA and SS. Materials and Methods. Donor-matched chondrocytes and synoviocytes were obtained from KL 3–4 OA patients undergoing total knee replacement. At passage 4, inflammation was promoted with 1 ng/ml IL-1B for 48 hours in absence and presence of Diart. ®. at 1:3 dilution rate. Nitric oxide (NO) from cell culture supernatant was measured by Griess reaction. Mitochondrial and cytoplasmatic ROS evaluation was assessed by flow cytometry with MitoSox and dichlorodihydrofluorescein diacetate (DCFDA) assays. Gene expression of inflammation/oxidative stress-related transcripts (MMP1/MMP3/INOS/COX2) was evaluated by qRT-PCR using TBP as reference. Results. NO was detected only in inflamed chondrocytes and Diart® was able to abolish its levels. NO was not detected in synoviocytes in all conditions. IL-1B reduced both cytoplasmic (−66%) and mitochondrial (−68%) ROS in chondrocytes, with Diart® partially restoring (+40%) mitochondrial levels. In synoviocytes, IL-1B did not alter ROS, with Diart® modestly increasing (+27%) mitochondrial levels. Inflammation was able to increase transcript levels of all tested markers, with the exception of INOS in synoviocytes. In chondrocytes, Diart® significantly (p < 0.05) reduced COX2 (−75%) and MMP1 (−33%). In synoviocytes, Diart® significantly reduced COX2 (−77%) and MMP3 (−84%), with MMP1 53% decreased albeit without reaching statistical significance. Conclusions. Diart. ®. biochemical and physiologic properties in the tested in vitro model of inflammation on donor-matched chondrocytes and synoviocytes allowed reducing inflammation and oxidative stress-related markers, prompting the use of this combination as successful strategy to manage OA-related symptoms

Osteoarthritis (OA) of the spine and diarthrodial joints is by far the most common cause of chronic disability in people over 50 years of age. The disease has a striking impact on quality of life and represents an enormous societal and economic cost, a burden that will increase greatly as populations age. OA is a complex condition with broad pathology. Damage to the articular cartilage is a consistent feature, accompanied by changes to the subchondral bone and synovium. Progression of the disease involves further degeneration of the articular cartilage, damage to the underlying bone and morphological changes that include subchondral bone thickening, development of cysts, osteophytes and inflammation of the synovium. Enhanced production of proinflammatory cytokines and matrix metalloproteinases accelerates degradation of the articular cartilage. It is striking that no approved pharmacological intervention, biological therapy or procedure prevents the progressive destruction of the OA joint. All current treatments, without exception, produce symptomatic rather than regenerative results. While there have been some exciting developments in the search for OA treatments in the last decade, including matrix metalloproteinase inhibitors, anti-TNF and anti-IL1 drugs for example, none of these has to date emerged as an effective medicinal product. There is thus an urgent and compelling need to identify, validate and test new biological therapeutics. Stromal cell therapy represents one such compelling approach. The results from several early clinical studies have indicated that this approach holds a great deal of promise for the treatment of OA. Most studies have involved direct intraarticular injection of a suspension of mesenchymal stromal cells (MSCs) for treatment of knee OA. Results from a number of controlled patient studies have suggested that this treatment results in an effective repair response. Although data regarding mechanism of action are limited, it appears that the cells have an anti-inflammatory effect, possibly targeting cells within the synovium, rather than a direct cartilage repair effect. Several recent reports have highlighted a dramatic and sustained response in patients receiving MSC treatment. For example, allogeneic expanded adipose-derived MSCs have been shown to be safe and effective in the treatment of complex perianal fistulas in Crohn's disease. Also, allogeneic bone marrow-derived MSCs has a been shown to have a positive effect in pediatric acute graft versus host disease. These observations point to a mechanism of action that involves host immunomodulation, but this needs further examination. Within the field of musculoskeletal disease effective translation of MSC technology has been hindered by a lack of randomized controlled patient studies, severe inconsistencies regarding the preparation and characterization of the cell product, and an incomplete understanding of the therapeutic mechanism. Direct to consumer clinics have flourished in some countries, providing cell treatments to OA patients. Most or all of these utilize unexpanded cell fractions from marrow or fat without even rudimentary product characterization and may report an exaggerated clinical outcome. Data from these clinics is not likely to yield information that will be useful. In fact, a recent systemic review of clinical trials involving MSC treatment in OA indicated that only a limited number of studies provided high quality evidence and long term follow up. Many suffered from a lack of consistency, including a diversity of methods for MSC preparation, and thus did not contribute to a supporting evidence base. There is a compelling need to provide clear and unambiguous clinical proof of concept relating to MSC treatment for OA. The ADIPOA2 study, currently active in Europe, will go some way towards achieving this. This is a 150 patient, phase 2b study designed to to assess the efficacy of a single injection of autologous adipose-derived MSCs in the treatment of mild to moderate OA of the knee, active and unresponsive to conservative therapy for at least 12 months

Introduction and Objective. The use of microfragmented adipose tissue (mFAT) for the treatment of musculoskeletal disorders, especially osteoarthritis, is gaining popularity following the positive results reported in recent case series and clinical trials. The purpose of this study is to characterize mFAT in terms of structure, cell content and secretome (i.e. protein and microvescicles released as paracrine mediators), and to compare it with unprocessed lipoaspirate tissue, in order to understand the possible mechanisms of action and the benefit derived from tissue processing. Materials and Methods. Unprocessed lipoaspirate (LA) and mFAT were obtained from 7 donors. Each tissue sample was divided in four aliquots: A) fixed in formalin for histological evaluation; B) enzymatically digested to harvest cells with the exclusion of adipocytes; C) cultured for 24 hours in serum-free DMEM to harvest secretome; D) freshly frozen for proteomic evaluation. Hematoxylin and eosin staning, as well as immunohistochemistry for CD31, CD90, CD146 were performed on aliquot A. Cell count, viability, senescence and immunophenotype were assessed on aliquot B. Culture medium from aliquot C was collected and used for proteomic analysis and micro-RNA extraction and quantitation from extracellular vesicles. Aliquot D was lysed, protein were extracted and analyzed using a high-throughput proteomic approach. Results. Histological investigations showed a lower red blood cell content in mFAT with respect to LA, while the presence of blood vessels (CD31+), stromal cells (CD90) and pericytes (CD146) was similar in all samples. These results were confirmed by flow cytometry, with reduction of erythrocytes (CD235a+) by 76% and reduction of lymphocytes (CD45+) by 79% in mFAT compared to LA. Otherwise, the proportions of stromal cells, pericytes and endothelial cells in LA and mFAT remained comparable. The percentage of senescent cells resulted similar before and after tissue processing, with very low values (< 5%). The analysis of the miRNAs contained in the extracellular vesicles in culture media identified 376 miRNAs in LA secretome and 381 in mFAT secretome. A high correlation in the expression of these miRNAs within subjects (LA and mFAT of each donor) was observed (R2> 0.8), indicating that processing in mFAT does not significantly alter the portfolio of miRNAs associated with extracellular vesicles. Proteomic analysis of secretome revealed that 217 proteins significantly differ between LA and mFAT. In particular, protein associated with acute phase were less represented in mFAT secretome, while intracellular proteins were more frequent. Proteomic analysis of tissues demonstrated a reduction of protein related to extracellular matrix and of proteins closely related to peripheral blood contamination in mFAT with respect to LA. Conclusions. Taken together, these results suggest that processing of LA into mFAT allow for removal of blood elements, in terms of red blood cells, lymphocytes, acute phase and complement system proteins, and for the reduction of extracellular matrix components. Otherwise, tissue structure, cell populations, cell viability and senescence are not influenced by tissue processing. Then, microfragmentation process represents a safe and efficient method for the application of adipose tissue properties to musculoskeletal disorders, allowing for the maintenance of all the effector elements for tissue regeneration while removing possible detrimental agents such as inflammatory mediators

Abstract. Objectives. Review the evidence of low intensity pulsed ultrasound (LIPUS) for fracture non-union treatment and the potential to treat fractures in patients with co-morbidities at risk of fracture non-union. Methods. Data was gathered from both animal and human studies of fracture repair to provide an overview of the LIPUS in bone healing applications to provide in-depth evidence to substantiate the use in treatment of non-union fractures and to propose a scientific rational to develop a clinical development programme. Results. LIPUS is an effective method for treating fracture non-union, with most studies showing heal rates in the mid 80%. In the UK NICE has published MTG-12 guidance for non-union treatment, which demonstrates that LIPUS is an effective and cost effective method as an alternative to surgery to treat non-union fractures. Basic science studies and evaluation of clinical trial data has led to the understanding that LIPUS can mitigate co-morbidities related to failure of bone healing such as diabetes, advancing age and tobacco use. Future clinical trials will evaluate the use of LIPUS in acute fractures in patients with high risk of low bone healing capacity to prevent the development of a non-union. As with all medical treatments, LIPUS for fracture repair needs to be used appropriately, with poorly fixed fractures or large fracture gaps, being unsuitable for LIPUS treatment. In addition, considerations such as targeting the fracture site in deep-seated bones and clinician / patient engagement to ensure good compliant usage are vital factors to ensure good clinical outcomes. Conclusion. Using basic science research, a thorough knowledge of the mechanism of action has been established, which has elucidated that co-morbidities related to the development of fracture non-union can be mitigated by the LIPUS technology. A pragmatic clinical trial in the United States is currently ongoing to test these hypothesises clinically. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Abstract. Objectives. A promising therapy for early osteoarthritis (OA) is the transplantation of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). The synovial fluid (SF) from a pre-clinical ovine model treated with hUC-MSCs has been profiled using proteomics and bioinformatics to elucidate potential mechanisms of therapeutic effect. Methods. Four weeks after a medial meniscus transection surgery, sheep were injected with 10. 7. hUC-MSCs in Phosphate Buffered Saline (PBS) or PBS only (n=7) and sacrificed at 12 weeks. SF was normalised for protein abundance (ProteoMiner. TM. ) and analysed using label-free quantitation proteomics. Bioinformatics analyses (Ingenuity Pathway Analysis (IPA) and STRING) were used to assess differentially regulated functions from the proteomic data. Human orthologues were identified for the ovine proteins using UniProt and DAVID resources and proteins that were ≥±1.3 fold differentially abundant between treatment groups, were included in the bioinformatics analyses. Results. hUC-MSC treated animals demonstrated significantly less joint space narrowing. Nineteen SF proteins were differentially abundant in treated cf. control sheep (FC±2.0; p<0.05). Biglycan (a small leucine-rich proteoglycan of the cartilage extracellular matrix) abundance was increased by 2.1 fold in treated compared to untreated sheep (p=0.024). IPA indicated that lipid synthesis (z-score=1.772; p=0.00267) and immune cell migration pathways (cell movement of mononuclear leukocytes: z-score=1.761; p=0.00259), amongst others, were likely to be activated in the treated sheep. Conversely, tissue damage (z-score=−2; p=0.00019), senescence (z-score=−1.981; p=0.00007) and necrosis (z-score=−1.728; p=0.00829) associated pathways as well as inflammation (z-score=−1.718; p=0.00057) and vascular permeability (z-score=−1.698; p=0.00002) were likely to be inhibited in treated cf. untreated sheep. Conclusions. hUC-MSC treatment prevented/delayed OA progression, demonstrated via a reduction in joint space narrowing. SF proteome bioinformatics revealed potential mechanisms of therapeutic action related to immunomodulation and the inhibition of multiple cell death, and tissue damage associated pathways. Further, a potential predicted upregulation in lipid synthesis in treated sheep represents a novel mechanism warranting further investigation. Additional work is required to validate these discovery phase proteomic findings in studies which specifically target and manipulate the proposed mechanisms highlighted. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Tendon injuries in both the human and horse represent a challenge due to persistent inflammation combined with inadequate reparative cells and a poorly organised extracellular matrix. The potential of mesenchymal stem cells (MSCs) in regenerating tendon injuries remains to be fully realised. The main mechanism of action by MSCs is considered to be primarily mediated via paracrine mechanisms. This may involve the production and release of extracellular vesicles (EVs) by stem cells with a sub-fraction of these EVs (<100 nm diameter) called exosomes that appear to be the main paracrine effectors. EVs can be readily prepared from MSCs and offer a clinically relevant therapy. However, EVs for tendon repair need to be fully characterised. The horse represents a highly relevant model of tendon and ligament injuries as it shares many features of mechanical loading, function and aetiopathology with the human. We have isolated and characterised EVs from equine MSCs for modulating tendon cell phenotype in an in vitro tendon injury model using IL-1ß. EVs can be isolated from IL-1ß stimulated MSCs although their levels are not significantly increased over controls suggesting that the nature of the stimulated EV cargo may be more important than absolute levels of released EVs

Low back pain (LBP), caused by intervertebral disc (IVD) degeneration represents one of the most significant socioeconomic conditions facing Western economies. Novel regenerative therapies, however, have the potential to restore function and relieve pain. We have previously shown that stimulation of adipose-derived stem cells (ASCs) with growth differentiation factor-6 (GDF6) promotes differentiation to nucleus pulposus (NP) cells of the IVD, offering a potential treatment for LBP. The aims of this study were to i) elucidate GDF6 cell surface receptor profile and signalling pathways to better understand mechanism of action; and (ii) develop a microparticle (MP) delivery system for GDF6 stimulation of ASCs. GDF6 receptor expression by ASCs (N=6) was profiled through western blot, immunofluorescence (IF) and flow cytometry. Signal transduction through Smad1/5/9 and non-Smad pathways following GDF6 (100ng/ml) stimulation was assessed using western blotting and confirmed using pathway specific blockers and type II receptor sub-unit knockdown using CRISPR. Release kinetics of GDF6 from MPs was calculated (BCA assay, ELISAs) and ASC differentiation to NP cells was assessed. BMPR profiling revealed high BMPR2 expression on ASCs. GDF6 stimulation of ASCs resulted in significant increases in Smad1/5/9 and Erk phosphorylation, but not p38 signalling. Blocking GDF6 signalling confirmed differentiation to NP cells required Smad phosphorylation, but not Erk. GDF6 release from MPs was controlled over 14days in vitro and demonstrated comparable NP-like differentiation to exogenous GDF6 delivery. This study elucidates the signalling mechanisms responsible for GDF6-induced ASC differentiation to NP cells and also demonstrates an effective and controllable release vehicle for GDF6

In England and Wales in 2012 over 160,000 primary total hip and knee replacements were performed with 57% of hip replacements utilising uncemented prostheses. The main cause of failure, affecting approximately 10% of patients, is aseptic loosening. Previous research has found that functionalising titanium with lysophosphatidic acid (LPA) induces an increase in human osteoblast maturation on the implant surface through co-operation with active metabolites of vitamin D3. This feature, the small size of the LPS molecule and its affinity to readily bind to titanium and hydroxylapatite makes it an especially desirable molecule for bone biomaterials. Nevertheless biomaterials that also demonstrate anti-microbial properties are highly desirable. To test the antimicrobial efficacy of the LPA-functionalised titanium, a clinical isolate of Staphylococcus aureus, obtained from an infected revision surgery, was cultured on the surface of titanium discs functionalised with 0, 0.1. 0.5, 1, 2 and 5μM LPA. Bacterial adhesion was quantified at 1, 2, 6, 12 and 24 hours by live/dead counts and biofilm mass quantified by crystal violet staining after 24, 48, 72 and 96 hours culture. To elucidate the mechanisms of action of LPA, proteomic analysis of adhered bacteria was performed using SDS-PAGE and Western blots. 500nM to 1μM LPA were the optimum concentrations to significantly inhibit bacterial adhesion (ANOVA, p<0.001). These concentrations also reduced biofilm mass on the surface of the titanium. Proteomic analysis highlighted an increase in low molecular weight proteins as a result of optimal LPA surface concentrations. Fatty acid chains as found in LPA have previously been associated with causing leakage of low molecular weight proteins through increased cell membrane permeability. LPA coatings have the potential to enhance implant osseointegration whilst simultaneously reducing bacterial attachment. This technology may reduce both septic and aseptic failure of cementless joint prostheses, ultimately prolonging implant longevity and patient quality of life

Intermittent parathyroid hormone 1–34 (teriparatide) is the N-fragment terminal of the intact hormone, currently in clinical use to treat osteoporosis. Unlike anti-catabolic agents such as bisphosphonates, PTH 1–34 not only affects the osteoclast, but also up regulates bone formation via both modelling and remodelling mechanisms. The actions of iPTH on mesenchymal stem cell differentiation (MSCs) may underpin a further method in the treatment of osteoporosis specifically, and for fracture healing in general. Stem cells from older female osteoporotic animals have reduced activity and poorer osteogenic potential; additionally, their migration to and retention at sites of increased bone turnover are reduced in comparison to cells from younger animals. The aim of this study was to isolate bone marrow derived MSCs from both young Wild Type (WT) and ovarectomized senile (OVX) rats, then to investigate and compare the effect of pulsatile and continuous PTH administration on migration to SDF-1, proliferation and osteogenic differentiation. MSCs were harvested from the femora of 6–9week Wistar rats, and from 10–13month ovarectomized rats with established osteopenia. Cells were cultured with 25, 50 and 100nmMol of PTH 1–34 added to osteogenic media either continuously or in a pulsatile fashion for 6 hours in every 72hour cycle. ALP and Alizarin Red were used to assess the optimal concentration of PTH for osteogenic differentiation. Subsequently, proliferation was assessed with Alamar Blue and cells were seeded in a Boyden chamber to quantify the migration to SDF-1. As the data was parametric a student t-test was used to analyse results, and a p value < 0.05 was considered significant. ALP and Alizarin Red parameters were significantly increased for both WT and OVX groups at 50nmMol of pulsatile PTH in comparison to groups cultured in 25 or 100nmMol. Continuous administration at all concentrations led to reduced calcium phosphate deposition by day 21 in all groups. Interestingly, in comparison to cells cultured in osteogenic media, 50nmMol of pulsatile PTH lead to statistically significant higher ALP and Alizarin Red measurements up to day 10 and 14 respectively in WT cells, and days 10 and 21 in OVX cells. The proliferation rate normalised against DNA was similar for both OVX and WT rats at all-time points. PTH administration did not effect cell proliferation in any group. WT MSCs not only had improved osteogenic differentiation, but also showed increased migration to SDF-1 in comparison to OVX groups. Pulsatile PTH led to further increases in migration of both OVX and WT cells. Intermittent PTH increases the osteogenic diffrentiation and migration of MSCs from both young and ovarectomised rats, though importantly this effect is not dose dependent. Ultimately, the role of PTH 1–34 on MSCs may lead to improved bone formation and cell homing capacity-particularly in the context of osteoporosis

The exact action of the Peroneus Longus muscle on the foot is not fully understood. It is involved in a number of pathological processes like tendonitis, tenosynovitis, chronic rupture and neurological conditions. It is described as having a consistent insertion to the base of the first metatarsal, but there have also been reports of significant variations and additional slips. Our aim was to further clarify the anatomy of the main insertion of the Peroneus Longus tendon and to describe the site and frequency of other variable insertion slips. The course of the distal peroneus longus tendon and its variable insertion was dissected in 20 embalmed, cadaveric specimens. The surface area of the main insertion footprint was measured using an Immersion Digital Microscibe and 3D mapping software. The site and frequency of the other variable insertion slips is presented. There was a consistent, main insertion to the infero-lateral aspect of the first metatarsal in all specimens. The surface area of this insertion was found to be proportional to the length of the foot. The insertion in males was found to be significantly larger than females. The most frequent additional slip was to the medial cuneiform. Other less frequent insertion slips were present to the lesser metatarsals. The main footprint of the Peroneus Longus tendon is on the first metatarsal. There appears to an additional slip to the medial cuneiform frequently. Although we are unsure about the significance of these additional slips, we hope it will lead to a better understanding of the mechanism of action of this muscle and its role both in the normal and pathological foot

Summary. This study describes the use of a quasi-static, 6DOF knee loading simulator using cadaveric specimens. Muscle force profiles yield repeatable results. Intra-articular pressure and contact area are dependent on loading condition and ACL integrity. Introduction. Abnormal contact mechanics of the tibiofemoral joint is believed to influence the development and progression of joint derangements. As such, understanding the factors that regulate joint stability may provide insight into the underlying injury mechanisms. Muscle action is believed to be the most important factor since it is the only dynamic regulator of joint stability. Furthermore, abnormal muscle control has been experimentally linked to the development of OA [Herzog, 2007] and in vivo ACL strain [Fleming, 2001]. However, the individual contributions to knee joint contact mechanics remain unclear. Thus, the purpose of this study was to examine the effects of individual muscle contributions on the tibiofemoral contact mechanics using an in-vitro experimental protocol. Methodology. Contact mechanics of 6 fresh frozen cadaver knee specimens were evaluated using the UofO Oxford knee loading device. Various combinations of quadriceps-hamstring co-contraction ratios were applied to the knee while it was “suspended” between the hip and foot components of the device. Loads of six muscle groups were computed using a hill-type musculoskeletal model [Buchanan, 2004]. Simulated ground reaction forces were also applied to the knee to represent force profiles of weight acceptance during gait as it has been shown to produce peak knee joint force in the gait cycle [Shelburne et al., 2006]. For respective medial and lateral joint compartments, the mean contact area (MC-CA and LC-CA), mean contact pressure (MC-CP and LC-CP), peak pressure (MC-PP and LC-PP), and centre of force displacement (MC-COFD and LC-COFD) were determined using a 4011 piezoelectric sensor form Tekscan (Tekscan Inc. Boston, MA). Additionally, the ACL was resected and measurements were repeated. Pearson correlations (r) examined the reliability of measurements as well as the effect an ACL transection on articular loads. Results. Positive correlations were computed for the following: COFD with intact ACL (r=0.99), COFD with resected ACL (r=0.82), MC-COFD pre vs. post ACL- resection (0.91). Furthermore, preliminary results indicated a positive correlation between MC-CA and ACL integrity (r=0.97). Discussion. The repeatability of the measured dependant variables validates the use of the knee-loading device. Interestingly, contact mechanics are more variable post ACL resection for a given muscle loading condition, indicating a decrease in knee joint stability. Also, the COFD is dependent on the different ratios of muscle loads applied to the knee, which demonstrates the importance of muscle action to the modulation of contact forces

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

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mass and deterioration of bone microarchitecture, which results in increased bone fragility and fracture risk. Casein kinase 2-interacting protein-1 (CKIP-1) is a protein that plays an important role in regulation of bone formation. The effect of CKIP-1 on bone formation is mainly mediated through negative regulation of the bone morphogenetic protein pathway. In addition, CKIP-1 has an important role in the progression of osteoporosis. This review provides a summary of the recent studies on the role of CKIP-1 in osteoporosis development and treatment.

Cite this article: X. Peng, X. Wu, J. Zhang, G. Zhang, G. Li, X. Pan. The role of CKIP-1 in osteoporosis development and treatment. Bone Joint Res 2018;7:173–178. DOI: 10.1302/2046-3758.72.BJR-2017-0172.R1.