Stem cells represent an exciting biological therapy for the management of many musculoskeletal tissues that suffer degenerative disease and/or where the reparative process results in non-functional tissue (‘failed healing’). The original hypothesis was that implanted cells would differentiate into the target tissue cell type and synthesise new matrix. However, this has been little evidence that this happens in live animals compared to the laboratory, and more recent theories have focussed on the immunomodulatory effects via the release of paracrine factors that can still improve the outcome, especially since inflammation is now considered one of the central processes that drive poor tendon healing. Because of the initial ‘soft’ regulatory environment for the use of stem cells in domestic mammals, bone and fat-derived stem cells quickly established themselves as a useful treatment for naturally occurring musculoskeletal diseases in the horse more than 20 years ago (Smith, Korda et al. 2003). Since the tendinopathy in the horse has many similarities to human tendinopathy, we propose that the following challenges and, the lessons learnt, in this journey are highly relevant to the development of stem cells therapies for human tendinopathy:. Source – while MSCs can be recovered from many tissues, the predominant sources for autologous MSCs have been bone and fat. Other sources, including blood, amnion, synovium, and dental pulp have also been commercialised for allogenic treatments. Preparation – ex vivo culture requires transport from a licensed laboratory while ‘minimally manipulated’ preparations can be prepared patient-side. Cells also need a vehicle for transport and implantation. Delivery – transport of cells from the laboratory to the clinic for autologous ex vivo culture techniques; implantation technique (usually by ultrasound-guided injection to minimise damage to the cells (or, more rarely, incorporated into a scaffold). They can also be delivered by regional perfusion via venous or arterial routes. Retention – relatively poor although small numbers of cells do survive for at least 5 months. Immediate loss to the lungs if the cells are administered via vascular routes. Synovially administered cells do not engraft into tendon. Adverse effects – very safe although needle tracts often visible (but do not seen to adversely affect the outcome). Allogenic cells require careful characterisation for MHC Class II antigens to avoid anaphylaxis or reduced efficacy. Appropriate injuries to treat – requires a contained lesion when administered via intra-lesional injection. Intrasynovial tendon lesions are more often associated with surface defects and are therefore less appropriate for treatment. Earlier treatment appears to be more effective than delayed, when implantation by injection is more challenging. Efficacy - beneficial effects shown at both tissue and whole animal (clinical outcome) level in naturally-occurring equine tendinopathy using bone marrow-derived autologous MSCs Recent (licenced) allogenic MSC treatment has shown equivalent efficacy while intra-synovial administration of MSCs is ineffective for open intra-synovial tendon lesions. Regulatory hurdles – these have been lighter for veterinary treatments which has facilitated their development. There has been greater regulation of commercial allogenic MSC preparations which have required EMA marketing authorisation

Current therapies for intervertebral disc degeneration are aimed at treating the pathologic and disabling conditions arising from discopathy rather than directly treating the underlying problem of disc degeneration. Our group is exploring the potential of cell therapy to repopulate the disc and stop the progressive loss of proteoglycans. Stem cells appear to be excellent candidates for this purpose, based on their ability to differentiate along multiple connective tissue lineages. The purpose of this study is to investigate the interaction between stem cells and nucleus polposus cells to test the feasibility of stem cell therapy for the treatment of disc degeneration. Human nucleus polposus cells (NPCs) were isolated from patients undergoing disc surgery and were co-cultured for 2 weeks with muscle-derived stem cells (MdSCs) from 3-week-old mdx mice in monolayer culture system at different ratio with or without added TGF-β1. Each well contained an admixture of cells with NPC-to-SC ratios of 0:100, 25:75, 50:50, 75:25, and 100:0. Proteoglycan synthesis and DNA content were measured. Co-culturing of NPCs with MdSCs in the monolayer culture system resulted in vigorous increases in proteoglycans synthesis as compared with NPCs alone and MdSCs alone both with and without TGF-β1. The increases were on the 200% for an NPC-to-MDSC ratio of 75:25. Addition of TGF-β1 to the NPC and MDSC co-cultures resulted in further increases up to 400%. DNA content also increased with co-culture. The data from this study show that there is a synergistic effect between stem cells and nNPC resulting in upregulated proteoglycan synthesis in vitro. The observed benefits of co-culture might be due either to stem cell plasticity, the stem cells trans-differentiation towards chondrocyte-like cells, or the stimulation of NPC by agents synthesised by stem cells or other mechanisms. Elucidation of the precise mechanisms of action may permit development of strategies to optimise the synergistic effects in vivo. These results support the feasibility of developing a stem cell therapy approach to treat and prevent intervertebral disc degeneration

Objectives. Distraction osteogenesis (DO) mobilises bone regenerative potential and avoids the complications of other treatments such as bone graft. The major disadvantage of DO is the length of time required for bone consolidation. Mesenchymal stem cells (MSCs) have been used to promote bone formation with some good results. Methods. We hereby review the published literature on the use of MSCs in promoting bone consolidation during DO. Results. Studies differed in animal type (mice, rabbit, dog, sheep), bone type (femur, tibia, skull), DO protocols and cell transplantation methods. Conclusion. The majority of studies reported that the transplantation of MSCs enhanced bone consolidation or formation in DO. Many questions relating to animal model, DO protocol and cell transplantation regime remain to be further investigated. Clinical trials are needed to test and confirm these findings from animal studies. Cite this article: Y. Yang, S. Lin, B. Wang, W. Gu, G. Li. Stem cell therapy for enhancement of bone consolidation in distraction osteogenesis: A contemporary review of experimental studies. Bone Joint Res 2017;6:385–390. DOI: 10.1302/2046-3758.66.BJR-2017-0023

Optimal treatment for symptomatic talus Osteochondral Lesions (OCLs) where primary surgical techniques have failed has not been established. Recent advances have focussed on biological repair such as Autologous Chondrocyte Implantation (ACI) however funding for this treatment is limited. Stem cell therapy in the ankle has not been assessed. The purpose of this pilot study was to evaluate the safety and efficacy of stem cell therapy in the treatment of ankle OCLs. The study was approved by the new procedures committee. Between January 2015 and December 2016, 26 patients, mean age of 36 years (range 16–58 years) with persisting disabling symptoms underwent Complete Cartilage Regeneration (CCR) using stem cells for failed primary treatment for ankle OCLs. Treatment involved iliac crest bone marrow aspiration, centrifugation to obtain bone marrow concentrate (BMC), and then injection of the BMC combined with hyaluronic acid into the OCL. Any necessary additional procedures, e.g. bone grafting or lateral ligament reconstruction were also undertaken. In 18 patients the lesion was on the medial talar dome, in 5 the lateral talar dome, 2 multiple, 1 tibial plafond. The Manchester-Oxford Foot Questionnaire (MOXFQ) was utilised to assess outcome. Average pre-operative MOXFQ scores were Walking dimension −78, Pain dimension − 65, and Social dimension − 64.2. Average 3 month post-operative MOXFQ scores were Walking − 54.8, Pain − 35.4, Social − 38.9. Average 6 month post-operative MOXFQ scores were Walking − 34.4, Pain − 35.4, Social − 28. Two patients from the beginning of the series had AOFAS scores only which improved from an average of 55 pre-operatively to 76 post-operatively. No early complications were noted. We conclude that CCR treatment is a safe treatment for talus OCLs in patients who have failed primary treatment. The procedure avoids two-stage surgery of ACI in some patients without large cysts. The early clinical outcome is favourable with no complications noted. Longer term follow-up is required

Introduction: Current therapies for degenerative disc disease (DDD) are aimed at treating the pathologic and disabling conditions arising from DDD rather than directly treating the underlying problem of disc degeneration. Our group are exploring the potential of Cell Therapy to repopulate the disc and stopping the progressive loss of proteoglycans. Stem cells appear to be excellent candidates for this purpose, based on their ability to differentiate along multiple connective tissue lineages. The purpose of this study is to investigate the in-vitro interaction between muscle-deroved stem cells (MdSC) and nucleus polposus cells (NPCs) and to determine in-vivo viability of mesenchymal stem cell (MSC) in the harsh environment of the IVD. Materials and Methods: (1) Human NPCs were isolated from patients undergoing disc surgery and were co-cultured for 2 weeks with MdSCs from 3-wk-old mdx mice and in monolayer culture system at different ratios of 0:100, 25:75, 50:50, 75:25, 100:0. Proteoglycan synthesis and DNA content were measured. (2) Rabbit mesenchymal stem cells were isolated from bone marrow and tagged with a retrovirus delivered LacZ reporter gene for tracking. MSCs were then injected into a healthy rabbit IVD via 30G needle. Rabbits were sacrificed at postoperatively at 3, 6, 12 and 24 weeks. Histological analysis for MSC viability was performed. Results: (1) Co-culturing of NPCs with MdSCs in the monolayer culture system resulted in vigorous increases in proteoglycans synthesis as compared with NPCs alone. The increases were on the 200% for an NPC-to-MDSC ratio of 75:25. DNA content also increased with co-culture. (2) Histological examination revealed presence of MSCs expressing LacZ without apparent decrease in numbers or diminishment of protein production. Conclusion: The data from this study show that there is a synergistic effect between stem cells and nucleus pulposus cells resulting in upregulated proteoglycan synthesis in-vitro. Mesenchymal stem cells remain viable and continue to express an ex vivo transduced protein without appreciable cell loss for up to 24 weeks post transplantation into the rabbit IVD. These results suggest that MSCs can survive in the harsh environment of the IVD and may favourably modify ECM production. These studies support the feasibility of developing a stem cell therapy approach for DDD

The use of mesenchymal stem cell (MSCs) for intervertebral disc (IVD) regeneration has been extensively explored in the last two decades. MSCs are potent cell types that can be easily and safely harvested due to their abundancy and availability. Moreover, they are characterized by the capacity to differentiate towards IVD cells as well as release growth factors to support resident cell metabolism and recruit local progenitor cells to induce endogenous repair of degenerated IVDs. This talk will outline the characteristics of the main MSC sources and their effect towards IVD regeneration based on available preclinical and clinical evidence. In addition, innovative aspects of MSC-derived cell-free therapies will also be discussed.

Aims

A growing number of fractures progress to delayed or nonunion, causing significant morbidity and socioeconomic impact. Localized delivery of stem cells and subcutaneous parathyroid hormone (PTH) has been shown individually to accelerate bony regeneration. This study aimed to combine the therapies with the aim of upregulating fracture healing.

Osteoarthritis (OA) is a degenerative disease with a strong inflammatory component. Intra-articular (IA) injections of mesenchymal stem cells (MSCs) modulate local inflammation, although the lack of engraftment suggests that they undergo apoptosis. The aim of this study is to investigate the fate of IA-delivered MSCs in an animal model of OA and to assess the role of apoptosis in vitro. Collagenase-induced OA (CIOA) was performed on C57BL/6 mice and 2×10∧5 GFP+ MSCs were IA-injected in the animals. 3 days later, knee joints were digested into a single-cell suspension and MSCs retrieved by cell sorting. Conditioned medium (CM) of retrieved cells was tested on murine macrophages and cytokine secretion was measured. Apoptosis of MSCs was induced in vitro with staurosporine (STS) and evaluated by Annexin V/Sytox Blue staining; activation of caspases was measured by FLICA assays. Murine lymphocytes were cocultured with apoptotic MSCs and their proliferation measured by quantification of Cell Trace Violet. 1.63% of injected cells were retrieved and proliferated in culture. Their CM significantly modulated activation of macrophages, with greater effects from OA-induced MSCs. STS induced apoptosis with activation of Caspase 3/7. Apoptotic MSCs significantly prevented the proliferation of murine lymphocytes. MSCs can be administered and retrieved from murine knees. Retrieval yield is low, consistent with previous studies. MSCs were licensed from the OA joint to produce an immunosuppressive milieu that modulated macrophages ex vivo. In vitro, apoptosis increased the immunomodulatory potential of MSCs. This suggests that apoptosis may contribute to the therapeutic effects of MSCs in OA.

Mesenchymal stem cells (MSCs) are exciting candidates for cellular repopulation and repair in intervertebral disc degeneration (IDD). Our purpose is to investigate the interaction between MSCs and nucleus polposus cells (NPCs) and to determine viability of MSC in the intervertebral disc (IVD).

Human NPCs and hMSCs were co-cultured in pellet system at different ratios. Proteoglycans were measured and normalized with DNA content. Histological analysis were also performed. Rabbit MSCs from bone marrow were trasduced with LacZ reporter gene and were injected into a rabbit IVD. Rabbits were sacrificed postoperatively at 3, 6, 12 and 24 weeks. Histological analysis was performed.

Co-culturing of hNPCs with hMSCs resulted in increases proteoglycans as compared with hNPCs alone. Histological examination of the injected IVDs revealed presence of MSCs without apparent decrease in numbers or diminishment of protein production at 3, 6, 12 and 24 weeks.

The data from this study show that there is a synergistic effect between MSCs and NPCs resulting in upregulated proteoglycan synthesis in-vitro. MSC remain viable and continue to express an ex-vivo transduced protein for up to 24 weeks. These results suggest that MSCs can survive in the harsh environment of the IVD and may favourably modify ECM production.

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases

Aims. The value of core decompression (CD) in the treatment of osteonecrosis of the femoral head (ONFH) remains controversial. We conducted a systematic review and meta-analysis to evaluate whether CD combined with other treatments could improve the clinical and radiological outcomes of ONFH patients compared with CD alone. Methods. We searched the PubMed, Embase, Web of Science, and Cochrane Library databases until June 2020. All randomized controlled trials (RCTs) and clinical controlled trials (CCTs) comparing CD alone and CD combined with other measures (CD + cell therapy, CD + bone grafting, CD + porous tantalum rod, etc.) for the treatment of ONFH were considered eligible for inclusion. The primary outcomes of interest were Harris Hip Score (HHS), ONFH stage progression, structural failure (collapse) of the femoral head, and conversion to total hip arthroplasty (THA). The pooled data were analyzed using Review Manager 5.3 software. Results. A total of 20 studies with 2,123 hips were included (CD alone = 768, CD combined with other treatments = 1,355). The combination of CD with other therapeutic interventions resulted in a higher HHS (mean difference (MD) = 6.46, 95% confidence interval (CI) = 2.10 to 10.83, p = 0.004) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score (MD = −10.92, 95% CI = -21.41 to -4.03, p = 0.040) and a lower visual analogue scale (VAS) score (MD = −0.99, 95% CI = -1.56 to -0.42, p < 0.001) than CD alone. For the rates of disease stage progression, 91 (20%) progressed in the intervention group compared to 146 (36%) in the control group (odds ratio (OR) = 0.32, 95% CI = 0.16 to 0.64, p = 0.001). In addition, the intervention group had a more significant advantage in delaying femoral head progression to the collapsed stage (OR = 0.32, 95% CI = 0.17 to 0.61, p < 0.001) and reducing the odds of conversion to THA (OR = 0.35, 95% CI = 0.23 to 0.55, p < 0.001) compared to the control group. There were no serious adverse events in either group. Subgroup analysis showed that the addition of cell therapy significantly improved clinical and radiological outcomes compared to CD alone, and this approach appeared to be more effective than other therapies, particularly in precollapse (stage I to II) ONFH patients. Conclusion. There was marked heterogeneity in the studies. There is a trend towards improved clinical outcomes with the addition of stem cell therapy to CD. Cite this article: Bone Joint Res 2021;10(7):445–458

Stem cell therapy for the intervertebral disc (IVD) is highly debated but holds great promises. From previous studies, it is known that notochordal cells are highly regenerative and may stimulate other differentiated cells to produce more matrix. Lately, a particular tissue-specific progenitor cell population has been identified in the centre of the intervertebral disc (IVD. The current hope is that these nucleus pulposus progenitor cells (NPPC) could play a particular role in IVD regeneration. Current evidence confirms the presence of these cells in murine, canine, bovine and in the human fetal/surgical samples. Noteworthy, one of the main markers to identify these cells, i.e., Tie2, is a typical marker for endothelial cells. Thus, it is not very clear what their origin and their role might be in the context of developmental biology. In human surgical specimens, their presence is, even more, obscured depending on the donor's age and the condition of the IVD and other yet unknown factors. Here, I revisit the recent literature on regenerative cells identified for the IVD in the past decades. Current evidence how these NPPC can be isolated and detected in various species and tissues will be recapitulated. Future directions will be provided on how these progenitor cells could be used for regenerative medicine and tissue engineering

Introduction. The use of stem cell and platelet-rich plasma (PRP) injections for knee osteoarthritis (OA) is extremely controversial and at best experimental. These treatments are being given to patients across the nation for “cash only payments”. Our objectives were (1) to determine the proportion of board certified orthopedic surgeons who offer stem cell or PRP treatment for knee OA, (2) how much the practices charge for those treatments and (3) if members of the knee society use these therapies. Methods. Board certified orthopedic surgeons’ offices in our county were identified by their AAOS active membership. Knee society membership roll was also utilized. Offices were contacted by telephone and presented with a hypothetical patient with end stage knee osteoarthritis searching for specific treatment (stem cells or PRP injections). T-test was used to compare the Dade county board certified orthopedists to knee society members. Results. A total of 186 board certified orthopedic surgeons’ offices were contacted. 17.6% of all contacted orthopedics offices offered PRP and 12.5% offered stem cell treatments. 61.2% of the offices were transparent on the pricing of PRP while 31.8% gave a price for stem cell therapy. The remaining practices stated that pricing would be “determined or discussed” during a scheduled visit. Mean cost for a PRP injection was $887 (SE 101; range: $350–$1700) and for a stem cell injection was $2800 (SE 852; range: $1000–$6000). Usage of these therapies amongst general AAOS members and Knee Society members was found to be significantly different for both PRP and stem cells (17% vs. 10%; p<0.001 and 26% vs. 13%; p<0.001, respectively). No practice had a “free” research protocol to study the treatments. Conclusions. Biological injectables as a treatment for knee OA has theoretical potential promise in the management of arthritis but continues to be at best investigational. Knee Society members demonstrated significantly more caution using these treatments

The December 2014 Foot & Ankle Roundup360 looks at: Charcot feet, biomarkers and diabetes; weight bearing following Achilles tendon rupture; endobuttons and mal-reduced diastasis; evidence for stem cell therapies in osteochondral lesions of the talus; syndesmosis fixation in SER ankle fractures; and self-reporting for foot and ankle outcomes

Objectives . Rotator cuff tears are among the most common and debilitating upper extremity injuries. Chronic cuff tears result in atrophy and an infiltration of fat into the muscle, a condition commonly referred to as ‘fatty degeneration’. While stem cell therapies hold promise for the treatment of cuff tears, a suitable immunodeficient animal model that could be used to study human or other xenograft-based therapies for the treatment of rotator cuff injuries had not previously been identified. Methods . A full-thickness, massive supraspinatus and infraspinatus tear was induced in adult T-cell deficient rats. We hypothesised that, compared with controls, 28 days after inducing a tear we would observe a decrease in muscle force production, an accumulation of type IIB fibres, and an upregulation in the expression of genes involved with muscle atrophy, fibrosis and inflammation. Results . Chronic cuff tears in nude rats resulted in a 30% to 40% decrease in muscle mass, a 23% reduction in production of muscle force, and an induction of genes that regulate atrophy, fibrosis, lipid accumulation, inflammation and macrophage recruitment. Marked large lipid droplet accumulation was also present. Conclusions . The extent of degenerative changes in nude rats was similar to what was observed in T-cell competent rats. T cells may not play an important role in regulating muscle degeneration following chronic muscle unloading. The general similarities between nude and T-cell competent rats suggest the nude rat is likely an appropriate preclinical model for the study of xenografts that have the potential to enhance the treatment of chronically torn rotator cuff muscles. Cite this article: Bone Joint Res 2014;3:262–72

A significant number of fractures develop non-union. Stem cell therapy may be beneficial in their treatment, however this requires acquisition, culture and delivery of stem cells. Stem cell homing and migration is regulated through SDF-1 and its receptor CXCR4. Studies have demonstrated endogenous mobilisation of different populations of stem and progenitor cells by administering growth factors with a pharmacological antagonist of CXCR4, AMD3100. This may therefore be a means to improve compromised fracture healing. A 1.5mm femoral osteotomy in adult female Wistar rats was stabilised with an external skeletal fixator. After osteotomy, saline/PBS (P) VEGF (V), IGF-1 (I) or GCSF (G) (100ug/kg, 0.5ml/100g i.p.), were administered daily for 4 days. On day 5, a single 5mg/kg i.p. dose of AMD3100 was given. Control group (C) did not receive growth factors or AMD 3100. At 5 weeks, the femur was retrieved and microCT scanned. Compared to group C (n=7), group P (n=5) had a significant increase in bone volume (P=0.01) 8.9±2.2um∧3 (control 4.3±3.1um∧3) and trabecular thickness (P=0.03). Group I (n=6) also had a significant increase in bone volume (P=0.035) 5.1±4.2um∧3 and trabecular thickness 0.062±0.008um (control 0.042±0.01um) (P=0.01). Group V (n=8), showed a non-significant increase in bone volume; 5.22±1.7um∧3 and trabecular thickness 0.048±0.007um. Group G (n=5) showed a significant decrease in bone volume (2.5±2.6um∧3) (P=0.048). AMD3100 alone and IgF1-AMD3100, showed the greatest increase in bone formation, presumably through mobilisation of beneficial combinations of stem and progenitor cells. GCSF-AMD3100, which is expected to mobilise hematopoietic progenitors inhibited bone healing

Background. Stem cell therapy has been suggested as a potential regenerative strategy to treat IVD degeneration and GDF6 has been shown to differentiate adipose-derived stem cells (ASCs) into an NP-like phenotype. However, for clinical translation, a delivery system is required to ensure controlled and sustained GDF6 release. This study aimed to investigate the encapsulation of GDF6 inside novel microparticles (MPs) to control delivery and assess the effect of the released GDF6 on NP-like differentiation of human ASCs. Methods. GDF6 release from PLGA-PEG-PLGA MPs over 14 days was determined using BCA and ELISA. The effect of MP loading density on collagen gel formation was assessed through SEM and histological staining. ASCs were cultured in collagen hydrogels for 14 days with GDF6 delivered exogenously or via microspheres. ASC differentiation was assessed by qPCR for NP markers, glycosaminoglycan production (DMMB) and immunohistochemistry. Results. GDF6 release from MPs was controlled over 14 days equivalently to exogenous addition. SEM and histology confirmed that MPs were distributed throughout gels and that gel formation was not disrupted. In 3D cultures, GDF6 release from microspheres elicited equivalent ASC differentiation and NP-like matrix formation compared to exogenous delivery in media, indicating activity was not affected by MP encapsulation. Conclusions. This study demonstrates the effective encapsulation and controlled delivery of GDF6, which was able to maintain its activity and induce ASC differentiation into an NP-like phenotype and production of an NP-like ECM. Delivery of GDF6 microspheres in combination with ASCs is a promising strategy for IVD regeneration and treatment of back pain. Conflicts of interest. No conflicts of interest. Sources of funding. We would like to acknowledge UKRMP Acellular Hub, MRC, NIHR Musculoskeletal BRU and The Rosetrees Trust for funding this research

Background. Currently, there is a focus on the development of cell based therapies to treat intervertebral disc (IVD) degeneration, particularly for regenerating/repairing the central region, the nucleus pulposus (NP). Recently, we demonstrated that GDF6 promotes NP-like differentiation in mesenchymal stem cells (MSCs). However, bone marrow- (BM-MSCs) and adipose- (Ad-MSCs) showed differential responses to GDF6, with Ad-MSCs adopting a more NP-like phenotype. Here, we investigated GDF6 signalling in BM-MSCs and Ad-MSCs, with the aim to improve future IVD stem cell therapies. Methods. GDF6 receptor expression in patient-matched BM-MSCs and Ad-MSCs (N=6) was profiled through western blot and immunocytochemistry (ICC). GDF6 signal transduction was investigated through stimulation with 100 ng ml. −1. GDF6 for defined time periods. Subsequently smad1/5/9 phosphorylation and alternative non-smad pathway activation (phospho-p38; phospho-Erk1/2) was analysed (western blot, ELISA). Their role in inducing NP-like gene expression in Ad-MSCs was examined through pathway specific inhibitors. Results. Western blot and ICC established that BMPR profiles differed between MSC populations; specifically, BMPR2 (a GDF6 receptor) expression, was significantly higher in Ad-MSCs (p<0.05). ELISA and western blot analysis showed that smad1/5/9 phosphorylation was significantly higher in Ad-MSCs following GDF6 stimulation (p<0.05). GDF6 stimulation also phosphorylated p38 and Erk1/2 pathways. Blocking of both smad and non-smad pathways resulted in variation of GDF6 induced NP-like gene expression. Conclusions. The upregulation of BMPR2 in Ad-MSCs and corresponding differences in smad1/5/9 and non-smad pathway phosphorylation in response to GDF6 indicates an enhanced discogenic potential in Ad-MSCs, suggesting they may be more suitable for GDF6 mediated cellular IVD regeneration. Conflicts of interest. No conflicts of interest. Sources of funding. We would like to acknowledge UKRMP Acellular Hub, MRC, NIHR Musculoskeletal BRU and The Rosetrees Trust for funding this research

Adipose derived mesenchymal stromal cells (ASC) are adult stem cells exhibiting functional properties that have open the way for cell-based clinical therapies. Primarily, their capacity of multilineage differentiation has been explored in a number of strategies for skeletal tissue regeneration. More recently, MSCs have been reported to exhibit immunosuppressive as well as healing capacities, to improve angiogenesis and prevent apoptosis or fibrosis through the secretion of paracrine mediators. Among the degenerative diseases associated with aging, osteoarthritis is the most common pathology and affects 16% of the female population over 65 years. Up to now, no therapeutic option exists to obtain a sustainable improvement of joint function beside knee arthroplasty. This prompted us to propose adipose derived stem cells as a possible cell therapy. We performed pre-clinical models of osteoarthritis and showed that a local injection of ASC showed a reduction of synovitis, reduction of osteophytes, joint stabilization, reducing the score of cartilage lesions. This work was completed by toxicology data showing the excellent tolerance of the local injection of ADSC and biodistribution showing the persistence of cells after 6 months in murine models. The aim of the ADIPOA trial is to demonstrate the efficacy of adipose derived stem cells therapy in knee osteoarthritis (OA) in a phase 2/3 controlled multicenter study controlled against standard of care. Safety and feasibility as well as dose response was previously assessed in the ADIPOA FP7 project. The bi-centric phase I clinical trial in Montpellier (France) and Würzburg (Germany) included 18 patients with moderate to severe knee OA, each patient received a single injection of autologous ADSC, in a open scale up dose trial, starting form 2 10 6 cells to 50 106 cells. The 107 dose appears to be well tolerated and showed preliminary response in terms of decreasing local inflammation. This first study confirmed the feasibility and safety of local injection of ADSC in knee OA and suggested the most effective dose (107 autologous ADSC). This work constituted a significant step forward treating this disease with ADSC to demonstrate safety of the procedure. we conduct a prospective multicenter randomized Phase 2/3 study with 86 patients with moderate to severe knee OA to demonstrate superiority of stem cell-based therapy compared to standard of care (SOC) in terms in reduction in clinical symptoms (WOMAC score) and structural benefit (assessed by T1rhoMRI that allow quantification of cartilage proteoglycan content). This project will offer EU a unique leadership in OA with strong positions in EU and US due to patents and quality of the methodology to demonstrate efficiency of ADSC. ADIPOA brings together a unique combination of expertises and leaders in clinical rheumatology, MRI specialists, Stem cell Institutes, national GMP grade adipose derived stem cell production platform (ECELLFRANCE) and SME specialized in cell therapy trials in the EU. The production of the cells will be granted to EFS through ECELLFRANCE national platform, which have the GMP facility and will work as a contracting manufacturing organization. The expertise, leadership and critical mass achieved by this Consortium should enable breakthroughs in ASC engineering directly amenable for clinical applications in OA