Osteoarthritis (OA) is the most common type of arthritis and causes a significant deterioration in patients’ quality of life. The high prevalence of OA as well as the current lack of disease-modifying drugs led to a rise in regenerative medicine efforts. The hope is that this will provide a treatment modality with the ability to alter the course of OA via structural modifications of damaged articular cartilage (AC). Regenerative therapy in OA starts with the concept that administered cells may engraft to a lesion site and differentiate into chondrocytes. However, recent studies show that cells, particularly when injected in suspension, rapidly undergo apoptosis after exerting a transient paracrine effect. If the injected stem cells do not lead to structural improvements of a diseased joint, the high cost of cell therapy for OA cannot be justified, particularly when compared with other injection therapeutics such as corticosteroids and hyaluronic acid. Long-term survival of implanted cells that offer prolonged paracrine effects or possible engraftment is essential for a successful cell therapy that will offer durable structural improvements. In this talk, the history and current status of regenerative therapy in OA are summarized along with the conceptual strategy and future directionsfor a successful regenerative therapy that can provide structural modifications in OA

The use of mesenchymal stromal cells (MSCs) in regenerative medicine and tissue engineering is well established, given their properties of self-renewal and differentiation. However, several studies have shown that these properties diminish with age, and understanding the pathways involved are important to provide regenerative therapies in an ageing population. In this PRISMA systematic review, we investigated the effects of chronological donor ageing on the senescence of MSCs. We identified 3023 studies after searching four databases including PubMed, Web of Science, Cochrane, and Medline. Nine studies met the inclusion and exclusion criteria and were included in the final analyses. These studies showed an increase in the expression of p21, p53, p16, ROS, and NF- B with chronological age. This implies an activated DNA damage response (DDR), as well as increased levels of stress and inflammation in the MSCs of older donors. Additionally, highlighting the effects of an activated DDR in cells from older donors, a decrease in the expression of proliferative markers including Ki67, MAPK pathway elements, and Wnt/ -catenin pathway elements was observed. Furthermore, we found an increase in the levels of SA- -galactosidase, a specific marker of cellular senescence. Together, these findings support an association between chronological age and MSC senescence. The precise threshold for chronological age where the reported changes become significant is yet to be defined and should form the basis for further scientific investigations. The outcomes of this review should direct further investigations into reversing the biological effects of chronological age on the MSC senescence phenotype

Little information exists when using cell viability assays to evaluate cells within whole tissue, particularly specific types such as the intervertebral disc (IVD). When comparing the reported methodologies and the protocols issued by manufacturers, the processing, working times, and dye concentrations vary significantly, making the assay's reproducibility a costly and time-consuming trial and error process. This study aims to develop a detailed step-by-step cell viability assay protocol for evaluating IVD tissue. IVDs were harvested from bovine tails (n=8) and processed at day 0 and after 7 days of culture. Nucleus pulposus (NP) and the annulus fibrosus (AF) 3 mm cuts were incubated at room temperature (26˚C) with a Viability/Cytotoxicity Kit containing Calcein AM and Ethidium Ethidium homodimer-1 for 2 hr, followed by flash freezing in liquid nitrogen. Thirty µm sections were placed in glass slides and sealed with nail varnish or Antifade Mounting Medium. The IVD tissue was imaged within the next 4h after freezing using an inverted confocal laser-scanning microscope equipped with 488 and 543 nm laser lines. Cell viability at day 0 (NP: 92±9.6 % and AF:80±14.0%) and day 7 (NP: 91±7.9% and AF:76±20%) was successfully maintained and evaluated. The incubation time required is dependent on the working temperatures and tissue thickness. The calcein-AM dye will not be retained in the cells for more than four hours. The specimen preparation and culturing protocol have demonstrated good cell viability at day 0 and after seven days of culture. Processing times and sample preparation play an essential role as the cell viability components in most kits hydrolyse or photobleach quickly. A step-by-step replicable protocol for evaluating the cell viability in IVD will facilitate the evaluation of cell and toxicity-related outcomes of biomechanical testing protocols and IVD regenerative therapies

Low back pain resulting from Interertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect. However, their behaviour in the harsh degenerate environment is unknown. Porcine NC cells (pNCs), and Human NP cells from degenerate IVDs were cultured in alginate beads to maintain phenotype. Cells were cultured alone or in combination, or co-stimulated with notochordal cell condition media (NCCM), in media to mimic the healthy and degenerate disc environment, together with controls for up to 1 week. Following culture viability, qPCR and proteomic analysis using Digiwest was performed. A small increase in pNC cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in pNCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Human NP cells cultured with NCCM, showed a decrease in IL-8 production compared to human NP cells alone when cultured in healthy media. However, gene expression analysis (ACAN, VEGF, MMP3 and IL-1β) demonstrated no significant difference between NP only and NP+NCCM groups. Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The potential use of NC cell sources for regenerative therapies can then be translated to investigate the potential use of iPSCs differentiated into NC cells as a regenerative cell source

In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFβ) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair

Chronic Achilles tendinopathy is characterised by sub-acute inflammation with pro-inflammatory type 1 macrophages (M1), tissue degeneration and consequent partial or total tendon injury. Control of the inflammatory response and M1-to-M2 macrophage polarisation can favour tendon healing both directly and indirectly, by allowing for the regenerative process driven by local mesenchymal stem cells. Ten patients (3 females and 7 males aged between 32 and 71 years old) with partial Achilles tendon injury were treated with injections of autologous peripheral blood mononuclear cells (PB-MNCs). The cell concentrate was obtained from 100-120 cc of each patient's blood with a selective point-of-care filtration system. PB-MNCs remained trapped in the filter and were injected immediately after sampling. Around 60% of the PB-MNC concentrate was injected directly into the injured area, while the remaining 40% was injected in smaller amounts into the surrounding parts of the Achilles tendon affected by tendinosis. All patients were evaluated both clinically with the help of the American Orthopaedic Foot & Ankle Society (AOFAS) scale, and radiologically (MRI examination) at baseline and 2 months after the PB-MNC injection. A clinical reassessment with the AOFAS scale was also performed 6 months after the intervention. The rehabilitation protocol implied full weight-bearing walking immediately after the procedure, light physical activity 3-4 days after the injection, and physiotherapist-assisted stretching exercises and eccentric training. In all patients, functional and radiological signs of tendon healing processes were detected as early as 2 months after a single treatment and the AOFAS scale rose from the initial mean value of 37.5 (baseline) to 85.4 (6 months). Our preliminary results indicate that regenerative therapies with PB-MNCs can prove useful for partial Achilles tendon injuries as a valid alternative to surgical options, especially when other conservative approaches have failed. Advantages of this therapy include rapid execution, no need for an operating theatre, easy reproducibility, quick recovery and good tolerability regardless of the patient's age (the procedure is not to be performed in subjects who are below 18 years old). Further studies on the topic are recommended to confirm these observations

Abstract. Objectives. In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Methods. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. Results. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFa) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Conclusions. Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair. 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

Osteoarthritis (OA) is a degenerative disease that lacks regenerative treatment options. Current research focuses on mesenchymal stem cells (MSCs) and Platelet-Rich Plasma (PRP) as regenerative therapies, but extracellular vesicles (EVs) have shown to be more advantageous. This study compares the regenerative potential of human umbilical cord MSC-derived EVs (cEVs) and platelet-derived EVs (pEVs) in ex vivo and in vivo OA models. In the ex vivo study, OA conditions were induced in human cartilage explants, which were then treated either with pEVs or cEVs. Results showed a higher content of DNA and collagen in the pEVs group compared to control and cEVs groups, suggesting that pEVs could be a potential alternative to cEVs. In the in vivo study, an OA model was established in the knee joints of rats through MIA (monoiodoacetate) injection and then treated either with pEVs or cEVs. Results showed that pEVs-treated knee joints had better subchondral bone integrity and greater OA reversion, particularly in female rats, indicating that pEVs are a viable regeneration treatment for OA and outperform cEVs in terms of efficacy. Overall, the study demonstrates the potential of EVs as a regenerative treatment for OA, with pEVs showing promising results in both ex vivo and in vivo models. The use of pEVs in clinical practice could provide a faster path to translation due to the established use of platelet concentrates in therapeutics. However, further studies are needed to fully evaluate the potential of pEVs for OA treatment and to elucidate the mechanisms behind their regenerative effects. Acknowledgments: The authors thank Dr Fernando Hierro (UIB) for their technical contribution with TEM, Mª Trinidad García (UIB) for the access to radioactivity facilities, Aina Arbós (IUNICS) for her contribution in the histology staining, María Tortosa (IdISBa) for her assistance with the animal care and ADEMA School of Dentistry for the access to the cone beam computed tomography (CBCT). Funding: This research was funded by Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (MS16/00124; CP16/00124), PROGRAMA JUNIOR del proyecto TALENT PLUS, construyendo SALUD, generando VALOR (JUNIOR01/18), financed by the sustainable tourism tax of the Balearic Islands; the Direcció General d'Investigació and Conselleria d'Investigació, Govern Balear (FPI/2046/2017); the Mecanisme de Recuperació i Resiliència, intended to execute research projects of «Noves polítiques públiques per a un mercat de treball dinàmic, resilient i inclusiu», collected in Pla de Recuperació, Transformació i Resiliència, financed by European Union-Next Generation EU and driven by SOIB and Conselleria de Fons Europeus, Universitat i Cultura i la Conselleria de Model Econòmic, Turisme i Treball (NG0421) and the grant SYN20/03 from IdISBa

The World Health Organisation (WHO) has included low back pain in its list of twelve priority diseases. Notably, Degenerative disc disease (DDD) presents a large, unmet medical need which results in a disabling loss of mechanical function. Today, no efficient therapy is available. Chronic cases often receive surgery, which may lead to biomechanical problems and accelerated degeneration of adjacent segments. Our consortium partners have developed and studied mesenchymal stem cell-based, regenerative therapies trials. In previous phase 2 trial, patients exhibited rapid and progressive improvement of functional and pain indexes after 1 year with no significant side effects. To develop the world's first rigorously proven, effective treatment of DDD, EUROSPINE aims to assess, via a multicentre, randomized, controlled, phase 2b clinical trial including 112 patients with DDD, the efficacy of an allogenic intervertebral mesenchymal stem cell (MSC)-based therapy. This innovative therapy aims to rapidly and sustainably (at least 24 months) reduce pain and disability. In addition, the consortium aims to provide new knowledge on immune response & safety associated with allogeneic BM-MSC intradiscal injection. This simple procedure would be cost-effective, minimally invasive, and standardised. At the end of the RESPINE trial, we aim to propose a broadly available and clinically applicable treatment for DDD, marketed by European SMEs

Abstract. Focal articular cartilage defects do not heal and, left untreated, progress to more widespread degenerative changes. A promising new approach for the repair of articular cartilage defects is the application of cell-based regenerative therapies using mesenchymal stromal cells (MSCs). MSCs are however present in a number of tissues and studies suggest that they vary in their proliferation, cell surface characterisation and differentiation. As the phenotypic properties of MSCs vary depending on tissue source, a systematic comparison of the transcriptomic signature would allow a better understanding of these differences between tissues, and allow the identification of markers specific to a MSC source that is best suited for clinical application. Tissue was used from patients undergoing total knee replacement surgery for osteoarthritis following ethical approval and informed consent. MSCs were isolated from bone, cartilage, synovium and infrapatellar fat pad. MSC number and expansion were quantified. Following expansion in culture, MSCs were characterised using flow cytometry with several cell surface markers; the cells from all sources were positive for CD44, CD90 and CD105. Their differentiation potential was assessed through tri-lineage differentiation assays. In addition, bulk mRNA-sequencing was used to determine the transcriptomic signatures. Differentially expressed (DE) genes were predicted. An enrichment analysis focused on the DE genes, against GO and pathway databases (KEGG and Reactome) was performed; protein-protein interaction networks were also inferred (Metascape, Reactome, Cytoscape). Optimal sourcing of MSCs will amplify their cartilage regeneration potential. This is imperative for assessing future therapeutic transplantation to maximise the chance of successful cartilage repair. A better understanding of differences in MSCs from various sources has implications beyond cartilage repair

The clinical translation of regenerative therapies, whether in the form of mesenchymal cells, macromolecules or small molecules, is hampered by several factors: the poor retention and short biological half-life of the therapeutic agent, the adverse side effects from systemic delivery, and difficulties with the administration of multiple doses to a target site. We report the development and application of a therapeutic reservoir device that enables sustained and repeated administration of small molecules, macromolecules and cells directly to organs and tissues of interest via a polymer-based reservoir connected to a subcutaneous port. In a myocardial infarct rodent model, we show that repeated administration of cells over a four-week period using the reservoir provided functional compared to a single injection of cells and to no treatment. Recent advances of the system include a multi-port and multi-reservoir system that can be tailored to cargo and application need. The pre-clinical use of our therapeutic reservoir as a research model may enable insights into regenerative orthopaedic therapy, particularly those therapies that require multi-dose approaches

The HIPGEN study funded under EU Horizon 2020 (Grant 7792939) has the aim to investigate the potential of the first regenerative cell therapy for the improvement of recovery after muscle injury in hip fracture patients. For this aim we intramuscularly injected placental derived mesenchymal stromal cells during hip fracture arthroplasty. Despite not having reached the primary endpoint, which was the Short Physical Performance Battery, we could observe an increase in abductor muscle strength and a faster return to balance looking at symmetry in insole measurements during follow up

Material-based strategies seek to engineer synthetic microenvironments that mimic the characteristics of physiological extracellular matrices for applications in regenerative therapies, including bone repair and regeneration. In our group, we identified a specific chemistry, poly(ethyl acrylate) (PEA), able to induce the organization of fibronectin (FN), upon adsorption of the protein, into fibrillar networks similar to the physiological ones, leading to enhanced cellular response, in terms of cell adhesion and differentiation. In this work, we exploit these FN networks to capture and present growth factors (GF) in combination with the integrin binding domain of FN during bone tissue healing. Fibrillar conformation of FN adsorbed on PEA favors the simultaneous availability of the GF binding domain (FNIII12–14) next to the integrin binding region (FNIII9–10), compared to poly(methyl acrylate) (PMA), a material with similar chemistry, where FN adopts a globular conformation. The combined exposure of specific adhesive sequences recognized by integrins and GF binding domains was found to improve the osteogenic differentiation of mesenchymal stem cells. A higher expression of bone proteins was found when BMP2 is bound or sequestered on the material surface versus its administration in the culture media in vitro. The potential of this system as recruiter of GFs was also investigated in a critical-size bone segmental defect in mouse. The synergistic integrin-GF signalling, induced by fibrillar FN, promoted bone formation in vivo with lower BMP2 doses than current technologies. Furthermore, we optimized the system for its potential use in translational research, seeking to address the clinical need of using biocompatible and biodegradable material implants. Polycaprolactone scaffolds were synthesized and coated with a thin layer of plasma- polymerized PEA that recruits and efficiently presents GF during healing of critical size defects. The material-driven FN fibrillogenesis provides a new strategy to efficiently reduce the GF doses administrated in bone regenerative therapies

While new biomaterials for regenerative therapies are being reported in the literature, clinical translation is slow. Existing regenerative approaches rely on high doses of growth factors, such as BMP-2 in bone regeneration, which can cause serious side effects. We describe an ultra-low-dose growth factor technology yielding high bioactivity based on a simple polymer, poly (ethyl acrylate) (PEA), and report its translation to a clinical veterinary setting. This polymer-based technology triggers spontaneous fibronectin organization and stimulates growth factor signaling, enabling synergistic integrin and BMP-2 receptor activation in mesenchymal stem cells. To translate this technology, we use plasma-polymerized PEA on 2D and 3D substrates to enhance cell signaling in vitro, showing the complete healing of a critical-size bone injury in mice in vivo. We demonstrate its safety and efficacy in a Münsterländer dog with a non-healing humerus fracture, establishing the clinical translation of advanced ultra-low-dose growth factor treatment

Cells with stem/progenitor characteristics can be isolated from articular cartilage and may have utility in cartilage repair and regeneration therapies. Unlike other adult cell types with differentiation capabilities, clonal chondroprogenitors differentiate into cartilage that resembles stable cartilage rather than endochondral cartilage. We have isolated a large series of chondroprogenitor clones from normal human articular cartilage from individuals of one to forty-five years of age and characterized them with known and novel markers. The clones were isolated separately from different zones of the articular cartilage. As first reported by others, the cloneable cells were mainly found in the upper zones. However, there are clones with chondroprogenitor status in the deeper zones, albeit at far lower frequency. These deep zone clones have different characteristics to those from the upper zones. We have used selected clones to re-engineer stable cartilage with use of the right environmental conditions (growth factors, oxygen level etc)

Low back pain is thought to relate to intervertebral disc (IVD) degeneration. Although the mechanisms have not been clearly identified, exhaustion of nucleus pulposus cells and their producing matrix is regarded as one cause. The matrix of the IVD is continuously replenished and remodeled by tissue-specialized cells and are crucial in supporting the IVD function. However, due to aging, trauma, and genetic and lifestyle factors, the cells can lose their potency and viability, thereby limiting their collective matrix production capacity. We have discovered the link between loss of angiopoietin-1 receptor (Tie2)-positive human NP progenitor cells (NPPC) and IVD degeneration. Tie2+ cells were characterized as undifferentiated cells with multipotency and possessing high self-renewal abilities. Thus we and others have proposed Tie2+ NPPC as a potent cell source for regenerative cell therapies against IVD degeneration. However, their utilization is hindered by low Tie2-expressing cell yields from NP tissue, in particular from commonly available older and degenerated tissue sources. Moreover, NPPC show a rapid Tie2 decrease due to cell differentiation as part of standard culture processes. As such, a need exists to optimize or develop new culture methods that enable the maintenance of Tie2-expressing NPPC. Trials to overcome these difficulties will be shared

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

Current medical treatments for IVD degeneration rely on conservative therapies or surgery. Surgical treatments (e.g. spinal fusion,) have shown satisfactory results in alleviating pain, but long-term clinical outcomes remain poor. Thus, there is an urgent need for alternative cell based regenerative therapies focussed on correcting the underlying pathogenesis of IVD degeneration. However, for these to be successful an appropriate cell source for implantation, together with a suitable growth factor to direct cell differentiation and formation of a functional matrix must be identified. Additionally, extensive in vitro studies are needed to establish and support further pre-clinical and potential commercial development. We have demonstrated that stimulation of both BM-MSCs and AD-MSCs with GDF6 results in improved differentiation to a nucleus pulposus (NP)-like phenotype and synthesis of proteoglycan rich matrix with micromechanical properties akin to the healthy IVD. Significantly, these studies have highlighted that AD-MSCs are the more appropriate cell source. Furthermore, our studies have shown hat GDF6 has anabolic effects on degenerate human NP cells, stimulating adoption of a more normal NP phenotype and increasing appropriate atrix synthesis. This suggests that delivery of GDF6 as part of an MSC-based therapy may be beneficial both in directing lineage-specific MSC differentiation, but also in restoring a more anabolic phenotype in native NP cells, thereby having a dual regenerative effect

The selection of a proper material to be used as a scaffold or as a hydrogel to support, hold or encapsulate cells is both a critical and a difficult choice that will determine the success of failure of any tissue engineering and regenerative medicine (TERM) strategy. We believe that the use of natural origin polymers, including a wide range of marine origin materials, is the best option for many different approaches that allow for the regeneration of different tissues. In addition to the selection of appropriate material systems it is of outmost importance the development of processing methodologies that allow for the production of adequate scaffolds/matrices, in many cases incorporating bioactive/differentiation agents in their structures. An adequate cell source should be selected. In many cases efficient cell isolation, expansion and differentiation, and in many cases the selection of a specific sub-population, methodologies should be developed and optimized. We have been using different human cell sources namely: mesenchymal stem cells from bone marrow, mesenchymal stem cells from human adipose tissue, human cells from amniotic fluids and membranes and cells obtained from human umbilical cords. The development of dynamic ways to culture the cells and of distinct ways to stimulate their differentiation in 3D environments, as well as the use of nano-based systems to induce their differentiation and internalization into cells, is also a key part of some of the strategies that are being developed in our research group. The potential of each combination materials/cells, to be used to develop novel useful regeneration therapies will be discussed. The use of different cells and their interactions with different natural origin degradable scaffolds and smart hydrogels will be described. Several examples of TERM strategies to regenerate different types of musculoskeletal tissues will be presented. Relevance to orthopaedics will be highlighted

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