Human synovium harbours macrophages and T-cells that secrete inflammatory cytokines, stimulating chondrocytes to release proteinases like aggrecanases and matrix metalloproteinases (MMPs) during the development of Osteoarthritis (OA). Inflammation of the synovium is a key feature of OA, linked to several clinical symptoms and the disease progression. As a prelude to testing in an OA mouse model, we have used the tetracycline system (Tet) to modify mouse mesenchymal stem cells (mMSCs) to over-express viral interleukin 10 (vIL10), an anti-inflammatory cytokine, to modulate the osteoarthritic environment and prevent disease development. MSCs isolated from the marrow of C57BL/6J mice expressed CD90.2, SCA-1, CD105, CD140a, and were negative for CD34, CD45 and CD11b by flow cytometry. Adenoviral transduction of MSCs carrying CMVIL10 and TetON as test, and untransduced, AdNull and TetOFF as negative controls was successful and tightly controlled vIL10 production was demonstrated by CMVIL10 and TetON MSCs using a vIL10 ELISA kit. Co-incubation of vIL10MSC CM with lipopolysaccharide activated bone-marrow derived murine macrophages (BMDMs) resulted in reduction of TNF-α, IL-6 levels and elevated production of IL-10 by ELISA and high iNOS release by Griess assay. Co-culture of active macrophages with TetON MSCs, resulted in polarisation of macrophage cell population from M1 to M2 phase, with decrease in pro-inflammatory MHC-II (M1 marker) and increase in regulatory CD206 (M2 marker) expression over time. The PCR profiler array on MSC CM treated BMDMs, also showed changes in gene expression of critical pro-inflammatory cytokines and receptors involved in the TLR4 pathway. The biscistronic TetON transduced MSCs proved to be most immuno-suppressive and therefore feasible as efficient anti-inflammatory therapy that can utilised in vivo.

Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells. For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed. We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored

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

Biomaterials with mechanical or biological competence are ubiquitous in musculoskeletal disorders, and understanding the inflammatory response they trigger is key to guide tissue regeneration. While macrophage role has been widely investigated, immune response is regulated by other immune cells, including neutrophils, the most abundant leukocyte in human blood. As first responders to injury, infection or material implantation, neutrophils recruit other immune cells, and therefore influence the onset and resolution of chronic inflammation, and macrophage polarization. This response depends on the physical and chemical properties of the biomaterials, among other factors. In this study we report an in vitro culture model to describe the most important neutrophil functions in relation to tissue repair. We identified neutrophil survival and death, neutrophils extracellular trap formation, release of reactive oxygen species and degranulation with cytokines release as key functions and introduced a corresponding array of assays. These tests were suitable to identify clear differences in the response by neutrophils that were cultured on material of different origin, stiffness and chemical composition. Overall, substrates from biopolymers of natural origin resulted in increased survival, less neutrophil extracellular trap formation, and more reactive oxygen species production than synthetic polymers. Within the range of mechanical properties explored (storage modulus below 5 k Pa), storage modulus of covalently crosslinked hyaluronic acid hydrogels did not significantly alter neutrophils response, whereas polyvinyl alcohol gels of matching mechanical properties displayed a response indicating increased activation. Additionally, we present the effect of material stiffness, charge, coating and culture conditions in the measured neutrophils response. Further studies are needed to correlate the neutrophil response to tissue healing. By deciphering how neutrophils initiate and modulate the immune response to material implantation, we aim at introducing new principles to design immunomodulatory biomaterials for musculoskeletal disorders. Acknowledgments. This work was supported by the AO Foundation, AO CMF, grant AOCMF-21-04S

Abstract. Purpose. It is becoming apparent that mesenchymal stem cells (MSCs) do not directly contribute to mesenchymal tissue regeneration. Pre-clinical attempts to repair large bone defects in big animal models have been hampered by poor MSCs survival after implantation which impedes their direct or indirect effects. Based on previous work, we hypothesized that a venous axial vascularization of the scaffold supporting MSCs or their combination with fresh bone marrow (BM) aspirate would improve their in vivo survival. Methods. Cross-shape profile tubular microporous monetite implants (12mm long, 5mm large) as two longitudinal halves were produced by 3D powder printing. They were implanted around the femoral veins of Wistar rats and loaded with 1mL of BM aspirate either alone or supplemented by 10. 7. MSCs. This was compared with BM-free scaffolds loaded only with 10. 7. MSCs. After 8 weeks bone formation were investigated by micro-CT, scanning electron microscopy, histology and immunohistochemistry. Results. Little bone formation was observed within the scaffold when it was only loaded with MSCs surprisingly. Coupling MSCs, autologous BM and venous perfusion of the scaffold led to a higher volume of new bone than BM alone suggesting that MSCs augmented the bone formation capacity of BM aspirate or enhanced its survival post implantation. Conclusion. Subcutaneous bone formation within 3D-printed implant that mixed of BM with or without MSCs was successfully achieved for the first time by venous perfusion. The inability of MSCs to form differentiated tissues by their own was confirmed in this study; however, contact between MSCs and BM cells and MSCs paracrine secretome (e.g., cytokines, chemokines, extracellular vesicles) may have induced immunomodulatory effects (e.g., macrophages polarization, Treg cells) that triggered bone formation. This approach, if translatable to large animal models, offers immediate clinical value as well as an insight into the role of immune system in tissue regeneration. 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