Osteoarthritis (OA) is an inflammatory condition still lacking effective treatments. Mesenchymal stem/stromal cells (MSCs) have been successfully employed in pre-clinical models aiming to resurface the degenerated cartilage. In early-phase clinical trials, intra-articular (IA) administration of MSCs leads to pain reduction and cartilage protection or healing. However, the consistent lack of engraftment indicates that the observed effect is delivered through a “hit-and-run” mechanism, by a temporal release of paracrine molecules. MSCs express a variety of chemokines and cytokines that aid in repair of degraded tissue, restoration of normal tissue metabolism and, most importantly, counteracting inflammation. Secretion of therapeutic factors is increased upon licensing by inflammatory signals or apoptosis, induced by the host immune system. Trophic effectors are released as soluble molecules or carried by extracellular vesicles (ECVs). This review provides an overview of the functions and mechanisms of MSC-secreted molecules found to be upregulated in models of OA, whether using in vitro or in vivo models.

Purpose: Substance P (SP) release and binding to NK-1 produces pain transmission. Pain from arthritis is expressed as both tenderness and as loss of function. Different arthritis pain models express pain differently. This study evaluated the relationship of dorsal root ganglion (DRG) SP expression to pain using both measures of tenderness and function in 3 different models of arthritis pain. Neurotoxins (NT) that prevent release of SP, such as onabotulinum toxin (BoNT/A), and those that deplete SP, such as vanilloids (VAN), can produce analgesia in these models. We correlated effects on SP expression in the DRG with analgesic responses. Methods: C57/Bl6 male mice received intra-articular (IA) carrageenan, Complete Freund's Adjuvant (CFA) or Collagenase (COL) to produce acute inflammatory, chronic inflammatory or chronic noninflammatory arthritis respectively. IA therapies were given at appropriate intervals before examination. Twelve-week-old mice were examined after arthritis induction. Evoked (tenderness) and spontaneous (functional) pain was quantitated. DRGs were harvested for immunohistochemistry (IHC) after pain assessment. SP expression was measured as % DRG neurons expressing SP. Results: Both Evoked and Spontaneous pain in arthritic and naïve mice

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Purpose: Mesenchymal stem cells (MSCs) enhance regeneration when locally delivered in pre-clinical models of osteoarthritis (OA). Their mechanism of action, however, is still poorly understood. Preliminary data show low engraftment of GFP-expressing syngeneic MSCs in mouse knee joints, consistent with published data using molecular techniques. We hypothesise that implanted cells undergo apoptosis or cell death. There is growing evidence that apoptotic MSCs exert an antiinflammatory effect, by release of immunosuppressive cytokines and, indirectly, through phagocytes. This study aims to investigate the immunomodulatory potential of apoptotic MSCs in vitro, to correlate with in vitro results in a mouse model of OA. Methods: Collagenase-induced OA (CIOA) was performed on C57BL/6 mice and 2x10 5 GFP-expressing MSCs were intra-articularly (IA)-delivered in the animals. Knee joints were harvested 3 days post-IA-injection and digested into a single cell suspension before sorting implanted GFPþ MSCs using the BD FACS Aria II. Retrieved cells were expanded in culture and characterised by cell surface markers and differentiation. Their immunosuppressive capacity was tested in vitro on murine lymphocytes and bone marrow-derived macrophages (BMDM). Apoptosis of MSCs was induced in vitro by treatment with Staurosporine (STS), while necrosis was produced by heat treatment (90 C, 5 min). Apoptosis was validated by pre-treating the cells with the pancaspase inhibitor Q-VD-OPH. All treatments were evaluated by flow cytometry using anti-Annexin V/Sytox Blue staining; activation of caspases was measured by FLICA assays. Apoptotic MSCs were co-cultured with activated murine splenocytes for 3 days. Suppression of proliferation was measured by quantification of Cell Trace Violet and expression of CD25. Conditioned media from apoptotic and necrotic MSCs were tested on LPS-activated BMDM and expression of pro-and anti-inflammatory molecules was assessed by ELISA (IL-6, IL-10, TGF-b, PGE2) and Griess assay (NO). Results: On average, 1.63% of the injected cells were successfully retrieved by cell sorting, with 82.1% sample purity. Sorted cells proliferated in culture retaining MSC-like morphology, expression of cell surface markers and differentiation potential. Retrieved cells have an increased immunosuppressive potential as observed on activated BMDMs and lymphocytes. STS successfully induced apoptosis, with activation of Caspase 3/7; Q-VD-OPH completely inhibited apoptosis, confirming the involvement of caspases. Necrotic, but most strikingly apoptotic MSCs, significantly prevented the proliferation and activation of murine splenocytes (Fig. 1). BMDM were also induced towards an anti-inflammatory phenotype, as shown by their secretory profile. Conclusions: MSCs can be administered and retrieved from murine knee joints and remain viable for characterisation. Retrieval yield is low, consistent with previous studies. IA-injected MSCs may contribute to local modulation of joint inflammation. Engrafted MSCs retrieved from the OA joint were ...

Purpose: Osteoarthritis (OA) is a major health issue worldwide due to the increasing age of active population and the impact on patients' daily life. Cartilage and synovial alterations are important factors involved in OA progression. Despite the advancements in this field, there is a largely unmet medical need for durable disease-modifying treatments. Cellbased therapies have become thriving therapeutic options for OA but nevertheless the choice of the suitable cell candidate is a source of debate. The purpose of this work was aimed to compare the regenerative potential of chondrocytes versus Mesenchymal Stem Cells (MSC), grown onto a hyaluronan-based scaffold (Hyaff-11) in counteracting OA progression in an experimental OA rabbit model at short and long-term follow-ups. Methods: Adult male New Zealand rabbits were enrolled in this study according to European and Italian laws for animal experimentation. A mild grade of OA was induced surgically through bilateral Anterior Cruciate Ligament Transection at 8 weeks. Chondrocytes and MSC were isolated from rabbit articular cartilage and bone marrow respectively, grown in monolayer cultures, seeded onto Hyaff-11 (HA) and transplanted after OA onset. Animals were divided into two groups: 1) chondrocyte-seeded HA (Chondro-HA) and 2) MSC-seeded HA (MSC-HA). Empty HA was used as control. Animals were euthanized at 3 and 6 months. Cartilage and synovia were assessed by histology supported by modified Mankin score and histomorphometric analyses. Protein expression of collagen type I, II and X, MMP-1, MMP-3 and MMP-13 were assessed by immunohistochemistry. The General Linear Model with Sidak correction was used to compare the two cell-based therapies. Results: At 3 months, chondro-HA group displayed an active contribution in reducing OA changes especially on cartilage with an improvement of tissue morphology, extracellular matrix organization and cell distribution compared to MSC-HA-one (p<0.05); the effects on synovia were less pronounced. Further analyses confirmed these findings with a decrease of MMP-3, MMP-13 and TNF-a mainly in cartilage (p<0.05). A reduction of hypertrophic processes was evident in cartilage for both cell treatments compared to HA (p<0.05). At 6 months, MSC-HA therapy displayed the best macroscopic and histological findings on cartilage and synovial tissues than chondro-HA-one (p<0.05). This last displayed some endochondral ossification processes and thickening of the subchondral bone plate in femur and hyperplasia and fibrous reactions in synovia. Moreover, a decreased presence of MMP-1, -3, -13, TNF-a and some hypertrophic markers was evident for MSC-HA therapy at both tissue level. Conversely, an increase of collagen X and alkaline phosphatase was noticed for chondro-HA therapy at long-term follow-