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.

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.