To define if adipose mesenchymal stromal cell (ASC) treatment mediated switching of the pro-inflammatory profile of M1-like macrophages as a means to develop a tailored in vitro efficacy/potency test. We firstly performed immunohistochemical analysis of CD68, CD80 (M1-like) and CD206 (M2-like) macrophages in osteoarthritic (OA) synovial tissue. ASC were co-cultured in contact and in transwell with activated (GM-CSF + IFNγ)-M1 macrophages. We analyzed IL1β, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9, IL10, CD163 and CD206 by qRT-PCR or immunoassays. Prostaglandin E2 (PGE2) blocking experiments were performed using PGE2 receptor antagonist. In moderate grade OA synovium we did not always find a higher percentage of CD80 with respect to CD206. M1-like-activated macrophage factors IL1β, TNFα, IL6, MIP1α/CCL3, S100A8 and S100A9 were down-modulated both in contact and in transwell by ASC. However, in both systems ASC induced the typical M2-like macrophage markers IL10, CD163 and CD206. Activated-M1-like macrophages pre-treated with PGE2 receptor antagonist failed to decrease secretion of TNFα, IL6 and to increase that of IL10, CD163 and CD206 when co-cultured with ASC confirming a PGE2 specific role. We demonstrated that ASC are responsible for the switching of activated-M1-like inflammatory macrophages to a M2-like phenotype, mainly through PGE2. This evidenced that activated-M1-like macrophages may represent a relevant cell model to test the efficacy/potency of ASC and suggests a specific role of ASC as important determinants in therapeutic dampening of synovial inflammation in OA.

The aim of the study was to characterize synovial cells from OA synovium with low-grade and moderate-grade synovitis and to define the role of synovial macrophages in cell culture. Synovial tissue explants were analyzed for the expression of typical markers of synovial fibroblasts (SF), synovial macrophages (SM) and endothelial cells. Synovial cells at passage 1 (p.1) and 5 (p.5) were analyzed for different phenotypical markers by flow cytometric analysis, inflammatory factors by multiplex immunoassay, anabolic and degradative factors by qRT-PCR. P.1 and p.5 synovial cells as different cell models were co-cultured with adipose stem cells (ASC) to define SM effects. Synovial tissue showed a higher percentage of CD68 marker in moderate compared with low-grade synovitis. Isolated synovial cells at p.1 were positive to typical markers of SM (CD14, CD16, CD68, CD80 and CD163) and SF (CD55, CD73, CD90, CD105, CD106), whereas p.5 synovial cells were positive only to SF markers and showed a higher percentage of CD55 and CD106. At p.1 synovial cells released a significantly higher amount of all inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and some anabolic (IL10) factors than those of p.5. Moreover, p.1 synovial cells also expressed a higher amount of some degradative factors (MMP13, S100A8, S100A9) than p.5 synovial cells. Co-culture experiments showed that the amount of SM in p.1 synovial cells differently induced or down-modulated some of the inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and degradative factors (ADAMTS5, MMP13, S100A8, S100A9). We found that p.1 (mix of SM and SF) and p.5 (only SF) synovial cells represent two cell models that effectively reproduce the low- or moderate-grade synovitis environment. The presence of SM in culture specifically induces the modulation of the different factors analyzed, confirming that SM are key effector cells.

Hydrogen sulfide (H₂S) is a gasotransmitter known to regulate bone formation and bone mass in unperturbed mice. However, it is presently unknown whether H₂S plays a role in pathologic bone loss. Here we show that ovariectomy (ovx), a model of postmenopausal bone loss, decreases serum H₂S levels and the bone marrow (BM) levels of two key H₂S‐generating enzymes, cystathione β‐synthase (CBS) and cystathione γ‐lyase (CSE). Treatment with the H₂S‐donor GYY4137 (GYY) normalizes serum H₂S in ovx mice, increases bone formation, and completely prevents the loss of trabecular bone induced by ovx. Mechanistic studies revealed that GYY increases murine osteoblastogenesis by activating Wnt signaling through increased production of the Wnt ligands Wnt16, Wnt2b, Wnt6, and Wnt10b in the BM. Moreover, in vitro treatment with 17β‐estradiol upregulates the expression of CBS and CSE in human BM stromal cells (hSCs), whereas an H₂S‐releasing drug induces osteogenic differentiation of hSCs. In summary, regulation of H₂S levels is a novel mechanism by which estrogen stimulates osteoblastogenesis and bone formation in mice and human cells. Blunted production of H₂S contributes to ovx‐induced bone loss in mice by limiting the compensatory increase in bone formation elicited by ovx. Restoration of H₂S levels is a potential novel therapeutic approach for postmenopausal osteoporosis. © 2015 American Society for Bone and Mineral Research.

The importance of hydrogen sulfide (H₂S) in the regulation of multiple physiological functions has been clearly recognized in the over 20 years since it was first identified as a novel gasotransmitter. In bone tissue H₂S exerts a cytoprotective effect and promotes bone formation. Just recently, the scientific community has begun to appreciate its role as a therapeutic agent in bone pathologies. Pharmacological administration of H₂S achieved encouraging results in preclinical studies in the treatment of systemic bone diseases, such as osteoporosis; however, a local delivery of H₂S at sites of bone damage may provide additional opportunities of treatment. Here, we highlight how H₂S stimulates multiple signaling pathways involved in various stages of the processes of bone repair. Moreover, we discuss how material science and chemistry have recently developed biomaterials and H₂S-donors with improved features, laying the ground for the development of H₂S-releasing devices for bone regenerative medicine. This review is intended to give a state-of-the-art description of the pro-regenerative properties of H₂S, with a focus on bone tissue, and to discuss the potential of H₂S-releasing scaffolds as a support for bone repair.