A large part of mesenchymal stromal cell (MSC) regenerative and immunomodulatory action is mediated by paracrine signaling. Hence, an increasing body of evidence acknowledges the potential of MSC secretome in a variety of preclinical and clinical scenarios. Mid-term serum deprivation is a common approach in the pipeline of MSC secretome production. Nevertheless, up to now, little is known about the impact of this procedure on the metabolic status of donor cells. Here, through untargeted differential metabolomics, we revealed an impairment of mitochondrial metabolism in adipose-derived MSCs exposed for 72 h to serum deprivation. This evidence was further confirmed by the significant accumulation of reactive oxygen species and the reduction of succinate dehydrogenase activity. Probably as a repair mechanism, an upregulation of mitochondrial superoxide dismutase was also induced. Of note, the analysis of mitochondrial functionality indicated that, despite a significant reduction of basal respiration and ATP production, serum-starved MSCs still responded to changes in energy demand. This metabolic phenotype correlates with the obtained evidence of mitochondrial elongation and branching upon starvation.

Analytical advancements in lipidomics have enabled large-scale investigations of lipid biology. Herein, we focused on four bioactive lipid families, namely polyunsaturated fatty acids, eicosanoids, endocannabinoids, and N-acylethanolamines, and their involvement in the mesenchymal stem cells (MSC)-related inflammatory scenario. Since MSC secretome may represent a valid therapeutic alternative, here, the complete secretome and its vesicular component from adipose- and bone marrow-derived MSC and dermal fibroblasts were characterized by targeted mass spectrometry lipidomics. The 2-arachidonoylglycerol (2AG) and the palmitoylethanolamide (PEA), previously quantified in the MSC’s secretome, were further investigated by assessing hypothetical effects in an in vitro model of osteoarthritis (OA) based on human primary articular chondrocytes (CH) stimulated with tumor necrosis factor alpha (TNFα). TNFα enhances the release of the inflammatory lipid prostaglandin E2 (PGE2), and an additional increment was observed when CH were treated with both TNFα and 2AG. In contrast, PEA downmodulates the PGE2 release to the levels of unstimulated CH suggesting a protective effect. TNFα also increases the expression of cyclooxygenase 2 (COX2), in particular when combined with 2AG, while PEA partly blunts TNFα-induced COX2 expression. In addition, TNFα-stimulated CH produce significantly higher levels of the inflammatory mediator nitric oxide (NO) both in the presence and in the absence of 2AG, and PEA was able to partially reduce NO release. Our results show a first partial lipidomic profile of MSC and DF secretome and suggest a possible implication of bioactive lipids in the OA scenario and in the future use of these cell-free products as innovative therapeutics.

The adipose-derived stem cell (ADSC) secretome is widely studied for its immunomodulatory and regenerative properties, yet its potential in maxillofacial medicine remains largely underexplored. This review takes a composition-driven approach, beginning with a list of chemokines, cytokines, receptors, and inflammatory and growth factors quantified in the ADSC secretome to infer its potential applications in this medical field. First, a review of the literature confirmed the presence of 107 bioactive factors in the secretome of ADSCs or other types of mesenchymal stem cells. This list was then analyzed using the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) software, revealing 844 enriched biological processes. From these, key processes were categorized into three major clinical application areas: immunoregulation (73 factors), bone regeneration (13 factors), and wound healing and soft tissue regeneration (27 factors), with several factors relevant to more than one area. The most relevant molecules were discussed in the context of existing literature to explore their therapeutic potential based on available evidence. Among these, TGFB1, IL10, and CSF2 have been shown to modulate immune and inflammatory responses, while OPG, IL6, HGF, and TIMP1 contribute to bone regeneration and tissue repair. Although the ADSC secretome holds great promise in oral and maxillofacial medicine, further research is needed to optimize its application and validate its clinical efficacy.

Connexin 43 (Cx43) exerts pivotal functions in articular chondrocytes (CH). It is involved in the communication among cells and between cells and the extracellular environment, and it contributes to the maintenance of the correct cell phenotype. The pro-inflammatory cytokine TNFα induces a reduction in Cx43 expression in CH. Here, we studied the dynamics of this decrease in expression. We evaluated Cx43 protein and gene expression and the involvement of C-terminal domain (CTD) cleavage and proteasomal degradation. Treatments able to counteract TNFα action were also examined, together with Gap Junction (GJ) functionality and Cx43 localization. TNFα induced a significant reduction in Cx43 expression already at day 1, and the down modulation reached a peak at day 3 (−46%). The decrease was linked to neither gene expression modulation nor CTD cleavage. Differently, the proteasome inhibitor MG132 reverted TNFα effect, indicating the involvement of proteasomal degradation in Cx43 reduction. In addition, the co-treatment with the anabolic factor TGF-β1 restored Cx43 levels. Cx43 decrease occurred both at the membrane level, where it partially influenced GJ communication, and in the nucleus. In conclusion, TNFα induced a rapid and lasting reduction in Cx43 expression mostly via the proteasome. The down modulation could be reverted by cartilage-protective factors such as MG132 and TGF-β1. These findings suggest a possible involvement of Cx43 perturbation during joint inflammation.

Osteoarthritis (OA) is a highly prevalent joint disease still lacking effective treatments. Its multifactorial etiology hampers the development of relevant preclinical models to evaluate innovative therapeutic solutions. In the last decade, the potential of Mesenchymal Stem Cell (MSC) secretome, or conditioned medium (CM), has emerged as an alternative to cell therapy. Here, we investigated the effects of the CM from adipose MSCs (ASCs), accounting for both soluble factors and extracellular vesicles, on human osteochondral explants. Biopsies, isolated from total knee replacement surgery, were cultured without additional treatment or with the CM from 10⁶ ASCs, both in the absence and in the presence of 10 ng/mL TNFα. Tissue viability and several OA-related hallmarks were monitored at 1, 3 and 6 days. Specimen viability was maintained over culture. After 3 days, TNFα induced the enhancement of matrix metalloproteinase activity and glycosaminoglycan release, both efficiently counteracted by CM. The screening of inflammatory lipids, proteases and cytokines outlined interesting modulations, driving the attention to new players in the OA process. Here, we confirmed the promising beneficial action of ASC secretome in the OA context and profiled several bioactive factors involved in its progression, in the perspective of accelerating an answer to its unmet clinical needs.

Osteoarthritis (OA), a chronic inflammatory joint disorder, still lacks effective therapeutic interventions. Consequently, the development of convenient experimental models is crucial. Recently, research has focused on the plasticity of Mesenchymal Stem/stromal Cells, particularly adipose-derived ones (ASCs), in halting OA progression. This study investigates the therapeutic potential of a cell-free approach, ASC-derived conditioned medium (CM), in reversing cytokine-induced OA markers in an ex vivo model of human cartilage explants. 4 mm cartilage punches, derived from the femoral heads of patients undergoing total hip replacement, were treated with 10 ng/ml TNFα, 1 ng/ml IL-1β, or a combination of both, over a 3-day period. Analysis of OA-related markers, such as MMP activity, the release of NO and GAGs, and the expression of PTGS2, allowed for the selection of the most effective inflammatory stimulus. Subsequently, explants challenged with TNFα+IL-1β were exposed to CM, consisting of a pool of concentrated supernatants from 72-h cultured ASCs, in order to evaluate its effect on cartilage catabolism and inflammation. The 3-day treatment with both 10ng/ml TNFα and 1ng/ml IL-1β significantly increased MMP activity and NO release, without affecting GAG release. The addition of CM significantly downregulated the abnormal MMP activity induced by the inflammatory stimuli, while also mildly reducing MMP3, MMP13, and PTGS2 gene expression. Finally, SOX9 and COL2A1 were downregulated by the cytokines, and further decreased by CM. The proposed cartilage explant model offers encouraging evidence of the therapeutic potential of ASC-derived CM against OA, and it could serve as a convenient ex vivo platform for drug screening.