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Equine muscle-derived mesenchymal stem cells protect cartilage explants in osteoarthritis model: secretome characterization

ABSTRACT: Background: Osteoarthritis is a highly prevalent joint degenerative disease for which therapeutic treatments are limited or invasive. Cell therapy based on mesenchymal stem/stromal cells (MSCs) is therefore seen as a promising approach for this disease, in both human and horses. Objective: As the regenerative potential of MSCs is mainly conferred by paracrine function, the goal of this study is to characterize the secretome of muscle-derived MSCs (mdMSCs) in an in vitro model of OA to evaluate the clinical interest of mdMSCs as cell therapy for joint diseases like osteoarthritis. Methods: An equine osteoarthritis model composed of cartilage explants exposed to pro-inflammatory cytokines was first developed. Then, the effects of mdMSC coculture on cartilage explant were studied by measuring the glycosaminoglycan release and the NO2- production. To identify the underlying molecular actors, SILAC-based secretome analyses were conducted, in the presence of serum. The relative abundance of highly sequenced proteins was finally confirmed by western blot. Results: Co-culture with muscle-derived MSCs decreases the cytokine-induced glycosaminoglycan release by cartilage explants, suggesting a protecting effect of mdMSCs. Among the 52 equine proteins sequenced in the co-culture medium, the abundance of decorin and matrix metalloproteinase 3 was modified, as confirmed by western blot analyses. Conclusions: These results suggest that muscle-derived MSCs could reduce the catabolic effect of TNF and IL-1 on cartilage explant by decreasing the secretion and activity of Matrix Metalloproteinase 3 and increasing the decorin secretion

INSTRUMENT(S): Bruker Daltonics timsTOF series

ORGANISM(S): Equus Caballus (horse)

TISSUE(S): Cell Culture, Mesenchymal Cell

DISEASE(S): Osteoarthritis

SUBMITTER: Marc Dieu

LAB HEAD: Patricia RENARD

PROVIDER: PXD031924 | Pride | 2022-11-08

REPOSITORIES: Pride

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Project description:Successfully replacing damaged cartilage with tissue-engineered constructs requires integration with the host tissue and could benefit from leveraging the native tissue's intrinsic healing capacity; however, efforts are limited by a poor understanding of how cartilage repairs minor defects. Here, we investigated the conditions that foster natural cartilage tissue repair to identify strategies that might be exploited to enhance the integration of engineered/ grafted cartilage with host tissue. We damaged porcine articular cartilage explants and using a combination of pulsed SILAC-based proteomics, ultrastructural imaging, and catabolic enzyme blocking strategies reveal that integration of damaged cartilage surfaces is not driven by neo-matrix synthesis, but rather local depletion of proteoglycans. ADAMTS4 expression and activity are upregulated in injured cartilage explants, but integration could be reduced by inhibiting metalloproteinase activity with TIMP3. These observations suggest that catabolic enzyme-mediated proteoglycan depletion likely allows existing collagen fibrils to undergo cross-linking, fibrillogenesis, or entanglement, driving integration. Catabolic enzymes are often considered pathophysiological markers of osteoarthritis. Our findings suggest that damage-induced upregulation of metalloproteinase activity may be a part of a healing response that tips towards tissue destruction under pathological conditions and in osteoarthritis, but could also be harnessed in tissue engineering strategies to mediate repair. Statement of significance: Cartilage tissue engineering strategies require graft integration with the surrounding tissue; however, how the native tissue repairs minor injuries is poorly understood. We applied pulsed SILACbased proteomics, ultrastructural imaging, and catabolic enzyme blocking strategies to a porcine cartilage explant model and found that integration of damaged cartilage surfaces is driven by catabolic enzyme-mediated local depletion of proteoglycans. Although catabolic enzymes have been implicated in cartilage destruction in osteoarthritis, our findings suggest that damage-induced upregulation of metalloproteinase activity may be a part of a healing response that tips towards tissue destruction under pathological conditions. They also suggest that this natural cartilage tissue repair process could be harnessed in tissue engineering strategies to enhance the integration of engineered cartilage with host tissue.

Project description:Fetal cartilage fully regenerates following injury while in adult mammals cartilage injury leads to osteoarthritis (OA). OA is characterized by cartilage breakdown and joint inflammation and associated with significant pain and socioeconomic costs. As no clinically satisfactory treatment is available to date, disease-modifying therapies aimed to achieve cartilage regeneration are urgently required. The inherent regeneration potential of fetal individuals may hold answers to this unmet need. Therefore, to characterize the differences in fetal and adult response to cartilage injury, we carried out histology and comprehensive proteome analyses on fetal (day 80/150-day gestation) and adult cartilage samples one (fetal samples) and three (adult and fetal samples) days after surgical induction of a full-thickness cartilage lesion. In addition, proteins secreted by inflamed fetal MSCs in vitro were compared with the in vivo response to injury to evaluate their therapeutic potential. Histology of synovial samples revealed the presence of neutrophils one day post injury (p.i.) and an influx of macrophages into the subsynovial tissue on day 3 p.i. in fetal samples. In contrast, adult synovial samples showed invasion of neutrophils on day 3 p.i. Activation and migration of Iba1+- macrophages of the synovial lining was observed both in fetal and adult animals. Comparative mass spectrometry revealed 57 proteins significantly up-regulated (> 2FC, FDR<0.05), and 67 proteins significantly down-regulated (<-2 FC) upon injury in adults. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult cartilage following injury compared to fetal sheep. In contrast, several immunomodulating proteins and growth factors were significantly higher expressed in the fetus than the adult. Comparison of the in vitro MSCs with the in vivo fetal proteome revealed shared upregulation of 17 proteins, which were considered to be of potential therapeutic interest. The results of this study support our molecular understanding of successful fetal cartilage healing and new therapeutic strategies to induce regeneration in adult articular cartilage by modulating the inflammatory environment. The shared protein upregulation in fetal cartilage in vivo and in fetal MSCS during in vitro inflammation supports the possible therapeutic potential of these factors in specific and fetal MSCs in general.

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Equine muscle-derived mesenchymal stem cells protect cartilage explants in osteoarthritis model: secretome characterization

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