In the context of regenerative medicine for the treatment of musculoskeletal pathologies mesenchymal stromal cells (MSCs) have shown good results thanks to secretion of therapeutic factors, both free and conveyed within the extracellular vesicles (EV), which in their totality constitute the “secretome”. The portfolio and biological activity of these molecules can be modulated by both The data showed that more than 50 cytokines / chemokines and more than 200 EV-microRNAs are detectable at various intensity levels in ASCs secretomes. The majority of the most abundantly present molecules are involved in the remodelling of the extracellular matrix and in the homeostasis and chemotaxis of inflammatory cells including macrophages, which in OA are often characterized by an M1 inflammatory polarization, promoting their transition to an M2 anti-inflammatory phenotype. Inflammatory priming with IFNγ and synovial fluid-like conditions were able to further increase the ability of the secretome to interact with inflammatory cells and modulate their migration. Finally, the penetration of the EVs in the cartilage explants resulted a rapid process, which begins a few minutes after administration of the EVs that are able to reach a depth of 30-40 μm in 5 hours. The same capacity for interaction was also verified in chondrocytes and synoviocytes isolated from the cartilage and synovial membrane of OA patients. Thanks to the soluble factors and EV-microRNAs, the ASCs secretome has shown a strong propensity to modulate the inflammatory and degenerative processes that characterize OA. The inflammatory pre-conditioning through high concentrations of inflammatory molecules or in conditions similar to the synovial fluid of OA patients was able to increase this capacity by increasing their chemotactic power. The microscopy data also support the hypothesis of the ability of MSC-EVs to influence the chondrocytes residing in the ECM of the cartilage and the synovial cells of the synovial membrane through active interaction and the release of their therapeutic content.
The use of microfragmented adipose tissue (mFAT) for the treatment of musculoskeletal disorders, especially osteoarthritis, is gaining popularity following the positive results reported in recent case series and clinical trials. The purpose of this study is to characterize mFAT in terms of structure, cell content and secretome (i.e. protein and microvescicles released as paracrine mediators), and to compare it with unprocessed lipoaspirate tissue, in order to understand the possible mechanisms of action and the benefit derived from tissue processing. Unprocessed lipoaspirate (LA) and mFAT were obtained from 7 donors. Each tissue sample was divided in four aliquots: A) fixed in formalin for histological evaluation; B) enzymatically digested to harvest cells with the exclusion of adipocytes; C) cultured for 24 hours in serum-free DMEM to harvest secretome; D) freshly frozen for proteomic evaluation. Hematoxylin and eosin staning, as well as immunohistochemistry for CD31, CD90, CD146 were performed on aliquot A. Cell count, viability, senescence and immunophenotype were assessed on aliquot B. Culture medium from aliquot C was collected and used for proteomic analysis and micro-RNA extraction and quantitation from extracellular vesicles. Aliquot D was lysed, protein were extracted and analyzed using a high-throughput proteomic approach.Introduction and Objective
Materials and Methods
Hyaluronic acid (HA) is an effective option for the treatment of osteoarthritis (OA) patients due to several properties such as normalization of the mechanical and rheological properties of the synovial fluid and amelioration of OA symptoms and joints function by promoting cartilage nutrition. Since OA progression is also significantly related to oxidative stress and reactive oxygen species (ROS), sodium succinate (SS) is envisioned as a promising compound for cartilage treatment by providing antioxidant defense able to normalize intracellular metabolism and tissue respiration via mitochondrial mechanism of action. The scope of this study was to investigate on an in vitro inflammatory model the efficacy of Diart® product, a combination of HA and SS. Donor-matched chondrocytes and synoviocytes were obtained from KL 3–4 OA patients undergoing total knee replacement. At passage 4, inflammation was promoted with 1 ng/ml IL-1B for 48 hours in absence and presence of Diart® at 1:3 dilution rate. Nitric oxide (NO) from cell culture supernatant was measured by Griess reaction. Mitochondrial and cytoplasmatic ROS evaluation was assessed by flow cytometry with MitoSox and dichlorodihydrofluorescein diacetate (DCFDA) assays. Gene expression of inflammation/oxidative stress-related transcripts (MMP1/MMP3/INOS/COX2) was evaluated by qRT-PCR using TBP as reference.Introduction and Objective
Materials and Methods
The donor-matched comparison between mesenchymal stem cells from knee infrapatellar and subcutaneous adipose tissue revealed their preferential commitment towards the chondrogenic and osteogenic lineage, respectively. These peculiarities could be relevant for the development of successful bone and cartilage cell-based applications. Mesenchymal stem cells (MSCs) have been proposed in bone and cartilage tissue engineering applications as an alternative to terminally differentiated cells. In the present study we characterised and performed a donor-matched comparison between MSCs resident within the infrapatellar fat pad (IFP-MSCs) and the knee subcutaneous adipose tissue (ASCs) of osteoarthritic patients. These two fat depots, indeed, can be considered appealing candidates for orthopaedic cell-based therapies since they are highly accessible during knee surgery.Summary
Introduction
