Background: Mesenchymal stem cells (MSCs) secrete a cocktail of growth factors and cytokines, which could promote tissue regeneration and wound healing. Therefore, in clinical practice, post-culture MSC supernatant treatment could be a more attractive alternative to autologous stem cell transplantation. In this study, we compared the regenerative properties of supernatants harvested from four newly established human adipose tissue mesenchymal stem cell lines (HATMSCs) derived from chronic wound patients or healthy donors. Methods: HATMSC supernatants were produced in a serum-free medium under hypoxia and their content was analyzed by a human angiogenesis antibody array. The regenerative effect of HATMSCs supernatants was investigated in an in vitro model of chronic wound, where cells originating from human skin, such as microvascular endothelial cells (HSkMEC.2), keratinocytes (HaCaT), and fibroblasts (MSU-1.1), were cultured in serum-free and oxygen-reduced conditions. The effect of supernatant treatment was evaluated using an MTT assay and light microscopy. In addition, fibroblasts and HATMSCs were labeled with PKH67 and PKH26 dye, respectively, and the effect of supernatant treatment was compared to that obtained when fibroblasts and HATMSCs were co-cultured, using flow cytometry and fluorescent microscopy. Results: A wide panel of angiogenesis-associated cytokines such as angiogenin, growth-regulated oncogene (GRO), interleukin-6 and 8 (IL-6, IL-8), vascular endothelial growth factor (VEGF), insulin growth factor 1 (IGF-1), and matrix metalloproteinase (MMP) were found in all tested HATMSCs supernatants. Moreover, supernatant treatment significantly enhanced the survival of fibroblasts, endothelial cells, and keratinocytes in our chronic wound model in vitro. Importantly, we have shown that in in vitro settings, HATMSC supernatant treatment results in superior fibroblast proliferation than in the case of co-culture with HATMSCs. Conclusions: Our results suggest that therapy based on bioactive factors released by the immortalized atMSC into supernatant has important effect on skin-derived cell proliferation and might preclude the need for a more expensive and difficult cell therapy approach to improve chronic wound healing.

Mesenchymal stem cells (MSCs) can improve chronic wound healing; however, recent studies suggest that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells, referred to as the MSC secretome. To overcome difficulties related to the translation of cell therapy into clinical use such as efficacy, safety and cost, we propose a hydrogel loaded with a secretome from the recently established human adipose tissue mesenchymal stem cell line (HATMSC2) as a potential treatment for chronic wounds. Biocompatibility and biological activity of hydrogel-released HATMSC2 supernatant were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR. The results demonstrated that the therapeutic effect of the HATMSC2-produced factors is maintained following incorporation into collagen hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. In addition, HATMSC2 supernatant revealed antimicrobial activity, and which therefore, in combination with the hydrogel has a potential to be used as advanced wound-healing dressing.

For twenty-five years, attempts have been made to use MSCs in the treatment of various diseases due to their regenerative and immunomodulatory properties. However, the results are not satisfactory. Assuming that MSCs can be replaced in some therapies by the active factors they produce, the immortalized MSCs line was established from human adipose tissue (HATMSC1) to produce conditioned media and test its regenerative potential in vitro in terms of possible clinical application. The production of biologically active factors by primary MSCs was lower compared to the HATMSC1 cell line and several factors were produced only by the cell line. It has been shown that an HATMSC1-conditioned medium increases the proliferation of various cell types, augments the adhesion of cells and improves endothelial cell function. It was found that hypoxia during culture resulted in an augmentation in the pro-angiogenic factors production, such as VEGF, IL-8, Angiogenin and MCP-1. The immunomodulatory factors caused an increase in the production of GM-CSF, IL-5, IL-6, MCP-1, RANTES and IL-8. These data suggest that these factors, produced under different culture conditions, could be used for different medical conditions, such as in regenerative medicine, when an increased concentration of pro-angiogenic factors may be beneficial, or in inflammatory diseases with conditioned media with a high concentration of immunomodulatory factors.

Cell-free therapy is gaining increasing interest among researchers as an alternative to mesenchymal stem/stromal cell (MSC) therapy. Since the therapeutic effects of MSCs rely predominantly on their paracrine activity, the use of their secretome as a therapeutic agent in broadly defined regenerative medicine, including cardiac regeneration, appears to be a rational approach. In this review, we discuss recent studies that employed secretomes derived from various types of MSCs in cardiomyocyte regeneration following myocardial infarction (MI). Special attention is given to the protein components of the secretome, which may drive tissue repair, and methods of priming the MSC to achieve secretome composition tailored for heart regeneration. Finally, we summarize recent preclinical findings on the effects of MSC secretomes on cardiomyocyte regeneration.

Research into bioprosthetic heart valve (BHV) materials seeks to enhance durability and biocompatibility, particularly by mitigating vascular calcification (VC). VC entails hydroxyapatite deposition in blood vessel walls through vascular smooth muscle cells (VSMCs) phenotypic switching to osteogenic differentiation, driven by pro-calcific signals that might be inhibited by fetuin-A. To our knowledge, no prior studies have reported fetuin-coated bovine vein tissue for this purpose. This study aimed to evaluate the cytotoxicity, biocompatibility, and anti-calcification potential of fetuin-coated biomaterial. The effectiveness of fetuin coating on biomaterial was characterized using photoluminescence (PL), FTIR and Raman spectroscopy, while fetuin release was quantified. Biocompatibility was evaluated in vitro on VSMCs and human iliac artery endothelial cells (HIAE-78T) and in vivo by subcutaneous implantation in Wistar rats. The effects of fetuin were investigated in two in vitro calcification models based on 2 mM inorganic phosphate, or 10 mM β-glycerophosphate in an organic model. The induction of osteogenic markers such as osterix, bone morphogenic protein 2, and collagen type 1, osteocalcin and osteopontin, was assessed at both the mRNA and protein levels. Detoxification of glutaraldehyde-fixed biomaterial using glycine and NaBH₄ eliminated cytotoxicity, markedly enhancing biocompatibility toward VSMCs, HIAE-78T and subcutaneous rat implants, which showed no acute rejection and preserved structural integrity. Fetuin coating, with ~30% sustained release over three weeks, significantly inhibited mineralization on non-cellular and organic VSMCs models by decreasing mineral deposition, osteogenic markers expression. These results position fetuin-coated bovine vein biomaterial as a novel, promising anti-calcification strategy enhancing long-term durability and cardiac valves safety.