Cellular In Vitro Responses Induced by Human Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Obtained from Suspension Culture.

Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-ß and IL-1ß was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.

Functionalized Hydrogels for Cartilage Repair: The Value of Secretome-Instructive Signaling.

Cartilage repair has been a challenge in the medical field for many years. Although treatments that alleviate pain and injury are available, none can effectively regenerate the cartilage. Currently, regenerative medicine and tissue engineering are among the developed strategies to treat cartilage injury. The use of stem cells, associated or not with scaffolds, has shown potential in cartilage regeneration. However, it is currently known that the effect of stem cells occurs mainly through the secretion of paracrine factors that act on local cells. In this review, we will address the use of the secretome-a set of bioactive factors (soluble factors and extracellular vesicles) secreted by the cells-of mesenchymal stem cells as a treatment for cartilage regeneration. We will also discuss methodologies for priming the secretome to enhance the chondroregenerative potential. In addition, considering the difficulty of delivering therapies to the injured cartilage site, we will address works that use hydrogels functionalized with growth factors and secretome components. We aim to show that secretome-functionalized hydrogels can be an exciting approach to cell-free cartilage repair therapy.

Bioactive decellularized extracellular matrix-based hydrogel supports human adipose tissue-derived stem cell maintenance and fibrocartilage phenotype.

Articular cartilage is a highly specialized tissue able to tolerate physical stress. However, its capacity for restoration is restricted, and injuries to the cartilage do not recover spontaneously. Interest in mesenchymal stem cells derived from human adipose tissue (hASCs) is growing due to their potential to improve tissue healing and recovery. Decellularized extracellular matrix (dECM)-based hydrogels combined with hASCs could serve as an interface for studying behavior and differentiation properties in a cartilage microenvironment. In the present study, we described the behavior of hASCs cultured in a commercial dECM MatriXpec™. The structural microtopography of MatriXpec™ was analyzed by scanning electron micrography, and its protein composition was accessed by mass spectrometry. The protein composition of MatriXpec™ is mainly represented by collagen proteins, building its fibrous ultrastructure. hASCs were cultured three-dimensionally (3D) on MatriXpec™ to perform cell viability, growth, and cartilage differentiation analysis. We showed that MatriXpec™ could be loaded with hASCs and that it supports cell maintenance for several days. We observed that the three-dimensional ultrastructure of the biomaterial is composed of nanofibers, and its protein composition reflects the tissue from which it was harvested. Finally, we showed that the molecular cues from the hydrogel are biologically active as these influence cell behavior and differentiation phenotype, increasing the expression of fibrocartilage-related genes such as SOX9, COL1, COL10, and MMP13. MatriXpec™ hydrogel can be used as an interface for 3D hASCs culture studies as it maintains cell viability and supports its differentiation process.

Evaluation of a Peptide Hydrogel as a Chondro-Instructive Three-Dimensional Microenvironment.

Articular cartilage injuries are inherently irreversible, even with the advancement in current therapeutic options. Alternative approaches, such as the use of mesenchymal stem/stromal cells (MSCs) and tissue engineering techniques, have gained prominence. MSCs represent an ideal source of cells due to their low immunogenicity, paracrine activity, and ability to differentiate. Among biomaterials, self-assembling peptide hydrogels (SAPH) are interesting given their characteristics such as good biocompatibility and tunable properties. Herein we associate human adipose-derived stem cells (hASCs) with a commercial SAPH, Puramatrix™, to evaluate how this three-dimensional microenvironment affects cell behavior and its ability to undergo chondrogenic differentiation. We demonstrate that the Puramatrix™ hydrogel comprises a highly porous matrix permissible for hASC adhesion and in vitro expansion. The morphology and cell growth dynamics of hASCs were affected when cultured on the hydrogel but had minimal alteration in their immunophenotype. Interestingly, hASCs spontaneously formed cell aggregates throughout culturing. Analysis of glycosaminoglycan production and gene expression revealed a noteworthy and donor-dependent trend suggesting that Puramatrix™ hydrogel may have a natural capacity to support the chondrogenic differentiation of hASCs. Altogether, the results provide a more comprehensive understanding of the potential applications and limitations of the Puramatrix™ hydrogel in developing functional cartilage tissue constructs.