The secretome of mesenchymal stromal cells (MSCs) derived from different tissue sources is considered an innovative therapeutic tool for regenerative medicine. Although adipose tissue-and bone marrow-derived MSCs (ADSCs and BMSCs, respectively) share many biological features, the different tissue origins can be mirrored by variations in their secretory profile, and in particular in the secreted extracellular vesicles (EVs). In this study, we carried out a detailed and comparative characterization of middle- and small-sized EVs (mEVs and sEVs, respectively) released by either ADSCs or BMSCs. Their involvement in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics. Although EVs separated from both cell sources presented similar characteristics in terms of size, concentration, and marker expression, they exhibited different characteristics in terms of protein content and functional effects. ADSC-EVs overexpressed pro-angiogenic factors in comparison to the BMSC-counterpart, and, consequently, they were able to induce a significant increase in endothelial cord outgrowth. On the other hand, BMSC-EVs contained a higher amount of pro-differentiation and chemotactic proteins, and they were able to prompt growth plate organization. The present study highlights the importance of selecting the appropriate cell source of EVs for targeted therapeutic applications.

Regenerative strategies for human articular cartilage are still challenging despite the presence of resident progenitor cell population. Today, many efforts in the field of regenerative medicine focus on the use of platelet derivatives due to their ability to reactivate endogenous mechanisms supporting tissue repair. While their use in orthopedics continues, mechanisms of action and efficacy need further characterization. We describe that the platelet lysate (PL) is able to activate chondro-progenitor cells in a terminally differentiated cartilage tissue. Primary cultures of human articular chondrocytes (ACs) and cartilage explants were set up from donor hip joint biopsies and were treated in vitro with PL. PL recruited a chondro-progenitors (CPCs)-enriched population from ex vivo cartilage culture, that showed high proliferation rate, clonogenicity and nestin expression. CPCs were positive for in vitro tri-lineage differentiation and formed hyaline cartilage-like tissue in vivo without hypertrophic fate. Moreover, the secretory profile of CPCs was analyzed, together with their migratory capabilities. Some CPC-features were also induced in PL-treated ACs compared to fetal bovine serum (FBS)-control ACs. PL treatment of human articular cartilage activates a stem cell niche responsive to injury. These facts can improve the PL therapeutic efficacy in cartilage applications.

Background: Mesenchymal stem/stromal cells (MSC) have been employed successfully in immunotherapy and regenerative medicine, but their therapeutic potential is reduced considerably by the ischemic environment that exists after transplantation. The assumption that preconditioning MSC to promote quiescence may result in increased survival and regenerative potential upon transplantation is gaining popularity. Methods: The purpose of this work was to evaluate the anti-inflammatory and regenerative effects of human bone marrow MSC (hBM-MSC) and their extracellular vesicles (EVs) grown and isolated in a serum-free medium, as compared to starved hBM-MSC (preconditioned) in streptozotocin-induced diabetic fractured male C57BL/6J mice. Results: Blood samples taken four hours and five days after injection revealed that cells, whether starved or not, generated similar plasma levels of inflammatory-related cytokines but lower levels than animals treated with EVs. Nonetheless, starved cells prompted the highest production of IL-17, IL-6, IL-13, eotaxin and keratinocyte-derived chemokines and induced an earlier soft callus formation and mineralization of the fracture site compared to EVs and regularly fed cells five days after administration. Conclusions: Preconditioning may be crucial for refining and defining new criteria for future MSC therapies. Additionally, the elucidation of mechanisms underpinning an MSC’s survival/adaptive processes may result in increased cell survival and enhanced therapeutic efficacy following transplantation.

Human gut is crucial for digestion, drug absorption, and overall health; however traditional in vitro and animal models struggle to accurately replicate its complex mechanisms. This study introduces an innovative gut‐on‐chip based on the MIVO millifluidic device, designed to faithfully replicate the human intestinal environment. CaCo‐2 and HT‐29 cells were co‐cultured under different ratio under dynamic flow conditions, resembling the bloodstream. Intestinal tissue differentiation was assessed through Trans‐epithelial electrical resistance (TEER) measurements, Zonula Occludens‐1, and Alcian blue staining. After model establishment, a second dynamic flow was applied on the apical side recapitulating the intestinal lumen niche. The dynamic culture conditions significantly reduced cell maturation time, obtaining a differentiated intestinal layer within 7‐10 days, compared to 21 days of static culture. In addition, CaCo‐2:HT‐29 co‐cultures enables to finely tune the mucus thicknesses and barrier function, essential for studying specific conditions. Furthermore, the introduction of a double apical‐basal flow system recapitulated intestinal permeability characteristics more closely resembling those observed in vivo. The Double‐Flow millifluidic Gut‐on‐Chip described and successfully validated enables to cross‐correlate the barrier function of the epithelial layer with the CaCo‐2:HT‐29 cells ratios, finally providing a predictive model useful for drug development and disease modelling.

Bullous pemphigoid (BP) is an autoimmune blistering disease that targets the haemidesmosomal proteins, mainly BP180. Extracellular vesicles (EVs) have been demonstrated to carry tissue-specific autoantigens in the setting of autoimmune diseases and transplant organ rejection; this phenomenon was demonstrated to have pathogenic implications in autoimmune diseases and to correlate with transplant rejection severity. The purpose of this study was to identify the presence of BP targeted autoantigens in blister fluid derived EVs. We isolated, by size exclusion chromatography, EVs derived from blisters of BP-patients and from suction blisters of healthy donors. EV characterization was performed by flow cytometry and nanoparticle tracking analysis. Western blot analysis was used to investigate the presence of autoantigens. A suspension enriched in EVs was efficiently obtained from blister fluid from patients and healthy donors. EV-enriched fractions were enriched in particles with a size distribution characterizing small-EVs (main peak was present at 94.5 nm). BP180 was found, by western blot analysis, in EVs derived from blister fluid of 3 out 6 BP patients and in none of EVs isolated from suction blister fluid of healthy donors. BP230 and Dsg1 were not detectable in EVs of any of the samples. No specific clinical characteristics seemed to correlate to the presence of BP180 in EVs. The discovery of BP180 in EVs derived from blister fluid might help understanding BP pathogenesis.

Gastroesophageal reflux disease (GERD) is a common gastrointestinal disorder, characterized by the retrograde flow of gastric contents into the esophagus, causing symptoms such as heartburn and regurgitation. Although many patients with symptomatic GERD show no visible mucosal injury, impairment of its integrity at microscopic level is considered a relevant factor contributing to symptom generation and inflammation. In this scenario, protection of esophageal mucosa by topical agents has become a potential therapeutic approach. We investigated the protective and regenerative effects of a new natural formulation (GSE Reflusolve Rapid), using a dynamic culture based on MIVO® esophagus-on-chip platform, compared to an alginate/carbonate-based product. Results demonstrate that both products preserved esophageal integrity following acid stimulation, mitigating transepithelial electrical resistance (TEER) decline and upregulating MUC5B expression. Moreover, the natural formulation showed a significant reduction in IL-8 levels (p < 0.05) in contrast to the alginate-based formulation, despite the lack of a buffer action. TEER measurement, FITC-dextran permeability assay and histological analysis showed also the reparative properties of both mucosal protectants after acidic insult. In particular, the natural formulation showed a more pronounced and uniform expression of MUC5B throughout the tissue. These findings highlight that GSE Reflusolve Rapid may be a valid alternative to control long-term GERD-symptoms.