A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate.

Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.

3D printed dental implants with a porous structure: The in vitro response of osteoblasts, fibroblasts, mesenchymal stem cells, and monocytes.

AIMS: The first aim of this study was to characterize the surface topography of a novel 3D-printed dental implant at the micro- and macro-level. Its second aim was to evaluate the osteogenic, angiogenic, and immunogenic responses of human oral osteoblasts (hOBs), gingival fibroblasts (hGFs), mesenchymal stem cells (hAD-MSCs), and monocytes to this novel implant surface. METHODS: A 3D-printed Ti-6Al-4 V implant was produced by selective laser melting and subjected to organic acid etching (TEST). It was then compared to a machined surface (CTRL). Its biological properties were evaluated via cell proliferation assays, morphological observations, gene expression analyses, mineralization assessments, and collagen quantifications. RESULTS: Scanning electron microscopy analysis showed that the TEST group was characterized by a highly interconnected porous architecture and a roughed surface. The morphological observations showed good adhesion of cells cultured on the TEST surface, with a significant increase in hOB growth. Similarly, the gene expression analysis showed significantly higher levels of osseointegration biomarkers. Picrosirius staining showed a slight increase in collagen production in the TEST group compared to the CTRL group. hAD-MSCs showed an increase in endothelial and osteogenic commitment-related markers. Monocytes showed increased mRNA synthesis related to the M2 (anti-inflammatory) macrophagic phenotype. CONCLUSIONS: Considering the higher interaction with hOBs, hGFs, hAD-MSCs, and monocytes, the prepared 3D-printed implant could be used for future clinical applications. CLINICAL RELEVANCE: This study demonstrated the excellent biological response of various cells to the porous surface of the novel 3D-printed implant.

Post-Operative Delirium and Cognitive Dysfunction in Aged Patients Undergoing Cardiac Surgery: A Randomized Comparison between Two Blood Oxygenators.

In elderly patients undergoing cardiac surgery, extracorporeal circulation affects the incidence of post-operative delirium and cognitive impairment with an impact on quality of life and mortality. In this study, a new oxygenator system (RemoweLL 2) was tested against a conventional system to assess its efficacy in reducing the onset of postoperative delirium and cognitive dysfunction and the levels of serum inflammatory markers. A total of 154 patients (>65 y.o.) undergoing cardiopulmonary bypass (CPB) were enrolled and randomly assigned to oxygenator RemoweLL 2 (n = 81) or to gold standard device Inspire (n = 73) between September 2019 and March 2022. The aims of the study were to assess the incidence of delirium and the cognitive decline by neuropsychiatric tests and the MoCa test intra-hospital and at 6 months after CPB. Inflammation biomarkers in both groups were also evaluated. Before the CPB, the experimental groups were comparable for all variables. After CPB, the incidence of severe post-operative delirium showed a better trend (p = 0.093) in patients assigned to RemoweLL 2 (16.0%) versus Inspire (26.0%). Differences in enolase levels (p = 0.049), white blood cells (p = 0.006), and neutrophils (p = 0.003) in favor of RemoweLL 2 were also found. The use of novel and better construction technologies in CPB oxygenator devices results in measurable better neurocognitive and neurological outcomes in the elderly population undergoing CPB.

Mitochondrial Metabolism and EV Cargo of Endothelial Cells Is Affected in Presence of EVs Derived from MSCs on Which HIF Is Activated.

Small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) have attracted growing interest as a possible novel therapeutic agent for the management of different cardiovascular diseases (CVDs). Hypoxia significantly enhances the secretion of angiogenic mediators from MSCs as well as sEVs. The iron-chelating deferoxamine mesylate (DFO) is a stabilizer of hypoxia-inducible factor 1 and consequently used as a substitute for environmental hypoxia. The improved regenerative potential of DFO-treated MSCs has been attributed to the increased release of angiogenic factors, but whether this effect is also mediated by the secreted sEVs has not yet been investigated. In this study, we treated adipose-derived stem cells (ASCs) with a nontoxic dose of DFO to harvest sEVs (DFO-sEVs). Human umbilical vein endothelial cells (HUVECs) treated with DFO-sEVs underwent mRNA sequencing and miRNA profiling of sEV cargo (HUVEC-sEVs). The transcriptomes revealed the upregulation of mitochondrial genes linked to oxidative phosphorylation. Functional enrichment analysis on miRNAs of HUVEC-sEVs showed a connection with the signaling pathways of cell proliferation and angiogenesis. In conclusion, mesenchymal cells treated with DFO release sEVs that induce in the recipient endothelial cells molecular pathways and biological processes strongly linked to proliferation and angiogenesis.

Playing with Biophysics: How a Symphony of Different Electromagnetic Fields Acts to Reduce the Inflammation in Diabetic Derived Cells.

Several factors, such as ischemia, infection and skin injury impair the wound healing process. One common pathway in all these processes is related to the reactive oxygen species (ROS), whose production plays a vital role in wound healing. In this view, several strategies have been developed to stimulate the activation of the antioxidative system, thereby reducing the damage related to oxidative stress and improving wound healing. For this purpose, complex magnetic fields (CMFs) are used in this work on fibroblast and monocyte cultures derived from diabetic patients in order to evaluate their influence on the ROS production and related wound healing properties. Biocompatibility, cytotoxicity, mitochondrial ROS production and gene expression have been evaluated. The results confirm the complete biocompatibility of the treatment and the lack of side effects on cell physiology following the ISO standard indication. Moreover, the results confirm that the CMF treatment induced a reduction in the ROS production, an increase in the macrophage M2 anti-inflammatory phenotype through the activation of miRNA 5591, a reduction in inflammatory cytokines, such as interleukin-1 (IL-1) and IL-6, an increase in anti-inflammatory ones, such as IL-10 and IL-12 and an increase in the markers related to improved wound healing such as collagen type I and integrins. In conclusion, our findings encourage the use of CMFs for the treatment of diabetic foot.

Methods to isolate adipose tissue-derived stem cells.

The use of adipose tissue has seen increasing interest in recent years for treating plastic surgery defects and for regenerative medicine applications. Adipose tissue is considered an optimal source of stem cells, as it contains more multipotent cells than bone marrow for the same volume. The adipose tissue-derived stem cells (ADSCs), isolated from the heterogeneous stromal vascular fraction (SVF), possess self-renewal properties and multilineage differentiation potential. In addition, adipose tissue can be obtained with less invasive procedures and patient morbidity than other tissue. For these reasons, numerous enzymatic, and non-enzymatic isolation methods have been developed over the years. The traditional method for isolation and culture of primary ADSCs from adipose tissue relies on enzymatic digestion with collagenase, followed by multiple steps of centrifugation. Alternative non-enzymatic isolation methods are based closed, sterile, and safe isolation processes that differ from each other for parameters such as the centrifugation force, pressure, filtration, and washing. Despite the existence of this multitude of systems, the best isolation method has not been identified to date. Therefore, the great challenge remains the achievement of the standardization of cellular products to allow the comparability between clinical studies and trials.

Exosomes of mesenchymal stem cells delivered from methacrylated hyaluronic acid patch improve the regenerative properties of endothelial and dermal cells.

Wound care management urgently needs the development of innovative smart wound dressings. The complexity of the wound often requires the use of personalized medication and the advent of three-dimensional (3D) bioprinting fits strongly with this need. In this view, in the present work a methacrylated hyaluronic acid (MeHA) bioink was tested for the fabrication of advanced smart patches as a delivery system of exosomes derived from human mesenchymal stem cells (hMSC-EXOs) suitable for wound healing purposes. MeHA patches were realized by 3D bioprinting technique and they were loaded with hMSC-EXOs. The 3D printed MeHA patches revealed improved mechanical performance, appropriate swelling ratio, extended degradation time, and suitable biocompatibility. Furthermore, MeHA patches loaded with hMSC-EXOs improved the proliferation, migration, angiogenic ability, and expression of specific markers related to wound healing process in human fibroblasts and human endothelial cells.

Biomaterials for Regenerative Medicine in Italy: Brief State of the Art of the Principal Research Centers.

Regenerative medicine is the branch of medicine that effectively uses stem cell therapy and tissue engineering strategies to guide the healing or replacement of damaged tissues or organs. A crucial element is undoubtedly the biomaterial that guides biological events to restore tissue continuity. The polymers, natural or synthetic, find wide application thanks to their great adaptability. In fact, they can be used as principal components, coatings or vehicles to functionalize several biomaterials. There are many leading centers for the research and development of biomaterials in Italy. The aim of this review is to provide an overview of the current state of the art on polymer research for regenerative medicine purposes. The last five years of scientific production of the main Italian research centers has been screened to analyze the current advancement in tissue engineering in order to highlight inputs for the development of novel biomaterials and strategies.

Biological Characterization of Human Autologous Pericardium Treated with the Ozaki Procedure for Aortic Valve Reconstruction.

BACKGROUND: The Ozaki procedure is an innovative surgical technique aiming at reconstructing aortic valves with human autologous pericardium. Even if this procedure is widely used, a comprehensive biological characterization of the glutaraldehyde (GA)-fixed pericardial tissue is still missing. METHODS: Morphological analysis was performed to assess the general organization of pericardium subjected to the Ozaki procedure (post-Ozaki) in comparison to native tissue (pre-Ozaki). The effect of GA treatment on cell viability and nuclear morphology was then investigated in whole biopsies and a cytotoxicity assay was executed to assess the biocompatibility of pericardium. Finally, human umbilical vein endothelial cells were seeded on post-Ozaki samples to evaluate the influence of GA in modulating the endothelialization ability in vitro and the production of pro-inflammatory mediators. RESULTS: The Ozaki procedure alters the arrangement of collagen and elastic fibers in the extracellular matrix and results in a significant reduction in cell viability compared to native tissue. GA treatment, however, is not cytotoxic to murine fibroblasts as compared to a commercially available bovine pericardium membrane. In addition, in in vitro experiments of endothelial cell adhesion, no difference in the inflammatory mediators with respect to the commercial patch was found. CONCLUSIONS: The Ozaki procedure, despite alteration of ECM organization and cell devitalization, allows for the establishment of a noncytotoxic environment in which endothelial cell repopulation occurs.

Elastomeric Cardiowrap Scaffolds Functionalized with Mesenchymal Stem Cells-Derived Exosomes Induce a Positive Modulation in the Inflammatory and Wound Healing Response of Mesenchymal Stem Cell and Macrophage.

A challenge in contractile restoration of myocardial scars is one of the principal aims in cardiovascular surgery. Recently, a new potent biological tool used within healing processes is represented by exosomes derived from mesenchymal stem cells (MSCs). These cells are the well-known extracellular nanovesicles released from cells to facilitate cell function and communication. In this work, a combination of elastomeric membranes and exosomes was obtained and tested as a bioimplant. Mesenchymal stem cells (MSCs) and macrophages were seeded into the scaffold (polycaprolactone) and filled with exosomes derived from MSCs. Cells were tested for proliferation with an MTT test, and for wound healing properties and macrophage polarization by gene expression. Moreover, morphological analyses of their ability to colonize the scaffolds surfaces have been further evaluated. Results confirm that exosomes were easily entrapped onto the surface of the elastomeric scaffolds, increasing the wound healing properties and collagen type I and vitronectin of the MSC, and improving the M2 phenotype of the macrophages, mainly thanks to the increase in miRNA124 and decrease in miRNA 125. We can conclude that the enrichment of elastomeric scaffolds functionalized with exosomes is as an effective strategy to improve myocardial regeneration.

Electrospun PCL-Based Vascular Grafts: In Vitro Tests.

BACKGROUND: Electrospun fibers have attracted a lot of attention from researchers due to their several characteristics, such as a very thin diameter, three-dimensional topography, large surface area, flexible surface, good mechanical characteristics, suitable for widespread applications. Indeed, electro-spinning offers many benefits, such as great surface-to-volume ratio, adjustable porosity, and the ability of imitating the tissue extra-cellular matrix. METHODS: we processed Poly ε-caprolactone (PCL) via electrospinning for the production of bilayered tubular scaffolds for vascular tissue engineering application. Endothelial cells and fibroblasts were seeded into the two side of the scaffolds: endothelial cells onto the inner side composed of PCL/Gelatin fibers able to mimic the inner surface of the vessels, and fibroblasts onto the outer side only exposing PCL fibers. Extracellular matrix production and organization has been performed by means of classical immunofluorescence against collagen type I fibers, Scanning Electron-Microscopy (SEM) has been performed in order to evaluated ultrastructural morphology, gene expression by means gene expression has been performed to evaluate the phenotype of endothelial cells and fibroblasts. RESULTS AND CONCLUSION: results confirmed that both cells population are able to conserve their phenotype colonizing the surface supporting the hypothesis that PCL scaffolds based on electrospun fibers should be a good candidate for vascular surgery.

Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

Citrate, generated in the mitochondria, is a key metabolite that might link metabolism with signaling, chromatin structure and transcription to orchestrate mesenchymal stem cells (MSCs) fate determination. Based on a detailed morphological analysis of 3D reconstruction of mitochondria and nuclei in single cells, we identified contact sites between these organelles that drastically increase in volume and number during the early stage of mesenchymal stem cell differentiation. These contact sites create a microdomain that facilitates exchange of signals from mitochondria to the nucleus. Interestingly, we found that the citrate derived from mitochondria is necessary for osteogenic lineage determination. Indeed, inhibition of the citrate transporter system dramatically affected osteogenesis, reduced citrate levels that could be converted in α-ketoglutarate, and consequently affected epigenetic marker H3K9me3 associated with the osteogenesis differentiation process. These findings highlight that mitochondrial metabolites play key regulatory roles in the MSCs differentiation process. Further in-depth investigation is needed to provide novel therapeutic strategies in the field of regenerative medicine.

Albumin-impregnated bone granules modulate the interactions between mesenchymal stem cells and monocytes under in vitro inflammatory conditions.

Bone regeneration around newly implanted biomaterials is a complex process, which in its early phases involves the interactions between Mesenchymal Stem Cells (MSCs) and immune cells. The response of these cells to the biomaterial depends both on the local microenvironment and on the characteristics of the inserted bone substitute. In this work, bone allografts impregnated with albumin are loaded with a co-culture of human MSCs and monocytes; bone granules without albumin are used for comparison. Co-cultures are contextually treated with pro-inflammatory cytokines to simulate the inflammatory milieu naturally present during the bone regeneration process. As revealed by microscopic images, albumin-impregnated bone granules promote adhesion and interactions between cells populations. Compared to control granules, albumin coating diminishes reactive species production by cells. This reduced oxidative stress may be attributable to antioxidant properties of albumin, and it is also reflected in the mitigated gene expression of mitochondrial electron transport chain complexes, where most intracellular reactive molecules are generated. MSCs-monocytes co-cultured onto albumin-impregnated bone granules additionally release higher amounts of immunomodulatory cytokines and growth factors. In summary, this work demonstrates that impregnation of bone granules with albumin positively modulates the interactions between MSCs and immune cells, consequently influencing their mutual activities and immunomodulatory functions.

Hyperbaric Oxygen Therapy Improves the Osteogenic and Vasculogenic Properties of Mesenchymal Stem Cells in the Presence of Inflammation In Vitro.

Hyperbaric oxygen (HBO) therapy has been reported to be beneficial for treating many conditions of inflammation-associated bone loss. The aim of this work was to in vitro investigate the effect of HBO in the course of osteogenesis of human Mesenchymal Stem Cells (MSCs) grown in a simulated pro-inflammatory environment. Cells were cultured with osteogenic differentiation factors in the presence or not of the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α), and simultaneously exposed daily for 60 min, and up to 21 days, at 2,4 atmosphere absolute (ATA) and 100% O2. To elucidate osteogenic differentiation-dependent effects, cells were additionally pre-committed prior to treatments. Cell metabolic activity was evaluated by means of the MTT assay and DNA content quantification, whereas osteogenic and vasculogenic differentiation was assessed by quantification of extracellular calcium deposition and gene expression analysis. Metabolic activity and osteogenic properties of cells did not differ between HBO, high pressure (HB) alone, or high oxygen (HO) alone and control if cells were pre-differentiated to the osteogenic lineage. In contrast, when treatments started contextually to the osteogenic differentiation of the cells, a significant reduction in cell metabolic activity first, and in mineral deposition at later time points, were observed in the HBO-treated group. Interestingly, TNF-α supplementation determined a significant improvement in the osteogenic capacity of cells subjected to HBO, which was not observed in TNF-α-treated cells exposed to HB or HO alone. This study suggests that exposure of osteogenic-differentiating MSCs to HBO under in vitro simulated inflammatory conditions enhances differentiation towards the osteogenic phenotype, providing evidence of the potential application of HBO in all those processes requiring bone regeneration.

Characterization of Dermal Stem Cells of Diabetic Patients.

Diabetic foot ulcers (DFUs) are lesions that involve loss of epithelium and dermis, sometimes involving deep structures, compartments, and bones. The aim of this work is to investigate the innate regenerative properties of dermal tissue around ulcers by the identification and analysis of resident dermal stem cells (DSCs). Dermal samples were taken at the edge of DFUs, and genes related to the wound healing process were analyzed by the real-time PCR array. The DSCs were isolated and analyzed by immunofluorescence, flow cytometry, and real-time PCR array to define their stemness properties. The gene expression profile of dermal tissue showed a dysregulation in growth factors, metalloproteinases, collagens, and integrins involved in the wound healing process. In the basal condition, diabetic DSCs adhered on the culture plate with spindle-shaped fibroblast-like morphology. They were positive to the mesenchymal stem cells markers CD44, CD73, CD90, and CD105, but negative for the hematopoietic markers CD14, CD34, CD45, and HLA-DR. In diabetic DSCs, the transcription of genes related to self-renewal and cell division were equivalent to that in normal DSCs. However, the expression of CCNA2, CCND2, CDK1, ALDH1A1, and ABCG2 was downregulated compared with that of normal DSCs. These genes are also related to cell cycle progression and stem cell maintenance. Further investigation will improve the understanding of the molecular mechanisms by which these genes together govern cell proliferation, revealing new strategies useful for future treatment of DFUs.

Bovine pericardium membrane as new tool for mesenchymal stem cells commitment.

Acellular matrices are widespread biomaterials used in surgical practice as tissue reinforcement and anatomical support to favor tissue regeneration. It is clear that a fundamental role in the regeneration of tissue is played by cell-material interaction. In this work, the interaction between a bovine pericardium membrane and human adult stem cells was investigated by microscopy analysis and gene expression analysis. Parallel cell cultures were prepared on the pericardium membrane or tissue culture plate. They were incubated in basal growth medium or in adipogenic differentiation medium to perform experiments on the seventh and the 14th day of culture. Results demonstrated that the membrane allows cell viability, adhesion, and proliferation of human stem cells. During adipogenic commitment on the membrane, the accumulation of cytoplasmatic lipid droplets and the expression of adipogenic gene PPARG, CEBPA, GLUT4, FABP4, and ADIPOQ were detected. Concurrently, a downregulation of mesenchymal stem cell gene CD29, CD90, and CD105 was detected. In basal medium, the adipogenic gene expression was upregulated, whereas the mesenchymal markers were indifferently expressed. These findings suggest that the bovine pericardium membrane is a biocompatible matrix and that their rough surface allows cell adhesion, spreading, and proliferation. The surface morphology activates mechanochemical signals that stimulate the adipogenic commitment of stem cells in basal medium and potentiate their commitment in adipogenic differentiation medium.

Effects of novel antidepressant drugs on mesenchymal stem cell physiology.

It is known that users of psychotropic drugs often have weight gain, adverse effects on bone mineral density and osteoporosis, but the molecular basis for these side effects is poorly understood. The aim of this study is to evaluate the effects in vitro of duloxetine (a serotonin and norepinephrine reuptake inhibitor) and fluoxetine (a selective serotonin reuptake inhibitor) on the physiology of human adult stem cells. Adipose-derived stem cells (ADSCs) were isolated and characterized investigating phenotype morphology, expression and frequency of surface markers. Then, a non-toxic concentration of duloxetine and fluoxetine was selected to treat cells during adipogenic and osteogenic differentiation. Stemness properties and the differentiation potential of drug-treated cells were investigated by the quantification of adipogenic and osteogenic markers gene expression and histological staining. The collected data showed that the administration of a daily non-toxic dose of duloxetine and fluoxetine has not directly influenced ADSCs proliferation and their stemness properties. The treatment with duloxetine or fluoxetine did not lead to morphological alterations during adipogenic or osteogenic commitment. However, treatments with the antidepressant showed a slight difference in adipogenic gene expression timing. Furthermore, duloxetine treatment caused an advance in gene expression of early and late osteogenic markers. Fluoxetine instead caused an increase in expression of osteogenic genes compared to untreated cells. In contrast, in pre-differentiated cells, the daily treatment with duloxetine or fluoxetine did not alter the expression profile of adipogenic and osteogenic differentiation. In conclusion, a non-toxic concentration of duloxetine and fluoxetine does not alter the stemness properties of ADSCs and does not prevent the commitment of pre-differentiated ADSCs in adipocytes or osteocyte. Probably, the weight gain and osteoporotic effects associated with the use of psychotropic drugs could be closely related to the direct action of serotonin.

Regulation of PKCß levels and autophagy by PML is essential for high-glucose-dependent mesenchymal stem cell adipogenesis.

BACKGROUND/OBJECTIVES: Obesity is a complex disease characterized by the accumulation of excess body fat, which is caused by an increase in adipose cell size and number. The major source of adipocytes comes from mesenchymal stem cells (MSCs), although their roles in obesity remain unclear. An understanding of the mechanisms, regulation, and outcomes of adipogenesis is crucial for the development of new treatments for obesity-related diseases. Recently an unexpected role for the tumor suppressor promyelocytic leukemia protein (PML) in hematopoietic stem cell biology and metabolism regulation has come to light, but its role in MSC biology remains unknown. Here, we investigated the molecular pathway underlying the role of PML in the control of adipogenic MSC differentiation. SUBJECTS/METHODS: Muscle-derived stem cells (MDSCs) and adipose-derived stem cells (ADSCs) obtained from mice and voluntary patients (as a source of MSCs) were cultured in the presence of high glucose (HG) concentration, a nutrient stress condition known to promote MSCs differentiation into mature adipocytes and the adipogenic potential of PML was assessed. RESULTS: PML is essential for a correct HG-dependent adipogenic differentiation, and the enhancement of PML levels is fundamental during adipogenesis. Increased PML expression enables the upregulation of protein kinase Cß (PKCß), which, in turn, by controlling autophagy levels permits an increase in peroxisome proliferator-activated receptor γ (PPARγ) that leads the adipogenic differentiation. Therefore, genetic and pharmacological depletion of PML prevents PKCß expression, and by increasing autophagy levels, impairs the MSCs adipogenic differentiation. Human ADSCs isolated from overweight patients displayed increased PML and PKCß levels compared to those found in normal weight individuals, indicating that the PML-PKCß pathway is directly involved in the enhancement of adipogenesis and human metabolism. CONCLUSIONS: The new link found among PML, PKCß, and autophagy opens new therapeutic avenues for diseases characterized by an imbalance in the MSCs differentiation process, such as metabolic syndromes and cancer.

In Vitro Effect of Bromelain on the Regenerative Properties of Mesenchymal Stem Cells.

BACKGROUND: Bromelain belongs to a group of protein-digesting enzymes obtained commercially from the fruit or stem of pineapple. Several studies demonstrated that bromelain exhibits various fibrinolytic, anti-edematous, antithrombotic, and anti-inflammatory activities supporting its application for many therapeutic benefits. The aim of this study was to analyze the effects of bromelain on the pro-wound healing activities and the regenerative properties of mesenchymal stem cells. METHODS: Mesenchymal stem cells were treated in vitro with bromelain alone or combined with dexamethasone sodium phosphate. Real-time polymerase chain reaction was performed to profile the expression of extracellular matrix components and remodeling enzymes, and cytokines. RESULTS: The combination of bromelain and dexamethasone sodium phosphate induced a great activation of mesenchymal stem cells with an increase in hyaluronan and collagen production and anti-inflammatory cytokines release. CONCLUSION: Based on the results of this in vitro study, the combined use of bromelain and dexamethasone sodium phosphate stimulated the pro-wound healing activities and the regenerative properties of mesenchymal stem cells better than bromelain and dexamethasone alone.

Therapeutic Potential of Autologous Adipose-Derived Stem Cells for the Treatment of Liver Disease.

Currently, the most effective therapy for liver diseases is liver transplantation, but its use is limited by organ donor shortage, economic reasons, and the requirement for lifelong immunosuppression. Mesenchymal stem cell (MSC) transplantation represents a promising alternative for treating liver pathologies in both human and veterinary medicine. Interestingly, these pathologies appear with a common clinical and pathological profile in the human and canine species; as a consequence, dogs may be a spontaneous model for clinical investigations in humans. The aim of this work was to characterize canine adipose-derived MSCs (cADSCs) and compare them to their human counterpart (hADSCs) in order to support the application of the canine model in cell-based therapy of liver diseases. Both cADSCs and hADSCs were successfully isolated from adipose tissue samples. The two cell populations shared a common fibroblast-like morphology, expression of stemness surface markers, and proliferation rate. When examining multilineage differentiation abilities, cADSCs showed lower adipogenic potential and higher osteogenic differentiation than human cells. Both cell populations retained high viability when kept in PBS at controlled temperature and up to 72 h, indicating the possibility of short-term storage and transportation. In addition, we evaluated the efficacy of autologous ADSCs transplantation in dogs with liver diseases. All animals exhibited significantly improved liver function, as evidenced by lower liver biomarkers levels measured after cells transplantation and evaluation of cytological specimens. These beneficial effects seem to be related to the immunomodulatory properties of stem cells. We therefore believe that such an approach could be a starting point for translating the results to the human clinical practice in future.