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Olive tree (Olea europaea) leaf extract (OELE) has important antioxidant and anti-inflammatory properties, supporting its use in human clinical practice. We recently designed an amorphous hydrogel called EHO-85 (EHO indicates olive leaf extract in Spanish) containing OELE for skin ulcer treatments. Yet, its effectiveness has not been previously compared with other products used in routine clinical practice. This is necessary to evaluate its potential translation to the human clinic. Thus, in this study, the effect of EHO-85 on healing was evaluated in comparison with treatments containing Indian/Asiatic pennywort (Centella asiatica), hyaluronic acid, or dexpanthenol in a rat model. The speed of wound closure and histological parameters after seven and 14 days were analyzed. All treatments accelerated wound closure, but there were differences between them. Dexpanthenol after seven days produced the highest epithelialization and the lowest inflammation and vascularization. EHO-85 also promoted epithelialization and reduced vascularization. After 14 days, wounds treated with EHO-85 showed less inflammation and higher levels of collagen in the extracellular matrix. This indicates a higher degree of maturity in the regenerated tissue. In conclusion, the effect of EHO-85 on healing was equal to or superior to that of other treatments routinely used in human clinical practice. Therefore, these results, together with previous data on the effects of this hydrogel on ulcer healing in humans, indicate that EHO-85 is a suitable, low-cost, and efficient therapeutic option for wound healing.

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Bone metabolism is regulated by osteoblasts, osteoclasts, osteocytes, and stem cells. Pathologies such as osteoporosis, osteoarthritis, osteonecrosis, and traumatic fractures require effective treatments that favor bone formation and regeneration. Among these, cell therapy based on mesenchymal stem cells (MSC) has been proposed. MSC are osteoprogenitors, but their regenerative activity depends in part on their paracrine properties. These are mainly mediated by extracellular vesicle (EV) secretion. EV modulates regenerative processes such as inflammation, angiogenesis, cell proliferation, migration, and differentiation. Thus, MSC-EV are currently an important tool for the development of cell-free therapies in regenerative medicine. This review describes the current knowledge of the effects of MSC-EV in the different phases of bone regeneration. MSC-EV has been used by intravenous injection, directly or in combination with different types of biomaterials, in preclinical models of bone diseases. They have shown great clinical potential in regenerative medicine applied to bone. These findings should be confirmed through standardization of protocols, a better understanding of the mechanisms of action, and appropriate clinical trials. All that will allow the translation of such cell-free therapy to human clinic applications.

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DPP4 may play a relevant role in MSC differentiation into osteoblasts or adipocytes. Dipeptidyl peptidase 4 (DPP4) inhibitors (DPP4i), such as sitagliptin and vildagliptin, are used as antidiabetic drugs. However, vildagliptin is not a specific DPP4i and also inhibits DPP8/9, which is involved in energy metabolism and immune regulation. The aim of this study is to evaluate how sitagliptin, vildagliptin or 1G244 (a DPP8/9 specific inhibitor) may influence cell viability, as well as osteogenic and adipogenic differentiation in human mesenchymal stem cells (MSC). Viability, apoptosis, osteoblastogenesis and adipogenesis markers, as well as protein synthesis of ß-catenin, were studied in MSC cultures induced to differentiate into osteoblasts or adipocytes in the presence or absence of sitagliptin, vildagliptin or 1G244. The two tested DPP4i did not affect MSC viability, but 1G244 significantly decreased it in MSC and osteoblast-induced cells. Additionally, 1G244 and vildagliptin inhibited osteogenesis and adipogenesis, unlike sitagliptin. Therefore, inhibition of DPP4 did not affect MSC viability and differentiation, whereas inhibition of DPP8/9 negatively affected MSC. To the best of our knowledge, these results show for the first time that DPP8/9 have an important role in the viability and differentiation of human MSC. This data can be considered for human clinical use of drugs affecting DPP8/9 activity.

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Objetivos: las células madre mesenquimales (MSC) se caracterizan por su actividad antiinflamatoria, inmunosupresora y sucapacidad de diferenciación. Esto las convierten en una interesante herramienta terapéutica en terapia celular y medicinaregenerativa. En parte, el efecto terapéutico de las MSC, está mediado por la secreción de vesículas extracelulares (EV). Elprecondicionamiento en hipoxia de las MSC puede mejorar la capacidad regenerativa de las EV secretadas. En este con-texto, el objetivo del estudio ha sido evaluar si EV derivadas de MSC humanas cultivadas en hipoxia y normoxia afectan ala osteoblastogénesis y adipogénesis de las MSC.Material y métodos: se aislaron EV de MSC mantenidas 48 h en normoxia o hipoxia (3 % O2) mediante ultrafiltración ycromatografía de exclusión por tamaño. Las EV fueron caracterizadas por “Western blot”, microscopía electrónica y análisisde seguimiento de nanopartículas. En cultivos de MSC se evaluó el efecto de las EV sobre la viabilidad por ensayo con MTT,la migración por “Oris assay” y la diferenciación a osteoblastos y adipocitos.Resultados: las EV aumentaron la viabilidad y migración, pero no hubo diferencias entre las derivadas de normoxia ehipoxia. Las EV, principalmente las derivadas de hipoxia, aumentaron la mineralización y la expresión de genes osteoblás-ticos. Sin embargo, no afectaron significativamente a la adipogénesis.Conclusiones: las EV derivadas de MSC en hipoxia no afectan a la adipogénesis, pero tienen una mayor capacidad de inducirla osteoblastogénesis. Por lo tanto, podrían potencialmente ser utilizadas en terapias de regeneración ósea y tratamientosde patologías óseas como la osteoporosis.(AU)

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The use of mesenchymal stem-cells (MSC) in cell therapy has received considerable attention because of their properties. These properties include high expansion and differentiation in vitro, low immunogenicity, and modulation of biological processes, such as inflammation, angiogenesis and hematopoiesis. Curiously, the regenerative effect of MSC is partly due to their paracrine activity. This has prompted numerous studies, to investigate the therapeutic potential of their secretome in general, and specifically their extracellular vesicles (EV). The latter contain proteins, lipids, nucleic acids, and other metabolites, which can cause physiological changes when released into recipient cells. Interestingly, contents of EV can be modulated by preconditioning MSC under different culture conditions. Among them, exposure to hypoxia stands out; these cells respond by activating hypoxia-inducible factor (HIF) at low O2 concentrations. HIF has direct and indirect pleiotropic effects, modulating expression of hundreds of genes involved in processes such as inflammation, migration, proliferation, differentiation, angiogenesis, metabolism, and cell apoptosis. Expression of these genes is reflected in the contents of secreted EV. Interestingly, numerous studies show that MSC-derived EV conditioned under hypoxia have a higher regenerative capacity than those obtained under normoxia. In this review, we show the implications of hypoxia responses in relation to tissue regeneration. In addition, hypoxia preconditioning of MSC is being evaluated as a very attractive strategy for isolation of EV, with a high potential for clinical use in regenerative medicine that can be applied to different pathologies.

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Dipeptidyl peptidase IV (DPP4) is a ubiquitous protease that can be found in membrane-anchored or soluble form. Incretins are one of the main DPP4 substrates. These hormones regulate glucose levels, by stimulating insulin secretion and decreasing glucagon production. Because DPP4 levels are high in diabetes, DPP4 inhibitor (DPP4i) drugs derived from gliptin are widespread used as hypoglycemic agents for its treatment. However, as DPP4 recognizes other substrates such as chemokines, growth factors and neuropeptides, pleiotropic effects have been observed in patients treated with DPP4i. Several of these substrates are part of the stem-cell niche. Thus, they may affect different physiological aspects of mesenchymal stem-cells (MSC). They include viability, differentiation, mobilization and immune response. MSC are involved in tissue homeostasis and regeneration under both physiological and pathological conditions. Therefore, such cells and their secretomes have a high clinical potential in regenerative medicine. In this context, DPP4 activity may modulate different aspects of MSC regenerative capacity. Therefore, the aim of this review is to analyze the effect of different DPP4 substrates on MSC. Likewise, how the regulation of DPP4 activity by DPP4i can be applied in regenerative medicine. That includes treatment of cardiovascular and bone pathologies, cutaneous ulcers, organ transplantation and pancreatic beta-cell regeneration, among others. Thus, DPP4i has an important clinical potential as a complement to therapeutic strategies in regenerative medicine. They involve enhancing the differentiation, immunomodulation and mobilization capacity of MSC for regenerative purposes.

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Phloretin (a flavonoid abundant in apple), has antioxidant, anti-inflammatory, and glucose-transporter inhibitory properties. Thus, it has interesting pharmacological and nutraceutical potential. Bone-marrow mesenchymal stem cells (MSC) have high differentiation capacity, being essential for maintaining homeostasis and regenerative capacity in the organism. Yet, they preferentially differentiate into adipocytes instead of osteoblasts with aging. This has a negative impact on bone turnover, remodeling, and formation. We have evaluated the effects of phloretin on human adipogenesis, analyzing MSC induced to differentiate into adipocytes. Expression of adipogenic genes, as well as genes encoding OPG and RANKL (involved in osteoclastogenesis), protein synthesis, lipid-droplets formation, and apoptosis, were studied. Results showed that 10 and 20 µM phloretin inhibited adipogenesis. This effect was mediated by increasing beta-catenin, as well as increasing apoptosis in adipocytes, at late stages of differentiation. In addition, this chemical increased OPG gene expression and OPG/RANKL ratio in adipocytes. These results suggest that this flavonoid (including phloretin-rich foods) has interesting potential for clinical and regenerative-medicine applications. Thus, such chemicals could be used to counteract obesity and prevent bone-marrow adiposity. That is particularly useful to protect bone mass and treat diseases like osteoporosis, which is an epidemic worldwide.

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BACKGROUND: Mesenchymal stem cells (MSC) of bone marrow are the progenitor of osteoblasts and adipocytes. MSC tend to differentiate into adipocytes, instead of osteoblasts, with aging. This favors the loss of bone mass and development of osteoporosis. Hypoxia induces hypoxia inducible factor 1α gene encoding transcription factor, which regulates the expression of genes related to energy metabolism and angiogenesis. That allows a better adaptation to low O2 conditions. Sustained hypoxia has negative effects on bone metabolism, favoring bone resorption. Yet, surprisingly, cyclic hypoxia (CH), short times of hypoxia followed by long times in normoxia, can modulate MSC differentiation and improve bone health in aging. AIM: To evaluate the CH effect on MSC differentiation, and whether it improves bone mineral density in elderly. METHODS: MSC cultures were induced to differentiate into osteoblasts or adipocytes, in CH (3% O2 for 1, 2 or 4 h, 4 d a week). Extracellular-matrix mineralization and lipid-droplet formation were studied in MSC induced to differentiate into osteoblast or adipocytes, respectively. In addition, gene expression of marker genes, for osteogenesis or adipogenesis, have been quantified by quantitative real time polymerase chain reaction. The in vivo studies with elderly (> 75 years old; n = 10) were carried out in a hypoxia chamber, simulating an altitude of 2500 m above sea level, or in normoxia, for 18 wk (36 CH sessions of 16 min each). Percentages of fat mass and bone mineral density from whole body, trunk and right proximal femur (femoral, femoral neck and trochanter) were assessed, using dual-energy X-ray absorptiometry. RESULTS: CH (4 h of hypoxic exposure) inhibited extracellular matrix mineralization and lipid-droplet formation in MSC induced to differentiate into osteoblasts or adipocytes, respectively. However, both parameters were not significantly affected by the other shorter hypoxia times assessed. The longest periods of hypoxia downregulated the expression of genes related to extracellular matrix formation, in MSC induced to differentiate into osteoblasts. Interestingly, osteocalcin (associated to energy metabolism) was upregulated. Vascular endothelial growth factor an expression and low-density lipoprotein receptor related protein 5/6/dickkopf Wnt signaling pathway inhibitor 1 (associated to Wnt/ß-catenin pathway activation) increased in osteoblasts. Yet, they decreased in adipocytes after CH treatments, mainly with the longest hypoxia times. However, the same CH treatments increased the osteoprotegerin/receptor activator for nuclear factor kappa B ligand ratio in both cell types. An increase in total bone mineral density was observed in elderly people exposed to CH, but not in specific regions. The percentage of fat did not vary between groups. CONCLUSION: CH may have positive effects on bone health in the elderly, due to its possible inhibitory effect on bone resorption, by increasing the osteoprotegerin / receptor activator for nuclear factor kappa B ligand ratio.