Gabriel Dorado

Background/Objectives: Obesity is a major public health problem and a risk factor for numerous diseases. Furthermore, the high prevalence of 25-hydroxyvitamin D (25(OH)D) deficiency in obese individuals promotes a state of persistent inflammation and contributes to the development of hard-to-heal skin wounds. Although calcifediol is effective in correcting this deficiency, its effect on cutaneous wound healing in obesity has not been fully established. Thus, the objective of this work has been to evaluate the impact of calcifediol treatment on wound healing in an animal model of obesity with vitamin D deficiency. Methods: Wistar rats fed a high-fat, vitamin D-deficient diet were subjected to dorsal skin wounds. One group received calcifediol treatment for 14 days. Serum 25(OH)D levels, wound closure rate, histological parameters, and molecular markers related to inflammation were analyzed. Results: Calcifediol significantly increased serum 25(OH)D levels and accelerated wound closure compared to untreated animals. It also improved re-epithelialization, reduced the depth and extent of the lesions, and promoted collagen deposition. Furthermore, it increased the expression of anti-inflammatory markers (IL-10 and CD163) and decreased the infiltration of CD68+ macrophages, indicating a more efficient resolution of the inflammatory response. Conclusions: Calcifediol supplementation improves cutaneous wound healing in obesity with 25(OH)D deficiency by accelerating wound closure, promoting extracellular matrix remodeling, and modulating the inflammatory response, supporting its use as a complementary strategy to enhance tissue repair in conditions of obesity and vitamin D deficiency.

The evaluation of the use of extracellular vesicles (derived from different cellular sources and mammalian fluids) in regenerative medicine has produced interesting results. This includes their great potential for the treatment of chronic skin ulcers, which is related to their effects on migration, proliferation, inflammation and angiogenesis, among other processes. However, large-scale production of mammalian extracellular vesicles may be limited by the need to maintain cell cultures continuously, without losing their ability to secrete extracellular vesicles with regenerative capacity. This may require complex and expensive infrastructures. It is therefore necessary to identify other possible, more efficient alternatives that can be easily transferred to clinical practice. Among these substitutes are plant-derived extracellular vesicles (PDEVs). Fortunately, they resemble those of mammals, playing a role in cell communications. As expected, their compositions depend on source tissues and the physiological conditions of the plants. They may carry numerous molecules with high biological activity. Interestingly, PDEVs are easy to obtain on a large scale, have good stability and are less immunogenic than mammalian-derived EVs. Numerous preclinical studies indicate that they can enhance chronic-wound healing through their immunomodulatory and angiogenic effects, among others. Thus, this review aims to describe the current state of knowledge on the potential therapeutic use of PDEVs in wound healing. It also describes the methods of obtaining and applying them, as well as regenerative processes in which they may intervene. The information provided shows the need to continue advancing knowledge about the production, isolation and mechanisms of action of PDEVs. This will allow new effective therapeutic strategies for the treatment of chronic cutaneous ulcers to be developed.

Three-dimensional bioprinting is a new advance in tissue engineering and regenerative medicine. Bioprinting allows manufacturing three-dimensional (3D) structures that mimic tissues or organs. The bioinks used are mainly made of natural or synthetic polymers that must be biocompatible, printable, and biodegradable. These bioinks may incorporate progenitor cells, favoring graft implantation and regeneration of injured tissues. However, the natures of biomaterials, bioprinting processes, a lack of vascularization, and immune responses are factors that limit the viability and functionality of implanted cells and the regeneration of damaged tissues. These limitations can be addressed by incorporating extracellular vesicles (EV) into bioinks. Indeed, EV from progenitor cells may have regenerative capacities, being similar to those of their source cells. Therefore, their combinations with biomaterials can be used in cell-free therapies. Likewise, they can complement the manufacture of bioinks by increasing the viability, differentiation, and regenerative ability of incorporated cells. Thus, the main objective of this review is to show how the use of 3D bioprinting technology can be used for the application of EV in regenerative medicine by incorporating these nanovesicles into hydrogels used as bioinks. To this end, the latest advances derived from in vitro and in vivo studies have been described. Together, these studies show the high therapeutic potential of this strategy in regenerative medicine.

Background: The prevention and treatment of bone loss and osteoporotic fractures is a public health challenge. Combined with normobaric hypoxia, whole-body vibration has a high clinic potential in bone health and body composition. The effect of this therapy may be mediated by its action on bone marrow mesenchymal stem cells (MSCs). Objectives: Evaluate the effects of cyclic low-vibration stimuli and/or hypoxia on bone marrow-derived human MSC differentiation. Methods: MSCs were exposed four days per week, two hours/day, to hypoxia (3% O₂) and/or vibration before they were induced to differentiate or during differentiation into osteoblasts or adipocytes. Gene and protein expression of osteoblastic, adipogenic, and cytoskeletal markers were studied, as well as extracellular matrix mineralization and lipid accumulation. Results: early osteoblastic markers increased in undifferentiated MSCs, pretreated in hypoxia and vibration. This pretreatment also increased mRNA levels of osteoblastic genes and beta-catenin protein in the early stages of differentiation into osteoblasts without increasing mineralization. When MSCs were exposed to vibration under hypoxia or normoxia during osteoblastic differentiation, mineralization increased with respect to cultures without vibrational stimuli. In MSCs differentiated into adipocytes, both in those pretreated as well as exposed to different conditions during differentiation, lipid formation decreased. Changes in adipogenic gene expression and increased beta-catenin protein were observed in cultures treated during differentiation. Conclusions: Exposure to cyclic hypoxia in combination with low-intensity vibratory stimuli had positive effects on osteoblastic differentiation and negative ones on adipogenesis of bone marrow-derived MSCs. These results suggest that in elderly or frail people with difficulty performing physical activity, exposure to normobaric cyclic hypoxia and low-density vibratory stimuli could improve bone metabolism and health.

Chronic wounds, especially those that are hard-to-heal, constitute a serious public-health problem. Although progress has been made in the development of wound dressings for healing, there is little high-quality evidence of their efficacy, with no evidence of superiority in the use of one hydrogel over another. To evaluate the superiority of a hydrogel (EHO-85), containing Olea europaea leaf extract (OELE), over a standard hydrogel (SH), the promotion and/or improvement of healing of difficult-to-heal wounds was compared in a prospective, parallel-group multicenter, randomized, observer-blinded, controlled trial (“MACAON”). Non-hospitalized patients with pressure, venous or diabetic foot-ulcers difficult-to-heal were recruited and treated with standard care, and EHO-85 (n = 35) or VariHesive (n = 34) as SH. Wound-area reduction (WAR; percentage) and healing rate (HR; mm²/day) were measured. EHO-85 showed a statistically significant superior effect over VariHesive. At the end of the follow-up period, the relative WAR decreased by 51.6% vs. 18.9% (p < 0.001), with a HR mean of 10.5 ± 5.7 vs. 1.0 ± 7.5 mm²/day (p = 0.036). EHO-85 superiority is probably based on its optimal ability to balance the ulcer bed, by modulating pH and oxidative stress. That complements the wetting and barrier functions, characteristics of conventional hydrogels. These results support the use of EHO-85 dressing, for treatment of hard-to-heal ulcers. Trial Registration AEMPS:PS/CR623/17/CE.

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.

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 β-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.

Many advanced wound healing dressings exist, but there is little high-quality evidence to support them. To determine the performance of a novel amorphous hydrogel (EHO-85) in relation to its application, we compared its rheological properties with those of other standard hydrogels (SH), and we assessed the induction of acceleration of the early stages of wound healing as a secondary objective of a prospective, multicenter, randomized, observer-blinded, controlled trial. The patients were recruited if they had pressure, venous, or diabetic foot ulcers and were treated with EHO-85 (n = 103) or VariHesive^® (SH) (n = 92), and their response was assessed by intention-to-treat as wound area reduction (WAR (%)) and healing rate (HR mm²/day) in the second and fourth weeks of treatment. Results: EHO-85 had the highest shear thinning and G′/G″ ratio, the lowest viscous modulus, G″, and relatively low cohesive energy; EHO-85 had a significantly superior effect over SH in WAR and HR, accelerating wound healing in the second and fourth weeks of application (p: 0.002). This superiority is likely based on its optimal moisturizing capacity and excellent pH-lowering and antioxidant properties. In addition, the distinct shear thinning of EHO-85 facilitates spreading by gentle hand pressure, making it easier to apply to wounds. These rheological properties contribute to its improved performance.

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.

Hard-to-heal chronic wounds associated with aging and high-prevalence pathologies, such as diabetes, are a global health problem. Therefore, it is necessary to advance effective treatments to accelerate wound healing. Among these potential treatments are new therapies based on mesenchymal stem cells (MSC) and their secretomes, including extracellular vesicles (EV). They have an important therapeutic potential for the treatment of chronic ulcers, due to their immunomodulatory activity, as well as their ability to induce angiogenesis, cell proliferation and cell migration. The use of MSC-derived EV in regenerative medicine involves cell-free therapies that decrease risks associated with cell therapies, such as the potential development of tumors. However, the short half-life of MSC-EV is a limitation for their clinical use. A therapeutic strategy to increase the regenerative efficiency of EV in wounds is to encapsulate them in biomaterials. The latter must protect and progressively release EV in damaged tissues, optimizing healing. Biomaterials that can be used include hydrogels. These, in addition to acting as a vehicle for sustained application of EV, can create favorable environments for wound healing. Thus, the aim of this review is to critically describe the latest advances in the development of such therapeutic strategies. It highlights the significance and clinical potential of these new therapies, as well as the need to develop clinical trials, to ascertain their performance.