Extracellular vesicle and soluble fractions of adipose tissue-derived mesenchymal stem cells secretome induce inflammatory cytokines modulation in an in vitro model of discogenic pain

.BACKGROUND CONTEXT: Mesenchymal Stem Cells (MSCs) secretome or conditioned medium (CM) is a complex cocktail of different molecules, some of which, particularly those con- tained in extracellular vesicles, already have proven therapeutic applications. PURPOSE: CM may well represent promising therapy for discogenic pain and the intention of this work is to assess its therapeutic potential using an in vitro model of this condition. STUDY DESIGN: This is an experimental study. METHODS: Our in vitro model comprised Nucleus Pulposus (NP) and Annulus Fibrosus (AF) cells inflamed with TNF. To assess the potential therapeutic value of CM and its components, extracellular vesicles (EVs) and soluble culture fraction (SF), cell inflammation took place under 3 different conditions: either in the presence of whole CM, isolated EVs or SF, and concentrations of pro-inflammatory cytokines, metalloproteinases (MMPs) and neurotrophic factors produced in all 3 cases were compared. RESULTS: In the presence of whole CM, both in vitro gene expression by the NP and AF test cells and analysis of their protein content showed high modulatory effects on inflammation and MMP inhibition. The presence of EVs and SF showed similar but much smaller effects, and this was par- ticularly marked in the case of NP cells. CONCLUSIONS: Our results show that, compared to EVs and SF, the presence of whole CM has the greatest positive effect on the modulation of pro-inflammatory and catabolic factors. These observations suggest that CM could protect against inflammation and the resulting intervertebral disc (IVD) degeneration that leads to discogenic pain. CLINICAL SIGNIFICANCE: Many patients' expectations are not met by current non-operative and surgical treatments for discogenic low back pain. We propose the use of the MSCs secretomeS

Application of adipose-derived mesenchymal stem cells in an in vivo model of peripheral nerve damage

[EN] Background: Neuropathic pain is one of the most difficult to treat chronic pain syndromes. It has significant effects on patients’ quality of life and substantially adds to the burden of direct and indirect medical costs. There is a critical need to improve therapies for peripheral nerve regeneration. The aim of this study is to address this issue by performing a detailed analysis of the therapeutic benefits of two treatment options: adipose tissue derived-mesenchymal stem cells (ASCs) and ASC-conditioned medium (CM). Methods: To this end, we used an in vivo rat sciatic nerve damage model to investigate the molecular mechanisms involved in the myelinating capacity of ASCs and CM. Furthermore, effect of TNF and CM on Schwann cells (SCs) was evaluated. For our in vivo model, biomaterial surgical implants containing TNF were used to induce peripheral neuropathy in rats. Damaged nerves were also treated with either ASCs or CM and molecular methods were used to collect evidence of nerve regeneration. Post-operatively, rats were subjected to walking track analysis and their sciatic functional index was evaluated. Morphological data was gathered through transmission electron microscopy (TEM) of sciatic nerves harvested from the experimental rats. We also evaluated the effect of TNF on Schwann cells (SCs) in vitro. Genes and their correspondent proteins associated with nerve regeneration were analyzed by qPCR, western blot, and confocal microscopy. Results: Our data suggests that both ASCs and CM are potentially beneficial treatments for promoting myelination and axonal regeneration. After TNF-induced nerve damage we observed an upregulation of c-Jun along with a downregulation of Krox-20 myelin-associated transcription factor. However, when CM was added to TNF-treated nerves the opposite effect occurred and also resulted in increased expression of myelin-related genes and their corresponding proteins. Conclusion: Findings from our in vivo model showed that both ASCs and CM aided the regeneration of axonal myelin sheaths and the remodeling of peripheral nerve morphologySIThis study was supported by the Fundación Leonesa ProNeurociencias. The AW’s Lab was supported by AEI/FEDER, EU (RTI2018-097503-B-I00) and the European Community’s H2020 Framework Program ERC Consolidator Grant (865157- MYERIBO

Regenerative Medicine Applied to Musculoskeletal Diseases in Equines: A Systematic Review

[EN] Musculoskeletal injuries in horses have a great economic impact, predominantly affecting tendons, ligaments, and cartilage, which have limited natural regeneration. Cell therapy, which uses mesenchymal stem cells due to their tissue differentiation properties and anti-inflammatory and immunoregulatory effects, aims to restore damaged tissue. In this manuscript, we performed a systematic review using the Parsifal tool, searching the PubMed and Web of Science databases for articles on regenerative medicine for equine musculoskeletal injuries. Our review covers 17 experimental clinical studies categorized by the therapeutic approach used: platelet-rich plasma, conditioned autologous serum, mesenchymal stem cells, and secretome. These therapies reduce healing time, promote regeneration of fibrocartilaginous tissue, improve cellular organization, and improve joint functionality and sustainability. In conclusion, regenerative therapies using platelet-rich plasma, conditioned autologous serum, equine mesenchymal stem cells, and the emerging field of the secretome represent a promising and highly effective approach for the treatment of joint pathologies in horses, implying a valuable advance in equine healthcare.SIThis research received no external funding

Access to information and degree of community awareness of preventive health measures in the face of covid-19 in Spain

[EN] The COVID-19 pandemic is posing a major health crisis. Spanish legislation establishes the mandatory use of masks and the implementation of hygienic measures such as hand washing and physical distancing. The aim of this study is to describe access to information and the level of community knowledge/adoption about the preventive measures proposed by the Spanish health authorities in response to the COVID-19 pandemic and to analyze the influence of socio-demographic factors in compliance among people over 18 years of age resident in Spain. An observational, descriptive and transversal study was conducted. Data was collected on sociodemographic variables, access to information and the degree of knowledge/adoption about the preventive measures: use of masks, hand hygiene and physical distancing. A total of 1811 people participated. The average age was 45.1 ± 15.1 years, predominantly female (69.3%), from an urban geographical area (74%), with a higher education level of 53.2%. Most of the respondents (57.5%) are or live with people at risk. The main access to information on preventive measures was from secondary sources (49.2%), with television being the main medium; 72.3% think that there are some difficulties in accessing information, while 8.7% of the participants do not consider the use of masks to be useful. As regards the choice of type of mask, the majority of people (44.8%) opt for the surgical variety; 88.5% of respondents believe that the physical distancing established is at least 1.5 m. This study confirmed that socio-demographic factors influence compliance with or the degree of knowledge/adoption of the preventive measures proposed to combat the COVID-19 pandemic and has made it possible to ascertain the sectors of the population with the greatest deficiencies in this respect. It shows the importance of implementing health information and education systems in the community, and it is advisable to promote specific programs aimed at men, people living in rural areas and people with a low level of education.S

Influence of chemistry and fiber diameter of electrospun PLA, PCL and their blend membranes, intended as cell supports, on their biological behavior

.The prevalence of osteoarthritis, a degenerative cartilage disease that causes joint surface erosion and loss of mobility, emphasizes the need of producing a functional articular cartilage replacement. Tissue engineering has been the focus of recent research as a possible strategy for cartilage regeneration and repair. The most widely used technique for the manufacture of nanofibers is polymer electrospinning. Polylactic acid (PLA) and polycaprolactone (PCL) have been proved particularly suitable for nanofiber preparation, with many biomedical applications. The main aim of this work was to evaluate the behavior of adipose tissue-derived mesenchymal stem cells (ASCs) cultured on biomaterials of PLA, PCL and a combination of both (PLA/PCL), manufactured by electrospinning. We analyzed the bioactive properties of these cells in cultures on them, in terms of proliferation, adhesion, morphology, viability and differentiation. In addition, the influence of the thickness of the fibers in each biomaterial on these cellular characteristics was evaluated for their use in Cartilage Regenerative Medicine applications to promote chondrogenic differentiation. Depending on the parameter assessed, different results were obtained on each biomaterial. Using both polymers successful results on cellular viability were obtained, although in the case of PCL the cellular response in all the experiments was significantly better. As for the blends, positive outcomes were obtained, but they did not overtake the characteristics of PCL. Interestingly, ASCs were able to differentiate into chondrocytes without adding specific chondrogenic media in the three biomaterials tested. Moreover, a marked cell differentiation on PCL with 1.8 μm-fiber diameter and PLA/PCL blends was observed. These findings may play a key role in cartilage Regenerative Medicine and Tissue Engineering.S

Aplicación de herramientas de realidad virtual en el aprendizaje de Anatomía en Ciencias de la salud

[ES] En este proyecto de Innovación Docente pretendemos diseñar, implementar y evaluar una propuesta didáctica basada en la realidad virtual como método de aprendizaje práctico en la asignatura de Anatomía Humana de primer curso del Grado en Podología y Enfermería. El objetivo principal es valorar la contribución del innovador método de realidad virtual como estrategia docente, para mejorar el logro de competencias en la asignatura “Anatomía Humana”. La utilización de la realidad virtual es una metodología innovadora, ampliamente utilizada en la actualidad, pero en el ámbito de la enseñanza de Anatomía humana es limitado. Los alumnos matriculados en la asignatura de Anatomía Humana del grado en Podología y de Enfermería de la Universidad de León utilizaron las gafas virtuales para visualizar varios vídeos seleccionados por los profesores. Posteriormente, los alumnos realizaron una encuesta de satisfacción de la actividad realizada. La RV puede ayudar en la enseñanza de la Anatomía, haciendo complementar las clases, los libros y los laboratorios, proporcionando una herramienta muy atractiva entre los métodos de enseñanza de la Anatomía. Utilizando la realidad virtual, el alumno se introduce en la anatomía de una forma inmersiva, le permite visualizar las distintas estructuras anatómicas desde una perspectiva totalmente diferente. Los resultados obtenidos en estas sesiones mostraron que la totalidad de los alumnos quedaron satisfechos con la experiencia y por tanto permite adoptar nuevas metodologías eficaces que complementen a la docencia tradicional de la Anatomía humana

Anti-inflammatory and regenerative effects of MKARE® Eggshell Membrane: An in vitro osteoarthritis model and placebo-controlled clinical study

[EN]MKARE®, a 100% natural ingredient derived from fresh eggshell membrane (ESM), has a rich composition in bioactive compounds like collagen, hyaluronic acid, and elastin. These components are beneficial for managing osteoarthritis (OA) due to their anti-inflammatory and regenerative properties. Highlighting the significance of freshness, our research has shown that the effectiveness of MKARE® is higher than that of other commercial products based on ESM that have been stored for several days at room temperature, losing their bioactive compounds. This study explores the MKARE® anti-inflammatory capacity through an in vitro and clinical analyses, demonstrating its ability to alleviate OA symptoms and improve joint health. This underscores the crucial role of freshness in optimizing the therapeutic benefits.S

Culture of Differentiated Adult Rabbit Auricular Chondrocytes

P. 54Chondrocytes dedifferentiate to a fibroblast-like phenotype on plastic surfaces. Dedifferentiation is reversible if these cells are then cultured embedded in gels as alginate, agarose or collagen. Chondrocytes cultured in suspension on a nonadherent surface are also known to form aggregates of differentiated cells. The knowledge of chondrocyte behavior in culture is relevant for tissue engineering purposes. In this report we describe a simple method to culture differentiated or redifferentiated rabbit auricular chondrocytes on plastic surfaces with a stable phenotype. When chondrocyte aggregates formed in suspension are next seeded on plastic surfaces, most of them attach to the plastic as round or polygonal cells, and this morphological differentiation, confirmed by the presence of type II collagen, is stable for long culture periods. We also report that the addition of aggregates to monolayer cultures of dedifferentiated chondrocytes results in their redifferentiation, as is shown by their morphological changes and the synthesis of type II collagen. Therefore, this simple method can be useful for the study of chondrocyte behavior on plastic surfaces and for redifferentiating previously proliferated chondrocytes in tissue engineering techniques. Furthermore, these results demonstrate that, in addition to culture conditions such as cell isolation method or cell-density, chondrocyte behavior on plastic depends on the presence or absence of aggregates resulting from the dissociation processS

Study on Viability and Chondrogenic Differentiation of Cryopreserved Adipose Tissue-Derived Mesenchymal stromal Cells for future use in regenerative medicine

P. 256–263Adipose-derived mesenchymal stromal cells are promising as a regenerative therapy tool for defective tissues in mesenchymal lineage, including fat, bone, cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation is an essential fundamental technology. The aim of this study is to define an adequate protocol for the cryopreservation of adipose-derived mesenchymal stromal cells, by comparing various protocols so as to determine the effects of cryopreservation on viability and chondrogenic differentiation potential of adipose-derived stem cells upon freeze-thawing of AT-MSCs colonies cryopreserved with standard and modified protocols, using flow cytometry and confocal microscopy. The study concludes that adipose-derived mesenchymal stromal cells could be long-term cryopreserved without any loss of their proliferative or differentiation potentialS

Immunomodulation of mesenchymal stem cells in discogenic pain

13 p.BACKGROUND CONTEXT: Back pain is a highly prevalent health problem in the world today and has a great economic impact on health-care budgets. Intervertebral disc (IVD) degeneration has been identified as a main cause of back pain. Inflammatory cytokines produced by macrophages or disc cells in an inflammatory environment play an important role in painful progressive degeneration of IVD. Mesenchymal stem cells (MSCs) have shown to have immunosuppressive and antiinflammatory properties. Mesenchymal stem cells express a variety of chemokines and cytokines receptors having tropism to inflammation sites. PURPOSE: This study aimed to develop an in vitro controlled and standardized model of inflammation and degeneration of IVD with rat cells and to evaluate the protective and immunomodulatory effect of conditioned medium (CM) from the culture of MSCs to improve the conditions presented in herniated disc and discogenic pain processes. STUDY DESIGN: This is an experimental study. METHODS: In this study, an in vitro model of inflammation and degeneration of IVD has been developed, as well as the effectiveness of CM from the culture of MSCs. RESULTS: Conditioned medium from MSCs downregulated the expression of various proinflammatory cytokines produced in the pathogenesis of discogenic pain such as interleukin (IL)-1β, IL-6, IL-17, and tumor necrosis factor (TNF). CONCLUSION: Mesenchymal stem cells represent a promising alternative strategy in the treatment of IVD degeneration inasmuch as there is currently no treatment which leads to a complete remission of long-term pain in the absence of drug