2. Universidad Cardenal Herrera-CEU

Background: Osteoarthritis (OA) is a joint disease characterized by cartilage degradation, low-grade synovitis and subchondral bone alterations. In the damaged joint, there is a progressive increase of oxidative stress leading to disruption of chondrocyte homeostasis. The modulation of oxidative stress could control the expression of inflammatory and catabolic mediators involved in OA. We have previously demonstrated that extracellular vesicles (EVs) present in the secretome of human mesenchymal stem cells from adipose tissue (AD-MSCs) exert antiinflammatory and anti-catabolic effects in OA chondrocytes. In the current work, we have investigated whether AD-MSC EVs could regulate oxidative stress in OA chondrocytes as well as the possible contribution of peroxiredoxin 6 (Prdx6). Methods: Microvesicles (MV) and exosomes (EX) were isolated from AD-MSC conditioned medium by differential centrifugation with size filtration. The size and concentration of EVs were determined by resistive pulse sensing. OA chondrocytes were isolated from knee articular cartilage of advanced OA patients. 4-Hydroxynonenal adducts, IL-6 and MMP-13 were determined by enzyme-linked immunosorbent assay. Expression of Prdx6 and autophagic markers was assessed by immunofluorescence and Western blotting. Prdx6 was downregulated in AD-MSCs by transfection with a specific siRNA. Results: MV and to a lesser extent EX significantly reduced the production of oxidative stress in OA chondrocytes stimulated with IL-1β. Treatment with MV resulted in a dramatic upregulation of Prdx6. MV also enhanced the expression of autophagy marker LC3B. We downregulated Prdx6 in AD-MSCs by using a specific siRNA and then MV were isolated. These Prdx6-silenced MV failed to modify oxidative stress and the expression of autophagy markers. We also assessed the possible contribution of Prdx6 to the effects of MV on IL-6 and MMP-13 production. The reduction in the levels of both mediators induced by MV was partly reverted after Prdx6 silencing. Conclusion: Our results indicate that EVs from AD-MSCs regulate the production of oxidative stress in OA chondrocytes during inflammation. Prdx6 may mediate the antioxidant and protective effects of MV. The translational potential of this article: This study gives insight into the protective properties of EVs from AD-MSCs in OA chondrocytes. Our findings support the development of novel therapies based on EVs to prevent or treat cartilage degradation.

Background/Aims: Chronic inflammation contributes to cartilage degeneration during the progression of osteoarthritis (OA). Adipose tissue-derived mesenchymal stem cells (ADMSC) show great potential to treat inflammatory and degradative processes in OA and have demonstrated paracrine effects in chondrocytes. In the present work, we have isolated and characterized the extracellular vesicles from human AD-MSC to investigate their role in the chondroprotective actions of these cells. Methods: AD-MSC were isolated by collagenase treatment from adipose tissue from healthy individuals subjected to abdominal lipectomy surgery. Microvesicles and exosomes were obtained from conditioned medium by filtration and differential centrifugation. Chondrocytes from OA patients were used in primary culture and stimulated with 10 ng/ml interleukin(IL)-1β in the presence or absence of AD-MSC microvesicles, exosomes or conditioned medium. Protein expression was investigated by ELISA and immunofluorescence, transcription factor-DNA binding by ELISA, gene expression by real-time PCR, prostaglandin E2 (PGE2) by radioimmunoassay, and matrix metalloproteinase (MMP) activity and nitric oxide (NO) production by fluorometry. Results: In OA chondrocytes stimulated with IL-1β, microvesicles and exosomes reduced the production of inflammatory mediators tumor necrosis factor-α, IL-6, PGE2 and NO. The downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 would lead to the decreased PGE2 production while the effect on NO could depend on the reduction of inducible nitric oxide synthase expression. Treatment of OA chondrocytes with extracellular vesicles also decreased the release of MMP activity and MMP-13 expression whereas the production of the anti-inflammatory cytokine IL-10 and the expression of collagen II were significantly enhanced. The reduction of inflammatory and catabolic mediators could be the consequence of a lower activation of nuclear factor-κB and activator protein-1. The upregulation of annexin A1 specially in MV may contribute to the anti-inflammatory and chondroprotective effects of AD-MSC. Conclusions: Our data support the interest of AD-MSC extracellular vesicles to develop new therapeutic approaches in joint conditions.

La Osteoartrosis (OA) es una de las enfermedades con más prevalencia y morbilidad en la sociedad actual, tanto en medicina humana como en medicina veterinaria, debido sobre todo al aumento de la esperanza de vida y a la prevalencia creciente de la obesidad. Además por su gravedad e irreversibilidad, cada vez existe un mayor interés y empeño en tratar de encontrar tratamientos que alivien los síntomas y retrasen la evolución de la patología. En los últimos años, se han buscado alternativas a los tratamientos convencionales ya que estos últimos únicamente palían la sintomatología de la enfermedad sin detener la progresión de la misma. Entre estas nuevas terapias englobadas dentro de la medicina regenerativa, encontramos el Plasma Rico en Factores de Crecimiento (PRGF) y las células mesenquimales, concretamente las obtenidas a partir del tejido adiposo (CMG). El objetivo del presente trabajo de Tesis Doctoral ha sido la evaluación del efecto de las CMG y del PRGF, así como de la combinación de ambos, en el tratamiento de la Enfermedad degenerativa articular (EDA), utilizando para ello un modelo clínico de la especie canina. Para llevar a cabo el estudio se seleccionaron 49 animales de la especie canina, con EDA en las articulaciones del codo, la cadera o la rodilla. A estos animales se les realizó previamente una exploración física completa, además de una minuciosa exploración traumatológica, y una analítica sanguínea para descartar otras enfermedades concomitantes. Una vez comprobado que todos los animales únicamente padecían EDA, se dividieron aleatoriamente en tres grupos de estudio, dependiendo del tratamiento aplicado (CMG, CMG+PRGF y PRGF), habiendo un total de 25 articulaciones en el grupo de CMG, 23 en el grupo de CMG+PRGF, y 47 en el grupo de PRGF. A continuación, tras la obtención del PRGF y de las CMG se realizó una infiltración intraarticular estándar a cada uno de los animales dependiendo del grupo asignado. Además se estudió la viabilidad de las células madre evaluando las características y naturaleza de estas células, y determinando los efectos de concentraciones crecientes de PRGF en la proliferación de las mismas. Posteriormente, a tiempo basal (antes de aplicar los tratamientos), uno, tres y seis meses tras a la aplicación de los mismos se les realizó a cada uno de los pacientes una evaluación de las variables del estudio entre las que se encuentran las variables de la escala bioarth (grado radiológico de artrosis, limitación funcional y movilidad articular), un cuestionario sobre la satisfacción ante el tratamiento, la valoración del dolor mediante la escala analógica visual (VAS) tanto por parte del propietario, como del veterinario y finalmente la extracción de suero para el posterior análisis de biomarcadores. Además, en los animales de las Palmas de Gran Canaria, se llevó a cabo en cada una de las revisiones un análisis cinético de las extremidades afectadas mediante la plataforma de fuerza. En el análisis de la viabilidad celular, además de obtener un crecimiento exponencial de las muestras con una curva proliferativa definida en un cultivo celular que contenía suero bovino fetal, observamos que los factores de crecimiento contenidos en PRGF pueden también ser suficientes para mejorar el rendimiento de las CMG en cultivos celulares. Tras el análisis estadístico de las variables contenidas en la escala bioarth, observamos que el grado de artrosis se mantuvo constante a lo largo del estudio en todos los grupos, sin existir diferencias significativas entre ellos. La limitación funcional mejoró de manera significativa en los tres grupos obteniendo mejores resultados a los 6 meses en los grupos tratados con CMG y con CMG+PRGF, lo mismo que ocurrió con la movilidad articular. En cuanto a la valoración del dolor mediante la VAS, tanto por parte del propietario como del veterinario, existió una disminución significativa del dolor que presentaban los pacientes desde el primer mes post tratamiento manteniéndose hasta los 6 meses posteriores a la aplicación de los mismos, obteniendo resultados significativamente más favorables en el grupo tratado con CMG+PRGF al finalizar el estudio. Respecto a las preguntas realizadas a los propietarios para determinar el grado de satisfacción que presentaban ante el tratamiento aplicado a sus mascotas, ya desde el primer mes post tratamiento se obtuvieron unos resultados muy satisfactorios, que se mantuvieron hasta el final del estudio en los 3 grupos de estudio. En el estudio de los biomarcadores analizados en el suero de los pacientes, observamos que tanto en el grupo de CMG como en el grupo de CMG+PRGF, existió un aumento progresivo y significativo del ácido hialurónico (HA) en el tiempo, no observándose estos cambios en el grupo tratado con PRGF. Resultados similares se obtuvieron con el C2C, en el cual también observamos tanto en el grupo de CMG como en el de CMG+PRGF una disminución progresiva de este biomarcador en el tiempo, sin existir cambios significativos en el grupo de PRGF. No existieron diferencias entre grupos en ninguno de los tiempos de estudio valorados. Por último, en el estudio de la plataforma de fuerza realizado en los animales de las Palmas de Gran Canaria se observó una mejora progresiva en las variables valoradas en el análisis cinético, alcanzando a los 6 meses de estudio los resultados significativamente más favorables en el grupo de estudio valorado. Tras los resultados logrados en el estudio, podemos decir que la terapia con CMG y PRGF, así como la combinación de ambos tratamientos, son una nueva vía de tratamiento que intenta conseguir una reparación lo más similar al cartílago nativo, sin necesidad de optar por técnicas más agresivas y que causen un daño mayor al organismo. Hay que destacar que en determinados parámetros existen diferencias entre el grupo tratado únicamente con PRGF y los tratados con CMG y la combinación de CMG con PRGF. Nuestra sospecha es que el uso de PRGF en inyección única produce inicialmente una mejora clara del paciente, cuyo efecto está mas limitado en el tiempo, por eso sería recomendable realizar una infiltración seriada de este tratamiento cuando queramos utilizarlo como terapia para la OA. Respecto a las diferencias entre el uso únicamente de CMG, o el uso de una combinación de CMG junto con PRGF, en esta tesis doctoral no hemos obtenido muchas diferencias significativas entre ambos grupos, aunque pensamos que esto es debido a la duración del estudio, ya que si el estudio se hubiese prolongado durante más tiempo, habríamos obtenido resultados con diferencias significativas obteniendo unos valores más favorables en el grupo tratado con CMG+PRGF debido al efecto sinérgico que tienes estos tratamientos. Osteoarthritis (OA) is one of the most prevalent diseases in today's society, both in human medicine and veterinary medicine, mainly due to increased life expectancy and the growing prevalence of obesity. Besides its severity and irreversibility, an increasing interest and commitment exists in trying to find treatments to alleviate symptoms and delay the evolution of the disease. In recent years, alternatives to conventional treatments have been sought after because the latter only palliates the symptoms of disease without halting its progression. Among these new therapies included within regenerative medicine, are Plasma Rich in Growth Factors (PRGF) and mesenchymal cells, particularly those derived from adipose tissue (ADMSC). The aim of this Thesis has been the evaluation of the effect of ADMSC and PRGF, as well as their combination in the treatment of degenerative joint disease (DJD), using a canine species clinical model. Forty nine animals were selected, with DJD in the elbow, hip or knee. A complete physical examination was performed on these animals previously, and a thorough orthopedic examination, and blood tests to rule out other comorbidities. Once verified that all animals were only suffering from DJD, they were randomly divided into three treatment groups, (ADMSC, ADMSC+PRGF and PRGF), having a total of 25 joints in the ADMSC group, 23 in the ADMSC+PRGF group and 47 in the PRGF group. Then, after obtaining the PRGF and ADMSC a standard intra-articular injection was performed. The viability of the stem cells was also studied, evaluating the characteristics and nature of these cells and determining the effects of the increasing concentrations of PRGF in proliferation. Patients were assessed, at baseline (before applying the treatments) and one, three and six months after the application studying variables including Bioath scale (degree of radiological osteoarthritis, functional limitation and joint mobility), questionnaire on satisfaction with the treatment, pain assessment by visual analogue scale (VAS) both by the owner and the Vet and finally serum extraction for subsequent analysis of biomarkers. Also in the animals at Palmas de Gran Canaria, a kinetic analysis of the affected limbs by force platform was performed at each follow up. In the cell viability analysis , in addition to obtaining an exponential growth in the samples with a proliferative curve defined in a cell culture containing fetal bovine serum, we observed that the growth factors contained in PRGF may also be sufficient to improve the performance of the AMSC cultures. After statistical analysis of the variables in the BIOARTH scale, we noted that the degree of osteoarthritis remained constant throughout the study in all groups, with no significant differences between them. Functional limitation improved significantly in all three groups with better results at 6 months in the groups treated with AMSC and ADMSC+PRGF, and the same happened with joint mobility. Regarding the questions owners were asked to determine their satisfaction with the treatment applied to their pets, from the first month post treatment very satisfactory results were observed, which remained constant throughout the study. As for the VAS assessment, by both the owner and vet, there was a significant decrease in pain in the patients from the first month after treatment which remained until 6 months after the application thereof, obtaining significantly more favorable results in the group treated with AMDSC+PRGF group up to the end of the study. With reference to the study of the markers in the serum, we observed that in both the ADMSC and ADMSC+PRGF, there was a progressive and significant increase of hyaluronic acid (HA) at the time and these changes were not observed in the group treated with PRGF alone. Similar results were obtained with the C2C, which we also observed in both the ADMSC group and the ADMSC+PRGF group showing a progressive decrease of this biomarker over time and existed without significant changes in the PRGF group. There were no differences between groups at any of the study times. Finally, the force platform evaluation conducted in animals at Las Palmas de Gran Canaria, progressive improvement was observed in the variables assessed in the kinetic analysis, reaching significantly more favorable results in the study group evaluated at 6 months after treatment. Following the results achieved in the study, we can say that therapy with ADMSC and PRGF, and the combination of both treatments, are a novel treatment attempting to achieve repair as similar to native cartilage without opting for more aggressive techniques and cause further damage to the body. It is important to note that there are differences between certain parameters in the group treated with PRGF alone and that treated with ADMSC and the combination of ADMSC with PRGF. We suspect that using a single PRGF injection initially produces a clear improvement for the patient, the effect is more limited in time, so it would be advisable to perform a serie of injections with this treatment when used as a therapy for OA. Regarding the differences between the use of only ADMSC, or using a combination of ADMSC with PRGF, in this thesis we have not observed any significant differences between the groups, although we think that this is due to the duration of the study and believe that had the studied continued for longer, significant differences would have been obtained with more favorable values in the group treated with ADMSC+PRGF due to the synergistic effect of these treatments.