1. Investigación

Osteoarthritis (OA) is the most common articular disease in adults and has a current prevalence of 12% in the population over 65 years old. This chronic disease causes damage to articular cartilage and synovial joints, causing pain and leading to a negative impact on patients’ function, decreasing quality of life. There are many limitations regarding OA conventional therapies—pharmacological therapy can cause gastrointestinal, renal, and cardiac adverse effects, and some of them could even be a threat to life. On the other hand, surgical options, such as microfracture, have been used for the last 20 years, but hyaline cartilage has a limited regeneration capacity. In recent years, the interest in new therapies, such as cell-based and cell-free therapies, has been considerably increasing. The purpose of this review is to describe and compare bioregenerative therapies’ efficacy for OA, with particular emphasis on the use of mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP). In OA, these therapies might be an alternative and less invasive treatment than surgery, and a more effective option than conventional therapies.

Chronic musculoskeletal (MSK) pain is one of the most common medical complaintsworldwide and musculoskeletal injuries have an enormous social and economical impact. Currentpharmacological and surgical treatments aim to relief pain and restore function; however,unsatiscactory outcomes are commonly reported. In order to find an accurate treatment to suchpathologies, over the last years, there has been a significantly increasing interest in cellular therapies,such as adipose-derived mesenchymal stem cells (AMSCs). These cells represent a relatively newstrategy in regenerative medicine, with many potential applications, especially regarding MSKdisorders, and preclinical and clinical studies have demonstrated their efficacy in muscle, tendon,bone and cartilage regeneration. Nevertheless, several worries about their safety and side effects atlong-term remain unsolved. This article aims to review the current state of AMSCs therapy in thetreatment of several MSK diseases and their clinical applications in veterinary and human medicine.

The aim of this study was to evaluate the use of serum type II collagen cleavage epitope and serum hyaluronic acid as biomarkers for treatment monitoring in osteoarthritic dogs. For this purpose, a treatment model based on mesenchymal stem cells derived from adipose tissue combined with plasma rich in growth factors was used. This clinical study included 10 dogs with hip osteoarthritis. Both analytes were measured in serum at baseline, just before applying the treatment, and 1, 3, and 6 months after treatment. These results were compared with those obtained from force plate analysis using the same animals during the same study period. Levels of type II collagen cleavage epitope decreased and those of hyaluronic acid increased with clinical improvement objectively verified via force plate analysis, suggesting these two biomarkers could be effective as indicators of clinical development of joint disease in dogs.

Background: Regenerative medicine using Mesenchymal Stem Cells (MSC) alone or combined with Plasma Rich in Growth Factors (PRGF) is a rapidly growing area of clinical research and is currently also being used to treat osteoarthritis (OA). Force platform analysis has been consistently used to verify and quantify the efficacy of different therapeutic strategies for the treatment of OA in dogs including MSC associated to PRGF, but never with AD-MSC alone. The aim of this study was to use a force platform to measure the efficacy of intraarticular ADMSC administration for limb function improvement in dogs with severe OA. Results: Ten lame dogs with severe hip OA and a control group of 5 sound dogs were used for this study. Results were statistically analyzed to detect a significant increase in peak vertical force (PVF) and vertical impulse (VI) in treated dogs. Mean values of PVF and VI were significantly improved within the first three months post-treatment in the OA group, increasing 9% and 2.5% body weight, respectively, at day 30. After this, the effect seems to decrease reaching initial values. Conclusion: Intraarticular ADMSC therapy objectively improved limb function in dogs with hip OA. The duration of maximal effect was less than 3 months.

Background: Adipose-derived mesenchymal stem cell (ADMSC) therapy in regenerative medicine is a rapidly growing area of research and is currently also being used to treat osteoarthritis (OA). Force platform analysis has been consistently used to verify the efficacy of different therapeutic strategies for the treatment of OA in dogs, but never with AD-MSC. The aim of this study was to use a force platform to measure the efficacy of intraarticular ADMSC administration for limb function improvement in dogs with severe OA. Results: Eight lame dogs with severe hip OA and a control group of 5 sound dogs were used for this study. Results were statistically analyzed to detect a significant increase in peak vertical force (PVF) and vertical impulse (VI) in treated dogs. Mean values of PVF and VI were significantly improved after treatment of the OA groups, reaching 53.02% and 14.84% of body weight, respectively, at day 180, compared with only 43.56% and 12.16% at day 0. Conclusion: This study objectively demonstrated that intraarticular ADMSC therapy resulted in reduced lameness due to OA.

Background: Subjective pain assessment scales have been widely used for assessing lameness in response to pain, but the accuracy of these scales has been questioned. To assess scale accuracy, 10 lame, presa Canario dogs with osteoarthritis (OA) associated with bilateral hip dysplasia were first treated with mesenchymal stem cells. Then, potential lameness improvement was analyzed using two pain scales (Bioarth and visual analog scale). These data were compared with similar data collected using a force platform with the same animals during a period of 6 months after treatment. Results: The F test for intraclass correlation showed that concordance in pain/lameness scores between the 2 measuring methodologies was not significant (P value ≥ 0.9213; 95 % confidence interval, –0.56, 0.11). Although subjective pain assessment showed improvement after 6 months, force platform data demonstrated those same animals had returned to the initial lameness state. Conclusion: Use of pain assessment scales to measure lameness associated with OA did not have great accuracy and concordance when compared with quantitative force platform gait analysis.

The overall increase of chronic degenerative diseases associated with ageing makes wound care a tremendous socioeconomic burden. Thus, there is a growing need to develop novel wound healing therapies to improve cutaneous wound healing. The use of regenerative therapies is becoming increasingly popular due to the low-invasive procedures needed to apply them. Platelet-rich plasma (PRP) is gaining interest due to its potential to stimulate and accelerate the wound healing process. The cytokines and growth factors forming PRP play a crucial role in the healing process. This article reviews the emerging field of skin wound regenerative therapies with particular emphasis on PRP and the role of growth factors in the wound healing process.

Osteoarthritis (OA) is a major cause of disability in elderly population around the world. More than one-third of people over 65 years old shows either clinical or radiological evidence of OA. There is no effective treatment for this degenerative disease, due to the limited capacity for spontaneous cartilage regeneration. Regarding the use of regenerative therapies, it has been reported that one option to restore degenerated cartilage are adipose-derived mesenchymal stem cells (ASCs). The purpose of this review is to describe and compare the efficacy of ASCs versus other therapies in OA. Methods: Recent studies have shown that ASCs exert paracrine effects protecting against degenerative changes in chondrocytes. According to the above, we have carried out a review of the literature using a combination of osteoarthritis, stem cells, and regenerative therapies as keywords. Results: Conventional pharmacological therapies for OA treatment are considered before the surgical option, however, they do not stop the progression of the disease. Moreover, total joint replacement is not recommended for patients under 55 years, and high tibia osteotomy (HTO) is a viable solution to address lower limb malalignment with concomitant OA, but some complications have been described. In recent years, the use of mesenchymal stem cells (MSCs) as a treatment strategy for OA is increasing considerably, thanks to their capacity to improve symptoms together with joint functionality and, therefore, the patients’ quality of life. Conclusions: ASC therapy has a positive effect on patients with OA, although there is limited evidence and little long-term follow-up.

Background: The use of Plasma Rich in Growth Factors (PRGF) and Adipose Derived Mesenchymal Stem Cells (ASCs) are today extensively studied in the field of regenerative medicine. In recent years, human and veterinary medicine prefer to avoid using traumatic techniques and choose low or non-invasive procedures. The objective of this study was to evaluate the efficacy of PRGF, ASCs and the combination of both in wound healing of full-thickness skin defects in rabbits. With this purpose, a total of 144 rabbits were used for this study. The animals were divided in three study groups of 48 rabbits each depending on the administered treatment: PRGF, ASCs, and PGRF+ASCs. Two wounds of 8 mm of diameter and separated from each other by 20 mm were created on the back of each rabbit: the first was treated with saline solution, and the second with the treatment assigned for each group. Macroscopic and microscopic evolution of wounds was assessed at 1, 2, 3, 5, 7 and 10 days post-surgery. With this aim, 8 animals from each treatment group and at each study time were euthanized to collect wounds for histopathological study. Results: Wounds treated with PRGF, ASCs and PRGF+ASCs showed significant higher wound healing and epithelialization rates, more natural aesthetic appearance, significant lower inflammatory response, significant higher collagen deposition and angiogenesis compared with control wounds. The combined treatment PRGF +ASCs showed a significant faster cutaneous wound healing process. Conclusions: The combined treatment PRGF+ASCs showed the best results, suggesting this is the best choice to enhance wound healing and improve aesthetic results in acute wounds.

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.