UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE ODONTOLOGÍA Departamento de Especialidades Clínicas Odontológicas TESIS DOCTORAL Terapia Celular en Regeneración Periodontal Cell Therapy in Periodontal Regeneration MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Silvia Nerea Sánchez Pérez Directores Prof. Dr. Mariano Sanz Alonso Dr. Jose Alberto García Sanz Madrid 2021 Cell Therapy in Periodontal Regereration 1 PhD Thesis of Nerea Sánchez Pérez 2 UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE ODONTOLOGÍA Departamento de Especialidades Clínicas Odontológicas TESIS DOCTORAL Terapia Celular en Regeneración Periodontal Cell Therapy in Periodontal Regeneration MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Silvia Nerea Sánchez Pérez Directores Prof. Dr. Mariano Sanz Alonso Dr. Jose Alberto García Sanz Madrid 2021 © Nerea Sánchez Pérez, 2021 Cell Therapy in Periodontal Regereration 3 Dedicado a mi familia PhD Thesis of Nerea Sánchez Pérez 4 AGRADECIMIENTOS En primer lugar me gustaría dar un agradecimiento especial a todas aquellas personas que han participado en la realización de estos estudios por su colaboración, apoyo y ayuda. A mis directores de tesis, los Dres. Mariano Sanz y Jose Alberto García Sanz. Al Dr. Mariano Sanz, por transmitirme su amor por la Periodoncia en mis años de formación postgraduada, por ser para mi un ejemplo a seguir en el mundo académico, y por haberme estimulado a buscar la excelencia en mi trabajo. Gracias también por haberme dado la oportunidad de participar en estos proyectos de investigación y por seguir confiando en mí. Al Dr. Jose Alberto García Sanz, por acogerme durante 3 cursos sucesivos en su laboratorio en el Centro de Investigaciones Biológicas-Margarita Salas y por su generosidad para enseñarme tantas cosas en el ámbito de los cultivos celulares y los trabajos con células mesenquimales. He aprendido muchísimo, gracias por permitirme trabajar contigo. A cada una de las personas que han participado activamente en este proyecto: Prof. David Herrera, gracias por haberme ayudado y apoyado durante el desarrollo de estos trabajos, por tu paciencia con los emails, porque me has enseñado mucho hasta con las correcciones. Sin duda, me siendo muy agradecida por todo lo que he aprendido en el Master de Periodoncia y por haber confiado en mi para la docencia en grado y en los cursos de formación continua. Prof. Elena Figuero, en primer lugar, porque, allá por 2008, cuando fuiste mi tutora en la asignatura “Terapéutica Periodontal Compleja”, me estimulaste, aunque no te dieras cuenta, para aplicar al Master de Periodoncia (la mejor decisión que pude tomar en aquel momento). En segundo lugar, gracias por haberme enseñado prácticamente todo lo que he necesitado para hacer el análisis de datos de esta tesis doctoral en tu curso de Metodología de la Investigación. También quiero reseñar tu apoyo todos estos años en el ámbito docente. Cell Therapy in Periodontal Regereration 5 A Ludovica Fierravanti, mi compañera en el ensayo clínico de terapia celular, tan trabajadora y responsable como brillante. Gracias por todo el esfuerzo. Finalmente lo conseguimos. Al Dr. Javier Nuñez, Dr. Fabio Vignoletti, Dr. Ignacio Sanz Martín, Silvia Santamaría y María González Zamora, gracias por ser mis compañeros en estos proyectos de investigación, por acompañarme en esta experiencia. Agradecer a los profesores que han contribuido a mi formación postgraduada en el Master de Periodoncia de la Universidad Complutense de Madrid, de los que tanto he aprendido durante el master. Agradecimiento especial al Dr. Antonio Bujaldón, por su amistad, su apoyo y por haber depositado su confianza en mí en numerosas ocasiones. Al Dr. Luis Blanco Jerez por sembrar en mi la inquietud por la investigación, estimular mi trabajo en el ámbito docente y por su amistad todos estos años. Mis inicios en el mundo de los cultivos celulares y las células mesenquimales fueron gracias a tí. Mención al Ministerio de Educación por haberme otorgado una ayuda predoctoral para la Formación de Profesorado Universitario. También me gustaría transmitirles mi agradecimiento de manera muy especiar a las personas que en el ámbito personal me han animado para seguirme desarrollando en el ámbito profesional y me han guiado cada día para crecer como persona. A Carmen, Merche, María y Estefi, por haber sido mis compañeras y mi sostén durante mis años de formación postgraduada, porque todos esos momentos de estudio en el máster se hiceron mucho más llevaderos con ellas y porque en ellas encontré unas excelentes amigas. A Nagore, Edu, Ana, Paula, Maria Riobóo y al resto de mis colegas del Máster de Periodoncia con los que ahora comparto mis horas de docencia en el Grado en Odontología, porque es una suerte trabajar con compañeros así y porque su capacidad de trabajo y disciplina me motivan para seguir esforzándome y aprendiendo cada día. A Beatriz Hernando y al resto de mis amigas de la carrera, por su amistad y cariño. PhD Thesis of Nerea Sánchez Pérez 6 Y por último, mi mayor agradecimiento va dirigido a las personas a las que dedico esta tesis doctoral: A mis padres, por su amor incondicional, por su amparo en todos los aspectos de mi vida, por estar siempre a mi lado, por ser un ejemplo desde que era una niña. Gracias papá por tu energía y estímulo insaciable para los nuevos proyectos; gracias mamá por transmitirme tu amor por la docencia y tu paciencia. Gracias por alentarme para hacer este trabajo. A mi hermana Marta, a Lucía y al resto de mi familia, por su apoyo y confianza, no solo en mi vida personal, sino también, en lo profesional. Tengo mucha suerte de teneros. A César, mi marido, por compartir su vida conmigo y hacerme feliz cada día. Su amor, paciencia y ánimos han sido el motor que me ha empujado para continuar. Por ser mi compañero de viaje y un ejemplo de bondad y empatía. Cell Therapy in Periodontal Regereration 7 PREFACE The present PhD Thesis has been the result of the research works developed thanks to a Scholarship for the Training of Research and Teaching Personnel (FPU) from the Spanish Ministry of Education (4 years duration) under the direction of Professor Mariano Sanz and a Research Stay in the Department of Periodontics and Oral Surgery, University of Michigan, USA (July 1 to October 15, 2014) under the direction of Professor Hector Rios. This Thesis consists of the following three publications: Study 1. Santamaría S, Sánchez N, Sanz M, & García-Sanz JA. (2017). Comparison of periodontal ligament and gingiva-derived mesenchymal stem cells for regenerative therapies. Clinical Oral Investigations 21(4), 1095-1102. Study 2. Núñez J, Sánchez N, Vignoletti F, Sanz-Martin I, Caffesse R, Santamaría S, García- Sanz JA, Sanz M. (2018). Cell therapy with allogenic canine periodontal ligament-derived cells in periodontal regeneration of critical size defects. Journal of Clinical Periodontology 45(4):453-461. Study 3. Sánchez N, Fierravanti L, Núñez J, Vignoletti F, González-Zamora M, Santamaría S, Suárez-Sancho S, Fernández-Santos ME, Figuero E, Herrera D, García-Sanz JA, Sanz M. (2020). Periodontal regeneration using a xenogeneic bone substitute seeded with autologous periodontal ligament-derived mesenchymal stem cells: A 12-month quasi- randomized controlled pilot clinical trial. Journal of Clinical Periodontology 47(11):1391- 1402. PhD Thesis of Nerea Sánchez Pérez 8 Abbreviations AEMPS: Spanish Medicines Agency (“Agencia Española de Medicamentos y Productos Sanitarios”) ADSCs: Adipose tissue-derived stem cells β-TCP: β-Tricalcium Phosphate BMSCs: Bone Marrow-derived Mesenchymal stromal cells BOP: Bleeding on probing CAL: Clinical attachment level CCT: Controlled clinical trial DBBM: Demineralized bovine bone mineral DNA: Deoxyribonucleic acid DPSCs: Dental pulp stem cells RCT: randomized controlled clinical trial EMD: Enamel matrix derivatives FISH: Fluorescence in situ hybridization FDA: American Food and Drug Administration GFP: Green Fluorescent Protein GMSCs: gingiva-derived mesenchymal stromal cells GTR: Guided tissue regeneration ISCT: International Society for Cell Therapy JCR: Journal Citation Report MIS: Minimally invasive surgery MIST: modification of the Minimally invasive surgery technique M-MIST: modification of the former technique MSCs: Mesenchymal stem cells or mesenchymal stromal cells MTT: Dimethylthiazol PDL-MSCs: Periodontal ligament-derived mesenchymal stromal cells P. gingivalis: Porphyromonas gingivalis PRP: Platelet-rich plasma PPD: Probing pocket depth rhFGF-2: Recombinant Human Fibroblast Growth Factor -2 rhPDGF-BB: Recombinant Human Platelet Derived Growth Factor-BB SD: Standard deviation SFA: Single-Flap Approach Cell Therapy in Periodontal Regereration 9 Table of Contents I. SUMMARY 10 RESUMEN 12 II. INTRODUCTION 14 Periodontitis 14 Treatment of Periodontitis 16 Periodontal Regeneration: concept and technologies 17 Efficacy and determining factors in periodontal regeneration 20 New approaches in regenerative treatments: cell therapies. 24 Application of cell therapy with stromal mesenchymal stem cells in periodontal regeneration. 29 III. JUSTIFICATION 32 IV. HYPOTHESIS 32 V. OBJECTIVES 33 VI. MATERIAL AND METHODS. RESULTS 36 Study 1. Santamaría S, Sánchez N, Sanz M, & García-Sanz JA. (2017). Comparison of periodontal ligament and gingiva-derived mesenchymal stem cells for regenerative therapies. Clinical Oral Investigations 21(4), 1095-1102. 36 Study 2. Núñez J, Sánchez N, Vignoletti F, Sanz-Martin I, Caffesse R, Santamaría S, García-Sanz JA, Sanz M. (2018). Cell therapy with allogenic canine periodontal ligament-derived cells in periodontal regeneration of critical size defects. Journal of Clinical Periodontology 45(4):453- 461. 36 Study 3. Sánchez N, Fierravanti L, Núñez J, Vignoletti F, González-Zamora M, Santamaría S, Suárez-Sancho S, Fernández-Santos ME, Figuero E, Herrera D, García-Sanz JA, Sanz M. (2020). Periodontal regeneration using a xenogeneic bone substitute seeded with autologous periodontal ligament-derived mesenchymal stem cells: A 12-month quasi-randomized controlled pilot clinical trial. Journal of Clinical Periodontology 47(11):1391-1402. 36 VII. DISCUSSION 40 VIII. CONCLUSIONS 59 IX. REFERENCES 60 X. FIGURES AND TABLES 78 PhD Thesis of Nerea Sánchez Pérez 10 I. SUMMARY Introduction. Current techniques for periodontal regeneration have demonstrated successful results in the treatment of defects with a specific morphology. Nevertheless, most periodontal defects produced by the disease do not have predictable regenerative outcomes. As a result, new treatment approaches have been proposed throughout the last decades, in order to improve the predictability of conventional strategies. The application of undifferentiated multipotent adult cells, the “mesenchymal stromal cells”, has emerged in periodontology and has been the focus of a large number of preclinical and clinical studies. Aims. The goals of these investigations were to compare the genomic stability, preclinical in vivo safety, and the ex vivo proliferative potentials of human periodontal ligament- and gingiva-derived mesenchymal stem cells in optimal and suboptimal culture conditions (Study 1), to evaluate the regenerative capacity of allogeneic periodontal ligament-derived mesenchymal cells in a critical-size periodontal defect model (Study 2) and to analyse the safety and efficacy of a cell therapy clinical protocol based on the application of autologous periodontal ligament-derived mesenchymal stromal cells for the treatment of 1 and 2-wall intrabony defects in patients suffering from periodontitis (Study 3). Material and Methods. Study 1: in this first work, an ex vivo investigation, periodontal ligament samples were obtained from extracted teeth with both an intact and a reduced periodontium. Gingival connective tissue samples were also isolated from the palate of four adult patients. Then, all the samples were digested and processed for ex vivo expansion in culture. Experiments for assessing the mesenchymal phenotype of the three cell populations confirmed adherence to the plastic flask, expression of immunohistochemical markers of mesenchymal cells and osteogenic, adipogenic and chondrogenic differentiation. In addition, periodontal ligament and gingiva-derived mesenchymal cells showed genomic stability, by using microarrays-based assays, and lack of tumorigenic potential six months after subcutaneous injection in immunodeficient mice. When proliferation rates of both cell populations were analyzed, no differences were observed between them in standard culture conditions; however, when subjected to suboptimal culture conditions, gingiva- derived mesenchymal cells exhibited significantly superior results. Cell Therapy in Periodontal Regereration 11 Study 2: in this preclinical investigation in 9 Beagle dogs, critical size 6-mm supra-alveolar periodontal defects were artificially created around the third and fourth premolars. After defects chronification, they were assigned, according to a randomized allocation, either to a test treatment group consisting of the use of allogeneic periodontal ligament- derived mesenchymal stromal cells seeded in hydroxyapatite-collagen scaffolds, or the control group (scaffold without cells). The histological analysis, performed 3 months after the intervention, didn´t show statistically significant differences between both treatment modalities in terms of new-formed cementum and total dimensions of periodontal regeneration. Study 3: this last study is a 12-month quasi-randomized double-blinded controlled pilot clinical trial with a parallel groups design that included 20 patients with 1 and 2-wall intrabony defects. Subjects were assigned a test treatment, consisting of the application of autologous periodontal ligament-derived mesenchymal stromal cells embedded in the same scaffold as in the previous preclinical study, and a control therapy, consisting of the use of the scaffolds without cells. The results showed that the use of autologous periodontal ligament-derived mesenchymal cells as therapeutic strategy in periodontal regeneration in humans is safe. Regarding the efficacy outcome variables, the test group exhibited a positive trend towards a greater clinical attachment level gain and a probing pocket depth reduction than the control group, although the differences between them were not statistically significant. Conclusions. On the basis of the preclinical and clinical data provided by these three investigations, we can say that the use of mesenchymal stromal cells in periodontal regeneration is safe. Nevertheless, the added beneficial effect of cell therapy in terms of clinical variables could not be demonstrated in this work. KEY WORDS: periodontal regeneration, stem cells, mesenchymal stem cells, tissue engineering, cell therapy. PhD Thesis of Nerea Sánchez Pérez 12 RESUMEN Introducción. Las técnicas actuales para la regeneración del periodonto son eficaces en defectos con una morfología específica. Sin embargo, la gran mayoría de defectos óseos ocasionados por la periodontitis, no disponen de un enfoque regenerativo predecible. Por ello, a lo largo de las últimas décadas, se ha promovido la búsqueda de nuevos enfoques de tratamiento con el objetivo de mejorar la predictibilidad de las estrategias convencionales. Por ello, la aplicación de células adultas multipotentes indiferenciadas, las llamadas “células mesenquimales estromales”, se ha abierto paso en periodoncia, y ha sido objeto de estudio en experimentación preclínica y clínica. Objetivos. Los objetivos de esta serie de trabajos fueron comparar, la estabilidad genómica, la seguridad preclínica in vivo y los potenciales de proliferación ex vivo de células mesenquimales humanas derivadas de ligamento periodontal y células mesenquimales procedentes de tejido conectivo gingival en condiciones óptimas y subóptimas de cultivo (Estudio 1), evaluar el potencial regenerativo de células mesenquimales alogénicas de ligamento periodontal en un modelo preclínico de defecto crítico supracrestal (Estudio 2) y analizar la seguridad y eficacia de un protocolo clínico de terapia celular, basado en la aplicación de células mesenquimales autólogas de ligamento periodontal en defectos intraóseos de 1 y 2 paredes, en pacientes afectados por periodontitis (Estudio 3). Material y Método. Estudio 1: este primer estudio de la serie, una investigación ex vivo, se obtuvieron muestras de ligamento periodontal, tanto de dientes extraídos con un periodonto intacto, como de dientes extraídos con un periodonto reducido, así como muestras de tejido conectivo gingival palatino, que, a continuación se digirieron y procesaron para su expansión en cultivo ex vivo. Se realizaron los experimentos para la confirmación del fenotipo mesenquimal de los tres tipos celulares, exhibiendo en todos los casos, adherencia al frasco de cultivo, expresión de la batería de marcadores inmunohistoquímicos típicos de célula mesenquimal y diferenciación osteogénica, adipogénica y condrogénica. Asimismo, se corroboró la estabilidad genómica de las células mesenquimales de encía mediante “microarrays” y la ausencia de potencial tumorigénico de las células mesenquimales de encía y de ligamento periodontal, 6 meses tras su inyección subcutánea en ratones inmunodeficientes. Cuando se analizó la tasa de proliferación celular en ambos tipos celulares, no se observaron diferencias en condiciones estándar de cultivo; sin embargo, la proliferación de las células de encía fue Cell Therapy in Periodontal Regereration 13 superior a las de ligamento periodontal cuando se emplearon unas condiciones subóptimas de cultivo. Estudio 2: para la realización de este estudio preclínico en 9 perros Beagle, se crearon artificialmente defectos críticos supracrestales de 6 mm alrededor de los terceros y cuartos premolares, que, tras su cronificación, se trataron, de acuerdo a la asignación aleatoria, bien con una terapia test, basada en el uso de células mesenquimales alogénicas de ligamento periodontal sembradas en matrices de hidroxiapatita–colágeno, o bien con la terapia control, consistente en la aplicación de la matriz sin células. El análisis histológico, realizado a los 3 meses de la intervención, reveló la ausencia de diferencias estadísticamente significativas entre los dos tratamientos en cuanto a niveles de cemento neoformado y dimensiones totales de la regeneración periodontal obtenida. Estudio 3: este último estudio de la serie, es un ensayo clínico piloto controlado cuasi- aleatorizado de grupos paralelos a doble ciego, de 12 meses de duración, que incluyó 20 pacientes con defectos intraóseos de 1 y 2 paredes, a los que se les asignó a un tratamiento test, basado en la aplicación de células mesenquimales autólogas de ligamento periodontal, embebidas en la misma matriz que en el estudio anterior, y una terapia control, consistente en la utilización de la matriz sin células. Los resultados mostraron que el uso de las células mesenquimales de ligamento periodontal autólogas como terapia en regeneración periodontal en humanos es seguro. En cuanto a los parámetros de eficacia, aunque se observó una tendencia positiva del grupo test en cuanto a una mayor ganancia en el nivel de inserción y una mayor reducción de la profundidad de sondaje que el grupo control, las diferencias entre los grupos no fueron estadísticamente significativas. Conclusiones. Tras la evaluación de los tres estudios, podemos decir que el uso de células mesenquimales estromales en regeneración periodontal constituye una terapia segura. Sin embargo, su beneficio añadido a nivel clínico no ha podido ser demostrado en este trabajo. PALABRAS CLAVE: regeneración periodontal, células madre, células mesenquimales estromales, ingeniería tisular, terapia celular. PhD Thesis of Nerea Sánchez Pérez 14 II. INTRODUCTION Periodontitis Periodontitis is a multifactorial chronic inflammatory disease with an infectious nature characterized by a progressive destruction of the periodontal tissues (gingiva, periodontal ligament, root cementum and alveolar bone) due to an imbalance in the homeostasis between the subgingival microbiota and the host immune-inflammatory response (Bartold, McCulloch et al. 2000). The current accepted etiological model of the pathogenesis of periodontitis consists of the presence of subgingival pathogenic bacteria organized in biofilms, which activate the immunoinflammatory mechanisms responsible for bone and connective remodeling and ultimately results in periodontal destruction (Page, Offenbacher et al. 1997). These destructive processes, in turn, are modulated by certain environmental and/or genetic factors that determine the phenotype of periodontitis (Kornman 2008). Until recently, the prevailing paradigm was that specific microorganisms were the main etiological agent of periodontitis, especially those with a strong association with this disease, such as those from the "red complex", such as Porphyromonas gingivalis (P. gingivalis), Treponema denticola and Tannerella forsythia (Socransky, Haffajee et al. 1998). However, scientific advances in recent decades have confirmed that the disruption in the homeostasis between microorganisms and the host is responsible for the destructive process that characterizes periodontitis (Dewhirst, Chen et al. 2010). The subgingival microbiota dysbiosis is mediated by keystone pathogens such as P. gingivalis (Hajishengallis, Liang et al. 2011), which induce alterations in the host mechanisms of defense, perturbing the growth and development of the subgingival biofilm, which triggers balance alterations between the bacterial ecosystem and the host, resulting in destructive chronic inflammatory processes (Hajishengallis 2014, Hajishengallis and Lamont 2014). Tissue destruction observed in periodontitis histopathologically results in the formation of the “periodontal pocket”, produced as a consequence of the connective tissue fibers attachment loss, the apical migration of the junctional epithelium and the alveolar bone resorption (Schroeder and Lindhe 1980, Bosshardt 2018). The resulting osseous lesions represent, therefore, the anatomical consequence of the apical progression of periodontitis (Papapanou and Tonetti 2000). Depending on the location of the bottom of the periodontal pocket with regards to the bone crest, the osseous lesions are classified in suprabony defects (bottom of the pocket coronal to the crest), infrabony (bottom of the Cell Therapy in Periodontal Regereration 15 pocket apical to the crest) (Goldman and Cohen 1958), or interradicular, when bone loss affects the interradicular aspect of multirradicular teeth (Papapanou and Tonetti 2000). Infrabony defects, in turn, are categorized in “craters”, if two teeth are affected, and “intrabony”, if one tooth is affected; the classification of the former depends on the number of walls and is categorized in 1, 2, and 3-wall intrabony defects and in combined lesions (Papapanou and Tonetti 2000). Severe periodontitis is a very prevalent disease. According to a systematic review with epidemiological data from 37 countries, it is the sixth most prevalent condition in the world, affecting approximately 11% of the adult population (Kassebaum, Bernabe et al. 2014). The prevalence of the disease increases with age, exhibiting a peak in incidence at around 40 years of age; it is widely distributed around the world and has important public health implications (Genco and Sanz 2020). In particular, in Spain, an oral health survey performed in 2015 estimated that around 23% and 10% of the young adults aged 35-44 years old suffered a clinical attachment level (CAL) loss of 4-5 mm and ≥6 mm, respectively. The CAL loss prevalence for the 65-74 years cohort group was slightly higher, since 39,5% of subjects lost 4-5 mm and 31.4% lost ≥6 mm (Bravo Pérez, Almerich Silla et al. 2016). This data demonstrate the relevance of treating the disease to avoid the damage of periodontal supporting structures and ultimately, tooth loss, what consequently worsen the quality of life of the population (Llodra Calvo, Oliver et al. 2012). Besides, periodontitis is related to several conditions and systemic diseases via the activation of the host inflammatory response by the periodontopahogenic microorganisms and the entrance of bacteria in the blood stream (Sanz, Ceriello et al. 2018, Schenkein, Papapanou et al. 2020). Scientific evidence has confirmed that periodontitis increases the risk of suffering from cardiovascular diseases, diabetes mellitus and pregnancy complications, among others (Madianos, Bobetsis et al. 2013, Sanz, Ceriello et al. 2018, Sanz, Marco Del Castillo et al. 2020). Furthermore, the treatment of periodontitis has an important role in achieving significant clinical improvements in some of these diseases, or, at least, in the promotion of relevant changes in the biomarkers associated with the disease (Chapple, Genco et al. 2013, Sanz, Kornman et al. 2013, Sanz, Marco Del Castillo et al. 2020). Therefore, the treatment of periodontitis is not only fundamental to promote oral health, but also to prevent and improve the specific systemic conditions that affect the general health in the long term (Genco and Sanz 2020). Given the importance of conducting an appropriate clinical diagnosis to perform the best available treatment of periodontitis, in 2017, experts from the European Federation of PhD Thesis of Nerea Sánchez Pérez 16 Periodontology and from the American Academy of Periodontology met in Chicago (World Workshop in Periodontology) to create a new classification scheme for Periodontal and Peri-Implant Diseases and Conditions (Caton, Armitage et al. 2018, Papapanou, Sanz et al. 2018). In this meeting, a new classification of Periodontitis based on Stages, related to the severity of the disease and Grades, related to its progression rate, the anticipated treatment response and the effects on systemic health. (Papapanou, Sanz et al. 2018). Within this new classification, advanced periodontitis would be included within Stages III and IV, which coincide with some of their severity criteria, such as interproximal CAL of at least 5 mm, locations with probing pocket depths (PPD) of at least 6 mm, the presence of class II and III furcation lesions and vertical bone defects and radiographic bone loss from the middle third to the apical third of the root. However, Stages III and IV differ in the number of teeth lost due to periodontitis, considering Stage III if less than 5 teeth have been lost and IV if at least 5 teeth have been lost due to the disease, which will require a multidisciplinary treatment and complete oral rehabilitation (Papapanou, Sanz et al. 2018). Treatment of Periodontitis The incorporation of the aforementioned new Classification of Periodontitis, aimed at linking each category of the disease with certain prevention and treatment approaches, has led to the development of Clinical Practice Guidelines based on the available evidence to help the clinician in the treatment of the Stage I-III periodontitis (Sanz, Herrera et al. 2020). According to these Guidelines, once the diagnosis of the disease has been made, therapy must be carried out incrementally, following treatment steps, which include several interventions. The first "step" of treatment is not only aimed at controlling the risk factors for periodontitis, but also at achieving the removal of the supragingival biofilm, by both the patient and the professional, an essential aspect for the control of the disease (Chapple, Van der Weijden et al. 2015, Carra, Detzen et al. 2020, Ramseier, Woelber et al. 2020). The “step” 2 includes interventions for the reduction/elimination of subgingival biofilm and calculus, and also the use of several physical and chemical agents, host modulators, and systemic and local antimicrobials (Donos, Calciolari et al. 2020, Herrera, Matesanz et al. 2020, Suvan, Leira et al. 2020, Teughels, Feres et al. 2020). According to the most recent evidence, subgingival mechanical instrumentation constitutes the fundamental strategy for the control of the disease in subjects with periodontitis; it may be performed with either hand or sonic/ultrasonic instruments and with either traditional quadrant-wise or full mouth delivery within 24 h (Suvan, Leira et al. 2020). In addition, Cell Therapy in Periodontal Regereration 17 this non-surgical periodontal therapy promotes significant improvements in terms of CAL gain and PPD and bleeding on probing (BOP) reduction (Van der Weijden and Timmerman 2002, Suvan 2005, Suvan, Leira et al. 2020). Non-surgical subgingival mechanical treatment allows the management of most forms of periodontitis (Suvan 2005); however, there are locations that may not resolve after this initial therapy due to the anatomical and microbiological limitations of mechanical treatment, as well as the exposure to certain environmental factors that may worsen the response to treatment (Waerhaug 1978, Kalkwarf, Kaldahl et al. 1988, Renvert, Nilveus et al. 1990, Renvert, Wikstrom et al. 1990, Tomasi, Leyland et al. 2007). The areas with residual PPD≥6mm after non-surgical periodontal treatment are locations at a higher risk of disease progression and tooth loss during supporting periodontal therapy (Claffey and Egelberg 1995, Matuliene, Pjetursson et al. 2008). The “step” 3 of therapy is aimed at treating those locations which do not respond appropriately to non-surgical treatment, such as areas with PPD≥4 mm with BOP or the presence of deep periodontal pockets≥ 6 mm (Sanz, Herrera et al. 2020). In these cases, either repeated subgingival instrumentation with or without adjunctive therapies or access, resective or regenerative periodontal surgery is recommended, with the aim of gaining access to subgingival instrumentation and managing bone lesions that create complexity with regards to the treatment of periodontitis (furcation and intraosseous defects) (Jepsen, Gennai et al. 2020, Nibali, Koidou et al. 2020, Polak, Wilensky et al. 2020, Sanz-Sanchez, Montero et al. 2020). Finally, it is necessary to include all the patients treated for periodontitis in a supportive periodontal care program (fourth "step"), with the aim of achieving the stability of the periodontal tissues over time, by scheduling periodical visits, according to the patient´s needs, in order to monitor and carry out mainly preventive interventions, although, sometimes therapeutic interventions are needed (Sanz, Herrera et al. 2020). Periodontal Regeneration: concept and technologies Conventional treatment strategies for periodontitis, focused on infection control, fail to completely restore the supporting periodontal tissues (Caton, Nyman et al. 1980). Regenerative periodontal therapy is the ideal treatment to recover the tissues damaged by the disease, since it enables, at least partially, the restitutio ad integrum of the tooth supporting structures: root cementum, periodontal ligament and alveolar bone (Nyman, Gottlow et al. 1982). Periodontal regeneration also allows the formation of a new PhD Thesis of Nerea Sánchez Pérez 18 connective tissue attachment, with well-oriented collagen fibers inserted into the newly formed root cementum (Isidor, Karring et al. 1985). Already in the 70s, the authorship of the cells responsible for the regenerative process started to be debated (Karring, Nyman et al. 1980, Nyman, Karring et al. 1980, Nyman, Gottlow et al. 1982, Nyman, Lindhe et al. 1982). Melcher proposed his hypothesis based on the fact that the cell type that repopulated the root surface after the periodontal surgery would determine the nature of the attachment formed on the root surface (Melcher 1976). In fact, histological studies that analysed healing after traditional periodontal surgery procedures revealed that the gingival epithelium cells were the ones that first reached the surface devoid of periodontal ligament and formed a long junctional epithelium, inhibiting a connective tissue attachment (Listgarten and Rosenberg 1979, Caton, Nyman et al. 1980). At the University of Gothenburg, Sweden, a series of experimental studies were carried out in dogs and monkeys, with the aim of evaluating the healing on the root surface according to the type of cells from the periodontium that repopulated the wound. Therefore, they experimentally induced periodontitis, carried out the removal of the periodontal ligament and cementum and created different models to study the colonization of the root surface by cells from the different compartments of the periodontium (Karring, Nyman et al. 1980, Nyman, Karring et al. 1980, Nyman, Gottlow et al. 1982). In one of the first works, the roots, devoid of periodontal ligament, were extracted, scraped and then reimplanted in bone cavities prepared in edentulous areas, so that the root surface was in contact with the bone. Colonization by bone line cells revealed, at 3 months, a healing characterized by repair phenomena, mainly root resorption and ankylosis (Karring, Nyman et al. 1980). In a second study, the roots were treated in a similar way and placed horizontally in bone concavities, so that half of their surface was covered by the flap and, therefore, in intimate contact with the cells of the connective tissue. At 3 months, histological analysis showed that the root portion colonized by gingival connective tissue cells presented connective tissue fibers parallel to the root surface without attachment to the tooth and root resorption phenomena in most cases (Nyman, Karring et al. 1980). Finally, in a later work in which, preference was given to the cells from the periodontal ligament to repopulate the wound, a "Millipore" cellulose acetate filter was placed after the periodontal ligament and cementum removal in order to avoid contact of the flap with the root surface during healing. In this case, at 6 months, the histological analysis demonstrated the formation of new cementum and new collagen fibers inserted in the area coronal to the notch, indicating that a new periodontal attachment had been created on the root surface (Nyman, Gottlow et al. 1982). These Cell Therapy in Periodontal Regereration 19 studies, therefore, demonstrated that the only cells with the capacity to regenerate the lost periodontal attachment were those from the periodontal ligament of the tooth (Karring, Nyman et al. 1980, Nyman, Karring et al. 1980, Nyman, Gottlow et al. 1982). Since then, multiple technologies have been studied to achieve the regeneration of cementum, periodontal ligament and alveolar bone, including guided tissue regeneration (GTR) with barrier membranes, both resorbable and non-resorbable, but also the use of biologically active agents, bone replacement grafts and combinations of the above (Cortellini and Tonetti 2015). Within the group of biologically active agents, the one supported probably by the strongest scientific evidence is the use of Enamel Matrix Proteins or Enamel Matrix Derivatives (EMD) (Hammarstrom, Heijl et al. 1997, Heijl, Heden et al. 1997, Sculean, Donos et al. 1999, Froum, Weinberg et al. 2001, Tonetti, Lang et al. 2002, Silvestri, Sartori et al. 2003, Jepsen, Heinz et al. 2004, Sanz, Tonetti et al. 2004, Esposito, Grusovin et al. 2009). This modality of periodontal regeneration aims to mimic the events that occur naturally during the development of the dental root, by which the cells of the epithelial sheath of Hertwig, located in the apical portion of the enamel organ, secrete and deposit a series of proteins derived from the enamel (amelogenin, tuftelin, etc.) that stimulate the differentiation of cementoblasts and the synthesis of root cementum (Hammarstrom 1997, Hammarstrom, Heijl et al. 1997). Currently, they are available as a purified acid extract of porcine origin (Emdogain®), a product supported by relevant histological evidence regarding the formation of new cementum with inserted collagen fibers and new alveolar bone (Hammarstrom, Heijl et al. 1997, Sculean, Donos et al. 1999). Another strongly studied biologically active agent, whose periodontal regenerative capacity has been demonstrated in various clinical studies, is the human recombinant platelet-derived growth factor, in its BB form (rhPDGF-BB) (Nevins, Camelo et al. 2003, Nevins, Giannobile et al. 2005, Nevins, Kao et al. 2013), a protein secreted by platelets during the early phase of fracture repair, present in the bone matrix, with the potential to stimulate type I collagen synthesis by the osteoblasts and healing through chemotactic and mitogenic activities (Andrew, Hoyland et al. 1995). The application of rhPDGF-BB for periodontal regeneration has been approved by the American Food and Drug Administration (FDA) for the treatment of intrabony defects and furcation lesions, so this factor is commonly used in the clinical practice in the United States (Suarez-Lopez Del Amo, Monje et al. 2015), usually in combination with bone grafts such as lyophilized demineralized bone allograft and β-tricalcium phosphate (β-TCP) (Nevins, Camelo et al. 2003, Nevins, Kao et al. 2013). PhD Thesis of Nerea Sánchez Pérez 20 Another agent whose use as periodontal regenerative therapy is approved in some countries is the recombinant human fibroblast growth factor-2 (rhFGF-2), which belongs to a family of proteins involved in the proliferation, migration and differentiation of periodontal ligament cells and the production of extracellular matrix (Kitamura, Akamatsu et al. 2011). This factor has also shown histological evidence of periodontal regeneration when applied in several preclinical models that reproduced class II furcation lesions and intrabony defects, demonstrating superiority of the results with respect to the control group (Takayama, Murakami et al. 2001, Oortgiesen, Walboomers et al. 2014, Nagayasu- Tanaka, Anzai et al. 2015). Most of these previously described technologies have been combined with different types of bone grafts in order to act as a matrix or scaffold for cell growth and to facilitate the maintenance of the space and the stability of the blood clot (Velasquez-Plata, Scheyer et al. 2002, Reynolds, Aichelmann-Reidy et al. 2003, Nevins, Giannobile et al. 2005, Tu, Needleman et al. 2012, Oortgiesen, Walboomers et al. 2014). As single therapy, the application of bone grafts (autologous, xenogeneic, allogeneic and synthetic) has been evaluated in the management of intrabony defects and furcation defects, but only some of these materials, such as demineralized freeze-dried bone allografts, have scientific evidence that supports the formation of new bone, cementum and periodontal ligament after its use in intrabony defects (Bowers, Chadroff et al. 1989, Hoidal, Grimard et al. 2008, Kao, Nares et al. 2015). Despite the large number of biomaterials and agents that have been evaluated in the regeneration of the periodontium, not all these strategies have the same evidence in terms of efficacy in the creation of a new connective tissue attachment (Sanz and Giovannoli 2000, Jepsen, Eberhard et al. 2002, Sculean, Nikolidakis et al. 2008, Sculean, Nikolidakis et al. 2015). In addition, there are several determining factors that may influence the result of the regenerative treatment and the long-term stability of the results (Tonetti, Pini-Prato et al. 1993, Tonetti, Pini-Prato et al. 1995, Tonetti, Prato et al. 1996). Efficacy and determining factors in periodontal regeneration The latest systematic reviews of randomized controlled clinical trials (RCT) have shown that strategies based on GTR and on the application of Enamel Matrix Derivatives would present an added clinical benefit of 1.43 mm (0.76-2.22) and 1.27 mm (0.79-1.74mm) respectively, in terms of CAL gain with respect to the access flap surgery (Jepsen, Gennai et al. 2020, Nibali, Koidou et al. 2020). Furthermore, according to these reviews, the Cell Therapy in Periodontal Regereration 21 combination of either of the two strategies with deproteinized bovine bone mineral would improve the results (Jepsen, Gennai et al. 2020, Nibali, Koidou et al. 2020). Several studies have compared the efficacy of these regenerative materials in the treatment of periodontal defects (Silvestri, Sartori et al. 2003, Jepsen, Heinz et al. 2004, Sanz, Tonetti et al. 2004, Siciliano, Andreuccetti et al. 2011). A multicenter parallel-group RCT that evaluated the results obtained with GTR with resorbable membranes and EMD in the treatment of intrabony defects, observed that 12 months after the intervention, there was no superiority of one treatment group over the other, in terms of CAL gain, the primary outcome variable of the study. However, GTR therapy exhibited a higher number of postsurgical complications (100%), mainly related to membrane exposure, compared to 6% of complications derived from the use of EMD (Sanz, Tonetti et al. 2004). Recent systematic reviews with meta-analysis have also assessed the application of rhPDGF-BB and rhFGF-2 (Kitamura, Akamatsu et al. 2011, Murakami 2011, Nevins, Kao et al. 2013) and have suggested a superiority of both molecules, with respect to the control group (biomaterial alone), in the treatment of intrabony defects (Khoshkam, Chan et al. 2015, Li, Yu et al. 2017). In addition, significant therapeutic benefits have been observed in terms of CAL gain and bone fill for rhPDGF-BB, and CAL gain for rhFGF-2 (Khoshkam, Chan et al. 2015, Li, Yu et al. 2017). The evidence supports that certain factors from the patient, the type of defect and the surgical technique determine the result of the regeneration and influence its success or failure (Tonetti, Pini-Prato et al. 1993, Tonetti, Prato et al. 1996). Among the prognostic factors related to the patient, the plaque control would influence the result of the regenerative procedure in a "dose-dependent" manner: the better the plaque control, the greater the CAL gain (Cortellini, Pini-Prato et al. 1994). On the contrary, the persistence of subgingival infection by P. gingivalis and an irregular and infrequent supportive periodontal therapy, would be associated with a lower CAL gain and with the loss of the clinical benefit achieved after regenerative treatment (Cortellini, Pini-Prato et al. 1994, Ehmke, Rudiger et al. 2003). On the other hand, active smoking seems to be another predictor strongly associated with a poor regenerative prognosis. Smokers may present a lower CAL gain, in a dose-dependent manner (the higher the consumption, the more unfavorable results), and a poorer tissue healing after periodontal regeneration (Tonetti, Pini-Prato et al. 1995, Mayfield, Soderholm et al. 1998). PhD Thesis of Nerea Sánchez Pérez 22 Regarding the defect factors, many studies have analysed the efficacy of regenerative periodontal treatment in different types of periodontal defects (Pontoriero, Lindhe et al. 1988, Pontoriero, Lindhe et al. 1989, Metzler, Seamons et al. 1991, Cortellini, Carnevale et al. 1998, Siciliano, Andreuccetti et al. 2011). However, only a few of these defects have been associated with predictable clinical outcomes after the regenerative procedure: intrabony defects and class-II mandibular furcation lesions (Jepsen, Gennai et al. 2020, Nibali, Koidou et al. 2020). Furthermore, the morphology of the defect plays an important role in wound healing, particularly for intrabony defects. The width of the intraosseous component, evaluated by means of the radiographic defect angle, is also an important prognostic factor according to the scientific literature, both for the GTR strategies and for the use of EMD, so that wider defects would be associated with lower CAL and bone level gains than narrower intrabony lesions (Tonetti, Pini-Prato et al. 1993, Tsitoura, Tucker et al. 2004). A retrospective study that analysed radiographs from teeth with intrabony defects, before and after regenerative treatment, reported that the probability of obtaining a CAL greater than 3mm in defects treated with EMD was 2.46 times greater when the radiographic defect angle was ≤ 22º than when it was ≥ 36º (Tsitoura, Tucker et al. 2004). In addition, the number of walls of the defect has also been considered as a factor that could affect the prognosis of the treatment, in a way that the greater the number of residual walls, the greater the CAL gain (Tonetti, Lang et al. 2002, Silvestri, Sartori et al. 2003). In a multicenter RCT, in which the use of EMD was compared with access flap surgery, it was observed that 3-wall intrabony defects were 269% more likely to exhibit at least 3 mm of CAL gain than one-wall intraosseous defects (Tonetti, Lang et al. 2002). However, it has been demonstrated that this unfavorable trend linked to less supportive intrabony defects is relevant when the materials used do not properly maintain the space for the regeneration, such as EMD or resorbable membranes as single elements; If these biomaterials are combined with a filling material or if a self-supporting membrane is used, the negative impact of the morphology of the defect decreases (Tonetti, Prato et al. 1996, Linares, Cortellini et al. 2006). Some studies have indicated that the total depth of the intraosseous component would significantly affect the amount of tissue gained after GTR (Tonetti, Pini-Prato et al. 1993). However, in a subsequent multicenter study, in which a subgroup analysis was performed according to the initial depth of the infrabony component of the defect, it was observed that the potential for the regeneration with GTR was similar in shallow and deep defects, Cell Therapy in Periodontal Regereration 23 indication that baseline defect depth was not a prognostic factor for the regeneration: although deep defects treated with GTR exhibited a higher linear CAL gain than shallow defects (< 3mm), when CAL gain was expressed as a percentage of the initial depth, the results were similar in both types of defect (Cortellini, Carnevale et al. 1998). Regarding the tooth, the evidence has shown that the endodontic status and the tooth mobility are relevant factors that affect the prognosis of the regenerative procedure (Cortellini and Tonetti 2015). If root canal therapy was properly performed with a good apical sealing, the endodontic status would not negatively influence healing and long-term stability of the results (Cortellini and Tonetti 2001). The mobility of the tooth in the initial visit has been related, in a dose-dependent manner, with an unfavorable result of regeneration (Cortellini, Tonetti et al. 2001). However, a retrospective study that analysed data from three RCTs, focused on the regeneration of intrabony defects, observed that, when the changes in clinical parameters after the regeneration were compared in teeth with physiological mobility and teeth with mobility ≤1 mm, no statistically significant differences were found (Trejo and Weltman 2004). Therefore, according to the scientific evidence, splinting teeth with mobility >1 mm prior to the regenerative procedure would be recommended (Cortellini and Tonetti 2015). The factors related to the surgical technique are also relevant for the healing process and the prognosis of the regeneration (Tonetti, Pini-Prato et al. 1993). The use of minimally invasive techniques with the aim of promoting space maintenance and blood clot stability, and preserving the integrity of the soft tissues are key aspects needed to achieve optimal results in periodontal regeneration approaches (Cortellini and Tonetti 2015). Papilla preservation techniques have been widely used over the last 25 years and have been associated with a significant improvement in clinical parameters and better preservation of the aesthetics of the surgical area (Cortellini, Prato et al. 1995, Cortellini, Prato et al. 1999, Cortellini and Tonetti 2015, Carra, Detzen et al. 2020). In particular, the aesthetic component related to the surgical treatment of intrabony defects in anterior teeth has stimulated the adaptation of mucogingival surgery techniques (Zucchelli and De Sanctis 2000) to the management of teeth subjected to periodontal regeneration, and even the design of apical approaches without incisions at the level of the papillas (Moreno Rodriguez, Ortiz Ruiz et al. 2019). Zucchelli and De Sanctis described a surgical technique to minimize post-surgery gingival recession by combining the simplified papilla preservation technique at the defect level and the coronally advanced flap of the adjacent PhD Thesis of Nerea Sánchez Pérez 24 teeth, thereby avoiding statistically and clinically significant changes in the soft tissues (Zucchelli and De Sanctis 2008). Strategies with a minimally invasive surgical approach (Harrel and Rees 1995), like the Minimally invasive surgical technique (MIST) (Cortellini and Tonetti 2007), a modification thereof, the modified MIST technique (M-MIST) (Cortellini, Pini-Prato et al. 2009, Cortellini and Tonetti 2009) or the Single-Flap approach (SFA) (Trombelli, Farina et al. 2009) have enabled the reduction of postoperative gingival recession and maximize CAL gain, by reducing flap raising and stimulating primary wound closure, blood clot stability and space maintenance (Cortellini 2012). In fact, the relevance of microsurgical techniques with a minimally invasive approach and delicate soft tissue management could be greater than even the regeneration material used, according to a clinical trial that revealed the absence of significant differences in the clinical and radiographic parameters after comparing M-MIST with and without regeneration material (Cortellini and Tonetti 2011). New approaches in regenerative treatments: cell therapies. According to the Commission Directive 2009/120/EC of 14 September 2009 amending Directive 2001/83/EC of the European Parliament and of the Council on the Community code relating to medicinal products for human use as regards advanced therapy medicinal products, a somatic cell therapy medicinal product is a biological medicinal product that contains i) cells or tissues (or is constituted by them), which have been subjected to substantial manipulation, in such a way that their biological characteristics, physiological functions or structural properties relevant to the intended clinical use have been altered, ii) cells or tissues that are not intended to be used for the same essential function in the recipient and in the donor (European Union 2009). They are included within advanced therapy drugs, according to Regulation (EC) No. 1394/2007, together with gene therapy, tissue engineered products and combined advanced therapy products (European Parliament 2007). Furthermore, cell therapy medicinal products "have properties to be used by humans, or administered to human beings, in order to treat, prevent or diagnose a disease through the pharmacological, immunological or metabolic action of their cells or tissues" (European Union 2009). Over the last decades, the concept of cell therapy has been introduced as a new technology for the regeneration of organs and tissues, and has been extensively investigated in multiple fields of medicine, such as cardiology or traumatology, among many others (Jeong, Yim et al. 2018, Nakamura and Murry 2019, Fernandes, Cortez de SantAnna et al. Cell Therapy in Periodontal Regereration 25 2020). The objective of Regenerative Cell Therapy is to restore the function of damaged organs and tissues as a result of traumatic injuries or chronic degenerative diseases, through the transplantation of cellular precursors that differentiate to the cell populations of the damaged tissue or that, through the secretion of chemical mediators or growth factors, are capable of stimulating the proliferation and differentiation of resident progenitor cells (Garcia-Gomez, Elvira et al. 2010, Han, Menicanin et al. 2014, Golpanian, Wolf et al. 2016). The key components of these therapeutic strategies are cell populations with proliferative potential, three-dimensional matrixes that act as scaffolds for cell growth or vehicles for the release of cells or bioactive molecules, and lastly, growth factors or biological agents, which stimulate cell proliferation in the receptor area (Han, Menicanin et al. 2014, Sánchez, Ríos et al. 2016). The cells with the highest proliferative capacity are the so-called “stem cells”, since they are the most undifferentiated progenitors, characterized by their potential for self- renewal, extensive proliferation and differentiation into several cell types (Garcia-Gomez, Elvira et al. 2010). There are mainly two categories of stem cells according to their origin and differentiation capacity: “embryonic stem cells”, which are pluripotent (differentiation capacity to cells of the three embryonic layers) and adult or postnatal stem cells, located in the majority of the tissues of the adult individual, since they have important roles in tissue homeostasis and repair, and with multipotency or differentiation capacity to a smaller number of cell lines (differentiation to cells of the same embryonic layer) (Hynes, Menicanin et al. 2012). The absence of ethical-legal conditions and tumorigenic potential of adult stem cells (King and Perrin 2014), has led this population to lead the cell-based therapeutics of the 21st century. In particular, one type of adult cells, the “hematopoietic stem cells”, has been used for decades as a therapeutic tool in bone marrow transplants (Thomas, Lochte et al. 1957). The other type of adult stem cells, the "Mesenchymal Stem Cells" or, following the terminology recommended by the International Society for Cell Therapy (ISCT), the "Mesenchymal Stromal Cells" (MSCs) (Horwitz, Le Blanc et al. 2005) were later isolated, in 1970, from bone marrow aspirates, as a subpopulation of multipotent cells that, in culture, formed adherent clonogenic cell clusters of fibroblast-like cells (Friedenstein, Chailakhjan et al. 1970). This cell population has now become the protagonist of cell-based regenerative medicine strategies (Golpanian, Wolf et al. 2016, Goldberg, Mitchell et al. 2017, Goncalves, Rodrigues et al. 2017, Jeong, Yim et al. 2018, Chen, Xu et al. 2019, Fisher, PhD Thesis of Nerea Sánchez Pérez 26 Cutler et al. 2019, Nunez, Vignoletti et al. 2019, Fernandes, Cortez de SantAnna et al. 2020). MSCs have not only been isolated from the bone marrow, but also from many other adult tissues, both extraoral and intraoral (Friedenstein, Chailakhjan et al. 1970, Gronthos, Mankani et al. 2000, Miura, Gronthos et al. 2003, Seo, Miura et al. 2004, Zhang, Shi et al. 2009). Among the extraoral sources of isolation, besides the bone marrow, MSCs can be obtained from the umbilical cord or the adipose tissue (ADSCs) (Mareschi, Biasin et al. 2001, Zuk, Zhu et al. 2001), and among the intraoral, the periodontal ligament (PDL- MSCs), the dental pulp of permanent (DPSCs) and deciduous teeth, the gingiva, the alveolar bone, the apical papilla or the dental follicle has been common isolation sources (Gronthos, Mankani et al. 2000, Miura, Gronthos et al. 2003, Seo, Miura et al. 2004, Matsubara, Suardita et al. 2005, Morsczeck, Gotz et al. 2005, Sonoyama, Liu et al. 2006, Zhang, Shi et al. 2009, Park, Kim et al. 2011). Since the isolation of MSCs in the 1970s, many publications have investigated in animal models, as well as in humans, the effect of transplanting MSCs as regenerative therapies in different fields of biomedicine (Zhang, Shi et al. 2009, Scuteri, Miloso et al. 2011, Golpanian, Wolf et al. 2016, Goldberg, Mitchell et al. 2017, Tassi, Sergio et al. 2017, Oka, Miyabe et al. 2018, Fisher, Cutler et al. 2019, Fernandes, Cortez de SantAnna et al. 2020). However, it was not until 2006, when the International Society for Cell Therapy established the minimum criteria to define human MSCs in laboratory research and preclinical studies (Dominici, Le Blanc et al. 2006). In this way, a general consensus would allow to homogenize, in a universal way, the minimum characteristics of the MSCs and therefore, enable us to compare studies carried out by different research groups. According to this consensus, the requirements that a cell population must exhibit to be considered as MSCs are the following: i) adherence to the plastic flask under standard culture conditions, ii) expression of cells surface markers of mesenchymal phenotype, for at least 95% of the cell population (CD90+, CD73+, CD105+) and absence (≤ 2% positive) of expression of CD34, CD45, CD79α or CD 19, CD14 or CD11b and HLA-DR class II, by flow cytometry and iii) differentiation capacity to osteoblasts, adipocytes and chondrocytes under in vitro differentiation conditions, demonstrated by cell culture staining (Dominici, Le Blanc et al. 2006). In addition, for their final goal, which is the transplantation of MSCs into humans, the safety of the expanded MSCs must be guaranteed, by analysing their genomic stability and confirming the absence of tumorigenic potential when transplanted into experimental animals (Barkholt, Flory et al. 2013, Neri 2019). Cell Therapy in Periodontal Regereration 27 The strategies using MSCs in the context of regenerative therapies aim to utilize the privileged potential of these cells to stimulate the biological processes that lead to tissue regeneration (Monsarrat, Vergnes et al. 2014). Several mechanisms have been described by which MSCs could exert their therapeutic effects (Liechty, MacKenzie et al. 2000, Kamihata, Matsubara et al. 2001, Muhammad, Nordin et al. 2018). Several in vivo studies have demonstrated the differentiation capacity of these undifferentiated progenitors towards cells of the receptor tissue (Liechty, MacKenzie et al. 2000, Kamihata, Matsubara et al. 2001). In fact, there is histological evidence that corroborates that MSCs labelled with Green Fluorescent Protein (GFP), prior to their transplantation, were present in the new formed tissues several weeks after their introduction in the receptor area (Wen, Lan et al. 2012, Yamada, Nakamura et al. 2013). For a long time it was thought that the tissue of origin where these cells were isolated could predispose the cell lineage towards which the MSCs differentiated (Kwon, Kim et al. 2016). However, the evidence supporting the hypothesis that the tissue of origin regulates the epigenetics of cells is limited (Ozkul and Galderisi 2016). On the other hand, it has also been suggested that MSCs could exhibit a property known as "cellular plasticity", by which they could differentiate towards other cellular phenotypes other than those of their tissue of origin, thus being considered pluripotent cells (Quesenberry, Abedi et al. 2004). Several investigations have evaluated this potential, analysing whether these "transdifferentiated" cells were actually capable of carrying out the same functions as the cells of the tissue into which they had differentiated (Scuteri, Miloso et al. 2011). The first protocols were based on the differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) into neurons, for the regeneration of the nervous system (Woodbury, Schwarz et al. 2000). Later, other investigations applied epigenetic modifiers and neuronal induction signals to differentiate mesenchymal cells into neuron-like cells; however, although the morphology and characteristics of these cells could suggest that they were really neurons, the evidence supporting their functionality was still weak (Alexanian 2015, Hernandez, Jimenez-Luna et al. 2020). The capacity of MSCs for multidifferentiation and permanence in the grafted tissue seems to be low (von Bahr, Batsis et al. 2012); Nevertheless, MSCs are extremely powerful tools thanks to another increasingly important mechanism: their paracrine potential. MSCs would exert, through the release of soluble bioactive molecules, such as anti-inflammatory cytokines, trophic molecules and anti-apoptotic factors, several effects on the cells of the recipient tissue, in such a way that the response of the immune system cells would be PhD Thesis of Nerea Sánchez Pérez 28 modified and the proliferation and differentiation of the resident progenitor cells would be stimulated (Wang, Chen et al. 2014). Recently, it has been described how the release of exosomes and microvesicles by MSCs could significantly contribute to tissue regeneration (Phinney and Pittenger 2017). This paracrine potential, based on the stimulation of the damaged tissue's own capacity for self-renewal by the transplanted cells, has also been tested using the so-called “conditioned medium”, that is, the culture medium where the cells have expanded (Nagata, Iwasaki et al. 2017, Vizoso, Eiro et al. 2017, Muhammad, Nordin et al. 2018). With this strategy, many growth factors and bioactive molecules secreted by the cells (their “Secretome”), during their ex vivo culture, would exert their trophic activity when the enriched culture medium was applied directly to the target tissues, without the need to introduce living cells and therefore, without the constraints associated with cell transplantation (Nagata, Iwasaki et al. 2017, Muhammad, Nordin et al. 2018, El Moshy, Radwan et al. 2020). Scientific evidence has shown that MSCs cells also exhibit important immunomodulatory properties, in relation to their paracrine potential, since they reduce the synthesis of regulatory T lymphocytes and suppress the proliferation of activated T lymphocytes, B cells, "Natural Killers" (NK) cells, dendritic cells and neutrophils, and they decrease the production of pro-inflammatory cytokines as well (Wada, Gronthos et al. 2013, Leyendecker, Pinheiro et al. 2018). This property has allowed the allogeneic transplantation of MSCs and the development of autoimmune diseases and graft-versus- host disease therapies (Garcia-Gomez, Elvira et al. 2010, Hernandez-Monjaraz, Santiago- Osorio et al. 2018, Fisher, Cutler et al. 2019). Similarly, these cells have demonstrated the ability to migrate to areas with tissue damage after intravenous infusion, orchestrated by the stimulation of certain chemokines and integrins, which is known as “homing”, a property that makes them interesting candidates for remote regenerative treatments, such as the regeneration of damaged myocardium after a heart attack, or for the treatment of cancer, through modified exosomes that release antitumor agents (Golpanian, Wolf et al. 2016, Vakhshiteh, Atyabi et al. 2019). Another of the main advantages provided by mesenchymal cells therapies is that their isolation is carried out by using minimally invasive procedures, with less morbidity than the conventional regenerative treatments (Stanovici, Le Nail et al. 2016). Therefore, the application of these therapies is of particular interest, for example, in the treatment of severe atrophies of the alveolar ridge in the maxillary bones where oral rehabilitation with dental implants is required (Shanbhag, Suliman et al. 2019). In these clinical situations, the Cell Therapy in Periodontal Regereration 29 therapeutic “gold standard” is based on the use of autologous grafts that not only entail great morbidity, but also limitations derived from a reduced availability of the donor area (Bianchi, Felice et al. 2008, Dahlin and Johansson 2011). In addition to a lower morbidity, the use of MSCs would also improve the prospects for the availability of the transplanted material, since cell counts can be easily monitored during ex vivo expansion (Stanovici, Le Nail et al. 2016, Shanbhag, Suliman et al. 2019). Therefore, thanks to the interesting properties of these cells, MSCs have been studied in many areas of medicine, in the management of autoimmune diseases, in the treatment of cancer, but also in the regeneration of damaged tissues as the periodontium in subjects suffering from periodontitis. Application of cell therapy with stromal mesenchymal stem cells in periodontal regeneration. During the last decades, many preclinical studies based on the use of MSCs in periodontal bioengineering have been published. These investigations have evaluated the application of cells with proliferative and paracrine potential in combination with three-dimensional scaffolds, with the aim of not only maintaining the stability of the blood clot and the space for the regeneration, but also to stimulate the regenerative process and improve the results of therapy in comparison to conventional strategies (Nunez, Vignoletti et al. 2019). The first preclinical investigations evaluating the transplantation of ex vivo expanded MSCs into in vivo periodontal lesions were published in the 1990s (Lang, Schuler et al. 1998). Since then, many preclinical studies have intended to analyse, on the one hand, the safety of the use of expanded cells, and on the other, to compare, through histological analysis, the formation of new cementum, connective tissue attachment and bone between the cell-treated group and the cell-free control group (Monsarrat, Vergnes et al. 2014, Tassi, Sergio et al. 2017). Mesenchymal cells from different origins, such as bone marrow (Paknejad, Eslaminejad et al. 2015), adipose tissue, (Tobita, Uysal et al. 2013), dental pulp of permanent and deciduous teeth (Fu, Jin et al. 2014), periodontal ligament (Nunez, Sanz- Blasco et al. 2012, Iwasaki, Komaki et al. 2014) or gingival connective tissue (Fawzy El- Sayed, Mekhemar et al. 2015), among other sources, have been transplanted into experimentally created periodontal defects. The cells, mainly from an autologous and intraoral origin, have mostly been tested in experimental models such as fenestration, infrabony defect and furcation lesions (Bright, Hynes et al. 2014, Monsarrat, Vergnes et al. 2014). All these investigations have revealed that the use of cell therapy is safe and most PhD Thesis of Nerea Sánchez Pérez 30 of them have shown successful histological results of the regenerative process mediated by ex vivo expanded MSCs (Du, Shan et al. 2014, Fu, Jin et al. 2014, Guo, He et al. 2014, Han, Menicanin et al. 2014, Iwasaki, Komaki et al. 2014, Cai, Yang et al. 2015, Fawzy El-Sayed, Mekhemar et al. 2015, Nagahara, Yoshimatsu et al. 2015, Paknejad, Eslaminejad et al. 2015). This has promoted the design of various studies in humans to assess the safety and clinical efficacy of MSCs as therapeutic tools in periodontal regeneration. Over the last 15 years, several clinical studies have been published, most of them, case series and case reports with follow-ups of up to 36 months (Yamada, Ueda et al. 2006, Yamada, Nakamura et al. 2013, Baba, Yamada et al. 2016, Li, Zhao et al. 2016, Hernandez- Monjaraz, Santiago-Osorio et al. 2018, Iwata, Yamato et al. 2018), but also some controlled clinical trials (CCTs) (d'Aquino, De Rosa et al. 2009, Dhote, Charde et al. 2015, Chen, Gao et al. 2016). All these publications agreed that MSCs-based therapies for periodontal regeneration are safe. To date, there are only three CCTs that have evaluated the effect of MSCs therapy on the functional regeneration of damaged periodontal tissues (d'Aquino, De Rosa et al. 2009, Dhote, Charde et al. 2015, Chen, Gao et al. 2016). These are all 12- month studies, which initially included between 20 and 30 patients with periodontitis and at least one tooth with a deep intraosseous defect. In the first published CCT, with a split- mouth design, collagen sponges with autologous MSCs derived from adult dental pulp were transplanted into defects located in the distal aspect of the lower second molars after the extraction of the impacted third molars (d'Aquino, De Rosa et al. 2009). The results derived from the evaluation of the 7 patients who completed the follow-up revealed that all defects in the group with MSCs achieved at least 70% bone regeneration with respect to the initial dimensions of the defect, while half of the defects in which the control treatment (collagen sponge without cells) was used did not show regeneration or showed bone regeneration of 30% of the initial dimensions of the defect (d'Aquino, De Rosa et al. 2009). In the second published trial, a parallel-group randomized study, a greater PPD reduction and a greater CAL gain were obtained, with statistically significant differences between the group in which the defects had been treated with umbilical cord allogeneic MSCs seeded in a β-TCP matrix with PDGF-BB (4.50 ± 1.08 mm and 3.91 ± 1.37 mm, for PPD reduction and CAL gain respectively), and the control group, which consisted of the use of an access surgery without biomaterials or growth factors (3.50 ± 0.90 mm and 2.08 ± 0.90 mm; p <0.05) (Dhote, Charde et al. 2015). In the most recently published parallel-group RCT, the transplantation of autologous periodontal ligament-derived MSCs embedded in deproteinized bovine bone mineral was carried out in 21 intrabony defects and was compared with the introduction of the scaffold without cells in 20 defects. Twelve months Cell Therapy in Periodontal Regereration 31 later, the results showed significant improvements in both groups, in terms of radiographic bone fill, the main outcome variable in terms of efficacy; unlike the two previous RCTs, no statistically significant differences were detected between the test and the control group (Chen, Gao et al. 2016). PhD Thesis of Nerea Sánchez Pérez 32 III. JUSTIFICATION Regenerative periodontal treatment of three-wall intrabony defects and mandibular class- II furcation lesions in patients with periodontitis has shown predictable results according to the available scientific evidence. However, one and two-wall intrabony defects and class-III furcation and supracrestal lesions, despite their high prevalence, do not still have a predictable regenerative treatment. The management of these unfavourable defects could extensively benefit from cell therapy approaches thanks to the proliferative and paracrine potential of mesenchymal stem cells, and the functional and structural support, space maintenance and blood clot stability provided by the three-dimensional scaffolds where the cells are embedded. Therefore, a cell therapy-based approach could increase the possibility of improving the regenerative results. IV. HYPOTHESIS The general hypothesis of this work is that the use of mesenchymal cells in periodontal regenerative therapy is safe and that it may provide additional clinical benefits with respect to the use of a cell-free control therapy in periodontal defects that to date, do not have a predictable treatment with conventional regenerative therapies. Cell Therapy in Periodontal Regereration 33 V. OBJECTIVES The general objective of this work is to evaluate the safety and efficacy of mesenchymal stromal cell therapy for periodontal regeneration. For the validation of the hypothesis and general objective of this work, three independent investigations have been carried out, each one with its justification, hypotheses and specific objectives: Study 1. Santamaría S, Sánchez N, Sanz M, & García-Sanz JA. (2017). Comparison of periodontal ligament and gingiva-derived mesenchymal stem cells for regenerative therapies. Clinical Oral Investigations 21(4), 1095-1102. Justification MSCs derived from gingival connective tissue may provide substantial advantages as candidates for periodontal regenerative strategies (i.e. ease of obtention and fast healing of the donor area); however, their use must be supported by rigorous ex vivo studies that confirm their mesenchymal phenotype and safety after culture expansion and evaluate its proliferative potential in a microenvironment that reproduces the conditions of the periodontal lesion. Specific hypothesis MSCs derived from gingival connective tissue exhibit similar characteristics to MSCs derived from the periodontal ligament in terms of genomic stability and in vivo safety, mesenchymal phenotype, and proliferation rates under optimal and suboptimal growth conditions. Specific aim To confirm the genomic stability after ex vivo culture of MSCs derived from gingival connective tissue and periodontal ligament, their safety after in vivo transplantation in a small experimental animal model, and their mesenchymal phenotype. Likewise, to compare the proliferation potential of these cell populations under standard and suboptimal culture conditions. PhD Thesis of Nerea Sánchez Pérez 34 Study 2. Núñez J, Sánchez N, Vignoletti F, Sanz-Martin I, Caffesse R, Santamaría S, García- Sanz JA, Sanz M. (2018). Cell therapy with allogenic canine periodontal ligament-derived cells in periodontal regeneration of critical size defects. Journal of Clinical Periodontology 45(4):453-461. Justification The possibility of achieving periodontal regeneration in supracrestal defects has been poorly investigated in in vivo experimental models. Besides, no study has evaluated the impact of cell therapy with periodontal ligament MSCs in this type of preclinical model. Specific hypothesis The transplantation of ex vivo expanded allogeneic periodontal ligament MSCs in combination with hydroxyapatite-collagen scaffolds in experimentally created critical-size supracrestal periodontal defects in Beagle dogs will promote a greater extension of the periodontal regeneration, according to the histological analysis, when compared with the use of the scaffold without cells. Specific objectives To evaluate the regenerative potential of the application of allogeneic MSCs combined with a three-dimensional hydroxyapatite-collagen scaffold in a preclinical in vivo model of critical-size supracrestal periodontal defect in comparison to the same scaffold without the cells. The efficacy of this therapy will be histologically and histomorphometrically evaluated. Cell Therapy in Periodontal Regereration 35 Study 3. Sánchez N, Fierravanti L, Núñez J, Vignoletti F, González-Zamora M, Santamaría S, Suárez-Sancho S, Fernández-Santos ME, Figuero E, Herrera D, García-Sanz JA, Sanz M. (2020). Periodontal regeneration using a xenogeneic bone substitute seeded with autologous periodontal ligament-derived mesenchymal stem cells: A 12-month quasi-randomized controlled pilot clinical trial. Journal of Clinical Periodontology 47(11):1391-1402. Justification Despite the large number of preclinical investigations that have demonstrated the safety and efficacy of the use of MSCs in periodontal regeneration, there are no clinical studies that confirm that MSCs therapies are predictable and safe, especially in lesions that have showed unfavorable results in standard regenerative technologies. Specific hypothesis A cell therapy protocol based on the use of autologous periodontal ligament MSCs, expanded ex vivo and embedded in hydroxyapatite-collagen scaffolds for the regenerative treatment of 1 and 2-wall intrabony defects in patients with periodontitis, will be a safe treatment and will provide an added clinical benefit with respect to the scaffold alone. Specific Objectives To evaluate the safety and clinical efficacy of a cell therapy protocol based on the transplantation of ex vivo expanded autologous periodontal ligament MSCs embedded in a xenogeneic hydroxyapatite-collagen scaffold for the treatment of one and two-wall intraosseous periodontal defects, in comparison with a control treatment using the same scaffold without cells. PhD Thesis of Nerea Sánchez Pérez 36 VI. MATERIAL AND METHODS. RESULTS The material and methods and the results of the three investigations of this doctoral thesis have been independently published in impact factor scientific journals (first Quartile-Q1) included in the Journal Citation Report (JCR): Study 1. Santamaría S, Sánchez N, Sanz M, & García-Sanz JA. (2017). Comparison of periodontal ligament and gingiva-derived mesenchymal stem cells for regenerative therapies. Clinical Oral Investigations 21(4), 1095-1102. Study 2. Núñez J, Sánchez N, Vignoletti F, Sanz-Martin I, Caffesse R, Santamaría S, García- Sanz JA, Sanz M. (2018). Cell therapy with allogenic canine periodontal ligament-derived cells in periodontal regeneration of critical size defects. Journal of Clinical Periodontology 45(4):453-461. Study 3. Sánchez N, Fierravanti L, Núñez J, Vignoletti F, González-Zamora M, Santamaría S, Suárez-Sancho S, Fernández-Santos ME, Figuero E, Herrera D, García-Sanz JA, Sanz M. (2020). Periodontal regeneration using a xenogeneic bone substitute seeded with autologous periodontal ligament-derived mesenchymal stem cells: A 12-month quasi- randomized controlled pilot clinical trial. Journal of Clinical Periodontology 47(11):1391- 1402. Cell Therapy in Periodontal Regereration 37 STUDY 1. Santamaría S, Sánchez N, Sanz M, & García-Sanz JA. (2017). Comparison of periodontal ligament and gingiva-derived mesenchymal stem cells for regenerative therapies. Clinical Oral Investigations 21(4), 1095-1102. Abstract Objectives: Tissue-engineering therapies using undifferentiated mesenchymal cells (MSCs) from intra-oral origin have been tested in experimental animals. This experimental study compared the characteristics of undifferentiated mesenchymal stem cells from either periodontal ligament or gingival origin, aiming to establish the basis for the future use of these cells on regenerative therapies. Materials and methods: Gingiva-derived mesenchymal stem cells (GMSCs) were obtained from de-epithelialized gingival biopsies, enzymatically digested and expanded in conditions of exponential growth. Their growth characteristics, phenotype, and differentiation ability were compared with those of periodontal ligament-derived mesenchymal stem cells (PDL-MSCs). Results: Both periodontal ligament- and gingiva-derived cells displayed a MSC-like phenotype and were able to differentiate into osteoblasts, chondroblasts, and adipocytes. These cells were genetically stable following in vitro expansion and did not generate tumours when implanted in immunocompromised mice. Furthermore, under suboptimal growth conditions, GMSCs proliferated with higher rates than PDLMSCs. Conclusions: Stem cells derived from gingival biopsies represent bona fide MSCs and have demonstrated genetic stability and lack of tumorigenicity. Clinical relevance: Gingiva-derived MSCs may represent an accessible source of mesenchymal stem cells to be used in future periodontal regenerative therapies. Keywords: Mesenchymal stem cells, Tissue engineering, Periodontal ligament, Gingival, Periodontal regeneration. ORIGINAL ARTICLE Received: 26 January 2016 /Accepted: 1 June 2016 /Published online: 6 June 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract Objectives Tissue-engineering therapies using undifferentiat- ed mesenchymal cells (MSCs) from intra-oral origin have been tested in experimental animals. This experimental study compared the characteristics of undifferentiated mesenchymal stem cells from either periodontal ligament or gingival origin, aiming to establish the basis for the future use of these cells on regenerative therapies. Materials and methods Gingiva-derived mesenchymal stem cells (GMSCs) were obtained from de-epithelialized gingival biopsies, enzymatically digested and expanded in conditions of exponential growth. Their growth characteristics, pheno- type, and differentiation ability were compared with those of periodontal ligament-derived mesenchymal stem cells (PDLMSCs). Results Both periodontal ligament- and gingiva-derived cells displayed aMSC-like phenotype and were able to differentiate into osteoblasts, chondroblasts, and adipocytes. These cells were genetically stable following in vitro expansion and did not generate tumors when implanted in immunocompromised mice. Furthermore, under suboptimal growth conditions, GMSCs proliferated with higher rates than PDLMSCs. Conclusions Stem cells derived from gingival biopsies repre- sent bona fide MSCs and have demonstrated genetic stability and lack of tumorigenicity. Clinical relevance Gingiva-derived MSCs may represent an accessible source of messenchymal stem cells to be used in future periodontal regenerative therapies. Keywords Mesenchymal stem cells . Tissue engineering . Periodontal ligament . Gingival . Periodontal regeneration Introduction Periodontitis is a chronic inflammatory disease of bacterial etiology, affecting a high percentage of the worldwide adult population [1]. This disease initiates and progresses in suscep- tible individuals by periodontal tissue destruction, as a conse- quence of the chronic host inflammatory immune response against pathogenic bacteria residing in the dental biofilm. Current treatment is based on bacterial biofilm removal. With these therapies, the disease process is arrested and the long-term maintenance of periodontal health can be achieved, but re-establishment of the original anatomy of the periodontal apparatus is, however, unlikely to occur [2]. Regenerative approaches are based on either using bioactive agents (as enamel matrix derivatives), which promote new cementum formation and periodontal attachment [3–5], or by placing barrier membranes to prevent overgrowth of epithelial cells from populating bone/PDL spaces (guided tissue regenera- tion). Both approaches have demonstrated efficacy in the re- generation of intrabony periodontal defects, but not in suprabony lesions, which are the most frequently affected. Therefore, these therapies fail most of the time to achieve a true regenerative outcome. This underlies the demand for more effective therapies in the management of this chronic inflammatory condition and allows envisaging the successful use in a near future of tissue-engineering approaches using * Jose A. Garcia-Sanz jasanz@cib.csic.es 1 Centro de Investigaciones Biologicas (CIB-CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain 2 ETEP Research Group, Faculty of Odontology, Universidad Complutense de Madrid (UCM), Madrid, Spain Clin Oral Invest (2017) 21:1095–1102 DOI 10.1007/s00784-016-1867-3 Comparison of periodontal ligament and gingiva-derived mesenchymal stem cells for regenerative therapies Silvia Santamaría1,2 & Nerea Sanchez2 & Mariano Sanz2 & Jose A. Garcia-Sanz1 http://crossmark.crossref.org/dialog/?doi=10.1007/s00784-016-1867-3&domain=pdf https://pubmed.ncbi.nlm.nih.gov/27270903/ PhD Thesis of Nerea Sánchez Pérez 38 STUDY 2. Núñez J, Sánchez N, Vignoletti F, Sanz-Martin I, Caffesse R, Santamaría S, García-Sanz JA, Sanz M. (2018). Cell therapy with allogenic canine periodontal ligament-derived cells in periodontal regeneration of critical size defects. Journal of Clinical Periodontology 45(4):453-461. Abstract Aim: The objective of this in vivo experimental study was to evaluate the regenerative potential of a cell therapy combining allogeneic periodontal ligament-derived cells within a xenogeneic bone substitute in a preclinical model of critical-size defects. Methods: In nine beagle dogs, critical-size 6-mm supra-alveolar periodontal defects were created around the PIII and PIV. The resulting supra-alveolar defects were randomly treated with either 1.4 x 106 allogeneic canine periodontal ligament-derived cells seeded on de-proteinized bovine bone mineral with 10% collagen (DBBM-C) (test group) or DBBM-C without cells (control group). Specimens were obtained at 3 months, and histological outcomes were studied. Results: The histological analysis showed that total furcation closure occurred very seldom in both groups, being the extent of periodontal regeneration located in the apical third of the defect. The calculated amount of periodontal regeneration at the furcation area was comparable in both the test and control groups (1.93 ± 1.14 mm (17%) versus 2.35 ± 1.74 mm (22%), respectively (p=0.37). Similarly, there were no significant differences in the amount of new cementum formation 4.49 ± 1.56 mm (41%) versus 4.97 ± 1.05 mm (47%), respectively (p=0.45). Conclusions: This experimental study was unable to demonstrate the added value of allogeneic cell therapy in supra-crestal periodontal regeneration. Keywords: bone remodelling/regeneration, cell biology, cementum, periodontal ligament, regenerative medicine J Clin Periodontol. 2018;45:453–461. wileyonlinelibrary.com/journal/jcpe  |  453© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1  | INTRODUCTION The periodontal ligament (PDL) contains mesenchymal stem cells (MSCs) responsible for the maintenance and regeneration of peri- odontal tissues. These cells, termed PDLSCs, were first isolated by Seo et al. (2004), and their use as cell therapy has been evaluated in experimental studies (Núñez et al., 2010) demonstrating regenerative potential when seeded in a collagen sponge and implanted in a three- wall self- contained intrabony periodontal defect model in beagle dogs (Nuñez et al., 2012). Other attempts to promote periodontal regen- eration by bio- engineered approaches have used collagen sponges mixed with recombinant human bone morphogenetic protein- 2 in three- wall intrabony defects (Choi et al., 2002) and growth/differenti- ation factor- 5 in one- wall intrabony periodontal defects (Kim, Wikesjö, et al., 2009). Collagen sponges as scaffold biomaterial, however, were unable to provide space maintenance in non- contained periodontal lesions. Recent investigations have used the furcation experimental model as a challenging model to evaluate the potential of cell thera- pies for periodontal regeneration, either using autologous periodontal ligament cells (Suaid et al., 2012) or embryonic stem cells (Yang et al., 2013). These studies demonstrated the capability of this cell therapy in regenerating the furcation lesion, although without achieving a Accepted: 21 December 2017 DOI: 10.1111/jcpe.12863 A N I M A L E X P E R I M E N T Cell therapy with allogenic canine periodontal ligament- derived cells in periodontal regeneration of critical size defects Javier Nuñez1  | Nerea Sanchez1 | Fabio Vignoletti1 | Ignacio Sanz-Martin1  |  Raul Caffesse1 | Silvia Santamaria2 | Jose A. Garcia-Sanz2 | Mariano Sanz1 1Section of Periodontology, Faculty of Odontology, University Complutense of Madrid, Madrid, Spain 2Department of Cellular and Molecular Medicine, Centro de Investigaciones Biologicas (CIB-CSIC), Madrid, Spain Correspondence Mariano Sanz, Facultad de Odontología - Universidad Complutense de Madrid, Madrid, Spain. Email: marianosanz@odon.ucm.es Funding information This study was partially supported through an Osteology Foundation Advanced Research Grant (10- 012). Abstract Aim: The objective of this in vivo experimental study to evaluate the regenerative potential of a cell therapy combining allogenic periodontal ligament- derived cells within a xenogeneic bone substitute in a similar experimental model. Methods: In nine beagle dogs, critical size 6- mm supra- alveolar periodontal defects were created around the PIII and PIV. The resulting supra- alveolar defects were ran- domly treated with either 1.4 × 106 allogenic canine periodontal ligament- derived cells seeded on de- proteinized bovine bone mineral with 10% collagen (DBBM- C) (test group) or DBBM- C without cells (control group). Specimens were obtained at 3 months, and histological outcomes were studied. Results: The histological analysis showed that total furcation closure occurred very seldom in both groups, being the extent of periodontal regeneration located in the apical third of the defect. The calculated amount of periodontal regeneration at the furcation area was comparable in both the test and control groups (1.93 ± 1.14 mm (17%) versus 2.35 ± 1.74 mm (22%), respectively (p = .37). Similarly, there were no sig- nificant differences in the amount of new cementum formation 4.49 ± 1.56 mm (41%) versus 4.97 ± 1.05 mm (47%), respectively (p = .45). Conclusions: This experimental study was unable to demonstrate the added value of allogenic cell therapy in supra- crestal periodontal regeneration. K E Y W O R D S bone remodelling/regeneration, cell biology, cementum, periodontal ligament, regenerative medicine www.wileyonlinelibrary.com/journal/jcpe http://orcid.org/0000-0002-9361-3273 http://orcid.org/0000-0001-7037-1163 mailto:marianosanz@odon.ucm.es https://pubmed.ncbi.nlm.nih.gov/29288504/ Cell Therapy in Periodontal Regereration 39 STUDY 3. Sánchez N, Fierravanti L, Núñez J, Vignoletti F, González-Zamora M, Santamaría S, Suárez- Sancho S, Fernández-Santos ME, Figuero E, Herrera D, García-Sanz JA, Sanz M. (2020). Periodontal regeneration using a xenogeneic bone substitute seeded with autologous periodontal ligament-derived mesenchymal stem cells: A 12-month quasi-randomized controlled pilot clinical trial. Journal of Clinical Periodontology 47(11):1391-1402. Abstract Aim: To evaluate the safety and efficacy of autologous periodontal ligament-derived mesenchymal stem cells (PDL-MSCs) embedded in a xenogeneic bone substitute (XBS) for the regenerative treatment of intra-bony periodontal defects. Material and Methods. This quasi-randomized controlled pilot phase II clinical trial included patients requiring a tooth extraction and presence of one intra-bony lesion (1-2 walls). Patients were allocated to either the experimental (XBS + 10x106 PDL- MSCs/100mg) or the control group (XBS). Clinical and radiographical parameters were recorded at baseline, 6, 9 and 12 months. The presence of adverse events was also evaluated. Chi-square, Student`s t-test, U-Mann Whitney, repeated-measures ANOVA and regression models were used. Results. Twenty patients were included. No serious adverse events were reported. Patients in the experimental group (n=9) showed greater clinical attachment level (CAL) gain [1.44, standard deviation (SD)=1.87] and probing pocket depth (PPD) reduction (2.33, SD=1.32) than the control group (n=10; CAL gain=0.88, SD=1.68, and PPD reduction=2.10, SD=2.46), without statistically significant differences. Conclusion. The application of PDL-MSCs to XBS for the treatment of one-two wall intra- bony lesions was safe and resulted in low postoperative morbidity and appropriate healing, although its additional benefit, when compared with the XBS alone, was not demonstrated. (ISRCTN 13093912) Keywords: cell therapy, mesenchymal stem cells, periodontal ligament, periodontal regeneration, tissue engineering. J. Clin. Periodontol. 2020;47:1391–1402.  | 1391wileyonlinelibrary.com/journal/jcpe Received: 5 June 2020  | Revised: 25 August 2020  | Accepted: 5 September 2020 DOI: 10.1111/jcpe.13368 O R I G I N A L A R T I C L E C L I N I C A L P E R I O D O N T O L O G Y Periodontal regeneration using a xenogeneic bone substitute seeded with autologous periodontal ligament-derived mesenchymal stem cells: A 12-month quasi-randomized controlled pilot clinical trial Nerea Sánchez1  | Ludovica Fierravanti1 | Javier Núñez1  | Fabio Vignoletti1 | María González-Zamora1 | Silvia Santamaría2 | Susana Suárez-Sancho3 | María Eugenia Fernández-Santos3 | Elena Figuero1  | David Herrera1  | Jose A. García-Sanz2 | Mariano Sanz1 © 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Trial registration: ISRCTN 13093912 1ETEP (Etiology and Therapy of Periodontal and Peri-implant Diseases) Research Group, University Complutense, Madrid, Spain 2Margarita Salas Center for Biological Research (CIB-CSIC), Madrid, Spain 3GMP-Cell Production Unit, Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Red de Terapia Celular (TERCEL) and CIBER Cardiovascular (CIBERCV), Instituto de Salud Carlos III, Madrid, Spain Correspondence Mariano Sanz, ETEP (Etiology and Therapy of Periodontal and Peri-implant Diseases) Research Group, Faculty of Odontology, University Complutense of Madrid, Plaza Ramón y Cajal s/n (Ciudad Universitaria) , 28040 Madrid, Spain. Email: marsan@ucm.es Funding information This study received funding from the Spanish Ministry of Health (EC10-095) to MS. Abstract Aim: To evaluate the safety and efficacy of autologous periodontal ligament-derived mesenchymal stem cells (PDL-MSCs) embedded in a xenogeneic bone substitute (XBS) for the regenerative treatment of intra-bony periodontal defects. Material and Methods: This quasi-randomized controlled pilot phase II clinical trial in- cluded patients requiring a tooth extraction and presence of one intra-bony lesion (1–2 walls). Patients were allocated to either the experimental (XBS + 10 × 106 PDL- MSCs/100 mg) or the control group (XBS). Clinical and radiographical parameters were recorded at baseline, 6, 9 and 12 months. The presence of adverse events was also evaluated. Chi-square, Student's t test, Mann–Whitney U, repeated-measures ANOVA and regression models were used. Results: Twenty patients were included. No serious adverse events were reported. Patients in the experimental group (n = 9) showed greater clinical attachment level (CAL) gain (1.44, standard deviation [SD] = 1.87) and probing pocket depth (PPD) re- duction (2.33, SD = 1.32) than the control group (n = 10; CAL gain = 0.88, SD = 1.68, and PPD reduction = 2.10, SD = 2.46), without statistically significant differences. Conclusion: The application of PDL-MSCs to XBS for the treatment of one- to two-wall intra-bony lesions was safe and resulted in low postoperative morbidity and appropri- ate healing, although its additional benefit, when compared with the XBS alone, was not demonstrated. K E Y W O R D S cell therapy, mesenchymal stem cells, periodontal ligament, periodontal regeneration, tissue engineering www.wileyonlinelibrary.com/journal/jcpe https://orcid.org/0000-0003-1681-428X https://orcid.org/0000-0002-9361-3273 https://orcid.org/0000-0002-3129-1416 https://orcid.org/0000-0002-5554-2777 mailto: https://orcid.org/0000-0002-6293-5755 mailto:marsan@ucm.es http://crossmark.crossref.org/dialog/?doi=10.1111%2Fjcpe.13368&domain=pdf&date_stamp=2020-10-26 https://pubmed.ncbi.nlm.nih.gov/32946590/ PhD Thesis of Nerea Sánchez Pérez 40 VII. DISCUSSION STUDY 1. Santamaría S, Sánchez N, Sanz M, & García-Sanz JA. (2017). Comparison of periodontal ligament and gingiva-derived mesenchymal stem cells for regenerative therapies. Clinical Oral Investigations 21(4), 1095-1102. The objective of this first work was to confirm the mesenchymal nature of various types of cells from intraoral origin and to verify their safety with the ultimate goal of future application in periodontal regeneration clinical investigations. First of all, cells were isolated from human gingival connective tissue, periodontal ligament from impacted third molars from patients with an intact periodontium and periodontal ligament from extracted teeth due to a hopeless prognosis from patients with a reduced periodontium. Then, the minimal criteria for defining human MSCs proposed by Mesenchymal and Tissue Stem Cell Committee of the ISCT were tested: 1) plastic-adherence when maintained in standard culture conditions, 2) expression of specific surface antigens of MSCs and 3) multipotent differentiation potential (Dominici, Le Blanc et al. 2006). Regarding the first criterion of the ISCT, in this study, the three cell populations confirmed plastic-adherence after the first passage, with no significant differences between the groups. The evaluation of the phenotypic markers of MSCs by flow cytometry (criterion 2) was carried out with cells from the second passage, which resulted in the expression of CD73, CD90 and CD105 and the absence of expression of CD34, CD45, HLA-DR, CD11b and CD19 in more than 97% of the cells of the three cell populations, suggesting homogeneous phenotypic characteristics. Regarding criterion 3, the three groups exhibited trilineage differentiation potential to bone, cartilage and adipose tissue, when cells from ≥ passage 3 were subjected to the specific differentiation media, unlike the control groups, which were not subjected to any differentiation medium and consequently, did not show any sign of differentiation. The three populations showed similar results, with no significant differences in terms of signal intensity or number of differentiated cells (Santamaria, Sanchez et al. 2017). As in our work, other investigations have tried to isolate human PDL-MSCs from teeth with an intact (Iwasaki, Komaki et al. 2014, Liu, Zhao et al. 2020) and reduced periodontium , as well as GMSCs (Yu, Ge et al. 2013, Tomasello, Mauceri et al. 2017, Abedian, Jenabian et al. 2020) for its application in regenerative therapies. In all of them, an attempt was made to confirm that the isolated cell population had a mesenchymal nature, but unlike our study, in none of these investigations, all the minimum requirements established by the ISCT for Cell Therapy in Periodontal Regereration 41 human MSCs was satisfied. Iwasaki et al. confirmed plastic-adherence and the differentiation potential of human PDL-MSCs to bone, cartilage and adipose tissue, but they carried out a partial analysis of the mesenchymal cell-specific surface markers, since they confirmed that the cells were CD90+, CD73+, CD105+ y CD45-, but did not evaluate any more of the required antigens (Iwasaki, Komaki et al. 2014). Similarly, Chen et al. isolated periodontal ligament cells from the third molars of subjects with periodontitis, which were subjected to adipogenic and osteogenic differentiation, but not to chondrogenic differentiation. Although they verified that the cells were CD105+, CD90+ y CD45-, the rest of the markers recommended by the ISCT consensus were not analysed prior to their transplantation in humans (Chen, Gao et al. 2016). Therefore, although all these studies tried to validate the mesenchymal phenotype of the cells, most of these investigations, despite having been carried out after the publication of this consensus, have not used these criteria or performed a partial analysis of them (Yu, Ge et al. 2013, Iwasaki, Komaki et al. 2014, Chen, Gao et al. 2016, Tang, Xia et al. 2016, Abedian, Jenabian et al. 2020, Liu, Zhao et al. 2020) a circumstance that occurs not only with PDL- MSCs and GMSCs, but also when ex vivo expanded putative MSCs of other origins are used (Fournier, Ferre et al. 2010, Park, Kim et al. 2011, Zhang, Nguyen et al. 2012, Dhote, Charde et al. 2015, Chen, Gao et al. 2016, Ducret, Fabre et al. 2016). It could be argued that the ISCT recognizes that some flexibility with respect to the minimal criteria for defining MSCs must be given, since, under certain conditions, there are negative markers, such as HLA, which could be expressed by MSCs upon stimulation with cytokines, so even though positivity for HLA class II molecule was observed, a certain cell population could still be considered as mesenchymal if the rest of the criteria were met (Dominici, Le Blanc et al. 2006). The appropriateness of the negative status of CD34 has also been intensively debated, since some research groups have indicated that MSCs isolated from adipose tissue could express this marker at the time of cell isolation, but it could be lost during cell expansion (Quirici, Scavullo et al. 2010). Therefore, it seems that the expression of MSCs markers could substantially vary depending on the source of cell isolation and the experimental conditions. A multitude of in vivo investigations, both in animals and in humans, have shown that, for the periodontal tissues to regenerate, the formation of a thin layer of mineralized tissue on the root surface (new cementum), as a synthetic product of the cementoblasts, is necessary (Sculean, Donos et al. 1999, Graziani, Laurell et al. 2005). In addition, in periodontal regeneration procedures, undifferentiated cells from the remaining PhD Thesis of Nerea Sánchez Pérez 42 periodontal ligament are responsible for the differentiation into cementoblasts, osteoblasts and fibroblasts, which synthetize the new tissues (Nyman, Gottlow et al. 1982, Nyman, Lindhe et al. 1982). Since the in vivo formation of the tooth-supporting structures is known to originate from undifferentiated stem cell populations present in the periodontal ligament, preliminary investigations with MSCs focused on reproducing this biological effect by introducing ex-vivo expanded periodontal ligament cells into periodontal lesions in different animal models (Boyko, Melcher et al. 1981, Lang, Schuler et al. 1995). Later on, it was found that, not only the undifferentiated stem cells of the periodontal ligament expanded in culture could regenerate the periodontal attachment apparatus (Seo, Miura et al. 2004), but also other MSCs populations from other extra and intraoral adult tissues, such as bone marrow, adipose tissue, dental pulp or gingiva (Tobita, Uysal et al. 2013, Fu, Jin et al. 2014, Fawzy El-Sayed, Mekhemar et al. 2015, Paknejad, Eslaminejad et al. 2015) For this reason, with this first work, we have tried to evaluate, not only the phenotypic and proliferative characteristics of cells from the periodontal ligament, but also, the properties of the cells obtained from gingival connective tissue, a source with great advantages due to their ease of isolation and lower morbidity in comparison with cells from other origins (Zhang, Shi et al. 2009). An important characteristic of MSCs is that, unlike differentiated cells, they have a huge proliferative capacity, that is not limited to a specific number of cellular mitoses (Caplan 1991). According to the available evidence, these cells would be capable of being renewed for long periods of time without significant changes in their properties. However, a question that may arise is whether a large number of cellular mitoses and the ex vivo manipulation of the cells could induce the accumulation of mutations and consequently alterations in the cell's genome. It has been reported that in vitro expansion, including that of stem cells, could reduce the replicative potential and multipotency, lead to senescence, and decrease the efficiency of deoxyribonucleic acid (DNA) polymerase and DNA repair, which could result in the accumulation of genetic material damage (Bonab, Alimoghaddam et al. 2006, Neri, Bourin et al. 2013). These alterations could induce, on the one hand, senescence and arrest of the proliferation of MSCs (the most frequent), and on the other, genomic instability with the consequent increase in the risk of transformation, resulting in both cases, in an alteration not only of the proliferative, differentiation, immunomodulatory and homing therapeutic efficacy of the cells, but also in questionable Cell Therapy in Periodontal Regereration 43 patient safety (Neri 2019). For this reason, it is very important that prior to its application in humans, a precise analysis of the genomic stability of the ex vivo expanded MSCs is carried out, and for this reason, in the first work of this doctoral thesis, we have evaluated their genomic stability by hybridizing the DNA of the expanded cells with human DNA chips ("DNA microarrays") and comparing the results with a reference database. Analysis of the evaluated samples confirmed the absence of duplications and deletions, corroborating the genomic stability of the expanded GMSCs. Our results agree with other investigations that have analysed the safety of expanded cells by using other techniques, such as karyotype analysis (study of human cells chromosomes, both autosomes and sex chromosomes), the microsatellites instability analysis (test for evaluating the ability of a cell to repair mistakes in DNA replication, as the presence of insertion or deletion errors at microsatellite repeat sequences, which are particularly sensitive to transcription errors), or fluorescent hybridization analysis (FISH) (“mapping” of the genetic material of the cells of an individual, which allows the localization of specific DNA sequences in a chromosome by means of specific fluorescent probes) (Bernardo, Zaffaroni et al. 2007, Tomar, Srivastava et al. 2010, Roselli, Lazzati et al. 2013, Kim, Im et al. 2015, Li, Feng et al. 2017). In a study in which cells from gingival connective tissue from healthy volunteers were expanded ex vivo, it was observed that the cells exhibited a normal karyotype in both passage 5 and passage 16 and that, in addition, telomerase activity was maintained both in passage 6 and in 13 (Tomar, Srivastava et al. 2010). Similar results were obtained in another investigation in which they wanted to compare the effect of the cryopreservation of PDL-MSCs; it was observed that this process did not alter either the viability or the chromosomal stability of the cryopreserved cells (Li, Feng et al. 2017). However, in another study, in which BMSCs were analysed for long periods of time in culture, using cytogenetic analysis, mixed lymphocyte reactions, proteomics and gene expression analysis, it was observed that, after passage 4, the immunomodulatory and cell differentiation functions were maintained, but the cells exhibited chromosomal variability, therefore recommending the use of MSCs of up to passage 4 for their application in cell therapy (Binato, de Souza Fernandez et al. 2013). Therefore, although many publications support the safety of cells expanded in culture, to use cells in early passages would be advisable for in vivo transplantation. Another aspect, closely related to genomic instability, is the tumorigenic potential of MSCs after their expansion in vitro. The absence of tumour formation when cells are transplanted in vivo must be demonstrated prior to their clinical use (Neri 2019). Therefore, in this first work, we carried out the analysis of the tumorigenicity of GMSCs PhD Thesis of Nerea Sánchez Pérez 44 and PDL-MSCs expanded in culture, by means of the subcutaneous injection of 2 x 106 cells in the flank region of immunodeficient mice and their evaluation during a period of six months. The results of the experiment did not reveal signs of migration of the cells to areas other than the injection site or the formation of teratomas during the 6-month follow-up. Our results agree with those of other investigations that have also demonstrated the absence of tumorigenic potential by MSCs of different origins (umbilical cord, bone marrow, gingiva and pulp of primary teeth) after their injection in immunodeficient mice, even using cells in passage 15 (Chen, Yue et al. 2014, Park, Kim et al. 2016, Li, Xu et al. 2018). As in our study, another investigation reported the absence of important adverse events in the major organs (heart, liver, spleen and kidneys) after the introduction of GMSCs in different animal models (Zhao, Wang et al. 2019). These results support the safety of using MSCs expanded in culture. In the first work of this doctoral thesis, we carried out a series of experiments that not only validated the phenotype of MSCs and confirmed their ex vivo safety, but were also focused on determining whether PDL-MSCs and GMSCs were suitable candidates for regenerative therapeutic interventions in the periodontum. For this purpose, we tried to reproduce in vitro the conditions to which these cells are subjected after their implantation in a periodontal defect in vivo, that is, to simulate a most unfavorable micro- environment, with fewer nutrients and more waste products. To this end, we subjected the cells to culture medium change every 7 days (instead of the standard change pattern for fresh medium every 3-4 days) and determine the cell proliferation rates. Before carrying out this experiment, in order to have the data on the reference proliferation rate under optimal culture conditions, we performed the standard cell expansion protocol for both types of cells over a period of 20 days; as a result, we observed that both cell populations increased exponentially, from a cell count of 105, at the time of cell seeding, to a cell count of 107, without significant differences in terms of proliferation rate between PDL-MSCs and GMSCs. However, when subjected to suboptimal culture conditions (change of medium every 7 days), after 14, 21 and 28 days, GMSCs exhibited a higher cell count than PDL-MSCs, with statistically significant differences. These results agree with other studies that have also compared the proliferative potential of PDL-MSCs and GMSCs, and have confirmed a higher proliferation rate of GMSCs, but under standard culture conditions (Yang, Gao et al. 2013, Abedian, Jenabian et al. 2020). This data suggest that GMSCs could be better candidates than PDL-MSCs for regenerative therapies, as they have superior proliferation potential, even under suboptimal culture conditions. Cell Therapy in Periodontal Regereration 45 STUDY 2. Núñez J, Sánchez N, Vignoletti F, Sanz-Martin I, Caffesse R, Santamaría S, García-Sanz JA, Sanz M. (2018). Cell therapy with allogenic canine periodontal ligament-derived cells in periodontal regeneration of critical size defects. Journal of Clinical Periodontology 45(4):453-461. The second work of this doctoral thesis is a preclinical study in Beagle dogs, in which the preclinical safety and efficacy of allogeneic PDL-MSCs seeded in xenogeneic scaffolds made of demineralized bovine bone mineral (DBBM)/10% collagen were evaluated in a critical- size supracrestal periodontal defect model with a class-III furcation lesion, a type of defect that currently, is highly unpredictable with conventional periodontal regenerative technologies. Regarding the safety of the experimental therapy, the transplanted cellular material did not show signs of infection or tumour formation in any case. Likewise, despite using allogeneic cells, there was no reaction consistent with graft rejection or graft versus host disease, thus ratifying the immunomodulatory properties of MSCs, which are capable of suppressing/reducing the functions of the immune system, enabling the safe transplantation of allogeneic cells (Wada, Gronthos et al. 2013). These results are supported by another investigation in which allogeneic and autologous PDL-MSCs together with β-TCP/collagen scaffolds were transplanted in the same critical periodontal defect model; in this study, serum concentrations of C-reactive protein, γ-interferon, IL-10 and CD30 were very similar in the group of dogs treated with allogeneic cells and in the group treated with autologous cells (p> 0.05) (Tsumanuma, Iwata et al. 2016). The vast majority of preclinical studies published in vivo in which MSCs are utilized as therapeutic tool in different models of periodontal defects agree with these results, since they have shown the absence of important adverse events, such as immunological reactions to the grafted material or infection (Tsumanuma, Iwata et al. 2011, Nunez, Sanz- Blasco et al. 2012, Iwasaki, Komaki et al. 2014, Monsarrat, Vergnes et al. 2014, Paknejad, Eslaminejad et al. 2015, Tassi, Sergio et al. 2017). According to a systematic review, complications such as ankylosis or root resorption have been described in the recipient area in several studies, but without significant differences concerning the number of samples that exhibited these adverse events in the test group and the control group (Monsarrat, Vergnes et al. 2014); consequently, these complications would not be specific to MSCs therapies and, therefore, cell therapy with autologous and allogeneic MSCs can be considered safe according to preclinical data. PhD Thesis of Nerea Sánchez Pérez 46 In our work, regarding wound healing after the procedure, the soft tissues showed health conditions without signs of visible inflammation throughout the 12 weeks of healing. However, we did observe soft tissue recession in the furcation area, both in the test (1.4 x 106 PDL-MSCs/matrix) and in the control group (matrix without cells), which led to an early exposure of the furcation fornix and probably, a progressive exfoliation of the regeneration material. Nevertheless, in the other study published with a design similar to ours, in a supracrestal defect model, no such retraction of the soft tissue was observed throughout the 8 weeks of follow-up, except in one specimen from the control group (Tsumanuma, Iwata et al. 2016). It is necessary to bear in mind that the methodology used in this last study is not the same as that described in the original publication on the creation and treatment of critical supracrestal defects (Wikesjo, Kean et al. 1994), since after the surgical creation of the defects, they were not chronified to emulate the lesions that occur naturally in periodontitis, that is, the flaps were not repositioned apically, nor were they sutured in a position immediately coronal to the new location of the reduced bone; in the same way, they were not subjected to bacterial plaque or calculus. On the contrary, immediately after bone removal and cementum scraping, the regenerative procedure was carried out, without any period of inflammation or chronification of the defect that would lead to soft tissue recession. This design, therefore, would greatly favor primary wound closure during the early healing period, so it could not be considered as a model of critical-sized supracrestal defect (Tsumanuma, Iwata et al. 2016). Several preclinical studies have shown successful results of the regenerative process mediated by ex vivo expanded MSCs (Bright, Hynes et al. 2014, Tassi, Sergio et al. 2017). Table 1 shows the design of the most recent preclinical investigations that have histologically evaluated the transplantation of MSCs from different tissue sources in periodontal defects, including the second work of this doctoral thesis (Hu, Cao et al. 2016, Liu, Yin et al. 2016, Basan, Welly et al. 2017, Guo, Kang et al. 2017, Takewaki, Kajiya et al. 2017, Nunez, Sanchez et al. 2018, Li, Han et al. 2019, Li, Nan et al. 2019, Rezaei, Jamshidi et al. 2019, Venkataiah, Handa et al. 2019). Regarding the transplanted medical device, most preclinical experimentation in periodontal regeneration has utilized ex vivo expanded MSCs seeded in three-dimensional scaffolds (Nunez, Sanz-Blasco et al. 2012, Liu, Yin et al. 2016, Basan, Welly et al. 2017, Rezaei, Jamshidi et al. 2019, Venkataiah, Handa et al. 2019); however, other approaches have also been promoted without the use of a scaffold (Hu, Cao et al. 2016, Li, Han et al. 2019) or even strategies with expansion systems different from the conventional method, such as the "cell sheets" technique; this approach does not use proteolytic enzymes such as trypsin to detach the cells from the surface of the flask, but Cell Therapy in Periodontal Regereration 47 allows the ex vivo expansion of cell layers that maintain, unlike standard techniques, their endogenous extracellular matrix, with its growth factors and fibronectin molecules (Guo, Kang et al. 2017, Takewaki, Kajiya et al. 2017). With this technology, when cells reach an optimal confluence (hyperconfluence usually), they can be detached by using temperature-responsive culture dishes or other method that allows intact cell layers, and transplanted directly into the defect without the need of a three-dimensional matrix (Iwata, Washio et al. 2015). From the culture of multilayer cell sheets, it is also possible to manufacture "cell pellets" or “micro-tissues”, which are three-dimensional aggregates that, according to the literature, could increase the secretion of endogenous extracellular matrix and exhibit improved mechanical properties and cell viability (Guo, He et al. 2014). Regarding the method of application of the cellular device, in our study, the graft was introduced into the defect after opening a full-thickness flap and debridement of the lesion, as in most recent studies (Table 1) (Liu, Zheng et al. 2008, Basan, Welly et al. 2017, Takewaki, Kajiya et al. 2017). However, some authors have employed a less invasive method, such as the subperiosteal injection (Li, Han et al. 2019). Hu et al. compared the regenerative efficacy of human DPSCs injections obtained by the conventional expansion method with grafted DPSCs cultured by using the cell sheet technique and transplanted after flap elevation for the treatment of experimental 3-wall intrabony defects in mini- pigs. Four months later, the histological analysis revealed the presence of newly formed alveolar bone and Sharpey’s fibers attached into the new layers of cementum in both groups. The histometric study revealed that the height of the newly formed bone in the group of DPSCs grafted into the defect (4.5 ± 0.3mm) was greater than that of the subperiosteal injection (3.8 ± 0.5mm), although without statistically significant differences. This slight superiority does not necessarily have to be attributable to the method of cell releasing, but also to other factors related to the study design such as the supplementation with Vitamin C of the culture medium in the grafted group (Hu, Cao et al. 2016). The culture methods in which the extracellular matrix is maintained, such as cell sheets or aggregates or pellets, is increasingly being used in preclinical studies of periodontal regeneration (Hu, Cao et al. 2016, Guo, Kang et al. 2017, Takewaki, Kajiya et al. 2017). Guo et al. observed that the treatment of 2-wall intrabony defects in Beagle dogs with allogeneic dental follicle-derived mesenchymal cells sheets exhibited better results, regarding the dimensions of new cementum (5.16 ± 0.23) and newly formed bone (4.67 ± 0.35) than allogeneic PDL-MSCs sheets therapy (3.84 ± 0.30 mm and 3.42 ± 0.26 mm respectively). Although complete regeneration occurred in the group of cells derived from PhD Thesis of Nerea Sánchez Pérez 48 the dental follicle and partial in that of PDL-MSCs, the differences found at 3 months after surgery were not statistically significant (Guo, Kang et al. 2017). Despite the increasing use of these methods that do not utilize three-dimensional matrices, recent preclinical studies have employed scaffold-based approaches, such as fibrin gel (Rezaei, Jamshidi et al. 2019, Venkataiah, Handa et al. 2019), collagen-based materials (membranes, matrices and powder) (Basan, Welly et al. 2017) and biomaterials composed of hydroxyapatite and/or calcium phosphates together with collagen (Liu, Yin et al. 2016, Basan, Welly et al. 2017, Nunez, Sanchez et al. 2018). In the second study of this PhD thesis, three months after the regenerative procedure with either PDL- MSCs/DBBM-10% collagen scaffold (test group) or the composite scaffold alone (control group), the results of the histological study revealed periodontal regeneration in both groups, in particular in the apical aspect of the furcation, where new cementum, periodontal ligament and alveolar bone were found, as well as new collagen fibers functionally oriented and attached into the cementum coronal to the alveolar bone. However, when the histomorphometric analysis was carried out, it was observed that, in the samples in which the test treatment had been used, the amount of regeneration achieved in the furcation area was slightly lower (1.93 ± 1.14 mm; 17%) than those in which the control treatment had been employed (2.35 ± 1.74 mm; 22%), but without statistically significant differences (p>0.05). Nor were any statistically significant differences found between the groups regarding other histometric variables, such as the amount of newly formed cementum or the dimensions of the root surface covered by epithelium and connective tissue at the level of the furcation and the interproximal area of the supracrestal defects. Conversely, Tsumanuma et al., using the same cell type (allogeneic PDL-MSCs) and also a supracrestal defect model, did find a greater regeneration of the cementum in the test group (around 60%) than in the control group without cells (40%), with statistically significant differences between both groups. These results must be taken with caution, since the design of this study presents substantial differences with respect to ours; As we have previously mentioned regarding the publication of Tsumanuma et al., in addition to not reproducing a supracrestal defect of a real critical size but rather a defect with a greater capacity for self-regeneration, they performed a guided tissue regeneration procedure by using a resorbable membrane fixed by bone pins to cover the cells/phosphocalcic-collagen matrix. This could have favored the stability of the blood clot and the biomaterial retention time throughout the healing period (Tsumanuma, Iwata et al. 2016). Nevertheless, in our study, most of the biomaterial was lost (only 5% remaining at 3 months) in both the test and control groups, possibly due to Cell Therapy in Periodontal Regereration 49 the lack of the space maintenance provided by the scaffold and due to the significant recession suffered during the healing period. Therefore, this model could not have provided the mechanical and functional characteristics necessary to evaluate the biological activity of the cells in the treatment of this large critical-size defect. Despite our results, several systematic reviews of preclinical investigations with MSCs in periodontal regeneration, which have not only included experimentally created supracrestal defects, but also intraosseous defects, dehiscence-type defects and fenestration and furcation lesions, have indicated that the application of MSCs from different sources would provide beneficial effects on the regenerative periodontal process (Bright, Hynes et al. 2014, Tassi, Sergio et al. 2017). The literature supports that MSCs of different origins (bone marrow, gingiva, periodontal ligament, adipose tissue, dental pulp, dental follicle, and alveolar bone) are capable of producing cementum-like tissue, alveolar bone and periodontal ligament, when introduced into periodontal defects in vivo (Seo, Miura et al. 2004, Zhao, Jin et al. 2004, Liu, Zheng et al. 2008, Tsumanuma, Iwata et al. 2011, Nunez, Sanz-Blasco et al. 2012, Tobita and Mizuno 2013, Iwasaki, Komaki et al. 2014, Takewaki, Kajiya et al. 2017, Rezaei, Jamshidi et al. 2019, Venkataiah, Handa et al. 2019). In addition, the adjuvant use of MSCs could improve the regenerative results of defects treated with membranes and bone substitutes (Tassi, Sergio et al. 2017), so they would provide an added benefit over many biomaterials available nowadays. Therefore, the translational research published to date regarding the safety and efficacy of MSCs therapies justifies the design of clinical trials for evaluating these advanced therapies in humans. PhD Thesis of Nerea Sánchez Pérez 50 STUDY 3. Sánchez N, Fierravanti L, Núñez J, Vignoletti F, González-Zamora M, Santamaría S, Suárez-Sancho S, Fernández-Santos ME, Figuero E, Herrera D, García-Sanz JA, Sanz M. (2020). Periodontal regeneration using a xenogeneic bone substitute seeded with autologous periodontal ligament-derived mesenchymal stem cells: A 12-month quasi-randomized controlled pilot clinical trial. Journal of Clinical Periodontology 47(11):1391-1402. The objective of this controlled clinical trial on 20 patients with periodontitis was to determine the safety and efficacy in humans of a protocol that compared the use of autologous mesenchymal cells (10x106 PDL-MSCs) seeded in three-dimensional scaffolds (100 mg of DBBM/10% collagen) and the use of the same scaffold (control) without cells in the treatment of intrabony defects. The general hypothesis of this work was that the use of mesenchymal cells as a therapeutic tool in the treatment of periodontal defects was safe and that it could provide a significant added clinical benefit with respect to control therapy. First, a series of experiments were carried out to ensure the safety of the transplanted cells. For this purpose, the absence of mycoplasma contamination was corroborated and the genomic stability of the cells in passage 3 was confirmed by comparative genomic hybridization. Using this cytogenetic technique, a large, high-resolution study of the genome with respect to copy number variations was performed to detect aneuploidies, microdelections/microduplications, and subtelomeric chromosomal alterations in this cell population in comparison to a reference sample by oligonucleotide array comparative genomic hybridization (Shinawi and Cheung 2008). Subsequently, once the regenerative surgery had been performed, the presence of adverse events was carefully monitored at each of the post-surgical follow-up visits by extra and intraoral visual inspection and all patients were instructed to notify any symptom of disease or systemic disturbance to the members of the surgical team to be taken into account in the safety analysis. The trial results showed adverse events similar to those commonly reported after a conventional surgical periodontal procedure. The most common reactions were the presence of mild- moderate pain during the first week after the intervention and mild dentin hypersensitivity in the successive weeks, both in the test and control groups. The clinical studies, published to date, in which MSCs are used for the regeneration of periodontal defects, are shown in Table 2. As in our study, these investigations, which have follow-up periods ranging from 6 to 36 months, agree that periodontal regeneration with autologous Cell Therapy in Periodontal Regereration 51 or allogeneic MSCs expanded in culture is safe (Yamada, Nakamura et al. 2013, Baba, Yamada et al. 2016, Li, Zhao et al. 2016, Hernandez-Monjaraz, Santiago-Osorio et al. 2018, Iwata, Yamato et al. 2018). In these studies, in addition to mild-moderate pain and dentin hypersensitivity, already reported in our trial, other complications are described, also common after any periodontal surgery, such as mild-moderate inflammation, gingival redness, hematoma, bleeding in the surgical area or angular cheilitis (Baba, Yamada et al. 2016, Chen, Gao et al. 2016, Iwata, Yamato et al. 2018). Only one study described severe pain in a patient who had also referred it in two previous periodontal surgeries (Iwata, Yamato et al. 2018). In our work, unlike the previous clinical studies, we evaluated pain as a quantitative variable, recording the number of anti-inflammatory drugs taken by the subjects the first week after surgery. The data analysis revealed that the anti-inflammatory drugs ingested by the patients in the test group (4.6 ± 4.2) were somewhat higher than those taken by the control group (2.4 ± 3.2), although the differences were not statistically significant (p <0.05). In addition to the extra and intraoral visual inspection and the symptoms reported by the subjects included in the studies, some investigations have carried out blood and urine tests, showing the absence of significant changes in the concentrations of immunoglobulin A, M and G, complement proteins C3 and C4, general serology and flow cytometry before the intervention and several months after periodontal surgery (Baba, Yamada et al. 2016, Chen, Gao et al. 2016, Hernandez-Monjaraz, Santiago-Osorio et al. 2018). In this third study, the well-being and quality of life of the patient before and after the surgical procedure, were also assessed using the OHIP-14Sp questionnaire, comparing the results obtained at baseline, 15 days after the intervention and at the end of follow-up (12 months). Although without statistically significant differences, the subjects in the control group reported a better oral quality of life at 15 days (4.2 ± 2.9) and at 12 months after the intervention (2.7 ± 3.4) than the patients in the test group treated with cells (5.7 ± 3.1; and 7.0 ± 10.73; p> 0.05, respectively), probably because more patients in the test group showed worse healing one week after the surgery (two patients showed some necrosis in the interdental papilla associated with the defect in the test group Vs. one patient in the control group, p> 0.05). The satisfaction rate with the aesthetic appearance of the surgical area at the end of the follow-up was also assessed using the VAS scale, which was also slightly lower in the test group (7.38 ± 2.9) than in the control group (8.80 ± 2.82), with no statistically significant differences (p> 0.05). In the rest of the clinical studies evaluating cell therapy in periodontal regeneration, shown in Table 2, no patient-based outcome variables were analysed. PhD Thesis of Nerea Sánchez Pérez 52 The approval of the research project by the Spanish Medicines Agency (AEMPS) that has given rise to this third publication of the doctoral thesis, was obtained in 2014, at which time only three clinical studies that evaluated the impact of the application of autologous MSCs in the treatment of periodontal defects had been published (Yamada, Ueda et al. 2006, d'Aquino, De Rosa et al. 2009, Yamada, Nakamura et al. 2013) (Table 2). Two of them, conducted by the same Japanese research group, did not have a design that provided consistent evidence (a case report and a case series), but revealed that the introduction of iliac crest-derived BMSCs along with platelet rich plasma (PRP) in periodontal defects resulted in a significant improvement in clinical and radiological variables (Yamada, Ueda et al. 2006). The other investigation, in this case, a 12-month controlled clinical trial, showed that the use of DPSCs seeded on collagen sponges promoted the complete regeneration of supraalveolar defects secondary to mandibular third molar impaction, unlike those treated with a collagen sponge (control group) (d'Aquino, De Rosa et al. 2009). Since then, more studies have been published (Table 2), two clinical trials, two case series and two case reports, with follow-ups ranging from 6 to 36 months, which have reported that the use of autologous or allogeneic MSCs, for the treatment of intrabony lesions, circumferential defects and furcation defects, seems to significantly improve the clinical and radiological outcome variables (Dhote, Charde et al. 2015, Baba, Yamada et al. 2016, Chen, Gao et al. 2016, Li, Zhao et al. 2016, Hernandez-Monjaraz, Santiago-Osorio et al. 2018, Iwata, Yamato et al. 2018). Together with our work, there are only three controlled clinical trials published to date that have evaluated the effect of cell therapy with MSCs on the functional regeneration of the lost periodontal tissues (Dhote, Charde et al. 2015, Chen, Gao et al. 2016). All three studies are clinical trials with a follow-up of 12 months, which initially included 20-30 patients with periodontitis and who presented at least one deep intrabony defect. In our trial, a periodontal regeneration procedure was carried out using a coronally advanced flap approach, comparing a test group (autologous PDL-MSCs/hydroxyapatite-collagen matrices) and a control group, based on the use of the same scaffold without cells. Intergroup comparisons revealed that the test group showed a greater CAL gain (1.44 ± 1.87 mm) and a greater PPD reduction (2.33 ± 1.32 mm) than the control group (0.80 ± 1.98 mm and 2.10 ± 2.46 mm, respectively), without statistically significant differences between the groups (p>0.05). A greater CAL gain (4.50 ± 1.08 mm) and greater PPD reduction (3.91 ± 1.37 mm) were also achieved in those defects treated with the test therapy versus the control therapy (3.50 ± 0.90 mm and 2.08 ± 0.90 mm, respectively), in other clinical trial, in which, unlike our study, the differences between groups were Cell Therapy in Periodontal Regereration 53 statistically significant (Dhote, Charde et al. 2015). It is important to note that in this last study, the magnitude of the improvement achieved was greater than in our publication, but it is also necessary to take into account that the effect of the test group was not only due to the potential of the MSCs embedded in the matrix, but to the tissue-inducing capacity of rhPDGF that was also part of the experimental treatment (Dhote, Charde et al. 2015). It could also be said that in our study, only patients with one and two-wall intrabony defects were selected, lesions that, according to the literature, would present a worse response to regenerative treatment than three-wall defects (Tonetti, Pini-Prato et al. 1993); However, Dhote et al., included all types of intrabony defects and did not stratify the results of the clinical variables according to the number of walls, reporting together the mean values of all defects. On the other hand, in the clinical trial by Chen et al., with a shorter evaluation period (6 months), a trend contrary to the two previous studies with respect to the CAL change was observed, since the test group (PDL -MSCs seeded in a DBBM scaffold) exhibited a slightly lower CAL gain than the control group (scaffold without cells) (p> 0.05) (Chen, Gao et al. 2016). With respect to another of the clinical variables, the changes in gingival recession between baseline and the end of follow-up, in our work we observed an increase in recession dimensions in both groups of approximately 1 mm (at 12 months) (Sanchez, Fierravanti et al. 2020), a similar trend to the one found in the Chinese group study at 6 months (Chen, Gao et al. 2016). Regarding the radiological outcome variables, Chen et al., established changes in the radiographic bone level between baseline and 12 months, as their primary outcome variable, data with which they could not show an added benefit of the test group versus the control group. Dhote et al. did find a statistically significant radiographic improvement in the trial, showing a greater defect reduction in the test group (3.50 ± 0.67 mm) than in the control group (1.83 ± 0.38 mm) (Dhote, Charde et al. 2015, Chen, Gao et al. 2016). Despite the absence of statistical significance, in our pilot clinical trial, it was suggested that the test treatment was superior to the control therapy with respect to the mean CAL gain and PPD reduction 12 months after the procedure. Besides, a greater proportion of sites with a CAL gain ≥2 mm and ≥3 mm were found in the test group (56% and 33%) compared to the control (40% and 20%); therefore, we can say that this cell therapy protocol could be a promising treatment, amenable to further research with an improved design and a greater sample size that enables a higher study power to detect significant differences between the treatment groups. PhD Thesis of Nerea Sánchez Pérez 54 Other modalities of cell therapy for the regeneration of the periodontium in humans In addition to MSCs, other cell populations have been utilized to promote the regeneration of the damaged cementum, periodontal ligament and alveolar bone, especially approaches based on the use of the so-called “whole tissue fractions containing MSCs” (Akbay, Baran et al. 2005, Aimetti, Ferrarotti et al. 2014, Aimetti, Ferrarotti et al. 2018, Ferrarotti, Romano et al. 2018). Unlike strategies that employ expanded undifferentiated MSCs, these other approaches do not require cell culture (Novello, Debouche et al. 2020). They are based on the transplantation of autologous tissue samples, mainly the periodontal ligament (Akbay, Baran et al. 2005, Shalini and Vandana 2018) and the dental pulp of permanent teeth (Aimetti, Ferrarotti et al. 2014, Aimetti, Ferrarotti et al. 2015, Aimetti, Ferrarotti et al. 2018, Ferrarotti, Romano et al. 2018), with minimal manipulation and no ex vivo expansion (Shanbhag, Suliman et al. 2019). Two different approaches of "whole tissue fractions containing MSCs" can be distinguished. In one of them, the tissue sample, immediately after its isolation, is directly transplanted into the periodontal defect without any type of manipulation (Akbay, Baran et al. 2005, Kl, Ryana et al. 2017, Shalini and Vandana 2018). In the second, once the sample is isolated, it is mechanically disaggregated with special devices to achieve the so-called "micro-grafts rich in different cell fractions, with MSCs among them" (Aimetti, Ferrarotti et al. 2014, Aimetti, Ferrarotti et al. 2015, Aimetti, Ferrarotti et al. 2018, Ferrarotti, Romano et al. 2018). An advantage of these strategies is the preservation of the extracellular matrix along with other cell fractions that are routinely discarded in ex vivo MSCs culture procedures. The hypothesis being considered is that these cells and the tissue fractions could have important functions in maintaining the niche/microenvironment in which stem cells exert their biological activity (Aimetti, Ferrarotti et al. 2018, Shalini and Vandana 2018). However, whole tissue fractions contain not only MSCs, but also other progenitors and cell populations that, although they do not possess the inducing potential of cell differentiation for regeneration, such as monocytes and other hematopoietic cells, can release growth factors and signalling molecules, which, in their physiological proportions, can stimulate the regenerative process (Jager, Herten et al. 2011, Fraser, Hicok et al. 2014). Another advantage of whole tissue fractions without ex vivo expansion is that all procedures are carried out in the dental chair in the same session, so they are more economical, since no specific technical personnel or manipulation is required in a cleanroom. Besides, all regulatory activities and lengthy approval procedures by the National Drug Agencies that Cell Therapy in Periodontal Regereration 55 govern ex vivo manipulation of mesenchymal cells for their subsequent transplantation into humans are avoided. This cell therapy based on the use of whole tissue fractions has been evaluated in clinical studies with different experimental designs (Table 3). In the first published clinical report in which tissue samples were used without any manipulation, periodontal ligament grafts, obtained by scraping the middle third of the root of third molars extracted with a healthy periodontium, were immediately transplanted into class II-furcation lesions in mandibular molars (Akbay, Baran et al. 2005). When, at 6 months, this treatment group was compared with the control group, which used only the coronal advancement flaps, the test group exhibited a greater CAL gain and PPD reduction (p <0.05). A RCT with a 12-month follow- up used a protocol called by the authors "Autologous Periodontal Stem Cells Assistance in in periodontal regeneration technique (SAI-PRT)" in the test group, by which the isolated periodontal ligament scraped from the root surface and the socket walls was combined with a gelatine sponge, and compared with an open flap debridement (control group) for the treatment of intrabony defects (Kl, Ryana et al. 2017, Shalini and Vandana 2018). This study also reported significant PPD reductions and CAL gains in the test group (Shalini and Vandana 2018). In another protocol, the isolated tissue (in this case, fresh tissue from the pulp obtained immediately after the extraction of the tooth), was subjected to mechanical disaggregation with a special device to obtain the so-called “micro-grafts”, which were then filtered, embedded in collagen sponges and transplanted into the defects (Aimetti, Ferrarotti et al. 2014). Publications of case reports that have utilized this approach have shown clinical and radiographic benefits in the treatment of non-supporting intrabony defects (Aimetti, Ferrarotti et al. 2014, Aimetti, Ferrarotti et al. 2015, Aimetti, Ferrarotti et al. 2018). Recently, a RCT compared the MIST technique with dental pulp micro-grafts seeded on a collagen sponge (test) with the same technique consisted of the matrix alone (control) for the treatment of deep intrabony defects (Ferrarotti, Romano et al. 2018). Twelve months later, the results revealed that the application of this cell therapy significantly improved the clinical and radiographic parameters, suggesting that this strategy could be a valid and simple strategy for periodontal regeneration (Ferrarotti, Romano et al. 2018). Despite all the potential advantages that whole tissue fractions with putative MSCs content seem to have, to date, there is no histological evidence on periodontal regeneration from preclinical studies, so we cannot reliably say that it stimulates the synthesis of a new connective tissue attachment, new cementum, alveolar bone and periodontal ligament PhD Thesis of Nerea Sánchez Pérez 56 after transplantation, so we cannot yet say that true periodontal regeneration occurs (Nagata, de Campos et al. 2014) Strengths of this doctoral thesis The use of culture-expanded mesenchymal cells is one of the most promising approaches in research today, with an exponential increase in the number of publications on cell therapy with this source in medicine. In this doctoral thesis, it has become clear that cell therapy with stromal mesenchymal cells derived from the periodontal ligament is safe, a key point for all subsequent research in humans. The clinical trial carried out as part of this doctoral thesis is one of only 4 controlled clinical trials on cell therapy with MSCs in periodontal regeneration published in the world. In this study, unlike other clinical trials, we performed a patient-based analysis and not a defect-based analysis. In addition, we observed that the group that received the cell transplant obtained superior clinical improvements in comparison to the control group (transplant without cells), although these differences were not statistically significant, probably due to the low power of the trial since it was a pilot study including only 20 patients. Limitations of the studies included in this doctoral thesis Regarding the preclinical work, the main drawback that prevented a true analysis regarding the impact of cell therapy on the regeneration of periodontal defects was the alterations of the soft tissues during the healing process. According to scientific evidence, the stability of the blood clot and the maintenance of the regeneration space are crucial aspects for an adequate cellular response that promotes tissue healing. Due to tissue dehiscence and exposure of the hydroxyapatite-collagen matrices to the oral environment, contamination by oral bacteria, the absence of space maintenance with the consequent collapse of soft tissues, probably prevented the MSCs from fully deploying its paracrine, and tissue-inductive biological potential. Another limitation of this preclinical research is that the transplanted MSCs were not labelled during their culture. Therefore, to analyse the cellular viability after the procedure in the subsequent histological analysis and to evaluate whether the newly formed tissues came from the differentiation of the implanted cells or from endogenous cells that had migrated to the site of damage in response to the biochemical signals released by the implanted cells was not possible. One of the great limitations of the clinical trial is its low power (12.2%), which could have significantly reduced the chances of detecting a statistically significant added clinical Cell Therapy in Periodontal Regereration 57 benefit by the test group with respect to the main outcome variable. The power calculation, performed at the end of the study (posthoc), using an effect magnitude of 0.645 regarding the changes in the main outcome variable (CAL gain) between baseline and 12 months, a sample size of 9 and 10 patients (test and control groups, respectively) and an alpha error of 5%, determined that the power of the study was 12.2%. This investigation was designed as a pilot study and, therefore, a sample size calculation was not carried out. Another major limitation of this study was the absence of randomized allocation of patients to the test and control group. Initially, the study was designed as a parallel-group randomized controlled clinical trial, that is, only the teeth from the patients allocated to the test group were going to be transported to the cleanroom for the isolation of the periodontal ligament sample and cell expansion; However, given the failure of cell proliferation in the samples isolated from the first patients of the test group, it was decided to initiate the cell culture process of the samples from all the subjects, and make the treatment group assignment according to cell growth: if cells proliferated, the subject was allocated to the test group. The main drawback of this modification is that it entails a selection bias, which reduces the strength of the study. Future prospects for clinical research with MSCs Clinical research with mesenchymal cells should be implemented with new randomized controlled clinical trials, with longer follow-up periods, a larger number of included subjects and robust designs. New studies should evaluate periodontal clinical parameters at the final visit and carry out data analysis based on the patient rather than the site. Ideally, the control group should be identical to the test group except for the use of cells, and the test group should not include any growth factor or biologically active agent in order to objectively analyse the impact of cell therapy. Additional treatment groups could be added to the study design (MSCs plus factors or bioactive molecules) but independently from the main test group (MSCs), and taking into account that, the greater the number of groups, the larger the sample size must be for each group to avoid underpowered studies. Furthermore, issues such as the cost-effectiveness of cell therapies are considerations that should be taken into account for future research. In this regard, the use of allogeneic cells from mesenchymal cell banks could facilitate the performance of randomized clinical trials, since, on the one hand, the morbidity associated with autologous tissue isolation and the possibilities of cell expansion failure would be avoided, and on the other hand, the costs associated with cell culture would be lowered. PhD Thesis of Nerea Sánchez Pérez 58 In addition, the use of conditioned medium, the medium where the cells have been cultured, where their growth factors and bioactive molecules have been released, is a therapeutic tool that should also be considered, since it would promote the paracrine potential characteristic of MSCs without the need of transplanting cells into a subject. This would possibly simplify the authorizations required by the National Drug Agencies since no genetic material is introduced, and the possibilities of mutations and tumour formations are lower. An unexplored field, of great interest, in light of the favorable clinical results, the absence of special regulations, low cost and the simplicity of the procedure, would be the design of preclinical studies to histologically evaluate the benefits of the use of whole tissue fractions and the micrografts containing MSCs. Likewise, clinical trials comparing these chair-side strategies with expanded MSCs approaches would be of great interest due to the lack of publications. Cell Therapy in Periodontal Regereration 59 VIII. CONCLUSIONS 1. The Periodontal ligament and gingival connective tissue-derived cells used in our first work can be considered mesenchymal stromal cells, since they demonstrate compliance with the minimum criteria described by the ISCT. These cells showed genomic stability after ex vivo expansion and absence of tumorigenic potential in experimental animals after 6 months. When subjected to suboptimal culture conditions, gingival connective tissue- derived mesenchymal cells showed a significantly higher proliferation rate than those isolated from the periodontal ligament. 2. The use of the combination allogeneic periodontal ligament-derived mesenchymal cells/xenogeneic scaffolds in the regenerative treatment of critical-size supracrestal periodontal defects in a preclinical model in Beagles dogs is safe. However, the regenerative capacity of this cell therapy was limited to the apical third of the defects and did not show statistically significant differences with the control group (scaffold without cells) in terms of the extent of periodontal regeneration and new cementum formation. 3. The application in patients of autologous periodontal ligament-derived MSCs embedded in a xenogeneic hydroxyapatite-collagen scaffold for the regenerative treatment of intrabony defects is safe, and shows adverse events similar to those of any other conventional periodontal surgery. Although an added clinical benefit could not be demonstrated in terms of clinical attachment level gain and probing pocket depth reduction, a significant trend was observed in favor of the test group (with cells) compared to the control group (without cells) 12 months after the intervention. 4. The results of the three investigations included in this doctoral thesis indicate that the application of mesenchymal stromal cells in periodontal regeneration is safe. 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(China) Minipigs (n=12)/ 3 months 3-wall Intrabony defects I) DPSCs injection II) DPSCs-VitC sheets (XEN) NaCl injection Grafting / Subperiosteal injection Basan et al. (Germany) Minipigs (n=15)/ 4 months Class II furcation defects PDL-MSCs/CP + SMem (AUT) b) CP + SMem c) CMem/HA-TCP + GF + SMem d) CMem + SMem e) CM+ SMem f) CM + GF + SMem d) Empty + SMem Grafting Takewaki et al. (Japan) Beagle Dogs (n=7)/ 3 months Class III furcation defects BMSCs sheets + osteogenic media (AUT) Flap Surgery Grafting Guo et al. (China) Beagle Dogs (n=5)/ 3 months 2-wall Intrabony defects DFSCs sheets PDL-MSCs sheets (ALO) - Grafting • Núñez et al. (Spain) Beagle Dogs (n=9)/ 3 months Critical-size supracrestal defects PDL-MSCs/DBBM- Collagen (ALO)/ DBBM-Collagen Grafting Venkataiah et al. (Japan) Minipigs (n=4)/ 1 month Furcation defects ADSCs/fibrin gel (AUT or ALO) Fibrin Gel Grafting Rezaei et al. (Iran) Mongrel Dogs (n=5) 2 months Class II furcation defects *BMSCs/fibrin glue/PRP *BMSCs/ fibrin glue (AUT) a) PRP + fibrin glue b) Fibrin glue Grafting Cell Therapy in Periodontal Regereration 79 Li et al. 2019 (China) Minipigs (n=9)/ 3 months Defects in both sizes of mesial root of 1st MB molar I) SCAPs II) SCAPs/SFRP2 Saline Subperiosteal injection Li et al. 2019 (China) Minipigs (n=6)/ 3 months Class II furcation defects DPSCs-IPs /β-TCP (AUT) β-TCP Grafting GF: growth factor; BMSCs: bone marrow-derived mesenchymal stem cells; DPSCs: dental pulp-derived mesenchymal stem cells; PDL-MSCs: periodontal ligament-derived mesenchymal stem cells; DFSCs: dental follicle-derived mesenchymal stem cells; ADSCs: adipose tissue-derived mesenchymal stem cells; SCAPs: apical papilla-derived mesenchymal stem cells; DPSCs- IPs: mesenchymal stem cells derived from inflammatory dental pulp tissue; AUT: autologous; XEN: xenogeneic; ALO: allogeneic; HA: hydroxyapatite; VitC: Vitamin C; CP: collagen powder; SMem: covered by semi-permeable membrane; DBBM: demineralized bovine bone mineral; NaCl: sodium chloride; CMem: collagen membrane; CM: collagen matrix; PRP: platelet- rich plasma; SFRPs: secreted frizzled related proteins; CMem/HA-TCP: collagen membrane/hydroxyapatite-tricalcium phosphate; β-TCP: beta-tricalcium phosphate; MB: mandibular. PhD Thesis of Nerea Sánchez Pérez 80 Table 2. Design of the clinical trials evaluating MSCs for periodontal regeneration. Author (Country) Design (Follow up) n (final) Test (CELLS ORIGIN) Control Type of defect Yamada et al. (Japan) CR (12 months) 1 BMSCs (AUT)/PRP - Intrabony defect D´Aquino et al. (Italy) Split-mouth CT (12 months) 7 DPSCs (AUT)/collagen sponge Collagen sponge Supracrestal defect secondary to an impacted MB third molar Yamada et al. (Japan) CS (6 months) 17 BMSCs (AUT)/PRP - Not reported Dhote et al. (India) Parallel-group RCT (12 months) 20 Cord Blood MSCs (ALO)/β-TCP/PDGF- BB Flap surgery Intrabony defects Chen et al. (China) Parallel-group RCT (12 months) 30 PDL-MSCs sheets (AUT)/DBBM DBBM Intrabony defects Baba et al. (Japan) CS (36 months) 10 BMSCs (AUT)/PRP/PLA - 1, 2, 3-wall Intrabony defects Li et al. (China) CR (9 months) 2 DPSCs-IPs (AUT)/ β- TCP Furcation Defects Iwata et al. (Japan) CS (6 months) 10 PDL-MSCs sheets (AUT)/PGA/β-TCP - 1, 2, 3-wall Intrabony, horizontal and circumferential defects Hernández- Monjaraz et al. (Mexico) CR (6 months) 1 DPSCs (ALO)/collagen/PVP/ Non-resorb. Membrane - Circumferential defects CR: case report; CT: controlled trial; CS: case series; RCT: randomized controlled clinical trial; BMSCs: bone marrow-derived mesenchymal stem cells; DPSCs: dental pulp-derived mesenchymal stem cells; MSCs: mesenchymal stem cells; PDL-MSCs: periodontal ligament-derived mesenchymal stem cells; AUT: autologous; ALO: allogeneic; PRP: platelet-rich plasma; β-TCP: beta-tricalcium phosphate; PDGF-BB: platelet-derived growth factor-BB; DBBM: demineralized bovine bone mineral; PLA: poly-lactide acid; DPSCs-IPs: mesenchymal stem cells derived from inflammatory dental pulp tissue; PGA: poly-glycolide acid; PVP: polyvinylpyrrolidone sponge; resorb.: resorbable; MB: mandibular. Cell Therapy in Periodontal Regereration 81 Table 3. Design of the clinical trials evaluating whole tissue fractions for periodontal regeneration. Author (Country) Design (Follow up) n (final) Test (CELLS ORIGIN) Control Grafts processing Periodontal defect Akbay et al. (Turkey) Split-mouth RCT (6 months) 10 PDL grafts (AUT) Flap Surgery None Class-II MB furcation defect Aimetti et al. (Italy) CR (12 months) 1 DPSCs (AUT)/Collagen sponge - Mechanical Dissociation (Medimax System) 1, 2-wall Intrabony defects Aimetti et al. (Italy) CR (12 months) 4 DPSCs (AUT)/ Collagen sponge - Mechanical Dissociation (Medimachine System) 1 or 2-wall Intrabony defects Kl et al. (India) CR (12 months) 1 PDL grafts (AUT)/ Gelatin sponge None Intrabony defect Aimetti et al. (Italy) CS (12 months) 11 Micro-grafts rich in DPSCs (AUT)/ Collagen sponge - Mechanical Dissociation (Rigenera System) Intrabony defects Ferrarotti et al. (Italy) Parallel- group RCT (12 months) 29 Micro-grafts rich in DPSCs (AUT)/ Collagen sponge Collagen Sponge Mechanical Dissociation (Rigenera System) Intrabony defects Shalini et al. (India) Parallel- group RCT (12 months) 28 PDL grafts (AUT) Flap Surgery None Intrabony defects RCT: randomized controlled clinical trial; CR: case report; CS: case series; PDL: periodontal ligament; DPSCs: dental pulp- derived mesenchymal stem cells; AUT: autologous; MB: mandibular. PhD Thesis of Nerea Sánchez Pérez 82 Tesis Silvia Nerea Sánchez Pérez PORTADA PREFACE ABBREVIATIONS TABLE OF CONTENTS I. SUMMARY RESUMEN II. INTRODUCTION III. JUSTIFICATION IV. HYPOTHESIS V. OBJECTIVES VI. MATERIAL AND METHODS. RESULTS Comparison of periodontal ligament and gingiva-derivedmesenchymal stem cells for regenerative therapies Cell therapy with allogenic canine periodontal ligament-derivedcells in periodontal regeneration of critical size defects Periodontal regeneration using a xenogeneic bone substituteseeded with autologous periodontal ligament-derivedmesenchymal stem cells: A 12-month quasi-randomizedcontrolled pilot clinical trial VII. DISCUSSION VIII. CONCLUSIONS IX. REFERENCES X. FIGURES Y TABLES