Academic literature on the topic 'Regenerative endodontics'

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária<br>Em Medicina Dentária a taxa de incidência de patologia pulpar é elevada. Atualmente o tratamento realizado nestes casos passa pelo tratamento endodôntico não cirúrgico e obturação do sistema tridimensional de canais do dente permanente e maturo necrosado. Quando o tecido pulpar sofre necrose o prognóstico do dente está comprometido. Até então a abordagem terapêutica nestes casos passa por tratamento dos canais radiculares no caso de dentes com ápices maduros, apexificação em dentes com ápice imaturo ou extração. Apesar de estas modalidades terapêuticas apresentarem elevadas taxas de sucesso, a presença de uma polpa vital é fundamental para manter a homeostase e longevidade da peça dentária. Uma forma de terapia ideal pode consistir em abordagens regenerativas que consistem na remoção e substituição da polpa danificada ou necrosada por tecido pulpar saudável de forma a revitalizar o dente. Para atingir este objetivo os investigadores têm estudado diversas técnicas: revascularização por coágulo sanguíneo, terapia com células estaminais, implantação pulpar, implantação de scaffold, impressão tridimensional celular, scaffold injetável e terapia genética. Este tipo de tratamentos envolve combinação de desinfeção, desbridamento do canal afetado, utilização de células estaminais adultas, scaffolds e fatores de crescimento. Pode ser necessário também o alargamento apical para permitir a revascularização. Com esta revisão pretende-se compreender melhor o procedimento de revascularização pulpar no geral, vantagens e desvantagens, sua aplicabilidade na prática clinica diária e aferir sobre os resultados obtidos na literatura. Apesar de os desafios para a introdução das técnicas regenerativas como tratamento alternativo de dentes necrosados serem substancias, estas podem ser benéficas pois apresentam a possibilidade de restabelecer a funcionalidade pulpar, tornando a polpa vital capaz de promover a correta maturação apical. A inexistência de um protocolo fiável de atuação que permita a criação das condições necessárias para que ocorra a revitalização do tecido torna este tratamento pouco utilizado como tratamento alternativo na prática clinica. Assim, mais estudos são necessários para que futuramente seja possível elaborar um protocolo que possibilite a introdução das técnicas de revascularização pulpar na prática clinica diária. Dentistry in the incidence rate of pulpal pathology is high. Currently the treatment performed in these cases through the nonsurgical endodontic treatment and obturation of the three-dimensional system of permanent tooth and necrosed mature channels. When the pulp tissue undergoes necrosis prognosis of the tooth is compromised. Until then the therapeutic approach in these cases involves treatment of root canals in the case of teeth with mature apices, apexificação in teeth with immature apex or extraction. Despite these therapeutic modalities have high success rates, the presence of a vital pulp is essential to maintain homeostasis and longevity of tooth number. An ideal form of therapy might consist of regenerative approaches involving the removal and replacement of damaged or necrotic pulp by healthy pulp tissue to revitalize the tooth. To achieve this goal researchers have studied various techniques: revascularization by a blood clot, stem cell therapy, pulp, implantation of scaffold, cell dimensional printing, injectable scaffold and gene therapy. This type of treatment involves combination of disinfection, debridement of the affected channel, use of adult stem cells, scaffolds and growth factors. You may also need the apical enlargement to allow revascularization. With this revision is intended to better understand the procedure of pulp revascularization in general, advantages and disadvantages, its applicability in daily clinical practice and benchmark the results obtained in the literature. Although the challenges for the introduction of regenerative techniques as an alternative treatment of necrotic teeth are substances, these can be beneficial since they have the ability to restore functionality pulp making the vital pulp capable of promoting correct apical maturation. The absence of a reliable protocol operation that allows the creation of conditions necessary for the revitalization of the fabric makes this treatment occurs rarely used as an alternative treatment in clinical practice. Thus, further studies are needed, in future be possible to develop a protocol which would include the pulp revascularization in endodontics.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária<br>Introdução: O endodontista é responsável pelo diagnóstico e tratamento de patologias da polpa e deve, sempre que possível, manter a sua vitalidade. O conhecimento pulpar é essencial para o desenvolvimento de um plano de tratamento adequado. O sucesso do tratamento depende não só dos materiais e instrumentos disponíveis, como da aptidão do clínico e colaboração do paciente. Objetivo: Recolha bibliográfica sobre o estado atual da endodontia regeneradora para dentes permanentes imaturos necrosados, abordando as principais vantagens e limitações dos procedimentos clínicos disponíveis, com ênfase nos princípios biológicos da técnica de apexificação. Materiais e métodos: Para tal, realizou-se uma pesquisa bibliográfica nos principais motores de busca (PubMed, B-On e Science Direct), como também em vários livros científicos, utilizando palavras-chave como: “Regenerative Endodontics”, “Treatment Outcome” e “Stem Cells”, que foram associadas de várias formas. Desta pesquisa, efetuada entre Março e Junho de 2015, foram escolhidos 57 artigos em inglês, dos quais foram utilizados 38. Além dos artigos, foram analisados cerca de 10 livros, dos quais foram utilizados 5. Resultados: Vários fatores, como o tipo de caso, prática do clínico e disponibilidade de instrumentos e materiais, fazem variar as taxas de sucesso das terapias endodônticas. Normalmente, os tratamentos são eficientes no que concerne à eliminação da dor e controlo de infeção. Conclusões: Esta nova técnica aparenta vir a ser uma boa ferramenta clínica e antevê-se uma potencial opção com perspetivas promissoras. No entanto, conclui-se que mais estudos clínicos deverão ser efetuados, com vista a proporcionar um melhor entendimento sobre este tipo de terapia, para assim se desenvolverem métodos que consigam ultrapassar as presentes dificuldades, que tornam a Endodontia Regeneradora tão controversa e imprevisível.<br>Introduction: The endodontist is responsible for the diagnosis and treatment of the dental pulp related pathologies and he must, whenever possible, keep its vitality. The learning of the pulp is essential for the development of an adequate treatment. Its success relies upon both the treatment and available tools, and the practitioner’s aptitude combined with the patient collaboration. Objective: Bibliographic research and collection about the present state of regenerative endodontics for permanent necrotic immature teeth, addressing its main advantages and the available clinical procedures limitations, emphasizing the biological principles of the apexification technics. Materials and methods: In order to do so, a bibliographic research was made with the help of the main web search engines (PubMed, B-on and Science Direct), and in various scientific books as well, using key-words such as: “Regenerative Endodontics”, “Treatment Outcome” and “Stem Cells” which were assembled in several ways. From this research, conducted between March and June of 2015, 57 articles in English were chosen of which 38 were used. In addition to that, about 10 books were analyzed of which 5 were used. Results: Various factors such as the type of case, the practitioner’s experience and the availability of tools and materials, result in the variation of the success rates of the endodontic therapies. Ordinarily the treatments are effective concerning pain elimination and infection control. Conclusions: This new technique could become a valuable clinical tool, as it is an option which has bright future prespectives. However, in conclusion more clinical studies should be accomplished in order to clearer understand this kind of therapy and so that the methods which can surpass the current difficulties can be developed, since these problems make Regenerative Endodontics quite controversial and unpredictable.

Background: Endodontic management of immature necrotic permanent teeth is currently undergoing a paradigm shift from the conventional apexification and apical plug techniques to biological based regenerative procedures. The shift towards a tissue engineered clinical based procedure is highly attractive. However, the ideal scaffold to support pulp-dentine complex regeneration is yet to be identified. Aim: The primary aim of this work was to develop, characterise and assess the biocompatibility of an acellular extracellular matrix scaffold developed through decellularisation of rat and human dental pulp tissues. The secondary aim was to assess the ability of the scaffold to support human dental pulp stem cells attachment, viability, and differentiation. Methods: Following ethical approval, rat and human dental pulps were retrieved and decellularised. The efficiency of the decellularised protocol was assessed using histology and immunohistochemistry staining methods, scanning electron microscopy, and DNA quantification assays. Contact and extract cytotoxicity assays were performed to determine the biocompatibility of the developed scaffold. Decellularised scaffolds were recellularised with human dental pulp stem cells and cell viability was assessed, for up to 14 days, in culture. Expression of odontoblastic markers and molecular proteins within the recellularised scaffold were also investigated. Quantitative data were analysed using Student’s t-test and one-way analysis of variance using GraphPad Prism(Version 6). Results: Assessment of decellularised scaffolds revealed an acellular matrix with preservation of the connective tissue architecture and composition. Acellular scaffolds were biocompatible with normal cell growth in direct contact with the acellular scaffold. No difference in cellular activity was found following incubation in acellular scaffold extracts (p > 0.05). Live/Dead® confocal imaging showed high majority of viable cells. Furthermore, the scaffold was able to support human dental pulp stem cells viability and attachment following recellularisation. Immunolabelling of dental pulp stem cells within the recellularised scaffold revealed a positive expression against several tested odontoblastic markers and molecular proteins. Conclusion: The decellularisation protocol used showed promising results following decellularisation of rat and human dental pulp tissues in terms of developing an acellular biological scaffold with preserved extracellular structural components required for tissue specific regeneration.

Trabalho apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária<br>Nos últimos anos têm-se realizado pesquisas sobre a utilidade de células estaminais em diversas áreas da saúde. Estes avanços científicos na área da bioengenharia tecidual têm levado ao desenvolvimento de novas terapias e curas de doenças que, até aos dias de hoje, não teriam cura. Recentes pesquisas têm revelado que os dentes são ótimas fontes de células estaminais. Tanto os dentes decíduos como permanentes têm células estaminais com grande potencial proliferativo e apresentam as caraterísticas de células estaminais: capacidade de autorrenovação e diferenciação em várias linhagens celulares. Mediante este contexto, o objetivo geral desta monografia foi realizar uma revisão de literatura em foco para atualizar o conhecimento acerca das células estaminais e a sua aplicação em medicina dentária. Tendo como objetivos específicos: analisar as células estaminais dentárias, ampliando o conhecimento sobre as possibilidades do seu potencial regenerativo, como fonte de células estaminais e sistematizar os conhecimentos, avanços científicos, limitações e perspetivas relativas à aplicação de células estaminais dentárias. Com esta revisão bibliográfica, é possível esclarecer determinadas dúvidas sobre células estaminais. O futuro passará em usar estas células como tratamento terapêutico, de maneira a regenerar muitos tecidos, fazendo com que os órgãos lesados possam voltar à sua vitalidade completamente saudável. As células estaminais são, assim, de uma enorme utilidade. Stem cells have been the target of abundant research focusing their potential in regenerative medicine. Scientific advances in bioengineered tissue has allowed the development of new therapies for diseases that, until recently, would not be possible. Recent researches have revealed teeth as great sources of stem cells. Both deciduous or permanent teeth have stem cells with high proliferative potential, exhibiting the characteristics of other studied stem cells: self-renewal capacity and differentiation into various cell lines. Under this context, the general objective of this thesis was to conduct a literature review focused on the stem cells knowledge and its application in dentistry. Having specific objectives: the study the dental stem cells, considering their regenerative potential as a source of stem cells and systematize the knowledge, scientific advances, limitations and prospects for the application of stem cells decay. This literature review, enables the clarification of certain doubts about stem cells. The future use of these cells as a therapeutic treatment in order to regenerate different tissues, causing the damaged organs can return to its vitality completely healthy. The stem cells are therefore extremely useful.

Les Traitements Endodontiques Régénératifs (TER) des dents immatures sont une alternative au traitement endodontique classique. Ces procédures s’appliquent après une désinfection et une préparation mécanique du canal radiculaire. Suite au saignement du périapex le sang remonte dans le canal en créant un caillot. Après sa réorganisation en un tissu conjonctif vascularisé, il peut remplacer le tissu endommagé et poursuivre l’apexogénèse. Les résultats des TER sont encourageants mais pas constants dû à une désinfection incomplète, une variabilité des cellules et des facteurs de croissance du sang, des propriétés mécaniques faibles et une dégradation rapide du caillot. Afin d’améliorer ces procédures, cette thèse a thèse a été conduite sur trois axes en ayant pour objectif de développer deux biomatériaux, un résorbable sous la forme de gel injectable et un non-résorbable.1)Mise au point et caractérisation d’un hydrogel à base de Fibrinogène et de sang.Afin d’améliorer les propriétés mécaniques et biologiques du caillot sanguin nous l’avons enrichi en fibrinogène. Du sang humain a été mixé avec du Fibrinogène(Fbg) dilué dans du milieu de culture cellulaire. Plusieurs concentrations des composants ont été testées. Les propriétés mécaniques d’hydrogels et de la pulpe dentaire ont été mesures par microscopie à force atomique et des mesures rhéologiques de cisaillement à faible amplitude. Le taux de dégradation et de gonflement des hydrogels a été évalué. La survie et la prolifération cellulaire ont été évaluées par la microscopie à fluorescence. Le changement de la structure fibrillaire de l’hydrogel a été étudié par la microscopie multiphotonique (MPM). Pour évaluer les propriétés angiogéniques du gel à différentes concentrations de Fbg, un modèle ex vivo basé sur l’utilisation des anneaux de l’aorte de rats a été utilisé. Avec cette première série d’expériences nous avons défini un hydrogel avec des propriétés biomécaniques similaires à la pulpe dentaire, prometteuses pour son utilisation dans le cadre de la revitalisation pulpaire.2)Comparaison histologique entre l’hydrogel à base Fbg-sang et la pulpe dentaire humaine. Nous avons injecté l’hydrogel Fbg-sang avec des cellules de la pulpe dentaire (DPCs) dans la racine de dents humaines extraites et observé le comportement cellulaire et la maturation du tissu néoformé après 4 semaines ex vivo, en comparant l’histologie de l’hydrogel avec celle de la pulpe dentaire humaine. La caractérisation du tissu néoformé, ainsi que de la pulpe de dents saines a été réalisée par: Histologie classique, immunohistochimie pour détecter des marqueurs périvasculaires (CD31, αSMA et Collagène de type IV) et la MPM.Le caractère du tissu obtenu à l’intérieur de la racine est un tissu de réparation présenté sous forme d’un réseau fibreux contenant des cellules. Ce tissu a une architecture semblable à celle de la pulpe dentaire en vue de la disposition cellulaire et de la formation des nouveaux vaisseaux sanguins.3) Nous avons étudié les propriétés biologiques d’un matériau non résorbable et bioactive qui sont dues à la fonctionnalisation de sa surface par un revêtement organique-inorganique. Ce matériau pourrait être utilisé comme une matrice dans TER. L’effet du revêtement dans les DPCs a été évalué in vitro(MTT, ALP, RT-qPCR )et ex vivo en combinant l’utilisation du gel de Fbg-sang avec le matériau bioactif à l’intérieur de la racine. Le tissu formé à l’intérieur du canal a été évalué par histologie classique(He-Eo) et MPM. Les résultats montrent que les surfaces avec revêtement bioactive stimulent la maturation et la différenciation cellulaire. Le modèle ex vivo détecte la formation d’un tissu fibreux organisé et attaché à la surface du matériau avec revêtement qui suggère la bio-intégration au tissu formé par le gel.Les biomatériaux développés au cours de cette thèse sont prometteurs et originaux pour leur application dans le domaine du traitement régénératif endodontique<br>Regenerative Endodontic Treatment (RET) of necrotic teeth is an alternative to conventional endodontic treatment. Clinical protocol for RET is based on evoked periapical bleeding trough root apex, followed by blood clot formation. After reorganization of the clot a vascularized connective tissue could be formed to replace the damaged pulp and continue apexogenesis.RET on immature necrotic teeth gave encouraging results but they are not consistent. Lack of reproducibility may be due to incomplete root canal disinfection; variability of cells and growth factors of blood and poor mechanical properties and rapid degradation of the blood clot. To improve these procedures, the objective of this thesis was to develop two types of biomaterials, one resorbable - presented like an injectable hydrogel and one non-resorbable bioactive material.The thesis was conducted on 3 main axes: 1) Development and characterization of an Fibrinogen-blood contained hydrogel; 2) Histological comparison between the fibrinogen-blood gel and human dental pulp and 3) Bioactive non resorbable materialIn the first part of the thesis we mixed different concentration of Fibrinogen (Fbg) and of peripheral whole blood to obtain different Fbg-blood hydrogels. Atomic Force Microscopy and small amplitude oscillatory shear rheological measurements were used to characterize their mechanical properties. Swollen and degradation rate were evaluated by weigh changes. Biological properties were evaluated by following dental pulp cells (DPCs) behavior after incubation in the gel up to 3 weeks. Angiogenic potential was assessed by ex vivo rat aortic ring assay. Finally we selected the hydrogel composition which presents biomechanical properties close to human dental pulp with that represents the best environment for DPCs proliferation and possess angiogenic properties.In the second stage, the gel was injected inside endodontically pre-treated root canals for 4 weeks and cultured under laboratory conditions. Characterization of the newly formed tissue was made by histology (H-E and Masson Trichrome), immunohistochemistry (IHC) for perivascular markers detection (CD31, αSMA and Collagen IV) and multiphoton microscopy (MPM: 2- photon excited fluorescence (2-PEF) and second harmonic generation (SHG)). Comparison with human dental pulp was performed. An extracellular matrix mostly composed by autofluorescent fibers (elastin or fibronectin) was detected. Fibers and cell disposition are similar to dental pulp. Positive marked zones to CD31, αSMA and Col IV suggests neo-vessels formation.The following part of the thesis consists on the evaluation of biological properties of a bioactive non resorbable material that could be used as a scaffold for RET. DPC proliferation and differentiation on functionalized non resorbable material surfaces was proved by biological tests (MTT, ALP, RT-qPCR). Surface of the bioactive non resorbable material enhances DPCs maturation and differentiation.This material was also placed inside root canal in vitro and ex vivo. For the in vitro model we injected DPCs after the placement of a bioactive non resorbable material inside the root canal of extracted human teeth. For the ex vivo model functionalized or non-functionalized non resorbable scaffold were placed inside root canals or plastic tips after Fbg-blood gel injection. Samples were incubated for 4 weeks and analyzed by scanning electron microscopy (SEM), multiphoton microscopy (MPM) and classical histological staining (H-E). Bio-integration of functionalized non resorbable scaffold with newly formed tissue and organized fiber formation with a longitudinally disposition was proved by histology and MPM.Developed biomaterials and models show encouraging results, placing them as original approaches in regenerative endodontic field. Next stages for validation of obtained results concerns in vivo application of novel biomaterials with a farther finality of establishment of clinical protocols