"The aim of this PhD thesis was to study experimental approaches for revitalization of necrotic teeth. Revitalization, also known as regenerative endodontic procedures (REPs), is a relatively new treatment for necrotic teeth which tries to regenerate the dentine-pulp complex instead of obturating the root canal with biologically inert materials (root canal treatment). Until very recently, the most reliable option for the treatment of immature necrotic teeth was apexification followed by root canal treatment. However, endodontically treated teeth remain devitalized throughout the patient's lifetime and therefore defenceless to new caries lesions, as the absence of pulp implies the lack of tooth immune mechanisms. On the contrary, the regeneration of the dentine-pulp complex allows further root development and aims to recover the natural immune and secretory system of the pulp, making teeth more resistant to future lesions or traumatisms.The therapy was developed to treat necrotic immature teeth (i.e. those that have not completed their root development). Clinically, the outcomes can be considered successful since there is a resolution of the symptomatology, healing of the apical pathosis and further root development in most cases. However, histological analysis has demonstrated that the tissues formed after the therapy are reparative tissues - such as cementum-like tissue - instead of dentine, as well as an unorganized connective tissue, instead of pulp with its characteristic odontoblast layer. Currently, numerous efforts are being made to shed light on the clinical and biological aspects involved in the regeneration of pulp.Chapter 1: As previously said, evidence shows that no dentine but reparative tissues (cementum-like tissue) are responsible for the root development after regenerative endodontics. As cementum is less hard and less elastic than dentine, the question arises whether a root with apposition of cementum can endure mechanical stress similarly to roots completed by dentine. Thus, one of the objectives of this thesis was to compare the biomechanical performance of cementum- and dentine-reinforced teeth, and therefore to evaluate the biomechanical advantages of dentine regeneration after regenerative endodontics. We developted a finite element model of cementum- and dentinereinformed teeth and studied the stress distribution after the simulation of biting, trauma and orthodontic movement. The results showed that apposition of hard tissue (whether cementum or dentine) after REPs reduces mechanical stress on 17 immature teeth and, more important, that the formation of dentine is advantageous because it, unlike cementum, facilitates an even stress distribution throughout the root. As far as we know, ours was the first study showing the biomechanical advantages of dentine regeneration.Chapter 2: Odontoblasts are post-mitotic cells that secrete dentine. The isolation and culture of odontoblasts may open numerous possibilities to study this cell type under standardized conditions, shedding light on their roles in dentine formation, immune defence and transmission of external stimuli. We evaluated different protocols of enzymatic treatment to isolate primary odontoblasts from human molars. The results showed that, regardless of the enzymatic solution used, odontoblasts in culture did not remain viable after 24 h. Additionally, we identified increased expression of nestin (NE), bone sialoprotein (BSP) and dentine matrix acidic phosphoprotein 1 (DMP1) in the odontoblast layer compared to pulp fibroblasts. Though primary odontoblasts can still not be cultivated after isolation, characteristic genes were identified to differentiate odontoblasts from pulp fibroblasts.Chapter 3: We analysed the effects of autologous platelet concentrates (APCs) in the clinical and histological outcomes of the therapy and the different clinical protocols clinically used through systematic reviews. The results indicated that APCs improve the clinical and radiographic outcomes of regenerative endodontics since the teeth treated with APCs achieved significantly better thickening of the dentine walls and root lengthening. However, true regeneration of pulp was not achieved with the addition of platelet concentrates, which only stimulated tissue repair. Additionally, most of the studies did not follow a standard clinical protocol for regenerative endodontic therapy and used irritant and intracanal medicaments that are cytotoxic and affect the differentiation and adherence of the stem cells.Chapters 4 and 5: As will be mentioned in detail, a small apical foramen acts as a physical barrier that hinders tissue ingrowth into the root canal and therefore reduces the possibility of revitalization of mature teeth. We studied different methods for apical foramen enlargement of mature teeth as a basis to apply it in a further animal study. We analysed manual instrumentation at different working lengths and apicoectomy on extracted human teeth and in situ teeth. We concluded that apicoectomy is not an effective technique for apical foramen enlargement and therefore should not be used for that purpose. Instrumentation 18 0.5mm beyond the apex resulted in the most effective technique. Later, we performed an animal study and evaluated pulp tissue regeneration/repair in mature teeth and the differentiation of the stem cells from the periapical tissues into odontoblast-like cells by adding preameloblast-conditioned medium. Preameloblast-conditioned medium was applied in pulpectomized ferret canines, whose apical foramina were enlarged using the previously developed method. We observed vascularized connective tissue occupying the apical third of the canal space in 50% of the teeth, showing the potential of revascularization of mature teeth. However, no odontoblast-like cells were observed showing that in vivo odontoblast-like differentiation of stem cells is still not possible with the tested technique.Chapter 6: Finally, we present here the preliminary data of characterization and odontoblast-like differentiation of amnion epithelial cells. Human amnion epithelial cells (hAECs) express pluripotent stem cell markers and have been proven to differentiate in cells of the three embryologic layers. However, as far as we know, these are the first experiments that have proved the potential of odontoblast-like differentiation of these cells in vitro. To induce the odontoblast-like differentiation, we seeded hAECs over dentine disks treated with EDTA and evaluated the morphological characteristic of cells. We observed that hAECs present a characteristic odontoblast-like morphology, with cytoplasmic processes located in dentinal tubuli, after 48 h. Further studies will be carried out with known concentrations of dentine matrix proteins and qPCR." -- TDX.

This book provides a detailed update on our knowledge of dental pulp and regenerative approaches to therapy. It is divided into three parts. The pulp components are first described, covering pulp cells, extracellular matrix, vascularization and innervation as well as pulp development and aging. The second part is devoted to pulp pathology and includes descriptions of the differences between reactionary and reparative dentin, the genetic alterations leading to dentinogenesis imperfecta and dentin dysplasia, the pulp reaction to dental materials, adverse impacts of bisphenol A and the effects of fluorosis, dioxin and other toxic agents. The final part of the book focuses on pulp repair and regeneration. It includes descriptions of various in vitro and in vivo (animal) experimental approaches, definition of the pulp stem cells with special focus on the stem cell niches, discussion of the regeneration of a living pulp and information on new strategies that induce pulp mineralization.

Sequential and reciprocal interactions between oral epithelial and cranial neural crest-derived mesenchymal cells give rise to the teeth and periodontium. Teeth are vital organs containing a rich number of blood vessels and nerve fibers within the dental pulp and periodontium. Teeth are composed by unique and specific collagenous (dentin, fibrillar cementum) and non-collagenous (enamel) highly mineralized extracellular matrices. Alveolar bone is another collagenous hard tissue that supports tooth stability and function through its close interaction with the periodontal ligament. Dental hard tissues are often damaged after infection or traumatic injuries that lead to the partial or complete destruction of the functional dental and supportive tissues. Well-established protocols are routinely used in dental clinics for the restoration or replacement of the damaged tooth and alveolar bone areas. Recent progress in the fields of cell biology, tissue engineering, and nanotechnology offers promising opportunities to repair damaged or missing dental tissues. Indeed, pulp and periodontal tissue regeneration is progressing rapidly with the application of stem cells, biodegradable scaffolds, and growth factors. Furthermore, methods that enable partial dental hard tissue repair and regeneration are being evaluated with variable degrees of success. However, these cell-based therapies are still incipient and many issues need to be addressed before any clinical application. The understanding of tooth and periodontal tissues formation would be beneficial for improving regenerative attempts in dental clinics. In the present e-book we have covered the various aspects dealing with dental and periodontal tissues physiology and regeneration in 6 chapters: 1. General principles on the use of stem cells for regenerating craniofacial and dental tissues 2. The roles of nerves, vessels and stem cell niches in tissue regeneration 3. Dental pulp regeneration and mechanisms of various odontoblast functions 4. Dental root and periodontal physiology, pathology and regeneration 5. Physiology and regeneration of the bone using various scaffolds and stem cell populations 6. Physiology, pathology and regeneration of enamel using dental epithelial stem cells

The translation of dental pulp stem cells (DPSC) transplantation into clinic practice is hindered by the Good Manufacturing Practice (GMP) approval for in vitro autologous DPSC expansion. In order to circumvent the laboratory procedure, we proposed an unprecedented approach for regenerative endodontics - direct dental pulp tissue grafting. This study has demonstrated that cells directly migrating out from dental pulp tissue explantation (DPTE), functioning as the putative cell source in our approach for tissue regeneration, exhibits stem cell properties such as elongated lifespan as well as multi-potent differentiation in vitro as equivalent to DPSC. Additionally, our data from ex vivo tissue engineering with either porous biodegradable scaffold (poly-L-Lactic acid) or injectable self-assembly scaffold (PuraMatrix Hydrogel) suggests that dental pulp tissue fragments are survivable and proliferative in three-dimensional culture, generating stem cell population with odontogenic differentiation potential as well as mineralization capacity. Consequently, this preliminary investigation has established the fundamental rationale for the utilization of direct pulp tissue grafting in pulp-dentin complex engineering, charting out a blueprint for subsequent studies of a brand-new technique for regenerative endodontics.

Tissue Engineering and Regeneration in Dentistry: Current Strategies presents a thorough update on the current advances, methods and understanding in tissue engineering in dentistry. It offers invaluable tools, case studies, and methodologies for undertaking research, including important biological and practical considerations to facilitate successful migration of research from the bench to the clinic. Offers detailed coverage of the basic underlying principles and scientific evidence, and includes protocols to highlight practical applications Written by an internationally renowned team of expert contributors A must-have read for researchers and specialist clinicians in tissue engineering, oral biology, dental materials science, periodontology and oral surgery

This dissertation, "A Lab-centred Approach for Initiating Early Stage Regeneration of Dental Pulp Using Specialised Alginate Biomimetic Microenvironments" by Manasi, Bhoj, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: A living, self-supporting pulp tissue replacement for patients remains a considerable bioengineering challenge. As yet there is no method for engineering a self-sustaining living pulp tissue that can be successfully transplanted into the patient and have any beneficial clinical outcomes. The main reason for this failure is the inability to reconstitute a functional blood vessel tubular network within the engineered pulp tissue. In addition, it has yet not been possible to engineer the various anatomically distinct structures and architectures of the healthy pulp tissue. The idea is that a bio-engineered pulp-like structure prior to transplantation will accelerate integration and reduce the potential for complications. Recently new strides are being taken to make a functioning living pulp tissue using the latest step by step enhancements that are occurring in biomimetic molecular engineering. The use of this basic tissue engineering processes of living organic matter in fabrication is ensuring that prospective lab-made tissue products are biologically compatible. Systems built into biomaterials involving self-assembly, feedback mechanisms will lead to realistic tissue replicates. The new methods include, fabricating biomimetic tissue frameworks with an array of keystone biochemical cues and structural cues. Alternatively others are suggesting that it is feasible to stimulate natural repair using small molecules and temporary frameworks together or independently. A typical tissue engineering strategy that also promises to work is to combine mixed cell populations and growth factors in a scaffold microenvironment. The hypothesis is that recreating an early stage tissue microenvironment-one that resembles the postnatal constitution that will initiate the autonomous reconstruction of native pulp tissue in vitro. As a result, a coherent self-sustaining and self-supporting tissue construct is prepared for rapid integration and remodeling. Our purpose, in this study is to create the formative elements for pulp tissue that go on to develop and regenerate into a functional living pulp tissue. The objective were firstly, to combine dental mesenchymal stem cells and human umbilical vein endothelial cells with limited key growth factors within a space filling the aalginate hydrogel. Secondly, to test the capacity of these encapsulated stem cells for proliferationon, and lastly, measure the capture level for soluble bio-chemicals and test the capacity of the system to release growth factors in a slow, continuous fashion. These are critical performance issues for building pulp de novo. There are more to be tackled in later stages of developing this technology. An alginate-RGD conjugated gel was used to encapsulate adult dental pulp mesenchymal stem cells and human umbilical vein endothelial cells (HUVEC) in the ratio of 1:1. It also served as a means for the targeted delivery of two key growth factors 〖VEGF〗 DEGREES121 (Vascular endothelial growth factor) and FGF-2 (Fibroblastic growth factor) towards the population of cells. In the experiments we varied the combinations of these four components and results with SEM and confocal microscopy show uniform cell distribution and viable cells in all test and control groups. There were significant differences between the groups with no growth factors and groups in which growth factors were added. Interestingly, c

Novel injectable materials for non-invasive surgical procedures are becoming increasingly popular. An advantage of these materials include easy deliverability into the body, however the suitability of their mechanical properties must also be carefully considered. Injectable biomaterials covers the materials, properties and biomedical applications of injectable materials, as well as novel developments in the technology.Part one focuses on materials and properties, with chapters covering the design of injectable biomaterials as well as their rheological properties and the mechanical properties of injectable polymers and composites. Part two covers the clinical applications of injectable biomaterials, including chapters on drug delivery, tissue engineering and orthopaedic applications as well as injectable materials for gene delivery systems. In part three, existing and developing technologies are discussed. Chapters in this part cover such topics as environmentally responsive biomaterials, injectable nanotechnology, injectable biodegradable materials and biocompatibility. There are also chapters focusing on troubleshooting and potential future applications of injectable biomaterials.With its distinguished editor and international team of contributors, Injectable biomaterials is a standard reference for materials scientists and researchers working in the biomaterials industry, as well as those with an academic interest in the subject. It will also be beneficial to clinicians. Comprehensively examines the materials, properties and biomedical applications of injectable materials, as well as novel developments in the technology Reviews the design of injectable biomaterials as well as their rheological properties and the mechanical properties of injectable polymers and composites Explores clinical applications of injectable biomaterials, including drug delivery, tissue engineering, orthopaedic applications and injectable materials for gene delivery systems