The literature on "dental stem cells" from 2002 to 2009 is composed of 936 works, mainly basic research articles, 159 reviews, 1 clinical study and only 6 case reports concerning endodontics and periodontology. After the year 2009 (1) the interest on this research topic has increased leading to 5177 new articles, including 757 reviews on different stem cell types, biomaterials and the possibility of "banking". The 46 clinical trials published in the last 9 years are clinical applications in periodontics and endodontics. However, still no definitive clinical guidelines are available (2,3). Regenerative therapy in endodontics, through the use of dental stem cells, is still an open question (4). The idea of healing the pulp instead of treating it by means of a conventional endodontic therapy is fascinating, but the current experimental protocol is limited to immature permanent teeth with pulp necrosis, which are a minority of endodontic treatments. Since 2005, with the first stem cell bank (Hiroshima University "Three Brackets") (5), attention has also been paid to the creation of private or university centres where it is possible to preserve autologous dental stem cells. In particular, due to the ease and natural availability, the interest is directed to SHED, Stem cells from Human Exfoliated Deciduous teeth (6). SHED are easy to collect, have excellent differentiation potential and, most importantly, are more cost-effective if compared with umbilical cord cells (5). For these reasons in 2008 the Norwegian Institute of Public Health and the University of Bergen started to collect and store the exfoliated teeth of 100,000 children. To date there are several collection centres in the northern emisphere: USA (Bioeden, Stem Save, Store a Tooth), Europe (Bergen, Future Health), India (Stemade biotech), Japan (Teeth Bank, Advanced Center of Tissue Engineering, Hiroshima University and Nogoya University), Taipai (Taipai Medical University), and recently China (National Dental Stem Cells Bank) (7). Dental stem cells are readily and easily available and are a promising resource not just in dentistry but for regenerative medicine in general (8). This is confirmed by the literature, since 25% of papers on stem cells involve the study and dissemination of research on dental tissues-derived stem cells. Currently there are only a few registered clinical trials for stem cell applications in dentistry and the results are still unavailable. To date, there are no dental treatments involving harvested stem cells but this is definitely an emerging science that might lead to important outcomes in the future.

Stem cells transplantation is the main method of tissue engineering regeneration treatment, the viability and therapeutic efficiency are limited. Scaffold materials also play an important role in tissue engineering, whereas there are still many limitations, such as rejection and toxic side effects caused by scaffold materials. Cell sheet engineering is a scaffold-free tissue technology, which avoids the side effects of traditional scaffolds and maximizes the function of stem cells. It is increasingly being used in the field of tissue regenerative medicine. Dental-derived mesenchymal stem cells (DMSCs) are multipotent cells that exist in various dental tissues and can be used in stem cell-based therapy, which is impactful in regenerative medicine. Emerging evidences show that cell sheets derived from DMSCs have better effects in the field of regenerative medicine applications. Extracellular matrix (ECM) is the main component of cell sheets, which is a dynamic repository of signalling biological molecules and has a variety of biological functions and may play an important role in the application of cell sheets. In this review, we summarized the application status, mechanisms that sheets and ECM may play and future prospect of DMSC sheets on regeneration medicine.

Dental tissue-derived stem cells (DSCs) provide an easy, accessible, relatively noninvasive promising source of adult stem cells (ASCs), which brought encouraging prospective for their clinical applications. DSCs provide a perfect opportunity to apply for a patient's own ASC, which poses a low risk of immune rejection. However, problems associated with the long-term culture of stem cells, including loss of proliferation and differentiation capacities, senescence, genetic instability, and the possibility of microbial contamination, make cell banking necessary. With the rapid development of advanced cryopreservation technology, various international DSC banks have been established for both research and clinical applications around the world. However, few studies have been published that provide step-by-step guidance on DSCs isolation and banking methods. The purpose of this review is to present protocols and technical details for all steps of cryopreserved DSCs, from donor selection, isolation, cryopreservation, to characterization and quality control. Here, the emphasis is on presenting practical principles in accordance with the available valid guidelines.

Currently regeneration of tooth and periodontal damage still remains great challenge. Stem cell-based tissue engineering raised novel therapeutic strategies for tooth and periodontal repair. Stem cells for tooth and periodontal regeneration include dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), stem cells from the dental apical papilla (SCAPs), and stem cells from human exfoliated deciduous teeth (SHEDs), dental follicle stem cells (DFSCs), dental epithelial stem cells (DESCs), bone marrow mesenchymal stem cells (BMMSCs), adipose-derived stem cells (ADSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). To date, substantial advances have been made in stem cell-based tooth and periodontal regeneration, including dentin-pulp, whole tooth, bioroot and periodontal regeneration. Translational investigations have been performed such as dental stem cell banking and clinical trials. In this review, we present strategies for stem cell-based tissue engineering for tooth and periodontal repair, and the translational studies.

Tooth defects are an extremely common health condition that affects millions of individuals. Currently used dental repair treatments include fillings for caries, endodontic treatment for pulp necrosis, and dental implants to replace missing teeth, all of which rely on the use of synthetic materials. By contrast, the fields of tissue engineering and regenerative medicine and dentistry (TERMD) use biologically based therapeutic strategies for vital tissue regeneration, and thus have the potential to regenerate living tissues. Methods to create bioengineered replacement teeth benefit from a detailed understanding of the molecular signaling networks regulating natural tooth development. We discuss how key signaling pathways regulating natural tooth development are being exploited for applications in TERMD approaches for vital tooth regeneration.

The teeth are highly differentiated chewing organs formed by the development of tooth germ tissue located in the jaw and consist of the enamel, dentin, cementum, pulp, and periodontal tissue. Moreover, the teeth have a complicated regulatory mechanism, special histologic origin, diverse structure, and important function in mastication, articulation, and aesthetics. These characteristics, to a certain extent, greatly complicate the research in tooth regeneration. Recently, new ideas for tooth and tissue regeneration have begun to appear with rapid developments in the theories and technologies in tissue engineering. Numerous types of stem cells have been isolated from dental tissue, such as dental pulp stem cells (DPSCs), stem cells isolated from human pulp of exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), stem cells from apical papilla (SCAPs), and dental follicle cells (DFCs). All these cells can regenerate the tissue of tooth. This review outlines the cell types and strategies of stem cell therapy applied in tooth regeneration, in order to provide theoretical basis for clinical treatments.

BACKGROUND:Unlike many vertebrates with continuous dental replacement, mammals have a maximum of two dental generations. Due to the absence of dental replacement in the laboratory mouse, the mechanisms of the mammalian tooth replacement system are poorly known. In this study, we use the European rabbit as a model for mammalian tooth development and replacement. RESULTS:We provide data on some key regulators of tooth development. We detected the presence of SOX2 in both the replacement dental lamina and the rudimentary successional dental lamina of unreplaced molars, indicating that SOX2 may not be sufficient to initiate and maintain tooth replacement. We showed that Shh does not seem to be directly involved in tooth replacement. The transient presence of the rudimentary successional dental lamina in the molar allowed us to identify genes that could be essential for the initiation or the maintenance of tooth replacement. Hence, the locations of Sostdc1, RUNX2, and LEF1 vary between the deciduous premolar, the replacement premolar, and the molar, indicating possible roles in tooth replacement. CONCLUSION:According to our observations, initiation and the maintenance of tooth replacement correlate with the presence of LEF1 cells and the absence of both mesenchymal RUNX2 and epithelial Sostdc1 cells.

Cyclical renewal of integumentary organs, including hair, feathers, and teeth occurs throughout an organism's lifetime. Transition from the resting to the initiation stage is critical for each cycle, but the mechanism remains largely unknown. Humans have two sets of dentitions-deciduous and permanent-and tooth replacement occurs only once. Prior to eruption of the permanent tooth (PT), the successional dental lamina (SDL) of the PT can be detected as early as the embryonic stage, even though it then takes about 6-12 years for the SDL to develop to late bell stage. Little is known about the mechanism by which resting SDL transitions into the initiation stage inside the mandible. As a large mammal, the miniature pig, which is also a diphyodont, was a suitable model for our recent study (EMBO J (2020)39: e102374). Using this model, we found that the SDL of PT did not begin the transition into the bud stage until the deciduous tooth (DT) began to erupt.

Although the dental lamina of permanent teeth in human being has been developed as early as the embryo stage, the replacement of the deciduous teeth by permanent teeth does not take place untill the age of 6 to 12 years old. The molecular mechanism of the initiation of permanent teeth is still unclear. The rodent species are usually used for the tooth development research in the past. However, this animal model is not suitable for the tooth replacement study because of the absence of tooth replacement in rodents. After 10 years of efforts, our team has established the animal model of miniature pig for tooth replacement research. Using this model, we firstly defined the spatiotemporal pattern of teeth replacement. In the further mechanism research, results showed that the growing rate of the deciduous teeth was faster than that of the surrounding alveolar bone, and biomechanical stress inside mandible was generated due to the fast growth of deciduous teeth. The stress might up-regulate the signal of Runt-related transcription factor 2 (RUNX2)-Wnt pathway in the mesenchyme between the deciduous and permanent teeth, sustain the successional dental lamina at the resting stage and inhibit the development of permanent teeth. A similar expression pattern was also found in the mesenchyme between the deciduous and permanent teeth in human. Our findings demonstrated that the eruption of deciduous tooth released the stress inside mandible, thus induced the "Wnt translocation" from the mesenchyme into the epithelium of permanent counterpart and therefore initiated the development of permanent teeth. The underlying mechanism of the replacement of deciduous teeth by permanent teeth is the regulation of biomechanical stress throughout the initiation process. Based on the findings, we proposed the theory of "biomechanical stress regulation of the tooth replacement" . The replacement pattern and regulatory mechanism provide a scientific foundation for the organ development and regeneration by regulating the biomechanical stress and Wnt pathway in the future.