METTL3 enhances dentinogenesis differentiation of dental pulp stem cells via increasing GDF6 and STC1 mRNA stability.

BACKGROUND: The dentinogenesis differentiation of dental pulp stem cells (DPSCs) is controlled by the spatio-temporal expression of differentiation related genes. RNA N6-methyladenosine (m6A) methylation, one of the most abundant internal epigenetic modification in mRNA, influences various events in RNA processing, stem cell pluripotency and differentiation. Methyltransferase like 3 (METTL3), one of the essential regulators, involves in the process of dentin formation and root development, while mechanism of METTL3-mediated RNA m6A methylation in DPSC dentinogenesis differentiation is still unclear. METHODS: Immunofluorescence staining and MeRIP-seq were performed to establish m6A modification profile in dentinogenesis differentiation. Lentivirus were used to knockdown or overexpression of METTL3. The dentinogenesis differentiation was analyzed by alkaline phosphatase, alizarin red staining and real time RT-PCR. RNA stability assay was determined by actinomycin D. A direct pulp capping model was established with rat molars to reveal the role of METTL3 in tertiary dentin formation. RESULTS: Dynamic characteristics of RNA m6A methylation in dentinogenesis differentiation were demonstrated by MeRIP-seq. Methyltransferases (METTL3 and METTL14) and demethylases (FTO and ALKBH5) were gradually up-regulated during dentinogenesis process. Methyltransferase METTL3 was selected for further study. Knockdown of METTL3 impaired the DPSCs dentinogenesis differentiation, and overexpression of METTL3 promoted the differentiation. METTL3-mediated m6A regulated the mRNA stabiliy of GDF6 and STC1. Furthermore, overexpression of METTL3 promoted tertiary dentin formation in direct pulp capping model. CONCLUSION: The modification of m6A showed dynamic characteristics during DPSCs dentinogenesis differentiation. METTL3-mediated m6A regulated in dentinogenesis differentiation through affecting the mRNA stability of GDF6 and STC1. METTL3 overexpression promoted tertiary dentin formation in vitro, suggesting its promising application in vital pulp therapy (VPT).

Epigenetics in developmental defects of enamel: A scoping review.

OBJECTIVES: The significant role of epigenetics has been revealed in normal enamel formation process and occurrence of developmental defects. This presented literature is aiming at summarizing the regulatory function of epigenetics in physiological amelogenesis process and reviewing the epigenetic mechanisms in occurrence of developmental defects of enamel (DDE), so as to provide biological foundation evidence to support early predication and clinical management of DDE. METHOD: An extensive literature review was conducted using electronic databases MEDLINE (through PubMed), Web of Science and EMBASE up to November 30, 2022. Studies about epigenetic effects on enamel tissue or cells associated with amelogenesis, including in vivo studies using human or animal models, and in vitro studies, are selected. RESULTS: A total of 22 studies were included. Epigenetic factors or effects specifically activate or silence certain genes, which may regulate related biological activities including cell proliferation, cell differentiation, enamel secretion, and mineralization during the process of amelogenesis. Once the status of epigenetic modification is altered, the quantity and quality of enamel may both be disturbed, which can finally result in DDE. CONCLUSION: Epigenetics plays a noteworthy role of regulating the amelogenesis process and DDE potentially by altering the expression levels of genes related to enamel formation, providing a new perspective of early predication and clinical management of DDE.

Metabolic-epigenetic nexus in regulation of stem cell fate.

Stem cell fate determination is one of the central questions in stem cell biology, and although its regulation has been studied at genomic and proteomic levels, a variety of biological activities in cells occur at the metabolic level. Metabolomics studies have established the metabolome during stem cell differentiation and have revealed the role of metabolites in stem cell fate determination. While metabolism is considered to play a biological regulatory role as an energy source, recent studies have suggested the nexus between metabolism and epigenetics because several metabolites function as cofactors and substrates in epigenetic mechanisms, including histone modification, DNA methylation, and microRNAs. Additionally, the epigenetic modification is sensitive to the dynamic metabolites and consequently leads to changes in transcription. The nexus between metabolism and epigenetics proposes a novel stem cell-based therapeutic strategy through manipulating metabolites. In the present review, we summarize the possible nexus between metabolic and epigenetic regulation in stem cell fate determination, and discuss the potential preventive and therapeutic strategies via targeting metabolites.

Epigenetic regulation of dental pulp stem cells and its potential in regenerative endodontics.

Regenerative endodontics (RE) therapy means physiologically replacing damaged pulp tissue and regaining functional dentin-pulp complex. Current clinical RE procedures recruit endogenous stem cells from the apical papilla, periodontal tissue, bone marrow and peripheral blood, with or without application of scaffolds and growth factors in the root canal space, resulting in cementum-like and bone-like tissue formation. Without the involvement of dental pulp stem cells (DPSCs), it is unlikely that functional pulp regeneration can be achieved, even though acceptable repair can be acquired. DPSCs, due to their specific odontogenic potential, high proliferation, neurovascular property, and easy accessibility, are considered as the most eligible cell source for dentin-pulp regeneration. The regenerative potential of DPSCs has been demonstrated by recent clinical progress. DPSC transplantation following pulpectomy has successfully reconstructed neurovascularized pulp that simulates the physiological structure of natural pulp. The self-renewal, proliferation, and odontogenic differentiation of DPSCs are under the control of a cascade of transcription factors. Over recent decades, epigenetic modulations implicating histone modifications, DNA methylation, and noncoding (nc)RNAs have manifested as a new layer of gene regulation. These modulations exhibit a profound effect on the cellular activities of DPSCs. In this review, we offer an overview about epigenetic regulation of the fate of DPSCs; in particular, on the proliferation, odontogenic differentiation, angiogenesis, and neurogenesis. We emphasize recent discoveries of epigenetic molecules that can alter DPSC status and promote pulp regeneration through manipulation over epigenetic profiles.

Roles of Dental Mesenchymal Stem Cells in the Management of Immature Necrotic Permanent Teeth.

Dental caries and trauma always lead to pulp necrosis and subsequent root development arrest of young permanent teeth. The traditional treatment, apexification, with the absence of further root formation, results in abnormal root morphology and compromises long-term prognosis. Regeneration endodontics procedures (REPs) have been developed and considered as an alternative strategy for management of immature permanent teeth with pulpal necrosis, including cell-free and cell-based REPs. Cell-free REPs, including revascularization and cell homing with molecules recruiting endogenous mesenchymal stem cells (MSCs), have been widely applied in clinical treatment, showing optimistic periapical lesion healing and continued root development. However, the regenerated pulp-dentin complex is still absent in these cases. Dental MSCs, as one of the essentials of tissue engineering, are vital seed cells in regenerative medicine. Dental MSC-based REPs have presented promising potential with pulp-dentin regeneration in large animal studies and clinical trials via cell transplantation. In the present review, we summarize current understanding of the biological basis of clinical treatments for immature necrotic permanent teeth and the roles of dental MSCs during this process and update the progress of MSC-based REPs in the administration of immature necrotic permanent teeth.

New insight into dental epithelial stem cells: Identification, regulation, and function in tooth homeostasis and repair.

Tooth enamel, a highly mineralized tissue covering the outermost area of teeth, is always damaged by dental caries or trauma. Tooth enamel rarely repairs or renews itself, due to the loss of ameloblasts and dental epithelial stem cells (DESCs) once the tooth erupts. Unlike human teeth, mouse incisors grow continuously due to the presence of DESCs that generate enamel-producing ameloblasts and other supporting dental epithelial lineages. The ready accessibility of mouse DESCs and wide availability of related transgenic mouse lines make mouse incisors an excellent model to examine the identity and heterogeneity of dental epithelial stem/progenitor cells; explore the regulatory mechanisms underlying enamel formation; and help answer the open question regarding the therapeutic development of enamel engineering. In the present review, we update the current understanding about the identification of DESCs in mouse incisors and summarize the regulatory mechanisms of enamel formation driven by DESCs. The roles of DESCs during homeostasis and repair are also discussed, which should improve our knowledge regarding enamel tissue engineering.

Dental Tissue-Derived Human Mesenchymal Stem Cells and Their Potential in Therapeutic Application.

Human mesenchymal stem cells (hMSCs) are multipotent cells, which exhibit plastic adherence, express specific cell surface marker spectrum, and have multi-lineage differentiation potential. These cells can be obtained from multiple tissues. Dental tissue-derived hMSCs (dental MSCs) possess the ability to give rise to mesodermal lineage (osteocytes, adipocytes, and chondrocytes), ectodermal lineage (neurocytes), and endodermal lineages (hepatocytes). Dental MSCs were first isolated from dental pulp of the extracted third molar and till now they have been purified from various dental tissues, including pulp tissue of permanent teeth and exfoliated deciduous teeth, apical papilla, periodontal ligament, gingiva, dental follicle, tooth germ, and alveolar bone. Dental MSCs are not only easily accessible but are also expandable in vitro with relative genomic stability for a long period of time. Moreover, dental MSCs have exhibited immunomodulatory properties by secreting cytokines. Easy accessibility, multi-lineage differentiation potential, and immunomodulatory effects make dental MSCs distinct from the other hMSCs and an effective tool in stem cell-based therapy. Several preclinical studies and clinical trials have been performed using dental MSCs in the treatment of multiple ailments, ranging from dental diseases to nondental diseases. The present review has summarized dental MSC sources, multi-lineage differentiation capacities, immunomodulatory features, its potential in the treatment of diseases, and its application in both preclinical studies and clinical trials. The regenerative therapeutic strategies in dental medicine have also been discussed.

Epiregulin enhances odontoblastic differentiation of dental pulp stem cells via activating MAPK signalling pathway.

OBJECTIVES: The odontoblastic differentiation of dental pulp stem cells (DPSCs) contributes to tertiary dentin formation. Our previous study indicated that epiregulin (EREG) enhanced odontogenesis potential of dental pulp. Here, we explored the effects of EREG during DPSC odontoblastic differentiation. METHODS: The changes in EREG were detected during tertiary dentin formation. DPSCs were treated with recombinant human EREG (rhEREG), EREG receptor inhibitor gefitinib and short hairpin RNAs. The odontoblastic differentiation was assessed with ALP staining, ALP activity assay, alizarin red S staining and real-time RT-PCR of DSPP, OCN, RUNX2 and OSX. Western blot was conducted to examine the levels of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2). The expression of EREG and odontoblastic differentiation-related markers was investigated in human dental pulp from teeth with deep caries and healthy teeth. RESULTS: Epiregulin was upregulated during tertiary dentin formation. rhEREG enhanced the odontoblastic differentiation of DPSCs following upregulated p38 MAPK and Erk1/2 phosphorylation, but not JNK, whereas depletion of EREG suppressed DPSC differentiation. Gefitinib decreased odontoblastic differentiation with decreased phosphorylation of p38 MAPK and Erk1/2. And suppression of p38 MAPK and Erk1/2 pathways attenuated DPSC differentiation. In human dental pulp tissue, EREG upregulation in deep caries correlates with odontoblastic differentiation enhancement. CONCLUSION: Epiregulin is released during tertiary dentin formation. And EREG enhanced DPSC odontoblastic differentiation via MAPK pathways.

The Origin and Identification of Mesenchymal Stem Cells in Teeth: from Odontogenic to Non-odontogenic.

BACKGROUND: Mesenchymal stem cells (MSCs) in teeth have been exploited as vital seed cells for stem cell-based dental medicine. To date, several mesenchymal stem cell populations originated from odontogenic tissue have been isolated and characterized by their expression of MSC surface markers and capacity of multi-lineage differentiation, including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP) and so on. However, their identity in vivo remains elusive, which hinders further understanding of their application in stem cell-based tooth regeneration. Label retaining and lineage tracing analyses, which serve as gold standards for identification of stem cells in vivo, provide feasibility for identifying MSCs in teeth. OBJECTIVES: In this review, we will discuss the issues of MSCs, including the origin and identification of both odontogenic and non-odontogenic MSCs, and address the role of nerve-derived Sonic hedgehog (Shh) in the regulation of MSCs in the neurovascular bundle (NVB). CONCLUSION: Based on label retaining and lineage tracing analyses, latest studies have found new populations of non-odontogenic MSCs in teeth, periarterial-derived and glial-derived, regulated by the Shh derived from nerves in the NVB, which provides a new hope for tooth regeneration.

Epigenetic Regulation of Gene Expression in Epithelial Stem Cells Fate.

BACKGROUND: Epithelial tissues have the ability to self-renew throughout animal's life due to the presence of the epithelial stem cells. Except for complicated genes regulation, the fate of epithelial stem cells is also regulated by the epigenetics, including DNA methylation, histone modification and microRNAs, which are emerging as vital elements of epigenetic regulation for epithelial stem cells self-renewal and differentiation. However, the mechanisms underlying these are still poorly understood. OBJECTIVE: In this review, we focus on the epigenetic regulation of gene expression in epithelial stem cells fate, using intestinal and epidermal stem cells as models. Meanwhile, a brief description of recent research about the possible impact of network regulation in epithelial stem cell-based amelogenesis by epigenetic regulation is therefore, being discussed. CONCLUSION: Epigenetic modification plays a vital role in the epithelial stem cells fate choice through the gene expression. The interaction between epigenetic modification and molecular signaling in epithelial stem cells fate choice still needs further exploration.

Mesenchymal Stem Cells for Cartilage Regeneration of TMJ Osteoarthritis.

Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease, characterized by progressive cartilage degradation, subchondral bone remodeling, synovitis, and chronic pain. Due to the limited self-healing capacity in condylar cartilage, traditional clinical treatments have limited symptom-modifying and structure-modifying effects to restore impaired cartilage as well as other TMJ tissues. In recent years, stem cell-based therapy has raised much attention as an alternative approach towards tissue repair and regeneration. Mesenchymal stem cells (MSCs), derived from the bone marrow, synovium, and even umbilical cord, play a role as seed cells for the cartilage regeneration of TMJ OA. MSCs possess multilineage differentiation potential, including chondrogenic differentiation as well as osteogenic differentiation. In addition, the trophic modulations of MSCs exert anti-inflammatory and immunomodulatory effects under aberrant conditions. Furthermore, MSCs combined with appropriate scaffolds can form cartilaginous or even osseous compartments to repair damaged tissue and impaired function of TMJ. In this review, we will briefly discuss the pathogenesis of cartilage degeneration in TMJ OA and emphasize the potential sources of MSCs and novel approaches for the cartilage regeneration of TMJ OA, particularly focusing on the MSC-based therapy and tissue engineering.

Symptoms and signs of temporomandibular disorders in patients with knee osteoarthritis.

OBJECTIVES: Both temporomandibular disorder (TMD) and knee osteoarthritis (KOA) are prevalent joint diseases; however, an association between them has not been reported. Therefore, this study investigated the prevalence of the specific symptoms and signs of TMDs (SSTs) in patients with KOA. METHODS: In total, 200 patients with KOA and 150 healthy individuals were recruited. The prevalence of specific SSTs in patients with mild or severe KOA was compared with the prevalence of specific SSTs in the control group and the results were analysed using a chi-square test. Logistic regression was used to adjust for potential confounders, such as gender and age. RESULTS: The prevalence of 'impaired range of jaw movement (IRM)' was 63.6% (n = 77) in the mild KOA group and 62.4% (n = 117) in the severe KOA group; the values for both KOA groups were significantly higher than that for the non-OA control group (34.7%, n = 144; P < 0.017). In addition, 54.7% of the patients with severe KOA reported 'impaired temporomandibular joint (TMJ) function', a value significantly higher than that of the control group (39.6%, P < 0.017). No significant differences between groups were found for other SSTs. CONCLUSIONS: Patients with KOA might be more likely to experience SSTs, such as IRM and impaired TMJ function.

Phenotypic research on senile osteoporosis caused by SIRT6 deficiency.

Osteoporosis is a serious public bone metabolic disease. However, the mechanisms underlying bone loss combined with ageing, which is known as senile osteoporosis, remains unknown. Here we show the detailed phenotype of this disease caused by SIRT6 knock out (KO) in mice. To the best of our knowledge, this is the first study to reveal that SIRT6 is expressed in both bone marrow stroma cells and bone-related cells in both mouse and human models, which suggests that SIRT6 is an important regulator in bone metabolism. SIRT6-KO mice exhibit a significant decrease in body weight and remarkable dwarfism. The skeleton of the SIRT6-KO mouse is deficient in cartilage and mineralized bone tissue. Moreover, the osteocalcin concentration in blood is lower, which suggests that bone mass is markedly lost. Besides, the tartrate-resistant acid phosphatase 5b (TRAP5b) concentration is much higher, which suggests that bone resorption is overactive. Both trabecular and cortical bones exhibit severe osteopenia, and the bone mineral density is decreased. Moreover, double-labelling analysis shows that bone formation is much slower. To determine whether SIRT6 directly regulates bone metabolism, we cultured primary bone marrow stromal cells for osteogenesis and osteoclastogenesis separately to avoid indirect interference in vivo responses such as inflammation. Taken together, these results show that SIRT6 can directly regulate osteoblast proliferation and differentiation, resulting in attenuation in mineralization. Furthermore, SIRT6 can directly regulate osteoclast differentiation and results in a higher number of small osteoclasts, which may be related to overactive bone resorption.