Tooth eruption is a localised event whereby the signals for eruption for a given tooth are synthesised in the dental follicle of that tooth with a possible cross talk of signals coming from the adjacent stellate reticulum. The eruption process requires alveolar bone resorption that is primarily regulated by the dental follicle. This is reflected by the fact that failures of eruption often can be traced to either osteoclast deficiencies or to dental follicle abnormalities. Recent advances in application of molecular techniques to animal models allowed for better understanding of gene regulatory events involved in the physiology of tooth eruption. This article attempts to consolidate and organise the facts that offshoot from animal studies.
Tooth Eruption ; Dental Sac ; Molecular Biology

OBJECTIVES: To summarize the open-eruption technique of impacted anterior maxillary teeth, this study reports a technically improved operation on surgical exposure based on dental follicles and evaluates post-treatment periodontal health considering the effect of dental follicles. METHODS: Patients who underwent open-eruption technique with unilateral labially impacted maxillary central incisors were selected. The impacted teeth were assigned to the experimental group, and the contralateral unimpacted maxillary central incisors were assigned to the control group. In the surgical exposure, the new technique makes use of dental follicles to manage the soft tissue, so as to preserve soft tissue for better aesthetic results and healthier periodontal tissue. Tooth length, root length, alveolar bone loss, and alveolar bone thickness were recorded after the therapy. RESULTS: A total of 17 patients with unilateral maxillary central incisor impaction were successfully treated. The tooth length and root length of the two groups showed a statistically significant difference between the impacted and homonym teeth, with a shorter length in the impacted tooth (P<0.05). More labial alveolar bone loss was found in the experimental group compared with that in the control group (P<0.05). The outcomes of the cementoenamel junction width, pa- latal alveolar bone loss, and alveolar bone thickness did not indicate statistical significance between the experimental and control groups (P>0.05). CONCLUSIONS In the surgical exposure, the new technique uses dental follicles to manage the soft tissue and preserve it for better aesthetic results and healthier periodontal tissues.
Humans ; Tooth, Impacted/surgery* ; Incisor ; Alveolar Bone Loss/diagnostic imaging* ; Tooth Root ; Dental Sac ; Maxilla/surgery* ; Esthetics, Dental

A splicing mutation in VPS4B can cause dentin dysplasia type I (DD-I), a hereditary autosomal-dominant disorder characterized by rootless teeth, the etiology of which is genetically heterogeneous. In our study, dental follicle cells (DFCs) were isolated and cultured from a patient with DD-I and compared with those from an age-matched, healthy control. In a previous study, this DD-I patient was confirmed to have a loss-of-function splicing mutation in VPS4B (IVS7 + 46C > G). The results from this study showed that the isolated DFCs were vimentin-positive and CK14-negative, indicating that the isolated cells were derived from the mesenchyme. DFCs harboring the VPS4B mutation had a significantly higher proliferation rate from day 3 to day 8 than control DFCs, indicating that VPS4B is involved in cell proliferation. The cells were then replenished with osteogenic medium to investigate how the VPS4B mutation affected osteogenic differentiation. Induction of osteogenesis, detected by alizarin red and alkaline phosphatase staining in vitro, was decreased in the DFCs from the DD-I patient compared to the control DFCs. Furthermore, we also found that the VPS4B mutation in the DD-I patient downregulated the expression of osteoblast-related genes, such as ALP, BSP, OCN, RUNX2, and their encoded proteins. These outcomes confirmed that the DD-I-associated VPS4B mutation could decrease the capacity of DFCs to differentiate during the mineralization process and may also impair physiological root formation and bone remodeling. This might provide valuable insights and implications for exploring the pathological mechanisms underlying DD-I root development.
ATPases Associated with Diverse Cellular Activities ; genetics ; Case-Control Studies ; Cell Differentiation ; genetics ; Cells, Cultured ; Dental Sac ; cytology ; Dentin Dysplasia ; genetics ; pathology ; physiopathology ; Endosomal Sorting Complexes Required for Transport ; genetics ; Humans ; Mutation ; genetics ; Osteogenesis ; genetics ; RNA Splicing ; genetics

As a member of the AFF (AF4/FMR2) family, AFF4 is a transcription elongation factor that is a component of the super elongation complex. AFF4 serves as a scaffolding protein that connects transcription factors and promotes gene transcription through elongation and chromatin remodelling. Here, we investigated the effect of AFF4 on human dental follicle cells (DFCs) in osteogenic differentiation. In this study, we found that small interfering RNA-mediated depletion of AFF4 resulted in decreased alkaline phosphatase (ALP) activity and impaired mineralization. In addition, the expression of osteogenic-related genes (DLX5, SP7, RUNX2 and BGLAP) was significantly downregulated. In contrast, lentivirus-mediated overexpression of AFF4 significantly enhanced the osteogenic potential of human DFCs. Mechanistically, we found that both the mRNA and protein levels of ALKBH1, a critical regulator of epigenetics, changed in accordance with AFF4 expression levels. Overexpression of ALKBH1 in AFF4-depleted DFCs partially rescued the impairment of osteogenic differentiation. Our data indicated that AFF4 promoted the osteogenic differentiation of DFCs by upregulating the transcription of ALKBH1.
Biomarkers ; metabolism ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; drug effects ; metabolism ; Gene Expression Regulation ; Humans ; Osteogenesis ; genetics ; Repressor Proteins ; Transcription Factors ; genetics ; metabolism ; Transcriptional Elongation Factors ; metabolism

BACKGROUND: Enhancement and maintenance of the stemness of mesenchymal stem cells (MSCs) is one of the most important factors contributing to the successful in vivo therapeutic application of these cells. In this regard, three-dimensional (3D) spheroid formation has been developed as reliable method for increasing the pluripotency of MSCs. Moreover, using a new protocol, we have previously shown that dental tissues of extracted wisdom teeth can be effectively cryopreserved for subsequent use as a source of autologous stem cells. The main purpose of this study is to analyze the stemness and in vitro osteogenic differentiation potential of 3D spheroid dental MSCs compared with conventional monolayer cultured MSCs. METHODS: In this study, MSC-characterized stem cells were isolated and cultured from long-term cryopreserved dental follicles (hDFSCs), and then 2D hDFSCs were cultured under 3D spheroid-forming conditions using a newly designed microchip dish. The spheroids (3D hDFSCs) thus produced were investigated and characterized with respect to stemness, MSC marker expression, apoptosis, cell cycle analysis, extracellular matrix (ECM) production, and osteogenic and adipogenic differentiation properties. RESULTS: In terms of MSC and senescence markers, spheroid cells showed no difference when compared with 2D hDFSCs; however, 3D hDFSCs were observed to have a higher proportion of cell cycle arrest and a larger number of apoptotic cells. Moreover, spheroids showed substantially increased levels of pluripotency marker (early transcription factors) and ECM protein expression. Compared with 2D hDFSCs, there was also a notable enhancement in the osteogenic induction potential of spheroids, although no differences were observed with respect to in vitro adipogenesis. CONCLUSION: To the best of our knowledge, this is the first study to demonstrate the application of a spheroid culture system for dental follicle-derived stem cells using a microchip dish. Although further studies are needed, including in vivo transplantation, the results obtained in this study indicate that spheroid hDFSCs derived from cryopreserved dental follicle tissues could be used as a valuable source of autologous stem cells for bone tissue regeneration.
Adipogenesis ; Aging ; Apoptosis ; Bone and Bones ; Cell Cycle ; Cell Cycle Checkpoints ; Dental Sac ; Extracellular Matrix ; Humans ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Methods ; Molar, Third ; Osteogenesis ; Regeneration ; Stem Cells

Bone formation is important for the reconstruction of bone-related structures in areas that have been damaged by inflammation. Inflammatory conditions such as those that occur in patients with rheumatoid arthritis, cystic fibrosis, and periodontitis have been shown to inhibit osteoblastic differentiation. This study focussed on dental follicle stem cells (DFSCs), which are found in developing tooth germ and participate in the reconstruction of alveolar bone and periodontal tissue in periodontal disease. After bacterial infection of inflamed dental tissue, the destruction of bone was observed. Currently, little is known about the relationship between the inflammatory environment and bone formation. Osteogenic differentiation of inflamed DFSCs resulted in decreased alkaline phosphatase (ALP) activity and alizarin red S staining compared to normal DFSCs. Additionally, in vivo transplantation of inflamed and normal DFSCs demonstrated severe impairment of osteogenesis by inflamed DFSCs. Protein profile analysis via liquid chromatography coupled with tandem mass spectrometry was performed to analyse the differences in protein expression in inflamed and normal tissue. Comparison of inflamed and normal DFSCs showed significant changes in the level of expression of transforming growth factor (TGF)-β2. Porphyromonas gingivalis (P.g.)-derived lipopolysaccharide (LPS) was used to create in vitro inflammatory conditions similar to periodontitis. The osteogenic differentiation of LPS-treated DFSCs was suppressed, and the cells displayed low levels of TGF-β1 and high levels of TGF-β2. DFSCs treated with TGF-β2 inhibitors showed significant increases in alizarin red S staining and ALP activity. TGF-β1 expression was also increased after inhibition of TGF-β2. By examining inflamed DFSCs and LPS-triggered DFSCs, these studies showed both clinically and experimentally that the increase in TGF-β2 levels that occurs under inflammatory conditions inhibits bone formation.
Adolescent ; Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Dental Sac ; cytology ; metabolism ; Down-Regulation ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunohistochemistry ; Male ; Mass Spectrometry ; Mice ; Nitric Oxide ; metabolism ; Osteogenesis ; drug effects ; Polymerase Chain Reaction ; Staining and Labeling ; Stem Cells ; cytology ; metabolism ; Transforming Growth Factor beta2 ; pharmacology ; Young Adult

OBJECTIVE: This study aims to investigate the effect of neurotrophin 3 (NT-3) on the osteogenic differentiation of human dental follicle cells (hDFCs). METHODS: hDFCs were isolated and cultured in vitro. Immunocytochemical staining was used to identify the origin of hDFCs. The effects of different NT-3 concentrations on hDFCs proliferation were detected by using CCK-8 assay. The alkaline phosphatase (ALP) activities and mRNA expression levels of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) were determined to investigate the effects of NT-3 on hDFCs osteogenesis. The difference in the number of mineralized nodules was detected using alizarin red staining. RESULTS: Vimentin and cytokeratin staining results showed that hDFCs originated from the mesenchymal cells. NT-3 exerted no evident effect on hDFCs proliferation. The ALP activity and the BMP-2 and OCN mRNA expression levels of hDFCs were significantly improved under treatment with different NT-3 concentrations (25, 50, and 100 ng·mL ⁻¹) compared with those in the control group. BMP-2 and OCN mRNA relative expression levels of hDFCs reached the highest when the NT-3 concentration was 100 ng·mL ⁻¹. The number of mineralized nodules reached the maximum when the hDFCs were treated with 50 and 100 ng·mL ⁻¹ NT-3. CONCLUSIONS Appropriate mass concentration of NT-3 can promote the osteogenic differentiation of hDFCs.
Alkaline Phosphatase ; Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; Humans ; Mesenchymal Stem Cells ; Neurotrophin 3 ; pharmacology ; Osteocalcin ; metabolism ; Osteogenesis

Root resorption of the permanent maxillary incisors can occur due to ectopic eruption of the permanent canines. Severe root resorption threatens the long-term survival of the affected incisors. The aim of the present study was to identify risk factors for root resorption of the maxillary incisors associated with impacted maxillary canines. In the present study, we retrospectively analyzed the Cone-Beam Computed Tomography (CBCT) scans of 65 children and adolescents with ectopically erupting maxillary canines (total of 88 impacted canines). Root resorption of central incisors was significantly associated with the mesiodistal position and root development of the adjacent canine. Root resorption of lateral incisors was significantly associated with sex, age, and the buccolingual and vertical position of the adjacent canine. However, enlargement of the dental follicle was not significantly associated with root resorption of adjacent incisors. Although incisor resorption is difficult to diagnose and predict, our findings suggest that changes in the dental follicles of the erupting maxillary canines do not cause resorption of the adjacent permanent incisors. CBCT should be utilized to ensure early diagnosis of impacted canines and precise evaluation of incisor root resorption.
Adolescent ; Child ; Cone-Beam Computed Tomography ; Dental Sac ; Early Diagnosis ; Humans ; Incisor ; Retrospective Studies ; Risk Factors ; Root Resorption ; Tooth, Impacted

OBJECTIVEThis study aimed to investigate the effects of hypoxia on the characteristics of human dental follicle cells (hDFCs).

METHODSThe tissue explant collagenase method was used to isolate hDFCs from young permanent teeth. The immunofluorescence technique was used to detect cell surface markers, and the multi-differentiation potential was detected by multilineage differentiation induction assay. Then, the hypoxic microenvironment was physically mimicked, and the cells were divided into the normoxia group (20%O₂) and the hypoxia group (2%O₂). The effects of hypoxia on cell migration and proliferation were examined by Transwell chamber test and CCK-8 assay, respectively. The gene and protein expression levels of stemness-related markers at both oxygen concentrations were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. After osteogenic induction of both groups, qRT-PCR was performed to evaluate the osteogenesis-related gene, and alizarin red staining was used to assess the formation of mineralized nodules.

RESULTSWith the multi-differentiation capacity of osteogenic cells, adipogenic cells, and nerves, hDFCs demonstrate strong stem cell characteristics and possess the criteria of mesenchymal stem cells, which can meet the requirements of seed cells in dental tissue engineering. Hypoxia was conducive to the maintenance of hDFC stemness. Hypoxia promoted the migration and proliferation of hDFCs. The hDFCs were induced to osteogenic differentiation under hypoxic conditions, thereby enhancing osteogenesis.

CONCLUSIONSHypoxic microenvironment plays an important role in maintaining the stemness and promoting the proliferation, migration, and differentiation of hDFCs. Thus, this microenvironment could also serve several important functions in future clinical applications.

Cell Differentiation ; Cell Hypoxia ; Cell Movement ; Dental Sac ; Humans ; Mesenchymal Stromal Cells ; Osteogenesis ; Real-Time Polymerase Chain Reaction ; Stem Cells

OBJECTIVETo observe the expression of wingless-type MMTV integration site family, member 5A (Wnt5A)/receptor tyrosine kinase-like orphan receptor 2 (Ror2) signal in the dental follicle cells during the normal eruption of the teeth as well as to explore the relationship between the expression of dental follicle cells and the formation of mature osteoclasts and eruption of the teeth.

METHODSThe mandibulars of 1-13 d old SD rats were separated to observe the growth and develop-ment of the teeth and alveolar bone through hematoxylin-eosin(HE) staining. Ror2 and Wnt5A expressions in rat dental follicle were also observed through immunohistochemistry. Dental follicle cells from the lower first intact molar germs of 5-6-day old SD rats were separated and cultured.

RESULTSOn the second day after birth, the dental follicle began to differentiate into periodontal tissues, but no obvious changes were observed in the alveolar bone one to three days after birth. On the fourth day, the number of osteoclasts increased significantly. The results of immunohistochemistry showed that Wnt5A was not significantly expressed in rat dental follicle tissues before the fourth day, but positive expression was expressed in the next day and continued to express to thirteenth days. Ror2 was expressed in the rat dental follicle at postnatal days 1-3, but weak expression was found in days 4-13.

CONCLUSIONSWnt5A and Ror2 expressions in the process of tooth eruption have specific time distributions, suggesting that these expressions may participate in the regulation of the eruption of the teeth.

Animals ; Dental Sac ; Molar ; Osteoclasts ; Periodontium ; Rats ; Rats, Sprague-Dawley ; Receptor Tyrosine Kinase-like Orphan Receptors ; Tooth Eruption ; Wnt Proteins ; Wnt-5a Protein