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Transcribed ultra-conserved regions (T-UCRs) are a new class of long non-coding RNA molecules transcribed from ultra-conserved regions (UCRs) of the human genome. T-UCRs are extremely conserved in the human, rat, and mouse genomes. Deletions of genomic areas containing UCRs resulted in live mice that developed without distinguishable phenotypes, implying that T-UCRs are involved in developmental processes. In addition, there is increasing evidence that dental follicle tissues exhibit various cellular alterations involving deregulation of protein-coding genes and non-coding RNAs. Accordingly, the main objective of the present study was to determine the clinical significance and distinct expression signatures of non-coding RNA molecules transcribed from ultra-conserved regions in dental follicle tissues of impacted mandibular third molars.From March 2021 to December 2021, a total of 42 patients who referred to clinic of oral and maxillofacial surgery department with the indications of impacted mandibular third molar extraction from 38th and 48th positions were enrolled for the study. For the analysis of T-UCR expression levels, real-time quantitative reverse transcription PCR method was used.Findings of the present study indicated that T-UCRs are distinctly expressed in dental follicle tissues of impacted mandibular third molars. The expression of uc.38, uc.112, and uc.338 was found to be significantly increased in the dental follicles of impacted mandibular third molars, indicating a clinical significance of these molecules. In addition, no differences in T-UCR expression were found as a function of demographic characteristics.Collectively, transcribed ultra-conserved elements, such as uc.38, uc.112, and uc.338, are considerably deregulated in the dental follicle tissues of impacted mandibular third molars and might be responsible for the molecular changes acquired by dental follicle tissues of impacted mandibular third molars.

Dental follicle (DF) is made up of mesenchymal cells and fibers surrounding the enamel organ of a developing tooth. It has been shown that cystic and neoplastic lesions can develop from the pericoronal follicles of impacted third molars (ITMs). But the molecular transformation of DF tissues has not yet been uncovered and remains elusive. Accordingly, in the present study, we aimed to investigate the differential expression of lncRNA genes in DF tissues associated with asymptomatic impacted mandibular third molars (IMTMs) that do not show pathological pericoronal radiolucency in radiographic examination.A total of 30 patients with unilateral mesioangular IMTMs were enrolled for the study. The expressions of lncRNA genes were determined in the DF and healthy gingival tissues obtained from study patients. For the determination of lncRNA expression levels, RNA was isolated from the obtained tissues, converted to cDNA samples, and analyzed by quantitative real-time PCR method.As a result, we found that the gene expression of MEG3 was increased about 10-fold in DF tissues compared to healthy gingival tissues (p 0.0001). In addition, NORAD expression was found to be upregulated 4.2-fold (p = 0.0002) in DF tissues. Also, expression level of MALAT1 was found to be decreased 1.24-fold (p = 0.584) and TP73-AS1 increased 2.6-fold (p = 0.093) in DF tissues compared to healthy gingival tissues.Consequently, present findings suggest that differentially expressed lncRNAs in DFs might be associated with the various levels of cellular events including osteogenic differentiation, DNA damage, and the transformation into odontogenic pathology.Expression levels of MEG3 and NORAD lncRNA molecules may guide clinicians in the evaluation of asymptomatic ITM dental follicles that cannot be determined radiologically and during extraction of these teeth for prophylactic purposes.

Objectives The shapes of gubernaculum tracts (GTs) in molars as accessional teeth remain unidentified. To elucidate imaging peculiarities of GTs in molars with aging on multidetector‐row computed tomography (MDCT). Material and methods This retrospective study was conducted using CT images, including maxillary and mandibular molars, with no abnormal findings from 239 patients. Shapes of alveolar bone, GTs, and dental sacs of the maxillary and mandibular molars were analyzed multi‐sectionally. Correlations between 2‐ and 3‐dimensional imaging figures of GTs in molars and chronological age or stage of molar formation were analyzed. Results Some forms of GTs in maxillary and mandibular third molars were observed. In the early stage, GTs were visualized as bone defect lines on the dentition and grooves on the mesial alveolar crest continuous with the dental sac to mesial tooth bud. GTs of the third molar formed a J‐shape in maxillary teeth and Y‐shape in mandibular teeth in the middle stage, as alveolar bone around the GT developed. In the mature stage, the course of the GT changed to straight and perpendicular. Some GT forms were also identified in first and second molars. Significant correlations were found between GT alterations and chronological age or stage of molar formation. Moreover, tracts continuing from the distal side of mandibular third molars were detected. Conclusions This paper describes the peculiarities and process of progression for GTs in molars, and the existence of tracts continuing from the distal side of mandibular third molars, unlikely dentition with deciduous predecessors. These preliminary data should prove beneficial for studies focusing on GTs in molars.

Background The present study aimed to report a technically improved operation on the surgical exposure of labially impacted maxillary canine, elaborating the management of soft tissue to achieve better aesthetic results, and post-treatment periodontal health. Methods Patients sought orthodontic treatment with unilateral labially impacted maxillary canines were selected in this study. The impacted teeth were assigned to the experimental group and contralateral unimpacted canines were assigned to the control group. The impacted canines were surgically exposed with dissected dental follicle (DF) stitching to muscle and mucosa surrounding the crowns. The gingival index (GI), probing depth (PD), the width of the keratinized gingiva (WKG), gingival scars (GS), bone loss (BL), and apical root resorption (ARR) were recorded after the removal of the fixed appliance. A two-sample t-test was used for independent samples for parametric variables. Results A total of 24 patients with unilateral maxillary canine impaction were successfully treated. The outcomes of GI, WKG, GS, BL, and ARR did not indicate statistical significance between the experimental group and the control group. Conclusions The preservation of DF promotes soft tissue management in combined surgical and orthodontic treatment of labially impacted maxillary canine to achieve better periodontal status. Trial Registration Chinese Clinical Trial Registry ChiCTR2000029091, 2020-01-12.

Background Dental follicle stem cells (DFSCs) show mesenchymal stem cell properties with the potential for alveolar bone regeneration. Stem cell properties can be impaired by reactive oxygen species (ROS), prompting us to examine the importance of scavenging ROS for stem cell-based tissue regeneration. This study aimed to investigate the effect and mechanism of N-acetylcysteine (NAC), a promising antioxidant, on the properties of DFSCs and DFSC-based alveolar bone regeneration. Methods DFSCs were cultured in media supplemented with different concentrations of NAC (0–10 mM). Cytologic experiments, RNA-sequencing and antioxidant assays were performed in vitro in human DFSCs (hDFSCs). Rat maxillary first molar extraction models were constructed, histological and radiological examinations were performed at day 7 post-surgery to investigate alveolar bone regeneration in tooth extraction sockets after local transplantation of NAC, rat DFSCs (rDFSCs) or NAC-treated rDFSCs. Results 5 mM NAC-treated hDFSCs exhibited better proliferation, less senescent rate, higher stem cell-specific marker and immune-related factor expression with the strongest osteogenic differentiation; other concentrations were also beneficial for maintaining stem cell properties. RNA-sequencing identified 803 differentially expressed genes between hDFSCs with and without 5 mM NAC. “Developmental process (GO:0032502)” was prominent, bioinformatic analysis of 394 involved genes revealed functional and pathway enrichment of ossification and PI3K/AKT pathway, respectively. Furthermore, after NAC treatment, the reduction of ROS levels (ROS, superoxide, hydrogen peroxide), the induction of antioxidant levels (glutathione, catalase, superoxide dismutase), the upregulation of PI3K/AKT signaling (PI3K-p110, PI3K-p85, AKT, phosphorylated-PI3K-p85, phosphorylated-AKT) and the rebound of ROS level upon PI3K/AKT inhibition were showed. Local transplantation of NAC, rDFSCs or NAC-treated rDFSCs was safe and promoted oral socket bone formation after tooth extraction, with application of NAC-treated rDFSCs possessing the best effect. Conclusions The proper concentration of NAC enhances DFSC properties, especially osteogenesis, via PI3K/AKT/ROS signaling, and offers clinical potential for stem cell-based alveolar bone regeneration.

Objectives The indication for removal of asymptomatic fully impacted third molars is still controversial. In this study, radiological and histological investigation of the dental follicle of asymptomatic impacted mandibular third molars was performed, aiming to provide a reference for clinical prophylactic extraction of these teeth. Methods Patients with impacted mandibular third molars were included and the maximum width of the dental follicle around the crown was measured in horizontal, sagittal and coronal sections by cone beam computed tomography. The dental follicles were stained with haematoxylin-eosin, analysed by a pathologist and classified as normal, inflammatory or cystic. A Chi-squared test was used to analyse the association of the incidence of inflammation and cysts with the clinical variables of the impacted mandibular third molars. Results Thirty-seven samples were normal dental follicles; 52 samples showed inflammatory infiltration with an incidence of 57.14%; 2 samples with a maximum dental follicle width of 2–3 mm were diagnosed as odontogenic cysts, and the incidence was 2.20%. There was no significant difference in the incidence of inflammatory and cystic dental follicles between males and females, or between different age groups (P > 0.05). With an increase of the maximum width of the dental follicle, there was a rise in the incidence and degree of infiltration of chronic nonspecific inflammation. Conclusion Asymptomatic impacted mandibular third molars tend to be extracted, especially for teeth with a 2–3 mm maximum width of the dental follicle on radiological examination.

Dental stem/progenitor cells are a promising cell sources for alveolar bone (AB) regeneration because of their same embryonic origin and superior osteogenic potential. However, their molecular processes during osteogenic differentiation remain unclear. The objective of this study was to identify the responsiveness of dental follicle cells (DFCs) and AB marrow-derived mesenchymal stem cells (ABM-MSCs) to recombinant human bone morphogenetic protein-2 (rhBMP-2). These cells expressed vimentin and MSC markers and did not express cytokeratin and hematopoietic stem cell markers and showed multilineage differentiation potential under specific culture conditions. DFCs exhibited higher proliferation and colony-forming unit-fibroblast efficiency than ABM-MSCs; rhBMP-2 induced DFCs to differentiate toward a cementoblast/osteoblast phenotype and ABM-MSCs to differentiate only toward a osteoblast phenotype; and rhBMP-2-induced DFCs exhibited higher osteogenic differentiation potential than ABM-MSCs. These cells adhered, grew, and produced extracellular matrix on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA). During a 14-day culture on nHAC/PLA, the extracellular alkaline phosphatase (ALP) activity of DFCs decreased gradually and that of ABM-MSCs increased gradually; rhBMP-2 enhanced their extracellular ALP activity, intracellular osteocalcin (OCN), and osteopontin (OPN) protein expression; and DFCs exhibited higher extracellular ALP activity and intracellular OCN protein expression than ABM-MSCs. When implanted subcutaneously in severe combined immunodeficient mice for 3 months, DFCs+nHAC/PLA+rhBMP-2 obtained higher percentage of bone formation area, OCN, and cementum attachment protein expression and lower OPN expression than ABM-MSCs+nHAC/PLA+rhBMP-2. These results showed that DFCs possessed superior proliferation and osteogenic differentiation potential in vitro, and formed higher quantity and quality bones in vivo. It suggested that DFCs might exhibit a more sensitive responsiveness to rhBMP-2, so that DFCs enter a relatively mature stage of osteogenic differentiation earlier than ABM-MSCs after rhBMP-2 induction. The findings imply that these dental stem/progenitor cells are alternative sources for AB engineering in regenerative medicine, and developing dental tissue may provide better source for stem/progenitor cells.

Background Human dental follicle cells (DFCs) are the precursor cells of the periodontium with a high potential for regenerative therapies of (alveolar) bone. However, the molecular mechanisms of osteogenic differentiation are inadequately understood. Classical isoforms of protein kinase C (PKC) are reported to inhibit osteogenesis of stem/precursor cells. This study evaluated the role of classical PKCs and potential downstream targets on the osteogenic differentiation of DFCs. Methods DFCs were osteogenic differentiated with dexamethasone or bone morphogenetic protein 2 (BMP2). Expression of PKC and potential upstream/downstream regulators was manipulated using activators, inhibitors, and small interfering ribonucleic acid (siRNA). Expression of proteins was examined by Western blot analysis, while the activation levels of enzymes and transcription factors were examined by their phosphorylation states or by specific activation assays. Expression levels of osteogenic markers were examined by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) analysis. Activity of alkaline phosphatase (ALP) and accumulation of calcium nodules by Alizarin Red staining were measured as indicators of mineralization. Results Classical PKCs like PKCα inhibit the osteogenic differentiation of DFCs, but do not interfere with the induction of differentiation. Inhibition of classical PKCs by Gö6976 enhanced activity of Akt after osteogenic induction. Akt was also regulated during differentiation and especially disturbed BMP2-induced mineralization. The PKC/Akt axis was further shown to regulate the canonical Wnt signaling pathway and eventually nuclear expression of active β-catenin during dexamethasone-induced osteogenesis. Moreover, the nuclear factor “kappa-light-chain-enhancer” of activated B cells (NF-κB) pathway is regulated during osteogenic differentiation of DFCs and via the PKC/Akt axis and disturbs the mineralization. Upstream, parathyroid hormone-related protein (PTHrP) sustained the activity of PKC, while Wnt5a inhibited it. Conclusions Our results demonstrate that classical PKCs like PKCα and Akt regulate the osteogenic differentiation of DFCs partly via both β-catenin and NF-κB.

Inflammatory cytokines such as interleukin-1α (IL-1α) are increased in teeth with periapical lesions. Primary teeth with periapical lesions have a propensity for accelerated eruption of the successors. In this study, we asked whether increased levels of IL-1α in the dental follicle (DF) occurring as the result of periapical lesions promote tooth eruption, possibly due to enhanced osteoclastic remodeling of DF cells (DFCs). To this end, we studied the effect and possible mechanism of IL-1α on osteogenic differentiation, osteoclastogenic activity, and matrix remodeling of DFCs. Results demonstrated that DFCs cultured with IL-1α exhibited reduced osteogenic capacity, higher osteoclastogenic activity, and stronger invasive ability. Phosphorylation of JNK and p38 was upregulated, and pretreatment with SB203580 and SP600125 reversed the effect of IL-1α on DFCs. Neonatal rats subjected to subcutaneous injection of an IL-1 receptor antagonist exhibited a reduced number in activated osteoclasts, increased expression of alkaline phosphatase and osteopontin, and delayed tooth eruption. These data support our hypothesis that increased IL-1α cytokine levels as they occur during periodontal and periapical inflammation cause osteoclastic remodeling of the alveolar socket as a requirement for tooth eruption and thus may indirectly promote the vertical eruption of teeth toward the occlusal plane.

Background and purpose Tooth eruption is a complicated process regulated by the dental follicles (DF). Our recent study discovered that tooth eruption was inhibited upon injection of bleomycin into DF. However, the mechanisms were unknown. Experimental approach Human dental follicle cells (hDFCs) were treated by bleomycin or exogenous TGF-β1 or transfected by plasmids loading SMAD7 or shRNA targeting SMAD7, followed by osteogenesis induction assay and signalling analysis. Human fresh DF tissues and Wistar rats were used to further confirm bleomycin function. Key results Bleomycin decreased expression of RUNX2 and osteogenic genes in hDFCs, reducing osteogenic capacity. TGF-β1 expression was up-regulated in bleomycin-treated hDFCs. The effects of exogenous TGF-β1 were similar to those of bleomycin in hDFCs. Additionally, compared to SMAD2/3, SMAD7 expression increased more in bleomycin- or TGF-β1-treated hDFCs. Overexpression of SMAD7 likewise significantly decreased RUNX2 expression and osteogenic capacity of hDFCs. Knockdown of SMAD7 markedly attenuated the inhibitory effects of bleomycin and TGF-β1 on osteogenic capacity and RUNX2 expression of hDFCs. Most importantly, changes in TGF-β1, SMAD7, and RUNX2 expressions were similar in the DF of rats and humans treated with bleomycin. Conclusion and implications SMAD7 was a negative regulator of osteogenic differentiation in DFCs through suppressing RUNX2 expression. Bleomycin or TGF-β1 inhibited osteogenic differentiation of DFCs via a TGF-β1/SMAD7/RUNX2 pathway. Our findings might be beneficial for enhancing the osteogenic activity of DFCs or inhibiting the eruption of undesirable teeth.

Ameloblastomas are epithelial odontogenic tumours that, although benign, are locally invasive and may exhibit aggressive behaviour. In the tumour microenvironment, the concentration of oxygen is reduced, which leads to intratumoral hypoxia. Under hypoxia, the crosstalk between the HIF-1α, MMP-2, VEGF, and VEGFR-2 proteins has been associated with hypoxia-induced angiogenesis, leading to tumour progression and increased invasiveness. This work showcases 24 ameloblastoma cases, 10 calcifying odontogenic cysts, and 9 dental follicles, used to investigate the expression of these proteins by immunohistochemistry. The anti-HIF-1α, anti-MMP-2, anti-VEGF, and anti-VEGFR-2 primary antibodies are used in this work. The results have been expressed by the mean grey value after immunostaining in images acquired with an objective of 40×. The ameloblastoma samples showed higher immunoexpression of HIF-1α, MMP-2, VEGF, and VEGFR-2 when compared to the dental follicles and calcifying odontogenic cysts. Ameloblastomas show a higher degree of expression of proteins associated with intratumoral hypoxia and proangiogenic proteins, which indicates the possible role of these proteins in the biological behaviour of this tumour.

The dental follicle is part of the tooth germ, and isolated stem cells from this tissue (dental follicle cells; DFCs) are considered, for example, for regenerative medicine and immunotherapies. However somatic stem cells can also improve pharmaceutical research. Cell proliferation is limited by the induction of senescence, which, while reducing the therapeutic potential of DFCs for cell therapy, can also be used to study aging processes at the cellular level that can be used to test anti-aging pharmaceuticals. Unfortunately, very little is known about cellular senescence in DFCs. This review presents current knowledge about cellular senescence in DFCs.

Lipopolysaccharide (LPS)-induced inflammatory microenvironment can enhance the dental follicle cells (DFCs) proliferation, differentiation, and adhesion abilities beneficial to periodontal regeneration, which possibly attributes the success to exosomes according to recent studies. This study aimed to investigate the therapeutic efficacy and underlying mechanisms of LPS-preconditioned DFC-derived small extracellular vesicles (sEVs), which enriched exosomes for periodontal regeneration in an inflammatory microenvironment. LPS preconditioning could significantly increase the secretion of sEVs derived from DFCs. Both LPS-preconditioned dental follicle cell-derived sEV (L-D-sEV) and DFC-derived sEV (D-sEV) promoted the proliferation of periodontal ligament cells from periodontitis (p-PDLCs) with a dose-dependent and saturable manner and also enhanced the migration and differentiation of p-PDLCs. Furthermore, L-D-sEV showed a modest benefit than D-sEV to promote p-PDLCs differentiation. In vivo, an L-D-sEV-loaded hydrogel applied in the treatment of periodontitis was beneficial to repair lost alveolar bone in the early stage of treatment and to maintain the level of alveolar bone in the late stage of treatment in experimental periodontitis rats, which could partly decrease the expression of the RANKL/OPG ratio. In conclusion, L-D-sEV was beneficial to p-PDLCs forming an integrity periodontal tissue. The biological injectable L-D-sEV-loaded hydrogel could be used as a treatment method for experimental periodontitis in rats, promoting periodontal tissue regeneration and providing a new alternative cell therapy method for periodontal tissue regeneration.

Background Pulpitis is a common dental disease characterized by sustained inflammation and impaired pulp self-repair. Mesenchymal stem cell-based minimally invasive vital pulp therapy (MSC-miVPT) is a potential treatment method, but its application is limited by the difficulty in acquiring MSCs. We recently revealed the immunomodulatory effects of rat dental follicle stem cells (rDFSCs) on acute lung injury. The present study focused on the paracrine effects of rDFSCs on the inflammation and regeneration of rat injured dental pulp to detect whether DFSCs are a potential candidate for MSC-miVPT. Methods Conditioned medium from rDFSCs (rDFSC-CM) was applied to lipopolysaccharide (LPS)-induced inflammatory rat dental pulp cells (rDPCs). The inflammation and regeneration of rDPCs were detected by RT-qPCR, Western blotting, immunofluorescence staining, Cell Counting Kit-8 (CCK-8) assay, flow cytometry, wound-healing assay, and Masson’s staining. The effects of rDFSC-CM on inflamed rat dental pulp were further evaluated by hematoxylin-eosin and immunohistochemical staining. Results rDFSC-CM downregulated the ERK1/2 and NF-κB signaling pathways, which resulted in suppression of the expression of IL-1β, IL-6, and TNF-α and promotion of the expression of IL-4 and TGF-β, and these findings lead to the attenuation of rDPC inflammation. rDFSC-CM enhanced the in vitro proliferation, migration, and odontogenic differentiation of inflammatory rDPCs and their in vivo ectopic dentinogenesis. Furthermore, rDFSC-CM inhibited inflammatory cell infiltration in rat pulpitis and triggered Runx2 expression in some of the odontoblast-like cells surrounding the injured site, and these effects were conducive to the repair of inflamed dental pulp. Conclusions rDFSC-CM exhibits therapeutic potential by rescuing the regeneration of the inflamed rat dental pulp through an immunomodulatory mechanism, indicating the application prospects of DFSCs in biological regenerative endodontics.

Replicative senescence causes a reduced osteogenic differentiation potential of senescent dental follicle cells (DFCs). The transcription factor p53 is often involved in the induction of cellular senescence, but little is known about its role in DFCs. This study examined for the first time the role of p53 compared to its pro-proliferative antagonist E2F-1 in terms of osteogenic differentiation potential and induction of senescence. Protein expression of E2F-1 decreased during cell aging, while p53 was expressed constitutively. Gene silencing of E2F1 (E2F-1) inhibited the proliferation rate of DFCs and increased the induction of cellular senescence. The induction of cellular senescence is regulated independently of the gene expression of TP53 (p53), since its gene expression depends on the expression of E2F1. Moreover, gene silencing of TP53 induced E2F1 gene expression and increased cell proliferation, but did not affect the rate of induction of cellular senescence. TP53 knockdown further induced the alkaline phosphatase and mineralization in DFCs. However, the simultaneous silencing of TP53 and E2F1 did not inhibit the inductive effect of TP53 knockdown on osteogenic differentiation, indicating that this effect is independent of E2F-1. In summary, our results suggest that p53 inhibits osteogenic differentiation and cell proliferation in senescent DFCs, but is not significantly involved in senescence induction.

Background Pericoronal radiolucent lesions are a common radiographic finding, but it is rare that they occur in multiple forms. Multiple calcifying hyperplastic dental follicles (MCHDF) are entities with few cases described to date; nevertheless, they appear to have a very particular phenotypic pattern. Cases presentation Case 1: A 10-year-old male was evaluated radiographically, revealing four impacted canines, each accompanied by unilocular pericoronal radiolucency. Case 2: A 16-year-old male was planning orthodontic treatment; following his radiological evaluation all third molars were found to be accompanied with pericoronal radiolucencies. Enucleation, and third molar removal along with the pericoronal tissue were the respective treatments. Microscopically, in both cases, the specimens shown odontogenic epithelium, and type I and II calcifications in the hyperplastic follicles, all these characteristics were consistent with MCHDF. Conclusion Although MCHDF are a rare entity, they must be considered in the differential diagnosis of multiple pericoronal lesions. Under the light of the current evidence, the histological findings may be relatively heterogeneous, but their integration with both the clinical data, which are apparently particular, and with the radiographic characteristics, can lead to a definitive diagnosis.

Dental follicles are necessary for tooth eruption, surround the enamel organ and dental papilla, and regulate both the formation and resorption of alveolar bone. Dental follicle progenitor cells (DFPCs), which are stem cells found in dental follicles, differentiate into different kinds of cells that are necessary for tooth formation and eruption. Runt‐related transcription factor 2 (Runx2) is a transcription factor that is essential for osteoblasts and osteoclasts differentiation, as well as bone remodeling. Mutation of Runx2 causing cleidocranial dysplasia negatively affects osteogenesis and the osteoclastic ability of dental follicles, resulting in tooth eruption difficulties. Among a variety of cells and molecules, Nel-like molecule type 1 (Nell-1) plays an important role in neural crest-derived tissues and is strongly expressed in dental follicles. Nell-1 was originally identified in pathologically fused and fusing sutures of patients with unilateral coronal synostosis, and it plays indispensable roles in bone remodeling, including roles in osteoblast differentiation, bone formation and regeneration, craniofacial skeleton development, and the differentiation of many kinds of stem cells. Runx2 was proven to directly target the Nell-1 gene and regulate its expression. These studies suggested that Runx2/Nell-1 axis may play an important role in the process of tooth eruption by affecting DFPCs. Studies on short and long regulatory noncoding RNAs have revealed the complexity of RNA-mediated regulation of gene expression at the posttranscriptional level. This ceRNA network participates in the regulation of Runx2 and Nell-1 gene expression in a complex way. However, non-study indicated the potential connection between Runx2 and Nell-1, and further researches are still needed.

Background Treatments based on stem cell-derived small extracellular vesicles (sEVs) have been explored as an alternative to stem cell transplantation-based therapies in periodontal regeneration. Dental follicle stem cells (DFSCs) have shown great potential for regenerative medicine applications. However, it is unclear whether sEVs derived from DFSCs (DFSCs-sEVs) could be used in periodontal regeneration. This study investigates whether DFSCs-sEVs could regenerate damaged periodontal tissue and the potential underlying mechanism. Methods DFSCs-sEVs were isolated and identified, and periodontal ligament stem cells (PDLSCs) were cocultured with the isolated sEVs. The effect of DFSCs-sEVs on the biological behaviour of PDLSCs was examined using EdU assay, CCK-8 assay, cell cycle analysis, wound healing, alizarin red staining, qRT-PCR, and western blot analysis. RNA sequencing and functional enrichment analysis were used to detect the signal pathway involved in the effect of DFSCs-sEVs on PDLSCs. PDLSCs were pretreated with ERK1/2 or p38 MAPK inhibitors to investigate the possible involvement of the ERK1/2 and p38 MAPK pathways. Additionally, DFSCs-sEVs were combined with collagen sponges and transplanted into the periodontal defects in SD rats, and then, pathological changes in periodontal tissue were examined using haematoxylin and eosin (HE) staining and micro-CT. Results PDLSCs could internalize DFSCs-sEVs, thereby enhancing the proliferation assessed using EdU assay, CCK-8 assay and cell cycle analysis. DFSCs-sEVs significantly enhanced the migration of PDLSCs. DFSCs-sEVs promoted osteogenic differentiation of PDLSCs, showing deep Alizarin red staining, upregulated osteogenic genes (RUNX2, BSP, COL1), and upregulated protein expression (RUNX2, BSP, COL1, ALP). We found that p38 MAPK signalling was activated via phosphorylation. Inhibition of this signalling pathway with a specific inhibitor (SB202190) partially weakened the enhanced proliferation. After DFSCs-sEVs transplantation, new periodontal ligament-like structures and bone formation were observed in the damaged periodontal area in rats. Labelled DFSCs-sEVs were observed in the newly formed periodontal ligament and soft tissue of the defect area. Conclusions Our study demonstrated that DFSCs-sEVs promoted periodontal tissue regeneration by promoting the proliferation, migration, and osteogenic differentiation of PDLSCs. The effect of DFSCs-sEVs in promoting PDLSCs proliferation may be partially attributed to the activation of p38 MAPK signalling pathway. DFSCs-sEVs provide us with a novel strategy for periodontal regeneration in the future.

Background The regeneration of bone loss that occurs after periodontal diseases is a significant challenge in clinical dentistry. Extracellular vesicles (EVs)-based cell-free regenerative therapies represent a promising alternative for traditional treatments. Developmental biology suggests matrix vesicles (MVs), a subtype of EVs, contain mineralizing-related biomolecules and play an important role in osteogenesis. Thus, we explore the therapeutic benefits and expect to find an optimized strategy for MV application. Methods Healthy human dental follicle cells (DFCs) were cultured with the osteogenic medium to generate MVs. Media MVs (MMVs) were isolated from culture supernatant, and collagenase-released MVs (CRMVs) were acquired from collagenase-digested cell suspension. We compared the biological features of the two MVs and investigated their induction of cell proliferation, migration, mineralization, and the modulation of osteogenic genes expression. Furthermore, we investigated the long-term regenerative capacity of MMVs and CRMVs in an alveolar bone defect rat model. Results We found that both DFC-derived MMVs and CRMVs effectively improved the proliferation, migration, and osteogenic differentiation of DFCs. Notably, CRMVs showed better bone regeneration capabilities. Compared to MMVs, CRMVs-induced DFCs exhibited increased synthesis of osteogenic marker proteins including ALP, OCN, OPN, and MMP-2. In the treatment of murine alveolar bone defects, CRMV-loaded collagen scaffold brought more significant therapeutic outcomes with less unhealing areas and more mature bone tissues in comparison with MMVs and acquired the effects resembling DFCs-based treatment. Furthermore, the western blotting results demonstrated the activation of the PLC/PKC/MAPK pathway in CRMVs-induced DFCs, while this cascade was inhibited by MMVs. Conclusions In summary, our findings revealed a novel cell-free regenerative therapy for repairing alveolar bone defects by specific MV subtypes and suggest that PLC/PKC/MAPK pathways contribute to MVs-mediated alveolar bone regeneration.

Among all the adult stem cells, odontogenic stem cells inherit the characterization of neurogenic potential of their precursor ones-the cranial crest cells. Dental follicle cells (DFCs), one of the special kind of odontogenic stem cells, are raising interest in applying to regenerative medicine for they possess multi-differentiation potential, relatively free access and ethic-friendly characteristic. Parkinson's disease (PD), as one of the common neurodegenerative disorders, affects about 0.3% of the general population. Stem cell therapies are thought to be effective to treat it. Aiming at tackling ethical-concernings, confined sources and practically applicational limits, we made use of dopaminergic neurongenic differentiation potential of the DFCs and dedicated every effort to applying them as promising cell source for treating PD. Dental follicle cells were cultured from human dental follicle tissues collected from 12 to 18-year-old teenagers' completely impacted third molars. Our data demonstrated that hDFCs were expressing mesenchymal stem cell-associated surface markers, and possessed the ability of osteogenic, adipogenic and neurogenic differentiation in vitro. Additionally, hDFCs formed neuron-like cells in vitro and in vivo, as well as expressing dopaminergic-neuronogenic marker-TH. Moreover, hDFCs survived in the transplanted areas of the Parkinson's disease model of mouse over six weeks post-surgery, and the number of TH-positive DFCs in the DFCs-Grafted group surpassed its counterpart of the MPTP group with statistically significant difference. This study indicated that hDFCs might be a promising source of dopaminergic neurons for functional transplantation, and encouraged further detailed studies on the potential of hDFCs for treating PD.

Insight into human tooth epithelial stem cells and their biology is sparse. Tissue-derived organoid models typically replicate the tissue's epithelial stem cell compartment. Here, we developed a first-in-time epithelial organoid model starting from human tooth. Dental follicle (DF) tissue, isolated from unerupted wisdom teeth, efficiently generated epithelial organoids that were long-term expandable. The organoids displayed a tooth epithelial stemness phenotype similar to the DF's epithelial cell rests of Malassez (ERM), a compartment containing dental epithelial stem cells. Single-cell transcriptomics reinforced this organoid-ERM congruence, and uncovered novel, mouse-mirroring stem cell features. Exposure of the organoids to epidermal growth factor induced transient proliferation and eventual epithelial-mesenchymal transition, highly mimicking events taking place in the ERM in vivo. Moreover, the ERM stemness organoids were able to unfold an ameloblast differentiation process, further enhanced by transforming growth factor-beta (TGF beta) and abrogated by TGF beta receptor inhibition, thereby reproducing TGF beta's known key position in amelogenesis. Interestingly, by creating a mesenchymal-epithelial composite organoid (assembloid) model, we demonstrated that the presence of dental mesenchymal cells (i.e. pulp stem cells) triggered ameloblast differentiation in the epithelial stem cells, thus replicating the known importance of mesenchyme-epithelium interaction in tooth development and amelogenesis. Also here, differentiation was abrogated by TGF beta receptor inhibition. Together, we developed novel organoid models empowering the exploration of human tooth epithelial stem cell biology and function as well as their interplay with dental mesenchyme, all at present only poorly defined in humans. Moreover, the new models may pave the way to future tooth-regenerative perspectives. This work was supported by grants from KU Leuven (Research Fund) and Fund for Scientific Research (FWO) Flanders. L.H. is an FWO PhD Fellow (1S84718N). Acknowledgements We are grateful to all staff members of the Oral and Maxillofacial Surgery (MKA) of UZ Leuven, as well as the patients, for their invaluable help and contribution to collect freshly extracted third molars. We would also like to thank Dr. Reinhilde Jacobs and Dr. Elisabeth Tijskens for their help with sample collection. We thank Dr. Diether Lambrechts’ group for their technical assistance in 10× Genomics. Computational resources for scRNA-seq analysis were provided by ‘Vlaams Supercomputer Centrum’ (VSC), managed by the Fund for Scientific Research (FWO)—Flanders (Belgium). We acknowledge the use of the TEM platforms at VIB-KU Leuven, Biomedical Research Institute (BIOMED) UHasselt, and Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). We would also like to thank Dr. Ronald Driesen (UHasselt) for help with CTOF analysis, and Dr. Marianne Carlon’s group (particularly Liesbeth De Keersmaecker; KU Leuven) for guiding and assisting in lentiviral transduction. We are grateful to Jeanine Santermans and Marc Jans (UHasselt) for their technical assistance in TEM and staining analyses. We also would like to thank Reena Chinnaraj and Vera Dermesrobian (FACS Core, KU Leuven) for their help with FACS experiments.

Human dental follicle cells (DFCs) as periodontal progenitor cells are used for studies and research in regenerative medicine and not only in dentistry. Even if innovative regenerative therapies in medicine are often considered the main research area for dental stem cells, these cells are also very useful in basic research and here, for example, for the elucidation of molecular processes in the differentiation into mineralizing cells. This article summarizes the molecular mechanisms driving osteogenic differentiation of DFCs. The positive feedback loop of bone morphogenetic protein (BMP) 2 and homeobox protein DLX3 and a signaling pathway associated with protein kinase B (AKT) and protein kinase C (PKC) are presented and further insights related to other signaling pathways such as the WNT signaling pathway are explained. Subsequently, some works are presented that have investigated epigenetic modifications and non-coding ncRNAs and their connection with the osteogenic differentiation of DFCs. In addition, studies are presented that have shown the influence of extracellular matrix molecules or fundamental biological processes such as cellular senescence on osteogenic differentiation. The putative role of factors associated with inflammatory processes, such as interleukin 8, in osteogenic differentiation is also briefly discussed. This article summarizes the most important insights into the mechanisms of osteogenic differentiation in DFCs and is intended to be a small help in the direction of new research projects in this area.

Objective Dental mesenchymal stem cells (MSCs) are potential for use in tissue regeneration in inflammatory diseases due to their rapid proliferating, multilineage differentiation, and strong anti-inflammatory features. In the present study, immunoregulatory and glandular tissue regeneration effects of the dental follicle (DF)MSCs in Sjögren’s Syndrome (SS) were investigated. Methods Dental follicle (DF) tissues were obtained from healthy individuals during tooth extraction, tissues were digested enzymatically and DFMSCs were cultured until the third passage. DFMSCs were labeled with Quantum dot 655 for cell tracking analysis. The induction of the SS mouse model was performed by the injection of Ro60-273-289 peptide intraperitoneally. DFMSCs were injected intraperitoneally, or into submandibular, or lacrimal glands. Splenocytes were analyzed for intracellular cytokine (IFN-γ, IL-17, IL-10) secretion in T helper cells, lymphocyte proliferation, and B lymphocyte subsets. Histologic analysis was done for submandibular and lacrimal glands with hematoxylin-eosin staining for morphologic examination. Results The systemic injection of DFMSCs significantly reduced intracellular IFN-γ and IL-17 secreting CD4+ T cells in splenocytes (p Conclusion DFMSCs have the potential for the regulation of Th1, Th17, and Treg balance in SS, and ameliorate glandular dysfunction. DFMSCs can be a beneficial therapeutic application for SS.

Yanli Huang,1– 3 Qian Liu,1,2,4 Li Liu,1,2,4 Fangjun Huo,1,2 Shujuan Guo,1,2,4 Weidong Tian1,3 1State Key Laboratory of Oral Disease & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Oral Regenerative Medicine, West China School of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Engineering Research Center of Oral Translational Medicine, Ministry of Education, West China School of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of China; 3Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, 610041, People’s Republic of China; 4Department of Periodontics, West China School of Stomatology, Sichuan University, Chengdu, Sichuan, 610041, People’s Republic of ChinaCorrespondence: Shujuan Guo; Weidong Tian, Tel/Fax +86 028 8550 3499, Email guo_shujuan@126.com; drtwd@sina.comPurpose: Lipopolysaccharide (LPS) pretreatment can enhance the therapeutic effect of dental follicle stem cells-derived small extracellular vesicles (DFC-sEV) for periodontitis, and this study aimed to investigate the underlying mechanisms and clinical application Of LPS-preconditioned DFC-sEV in periodontitis.Methods: The protein spectrum of DFC-sEV before and after LPS pretreatment was determined by liquid chromatography-tandem mass spectrometry and bioinformatic analysis. Their effects on inflammatory periodontal ligament stem cells (PDLSCs) and macrophages were investigated for cell proliferation, migration, type 2 macrophage (M2) polarization, and intracellular reactive oxygen species (ROS) levels separately. In addition, the regulation of ROS/Jun amino-terminal kinases (JNK) and ROS/extracellular signal-related kinases (ERK) signaling by LPS-preconditioned DFC-sEV was also studied to reveal the antioxidant mechanism. In vivo, two kinds of DFC-sEV loaded with 0.2% hyaluronic acid (HA) gel were applied for canine periodontitis to evaluate the therapeutic potential.Results: The proteomic analysis showed that thirty-eight proteins were differentially expressed in LPS-preconditioned DFC-sEV, and interestingly, the highly expressed proteins were mainly involved in antioxidant and enzyme-regulating activities. In addition to promoting PDLSCs and macrophage proliferation, LPS-preconditioned DFC-sEV inhibited intracellular ROS as an antioxidant. It reduced the RANKL/OPG ratio of PDLSCs by inhibiting ROS/JNK signaling under inflammatory conditions and promoted macrophages to polarize toward the M2 phenotype via ROS/ERK signaling. Furthermore, LPS-preconditioned DFC-sEV loaded with the HA injectable system could sustainably release sEV and enhance the therapeutic efficacy for periodontitis in canines.Conclusion: LPS-preconditioned DFC-sEV could be effectively used as an auxiliary method for periodontitis treatment via antioxidant effects in a subgingival environment, and loading it with HA is feasible and effective for clinical applications.Keywords: small extracellular vesicle, dental follicle stem cell, periodontitis, reactive oxygen species, antioxidant effect

This study experimentally substantiates that the micromechanical compatibility between cell and substrate is essential for cells to achieve energetically favorable mechanotransduction that directs phenotypic transitions. The argument for this compatibility is based on a thermodynamic model that suggests that the response of cells to their substrate mechanical environment is a consequence of the interchange between forms of energy governing the cell-substrate interaction. Experimental validation for the model has been carried out by investigating the osteogenic differentiation of dental follicle stem cells (DFSCs) seeded on electrospun fibrous scaffolds. Electrospinning of blends containing polycaprolactone (PCL) and silk fibroin (SF) with varying composition of cellulose nanocrystals (CNCs) resulted in three-dimensional (3D) fibrous scaffolds with bimodal distribution of fiber diameter, which provides both macroscopically stiff and microscopically compliant scaffolds for cells without affecting the surface chemical functionality of scaffolds. Atomic force microscopy (AFM) with a colloidal probe and single-cell force spectroscopy were used to characterize cell stiffness and scaffold stiffness on the cellular level, as well as cell-scaffold adhesive interaction (chemical functionality). This study has successfully varied scaffold mechanical properties without affecting their surface chemistry. In vitro tests indicate that the micromechanical compatibility between cells and scaffolds has been significantly correlated with mechanosensitive gene expression markers and osteogenic differentiation markers of DFSCs. The agreement between experimental observations and the thermodynamic model affirms that the cellular response to the mechanical environment, though biological in nature, follows the laws of the energy interchange to achieve its self-regulating behavior. More importantly, this study provides systematic evidence, through extensive and rigorous experimental studies, for the first time that rationalizes that micromechanical compatibility is indeed important to the efficacy of regenerative medicine.

This study aimed at exploring a new tooth development evaluation method for age assessment and investigating the dynamic alteration and potential trend of tooth development by orthopantomograms (OPGs), in order to provide references for tooth development prediction and forensic purpose.A total of 132 OPGs of children aged 3-8 years were collected. The developmental stages of the permanent mandibular second molar (M2)were evaluated by experienced examiners according to the Nolla method and Mimics software, respectively. Quantitative analysis of the ratio of the radiopaque calcified area to the dental follicle (RCA/DF) in different stages, ages, sexes and quadrants were evaluated and compared via descriptive statistics and Spearman's correlation coefficient analysis.There was a strong, positive correlation between the examiners' evaluation and mimics analysis results. With the age increased, the Nolla stage of M2 observed by OPGs increased, and the RCA/DF showed increased trends both in males and females. There were significant differences of the RCA/DF of the M2 at various ages. The tooth calcification development of female was 9.08% earlier than that of male between 3 and 8 years old. However, teeth of male seemed to develop faster than that of female during this period. There was no significant difference between left and right quadrant either according to the Nolla stage or RCA/DF.The RCA/DF value obtained from OPGs of the developmental mandibular second permanent molars could be used as a reliable indicator for tooth maturity and age estimation in children aged 3-8 years.Age assessment based on radiographs is considered as a reliable and efficient indicator for judging different types of malocclusion, making suitable orthodontic treatment plan, deciding the extraction time of retained deciduous teeth in clinic practice.

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while α-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells. The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

Impacted third molars are commonly seen in teenagers and young adults and can cause considerable suffering. Preventing eruption of the third molars can reduce pain at the source. Our previous study has shown that dexamethasone (DEX) at a certain concentration can prevent the eruption of third molars without damaging alveolar bone in Sprague-Dawley (SD) rats, but the relevant molecular mechanisms need to be explored. This study aimed to explore the effects of high concentrations of DEX on osteogenic signaling pathways, including BMP/Smad and Wnt/β-catenin pathways, in rat dental follicle cells (rDFCs) and to elucidate the possible mechanisms. The results showed that BMP7 induced osteogenic differentiation by increasing the activity of ALP and the protein levels of OPN in rDFCs. DEX decreased endogenous BMP7 and phosphorylated Smad1/5/8 expression as well as BMP7-induced osteogenic differentiation. DEX also reduced the mRNA and protein levels of β-catenin by enhancing the expression of GSK-3β. In addition, regardless of DEX intervention, overexpression of BMP7 promoted the expression of β-catenin, while knockdown of BMP7 attenuated it. Further investigation revealed that overexpression of BMP7 attenuated the DEX-mediated inhibition of AKT and GSK-3β phosphorylation, but knockdown of BMP7 exerted the opposite effects. This study suggests that high concentrations of DEX may inhibit the expression of β-catenin via the PI3K/AKT/GSK-3β pathway in a manner mediated by BMP7. The findings further illustrate the possible molecular mechanisms by which DEX prevents tooth development.

OBJECTIVES: Primary failure of eruption (PFE) is a genetic disorder exhibiting the cessation of tooth eruption. Loss-of-function mutations in parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor (PTH/PTHrP receptor, PPR) were reported as the underlying cause of this disorder in humans. We showed in a PFE mouse model that PTHrP-PPR signaling is responsible for normal dental follicle cell differentiation and tooth eruption. However, the mechanism underlying the eruption defect in PFE remains undefined. In this descriptive study, we aim to chronologically observe tooth eruption and root formation of mouse PFE molars through 3D microCT analyses. SETTING AND SAMPLE POPULATION: Two individuals with PFE were recruited at Showa University. A mouse PFE model was generated by deleting PPR specifically in PTHrP-expressing dental follicle and divided into three groups, PPR(fl/fl);R26R(tdTomato/+)(Control), PTHrP-creER;PPR(fl/+);R26R(tdTomato/+)(cHet), and PTHrP-creER;PRR(fl/fl);R26R(tdTomato/+)(cKO). MATERIALS AND METHODS: Images from human PFE subjects were acquired by CBCT. All groups of mouse samples were studied at postnatal days 14, 25, 91, and 182 after a tamoxifen pulse at P3, and superimposition of 3D microCT images among three groups was rendered. RESULTS: Mouse and human PFE molars exhibited a similar presentation in the 3D CT analyses. The quantitative analysis in mice demonstrated a statistically significant decrease in the eruption height of cKO first and second molars compared to other groups after postnatal day 25. Additionally, cKO molars demonstrated significantly shortened roots with dilacerations associated with the reduced interradicular bone height. CONCLUSIONS: Mouse PFE molars erupt at a much slower rate compared to normal molars, associated with shortened and dilacerated roots and defective interradicular bones.