Dental Follicle Research Articles

Periostin plays a key role in maintaining the osteogenic abilities of dental follicle stem cells in the inflammatory microenvironment

ObjectiveThis study aimed to explore the effect of periostin in the osteogenic abilities of dental follicle stem cells (DFSCs) and DFSC sheets in the inflammatory microenvironment. DesignDFSCs were isolated from dental follicles and identified. A lentiviral vector was used to knock down periostin in DFSCs. 250 ng/ml lipopolysaccharide from Porphyromonas gingivalis (P.g-LPS) was used to construct the inflammatory microenvironment. Osteogenic differentiation was evaluated by alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot. The formation of extracellular matrix was assessed by qRT-PCR and immunofluorescence. The expressions of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) were measured by western blot. ResultsKnockdown of periostin inhibited osteogenic differentiation and promoted adipogenic differentiation of DFSCs. In an inflammatory microenvironment, knockdown of periostin attenuated the proliferation and osteogenic differentiation of DFSCs. Knockdown of periostin inhibited the formation of extracellular matrix collagen I (COL-I), fibronectin, and laminin in DFSC sheets, but did not affect the expression of osteogenesis-related markers alkaline phosphatase (ALP) and osteocalcin (OCN). In the inflammatory microenvironment, knocking down periostin inhibited the expression of OCN and OPG in DFSC sheets, and promoted the expression of RANKL. ConclusionPeriostin played a key role in maintaining the osteogenic abilities of DFSCs and DFSC sheets in the inflammatory microenvironment and might be an important molecule in the process of DFSCs coping with inflammatory microenvironment and promoting periodontal tissues regeneration.

TRPM7 is Involved in the Regulation of Proliferation, Migration and Osteogenic Differentiation of Human Dental Follicle Cells.

Dental follicle cells (DFCs) are promising candidates for tissue engineering. However, the molecular mechanisms that regulate the biological characteristics of DFCs are still unclear. Transient receptor potential melastatin 7 (TRPM7) is a Ca2+- and Mg2+-permeable cation channel. The aim of this study was to determine the impact of TRPM7 on the proliferation, migration and osteogenic differentiation of DFCs. PCR, Western blotting, Immunocytochemical staining and Patch clamp methods were used to identify the gene and protein expression of TRPM7 in DFCs. DFCs were infected with lentiviruses that expressed either TRPM7 specific shRNA or scrambled non-effective shRNA to investigate its functional role. Cell proliferation and migration were assessed using Cell Counting Kit-8 assays and transwell cell culture chambers separately. Cell osteogenic differentiation were determined by ALP assay kit and Alizarin Red staining. Gene and protein expression of TRPM7 were detected in DFCs, but not of TRPM6, which is a closely related channel with similar function. In the absence of Mg2+, typical whole cell TRPM7-like currents were recorded by patch clamp. These were inhibited by low concentrations of 2-APB, but activated by high concentrations of 2-APB. Functional studies demonstrated that suppression of TRPM7 expression inhibited the proliferation and migration of DFCs, and promoted their osteogenic differentiation. Furthermore, Mg2+ deficiency mimicked the effects of TRPM7 knockdown in terms of osteogenic differentiation of DFCs. These results demonstrate that TRPM7 is involved in regulating the proliferation, migration and osteogenic differentiation of DFCs.

Pediatric Oral Pathology: Odontogenic Cysts.

Cysts encountered in the head and neck typically arise from epithelium that would normally be programmed to form teeth or tooth-supporting structures (odontogenic epithelium). These cysts come with a confusing array of similar-sounding names and histopathologic features that are sometimes shared between conditions. Here we describe and contrast the relatively-common lesions: hyperplastic dental follicle, dentigerous cyst, radicular cyst, buccal bifurcation cyst, odontogenic keratocyst, glandular odontogenic cyst, and the less-common gingival cyst of the new-born and thyroglossal duct cyst. The goal of this review is to help clarify and simplify these lesions for the general pathologist, pediatric pathologist, and surgeon.

DLP 3D printing of high-resolution root scaffold with bionic bioactivity and biomechanics for personalized bio-root regeneration

Digital light projection (DLP) printing of hydroxyapatite (HAp) bioceramic provides a promising strategy for fabrication of complex personalized bio-tooth root scaffold with high-resolution. However, it is still a challenge to fabricate bionic bio-tooth root with satisfied bioactivity and biomechanics. This research studied the HAp-based bioceramic scaffold with bionic bioactivity and biomechanics for personalized bio-root regeneration. Compared to natural decellularized dentine (NDD) scaffolds with unitary shape and restricted mechanical properties, those DLP printing bio-tooth roots with natural size, high precision appearance, excellent structure, and a smooth surface were successfully manufactured, which met various shape and structure requirements for personalized bio-tooth regeneration. Moreover, the bioceramic sintering at 1250 °C enhanced the physicochemical properties of HAp and exhibited good elastic modulus (11.72 ± 0.53 GPa), which was almost twice of early NDD (4.76 ± 0.75 GPa). To further improve the surface activity of sintered biomimetic, the nano-HAw (nano-hydroxyapatite whiskers) coating deposited by hydrothermal treatment increased the mechanical properties and surface hydrophilicity, which indicated positive effects on dental follicle stem cells (DFSCs)’ proliferation and enhanced the DFSCs osteoblastic differentiation in vitro. Subcutaneous transplantation in nude mice and in-situ transplantation in rat alveolar fossa proved that the nano-HAw-containing scaffold could promote the DFSCs differentiate into periodontal ligament-like enthesis formation. In conclusion, by combining the optimized sintering temperature and modified nano-HAw interface through hydrothermal treatment, the DLP-printing of HAp-based bioceramic with favorable bioactivity and biomechanics is a promising candidate for personalized bio-root regeneration.

In Vitro and In Vivo Evaluation of a Polycaprolactone (PCL)/Polylactic-Co-Glycolic Acid (PLGA) (80:20) Scaffold for Improved Treatment of Chondral (Cartilage) Injuries.

Articular cartilage is a specialized tissue that provides a smooth surface for joint movement and load transmission. Unfortunately, it has limited regenerative capacity. Tissue engineering, combining different cell types, scaffolds, growth factors, and physical stimulation has become an alternative for repairing and regenerating articular cartilage. Dental Follicle Mesenchymal Stem Cells (DFMSCs) are attractive candidates for cartilage tissue engineering because of their ability to differentiate into chondrocytes, on the other hand, the polymers blend like Polycaprolactone (PCL) and Poly Lactic-co-Glycolic Acid (PLGA) have shown promise given their mechanical properties and biocompatibility. In this work, the physicochemical properties of polymer blends were evaluated by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM) and were positive for both techniques. The DFMSCs demonstrated stemness by flow cytometry. The scaffold showed to be a non-toxic effect when we evaluated it with Alamar blue, and the samples were analyzed using SEM and phalloidin staining to evaluate cell adhesion to the scaffold. The synthesis of glycosaminoglycans was positive on the construct in vitro. Finally, the PCL/PLGA scaffold showed a better repair capacity than two commercial compounds, when tested in a chondral defect rat model. These results suggest that the PCL/PLGA (80:20) scaffold may be suitable for applications in the tissue engineering of articular hyaline cartilage.

Single-cell transcriptome analysis reveals development atlas of mouse molar pulp cells

Objective: Single-cell RNA sequencing (scRNA-seq) was used to analyze the developing mouse molars, in order to construct a spatiotemporal development atlas of pulp cells, and further to reveal the developmental process and regulatory mechanism of tooth development. Methods: Ten mandibular first molars from C57BL/6 mice in postnatal day (PN) 0 and 3 were respectively dissected and digested to obtain single-cell suspensions. scRNA-seq was performed on 10× Genomics platform. PN 7 mouse molar scRNA-seq data were obtained from our previous study. PN 0, 3, and 7 scRNA-seq data were integrated for following analysis. The initial quality control, mapping and single cell expression matrix construction were performed by Cell Ranger. Quality control, standardization, dimensional reduction and cluster analysis were performed by using Seurat. Monocle was used to generate the pseudotime trajectory. Scillus was used to perform gene ontology analysis. In order to detect the spatiotemporal change of different population of pulp cells, the marker genes of each cluster were demonstrated by RNAscope in situ hybridization. Results: There were twenty-six cell clusters within mouse molars, which were identified as eight different cell types, including dental pulp cells, dental follicle cells, epithelial cells, immune cells, endothelial cells, perivascular cells, glial cells and erythrocytes. We further re-clustered and analyzed dental pulp cells. Cluster 0 were mature pulp cells, which located at the upper portion of crown. The main functions of cluster 0 were osteogenesis and extracellular structure organization. Cluster 1 were apical papilla cells, which located at the apical part of roots, whose main functions were extracellular structure organization and organ development. Cluster 2 were cycling cells, which were actively proliferated, resided in the lower portion of the crown. Cluster 3 and 4 were preodontoblasts and odontoblasts, respectively. Their functions were closely related to biomineralization. The proportion of mature pulp cells increased with the development process, while the proportion of cycling cells and odontoblast lineage decreased. According to the expression pattern of marker genes of each cluster, we constructed a cell atlas of dental pulp. Pseudotime trajectory analysis found there were two development trajectories within dental pulp. They both started from SPARC related modular calcium binding 2 (Smoc2)+ dental papilla cells, then went through DNA topoisomerase Ⅱ alpha (Top2a)+ cycling cells, and finally divided into coxsackie virus and adenovirus receptor (Cxadr)+ mature pulp cells or dentin sialophosphoprotein (Dspp)+ odontoblasts two lineages. Conclusions: scRNA-seq could fully discover the intercellular heterogeneity of cells on transcriptome level, which provides a powerful tool to study the process and regulatory mechanism of organ development.

RUNX2 mutation inhibits the cellular senescence of dental follicle cells via ERK signalling pathway.

The aim of this study was to explore the regulatory effect of RUNX2 mutation on dental follicle cells (DFCs) senescence and clarify the underlying mechanism. This study aimed to explore the basis for a novel mechanism of delayed permanent tooth eruption in cleidocranial dysplasia (CCD) patients. Dental follicles were collected from a CCD patient and healthy controls. Senescence-associated β-galactosidase (SA-β-gal) staining, Ki67 staining, cell cycle assays, and senescence-related gene and protein expression assays were performed to assess DFCs senescence. Western blotting was performed to detect the activation of mitogen-activated protein kinase (MAPK) signalling pathways, and the molecular mechanism underlying RUNX2 regulating in DFCs senescence was explored. RUNX2 mutation inhibited the cellular senescence of DFCs from the CCD patient compared with healthy controls. Ki67 staining showed that mutant RUNX2 promoted DFCs proliferation, and cell cycle assays revealed that the healthy control-derived DFCs arrested at G1 phase. RUNX2 mutation significantly downregulated senescence-associated gene and protein expression. RUNX2 mutation suppressed ERK signalling pathway activation, an ERK inhibitor decreased healthy control-derived DFCs senescence, and an ERK activator promoted CCD patient-derived DFCs senescence. RUNX2 mutation delayed DFCs senescence through the ERK signalling pathway, which may be responsible for delayed permanent tooth eruption in CCD patients.

LncRNA HOTAIRM1 promotes dental follicle stem cell-mediated bone regeneration by regulating HIF-1α/KDM6/EZH2/H3K27me3 axis.

Large bone defect reconstruction undergoes hypoxia and remains a major practical challenge. Bone tissue engineering with a more promising stem cell source facilitates the development of better therapeutic outcomes. Human dental follicle stem cells (hDFSCs) with superior multipotency, osteogenic capacity, and accessibility have been proven a promising cell source for bone regeneration. We previously identified a novel long noncoding RNA (lncRNA), HOTAIRM1, to be highly expressed in hDFSCs. Here we found that HOTAIRM1 overexpressed hDFSCs promoted bone regeneration in rat critical-size calvarial defect model. Mechanically, HOTAIRM1 was induced in hDFSCs under hypoxic conditions and activated HIF-1α. RNA-sequencing analysis indicated that HOTAIRM1 upregulated oxygen-sensing histone demethylases KDM6A/B and suppressed methyltransferase EZH2 via targeting HIF-1α. The osteogenic differentiation of hDFSCs was accompanied with demethylation of H3K27, and HOTAIRM1 overexpression decreased the distribution of H3K27me3 in osteogenic genes, including ALP, M-CSF, Wnt-3a, Wnt-5a, Wnt-7a, and β-catenin, thus promoted their transcription. Our study provided evidence that HOTAIRM1 upregulated KDM6A/B and inhibited EZH2 in a HIF-1α dependent manner to enhance the osteogenesis of hDFSCs. HOTAIRM1-mediated hDFSCs may serve as a promising therapeutic approach to promote bone regeneration in clinical practice.

Impact of primary molars with periapical disease on permanent successors: A retrospective radiographic study

ObjectiveTo clarify the effects on the development, position and morphology of the permanent successors of primary molars affected by apical periodontitis (AP). MethodA total of 132 panoramic radiographs of children aged from 4 to 10 were screened out and a total of 159 mandibular second primary molars with chronic apical periodontitis（AP）(93 males and 66 females) were analyzed. The maturation values of permanent successors were interpreted and scored according to Nolla's method and compared with normal ones'. The proportion of abnormalities in the morphology and orientation of permanent successors were counted, and the differences between men and women was analyzed. The distribution of various abnormalities in different age groups was also analyzed. ResultThere were significant differences in development of permanent successors in this study compared with the normal ones' in all age groups, among which the differences were statistically significant in males aged in 4,5,7 groups and females aged in 4,6 (P < 0.05). The proportions of permanent successors involved with dental follicle broken, malposition and malformation were 78.94%, 42.1%, 8.42% and 82.50%, 38.75%, 15.00%, respectively, with no gender difference. And the highest proportion of these three were all found in 9 years old age group. ConclusionAP of primary teeth can lead to accelerated or delayed development of permanent successors to some extent, and may also lead to changes in their shape and direction.

Clinical application prospects and transformation value of dental follicle stem cells in oral and neurological diseases.

Since dental pulp stem cells (DPSCs) were first reported, six types of dental SCs (DSCs) have been isolated and identified. DSCs originating from the craniofacial neural crest exhibit dental-like tissue differentiation potential and neuro-ectodermal features. As a member of DSCs, dental follicle SCs (DFSCs) are the only cell type obtained at the early developing stage of the tooth prior to eruption. Dental follicle tissue has the distinct advantage of large tissue volume compared with other dental tissues, which is a prerequisite for obtaining a sufficient number of cells to meet the needs of clinical applications. Furthermore, DFSCs exhibit a significantly higher cell proliferation rate, higher colony-formation capacity, and more primitive and better anti-inflammatory effects than other DSCs. In this respect, DFSCs have the potential to be of great clinical significance and translational value in oral and neurological diseases, with natural advantages based on their origin. Lastly, cryopreservation preserves the biological properties of DFSCs and enables them to be used as off-shelf products for clinical applications. This review summarizes and comments on the properties, application potential, and clinical transformation value of DFSCs, thereby inspiring novel perspectives in the future treatment of oral and neurological diseases.

The Emerging Biological Functions of Exosomes from Dental Tissue-Derived Mesenchymal Stem Cells.

Exosomes are one kind of small-cell extracellular membranous vesicles that can regulate intercellular communication and give rise to mediating the biological behaviors of cells, involving in tissue formation, repair, the modulation of inflammation, and nerve regeneration. The abundant kinds of cells can secret exosomes, among them, mesenchymal stem cells (MSCs) are very perfect cells for mass production of exosomes. Dental tissue-derived mesenchymal stem cells (DT-MSCs), including dental pulp stem cells, stem cells from exfoliated deciduous teeth, stem cells from apical papilla, stem cells from human periodontal ligament (PDLSCs), gingiva-derived mesenchymal stem cells, dental follicle stem cells, tooth germ stem cells, and alveolar bone-derived mesenchymal stem cells, are now known as a potent tool in the area of cell regeneration and therapy, more importantly, DT-MSCs can also release numerous types of exosomes, participating in the biological functions of cells. Hence, we briefly depict the characteristics of exosomes, give a detailed description of the biological functions and clinical application in some respects of exosomes from DT-MSCs through systematically reviewing the latest evidence, and provide a rationale for their use as tools for potential application in tissue engineering.

ASPECTS OF ETIOLOGY, DIAGNOSIS AND TREATMENT OF PREERUPTIVE INTRACORONAL RESORPTION IN CHILDREN (LITERATURE REVIEW)

The purpose of this work was to study the literature on research papers by domestic and foreign authors describing pre-eruption intracoronal resorption. The review discusses issues that indicate the presence of a resorption process in permanent teeth before their eruption. This lesion of the hard tissues of the teeth is rare, and the etiology of this process is not fully understood. The processes of tooth resorption can be observed outside on the root surface or inside the pulp chamber, and if a permanent tooth is involved in this process, this indicates the development of pathological resorption. Along with dental caries and periodontal disease in childhood, processes of resorption of coronal dentin tissue in unerupted permanent teeth can be observed. Some authors describe internal resorption, ectopic positioning, external resorption, and origin from developmental pits on the occlusal surface as etiological factors for preeruptive intracoronal resorption, but other authors suggest that the etiology of this lesion remains poorly understood. The main diagnostic method is an x-ray examination, in which preeruptive intracoronal resorption is accidentally detected and other research methods are additionally used. Histological examination reveals osteoclasts, multinucleated giant cells, macrophages, and serrated edges in this lesion, which may possibly enter the developing tooth from the surrounding bone or through breaks in the dental follicle or enamel epithelium. With a small lesion and if the tooth is close to eruption, periodic X-ray control is necessary, and after eruption, when occlusal access is reached, the defect is restored in order to minimize the attachment of the carious process.

Histologic Features of External Cervical Resorption Affecting Impacted Maxillary Canines: A Report of 2 Cases

Reports on the histopathologic features of external cervical resorption (ECR) in unerupted teeth are scarce. This article reports on 2 maxillary impacted canines from different patients that showed ECR lesions and were surgically removed and histologically evaluated. Case 1 showed symptoms associated with oral communication of the dental follicle and pulpal exposure. Radiographs and cone-beam computed tomographic imaging showed that resorption affected a large part of the root and the crown. Case 2 was asymptomatic, and the resorption cavity was restricted to the tooth crown. Both teeth were extracted and subjected to histologic processing and analysis. In case number 1, the coronal pulp was necrotic and infected, showing areas of exposure to the resorption channels. The apical pulp was vital and uninflamed. The pulp space was surrounded by a continuous pericanalar resorption-resistant sheet. Part of the resorbed area of dentin was occupied by inflamed granulation tissue. More apically, a network of trabecular bone was present in the resorbed area. In case number 2, the pulp around the resorbed area was uninflamed and vital. A large fraction of the lost dentin was replaced by trabecular bone, closely adapted to the irregularly resorbed dentin surface, and no inflammatory cells. Our findings indicate that impacted teeth with ECR may remain asymptomatic until associated with infection. Histologic features of resorption in impacted teeth were similar to those in erupted teeth. In the case associated with infection, the resorbed area was occupied by inflamed tissue and newly formed bone. In the case with no infection, the resorption area was filled with bone undergoing a remodeling process.

Dental Follicle Stem Cells Promote Periodontal Regeneration through Periostin-Mediated Macrophage Infiltration and Reprogramming in an Inflammatory Microenvironment.

Dental follicle stem cells (DFSCs) have been verified to promote periodontal regeneration in an inflammatory microenvironment. When coping with inflammatory stimulation, DFSCs highly express periostin, a bioactive molecule closely related to periodontal homeostasis. It is worth exploring whether and how periostin plays a role in the promotion of periodontal regeneration by DFSCs. By tracking the fate of DFSCs, it was found that DFSCs significantly contributed to periodontal regeneration in rat periodontal defects while they had a low survival rate. They highly expressed periostin and improved the immune microenvironment in the defect area, especially via the recruitment and reprogramming of macrophages. Silencing periostin attenuated the effects of DFSCs in promoting periodontal regeneration and regulating macrophages. Recombinant human periostin (rhPeriostin) could not only directly promote macrophage reprogramming through the integrin αM/phosphorylated extracellular signal-regulated kinase (p-Erk)/Erk signaling pathway, but it also exhibited the potential to promote periodontal regeneration in rats when loaded in a collagen matrix. These results indicated that periostin is actively involved in the process by which DFSCs promote periodontal regeneration through the regulation of macrophages and is a promising molecular agent to promote periodontal regeneration. This study provides new insight into the mechanism by which DFSCs promote periodontal regeneration and suggests a new approach for periodontal regeneration therapy.

Role of Collagen Fibers in Predicting the Biologic Behavior of Dentigerous Cyst, Normal Dental Follicle, and Hyperplastic Dental Follicle Using Special Stains

Role of Collagen Fibers in Predicting the Biologic Behavior of Dentigerous Cyst, Normal Dental Follicle, and Hyperplastic Dental Follicle Using Special Stains

DNA protein kinase promotes cellular senescence in dental follicle cells

ObjectiveShort telomeres and genomic DNA damage are causes of cellular senescence in dental follicle cells (DFCs). DesignThis study examined the role of the DNA damage response (DDR) during cellular senescence of DFCs by β-galactosidase activity and DNA damage by comet assay. Expression of genes/proteins was determined by Western Blots and reverse transcription-quantitative polymerase chain reaction, while glycolysis was enzymatically estimated. Cell cycle stages and reactive oxygen species (ROS) were investigated by flow cytometry. ResultsDuring the induction of cellular senescence gene expression of DDR genes were down-regulated, while DNA double-strand breaks occurred at the same time. Furthermore, inhibition of DNA protein kinase (DNA-PK) reduced senescence and ROS, both of which are associated with cellular senescence. In contrast, while these data suggest that inhibition of DDR is associated with the induction of cellular senescence, inhibition of DNA-PK did not result in renewal of DFCs, as inhibition resulted in typical features of depleted cells such as increased cell size and reduced cell proliferation rate. DNA-PK repression inhibited both osteogenic differentiation potential and glycolysis, which are typical features of cellular exhaustion. Moreover, DNA-PK affects cellular senescence via activation of AKT1 (protein kinase B). ConclusionOur results suggest that DNA-PK promotes cellular senescence, but DFCs may control the induction of cellular senescence via down-regulation of DDR genes. However, we also showed that inhibition of DNA-PK cannot renew senescent DFCs

Transcriptome landscape comparison of periodontium in developmental and renewal stages.

Periodontium regeneration remains a significant challenge in clinics and research, and it is essential to understand the stage-specific biological process in situ. However, differing findings have been reported, and the mechanism has yet to be elucidated. The periodontium of adult mice molars is considered to be stable remodeling tissue. At the same time, the continuously growing incisors and the developing dental follicle (DF) of postnatal mice highly represent fast remodeling tissue. In this study, we attempted to explore different clues of temporal and spatial comparisons to provide improved references for periodontal regeneration. Periodontal tissues from the developing periodontium (DeP) of postnatal mice, and continuously growing periodontium (CgP) and stable remodeling periodontium (ReP) of adult mice were isolated and compared using RNA sequencing. Based on the Dep and CgP separately compared with the ReP, differentially expressed genes and signaling pathways were analyzed using GO, KEGG databases, and Ingenuity Pathway Analysis (IPA). The results and validation were obtained by immunofluorescence staining and RT-PCR assays. Data were expressed as means ± standard deviation (SD) and analyzed by GraphPad Prism 8 software package, and one-way ANOVA was used to test multiple groups. Principal component analysis showed that the three groups of periodontal tissue were successfully isolated and had distinct expression profiles. A total of 792 and 612 DEGs were identified in the DeP and CgP groups compared with the ReP. Upregulated DEGs in the DeP were closely related to developmental processes, while the CgP showed significantly enhanced cellular energy metabolism. The DeP and CgP showed a common downregulation of the immune response, with activation, migration, and recruitment of immune cells. IPA and further validation jointly suggested that the MyD88/p38 MAPK pathway played an essential regulatory role in periodontium remodeling. Tissue development, energy metabolism, and immune response were critical regulatory processes during periodontal remodeling. Developmental and adult stages of periodontal remodeling showed different expression patterns. These results contribute to a deeper understanding of periodontal development and remodeling and may provide references for periodontal regeneration.

The role of cell division cycle 42 in root development of tooth and the involved mechanisms

Objective: To explore the effect of cell division cycle 42 (CDC42) on root development and its regulation on cell proliferation and migration in Hertwig's epithelial root sheath (HERS). Methods: Trace the spatiotemporal expression of CDC42 in root development process [postnatal day 5 (P5), P7, P14] through immunofluorescence staining. Nine eight-week-old C57BL/6J male mice were randomly divided into 3 groups using a simple random sample method (n=3 in each group). P3 tooth germ was cultured in air-liquid system for 1 day and then transplanted to renal capsule each to observe tooth root development. The control group implanted tooth germ only. The phosphate buffered saline (PBS) group implanted tooth germ and gel beads soaked with PBS, while the ML141 group implanted tooth germ and gel beads soaked with CDC42 inhibitor (ML141). Cdc42 in HERS cells was inhibited via lentivirus transfection. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and scratch assay were performed. The distribution of Golgi apparatus (GM130) and cytoskeleton (F-actin) in migrated cells were mapped via immunofluorescence staining. Results: CDC42 was expressed in epithelial cells of HERS, polarized ameloblasts and odontoblasts, as well as adjacent dental papilla and dental follicle cells during tooth root development. The root length of the ML141 group [(0.61±0.09) mm] was substantially shorter than that of control group [(1.03±0.19) mm, P=0.007] and PBS group [(0.98±0.10) mm, P=0.021] according to the data of renal capsule transplantation. After lentiviral transfection, the relative expression of Cdc42 in knockdown group (0.31±0.33) was significantly lower than that in control group (1.05±0.08) (t=15.38, P<0.001), demonstrating the knockdown efficiency closed to 70%. Cell viabilities were significantly inhibited in knockdown group (0.87±0.04, 0.96±0.10, 0.59±0.06, respectively) compared with those in control group (1.09±0.13, 1.55±0.32, 1.10±0.09, respectively) after 3, 4 and 5 days (t=3.16, P=0.016; t=4.23, P=0.002; t=5.08, P<0.01), and the cell proliferation ability in knockdown group [(1.65±0.64)%] also decreased than that in the control group [(4.02±1.12)%](t=5.21, P<0.001). In addition, the cell migration rates after 24 and 48 h [(45.1±4.2)%, (56.4±8.3)%] in knockdown group were obviously lower than those in the control group [(63.8±7.4)%, (80.2±7.8)%] (t=3.78, P=0.019; t=3.62, P=0.023). After Cdc42 was knocked down, Golgi apparatus distributed along the nucleus while behaved oriented in the control group. Conclusions: CDC42 plays an important role in the regulation of root length during root development, which may mediate root elongation by affecting the migration and proliferation of HERS cells.

Crucial Triad in Pulp-Dentin Complex Regeneration: Dental Stem Cells, Scaffolds, and Signaling Molecules

BACKGROUND: Pulp damage can lead to dentinogenesis impairment, irreversible pulpitis, or pulp necrosis. Despite being the most used endodontic procedure to treat damaged pulp, root canal therapy only results in nonvital teeth which are prone to fractures and secondary infection. Pulp-dentin regeneration has a potential to regenerate structure similar to normal pulp-dentin complex, and can be achieved by combining dental stem cells, scaffold, and signaling molecules. This article reviews the role of various types of dental stem cells, scaffolds, signaling molecules, and their combinations in regenerating pulp-dentin complex.CONTENT: Dental pulp stem cell (DPSC), stem cell from human exfoliated deciduous teeth (SHED), and dental follicle stem cell (DFSC) were reported to regenerate pulp-dentin complex in situ. SHED might be more promising than DPSCs and DFSCs for regenerating pulp-dentin complex, since SHED have a higher proliferation potential and higher expression levels of signaling molecules. Scaffolds have characteristics resembling extracellular matrix, thus providing a suitable microenvironment for transplanted dental stem cells. To accelerate the regeneration process, exogenous signaling molecules are often delivered together with dental stem cells. Scaffolds and signaling molecules have different regenerative potential, including induction of cell proliferation and migration, formation of pulp- and/or dentin-like tissue, as well as angiogenesis and neurogenesis promotion.SUMMARY: Combinations of dental stem cells, scaffold, and signaling molecules are important to achieve the functional pulp-dentin complex formation. Current trends and future directions on regenerative endodontics should be explored. The right combination of dental stem cells, scaffold, and signaling molecules could be determined based on the patients’ characteristics. Incomplete pulp-dentin regeneration could be overcome by applying dental stem cells, scaffold, and/or signaling molecules in multiple visits.KEYWORDS: pulp-dentin regeneration, regenerative endodontics, dental stem cells, scaffold, signaling molecules

Exposure of suckling rats to hexavalent chromium (CrVI) alters bone formation at the base of the alveolus causing a delay in tooth eruption

ObjectivesHexavalent chromium (CrVI)-exposure of suckling rats has been shown to delay tooth eruption. However, the effects of CrVI-exposure on bone formation at the base of the alveolus, which provides a motive force for tooth eruption in the early stages of the process, remain unknown. The present work sought to evaluate the effect of CrVI-exposure on bone formation at the base of the alveolus during the intraosseous stage of tooth eruption in suckling rats, using histomorphometric and immunohistochemical studies. MethodsExperimental animals received 12.5 mg/kg-bw/day of potassium dichromate dissolved in saline solution by gavage starting on day 4 of the experiment; controls similarly received an equivalent volume of saline. All the animals were euthanized at the age of 9 days. The base of the developing alveolus at the level of the mesial root of the first lower molar was analyzed histomorphometrically and immunohistochemically. Data were statistically analyzed using student's t-test, with statistical significance set at p < 0.05. ResultsCrVI-exposed animals showed lower bone volume and height at the base of the alveolus, a significant preponderance of bone rest on the surface adjacent to the dental follicle, and a significantly lower percentage of positive Runt-related transcription factor-2 (RUNX2+) osteoblasts and a significantly higher number of mesenchymal-like RUNX2+ cells at the latter site. ConclusionCrVI-exposure during lactation affects bone formation at the base of the developing alveolus, delaying tooth eruption. These findings underscore the importance of controlling drinking water levels of toxic substances since their effects can alter the growth and development of individuals exposed during early childhood.