Heat Shock Proteins in Tooth Development and Injury Repair.

Heat shock proteins (HSPs) are a class of molecular chaperones with expression increased in response to heat or other stresses. HSPs regulate cell homeostasis by modulating the folding and maturation of intracellular proteins. Tooth development is a complex process that involves many cell activities. During tooth preparation or trauma, teeth can be damaged. The damaged teeth start their repair process by remineralizing and regenerating tissue. During tooth development and injury repair, different HSPs have different expression patterns and play a special role in odontoblast differentiation and ameloblast secretion by mediating signaling pathways or participating in protein transport. This review explores the expression patterns and potential mechanisms of HSPs, particularly HSP25, HSP60 and HSP70, in tooth development and injury repair.

Mediator1 involved in functional integration of Smad3 and Notch1 promoting enamel mineralization.

Enamel hypoplasia is a tooth development defection due to the disruption of enamel matrix mineralization, manifesting as chalky white phenotype. Multiple genes may be involved in this tooth agenesis. It has been proved that ablation of coactivator Mediator1 (Med1) switches the cell fate of dental epithelia, resulting in abnormal tooth development via Notch1 signaling. Smad3 (-/-) mice displays the similar chalky white incisors. However, the expression of Smad3 in Med1 ablation mice and the impact of Med1 on functional integration between Smad3 and Notch1 remains unclear. Cre-loxP-based C57/BL6 mice with epithelial-specific Med1 knockout (Med1 KO) backgrounds were generated. Mandibles and dental epithelial stem cells (DE-SCs) from incisors cervical loop (CL) were isolated from wild-type (CON) mice and Med1 KO mice. Transcriptome sequencing was used to analyze the differences of CL tissue between KO and CON mice. The results revealed the enrichment of TGF-ß signaling pathway. qRT-PCR and western blot were performed to show the gene and protein expression of Smad3, pSmad3, Notch1 and NICD, the key regulators of TGF-ß and Notch1 signaling pathway. Expression of Notch1 and Smad3 was confirmed to be down-regulated in Med1 KO cells. Using activators of Smad3 and Notch1 on Med1 KO cells, both pSmad3 and NICD were rescued. Moreover, adding inhibitors and activators of Smad3 and Notch1 to cells of CON groups respectively, the protein expressions of Smad3, pSmad3, Notch1 and NICD were synergistically affected. In summary, Med1 participates in the functional integration of Smad3 and Notch1, thus promoting enamel mineralization.

MED1 Ablation Promotes Oral Mucosal Wound Healing via JNK Signaling Pathway.

Mediator complex subunit 1 (MED1) is a coactivator of multiple transcription factors and plays a key role in regulating epidermal homeostasis as well as skin wound healing. It is unknown, however, whether it plays a role in healing oral mucosal wounds. In this study, we investigate MED1's functional effects on oral mucosal wound healing and its underlying mechanism. The epithelial-specific MED1 null (Med1epi-/-) mice were established using the Cre-loxP system with C57/BL6 background. A 3 mm diameter wound was made in the cheek mucosa of the 8-week-old mice. In vivo experiments were conducted using HE staining and immunostaining with Ki67 and uPAR antibodies. The in vitro study used lentiviral transduction, scratch assays, qRT-PCR, and Western blotting to reveal the underlying mechanisms. The results showed that ablation of MED1 accelerated oral mucosal wound healing in 8-week-old mice. As a result of ablation of MED1, Activin A/Follistatin expression was altered, resulting in an activation of the JNK/c-Jun pathway. Similarly, knockdown of MED1 enhanced the proliferation and migration of keratinocytes in vitro, promoting re-epithelialization, which accelerates the healing of oral mucosal wounds. Our study reveals a novel role for MED1 in oral keratinocytes, providing a new molecular therapeutic target for accelerated wound healing.

Type-2 epithelial-mesenchymal transition in oral mucosal nonneoplastic diseases.

The oral mucosa is a membranous structure comprising epithelial and connective tissue that covers the oral cavity. The oral mucosa is the first immune barrier to protect the body against pathogens for systemic protection. It is frequently exposed to mechanical abrasion, chemical erosion, and pathogenic invasion, resulting in oral mucosal lesions, particularly inflammatory diseases. Epithelial-mesenchymal transition (EMT) is a crucial biological process in the pathogenesis of oral mucosal disorders, which are classified into three types (types 1, 2, and 3) based on their physiological consequences. Among these, type-2 EMT is crucial in wound repair, organ fibrosis, and tissue regeneration. It causes infectious and dis-infectious immunological diseases, such as oral lichen planus (OLP), oral leukoplakia, oral submucosal fibrosis, and other precancerous lesions. However, the mechanism and cognition between type-2 EMT and oral mucosal inflammatory disorders remain unknown. This review first provides a comprehensive evaluation of type-2 EMT in chronically inflammatory oral mucosal disorders. The aim is to lay a foundation for future research and suggest potential treatments.

N-acetylcysteine regulates dental follicle stem cell osteogenesis and alveolar bone repair via ROS scavenging.

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.

Scientometric Analysis of Dental Implant Research over the Past 10 Years and Future Research Trends.

BACKGROUND: We conducted a bibliometrics analysis to explore the recent trends in dental implant research which could help researchers have a clear grasp of the relevant research hotspots and prospects. Material and Methods. Altogether, 15,770 articles on dental implants, from January 1, 2010, to October 31, 2019, were selected from the Web of Science Core Collection. We used BICOMB software to extract the high-frequency MeSH terms and construct binary and coword matrices. gCLUTO software was used for biclustering and visual analysis, Ucinet 6 software for social network analysis, SCIMAT software for strategic diagram building, Citespace 5.5 software to form timeline visualization, and VOSviewer software, eventually, for bibliometrics cocitation network. RESULTS: Altogether, 72 high-frequency keywords were extracted from the selected articles and 4 clusters and 7 subcategories were identified through biclustering analysis in the dental implant research field. The use of the strategic diagram also enabled us to find the research hotspot and development trends. CONCLUSIONS: The survival rate of dental implants and subsequent restoration have always been the core focus of research. Sinus floor elevation and guided bone regeneration are worthy of constant exploration owing to their reliability. With continuous improvement in technology, immediate loading could become a future research hot spot.

Radial P34HB Electrospun Fiber: A Scaffold for Bone Tissue Engineering.

Finding suitable scaffold material is always an enormous challenge in the study of bone tissue engineering. Designing and preparing bone scaffolds with biomimetic properties is also a difficult problem for bone reparation projects. This project intends to fabricate radial bio-plasticpoly-3-hydroxybutyrate-4-hydroxybutyrate (P34HB) electrospun fibers scaffold that mimic structural, compositional and stiffness properties via an electro spinning technique. The surface morphology, hydrophilicity of the radial fibers scaffold were tested, a contact angle meter and a universal material tester. Bone marrow mesenchymal stem cell (BMSC) morphologies on radial P34HB electrospun fibers scaffold were observed after cell culture under fluorescence microscopy. Tests of cell viability on radial P34HB electrospun fibers scaffold were conducted. We further tested the osteogenic differentiation ability of radial fibers scaffold. These results showed that radial P34HB electrospun fibers scaffold have good biosafety, biocompatibility and osteogenic induction. The radial structure of the scaffold also has a strong effect on the induction of bone formation. Moreover, the structure could also improve the bone contact area of the implant and increase the locking and fixation between the implant and the bone. We plan to apply this procedure to animal experiments for bone defect repair in further research.

[Comparison of two types of T-shaped coronectomy applied in extraction of impacted mandibular third molars].

PURPOSE: To compare the clinical efficiency of two types of T-shaped coronectomy applied in extraction of impacted mandibular third molars. METHODS：One hundred and twenty patients meeting with the criteria were randomly divided into 2 groups. Two types of T-shaped coronectomy were applied in extraction of impacted mandibular third molars. Indexes including operation time, intraoperative anxiety, postoperative pain and limitation of mouth opening were recorded and analyzed with SPSS 22.0 software package. RESULTS: Patients with transverse method experienced more time in operation [(17.41±3.72) vs (15.22±2.53) min], with less intraoperative anxiety (P<0.05). There was no significant difference in postoperative pain and limitation of mouth opening(P>0.05) between two methods. CONCLUSIONS: Transverse T-shaped method can alleviate intraoperative anxiety, while longitudinal method is more conducive to shortening operation time.

[Diagonal root separation method for extracting impacted double-root mandibular third molars].

Double-root mandibular third molars are the common types of mandibular third molars that are prone to impac-tion. Based on the summary of clinical cases and experience, the commonly used crown-root separation technique is improved as diagonal root separation technique that divides the roots by the line connecting the distal axis angle to the root furcation. This new method improves the efficiency of the operation and reduces surgical trauma. This paper details the surgical method, minimally invasive mechanics, and key points of this technique, compares the technique with the crown-root separation tech-nique, and discusses their joint applications.

P34HB electrospun fibres promote bone regeneration in vivo.

OBJECTIVE: Bone tissue engineering was introduced in 1995 and provides a new way to reconstruct bone and repair bone defects. However, the design and fabrication of suitable bionic bone scaffolds are still challenging, and the ideal scaffolds in bone tissue engineering should have a three-dimensional porous network, good biocompatibility, excellent biodegradability and so on. The purpose of our research was to investigate whether a bioplasticpoly3-hydroxybutyrate4-hydroxybutyrate (P34HB) electrospun fibre scaffold is conducive to the repair of bone defects, and whether it is a potential scaffold for bone tissue engineering. MATERIALS AND METHODS: The P34HB electrospun fibre scaffolds were prepared by electrospinning technology, and the surface morphology, hydrophilicity, mechanical properties and cytological behaviour of the scaffolds were tested. Furthermore, a calvarial defect model was created in rats, and through layer-by-layer paper-stacking technology, the P34HB electrospun fibre scaffolds were implanted into the calvarial defect area and their effect on bone repair was evaluated. RESULTS: The results showed that the P34HB electrospun fibre scaffolds are interwoven with several fibres and have good porosity, physical properties and chemical properties and can promote cell adhesion and proliferation with no cytotoxicity in vitro. In addition, the P34HB electrospun fibre scaffolds can promote the repair of calvarial defects in vivo. CONCLUSIONS: These results demonstrated that the P34HB electrospun fibre scaffold has a three-dimensional porous network with good biocompatibility, excellent biosafety and ability for bone regeneration and repair; thus, the P34HB electrospun fibre scaffold is a potential scaffold for bone tissue engineering.

KDM6A promotes chondrogenic differentiation of periodontal ligament stem cells by demethylation of SOX9.

OBJECTIVES: KDM6A has been demonstrated critical in the regulation of cell fates. However, whether KDM6A is involved in cartilage formation remains unclear. In this study, we investigated the role of KDM6A in chondrogenic differentiation of PDLSCs, as well as the underlying epigenetic mechanisms. METHODS: KDM6A shRNA was transfected into PDLSCs by lentivirus. The chondrogenic differentiation potential of PDLSCs was assessed by Alcian blue staining. Immunofluorescence was performed to demonstrate H3K27me3 and H3K4me3 levels during chondrogenesis. SOX9, Col2a1, ACAN and miRNAs (miR-29a, miR-204, miR-211) were detected by real-time RT-PCR. Western blot was performed to evaluate SOX9, H3K27me3 and H3K4me3. RESULTS: The production of proteoglycans in PDLSCs was decreased after knockdown of KDM6A. Depletion of KDM6A inhibited the expression of SOX9, Col2a1, ACAN and resulted in increased H3K27me3 and decreased H3K4me3 levels. EZH2 inhibitor rescued the chondrogenic potential of PDLSCs after knockdown of KDM6A by regulating H3K27me3. Additionally, miR-29a, miR-204 and miR-211 were also involved in the process of PDLSCs chondrogenesis. CONCLUSIONS: KDM6A is required in chondrogenic differentiation of PDLSCs by demethylation of H3K27me3, and EZH2 inhibitor could rescue chondrogenesis of PDLSCs after knockdown of KDM6A. It could be inferred that upregulation of KDM6A or application of EZH2 inhibitor might improve mesenchymal stem cell mediated cartilage regeneration in inflammatory tissue destruction such as osteoarthritis.

Research Progress of the Types and Preparation Techniques of Scaffold Materials in Cartilage Tissue Engineering.

The management of chondral defects has been a challenge for a long time because of the poor self-healing capacity of articular cartilage. Many approaches ranging from symptomatic treatment to structural cartilage regeneration are not that successful with very limited satisfactory results. Chondral defects caused by tumor, trauma, infection, congenital malformations are very common in clinical trials. It seriously affects the patient's physical function and quality of life. The appearance of cartilage tissue engineering has brought good news for cartilage defect repair. Through this review, we are aimed at reviewing the progress of the types and preparation techniques of scaffold materials in cartilage tissue engineering.

Tumorigenicity analysis of heterogeneous dental stem cells and its self-modification for chromosome instability.

Heterogeneity demonstrates that stem cells are constituted by several sub-clones in various differentiation states. The heterogeneous state is maintained by cross-talk among sub-clones, thereby ensuring stem cell adaption. In this study, we investigated the roles of heterogeneity on genetic stability. Three sub-clones (DF2, DF8 and DF18) were isolated from heterogeneous dental stem cells (DSCs), and were proved to be chromosome instability (CIN) after long term expansion. Cell apoptosis were not detected in sub-clones, which exhibited strong tumorigenesis tendency, coupled with weak expression of p53 and aberrant ultra-structure. However, 3 sub-clones did not overexpress tumor related markers or induce tumorigenesis in vivo. The mixed-culture study suggested that 3-clone-mixed culturing cells (DF1) presented apparent decrease in the ratio of aneuploidy. The screening experiment further proved that 3 sub-clones functioned separately in this modification procedure but only mixed culturing all 3 sub-clones, simulated heterogeneous microenvironment, could achieve complete modification. Additionally, osteogenesis capability of 3 sub-clones was partially influenced by CIN while DSCs still kept stronger osteogenesis than sub-clones. These results suggested aberrant sub-clones isolated from heterogeneous DSCs were not tumorigenesis and could modify CIN by cross-talk among themselves, indicating that the heterogeneity played a key role in maintaining genetic stability and differentiation capability in dental stem cells.

Restoration of critical-size defects in the rabbit mandible using porous nanohydroxyapatite-polyamide scaffolds.

Composite nanohydroxyapatite/polyamide (n-HA/PA) biomaterials have been indicated for bone defect reconstruction, where PA is added to enhance the toughness of n-HA. However, a comprehensive understanding of the biological performance of this implant material remains to be determined. In this study, the biological activity of n-HA/PA biomaterials was characterized in vitro by assessing the growth of bone marrow stromal cells (BMSCs), and in an in vivo rabbit model. To evaluate the n-HA/PA performance under different osteogenic conditions in vivo, implants were inserted to critical-size bone defects in the angle and body of the rabbit mandible. To determine the necessity of ectogenic BMSC-n-HA/PA hybrids at different implantation sites, both raw n-HA/PA materials and BMSC-seeded n-HA/PA hybrids were implanted. Bone formation was detected by radiology and histological studies. The results showed that n-HA/PA composites had great bioactivity, demonstrating significant BMSC proliferation, active alkaline phosphatase secretion, and stimulating the expression of osteogenic proteins (bone morphogenetic protein 2 [BMP2], osteoprotegerin [OPG], osteopontin [OPN], collagen type I [Col I], and osteocalcin [OCN]), in comparison to the control (polyethylene). At marrow-rich implantation sites (mandibular body), the amount of new bone formation was significant, but was not enhanced by the presence of BMSCs in the BMSC-n-HA/PA hybrids. However, the BMSC-n-HA/PA hybrids were essential for promoting bone formation in marrow-poor sites (mandibular angle). In conclusion, n-HA/PA biomaterials, which offer the advantage of enhanced mechanical performance over n-HA, exhibit significant bioactivity, including the capacity for bone regeneration at marrow-poor sites when implanted in combination with BMSCs.