The Role of Autophagy on Osteogenesis of Dental Follicle Cells Under Inflammatory Microenvironment.

OBJECTIVE: This study investigated the role of autophagy on osteogenesis of DFCs under inflammatory microenvironment during tooth eruption. METHODS: DFCs were isolated and identified. Lipopolysaccharide (LPS) was used to construct the inflammatory microenvironment in vitro and in vivo. Cell viability was examined by CCK-8 assay. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining. The gene and protein levels were examined using qRT-PCR and western blot analysis, respectively. We observed the process of tooth eruption after local LPS injection by micro-CT and HE staining. Osteogenesis and autophagy were monitored through qRT-PCR, western blot and histological staining of specific markers. RESULTS: LPS at the indicated concentrations did not produce toxic effects on DFCs, and significantly promoted the inflammatory gene expression. LPS inhibited osteogenic differentiation and activated autophagy in DFCs. Blocking autophagy with 3-MA reversed the expression of osteogenic markers in LPS-treated DFCs. Additionally, the eruption of LPS-treated teeth was accelerated and their DFs exhibited an increased expression of TNF-α and Beclin1, and decreased expression of ALP and RUNX2. CONCLUSIONS: Autophagy was involved in the suppression of the DFCs osteogenesis in an LPS-induced inflammatory condition, suggesting the pivotal role of autophagy in inflammation-induced premature tooth eruption.

4-Methylumbelliferone promotes the migration and odontogenetic differentiation of human dental pulp stem cells exposed to lipopolysaccharide in vitro.

Hyaluronic acid (HA), a major component of the extracellular matrix, is essential to inflammatory regulation. 4-Methylumbelliferone (4-mu), as the specific inhibitor of HA synthesis, is an anti-inflammatory in multiple systems. However, there have been no studies, to our knowledge, regarding 4-mu treatment in pulp inflammation. Therefore, the purpose of this study was to investigate the effects of 4-mu on biological behaviors in human dental pulp stem cells (hDPSCs) exposed to lipopolysaccharide (LPS) in vitro. hDPSCs were exposed to LPS to construct the inflammation model in vitro. Immunocytochemistry, quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, scratch/Transwell assay, and alizarin red staining/alkaline phosphatase staining were selected to explore the effect of 4-mu on the expression of inflammatory factors, cell proliferation, cell migration, and the odontogenic differentiation ability of hDPSCs. LPS stimulated hDPSCs to highly express the related inflammatory factors and CD44 (the major HA receptor), which were all inhibited by 0.1 mM of 4-mu. In addition, the cell proliferation ability of hDPSCs was suppressed by 4-mu, while cell migration and odontogenic differentiation abilities were significantly improved under inflammation. In conclusion, 4-mu suppressed inflammatory cytokines in inflamed hDPSCs and had a positive effect on the migration and odontogenic differentiation of hDPSCs.

A novel mutation of adenomatous polyposis coli (APC) gene results in the formation of supernumerary teeth.

Supernumerary teeth are teeth that are present in addition to normal teeth. Although several hypotheses and some molecular signalling pathways explain the formation of supernumerary teeth, but their exact disease pathogenesis is unknown. To study the molecular mechanisms of supernumerary tooth-related syndrome (Gardner syndrome), a deeper understanding of the aetiology of supernumerary teeth and the associated syndrome is needed, with the goal of inhibiting disease inheritance via prenatal diagnosis. We recruited a Chinese family with Gardner syndrome. Haematoxylin and eosin staining of supernumerary teeth and colonic polyp lesion biopsies revealed that these patients exhibited significant pathological characteristics. APC gene mutations were detected by PCR and direct sequencing. We revealed the pathological pathway involved in human supernumerary tooth development and the mouse tooth germ development expression profile by RNA sequencing (RNA-seq). Sequencing analysis revealed that an APC gene mutation in exon 15, namely 4292-4293-Del GA, caused Gardner syndrome in this family. This mutation not only initiated the various manifestations typical of Gardner syndrome but also resulted in odontoma and supernumerary teeth in this case. Furthermore, RNA-seq analysis of human supernumerary teeth suggests that the APC gene is the key gene involved in the development of supernumerary teeth in humans. The mouse tooth germ development expression profile shows that the APC gene plays an important role in tooth germ development. We identified a new mutation in the APC gene that results in supernumerary teeth in association with Gardner syndrome. This information may shed light on the molecular pathogenesis of supernumerary teeth. Gene-based diagnosis and gene therapy for supernumerary teeth may become available in the future, and our study provides a high-resolution reference for treating other syndromes associated with supernumerary teeth.

Concise reviews: Characteristics and potential applications of human dental tissue-derived mesenchymal stem cells.

Recently, numerous types of human dental tissue-derived mesenchymal stem cells (MSCs) have been isolated and characterized, including dental pulp stem cells, stem cells from exfoliated deciduous teeth, periodontal ligament stem cells, dental follicle progenitor cells, alveolar bone-derived MSCs, stem cells from apical papilla, tooth germ progenitor cells, and gingival MSCs. All these MSC-like cells exhibit self-renewal, multilineage differentiation potential, and immunomodulatory properties. Several studies have demonstrated the potential advantages of dental stem cell-based approaches for regenerative treatments and immunotherapies. This review outlines the properties of various dental MSC-like populations and the progress toward their use in regenerative therapy. Several dental stem cell banks worldwide are also introduced, with a view toward future clinical application.

Effect of Eruption Status of the Mandibular Third Molar on Distal Caries in the Adjacent Second Molar.

PURPOSE: To analyze the effect of the eruption status of the mandibular third molar (MTM) on distal caries in the mandibular second molar (MSM) by cone-beam computed tomography (CBCT). MATERIALS AND METHODS: Five hundred CBCT images of MTMs from 469 patients were evaluated. Presence of distal caries in MSMs, impaction depths and angulations of MTMs, cementoenamel junction (CEJ) distances between distal MSMs and mesial MTMs, presence of pericoronitis in MTMs, and patient characteristics (age and gender) were assessed. Data were analyzed by χ(2) test, univariate and multivariate logistic regression analyses, and Spearman correlation analysis. Descriptive and bivariate statistics were computed and the P value was set at .05. RESULTS: The overall prevalence of distal caries in the MSM was 52.0%. According to the classification of Pell and Gregory, position A was the impaction depth at which most distal caries in MSMs were present (P = .036). For angulation of the MTM, when mesial angulations were 43° to 73°, MSMs developed more distal caries (P < .0001). For the CEJ distance between the distal MSM and the mesial MTM, when distances ranged from 6 to 15 mm, distal caries in MSMs occurred more frequently (6 to 8 mm, P < .0001; 8 to 15 mm, P = .037). Furthermore, there was a linear correlation between angulation of the MTM and the CEJ distance between the distal MSM and the mesial MTM (P < .0001). CONCLUSIONS: Impaction depth and angulation of the MTM are associated with distal caries in the MSM. Angulation of the MTM is more stable and reliable than the CEJ distance between the distal MSM and the mesial MTM for the estimation of risk factors related to the MTM.

Current approaches to the diagnosis and treatment of white sponge nevus.

White sponge nevus (WSN) in the oral mucosa is a rare autosomal dominant genetic disease. The involved mucosa is white or greyish, thickened, folded and spongy. The genes associated with WSN include mutant cytokeratin keratin 4 (KRT4) and keratin 13 (KRT13). In recent years, new cases of WSN and associated mutations have been reported. Here, we summarise the recent progress in our understanding of WSN, including clinical reports, genetics, animal models, treatment, pathogenic mechanisms and future directions. Gene-based diagnosis and gene therapy for WSN may become available in the near future and could provide a reference and instruction for treating other KRT-associated diseases.

The effect of injectable platelet-rich fibrin and platelet-rich fibrin in regenerative endodontics: a comparative in vitro study.

OBJECTIVE: To explore the feasibility of injectable platelet-rich fibrin (i-PRF) in regenerative endodontics by comparing the effect of i-PRF and platelet-rich fibrin (PRF) on the biological behavior and angiogenesis of human stem cells from the apical papilla (SCAPs). METHODOLOGY: i-PRF and PRF were obtained from venous blood by two different centrifugation methods, followed by hematoxylin-eosin (HE) staining and scanning electron microscopy (SEM). Enzyme-linked immunosorbent assay (ELISA) was conducted to quantify the growth factors. SCAPs were cultured with different concentrations of i-PRF extract (i-PRFe) and PRF extract (PRFe), and the optimal concentrations were selected using the Cell Counting Kit-8 (CCK-8) assay. The cell proliferation and migration potentials of SCAPs were then observed using the CCK-8 and Transwell assays. Mineralization ability was detected by alizarin red staining (ARS), and angiogenesis ability was detected by tube formation assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the expression of genes related to mineralization and angiogenesis. The data were subjected to statistical analysis. RESULTS: i-PRF and PRF showed a similar three-dimensional fibrin structure, while i-PRF released a higher concentration of growth factors than PRF ( P <.05). 1/4× i-PRFe and 1/4× PRFe were selected as the optimal concentrations. The cell proliferation rate of the i-PRFe group was higher than that of the PRFe group ( P <.05), while no statistical difference was observed between them in terms of cell mitigation ( P >.05). More importantly, our results showed that i-PRFe had a stronger effect on SCAPs than PRFe in facilitating mineralization and angiogenesis, with the consistent result of RT-qPCR ( P <.05). CONCLUSION: This study revealed that i-PRF released a higher concentration of growth factors and was superior to PRF in promoting proliferation, mineralization and angiogenesis of SCAPs, which indicates that i-PRF could be a promising biological scaffold for application in pulp regeneration.

Hyaluronan degradation by HYAL2 is essential for odontoblastic differentiation and migration of mouse dental papilla cells.

The coordination between odontoblastic differentiation and directed cell migration of mesenchymal progenitors is necessary for regular dentin formation. The synthesis and degradation of hyaluronan (HA) in the extracellular matrix create a permissive niche that directly regulates cell behaviors. However, the role and mechanisms of HA degradation in dentin formation remain unknown. In this work, we present that HA digestion promotes odontoblastic differentiation and cell migration of mouse dental papilla cells (mDPCs). Hyaluronidase 2 (HYAL2) is responsible for promoting odontoblastic differentiation through degrading HA, while hyaluronidase 1 (HYAL1) exhibits negligible effect. Silencing Hyal2 generates an extracellular environment rich in HA, which attenuates F-actin and filopodium formation and in turn inhibits cell migration of mDPCs. In addition, activating PI3K/Akt signaling significantly rescues the effects of HA accumulation on cytodifferentiation. Taken together, the results confirm the contribution of HYAL2 to HA degradation in dentinogenesis and uncover the mechanism of the HYAL2-mediated HA degradation in regulating the odontoblastic differentiation and migration of mDPCs.

Experts consensus on management of tooth luxation and avulsion.

Traumatic dental injuries (TDIs) of teeth occur frequently in children and adolescents. TDIs that impact the periodontal tissues and alveolar tissue can be classified into concussion, subluxation, extrusive luxation, intrusive luxation, lateral luxation, and avulsion. In these TDIs, management of injured soft tissue, mainly periodontal ligament, and dental pulp, is crucial in maintaining the function and longevity of the injured teeth. Factors that need to be considered for management in laxation injuries include the maturation stage of the traumatic teeth, mobility, direction of displacement, distance of displacement, and whether there are alveolar fractures. In avulsion, the maturation stage of the permanent tooth, the out-socket time, storage media/condition of the avulsed tooth, and management of the PDL should also be considered. Especially, in this review, we have subdivided the immature tooth into the adolescent tooth (Nolla stage 9) and the very young tooth (Nolla stage 8 and below). This consensus paper aimed to discuss the impacts of those factors on the trauma management and prognosis of TDI to provide a streamlined guide for clinicians from clinical evaluation, diagnostic process, management plan decision, follow-up, and orthodontic treatment for tooth luxation and avulsion injuries.

Expert consensus on pediatric orthodontic therapies of malocclusions in children.

Malocclusion, identified by the World Health Organization (WHO) as one of three major oral diseases, profoundly impacts the dental-maxillofacial functions, facial esthetics, and long-term development of ~260 million children in China. Beyond its physical manifestations, malocclusion also significantly influences the psycho-social well-being of these children. Timely intervention in malocclusion can foster an environment conducive to dental-maxillofacial development and substantially decrease the incidence of malocclusion or reduce the severity and complexity of malocclusion in the permanent dentition, by mitigating the negative impact of abnormal environmental influences on the growth. Early orthodontic treatment encompasses accurate identification and treatment of dental and maxillofacial morphological and functional abnormalities during various stages of dental-maxillofacial development, ranging from fetal stages to the early permanent dentition phase. From an economic and societal standpoint, the urgency for effective early orthodontic treatments for malocclusions in childhood cannot be overstated, underlining its profound practical and social importance. This consensus paper discusses the characteristics and the detrimental effects of malocclusion in children, emphasizing critical need for early treatment. It elaborates on corresponding core principles and fundamental approaches in early orthodontics, proposing comprehensive guidance for preventive and interceptive orthodontic treatment, serving as a reference for clinicians engaged in early orthodontic treatment.

Spatial and Temporal Expression of Ectodysplasin-A Signaling Pathway Members During Mandibular Condylar Development in Postnatal Mice.

A growing body of evidence emerging supported that ectodysplasin-A (EDA) signaling pathway contributed to craniofacial development. However, their expression in condyle has not been elucidated yet. This study investigated the expression patterns of EDA, EDA receptor (EDAR), and EDAR-associated death domain (EDARADD) in condyle of postnatal mice. Histological staining and micro-computed tomography (CT) scanning showed that as endochondral ossification proceeded, the thickness of chondrocyte layer decreased, and the volume of mandibular condyle increased. Osteoclasts remained active throughout the condylar development. Immunohistochemistry staining demonstrated that EDA was expressed in almost all layers during the first 2 weeks after birth. EDA shifted from the mature and hypertrophic layers to fibrous and proliferating layers at postnatal 3 weeks. As condyle matured, the distribution of EDA tended to be limited to hypertrophic layer. The distribution patterns of EDAR and EDARADD were consistent with EDA, while the level of EDAR expression was slightly lower. mRNA expression levels of EDA signaling pathway-related components increased after birth. Furthermore, we evaluated the expression of EDA using ATDC5 in vitro. EDA increased during the late stage of chondrogenesis. These findings proved that EDA signaling pathway was involved in condylar development and acted as a regulatory factor in condylar maturation and differentiation.

Effects of immunoglobulin Y-loaded amorphous calcium phosphate on dentinal tubules occlusion and antibacterial activity.

Aim: This study aimed to evaluate the effects of immunoglobulin Y (IgY)-loaded amorphous calcium phosphate (ACP) (IgY@ACP) on dentinal tubule occlusion and antibacterial activity. Methodology: IgY@ACP was synthesized based on a biomimetic mineralization strategy. The structure was examined by transmission electron microscopy and Fourier transform infrared spectroscopy. The IgY release property was assessed in vitro. The cell biocompatibility of IgY@ACP was evaluated by CCK-8. The dentin disks were prepared using healthy human molars, and their dentinal tubules were exposed to EDTA. Subsequently, they were randomly selected and treated with or without IgY@ACP for 7 days. The tubule occlusion morphologies and newly formed layers were observed by scanning electron microscopy (SEM) and x-ray diffraction, respectively. To evaluate the acid resistance and abrasion resistance of IgY@ACP, dentin disks that were treated for 1 day were immersed in acid solution or subjected to a toothbrush. The antibacterial effects against Streptococcus mutans (S. mutans) were evaluated by colony-forming unit (CFU) counting, adhesion property assessment, and crystal violet and live/dead bacterial staining. Finally, the occlusion effect was evaluated in rat incisors in vivo. One-way analysis of variance (ANOVA) was performed for statistical analysis. The level of significance was set at 0.05. Results: IgY@ACP presented an amorphous phase with a nanosize (60-80 nm) and sustained release of protein within 48 h. The CCK-8 results showed that IgY@ACP had good biocompatibility. After treatment with IgY@ACP for 1 day, the majority of dentinal tubules were occluded by a 0.3-µm-thick mineralized layer. Seven days later, all dentinal tubules were occluded by mineralization with a thickness of 1.4 µm and a depth of 16 µm. The newly mineralized layer showed hydroxyapatite-like diffraction peaks. In addition, IgY@ACP had good acid and abrasion resistance. After treatment with IgY@ACP, the CFU counting and adhesion rate of S. mutans were significantly reduced, the crystal violet staining was lighter, and the S. mutans staining revealed more dead cells. Most importantly, IgY@ACP had a certain occluding property in rat incisors in vivo. Conclusion: IgY@ACP can effectively occlude dentinal tubules with acid-resistant stability and has prominent anti-S. mutans effects, rendering it a potentially suitable desensitization material in the clinic.

Expert consensus on clinical management for early childhood caries.

Early childhood caries (ECC) is the most prevalent chronic oral disease and one of the worldwide public health problems of great urgency for children. ECC can affect children's teeth, dentition, craniomaxillofacial, and general health and development. Therefore, through dental caries etiologies and caries risk assessment, patient-centered, personalized planning and a combination of prevention and treatment should be implemented in the clinical management for ECC. Periodic and continuous cycle management can only be accomplished with the cooperation of medical staff, children, and their guardians. This expert consensus will expound the clinical management of ECC in the following aspects: caries risk assessment, early clinical prevention, treatment strategies, and postoperative management.

Expert consensus on early childhood caries management.

Early childhood caries (ECC) is a significant chronic disease of childhood and a rising public health burden worldwide. ECC may cause a higher risk of new caries lesions in both primary and permanent dentition, affecting lifelong oral health. The occurrence of ECC has been closely related to the core microbiome change in the oral cavity, which may be influenced by diet habits, oral health management, fluoride use, and dental manipulations. So, it is essential to improve parental oral health and awareness of health care, to establish a dental home at the early stage of childhood, and make an individualized caries management plan. Dental interventions according to the minimally invasive concept should be carried out to treat dental caries. This expert consensus mainly discusses the etiology of ECC, caries-risk assessment of children, prevention and treatment plan of ECC, aiming to achieve lifelong oral health.

Experts' consensus on space management of mixed dentition.

The mixed dentition stage is the period between primary and permanent dentition. The following biological processes are complicated and variable: jaw growth, development of inherited permanent teeth embryo, physiological absorption of primary teeth, restoration of surrounding alveolar bones, and growth and function establishment of soft tissues. For the normal development of the jaw, the establishment of the good occlusion relationship, development, and function of soft tissue is very important, whether or not the primary teeth are normally replaced by the permanent teeth in the mixed dentition stage. The eruption space is linked to the normal replacement of primary and permanent teeth. The presence of a mixed dentition space results in the incidence and progression of malocclusion and impacts the normal growth and development of the occlusion, jaw, and face. Space management in the mixed dentition stage is a crucial means to prevent and reduce malocclusion. The following were discussed and analyzed: the possible space problems, why the size of the space was affected, the content that needs to be assessed, and the methods of space management in the mixed dentition that can be used to unify and standardize the management of mixed dentition. This paper was developed to serve as a guide for regulated space management during the mixed dentition period.

[Effect of DKK1 on the biological behaviors of dental pulp cells exposed to lipopolysaccharide].

PURPOSE: To detect the effect of DKK1 on biological behaviors of human dental pulp cells exposed to lipopolysaccharide (LPS). METHODS: The dental pulp cells were isolated and cultured by modified enzyme-tissue block method and identified by immunofluorescence staining. The effect of DKK1 on proliferation and migration of human dental pulp cells exposed to LPS were measured by cell counting kit (CCK-8) and Transwell assay. Meanwhile, the effect of DKK1 on differentiation of human dental cells exposed to LPS were studied by alizarin red staining and real-time PCR experiment, statistical analysis was performed using SPSS 20.0 software package. RESULTS: The results of immunofluorescence showed that the cultured cells were in consistent with the mesenchymal stem cells. The result of CCK-8 indicated that DKK1 had no significant effect on proliferation of dental pulp cells exposed to LPS; The result of transwell assay showed that DKK1 significantly promoted the cell migration of dental pulp cells exposed to LPS. The results of Alizarin red staining and real-time PCR revealed that DKK1 could promote cytodifferentiation of dental pulp cells exposed to LPS. CONCLUSIONS: DKK1 promotes the ability of cell migration and cytodifferentiation of LPS treated dental pulp cells, which may be resulted from inhibition of Wnt/ß-catenin pathway.

Potential apply of hydrogel-carried chlorhexidine and metronidazole in root canal disinfection.

Microorganisms may persist in the root canal system after root canal therapy (RCT). The purpose of this study was to explore the metronidazole (MTR)- and chlorhexidine (CHX)-loaded hydrogels as the potential application in intracanal medicaments for root canal disinfection. Ultraviolet cross-linked hydrogels (gGels) were synthesized by GelMA solution and photoinitiator, which were loaded with MTR (MTR@gGels) and CHX (CHX@gGels). gGels, MTR@gGels and CHX@gGels were characterized by scanning electron microscopy. The antimicrobial activity against E. faecalis, S. mutans and P. intermedia was assessed. Meanwhile, the biocompatibility of human dental pulp stem cells (hDPSCs) was evaluated. DCT, CCK-8, CFU and live/dead-stained biofilm results showed that the viability of E. faecalis, S. mutans and P. intermedia was significantly reduced in MTR@gGels and CHX@gGels in vitro. CCK-8 results showed considerable biocompatibility with hDPSCs. The filling and clearance of gGels in root canals were demonstrated in vivo. Therefore, MTR- and CHX-loaded hydrogels may be a potential application in intracanal medicaments for root canal disinfection.

China experts' consensus on preventive and interceptive orthodontic treatments of malocclusions of children.

Malocclusion is one of the three most common oral diseases reported by World Health Organization(WHO). In China, its incidence rate is rising. Malocclusion seriously affects the dental and maxillofacial function, facial appearance and growth development of nearly 260 million children in China, and what is more, it affects their physical and mental health development. Malocclusion occurrence is related to genetic and environmental factors. Early treatment of malocclusion can create a good dental and maxillofacial development environment, correct abnormal growth and control the adverse effects of abnormal genetic factors. It can effectively reduce the prevalence of children's malocclusion and enhance their physical and mental health. This is an urgent need from the economic perspective of our society, so it has great practical and social significance. Experts from the project group "standard diagnose and treatment protocols for early orthodontic intervention of malocclusions of children" which initiated by China National Health Institute of Hospital Administration wrote the "China Experts' Consensus on Preventive and Interceptive Orthodontic Treatments of Malocclusions of Children", which aims to guide and popularize the clinical practice, improve the clinical theory and practice level, and accelerate the disciplinary development of early treatment of children's malocclusion in China. The consensus elaborates the harmfulness of malocclusion and the necessity of early treatment, and brings up the principles and fundamental contents. Based on the law of dental and maxillofacial development, this paper puts forward the guiding suggestions of preventive and interceptive treatments in different stages of dental development ranging from fetus to early permanent dentition. It is a systematic project to promote and standardize the early treatment of malocclusion. Through scientific and comprehensive stratified clinical practice and professional training, the clinical system of early treatment of malocclusion in China will eventually be perfected, so as to comprehensively care for children's dental and maxillofacial health, and improve their oral and physical health in China.

The effect of injectable platelet-rich fibrin and platelet-rich fibrin in regenerative endodontics: a comparative in vitro study

Abstract Objective To explore the feasibility of injectable platelet-rich fibrin (i-PRF) in regenerative endodontics by comparing the effect of i-PRF and platelet-rich fibrin (PRF) on the biological behavior and angiogenesis of human stem cells from the apical papilla (SCAPs). Methodology i-PRF and PRF were obtained from venous blood by two different centrifugation methods, followed by hematoxylin-eosin (HE) staining and scanning electron microscopy (SEM). Enzyme-linked immunosorbent assay (ELISA) was conducted to quantify the growth factors. SCAPs were cultured with different concentrations of i-PRF extract (i-PRFe) and PRF extract (PRFe), and the optimal concentrations were selected using the Cell Counting Kit-8 (CCK-8) assay. The cell proliferation and migration potentials of SCAPs were then observed using the CCK-8 and Transwell assays. Mineralization ability was detected by alizarin red staining (ARS), and angiogenesis ability was detected by tube formation assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the expression of genes related to mineralization and angiogenesis. The data were subjected to statistical analysis. Results i-PRF and PRF showed a similar three-dimensional fibrin structure, while i-PRF released a higher concentration of growth factors than PRF ( P <.05). 1/4× i-PRFe and 1/4× PRFe were selected as the optimal concentrations. The cell proliferation rate of the i-PRFe group was higher than that of the PRFe group ( P <.05), while no statistical difference was observed between them in terms of cell mitigation ( P >.05). More importantly, our results showed that i-PRFe had a stronger effect on SCAPs than PRFe in facilitating mineralization and angiogenesis, with the consistent result of RT-qPCR ( P <.05). Conclusion This study revealed that i-PRF released a higher concentration of growth factors and was superior to PRF in promoting proliferation, mineralization and angiogenesis of SCAPs, which indicates that i-PRF could be a promising biological scaffold for application in pulp regeneration.

The inhibition of glycosaminoglycan incorporation influences the cell proliferation and cytodifferentiation in cultured embryonic mouse molars.

The extracellular matrix (ECM) contains a variety of complex macromolecules including proteoglycans (PGs) and glycosaminoglycans (GAGs). PG consists of a protein core with covalently attached carbohydrate side chains called GAGs. Several PGs, including versican, biglycan, decorin and syndecan are involved in odontogenesis while the role of GAGs in those PGs in this process remains unclarified. The purpose of this study was to investigate the influence of GAGs on tooth development. The mandibular first molars at early bell stage were cultivated with or without 4-methylumbelliferyl-ß-D-xyloside (Xyl-MU). The cultured tooth germs were metabolically labelled with [35S] Na2SO4, then PGs in tooth germs and cultured medium were extracted separately and analyzed by gel filtration. Morphological changes were evaluated on days 2, 4, 6, and histological changes were examined by hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). Related proteins and genes of cytodifferentiation were further examined by immunohistochemistry (IHC) and quantitive real-time PCR (qPCR) respectively. Meanwhile, BrdU incorporation assay was used to explore the effect of Xyl-MU on the cell proliferation of cultured tooth germs. The results demonstrated that the incorporation of GAGs to PGs in cultured tooth germs was heavily inhibited by Xyl-MU. Accompanied by the inhibition of GAGs incorporation, Xyl-MU altered tooth morphogenesis and delayed the differentiation of ameloblasts and odontoblasts. Proliferation of inner enamel epithelium (IEE) was also inhibited. Therefore, we draw a conclusion that the inhibition of GAGs incorporation influences the cell proliferation and cytodifferentiation in cultured embryonic mouse molars.