Developmental endothelial locus-1 promotes osteogenic differentiation and alveolar bone regeneration in experimental periodontitis with type 2 diabetes mellitus.

OBJECTIVES: This study sought to explore the role of developmental endothelial locus-1 (DEL-1) in osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and investigate the therapeutic effect of DEL-1 in ligature-induced experimental periodontitis with type 2 diabetes mellitus (T2DM). BACKGROUND: T2DM is a significant risk factor for periodontitis. Treatment modalities for periodontitis with T2DM are being explored. DEL-1 is a versatile protein that can modulate the different stages of inflammatory diseases including periodontitis. The direct effect of DEL-1 on osteogenic differentiation of PDLSCs in periodontitis with T2DM is poorly understood. METHODS: Primary hPDLSCs were isolated from periodontal ligament tissue and identified by flow cytometry. In osteogenesis experiments, alkaline phosphatase (ALP), Alizarin Red staining and western blot were used to assess the osteogenic effect of DEL-1 on hPDLSCs in high glucose and inflammation environments. The mouse model of ligature-induced experimental periodontitis was established. H&E and Masson's trichrome staining were used to assess the change of periodontal tissue after local periodontal injection of DEL-1. Immunohistochemical staining was used to evaluate osteogenic-related protein expression. RESULTS: hPDLSCs expressed mesenchymal stem cell (MSC)-specific surface markers and were negative for hematopoietic cell surface markers. hPDLSCs had the potential for multidirectional differentiation. DEL-1 could enhance the osteogenic differentiation of hPDLSCs in high glucose and inflammation environments, although it did not return to the control level. Histological staining showed that DEL-1 contributed to alveolar bone regeneration and osteogenic-related protein expression, but the degree of improvement in T2DM mice was lower than in non-T2DM mice. CONCLUSIONS: In summary, we demonstrated that DEL-1 could promote osteogenic differentiation of hPDLSCs in high glucose and inflammation environment and rescue alveolar bone loss in experimental periodontitis with T2DM, which could provide a novel therapeutic target for periodontitis with T2DM.

Lipotoxicity: The missing link between diabetes and periodontitis?

Lipotoxicity refers to the accumulation of lipids in tissues other than adipose tissue (body fat). It is one of the major pathophysiological mechanisms responsible for the progression of diabetes complications such as non-alcoholic fatty liver disease and diabetic nephropathy. Accumulating evidence indicates that lipotoxicity also contributes significantly to the toxic effects of diabetes on periodontitis. Therefore, we reviewed the current in vivo, in vitro, and clinical evidence of the detrimental effects of lipotoxicity on periodontitis, focusing on its molecular mechanisms, especially oxidative and endoplasmic reticulum stress, inflammation, ceramides, adipokines, and programmed cell death pathways. By elucidating potential therapeutic strategies targeting lipotoxicity and describing their associated mechanisms and clinical outcomes, including metformin, statins, liraglutide, adiponectin, and omega-3 PUFA, this review seeks to provide a more comprehensive and effective treatment framework against diabetes-associated periodontitis. Furthermore, the challenges and future research directions are proposed, aiming to contribute to a more profound understanding of the impact of lipotoxicity on periodontitis.

Comparative assessment of orthodontic clear aligner versus fixed appliance for anterior retraction: a finite element study.

BACKGROUND: The aim of this study is to conduct a comparative evaluation of different designs of clear aligners and examine the disparities between clear aligners and fixed appliances. METHODS: 3D digital models were created, consisting of a maxillary dentition without first premolars, maxilla, periodontal ligaments, attachments, micro-implant, 3D printed lingual retractor, brackets, archwire and clear aligner. The study involved the creation of five design models for clear aligner maxillary anterior internal retraction and one design model for fixed appliance maxillary anterior internal retraction, which were subsequently subjected to finite element analysis. These design models included: (1) Model C0 Control, (2) Model C1 Posterior Micro-implant, (3) Model C2 Anterior Micro-implant, (4) Model C3 Palatal Plate, (5) Model C4 Lingual Retractor, and (6) Model F0 Fixed Appliance. RESULTS: In the clear aligner models, a consistent pattern of tooth movement was observed. Notably, among all tested models, the modified clear aligner Model C3 exhibited the smallest differences in sagittal displacement of the crown-root of the central incisor, vertical displacement of the central incisor, sagittal displacement of the second premolar and second molar, as well as vertical displacement of posterior teeth. However, distinct variations in tooth movement trends were observed between the clear aligner models and the fixed appliance model. Furthermore, compared to the fixed appliance model, significant increases in tooth displacement were achieved with the use of clear aligner models. CONCLUSIONS: In the clear aligner models, the movement trend of the teeth remained consistent, but there were variations in the amount of tooth displacement. Overall, the Model C3 exhibited better torque control and provided greater protection for posterior anchorage teeth compared to the other four clear aligner models. On the other hand, the fixed appliance model provides superior anterior torque control and better protection of the posterior anchorage teeth compared to clear aligner models. The clear aligner approach and the fixed appliance approach still exhibit a disparity; nevertheless, this study offers a developmental direction and establishes a theoretical foundation for future non-invasive, aesthetically pleasing, comfortable, and efficient modalities of clear aligner treatment.

Automated detection of anterior crossbite on intraoral images and videos utilizing deep learning.

AIM: Malocclusion has emerged as a burgeoning global public health concern. Individuals with an anterior crossbite face an elevated risk of exhibiting characteristics such as a concave facial profile, negative overjet, and poor masticatory efficiency. In response to this issue, we proposed a convolutional neural network (CNN)-based model designed for the automated detection and classification of intraoral images and videos. MATERIALS AND METHODS: A total of 1865 intraoral images were included in this study, 1493 (80%) of which were allocated for training and 372 (20%) for testing the CNN. Additionally, we tested the models on 10 videos, spanning a cumulative duration of 124 seconds. To assess the performance of our predictions, metrics including accuracy, sensitivity, specificity, precision, F1-score, area under the precision-recall (AUPR) curve, and area under the receiver operating characteristic (ROC) curve (AUC) were employed. RESULTS: The trained model exhibited commendable classification performance, achieving an accuracy of 0.965 and an AUC of 0.986. Moreover, it demonstrated superior specificity (0.992 vs. 0.978 and 0.956, P < 0.05) in comparison to assessments by two orthodontists. Conversely, the CNN model displayed diminished sensitivity (0.89 vs. 0.96 and 0.92, P < 0.05) relative to the orthodontists. Notably, the CNN model accomplished a perfect classification rate, successfully identifying 100% of the videos in the test set. CONCLUSION: The deep learning (DL) model exhibited remarkable classification accuracy in identifying anterior crossbite through both intraoral images and videos. This proficiency holds the potential to expedite the detection of severe malocclusions, facilitating timely classification for appropriate treatment and, consequently, mitigating the risk of complications.

Advanced glycation end products impair bone marrow mesenchymal stem cells osteogenesis in periodontitis with diabetes via FTO-mediated N6-methyladenosine modification of sclerostin.

BACKGROUND: Diabetes mellitus (DM) and periodontitis are two prevalent diseases with mutual influence. Accumulation of advanced glycation end products (AGEs) in hyperglycemia may impair cell function and worsen periodontal conditions. N6-methyladenosine (m6A) is an important post-transcriptional modification in RNAs that regulates cell fate determinant and progression of diseases. However, whether m6A methylation participates in the process of periodontitis with diabetes is unclear. Thus, we aimed to investigate the effects of AGEs on bone marrow mesenchymal stem cells (BMSCs), elucidate the m6A modification mechanism in diabetes-associated periodontitis. METHODS: Periodontitis with diabetes were established by high-fat diet/streptozotocin injection and silk ligation. M6A modifications in alveolar bone were demonstrated by RNA immunoprecipitation sequence. BMSCs treated with AGEs, fat mass and obesity associated (FTO) protein knockdown and sclerostin (SOST) interference were evaluated by quantitative polymerase chain reaction, western blot, immunofluorescence, alkaline phosphatase and Alizarin red S staining. RESULTS: Diabetes damaged alveolar bone regeneration was validated in vivo. In vitro experiments showed AGEs inhibited BMSCs osteogenesis and influenced the FTO expression and m6A level in total RNA. FTO knockdown increased the m6A levels and reversed the AGE-induced inhibition of BMSCs differentiation. Mechanically, FTO regulated m6A modification on SOST transcripts, and AGEs affected the binding of FTO to SOST transcripts. FTO knockdown accelerated the degradation of SOST mRNA in presence of AGEs. Interference with SOST expression in AGE-treated BMSCs partially rescued the osteogenesis by activating Wnt Signaling. CONCLUSIONS: AGEs impaired BMSCs osteogenesis by regulating SOST in an m6A-dependent manner, presenting a promising method for bone regeneration treatment of periodontitis with diabetes.

Mechanical loading and autophagy: A study on the BoNT-A injection-induced condylar cartilage degeneration.

Botulinum toxin A (BoNT-A) has emerged as a treatment option for temporomandibular disorder (TMD). By injecting BoNT-A into the masseter muscle, it is possible to reduce mechanical loading on the temporomandibular joint (TMJ). However, numerous prior studies have indicated excessive reduction in mechanical loading can have detrimental effects on TMJ cartilage. This study proposes that autophagy, a process influenced by mechanical loading, could play a role in BoNT-A-induced mandibular condyle cartilage degeneration. To explore this hypothesis, we employed both BoNT-A injection and an excessive biting model to induce variations in mechanical loading on the condyle cartilage of C57BL/6 mice, thereby simulating an increase and decrease in mechanical loading, respectively. Results showed a significant reduction in cartilage thickness and downregulation of Runt-related transcription factor 2 (Runx2) expression in chondrocytes following BoNT-A injection. In vitro experiments demonstrated that the reduction of Runx2 expression in chondrocytes is associated with autophagy, possibly dependent on decreased YAP expression induced by low mechanical loading. This study reveals the potential involvement of the YAP/LC3/Runx2 signaling pathway in BoNT-A mediated mandibular condylar cartilage degeneration.

Oxidative stress-related biomarkers in chronic periodontitis patients with or without type 2 diabetes: A systematic review and meta-analysis.

OBJECTIVE: The purpose of this meta-analysis was to look at the differences in oxidative stress (OS) biomarkers between type 2 diabetes mellitus with chronic periodontitis (DMCP) and chronic periodontitis (CP) patients. BACKGROUND: Oxidative stress has been shown to be a key pathogenic component in DMCP. However, it is unclear whether oxidative stress levels differ in periodontitis patients with or without diabetes. METHOD: A systematic search was conducted on PubMed, Cochrane, and Embase databases. Studies of DMCP participants were used as the experimental group and CP participants were used as the control group. Results are expressed as mean effects. RESULTS: Of a total of 1989 articles, 19 met the inclusion criteria. We found the levels of catalase (CAT) levels were reduced in the DMCP group compared with the CP group. However, there was no significant difference in the levels of superoxide dismutase (SOD), total antioxidant capacity (TAOC) malondialdehyde (MDA), and glutathione (GSH) between the two groups. And high heterogeneity was observed in some of the studies evaluated. CONCLUSION: Despite the limitations of this study, our results support the theory that there is an association between T2DM and the levels of OS-related biomarkers, especially CAT, in CP subjects, suggesting that OS plays an important role in the pathogenesis and development of DMCP.

Increased serum α-tocopherol acetate mediated by gut microbiota ameliorates alveolar bone loss through the STAT3 signalling pathway in diabetic periodontitis.

AIM: To evaluate whether and how gut microbiota-meditated metabolites regulate alveolar bone homeostasis in diabetic periodontitis (DP). MATERIALS AND METHODS: Lactobacillus casei (L. casei) was employed as a positive modulator of gut microbiota in DP mice. The destruction of alveolar bone was evaluated. Untargeted metabolomics was conducted to screen out the pivotal metabolites. A co-housing experiment was conducted to determine the connection between the gut microbiota and alpha-tocopherol acetate (α-TA). α-TA was applied to DP mice to investigate its effect against alveolar bone loss. Human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (HGFs) were extracted for the in vitro experiment. Transcriptomic analysis and immunohistochemistry were performed to detect the major affected signalling pathways. RESULTS: Positive regulation of the gut microbiota significantly attenuated alveolar bone loss and increased the serum α-TA level. The alteration in gut microbiota composition could affect the serum α-T (the hydrolysates of α-TA) level. α-TA could alleviate alveolar bone destruction in DP mice and α-T exert beneficial effects on hPDLCs and HGFs. Mechanistically, the STAT3 signalling pathway was the pivotal pathway involved in the protective role of α-TA. CONCLUSIONS: The gut microbiota-α-TA-STAT3 axis plays an important role in the regulation of diabetic alveolar bone homeostasis.

SHED-derived exosomes improve the repair capacity and osteogenesis potential of hPDLCs.

OBJECTIVE: Exosomes secreted by stem cells are recognized as a critical component in tissue regeneration during stem cell-based therapy. Considering the limited sources and bone regeneration efficiency of human periodontal ligament cells (hPDLCs), we explored whether exosomes secreted by stem cells from human exfoliated deciduous teeth (SHED-exo) could improve the pluripotency and regenerative potential of hPDLCs. METHODS AND MATERIALS: In hPDLCs, cell proliferation, migration, cell cycle, apoptosis, and osteogenic differentiation were detected after cells were exposed to SHED-exo (SHED-exo group), blank (control group), or control supernatant without exo (Csup group), via CCK-8, scratch analysis, flow cytometric, real-time PCR, and so on. Exosomes sequencing was performed to compare and analyze miRNAs contented in SHED-exo and hPDLC-exo. RESULTS: As compared to control or Csup, SHED-exo significantly increased migration, apoptosis, and proliferation, promoted cell cycle transition from G1 to S phase in hPDLCs, and enhanced Runx2 expression and mineralization. In addition, it may be explained by the significant differences in miRNA contented in SHED-exo and hPDLC-exo. CONCLUSION: Exosomes from SHED can improve cell proliferation, migration, cell cycle, apoptosis, and osteogenic differentiation of hPDLCs, which highlights the therapeutic value of this bioactive component in the regeneration of periodontal tissues using hPDLCs in clinical practice.

Enhanced effect of a novel bioactive glass-ceramic for dental application.

OBJECTIVES: Dental caries is the most common chronic disease in humans, caused by the acid produced by the microflora in the mouth that dissolves the enamel minerals. Bioactive glass (BAG) has been used in various clinical applications due to its unique bioactive properties, such as bone graft substitutes and dental restorative composites. In this study, we introduce a novel bioactive glass-ceramic (NBGC) prepared through a sol-gel process under a water-free condition. MATERIALS AND METHODS: The anti-demineralization and remineralization effects of NBGC were evaluated by comparing the measurements of bovine enamel surface morphology, surface roughness, surface micro-hardness, enamel elements, and mineral content before and after related treatments with a commercial BAG. The antibacterial effect was characterized by minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). RESULTS: Results showed that NBGC had greater acid resistance and remineralization potential compared to commercial BAG. The fast formation of a hydroxy carbonate apatite (HCA) layer suggests efficient bioactivity. CLINICAL RELEVANCE: In addition to its antibacterial properties, NBGC shows promise as an ingredient in oral care products that can prevent demineralization and restore enamel.

Hyperglycemia Aggravates Periodontitis via Autophagy Impairment and ROS-Inflammasome-Mediated Macrophage Pyroptosis.

Macrophage pyroptosis drives the secretion of IL-1ß, which has been recently reported to be a featured salivary biomarker for discriminating periodontitis in the presence of diabetes. This study aimed to explore whether macrophage pyroptosis plays a role in the development of diabetes mellitus-periodontitis, as well as potential therapeutic strategies. By establishing a model of experimental diabetes mellitus-periodontitis in rats, we found that IL-1ß and gasdermin D were highly expressed, leading to aggravated destruction of periodontal tissue. MCC950, a potent and selective molecule inhibitor of the NLRP3 inflammasome, effectively inhibited macrophage pyroptosis and attenuated alveolar bone losses in diabetes mellitus-periodontitis. Consistently, in vitro, high glucose could induce macrophage pyroptosis and thus promoted IL-1ß production in macrophages stimulated by lipopolysaccharide. In addition, autophagy blockade by high glucose via the mTOR-ULK1 pathway led to severe oxidative stress response in macrophages stimulated by lipopolysaccharide. Activation of autophagy by rapamycin, clearance of mitochondrial ROS by mitoTEMPO, and inhibition of inflammasome by MCC950 could significantly reduce macrophage pyroptosis and IL-1ß secretion. Our study demonstrates that hyperglycemia promotes IL-1ß production and pyroptosis in macrophages suffered by periodontal microbial stimuli. Modulation of autophagy activity and specific targeting of the ROS-inflammasome pathway may offer promising therapeutic strategies to alleviate diabetes mellitus-periodontitis.

Effects of novel microimplant-assisted rapid palatal expanders manufactured by 3-dimensional printing technology: A finite element study.

INTRODUCTION: The expansion effects of several new microimplant-assisted rapid palatal expanders (MARPEs) manufactured by 3-dimensional printing technology were evaluated by finite element analysis (FEA). The aim was to identify a novel MARPE suitable for treating maxillary transverse deficiency. METHODS: The finite element model was established using MIMICS software (version 19.0; Materialise, Leuven, Belgium). First, the appropriate microimplant insertion characteristics were identified via FEA, and several MARPEs with the above insertion patterns were manufactured by 3-dimensional printing technology. Then, the stress distribution and displacement prediction of the 4 MARPEs and hyrax expander (model E) were evaluated via FEA: bone-borne (model A), bone-tooth-borne (model B), bone-mucous-borne (model C), bone-tooth-mucous-borne (model D). RESULTS: Monocortical microimplants perpendicular to the cortical bone on the coronal plane resulted in better expansion effects. Compared with a conventional hyrax expander, the orthopedic expansion of each of the 4 MARPEs was far larger, the parallelism was greater, and the posterior teeth tipping rate was lower. Among them, the expansion effects of models C and D were the best; the von Mises peak values on the surfaces of the microimplants were smaller than those of models A and B. CONCLUSIONS: This study may demonstrate that the 4 MARPEs obtained more advantageous orthopedic expansion effects than a hyrax expander. Models C and D obtained better biomechanical effects and had better primary stability. Overall, model D is the recommended expander for treating maxillary transverse deficiency because its structure acts like an implant guide and is beneficial for the accurate insertion of the microimplant.

The effects of lingual buttons, precision cuts, and patient-specific attachments during maxillary molar distalization with clear aligners: Comparison of finite element analysis.

INTRODUCTION: This study aimed to analyze the biomechanical effects of the combined use of clear aligners (CA) and auxiliaries (precision cuts, lingual buttons, and patient-specific attachments) on mesial tipping and extrusion of the premolars during maxillary molars distalization. METHODS: Three-dimensional finite element method was employed to simulate clinical scenarios of CA with different auxiliaries for molar distalization. As such, 200 g of distal force was applied to the microimplants from the notches, lingual buttons, and hooks. Orthodontic tooth movement and the hydrostatic pressure in the periodontal ligament were compared. RESULTS: Adding auxiliaries can provide the maxillary arch anchorage and promote the distal tipping of premolars and retroclination of maxillary incisors. In contrast, this effect was more pronounced in patient-specific attachment applications than in other types of auxiliaries. The independent application of the CA caused mesial tipping and extrusion of the premolar and also caused the incisor proclination. CONCLUSIONS: The anchorage loss caused by the CA alone could be alleviated with the assistance of auxiliaries. Notably, patient-specific attachments further reinforce the anchorage of the anterior arch by incorporating anchor teeth as 1 anchorage unit.

Oxidative stress state inhibits exosome secretion of hPDLCs through a specific mechanism mediated by PRMT1.

BACKGROUND AND OBJECTIVES: Periodontitis, the most common chronic inflammation characterized by persistent alveolar bone resorption in the periodontitis, affects almost half of the adult population worldwide. Oxidative stress is one of the pathophysiological mechanisms underlying periodontitis, which affects the occurrence and development of periodontitis. Exosomes are increasingly recognized as vehicles of intercellular communication and are closely related to periodontitis. However, the effects of oxidative stress on exosome secretion and the specific mechanisms remain elusive in human periodontal ligament cells (hPDLCs). The relationship between exosome secretion and the osteogenic differentiation of hPDLCs also needs to be investigated. METHODS: Isolated PDLSCs were identified using flow cytometry. Osteogenesis was measured using alizarin red staining and ALP staining. Expression of exosomal markers and PRMT1 was analyzed using western blot. Immunofluorescence was used to measure exosome uptake and the expression of EEA1. RESULTS: The secretion capacity of exosomes was markedly suppressed under oxidative stress. Protein arginine methyltransferase 1 (PRMT1) has been strongly associated with both oxidative stress and inflammation, and PRMT1 was significantly upregulated under oxidative stress conditions. Lentivirus-mediated overexpression of PRMT1 caused a significant reduction in the secretion of exosomes, but multivesicular bodies (MVBs) containing a large number of intraluminal vesicles (ILVs) were increased. Rab11a and Rab27a expression, which mediate MVBs fusion with cell membranes, decreased, although this phenomenon was restored after knocking down PRMT1 expression under oxidative stress. CONCLUSIONS: These results indicated that PRMT1 mediated a decrease in exosome secretion of hPDLCs. The decrease in Rab11a and Rab27a leads to a large accumulation of MVBs in cells and is one of the main reasons for impaired exosome secretion. The decrease in osteogenic differentiation of hPDLCs caused by H2 O2 may originate in part from the inhibition of exosome secretion.

Predicting the risk of dental implant loss using deep learning.

AIM: To investigate the feasibility of predicting dental implant loss risk with deep learning (DL) based on preoperative cone-beam computed tomography. MATERIALS AND METHODS: Six hundred and three patients who underwent implant surgery (279 high-risk patients who did and 324 low-risk patients who did not experience implant loss within 5 years) between January 2012 and January 2020 were enrolled. Three models, a logistic regression clinical model (CM) based on clinical features, a DL model based on radiography features, and an integrated model (IM) developed by combining CM with DL, were developed to predict the 5-year implant loss risk. The area under the receiver operating characteristic curve (AUC) was used to evaluate the model performance. Time to implant loss was considered for both groups, and Kaplan-Meier curves were created and compared by the log-rank test. RESULTS: The IM exhibited the best performance in predicting implant loss risk (AUC = 0.90, 95% confidence interval [CI] 0.84-0.95), followed by the DL model (AUC = 0.87, 95% CI 0.80-0.92) and the CM (AUC = 0.72, 95% CI 0.63-0.79). CONCLUSIONS: Our study offers preliminary evidence that both the DL model and the IM performed well in predicting implant fate within 5 years and thus may greatly facilitate implant practitioners in assessing preoperative risks.

C-reactive protein perturbs alveolar bone homeostasis: An experimental study of periodontitis and diabetes in the rat.

AIM: To explore the role of C-reactive protein (CRP) in periodontitis and diabetes and its mechanism in alveolar bone homeostasis. MATERIALS AND METHODS: In vivo, normal, and Crp knockout (KO) rats were randomly divided into control, diabetes, periodontitis, and diabetes and periodontitis groups, respectively. The diabetes model was established using a high-fat diet combined with streptozotocin injection. The periodontitis model was established by ligature combined with lipopolysaccharide (LPS) injection. Alveolar bones were analysed using micro-computed tomography, histology, and immunohistochemistry. In vitro, human periodontal ligament cells (hPDLCs) were treated with LPS and high glucose. CRP knockdown lentivirus or CRP overexpression adenovirus combined with a PI3K/AKT signalling inhibitor or agonist were used to explore the regulatory mechanism of CRP in osteogenesis and osteoclastogenesis of hPDLCs, as evidenced by alkaline phosphatase staining, Western blot, and quantitative polymerase chain reaction. RESULTS: In periodontitis and diabetes, CRP KO decreased the alveolar bone loss and the expression levels of osteoclastogenic markers, while increasing the expression levels of osteogenic markers. CRP constrained osteogenesis while promoting the osteoclastogenesis of hPDLCs via PI3K/AKT signalling under high glucose and pro-inflammatory conditions. CONCLUSIONS: CRP inhibits osteogenesis and promotes osteoclastogenesis via PI3K/AKT signalling under diabetic and pro-inflammatory conditions, thus perturbing alveolar bone homeostasis.

Development of a rat model for type 2 diabetes mellitus peri-implantitis: A preliminary study.

OBJECTIVE: To develop an in vivo model to simulate the complex internal environment of diabetic peri-implantitis (T2DM-PI) model for a better understanding of peri-implantitis in type 2 diabetic patients. MATERIALS AND METHODS: Maxillary first molars were extracted in Sprague-Dawley (SD) rats, and customized cone-shaped titanium implants were installed in the extraction sites. Thereafter, implants were uncovered and customized abutments were screwed into implants. A high-fat diet and a low-dose injection of streptozotocin were utilized to induce T2DM. Finally, LPS was locally injected in implant sulcus to induce peri-implantitis. RESULTS: In the present study, T2DM-PI model has been successfully established. Imaging analysis revealed that abundant inflammatory cells infiltrated in the soft tissue in T2DM-PI group with concomitant excessive secretion of inflammatory cytokines. Moreover, higher expression of MMP and increased number of osteoclasts led to collagen disintegration and bone resorption in T2DM-PI group. CONCLUSIONS: These results describe a novel rat model which stimulate T2DM-PI in vivo, characterized by overwhelming inflammatory response and bone resorption. This model has a potential to be used for investigation of initiation, progression and interventional therapy of T2DM-PI.

BMP9-initiated osteogenic/odontogenic differentiation of mouse tooth germ mesenchymal cells (TGMCS) requires Wnt/ß-catenin signalling activity.

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/ß-catenin signalling in BMP9-induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down ß-catenin expression. In vivo implantation assay revealed that while BMP9-stimulated iTGMCs induced robust formation of ectopic bone, knocking down ß-catenin expression in iTGMCs remarkably diminished BMP9-initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio-factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.

SHED-derived conditioned exosomes enhance the osteogenic differentiation of PDLSCs via Wnt and BMP signaling in vitro.

The exosomes from human exfoliated deciduous teeth (SHED-Exos) have exhibited potential therapeutic role in dental and oral disorders. The biological effects of exosomes largely depend on cellular origin and physiological status of donor cell. In the present study, we explored the influence of conditioned exosomes from SHED with osteogenic induction on periodontal ligament stem cells (PDLSCs) in vitro. Conditioned SHED-Exos from a 3-day osteogenic supernatant were applied during PDLSCs osteogenic differentiation. We found that conditioned SHED-Exos had no cytotoxicity on PDLSCs viability assessed by CCK-8 assay. These SHED-Exos promoted PDLSCs osteogenic differentiation with deep Alizarin red staining, high alkaline phosphatase (ALP) activity and upregulated osteogenic gene expression (RUNX2, OPN and OCN). We further found BMP/Smad signaling and Wnt/ß-catenin were activated by enhanced Smad1/5/8 phosphorylation and increased nuclear ß-catenin protein expression. Inhibiting these two signaling pathways with specific inhibitors (cardamonin and LDN193189) remarkably weakened the enhanced osteogenic differentiation. Furthermore, Wnt3a and BMP2 were upregulated in SHED and SHED-Exos. Silencing Wnt3a and BMP2 in SHED-Exos partially counteracts the enhanced osteogenic differentiation. Our findings indicate that conditioned SHED-Exos-enhanced PDLSCs osteogenic differentiation was partly due to its carrying Wnt3a and BMP2. These data provide new insights into the use of SHED-Exos in periodontitis-induced bone defects therapy.

LIPUS inhibited the expression of inflammatory factors and promoted the osteogenic differentiation capacity of hPDLCs by inhibiting the NF-κB signaling pathway.

BACKGROUND AND OBJECTIVES: As a chronic infectious disease, periodontitis could lead to tooth and bone loss. Low-intensity pulsed ultrasound (LIPUS) is a safe, noninvasive treatment method to effectively inhibit inflammation and promote bone differentiation. However, the application of LIPUS in curing periodontitis is still rare. Our study aimed to explore the ability of LIPUS to inhibit inflammatory factors and promote the osteogenic differentiation capacity of human periodontal ligament cells (hPDLCs), and its underlying mechanism. MATERIAL AND METHODS: Human periodontal ligament cells were obtained and cultured from the premolar tissue samples for experiments. First, hPDLCs were treated for 24 hours using lipopolysaccharide (LPS) and then exposed to LIPUS (10 mW/cm2 , 30 mW/cm2 , 60 mW/cm2 , and 90 mW/cm2 ) to determine the appropriate intensity to inhibit expression of the inflammatory factors interleukin-6 (IL-6) and interleukin-8 (IL-8) expression. The expression of IL-6 and IL-8 was detected by real-time PCR and enzyme-linked immunosorbent assay. The safety of the most appropriate intensity of LIPUS was tested by a cell counting kit 8 test and an apoptosis assay. Then, LPS-induced hPDLCs were treated in osteogenic medium for 7-21 days with or without LIPUS (90 mW/cm2 , 30 min/d) stimulation. The osteogenic genes RUNX2, OPN, OSX, and OCN were measured by real-time PCR. Additionally, osteogenic differentiation capacity was determined using alkaline phosphatase (ALP) staining, ALP activity analysis, and Alizarin red staining. The activity of the nuclear factor-kappa B (NF-κB) signaling pathway was determined by western blotting, real-time PCR, immunofluorescence, and pathway blockade assays. RESULTS: Lipopolysaccharide significantly upregulated the production and gene expression of IL-6 and IL-8, while LIPUS stimulation significantly inhibited IL-6 and IL-8 expression in an intensity-dependent manner. LIPUS (90 mW/cm2 ) was chosen as the most appropriate intensity, and there was no detrimental influence on cell proliferation and status with or without osteogenic medium. In addition, consecutive stimulation with LIPUS (90 mW/cm2 ) for 30 min/d for 7 days could also inhibit IL-6 and IL-8 gene expression, upregulate the expression of the osteogenesis-related genes RUNX2, OPN, OSX, and OCN, and promote osteogenic differentiation capacity in osteogenic medium in inflamed hPDLCs. The NF-κB signaling pathway was inhibited with LIPUS (90 mW/cm2 ) via inhibition of the phosphorylation of IκBα and the translocation of p65 into the nucleus in inflamed hPDLCs. Additional investigations of the NF-κB inhibitor, BAY 11-7082, revealed that LIPUS (90 mW/cm2 ) acted similarly to BAY 11-7802 to inhibit the NF-κB signaling pathway and increase osteogenesis-related genes and promote the osteogenic differentiation capacity of inflamed hPDLCs. CONCLUSION: Low-intensity pulsed ultrasound (90 mW/cm2 ) stimulation could be a safe method to inhibit IL-6 and IL-8 in hPDLCs by inhibiting the NF-κB signaling pathway. The effect of LIPUS (90 mW/cm2 ) and BAY 11-7082 on LPS-induced inflammation demonstrated that both of these agents were capable of promoting osteogenesis-related gene expression and osteogenic differentiation in hPDLCs, suggesting that the effect of LIPUS on the promotion of osteogenic activity could be mediated in part through its ability to inhibit the NF-κB signal pathway. Hence, LIPUS could be a potential therapeutic method to cure periodontitis.