Cyclic di-adenosine monophosphate regulates the osteogenic and adipogenic differentiation of hPDLSCs via MAPK and NF-κB signaling.

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that can be recognized by infected host cells and activate the immunoinflammatory response. The purpose of this study is to demonstrate the effect of c-di-AMP on the differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mechanisms. In the present study, we find that the gingival crevicular fluid (GCF) of patients with chronic periodontitis has a higher expression level of c-di-AMP than that of healthy people. In vitro, c-di-AMP influences the differentiation of hPDLSCs by upregulating Toll-like receptors (TLRs); specifically, it inhibits osteogenic differentiation by activating NF-κB and ERK/MAPK and promotes adipogenic differentiation through the NF-κB and p38/MAPK signaling pathways. Inhibitors of TLRs or activated pathways reduce the changes induced by c-di-AMP. Our results establish the potential correlation among bacterial c-di-AMP, periodontal tissue homeostasis and chronic periodontitis pathogenesis.

Cellular communication network factor 1 interlinks autophagy and ERK signaling to promote osteogenesis of periodontal ligament stem cells.

OBJECTIVES: To investigate the effects of cellular communication network factor 1 (CCN1), a critical matricellular protein, on alveolar bone regeneration, and to elucidate the underlying molecular mechanism. BACKGROUND: In the process of orthodontic tooth movement, bone deposition on the tension side of human periodontal ligament stem cells (hPDLSCs) ensured high efficiency and long-term stability of the treatment. The matricellular protein CCN1 is responsive to mechanical stimulation, exhibiting important tasks in bone homoeostasis. However, the role and mechanism of CCN1 on alveolar bone remodeling of hPDLSCs remains unclear. METHODS: The expression and distribution of CCN1 in rat periodontal ligament were detected by immunofluorescence staining and immunohistochemical staining. ELISA verified the secretion of CCN1 triggered by stretch loading. To examine the mineralization ability of hPDLSCs induced by CCN1, Western blotting, qRT-PCR, ARS, and ALP staining were performed. CCK-8 and cell migration assay were performed to detect the cell proliferation rate and the wound healing. PI3K/Akt, MAPK, and autophagy activation were examined via Western blotting and immunofluorescence. RESULTS: Mechanical stimuli induced the release of CCN1 into extracellular environment by hPDLSCs. Knockdown of CCN1 attenuated the osteogenesis of hPDLSCs while rhCCN1 enhanced the expression of Runx2, Col 1, ALPL, and promoted the mineralization nodule formation. CCN1 activated PI3K/Akt and ERK signaling, and blockage of PI3K/Akt signaling reversed the accelerated cell migration triggered by CCN1. The enhanced osteogenesis induced by CCN1 was abolished by ERK signaling inhibitor PD98059 or autophagy inhibitor 3-MA. Further investigation demonstrated PD98059 abrogated the activation of autophagy. CONCLUSION: This study demonstrated that CCN1 promotes osteogenesis in hPDLSCs via autophagy and MAPK/ERK pathway.

Relative effectiveness of facemask therapy with alternate maxillary expansion and constriction in the early treatment of Class III malocclusion.

INTRODUCTION: This study aimed to investigate the relative efficacy of maxillary protraction combined with a modified alternate rapid maxillary expansion and constriction (Alt-RAMEC) protocol compared with conventional protocols in the early orthopedic treatment of skeletal Class III malocclusion. METHODS: A sample of 39 patients was divided into 3 groups on the basis of different interventions. Conventional facemask (FM) with splint-type intraoral devices was performed in the FM group (7 males and 5 females; mean age, 9.53 ± 1.37 years). Maxillary expansion with an activation rate of 0.5 mm/d (twice a day) followed by FM therapy was applied in the rapid maxillary expansion group (RME/FM) (6 males and 6 females; mean age, 9.31 ± 1.60 years). In the Alt-RAMEC/FM group (7 males and 8 females; mean age, 10.01 ± 1.31 years), Alt-RAMEC was started simultaneously and throughout the entire course of maxillary protraction, with repetitive alternations between activation and deactivation of expanders (0.5 mm/d for 7 days). The patients in all groups were instructed to wear FMs for a minimum of 12 h/d. Pretreatment and posttreatment lateral cephalograms were all traced and measured. RESULTS: The Alt-RAMEC group showed statistically more significant maxillary advancement than other groups (A-VRP, 3.87 mm vs 3.04 mm [RME/FM], vs 2.04 mm [FM]; P <0.05). Analysis of variance did not reveal significant intergroup differences in palatal plane angulation changes (P >0.05). No pronounced mandibular clockwise rotations were noted in the Alt-RAMEC/FM group with distinct intergroup differences (P <0.05). There were more skeletal effects (88.7%) during overjet correction in the Alt-RAMEC/FM protocol. CONCLUSIONS: A combination of the modified Alt-RAMEC protocol with FM revealed more favorable skeletal effects compared with FM and RME/FM protocols in treating prepubertal patients with maxillary deficiency.

The role of EphB4/ephrinB2 signaling in root repair after orthodontically-induced root resorption.

INTRODUCTION: This study aimed to investigate the effect of EphB4/ephrinB2 signaling on orthodontically-induced root resorption repair and the possible molecular mechanism behind it. METHODS: Seventy-two 6-week-old male Wistar rats were randomly divided into 3 groups: blank control group, physiological regeneration group (PHY), and EphB4 inhibitor local injection group (INH). A root repair model was built on experimental rats of the PHY and INH groups. The animals in the INH groups received a daily periodontal local injection of EphB4 inhibitor NVP-BHG712, whereas the blank control group and PHY groups received only the vehicle. RESULTS: Histologic staining and microcomputed tomography analysis showed that root regeneration was inhibited in the INH group compared with the PHY group with a greater number of osteoclasts. Immunohistochemical staining showed active EphB4/ephrinB2 signaling activities during root regeneration. The cementogenesis-related factors cementum attachment protein, alkaline phosphatase, osteopontin, and runt-related transcription factor 2, and osteoclastic-related factors RANKL and osteoprotegerin were affected by regulated EphB4/ephrinB2 signaling. CONCLUSIONS: These findings demonstrated that the EphB4/ephrinB2 signaling might be a promising therapeutic target for novel therapeutic approaches to reduce orthodontically-induced root resorption through enhancement of cementogenesis.

AFF4 enhances odontogenic differentiation of human dental pulp cells.

AFF4 is a component of super elongation complex (SECs) and functions as a scaffold protein to bridge the transcription elongation factors. It is associated with leukemia, HIV transcription, and head neck cancer. However, its role in odontogenic differentiation of dental pulp cells (DPCs) is unclear. Here, we show the expression of AFF4 is increased during odontogenesis. Depletion of AFF4 in human DPCs leads to a decrease of alkaline phosphatase (ALP) activity, calcium mineralization and odontogenic-related genes expression. On the contrary, Lentivirus-mediated overexpression of AFF4 induces the odontogenic potential of DPCs. Mechanistically, we found AFF4 regulates the transcription of NFIC, a key factor for tooth root formation. Overexpression of NFIC successfully rescues the restricted differentiation of AFF4-depleted cells. Our data demonstrate that AFF4 serves as a previously unknown regulator of odontogenesis.

A Bayesian network meta-analysis of orthopaedic treatment in Class III malocclusion: Maxillary protraction with skeletal anchorage or a rapid maxillary expander.

To evaluate and compare the effectiveness of orthopaedic treatment for Class III malocclusions using skeletal anchorage or a rapid maxillary expander for maxillary protraction. Electronic databases, including PubMed, EMBASE, Cochrane Library and Web of Science, were searched for randomized controlled trials (RCTs) and non-randomized clinical trials (CCTs) for orthopaedic treatment of Class III malocclusions. Five interventions were studied: a facemask with a maxillary temporary anchorage device (MTAD), a bone-anchored rapid maxillary expansion (BARME), a rapid maxillary expansion (RME), an alternate rapid maxillary expansion and contraction (Alt-RAMEC), and a bone-anchored intermaxillary traction (BAIMT). Eight outcomes (SNA, SNB, ANB, overjet, SN-GoGn, ANS-Me, IMPA (L1-MP), and U1-PP) were statistically polled. We conducted network meta-analysis using R statistical software with the GeMTC package. Twenty-five studies met the inclusion criteria. Compared with the RME group, the Alt-RAMEC group (mean difference (MD): 1.3; 95% credibility interval (CrI): 0.26, 2.3) and MTAD group (MD: 0.85; 95% CrI: 0.065, 1.6) showed a better effect on ANB in CCTs. Regarding the vertical relationship, the BAIMT group (MD: -2.2; 95% CrI: -5.2, 0.73) showed a smaller effect regarding increasing the vertical dimension of ANS-Me. The RME, MTAD and Alt-RAMEC group showed a higher ability to decrease the angle of L1-MP. The Alt-RAMEC and MTAD protocol have a higher possibility to obtain a skeletal and tooth effect in sagittal relationships. The BAIMT protocol can acquire a better skeletal effect in sagittal relationships with less vertical and dental changes. More well-designed RCTs are needed to ensure that the conclusion is reliable.

Long non-coding RNA LncTUG1 regulates favourable compression force-induced cementocytes mineralization via PU.1/TLR4/SphK1 signalling.

Orthodontic tooth movement (OTM) is a highly coordinated biomechanical response to orthodontic forces with active remodelling of alveolar bone but minor root resorption. Such antiresorptive properties of root relate to cementocyte mineralization, the mechanisms of which remain largely unknown. This study used the microarray analysis to explore long non-coding ribonucleic acids involved in stress-induced cementocyte mineralization. Gain- and loss-of-function experiments, including Alkaline phosphatase (ALP) activity and Alizarin Red S staining, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence analyses of mineralization-associated factors, were conducted to verify long non-coding ribonucleic acids taurine-upregulated gene 1 (LncTUG1) regulation in stress-induced cementocyte mineralization, via targeting the Toll-like receptor 4 (TLR4)/SphK1 axis. The luciferase reporter assays, chromatin immunoprecipitation assays, RNA pull-down, RNA immunoprecipitation, and co-localization assays were performed to elucidate the interactions between LncTUG1, PU.1, and TLR4. Our findings indicated that LncTUG1 overexpression attenuated stress-induced cementocyte mineralization, while blocking the TLR4/SphK1 axis reversed the inhibitory effect of LncTUG1 on stress-induced cementocyte mineralization. The in vivo findings also confirmed the involvement of TLR4/SphK1 signalling in cementocyte mineralization during OTM. Mechanistically, LncTUG1 bound with PU.1 subsequently enhanced TLR4 promotor activity and thus transcriptionally elevated the expression of TLR4. In conclusion, our data revealed a critical role of LncTUG1 in regulating stress-induced cementocyte mineralization via PU.1/TLR4/SphK1 signalling, which might provide further insights for developing novel therapeutic strategies that could protect roots from resorption during OTM.

Photobiomodulation Therapy and Pulp-Regenerative Endodontics: A Narrative Review.

Regenerative endodontic procedures (REPs) were used to recover the dental pulp's vitality in order to avoid the undesirable outcomes of conventional endodontic treatment and to promote dentinal formation, especially for immature permanent teeth. Photobiomodulation therapy (PBMT) exhibits photobiological and photochemical effects for improving the root canal's environmental conditions by compensating for oxidative stress and increasing the blood supply to implanted stem cells and improving their survival. Basic research has revealed that PBMT can modulate human dental pulp stem cells' (hDPSCs) differentiation, proliferation, and activity, and subsequent tissue activation. However, many unclear points still remain regarding the mechanisms of action induced by PBMT in REPs. Therefore, in this review, we present the applications of laser and PBMT irradiation to the procedures of REPs and in endodontics. In addition, the effects of PBMT on the regenerative processes of hDPSCs are reviewed from biochemical and cytological perspectives on the basis of the available literature. Furthermore, we consider the feasibility of treatment in which PBMT irradiation is applied to stem cells, including dental pulp stem cells, and we discuss research that has reported on its effect.

LITTIP/Lgr6/HnRNPK complex regulates cementogenesis via Wnt signaling.

Orthodontically induced tooth root resorption (OIRR) is a serious complication during orthodontic treatment. Stimulating cementum repair is the fundamental approach for the treatment of OIRR. Parathyroid hormone (PTH) might be a potential therapeutic agent for OIRR, but its effects still lack direct evidence, and the underlying mechanisms remain unclear. This study aims to explore the potential involvement of long noncoding RNAs (lncRNAs) in mediating the anabolic effects of intermittent PTH and contributing to cementum repair, as identifying lncRNA-disease associations can provide valuable insights for disease diagnosis and treatment. Here, we showed that intermittent PTH regulates cell proliferation and mineralization in immortalized murine cementoblast OCCM-30 via the regulation of the Wnt pathway. In vivo, daily administration of PTH is sufficient to accelerate root regeneration by locally inhibiting Wnt/ß-catenin signaling. Through RNA microarray analysis, lncRNA LITTIP (LGR6 intergenic transcript under intermittent PTH) is identified as a key regulator of cementogenesis under intermittent PTH. Chromatin isolation by RNA purification (ChIRP) and RNA immunoprecipitation (RIP) assays revealed that LITTIP binds to mRNA of leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and heterogeneous nuclear ribonucleoprotein K (HnRNPK) protein. Further co-transfection experiments confirmed that LITTIP plays a structural role in the formation of the LITTIP/Lgr6/HnRNPK complex. Moreover, LITTIP is able to promote the expression of LGR6 via the RNA-binding protein HnRNPK. Collectively, our results indicate that the intermittent PTH administration accelerates root regeneration via inhibiting Wnt pathway. The lncRNA LITTIP is identified to negatively regulate cementogenesis, which activates Wnt/ß-catenin signaling via high expression of LGR6 promoted by HnRNPK.

Activation of the pattern recognition receptor NOD1 in periodontitis impairs the osteogenic capacity of human periodontal ligament stem cells via p38/MAPK signalling.

OBJECTIVES: Nucleotide oligomerization domain receptor 1 (NOD1) mediates host recognition of pathogenic bacteria in periodontium. However, the specific role of NOD1 in regulating osteogenesis is unclear. Therefore, this study focused on the activation status of NOD1 in periodontitis and its effect on the osteogenic capacity of human periodontal ligament stem cells (hPDLSCs) as well as the underlying mechanism. METHODS: Histological staining and Western blot were utilized to assess NOD1 expression in the periodontium of people with or without periodontitis. HPDLSCs were cultured under NOD1 agonist or antagonist treatment. Q-PCR and Western blot were employed to assess the expression of osteogenic marker genes and proteins. Alizarin red staining and alkaline phosphatase staining were used to determine the osteogenic capability of hPDLSCs. The activation of downstream signalling was determined and specific inhibitors were utilized to confirm the signalling pathway in NOD1-regulated osteogenesis. RESULTS: NOD1 expression is significantly elevated in periodontitis. With NOD1 activated by particular agonist tri-DAP, the osteogenic potential of hPDLSCs was impaired. NOD1 antagonist co-incubation partially restored the decreased osteogenesis in hPDLSCs. P38/MAPK was phosphorylated in tri-DAP-induced NOD1 activation. The inhibitor of p38 rescued the suppression of osteogenesis induced by tri-DAP in hPDLSCs. CONCLUSIONS: Our study revealed the expression status of NOD1 in periodontitis. Its activation greatly decreased the osteogenic capacity of hPDLSCs which was mediated by the phosphorylation of p38 downstream signalling.

Connective tissue growth factor promotes cementogenesis and cementum repair via Cx43/ß-catenin axis.

BACKGROUND: Orthodontic tooth movement inevitably induces cementum resorption, which is an urgent problem for orthodontists to confront. Human periodontal ligament stem cells (hPDLSCs) exert an important role in the orthodontic tooth movement and exhibit multidirectional differentiation ability in cementum regeneration. Connective tissue growth factor (CTGF) is an important extracellular matrix protein for bone homeostasis and cell differentiation. The purpose of our study was to explore the role of CTGF in cementum repair and cementogenesis and to elucidate its underlying mechanism. METHODS: A cementum defect model was established by tooth movement with heavy forces, and the cementum repair effect of CTGF was observed via micro-CT, HE staining and immunohistochemical staining. RT‒qPCR, western blotting (WB), alizarin red staining and ALP activity experiments verified the mineralization ability of hPDLSCs stimulated with CTGF. The expression of Cx43 in periodontal ligament cells was detected by WB and immunofluorescence (IF) experiments after CTGF stimulation in vivo and in vitro. Subsequently, the mineralization ability of hPDLSCs was observed after application of CTGF and the small interfering RNA Si-Cx43. Additionally, co-intervention via application of the small interfering RNA Si-CTGF and the Cx43 agonist ATRA in hPDLSCs was performed to deepen the mechanistic study. Next, WB, IF experiments and co-immunoprecipitation were conducted to confirm whether CTGF triggers the Cx43/ß-catenin axis to regulate cementoblast differentiation of hPDLSCs. RESULTS: Local oral administration of CTGF to the cementum defects in vivo facilitated cementum repair. CTGF facilitated the cementogenesis of hPDLSCs in a concentration-dependent manner. Cx43 acted as a downstream effector of CTGF to regulate cementoblast differentiation. Si-Cx43 reduced CTGF-induced cementoblast differentiation. The Cx43 agonist ATRA restored the low differentiation capacity induced by Si-CTGF. Further mechanistic studies showed that CTGF triggered the activation of ß-catenin in a dose-dependent manner. In addition, co-localization IF analysis and co-immunoprecipitation demonstrated that Cx43 interacted with ß-catenin at cell‒cell connections. Si-Cx43 attenuated the substantial expression of ß-catenin induced by CTGF. The Cx43 agonist reversed the inhibition of ß-catenin induced by Si-CTGF. IF demonstrated that the nuclear importation of ß-catenin was related to the immense expression of Cx43 at cell‒cell junctions. CONCLUSIONS: Taken together, these data demonstrate that CTGF promotes cementum repair and cementogenesis through activation of the Cx43/ß-catenin signalling axis.

Comprehensive assessment of tranexamic acid during orthognathic surgery: A systematic review and meta-analysis of randomized, controlled trials.

The objective of this study was to comprehensively assess the use of tranexamic acid (TXA) during orthognathic surgery. A systematic review and meta-analysis of randomized controlled trials addressing these issues were carried out. Three electronic databases, included PubMed, Web of Science, and Cochrane Library, were searched until April 30, 2018. Eligible studies were restricted to randomized, controlled trials (RCTs). Weighted mean differences (WMD) for blood loss, operation time, haematocrit, quality of surgical field, and odds ratio (OR) for transfusion rates were pooled for the included studies. Eight randomized, controlled trials were included for analysis. Compared with the control group, the TXA group showed a reduction in intraoperative blood loss of 165.03 ml (p < 0.00001; 95% CI, -200.93 to -129.13 ml), a reduction in the drop of haematocrit of 2.32 g/dl (p < 0.00001; 95% CI, -3.38 to -1.26 g/dl), and an improved quality of surgical field (p < 0.00001; MD, -1.01; 95% CI, -1.23 to -0.80). Tranexamic acid has a limited effect on reducing operative time (p < 0.00001; MD, -16.18 min; 95% CI, -19.60 to -12.75 min) and on decreasing the transfusion rates (p = 0.02; OR = 0.33; 95% CI, 0.13 to 0.83).