A comparison of osteogenic effect of newly manufactured calcium silicate-based sealers in vitro.

This study aimed to assess the effect of different calcium silicate-based root canal sealers (CSRS) on osteogenic effect in human periodontal ligament cells (hPDLCs). hPDLCs were cultured in a medium containing extract of 5 types of CSRS. The specimens were assessed by the cell cytotoxicity test, alkaline phosphatase staining, alizarin red S staining, quantitative real-time PCR, Western blot analysis, and enzyme-linked immunosorbent assay. The diluted concentrations of extracted solutions had no significant effect on the viability of hPDLCs. There was a statistically significant difference in the mRNA expression level of bone sialoprotein (BSP), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2) among some groups. The protein expressions of BSP, OCN, and RUNX2 were significantly higher in some groups compared to the control group. The CSRS did not interfere with the osteogenic differentiation of hPDLCs, compared to the control group. CSRS are shown to have biocompatibility and osteogenic differentiation effect on hPDLCs.

Irisin: A Myokine With Therapeutic And Diagnostic Potential In Clinical Dentistry.

Irisin is a novel adipomyokine which has extensive systemic and local effects in different tissues of the body. The scientific interest in understanding the physiological roles of irisin in the body has increased tremendously in the past few years due to its vast therapeutic potential in different fields of medicine. The current narrative review was planned to describe the molecular mechanisms by which irisin regulates oral hard and soft tissues. The information gleaned provided useful insights for future researchers to investigate newly discovered roles of irisin in craniofacial health and disease, and to explore the potential of irisin as a promising therapeutic and diagnostic agent in clinical dentistry.

Regulation of reactive oxygen species on the mitophagy of human periodontal ligament cells through the PINK1/Parkin pathway under starvation.

OBJECTIVES: This study aimed to explore the specific mechanism, mediated by the reactive oxygen species (ROS) and PINK1/Parkin pathway, of the mitochondrial autophagy of human periodontal ligament cells (hPDLCs) under starvation conditions. METHODS: hPDLCs were isolated and cultured from normal periodontal tissues. Earle's balanced salt solution (EBSS) was used to simulated a starvation environment and thus stimulate hPDLCs mitochondrial autophagy. N-Acetyl-L-cysteine (NAC) was used to inhibit ROS production to explore the role of ROS in hPDLC mitochondrial autophagy. Cyclosporin A was used to inhibit the PINK1/Parkin pathway to study the role of ROS and the PINK1/Parkin pathway in hPDLCs activation under starvation. The mitochondrial membrane potential was detected by flow cytometry with a JC-1 mitochondrial membrane potential detection kit. The morphological structure of mitochondria and the formation of mitochondrial autophagosome were observed by transmission electron microscopy. Mito tracker red cmxros and lyso tracker green staining were used to observe the localization of mitochondria and lysosomes. The formation intensity of ROS was detected with a DCFH-DA ROS fluorescent probe. The expression levels of mitochondrial autophagy genes (Tomm20 and Timm23) and the PINK1/Parkin pathway were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of mitochondrial autophagy proteins (Tomm20 and Timm23) and PINK1/Parkin protein were detected by Western blot. RESULTS: EBSS starvation for 30 min induced the strongest activation of hPDLCs mitochondrial autophagy, increased the expression of ROS, downregulated the expression of mitochondrial autophagy-related genes (Tomm20 and Timm23) (P<0.001), and upregulated the PINK1/Parkin pathway (P<0.001). After NACinhibited ROS production, mitochondrial autophagy was also inhibited. Meanwhile, the expression of Tomm20 and Timm23 was upregulated (P<0.001 and P<0.05), and the expression of the PINK1/parkin pathway (P<0.001 and P<0.05) was down regulated. When cyclosporin A inhibited the expression of the PINK1/Parkin pathway (P<0.05 and P<0.05), it reversed the mitochondrial autophagy of hPDLCs (P<0.001 and P<0.01) and also upregulated the expression of Tomm20 and Timm23 (P<0.001 and P<0.01). CONCLUSIONS: ROS enhanced the mitochondrial autophagy of hPDLCs primarily through the PINK1/Parkin pathway under starvation conditions.

FNDC5/irisin is expressed and regulated differently in human periodontal ligament cells, dental pulp stem cells and osteoblasts.

OBJECTIVE: To examine the expression and regulation of fibronectin type III domain-containing protein 5/irisin (FNDC5/irisin) in primary human periodontal ligament (hPDL) cells, dental pulp stem cells (hDPCs) and osteoblasts (hOBs). METHODS: FNDC5/irisin was identified in sections of paraffin embedded rat maxillae, cryo-sections of 3D cultured spheroids hPDL cells, hDPCs and hOBs, 2D cultured hPDL cells, hDPCs and hOBs by immunohistochemistry. The expression of FNDC5/irisin was identified by qPCR, followed by sequencing of the qPCR product. Regulation of FNDC5/irisin expression in hPDL cells, hDPCs and hOBs were evaluated after administration of different concentrations of irisin and all-trans retinoic acid (ATRA). qPCR and ELISA were used to identify expression and secretion of FNDC5/irisin in odontoblast-like differentiation of hDPCs. RESULTS: FNDC5/irisin was confirmed to be present in rat periodontium and dental pulp regions, as well as in 2D and 3D cultured hPDL cells, hDPCs and hOBs. BLAST analyses verified the generated nucleotide alignments matched human FNDC5/irisin. FNDC5/irisin gene expression was enhanced during odontoblast-like differentiation of hDPCs whereas the secretion of the protein was decreased compared to control. The protein signals in rat periodontal and pulpal tissues were higher than that of alveolar bone, and the expression of FNDC5/irisin was differently regulated by recombinant irisin and ATRA in hPDL cells and hDPCs compared to hOBs. CONCLUSIONS: FNDC5/irisin expression was verified in rodent periodontium and dental pulp, and in hPDL cells, hDPCs and hOBs. The FNDC5/irisin expression was regulated by recombinant irisin and ATRA. Finally, expression and secretion of FNDC5/irisin were affected during odontoblast-like differentiation of hDPCs.

MicroRNA expression profiling of nicotine-treated human periodontal ligament cells.

Cigarette smoking is a lifestyle-related risk factor involved in the causation and progression of periodontal disease. Nicotine is a key toxic component of tobacco. However, the mechanisms underlying nicotine-induced periodontitis have not yet been fully elucidated. The present study investigated the microRNA (miRNA) expression profile of human periodontal ligament cells (PDLCs) treated with nicotine. Using differential analysis of miRNA array data, several differentially expressed miRNAs were identified in nicotine-treated PDLCs. Quantitative real-time PCR was employed to verify the accuracy of the miRNA array, and the targets of these dysregulated miRNAs were further analyzed. Function and pathway enrichment of differentially expressed miRNAs suggested that several important signaling pathways, such as the Toll-like receptor signaling pathway, nicotine addiction, the transforming growth factor-beta signaling pathway, and the hypoxia inducible factor-1 signaling pathway, are potentially responsible for nicotine-induced periodontitis. This study has helped to clarify the epigenetic mechanisms of nicotine-induced periodontitis, highlighting novel biomarkers and therapeutic targets.

Force-induced decline of TEA domain family member 1 contributes to osteoclastogenesis via regulation of Osteoprotegerin.

OBJECTIVE: This study aims to investigate the responsiveness of transcription factor TEA domain family member 1 (TEAD1) to mechanical force and its impact on osteoclastogenesis as well as expression of Osteoprotegerin (OPG), an inhibitor for osteoclastogenesis playing crucial roles in mechanical stress-induced bone remodeling and orthodontic tooth movement (OTM). METHODS: We first analyzed the correlation between several transcription factors and OPG expression in human periodontal ligament cells (PDLCs). Then dynamic expression changes of TEAD1 with force application were analyzed due to its high correlation with OPG. Loss-of-function experiments were performed to demonstrate the role of TEAD1 in regulation of RANKL/OPG, as well as osteoclastogenesis by tartrate-resistant acid phosphatase (TRAP) staining. Combination of bioinformatics analyzes and chromatin immunoprecipitation assay was utilized to investigate occupancy of TEAD1 on the enhancer elements of OPG and the dynamic change in response to force stimuli. Involvement of Hippo signaling in regulation of OPG was further demonstrated by pharmacologic inhibitors of several components. RESULTS: Expression of TEAD1 highly correlates with that of OPG and decreases in response to mechanical force in human PDLCs. Knockdown of TEAD1 downregulates expression of OPG and promotes osteoclast differentiation. Mechanical force induced decreased binding of TEAD1 on an enhancer element ˜22 kilobases upstream of OPG promoter. OPG was also affected by pharmaceutical disruption of Hippo signaling pathway. CONCLUSIONS: TEAD1 is a novel mechano-responsive gene and plays an important role in force-induced osteoclastogenesis, which is dependent, as least partially, on transcriptional regulation of OPG.

Existence of ATP sensitive potassium currents on human periodontal ligament cells.

OBJECTIVE: Potassium channels of the ATP-sensitive family (KATP channel) are inhibited by increase in intracellular ATP. Electrophysiological studies have demonstrated that the kinetics and pharmacological properties of KATP channels vary among different tissues, suggesting structurally and functionally distinct types. There are studies showing human periodontal ligament (PDL) cells respond to mechanical stress by increasing ATP release, which participates in bone resorption or bone homeostasis. So, in this study we investigated the existence of KATP channel subunit and their single channel properties in human periodontal ligaments. MATERIALS & METHOD: The human PDL cells were isolated from healthy erupted third molar. For patch-clamp experiments, human PDL fibroblasts were seeded on 3.5cm plastic dishes. The inside-out patch clamp recordings were performed under voltage clamp mode. Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted to identify the channel subunits. All pair-wise comparisons were performed by Paired t-test. A P value <0.05 was considered significant. RESULTS: We observed mRNA transcripts for Kir6.1, Kir6.2 and Sur2B subuits in the human PDL cells. In inside-out patch mode, the single channel conductance was 163pS at symmetrical K+ concentration of 140mM and inward rectification was seen in ATP-free bath solution. The reversal potential of the currents was found to be 0mV at symmetrical concentration (140mM) of K+ in bath solution. The single channel currents were almost blocked by adding 5mM ATP in the bath solution. However, the currents were not blocked by 100µM glibenclamide, a subunit specific KATP channel blocker. CONCLUSIONS: These results indicate that human PDL cells express KATP channels subunit including Sur2B and Kir6.1 and Kir6.2 which are sensitive to ATP but insensitive to glibenclamide.

Activation of NLRP1 and NLRP3 inflammasomes contributed to cyclic stretch-induced pyroptosis and release of IL-1ß in human periodontal ligament cells.

Inflammasomes have been reported to be present in periodontal inflammatory tissue, but the exact role of inflammasomes in periodontal inflammatory reactions especially those related to mechanical stimulations has not been clarified. In this study, it was shown that cyclic stretch activated the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 1 and 3 (NLRP1 and NLRP3) inflammasomes and induced the release of IL-1ß and pyroptosis via a caspase-1-related mechanism in human periodontal ligament cells (HPDLCs). This study firstly demonstrated that activation of NLRP inflammasomes contributed to the stretch-induced inflammatory response in HPDLCs. As inflammasomes have been reported to be involved in both programmed cell death and inflammation, further studies are required to elucidate the exact roles and signaling pathway of inflammasomes in stretch-induced periodontal inflammation.

Effects of Luteolinl on the Proliferation and Differentiation in Human Periodontal Ligament Cells / 沈阳医学院学报

Objective:To evaluate the biological effects of Luteolinl on the proliferation and differentiation in human periodontal ligament cells (PDLC) . Methods:MTT, ALP kit and Q-PCR was used to detect the expression of osteogenesis related gene. Results:Luteolinl (100, 10, 1, 0.1, 0.01μmol/L) could increase the proliferation of PDLC, and there were significant differences compared with control group (P<0.05) . The ALP Kit results showed that Luteolinl could increase the ALP actiuty of PDLC (P<0.05) . The Q-PCR results showed that Luteolin could increase the expression of ALP and RUNX2 (P<0.05) . Conclution:At proper concentration,Luteolinl can increase the proliferation and differentiation of PDLC.

Screening of osteoanagenesis-active compounds from Scutellaria baicalensis Georgi by hPDLC/CMC-online-HPLC/MS.

In present study, an online analytical method using human periodontal ligament cell/cell membrane chromatography (hPDLC/CMC) combined with high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for direct recognition, separation, and identification of compounds for the first time from Scutellaria baicalensis Georgi (SBG) that are active on hPDLCs. Baicalein (BAI) and wogonin (WOG), which were identified as the active compounds of the ethyl ether extract of S. baicalensis Georgi (SBGEE), could bind to the same membrane receptor of hPDLC for simvastatin (SIM). Moreover, BAI (0.15-0.6 mg/L) and WOG (0.015-0.6 mg/L) had the capability to enhance cell proliferation, matrix calcification, and formation of calcified nodules, which are comparable to the activities of SIM (0.1 mg/L) in vitro. These observations are consistent with the K(A) of the various drugs. It is very important for the development of SBG used to treat periodontitis.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells / 药物分析学报

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in cultured human periodontal ligament (HPDL) cells. Methods Small interfering RNA (siRNA) eukaryotic expression vector targeted transforming growth factor βⅡ receptor (TGF-β RⅡ) was constructed and transfected into T cells. HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide (LPS) and baicalin. The obtained solution was divided into six groups according to the components (group Ⅰ: HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ: HPDL cells+LPS+T cells transfected with siRNA1; group Ⅲ: HPDL cells+LPS+T cells+baicalin; group Ⅳ: HPDL cells+LPS+T cells; group Ⅴ: HPDL cells+baicalin; group Ⅵ: HPDL cells) and was cultured for 48 hours. RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells. Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ (P<0.01) and higher than that in group Ⅲ (P<0.01); The ratio of RANKL/OPG in gronp Ⅲ was lower than that in group Ⅳ (P<0.01); the ratio of RANKL/ OPG in group Ⅳ was higher than that in group Ⅵ (P<0.01); the ratio of RANKL/OPG in group Ⅴ was lower than TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells / 西安交通大学学报·英文版

Objective: To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in cultured human periodontal ligament (HPDL) cells. Methods: Small interfering RNA (siRNA) eukaryotic expression vector targeted transforming growth factor βII receptor (TGF-β R II) was constructed and transfected into T cells. HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide (LPS) and baicalin. The obtained solution was divided into six groups according to the components (group I: HPDL cells+LPS+ T cells transfected with siRNA1 + baicalin; group II: HPDL cells+LPS+T cells transfected with siRNA1; group III: HPDL cells+LPS+T cells+baicalin; group IV: HPDL cells+LPS+T cells; group V: HPDL cells+baicalin; group VI: HPDL cells) and was cultured for 48 hours. RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells. Results: The ratio of RANKL/OPG in group I was lower than that in group II (P<0.01) and higher than that in group III (P<0.01); The ratio of RANKL/OPG in group III was lower than that in group IV (P<0.01); the ratio of RANKL/OPG in group IV was higher than that in group VI (P<0.01); the ratio of RANKL/OPG in group V was lower than that in group VI (P<0.05). Conclusion: Circled digit one Baicalin could decrease the ratio of RANKL/OPG in HPDL cells. Circled digit two The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells. Circled digit three Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells, but also through other pathways.

Effects of various NSAIDs on prostaglandin synthesis and cellular configuration of human periodontal ligament cells

The present study was designed to evaluate effects of the commonly used NSAIDs(acetaminophen, aspirin, and ibuprofen) on human periodontal ligament cells. Human periodontal ligament cells were grown from a cell line provided by Kyungpook National University. Effects of NSAIDs on the proliferation of human periodontal ligament cells were assessed using MTT assays. And then PGE2 concentrations were determined by ELISA and the changes of cellular configuration were found by electron micrograph. The results were as follows; 1. The MTT assay demonstrated that the commonly used NSAIDs(acetaminophen, aspirin, and ibuprofen) had not significant cytotoxic effect on human periodontal ligament cells. 2. NSAIDs inhibited the PGE2 synthesis of human periodontal ligament cells compared with the control group. These inhibitory effects had no significant differences with NSAID type and concentration. 3. Electron micrographs of human periodontal ligament cells treated with NSAIDs showed more narrow and irregular shape.

Evaluation of the radiopacity and cytotoxicity of resinous root canal sealers / 대한치과보존학회지

The aim of this study was to evaluate the radiopacity and cytotoxicity of three resin-based (AH 26, EZ fill and AD Seal), a zinc oxide-eugenol-based (ZOB Seal), and a calcium hydroxide-based (Sealapex) root canal sealers. Specimens, 10 mm in diameter and 1 mm in thickness, were radiographed simultaneously with an aluminum step wedge using occlusal films, according to ISO 6876/2001 standards. Radiographs were digitized, and the radiopacity of sealers was compared to the different thicknesses of the aluminum step wedge, using the Scion image software. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicity of each material was determined in immortalized human periodontal ligament (IPDL) cells. The results demonstrated that EZ fill was the most radiopaque sealer, while Sealapex was the least radiopaque (p 0.05). These results indicate that resin-based root canal sealer is more biocompatible and has advantage in terms of radiopacity.

Effect of continuously compressive pressure and human periodontal ligament cells on the differentiation of osteoclast-like cells in vitro / 西安交通大学学报(医学版)

Objective To study the effect of continuously compressive pressure(CCP) and human periodontal ligament cells(HPDLCs) on the differentiation of osteoclast-like cells(OLC) induced from umbilical cord blood cells in vitro,and to investigate the role of continuously compressive pressure and human periodontal ligament cells in alveolar bone rebuilding during orthordontic tooth movement.Methods Mononucleared cells of umbilical cord blood(HCMNCs) were separated by density gradient centrifugation,HPDLCs were isolated from human periodontal ligament by explanting enzymatic digestion with trypsin and collagenase.We also established transwell co-culture system with HCMNCs in the lower layer and HPDLCs in the upper layer.Group A: HCMNCs and HPDLCs were co-cultured with 150 kPa CCP for 1.5 hours on the model.Group B: only HCMNCs were cultured with the same CCP as Group A.Groups A'and B' were the respective control group of Groups A and B with no CCP exerted.The cell appearance was observed under the phase contrast microscope,and its identification was performed by histochemical analysis of tartrater-resistant acid phosphatase(TRAP).The capacity of bone resorption of the cell was assessed by lacunae forming ability on bone slice.Results HCMNCs in Group A began to fuse on the 2nd day,More positive multinucleated cells could be seen with TRAP staining and cortical bone pit formation on the 3rd day.Only a few multinucleated cells formed in the other groups,with no cortical bone pit formation.Conclusion HCMNCs can fuse into multinucleated OLC under CCP with the induction of HPDLCs.