M2 Macrophages regulate cementogenic differentiation of human periodontal ligament stem cells by modu-lating oxidant-antioxidant system and mitophagy / 实用口腔医学杂志

Objective:To investigate the effects of macrophage(Mφ)polarization on the cementogenic differentiation of human perio-dontal ligament stem cells(hPDLSCs)and the underlying mechanism.Methods:Human monocytic THP-1 cells were induced to M0,M1 and M2 Mφ subsets,then RPM1 1640 medium or supernatants of different Mφ phenotypes were mixed with an equal volume of ce-mentoblastic induction medium to generate conditioned mediums(CMs),and termed as CM-Control,CM-M0,CM-M1 and CM-M2,respectively.hPDLSCs were cultured with different CMs,and the hPDLSCs sheets were then wrapped around treated dentin matrix(TDM)to generate cell sheet/dentin complexes.The complexes were subcutaneously implanted into nude mice.The cementum-like tissue formation was evaluated by HE staining,immunofluorescent staining(IMF)and qRT-PCR were used to detect the expression level of cementogenic differentiation-related markers bone sialoprotein(BSP),cementum attachment protein(CAP)and cementum pro-tein-1(CEMP-1),oxidant-antioxidant system-related markers superoxide dismutase 1(SOD1)and nuclear factor erythroid 2-related factor 2(NRF2),mitophagy-related markers PTEN induced putative kinase 1(PINK1)and microtubule asso ciated proteins 1A/1B light chain 3(LC3).Results:In vivo,CM-M2-treated hPDLSCs(CM-M2)group formed more cementum-like tissues and expressed higher protein levels of CAP,CEMP-1,SOD1,PINK1 and LC3 than that in other groups.In vitro tests showed that,compared with CM-Control group,hPDLSCs incubated with CM-M2 increased the levels of BSP(P＜0.01),CAP(P＜0.001),CEMP-1(P＜0.01)and SOD1(P＜0.05),while no statistically significant difference was detected for NRF2(P＞0.05),and increasedthe expression of PINK1(P＜0.05).Conclusion:M2 Mφ regulate the cementogenic differentiation of hPDLSCs possibly via modulating oxidant-antioxidant system and mitophagy.

The influence of static mechanical strain on osteoclastogenic gene expression of HPDLSCs and PPDLSCs / 实用口腔医学杂志

Objective:To explore the influence of static mechanical strain(SMS)on the osteoclastogenic gene expression of healthy periodontal ligment stem cells(HPDLSCs)and periodontitis periodontal ligment stem cells(PPDLSCs).Methods:HPDLSCs and PP-DLSCs were respectively isolated and cultured by low density in vitro.The expression of mesenchymal stem cell markers were detected by flow cytometry.Then,6％,8％,10％,12％and 14％SMS were respectively loaded to the HPDLSCs and PPDLSCs by Flexcell Tension Unit,and the expression of RANKL and C-fos was detected by real time RT-PCR.Results:Both HPDLSCs and PPDLSCs strongly expressed the mesenchymal stem cell markers STRO-1,CD146,CD90 and CD29,and higher expression of the above markers was found in HPDLSCs compared with PPDLSCs(P＜0.05).The expression of RANKL and C-fos in PPDLSCs was more obvious than that in HPDLSCs without SMS loading(P＜0.05).For HPDLSCs,the SMS of 14％induced significant up-regulation of RANKL and C-fos(P＜0.05),while no alteration was confirmed for the above osteoclastogenic genes when the SMS≤ 12％(P＞O.05).In addition,the expression of RANKL and C-fos was up-regulated significantly in PPDLSCs when the SMS≥ 10％(P＜0.05),and the expression of the above genes was not activated when the SMS ≤8％.Conclusion:HPDLSCs and PPDLSCs response differently to SMS,and ex-cessive SMS may lead to enhanced expression of osteoclastogenic genes in PPDLSCs.

LncRNA SNHG4 regulates miR-152-3p during osteoblastic differentiation of periodontal ligament stem cells / 中国组织工程研究

BACKGROUND:Studies have shown that long non-coding RNA small nucleolar RNA host gene 4(lncRNA SNHG4)is involved in the progress of many inflammatory diseases,but the effect of lncRNA SNHG4 on the osteogenic differentiation of human periodontal ligament stem cells during the treatment of periodontitis is still unclear. OBJECTIVE:To investigate the effect of lncRNA SNHG4 on the osteogenic differentiation of human periodontal ligament stem cells by regulating miR-152-3p. METHODS:Human periodontal ligament stem cells were isolated from periodontal membranes of premolars extracted for orthodontic purposes.After human periodontal ligament stem cells were induced to differentiate into osteoblasts for 0,7,and 14 days,the expression levels of RUNX family transcription factor 2,osteocalcin mRNA,lncRNA SNHG4 and miR-152-3p in human periodontal ligament stem cells were detected by qRT-PCR.The third-generation human periodontal ligament stem cells were divided into the NC group,pcDNA group,pcDNA-SNHG4 group,inhibitor NC group,miR-152-3p inhibitor group,pcDNA-SNHG4+mimic NC group,and pcDNA-SNHG4+miR-152-3p mimic group.The expression of lncRNA SNHG4 and miR-152-3p in human periodontal ligament stem cells was detected by qRT-PCR.The proliferation of human periodontal ligament stem cells was detected by CCK-8 assay.Alkaline phosphatase activity was detected by colorimetry.The formation of mineralized nodules was detected by alizarin red staining.Western blot assay was used to detect the expression of RUNX family transcription factor 2,osteocalcin and alkaline phosphatase proteins.A double luciferase reporter gene experiment was applied to verify the relationship between lncRNA SNHG4 and miR-152-3p. RESULTS AND CONCLUSION:(1)The expression of RUNX family transcription factor 2,osteocalcin mRNA and lncRNA SNHG4 in human periodontal ligament stem cells after 7 and 14 days of osteogenic induction was higher than that after 0 days of osteogenic induction,while the expression of miR-152-3p was lower(P<0.05).(2)Overexpression of lncRNA SNHG4 or inhibition of miR-152-3p was able to enhance the proliferation of human periodontal ligament stem cells,the alkaline phosphatase activity,mineralized nodule formation,the expression of RUNX family transcription factor 2,osteocalcin,and alkaline phosphatase proteins(P<0.05).miR-152-3p mimic attenuated the promoting effect of overexpression of lncRNA SNHG4 on osteogenic differentiation of human periodontal ligament stem cells.LncRNA SNHG4 had a targeting relationship with miR-152-3p.(3)These findings indicate that overexpression of lncRNA SNHG4 may promote the osteogenic differentiation of human periodontal ligament stem cells by inhibiting miR-152-3p.

The role of reactive oxygen species in periodontitis and periodontal tissue regeneration / 中国组织工程研究

BACKGROUND:Reactive oxygen species is a double-edged sword in the development of periodontitis and the regeneration of periodontal tissue.Low concentration of reactive oxygen species induces the differentiation of periodontal fibroblasts,and excessive reactive oxygen species will cause damage to periodontal tissue.In the process of inflammation,the accumulation of reactive oxygen species in periodontal tissue induces damage to cells and tissues through a variety of signaling pathways or through redox reactions. OBJECTIVE:To review the double-edged sword effect of reactive oxygen species in periodontitis and periodontal tissue regeneration,thereby providing potential targets and treatment ideas for the clinical treatment of periodontitis and periodontal tissue regeneration. METHODS:Databases of CNKI and PubMed were searched for relevant articles published from April 1990 to April 2023 with the key words of"periodontal tissue engineering,periodontal defect,regeneration of periodontal tissue,chronic periodontitis,reactive oxygen species,oxidative stress,antioxidative stress,oxidative injuries,free radicals,reactive nitrogen species"in Chinese and English,respectively.By reading the titles and abstracts,repetitive studies or irrelevant literatures were excluded.Finally,77 articles were included for review. RESULTS AND CONCLUSION:Reactive oxygen species are a kind of free radicals with high reactivity.When bacteria invade,reactive oxygen species are released in large quantities by the respiratory explosion of neutrophils and play a double-edged sword role in the body through their redox reactions or as pleiotropic physiological signal transmitters.In periodontitis,low concentrations of reactive oxygen species can kill invading pathogenic bacteria,but high concentrations of reactive oxygen species promote the secretion of inflammatory factors through JNK,RANK,Wnt/β-Catenin and other pathways,promote immune damage or directly damage tissues through oxidative reactions or through other ways to aggravate periodontitis.In the process of periodontal tissue regeneration,low concentrations of reactive oxygen species can promote the proliferation and differentiation of periodontal ligament stem cells through Nrf2 and other pathways and can promote the secretion of vascular endothelial growth factor to promote vascular regeneration.This provides seeds and a nutrient environment for periodontal tissue regeneration,which is extremely important for promoting periodontal tissue regeneration.However,high concentrations of reactive oxygen species will reduce the activity of periodontal ligament stem cells and damage endothelial cells,which are not conducive to vascular regeneration.This will inhibit wound healing and periodontal tissue regeneration.Therefore,it is important to explore the role of reactive oxygen species in the development of periodontitis and periodontal tissue regeneration and to discover the potential mechanism of its action and to explore the appropriate concentration for its role in reducing periodontal inflammation and promoting periodontal tissue regeneration for the future treatment of periodontitis and periodontal tissue regeneration in clinical practice.Using reactive oxygen species as a target to explore ways to reduce periodontal inflammation and promote periodontal ligament stem cell activity and vascular regeneration may become a clinically effective method for treating periodontitis and promoting periodontal tissue regeneration.

Long non-coding RNA and periodontitis / 中国组织工程研究

BACKGROUND:In recent years,numerous studies have shown that long non-coding RNAs(lncRNAs)have an important role in the pathogenesis of periodontitis,including the immune response process and the biological activity and function of cells(periodontal stem cells and osteoblasts).Researchers attempt to regulate periodontal inflammation and periodontal regeneration by regulating lncRNA expression levels. OBJECTIVE:To review the progress of research on lncRNAs in periodontitis,thereby advancing the research of lncRNAs in periodontitis. METHODS:The first author searched PubMed,China National Knowledge Infrastructure(CNKI),Web of Science database,and WanFang Database for relevant literature published up to March 2023."Long non-coding RNA,lncRNA,periodontitis,periodontium,immunity,inflammation,periodontal ligament stem cells,osteoclasts,osteogenic differentiation,bone resorption,bone formation,recurrence,hypoxia,oxidative stress,static mechanical strain"were used as the Chinese search terms."lncRNA,periodontitis,periodontal,immunity,inflammation,periodontal membrane stem cells,osteoclasts,osteogenic differentiation,bone resorption,bone formation,recurrence,hypoxia,oxidative stress,static mechanical strain"were used as the English search terms.The title and abstract of each paper were read for initial screening,and 87 papers were finally selected for inductive analysis. RESULTS AND CONCLUSION:Periodontal pathogens stimulate the body,leading to immune imbalance that triggers inflammatory response and destroy periodontal tissue,and lncRNAs are involved in its regulatory mechanism.LncRNAs are involved in the pro-inflammatory regulation of periodontal ligament cells in an inflammatory environment,and their effects on osteoclast differentiation are regulated through ceRNA mechanism,which provides new clues for exploring the pathogenesis of periodontitis.For B cells and macrophages,lncRNAs can regulate the infiltration,cell activity and function of their subsets in periodontitis.LncRNAs participate in the immune response related to periodontitis mainly through two pattern recognition receptors,Toll-like receptors and NOD-like receptors,and nuclear factor-κB signaling pathway.To investigate whether lncRNAs can be used as a biomarker of periodontitis has great value in the diagnosis and prognosis of periodontitis.Animal experiments have demonstrated that the role of lncRNAs in periodontitis can be reversed by modulating the expression level of lncRNAs and thus lncRNAs act as an anti-inflammatory agent,which is of great value for the study of immunotherapy in periodontitis.The regulation of lncRNAs on periodontal ligament stem cells is mainly realized through endogenous competition mechanism and various signaling pathways,and its effects are influenced by various factors,such as inflammatory environment,mechanical strain,hypoxia and oxidative stress.Research on related mechanisms through these related factors provides new ideas for the treatment of periodontitis.

Research progress on immunomodulatory properties of periodontal ligament stem cells / 口腔疾病防治

@#Periodontal ligament stem cells (PDLSCs) have the potential for multidirectional differentiation and are the preferred seed cells for periodontal tissue regeneration. In recent years, a large number of studies have confirmed that PDLSCs also possess broad immunomodulatory properties. Therefore, in-depth exploration of their specific molecular mechanisms is of great significance for the treatment of periodontitis. The aim of this paper is to summarize the research progress on the regulation of PDLSCs on various immune cells and the effect of the inflammatory environment on the immune characteristics of PDLSCs to provide an important theoretical basis for the allotransplantation of PDLSCs and improve the therapeutic effect of periodontal tissue regeneration. Studies have shown that PDLSCs possess a certain degree of immunosuppressive effect on both innate and acquired immune cells, and inflammatory stimulation may lead to the impairment of the immunoregulatory properties of PDLSCs. However, current studies are mainly limited to in vitro cell tests and lack in-depth studies on the immunomodulatory effects of PDLSCs in vivo. In vivo studies based on cell lineage tracing and conditional gene knockout technology may become the main directions for future research.

Inhibition of lipopolysaccharide-induced NF-κB maintains osteogenesis of dental pulp and periodontal ligament stem cells

Abstract Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) can differentiate into osteoblasts, indicating that both are potential candidates for bone tissue engineering. Osteogenesis is influenced by many environmental factors, one of which is lipopolysaccharide (LPS). LPS-induced NF-κB activity affects the osteogenic potencies of different types of MSCs differently. This study evaluated the effect of LPS-induced NF-κB activity and its inhibition in DPSCs and PDLSCs. DPSCs and PDLSCs were cultured in an osteogenic medium, pretreated with/without NF-κB inhibitor Bay 11-7082, and treated with/without LPS. Alizarin red staining was performed to assess bone nodule formation, which was observed under an inverted light microscope. NF-κB and alkaline phosphatase (ALP) activities were measured to examine the effect of Bay 11-7082 pretreatment and LPS supplementation on osteogenic differentiation of DPSCs and PDLSCs. LPS significantly induced NF-κB activity (p = 0.000) and reduced ALP activity (p = 0.000), which inhibited bone nodule formation in DPSCs and PDLSCs. Bay 11-7082 inhibited LPS-induced NF-κB activity, and partially maintained ALP activity and osteogenic potency of LPS-supplemented DPSCs and PDLSCs. Thus, inhibition of LPS-induced NF-κB activity can maintain the osteogenic potency of DPSCs and PDLSCs.

Comparison of two obturation techniques on adhesion and proliferation of human periodontal ligament fibroblasts / Comparación de la adhesión y proliferación de fibroblastos periodontales humanos en dos técnicas de obturación endodóntica

The present study aimed to compare the adhesion and proliferation of human periodontal ligament fibroblasts (hPDL) in transverse sections of the teeth sealed with two different obturation techniques, BioRoot RCS/hydraulic obturation (HO) and AH-Plus/continuous-wave condensation (CWC). The techniques were tested using an in vitro model to simulate the interaction between periodontal tissues and the materials. The root canals were instrumented and sterilized. A total of 15 samples were obturated with BioRoot RCS/HO and 15 samples with AH-Plus/CWC. Then, roots were sectioned to obtain obturated teeth slices, and hPDL cells were seeded onto the root slices. The results were obtained at intervals of 4 and 24h for cell adhesion; and at 3,7,14, and 21 days for cell proliferation. Empty cell culture plates were use as controls. The cell adhesion was increased at 4 and 24h for both groups, with an increased response observed in the BioRoot RCS/HO group (p<0.05). The difference in cell proliferation was also found between experimental groups. After 14 days of culture, BioRoot RCS/HO group showed an increase response than control and AH-Plus/CWC groups (p<0.05), and after 21 days both groups behaved better than control group, with an increased response observed in the BioRoot RCS/HO group. This study demonstrated that both root canal sealers allow the attach and growth of periodontal ligament fibroblasts, with an increased biological response in the BioRoot RCS/HO group.

El presente estudio se enfocó en comparar la adhesión y proliferación de fibroblastos de ligamento periodontal humano (hPDL) en secciones transversales de raíces previamente obturadas con dos técnicas de obturación diferentes: obturación hidráulica empleando cono único de gutapercha y BioRoot RCS como sellador (HO), y obturación de condensación de onda continua y AH-Plus como sellador (CWC). Los selladores se usaron en un modelo in vitro que simula la interacción entre los tejidos periodontales y los materiales de obturación. Los conductos radiculares fueron instrumentados, esterilizados y obturados. La muestra se compuso de un total de 15 raíces con la técnica BioRoot RCS/HO y 15 raíces con la técnica AH-Plus/CWC. Las células de hPDL fueron sembradas en condiciones estándar de cultivo sobre las raíces seccionadas. Los resultados fueron obtenidos a intervalos de 4 y 24h para adhesión celular, y a los 3,5,7,14 y 21 días de cultivo para proliferación celular. La adhesión celular a las 4 y 24 horas mostró ser diferente para ambas técnicas en comparación con el grupo control, siendo más importante en el grupo BioRoot RCS/HO. La diferencia en la proliferación entre grupos se observó a los 14 días de cultivo, únicamente para el grupo BioRoot RCS/HO; Sin embargo para el día 21 ambas técnicas mostraron mayor proliferación celular que el grupo control, con mejor respuesta para el grupo BioRoot RCS/HO. Este estudio ha demostrado que ambos selladores de conductos permiten la adhesión y crecimiento de fibroblastos de ligamento periodontal, siendo el grupo BioRoot RCS/HO el que mostró mayor biocompatibilidad.

Research progress on ankylosis of primary molars / 口腔疾病防治

@#Ankylosis of primary molars is a kind of eruption abnormality of the teeth, where the periodontal membrane disappears, owing to a bony union between bone and root. Studies have shown that the common proportion of ankylosed primary molars is 1.3%~8.9% with an equal occurrence. In the primary dentition, the mandibular first primary molar is the most commonly affected tooth, while in the middle mixed dentition stage of development, the second primary molar is more affected. Its etiology may be related to genetics, signaling pathways of mineralization metabolism of local alveolar bone or cementum, cytokines secreted by epithelial rest cells of Malassez, and enhanced inflammatory reactions during physiological absorption of roots. Ankylosis of primary molars can be diagnosed by clinical symptoms and imaging and is classified as mild, moderate and severe according to the degree of infraocclusion. As it may cause a series of complications, such as occlusal disturbances, delayed exfoliation and incomplete alveolar process development, multidisciplinary treatment, including in the departments of pediatric dentistry, orthodontics, periodontics and prosthodontics, should be adopted, and long-term treatment is determined based on the patient's age, severity of infraocclusion, and presence of permanent teeth. This review summarizes the etiology, diagnosis, complications and treatment of ankylosed primary molars to provide a reference for the clinical diagnosis and treatment of decidual molar fixation.

Overview of the main biological mechanisms linked to changes in periodontal ligament stem cells and the inflammatory microenvironment / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Periodontitis is a complex chronic inflammatory disease. The invasion of pathogens induces the inflammatory microenvironment in periodontitis. Cell behavior changes in response to changes in the microenvironment, which in turn alters the local inflammatory microenvironment of the periodontium through factors secreted by cells. It has been confirmed that periodontal ligament stem cells (PDLSCs) are vital in the development of periodontal disease. Moreover, PDLSCs are the most effective cell type to be used for periodontium regeneration. This review focuses on changes in PDLSCs, their basic biological behavior, osteogenic differentiation, and drug effects caused by the inflammatory microenvironment, to provide a better understanding of the influence of these factors on periodontal tissue homeostasis. In addition, we discuss the underlying mechanism in detail behind the reciprocal responses of PDLSCs that affect the microenvironment.

Red light-emitting diode light mediated by the KEAP1-NRF2/HO-1 pathway induces osteogenic differentiation and attenuates the oxidative stress damage of human periodontal ligament stem cells induced by high glucose / 口腔疾病防治

Objective@# To explore the effects of red LED light mediated by the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (KEAP1-NRF2/HO-1) pathway on osteogenic differentiation and oxidative stress damage of human periodontal ligament stem cells (hPDLSCs) induced by high glucose, which provides a basis for the application of red light-emitting diode (LED) light in cell antioxidative damage.@*Methods@#hPDLSCs were identified by flow cytometric analysis, alkaline phosphatase (ALP) staining and Alizarin red-S staining; hPDLSCs were pretreated in a high glucose environment for 48 hours and irradiated with 1, 3, or 5 J/cm2 red LED light. A CCK-8 assay was performed to choose the radiant exposure that had the strongest effect on promoting the cell proliferation rate for subsequent experiments. hPDLSCs were divided into a control group, a high glucose group and a high glucose+light exposure group. ALP staining, ALP activity, Alizarin red-S staining and quantitative calcified nodules were used to detect the osteogenic differentiation of hPDLSCs; qRT-PCR and Western blot were used to detect the gene and protein expression levels of ALP, runt-related transcription factor 2 (RUNX2) and osterix (OSX); the relative mRNA expression levels of antioxidant enzyme-related genes superoxide dismutase 2 (SOD2) and catalase (CAT) in hPDLSCs were detected by qRT-PCR; reactive oxygen species (ROS) levels were detected by fluorescence microscopy and flow cytometry; the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels in cell supernatants were detected by ELISA; the NRF2-specific inhibitor ML385 was used to inhibit the NRF2 pathway; ALP staining and ALP activity were used to detect the markers of early osteogenic differentiation; qRT-PCR was used to detect the gene expression of ALP, RUNX2 and OSX; and the protein expression levels of KEAP1, NRF2 and HO-1 were detected by Western blot.@*Results @# Identified, and irradiant exposure of 5 J/cm2 was chosen for subsequent experiments. Red LED light irradiation (5 J/cm2) improved the osteogenic differentiation of hPDLSCs induced by high glucose (P<0.05), increased the mRNA and protein levels of ALP, RUNX2 and OSX (P<0.05), upregulated the mRNA expression levels of SOD2 and CAT (P<0.05), reduced the levels of ROS (P<0.05), and reduced TNF-α and IL-1β levels in the cell supernatants (P<0.05). When ML385 was added to inhibit the NRF2 pathway, the ALP activity of cells was decreased (P<0.05); the gene expression levels of ALP, RUNX2 and OSX were downregulated (P<0.05); the protein level of KEAP1 was upregulated (P<0.05); and the protein levels of NRF2 and HO-1 were downregulated (P<0.05)@*Conclusion@#Red LED light may promote the proliferation and osteoblastic differentiation of hPDLSCs induced by high glucose through the KEAP1-NRF2/HO-1 pathway and reduce the oxidative stress damage to hPDLSCs induced by high glucose.

Effect and mechanism of baicalin on the proliferation and migration of human periodontal ligament stem cells induced by lipopolysaccharide / 中国药房

OBJECTIVE To explore the regulatory effects of baicalin on the proliferation and migration of human periodontal ligament stem cells （hPDLSCs） induced by lipopolysaccharide （LPS） and Janus protein tyrosine kinase 2 （JAK2）/signal transduction and transcription activator 3 （STAT3） signaling pathways. METHODS hPDLSCs were divided into control group， LPS group， different concentration baicalin groups （0.1， 1 and 10 mg/L）. ELISA method and CCK-8 assay were used to determine the contents of cell inflammatory factors [interleukin 6 （IL-6）， IL-1β and tumor necrosis factor-α （TNF-α）] and cell viability， so as to screen the optimal concentration of baicalin for follow-up pathway validation experiments. The cells were then divided into control group， LPS group， optimal baicalin concentration group and inhibitor group （10 μg/mL LPS+1 mg/L baicalin +3 μmol/L JAK2/STAT3 pathway inhibitor AG490）. After treated for 24 h， the proliferation rate of hPDLSCs， apoptosis rate， migration rate， invasion cell number， mRNA and protein expressions of Cyclin D1 and caspase-3， the expression of JAK2/STAT3 pathway-related proteins were all detected. RESULTS According to cell inflammatory factors and cell viability， 1 mg/L was selected as the optimal concentration of baicalin. Compared with control group， cell proliferation rate， migration rate， invasion cell number， Cyclin D1 mRNA and protein expression were significantly decreased in LPS group， while cell apoptosis rate， caspase-3 mRNA and protein expression， p-JAK2 and p-STAT3 protein expression were significantly increased （P＜0.05）. After treated with 1 mg/L baicalin， the above indexes were reversed significantly （P＜0.05）. The improvement of above indexes in the inhibitor group was more obvious （P＜0.05）. CONCLUSIONS Baicalin can promote the proliferation， migration and invasion of LPS-induced hPDLSCs and inhibit their apoptosis and inflammation by blocking the JAK2/STAT3 pathway.

Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor‑α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells / 华西口腔医学杂志

OBJECTIVES@#This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients.@*METHODS@#HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco's modification of Eagle's medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm2) and C2 and D2 (energy density: 5.625 J/cm2). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay.@*RESULTS@#1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05).@*CONCLUSIONS@#1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm2. Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.

Effect of naringenin on the anti-inflammatory, vascularization, and osteogenesis differentiation of human periodontal ligament stem cells via the stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 signaling axis stimulated by lipopolysaccharide / 华西口腔医学杂志

OBJECTIVES@#This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism.@*METHODS@#Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6.@*RESULTS@#We observed that 10 μmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 μg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 μmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor).@*CONCLUSIONS@#Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.

Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress / 华西口腔医学杂志

OBJECTIVES@#This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs).@*METHODS@#hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs.@*RESULTS@#CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05).@*CONCLUSIONS@#The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.

Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment / 华西口腔医学杂志

OBJECTIVES@#This study aimed to clarify the effects of Foxp3 silencing on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLFs) in an inflammatory environment and on cell proliferation and invasiveness, as well as to explore the role of Foxp3 gene in the development of periodontitis.@*METHODS@#An small interfering RNA (siRNA) construct specific for Foxp3 was transfected into hPDLFs. Foxp3 silencing efficiency was verified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the siRNA with the optimum silencing effect of Foxp3 gene was screened. Using lipopolysaccharide to simulate an inflammatory environment in vitro, CCK-8 detected the effect of silencing Foxp3 on hPDLFs proliferation under inflammatory conditions. Wound-healing experiments and transwell assays were conducted to detect the effect of silencing Foxp3 on hPDLF migration under inflammatory conditions. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was detected by RT-PCR and Western blotting under inflammatory conditions.@*RESULTS@#After siRNA transfection, RT-PCR and Western blotting analyses showed that the expression of Foxp3 mRNA in the Foxp3-si3 group decreased significantly (t=21.03, P<0.000 1), and the protein expression of Foxp3 also decreased significantly (t=12.8, P<0.001). In the inflammatory environment, Foxp3 gene silencing had no significant effect on hPDLFs proliferation (P>0.05), and Foxp3 gene silencing promoted hPDLFs migration (P<0.05). Moreover, the expression of IL-6 and IL-8 increased (P<0.05).@*CONCLUSIONS@#In an inflammatory environment, Foxp3 gene silencing promoted hPDLFs migration but had no significant effect on hPDLFs proliferation. The expression of inflammatory factors expressed in hPDLFs increased after Foxp3 gene silencing, indicating that Foxp3 gene inhibited inflammation in periodontitis.

Viability of periodontal ligament cells in selected transport media for avulsed teeth: an in vitro study

To compare the viability of periodontal ligament (PDL) cells stored in Hanks Balanced Salt Solution (HBSS) with those in readily available transport media over a variable period of time. Methods: Periodontal ligament cells harvested from premolars freshly extracted for orthodontic reasons were cultured for exponential growth. The cells were exposed to egg white, evaporated milk, water and Hanks Balanced Salt Solution (HBSS) at room temperature. Their viability was evaluated after 30 minutes, 1 hour and 3 hours with the tetrazolium salt-based colorimetric assay (MTT assay). Statistical analysis was done using the IBM® SPSS version 23.0 software. Comparison between the Mean Optical Densities (MODs) of the cells stored in HBSS and other media at each time interval was done using the independent t test. Repeated measure ANOVA and Tukey post-hoc test were also carried out to compare the MOD of cells within each medium over time. The level of significance was set at p <0.05. Result: The PDL cells stored in egg white had higher MODs than those in HBSS at 30 minutes and 1 hour. Conversely, the MODs of the cells stored in milk and water were lower than those in HBSS at all the studied points. There was a significant difference in the viability of the cells stored in HBSS and water at all the time points (p<0.05). Conclusion: For up to an hour, egg white was found to perform better than HBSS in supporting the viability of PDL cell

Protease-activated receptor type 2 activation downregulates osteogenesis in periodontal ligament stem cells

Abstract Protease-activated receptor-2 (PAR2) is associated with the pathogenesis of many chronic diseases with inflammatory characteristics, including periodontitis. This study aimed to evaluate how the activation of PAR2 can affect the osteogenic activity of human periodontal ligament stem cells (PDLSCs) in vitro. PDLSCs collected from three subjects were treated in osteogenic medium for 2, 7, 14, and 21 days with trypsin (0.1 U/mL), PAR2 specific agonist peptide (SLIGRL-NH2) (100 nM), and PAR2 antagonist peptide (FSLLRY-NH2) (100 nM). Gene (RT-qPCR) expression and protein expression (ELISA) of osteogenic factors, bone metabolism, and inflammatory cytokines, cell proliferation, alkaline phosphatase (ALP) activity, alizarin red S staining, and supernatant concentration were assessed. Statistical analysis of the results with a significance level of 5% was performed. Activation of PAR2 led to decreases in cell proliferation and calcium deposition (p < 0.05), calcium concentration (p < 0.05), and ALP activity (p < 0.05). Additionally, PAR2 activation increased gene and protein expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) (p < 0.05) and significantly decreased the gene and protein expression of osteoprotegerin (p <0. 05). Considering the findings, the present study demonstrated PAR2 activation was able to decrease cell proliferation, decreased osteogenic activity of PDLSCs, and upregulated conditions for bone resorption. PAR2 may be considered a promising target in periodontal regenerative procedures.

Assessment of Proliferation, Clonogenic Assay, and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells Following Application of Orthodontic Forces

Context: The proliferation and differentiation of human periodontal ligament stem cells (hPDLSC) into other cell types are also mediated by mechanical stresses; they might offer therapeutic benefits in tissue regeneration and angiogenesis. Objectives: The study was planned to assess the proliferation, clonogenic potential, and osteogenic differentiation of human periodontal ligament stem cells (PDLSC) following the application of light and heavy orthodontic forces. Materials and Methods: A couple forces of 50 gm (light force) were applied on the 1st premolar on the one side and 250 gm (heavy force) on the contralateral side in the upper arch of patients requiring orthodontic treatment with extraction of all 1st premolars. After 30 days, periodontal tissues were scrapped from extracted teeth for the establishment of PDLSC in vitro. PDLC from the lower premolar teeth where no orthodontic force was applied acted as the control group. Morphology, viability, proliferating rate and population doubling time, clonogenicity, and alkaline phosphatase activity were analysed. Result: The osteogenic potential was confirmed by Alizarin red staining and the expression of the osteogenic markers by qRT?PCR. The morphology, growth kinetics, potency, and osteogenic lineage characteristics inferred the application of high force reduced the proliferative ability and osteogenesis of PDLSC, though the difference was not significant. Conclusion: The established PDLSCs demonstrated their MSC?like properties based on morphology, growth kinetics, colony forming ability, and AP activity. The culture?expanded PDLSCs showed their differentiation potential into osteocytes. The application of high force reduced the proliferative ability and osteogenesis of PDLSCs, variations were not significant.differentiation

An in vitro evaluation of ice apple water, Aloe vera, and propolis as a storage medium to preserve viability of human periodontal ligament fibroblasts

Background: A number of media that create the best possible conditions to maintain periodontal ligament (PDL) cell viability after dental avulsion have been reported. Aim: The aim of this study is to evaluate ice apple water (IAW), Aloe vera, and propolis as a storage medium to preserve the viability of human PDL fibroblasts. Methods: An in vitro comparative type of study was performed on a PDL cell culture model. PDL fibroblasts obtained from the roots of healthy premolars were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with ice apple water (IAW), 7% propolis extract (PE), 30% Aloe vera extract (AVE), positive control DMEM supplemented with fetal bovine serum, negative control (NC) without any agent, and incubated at 37°C for 1 h, 3 h, and 24 h. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay after every test period. Optical density was measured at a wavelength of 490 nm. Statistical Analysis Used: The effects of the test storage media were evaluated by one-way analysis of variance test, followed by post hoc Tukey's multiple comparison test (P < 0.05). Results: Seven percent PE demonstrated the highest capacity of maintaining PDL cell viability at 1 h and 24 h. IAW showed a statistically significantly lower percentage of viable cells at all three test periods as compared to 7% PE. After 3 h, 30% AVE demonstrated maximum viable cells. Conclusions: Within the limitations of this study, propolis at a concentration of 7% was the most effective medium for maintaining PDL cell viability.