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.

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.

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.

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.

Expression and distribution of calprotectin in healthy and inflamed periodontal tissues / 北京大学学报(医学版)

OBJECTIVE@#Calprotectin, the heterdimer of S100A8 and S100A9, is the major cytoplasmic protein of neutrophils, which is also expressed or induced in gingival epithelial cells, activated mononuclear macrophages and vascular endothelial cells. Calprotectin is intimately associated with the initiation and progression of periodontitis, but the in vivo expression patterns of calprotectin in healthy and inflamed periodontal tissue are not fully understood. To observe the expression, distribution and cellular localization of calprotectin in the samples of healthy periodontal tissues and experimental periodontitis tissues of Beagles and to explore their relationship with periodontal inflammation and possible effect.@*METHODS@#Experimental periodontitis model was established by ligation around the mandibular second molar of the Beagle dogs, while the contralateral teeth were healthy controls. Induction duration was 12 weeks, before the dogs were executed. Tissue specimens were demineralized and serial sections were made conventionally. The in vivo expression of calprotectin in the healthy and inflamed periodontal tissues were examined by immunohistochemistry. The in vitro expression of calprotectin in human primary gingival fibroblasts (GFs) and periodontal ligament (PDL) cells were detected by immunocytochemistry.@*RESULTS@#Immunohistochemistry analysis indicated that calprotectin was expressed in gingival epithelial cells and infiltrated neutrophils in the healthy periodontium within the gingival epithelium, S100A8/A9 was most strongly expressed in the junctional epithelium, followed by surface epithelium, and least expressed in the sulcular epithelium. The S100A8/A9 expression levels were sharply defined at the junction between the junctional epithelium and the sulcular epithelium. In periodontal inflammatory lesions, the expression level of calprotectin in sulcular epithelium and junctional epithelium was up-regulated than that in the healthy gingival epithelium. Calprotectin was inducibly expressed in fibroblast-like cells in gingival connective tissue and periodontal ligament tissue, microvascular endothelial cells (ECs) and bone marrow fibroblasts under inflammatory conditions. Additionally, the expression of calprotectin in primary human GFs and PDL cells was confirmed by immunnocytochemistry staining.@*CONCLUSION@#Constitutively expressed in neutrophils and gingival epithelial cells, and calprotectin might maintain the homeostasis and integrity of periodontium. Inflammation-induced expression of calprotectin in GFs, PDL cells, microvascular ECs and bone marrow fibroblasts might process anti-microbial function and promote leukocytes transmigration to defend the host against the microorganisms.

Exosomes derived from lipopolysaccharide-preconditioned dental folic cells regulate osteogenic differentiation of periodontal ligament cell in periodontitis / 口腔疾病防治

Objective@#The purpose of this study was to investigate the effect of different concentrations of exosomes (Exos) secreted from dental folic cells (DFCs) preconditioned with lipopolysaccharide (LPS) on the osteogenic differentiation ability of periodontal cells in periodontitis (p-PDLCs) in patients to provide a basis for the prevention and treatment of periodontal disease.@*Method @#Tissue block and enzyme digestion methods were used to culture DFCs and p-PDLCs. Exosomes were isolated from 250 ng/mL LPS-preconditioned DFCs 24 h later. The characteristics of exosomes were detected by transmission electron microscopy, particle size analysis and Western blotting. The effects of 10 μg/mL and 100 μg/mL exosomes on the osteogenic differentiation of p-PDLCs were detected by RT-PCR and Alizarin red staining.@*Results @# LPS-pretreated DFC-derived exosomes (L-Exos) are vesicle-like structures with a size between 30-100 nm that positively express CD63 and Alix. Compared with the control group, exosomes significantly upregulated Periostin, Col Ⅰ, and Col Ⅲ expression at 100 μg/mL (P < 0.05), while TGF- β1 was significantly upregulated at 10 μg/mL (P < 0.01). At 7 days after osteogenic induction, mineralized nodules were significantly more abundant in the exosome group than in the control group (P < 0.01), and the results were better at a concentration of 100 μg/mL (P < 0.01). @*Conclusion@# 100 μg/mL L-Exos are better than 10 μg/mL L-Exos in enhancing the osteogenic differentiation ability of p-PDLCs.

Astaxanthin inhibits inflammation of human periodontal ligament cells induced by lipopolysaccharide / 中南大学学报(医学版)

OBJECTIVES@#Human periodontal ligament cells (hPDLCs) are important source of periodontal tissue reconstruction. Under chronic inflammation, the multi-directional differentiation potential and chemotaxis in hPDLCs are decreased. Therefore, inhibiting inflammatory microenvironment and improving the functional characteristics of stem cells can better promote periodontal tissue reconstruction. This study was to investigate the effect of astaxanthin (AST) on lipopolysaccharide (LPS)-induced inflammation in hPDLCs and the underlying mechanisms.@*METHODS@#hPDLCs were isolated and cultured in vitro, and vimentin and keratin immunocytochemical staining were used to identify hPDLCs. CCK-8 assay was used to measure the effects of AST (1, 5, 10, 20, 50, 100, and 200 μmol/L) on proliferation of hPDLCs. Quantitative RT-PCR (RT-qPCR) and ELISA were used to measure the mRNA and protein expression of inflammatory factors (IL-6, IL-1β, and TNF-α) in the control (Con) group, the LPS group, and the LPS+AST (5, 10, 20, and 50 μmol/L) group. Western blotting was used to detect the protein expression of IKBα, phosphorylated IKBα (p-IKBα), and p65 in the Con group, the LPS group, the AST (20 μmol/L) group, and the LPS+AST (20 μmol/L) group. After 10 μmol/L PDTC treatment, the mRNA and protein expressions of IL-6, IL-1β, and TNF-α were detected by RT-qPCR and ELISA.@*RESULTS@#Cell morphology and immunocytochemical staining showed that the cells were in line with the characteristics of hPDLCs. Treatment with AST could promote the proliferation of hPDLCs, which reached the peak at 20 μmol/L. The mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS group were higher than those in the Con group (all @*CONCLUSIONS@#AST promotes the proliferation of hPDLCs, which is related to suppression of LPS-induced the secretion of inflammatory factors via inhibiting the activation of NF-κB signaling pathway.

Calcium sulfate-based bioactive cement for periodontal regeneration: An In Vitro study

Background and Objective: Various types of osteoconductive graft materials are used for the management of alveolar bone defects arising out of periodontal disease. Inorganic, self-setting, bioactive bone cements are suggested to be most appropriate because they can conformally fill the bone defect and resorb progressively along with the regeneration of the host site. A new calcium sulfate-based bioactive bone cement (BioCaS) is developed, having simplicity and effectiveness for bone grafting applications. The response of primary human periodontal ligament (hPDL) cells to this material is investigated through in vitro cell culture model so as to qualify it for the repair of periodontal infrabony defects. Method: The BioCaS was designed as powder-liquid combination with in-house synthesized high purity calcium sulfate hemihydrate incorporating hydrogen orthophosphate ions. hPDL cells were isolated, cultured and characterized using optimized primary cell culture techniques. The cytotoxicity and cytocompatibility of the BioCaS samples were evaluated using the hPDL cells, with hydroxyapatite ceramic material as control. Osteogenic differentiation of the hPDL cells in presence of BioCaS was also evaluated using Alizarin red staining, Alizarin red assay, Von Kossa staining and Masson's trichrome staining. Results: The primary cell culture techniques yielded a healthy population of periodontal ligament cells, with fibroblast morphology and characteristic marker expressions. The hPDL cells exhibited good viability, adhesion and spreading to the BioCaS cement in comparison to sintered hydroxyapatite. In addition, the cells differentiated to osteogenic lineage in the presence of the BioCaS cement, without extraneous osteogenic supplements, confirming the inherent bioactivity of the cement. Conclusion: The new BioCaS cement is a potential candidate for the repair of periodontal defects.

Research on the surface structure of a dentin matrix with complete demineralization and incomplete demineralization and the osteogenic property promotion of human periodontal ligament cells / 口腔疾病防治

Objective@# To analyze the different fabrication methods and surface structure of treated dentin matrix (TDM) and demineralized dentin matrix (DDM) and their diverse function on promoting the proliferation and osteogenic differential capability of human periodontal ligament cells (hPDLCs). This study provides a preliminary basis for the treatment of periodontal bone defects with bone substitutes from teeth.@*Methods@#TDM was made from human dentin matrices and demineralized incompletely by soaking in different concentrations of ethylene diamine tetra-acetic while DDM was made of human dentin matrices and demineralized completely by soaking in a hydrochloric acid solution followed by observation via SEM. The liquid extracts of TDM and DDM were collected according to the protocol of the International Standardization Organization (ISO 10993). Then, hPDLCs were divided into the following three groups: the TDM group (liquid extracts of TDM), the DDM group (liquid extracts of DDM), the control group (a-modified eagle medium with 10% fetal bovine serum), hPDLCs were cultured with liquid extracts of TDM or DDM, or a-modified eagle medium with 10% FBS). hPDLC proliferation was detected by a Cell Counting Kit-8 (CCK-8). The alkaline phosphatase (ALP) expression and calcified nodules of hPDLCs were tested.@*Results @#TDM obtained a preferable surface structure compared to DDM due to more sufficiently exposed dentinal tubules and looser fiber bundles of the intertubular and peritubular dentin. Both TDM and DDM promoted the proliferation of hPDLCs compared with the control group, and the proliferation of hPDLCs was significantly greater in the TDM group compared to the DDM group (F = 36.480, P < 0.05). The ALP activity of hPDLCs in the TDM group was higher than the DDM group. After a 14-day osteogenic induction, Alizarin red staining mineral nodes were observed in both groups; however, the TDM group displayed more calcified nodules than the DDM group.@*Conclusion@#The advantages of TDM including the surface structure, proliferation and osteogenic differentiation of hPDLCs, are more prominent than those of DDM, suggesting that TDM is a potential promising bone graft substitute in periodontal regeneration.

Effect of endoplasmic reticulum stress on the osteogenic differentiation of periodontal ligament cells under continuous static pressure / 口腔疾病防治

Objective@#To study the effect of continuous static pressure on the endoplasmic reticulum of human periodontal ligament cells (hPDLCs) and the mechanism of osteogenic differentiation.@*Methods@#hPDLCs cultured in vitro were subjected to 1 g/cm 2 of continuous compressive pressure (CCP) by custom-made, round, glass panes for 0, 2, 4, and 6 h, respectively. Alkaline phosphatase staining was used to detect osteogenic differentiation, and real-time quantitative PCR was used to detect the expression of protein kinase receptor-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), and transcription activation factor 4 (ATF-4). The 0 h loading group was the control group.@*Results@#After CCP treatment, the alkaline phosphatase staining of hPDLCs was blue-violet and significantly stronger than that of cells in the control group. The expression levels of PERK and ATF4 in the hPDLCs after CCP treatment were higher than those of cells in the control group (P < 0.05) and increased over time (P < 0.05). The expression of eIF2α was lower in the experimental groups than in the control group (P < 0.05) and decreased over time (P < 0.05).@*Conclusion @#Mechanical stimulation can activate ERS in hPDLCs, leading to enhanced PERK-eIF2α-ATF4 signaling and inducing osteogenic differentiation.

Construction of human telomerase reverse transcriptase periodontal ligament cell line mediated by adenovirus / 华西口腔医学杂志

OBJECTIVE@#This study aims to establish an effective and stable periodontal ligament cell line stably expressing human telomerase reverse transcriptase (hTERT) gene by using the adenovirus method.@*METHODS@#Polymerase chain reaction (PCR) was used to amplify the full length of hTERT gene to construct recombinant adenovirus plasmid pAd-pshuttle-cmv-hTERT. Packaged adenovirus particles were used for infection of human periodontal ligament cells. The expression levels of hTERT and osteogenic genes, such as alkaline phosphatase, Runt-related transcription factor 2, bone sialoprotein, osteocalcin, osteopontin, and collagen Ⅰ mRNA, were detected by quantitative real-time PCR (qRT-PCR). The ability of osteogenic differentiation was observed by alizarin red staining, and the cell proliferation was determined by CCK-8.@*RESULTS@#Adenovirus particles containing the hTERT gene were successfully constructed and infected with periodontal ligament cells. The infected cells were similar to normal periodontal ligament cells. The qRT-PCR results showed that hTERT and osteogenesis-associated genes were highly expressed in the periodontal ligament cell lines constructed by adenoviruses. Alizarin red staining showed that the periodontal ligament cell line had strong osteogenic differentiation capability. CCK-8 showed that the periodontal ligament cell line had strong proliferation capability.@*CONCLUSIONS@#The human periodontal ligament cell line with high efficiency and stable expression of hTERT was established by the adenovirus method, thereby providing an ideal cell line for studying the mechanism of periodontal regeneration.

Effects of inflammatory microenvironment mediated by macrophage on the proliferation and osteogenic differentiation of periodontal ligament cells / 口腔疾病防治

Objective@#The present study investigated the effects of the inflammatory microenvironment mediated by macrophages on the proliferation and osteogenic differentiation of periodontal ligament cells (PDLCs).@*Methods@#Conditioned medium containing inflammatory factors was collected following macrophage activation with 1 μg/mL lipopolysaccharide (LPS). PDLCs were isolated from healthy teeth and cultured in conditioned medium (LPS-CM group) or normal medium (control group), and the proliferation of PDLCs was detected using the MTT assay. The cells were cocultured with an osteogenic inducer for 3 d and 7 d, and the alkaline phosphatase (ALP) activity of PDLCs was detected using an ALP kit. The mRNA expression levels of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and collagen I (COL-I) were detected using real-time PCR, and the protein levels of RUNX2, OCN, and COL-I were detected using Western blotting. Mineralization nodules were observed using Alizarin red staining after osteoinduction for 14 d. @*Results@#The OD value of PDLCs in the LPS-CM group was lower than that in the control group (P < 0.05). The mRNA levels of RUNX2, OCN, and COL-I in the LPS-CM group were lower than those in the control group (P < 0.05). In addition to the OCN 3 d group (t = 2.75, P = 0.056), the protein expression of RUNX2, OCN, and COL-I in the LPS-CM group was lower than that in the control group (P < 0.05). However, the ALP activity of the LPS-CM group was higher than that of the control group, which was 1.58-fold greater (t = 5.91, P = 0.030) at 3 d and 1.29-fold greater (t = 6.01, P = 0.046) at 7 d. The number of calcified nodules in the LPS-CM group was significantly less than that in the control group (t = 8.63, P = 0.048). @*Conclusion@# The inflammatory microenvironment mediated by macrophages may inhibit the proliferation and osteogenic differentiation of PDLCs.

Influence of 3,3'-diindolylmethane on expression of inflammatory cytokines in periodontal ligament cells induced by lipopolysaccharide / 上海交通大学学报（医学版）

Objective: To investigate the effect of 3,3'-diindolylmethane (DIM) on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLCs) induced by lipopolysaccharide (LPS) and to study the related mechanism. Methods: hPDLCs were isolated and cultured, and CCK-8 method was used to detect the effect of DIM on the proliferation of hPDLCs. hPDLCs were randomly divided into 4 groups: blank group (without LPS and DIM), LPS group (10 μg/mL LPS), 10 μg/mL LPS+6.25 μg/mL DIM, 10 μg/mL LPS+12.50 μg/mL DIM. The cells of all groups were cultured for 12 h. The protein levels of TNF-α, IL-1β and IL-6 in supernatant were detected by enzyme linked immunosorbent assay. The change of mitogenactivated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways were detected by Western blotting. Results: The cell viability was not affected when the DIM concentration was less than 50 μmol/L (P>0.05). DIM at 6.25 and 12.50 μg/mL reduced the LPS-induced expression of TNF-α, IL-1β and IL-6 at protein levels (P<0.05). DIM inhibited the activation of the NF-κB signaling pathway. Conclusion: DIM can reduce the LPS-induced inflammatory cytokine expression in hPDLCs via restraining the activation of the NF-κB signaling pathway.

Anti-inflammation effects of psoralen and angelicin on human periodontal ligament cells in vitro / 上海交通大学学报（医学版）

Objective: To investigate the effects of psoralen and angelicin on inflammation cytokine expression of human periodontal ligament cells (hPDLCs). Methods: hPDLCs were primarily cultured using tissue explant method. Effects of psoralen and angelicin on the cell viability were tested by CCK-8 assay. hPDLCs were stimulated by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) after treatment with different concentrations of psoralen and angelicin for 2 h. mRNA expression of IL-1β and IL-8 were determined by real-time PCR. Enzyme-linked immunosorbent assay (ELISA) was used to measure the secretion of IL-1β and IL-8. Results: hPDLCs were cultured successfully by tissue explant method. Psoralen and angelicin (≤ 12.5 μg/mL) did not show significant effects on the cell viability of hPDLCs. Pg-LPS markedly elevated the mRNA expression of IL-1β and IL-8, which could be attenuated by psoralen and angelicin in a dose-dependent manner. Likewise, the up-regulated protein secretion of IL-1β and IL-8 could be significantly blocked by psoralen and angelicin. Conclusion: Psoralen and angelicin could attenuate the inflammatory response of hPDLCs induced by Pg-LPS. Therefore, psoralen and angelicin may act as natural agents to prevent and treat periodontitis.

microRNA-146a reverses the inhibitory effects of Porphyromonas gingivalis lipopolysaccharide on osteogenesis of human periodontal ligament cells / 中华口腔医学杂志

Objective@#To investigate the effects and mechanisms of microRNA-146a (miR-146a) on osteogenic differentiation of human periodontal ligament cells (hPDLC) stimulated by lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg).@*Methods@#hPDLC were cultured in vitro and induced to the phase of osteogenic differentiation. These cells were divided into five groups: non-osteogenic differentiation cells, osteogenic differentiation cells, osteogenic differentiation cells treated with Pg LPS, osteogenic differentiation cells treated with Pg LPS and miR-146a mimic, osteogenic differentiation cells treated with Pg LPS and miR-146a negative control. Osteogenic markers and mineralization were detected via quantitative real-time PCR (qPCR) and alizarin red staining, respectively. Meanwhile, non-radioactive transcription factor assay was applied to explore the nuclear activity of nuclear factor kappa B (NF-κB) p65 in nuclear extracts of hPDLC.@*Results@#Compared with cells of osteogenic differentiation in non-LPS-stimulated groups, Pg LPS could decrease the markers of osteogenic differentiation of hPDLC such as collagen Ⅰ (Col-Ⅰ), alkaline phosphatase (ALP), Runt-related transcription factor-2 (RUNX2) and osteocalcin (OCN) (P<0.05), inhibit mineralization, and stimulate NF-κB p65 nuclear activity expression (non-LPS stimulated group: 1.023±0.217, LPS stimulated group: 6.252±0.613, P=0.008). However, compared with cells in Pg LPS/miR-146a negative control group, miR-146a increased Col-Ⅰ (P=0.007) and OCN (P=0.049) mRNA expression, rather than ALP (P=0.167) and RUNX2 (P=0.580) at day 3; miR-146a also upregulated mRNA levels of Col-Ⅰ, ALP, RUNX2 and OCN (P<0.05) at day 7 and day 14, and enhance mineralization. Meanwhile, miR-146a mimic could decrease the nuclear activity of NF-κB p65 induced by Pg LPS in hPDLC (miR-146a: 2.427±0.354, negative control: 5.863±0.482, P=0.019).@*Conclusions@#miR-146a could reverse the inhibitory effects of Pg LPS on osteogenic differentiation of hPDLC through enhancing the expression of osteogenic markers and decreasing inflammatory pathway in hPDLC.

Influence of 3,3'-diindolylmethane on expression of inflammatory cytokines in periodontal ligament cells induced by lipopolysaccharide / 上海交通大学学报（医学版）

Objective·To investigate the effect of 3,3'-diindolylmethane (DIM) on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLCs) induced by lipopolysaccharide (LPS) and to study the related mechanism.Methyls· hPDLCs were isolated and cultured,and CCK-8 method was used to detect the effect of DIM on the proliferation of hPDLCs.hPDLCs were randomly divided into 4 groups:blank group (without LPS and DIM),LPS group (10 μg/mL LPS),10 μg/mL LPS+6.25 μg/mL DIM,10 μg/mL LPS+12.50 μg/mL DIM.The cells of all groups were cultured for 12 h.The protein levels of TNF-α,IL-1β and IL-6 in supernatant were detected by enzyme linked immunosorbent assay.The change of mitogenactivated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways were detected by Western blotting.Results· The cell viability was not affected when the DIM concentration was less than 50 μmol/L (P＞0.05).DIM at 6.25 and 12.50 μg/mL reduced the LPS-induced expression of TNF-α,IL-1β and IL-6 at protein levels (P＜0.05).DIM inhibited the activation of the NF-κB signaling pathway.Conclusion· DIM can reduce the LPS-induced inflammatory cytokine expression in hPDLCs via restraining the activation of the NF-κB signaling pathway.

Anti-inflammation effects of psoralen and angelicin on human periodontal ligament cells in vitro / 上海交通大学学报（医学版）

Objective·To investigate the effects of psoralen and angelicin on inflammation cytokine expression of human periodontal ligament cells (hPDLCs).Methods· hPDLCs were primarily cultured using tissue explant method.Effects ofpsoralen and angelicin on the cell viability were tested by CCK-8 assay,hPDLCs were stimulated by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) after treatment with different concentrations of psoralen and angelicin for 2 h.mRNA expression of IL-1β and IL-8 were determined by real-time PCR.Enzyme-linked immunosorbent assay (ELISA) was used to measure the secretion of IL-1β and IL-8.Results · hPDLCs were cultured successfully by tissue explant method.Psoralen and angelicin (≤ 12.5 μg/mL) did not show significant effects on the cell viability of hPDLCs.Pg-LPS markedly elevated the mRNA expression of IL-1β and IL-8,which could be attenuated by psoralen and angelicin in a dose-dependent manner.Likewise,the up-regulated protein secretion of IL-1β and IL-8 could be significantly blocked by psoralen and angelicin.Conclusion · Psoralen and angelicin could attenuate the inflammatory response of hPDLCs induced by Pg-LPS,Therefore,psoralen and angelicin may act as natural agents to prevent and treat periodontitis.

Protective effects of hydrogen-rich medium on lipopolysaccharides-induced injury in human periodontal ligament cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>In this study, lipopolysaccharides (LPS) was used to damage human periodontal ligament cells (hPDLCs) and consequently investigate the protective effects of hydrogen on reducing oxidative stress and cell apoptosis rate.</p><p><b>METHODS</b>hPDLCs were isolated, and then cultured with normal medium+1 μg·mL⁻¹ LPS or with hydrogen-rich medium+
1 μg·mL⁻¹ LPS. Cell proliferation activity was assessed using a cell counting kit-8 (CCK-8), and lactic dehydrogenase (LDH) release was also detected. The activities of superoxide dismutase (SOD) and catalase (CAT), and the level of malonaldehyde (MDA) in supernatants were also measured. Cell apoptosis was detected by flow cytometry at 24 h after LPS stimulation.</p><p><b>RESULTS</b>CCK-8 results showed that hydrogen could significantly improve hPDLCs growth and decrease cell apoptosis under LPS stimulation (P<0.05). However, no significant difference in LDH release was found between the two groups. The CAT levels significantly increased at 6 and 12 h in the hydrogen-rich medium as compared with the normal medium group (P<0.05, P<0.01, respectively). However, SOD levels were not significant different at each time point. At 6 h after LPS stimulation, the MDA levels in the cell supernatant of hydrogen-rich medium group were significantly reduced as compared with those in the normal medium group (P<0.05).</p><p><b>CONCLUSIONS</b>The hydrogen-rich medium can effectively improve hPDLCs proliferation activity and antioxidant capacity and reduce apoptosis and oxidative stress under LPS stimulation.</p>

Autotrasplante de un tercer molar inmaduro: reporte de un caso / Autotransplantation of an immature third molar: a case report

El autotrasplante se refiere a la transferencia quirúrgica de un dientede una posición a otra en el mismo individuo dentro de los alveolosde dientes extraídos o sitios preparados quirúrgicamente. El autotrasplante de un diente inmaduro puede ser una opción para reemplazarmolares con caries extensas en pacientes jóvenes como una alternativaal reemplazo de dientes con prótesis fi ja o implanto-soportada. Es unprocedimiento clínico con un índice de éxito de 98 por ciento cuando los dientes son trasplantados traumáticamente y el tiempo extraoral se mantiene al mínimo. El estado de desarrollo del diente determina ampliamente el potencial de reparación pulpar después del autotrasplante. Para obtener una pulpa vital en un diente autotrasplantado el foramen apical no debe medir menos de1 mm de diámetro. El área receptora debe ser 1-2 mm más grande y profunda que las medidas de las raíces donadoras parapreservar las células del ligamento periodontal, un óptimo contacto entreambas estructuras puede mejorar el suministro sanguíneo y los nivelesde nutrición de las células del ligamento periodontal, el cual puedeincrementar el éxito del autotrasplante. En este artículo se presenta un caso exitoso de autotrasplante de tercer molar inmaduro.

Autotransplantation refers to the surgical transfer of a tooth fromone position to another in the same individual onto extracted toothsockets or surgical prepared recipient sites. Autotransplantation of animmature teeth can be an option to replace extensive decayed molarsin young patients as an alternative to immediately replacing teeth withfi xed or implant-supported prosthesis. Autotransplantation is a clinicalprocedure with a success rate of 98% when teeth are transplantedatraumatically and when the extraoral time is keep to a minimum.The developmental stage of the tooth highly determines the potentialof pulpal repair after transplantation. To obtain a vital pulp in anautotransplanted tooth, the apical foramen should not be smaller than1 mm in diameter. The recipient area must be 1-2 mm larger and deeperthan the measurements of the donor roots to preserve the periodontalligament cells, an optimal contact between both structures can improvethe blood supply and the level of nutrition to the periodontal ligamentcells, which can increase the success of autotransplantation. In thispaper, we report one case of successful autotransplantation of animmature third molar.

A preliminary study on the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells / 医用生物力学

Objective To study the expression of caspases in stretch-induced apoptosis in human periodontal ligament cells (HPDLCs).Methods HPDLCs in vitro were subjected to mechanical stretch with 20％ strain for 6 h or 24 h.The apoptotic rates were analyzed by flow cytometry.The protein expression of caspase-3,-5,-7,-8 and -9 was detected by Western blotting,and the activity of caspase-3,-5,-8 and-9 was measured using colorimetric assay.Results Mechanical stretches with 20％ strain for 6 h and 24 h could induce apoptosis in HPDLCs.Compared with non-stretching control group,the protein expression level and activity of caspase-3,as well as the protein expression level of caspase-7 were up-regulated by 24 h-stretch.The protein expression level and activity of caspase-5,-8,-9 were up-regulated after stretches for 6 h and 24 h.Conclusions Mechanical stretch with 20％ strain can induce apoptosis in HPDLCs in vitro,with the activation of caspase-3,-5,-7,-8 and-9.