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.

Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts in vitro and promotion of tendon-bone healing in rabbits / 中国修复重建外科杂志

OBJECTIVE@#To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits.@*METHODS@#hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues.@*RESULTS@#Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups.@*CONCLUSION@#Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.

Research progress on the regulatory mechanism of estrogen in periodontal ligament cells repair and the reconstruction of periodontal tissue / 口腔疾病防治

@#Periodontium regeneration and repair is a controversial and difficult point in the treatment of periodontosis. The proliferation, differentiation, migration and adhesion of periodontal ligament cells and the dynamic relationship between periodontal ligament cells and their extracellular matrix proteins are the basis of periodontium morphological reconstruction, functional maintenance and tissue repair. This article reviews the mechanism of estrogen-regulated periodontal membrane fine repair and periodontal tissue reconstruction to provide the basis for follow-up research on the treatment of periodontitis and the promotion of periodontal tissue repair and reconstruction by exogenous estrogen-mediated periodontal membranes. Under the regulation of certain concentrations of estrogen, the proliferation and differentiation ability of periodontal ligament stem cells (PDLSCs) and bone mesenchymal stem cells (BMSCs) to other periodontal ligament cells were enhanced. At the same time, PDLSCs, BMSCs, human periodontal ligament fibroblasts (HPLFSs), osteoblasts and cementoblasts synthesized and secreted collagen I (COLI), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OCN) into the extracellular matrix. They interact with fibronectin (FN) and cementum attachment protein (CAP) in the extracellular matrix to form a variety of chain complexes and regulate each other, thus promoting the growth, migration, adhesion and fibrosis of periodontal ligament cells, repairing the collagen fiber skeleton of the periodontal ligament and adhering the two ends to the new cementum and the inherent alveolar bone.

Protective effect of low-level laser irradiation on lipopolysaccharide-mediated inflammatory injury of human periodontal ligament fibroblasts / 华西口腔医学杂志

OBJECTIVES@#To study the effect and mechanism of low-level laser irradiation (LLLI) on lipopolysaccharide (LPS)-induced inflammatory injury of human periodontal ligament fibroblasts (hPDLFs).@*METHODS@#hPDLFs were inoculated into well plates and randomly divided into the normal group, LPS group, and LPS+LLLI group. The cells in the normal group were cultured in conventional medium. The hPDLFs in the LPS and LPS+LLLI groups were cultured in RPMI1640 medium containing 1 mg·L@*RESULTS@#Compared with the normal group, the LPS group showed increased apoptosis rate of hPDLFs and intracellular free Ca@*CONCLUSIONS@#LLLI has a protective effect on the inflammatory injury of hPDLFs induced by LPS, and the effect is most obvious when the irradiation intensity is 4 J·cm

Periostin inhibits hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts p38 MAPK signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of periostin on hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts and the molecular mechanism involved.@*METHODS@# cultured human periodontal ligament fibroblasts were placed in an anaerobic gas-producing bag for hypoxia treatment for 48 h followed by treatment with periostin at low (25 ng/mL), moderate (50 ng/mL) or high (100 ng/mL) doses. MTT assay was used to measure the cell viability, and the cell apoptosis rate was determined using flow cytometry. The contents of IL-1β, IL-6 and TNF-α in the cells were determined with ELISA, and ROS levels were measured using a fluorescent plate reader. The intracellular SOD activity was detected using ELISA. The expressions of HIF-1α, P21, cyclin D1, Bax, cleaved caspase-3, Bcl-2, P38MAPK and p-p38 MAPK proteins in the cells were detected with Western blotting.@*RESULTS@#Hypoxia treatment significantly reduced the cell viability ( < 0.05), increased P21, Bax, and cleaved caspase-3 protein levels ( < 0.05), promoted cell apoptosis ( < 0.05), and decreased cyclin D1 and Bcl-2 protein levels ( < 0.05) in the cells. Compared with the hypoxic group, the cells treated with periostin at different concentrations showed significantly increased cell viability ( < 0.05) with significantly lowered apoptotic rates ( < 0.05) and decreased expression levels of Bax and cleaved caspase-3 ( < 0.05) but significantly increased expression levels of cyclin D1 and Bcl-2 ( < 0.05). Hypoxic exposure of the cells resulted in significantly increased expression levels of HIF-1α and p-p38 MAPK ( < 0.05) and increased levels of IL-1β, IL-6, TNF-α and ROS ( < 0.05) but decreased SOD activity ( < 0.05). Periostin treatment at different concentrations significantly lowered the expression levels of HIF-1α and p-p38 MAPK ( < 0.05) and the levels of IL-1β, IL-6, TNF-α and ROS ( < 0.05) and significantly increased SOD activity in the hypoxic cells ( < 0.05).@*CONCLUSIONS@#Periostin promotes the proliferation, inhibits apoptosis, enhances cellular antioxidant capacity, and reduces inflammatory damage in human periodontal ligament fibroblasts exposed to hypoxia possibly by inhibiting the activation of the p38 MAPK signaling pathway.

Effect of Milk Renewal on Cell Viability In Vitro at Different Time Frames

Abstract The purpose of this study was to evaluate if the renewal of milk as a storage medium, every 12, 24 and 48 h, is able to increase its ability to maintain human periodontal ligament fibroblasts (PDLF) viability over time. PDLF were soaked in Minimum Essential Medium at 37 °C (MEM-37) (positive control), tap water (Water) (negative control) and in skimmed milk (44 wells) at 5 °C and 20 °C. The skimmed milk was renewed every 12 h (Milk-12), 24 h (Milk-24) and 48 h (Milk-48) in 11 wells of each plate, and the milk in the remaining 11 wells of each plate was maintained in situ (not renewed milk) (NRM). After 24, 48, 72, 96 and 120 h, cell viability was determined by the tetrazolium salt-based colorimetric (MTT) assay. Data were statistically analyzed by Kruskal-Wallis, Scheffé and Mann-Whitney tests (a=5%). At 5 °C, only Milk-48 was significantly better than NRM. At 20 °C, NRM was more effective than Milk-12 and Milk-24 in all time periods. In relation to the temperature (5 °C or 20 °C), renewal of milk at 5 °C was better in maintaining cell viability than the renewal at 20 °C. In conclusion, the renewal of milk was able to increase its ability to maintain cell viability only when performed every 48 h in milk maintained at 5 °C.

Resumo O objetivo deste estudo foi avaliar se a renovação do leite, a cada 12, 24 e 48 h, é capaz de aumentar sua capacidade de manter a viabilidade de fibroblastos do ligamento periodontal humano (FLPH) ao longo do tempo. FLPH foram conservados em Meio Essencial Mínimo a 37 °C (MEM-37) (controle positivo), água da torneira (água) (controle negativo) e em leite desnatado (44 poços) a 5 °C e 20 °C. O leite desnatado foi renovado a cada 12 h (leite-12), 24 h (leite-24) e 48 h (leite-48) em 11 poços de cada placa, e em outros 11 poços de cada placa o leite foi deixado in situ (leite não renovado) (LNR). Depois de 24, 48, 72, 96 e 120 h, a viabilidade celular foi determinada pelo ensaio colorimétrico à base de sal tetrazólio (MTT). Os dados foram analisados estatisticamente pelos testes de Kruskal-Wallis, Scheffé e Mann-Whitney (α=5%). A 5 °C, somente o leite-48 foi significantemente melhor do que o LNR. A 20 °C, LNR foi mais efetivo do que o leite-12 e leite-24 em todos os períodos de tempo. Em relação à temperatura (5 °C ou 20 °C), a renovação do leite a 5 °C foi melhor na manutenção da viabilidade celular do que a renovação a 20 °C. Concluindo, a renovação do leite foi capaz de aumentar sua habilidade em manter a viabilidade celular apenas quando realizada a cada 48 h no leite mantido a 5 °C.

Effects of arecoline on apoptosis of human periodontal ligament fibroblasts / 口腔疾病防治

Objective @#To observe the effects of arecoline on the related protein expressions in the apoptosis of cultured human periodontal ligament fibroblasts (hPDLFs), such as p-JNK, p-p53, and Bcl-2.@*Methods @#hPDLFs were isolated from human periodontal ligament tissues and expanded in vitro. hPDLFs were treated with different concentrations of arecoline (0, 20, 40 and 80 μg/mL) for 12 h. The proliferative activities were evaluated by MTT. The expressions of p-JNK, p-p53, and Bcl-2 were determined by Western blot. @*Results@#Arecoline inhibited cell proliferation and induced apoptosis protein. The protein level of Bcl-2 was decreased, while p-JNK and p-p53 were increased (P < 0.05). The protein expressions were in a concentration-dependent manner with arecoline. @*Conclusion @#It demonstrates arecoline might contribute to the apoptosis of hPDLFs, and could destroy periodontal tissues.

Preparation and characteristics of drug loaded PLGA chitosan/nano-hydroxyapatite membrane for guided periodontal tissue regeneration in surgical implanting / 第二军医大学学报

Objective To prepare the porous drug-conlaining membrane by poly (ladic-co-glycolic acid) (PLGA)/ chitosan (CS)/nano-hydroxyapatitc for guided periodontal tissue regeneration in surgery, and lo evaluate its performance in vitro. Methods The samples were divided into four groups by different mass ratios of the PLGA lo CS (100: 0, 90: 10, 80: 20 and 70: 30). The PLGA/CS/nano-hydroxyapatitcporous films were prepared by freeze-drying process, wilh polyvinylpyrrolidone (PVP) used as porogcn. The best ratio was chosen by detecting the porosity, water absorption, mechanical properties and degradation of the films; and then il was used as drug carrier lo prepare membrane material for clindamycin controlled release. The morphology of membrane was observed by scan electronmicroscope, (he porosity was detected by anhydrous ethanol liquid displacement method, water absorption was determined by ratio of wet to dry weight of the film, (he wel mechanical performance was tested by electronic universal material testing, (he degradation was determined by weight loss and swelling degree, and the release character was investigated by ultraviolet spcctrophotometric method. In the in vitro experiments, periodontal ligament fibroblast cells (PLFs) were cultured in the membrane for 1-7 days, and cell proliferation was measured by CCK-8. Results The optimal porosity and degradation were found when the mass ratio of PLGA lo CS was 90: 10, wilh the porosity being (28. 66il. 35)%, water absorption being (108. 65 ± 2. 27) %, tensile strength being (2. 36 ± 0. 04) MPa, elongation at break of films being (203.64±3. 89)%, breaking power being (45. 98 ± 2. 46) N, and degradation being (17. 60 ± 0. 86)%. The maximum drug release was 150 μg • mL-1 • d-1, and the effective drug release concentration lasted for 15 d, which could promote the proliferation of PLFs. Conclusion The porous PLGA/CS/nano-hydroxyapatite film prepared in the present study has optimal porosity, its degradation in vitro fit well with the tissue growth, and can create and maintain a specific space for guided periodontal tissue regeneration, allowing for steady drug release for a certain period of time.

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

Antioxidant Effect of Eugenol in Human Periodontal Ligament Fibroblasts / 체질인류학회지

Periodontal disease (PD) is the most common osteolytic disease of alveolar bone, oral infection seen in humans worldwide. PD is a common, chronic immunoinflammatory disease initiated by a complex subgingival bacterial and results in the inflammatory destruction of periodontal tissues, including the alveolar bone periodontal ligament, and gingivae. The effects of eugenol on periodontal ligament fibroblasts (PDLF) cell under oxidative injury have not been fully studied. Despite many studies in regard to the antioxidant effect of eugenol, the protective effect of eugenol against oxidative damage to PDLF cell, as well as the relationship between eugenol and apoptosis, has not been investigated so far. The aim of this study was to assess the protective effect of eugenol against H2O2-induced oxidative stress in PDLF cell. Cell lines were separately grown as monolayers at 5% CO2 and 37degrees C humidified atmosphere using appropriate media supplemented with 10% fetal bovine serum, 2 mM glutamine and 100 microg/mL penicillin-streptomycin. DMEM/F12 was used as the culture medium for periodontal ligament fibroblast cells. The viability of the PDLF cells which induced by the different concentrations of H2O2 (control, 50, 100, 200, 400 microM) for 24 h was detected by MTT assay. Cell viability was significantly reduced in a H2O2-concentration dose-dependent manner. The mitochondria-dependent pathway of apoptosis is regulated by Bcl-xl family, such as the anti-apoptotic protein Bcl-xl, pro-apoptotic protein Bak. With H2O2 injury, the protein level of Bak was up-regulated while the protein level of Bcl-xl was down-regulated. In group treated H2O2 and eugenol, the ratio was reduced and the expression of Bak decreased at the same time, indicating that eugenol can attenuate apoptosis through mitochondrial related pathway in PDLF cells. Therefore, although the findings of this study are limited to an in vitro interpretation, we suggest that eugenol preconditioning may have a beneficial effect in the recovery of periodontal ligament from oxidative stress.

Glucosamine Inhibits Lipopolysaccharide-induced Inflammatory Responses in Human Periodontal Ligament Fibroblasts

Glucosamine is commonly taken by the elderly without prescription as a nutritional supplement to attenuate the progression or symptoms of osteoarthritis. Previous studies demonstrated that glucosamine shows anti-inflammatory effects in tissues such as blood vessels and the heart. However, there have been few reports about the effects of glucosamine on oral inflammatory diseases. Therefore, in this study, the effects of glucosamine on lipopolysaccharide (LPS)-induced inflammatory responses were investigated using human periodontal ligament fibroblasts (HPDLFs). HPDLFs were incubated in the presence and absence of glucosamine (10 mM) for 24 h, followed by treatment with E. coli LPS (100 ng/ml) or vehicle. Quantitative RT-PCR and ELISA results showed that LPS exposure significantly increased the levels of IL-6 and IL-8 mRNA and protein, while the effect was significantly suppressed by glucosamine treatment. Glucosamine did not attenuate, but slightly increased, the LPS-induced activation of mitogen activated kinases (ERK, p38, JNK). However, it suppressed the LPS-induced increase in the DNA binding affinity and transcriptional activity of NF-kappaB. These results suggest that glucosamine exerts anti-inflammatory effects on HPDLFs exposed to LPS via inhibition of NF-kappaB activity, necessitating further studies using animal periodontitis models.

The effect of tumor necrosis factor (TNF)-alpha to induce matrix metalloproteinase (MMPs) from the human dental pulp, gingival, and periodontal ligament cells / 대한치과보존학회지

OBJECTIVES: In the present study, three kinds of tissues cells (pulp, gingiva, and periodontal ligament) were investigated if those cells express MMP and TIMP when they were stimulated with neuropeptides (substance P, CGRP) or proinflammatory cytokine, TNF-alpha. MATERIALS AND METHODS: The cells cultured from human dental pulp (PF), gingiva (GF) and periodontal ligament were (PDLF) stimulated with Mock, SP, TNF-alpha, and CGRP for 24 hrs and 48 hrs. for an RNase protection assay and Enzyme Linked Immunosorbent Assay. Cells (PF, GF and PDLF) seeded in 100 mm culture dish were stimulated with SP (10(-5), 10(-8) M) or only with medium (Mock stimulation) for 4hrs and for 24 hrs for RNase Protection Assay, and they were stimulated with CGRP (10(-5) M) and TNF-alpha (2 ng/mL) for 24 hrs and with various concentraion of TNF-alpha (2, 10, and 100 ng/mL) for Rnase Protection Assay with a human MMP-1 probe set including MMP 1, 2, 8, 7, 8, 9, 12, and TIMP 2, 3. In addition, cells (PF, GF and PDLF) were stimulated with Mock and various concentraion of TNF-alpha(2, 10, and 100 ng/mL) for 24 hrs and with TNF-alpha(10 ng/mL) for 48 hrs, and the supernatents from the cells were collected for Enzyme Linked Immunosorbent Assay (ELISA) for MMP-1 and MMP-13. RESULTS: The expression of MMPs in PF, GF, PDLF after stimulation with SP and CGRP were not changed compared with Mock stimulation for 4 hrs and 24 hrs. The expression of MMP-1, -12, -13 24 hrs after stimulation with TNF-alpha were upregulated, however the expression of TIMP-3 in PF, GF, PDLF after stimulation with TNF-alpha were downregulated. TNF-alpha (2 ng/mL, 10 ng/mL, 100 ng/mL) increased MMP-1 and MMP-12 expression in PF dose dependently for 24 hrs. CONCLUSIONS: TNF-alpha in the area of inflammation may play an important role in regulating the remodeling of dentin, cementum, and alveolar bone.

Effect of platelet-rich plasma on human periodontal ligament fibroblasts' proliferation, migration and differentiation / 实用口腔医学杂志

Objective:To investigate the in vitro effects of platelet-rich plasma(PRP) on human periodontal ligament fibroblasts(PDLFs). Methods: Various concentrations of PRP (10, 50, 100, 200, 300, 500 ml/L) were applied to primary cultures of human PDLFs. MTT assays were utilized to assess cell proliferation ability. Migration was determined by assessing the cell response to a concentration gradient with Transwell chamber. Differentiation was assessed using alkaline phosphatase (ALP) kit. Results: A beneficial effect on proliferation was observed, especially in response to 200 ml/L PRP.PRP had stimulatory effects on the migration of human PDLFs. PRP facilitated differentiation of PDLFs. Conclusion: PRP can exert a positive effect on human PDLFs,but this effect is concentration specific, while higher concentrations is not necessary to result in optimal outcomes.

Experimental studies of hypoxia on the proliferation ability and ultrastructure of cultured human periodontal ligament fibroblasts / 实用口腔医学杂志

Objective: To observe the effect of hypoxia on changes of proliferation ability of cultured human periodontal ligament fibroblasts (HPLFS) in vitro. Methods: HPLFS were randomly divided into two groups (experimental group and control group) by different oxygen concentrations. The oxygen concentration of control group was 21%. The oxygen concentrations of experiment group were 10%, 5% and 2%. The proliferation ability of HPLFS was detected by MTT colorimetric assay. The cell ultrastructure was observed by transmission electron microscope (TEM). Results: MTT assay results showed that compared with the control group, at the 12 h and 24 h, cell proliferation was enhanced with the hypoxia degree. At 24 h, cell proliferation showed significant differences. At 48 h and 72 h, proliferation of the cultured HPLFS in severe hypoxia group reduced significantly. Observed by TEM, at 24 h, not only the number of mitochondria, rough endoplasmic reticulum but also cell process increased in the cultured HPLFS in severe hypoxia group. At 72 h, the number of lysosome increased and the cell structure degenerated. Conclusion: Long-time severe hypoxia may lower the repair and remodeling abilities of periodontium, which might be one of the important etiological factors of periodontal disease under condition of high altitude.

Effects of H2O2 and chlorhexidine on MMP-1, TIMP-1,2, Type 1 collagen, fibronectin and UNCL expressions in human periodontal ligament fibroblasts / 대한치주과학회지

PURPOSE: To evaulate the effects of chlorhexidine and H2O2 on matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase(TIMP-1, TIMP-2), Type 1 collagen, fibronectin and UNCL expressions in human periodontal ligament fibroblasts (hPDLF). MATERIALS AND METHODS: 1.2x10(-1)%, 1.2x10(-2)% and 1.2x10(-3)% CHX and 3x10(-3)%, 3x10(-4)% and 3x10(-5)% H2O2 and mixture of CHX and H2O2 were applied to hPDLF for 1 min and 30 min. The mRNA expressions of MMP-1, TIMP-1 and 2, Type 1 collagen, fibronectin and UNCL in hPDLF were analysed by RT-PCR. RESULTS: The result were as follows: 1. The expression of UNCL mRNA was higher than that of other mRNAs. 2. 1.2x10(-3) % CHX increased mRNA expressions of hPDLF as application time increased. 3. H2O2 lower than 3x10(-3) % increased expression of UNCL mRNA, and did not decrease mRNA expression of hPDLF. 4. hPDLF treatment with 1.2x10(-1) % CHX (with or without H2O2) resulted in no gene expression. 5. hPDLF treatment with 1.2x10(-2) % CHX (with or without H2O2) for 30 minutes resulted in no gene expression. CONCLUSION: Because low concentration of CHX and H2O2 increased UNCL mRNA expression of hPDLF, low concentraction of CHX and H2O2 may have an antioxidative effect.

Effects of H2O2 and ascorbic acid on TIMP-2, Type 1 collagen, and PDLs22 levels in human periodontal ligament fibroblasts / 대한치주과학회지

Reactive oxygen species (ROS) have been implicated in the pathogenesis of various diseases. And vitamin C has shown a protective effect for the tissues. The aim of this study was to evaluate the effects of H2O2 and ascorbic acid on matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase (TIMP: TIMP-1, TIMP-2), Type 1 collagen, fibronectin, and PDLs22 level in human periodontal ligament fibroblasts (hPDLF) via reverse transcription-polymerase chain reaction (RT-PCR). hPDLF was obtained from a healthy periodontium and cultured in Dulbecco's modified Eagles's medium plus 10% fetal bone serum. The concentration of ascorbic acid in hPDLF was 50 microgram/ml, and that of H2O2 in hPDLF was 0.03% and 0.00003%. Ascorbic acid only, H2O2 only and mixture of ascorbic acid and H2O2 were applied with hPDLF for 1-, 3-, and 30-min., respectively. The gene expression of MMP-1-, TIMP-1-, TIMP-2-, Type 1 collagen-, fibronectin-, and PDLs22-mRNA in hPDLF was analysed via RT-PCR. The results were as follows; 1. hPDLF in response to 30-min. incubation with 0.03% H2O2 did not show any gene expression. 2. In all the experimental groups, the gene expression of fibronectin mRNA showed the decreased tendency compared to control. 3. In all the experimental groups, the gene expression of TIMP-1 mRNA showed the tendency similar to control. 4. hPDLF in response to 30-min. incubation with 0.03% H2O2 and ascorbic acid increased mRNA induction for MMP-1. 5. In all the experimental groups, hPDLF increased mRNA induction for PDLs22, collagen type I, and TIMP-2 compared to control. Within the limited experiments, H2O2 and ascorbic acid increased mRNA induction for PDLs22, collagen type I, and TIMP-2 in hPDLF. More research will be needed in order to confirm the relative importance of the different roles of ROS and antioxidants in hPDLF from a periodontal regeneration or repair standpoint.

Effect of dexamethasone on proliferation of human periodontal ligament fibroblasts cultivated in vitro / 吉林大学学报(医学版)

Objective To investigate the effect of dexamethasone on proliferation of human periodontal ligament fibroblasts(hPDLF) cultivated in vitro,and search for optimal culture condition for hPDLF in vitro,and provide basis for further study on regeneration of periodontinum.Methods The hPDLF cultivated in vitro were divided into 5 groups and cultivated in 5 different culture media separately which all included 10% fetal bovine serium: ①control group(no DEX);②5 mg?L-1DEX;③10 mg?L-1 DEX;④20 mg?L-1 DEX;⑤50 mg?L-1 DEX.The proliferation of hPDLF was assyed by MTT method.The phase contrast microscope was used to observe the morphological changes of the cells.Results The PDLF appeared aggregation and cells on bottom of culture bottle showed squamae-shape after inoculated on plate with 24 hole for 3 d.There were more layers of smaller hPDLF with blunt prominency came forth after cultivated in DEX culture media.MTT method showed that DEX promoted the proliferation of hPDLF obviously compared with control group(P

Expression of mRNA for matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gingival and periodontal ligament fibroblasts treated with lipopolysaccharide from Prevotella intermedia / 대한치주과학회지

Matrix metalloproteinases (MMPs) are a family of host-derived proteolytic enzymes and implicated in the remodeling and degradation of extracellular matrix under both physiological and pathological conditions. Connective tissue degradation in periodontal diseases is thought to be due to excessive MMP activities over their specific inhibitors. The effects of lipopolysaccharide (LPS) from Prevotella intermedia, one of the major putative pathogens of periodontitis, on the expression of mRNA for MMPs and tissue inhibitors of metalloproteinases (TIMPs) in human gingival and periodontal ligament fibroblasts were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of mRNAs encoding MMP-1, -2, -3, -10, and -14 was increased in human gingival fibroblasts treated with P. intermedia LPS, whereas MMP-11 and TIMP-2 mRNA expression was decreased in these cells stimulated with LPS. P. intermedia LPS increased the MMP-1, -2, -10, -11, and -14 mRNA expression and decreased TIMP-1 and -2 mRNA expression in human periodontal ligament fibroblasts. These findings imply that P. intermedia LPS may play an important role in the connective tissue degradation in periodontitis.

A study of mutagenic effects of simulating static magnetic field produced by magnetic attachment on human periodontal ligament fibroblasts / 实用口腔医学杂志

0.05).Conclusion:It suggests that HPDLFs exposured to static magnetic field produced by magnetic attachment have little mutagenic effects on chromosomes and DNA.

Effects of roxithromycin on TNF-? and IL-6 release from periodontal ligment fibroblasts stimulated with lipopolysaccharide / 实用口腔医学杂志

Objective:To test the effects of roxithromycin(RM) on TNF? and IL-6 release from periodontal ligment fibroblasts(PDLCs) stimulated with lipopolysaccharide(LPS).Methods:PDLCs of passage 4~8 were cultured with RM at 2,20 and 200 ?g/ml respectively with 10 ?g/ml of LPS.The control cells were cultured with culture medium only.The cultures were continued for 3,6,12 and 24 hours respectively.ELISA method was used to measure TNF-? and IL-6 released into the culture medium from PDLCs in the different groups.Results:LPS increased both TNF-? and IL-6 release from PDLCs at all the time points(P