周期性牵张应力作用下KLF5对人牙周膜细胞增殖和成骨分化的影响

目的: 探讨周期性牵张应力(cyclic tensile stress, CTS) 作用下Kruppel样转录因子5 (Kruppel-like factor 5, KLF5) 在人牙周膜细胞 (human periodontal ligament cells, hPDLCs) 中的表达及其对 hPDLCs增殖和成骨分化的影响。方法: 酶解组织块法体外培养hPDLCs,对其施加形变率10%,频率0.5 Hz的周期性牵张应力1、4、8、12、24 h,采用RT-PCR 和Western印迹法检测细胞中KLF5 mRNA和蛋白的表达。转染siRNA（si-KLF5）至hPDLCs沉默KLF5表达,RT-PCR检测KLF5 mRNA的表达。同时,将过表达碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF、FGF2）的pcDNA3.1-FGF2转染至稳定转染si-KLF5的hPDLCs,加力8 h 后,以CCK8法检测各组细胞的增殖活性,碱性磷酸酶(alkaline phosphatase, ALP)试剂盒检测ALP活性,RT-PCR检测成骨分化因子Runx2和Osterix的mRNA表达,Western印迹法检测Runx2、Osterix、FGF2、GSK-3β、P-GSK-3β (ser 9)、β-catenin蛋白的表达。采用SPSS22.0 软件包对数据进行统计学分析。结果: 10% CTS刺激呈时间依赖性地诱导hPDLCs中KLF5 mRNA 和蛋白表达。si-KLF5转染可显著抑制10% CTS诱导的hPDLCs的增殖,降低其ALP活性,减少Runx2和Osterix的mRNA和蛋白表达,并抑制FGF2-GSK-3β/β-catenin信号通路的激活;而过表达FGF2可以部分逆转沉默KLF5对hPDLCs增殖和成骨分化的抑制效应。结论: 周期性牵张应力作用下,KLF5通过FGF2-GSK-3β/ /β-catenin信号通路影响人牙周膜细胞的增殖和成骨分化。     

Fluid Shear Stress Stimulates Osteogenic Differentiation of Human Periodontal Ligament Cells via the Extracellular Signal‐Regulated Kinase 1/2 and p38 Mitogen‐Activated Protein Kinase Signaling Pathways

Background: Fluid shear stress (FSS) is a major type of mechanical stress that is loaded on human periodontal ligament cells (hPDLCs) during mastication and orthodontic tooth movement. This study aims to clarify the effect of FSS on the osteogenic differentiation of hPDLCs and to further verify the involvement of mitogen‐activated protein kinase (MAPK) signaling in this process. Methods: After isolation and characterization, hPDLCs were subjected to 2‐hour FSS at 12 dynes/cm², and cell viability, osteogenic gene mRNA expression, alkaline phosphatase (ALP) activity, secretion of Type I collagen (COL‐I), and calcium deposition were assayed. The levels of phosphorylated p38 and phosphorylated extracellular signal‐regulated kinase 1/2 (ERK1/2) in response to FSS were detected by Western blot, and the involvement of ERK1/2 and p38 MAPK signaling pathways in hPDLC osteogenesis under FSS was investigated using the specific MAPK inhibitors U0126 (2Z,3Z)‐2,3‐bis[amino(2‐aminophenylthio)methylene]succinonitrile,ethanol) and SB203580 (4‐[4‐(4‐fluorophenyl)‐2‐(4‐[methylsulfinyl]phenyl)‐1H‐imidazol‐5‐yl]pyridine). Results: The application of FSS on hPDLCs induced an early morphologic change and rearrangement of filamentous actin. ALP activity, messenger RNA (mRNA) levels of osteogenic genes, COL‐I, and osteoid nodules were significantly increased by FSS. Moreover, ERK1/2 and p38 were activated in different ways after FSS exposure. U0126 and SB203580 completely blocked the FSS‐induced increases in ALP activity and osteogenic gene mRNA expression and osteoid nodules formation. Conclusions: FSS is an effective approach for stimulating osteogenic differentiation of hPDLCs. The ERK1/2 and p38 MAPK signaling pathways are involved in this cellular process.     

黄芩素对人牙周膜细胞增殖、RUNX2和BMP2表达的研究

目的探讨中药黄芩提取物黄芩素对人牙周膜细胞(hPDLCs)增殖和成骨基因RUNX2、BMP2表达的影响。方法原代培养hPDLCs,hPDLCs中分别加入浓度为1.25、2.50、5.00、10.00μmol/L的黄芩素作为实验组,空白组不加黄芩素。MTT法检测黄芩素对hPDLCs增殖的影响;试剂盒检测ALP的活性和表达;qRT-PCR研究黄芩素对hPDLCs RUNX2、BMP2表达的影响。结果较空白组,1.25~10.00μmol/L黄芩素作用于hPDLCs,其增殖活力略下降,但差异无统计学意义(P>0.05);黄芩素组ALP活性和表达上升(P<0.05),呈浓度依赖性;成骨相关基因RUNX2、BMP2 mRNA表达水平随时间延长,表达量持续上升,11 d上升最为显著(P<0.05),且10.00μmol/L黄芩素组显著高于其余浓度组(P<0.05)。结论黄芩素对hPDLCs骨向分化有促进作用,有可能作为牙周再生的选择之一。     

低氧对牙周膜成纤维细胞增殖和成骨分化的影响

目的:观察低氧对体外培养的人牙周膜成纤维细胞(periodontal ligament cells, PDLCs)增殖与成骨分化的影响。方法:体外培养鉴定人牙周膜成纤维细胞,分别用浓度为0、100、200、400 μmol/L的CoCl2作用于细胞,采用MTT法观察CoCl2对PDLCs增殖的影响,运用实时定量PCR和Western免疫印迹技术检测CoCl2模拟低氧对PDLCs成骨分化的影响。采用SPSS13.0软件包对数据进行统计学分析。结果:免疫组化染色结果证实,培养细胞为牙周膜成纤维细胞。200 μmol/L及400 μmol/L的CoCl2均能抑制PDLCs的增殖及碱性磷酸酶、RUNX2、Ⅰ型胶原的表达,并呈剂量依赖性。结论:氯化钴模拟的低氧环境对PDLCs增殖及成骨分化具有抑制作用。     

Effects of varied ionic calcium and phosphate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells in vitro

An S, Ling J, Gao Y, Xiao Y. Effects of varied ionic calcium and phosphate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells in vitro. J Periodont Res 2012; 47: 374–382. ©2011 John Wiley & Sons A/S Background and Objective: A number of bone‐filling materials containing calcium (Ca²⁺) and phosphate (P) ions have been used in the repair of periodontal bone defects; however, the effects that local release of Ca²⁺ and P ions has on biological reactions are not fully understood. In this study, we investigated the effects of various levels of Ca²⁺ and P ions on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells (hPDLCs). Material and Methods: The hPDLCs were obtained using an explant culture method. Defined concentrations and ratios of ionic Ca²⁺ to inorganic P were added to standard culture and osteogenic induction media. The ability of hPDLCs to proliferate in these growth media was assayed using the Cell Counting Kit‐8. Cell apoptosis was evaluated by the fluorescein isothiocyanate–annexin V/propidium iodide double‐staining method. Osteogenic differentiation and mineralization were investigated by morphological observations, alkaline phosphatase activity and Alizarin Red S/von Kossa staining. The mRNA expression of osteogenic related markers was analysed using RT‐PCR. Results: Within the ranges of Ca²⁺ and P ion concentrations tested, we observed that increased concentrations of Ca²⁺ and P ions enhanced cell proliferation and formation of mineralized matrix nodules, whereas alkaline phosphatase activity was reduced. The RT‐PCR results showed that elevated concentrations of Ca²⁺ and P ions led to a general increase of Runx2 mRNA expression and decreased alkaline phosphatase mRNA expression, but gave no clear trend on osteocalcin mRNA levels. Conclusion: The concentrations and ratios of Ca²⁺ and P ions could significantly influence proliferation, differentiation and mineralization of hPDLCs. Within the range of concentrations tested, we found that the combination of 9.0 mm Ca²⁺ ions and 4.5 mm P ions were the optimal concentrations for proliferation, differentiation and mineralization in hPDLCs.     

Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E(2) by hypoxia-inducible factor-1α up-regulation in human periodontal ligament cells

Kim Y‐S, Shin S‐I, Kang K‐L, Herr Y, Bae W‐J, Kim E‐C. Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E₂ by hypoxia‐inducible factor‐1α up‐regulation in human periodontal ligament cells. J Periodont Res 2012; 47: 719–728. © 2012 John Wiley & Sons A/S Background and Objective: Although hypoxia‐inducible factor 1α (HIF‐1α) is up‐regulated in the periodontal pockets of periodontitis patients, the expression and precise molecular mechanisms of HIF‐1α remain unknown in human periodontal ligament cells (PDLCs). The aim of this study was to explore the effects, as well as the signaling pathway, of nicotine and lipopolysaccharide (LPS) on the expression of HIF‐1α and on the production of its target genes, including cyclooxygenase‐2 (COX‐2)‐derived prostaglandin E₂ (PGE₂), MMP‐2 and MMP‐9 in PDLCs. Material and Methods: The expression of COX‐2 and HIF‐1α proteins was evaluated using western blotting. The production of PGE₂ and MMPs was evaluated using enzyme immunoassays and zymography, respectively. Results: LPS and nicotine synergistically induced the production of PGE₂, MMP‐2 and MMP‐9, and increased the expression of MMP‐2, MMP‐9, COX‐2 and HIF‐1α proteins. Inhibition of HIF‐1α activity by chetomin or knockdown of HIF1α gene expression by small interfering RNA markedly attenuated the production of LPS‐ and nicotine‐stimulated PGE₂ and MMPs, as well as the expression of COX‐2 and HIF‐1α. Furthermore, pretreatment with inhibitors of COX‐2, p38, extracellular signal‐regulated kinase, Jun N‐terminal kinase, protein kinase C, phosphatidylinositol 3‐kinase and nuclear factor‐kappaB decreased the expression of nicotine‐ and LPS‐induced HIF‐1α and COX‐2, as well as the activity of PGE₂ and MMPs. Conclusion: These data demonstrate novel mechanisms by which nicotine and LPS promote periodontal tissue destruction, and provide further evidence that HIF‐1α is a potential target in periodontal disease associated with smoking and dental plaque.     

Effects of adenoviral‐mediated coexpression of bone morphogenetic protein‐7 and insulin‐like growth factor‐1 on human periodontal ligament cells

Yang L, Zhang Y, Dong R, Peng L, Liu X, Wang Y, Cheng X. Effects of adenoviral‐mediated coexpression of bone morphogenetic protein‐7 and insulin‐like growth factor‐1 on human periodontal ligament cells. J Periodont Res 2010; 45: 532–540. © 2010 John Wiley & Sons A/S Background and Objective: Bone morphogenetic protein‐7 (BMP‐7) and insulin‐like growth factor‐1 (IGF‐1) are important in periodontal reconstruction. However, their synergistic effect in periodontal regeneration by gene delivery has not been reported. In this study, gene delivery of these two growth factors to human periodontal ligament cells (hPDLCs) was examined for its effects on cell proliferation and differentiation. Material and Methods: Recombinant adenoviruses containing both human BMP‐7 and IGF‐1 cDNA created by introducing the internal ribosome entry site (IRES) sequence were used to transfer the genes into hPDLCs. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and cell cycle analysis were used to observe their effects on cell proliferation, while alkaline phosphatase activity measurement, RT‐PCR and in vivo tests were conducted to investigate their effects on cell differentiation. Results: The proliferation of hPDLCs transduced by adenoviruses coexpressing BMP‐7 and IGF‐1 was suppressed while their differentiation ability was enhanced. There was a synergism of BMP‐7 and IGF‐1 in up‐regulating alkaline phosphatase activity and mRNA levels of collagen type I and Runx2. Implantation in vivo with scaffolds illustrated that the transduced cells exhibited osteogenic differentiation and formed bone‐like structures. Conclusion: The combined delivery of BMP‐7 and IGF‐1 genes using an IRES‐based strategy synergistically enhanced differentiation of hPDLCs. It is suggested that this could be a new potential method in gene therapy for periodontal reconstruction.     

孕酮对人牙周膜细胞增殖及成骨分化的调控作用

目的：通过细胞免疫组织化学方法检测人牙周膜细胞（hPDLCs）中孕激素受体（PR）的表达,研究孕酮在hPDLCs增殖及成骨分化过程中的作用。方法：原代培养hPDLCs,取第4～6代细胞用于实验。分为空白对照组,孕酮组,抑制剂组。用含有不同浓度（1,10,100 nM）孕酮的培养液培养hPDLCs 1,2,3,4,5,6,7 d,采用MTT法检测在孕酮作用下hPDLCs生长曲线的变化情况。检测普通培养液和成骨诱导培养液中的各组细胞在孕酮作用后茜素红染色钙化结节面积的变化。结果：PR在hPDLCs的胞质和胞核中均有表达,且在胞核的表达明显高于胞质。孕酮干预hPDLCs后,细胞的增殖高于对照组,孕激素受体抑制剂能明显抑制hPDLCs的生长。孕酮能够促进茜素红染色钙化结节的形成。在成骨诱导液中,孕酮同样有促进成骨的作用。与普通培养组相比较,孕酮对成骨诱导组的成骨促进作用更为明显。结论：人牙周膜细胞中有PR的表达,孕酮能够促进人牙周膜细胞的增殖和成骨分化。     

Hydrogen sulfide promotes osteogenic differentiation of human periodontal ligament cells via p38-MAPK signaling pathway under proper tension stimulation

ObjectiveHydrogen sulfide (H₂S), one of endogenous gaseous signalling molecules, can be induced by mechanical force stimulation on human periodontal ligament cells (hPDLCs). Little is known about the mechanism of H₂S on the osteogenic differentiation although previous studies have demonstrated that H₂S stimulated or inhibited osteoclastic differentiation. The present study was to investigate whether H₂S played a regulatory role in osteogenic differentiation of the periodontal tissue remodeling and the involvement of mitogen-activated protein kinase (MAPK) signaling in this process.DesignhPDLCs were applied with cycle tension force for 6 h, 12 h, 24 h or 48 h to select the optimal time for force application. Then the effects of H₂S on hPDLCs osteogenic differentiation were investigated. Signal-regulated kinases p38-MAPK and ERK activities with H₂S treatment were measured. Finally, specific MAPK inhibitors SB203580 and U0126 were employed to investigate the involvement of the two kinases in hPDLCs osteogenic differentiation with H₂S pre-treatment.ResultsTension stimulation promoted mRNA and protein expression of ALP, OCN and Runx2 in hPDLCs. The expression of ALP, OCN and Runx2 increased in a concentration-dependent manner with H₂S pre-treatment. Importantly, p38-MAPK and ERK were activated in different ways upon induction by H₂S. Furthermore, expression of Runx2, ALP and OCN, the osteogenic regulators, was reversed by SB203580 and U0126.ConclusionsH₂S could promote osteogenic differentiation of hPDLCs by activating p38-MAPK and ERK signaling pathways.     

Physiologic Levels of Endogenous Hydrogen Sulfide Maintain the Proliferation and Differentiation Capacity of Periodontal Ligament Stem Cells

Background: Many invading oral bacteria are known to produce considerable amounts of hydrogen sulfide (H₂S). The toxic activity of exogenous H₂S in periodontal tissue has been demonstrated, but the role of endogenous H₂S in the physiologic function of periodontal tissue remains poorly understood. The purpose of the present study is to investigate the biologic functions of H₂S in the proliferation and differentiation of human periodontal ligament stem cells (PDLSCs). Methods: PDLSCs were isolated from periodontal ligament tissues of periodontally healthy volunteers or patients with periodontitis. Immunocytochemical staining, flow cytometry, and Western blot analysis were used to examine the expression of H₂S‐synthesizing enzymes cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE). The proliferation capacity of PDLSCs was determined by cell counting kit‐8 assay, carboxyfluorescein succinimidyl ester analysis, and 5‐ethynyl‐2′‐deoxyuridine assay. The osteogenic potential of PDLSCs was tested using alkaline phosphatase staining, Alizarin Red staining, and in vivo transplantation experiments. Oil Red O staining was used to analyze adipogenic ability. Results: The results show that human PDLSCs express both CBS and CSE and produce H₂S. Blocking the generation of endogenous H₂S with CBS inhibitor hydroxylamine significantly attenuated PDLSC proliferation and reduced the osteogenic and adipogenic differentiation capacity of PDLSCs. In contrast, CSE inhibitor dl ‐propargylglycine had no effect on PDLSC function. Exogenous H₂S could inhibit the production of endogenous H₂S and impair PDLSC function in a dose‐dependent manner. Conclusion: Physiologic levels of endogenous H₂S maintain the proliferation and differentiation capacity of PDLSCs, and CBS may be the main source of endogenous H₂S in PDLSCs.     

The Proteasome Inhibitor Bortezomib Inhibits Inflammatory Response of Periodontal Ligament Cells and Ameliorates Experimental Periodontitis in Rats

Background: Periodontitis is a chronic inflammatory disease initiated by bacteria and their virulence factors. Bortezomib (BTZ) is the first proteasome inhibitor for clinical treatment of malignancies. Its anticancer activity is accompanied by an anti‐inflammatory effect. However, there are few reports about its anti‐inflammatory effect and underlying mechanism in periodontal disease, especially on human periodontal ligament cells (hPDLCs), which are considered a promising cell‐based therapy for treating periodontitis. Methods: hPDLCs were treated with lipopolysaccharide (LPS) and pretreated with BTZ. mRNA and protein levels of tumor necrosis factor (TNF)‐alpha, interleukin (IL)‐1β, IL‐6, and IL‐8 were determined. The anti‐inflammatory mechanism of BTZ was studied. Further, experimental rat periodontitis was induced with ligature and LPS injection, and simultaneously and locally treated with BTZ (three injections/week). Four weeks after treatment, microcomputed tomography, immunohistochemical, and histopathologic analyses were performed. Results: Bortezomib administration at safe concentrations (≤1 nM) inhibited production of proinflammatory cytokines in LPS‐stimulated hPDLCs via nuclear factor (NF)‐kappa B, p38/extracellular signal‐regulated kinase, and mitogen‐activated protein kinase/activator protein‐1 pathways. Moreover, in the LPS and ligature‐induced periodontitis rat model, BTZ suppressed expression of TNF‐α, IL‐1β, IL‐6, and IL‐8, reduced the ratio of receptor activator of NF‐κB ligand/osteoprotegerin, and prevented alveolar bone absorption. Conclusion: These findings demonstrate the anti‐inflammatory activity of BTZ against periodontal inflammatory response and present BTZ as a promising therapy for periodontal disease.