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.     

丹酚酸B对人牙周膜细胞成骨分化的影响

目的探讨中药丹参主要活性成分丹酚酸B（SalB）对人牙周膜细胞（hPDLCs）成骨分化的影响。方法取第3代hPDLCs进行实验,运用甲基噻唑基四唑（MTT）法检测不同浓度丹酚酸B对hPDLCs增殖活性影响,通过检测碱性磷酸酶（ALP）活性、矿化结节染色、骨钙素（OCN）mRNA表达来探讨丹酚酸B对hPDLCs成骨分化的影响。结果丹酚酸B对hPDLCs增殖活性无影响。当丹酚酸B浓度为0.5、1、5 μmol·L^-1时均能增加hPDLCs的ALP活性和OCN mRNA表达,与OIM组比较差异有统计学意义（P<0.05）;且当丹酚酸B浓度为0.5、1、5 μmol·L^-1时hPDLCs形成矿化结节数量明显高于OIM组。结论适宜浓度的丹酚酸B能有效促进hPDLCs成骨分化。     

镁离子对人牙周韧带细胞体外成骨能力的影响

目的:探究不同浓度Mg²⁺对hPDLCs成骨向分化的影响,为后续牙周组织再生实验选择合适的Mg²⁺注入浓度提供依据。方法:取P3-P5代hPDLCs于Mg²⁺浓度分别为0、10、15、25、35、50 mmol/L条件中常规培养,用CCK-8法检测hPDLCs增殖情况。成骨诱导后比较各组ALP活性差异及茜素红矿化结节着色情况,进行RT-qPCR检测成骨相关基因Runx2、ALP、Col1、OPN及Bglap的表达差异。采用SPSS16.0软件,运用单因素方差分析进行统计学分析。结果:10、15、25 mmol/L Mg²⁺浓度可促进细胞增殖并提高ALP活性,35、50 mmol/L Mg²⁺浓度抑制细胞增殖及ALP活性。结论:适宜浓度镁离子(0~25 mmol/L)可促进hPDLCs早期成骨分化并抑制矿化。     

褪黑素对人牙周膜细胞成骨分化能力的影响

目的 探讨褪黑素对人牙周膜细胞(hPDLCs)成骨分化能力的影响.方法 原代培养法成功提取hPDLCs后,加入不同浓度的褪黑素作为实验组,空白对照组不加褪黑素.MTT法检测不同浓度褪黑素对细胞增殖的影响,相关试剂盒检测碱性磷酸酶(ALP)的活性和表达后,采用茜素红S染色、Real-time PCR和Western Bl...     

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.     

High‐power,red‐light‐emitting diode irradiation enhances proliferation,osteogenic differentiation,and mineralization of human periodontal ligament stem cells via ERK signaling pathway

1 Background

Light‐emitting diode (LED) is attracting attention as a new light source for phototherapy. However, its effects on periodontal tissue regeneration remain unknown. The aim of this study was to examine the effects of high‐power, red LED irradiation on human periodontal ligament stem cells (PDLSCs), which play an important role in periodontal tissue regeneration.

2 Methods

PDLSCs were derived from adult human third molars. The light source was red LED (peak wavelength: 650 nm). Energy densities ranging from 0 to 10 J/cm² were tested to determine the optimal dose. PDLSC proliferation was measured using two parameters: live cell protease and ATP levels. After the cells were induced to differentiate, the effect of LED irradiation on osteogenic differentiation and mineralization was examined, with particular focus on the extracellular signal‐regulated kinase (ERK)1/2 signaling pathway using an ERK inhibitor (PD98059).

3 Results

LED irradiation at 8 J/cm² led to a significant increase in PDLSC proliferation and enhanced Runx2 and Osterix mRNA expression, Alkaline phosphatase activity, procollagen type I C‐peptide and osteocalcin production, calcium deposition, and alizarin red S staining. In addition, LED induced the activation of ERK1/2, and the effects of LED on PDLSC proliferation, differentiation, and mineralization could be suppressed by treatment with PD98059.

4 Conclusions

The results of this study show that 650‐nm high‐power, red, LED irradiation increases PDLSCs proliferation, and osteogenic differentiation and mineralization, mediated by ERK1/2 activation. These findings suggest that LED may be a useful tool for periodontal tissue regeneration.     

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。     

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。     

姜黄素通过调控Wnt通路改善牙周膜干细胞成骨分化能力

目的:探讨姜黄素对人牙周膜干细胞(PDLSCs)成骨分化的影响。方法:取第4~6代的PDLCs进行实验,用CCK-8实验检测姜黄素对PDLCs增殖活性影响,通过检测碱性磷酸酶(ALP)活性、矿化结节染色以及ALP、OCN和Runx2等成骨相关基因的表达水平来探讨姜黄素对牙周膜干细胞成骨分化能力的影响,同时检测姜黄素对经典Wnt通路及其配体的影响。结果:姜黄素对PDLSCs的增殖能力无影响。与对照组相比较,姜黄素能增加PDLSCs的ALP活性、上调ALP、OCN和Runx2等成骨相关基因的表达、增加矿化结节数量(P<0.05)。姜黄素能阻断Wnt信号通路,但加入Wnt信号通路激动剂(氯化锂)后,PDLSCs的成骨分化能力显著下降。结论:姜黄素能通过抑制Wnt信号通路,增强牙周膜干细胞成骨分化能力。     

Asiaticoside induces osteogenic differentiation of human periodontal ligament cells through the Wnt pathway

1 Background

Asiaticoside is a compound isolated from Herb Centella asiatica, which has been shown to promote osteogenic differentiation of human periodontal ligament (hPDL) cells. This study investigated the molecular mechanism underlying the asiaticoside‐induced osteogenic differentiation of hPDL cells.

2 Methods

hPDL cells were incubated with various concentrations of asiaticoside to test cell viability by MTT assay. The mRNA expression levels were analyzed by using quantitative real‐time polymerase chain reaction (PCR). Osteogenic differentiation was determined by alkaline phosphatase activity assay and alizarin red staining. The subcellular localization of β‐catenin was analyzed by both immunofluorescence and western blot.

3 Results

The results showed that asiaticoside had no effect on the cell viability at any of the tested concentrations. Real‐time PCR revealed that osterix (OSX) and dentin matrix protein1 (DMP1) mRNA were significantly enhanced by asiaticoside treatment. Alkaline phosphatase activity and in vitro mineralization were also significantly induced. Interestingly, asiaticoside dose‐dependently increased WNT3A mRNA expression, but not WNT5A and WNT10B. The activation of Wnt signaling was shown to result in nuclear accumulation of β‐catenin as evaluated by immunofluorescence staining and western blot analysis. Pre‐treatment with recombinant human Dickkopf1 (rhDKK1) inhibited asiaticoside‐induced β‐catenin nuclear translocation and osteoblast marker gene expression. Moreover, rhDKK1 attenuated asiaticoside‐induced DMP1 protein expression.

4 Conclusion

The data demonstrate that asiaticoside induces osteogenic differentiation of hPDL cells by activating the Wnt/β‐catenin signaling pathway. The findings suggest that asiaticoside could be used as a novel therapeutic drug for periodontal tissue regeneration.     

氟化钠对人牙周膜细胞增殖及矿化能力的影响

目的:观察氟化钠对体外培养的人牙周膜细胞增殖及矿化能力的影响,为氟添加入牙周组织工程药物中的应用提供依据.方法:原代培养并鉴定人牙周膜细胞,应用CCK8检测不同浓度NaF对hPDLCs增殖的影响,并筛选出4个浓度用于矿化实验.矿化条件下,将0、1×10-5、5×10-4和1×10-3 mol/L的NaF作用hPDLCs后,通过碱性磷酸酶(ALP)染色、茜素红染色和实时荧光定量PCR检测矿化能力及成骨相关基因的表达.采用SPSS20.0软件包对数据进行单因素方差分析.结果:5×10-5、1×10-4、5×10-4 mol/L的NaF均能促进hPDLCs增殖,且以5×10-4 mol/L效果最佳(P＜0.05).而1×104 mol/L的NaF碱性磷酸酶染色阳性面积最大、茜素红染色矿化结节数量最多(P＜0.05).RT-PCR结果根据时间、指标变化程度较大.结论:5×10-5、1×10-4、5×10-4 mol/L的NaF能促进hPDLCs的增殖能力,1×10-5 mol/L的NaF能提高hPDLCs的碱性磷酸酶活性及钙结节形成.     

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.