人非ATP酶依赖性调节颗粒13对人牙周膜细胞的调节作用

目的：探讨人非ATP酶依赖性调节颗粒13（hRpn13）通过泛素-蛋白酶体通路（UPP）途径对人牙周膜细胞（PDLC）的作用,从而揭示其对PDLC增殖、分化和功能的调节作用。方法通过转染敲除来改变PDLC中hRpn13的表达后,用MTT法观测细胞形态,4′,6-二脒基-2-苯基吲哚（DAPI）来检测细胞增殖情况,通过检测碱性磷酸酶（ALP）和Ⅰ型胶原纤维的分泌情况来检测细胞成骨分化情况,最后通过检测NF鄄κB受体活化子配体（RANKL）、骨保护素（OPG）、泛素化蛋白的改变来研究hRpn13对PDLC功能的调节作用及其可能机制。结果 hRpn13对成纤维细胞的增殖和ALP的活性,Ⅰ型胶原蛋白以及RANKL、OPG的表达起到了负性调节作用,同时hRpn13使泛素化蛋白聚集减少。结论 hRpn13可以通过影响UPP通路来调节成纤维细胞的增殖、分化和功能。     

重组腺病毒载体Ad5-hOPG-EGFP转染的Beagle犬牙周膜细胞成骨能力研究

目的    观察重组腺病毒载体 Ad5-hOPG-EGFP 转染 的Beagle 犬牙周膜细胞（ PDLCs）体内和体外的骨化能力。方法    将体外构建的携带人骨保护素（human osteoprotegerin,hOPG）基因的腺病毒载体Ad5-hOPG-EGFP转染Beagle犬PDLCs,通过体外Von Kossa染色实验、实时荧光定量PCR检测成骨指标的表达情况、酶联免疫检测RANKL/OPG的值及裸鼠体内胶原膜成骨实验比较转染组和对照组的成骨能力。结果    组织学和形态学结果显示,转染了重组腺病毒Ad5-hOPG-EGFP的Beagle犬PDLCs形成的矿化结节数目多且体积大、深染。转染组中犬碱性磷酸酶（alkaline phosphates,ALP）、骨钙素（osteocalcin,OC）、骨涎蛋白（bone sialoprotein,BSP）基因相对表达水平均高于空载体组和空白对照组（P＜0.05）。转染组中犬RANKL/OPG的值下降趋势最为明显。裸鼠体内实验中转染组胶原膜也体现出较好的成骨能力。结论    重组腺病毒介导的hOPG基因可以促进PDLCs成骨。     

Impact of titanium ions on osteoblast-, osteoclast- and gingival epithelial-like cells

PurposeTo investigate the effects of titanium (Ti) ions on the cell viability, the cell differentiation and the gene expressions related to bone resorption including Receptor Activator of NF-κB Ligand (RANKL) and Osteoprotegerin (OPG) in the tissues around dental implants, the osteoblast-, osteoclast-, and gingival epithelial-like cells were exposed to Ti ions.MethodsAn MTS assay was carried out to evaluate the viabilities of osteoblast-like MC3T3-E1, osteoclast-like RAW264.7 and epithelial cell-like GE-1 cells. The gene expressions in these cells were analyzed by the use of RT-PCR and real-time quantitative RT-PCR.ResultsTi ions in the concentration range 1–9 ppm had little effect on the viabilities of MC3T3-E1, RAW264.7 and GE-1, whereas 20 ppm Ti ions significantly decreased the viabilities of all cells. Analyses of RT-PCR and real-time quantitative RT-PCR data revealed that Ti ions at 9 ppm remarkably inhibited the expressions of Runx2, Osterix and type I collagen in MC3T3-E1. In RAW264.7, Ti ions showed no effects on the levels of mRNAs for TRAP and cathepsin K enhanced by RANKL. Ti ions at the range of 1–9 ppm showed no effects on the levels of mRNAs for RANKL and OPG in GE-1, while Ti ions at 9 ppm enhanced the expression of these genes in MC3T3-E1.ConclusionsThese results, taken together, suggested that Ti ions show the biological effects, both on the viabilities of osteoblast and osteoclast and on the differentiation of either the osteoblastic or osteoclastic cells, which may influence the prognosis of dental implants.     

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?? Objective    To observe the osteogenesis of periodontal ligament cells ??PDLCs?? with human osteoprotegerin??hOPG?? transfection in vivo and vitro. Methods    Ad5-hOPG-EGFP was constructed in vitro and transfected into canine PDLCs. Von Kossa staining?? Real-Time PCR?? ELISA and experiment in nude mice vivo were used to assess the osteogenic indices of hOPG-hPDLCs. Results    Morphologic and histological study demonstrated that hOPG-hPDLCs formed much more and larger mineralized nodules?? and expressed higher level of alkaline phosphates??ALP????osteocalcin??OC?? and bone sialoprotein??BSP?? ??P < 0.05??. RANKL/OPG ratio declined most in hOPG-PDLCs. Transfected collagen membrane group also reflected the better osteogenic ability in nude mice vivo experiment. Conclusion    OPG gene transfection can promote osteogenic ability of PDLCs.     

Chemical modification of an implant surface increases osteogenesis and simultaneously reduces osteoclastogenesis: an in vitro study

Objectives: The present study investigated the effect of a chemical modification of the SLA surface (SLActive surface) on human periodontal ligament (hPDL) cell (1) adhesion, (2) proliferation, (3) osteogenic differentiation (core binding factor α‐1 [Cbfa‐1], bone morphogenetic protein‐7 [BMP‐7] gene expression and alkaline phosphatase [ALP] activity) and (4) osteoclast formation and activity (osteoprotegerin [OPG] and receptor activator of nuclear factor‐κ B ligand [RANKL] gene expression). The above activities were based on the hypothesis that the expression of such molecules might be dependent on the characteristics of the implant surface. Material and methods: hPDL cells were isolated and characterized for their mesenchymal origin, fibroblastic and osteoblastic phenotype. hPDL cells were cultured on smooth, SLA and SLActive implant surfaces (chemically modified). Cell attachment and proliferation were assessed for 5, 24, 72 h, 5 and 7 days. Cbfa‐1, BMP‐7, OPG and RANKL gene expression was assessed by RT‐PCR and a colorimetric assay for ALP activity was applied. Results: hPDL cells grown on SLActive surfaces demonstrated increased proliferation rates (24 h, 5 and 7 days of the incubation period), and ALP activity was found to be significantly upregulated (5, 72 h and 7 days) as compared with the SLA surfaces. After 7 days of culture, the gene expression of BMP‐7, Cbfa‐1 and OPG by hPDL cells was significantly upregulated, while RANKL gene expression was significantly downregulated in response to the SLActive surface. Conclusion: Chemical modification of a previously roughened implant surface increases hPDL proliferation and upregulates osteoblastic differentiation. It can also suppress osteoclastogenesis regulating the RANKL–RANK–OPG axis. Hence, an osteoprotective microenvironment is created around chemically modified implants that may enhance osseointegration. To cite this article:
Mamalis AA, Markopoulou C, Vrotsos I, Koutsilirieris, M. Chemical modification of an implant surface increases osteogenesis and simultaneously reduces osteoclastogenesis: an in vitro study.
Clin. Oral Impl. Res. 22 , 2011; 619–626
doi: 10.1111/j.1600‐0501.2010.02027.x     

Interleukin-6 and soluble interleukin-6 receptor suppress osteoclastic differentiation by inducing PGE(2) production in chondrocytes

This study examined how interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6r) influence osteoclastic differentiation through the function of chondrocytes. Chondrocytes were cultured with or without IL-6 and/or sIL-6r in the presence or absence of NS398, a specific inhibitor of cyclooxygenase (COX)-2, for up to 28 days. Chondrocytes were also cultured with or without IL-6 and sIL-6r for 28 days, and the conditioned medium from cells cultured without IL-6 and sIL-6r was used to induce differentiation of RAW264.7 cells into osteoclast precursors. Osteoclastic differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) staining. Expression of osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), COX-2, and prostaglandin E(2) (PGE(2)) increased in cells exposed to IL-6 and sIL-6r, whereas expression of macrophage colony-stimulating factor (M-CSF) and bone resorption-related enzymes decreased. NS398 blocked the stimulatory/suppressive effects of IL-6 and sIL-6r on the expression of OPG, RANKL, and M-CSF. Fewer TRAP-positive multinucleated cells were detected after treatment with conditioned medium from IL-6- and sIL-6r-treated chondrocytes than after treatment with conditioned medium from untreated chondrocytes. These results suggest that IL-6 and sIL-6r interfere with osteoclast function through the involvement of chondrocytes. Specifically, they appear to suppress the differentiation of osteoclast precursors into osteoclasts by inducing chondrocytic PGE(2) production, which, in turn, increases OPG secretion and decreases M-CSF secretion by chondrocytes.     

生理性根吸收过程中乳牙牙髓干细胞通过α7nAChR调节破骨细胞分化能力的研究

目的:探讨生理性根吸收过程中乳牙牙髓干细胞(DDPSCs)通过α7亚型烟碱型乙酰胆碱受体(α7nAChR)对破骨分化能力的调节作用.方法:酶消化法和有限稀释法分离培养根吸收不同时期DDPSCs和恒牙牙髓干细胞(DPSCs).采用实时定量PCR和Western blot,检测各组细胞α7nAChR、RANKL、OPG的表达及其比值差异,以及阻断α7nAChR后RANKL、OPG的表达及其比值变化.结果:α7nAChR表达及RANKL/OPG比值在根吸收中期DDPSCs中明显升高(P＜0.05).阻断α7nAChR后,RANKL/OPG明显下降(P＜0.05).结论:在乳牙生理性根吸收过程中,α7nAChR通过上调自身表达,参与对RANKL/OPG的调节,影响DDPSCs的破骨分化能力.     

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。     

Td92, an outer membrane protein of Treponema denticola,induces osteoclastogenesis via prostaglandin E2‐mediated RANKL/osteoprotegerin regulation

Kim M, Jun H‐K, Choi B‐K, Cha J‐H, Yoo Y‐J. Td92, an outer membrane protein of Treponema denticola, induces osteoclastogenesis via prostaglandin E₂‐mediated RANKL/osteoprotegerin regulation. J Periodont Res 2010; 45: 772–779. © 2010 John Wiley & Sons A/S Background and Objective: Periodontitis is a chronic inflammatory disease of the periodontium that causes significant alveolar bone loss. Osteoclasts are bone‐resorbing multinucleated cells. Osteoblasts regulate osteoclast differentiation by expression of RANKL and osteoprotegerin (OPG). Td92 is a surface‐exposed outer membrane protein of Treponema denticola, a periodontopathogen. Although it has been demonstrated that Td92 acts as a stimulator of various proinflammatory mediators, the role of Td92 in alveolar bone resorption remains unclear. Therefore, in this study, we investigated the role of Td92 in bone resorption. Material and Methods: Mouse bone marrow cells were co‐cultured with calvariae‐derived osteoblasts in the presence or absence of Td92. Osteoclast formation was assessed by TRAP staining. Expressions of RANKL, osteoprotegerin (OPG) and prostaglandin E₂ (PGE₂) in osteoblasts were estimated by ELISA. Results: Td92 induced osteoclast formation in the co‐cultures. In the osteoblasts, RANKL and PGE₂ expressions were up‐regulated, whereas OPG expression was down‐regulated by Td92. The addition of OPG inhibited Td92‐induced osteoclast formation. The prostaglandin synthesis inhibitors NS398 and indomethacin were also shown to inhibit Td92‐induced osteoclast formation. The effects of Td92 on the expressions of RANKL, OPG and PGE₂ in osteoblasts were blocked by NS398 or indomethacin. Conclusion: These results suggest that Td92 promotes osteoclast formation through the regulation of RANKL and OPG production via a PGE₂‐dependent mechanism.     

miR-223调控人牙周膜成纤维细胞中RANKL表达的功能初探

目的：探讨miR?223对人牙周膜成纤维细胞中核因子?κB受体活化因子配体（ RANKL）的调控作用。方法：构建过表达miR?223的人牙周膜成纤维细胞,Western blot及实时定量RT?PCR检测人牙周膜成纤维细胞内RANKL和骨保护素（ OPG）的表达,计算RANKL／OPG比值的改变。结果：过表达miR?223的人牙周膜成纤维细胞,RANKL的表达明显下调,RANKL／OPG比值下降。结论：miR?223可调控RANKL的表达,破坏RANKL／OPG比例的平衡,改变破骨细胞的微环境,影响破骨细胞分化。     

Endogenous hydrogen sulfide is involved in osteogenic differentiation in human periodontal ligament cells

ObjectiveEndogenous hydrogen sulfide (H₂S) has recently emerged as an important intracellular gaseous signaling molecule within cellular systems. Endogenous H₂S is synthesized from l-cysteine via cystathionine β-synthase and cystathionine γ-lyase and it regulates multiple signaling pathways in mammalian cells. Indeed, aberrant H₂S levels have been linked to defects in bone formation in experimental mice. The aim of this study was to examine the potential production mechanism and function of endogenous H₂S within primary human periodontal ligament cells (PDLCs).DesignPrimary human PDLCs were obtained from donor molars with volunteer permission. Immunofluorescent labeling determined expression of the H₂S synthetase enzymes. These enzymes were inhibited with D,L-propargylglycine or hydroxylamine to examine the effects of H₂S signaling upon the osteogenic differentiation of PDLCs. Gene and protein expression levels of osteogenic markers in conjunction with ALP staining and activity and alizarin red S staining of calcium deposition were used to assay the progression of osteogenesis under different treatment conditions. Cultures were exposed to Wnt3a treatment to assess downstream signaling mechanisms.ResultsIn this study, we show that H₂S is produced by human PDLCs via the cystathionine β-synthase/cystathionine γ-lyase pathway to promote their osteogenic differentiation. These levels must be carefully maintained as excessive or deficient H₂S levels temper the observed osteogenic effect by inhibiting Wnt/β-catenin signaling.ConclusionsThese results demonstrate that optimal concentrations of endogenous H₂S must be maintained within PDLCs to promote osteogenic differentiation by activating the Wnt/β-catenin signaling cascade.     

Lactoferrin inhibits infection-related osteoclastogenesis without interrupting compressive force-related osteoclastogenesis

BackgroundControl of periodontal tissue inflammation during orthodontic treatment is very important in achieving a favourable therapeutic goal. We previously demonstrated that orally applied bovine lactoferrin (bLF) inhibited LPS-induced bone resorption but not orthodontic force-induced tooth movement in vivo. This study is designed to examine the underlying mechanism of it.MethodsWe examined the inhibitory effects of bLF on the expression of RANKL, OPG, TNF-α and COX-2 in osteoblasts loaded with compressive stress (CS) in comparison with LPS stimulated osteoblasts. Formation of osteoclasts was evaluated by co-culture system.ResultsBoth CS- and LPS-applications upregulated COX-2 and RANKL but downregulated OPG. TNF-α was upregulated in LPS-stimulated osteoblasts but downregulated in CS-loaded osteoblasts. NS398 (a specific inhibitor of COX-2) significantly inhibited CS-induced RANKL-upregulation but not LPS-induced RANKL upregulation, indicating a critical role of COX-2/PGE₂ pathway in CS-induced osteoclastogenesis. bLF significantly downregulated LPS-induced upregulation of RANKL and eliminated OPG suppression but not affected in CS-induced changes. Moreover, bLF significantly decreased LPS-induced osteoclast formation, whereas bLF had no effect on PGE₂-induced osteoclast formation.ConclusionsbLF can effectively suppress harmful bone destruction associated with periodontitis without inhibiting bone remodelling by CS-loading. Therefore, oral administration of bLF may be highly beneficial for control of periodontitis in orthodontic patients.     

富血小板纤维蛋白对体外培养的成骨细胞生物学特性的影响

目的探讨富血小板纤维蛋白（platelet-rich fibrin, PRF）对成骨细胞生物学特性的影响。方法体外培养成骨细胞MG63,实验组采用PRF,对照组为正常高糖DMEM培养液。以MTT法检测细胞增殖,碱性磷酸酶（ALP）染色法检测ALP活性,免疫组织化学法检测Ⅰ型胶原、骨保护素(OPG)及其配体(RANKL)的表达。采用SPSS17.0软件包对数据进行统计学分析。结果PRF能够促进细胞增殖、ALP分泌、Ⅰ型胶原和骨保护素的表达,对骨保护素配体的表达影响不明显。结论PRF能够促进成骨细胞增殖、分化及骨保护素的表达,PRF可能通过对骨保护素及其配体的调节,参与骨的重建过程。     

Comparison of the properties of human CD146+ and CD146− periodontal ligament cells in response to stimulation with tumour necrosis factor α

ObjectivesPeriodontal ligament stem cells (PDLSCs) can be used in periodontal regeneration. Tumour necrosis factor-alpha (TNF-α) participates in the regulation of cell proliferation, apoptosis, differentiation, and migration. However, whether TNF-α can affect the biological features of PDLSCs is still unclear. The objective of this study was to illustrate the biological effects (proliferation, apoptosis, osteogenesis and migration) of TNF-α on human CD146 positive periodontal ligament cells (CD146+PLDCs) and CD146 negative periodontal ligament cells (CD146?PDLCs).MethodsCD146 ± PDLCs were isolated from human PDLCs and analyzed using a fluorescence-activated cell sorter. The biological effects of TNF-α on CD146 ± PDLCs were evaluated by CCK-8 assay (proliferation), DAPI staining (apoptosis), alizarin red staining and alkaline phosphatase activities assay (osteogenesis), and wounding assay and transwell assay (migration).ResultsCD146+PDLCs, which expressed MSC surface markers CD105, CD90, CD73, CD44, and Stro-1, showed higher proliferative and osteogenic potential than CD146?PDLCs. TNF-α at a dose of 2.5 ng/ml was found to enhance both proliferation and osteogenesis in CD146+PDLCs. At 5 ng/ml, TNF-α promoted proliferation, osteogenesis, and apoptosis in CD146+PDLCs and enhanced osteogenesis in CD146?PDLCs. At 10 ng/ml, TNF-α only aggravated apoptosis in CD146+PDLCs. The migratory ability of both CD146+PDLCs and CD146?PDLCs was not altered by TNF-α.ConclusionsCD146+PDLCs were subpopulation of MSC. It showed greater proliferative and osteogenic potential than CD146?PDLCs. At low concentration, TNF-α was beneficial to CD146+PDLCs on proliferation and osteogenesis, and at high concentration it was detrimental. CD146?PDLCs were found to be less sensitive to TNF-α.     

Effect of hydrogen sulphide on the expression of osteoprotegerin and receptor activator of NF-κB ligand in human periodontal ligament cells induced by tension-force stimulation

ObjectiveHydrogen sulphide (H₂S) is an endogenous gaseous signalling molecule, the generation rate of which is affected by mechanical force in cells. Recently, it was reported that mechanical force plays an important role in some pathways such as osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) in human periodontal ligament cells (hPDLCs). Here, we investigated whether H₂S played a regulatory role within the periodontal remodelling process by addressing the expression level of OPG/RANKL in hPDLCs.DesignhPDLCs were first applied with tension force for 0–120 min to select the optimal time for force application. These cells were treated with H₂S for 24 h followed by stimulation with tension-force application. Cell proliferation and apoptosis were assessed by the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry analysis. For OPG, RANKL and cystathionine-γ-lyase (CSE), real-time polymerase chain reaction (PCR) was used to analyse the messenger RNA (mRNA) expression; enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of OPG and soluble RANKL (sRANKL).ResultsTension force promoted the mRNA expression of CSE and the optimal application time was 60 min. The expression of OPG was increased in a concentration-dependent manner by H₂S treatment. Importantly, the relative OPG/RANKL expression ratio was significantly increased upon induction by H₂S, an effect that was enhanced by tension-force application.ConclusionsH₂S could promote osteogenic differentiation by regulating the relative OPG/RANKL expression ratio of hPDLCs, which is enhanced by tension force. These findings may be valuable for understanding the mechanism of H₂S in the periodontal remodelling, especially in the process of tooth movement.     

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。