骨形成蛋白—2和成纤维细胞生长因子对人牙周膜细胞增殖的联合效应

目的：探讨重组人骨形成蛋白－2（rhBMP-2)和碱性成纤维细胞生长因子(bFGF) 单独或联合作用对人牙周膜细胞（PDLCs)增殖的影响。方法：体外培养人PDLCs,分别用不同浓度的rhBMP-2和bFGF单独或联合作用,用四唑盐比色法（MTT法）进行观察。结果：rhBMP-2和bFGF单独作用后PDLCs的增殖较对照组有明显的升高;而rhBMP-2与bFGF联合作用后PDLCs的增殖较各自单独作用有更明显的升高（P＜0．05）。结论：rhBMP-2与bFGF联合应用对于促进人PDLCs的增殖具有协同作用。     

Effect of prostaglandin E2 on recombinant human bone morphogenetic protein-2-stimulated osteoblastic differentiation in human periodontal ligament cells

Recombinant human (rh) bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic differentiation in cells isolated from human periodontal ligament (HPLC), and this action of rhBMP-2 may be modulated by prostaglandins (PGs), which are local regulatory factors in the bone metabolism. In the present study, we investigated the effect of prostaglandin E2 (PGE2) on rhBMP-2-stimulated osteoblastic differentiation in cultured HPLC. rhBMP-2 (500 ng/ml)-stimulated alkaline phosphatase (ALPase) activity was enhanced by simultaneous treatment with low concentrations (10(-10)-10(-8) M) of PGE2, whereas a high concentration (10(-6) M) of PGE2 suppressed it. rhBMP-2 did not induce cyclo-oxygenase-2 (COX-2) mRNA expression or subsequent PGE2 production, whereas it remarkably suppressed rhIL-1 beta-induced COX-2 mRNA expression and PGE2 production. The rhBMP-2 action on osteoblastic differentiation in HPLC was also enhanced by co-treatment with 0.25 to 25 ng/ml of rh interleukin-1 beta (IL-1 beta). The ALPase activity stimulated by simultaneous treatment with rhBMP-2 and rhIL-1 beta was partially inhibited by addition of 10(-6) M of indomethacin, which completely inhibited rhIL-1 beta-induced PGE2 production. These results reveal that PGE2 at different concentrations exerts a biphasic effect on BMP-2-stimulated osteoblastic differentiation in HPLC, BMP-2 inhibits IL-1 beta-induced PGE2 production through suppressing COX-2 expression, and the BMP-2-stimulated osteoblastic differentiation may be enhanced by the endogenous PGE2 induced by BMP-2 and IL-1 beta. These suggest that BMP-2 action on osteoblastic differentiation in HPLC may be modulated by PGE2 in autocrine and paracrine fashions.     

人牙周膜细胞中内源性骨形成蛋白的流式细胞仪分析

目的：定量检测和分析人牙周膜细胞（ＰＤＬＣ）表达内源性骨形成蛋白（ＢＭＰ）的情况。方法：应用ＢＭＰ单克隆抗体，通过流式细胞仪和免疫组化ＡＢＣ的方法双重判定。结果：在离体培养的人ＰＤＬＣ中有一半左右的细胞能表达ＢＭＰ。结论：牙周膜细胞具有一定的合成和分泌ＢＭＰ的能力；可以进一步认为人的牙周膜细胞是具有成骨潜能的，在牙周组织再生中有积极作用。     

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.     

Subculture affects the phenotypic expression of human periodontal ligament cells and their response to fibroblast growth factor-2 and bone morphogenetic protein-7 in vitro

Background and Objective: Although periodontal ligament cells display several osteoblastic traits, their phenotypic expression is still not well established. It remains a matter of debate whether they resemble a terminally differentiated cell type or an intermediate maturation state that potentially can be directed towards a fibroblastic or an osteoblastic phenotype. Material and Methods: To explore the characteristics of periodontal ligament cells in greater detail, fourth‐passage, sixth‐passage and eighth‐passage human periodontal ligament cells were cultured for up to 3 wk. Ki‐67, alkaline phosphatase, osteocalcin, osteoprotegerin and receptor activator of nuclear factor‐κB ligand (RANKL) mRNA expression was quantified by real‐time polymerase chain reaction. Furthermore, the cellular response to fibroblast growth factor‐2 and bone morphogenetic protein‐7 was examined in first‐passage and fourth‐passage cells. Dermal fibroblasts (1BR.3.G) and osteoblast‐like cells (MG63) served as reference cell lines. Results: Proliferation decreased over time and was highest in fourth‐passage cells. The expression of differentiation parameters, osteoprotegerin and RANKL increased with culture time and was higher in fourth‐passage cells than in cells of later passages. The RANKL/osteoprotegerin ratio increased steadily until day 21. Administration of fibroblast growth factor‐2 enhanced cell numbers in both passages, whereas alkaline phosphatase and osteocalcin production remained unchanged. By contrast, exposure of periodontal ligament cells to bone morphogenetic protein‐7 resulted in a reduction of cell number in the first and fourth passages, whereas the production of alkaline phosphatase and osteocalcin was enhanced. In dermal fibroblasts, differentiation parameters did not respond to both stimuli. MG63 cells behaved similarly to periodontal ligament cells. Conclusion: These results indicate that subculture affects the phenotypic expression of human periodontal ligament cells with respect to the characteristics that these cells share with osteoblasts. Furthermore, the periodontal ligament cell phenotype can be altered by fibroblastic and osteoblastic growth factors.     

Interactions of regenerative, inflammatory and biomechanical signals on bone morphogenetic protein-2 in periodontal ligament cells

Nokhbehsaim M, Deschner B, Winter J, Bourauel C, Rath B, Jäger A, Jepsen S, Deschner J. Interactions of regenerative, inflammatory and biomechanical signals on bone morphogenetic protein‐2 in periodontal ligament cells. J Periodont Res 2011; 46: 374–381.© 2011 John Wiley & Sons A/S Background and Objective: Regeneration of periodontal tissues by EMD remains a major challenge because a number of modifying factors are as yet unknown. The effects of EMD seem to be mediated, at least in part, by bone morphogenetic protein‐2 (BMP‐2). This in vitro study was performed to examine whether the effects of EMD on BMP‐2 activity are modulated by inflammatory and/or biomechanical signals. Material and Methods: Periodontal ligament cells were seeded on BioFlex^® plates and exposed to EMD under normal, inflammatory or biomechanical loading conditions for 1 and 6 d. In order to mimic proinflammatory or biomechanical loading conditions in vitro, cells were stimulated with interleukin‐1β (IL‐1β), which is increased at inflamed periodontal sites, and cyclic tensile strain of various magnitudes, respectively. The synthesis of BMP‐2, its receptors (BMPR‐1A, BMPR‐1B and BMPR‐2) and its inhibitors (follistatin, matrix gla protein and noggin) were analyzed using real‐time RT‐PCR and ELISA. Results: In EMD‐treated cells, BMP‐2 synthesis was increased significantly at 1 d. EMD also induced the expression of all BMP receptors, and of the BMP inhibitors follistatin and noggin. In general, IL‐1β and biomechanical loading neither down‐regulated BMP‐2 nor up‐regulated BMP inhibitors in EMD‐stimulated cells. However, IL‐1β and biomechanical loading, when applied for a longer time period, caused a down‐regulation of EMD‐induced BMP receptors. Conclusion: EMD induces not only BMP‐2, but also its receptors and inhibitors, in PDL cells. IL‐1β and biomechanical forces may counteract the beneficial effects of EMD on BMP‐2 activity via the down‐regulation of BMP receptors.     

骨形成蛋白-2和碱性成纤维细胞生长因子对牙周膜细胞碱性磷酸酶活性的联合效应

目的 :了解重组人骨形成蛋白 - 2 (rhBMP - 2 )和碱性成纤维细胞生长因子 (bFGF)单独和联合作用对人牙周膜细胞 (PDLC)碱性磷酸酶 (ALP)活性的影响。方法 :体外培养人PDLC ,分别用不同浓度的rhBMP- 2和bFGF单独或联合作用 ,用酶动力学方法检测PDLC的ALP活性。结果 :5 0～ 2 0 0 μg/L浓度的rhBMP - 2可显著增强人PDLC的ALP活性 (P <0 .0 1) ,而 10 μg/L浓度的bFGF可显著抑制人PDLC的ALP活性(P <0 .0 1) ,rhBMP - 2和bFGF联合作用仍可较明显地增强人PDLC的ALP活性 (P <0 .0 5 )。结论 :rhBMP - 2和bFGF联合应用可增强人PDLC的ALP活性     

胰岛素样生长因子-Ⅰ和骨形态发生蛋白-2对人牙周膜细胞增殖的作用

目的：重组人胰岛素样生长因子－Ｉ（ｒｈＩＧＦ－Ｉ）、重组人骨形态发生蛋白－２（ｒｈＢＭＰ－２）分别或联合应用对人牙周膜（ＰＤＬ）细胞增殖的影响。方法：采用组织块法体外培养人ＰＤＬ细胞,ＭＴＴ法测定ＰＤＬ细胞在不同生长因子刺激下的增殖情况。结果：ｒｈＩＧＦ－Ｉ、ｒｈＢＭＰ－２都可促进人ＰＤＬ细胞的增殖,这种促增殖作用呈一定的浓度依赖性,ｒｈＩＧＦ－Ｉ与ｒｈＢＭＰ－２联合应用对人ＰＤＬ细胞的增殖有协同作用,且与单独应用相比相差显著。结论：ｒｈＩＧＦ－Ｉ、ｒｈＢＭＰ－２可望作为牙周再生的生物活性介质,ｒｈＩＧＦ－Ｉ与ｒｈＢＭＰ－２联合应用对ＰＤＬ细胞的促增殖作用更强。     

IGF-1对骨髓基质细胞分泌BMP-2和VEGF的影响

目的 :研究不同浓度的胰岛素样生长因子 1(IGF -1)对骨髓基质细胞 (MSC)分泌骨形成蛋白 -2(BMP -2 )和血管内皮细胞生长因子 (VEGF)的影响。方法 :培养大鼠骨髓基质细胞 ,对第三代细胞分别用 5 0ng/ml,2 5ng/ml ,12 .5ng/ml,6.2 5ng/ml,3 .12 5ng/ml的IGF -1进行培养 ,以正常培养的MSC为对照组 ,分别于1、3、5、7d对MSC进行VEGF或BMP -2免疫组化观察。通过图像分析观测MSC分泌BMP -2和VEGF的变化。结果 :IGF -1对BMP -2的分泌有一定的促进作用。IGF -1可以明显促进MSC表达VEGF ,以 5 0ng/ml合成作用最明显 ,IGF -1促进MSC分泌VEGF和BMP -2具有时间及浓度依赖性。结论 :IGF -1可明显地促进MSC分泌BMP -2和VEGF。     

人牙周膜干细胞的分离鉴定及骨形态发生蛋白-2对其趋化效应的研究

目的研究人牙周膜干细胞（PDLSCs）对骨形态发生蛋白-2（BMP-2）的趋化反应。方法通过有限稀释法分离、培养人PDLSCs,利用免疫荧光染色检测人PDLSCs波形丝蛋白及干细胞表面标志物STRO-1的表达,检测人PDLSCs多向分化能力,通过克隆形成实验和5-溴-2-脱氧尿嘧啶核苷（BrdU）共培养的方法检测其干细胞特性。利用24孔的Transwell细胞培养室来检测人PDLSCs对BMP-2的趋化反应,光镜下计迁移至滤膜下侧面的不同视野的细胞数。结果人PDLSCs抗波形丝蛋白染色阳性,表达干细胞表面标志物STRO-1,体外诱导培养的人PDLSCs能够向成骨细胞和成脂细胞分化,具有较高的自我更新能力,并在体外呈克隆状生长。在100、200 ng·mL-1 BMP-2实验组,Transwell细胞培养室中迁移的细胞数目显著多于空白对照组（P<0.01）。结论BMP-2对人PDLSCs有趋化效应。     

Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein-2 on periodontal regeneration

Song D‐S, Park J‐C, Jung I‐H, Choi S‐H, Cho K‐S, Kim C‐K, Kim C‐S. Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein‐2 on periodontal regeneration. J Periodont Res 2011; 46: 193–203. © 2010 John Wiley & Sons A/S Background and Objective: Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP‐2 on the in vitro and in vivo biologic activity of well‐characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP‐2. Material and Methods: hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP‐2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8‐wk healing period. The effects of rhBMP‐2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP‐2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. Results: In the present study, rhBMP‐2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down‐regulated following treatment with rhBMP‐2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. Conclusion: In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP‐2 on cementum and PDL tissue regeneration by hPDLSCs.     

rhBMP-2对人牙周膜细胞骨桥蛋白表达的影响

目的　深入了解牙周膜细胞 (periodontalligamentcells,PDLC)的成骨样细胞特性 ,以及非胶原蛋白在牙周组织矿化中的作用。方法　在体外培养条件下 ,观察重组人骨形成蛋白 2(recombinanthumanbonemorphogeneticprotein 2 ,rhBMP 2 )作用前后是否对矿化相关蛋白之一的骨桥蛋白 (osteopontin ,OPN)在人PDLC中的存在及表达情况产生影响。在小玻片上培养第 5代PDLC ,分为加rhBMP 2 (5 0 μg/L)刺激的实验组和不加任何因子的空白对照组 ,以地高辛标记的OPNcDNA探针 ,采用原位杂交技术对PDLC中OPN的表达情况进行检测 ;同时做PBS替代探针和RNA酶预处理的方法学对照。结果　对照组、替代对照和RNA酶预处理的PDLC均显示为阴性反应 ,没有显示出OPN的阳性表达信号 ;实验组PDLC的胞浆中可见明显的OPN阳性反应 ,表达信号较强。结论　rhBMP 2的刺激作用可使PDLC表达出较强的OPN阳性信号 ;表明在一定条件和外界因子的诱导下 ,PDLC具有向成骨特性方向转变的潜能 ,对牙周组织再生有促进效应。     

人骨形成蛋白－2对人牙髓细胞增殖和分化的影响

目的：探讨骨形成蛋白－2（BMP－2）对体外培养的人牙髓细胞（DPCs）增殖和分化的影响。方法：体外培养人DPCs,分别与不同浓度的BMP－2（50、100、200 ng／mL）共同培养;分别检测各组细胞的增殖情况、碱性磷酸酶（alkaline phosphatase,ALP）活性、钙化结节形成量以及牙本质涎磷蛋白（DSPP）、牙本质基质蛋白－1（DMP－1）各成牙本质相关基因的表达水平。结果：50～200 ng／mL 的BMP－2对DPCs的增殖均无明显促进作用;但是,能呈剂量依赖性地提高细胞的ALP活性、促进钙化结节的形成、上调DSPP和DMP－1 mRNA的表达水平,各浓度BMP－2组均高于对照组（P ＜0．05）。结论：BMP－2对体外培养的人DPCs增殖无明显影响,但可明显促进DPCs的成牙本质细胞方向分化。     

Isolation and characterization of multipotent human periodontal ligament stem cells

BACKGROUND: Periodontal ligament (PDL) repair is thought to involve mesenchymal progenitor cells capable of forming fibroblasts, osteoblasts and cementoblasts. However, full characterization of PDL stem cell (SC) populations has not been achieved. OBJECTIVE: To isolate and characterize PDLSC and assess their capability to differentiate into bone, cartilage and adipose tissue. METHODS: Human PDL cells were stained for STRO-1, FACS sorted and expanded in culture. Human bone marrow SC (BMSC) served as a positive control. PDLSC and BMSC were cultured using standard conditions conducive for osteogenic, chondrogenic and adipogenic differentiation. Osteogenic induction was assayed using alizarine red S staining and expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP). Adipogenic induction was assayed using Oil Red O staining and the expression of PPAR gamma 2 (early) and LPL (late) adipogenic markers. Chondrogenic induction was assayed by collagen type II expression and toluidine blue staining. RESULTS: Human PDL tissue contains about 27% STRO-1 positive cells with 3% strongly positive. In osteogenic cultures ALP was observed by day-7 in BMSC and day-14 in PDLSC. BSP expression was detectable by day-7; with more intense staining in PDLSC cultures. In adipogenic cultures both cell populations showed positive Oil Red O staining by day-25 with PPAR gamma 2 and LPL expression. By day-21, both BMSC and PDLSC chondrogenic induced cultures expressed collagen type II and glycosaminoglycans. CONCLUSIONS: The PDL contains SC that have the potential to differentiate into osteoblasts, chondrocytes and adipocytes, comparable with previously characterized BMSC. This adult PDLSC population can be utilized for potential therapeutic procedures related to PDL regeneration.     

碱性成纤维细胞生长因子对牙周膜细胞表皮生长因子受体基因表达的影响

目的研究碱性成纤维细胞生长因子（bFGF）对人牙周膜细胞（PDLC）表达表皮生长因子受体（EGFR）的影响,探讨bFGF在牙周组织分化再生中的意义。方法体外原代培养人PDLC,有限稀释法形成单细胞克隆,用外源性bFGF刺激单细胞克隆,采用逆转录聚合酶链反应（RT-PCR）检测克隆细胞内EGFR基因表达的变化。结果bFGF促进人PDLC内EGFR mRNA的合成,并且随着质量浓度的增加促进作用增强。结论bFGF对EGFR的促进作用很可能是牙周炎损伤修复过程中一个重要的调节因素,为牙周组织分化再生提供部分理论基础。