BMP2/7异源二聚体与全反式维甲酸在体外细胞成骨中的作用
摘要
骨缺损在临床上十分普遍,而由先天骨发育不良或发育畸形以及肿瘤、炎症、外伤等原因造成的大面积以及极限骨缺损仍是正颌外科、口腔颌面外科以及口腔种植科医生所面临的严峻挑战之一。骨形态发生蛋白(BMP)是一组具有高度保守结构的二聚体蛋白,属于转移生长因子(TGF-)超家族成员,在促进骨再生研究的领域中是主要的细胞因子之一。尤其是BMP2同源二聚体和BMP7同源二聚体,先后得到FDA认证,可用于脊椎融合术、修复骨缺损、加速骨联合等方面的辅助治疗。BMP2/7异源二聚体较BMP同源二聚体有更高效的诱导成骨作用,其诱导的细胞ALP活性以及体内成骨量是相应BMP同源二聚体的数倍至数十倍。同时,BMP同源二聚体在破骨发生中也发挥了显著作用,刺激和调控破骨细胞分化,刺激破骨细胞的活性,促进成熟破骨细胞的骨吸收功能等。相比与BMP2和BMP7, BMP2/7能在显著较低的浓度起效诱导细胞成骨发生,并在较低浓度范围内达到与BMP2和BMP7在较高浓度范围内所达到的作用效果峰值；并且BMP2/7在诱导细胞破骨发生过程中并不存在这一特点,即在低浓度范围内促进破骨发生的程度仍然较低,因而可获得更大的成骨净效应。近期的动物实验建立小型猪颅顶骨的极限种植体周围骨缺损模型,通过micro-CT的扫描检测和新生骨小梁的数量、厚度、距离以及新骨结构模型等参数分析,BMP2/7诱导的新生骨组织量显著多于BMP2和BMP7组,并且BMP2/7诱导的新生骨组织结构最接近于缺损周边正常骨组织。
此外,有文献报道全反式维甲酸(all-trans retinoic acid, ATRA)1能够上调多种细胞,包括成骨细胞、成纤维细胞、骨髓基质细胞、干细胞等的成骨相关基因的表达,促进基质矿化。同时,也有研究报道指出ATRA与BMP2联合应用能够协同诱导脂肪源细胞的成骨分化,促进颅骨成骨细胞和软骨细胞的成骨分化,以及通过细胞介导的组织工程学技术促进体内的成骨细胞募集和骨组织改建更新。但亦有报道指出ATRA和BMP协同抑制细胞成骨分化并促进细胞成脂分化。故本实验着重研究ATRA与低剂量BMP2/7对不同种类细胞成骨分化的影响,及二者联合应用能否对细胞成骨分化产生协同促进作用,进一步有效促进成骨分化和新骨形成。
第一部分：BMP2/7异源二聚体与全反式维甲酸对小鼠原成骨细胞MC3T3-E1的作用
目的：
BMP2/7异源二聚体较BMP同源二聚体有更高效的诱导成骨作用,其诱导的细胞ALP活性以及体内成骨量是相应BMP同源二聚体的数倍至数十倍。同时,BMP同源二聚体在破骨发生中也发挥了显著作用,刺激和调控破骨细胞分化,刺激破骨细胞的活性,促进成熟破骨细胞的骨吸收功能等。相比与BMP2和BMP7,BMP2/7能在显著较低的浓度起效诱导细胞成骨发生,并在较低浓度范围内达到与BMP2和BMP7在较高浓度范围内所达到的作用效果峰值；并且BMP2/7在诱导细胞破骨发生过程中并不存在这一特点,即在低浓度范围内促进破骨发生的程度仍然较低,因而可获得更大的成骨净效应。全反式维甲酸(all-trans retinoic acid, ATRA)能够上调多种细胞,包括成骨细胞、成纤维细胞、骨髓基质细胞、干细胞等的成骨相关基因的表达,促进基质矿化。同时,也有研究报道指出ATRA与BMP2联合应用能够协同诱导脂肪源细胞的成骨分化,促进颅骨成骨细胞和软骨细胞的成骨分化,以及通过细胞介导的组织工程学技术促进体内的成骨细胞募集和骨组织改建更新。但亦有报道指出ATRA和BMP协同抑制细胞成骨分化并促进细胞成脂分化。本研究旨在探讨rhBMP2/7异源二聚体和ATRA分别对小鼠原成骨细胞MC3T3-E1的作用,二者之间是否存在协同诱导小鼠原成骨细胞MC3T3-E1成骨的作用及其体外诱导成骨发生的具体生物学功能特点。
材料和方法：
以不同浓度的rhBMP2/7异源二聚体(5ng/ml,50ng/ml)及ATRA (luM)作用于小鼠原成骨细胞MC3T3-E1,并设立对照组,检测rhBMP2/7和ATRA及二者联合应用在诱导成骨前体细胞MC3T3-E1成骨发生过程中的作用,检测成骨发生各阶段的相关指标。用荧光定量法检测细胞增殖后的细胞个数；用比色法检测细胞成骨分化早期指标碱性磷酸酶(ALP)活性；用ELISA方法检测成骨分化晚期指标细胞骨钙素(OCN)的分泌量；用RT-PCR法检测细胞成骨相关基因(ALP、 OCN、Coll、Runx2)的表达水平；用茜素红染色观察并计量细胞矿化诱导培养后的细胞外基质矿化结节面积及吸光度。
结果：
细胞因子刺激培养1天时,仅单纯50ng/ml BMP2/7组细胞数明显增加,而其他5组并未发现明显的抑制或促进细胞增殖的作用。第4天时,单纯ATRA对细胞增殖具有抑制作用,但ATRA对细胞增殖的这种抑制作用,可以通过添加5ng/ml或50ng/ml BMP2/7予以补偿。而5ng/ml或50ng/ml BMP2/7单独作用下,细胞增殖均较其他组显著。但ATRA未明显影响细胞ALP活性,无论是否添加ATRA,BMP2/7在任一时间点均可明显促进细胞ALP活性表达,并且具有浓度依赖性。ATRA对细胞OCN表达的影响较其对细胞增殖的影响相似,在第4天和第7天ATRA均能明显抑制细胞OCN表达,第4天时,5ng/ml BMP2/7即可完全拮抗ATRA对OCN表达所产生的抑制作用,使而二者联合应用时OCN表达水平与空白对照组OCN表达水平相近。然而在第7天时,其对ATRA所产生的拮抗作用有所减弱。总之,ATRA在任一时间点均可抑制BMP2/引起的OCN的表达。茜素红染色结果显示,luM ATRA可以明显抑制细胞外基质矿化。5ng/ml BMP2/7+ATRA组细胞外基质矿化面积明显低于空白对照组,而50ng/ml BMP2/7则可完全拮抗ATRA对细胞外基质矿化所产生的的抑制作用,二者联合应用时细胞外基质矿化面积约为空白对照组的2.5倍。单纯50ng/ml BMP2/7作用下细胞外基质矿化最为明显。基因研究结果显示在第一天,无论是否添加BMP2/7, ATRA均显著抑制Runx2基因的表达。然而,单纯ATRA则可在第4天和第7天时明显促进Runx2的基因表达。BMP2/7对Runx2基因表达的促进作用则表现为浓度依赖型。任一时间点时,ATRA均可明显抑制Ⅰ型胶原基因的表达,而5ng/ml BMP2/7在任一时间均可完全拮抗由ATRA产生的这种对Ⅰ型胶原基因表达的抑制作用。50ng/ml BMP2/7在第一天和第7天时亦可产生与5ng/ml BMP2/7相同的作用,并且在第四天时50ng/ml BMP2/7可明显促进Ⅰ型胶原基因的表达。与ALP活性表达不同的是,ATRA在任一时间点均抑制ALP基因的表达。5ng/ml和50ng/ml BMP2/7均能拮抗ATRA对细胞ALP基因所产生的抑制作用,并促进细胞ALP基因的表达。单纯BMP2/7对细胞OCN基因表达水平则具有明显促进作用,且具有一定的浓度依赖性,但在第4天和第7天ATRA均能明显抑制BMP2/7所诱导的OCN基因表达。任一时间点,单纯(?)ATRA均可明显抑制OCN基因表达,5ng/ml BMP2/7即可完全拮抗由ATRA所产生的抑制作用,而50ng/ml BMP2/7不仅可以完全拮抗ATRA所产生的拮抗作用,而且可明显促进细胞OCN基因的表达。无论是否添加ATRA, BMP2/7对细胞OCN基因表达的促进作用均呈现时间和浓度依赖性。而第4天和第7天时,ATRA均能明显抑制由5或50ng/ml BMP2/7所引起的OCN基因的表达。
结论：
ATRA抑制小鼠原成骨细胞MC3T3-E1的成骨分化,BMP2/7促进小鼠原成骨细胞MC3T3-E1的成骨分化,并呈现浓度依赖性；ATRA与BMP2/7间并不存在协同诱导小鼠原成骨细胞MC3T3-E1成骨分化的作用,而BMP2/7可拮抗ATRA对细胞成骨分化所产生的抑制作用并明显促进细胞成骨分化。
第二部分：BMP2/7异源二聚体与全反式维甲酸对SD大鼠BMSC成骨分化的作用
目的：
BMP2/7异源二聚体较BMP同源二聚体有更高效的诱导成骨作用,其诱导的细胞ALP活性以及体内成骨量是相应BMP同源二聚体的数倍至数十倍。同时,BMP同源二聚体在破骨发生中也发挥了显著作用,刺激和调控破骨细胞分化,刺激破骨细胞的活性,促进成熟破骨细胞的骨吸收功能等。相比与BMP2和BMP7, BMP2/7能在显著较低的浓度起效诱导细胞成骨发生,并在较低浓度范围内达到与BMP2和BMP7在较高浓度范围内所达到的作用效果峰值；并且BMP2/7在诱导细胞破骨发生过程中并不存在这一特点,即在低浓度范围内促进破骨发生的程度仍然较低,因而可获得更大的成骨净效应。全反式维甲酸(all-trans retinoic acid, ATRA)能够上调多种细胞,包括成骨细胞、成纤维细胞、骨髓基质细胞、干细胞等的成骨相关基因的表达,促进基质矿化。同时,也有研究报道指出ATRA与BMP2联合应用能够协同诱导脂肪源细胞的成骨分化,促进颅骨成骨细胞和软骨细胞的成骨分化,以及通过细胞介导的组织工程学技术促进体内的成骨细胞募集和骨组织改建更新。本研究旨在探讨rhBMP2/7异源二聚体和ATRA诱导大鼠原代BMSC成骨分化的作用,二者之间是否存在协同诱导小大鼠原代BMSC成骨分化作用及其体外诱导成骨发生的具体生物学功能特点。
材料和方法：
获取SD大鼠胫骨及股骨原代BMSCs,流失细胞仪鉴定细胞纯度后以不同浓度的rhBMP2/7异源二聚体(5ng/ml,50ng/ml)及ATRA (luM)作用于大鼠原代BMSC成骨分化,并设立对照组,检测rhBMP2/7和ATRA及二者联合应用在诱导大鼠原代BMSC成骨发生过程中的作用,检测成骨发生各阶段的相关指标。用荧光定量法检测细胞增殖后的各组DNA含量；用比色法检测细胞成骨分化早期指标碱性磷酸酶(ALP)活性；用ELISA方法检测成骨分化晚期指标细胞骨钙素(OCN)的分泌量；用实时荧光定量RT-PCR法检测细胞成骨相关基因(ALP、OCN、Runx2)的表达水平；用茜素红染色观察并计量细胞矿化诱导培养后的细胞外基质矿化结节面积。
结果：
单纯ATRA作用下,细胞增殖明显被抑制,第七天DNA含量仅较第四天略有提升,而BMP2/7可以对抗ATRA对细胞增殖产生的抑制作用,在第四天,ATRA联合应用5ng/ml及50ng/ml的BMP2/7较单独应用ATRA,均可以明显促进细胞增殖,而在第七天时,与单独应用ATRA相比,ATRA与50ng/ml BMP2/7联合应用亦使DNA含量明显增高。不同于ATRA和BMP2/7对细胞增殖的影响,ATRA和BMP2/7均能明显促进BMSC在第四天及第七天ALP活性表达。任一时间点,ATRA与BMP2/7(5ng/ml,50ng/ml)联合作用下,均可明显促进细胞ALP的活性表达。第七天,ATRA与50ng/ml协同促进BMSC ALP活性表达水平则达到空白组ALP活性表达水平的7.01倍。BMP2/7对细胞OCN水平具有促进作用,并随BMP2/7浓度的增加而增加。然而,任一时间点,单纯ATRA作用时明显抑制了OCN的表达。在ATRA作用下,BMP2/7对OCN表达水平的作用则表现为浓度和时间依赖性。细胞因子刺激培养14天后,仅BMP2/750ng/ml组出现基质矿化,细胞因子刺激培养21天后,空白对照组及单纯ATRA作用组仅可检测到极少量的钙盐沉积,可见单纯ATRA对BMSC细胞矿化仅有极小的影响。BMP2/7含或不含ATRA均能明显促进细胞基质矿化,并且细胞外基质矿化面积随BMP2/7浓度的增加而增加。ATRA明显下调5ng/ml BMP2/7所诱导的细胞外基质矿化,但这种抑制作用在50ng/ml BMP2/7时则并不存在。单纯5ng/ml BMP2/7或者ATRA刺激培养7天后才能明显促进Runx2基因的表达。50ng/ml BMP2/7在细胞因子刺激培养4天和7天时均能明显促进Runx2基因的表达,与单纯ATRA组及5ng/ml BMP2/7+ATRA组相比,50ng/ml BMP2/7+ATRA组则明显促进Runx2基因的表达。与ALP活性表达不同的是,在第4天时,ATRA并没有明显影响ALP基因的表达,在第7天时,ATRA对ALP基因的表达表现出明显的抑制作用。单纯5ng/ml BMP2/7和50ng/ml BMP2/7则在细胞因子刺激培养第7天时表现为促进作用。细胞因子刺激培养第4天时,与空白对照组相比,ATRA和任一浓度的BMP2/7共同作用均能明显促进ALP基因的表达。与第4天相比,ATRA和50ng/ml BMP2/7在第7天时明显促进细胞ALP基因表达。细胞因子刺激培养7天时,空白对照组及单纯BMP2/7作用组的细胞OCN基因表达水平较4天时明显上升,在第7天时,任何BMP2/7浓度(Ong/ml,5ng/ml,50ng/ml)下ATRA均能明显抑制细胞OCN基因表达水平,而这种抑制现在在细胞因子刺激培养4天时并未出现。
结论：
BMP2/7促进SD大鼠BMSCs成骨分化,并呈现浓度依赖性。BMP2/7和ATRA协同诱导SD大鼠BMSCs成骨早期分化,促进ALP活性表达,但ATRA抑制BMSC中晚期成骨分化,抑制细胞外基质矿化,而BMP2/7促进BMSC成骨分化,促进细胞外基质矿化,并呈浓度依赖性,ATRA可抑制低浓度BMP2/7诱导细胞成骨分化的能力,而相对高浓度BMP2/7诱导细胞成骨分化的能力不受ATRA影响。
Sufficient bone volume and adequate bone quality are common phenomenon in clinical, and large and limit bone defect which caused by congenital osteomyelodysplasia、tumor,phlegmonosis and injury are still large challenge to orthognathic surgery, oral and maxillofacial surgery and Oral implant technology doctors. Bone morphogenetic proteins(BMPs) a group of dimeric disulfide-linked polypeptide growth factors under transforming growth factor-β superfamily, are one of the paramount cytokines in promoting bone regeneration. BMP2and BMP7homodimer which can be used to spondylodesis, repair bone defect and accelerate bone joint. Compared with BMP homodimer, BMP2/7heterodimer is more effective to induce bone regeneration, the ALP activity and bone formation content in vivo induced by BMP2/7heterodimer are several times or dozens of times than that induce by BMP homodimer. BMP homodimer also take an important function in osteoclastogenesis, it stimulate and regulate osteoclastogenesis, regulate the activity of osteoclast and promote mature osteoclast bone resorption. Compared with BMP2and BMP7, rhBMP2/7heterodimer induced cell differentiation with significantly lower threshold concentrations but similar maximum effects. And this phenomenon was not occure in the process of osteoclastogenesis, and low concentration of BMP2/7can only induce weak osteoclastogenesis, so more bone can be reached. Animal experiment also test and verified this result, and the new bone induced by BMP2/7are more close to normal bone.
All-trans retinoic acid can induce osteoblastic gene expression in osteoblast, stem cells, fibroblast and bone marrow stromal cells, promote matrix mineralization. The effect of BMPs and ATRA is still controversial in inducing osteogenic differentiation. One opinion is that ATRA and the BMP signing pathway cooperate to inhibit osteogenesis and promote adipogenesis of mouse embryonic palate mesenchymal cells, while others found that retinoic acid cooperates with BMP2to induce osteoblastic differentiation of C2C12cells. In this study, we investigated the respective and the combinatory effects of ATRA and BMP2/7on the in vitro osteogenesis of different cell types.
Part1:The effect of rhBMP2/7and ATRA on the osteogenic differentiation of MC3T3-E1cells
Objectives:
Compared with BMP homodimer, BMP2/7heterodimer is more effective to induce bone regeneration, the ALP activity and bone formation content in vivo induced by BMP2/7heterodimer are several times or dozens of times than that induce by BMP homodimer. BMP homodimer also take an important function in osteoclastogenesis, it stimulate and regulate osteoclastogenesis, regulate the activity of osteoclast and promote mature osteoclast bone resorption. Compared with BMP2and BMP7, rhBMP2/7heterodimer induced cell differentiation with significantly lower threshold concentrations but similar maximum effects. And this phenomenon was not occure in the process of osteoclastogenesis, and low concentration of BMP2/7can only induce weak osteoclastogenesis, so more bone can be reached. All-trans retinoic acid can induce osteoblastic gene expression in osteoblast, stem cells, fibroblast and bone marrow stromal cells, promote matrix mineralization. The effect of BMPs and ATRA is still controversial in inducing osteogenic differentiation. One opinion is that ATRA and the BMP signing pathway cooperate to inhibit osteogenesis and promote adipogenesis of mouse embryonic palate mesenchymal cells, while others found that retinoic acid cooperates with BMP2to induce osteoblastic differentiation of C2C12cells. In this study, we investigated the respective and the combinatory effects of ATRA and BMP2/7on the in vitro osteogenesis of MC3T3-E1cells.
Materials and Methods:
The MC3T3-E1cells were dealed with different concentration of BMP2/7heterodimer and1uM ATRA, then we focused on the effect of BMP2/7heterodimer and ATRA on the osteogenic differentiation of MC3T3-E1cells, and tested the figures in different stages of differentiation. We use quantitative fluorescence method to determine the cell numbers; use the colorimetric method to test the ALP activity as an early indicators of osteogenesis of the cells; use the ELISA method to test the OCN concentration which secreted to the medium as late indicators of osteogenesis; use the Real time fluorescent quantitative method(RT-PCR) to test the expression of osteogenic genes such as ALP, OCN, Coll, Runx2gene; use the alizarin red staining method to observe and measure the calcified area.
Results:
On the1st day, a significant increase in the cell numbers was detected only under the treatment of no ATRA,50ng/ml BMP2/7. Neither significant enhancement nor inhibition in cell proliferation was detected in the other groups. On the4th day, ATRA alone significantly decreased the cell numbers, which could be completely restored by 5ng/ml or50ng/ml BMP2/7. In contrast,5ng/ml or50ng/ml BMP2/7alone could result in a significantly higher cell number than the other treatments. In contrast to the inhibitory effect of ATRA on cell proliferation, ATRA alone didn't significantly influence the ALP activity. BMP2/7significantly increased the ALP activity in a dose-dependent manner irrespective of ATRA at both time points. The effect of ATRA on OCN expression is similar to its effects on cell proliferation, ATRA alone could significantly inhibit the OCN expression on the4th and7th day. On the4th day,5ng/ml BMP2/7antagonized the inhibitory effect of ATRA and completely restored the OCN expression. However, on the7th day5ng/ml BMP2/7only restored a part of OCN expression. Albeit so, the BMP2/7induced OCN expression was significantly inhibited by ATRA at both time points. According to the result of alizarin red staining, we found that luM ATRA could significantly inhibit the matrix mineralization. The mineralization area in the group of1uM ATRA,5ng/ml BMP2/7was significantly lower than that in the control group(no ATRA, no BMP2/7). In contrast,50ng/ml BMP2/7could antagonize ATRA and significantly enhance the mineralization about2.5folds in comparison with the control group(no ATRA, no BMP2/7).50ng/ml BMP2/7alone resulted in the highest mineralization. On the lth day, Runx2expression was significantly suppressed by ATRA with or without BMP2/7. The expression of Runx2gene was, whereas, significantly enhanced by ATRA alone on the4th and7th day. BMP2/7significantly increased the expression of Runx2gene in a dose-dependent manner. The expression of Collagen la gene was significantly suppressed by ATRA alone at the three time points.5ng/ml BMP2/7could completely restore the expression of Collagen la gene that was inhibited by ATRA at all time points.50ng/ml BMP2/7could also restore the expression of Collagen Ia gene on the1st day and7th day, and significantly enhance it on the4th day. Different from the ALP activity, the expression of ALP gene was significantly suppressed by ATRA alone at all the three time points. In the presence of ATRA,5ng/ml and50ng/ml could significantly enhance ALP gene expression in comparison with the two groups without BMP2/7. The expression of OCN gene was also significant suppressed by ATRA alone at all the three time points. In the presence of ATRA,5ng/ml BMP2/7completely restored the expression of OCN gene, and50ng/ml could further significantly enhance the expression of OCN gene. BMP2/7could significantly enhance the gene expression of OCN in a time-course and dose-dependent manner irrespective of ATRA. At both time points, ATRA significantly inhibited the expression of OCN gene that was induced by5or50ng/ml BMP2/7.
Conclusions:
ATRA inhibited the osteogenic differentiation of MC3T3-E1cells, whereas BMP2/7induce the osteogenic differentiation of MC3T3-E1cells in a dose-dependent and time-course manner; ATRA and BMP2/7didn't cooperate to induce the osteogenic differentiation of MC3T3-E1cells, and BMP2/7could antagonize the inhibition of ATRA and promotes the osteoblastogenesis.
Part2:The effect of rhBMP2/7and ATRA on the osteogenic differentiation of bone marrow stromal cells
Objectives:
Compared with BMP homodimer, BMP2/7heterodimer is more effective to induce bone regeneration, the ALP activity and bone formation content in vivo induced by BMP2/7heterodimer are several times or dozens of times than that induce by BMP homodimer. BMP homodimer also take an important function in osteoclastogenesis, it stimulate and regulate osteoclastogenesis, regulate the activity of osteoclast and promote mature osteoclast bone resorption. Compared with BMP2and BMP7, rhBMP2/7heterodimer induced cell differentiation with significantly lower threshold concentrations but similar maximum effects. And this phenomenon was not occure in the process of osteoclastogenesis, and low concentration of BMP2/7can only induce weak osteoclastogenesis, so more bone can be reached. All-trans retinoic acid can induce osteoblastic gene expression in osteoblast, stem cells, fibroblast and bone marrow stromal cells, promote matrix mineralization. The effect of BMPs and ATRA is still controversial in inducing osteogenic differentiation. One opinion is that ATRA and the BMP signing pathway cooperate to inhibit osteogenesis and promote adipogenesis of mouse embryonic palate mesenchymal cells, while others found that retinoic acid cooperates with BMP2to induce osteoblastic differentiation of C2C12cells. In this study, we investigated the respective and the combinatory effects of ATRA and BMP2/7on the in vitro osteogenesis of rat bone marrow stromal cells.
Materials and Methods:
The rat bone marrow stromal cells were harvested from a4-week-old male Sprague-Dawley (SD) rats. We used flow cytometry to characterize the biomarkers of BMSC, then the cells were dealed with different concentration of BMP2/7heterodimer and luM ATRA, then we focused on the effect of BMP2/7heterodimer and ATRA on the osteogenic differentiation of rat bone marrow stromal cells, and tested the figures in different stages of differentiation. We use quantitative fluorescence method to determine the cell numbers; use the colorimetric method to test the ALP activity as an early indicators of osteogenesis of the cells; use the ELISA method to test the OCN concentration which secreted to the medium as late indicators of osteogenesis; use the Real time fluorescent quantitative method(RT-PCR) to test the expression of osteogenic genes such as ALP, OCN, Coll, Runx2gene; use the alizarin red staining method to observe and measure the calcified area.
Results:
ATRA alone could significantly inhibit the proliferation of BMSCs, resulting in only a significantly higher DNA amount from day4to day7. Interestingly, BMP2/7could antagonize the inhibitive effects of ATRA on cell proliferation. On day4, BMP2/7at both5ng/mL and50ng/mL resulted in a significantly higher DNA amount than ATRA alone. On day7, on50ng/mL BMP2/7was associated with a significantly higher DNA amount than ATRA alone. In comparison with the contrary effects of BMP2/7and ATRA on the proliferation, both cytokines significantly increased the ALP activity of BMSCs on day4and day7. At any time point, ATRA and BMP2/7(5ng/ml,50ng/ml) synergied to enhance the ALP expression of rat bone marrow stromal cells. ATRA and50ng/mL BMP2/7synergistically promoted (about7.01folds) in the ALP activity of BMSCs on both day4and day7. BMP2/7alone could significantly promote the late osteogenic differentiation marker--OCN in a dose-dependent manner on both day4and day7. In contrast, ATRA alone significantly inhibited OCN expression on both time points. In the presence of ATRA, the promoting effects of BMP2/7were in dose-dependent and time-dependent manners. After a14-day treatment, mineralization of BMSCs in cell matrix was only found in the presence50ng/mL BMP2/7. On day21, very mild calcium deposition could be detected in the two groups without BMP2/7. The influence of ATRA on the mineralization of BMSCs was very mild without the presence of BMP2/7. Regardless of ATRA, BMP2/7could significantly promote mineralization in a dose-dependent manner. ATRA could significantly downregulate the osteogenesis induced by5ng/mL BMP2/7. However, the inhibitive effect of ATRA was insignificant for50ng/mL BMP2/7.5ng/mL BMP2/7or ATRA only significantly enhanced the expression of Runx2gene only on day7. Without ATRA,50ng/mL BMP2/7could significantly enhance the expression ofRunx2gene on both day4(1.20-fold and1.22-fold) and day7(2.07-fold and1.61-fold) in comparison with0ng/mL BMP2/7and5ng/mL BMP2/7, respectively. Furthermore, ATRA and50ng/mL BMP2/7 significantly enhance the expression of Runx2gene on both day4(2.06-fold and2.23-fold) and day7(1.96-fold and2.02-fold) in comparison with0ng/mL BMP2/7and5ng/mL BMP2/7, respectively. Different from ALP activity, ATRA didn't significantly influence the expression of ALP gene on day4. Furthermore, ATRA significantly suppressed the expression of ALP gene on day7. Without ATRA,5ng/mL and50ng/mL BMP2/7enhanced the expression of ALP gene only on day7. On day4, ATRA and BMP2/7of both concentrations could significantly enhance the expression of ALP gene in comparison with the controls. On day7, ATRA and50ng/mL BMP further significantly increased the expression of the ALP gene in comparison with on day4. Without the presence of ATRA, a significant enhancement in the expression of OCN gene could be detected from day4to day7. A significant suppression of OCN gene expression by ATRA was detected on day7irrespective of BMP2/7, while such a suppressive effect of ATRA was not seen on day4.
Conclusions:
ATRA and BMP2/7synergistically induced the early osteogenesis of rat bone marrow stromal cells, and synergistically promoted the expression of ALP. But ATRA inhibited the late osteogenesis of rat bone marrow stromal cells and mineralization of BMSCs in cell matrix. BMP2/7promoted the osteogenesis of rat bone marrow stromal cells in a concentration-dependent manners. ATRA could inhibited the osteogensis induced by low concentration of BMP2/7, but it didn't influence the osteogenesis induced by high concentration of BMP2/7. In conclusion ATRA and BMP2/7showed both antagonistic and synergistic effects on the osteogenesis of BMSCs.
此外,有文献报道全反式维甲酸(all-trans retinoic acid, ATRA)1能够上调多种细胞,包括成骨细胞、成纤维细胞、骨髓基质细胞、干细胞等的成骨相关基因的表达,促进基质矿化。同时,也有研究报道指出ATRA与BMP2联合应用能够协同诱导脂肪源细胞的成骨分化,促进颅骨成骨细胞和软骨细胞的成骨分化,以及通过细胞介导的组织工程学技术促进体内的成骨细胞募集和骨组织改建更新。但亦有报道指出ATRA和BMP协同抑制细胞成骨分化并促进细胞成脂分化。故本实验着重研究ATRA与低剂量BMP2/7对不同种类细胞成骨分化的影响,及二者联合应用能否对细胞成骨分化产生协同促进作用,进一步有效促进成骨分化和新骨形成。
第一部分：BMP2/7异源二聚体与全反式维甲酸对小鼠原成骨细胞MC3T3-E1的作用
目的：
BMP2/7异源二聚体较BMP同源二聚体有更高效的诱导成骨作用,其诱导的细胞ALP活性以及体内成骨量是相应BMP同源二聚体的数倍至数十倍。同时,BMP同源二聚体在破骨发生中也发挥了显著作用,刺激和调控破骨细胞分化,刺激破骨细胞的活性,促进成熟破骨细胞的骨吸收功能等。相比与BMP2和BMP7,BMP2/7能在显著较低的浓度起效诱导细胞成骨发生,并在较低浓度范围内达到与BMP2和BMP7在较高浓度范围内所达到的作用效果峰值；并且BMP2/7在诱导细胞破骨发生过程中并不存在这一特点,即在低浓度范围内促进破骨发生的程度仍然较低,因而可获得更大的成骨净效应。全反式维甲酸(all-trans retinoic acid, ATRA)能够上调多种细胞,包括成骨细胞、成纤维细胞、骨髓基质细胞、干细胞等的成骨相关基因的表达,促进基质矿化。同时,也有研究报道指出ATRA与BMP2联合应用能够协同诱导脂肪源细胞的成骨分化,促进颅骨成骨细胞和软骨细胞的成骨分化,以及通过细胞介导的组织工程学技术促进体内的成骨细胞募集和骨组织改建更新。但亦有报道指出ATRA和BMP协同抑制细胞成骨分化并促进细胞成脂分化。本研究旨在探讨rhBMP2/7异源二聚体和ATRA分别对小鼠原成骨细胞MC3T3-E1的作用,二者之间是否存在协同诱导小鼠原成骨细胞MC3T3-E1成骨的作用及其体外诱导成骨发生的具体生物学功能特点。
材料和方法：
以不同浓度的rhBMP2/7异源二聚体(5ng/ml,50ng/ml)及ATRA (luM)作用于小鼠原成骨细胞MC3T3-E1,并设立对照组,检测rhBMP2/7和ATRA及二者联合应用在诱导成骨前体细胞MC3T3-E1成骨发生过程中的作用,检测成骨发生各阶段的相关指标。用荧光定量法检测细胞增殖后的细胞个数；用比色法检测细胞成骨分化早期指标碱性磷酸酶(ALP)活性；用ELISA方法检测成骨分化晚期指标细胞骨钙素(OCN)的分泌量；用RT-PCR法检测细胞成骨相关基因(ALP、 OCN、Coll、Runx2)的表达水平；用茜素红染色观察并计量细胞矿化诱导培养后的细胞外基质矿化结节面积及吸光度。
结果：
细胞因子刺激培养1天时,仅单纯50ng/ml BMP2/7组细胞数明显增加,而其他5组并未发现明显的抑制或促进细胞增殖的作用。第4天时,单纯ATRA对细胞增殖具有抑制作用,但ATRA对细胞增殖的这种抑制作用,可以通过添加5ng/ml或50ng/ml BMP2/7予以补偿。而5ng/ml或50ng/ml BMP2/7单独作用下,细胞增殖均较其他组显著。但ATRA未明显影响细胞ALP活性,无论是否添加ATRA,BMP2/7在任一时间点均可明显促进细胞ALP活性表达,并且具有浓度依赖性。ATRA对细胞OCN表达的影响较其对细胞增殖的影响相似,在第4天和第7天ATRA均能明显抑制细胞OCN表达,第4天时,5ng/ml BMP2/7即可完全拮抗ATRA对OCN表达所产生的抑制作用,使而二者联合应用时OCN表达水平与空白对照组OCN表达水平相近。然而在第7天时,其对ATRA所产生的拮抗作用有所减弱。总之,ATRA在任一时间点均可抑制BMP2/引起的OCN的表达。茜素红染色结果显示,luM ATRA可以明显抑制细胞外基质矿化。5ng/ml BMP2/7+ATRA组细胞外基质矿化面积明显低于空白对照组,而50ng/ml BMP2/7则可完全拮抗ATRA对细胞外基质矿化所产生的的抑制作用,二者联合应用时细胞外基质矿化面积约为空白对照组的2.5倍。单纯50ng/ml BMP2/7作用下细胞外基质矿化最为明显。基因研究结果显示在第一天,无论是否添加BMP2/7, ATRA均显著抑制Runx2基因的表达。然而,单纯ATRA则可在第4天和第7天时明显促进Runx2的基因表达。BMP2/7对Runx2基因表达的促进作用则表现为浓度依赖型。任一时间点时,ATRA均可明显抑制Ⅰ型胶原基因的表达,而5ng/ml BMP2/7在任一时间均可完全拮抗由ATRA产生的这种对Ⅰ型胶原基因表达的抑制作用。50ng/ml BMP2/7在第一天和第7天时亦可产生与5ng/ml BMP2/7相同的作用,并且在第四天时50ng/ml BMP2/7可明显促进Ⅰ型胶原基因的表达。与ALP活性表达不同的是,ATRA在任一时间点均抑制ALP基因的表达。5ng/ml和50ng/ml BMP2/7均能拮抗ATRA对细胞ALP基因所产生的抑制作用,并促进细胞ALP基因的表达。单纯BMP2/7对细胞OCN基因表达水平则具有明显促进作用,且具有一定的浓度依赖性,但在第4天和第7天ATRA均能明显抑制BMP2/7所诱导的OCN基因表达。任一时间点,单纯(?)ATRA均可明显抑制OCN基因表达,5ng/ml BMP2/7即可完全拮抗由ATRA所产生的抑制作用,而50ng/ml BMP2/7不仅可以完全拮抗ATRA所产生的拮抗作用,而且可明显促进细胞OCN基因的表达。无论是否添加ATRA, BMP2/7对细胞OCN基因表达的促进作用均呈现时间和浓度依赖性。而第4天和第7天时,ATRA均能明显抑制由5或50ng/ml BMP2/7所引起的OCN基因的表达。
结论：
ATRA抑制小鼠原成骨细胞MC3T3-E1的成骨分化,BMP2/7促进小鼠原成骨细胞MC3T3-E1的成骨分化,并呈现浓度依赖性；ATRA与BMP2/7间并不存在协同诱导小鼠原成骨细胞MC3T3-E1成骨分化的作用,而BMP2/7可拮抗ATRA对细胞成骨分化所产生的抑制作用并明显促进细胞成骨分化。
第二部分：BMP2/7异源二聚体与全反式维甲酸对SD大鼠BMSC成骨分化的作用
目的：
BMP2/7异源二聚体较BMP同源二聚体有更高效的诱导成骨作用,其诱导的细胞ALP活性以及体内成骨量是相应BMP同源二聚体的数倍至数十倍。同时,BMP同源二聚体在破骨发生中也发挥了显著作用,刺激和调控破骨细胞分化,刺激破骨细胞的活性,促进成熟破骨细胞的骨吸收功能等。相比与BMP2和BMP7, BMP2/7能在显著较低的浓度起效诱导细胞成骨发生,并在较低浓度范围内达到与BMP2和BMP7在较高浓度范围内所达到的作用效果峰值；并且BMP2/7在诱导细胞破骨发生过程中并不存在这一特点,即在低浓度范围内促进破骨发生的程度仍然较低,因而可获得更大的成骨净效应。全反式维甲酸(all-trans retinoic acid, ATRA)能够上调多种细胞,包括成骨细胞、成纤维细胞、骨髓基质细胞、干细胞等的成骨相关基因的表达,促进基质矿化。同时,也有研究报道指出ATRA与BMP2联合应用能够协同诱导脂肪源细胞的成骨分化,促进颅骨成骨细胞和软骨细胞的成骨分化,以及通过细胞介导的组织工程学技术促进体内的成骨细胞募集和骨组织改建更新。本研究旨在探讨rhBMP2/7异源二聚体和ATRA诱导大鼠原代BMSC成骨分化的作用,二者之间是否存在协同诱导小大鼠原代BMSC成骨分化作用及其体外诱导成骨发生的具体生物学功能特点。
材料和方法：
获取SD大鼠胫骨及股骨原代BMSCs,流失细胞仪鉴定细胞纯度后以不同浓度的rhBMP2/7异源二聚体(5ng/ml,50ng/ml)及ATRA (luM)作用于大鼠原代BMSC成骨分化,并设立对照组,检测rhBMP2/7和ATRA及二者联合应用在诱导大鼠原代BMSC成骨发生过程中的作用,检测成骨发生各阶段的相关指标。用荧光定量法检测细胞增殖后的各组DNA含量；用比色法检测细胞成骨分化早期指标碱性磷酸酶(ALP)活性；用ELISA方法检测成骨分化晚期指标细胞骨钙素(OCN)的分泌量；用实时荧光定量RT-PCR法检测细胞成骨相关基因(ALP、OCN、Runx2)的表达水平；用茜素红染色观察并计量细胞矿化诱导培养后的细胞外基质矿化结节面积。
结果：
单纯ATRA作用下,细胞增殖明显被抑制,第七天DNA含量仅较第四天略有提升,而BMP2/7可以对抗ATRA对细胞增殖产生的抑制作用,在第四天,ATRA联合应用5ng/ml及50ng/ml的BMP2/7较单独应用ATRA,均可以明显促进细胞增殖,而在第七天时,与单独应用ATRA相比,ATRA与50ng/ml BMP2/7联合应用亦使DNA含量明显增高。不同于ATRA和BMP2/7对细胞增殖的影响,ATRA和BMP2/7均能明显促进BMSC在第四天及第七天ALP活性表达。任一时间点,ATRA与BMP2/7(5ng/ml,50ng/ml)联合作用下,均可明显促进细胞ALP的活性表达。第七天,ATRA与50ng/ml协同促进BMSC ALP活性表达水平则达到空白组ALP活性表达水平的7.01倍。BMP2/7对细胞OCN水平具有促进作用,并随BMP2/7浓度的增加而增加。然而,任一时间点,单纯ATRA作用时明显抑制了OCN的表达。在ATRA作用下,BMP2/7对OCN表达水平的作用则表现为浓度和时间依赖性。细胞因子刺激培养14天后,仅BMP2/750ng/ml组出现基质矿化,细胞因子刺激培养21天后,空白对照组及单纯ATRA作用组仅可检测到极少量的钙盐沉积,可见单纯ATRA对BMSC细胞矿化仅有极小的影响。BMP2/7含或不含ATRA均能明显促进细胞基质矿化,并且细胞外基质矿化面积随BMP2/7浓度的增加而增加。ATRA明显下调5ng/ml BMP2/7所诱导的细胞外基质矿化,但这种抑制作用在50ng/ml BMP2/7时则并不存在。单纯5ng/ml BMP2/7或者ATRA刺激培养7天后才能明显促进Runx2基因的表达。50ng/ml BMP2/7在细胞因子刺激培养4天和7天时均能明显促进Runx2基因的表达,与单纯ATRA组及5ng/ml BMP2/7+ATRA组相比,50ng/ml BMP2/7+ATRA组则明显促进Runx2基因的表达。与ALP活性表达不同的是,在第4天时,ATRA并没有明显影响ALP基因的表达,在第7天时,ATRA对ALP基因的表达表现出明显的抑制作用。单纯5ng/ml BMP2/7和50ng/ml BMP2/7则在细胞因子刺激培养第7天时表现为促进作用。细胞因子刺激培养第4天时,与空白对照组相比,ATRA和任一浓度的BMP2/7共同作用均能明显促进ALP基因的表达。与第4天相比,ATRA和50ng/ml BMP2/7在第7天时明显促进细胞ALP基因表达。细胞因子刺激培养7天时,空白对照组及单纯BMP2/7作用组的细胞OCN基因表达水平较4天时明显上升,在第7天时,任何BMP2/7浓度(Ong/ml,5ng/ml,50ng/ml)下ATRA均能明显抑制细胞OCN基因表达水平,而这种抑制现在在细胞因子刺激培养4天时并未出现。
结论：
BMP2/7促进SD大鼠BMSCs成骨分化,并呈现浓度依赖性。BMP2/7和ATRA协同诱导SD大鼠BMSCs成骨早期分化,促进ALP活性表达,但ATRA抑制BMSC中晚期成骨分化,抑制细胞外基质矿化,而BMP2/7促进BMSC成骨分化,促进细胞外基质矿化,并呈浓度依赖性,ATRA可抑制低浓度BMP2/7诱导细胞成骨分化的能力,而相对高浓度BMP2/7诱导细胞成骨分化的能力不受ATRA影响。
Sufficient bone volume and adequate bone quality are common phenomenon in clinical, and large and limit bone defect which caused by congenital osteomyelodysplasia、tumor,phlegmonosis and injury are still large challenge to orthognathic surgery, oral and maxillofacial surgery and Oral implant technology doctors. Bone morphogenetic proteins(BMPs) a group of dimeric disulfide-linked polypeptide growth factors under transforming growth factor-β superfamily, are one of the paramount cytokines in promoting bone regeneration. BMP2and BMP7homodimer which can be used to spondylodesis, repair bone defect and accelerate bone joint. Compared with BMP homodimer, BMP2/7heterodimer is more effective to induce bone regeneration, the ALP activity and bone formation content in vivo induced by BMP2/7heterodimer are several times or dozens of times than that induce by BMP homodimer. BMP homodimer also take an important function in osteoclastogenesis, it stimulate and regulate osteoclastogenesis, regulate the activity of osteoclast and promote mature osteoclast bone resorption. Compared with BMP2and BMP7, rhBMP2/7heterodimer induced cell differentiation with significantly lower threshold concentrations but similar maximum effects. And this phenomenon was not occure in the process of osteoclastogenesis, and low concentration of BMP2/7can only induce weak osteoclastogenesis, so more bone can be reached. Animal experiment also test and verified this result, and the new bone induced by BMP2/7are more close to normal bone.
All-trans retinoic acid can induce osteoblastic gene expression in osteoblast, stem cells, fibroblast and bone marrow stromal cells, promote matrix mineralization. The effect of BMPs and ATRA is still controversial in inducing osteogenic differentiation. One opinion is that ATRA and the BMP signing pathway cooperate to inhibit osteogenesis and promote adipogenesis of mouse embryonic palate mesenchymal cells, while others found that retinoic acid cooperates with BMP2to induce osteoblastic differentiation of C2C12cells. In this study, we investigated the respective and the combinatory effects of ATRA and BMP2/7on the in vitro osteogenesis of different cell types.
Part1:The effect of rhBMP2/7and ATRA on the osteogenic differentiation of MC3T3-E1cells
Objectives:
Compared with BMP homodimer, BMP2/7heterodimer is more effective to induce bone regeneration, the ALP activity and bone formation content in vivo induced by BMP2/7heterodimer are several times or dozens of times than that induce by BMP homodimer. BMP homodimer also take an important function in osteoclastogenesis, it stimulate and regulate osteoclastogenesis, regulate the activity of osteoclast and promote mature osteoclast bone resorption. Compared with BMP2and BMP7, rhBMP2/7heterodimer induced cell differentiation with significantly lower threshold concentrations but similar maximum effects. And this phenomenon was not occure in the process of osteoclastogenesis, and low concentration of BMP2/7can only induce weak osteoclastogenesis, so more bone can be reached. All-trans retinoic acid can induce osteoblastic gene expression in osteoblast, stem cells, fibroblast and bone marrow stromal cells, promote matrix mineralization. The effect of BMPs and ATRA is still controversial in inducing osteogenic differentiation. One opinion is that ATRA and the BMP signing pathway cooperate to inhibit osteogenesis and promote adipogenesis of mouse embryonic palate mesenchymal cells, while others found that retinoic acid cooperates with BMP2to induce osteoblastic differentiation of C2C12cells. In this study, we investigated the respective and the combinatory effects of ATRA and BMP2/7on the in vitro osteogenesis of MC3T3-E1cells.
Materials and Methods:
The MC3T3-E1cells were dealed with different concentration of BMP2/7heterodimer and1uM ATRA, then we focused on the effect of BMP2/7heterodimer and ATRA on the osteogenic differentiation of MC3T3-E1cells, and tested the figures in different stages of differentiation. We use quantitative fluorescence method to determine the cell numbers; use the colorimetric method to test the ALP activity as an early indicators of osteogenesis of the cells; use the ELISA method to test the OCN concentration which secreted to the medium as late indicators of osteogenesis; use the Real time fluorescent quantitative method(RT-PCR) to test the expression of osteogenic genes such as ALP, OCN, Coll, Runx2gene; use the alizarin red staining method to observe and measure the calcified area.
Results:
On the1st day, a significant increase in the cell numbers was detected only under the treatment of no ATRA,50ng/ml BMP2/7. Neither significant enhancement nor inhibition in cell proliferation was detected in the other groups. On the4th day, ATRA alone significantly decreased the cell numbers, which could be completely restored by 5ng/ml or50ng/ml BMP2/7. In contrast,5ng/ml or50ng/ml BMP2/7alone could result in a significantly higher cell number than the other treatments. In contrast to the inhibitory effect of ATRA on cell proliferation, ATRA alone didn't significantly influence the ALP activity. BMP2/7significantly increased the ALP activity in a dose-dependent manner irrespective of ATRA at both time points. The effect of ATRA on OCN expression is similar to its effects on cell proliferation, ATRA alone could significantly inhibit the OCN expression on the4th and7th day. On the4th day,5ng/ml BMP2/7antagonized the inhibitory effect of ATRA and completely restored the OCN expression. However, on the7th day5ng/ml BMP2/7only restored a part of OCN expression. Albeit so, the BMP2/7induced OCN expression was significantly inhibited by ATRA at both time points. According to the result of alizarin red staining, we found that luM ATRA could significantly inhibit the matrix mineralization. The mineralization area in the group of1uM ATRA,5ng/ml BMP2/7was significantly lower than that in the control group(no ATRA, no BMP2/7). In contrast,50ng/ml BMP2/7could antagonize ATRA and significantly enhance the mineralization about2.5folds in comparison with the control group(no ATRA, no BMP2/7).50ng/ml BMP2/7alone resulted in the highest mineralization. On the lth day, Runx2expression was significantly suppressed by ATRA with or without BMP2/7. The expression of Runx2gene was, whereas, significantly enhanced by ATRA alone on the4th and7th day. BMP2/7significantly increased the expression of Runx2gene in a dose-dependent manner. The expression of Collagen la gene was significantly suppressed by ATRA alone at the three time points.5ng/ml BMP2/7could completely restore the expression of Collagen la gene that was inhibited by ATRA at all time points.50ng/ml BMP2/7could also restore the expression of Collagen Ia gene on the1st day and7th day, and significantly enhance it on the4th day. Different from the ALP activity, the expression of ALP gene was significantly suppressed by ATRA alone at all the three time points. In the presence of ATRA,5ng/ml and50ng/ml could significantly enhance ALP gene expression in comparison with the two groups without BMP2/7. The expression of OCN gene was also significant suppressed by ATRA alone at all the three time points. In the presence of ATRA,5ng/ml BMP2/7completely restored the expression of OCN gene, and50ng/ml could further significantly enhance the expression of OCN gene. BMP2/7could significantly enhance the gene expression of OCN in a time-course and dose-dependent manner irrespective of ATRA. At both time points, ATRA significantly inhibited the expression of OCN gene that was induced by5or50ng/ml BMP2/7.
Conclusions:
ATRA inhibited the osteogenic differentiation of MC3T3-E1cells, whereas BMP2/7induce the osteogenic differentiation of MC3T3-E1cells in a dose-dependent and time-course manner; ATRA and BMP2/7didn't cooperate to induce the osteogenic differentiation of MC3T3-E1cells, and BMP2/7could antagonize the inhibition of ATRA and promotes the osteoblastogenesis.
Part2:The effect of rhBMP2/7and ATRA on the osteogenic differentiation of bone marrow stromal cells
Objectives:
Compared with BMP homodimer, BMP2/7heterodimer is more effective to induce bone regeneration, the ALP activity and bone formation content in vivo induced by BMP2/7heterodimer are several times or dozens of times than that induce by BMP homodimer. BMP homodimer also take an important function in osteoclastogenesis, it stimulate and regulate osteoclastogenesis, regulate the activity of osteoclast and promote mature osteoclast bone resorption. Compared with BMP2and BMP7, rhBMP2/7heterodimer induced cell differentiation with significantly lower threshold concentrations but similar maximum effects. And this phenomenon was not occure in the process of osteoclastogenesis, and low concentration of BMP2/7can only induce weak osteoclastogenesis, so more bone can be reached. All-trans retinoic acid can induce osteoblastic gene expression in osteoblast, stem cells, fibroblast and bone marrow stromal cells, promote matrix mineralization. The effect of BMPs and ATRA is still controversial in inducing osteogenic differentiation. One opinion is that ATRA and the BMP signing pathway cooperate to inhibit osteogenesis and promote adipogenesis of mouse embryonic palate mesenchymal cells, while others found that retinoic acid cooperates with BMP2to induce osteoblastic differentiation of C2C12cells. In this study, we investigated the respective and the combinatory effects of ATRA and BMP2/7on the in vitro osteogenesis of rat bone marrow stromal cells.
Materials and Methods:
The rat bone marrow stromal cells were harvested from a4-week-old male Sprague-Dawley (SD) rats. We used flow cytometry to characterize the biomarkers of BMSC, then the cells were dealed with different concentration of BMP2/7heterodimer and luM ATRA, then we focused on the effect of BMP2/7heterodimer and ATRA on the osteogenic differentiation of rat bone marrow stromal cells, and tested the figures in different stages of differentiation. We use quantitative fluorescence method to determine the cell numbers; use the colorimetric method to test the ALP activity as an early indicators of osteogenesis of the cells; use the ELISA method to test the OCN concentration which secreted to the medium as late indicators of osteogenesis; use the Real time fluorescent quantitative method(RT-PCR) to test the expression of osteogenic genes such as ALP, OCN, Coll, Runx2gene; use the alizarin red staining method to observe and measure the calcified area.
Results:
ATRA alone could significantly inhibit the proliferation of BMSCs, resulting in only a significantly higher DNA amount from day4to day7. Interestingly, BMP2/7could antagonize the inhibitive effects of ATRA on cell proliferation. On day4, BMP2/7at both5ng/mL and50ng/mL resulted in a significantly higher DNA amount than ATRA alone. On day7, on50ng/mL BMP2/7was associated with a significantly higher DNA amount than ATRA alone. In comparison with the contrary effects of BMP2/7and ATRA on the proliferation, both cytokines significantly increased the ALP activity of BMSCs on day4and day7. At any time point, ATRA and BMP2/7(5ng/ml,50ng/ml) synergied to enhance the ALP expression of rat bone marrow stromal cells. ATRA and50ng/mL BMP2/7synergistically promoted (about7.01folds) in the ALP activity of BMSCs on both day4and day7. BMP2/7alone could significantly promote the late osteogenic differentiation marker--OCN in a dose-dependent manner on both day4and day7. In contrast, ATRA alone significantly inhibited OCN expression on both time points. In the presence of ATRA, the promoting effects of BMP2/7were in dose-dependent and time-dependent manners. After a14-day treatment, mineralization of BMSCs in cell matrix was only found in the presence50ng/mL BMP2/7. On day21, very mild calcium deposition could be detected in the two groups without BMP2/7. The influence of ATRA on the mineralization of BMSCs was very mild without the presence of BMP2/7. Regardless of ATRA, BMP2/7could significantly promote mineralization in a dose-dependent manner. ATRA could significantly downregulate the osteogenesis induced by5ng/mL BMP2/7. However, the inhibitive effect of ATRA was insignificant for50ng/mL BMP2/7.5ng/mL BMP2/7or ATRA only significantly enhanced the expression of Runx2gene only on day7. Without ATRA,50ng/mL BMP2/7could significantly enhance the expression ofRunx2gene on both day4(1.20-fold and1.22-fold) and day7(2.07-fold and1.61-fold) in comparison with0ng/mL BMP2/7and5ng/mL BMP2/7, respectively. Furthermore, ATRA and50ng/mL BMP2/7 significantly enhance the expression of Runx2gene on both day4(2.06-fold and2.23-fold) and day7(1.96-fold and2.02-fold) in comparison with0ng/mL BMP2/7and5ng/mL BMP2/7, respectively. Different from ALP activity, ATRA didn't significantly influence the expression of ALP gene on day4. Furthermore, ATRA significantly suppressed the expression of ALP gene on day7. Without ATRA,5ng/mL and50ng/mL BMP2/7enhanced the expression of ALP gene only on day7. On day4, ATRA and BMP2/7of both concentrations could significantly enhance the expression of ALP gene in comparison with the controls. On day7, ATRA and50ng/mL BMP further significantly increased the expression of the ALP gene in comparison with on day4. Without the presence of ATRA, a significant enhancement in the expression of OCN gene could be detected from day4to day7. A significant suppression of OCN gene expression by ATRA was detected on day7irrespective of BMP2/7, while such a suppressive effect of ATRA was not seen on day4.
Conclusions:
ATRA and BMP2/7synergistically induced the early osteogenesis of rat bone marrow stromal cells, and synergistically promoted the expression of ALP. But ATRA inhibited the late osteogenesis of rat bone marrow stromal cells and mineralization of BMSCs in cell matrix. BMP2/7promoted the osteogenesis of rat bone marrow stromal cells in a concentration-dependent manners. ATRA could inhibited the osteogensis induced by low concentration of BMP2/7, but it didn't influence the osteogenesis induced by high concentration of BMP2/7. In conclusion ATRA and BMP2/7showed both antagonistic and synergistic effects on the osteogenesis of BMSCs.
引文
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