骨形态发生蛋白（BMP）异源二聚体在诱导成骨发生及破骨发生的体外研究
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摘要
骨缺损在临床上十分普遍,而由多种原因造成的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(BMPs)是一组具有诱导异位成骨能力的二聚体蛋白,属于转移生长因子-p超家族成员,是促进骨再生研究领域的主要细胞因子之一。在极限骨缺损修复中,BMPs的介入治疗由于其具有相对便利的可操作性有望替代骨移植治疗。
第一部分
骨形态发生蛋白异源二聚体诱导成骨发生的体外比较研究
目的：BMP同源二聚体(如BMP2、BMP7)由于其过高的有效使用剂量而未能在临床普遍应用。BMP异源二聚体近年来被报道具有比相应同源二聚体更强的诱导骨形成作用。然而,BMP异源二聚体在一系列成骨发生细胞事件,如细胞趋化迁移、细胞增殖、成骨分化以及细胞基质外矿化等环节中的具体生物学作用尚不明确。本实验旨在采用纯化的rhBMP2/7异源二聚体以及相应的同源二聚体分别作用于成骨前体细胞,以此比较研究BMP异源二聚体在体外诱导成骨发生的具体生物学功能特点。材料和方法：通过时间梯度和浓度梯度系统性研究rhBMP2/7、rhBMP2、rhBMP7以及rhBMP2和rhBMP7的等比混合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：用实时定量细胞电感应／细胞迁移系统(RT-CES/CIM system)监测细胞趋化迁移运动；用荧光定量法检测细胞增殖后DNA含量；用比色法检测细胞成骨分化碱性磷酸酶(ALP)活性；用ELISA方法检测成骨细胞骨钙素(OCN)的分泌量；用实时荧光定量RT-PCR检测细胞成骨相关基因(ALP、OCN、Col1、Runx2)的表达水平；用茜素红染色观察并计量细胞矿化培养后的基质外矿化结节面积。
结果：RhBMP2/7异源二聚体在诱导成骨发生的各阶段均呈现与rhBMP同源二聚体相似的作用模式。RhBMP2/7诱导MC3T3-E1趋化迁移、细胞增殖和细胞成骨分化的起效浓度和最佳浓度是rhBMP同源二聚体的1／10～1／2,但其最大作用效果除了诱导细胞趋化迁移显著高于同源二聚体外,其余几项指标,包括ALP活性、OCN表达量均与rhBMP2的最大作用效果相近。此外,在较低浓度范围内(5-100ng/ml), rhBMP2/7异源二聚体在细胞成骨发生的早期阶段(第1-4天)相比与rhBMP同源二聚体的作用优势要显著强于细胞成骨发生的后阶段(第4～7天)。与以往的研究结果相似,rhBMP同源二聚体的等比混合并不能带来协同效果。茜素红钙化结节染色面积的比较突出表明了rhBMP2/7 (50ng/ml)在3周时即能有效促进细胞外基质矿化；而rhBMP同源二聚体组(50ng/ml)以及对照组均只有在4周时才能观察到矿化结节,并且,rhBMP2/7诱导的矿化面积是rhBMP2组的12倍,rhBMP7组的38倍。不管是rhBMP2/7还是rhBMP同源二聚体,在诱导细胞趋化迁移、增殖、ALP活性以及成骨相关基因表达上都呈现浓度-效应钟形曲线,即并非浓度越高,作用效果越好,提示了rhBMPs必须以适宜的浓度作用于细胞才能发挥其诱导细胞成骨发生各环节的最佳作用效果。
结论：RhBMP2/7异源二聚体在诱导细胞成骨发生的有效作用浓度低至5ng/ml,是相应同源二聚体的1／10,并于50ng/ml达到作用峰值平台期,其水平与相应同源二聚体在高浓度时获得的最高作用水平无显著性差异。该特点使得rhBMP2/7有替代rhBMP2应用于临床的潜力,并且符合生理规律的低水平缓慢释放能使rhBMP2/7在诱导成骨发生过程中发挥最佳效果。
第二部分
骨形态发生蛋白异源二聚体影响破骨发生的体外比较研究
目的：BMP同源二聚体能显著刺激破骨细胞的分化和功能,特别是当其以较高的临床使用剂量应用时。BMP异源二聚体由于其具有低浓度起效,有效作用浓度低等特点有望替代BMP2同源二聚体。但现有的文献报道尚未涉及BMP异源二聚体对破骨细胞的作用。BMP异源二聚体是否在破骨发生中也呈现低浓度起效的作用特点值得研究。本实验的目的在于比较研究BMP异源二聚体和相应的同源二聚体在影响细胞破骨发生中的作用。
材料与方法：通过时间梯度和浓度梯度系统性研究rhBMP2/7、rhBMP2、rhBMP7以及rhBMP2和rhBMP7的等比混合在影响RANKL (50ng/ml)诱导的破骨前体细胞RAW264.7破骨发生过程中的作用,检测破骨发生各阶段的相关指标：用荧光定量法检测细胞增殖后DNA含量；用比色法检测细胞破骨分化抗酒石酸酸性磷酸酶(TRAP)活性；用实时荧光定量RT-PCR检测细胞成骨相关基因(TRAP基因Acp5、降钙素受体基因Caclcr、蛋白组织酶K基因Ctsk)的表达水平；用TRAP染色观察并计量TRAP+多核细胞(TRAP+MNCs)的表面积、数目以及单位表面积；用破骨细胞活性分析板(OAAS)检测破骨细胞的钙盐吸收功能。
结果：经过3d的rhBMPs刺激,RANKL诱导的RAW264.7细胞增殖明显,并出现融合。RhBMP2/7的最有效促进增殖浓度是150ng／ml,rhBMP2、rhBMP7均是lOng/ml, rhBMP2+rhBMP7是100ng/ml,四组之间的最高DNA含量水平基本相近。经过4d的rhBMPs刺激,TRAP活性均被有所上调,并以100-150ng/ml时上调幅度最大。破骨相关基因的表达变化显示rhBMP2在200ng/ml时能有效促进Acp5基因的表达,而rhBMP2/7、rhBMP7无显著上调作用。Calcr的表达水平与RANKL对照组相比反而降低,特别是rhBMP2/7和rhBMP2在5ng/ml时以及rhBMP7在200ng／ml时。RhBMPs的额外加入对Ctsk表达水平的影响与RANKL对照组相比略有升降,但并无显著性差异。TRAP+MNCs最大表面积在150ng/ml的rhBMP2作用下获得,并显著高于150ng／ml的rhBMP2／7以及100ng/ml的rhBMP7.各组诱导的TRAP+MNCs数目的最高值基本相近,相应的TRAP+MNCs单位表面积以rhBMP2(200ng／ml)组最大。经过7d的rhBMPs刺激,所有实验组的破骨细胞在OAAS板上形成的钙盐吸收陷窝面积平均值均显著大于RANKL对照组,除了rhBMP7(150ng／ml)。比较这三组rhBMP的促进破骨细胞钙盐吸收的作用,获得最大吸收面积的组别依次为rhBMP2(150ng/ml), rhBMP2(50ng/ml)和rhBMP2/7 (150ng/ml),以及rhBMP7 (5ng/ml)。RhBMP2和rhBMP7的等比混合在各项指标的检测中并没有产生协同作用。
结论：RhBMP2/7、rhBMP2以及rhBMP7都能进一步调控经RANKL诱导的RAW264.7细胞的破骨发生。RhBMP2/7异源二聚体在其中呈现浓度依赖效应递增趋势,并且仅在高浓度时诱导破骨发生的最大作用效果稍低或接近于rhBMP2同源二聚体。以上结果提示,当rhBMP2/7低剂量应用时,仅能最小程度地刺激细胞破骨发生,因此在促进成骨发生的同时并不会带来显著的破骨吸收。
总结
RhBMP2/7异源二聚体在诱导细胞成骨发生的有效作用浓度低至5ng/ml,并于50ng／ml达到作用峰值平台期；而相应]hBMP2、rhBMP7同源二聚体的起效浓度为50ng／ml；两者在各自适宜浓度时诱导成骨发生的最大作用效果相近。
RhBMP2/7异源二聚体在诱导细胞破骨发生的过程中,在5-200ng/ml浓度范围内基本呈现浓度依赖效应递增趋势,在高浓度时诱导破骨发生的最大作用效果稍低或接近于rhBMP2同源二聚体。
基于以上两种特点,若将rhBMP2/7以低剂量应用,则能有效促进成骨发生,而同期破骨发生未被刺激或仅被微弱刺激,从而比rhBMP同源二聚体更有利于骨缺损区的新骨形成和种植体周围的骨整合。
The osseous restoration of voluminous bone defect remains a challenge in orthopedics, maxillofacial surgery and implantology. Bone tissue engineering including gene, cell, and cytokine therapies has been reported to substitute autografting and allografting. Among the three therapies, cytokine therapy is advantageous in safety, feasibility, and potential for nearest clinical application over the other two. Bone morphogenetic proteins (BMPs) a group of dimeric disulfide-linked polypeptide growth factors under transforming growth factor-P superfamily, are one of the paramount cytokines in promoting bone regeneration.
Part 1:
Direct Effect of RhBMP2/7 Heterodimer on Osteoblastogenesis in Vitro Compared to RhBMP2 and RhBMP7 Homodimers
Objectives:The indication of BMPs for clinical applications is limited due to their high effective doses. BMP heterodimers were exhibited to be more potent than and thus were potential substitutes for BMP homodimers. However, the effect of BMP heterodimers on the sequential osteoblastogenetic events such as chemotactic migration, proliferation, differentiation and mineralization remains uncovered. This study aims to delineate the bio-functional characteristics of rhBMP2/7 heterodimer in inducing osteoblastogenesis of MC3T3-E1 in vitro.
Materials and Methods:Time-course and dose-response studies were performed to compare the potencies of purified rhBMP2/7 heterodimer (R&D systems) versus rhBMP2 homodimer, rhBMP7 homodimer or their 1:1 mixture using a preosteoblast cell line (MC3T3-E1). Chemotactic effects were gauged using a real time cell electronic sensing/cell migration system (RT-CES/CIM system). Cell proliferation was examined by fluorometric quantification of cellular DNA content. Alkaline phosphatase (ALP) activities were investigated with a colorimetric assay kit. Osteocalcin (OCN) expressions were determined using an enzyme-linked immunosorbent assay (ELISA) kit. The gene expressions of ALP and OCN were detected by real-time RT-PCR analysis and normalized byβ-actin gene expression. Cell matrix mineralization was evaluated by alizarin red staining.
Results:RhBMP2/7 heterodimer induced the sequential dose-dependent biological activities of preosteoblast in the same pattern as homodimers. The threshold and optimal concentrations of rhBMP2/7 were significantly lower (1/10-1/2) than rhBMP2 or rhBMP7 in inducing chemotaxis, proliferation, differentiation and matrix mineralization of MC3T3-E1. The maximum levels in chemotaxis induced by rhBMP2/7 were significantly higher than the respective rhBMPs homodimers. However, ALP activity and OCN expression induced by rhBMP2/7 heterodimer were similar to those induced by the homodimers. Besides, at a lower concentration range (5-100ng/ml), rhBMP2/7 heterodimer exhibited more advantages in the early period (dayl-day4) than the later period (day4-day7) of differentiation when compared to homodimers. ALP and OCN gene expression also showed similar results. Consistent with previous studies, the 1:1 mixture of rhBMP2 and rhBMP7 did not show synergistic effect. Regarding to cell matrix mineralization, on day21, calcium deposition could only be detected in the group of 50ng/ml rhBMP2/7. Moreover, on day28, areas of calcium depositions induced by 50ng/ml rhBMP2/7 were 12-or 38-fold more than those induced by 50ng/ml rhBMP2 or 50ng/ml rhBMP7 respectively. Similar to the homodimers, the rhBMP2/7-induced dose-dependent responses in chemotaxis, proliferation, ALP activities and gene expressions showed bell-shape curves, in which overdose resulted in a descending trend. These findings strongly suggested that the specificity of rhBMP2/7 heterodimer, as an osteoblastogenesis-inducer, was lower-effective-concentration instead of higher-potency in comparison to the homodimers. In addition, the action of rhBMP2/7 heterodimer during the in vitro osteoblastogenesis indicated that the cellular events sequentially occurred with the concentration increasing. One event booms with the former one tapering although there were broad overlaps. Only the specifically appropriate concentration of rhBMP2/7 heterodimer could exert an optimal promoting effect on certain cellular event.
Conclusions:RhBMP2/7 heterodimer is an osteoblastogenesis-inducer that does not have a higher-potency but does have a lower-effective-concentration compared to rhBMP2 and rhBMP7 homodimers. This Specificity confers rhBMP2/7 heterodimer a promising clinical application potential over its respective homodimers. Besides, rhBMP2/7 should be delivered in a physiological-like manner to match the demand of each sequential osteoblastogenetic event.
Part 2:
Direct Effect of RhBMP2/7 Heterodimer on Rankl-stimulated Osteoclastogenesis in Vitro Compared to RhBMP2 and RhBMP7 Homodimers Objectives:Homodimeric recombinant human bone morphogenetic proteins (rhBMPs) can potentially over-stimulate osteoclastic activities for their high clinical effective doses. Heterodimeric BMPs could be ideal substitutes for their lower effective doses. However, the hypothesis has not been corroborated. We, hereby, explored the effects of rhBMP2/7 heterodimer on in vitro osteoclastogenesis in comparison to rhBMP2 and rhBMP7 homodimers.
Materials and Methods:Time-course and dose-response studies were performed on osteoblastogenesis of RANKL(50ng/ml)-stimulated RAW264.7 cells. Cell proliferation and differentiation were monitored using DNA content, tartrate-resistant acid phosphatase (TRAP) activity and relative gene expressions of calcitonin receptor (calcr), capthesin K and TRAP (acp5). The area of TRAP positive multinucleated osteoclasts (TRAP+MNCs) and the calcium phosphate resorption pit formed by active osteoclasts were histomorphometrically determined and statistically analyzed by ANOVA(p<0.05).
Results:After 3-day stimulation, the most effective concentration for stimulating preosteoclast proliferation was 150ng/ml for rhBMP2/7, lOng/ml for rhBMP2 or rhBMP7 with similar peak value. After 4-day stimulation, TRAP activity was increased by rhBMPs and the most stimulating effect occurred at 100-150ng/ml. Refer to relative gene expression, the peak value of acp5 occured when stimulated by 200ng/ml rhBMP2, while rhBMP2/7 and rhBMP7 had no significant effect. Interestingly, calcr expression decreased under the stimulation of rhBMPs, especially at 5ng/ml rhBMP2/rhBMP2/7, or 200ng/ml rhBMP7. No significant effect on cathepsin K expression could be detected for all the rhBMPs. The largest area of TRAP+MNCs was achieved by 150ng/ml rhBMP2 followed by 150ng/ml rhBMP2/7 or 100ng/ml rhBMP7. After 7-day stimulation, all the rhBMPs were shown to significantly promote pit formation except rhBMP7 at 150ng/ml. Of all the three different rhBMP groups, the largest pit area was observed at 150ng/ml rhBMP2 followed by 50ng/ml rhBMP2, 150ng/ml rhBMP2/7 and 5ng/ml rhBMP7. No synergistic effects were found between BMP2 and BMP7 homodimers for all parameters.
Conclusions:Osteoclastogenesis could be specifically modulated by rhBMPs. RhBMP2/7 heterodimer of lowest effective concentration for osteogenesis resulted in less osteoclastic activities than the respective rhBMP homodimers.
Summary
RhBMP2/7 heterodimer was proved in vitro to induce osteoblastogenesis at a concentration as low as 5ng/ml and could reach its effect plateau at 50ng/ml at which respective homodimers started to take effect in inducing osteoblastogenesis in vitro. Refer to rhBMP2/7's direct effect on rankl-stimulated osteoclastogenesis in vitro, a dose-dependent increasing manner was exhibited in the select concentration (5-200ng/ml) with peak level similar to respective homodimers.
Thus, these two specificities conferred rhBMP2/7 heterodimer a promising application value in vivo to enhace bone regeneration in critical bone defect and peri-implant osteointergration.
第一部分
骨形态发生蛋白异源二聚体诱导成骨发生的体外比较研究
目的：BMP同源二聚体(如BMP2、BMP7)由于其过高的有效使用剂量而未能在临床普遍应用。BMP异源二聚体近年来被报道具有比相应同源二聚体更强的诱导骨形成作用。然而,BMP异源二聚体在一系列成骨发生细胞事件,如细胞趋化迁移、细胞增殖、成骨分化以及细胞基质外矿化等环节中的具体生物学作用尚不明确。本实验旨在采用纯化的rhBMP2/7异源二聚体以及相应的同源二聚体分别作用于成骨前体细胞,以此比较研究BMP异源二聚体在体外诱导成骨发生的具体生物学功能特点。材料和方法：通过时间梯度和浓度梯度系统性研究rhBMP2/7、rhBMP2、rhBMP7以及rhBMP2和rhBMP7的等比混合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：用实时定量细胞电感应／细胞迁移系统(RT-CES/CIM system)监测细胞趋化迁移运动；用荧光定量法检测细胞增殖后DNA含量；用比色法检测细胞成骨分化碱性磷酸酶(ALP)活性；用ELISA方法检测成骨细胞骨钙素(OCN)的分泌量；用实时荧光定量RT-PCR检测细胞成骨相关基因(ALP、OCN、Col1、Runx2)的表达水平；用茜素红染色观察并计量细胞矿化培养后的基质外矿化结节面积。
结果：RhBMP2/7异源二聚体在诱导成骨发生的各阶段均呈现与rhBMP同源二聚体相似的作用模式。RhBMP2/7诱导MC3T3-E1趋化迁移、细胞增殖和细胞成骨分化的起效浓度和最佳浓度是rhBMP同源二聚体的1／10～1／2,但其最大作用效果除了诱导细胞趋化迁移显著高于同源二聚体外,其余几项指标,包括ALP活性、OCN表达量均与rhBMP2的最大作用效果相近。此外,在较低浓度范围内(5-100ng/ml), rhBMP2/7异源二聚体在细胞成骨发生的早期阶段(第1-4天)相比与rhBMP同源二聚体的作用优势要显著强于细胞成骨发生的后阶段(第4～7天)。与以往的研究结果相似,rhBMP同源二聚体的等比混合并不能带来协同效果。茜素红钙化结节染色面积的比较突出表明了rhBMP2/7 (50ng/ml)在3周时即能有效促进细胞外基质矿化；而rhBMP同源二聚体组(50ng/ml)以及对照组均只有在4周时才能观察到矿化结节,并且,rhBMP2/7诱导的矿化面积是rhBMP2组的12倍,rhBMP7组的38倍。不管是rhBMP2/7还是rhBMP同源二聚体,在诱导细胞趋化迁移、增殖、ALP活性以及成骨相关基因表达上都呈现浓度-效应钟形曲线,即并非浓度越高,作用效果越好,提示了rhBMPs必须以适宜的浓度作用于细胞才能发挥其诱导细胞成骨发生各环节的最佳作用效果。
结论：RhBMP2/7异源二聚体在诱导细胞成骨发生的有效作用浓度低至5ng/ml,是相应同源二聚体的1／10,并于50ng/ml达到作用峰值平台期,其水平与相应同源二聚体在高浓度时获得的最高作用水平无显著性差异。该特点使得rhBMP2/7有替代rhBMP2应用于临床的潜力,并且符合生理规律的低水平缓慢释放能使rhBMP2/7在诱导成骨发生过程中发挥最佳效果。
第二部分
骨形态发生蛋白异源二聚体影响破骨发生的体外比较研究
目的：BMP同源二聚体能显著刺激破骨细胞的分化和功能,特别是当其以较高的临床使用剂量应用时。BMP异源二聚体由于其具有低浓度起效,有效作用浓度低等特点有望替代BMP2同源二聚体。但现有的文献报道尚未涉及BMP异源二聚体对破骨细胞的作用。BMP异源二聚体是否在破骨发生中也呈现低浓度起效的作用特点值得研究。本实验的目的在于比较研究BMP异源二聚体和相应的同源二聚体在影响细胞破骨发生中的作用。
材料与方法：通过时间梯度和浓度梯度系统性研究rhBMP2/7、rhBMP2、rhBMP7以及rhBMP2和rhBMP7的等比混合在影响RANKL (50ng/ml)诱导的破骨前体细胞RAW264.7破骨发生过程中的作用,检测破骨发生各阶段的相关指标：用荧光定量法检测细胞增殖后DNA含量；用比色法检测细胞破骨分化抗酒石酸酸性磷酸酶(TRAP)活性；用实时荧光定量RT-PCR检测细胞成骨相关基因(TRAP基因Acp5、降钙素受体基因Caclcr、蛋白组织酶K基因Ctsk)的表达水平；用TRAP染色观察并计量TRAP+多核细胞(TRAP+MNCs)的表面积、数目以及单位表面积；用破骨细胞活性分析板(OAAS)检测破骨细胞的钙盐吸收功能。
结果：经过3d的rhBMPs刺激,RANKL诱导的RAW264.7细胞增殖明显,并出现融合。RhBMP2/7的最有效促进增殖浓度是150ng／ml,rhBMP2、rhBMP7均是lOng/ml, rhBMP2+rhBMP7是100ng/ml,四组之间的最高DNA含量水平基本相近。经过4d的rhBMPs刺激,TRAP活性均被有所上调,并以100-150ng/ml时上调幅度最大。破骨相关基因的表达变化显示rhBMP2在200ng/ml时能有效促进Acp5基因的表达,而rhBMP2/7、rhBMP7无显著上调作用。Calcr的表达水平与RANKL对照组相比反而降低,特别是rhBMP2/7和rhBMP2在5ng/ml时以及rhBMP7在200ng／ml时。RhBMPs的额外加入对Ctsk表达水平的影响与RANKL对照组相比略有升降,但并无显著性差异。TRAP+MNCs最大表面积在150ng/ml的rhBMP2作用下获得,并显著高于150ng／ml的rhBMP2／7以及100ng/ml的rhBMP7.各组诱导的TRAP+MNCs数目的最高值基本相近,相应的TRAP+MNCs单位表面积以rhBMP2(200ng／ml)组最大。经过7d的rhBMPs刺激,所有实验组的破骨细胞在OAAS板上形成的钙盐吸收陷窝面积平均值均显著大于RANKL对照组,除了rhBMP7(150ng／ml)。比较这三组rhBMP的促进破骨细胞钙盐吸收的作用,获得最大吸收面积的组别依次为rhBMP2(150ng/ml), rhBMP2(50ng/ml)和rhBMP2/7 (150ng/ml),以及rhBMP7 (5ng/ml)。RhBMP2和rhBMP7的等比混合在各项指标的检测中并没有产生协同作用。
结论：RhBMP2/7、rhBMP2以及rhBMP7都能进一步调控经RANKL诱导的RAW264.7细胞的破骨发生。RhBMP2/7异源二聚体在其中呈现浓度依赖效应递增趋势,并且仅在高浓度时诱导破骨发生的最大作用效果稍低或接近于rhBMP2同源二聚体。以上结果提示,当rhBMP2/7低剂量应用时,仅能最小程度地刺激细胞破骨发生,因此在促进成骨发生的同时并不会带来显著的破骨吸收。
总结
RhBMP2/7异源二聚体在诱导细胞成骨发生的有效作用浓度低至5ng/ml,并于50ng／ml达到作用峰值平台期；而相应]hBMP2、rhBMP7同源二聚体的起效浓度为50ng／ml；两者在各自适宜浓度时诱导成骨发生的最大作用效果相近。
RhBMP2/7异源二聚体在诱导细胞破骨发生的过程中,在5-200ng/ml浓度范围内基本呈现浓度依赖效应递增趋势,在高浓度时诱导破骨发生的最大作用效果稍低或接近于rhBMP2同源二聚体。
基于以上两种特点,若将rhBMP2/7以低剂量应用,则能有效促进成骨发生,而同期破骨发生未被刺激或仅被微弱刺激,从而比rhBMP同源二聚体更有利于骨缺损区的新骨形成和种植体周围的骨整合。
The osseous restoration of voluminous bone defect remains a challenge in orthopedics, maxillofacial surgery and implantology. Bone tissue engineering including gene, cell, and cytokine therapies has been reported to substitute autografting and allografting. Among the three therapies, cytokine therapy is advantageous in safety, feasibility, and potential for nearest clinical application over the other two. Bone morphogenetic proteins (BMPs) a group of dimeric disulfide-linked polypeptide growth factors under transforming growth factor-P superfamily, are one of the paramount cytokines in promoting bone regeneration.
Part 1:
Direct Effect of RhBMP2/7 Heterodimer on Osteoblastogenesis in Vitro Compared to RhBMP2 and RhBMP7 Homodimers
Objectives:The indication of BMPs for clinical applications is limited due to their high effective doses. BMP heterodimers were exhibited to be more potent than and thus were potential substitutes for BMP homodimers. However, the effect of BMP heterodimers on the sequential osteoblastogenetic events such as chemotactic migration, proliferation, differentiation and mineralization remains uncovered. This study aims to delineate the bio-functional characteristics of rhBMP2/7 heterodimer in inducing osteoblastogenesis of MC3T3-E1 in vitro.
Materials and Methods:Time-course and dose-response studies were performed to compare the potencies of purified rhBMP2/7 heterodimer (R&D systems) versus rhBMP2 homodimer, rhBMP7 homodimer or their 1:1 mixture using a preosteoblast cell line (MC3T3-E1). Chemotactic effects were gauged using a real time cell electronic sensing/cell migration system (RT-CES/CIM system). Cell proliferation was examined by fluorometric quantification of cellular DNA content. Alkaline phosphatase (ALP) activities were investigated with a colorimetric assay kit. Osteocalcin (OCN) expressions were determined using an enzyme-linked immunosorbent assay (ELISA) kit. The gene expressions of ALP and OCN were detected by real-time RT-PCR analysis and normalized byβ-actin gene expression. Cell matrix mineralization was evaluated by alizarin red staining.
Results:RhBMP2/7 heterodimer induced the sequential dose-dependent biological activities of preosteoblast in the same pattern as homodimers. The threshold and optimal concentrations of rhBMP2/7 were significantly lower (1/10-1/2) than rhBMP2 or rhBMP7 in inducing chemotaxis, proliferation, differentiation and matrix mineralization of MC3T3-E1. The maximum levels in chemotaxis induced by rhBMP2/7 were significantly higher than the respective rhBMPs homodimers. However, ALP activity and OCN expression induced by rhBMP2/7 heterodimer were similar to those induced by the homodimers. Besides, at a lower concentration range (5-100ng/ml), rhBMP2/7 heterodimer exhibited more advantages in the early period (dayl-day4) than the later period (day4-day7) of differentiation when compared to homodimers. ALP and OCN gene expression also showed similar results. Consistent with previous studies, the 1:1 mixture of rhBMP2 and rhBMP7 did not show synergistic effect. Regarding to cell matrix mineralization, on day21, calcium deposition could only be detected in the group of 50ng/ml rhBMP2/7. Moreover, on day28, areas of calcium depositions induced by 50ng/ml rhBMP2/7 were 12-or 38-fold more than those induced by 50ng/ml rhBMP2 or 50ng/ml rhBMP7 respectively. Similar to the homodimers, the rhBMP2/7-induced dose-dependent responses in chemotaxis, proliferation, ALP activities and gene expressions showed bell-shape curves, in which overdose resulted in a descending trend. These findings strongly suggested that the specificity of rhBMP2/7 heterodimer, as an osteoblastogenesis-inducer, was lower-effective-concentration instead of higher-potency in comparison to the homodimers. In addition, the action of rhBMP2/7 heterodimer during the in vitro osteoblastogenesis indicated that the cellular events sequentially occurred with the concentration increasing. One event booms with the former one tapering although there were broad overlaps. Only the specifically appropriate concentration of rhBMP2/7 heterodimer could exert an optimal promoting effect on certain cellular event.
Conclusions:RhBMP2/7 heterodimer is an osteoblastogenesis-inducer that does not have a higher-potency but does have a lower-effective-concentration compared to rhBMP2 and rhBMP7 homodimers. This Specificity confers rhBMP2/7 heterodimer a promising clinical application potential over its respective homodimers. Besides, rhBMP2/7 should be delivered in a physiological-like manner to match the demand of each sequential osteoblastogenetic event.
Part 2:
Direct Effect of RhBMP2/7 Heterodimer on Rankl-stimulated Osteoclastogenesis in Vitro Compared to RhBMP2 and RhBMP7 Homodimers Objectives:Homodimeric recombinant human bone morphogenetic proteins (rhBMPs) can potentially over-stimulate osteoclastic activities for their high clinical effective doses. Heterodimeric BMPs could be ideal substitutes for their lower effective doses. However, the hypothesis has not been corroborated. We, hereby, explored the effects of rhBMP2/7 heterodimer on in vitro osteoclastogenesis in comparison to rhBMP2 and rhBMP7 homodimers.
Materials and Methods:Time-course and dose-response studies were performed on osteoblastogenesis of RANKL(50ng/ml)-stimulated RAW264.7 cells. Cell proliferation and differentiation were monitored using DNA content, tartrate-resistant acid phosphatase (TRAP) activity and relative gene expressions of calcitonin receptor (calcr), capthesin K and TRAP (acp5). The area of TRAP positive multinucleated osteoclasts (TRAP+MNCs) and the calcium phosphate resorption pit formed by active osteoclasts were histomorphometrically determined and statistically analyzed by ANOVA(p<0.05).
Results:After 3-day stimulation, the most effective concentration for stimulating preosteoclast proliferation was 150ng/ml for rhBMP2/7, lOng/ml for rhBMP2 or rhBMP7 with similar peak value. After 4-day stimulation, TRAP activity was increased by rhBMPs and the most stimulating effect occurred at 100-150ng/ml. Refer to relative gene expression, the peak value of acp5 occured when stimulated by 200ng/ml rhBMP2, while rhBMP2/7 and rhBMP7 had no significant effect. Interestingly, calcr expression decreased under the stimulation of rhBMPs, especially at 5ng/ml rhBMP2/rhBMP2/7, or 200ng/ml rhBMP7. No significant effect on cathepsin K expression could be detected for all the rhBMPs. The largest area of TRAP+MNCs was achieved by 150ng/ml rhBMP2 followed by 150ng/ml rhBMP2/7 or 100ng/ml rhBMP7. After 7-day stimulation, all the rhBMPs were shown to significantly promote pit formation except rhBMP7 at 150ng/ml. Of all the three different rhBMP groups, the largest pit area was observed at 150ng/ml rhBMP2 followed by 50ng/ml rhBMP2, 150ng/ml rhBMP2/7 and 5ng/ml rhBMP7. No synergistic effects were found between BMP2 and BMP7 homodimers for all parameters.
Conclusions:Osteoclastogenesis could be specifically modulated by rhBMPs. RhBMP2/7 heterodimer of lowest effective concentration for osteogenesis resulted in less osteoclastic activities than the respective rhBMP homodimers.
Summary
RhBMP2/7 heterodimer was proved in vitro to induce osteoblastogenesis at a concentration as low as 5ng/ml and could reach its effect plateau at 50ng/ml at which respective homodimers started to take effect in inducing osteoblastogenesis in vitro. Refer to rhBMP2/7's direct effect on rankl-stimulated osteoclastogenesis in vitro, a dose-dependent increasing manner was exhibited in the select concentration (5-200ng/ml) with peak level similar to respective homodimers.
Thus, these two specificities conferred rhBMP2/7 heterodimer a promising application value in vivo to enhace bone regeneration in critical bone defect and peri-implant osteointergration.
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8. Toth JM, Boden SD, Burkus JK, Badura JM, Peckham SM, McKay WF. Short-term osteoclastic activity induced by locally high concentrations of recombinant human bone morphogenetic protein-2 in a cancellous bone environment. Spine (Phila Pa 1976) 2009 Mar 15;34(6):539-550.
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1. Boden SD, Kang J, Sandhu H, Heller JG. Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in humans:a prospective, randomized clinical pilot trial:2002 Volvo Award in clinical studies. Spine 2002 Dec 1;27(23):2662-2673.
2. Govender S, Csimma C, Genant HK, Valentin-Opran A, Amit Y, Arbel R, et al. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures:a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002 Dec;84-A(12):2123-2134.
3. Zlotolow DA, Vaccaro AR, Salamon ML, Albert TJ. The role of human bone morphogenetic proteins in spinal fusion. J Am Acad Orthop Surg 2000 Jan-Feb;8(1):3-9.
4. Kaneko H, Arakawa T, Mano H, Kaneda T, Ogasawara A, Nakagawa M, et al. Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts. Bone 2000 Oct;27(4):479-486.
5. Sampath TK, Coughlin JE, Whetstone RM, Banach D, Corbett C, Ridge RJ, et al. Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily. J Biol Chem 1990 Aug 5;265(22):13198-13205.
6. Aono A, Hazama M, Notoya K, Taketomi S, Yamasaki H, Tsukuda R, et al. Potent ectopic bone-inducing activity of bone morphogenetic protein-4/7 heterodimer. Biochem Biophys Res Commun 1995 May 25;210(3):670-677.
7. Israel DI, Nove J, Kerns KM, Kaufman RJ, Rosen V, Cox KA, et al. Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo. Growth Factors 1996; 13(3-4):291-300.
8. Zhu W, Rawlins BA, Boachie-Adjei O, Myers ER, Arimizu J, Choi E, et al. Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model. J Bone Miner Res 2004 Dec;19(12):2021-2032.
9. Franceschi RT, Yang S, Rutherford RB, Krebsbach PH, Zhao M, Wang D. Gene therapy approaches for bone regeneration. Cells Tissues Organs 2004;176(1-3):95-108.
10. Zhao M, Zhao Z, Koh JT, Jin TC, Franceschi RT. Combinatorial gene therapy for bone regeneration:Cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2,4, and 7. Journal of Cellular Biochemistry 2005 May 1;95(1):1-16.
11. Koh JT, Zhao Z, Wang Z, Lewis IS, Krebsbach PH, Franceschi RT. Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration. J Dent Res 2008 Sep;87(9):845-849.
12. Zhu W, Boachie-Adjei O, Rawlins B, Crystal RG, Hidaka C. A BMP2/7 fusion gene enhances osteoblastic differentiation in mesenchymal cells. Molecular Therapy 2004 May;9:S338-S338.
13. Zhu W, Kim J, Cheng C, Rawlins BA, Boachie-Adjei O, Crystal RG, et al. Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro. Bone 2006 Jul;39(1):61-71.
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16.孙奋勇,汪炬,董群伟,谢秋玲,戴云,inventor;暨南大学,assignee.一种BMP2／7异源二聚体结构类似物及其表达方法与应用.中国,2006 2006.3.24.
17. Wutzl A, Brozek W, Lernbass I, Rauner M, Hofbauer G, Schopper C, et al. Bone morphogenetic proteins 5 and 6 stimulate osteoclast generation. J Biomed Mater Res A 2006 Apr;77(1):75-83.
18. Nohe A, Hassel S, Ehrlich M, Neubauer F, Sebald W, Henis YI, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem 2002 Feb 15;277(7):5330-5338.
19. Yu PB, Beppu H, Kawai N, Li E, Bloch KD. Bone morphogenetic protein (BMP) type Ⅱ receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary artery smooth muscle cells. J Biol Chem 2005 Jul 1;280(26):24443-24450.
20. Lavery K, Swain P, Falb D, Alaoui-Ismaili MH. BMP-2/4 and BMP-6/7 differentially utilize cell surface receptors to induce osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells. J Biol Chem 2008 Jul 25;283(30):20948-20958.
21. Little SC, Mullins MC. Bone morphogenetic protein heterodimers assemble heteromeric type I receptor complexes to pattern the dorsoventral axis. Nat Cell Biol 2009 May;11(5):637-643.
22. Groppe J, Greenwald J, Wiater E, Rodriguez-Leon J, Economides AN, Kwiatkowski W, et al. Structural basis of BMP signalling inhibition by the cystine knot protein Noggin. Nature 2002 Dec 12;420(6916):636-642.
23. Gazzerro E, Du Z, Devlin RD, Rydziel S, Priest L, Economides AN, et al. Noggin arrests stromal cell differentiation in vitro. Bone 2003 Feb;32(2):111-119.
24. Groppe J, Greenwald J, Wiater E, Rodriguez-Leon J, Economides AN, Kwiatkowski W, et al. Structural basis of BMP signaling inhibition by Noggin, a novel twelve-membered cystine knot protein. J Bone Joint Surg Am 2003;85-A Suppl 3:52-58.
25. Shen ZJ, Nakamoto T, Tsuji K, Nifuji A, Miyazono K, Komori T, et al. Negative regulation of bone morphogenetic protein/Smad signaling by Cas-interacting zinc finger protein in osteoblasts. J Biol Chem 2002 Aug 16;277(33):29840-29846.
26.潘秋辉,杨松海,董群伟,孙奋勇.BMP2／7异源二聚体调控CIZ的表达与自身活性的关系.中国生物工程杂志2007；27(9)：14-18.
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15. Govender S, Csimma C, Genant HK, Valentin-Opran A, Amit Y, Arbel R, et al. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures:a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002 Dec;84-A(12):2123-2134.
16. Zlotolow DA, Vaccaro AR, Salamon ML, Albert TJ. The role of human bone morphogenetic proteins in spinal fusion. J Am Acad Orthop Surg 2000 Jan-Feb;8(1):3-9.
17. Kaneko H, Arakawa T, Mano H, Kaneda T, Ogasawara A, Nakagawa M, et al. Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts. Bone 2000 Oct;27(4):479-486.
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21. Zhao M, Zhao Z, Koh JT, Jin TC, Franceschi RT. Combinatorial gene therapy for bone regeneration:Cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2,4, and 7. Journal of Cellular Biochemistry 2005 May 1;95(1):1-16.
22. Zhu W, Kim J, Cheng C, Rawlins BA, Boachie-Adjei O, Crystal RG, et al. Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro. Bone 2006 Jul;39(1):61-71.
1. Boden SD, Kang J, Sandhu H, Heller JG. Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in humans:a prospective, randomized clinical pilot trial:2002 Volvo Award in clinical studies. Spine 2002 Dec 1;27(23):2662-2673.
2. Govender S, Csimma C, Genant HK, Valentin-Opran A, Amit Y, Arbel R, et al. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures:a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002 Dec;84-A(12):2123-2134.
3. Aono A, Hazama M, Notoya K, Taketomi S, Yamasaki H, Tsukuda R, et al. Potent ectopic bone-inducing activity of bone morphogenetic protein-4/7 heterodimer. Biochem Biophys Res Commun 1995 May 25;210(3):670-677.
4. Israel DI, Nove J, Kerns KM, Kaufman RJ, Rosen V, Cox KA, et al. Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo. Growth Factors 1996; 13(3-4):291-300.
5. Zhu W, Rawlins BA, Boachie-Adjei O, Myers ER, Arimizu J, Choi E, et al. Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model. J Bone Miner Res 2004 Dec;19(12):2021-2032.
6. Zhao M, Zhao Z, Koh JT, Jin TC, Franceschi RT. Combinatorial gene therapy for bone regeneration:Cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2,4, and 7. Journal of Cellular Biochemistry 2005 May 1;95(1):1-16.
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15. Pan Qiuhui YS, Dong Qunwei,Sun Fenyong. Relationship between the bioactivity of BMP2/7 heterodimers and its regulation of CIZ expression. China Biotechnology 2007;27(9):14-18.
16. Little SC, Mullins MC. Bone morphogenetic protein heterodimers assemble heteromeric type I receptor complexes to pattern the dorsoventral axis. Nat Cell Biol 2009 May;11(5):637-643.
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19. Nohe A, Hassel S, Ehrlich M, Neubauer F, Sebald W, Henis YI, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem 2002 Feb 15;277(7):5330-5338.
20. Yu PB, Beppu H, Kawai N, Li E, Bloch KD. Bone morphogenetic protein (BMP) type Ⅱ receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary artery smooth muscle cells. J Biol Chem 2005 Jul 1;280(26):24443-24450.
21. Lavery K, Swain P, Falb D, Alaoui-Ismaili MH. BMP-2/4 and BMP-6/7 differentially utilize cell surface receptors to induce osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells. J Biol Chem 2008 Jul 25;283(30):20948-20958.
22. Groppe J, Greenwald J, Wiater E, Rodriguez-Leon J, Economides AN, Kwiatkowski W, et al. Structural basis of BMP signalling inhibition by the cystine knot protein Noggin. Nature 2002 Dec 12;420(6916):636-642.
23. Gazzerro E, Du Z, Devlin RD, Rydziel S, Priest L, Economides AN, et al. Noggin arrests stromal cell differentiation in vitro. Bone 2003 Feb;32(2):111-119.
24. Groppe J, Greenwald J, Wiater E, Rodriguez-Leon J, Economides AN, Kwiatkowski W, et al. Structural basis of BMP signaling inhibition by Noggin, a novel twelve-membered cystine knot protein. J Bone Joint Surg Am 2003;85-A Suppl 3:52-58.
25. Zhu W, Kim J, Cheng C, Rawlins BA, Boachie-Adjei O, Crystal RG, et al. Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro. Bone 2006 Jul;39(1):61-71.
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27.潘秋辉,杨松海,董群伟,孙奋勇.BMP2／7异源二聚体调控CIZ的表达与自身活性的关系.中国生物工程杂志2007；27(9)：14-18.
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40. Koh JT, Zhao Z, Wang Z, Lewis IS, Krebsbach PH, Franceschi RT. Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration. J Dent Res 2008 Sep;87(9):845-849.
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