BMP-2 and TGF-β3 do not prevent spontaneous degeneration in rabbit disc explants but induce ossification of the annulus fibrosus
BMP-2 and TGF-β3 do not prevent spontaneous degeneration in rabbit disc explants but induce ossification of the annulus fibrosus
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DOI：
10.1007/s00586-012-2371-3
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发表时间：
2012-09-01
影响因子：
2.8
通讯作者：
Stoyanov, Jivko V.
中科院分区：
文献类型：
--
作者：
Haschtmann, Daniel;Ferguson, Stephen J.;Stoyanov, Jivko V.
目前正在研究不同的椎间盘再生方法。除了基因治疗和组织工程技术外，生长因子和分化因子的应用也具有广阔的前景。使用缩小的椎间盘模型（例如细胞或组织碎片培养物）的研究有效性有限，并且根据所采用的实验模型显示出有争议的结果。因此，本研究的目的是利用体外全器官椎间盘/终板培养系统探讨BMP-2和TGF-β3对椎间盘退变的影响。将兔椎间盘外植体在1μg/ml BMP-2或TGF-β3存在的DMEM/F12培养基中培养21天。通过 RT-qPCR 分析细胞核和纤维环的 I 型和 II 型胶原 (Col I/II)、聚集蛋白聚糖、胶原酶 (MMP-1/MMP-13) 的基因表达、骨和蛋白多糖特异性染色（von Kossa、甲苯胺蓝）的组织学变化以及细胞结构 (DNA) 和蛋白多糖含量的差异（阿新蓝结合测定）。结果表明，椎间盘蛋白多糖浓度随着时间的推移而降低TGF-β 3 和 BMP-2 组。在纤维环 (AF) 中，TGF-β 3 和 BMP-2 导致 Col I 和 II 型以及聚集蛋白聚糖基因表达上调。相反，MMP 基因受到抑制。在细胞核中，生长因子降低了聚集蛋白聚糖的基因表达，TGF-β3 抑制了自发的 Col I 上调，而 BMP-2 则降低了 Col II 的表达。大多数采样点对MMP-1和MMP-13的表达没有影响。然而，组织学证明了 TGF-β 3 和 BMP-2 诱导 AF 骨化。可以得出的结论是，两种生长因子在测试浓度下可能不适合再生整个椎间盘器官，但它们是单独或组合注射到疼痛椎间盘中以诱导骨融合（脊椎融合术）的有趣候选者。
Different approaches for disc regeneration are currently under investigation. Beside gene therapy and tissue engineering techniques, the application of growth and differentiation factors own promising potential. Studies using reduced intervertebral disc models, such as cell or tissue fragment cultures, have limited validity and show controversial results depending on the employed experimental model. Therefore, the goal of the current study was to investigate the effect of BMP-2 and TGF-beta 3 on intervertebral disc degeneration using an in vitro full-organ disc/endplate culture system.Intervertebral rabbit disc explants were cultured in the presence of 1 mu g/ml BMP-2 or TGF-beta 3 for 21 days in DMEM/F12 media. Nucleus and annulus were analyzed for gene expression of collagen type I and II (Col I/II), aggrecan, collagenases (MMP-1/MMP-13) with RT-qPCR, histological changes with bone and proteoglycan-specific staining (von Kossa, toluidine blue) and differences in cellularity (DNA) and proteoglycan content (alcian blue binding assay).The results demonstrate that disc proteoglycan concentration decreased with time in the TGF-beta 3 and BMP-2 groups. In the annulus fibrosus (AF), TGF-beta 3 and BMP-2 resulted in an up-regulation of Col I and type II, and of aggrecan gene expression. In contrast, MMP genes were inhibited. In the nucleus, the growth factors decreased gene expression of aggrecan and spontaneous Col I up-regulation was inhibited by TGF-beta 3, whereas expression of Col II was decreased with BMP-2. There was no effect on expression of MMP-1 and MMP-13 for most sampling points. However, TGF-beta 3 and BMP-2 induced ossification of the AF was demonstrated by histology.It can be concluded that both growth factors, at the tested concentrations, may not be suitable to regenerate the whole intervertebral disc organ but they are interesting candidates for being injected alone or in combination into a painful intervertebral disc to induce osseous fusion (spondylodesis).