Bone morphogenetic protein-2 facilitates expression of chondrogenic, not osteogenic, phenotype of human intervertebral disc cells
Bone morphogenetic protein-2 facilitates expression of chondrogenic, not osteogenic, phenotype of human intervertebral disc cells
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DOI：
10.1097/01.brs.0000101445.46487.16
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发表时间：
2003-12-15
期刊：
影响因子：
3
通讯作者：
Lee, HM
中科院分区：
文献类型：
--
作者：
Kim, DJ;Moon, SH;Lee, HM
研究设计.使用骨形态发生蛋白-2（BMP-2）和人椎间盘（IVD）细胞的体外实验。证明BMP-2对人IVD细胞中mRNA表达（I型胶原、II型胶原、聚集蛋白聚糖和骨钙素）、蛋白聚糖合成、碱性磷酸酶表达、骨结节形成的影响。BMP-2是一种强有力的骨诱导剂，是早期软骨形成和软骨表型维持的关键生长因子。BMP-2被证明在刺激关节软骨细胞和IVD细胞中的蛋白多糖合成中是有效的。然而，BMP-2对IVD细胞的作用，无论是软骨形成还是成骨形成，在转录水平和组织化学染色上都没有彻底阐明。收获人IVD并酶消化。然后将IVD细胞在藻酸盐珠中进行三维培养。取髂骨松质骨培养成骨细胞，进行组织化学染色。将BMP-2基因导入中国仓鼠卵巢细胞，经纯化后获得重组人BMP-2（rhBMP-2）。然后将IVD细胞培养物暴露于不同浓度的rhBMP-2。逆转录-聚合酶链反应的mRNA表达的聚集蛋白聚糖，胶原蛋白I型，胶原蛋白II型，和骨钙素进行。新合成的蛋白多糖通过在PD 10柱中SephadexG-25 M上S-35-硫酸盐掺入来测量。采用碱性磷酸酶和茜素红S染色分别检测成骨标志物和骨结节形成。在rhBMP-2处理的培养物中，与未处理对照相比，新合成的蛋白聚糖增加（300 ng/mL rhBMP-2为67%，1，500 ng/mL rhBMP-2为200%），聚集蛋白聚糖、I型胶原和II型胶原mRNA的表达上调。rhBMP-2在一定剂量和培养时间内均不上调骨钙素mRNA的表达。含有rhBMP-2的IVD细胞培养物在组织化学染色中未显示骨形成的证据，即，碱性磷酸酶和茜素红S染色，成骨细胞培养呈强阳性。rhBMP-2明显上调软骨形成成分的mRNA表达，也刺激蛋白多糖合成，而不表达成骨表型。本研究提示rhBMP-2可作为椎间盘基质再生的促合成剂。
Study Design. In vitro experiment using bone morphogenetic protein-2 (BMP-2) and human intervertebral disc (IVD) cells.Objectives. To demonstrate the effect of BMP-2 on mRNAs expression (collagen type I, collagen type II, aggrecan, and osteocalcin), proteoglycan synthesis, expression of alkaline phosphatase, bone nodule formation in human IVD cells.Summary of Background Data. BMP-2 was widely known as a powerful agent for osteoinduction and a crucial growth factor for early chondrogenesis and maintenance of cartilaginous phenotype. BMP-2 proved to be effective in stimulating proteoglycan synthesis in articular chondrocytes and IVD cells. Nevertheless, the effect of BMP-2 on IVD cells, whether chondrogenic or osteogenic, was not thoroughly elucidated in transcriptional level and histochemical stains.Materials and Methods. Human IVDs were harvested and enzymatically digested. Then IVD cells were cultured three-dimensionally in alginate beads. Osteoblasts were cultured from cancellous bone of ilium for histochemical stains. Recombinant human BMP-2 (rhBMP-2) was produced by Chinese hamster ovary cells after transduction of BMP-2 cDNA, then concentrated and purified. Then IVD cell cultures were exposed to various concentrations of rhBMP-2. Reverse transcription-polymerase chain reaction for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin was performed. Newly synthesized proteoglycan was measured by S-35-sulfate incorporation on Sephadex G-25 M in PD 10 columns. As a histochemical examination, alkaline phosphatase and Alizarin red-S stains were used to detect osteogenic marker and bone nodule formation, respectively.Results. In the rhBMP-2 treated cultures, there was increased newly synthesized proteoglycan (67% in 300 ng/mL and 200% in 1,500 ng/mL of rhBMP-2) and up-regulated expression of aggrecan, collagen type I, and collagen type II mRNA over untreated control. However, rhBMP-2 did not up-regulate expression of osteocalcin mRNA in the given dose and culture period. IVD cell cultures with rhBMP-2 showed no evidence of bone formation in histochemical stains, i.e., alkaline phosphatase and Alizarin red-S, while osteoblast culture exhibited strong positive stains.Conclusions. The rhBMP-2 clearly up-regulated mRNA expression of chondrogenic components and also stimulated proteoglycan synthesis without expression of osteogenic phenotype. Taken together, this study raise the possibility of rhBMP-2 can be anabolic agent for regenerating matrix of intervertebral disc.