颈椎间盘纤维环成纤维细胞成骨潜能的研究
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摘要
背景：颈椎椎间盘退变，导致椎间异常活动增加，椎体周缘的骨赘形成及脊柱韧带的退变、骨化是一种机体代偿保护性反应(再稳定)。但骨赘的具体来源及纤维环退变的病理过程尚无定论，有研究观点提出随着椎间盘的退变，纤维环成纤维细胞增生、化生为软骨细胞，其增殖越过椎间盘边缘进而产生软骨内骨化形成骨赘。即纤维环中成纤维细胞的退变、软骨化生是椎间盘退变的病理基础。
目的：本研究从实验动物颈椎椎间融合过程观察及体外纤维环成纤维细胞的成骨诱导培养来探讨纤维环成纤维细胞的成骨潜能，对比骨形态发生蛋白(rhBMP_2)和α—肿瘤坏死因子(TNF-α)对成纤维细胞成骨的不同影响，研究纤维环成纤维细胞成骨化生在颈椎病退变机制中的作用。
方法：(1)通过观察山羊颈椎椎间融合过程中椎间融合器内松质骨、纤维环组织等不同填充组织的组织学变化过程，以期了解成纤维细胞成骨潜能在体内骨融合过程中的作用机制；(2)利用体外细胞培养技术，建立实验山羊颈椎间盘纤维环成纤维细胞培养体系，并观察其在细胞因子rhBMP_2、TNF-α条件培养液诱导下的成骨表型变化，了解纤维环成纤维细胞的生物学特性；(3)利用体外细胞培养技术，建立颈椎病患者的颈椎间盘纤维环中成纤维细胞的培养体系，观察其在细胞因子rhBMP_2、TNF-α条件培养液诱导下的成骨潜能表现，以进一步揭示颈椎病的发病机理。
结果：(1)动物体内实验结果中X线片示实验山羊的内植颈椎椎间内固定器(CHTF)在整个实验过程中均在位，无松动、移位、脱落。6周时CHTF与椎体相对固定部位周围有骨组织生长，CHTF与椎体终板接触部位有成骨现象，骨桥形成。CT平扫示CHTF中空部位模糊。12周时X线片示终板部位及骨—金属界面上有较多高密度骨桥形成。CHTF内充填松质骨组术后6周组织学切片观察示新生软骨、骨小梁存在，术后12周新骨生成明显、层状堆积，原植入骨坏死；CHTF内充填松质骨+纤维环组纤维组织有坏死、少量纤维软骨，术后12周
原骨小梁、纤维环周围新生骨存在，新生软骨堆积;CHTF内充填纤维环组术后6
周组织学观察纤维组织内有纤维软骨、新生软骨存在，术后12周新生软骨存在;
CHTF内未填充组织(空白组)术后6周组织学观察无阳性染色结果，术后12周
有少量新生软骨。
(2)非条件培养组和条件培养组在传代的山羊纤维环成纤维细胞培养的各
时间点的细胞增殖活力(MTT)测定无明显差异(P>0 .05)，条件培养液对细胞增
殖无明显影响。 rhBMPZ、rhBMPZ+TNF一a条件培养组细胞趋化性生长明显，矿化
结节出现，经ARS染色，呈深红色钙阳性反应。各条件培养组的ALP活力经测定
均与空白对照组ALP活力有显著性差异(P<0.05);rhBMP:组与rhBMPZ+T NF一。
组的成纤维细胞的骨钙素分泌量均与空白对照组有显著性差异(P<0 .05)，而
TNF一a组则与空白对照组无显著性差异。条件培养组细胞I型胶原免疫细胞化
学染色均呈阳性。
(3)非条件培养组和条件培养组在传代的颈椎病患者纤维环成纤维细胞培
养的各时间点的细胞增殖活力(MTT)测定无明显差异(P>0 .05)，细胞增殖速度
较慢，条件培养液对细胞增殖无明显影响。rhBMPZ、rhBMPZ+TNF一。条件培养组
细胞趋化性生长明显，细胞呈漩涡状和复层生长，局部密度增高，可见有肉眼观呈
黑色点状矿化结节出现。各条件培养组的ALP活力经测定均与空白对照组ALP活
力有显著性差异(P<0 .05)， rhBMPZ+T NF一。组的成纤维细胞所形成的骨钙素分
泌量均与空白对照组有显著性差异(P<0.05)，而rhBMP:组、TNF一a组则与空
白对照组无显著性差异。条件培养组细胞I型胶原免疫细胞化学染色均呈阳性。
结论:(l)、颈椎间盘纤维环成纤维细胞存在成骨潜能，成骨形式可能是软
骨内化骨;CHTF椎间植骨融合最佳植骨材料是松质骨;椎间植骨融合过程中CHTF
内充填松质骨+纤维环，纤维环组织有成骨趋势;(2)、rhBMP一2对纤维环成纤
维细胞体外有明确的诱导成骨作用;(3)、纤维环成纤维细胞的骨化在颈椎退变
的病理过程中有重要作用。
Osteogenic potential of cervical intervertebral disc flbroblasts in
vivo and vitro: a histochemical and cell culture study
Summary of Background Data. Intervertebral disc degeneration such as anular protrusion and disc herniation is a common cause of cervical spondylosis myelopathy. Some studies confirm that proliferation of chondrocytes replace normal anular cells during disc degeneration.In other words, progressive ossification differentiation of flbroblasts in anular is the pathological basic of cervical spondylosis myelopathy.
Objectives. This study was conducted to explore the osteogenic potential of cervical intervertebral disc fibroblasts in vivo and vitro. To investigate the regulatory factors of rhBMP-2 and TNF- a on osteogenic phenotype of fibroblasts and discuss the condition that facilitates osteogenesis of fibroblasts.
Methods. (1) CHTF (cervical hollow threaded fixator) filled with different materials of cancellous bone and anulus fibrosus was applied to the fusion of the bone segments in experiment goats. X-ray films and CT were taken after operation to observe the stability and fusion of the segments. Pathological study of CHTF slice were applied to find the differentiation of annulus fibrosus.(2) To establish the annulus fibroblasts cell lines of experiment goats in vitro and the biologic specificity was found. The fibroblasts were grown in incubation in the media of rhBMP-2 and TNF-a for about three weeks , then,the marker for osteogenic features were investigated by biochemistry, histochemistry observations.(S) The fibroblasts were isolated , and purified from the annulus fibrosus of the cervical spondylosis myelopathy patients. In the same way, to explore the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts.
Results. (1). X-ray films and CT scans showed that the stability of the operated segment obtained by CHTF technique. The callus was found around the CHTF after 6 weeks of operation, especially in the position between the endplate of
the vertebrae and metal.
Pathological study of CHTF filled with cancellous bone showed new cartillage and bone trabecula after 6 weeks of operation. After 12 weeks of operation, new bone trabecula was obvious and nocrosis cancellous bone was found. Pathological study of CHTF filled with cancellous bone and anulus flbrosus showed nocrosis tissue and a little fibrous cartilage aound the fibous after 6 weeks of operation. At the point of 12 weeks, new bone trabecula and cartilage were found aound the old trabecula or fibous. The CHTF only filled with anulus fibrosus were found fibrous cartilage and new cartilage during the experiment. The CHTF filled with nothing only found a little fibrous cartilage afer 12 weeks of operation.
(2).It was found that rhBMPi and TNF-α had no effect on the proliferation of fibroblasts from both the experiment goats and the cervical spondylosis myelopathy patients. Using rhBMP2 or TNF-a could stimulate fibroblasts to secrete alkaline phosphatase and I collagen. It was found that the combined use of rhBMP2 and TNF-a or the single use of rhBMP2 could make fibroblasts to secrete osteocalin and the morphological changes of the fibroblasts were very obvious.The fibroblasts orientated themselves in a radiating pattern around the calcium granules.The fibroblasts were interwoven one on top of another in the form of multilayer structure and the extracellular matrix increased in size in forming larger nodules. Histochemical study of the nodules with specific new bone labeller (Alizarin red S) revealed positive reaction, denoting that the nodules produced by the fibroblasts were bone tissues.
Conclusions: The results point out clearly that rhBMP2 can induce the osteogenic potential of annulus fibroblasts in vitro. The osteogenic of annulus fibroblasts play an important role in the pathological changes of cervical spondylosis myelopathy.
目的：本研究从实验动物颈椎椎间融合过程观察及体外纤维环成纤维细胞的成骨诱导培养来探讨纤维环成纤维细胞的成骨潜能，对比骨形态发生蛋白(rhBMP_2)和α—肿瘤坏死因子(TNF-α)对成纤维细胞成骨的不同影响，研究纤维环成纤维细胞成骨化生在颈椎病退变机制中的作用。
方法：(1)通过观察山羊颈椎椎间融合过程中椎间融合器内松质骨、纤维环组织等不同填充组织的组织学变化过程，以期了解成纤维细胞成骨潜能在体内骨融合过程中的作用机制；(2)利用体外细胞培养技术，建立实验山羊颈椎间盘纤维环成纤维细胞培养体系，并观察其在细胞因子rhBMP_2、TNF-α条件培养液诱导下的成骨表型变化，了解纤维环成纤维细胞的生物学特性；(3)利用体外细胞培养技术，建立颈椎病患者的颈椎间盘纤维环中成纤维细胞的培养体系，观察其在细胞因子rhBMP_2、TNF-α条件培养液诱导下的成骨潜能表现，以进一步揭示颈椎病的发病机理。
结果：(1)动物体内实验结果中X线片示实验山羊的内植颈椎椎间内固定器(CHTF)在整个实验过程中均在位，无松动、移位、脱落。6周时CHTF与椎体相对固定部位周围有骨组织生长，CHTF与椎体终板接触部位有成骨现象，骨桥形成。CT平扫示CHTF中空部位模糊。12周时X线片示终板部位及骨—金属界面上有较多高密度骨桥形成。CHTF内充填松质骨组术后6周组织学切片观察示新生软骨、骨小梁存在，术后12周新骨生成明显、层状堆积，原植入骨坏死；CHTF内充填松质骨+纤维环组纤维组织有坏死、少量纤维软骨，术后12周
原骨小梁、纤维环周围新生骨存在，新生软骨堆积;CHTF内充填纤维环组术后6
周组织学观察纤维组织内有纤维软骨、新生软骨存在，术后12周新生软骨存在;
CHTF内未填充组织(空白组)术后6周组织学观察无阳性染色结果，术后12周
有少量新生软骨。
(2)非条件培养组和条件培养组在传代的山羊纤维环成纤维细胞培养的各
时间点的细胞增殖活力(MTT)测定无明显差异(P>0 .05)，条件培养液对细胞增
殖无明显影响。 rhBMPZ、rhBMPZ+TNF一a条件培养组细胞趋化性生长明显，矿化
结节出现，经ARS染色，呈深红色钙阳性反应。各条件培养组的ALP活力经测定
均与空白对照组ALP活力有显著性差异(P<0.05);rhBMP:组与rhBMPZ+T NF一。
组的成纤维细胞的骨钙素分泌量均与空白对照组有显著性差异(P<0 .05)，而
TNF一a组则与空白对照组无显著性差异。条件培养组细胞I型胶原免疫细胞化
学染色均呈阳性。
(3)非条件培养组和条件培养组在传代的颈椎病患者纤维环成纤维细胞培
养的各时间点的细胞增殖活力(MTT)测定无明显差异(P>0 .05)，细胞增殖速度
较慢，条件培养液对细胞增殖无明显影响。rhBMPZ、rhBMPZ+TNF一。条件培养组
细胞趋化性生长明显，细胞呈漩涡状和复层生长，局部密度增高，可见有肉眼观呈
黑色点状矿化结节出现。各条件培养组的ALP活力经测定均与空白对照组ALP活
力有显著性差异(P<0 .05)， rhBMPZ+T NF一。组的成纤维细胞所形成的骨钙素分
泌量均与空白对照组有显著性差异(P<0.05)，而rhBMP:组、TNF一a组则与空
白对照组无显著性差异。条件培养组细胞I型胶原免疫细胞化学染色均呈阳性。
结论:(l)、颈椎间盘纤维环成纤维细胞存在成骨潜能，成骨形式可能是软
骨内化骨;CHTF椎间植骨融合最佳植骨材料是松质骨;椎间植骨融合过程中CHTF
内充填松质骨+纤维环，纤维环组织有成骨趋势;(2)、rhBMP一2对纤维环成纤
维细胞体外有明确的诱导成骨作用;(3)、纤维环成纤维细胞的骨化在颈椎退变
的病理过程中有重要作用。
Osteogenic potential of cervical intervertebral disc flbroblasts in
vivo and vitro: a histochemical and cell culture study
Summary of Background Data. Intervertebral disc degeneration such as anular protrusion and disc herniation is a common cause of cervical spondylosis myelopathy. Some studies confirm that proliferation of chondrocytes replace normal anular cells during disc degeneration.In other words, progressive ossification differentiation of flbroblasts in anular is the pathological basic of cervical spondylosis myelopathy.
Objectives. This study was conducted to explore the osteogenic potential of cervical intervertebral disc fibroblasts in vivo and vitro. To investigate the regulatory factors of rhBMP-2 and TNF- a on osteogenic phenotype of fibroblasts and discuss the condition that facilitates osteogenesis of fibroblasts.
Methods. (1) CHTF (cervical hollow threaded fixator) filled with different materials of cancellous bone and anulus fibrosus was applied to the fusion of the bone segments in experiment goats. X-ray films and CT were taken after operation to observe the stability and fusion of the segments. Pathological study of CHTF slice were applied to find the differentiation of annulus fibrosus.(2) To establish the annulus fibroblasts cell lines of experiment goats in vitro and the biologic specificity was found. The fibroblasts were grown in incubation in the media of rhBMP-2 and TNF-a for about three weeks , then,the marker for osteogenic features were investigated by biochemistry, histochemistry observations.(S) The fibroblasts were isolated , and purified from the annulus fibrosus of the cervical spondylosis myelopathy patients. In the same way, to explore the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts.
Results. (1). X-ray films and CT scans showed that the stability of the operated segment obtained by CHTF technique. The callus was found around the CHTF after 6 weeks of operation, especially in the position between the endplate of
the vertebrae and metal.
Pathological study of CHTF filled with cancellous bone showed new cartillage and bone trabecula after 6 weeks of operation. After 12 weeks of operation, new bone trabecula was obvious and nocrosis cancellous bone was found. Pathological study of CHTF filled with cancellous bone and anulus flbrosus showed nocrosis tissue and a little fibrous cartilage aound the fibous after 6 weeks of operation. At the point of 12 weeks, new bone trabecula and cartilage were found aound the old trabecula or fibous. The CHTF only filled with anulus fibrosus were found fibrous cartilage and new cartilage during the experiment. The CHTF filled with nothing only found a little fibrous cartilage afer 12 weeks of operation.
(2).It was found that rhBMPi and TNF-α had no effect on the proliferation of fibroblasts from both the experiment goats and the cervical spondylosis myelopathy patients. Using rhBMP2 or TNF-a could stimulate fibroblasts to secrete alkaline phosphatase and I collagen. It was found that the combined use of rhBMP2 and TNF-a or the single use of rhBMP2 could make fibroblasts to secrete osteocalin and the morphological changes of the fibroblasts were very obvious.The fibroblasts orientated themselves in a radiating pattern around the calcium granules.The fibroblasts were interwoven one on top of another in the form of multilayer structure and the extracellular matrix increased in size in forming larger nodules. Histochemical study of the nodules with specific new bone labeller (Alizarin red S) revealed positive reaction, denoting that the nodules produced by the fibroblasts were bone tissues.
Conclusions: The results point out clearly that rhBMP2 can induce the osteogenic potential of annulus fibroblasts in vitro. The osteogenic of annulus fibroblasts play an important role in the pathological changes of cervical spondylosis myelopathy.
引文
1．赵定麟主编，现代骨科学．北京 第1版．科学出版社 2004．
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3．周秉文，丁梅，黄勇．腰椎后缘骨块的类型及发生机理．中国脊柱脊髓杂志，1999，19(1)：9．
4．彭宝淦，施杞．退变椎间盘的组织病理学变化．中国中医骨伤科杂志，1996，4(6)：225．
5．孙常太，黄公怡，李海生．髓核组织对椎间植骨融合的影响．中国脊柱脊髓杂志，2002，12(3)：198—200．
6．柴本甫，汤雪明，徐荣辉．家兔皮肤成纤维细胞体外成骨潜能的研究．中华外科杂志，1994，7(7)：443-445．
7．冯雪，陈富林，任卫红等．体外培养牙周膜成纤维细胞矿化能力的实验研究．华西口腔医学杂志，1998，16(4)：294-296．
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2．杨裕红，吴揭地．腰椎间盘突出伴椎间盘组织骨化11例报告[J]．中国脊柱脊髓杂志，1996，6(4)：194．
3．周秉文，丁梅，黄勇．腰椎后缘骨块的类型及发生机理．中国脊柱脊髓杂志，1999，19(1)：9．
4．彭宝淦，施杞．退变椎间盘的组织病理学变化．中国中医骨伤科杂志，1996，4(6)：225．
5．孙常太，黄公怡，李海生．髓核组织对椎间植骨融合的影响．中国脊柱脊髓杂志，2002，12(3)：198—200．
6．柴本甫，汤雪明，徐荣辉．家兔皮肤成纤维细胞体外成骨潜能的研究．中华外科杂志，1994，7(7)：443-445．
7．冯雪，陈富林，任卫红等．体外培养牙周膜成纤维细胞矿化能力的实验研究．华西口腔医学杂志，1998，16(4)：294-296．
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