椎间盘器官整体培养条件下髓核组织的变化
|
摘要
椎间盘退变是慢性下腰痛及脊柱功能损害的重要原因。由椎间盘退变可引起如椎间盘突出症、椎管狭窄症、脊柱滑脱症等一系列严重的临床疾患,90%以上的脊柱手术与椎间盘退变有关。
椎间盘退变的病因学十分复杂,危险因素颇多,我们对它的研究也在逐步的深入。有许多实验方法和实验模型被应用于研究椎间盘的功能,这些实验模型包括在体动物模型及细胞离体培养模型等。在动物模型中,椎间盘退变通常由力学或化学方法诱导,但它不利于探究与细胞及基质代谢有关的疾病和信号传导方面的问题；许多研究者利用体外细胞培养进行研究,但失去细胞基质的细胞会发生分化或丧失功能,达不到研究的目的。
如果能建立一种椎间盘器官整体培养的方法,就可以确保髓核细胞及其周围基质环境的完整,使细胞与细胞间、基质内部及细胞与基质间的相互作用成为可能,有利于细胞表型及功能的表达,还为研究基质间信号的传导和椎间盘对外界刺激的反应创造了一个实验平台,在椎间盘退变研究方面有一定优势,所以有必要椎间盘器官整体培养方法作相关探讨。
第一章体外培养椎间盘器官
目的：
对大鼠椎间盘包括上下软骨终板、纤维环及髓核组织进行整体培养,观察髓核组织的变化,探索椎间盘器官整体培养的实用技术。
方法：
1、取健康清洁级SD大鼠60只,5-6周龄,150g左右,无菌条件下完整取出腰段椎间盘器官(包括髓核组织、纤维环、上下软骨终板及少量附着于终板的松质骨)共350个,随机分为5组；
2、无菌条件下高渗肝素盐水冲洗椎间盘3次,2.5倍放大镜下进一步修整后置入12孔培养皿内,37℃、5%C02条件下培养,高渗DMEM／F12培养液通过每1000ml等渗培养液内加入3.72克NaCL获得,小牛血清浓度：20%,每天换液一次；
3、任选10个椎间盘器官,随机分为两组,对照组5个椎间盘在培养前置入液氮内浸泡3次,每次浸泡1分钟左右,随后在两组椎间盘的培养液内均加入浓度为10mg／ml的NBT液240μl,吹打混匀,培养8小时后取出两组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片观察；
4、任取75个椎间盘,随机分成5组,每组15个椎间盘,分别在培养的第0、3、7、14、21天取出1组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片观察；取出椎间盘前8小时在培养液内均加入浓度为10mg/ml的NBT液240μl,吹打混匀；
5、任取10个椎间盘,随机分为5组,每组2个椎间盘,分别在在培养的第0、3、7、14、21天取出1组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片,厚度4-6μm,免疫组织化学染色观察；
6、任取75个椎间盘,随机分成5组,每组15个椎间盘,分别在培养的第0、3、7、14、21天取出1组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片,厚度4-6μm,HE染色观察；
7、任取75个椎间盘,随机分成5组,每组15个椎间盘,分别在培养的第0、3、7、14、21天取出1组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片,厚度4-6μm,番红O染色观察；
8、任取100个椎间盘,随机分成4组,每组25个椎间盘,分别在培养的第0、3、7、14天取出1组椎间盘,高渗盐水冲洗3次,无菌条件下剖开椎间盘,每5个椎间盘取出1组髓核组织,每组重量约250μg,Trizol试剂裂解细胞,PCR测试基质蛋白表达。
结果：
1、可通过解剖获得完整无损的SD大鼠椎间盘器官,但软骨终板表面残留少量松质骨组织；
2、NBT可对存活椎间盘细胞着色,而对死亡椎间盘细胞不着色；
3、椎间盘器官体外培养两周内,髓核细胞成活率与对照组培养0天细胞成活率比较无统计学意义(P>0.05),培养21 d时成活率显著低于其余各时间点(P<0.01)：
4、器官培养各时间点免疫组织化学染色均显示髓核细胞内含大量Ⅱ型胶原；
5、椎间盘器官体外培养两周内,HE染色显示髓核组织结构完整,培养21天组髓核组织结构分解,细胞散在分布,完整性消失；
6、椎间盘器官体外培养两周内,番红O染色显示基质结构完整,培养21天组显示基质染色不均匀,完整性消失,图像分析结果显示：培养14天内各时间点灰度值比较无统计学意义(P>0.05),培养21天组与其他各时间点灰度值比较均有统计学意义(P<0.05)；
7、随着培养时间的延长,Ⅰ型胶原的mRNA表达呈上升趋势,而Ⅱ型胶原、核心蛋白聚糖和聚集蛋白聚糖的mRNA表达呈下降趋势,使用单因素方差分析结果显示各组间相比差异均有统计学意义。
结论：1、NBT染料可用于鉴别器官培养条件下细胞成活与否；2、利用高渗培养液可对椎间盘器官进行整体培养,为椎间盘生理与病理研究提供了一种理想模型
第二章皮下埋植培养大鼠椎间盘器官的可行性研究
目的：
体内皮下埋植培养椎间盘器官的可行性研究
方法：
1、取健康清洁级SD大鼠32只,5-6周龄,150g左右,无菌条件下完整取出腰段椎间盘器官(包括髓核组织、纤维环、上下软骨终板及少量附着于终板的松质骨)160个,随机分为6组；
2、无菌条件下高渗肝素盐水冲洗椎间盘3次,2.5倍放大镜下进一步修整,置入含高渗培养液的培养皿,搁置于CO2孵箱内备用；
3、取健康清洁级SD大鼠16只,5-6周龄,150克左右,5%水合氯醛麻醉,无菌条件下把已完整取出备用的椎间盘器官5个为1组埋植于麻醉大鼠背部皮下组织内,待大鼠苏醒后常规养殖；
4、于培养的第3、7天各任选3只大鼠取出椎间盘器官15个,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片,厚度4-6μm,番红O染色观察；
5、于培养的第3、7天各任选5只大鼠取出椎间盘器官25个,高渗盐水冲洗3次,无菌条件下剖开椎间盘,每5个椎间盘取出1组髓核组织,每组重量约250μg,Trizol试剂裂解细胞,PCR测试基质蛋白表达。
结果：
1、培养3、7天组番红O染色显示基质结构分解,染色极不均匀,图像分析结果显示培养0、3、7天组两两比较有统计学差异(P<0.01)；
2、随着培养时间的延长,Ⅰ型胶原的mRNA表达呈上升趋势,而Ⅱ型胶原、核心蛋白聚糖和聚集蛋白聚糖的mRNA表达呈下降趋势。使用单因素方差分析结果显示各组间相比差异均有统计学意义(P<0.01)
结论：
体内椎间盘器官培养的条件及理想部位尚需进一步探讨
Intervertebral disc degeneration is the important reason of low back pain and the damage of spine. Intervertebral disc degeneration could lead to disc herniation、spinal stenosis and spondylolishesis, etc. More than 90% spine operations are concerned with disc degeneration.
The etiology of disc degeneration is very complex, and our research on this topic is moving forward gradually. Many methods and models were used for the experiments including in vivo system and/or in vitro system. Disc degeneration could be induced by mechanical or chemical method, but it could not be used for the test about cell metabolism and signal conduction. Cell culture is a method of in vitro experiment, but the cell without matrix could not present its phenotype.
A system of organ culture should be built to make sure the integrity of intervertebral disc and the interaction between cells and matrix. It has advantage in cell culture and signal conduct.
Part 1 Organ culture of the whole intervertebral disc in vitro
Objective:
To find a practical method of culturing discs organ system by observing the changes of the nucleus pulposus after the whole intervertebral discs, including cartilage eng-plate nucleus,pulposus and anulus fibrosus, were cultivated.
Methods:
1、A total of 350 intervertebral discs were taken out completely from 60 healthy SD rats (about 150g) aged 5-6 weeks of clear grade and divided into 5 groups randomly. The whole intervertebral disc includes nucleus pulposus. annular fibrosus. cartilagineus end plate and little cancellated bone adhering end plate;
2、The discs were rinsed by high osmotic saline solution containing heparin for 3 times and trimmed with microscopic spectacles, then put into the culture plate. The whole intervertebral discs were cultured with high osmotic culture medium and this medium was changed once every day. The high osmotic culture medium was made by adding 3.72g NaCL to 1000ml DMEM/F12 culture medium. Culture condition is 37℃,5%CO2. The concentration of fetal bovine serum is 20%;
3、10 discs were selected randomly and divided into 5 groups randomly, and the 5 discs of control group were soaked in liquid nitrogen for 3 times(1 minute per time).240μl NBT was added into the culture medium. The discs were taken out after 8 hours, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, The discs were embedded in paraffin, then sectioned for observation.
4.75 discs were selected randomly and divided into 5 groups randomly and cultured in the medium. Each group has 15 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin, sectioned for observation. 240μl NBT was added into the culture medium 8 hours before the discs were taken out.
5、10 discs were selected randomly and divided into 5 groups randomly, then cultured in the medium. Each group has 2 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin and sectioned to 4-6μm for immunohistochemistry test;
6、75 discs were selected randomly and divided into 5 groups randomly, then cultured in the medium. Each group has 15 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin and sectioned to 4-6μm for HE stain;
7、75 discs were selected randomly and divided into 5 groups randomly, then cultured in the medium. Each group has 15 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin and sectioned to 4-6μm for safranineO stain;
8、100 discs were selected randomly and divided into 4 groups randomly, then cultured in the medium. Each group has 25 discs. The discs of one group were taken out respectively after 0,3,7 and 14 days, and rinsed by high osmotic saline solution for 3 times.250μg nucleus pulposus were taken out from 5 discs, then decomposed by Trizol for PCR.
Result:
1、The whole intervertebral disc organ could be dissected from the spine of SD rat except for little cancellated bone adhering end plate;
2、NBT could stain live disc cells, but couldn't stain dead cells;
3、The cell viability was not changed significantly at 14 day (P> 0.05) and was significantly lower at 21 days than at other time points (P< 0.01).
4、The immunohistochemistry staining results for collageⅡwere positive in nucleus pulposus cells at every time point.
5、HE staining showed that the tissue integrity and morphology of the whole intervertebral discs were not changed at 14 days
6、SafranineO staining showed no significant differences in the matrix grey scale (P> 0.05) at 0-14 days and significant differences between at 21 day and at 0-14 days (P< 0.05).
7、RT-PCR results showed that the mRNA of CollageⅠincreased with time, but the expressions of CollageⅡ, aggrecan and decorin decreased, showing statistically significant differences in the mRNA expressions of the matrix protein at each time point (P<0.05).
Conclusion:
1、NBT could be used to detect live disc cells;
2、Our system could be used to cultivate the whole intervertebral discs, it is an ideal model for further studies on physiology and pathology of intervertebral discs.
Part 2 Organ culture of the whole intervertebral disc in subcutaneous tissue of SD rat
Objective:
Is subcutaneous tissue of SD rat a good area for Organ culture of the whole intervertebral disc?
Methods:
1、A total of 160 intervertebral discs were taken out completely from 32 healthy SD rats (about 150g) aged 5-6 weeks of clear grade and divided into 6 groups randomly. The whole intervertebral disc includes nucleus pulposus、annular fibrosus、cartilagineus end plate and little cancellated bone adhering end plate;
2、The discs were rinsed by high osmotic saline solution containing heparin for 3 times and trimmed with microscopic spectacles, then put into the culture plate in culture condition for using later;
3、The discs were imbedded in the subcutaneous tissue of 16 healthy SD rats anesthetized by 10% Chloral Hydrate. And the SD rats were fed commonly after analepsia;
4、15 discs were taken out respectively from 3 SD rats after 3 and 7days randomly, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last the discs were embedded in paraffin and sectioned to 4-6μm for safranineO stain;
5、25 discs were taken out respectively after 3 and 7days randomly, and rinsed by high osmotic saline solution for 3 times.250ug nucleus pulposus were taken out from 5 discs, then decomposed by Trizol for PCR.
Result:
1、The architectonic of matrix was decomposed in safranineO stain at 3 and 7 day. The result of image analysis was different statistically at 0,3 and 7day (P< 0.01);
2、RT-PCR results showed that the mRNA of Collage I increased with time, but the expressions of Collage II, aggrecan and decorin decreased, showing statistically significant differences in the mRNA expressions of the matrix protein at each time point (P<0.05).
Conclusion:
The method and the location of culturing the whole intervertebral disc in the body should be discussed carefully later.
椎间盘退变的病因学十分复杂,危险因素颇多,我们对它的研究也在逐步的深入。有许多实验方法和实验模型被应用于研究椎间盘的功能,这些实验模型包括在体动物模型及细胞离体培养模型等。在动物模型中,椎间盘退变通常由力学或化学方法诱导,但它不利于探究与细胞及基质代谢有关的疾病和信号传导方面的问题；许多研究者利用体外细胞培养进行研究,但失去细胞基质的细胞会发生分化或丧失功能,达不到研究的目的。
如果能建立一种椎间盘器官整体培养的方法,就可以确保髓核细胞及其周围基质环境的完整,使细胞与细胞间、基质内部及细胞与基质间的相互作用成为可能,有利于细胞表型及功能的表达,还为研究基质间信号的传导和椎间盘对外界刺激的反应创造了一个实验平台,在椎间盘退变研究方面有一定优势,所以有必要椎间盘器官整体培养方法作相关探讨。
第一章体外培养椎间盘器官
目的：
对大鼠椎间盘包括上下软骨终板、纤维环及髓核组织进行整体培养,观察髓核组织的变化,探索椎间盘器官整体培养的实用技术。
方法：
1、取健康清洁级SD大鼠60只,5-6周龄,150g左右,无菌条件下完整取出腰段椎间盘器官(包括髓核组织、纤维环、上下软骨终板及少量附着于终板的松质骨)共350个,随机分为5组；
2、无菌条件下高渗肝素盐水冲洗椎间盘3次,2.5倍放大镜下进一步修整后置入12孔培养皿内,37℃、5%C02条件下培养,高渗DMEM／F12培养液通过每1000ml等渗培养液内加入3.72克NaCL获得,小牛血清浓度：20%,每天换液一次；
3、任选10个椎间盘器官,随机分为两组,对照组5个椎间盘在培养前置入液氮内浸泡3次,每次浸泡1分钟左右,随后在两组椎间盘的培养液内均加入浓度为10mg／ml的NBT液240μl,吹打混匀,培养8小时后取出两组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片观察；
4、任取75个椎间盘,随机分成5组,每组15个椎间盘,分别在培养的第0、3、7、14、21天取出1组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片观察；取出椎间盘前8小时在培养液内均加入浓度为10mg/ml的NBT液240μl,吹打混匀；
5、任取10个椎间盘,随机分为5组,每组2个椎间盘,分别在在培养的第0、3、7、14、21天取出1组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片,厚度4-6μm,免疫组织化学染色观察；
6、任取75个椎间盘,随机分成5组,每组15个椎间盘,分别在培养的第0、3、7、14、21天取出1组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片,厚度4-6μm,HE染色观察；
7、任取75个椎间盘,随机分成5组,每组15个椎间盘,分别在培养的第0、3、7、14、21天取出1组椎间盘,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片,厚度4-6μm,番红O染色观察；
8、任取100个椎间盘,随机分成4组,每组25个椎间盘,分别在培养的第0、3、7、14天取出1组椎间盘,高渗盐水冲洗3次,无菌条件下剖开椎间盘,每5个椎间盘取出1组髓核组织,每组重量约250μg,Trizol试剂裂解细胞,PCR测试基质蛋白表达。
结果：
1、可通过解剖获得完整无损的SD大鼠椎间盘器官,但软骨终板表面残留少量松质骨组织；
2、NBT可对存活椎间盘细胞着色,而对死亡椎间盘细胞不着色；
3、椎间盘器官体外培养两周内,髓核细胞成活率与对照组培养0天细胞成活率比较无统计学意义(P>0.05),培养21 d时成活率显著低于其余各时间点(P<0.01)：
4、器官培养各时间点免疫组织化学染色均显示髓核细胞内含大量Ⅱ型胶原；
5、椎间盘器官体外培养两周内,HE染色显示髓核组织结构完整,培养21天组髓核组织结构分解,细胞散在分布,完整性消失；
6、椎间盘器官体外培养两周内,番红O染色显示基质结构完整,培养21天组显示基质染色不均匀,完整性消失,图像分析结果显示：培养14天内各时间点灰度值比较无统计学意义(P>0.05),培养21天组与其他各时间点灰度值比较均有统计学意义(P<0.05)；
7、随着培养时间的延长,Ⅰ型胶原的mRNA表达呈上升趋势,而Ⅱ型胶原、核心蛋白聚糖和聚集蛋白聚糖的mRNA表达呈下降趋势,使用单因素方差分析结果显示各组间相比差异均有统计学意义。
结论：1、NBT染料可用于鉴别器官培养条件下细胞成活与否；2、利用高渗培养液可对椎间盘器官进行整体培养,为椎间盘生理与病理研究提供了一种理想模型
第二章皮下埋植培养大鼠椎间盘器官的可行性研究
目的：
体内皮下埋植培养椎间盘器官的可行性研究
方法：
1、取健康清洁级SD大鼠32只,5-6周龄,150g左右,无菌条件下完整取出腰段椎间盘器官(包括髓核组织、纤维环、上下软骨终板及少量附着于终板的松质骨)160个,随机分为6组；
2、无菌条件下高渗肝素盐水冲洗椎间盘3次,2.5倍放大镜下进一步修整,置入含高渗培养液的培养皿,搁置于CO2孵箱内备用；
3、取健康清洁级SD大鼠16只,5-6周龄,150克左右,5%水合氯醛麻醉,无菌条件下把已完整取出备用的椎间盘器官5个为1组埋植于麻醉大鼠背部皮下组织内,待大鼠苏醒后常规养殖；
4、于培养的第3、7天各任选3只大鼠取出椎间盘器官15个,高渗盐水冲洗3次,10%福尔马林固定48小时,石蜡包埋,切片,厚度4-6μm,番红O染色观察；
5、于培养的第3、7天各任选5只大鼠取出椎间盘器官25个,高渗盐水冲洗3次,无菌条件下剖开椎间盘,每5个椎间盘取出1组髓核组织,每组重量约250μg,Trizol试剂裂解细胞,PCR测试基质蛋白表达。
结果：
1、培养3、7天组番红O染色显示基质结构分解,染色极不均匀,图像分析结果显示培养0、3、7天组两两比较有统计学差异(P<0.01)；
2、随着培养时间的延长,Ⅰ型胶原的mRNA表达呈上升趋势,而Ⅱ型胶原、核心蛋白聚糖和聚集蛋白聚糖的mRNA表达呈下降趋势。使用单因素方差分析结果显示各组间相比差异均有统计学意义(P<0.01)
结论：
体内椎间盘器官培养的条件及理想部位尚需进一步探讨
Intervertebral disc degeneration is the important reason of low back pain and the damage of spine. Intervertebral disc degeneration could lead to disc herniation、spinal stenosis and spondylolishesis, etc. More than 90% spine operations are concerned with disc degeneration.
The etiology of disc degeneration is very complex, and our research on this topic is moving forward gradually. Many methods and models were used for the experiments including in vivo system and/or in vitro system. Disc degeneration could be induced by mechanical or chemical method, but it could not be used for the test about cell metabolism and signal conduction. Cell culture is a method of in vitro experiment, but the cell without matrix could not present its phenotype.
A system of organ culture should be built to make sure the integrity of intervertebral disc and the interaction between cells and matrix. It has advantage in cell culture and signal conduct.
Part 1 Organ culture of the whole intervertebral disc in vitro
Objective:
To find a practical method of culturing discs organ system by observing the changes of the nucleus pulposus after the whole intervertebral discs, including cartilage eng-plate nucleus,pulposus and anulus fibrosus, were cultivated.
Methods:
1、A total of 350 intervertebral discs were taken out completely from 60 healthy SD rats (about 150g) aged 5-6 weeks of clear grade and divided into 5 groups randomly. The whole intervertebral disc includes nucleus pulposus. annular fibrosus. cartilagineus end plate and little cancellated bone adhering end plate;
2、The discs were rinsed by high osmotic saline solution containing heparin for 3 times and trimmed with microscopic spectacles, then put into the culture plate. The whole intervertebral discs were cultured with high osmotic culture medium and this medium was changed once every day. The high osmotic culture medium was made by adding 3.72g NaCL to 1000ml DMEM/F12 culture medium. Culture condition is 37℃,5%CO2. The concentration of fetal bovine serum is 20%;
3、10 discs were selected randomly and divided into 5 groups randomly, and the 5 discs of control group were soaked in liquid nitrogen for 3 times(1 minute per time).240μl NBT was added into the culture medium. The discs were taken out after 8 hours, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, The discs were embedded in paraffin, then sectioned for observation.
4.75 discs were selected randomly and divided into 5 groups randomly and cultured in the medium. Each group has 15 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin, sectioned for observation. 240μl NBT was added into the culture medium 8 hours before the discs were taken out.
5、10 discs were selected randomly and divided into 5 groups randomly, then cultured in the medium. Each group has 2 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin and sectioned to 4-6μm for immunohistochemistry test;
6、75 discs were selected randomly and divided into 5 groups randomly, then cultured in the medium. Each group has 15 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin and sectioned to 4-6μm for HE stain;
7、75 discs were selected randomly and divided into 5 groups randomly, then cultured in the medium. Each group has 15 discs. The discs of one group were taken out respectively after 0,3,7,14 and 21 days, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last, the discs were embedded in paraffin and sectioned to 4-6μm for safranineO stain;
8、100 discs were selected randomly and divided into 4 groups randomly, then cultured in the medium. Each group has 25 discs. The discs of one group were taken out respectively after 0,3,7 and 14 days, and rinsed by high osmotic saline solution for 3 times.250μg nucleus pulposus were taken out from 5 discs, then decomposed by Trizol for PCR.
Result:
1、The whole intervertebral disc organ could be dissected from the spine of SD rat except for little cancellated bone adhering end plate;
2、NBT could stain live disc cells, but couldn't stain dead cells;
3、The cell viability was not changed significantly at 14 day (P> 0.05) and was significantly lower at 21 days than at other time points (P< 0.01).
4、The immunohistochemistry staining results for collageⅡwere positive in nucleus pulposus cells at every time point.
5、HE staining showed that the tissue integrity and morphology of the whole intervertebral discs were not changed at 14 days
6、SafranineO staining showed no significant differences in the matrix grey scale (P> 0.05) at 0-14 days and significant differences between at 21 day and at 0-14 days (P< 0.05).
7、RT-PCR results showed that the mRNA of CollageⅠincreased with time, but the expressions of CollageⅡ, aggrecan and decorin decreased, showing statistically significant differences in the mRNA expressions of the matrix protein at each time point (P<0.05).
Conclusion:
1、NBT could be used to detect live disc cells;
2、Our system could be used to cultivate the whole intervertebral discs, it is an ideal model for further studies on physiology and pathology of intervertebral discs.
Part 2 Organ culture of the whole intervertebral disc in subcutaneous tissue of SD rat
Objective:
Is subcutaneous tissue of SD rat a good area for Organ culture of the whole intervertebral disc?
Methods:
1、A total of 160 intervertebral discs were taken out completely from 32 healthy SD rats (about 150g) aged 5-6 weeks of clear grade and divided into 6 groups randomly. The whole intervertebral disc includes nucleus pulposus、annular fibrosus、cartilagineus end plate and little cancellated bone adhering end plate;
2、The discs were rinsed by high osmotic saline solution containing heparin for 3 times and trimmed with microscopic spectacles, then put into the culture plate in culture condition for using later;
3、The discs were imbedded in the subcutaneous tissue of 16 healthy SD rats anesthetized by 10% Chloral Hydrate. And the SD rats were fed commonly after analepsia;
4、15 discs were taken out respectively from 3 SD rats after 3 and 7days randomly, and rinsed by high osmotic saline solution for 3 times, then soaked in 10% neutral-buffered formalin for 48 hours. At last the discs were embedded in paraffin and sectioned to 4-6μm for safranineO stain;
5、25 discs were taken out respectively after 3 and 7days randomly, and rinsed by high osmotic saline solution for 3 times.250ug nucleus pulposus were taken out from 5 discs, then decomposed by Trizol for PCR.
Result:
1、The architectonic of matrix was decomposed in safranineO stain at 3 and 7 day. The result of image analysis was different statistically at 0,3 and 7day (P< 0.01);
2、RT-PCR results showed that the mRNA of Collage I increased with time, but the expressions of Collage II, aggrecan and decorin decreased, showing statistically significant differences in the mRNA expressions of the matrix protein at each time point (P<0.05).
Conclusion:
The method and the location of culturing the whole intervertebral disc in the body should be discussed carefully later.
引文
[1]Heinzelmann J, Enke U, Seyfarth L, et al.:Development of a human model to study homing behavior of immune cells into decidua and placental villi under ex vivo conditions. Am J Reprod Immunol 2009;61:19-25.
[2]Roughley PJ:Biology of intervertebral disc aging and degeneration: involvement of the extracellular matrix. Spine (Phila Pa 1976) 2004;29:2691-2699.
[3]Callaghan JP, McGill SM:Intervertebral disc herniation:studies on a porcine model exposed to highly repetitive flexion/extension motion with compressive force. Clin Biomech (Bristol, Avon) 2001;16:28-37.
[4]Lotz JC, Colliou OK, Chin JR, et al.:Compression-induced degeneration of the intervertebral disc:an in vivo mouse model and finite-element study. Spine (Phila Pa 1976) 1998;23:2493-2506.
[5]Kroeber MW, Unglaub F, Wang H, et al.:New in vivo animal model to create intervertebral disc degeneration and to investigate the effects of therapeutic strategies to stimulate disc regeneration. Spine (Phila Pa 1976) 2002;27:2684-2690.
[6]Unglaub F, Guehring T, Lorenz H, et al.:Effects of unisegmental disc compression on adjacent segments:an in vivo animal model. Eur Spine J 2005;14:949-955.
[7]Anderson DG, Izzo MW, Hall DJ, et al.:Comparative gene expression profiling of normal and degenerative discs:analysis of a rabbit annular laceration model. Spine (Phila Pa 1976) 2002;27:1291-1296.
[8]Cs-Szabo G, Ragasa-San Juan D, Turumella V, et al.:Changes in mRNA and protein levels of proteoglycans of the anulus fibrosus and nucleus pulposus during intervertebral disc degeneration. Spine (Phila Pa 1976) 2002;27:2212- 2219.
[9]Bradford DS, Cooper KM, Oegema TR, Jr.:Chymopapain, chemonucleolysis, and nucleus pulposus regeneration. J Bone Joint Surg Am 1983;65:1220-1231.
[10]Norcross JP, Lester GE, Weinhold P, Dahners LE:An in vivo model of degenerative disc disease. J Orthop Res 2003;21:183-188.
[11]Iatridis JC, Mente PL, Stokes IA, et al.:Compression-induced changes in intervertebral disc properties in a rat tail model. Spine (Phila Pa 1976) 1999;24: 996-1002.
[12]Stern S, Lindenhayn K, Perka C:Human intervertebral disc cell culture for disc disorders. Clin Orthop Relat Res 2004:238-244.
[13]Kasra M, Goel V, Martin J, et al.:Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells. J Orthop Res 2003;21:597-603.
[14]Olmarker K:Neovascularization and neoinnervation of subcutaneously placed nucleus pulposus and the inhibitory effects of certain drugs. Spine (Phila Pa 1976) 2005;30:1501-1504.
[15]Chen WH, Liu HY, Lo WC, et al.:Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma. Biomaterials 2009;30:5523-5533.
[16]Urban JP, Smith S, Fairbank JC:Nutrition of the intervertebral disc. Spine (Phila Pa 1976) 2004;29:2700-2709.
[17]Risbud MV, Izzo MW, Adams CS, et al.:An organ culture system for the study of the nucleus pulposus:description of the system and evaluation of the cells. Spine (Phila Pa 1976) 2003;28:2652-2658; discussion 2658-2659.
[18]Ariga K, Yonenobu K, Nakase T, et al.:Mechanical stress-induced apoptosis of endplate chondrocytes in organ-cultured mouse intervertebral discs:an ex vivo study. Spine (Phila Pa 1976) 2003;28:1528-1533.
[19]Lim TH, Ramakrishnan PS, Kurriger GL, et al.:Rat spinal motion segment in organ culture:a cell viability study. Spine (Phila Pa 1976) 2006;31:1291-1297; discussion 1298.
[20]Lee CR, Iatridis JC, Poveda L, Alini M:In vitro organ culture of the bovine intervertebral disc:effects of vertebral endplate and potential for mechanobiology studies. Spine (Phila Pa 1976) 2006;31:515-522.
[21]Haschtmann D, Stoyanov JV, Ettinger L, et al.:Establishment of a novel intervertebral disc/endplate culture model:analysis of an ex vivo in vitro whole-organ rabbit culture system. Spine (Phila Pa 1976) 2006;31:2918-2925.
[22]Gantenbein B, Grunhagen T, Lee CR, et al.:An in vitro organ culturing system for intervertebral disc explants with vertebral endplates:a feasibility study with ovine caudal discs. Spine (Phila Pa 1976) 2006;31:2665-2673.
[23]Korecki CL, Costi JJ, Iatridis JC:Needle puncture injury affects intervertebral disc mechanics and biology in an organ culture model. Spine (Phila Pa 1976) 2008;33:235-241.
[24]Richardson SM, Walker RV, Parker S, et al.:Intervertebral disc cell-mediated mesenchymal stem cell differentiation. Stem Cells 2006;24:707-716.
[25]Mavrogonatou E, Kletsas D:High osmolality activates the G1 and G2 cell cycle checkpoints and affects the DNA integrity of nucleus pulposus intervertebral disc cells triggering an enhanced DNA repair response. DNA Repair (Amst) 2009;8:930-943.
[26]Zhang Y, Phillips FM, Thonar EJ, et al.:Cell therapy using articular chondrocytes overexpressing BMP-7 or BMP-10 in a rabbit disc organ culture model. Spine (Phila Pa 1976) 2008;33:831-838.
[27]Larsson K, Rydevik B, Olmarker K:Disc related cytokines inhibit axonal outgrowth from dorsal root ganglion cells in vitro. Spine (Phila Pa 1976) 2005;30:621-624.
[28]Aoki Y, Akeda K, An H, et al.:Nerve fiber ingrowth into scar tissue formed following nucleus pulposus extrusion in the rabbit anular-puncture disc degeneration model:effects of depth of puncture. Spine (Phila Pa 1976) 2006;31:E774-780.
[1]Larson JW,3rd, Levicoff EA, Gilbertson LG, Kang JD:Biologic modification of animal models of intervertebral disc degeneration. J Bone Joint Surg Am 2006;88 Suppl 2:83-87.
[2]Roberts S, Menage J, Urban JP:Biochemical and structural properties of the cartilage end-plate and its relation to the intervertebral disc. Spine (Phila Pa 1976) 1989; 14:166-174.
[3]Chelberg MK, Banks GM, Geiger DF, Oegema TR, Jr.:Identification of heterogeneous cell populations in normal human intervertebral disc. J Anat 1995;186(Pt1):43-53.
[4]Marchand F, Ahmed AM:Investigation of the laminate structure of lumbar disc anulus fibrosus. Spine (Phila Pa 1976) 1990;15:402-410.
[5]Roberts S, Eisenstein SM, Menage J, et al.:Mechanoreceptors in intervertebral discs. Morphology, distribution, and neuropeptides. Spine (Phila Pa 1976) 1995;20:2645-2651.
[6]Donohue PJ, Jahnke MR, Blaha JD, Caterson B:Characterization of link protein(s) from human intervertebral-disc tissues. Biochem J 1988;251:739-747.
[7]Boos N, Weissbach S, Rohrbach H, et al.:Classification of age-related changes
in lumbar intervertebral discs:2002 Volvo Award in basic science. Spine (Phila Pa 1976) 2002;27:2631-2644.
[8]Johnson WE, Eisenstein SM, Roberts S:Cell cluster formation in degenerate lumbar intervertebral discs is associated with increased disc cell proliferation. Connect Tissue Res 2001;42:197-207.
[9]Suzuki A, Yamakawa M, Tsukamoto M:The adhesion molecules,1-selectin and sialyl lewis x, relate to the formation of the follicular dendritic cell-lymphocyte cluster in the mantle zone. Immunol Lett 2001;79:181-187.
[10]Roberts S, Caterson B, Menage J, et al.:Matrix metalloproteinases and aggrecanase:their role in disorders of the human intervertebral disc. Spine (Phila Pa 1976) 2000;25:3005-3013.
[11]Hilton RC, Ball J, Benn RT:Vertebral end-plate lesions (Schmorl's nodes) in the dorsolumbar spine. Ann Rheum Dis 1976;35:127-132.
[12]Matsuo H:[Venographic findings on changes in the lumbar anterior internal vertebral vein with regard to age]. Nippon Ika Daigaku Zasshi 1985;52:633-641.
[13]Twomey L, Taylor J:Age changes in lumbar intervertebral discs. Acta Orthop Scand 1985;56:496-499.
[14]Gruber HE, Ingram JA, Leslie K, et al.:Cell shape and gene expression in human intervertebral disc cells:in vitro tissue engineering studies. Biotech Histochem 2003;78:109-117.
[15]Johnson WE, Roberts S:Human intervertebral disc cell morphology and cytoskeletal composition:a preliminary study of regional variations in health and disease. J Anat 2003;203:605-612.
[16]Kim KW, Lim TH, Kim JG, et al.:The origin of chondrocytes in the nucleus pulposus and histologic findings associated with the transition of a notochordal
nucleus pulposus to a fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs. Spine (Phila Pa 1976) 2003;28:982-990.
[17]Andersson GB, An HS, Oegema TR, Jr., Setton LA:Directions for future research. J Bone Joint Surg Am 2006;88 Suppl 2:110-114.
[18]Eyre DR, Matsui Y, Wu JJ:Collagen polymorphisms of the intervertebral disc. Biochem Soc Trans 2002;30:844-848.
[19]Sztrolovics R, Alini M, Roughley PJ, Mort JS:Aggrecan degradation in human intervertebral disc and articular cartilage. Biochem J 1997;326 (Pt 1):235-241.
[20]Eyre DR, Wu JJ, Fernandes RJ, et al.:Recent developments in cartilage research:matrix biology of the collagen II/IX/XI heterofibril network. Biochem Soc Trans 2002;30:893-899.
[21]Xu L, Peng H, Wu D, et al.:Activation of the discoidin domain receptor 2 induces expression of matrix metalloproteinase 13 associated with osteoarthritis in mice. J Biol Chem 2005;280:548-555.
[22]Sjoberg A, Onnerfjord P, Morgelin M, et al.:The extracellular matrix and inflammation:fibromodulin activates the classical pathway of complement by directly binding Clq. J Biol Chem 2005;280:32301-32308.
[23]Holm S, Maroudas A, Urban JP, et al.:Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res 1981;8:101-119.
[24]Ishihara H, Urban JP:Effects of low oxygen concentrations and metabolic inhibitors on proteoglycan and protein synthesis rates in the intervertebral disc. J Orthop Res 1999;17:829-835.
[25]Zhang Y, An HS, Song S, et al.:Growth factor osteogenic protein-1:differing effects on cells from three distinct zones in the bovine intervertebral disc. Am J Phys Med Rehabil 2004;83:515-521.
[26]Bibby SR, Jones DA, Ripley RM, Urban JP:Metabolism of the intervertebral
disc:effects of low levels of oxygen, glucose, and pH on rates of energy metabolism of bovine nucleus pulposus cells. Spine (Phila Pa 1976) 2005;30:487-496.
[27]Lee RB, Wilkins RJ, Razaq S, Urban JP:The effect of mechanical stress on cartilage energy metabolism. Biorheology 2002;39:133-143.
[28]Razaq S, Wilkins RJ, Urban JP:The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus. Eur Spine J 2003;12:341-349.
[29]Bernick S, Cailliet R:Vertebral end-plate changes with aging of human vertebrae. Spine (Phila Pa 1976) 1982;7:97-102.
[30]Roberts S, Urban JP, Evans H, Eisenstein SM:Transport properties of the human cartilage endplate in relation to its composition and calcification. Spine (Phila Pa 1976) 1996;21:415-420.
[31]Urban MR, Fairbank JC, Etherington PJ, et al.:Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer. Spine (Phila Pa 1976) 2001;26:984-990.
[32]Bibby SR, Fairbank JC, Urban MR, Urban JP:Cell viability in scoliotic discs in relation to disc deformity and nutrient levels. Spine (Phila Pa 1976) 2002;27:2220-2228; discussion 2227-2228.
[33]Alini M, Roughley PJ, Antoniou J, et al.:A biological approach to treating disc degeneration:not for today, but maybe for tomorrow. Eur Spine J 2002; 11 Suppl 2:S215-220.
[34]Masuda K, Oegema TR, Jr., An HS:Growth factors and treatment of intervertebral disc degeneration. Spine (Phila Pa 1976) 2004;29:2757-2769.
[35]Shimer AL, Chadderdon RC, Gilbertson LG, Kang JD:Gene therapy approaches for intervertebral disc degeneration. Spine (Phila Pa 1976)
2004;29:2770-2778.
[36]Hamilton DJ, Seguin CA, Wang J, et al.:Formation of a nucleus pulposus-cartilage endplate construct in vitro. Biomaterials 2006;27:397-405.
[37]Grunhagen T, Wilde G, Soukane DM, et al.:Nutrient supply and intervertebral disc metabolism. J Bone Joint Surg Am 2006;88 Suppl 2:30-35.
[38]Paonessa KJ, Engler GL:Back pain and disability after Harrington rod fusion to the lumbar spine for scoliosis. Spine (Phila Pa 1976) 1992;17:S249-253.
[39]Urban JP, Maroudas A:Swelling of the intervertebral disc in vitro. Connect Tissue Res 1981;9:1-10.
[40]Iatridis JC, Mente PL, Stokes IA, et al.:Compression-induced changes in intervertebral disc properties in a rat tail model. Spine (Phila Pa 1976) 1999;24:996-1002.
[41]Ohshima H, Urban JP, Bergel DH:Effect of static load on matrix synthesis rates in the intervertebral disc measured in vitro by a new perfusion technique. J Orthop Res 1995; 13:22-29.
[42]Maclean JJ, Lee CR, Alini M, Iatridis JC:Anabolic and catabolic mRNA levels of the intervertebral disc vary with the magnitude and frequency of in vivo dynamic compression. J Orthop Res 2004;22:1193-1200.
[43]Walsh AJ, Lotz JC:Biological response of the intervertebral disc to dynamic loading. J Biomech 2004;37:329-337.
[44]Chen J, Yan W, Setton LA:Static compression induces zonal-specific changes in gene expression for extracellular matrix and cytoskeletal proteins in intervertebral disc cells in vitro. Matrix Biol 2004;22:573-583.
[45]Ishihara H, Warensjo K, Roberts S, Urban JP:Proteoglycan synthesis in the intervertebral disk nucleus:the role of extracellular osmolality. Am J Physiol 1997;272:C1499-1506.
[46]Pritchard S, Erickson GR, Guilak F:Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells:the role of the actin cytoskeleton. Biophys J 2002;83:2502-2510.
[47]Battie MC, Videman T:Lumbar disc degeneration:epidemiology and genetics. J Bone Joint Surg Am 2006;88 Suppl 2:3-9.
[48]Battie MC, Videman T, Gill K, et al.:1991 Volvo Award in clinical sciences. Smoking and lumbar intervertebral disc degeneration:an MRI study of identical twins. Spine (Phila Pa 1976) 1991;16:1015-1021.
[49]Battie MC, Videman T, Parent E:Lumbar disc degeneration:epidemiology and genetic influences. Spine (Phila Pa 1976) 2004;29:2679-2690.
[50]Videman T, Leppavuori J, Kaprio J, et al.:Intragenic polymorphisms of the vitamin D receptor gene associated with intervertebral disc degeneration. Spine (Phila Pa 1976) 1998;23:2477-2485.
[51]Frymoyer JW:Lumbar disk disease:epidemiology. Instr Course Lect 1992;41:217-223.
[52]Evans CH, Rosier RN:Molecular biology in orthopaedics:the advent of molecular orthopaedics. J Bone Joint Surg Am 2005;87:2550-2564.
[53]Evans C:Potential biologic therapies for the intervertebral disc. J Bone Joint Surg Am 2006;88 Suppl 2:95-98.
[54]Phillips FM, An H, Kang JD, et al.:Biologic treatment for intervertebral disc degeneration:summary statement. Spine (Phila Pa 1976) 2003;28:S99.
[55]Evans CH, Ghivizzani SC, Herndon JH, Robbins PD:Gene therapy for the treatment of musculoskeletal diseases. J Am Acad Orthop Surg 2005; 13:230-242.
[56]Kalota A, Shetzline SE, Gewirtz AM:Progress in the development of nucleic acid therapeutics for cancer. Cancer Biol Ther 2004;3:4-12.
[57]Higuchi M, Abe K, Kaneda K:Changes in the nucleus pulposus of the intervertebral disc in bipedal mice. A light and electron microscopic study. Clin Orthop Relat Res 1983:251-257.
[58]Scott NA, Harris PF, Bagnall KM:A morphological and histological study of the postnatal development of intervertebral discs in the lumbar spine of the rabbit. J Anat 1980;130:75-81.
[59]Trout JJ, Buckwalter JA, Moore KC, Landas SK:Ultrastructure of the human intervertebral disc. I. Changes in notochordal cells with age. Tissue Cell 1982;14:359-369.
[60]Risbud MV, Izzo MW, Adams CS, et al.:An organ culture system for the study of the nucleus pulposus:description of the system and evaluation of the cells. Spine (Phila Pa 1976) 2003;28:2652-2658; discussion 2658-2659.
[61]Gruber HE, Hanley EN, Jr.:Analysis of aging and degeneration of the human intervertebral disc. Comparison of surgical specimens with normal controls. Spine (Phila Pa 1976) 1998;23:751-757.
[62]Okuda S, Myoui A, Ariga K, et al.:Mechanisms of age-related decline in insulin-like growth factor-I dependent proteoglycan synthesis in rat intervertebral disc cells. Spine (Phila Pa 1976) 2001;26:2421-2426.
[63]Chiba K, Andersson GB, Masuda K, Thonar EJ:Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate. Spine (Phila Pa 1976) 1997;22:2885-2893.
[64]Gruber HE, Fisher EC, Jr., Desai B, et al.:Human intervertebral disc cells from the annulus:three-dimensional culture in agarose or alginate and responsiveness to TGF-betal. Exp Cell Res 1997;235:13-21.
[65]Aota Y, An HS, Homandberg G, et al.:Differential effects of fibronectin fragment on proteoglycan metabolism by intervertebral disc cells:a
comparison with articular chondrocytes. Spine (Phila Pa 1976) 2005;30:722-728.
[66]Thompson JP, Oegema TR, Jr., Bradford DS:Stimulation of mature canine intervertebral disc by growth factors. Spine (Phila Pa 1976) 1991; 16:253-260.
[67]Silberberg R, Aufdermaur M, Adler JH:Degeneration of the intervertebral disks and spondylosis in aging sand rats. Arch Pathol Lab Med 1979;103:231-235.
[68]Adler JH, Schoenbaum M, Silberberg R:Early onset of disk degeneration and spondylosis in sand rats (Psammomys obesus). Vet Pathol 1983;20:13-22.
[69]Gruber HE, Johnson T, Norton HJ, Hanley EN, Jr.:The sand rat model for disc degeneration:radiologic characterization of age-related changes: cross-sectional and prospective analyses. Spine (Phila Pa 1976) 2002;27:230-234.
[70]Masuda K, Aota Y, Muehleman C, et al.:A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture:correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976) 2005;30:5-14.
[71]Lim TH, Ramakrishnan PS, Kurriger GL, et al.:Rat spinal motion segment in organ culture:a cell viability study. Spine (Phila Pa 1976) 2006;31:1291-1297; discussion 1298.
[72]Lee CR, Iatridis JC, Poveda L, Alini M:In vitro organ culture of the bovine intervertebral disc:effects of vertebral endplate and potential for mechanobiology studies. Spine (Phila Pa 1976) 2006;31:515-522.
[73]Haschtmann D, Stoyanov JV, Ettinger L, et al.:Establishment of a novel intervertebral disc/endplate culture model:analysis of an ex vivo in vitro whole-organ rabbit culture system. Spine (Phila Pa 1976) 2006;31:2918-2925.
[74]Gantenbein B, Grunhagen T, Lee CR, et al.:An in vitro organ culturing system for intervertebral disc explants with vertebral endplates:a feasibility study with ovine caudal discs. Spine (Phila Pa 1976) 2006;31:2665-2673.
[75]Kurowski P, Kubo A:The relationship of degeneration of the intervertebral disc to mechanical loading conditions on lumbar vertebrae. Spine (Phila Pa 1976) 1986;11:726-731.
[76]Natarajan RN, Ke JH, Andersson GB:A model to study the disc degeneration process. Spine (Phila Pa 1976) 1994; 19:259-265.
[77]Haughton V:Imaging intervertebral disc degeneration. J Bone Joint Surg Am 2006;88Suppl 2:15-20.
[78]Hsu EW, Setton LA:Diffusion tensor microscopy of the intervertebral disc anulus fibrosus. Magn Reson Med 1999;41:992-999.
[79]Lin CS, Fertikh D, Davis B, et al.:2D CSI proton MR spectroscopy of human spinal vertebra:feasibility studies. J Magn Reson Imaging 2000; 11:287-293.
[80]Schellinger D, Lin CS, Hatipoglu HG, Fertikh D:Potential value of vertebral proton MR spectroscopy in determining bone weakness. AJNR Am J Neuroradiol 2001;22:1620-1627.
[2]Roughley PJ:Biology of intervertebral disc aging and degeneration: involvement of the extracellular matrix. Spine (Phila Pa 1976) 2004;29:2691-2699.
[3]Callaghan JP, McGill SM:Intervertebral disc herniation:studies on a porcine model exposed to highly repetitive flexion/extension motion with compressive force. Clin Biomech (Bristol, Avon) 2001;16:28-37.
[4]Lotz JC, Colliou OK, Chin JR, et al.:Compression-induced degeneration of the intervertebral disc:an in vivo mouse model and finite-element study. Spine (Phila Pa 1976) 1998;23:2493-2506.
[5]Kroeber MW, Unglaub F, Wang H, et al.:New in vivo animal model to create intervertebral disc degeneration and to investigate the effects of therapeutic strategies to stimulate disc regeneration. Spine (Phila Pa 1976) 2002;27:2684-2690.
[6]Unglaub F, Guehring T, Lorenz H, et al.:Effects of unisegmental disc compression on adjacent segments:an in vivo animal model. Eur Spine J 2005;14:949-955.
[7]Anderson DG, Izzo MW, Hall DJ, et al.:Comparative gene expression profiling of normal and degenerative discs:analysis of a rabbit annular laceration model. Spine (Phila Pa 1976) 2002;27:1291-1296.
[8]Cs-Szabo G, Ragasa-San Juan D, Turumella V, et al.:Changes in mRNA and protein levels of proteoglycans of the anulus fibrosus and nucleus pulposus during intervertebral disc degeneration. Spine (Phila Pa 1976) 2002;27:2212- 2219.
[9]Bradford DS, Cooper KM, Oegema TR, Jr.:Chymopapain, chemonucleolysis, and nucleus pulposus regeneration. J Bone Joint Surg Am 1983;65:1220-1231.
[10]Norcross JP, Lester GE, Weinhold P, Dahners LE:An in vivo model of degenerative disc disease. J Orthop Res 2003;21:183-188.
[11]Iatridis JC, Mente PL, Stokes IA, et al.:Compression-induced changes in intervertebral disc properties in a rat tail model. Spine (Phila Pa 1976) 1999;24: 996-1002.
[12]Stern S, Lindenhayn K, Perka C:Human intervertebral disc cell culture for disc disorders. Clin Orthop Relat Res 2004:238-244.
[13]Kasra M, Goel V, Martin J, et al.:Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells. J Orthop Res 2003;21:597-603.
[14]Olmarker K:Neovascularization and neoinnervation of subcutaneously placed nucleus pulposus and the inhibitory effects of certain drugs. Spine (Phila Pa 1976) 2005;30:1501-1504.
[15]Chen WH, Liu HY, Lo WC, et al.:Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma. Biomaterials 2009;30:5523-5533.
[16]Urban JP, Smith S, Fairbank JC:Nutrition of the intervertebral disc. Spine (Phila Pa 1976) 2004;29:2700-2709.
[17]Risbud MV, Izzo MW, Adams CS, et al.:An organ culture system for the study of the nucleus pulposus:description of the system and evaluation of the cells. Spine (Phila Pa 1976) 2003;28:2652-2658; discussion 2658-2659.
[18]Ariga K, Yonenobu K, Nakase T, et al.:Mechanical stress-induced apoptosis of endplate chondrocytes in organ-cultured mouse intervertebral discs:an ex vivo study. Spine (Phila Pa 1976) 2003;28:1528-1533.
[19]Lim TH, Ramakrishnan PS, Kurriger GL, et al.:Rat spinal motion segment in organ culture:a cell viability study. Spine (Phila Pa 1976) 2006;31:1291-1297; discussion 1298.
[20]Lee CR, Iatridis JC, Poveda L, Alini M:In vitro organ culture of the bovine intervertebral disc:effects of vertebral endplate and potential for mechanobiology studies. Spine (Phila Pa 1976) 2006;31:515-522.
[21]Haschtmann D, Stoyanov JV, Ettinger L, et al.:Establishment of a novel intervertebral disc/endplate culture model:analysis of an ex vivo in vitro whole-organ rabbit culture system. Spine (Phila Pa 1976) 2006;31:2918-2925.
[22]Gantenbein B, Grunhagen T, Lee CR, et al.:An in vitro organ culturing system for intervertebral disc explants with vertebral endplates:a feasibility study with ovine caudal discs. Spine (Phila Pa 1976) 2006;31:2665-2673.
[23]Korecki CL, Costi JJ, Iatridis JC:Needle puncture injury affects intervertebral disc mechanics and biology in an organ culture model. Spine (Phila Pa 1976) 2008;33:235-241.
[24]Richardson SM, Walker RV, Parker S, et al.:Intervertebral disc cell-mediated mesenchymal stem cell differentiation. Stem Cells 2006;24:707-716.
[25]Mavrogonatou E, Kletsas D:High osmolality activates the G1 and G2 cell cycle checkpoints and affects the DNA integrity of nucleus pulposus intervertebral disc cells triggering an enhanced DNA repair response. DNA Repair (Amst) 2009;8:930-943.
[26]Zhang Y, Phillips FM, Thonar EJ, et al.:Cell therapy using articular chondrocytes overexpressing BMP-7 or BMP-10 in a rabbit disc organ culture model. Spine (Phila Pa 1976) 2008;33:831-838.
[27]Larsson K, Rydevik B, Olmarker K:Disc related cytokines inhibit axonal outgrowth from dorsal root ganglion cells in vitro. Spine (Phila Pa 1976) 2005;30:621-624.
[28]Aoki Y, Akeda K, An H, et al.:Nerve fiber ingrowth into scar tissue formed following nucleus pulposus extrusion in the rabbit anular-puncture disc degeneration model:effects of depth of puncture. Spine (Phila Pa 1976) 2006;31:E774-780.
[1]Larson JW,3rd, Levicoff EA, Gilbertson LG, Kang JD:Biologic modification of animal models of intervertebral disc degeneration. J Bone Joint Surg Am 2006;88 Suppl 2:83-87.
[2]Roberts S, Menage J, Urban JP:Biochemical and structural properties of the cartilage end-plate and its relation to the intervertebral disc. Spine (Phila Pa 1976) 1989; 14:166-174.
[3]Chelberg MK, Banks GM, Geiger DF, Oegema TR, Jr.:Identification of heterogeneous cell populations in normal human intervertebral disc. J Anat 1995;186(Pt1):43-53.
[4]Marchand F, Ahmed AM:Investigation of the laminate structure of lumbar disc anulus fibrosus. Spine (Phila Pa 1976) 1990;15:402-410.
[5]Roberts S, Eisenstein SM, Menage J, et al.:Mechanoreceptors in intervertebral discs. Morphology, distribution, and neuropeptides. Spine (Phila Pa 1976) 1995;20:2645-2651.
[6]Donohue PJ, Jahnke MR, Blaha JD, Caterson B:Characterization of link protein(s) from human intervertebral-disc tissues. Biochem J 1988;251:739-747.
[7]Boos N, Weissbach S, Rohrbach H, et al.:Classification of age-related changes
in lumbar intervertebral discs:2002 Volvo Award in basic science. Spine (Phila Pa 1976) 2002;27:2631-2644.
[8]Johnson WE, Eisenstein SM, Roberts S:Cell cluster formation in degenerate lumbar intervertebral discs is associated with increased disc cell proliferation. Connect Tissue Res 2001;42:197-207.
[9]Suzuki A, Yamakawa M, Tsukamoto M:The adhesion molecules,1-selectin and sialyl lewis x, relate to the formation of the follicular dendritic cell-lymphocyte cluster in the mantle zone. Immunol Lett 2001;79:181-187.
[10]Roberts S, Caterson B, Menage J, et al.:Matrix metalloproteinases and aggrecanase:their role in disorders of the human intervertebral disc. Spine (Phila Pa 1976) 2000;25:3005-3013.
[11]Hilton RC, Ball J, Benn RT:Vertebral end-plate lesions (Schmorl's nodes) in the dorsolumbar spine. Ann Rheum Dis 1976;35:127-132.
[12]Matsuo H:[Venographic findings on changes in the lumbar anterior internal vertebral vein with regard to age]. Nippon Ika Daigaku Zasshi 1985;52:633-641.
[13]Twomey L, Taylor J:Age changes in lumbar intervertebral discs. Acta Orthop Scand 1985;56:496-499.
[14]Gruber HE, Ingram JA, Leslie K, et al.:Cell shape and gene expression in human intervertebral disc cells:in vitro tissue engineering studies. Biotech Histochem 2003;78:109-117.
[15]Johnson WE, Roberts S:Human intervertebral disc cell morphology and cytoskeletal composition:a preliminary study of regional variations in health and disease. J Anat 2003;203:605-612.
[16]Kim KW, Lim TH, Kim JG, et al.:The origin of chondrocytes in the nucleus pulposus and histologic findings associated with the transition of a notochordal
nucleus pulposus to a fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs. Spine (Phila Pa 1976) 2003;28:982-990.
[17]Andersson GB, An HS, Oegema TR, Jr., Setton LA:Directions for future research. J Bone Joint Surg Am 2006;88 Suppl 2:110-114.
[18]Eyre DR, Matsui Y, Wu JJ:Collagen polymorphisms of the intervertebral disc. Biochem Soc Trans 2002;30:844-848.
[19]Sztrolovics R, Alini M, Roughley PJ, Mort JS:Aggrecan degradation in human intervertebral disc and articular cartilage. Biochem J 1997;326 (Pt 1):235-241.
[20]Eyre DR, Wu JJ, Fernandes RJ, et al.:Recent developments in cartilage research:matrix biology of the collagen II/IX/XI heterofibril network. Biochem Soc Trans 2002;30:893-899.
[21]Xu L, Peng H, Wu D, et al.:Activation of the discoidin domain receptor 2 induces expression of matrix metalloproteinase 13 associated with osteoarthritis in mice. J Biol Chem 2005;280:548-555.
[22]Sjoberg A, Onnerfjord P, Morgelin M, et al.:The extracellular matrix and inflammation:fibromodulin activates the classical pathway of complement by directly binding Clq. J Biol Chem 2005;280:32301-32308.
[23]Holm S, Maroudas A, Urban JP, et al.:Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res 1981;8:101-119.
[24]Ishihara H, Urban JP:Effects of low oxygen concentrations and metabolic inhibitors on proteoglycan and protein synthesis rates in the intervertebral disc. J Orthop Res 1999;17:829-835.
[25]Zhang Y, An HS, Song S, et al.:Growth factor osteogenic protein-1:differing effects on cells from three distinct zones in the bovine intervertebral disc. Am J Phys Med Rehabil 2004;83:515-521.
[26]Bibby SR, Jones DA, Ripley RM, Urban JP:Metabolism of the intervertebral
disc:effects of low levels of oxygen, glucose, and pH on rates of energy metabolism of bovine nucleus pulposus cells. Spine (Phila Pa 1976) 2005;30:487-496.
[27]Lee RB, Wilkins RJ, Razaq S, Urban JP:The effect of mechanical stress on cartilage energy metabolism. Biorheology 2002;39:133-143.
[28]Razaq S, Wilkins RJ, Urban JP:The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus. Eur Spine J 2003;12:341-349.
[29]Bernick S, Cailliet R:Vertebral end-plate changes with aging of human vertebrae. Spine (Phila Pa 1976) 1982;7:97-102.
[30]Roberts S, Urban JP, Evans H, Eisenstein SM:Transport properties of the human cartilage endplate in relation to its composition and calcification. Spine (Phila Pa 1976) 1996;21:415-420.
[31]Urban MR, Fairbank JC, Etherington PJ, et al.:Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer. Spine (Phila Pa 1976) 2001;26:984-990.
[32]Bibby SR, Fairbank JC, Urban MR, Urban JP:Cell viability in scoliotic discs in relation to disc deformity and nutrient levels. Spine (Phila Pa 1976) 2002;27:2220-2228; discussion 2227-2228.
[33]Alini M, Roughley PJ, Antoniou J, et al.:A biological approach to treating disc degeneration:not for today, but maybe for tomorrow. Eur Spine J 2002; 11 Suppl 2:S215-220.
[34]Masuda K, Oegema TR, Jr., An HS:Growth factors and treatment of intervertebral disc degeneration. Spine (Phila Pa 1976) 2004;29:2757-2769.
[35]Shimer AL, Chadderdon RC, Gilbertson LG, Kang JD:Gene therapy approaches for intervertebral disc degeneration. Spine (Phila Pa 1976)
2004;29:2770-2778.
[36]Hamilton DJ, Seguin CA, Wang J, et al.:Formation of a nucleus pulposus-cartilage endplate construct in vitro. Biomaterials 2006;27:397-405.
[37]Grunhagen T, Wilde G, Soukane DM, et al.:Nutrient supply and intervertebral disc metabolism. J Bone Joint Surg Am 2006;88 Suppl 2:30-35.
[38]Paonessa KJ, Engler GL:Back pain and disability after Harrington rod fusion to the lumbar spine for scoliosis. Spine (Phila Pa 1976) 1992;17:S249-253.
[39]Urban JP, Maroudas A:Swelling of the intervertebral disc in vitro. Connect Tissue Res 1981;9:1-10.
[40]Iatridis JC, Mente PL, Stokes IA, et al.:Compression-induced changes in intervertebral disc properties in a rat tail model. Spine (Phila Pa 1976) 1999;24:996-1002.
[41]Ohshima H, Urban JP, Bergel DH:Effect of static load on matrix synthesis rates in the intervertebral disc measured in vitro by a new perfusion technique. J Orthop Res 1995; 13:22-29.
[42]Maclean JJ, Lee CR, Alini M, Iatridis JC:Anabolic and catabolic mRNA levels of the intervertebral disc vary with the magnitude and frequency of in vivo dynamic compression. J Orthop Res 2004;22:1193-1200.
[43]Walsh AJ, Lotz JC:Biological response of the intervertebral disc to dynamic loading. J Biomech 2004;37:329-337.
[44]Chen J, Yan W, Setton LA:Static compression induces zonal-specific changes in gene expression for extracellular matrix and cytoskeletal proteins in intervertebral disc cells in vitro. Matrix Biol 2004;22:573-583.
[45]Ishihara H, Warensjo K, Roberts S, Urban JP:Proteoglycan synthesis in the intervertebral disk nucleus:the role of extracellular osmolality. Am J Physiol 1997;272:C1499-1506.
[46]Pritchard S, Erickson GR, Guilak F:Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells:the role of the actin cytoskeleton. Biophys J 2002;83:2502-2510.
[47]Battie MC, Videman T:Lumbar disc degeneration:epidemiology and genetics. J Bone Joint Surg Am 2006;88 Suppl 2:3-9.
[48]Battie MC, Videman T, Gill K, et al.:1991 Volvo Award in clinical sciences. Smoking and lumbar intervertebral disc degeneration:an MRI study of identical twins. Spine (Phila Pa 1976) 1991;16:1015-1021.
[49]Battie MC, Videman T, Parent E:Lumbar disc degeneration:epidemiology and genetic influences. Spine (Phila Pa 1976) 2004;29:2679-2690.
[50]Videman T, Leppavuori J, Kaprio J, et al.:Intragenic polymorphisms of the vitamin D receptor gene associated with intervertebral disc degeneration. Spine (Phila Pa 1976) 1998;23:2477-2485.
[51]Frymoyer JW:Lumbar disk disease:epidemiology. Instr Course Lect 1992;41:217-223.
[52]Evans CH, Rosier RN:Molecular biology in orthopaedics:the advent of molecular orthopaedics. J Bone Joint Surg Am 2005;87:2550-2564.
[53]Evans C:Potential biologic therapies for the intervertebral disc. J Bone Joint Surg Am 2006;88 Suppl 2:95-98.
[54]Phillips FM, An H, Kang JD, et al.:Biologic treatment for intervertebral disc degeneration:summary statement. Spine (Phila Pa 1976) 2003;28:S99.
[55]Evans CH, Ghivizzani SC, Herndon JH, Robbins PD:Gene therapy for the treatment of musculoskeletal diseases. J Am Acad Orthop Surg 2005; 13:230-242.
[56]Kalota A, Shetzline SE, Gewirtz AM:Progress in the development of nucleic acid therapeutics for cancer. Cancer Biol Ther 2004;3:4-12.
[57]Higuchi M, Abe K, Kaneda K:Changes in the nucleus pulposus of the intervertebral disc in bipedal mice. A light and electron microscopic study. Clin Orthop Relat Res 1983:251-257.
[58]Scott NA, Harris PF, Bagnall KM:A morphological and histological study of the postnatal development of intervertebral discs in the lumbar spine of the rabbit. J Anat 1980;130:75-81.
[59]Trout JJ, Buckwalter JA, Moore KC, Landas SK:Ultrastructure of the human intervertebral disc. I. Changes in notochordal cells with age. Tissue Cell 1982;14:359-369.
[60]Risbud MV, Izzo MW, Adams CS, et al.:An organ culture system for the study of the nucleus pulposus:description of the system and evaluation of the cells. Spine (Phila Pa 1976) 2003;28:2652-2658; discussion 2658-2659.
[61]Gruber HE, Hanley EN, Jr.:Analysis of aging and degeneration of the human intervertebral disc. Comparison of surgical specimens with normal controls. Spine (Phila Pa 1976) 1998;23:751-757.
[62]Okuda S, Myoui A, Ariga K, et al.:Mechanisms of age-related decline in insulin-like growth factor-I dependent proteoglycan synthesis in rat intervertebral disc cells. Spine (Phila Pa 1976) 2001;26:2421-2426.
[63]Chiba K, Andersson GB, Masuda K, Thonar EJ:Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate. Spine (Phila Pa 1976) 1997;22:2885-2893.
[64]Gruber HE, Fisher EC, Jr., Desai B, et al.:Human intervertebral disc cells from the annulus:three-dimensional culture in agarose or alginate and responsiveness to TGF-betal. Exp Cell Res 1997;235:13-21.
[65]Aota Y, An HS, Homandberg G, et al.:Differential effects of fibronectin fragment on proteoglycan metabolism by intervertebral disc cells:a
comparison with articular chondrocytes. Spine (Phila Pa 1976) 2005;30:722-728.
[66]Thompson JP, Oegema TR, Jr., Bradford DS:Stimulation of mature canine intervertebral disc by growth factors. Spine (Phila Pa 1976) 1991; 16:253-260.
[67]Silberberg R, Aufdermaur M, Adler JH:Degeneration of the intervertebral disks and spondylosis in aging sand rats. Arch Pathol Lab Med 1979;103:231-235.
[68]Adler JH, Schoenbaum M, Silberberg R:Early onset of disk degeneration and spondylosis in sand rats (Psammomys obesus). Vet Pathol 1983;20:13-22.
[69]Gruber HE, Johnson T, Norton HJ, Hanley EN, Jr.:The sand rat model for disc degeneration:radiologic characterization of age-related changes: cross-sectional and prospective analyses. Spine (Phila Pa 1976) 2002;27:230-234.
[70]Masuda K, Aota Y, Muehleman C, et al.:A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture:correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976) 2005;30:5-14.
[71]Lim TH, Ramakrishnan PS, Kurriger GL, et al.:Rat spinal motion segment in organ culture:a cell viability study. Spine (Phila Pa 1976) 2006;31:1291-1297; discussion 1298.
[72]Lee CR, Iatridis JC, Poveda L, Alini M:In vitro organ culture of the bovine intervertebral disc:effects of vertebral endplate and potential for mechanobiology studies. Spine (Phila Pa 1976) 2006;31:515-522.
[73]Haschtmann D, Stoyanov JV, Ettinger L, et al.:Establishment of a novel intervertebral disc/endplate culture model:analysis of an ex vivo in vitro whole-organ rabbit culture system. Spine (Phila Pa 1976) 2006;31:2918-2925.
[74]Gantenbein B, Grunhagen T, Lee CR, et al.:An in vitro organ culturing system for intervertebral disc explants with vertebral endplates:a feasibility study with ovine caudal discs. Spine (Phila Pa 1976) 2006;31:2665-2673.
[75]Kurowski P, Kubo A:The relationship of degeneration of the intervertebral disc to mechanical loading conditions on lumbar vertebrae. Spine (Phila Pa 1976) 1986;11:726-731.
[76]Natarajan RN, Ke JH, Andersson GB:A model to study the disc degeneration process. Spine (Phila Pa 1976) 1994; 19:259-265.
[77]Haughton V:Imaging intervertebral disc degeneration. J Bone Joint Surg Am 2006;88Suppl 2:15-20.
[78]Hsu EW, Setton LA:Diffusion tensor microscopy of the intervertebral disc anulus fibrosus. Magn Reson Med 1999;41:992-999.
[79]Lin CS, Fertikh D, Davis B, et al.:2D CSI proton MR spectroscopy of human spinal vertebra:feasibility studies. J Magn Reson Imaging 2000; 11:287-293.
[80]Schellinger D, Lin CS, Hatipoglu HG, Fertikh D:Potential value of vertebral proton MR spectroscopy in determining bone weakness. AJNR Am J Neuroradiol 2001;22:1620-1627.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |