持续压力负荷离体兔脊柱运动节段软骨终板的应力变化
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摘要
背景:研究表明软骨组织的改变将会影响椎间盘的功能。目的:观察持续压力负荷对离体兔脊柱运动节段软骨终板的影响。方法:将16只新西兰白兔处死后在无菌条件下取出脊柱运动节段,随机分为对照组(无压力)与压力组(持续29.4N压力),利用离体加载和培养装置进行离体培养,于培养前及培养后第3,7,14天,取2组兔终板软骨组织各10个样本,苏木精-伊红染色进行组织学观察,免疫组织化学检测基质金属蛋白酶13、Ⅱ型胶原、蛋白多糖表达。结果与结论:①压力组培养至14d时椎间盘终板软骨组织中基质染色不均匀,数量减少,软骨组织有退变趋势,与对照组相比有明显改变;②培养14 d,与培养前相比压力组增强了终板内基质金属蛋白酶13蛋白的表达,基质金属蛋白酶13蛋白平均吸光度值显著高于对照组(P <0.05);③培养3 d,压力组Ⅱ型胶原平均吸光度值显著高于对照组(P<0.05);培养7,14d,压力组Ⅱ型胶原染色较培养前强度明显降低,但与对照组相比无显著差异;④培养3,7,14 d,2组蛋白多糖染色变浅(P <0.05);培养7 d压力组蛋白多糖平均吸光度值显著低于对照组(P <0.05);⑤结果说明,持续压力会导致离体培养椎间盘模型终板退变,这一结果会为椎间盘退变过程中软骨损伤及早期防治提供有意义的数据。
BACKGROUND: Changes of cartilage tissues have been shown to affect the function of intervertebral disc.OBJECTIVE: To observe the effect of continuous pressure load on the cartilage endplate of isolated rabbit spinal segment.METHODS: After the 16 New Zealand white rabbits were killed, the spinal movement segments were removed under the aseptic condition and randomly divided into control group(no pressure) and pressure group(continuous 29.4 N of pressure). The isolated culture was used in vitro and cultured in vitro. Before and after cultured for 3, 7 and 14 days, the 10 cartilage endplates in each group were removed for hematoxylin-eosin staining and expression of matrix metalloproteinase 13, collagen type II and proteoglycan were detected by immunohistochemistry, respectively.RESULTS AND CONCLUSION:(1) After 14 days of culture, the cartilage structure of the intervertebral disc endplate was almost intact, the number was reduced and tended to degenerate, which showed no significant changes compared with the control group.(2) The expression of matrix metalloproteinase 13 at 14 days after culture in the pressure group was increased compared with the baseline. The average absorbance value of matrix metalloproteinase 13 in the pressure group was significantly higher than that in the control group(P < 0.05).(3)The average absorbance value of collagen type II in the pressure group was significantly higher than that in the control group at 3 days after culture(P < 0.05). The expression of collage type II after 1 and 7 days of culture in the pressure group was significantly decreased compared with the baseline, which showed no significant difference from the control group.(4) The proteoglycan staining became slight in both groups at3, 7 and 14 days after culture(P < 0.05). The average absorbance value of proteoglycan in the pressure group was significantly higher than that in the control group at 7 days(P < 0.05).(5) In summary, continuous pressure will lead to degeneration of endplate in vitro culture, which provides evidence for the early prevention and treatment of cartilage injury in intervertebral disc degeneration.
BACKGROUND: Changes of cartilage tissues have been shown to affect the function of intervertebral disc.OBJECTIVE: To observe the effect of continuous pressure load on the cartilage endplate of isolated rabbit spinal segment.METHODS: After the 16 New Zealand white rabbits were killed, the spinal movement segments were removed under the aseptic condition and randomly divided into control group(no pressure) and pressure group(continuous 29.4 N of pressure). The isolated culture was used in vitro and cultured in vitro. Before and after cultured for 3, 7 and 14 days, the 10 cartilage endplates in each group were removed for hematoxylin-eosin staining and expression of matrix metalloproteinase 13, collagen type II and proteoglycan were detected by immunohistochemistry, respectively.RESULTS AND CONCLUSION:(1) After 14 days of culture, the cartilage structure of the intervertebral disc endplate was almost intact, the number was reduced and tended to degenerate, which showed no significant changes compared with the control group.(2) The expression of matrix metalloproteinase 13 at 14 days after culture in the pressure group was increased compared with the baseline. The average absorbance value of matrix metalloproteinase 13 in the pressure group was significantly higher than that in the control group(P < 0.05).(3)The average absorbance value of collagen type II in the pressure group was significantly higher than that in the control group at 3 days after culture(P < 0.05). The expression of collage type II after 1 and 7 days of culture in the pressure group was significantly decreased compared with the baseline, which showed no significant difference from the control group.(4) The proteoglycan staining became slight in both groups at3, 7 and 14 days after culture(P < 0.05). The average absorbance value of proteoglycan in the pressure group was significantly higher than that in the control group at 7 days(P < 0.05).(5) In summary, continuous pressure will lead to degeneration of endplate in vitro culture, which provides evidence for the early prevention and treatment of cartilage injury in intervertebral disc degeneration.
引文
[1]展嘉文,王尚全,朱立国,等.持续压力对离体培养兔脊柱运动节段终板内血管内皮生长因子及β-catenin的影响[J].中国中医骨伤科杂志,2018,26(6):1-5.
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[7]牛朋彦,熊伟,李锋,等.渗透压负荷对兔椎间盘器官培养模型的影响[J].中国脊柱脊髓杂志,2009,19(10):729-734.
[8]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(3):235-241.
[9]杨松波,高春华,庞晓东,等.椎间盘退变模型的研究进展[J].脊柱外科杂志,2012,10(5):235-241.
[10]展嘉文,朱立国,王尚全,等.血管内皮生长因子及其受体在离体培养椎体终板退变过程中的表达[J].中国中医骨伤科杂志,2018,26(7):7-10.
[11]冯敏山,展嘉文,朱立国,等.体外培养条件下兔脊柱运动节段髓核组织的变化[J].中国组织工程研究,2015,19(51):8241-8246.
[12]朱立国,展嘉文,冯敏山,等.兔椎间盘器官与脊柱运动节段离体培养条件下髓核组织的变化[J].中国骨伤,2015,28(9):824-831.
[13]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(PhilaPa 1976).2003;28(24):2652-2658.
[14]王鹏,伍骥,郑超.生物力学对椎间盘营养及其退变的影响[J].中国矫形外科杂志,2015,23(3):260-262.
[15]徐勇,蒋青.关节软骨细胞生物力学研究进展[J].江苏医药,2009,35(8):954-955.
[16]Plumb Mandy S,Treon Katrina,Aspden Richard M.Competing regulation of matrix biosynthesis by mechanical and IGF-1 signalling in elderly human articular cartilage in vitro.Biochim.Biophys.Acta.2006;1760(5):762-767.
[17]De Croos J NA,Dhaliwal SS,Grynpas MD et al.Cyclic compressive mechanical stimulation induces sequential catabolic and anabolic gene changes in chondrocytes resulting in increased extracellular matrix accumulation.Matrix Biol.2006;25(6):323-331.
[18]Hunter Christopher J,Imler Stacy M,Malaviya Prasanna,et al.Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels.Biomaterials.2002;23(4):1249-1259.
[19]Seol D,Choe H,Ramakrishnan PS,et al.Organ culture stability of the intervertebral disc:rat versus rabbit.J Orthop Res.2013;31(6):838-846.
[20]徐宏光,章平治,宋俊兴,等.退变大鼠椎间盘器官培养模型的建立及其意义[J].中国骨与关节外科,2012,5(3):233-237+197.
[21]丁亮,唐果,薛锋,等.压应力对人软骨终板细胞MMP-1、3、13和TIMP-1表达的影响研究[J].生物骨科材料与临床研究,2018,15(1):8-12.
[22]徐无忌,李悦,原超.六味地黄丸含药血清对椎间盘Ⅰ型和Ⅱ型胶原表达的影响[J].中国组织工程研究,2013,17(26):4857-4864.
[23]Roberts S,Urban JP,Evans H et al.Transport properties of the human cartilage endplate in relation to its composition and calcification.Spine.1996;21(4):415-420.
[2]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.
[3]Haschtmann D,Stoyanov JV,Gédet P,et al.Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro.Eur Spine J.2008;17(2):289-299.
[4]Gruber HE,Gordon B,Norton HJ,et al.Analysis of cell death and vertebral end plate bone mineral density in the annulus of the aging sand rat.Spine J.2008;8(3):475-481.
[5]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(24):2652-2658.
[6]Richardson SM,Walker RV,Parker S,et al.Intervertebral disccell mediated mesenchymal stem cell differentiation.Stem Cells.2006;24(3):707-716.
[7]牛朋彦,熊伟,李锋,等.渗透压负荷对兔椎间盘器官培养模型的影响[J].中国脊柱脊髓杂志,2009,19(10):729-734.
[8]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(3):235-241.
[9]杨松波,高春华,庞晓东,等.椎间盘退变模型的研究进展[J].脊柱外科杂志,2012,10(5):235-241.
[10]展嘉文,朱立国,王尚全,等.血管内皮生长因子及其受体在离体培养椎体终板退变过程中的表达[J].中国中医骨伤科杂志,2018,26(7):7-10.
[11]冯敏山,展嘉文,朱立国,等.体外培养条件下兔脊柱运动节段髓核组织的变化[J].中国组织工程研究,2015,19(51):8241-8246.
[12]朱立国,展嘉文,冯敏山,等.兔椎间盘器官与脊柱运动节段离体培养条件下髓核组织的变化[J].中国骨伤,2015,28(9):824-831.
[13]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(PhilaPa 1976).2003;28(24):2652-2658.
[14]王鹏,伍骥,郑超.生物力学对椎间盘营养及其退变的影响[J].中国矫形外科杂志,2015,23(3):260-262.
[15]徐勇,蒋青.关节软骨细胞生物力学研究进展[J].江苏医药,2009,35(8):954-955.
[16]Plumb Mandy S,Treon Katrina,Aspden Richard M.Competing regulation of matrix biosynthesis by mechanical and IGF-1 signalling in elderly human articular cartilage in vitro.Biochim.Biophys.Acta.2006;1760(5):762-767.
[17]De Croos J NA,Dhaliwal SS,Grynpas MD et al.Cyclic compressive mechanical stimulation induces sequential catabolic and anabolic gene changes in chondrocytes resulting in increased extracellular matrix accumulation.Matrix Biol.2006;25(6):323-331.
[18]Hunter Christopher J,Imler Stacy M,Malaviya Prasanna,et al.Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels.Biomaterials.2002;23(4):1249-1259.
[19]Seol D,Choe H,Ramakrishnan PS,et al.Organ culture stability of the intervertebral disc:rat versus rabbit.J Orthop Res.2013;31(6):838-846.
[20]徐宏光,章平治,宋俊兴,等.退变大鼠椎间盘器官培养模型的建立及其意义[J].中国骨与关节外科,2012,5(3):233-237+197.
[21]丁亮,唐果,薛锋,等.压应力对人软骨终板细胞MMP-1、3、13和TIMP-1表达的影响研究[J].生物骨科材料与临床研究,2018,15(1):8-12.
[22]徐无忌,李悦,原超.六味地黄丸含药血清对椎间盘Ⅰ型和Ⅱ型胶原表达的影响[J].中国组织工程研究,2013,17(26):4857-4864.
[23]Roberts S,Urban JP,Evans H et al.Transport properties of the human cartilage endplate in relation to its composition and calcification.Spine.1996;21(4):415-420.