Effect of ultrasound-guided needle-knife on matrix homeostasis of rabbit cartilaginous endplate

doi:10.12307/2025.647

Abstract

Abstract: BACKGROUND: Cartilaginous endplate stromal homeostasis is closely related to intervertebral disc degeneration. Acupotomy that releases posterior wall ligamentum flavum of the interforaminal orifice can improve the symptoms of patients with lumbar disc degeneration. Whether the mechanism of action is related to the regulation of cartilaginous endplate stromal homeostasis remains to be clarified.
OBJECTIVE: To investigate the effect of ultrasound-guided needle-knife relaxation of the internal orifice of the rabbit L7/S1 intervertebral foramina on the matrix homeostasis of the rabbit cartilage endplate.
METHODS: A total of 24 male New Zealand rabbits were randomly divided into a control group (n=6) and a modeling group (n=18). Lumbar disc degeneration modeling was performed by intermittent standing after transection of the supraspinous interspinous ligament for 12 weeks. After modeling, the modeling group was randomly divided into model group, needle-knife group and fake needle knife group, with 6 rabbits in each group. In the needle-knife group, the ultrasound-guided needle-knife released the ligamentum flavum posterior wall of the right intervertebral foramina, while in the fake needle-knife group, the needle-knife only reached the surface of the posterior wall ligamentum flavum of the intervertebral foramina, with a total of four interventions, each time at an interval of 1 week. No intervention was performed in the control and model groups. Thirty days after the intervention, the disc degeneration was observed by MRI. The morphology of L7/S1 intervertebral disc cartilaginous endplate, nucleus pulposus and annulus fibrosus were observed with hematoxylin-eosin and safranin O-fast green staining and was then scored. The expressions of type II collagen, proteoglycan and matrix metalloproteinase-13 in nucleus pulposus and cartilage endplate of L7/S1 intervertebral disc were detected by immunohistochemistry.
RESULTS AND CONCLUSION: MRI results showed that the relative gray value of L7/S1 nucleus pulposus was significantly lower in the model group than in the control group (P < 0.01), and higher in the needle-knife group than in the model group and the fake needle-knife group (P < 0.05). Hematoxylin-eosin and safranin O-fast green staining showed obvious degeneration of the nucleus pulposus and annulus fibrosus, thinning of the cartilaginous endplate, and obvious calcification at the junction with bone endplate in the model group. The morphology of the cartilaginous endplate and degeneration of the nucleus pulposus were improved in the needle-knife group compared with the model group. The morphological scores of the model group, needle-knife group and fake needle-knife group were significantly higher than those of control group (P < 0.01). The morphological score of the needle-knife group was significantly lower than that of the model group and fake needle-knife group (P < 0.01). Immunohistochemistry results showed that compared with the control group, the expressions of type II collagen and Aggrecan in the nucleus pulposus and cartilage endplate in the model group and fake needle-knife group were significantly decreased (P < 0.01), while the expression of matrix metalloproteinase-13 was significantly increased (P < 0.01). Compared with the model group and fake needle-knife group, the expressions of type II collagen and Aggrecan in the nucleus pulposus and cartilage endplate were increased in the needle-knife group (P < 0.05), while the expression of matrix metalloproteinase-13 was decreased significantly (P < 0.01). In conclusion, the relaxation of the interforaminal orifice by needle knife can regulate the matrix homeostasis of the rabbit cartilage endplate and improve the degeneration of the intervertebral disc.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: lumbar disc degeneration, cartilaginous endplate, needle knife, internal orifice of the intervertebral foramina, ultrasound

CLC Number:

Hu Binhan, Chen Can, Huang Maochang, Zhao Yu, Liu Junning, Niu Susheng, Zhang Yan. Effect of ultrasound-guided needle-knife on matrix homeostasis of rabbit cartilaginous endplate[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(18): 3758-3766.

Figures/Tables 5

2.1 实验动物一般情况 24只新西兰兔分为4组，各组兔实验过程中均无脱失。针刀组兔干预过程中未出现下肢抽动，进针点未见无色透明液体渗出。 2.2 各组椎间盘MRI检查结果 2.2.1 MRI显示对照组T2WI像可见髓核呈高亮信号，与纤维环、软骨终板界限清晰；模型组髓核呈灰-黑色，与纤维环、软骨终板界限模糊，纤维环超出椎体终板边缘，椎间盘高度较对照组下降；针刀组髓核亮度略高于模型组，与纤维环、软骨终板界限相对清晰，椎间盘高度较对照组下降，但高于模型组；假针刀组髓核亮度处于模型组与针刀组之间，髓核明显缩小，见图3A。 2.2.2 L7/S1椎间盘相对灰度值比较模型组、针刀组、假针刀组 L7/S1椎间盘相对灰度值均低于对照组(P < 0.01，P < 0.05，P < 0.01)；针刀组高于模型组、假针刀组(P < 0.05)；假针刀组稍高于模型组，但差异无显著性意义(P > 0.05)，见图3B。 2.3 各组椎间盘苏木精-伊红染色、番红O固绿染色及形态学评分结果干预结束30 d后，苏木精-伊红染色及番红O固绿染色结果显示，对照组L7/S1髓核呈椭圆形，有大量圆形空泡状的脊索细胞，基质呈凝胶状；纤维环呈规则的层状结构；软骨终板表层细胞呈椭圆形或梭形，深层细胞近圆形，沿椎间盘盘面方向整齐排列，其与骨性终板交界处含有大量毛细血管。模型组L7/S1髓核不规则，圆形空泡状的脊索细胞消失，出现大量散在的软骨样细胞，基质散在被番红浅染；内层纤维环内折明显，存在大量蛇形纤维，部分断裂，髓核突出至内层纤维环与外层纤维环间；软骨终板厚度不均，较对照组明显变薄，细胞排列紊乱，数量减少，深层近圆形的软骨细胞大量消失，与对照组相比，模型组软骨终板与骨性终板交界处出现钙化区，分隔软骨基质与血管。针刀组L7/S1髓核呈椭圆形，脊索细胞、软骨样细胞混杂，基质部分浓缩；内层纤维环部分纤维排列紊乱，髓核与纤维环界限相对清晰；软骨终板厚度较模型组均匀，细胞形态与排列较整齐，其与骨性终板交界处含有大量毛细血管。假针刀组L7/S1髓核呈椭圆形，脊索细胞数量较对照组大量减少，可见少量聚集于髓核中央区域圆形空泡状的脊索细胞，基质明显浓缩，可见大面积无细胞存在的基质区域；内层纤维环纤维呈蛇形，髓核与纤维环界限不清；软骨终板变薄，细胞排列紊乱，数量减少，软骨终板与血管间可见明显的钙化区。形态学评分比较显示，模型组、针刀组、假针刀组评分明显高于对照组(P < 0.01)；针刀组评分明显低于模型组、假针刀组(P < 0.01)；假针刀组评分低于模型组，但差异无显著性意义(P > 0.05)，见图4。 2.4 各组兔L7/S1椎间盘髓核Ⅱ型胶原、蛋白聚糖、基质金属蛋白酶13表达免疫组化显示，模型组和假针刀组髓核Ⅱ型胶原、蛋白聚糖阳性表达明显低于对照组(P < 0.01)，基质金属蛋白酶13阳性表达明显高于对照组(P < 0.01)。针刀组Ⅱ型胶原阳性表达明显高于模型组和假针刀组(P < 0.01)，低于对照组(P < 0.05)；蛋白聚糖阳性表达明显高于模型组和假针刀组(P < 0.01，P < 0.05)，低于对照组(P < 0.01)；基质金属蛋白酶13阳性表达明显低于模型组和假针刀组(P < 0.01)，略高于对照组，但差异无"

References

[1] SENCK S, TRIEB K, KASTNER J, et al. Visualization of intervertebral disc degeneration in a cadaveric human lumbar spine using microcomputed tomography. J Anat. 2020;236(2):243-251.
[2] MOHD II, TEOH SL, MOHD NN, et al. Discogenic Low Back Pain: Anatomy, Pathophysiology and Treatments of Intervertebral Disc Degeneration. Int J Mol Sci. 2022;24(1):208.
[3] 石翀,王海龙,邱祖云,等. 超声引导下针刀经皮松解黄韧带治疗腰椎间盘突出症的初步疗效观察[J]. 中日友好医院学报,2022, 36(3):178-179.
[4] 顾晞,甘宁.水针刀椎管内靶点治疗腰椎间盘突出症的疗效观察[J].云南中医学院学报,2022,45(3):31-34.
[5] VAN DER GRAAF JW, KROEZE RJ, BUCKENS C, et al. MRI image features with an evident relation to low back pain: a narrative review. Eur Spine J. 2023;32(5):1830-1841.
[6] BONNHEIM NB, WANG L, LAZAR AA, et al. The contributions of cartilage endplate composition and vertebral bone marrow fat to intervertebral disc degeneration in patients with chronic low back pain. Eur Spine J. 2022;31(7):1866-1872.
[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(23):2631-2644.
[8] LAKSTINS K, ARNOLD L, GUNSCH G, et al. Characterization of the human intervertebral disc cartilage endplate at the molecular, cell, and tissue levels. J Orthop Res. 2021;39(9):1898-1907.
[9] LAO YJ, XU TT, JIN HT, et al. Accumulated Spinal Axial Biomechanical Loading Induces Degeneration in Intervertebral Disc of Mice Lumbar Spine. Orthop Surg. 2018;10(1):56-63.
[10] ZHOU N, SHEN B, BAI C, et al. Nutritional deficiency induces nucleus pulposus cell apoptosis via the ATF4-PKM2-AKT signal axis. BMC Musculoskelet Disord. 2022;23(1):946.
[11] 赵宇. 超声引导下针刀松解兔腰椎肥厚黄韧带的作用及其对TGF-β1/Smad2通路的影响[D]. 福州:福建中医药大学,2023.
[12] 陈灿, 赵宇, 胡斌涵, 等. 超声引导下针刀松解黄韧带对兔退变腰椎间盘整合素α5、β1表达的影响[J]. 中国组织工程研究,2025,29(2):331-338.
[13] 赵兵营. 椎体终板生物力学特性研究[D]. 太原:太原理工大学, 2022.
[14] WONG J, SAMPSON S L, BELL-BRIONES H, et al. Nutrient supply and nucleus pulposus cell function: effects of the transport properties of the cartilage endplate and potential implications for intradiscal biologic therapy. Osteoarthritis Cartilage. 2019;27(6):956-964.
[15] 赵宇, 郭双, 陈灿, 等. 超声引导下针刀松解兔腰椎黄韧带的解剖学研究[J].中国医药导报,2023,20(13):4-8.
[16] RUAN D, HE Q, DING Y, et al. Intervertebral disc transplantation in the treatment of degenerative spine disease: a preliminary study. Lancet. 2007;369(9566):993-999.
[17] PFIRRMANN CW, METZDORF A, ZANETTI M, et al. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine (Phila Pa 1976). 2001;26(17):1873-1878.
[18] GULLBRAND SE, ASHINSKY BG, LAI A, et al. Development of a standardized histopathology scoring system for intervertebral disc degeneration and regeneration in rabbit models-An initiative of the ORSspine section. JOR Spine. 2021;4(2):e1147.
[19] 刘爽, 施杞, 杜文岚, 等. 基于筋骨平衡理论探讨椎旁肌病变与腰椎退变的关系[J].中医正骨,2024,36(2):60-64.
[20] 朱汉章. 针刀医学体系概论[J]. 中国工程科学,2006(7):1-15.
[21] 刘福水, 方婷, 周凡媛. 针刀“调筋治骨”法治疗颈椎病力学机制探讨[J]. 中华中医药学刊,2018,36(12):2862-2865.
[22] 林秀华, 刘存斌, 耿凯, 等. 超声引导下针刀松解黄韧带治疗腰椎间盘突出症的临床效果[J]. 中国医药导报,2022,19(9):157-160.
[23] 陈小峰, 曹晔, 浦建枫, 等. CT引导下水针刀靶向微创松解技术在腰椎间盘突出症治疗中的应用研究[J].介入放射学杂志,2019, 28(8):754-758.
[24] 欧林宏, 乔英杰, 张万里, 等. 李华东从椎间孔内外口靶点治疗腰椎间盘突出症经验[J].山东中医杂志,2022,41(8):891-894.
[25] 周俏吟,申毅锋,孙小洁,等.可视化针刀发展的机遇和挑战[J].中华中医药杂志,2020,35(10):4801-4804.
[26] BURT KG, VIOLA DC, LISIEWSKI LE, et al. An in vivo model of ligamentum flavum hypertrophy from early-stage inflammation to fibrosis. JOR Spine. 2023;6(3):e1260.
[27] ZHANG Y, LENART BA, LEE JK, et al. Histological features of endplates of the mammalian spine: from mice to men. Spine (Phila Pa 1976). 2014;39(5):E312-E317.
[28] BRENDLER J, WINTER K, LOCHHEAD P, et al. Histological differences between lumbar and tail intervertebral discs in mice. J Anat. 2022; 240(1):84-93.
[29] LI J, MA Y, JIAO Y, et al. Intervertebral Disc Degeneration and Low Back Pain Depends on Duration and Magnitude of Axial Compression. Oxid Med Cell Longev. 2022;2022:1045999.
[30] XU H, ZHANG Y, ZHANG Y, et al. A novel rat model of annulus fibrosus injury for intervertebral disc degeneration. Spine J. 2024;24(2): 373-386.
[31] QUAN H, ZUO X, HUAN Y, et al. A systematic morphology study on the effect of high glucose on intervertebral disc endplate degeneration in mice. Heliyon. 2023;9(2):e13295.
[32] FINE N, LIVELY S, SEGUIN CA, et al. Intervertebral disc degeneration and osteoarthritis: a common molecular disease spectrum. Nat Rev Rheumatol. 2023;19(3):136-152.
[33] VERES SP, ROBERTSON PA, BROOM ND. ISSLS prize winner: how loading rate influences disc failure mechanics: a microstructural assessment of internal disruption.Spine (Phila Pa 1976). Spine (Phila Pa 1976). 2010;35(21):1897-1908.
[34] 韩涛, 尹逊路, 展嘉文, 等. 补肾活血中药介导Wnt/β-catenin信号通路延缓椎间盘终板压力退变的机制研究[J].中国全科医学, 2021,24(35):4427-4436.
[35] ZHENG Q, WANG CD, SHAO S, et al. Intermittent cyclic mechanical compression promotes endplate chondrocytes degeneration by disturbing Nrf2/PINK1 signaling pathway-dependent mitophagy. Hum Cell. 2023;36(6):1978-1990.
[36] 范凯健, 吴菁, 李钦, 等.基质金属蛋白酶13在软骨重塑和关节炎中的研究进展[J].中国药理学通报,2018,34(5):607-611.
[37] HUANG YZ, ZHAO L, ZHU Y, et al. Interrupting TGF-β1/CCN2/integrin-α5β1 signaling alleviates high mechanical-stress caused chondrocyte fibrosis.Eur Rev Med Pharmacol Sci. 2021;25(3):1233-1241.