Histological changes of cervical disc tissue and underlying ossification mechanism in patients with cervical degenerative ossification

doi:10.12307/2022.696

Abstract

Abstract: BACKGROUND: Cervical degenerative ossification may aggravate nerve compression in patients with cervical degenerative disease, and also make the decompression procedure more challenging. However, the accompanied histological changes of the intervertebral disc and the underlying mechanism of degenerative ossification have not yet been documented.
OBJECTIVE: To explore the histological features of the cervical disc in patient with cervical degenerative ossification and the underlying mechanisms of local ossification.
METHODS: Patients with cervical spondylosis undergoing surgical treatment were selected from the clinical practice, and cervical disc samples were harvested during the surgical process. Based on preoperative cervical X-ray and computed tomography (CT) examinations, all the disc samples were divided into ossification group and non-ossification group. Hematoxylin-eosin staining, Masson trichrome staining and Safranin O-fast green staining were used to compare the histological differences between the two groups. Furthermore, for the expression of transforming growth factor-β1, p-Smad2 and p-Smad3 in the disc and osteophyte samples, a semi-quantitative immunohistochemistry method was used to compare the difference between the two groups.
RESULTS AND CONCLUSION: All the disc tissues could be clearly divided into the outer annulus fibrosus, the inner annulus fibrosus and the nucleus pulposus. Mature trabeculae and bone marrow cavities were detected in all ossification samples. Histologically, the number of disc cells in the ossification group was significantly higher than that of the non-ossification group (P < 0.05). However, the content of proteoglycan in the matrix was significantly lower than that in the non-ossification group (P < 0.05). Transforming growth factor β1/Smad2/3 was detected in all the disc and osteophyte tissues. Local content of transforming growth factor β1, p-Smad2 and p-Smad3 in the ossification group was significantly higher than that of the non-ossification group (P < 0.05). All the findings indicate that, compared with the non-ossification group, cervical degenerative ossification in patients in the ossification group is more severe. Higher expression of transforming growth factor β1 in the local microenvironment may promote the development of degenerative ossification. However, downstream-activated p-Smad2 and p-Smad3 may play different roles in the development of degenerative ossification.

Key words: cervical spondylosis, cervical intervertebral disc, intervertebral disc degeneration, degenerative ossification, osteophyte, histology, transforming growth factor β1, Smad2/3

CLC Number:

Xiong Yang, Yang Yingli, Gao Yushan, Wang Xiumei, Yang Yongdong, Zhao Dingyan, Zhao He, Li Chuanhong, Yang Kaitan, Yu Xing. Histological changes of cervical disc tissue and underlying ossification mechanism in patients with cervical degenerative ossification[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(18): 2868-2873.

Figures/Tables 4

苏木精-伊红染色结果显示(图2A，B及3A)，椎间盘内从外层纤维环到内层纤维环再到髓核各部分组织结构具有明显差异。由外到内，胶原纤维束排列由相互平行、致密逐渐变为紊乱交错、稀疏。同时，细胞数量逐渐减少。外层纤维环中细胞细长呈梭形，整体形态类似于成纤维细胞，排列多与胶原纤维走向平行或连接成串。内层纤维环中细胞大多散在分布，形态呈卵圆形或球形，整体类似于软骨细胞。髓核中细胞稀疏，部分软骨样细胞散在分布，另有部分聚集呈脊索样细胞，胞外基质中纤维结构模糊不清。成骨组骨赘中可见到成熟的骨结构，包括骨小梁、骨髓腔及骨陷窝，髓腔中可见成骨细胞及破骨细胞或其前体单核巨噬细胞。Masson三色染色结果显示(图2C，3C)，椎间盘内纤维及骨赘均呈蓝染，椎间盘内纤维排列由外到内亦具有减少趋势，结果同苏木精-伊红染色。改良番红O-固绿软骨染色可见(图2D， 3E)，外层纤维环以绿色为主，内层纤维环为红绿相间，髓核则以红色为主，反映出椎间盘基质中蛋白多糖主要分布于内层纤维环与髓核中。成骨组骨赘染色可见骨小梁呈绿色或灰绿色，骨髓腔中可见大量空泡，细胞外基质红绿相间，在近骨小梁处，可见红染、增生、肥大的类软骨细胞，这表明局部骨组织或通过软骨内化骨形成。半定量检测显示(图3B，D，F)，成骨组椎间盘各部分细胞数量均显著高于非成骨组，差异有显著性意义(外层纤维环中P < 0.001，内层纤维环及髓核中P=0.04，0.01)。椎间盘组织从外到内，胶原纤维含量逐渐减少，但组间比较差异无显著性意义(外层纤维环、内层纤维环及髓核中P=0.83，0.17，0.11)。椎间盘基质中蛋白多糖含量逐渐增加，组间比较可见，成骨组内层纤维环和髓核中蛋白多糖含量著低于非成骨组，差异有显著性意义(外层纤维环、内层纤维环及髓核中P=0.40，0.02，0.04)。 2.2 免疫组织化学染色所有退变椎间盘组织及骨赘中均可见转化生长因子β1、p-Smad2及p-Smad3的阳性表达，这表明转化生长因子β1/Smad2/3参与到了椎间盘的退变及骨赘的形成过程中。半定量检测显示，与非成骨组比较，成骨组外层纤维环和髓核中的转化生长因子β1含量显著更高，差异有显著性意义(外层纤维环、内层纤维环中P=0.001，0.09，髓核中P < 0.001)。成骨组内层纤维环中p-Smad2及外层纤维环中p-Smad3表达量均显著高于非成骨组，差异有显著性意义(p-Smad2：外层纤维环、内层纤维环及髓核中P=0.07，0.04，0.31；p-Smad3：外层纤维环、内层纤维环及髓核中P=0.02，0.15，0.28)，见图4，5。"

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