Biological characteristics of nucleus pulposus cells in cervical intervertebral discs with different Miyazaki grades

English Keywords:Cervical vertebra  Intervertebral disc  Nucleus pulposus cell  Miyazaki grade  Biological characteristics

English Abstract:

  【Abstract】 Objectives: To compare the biological characteristics of cervical nucleus pulposus cells in different Miyazaki grades in order to evaluate the reliability of the grading system. Methods: These 45 nucleus pulposus were collected from 36 patients (aged 41.2±4.5 years) undergone anterior cervical operation from December 2017 to July 2018 in our hospital, including 21 males and 15 females, and 27 cases of single segment surgery, and 9 cases of two segments surgery. The 45 nucleus pulposus were divided into 4 groups according to Miyazaki classification, grade Ⅱ (n=6), grade Ⅲ (n=10), grade Ⅳ (n=14) and grade Ⅴ (n=15). Tissue sections were stained with toluidine blue to observe the density and distribution of nucleus pulposus cells. Immunohistochemistry and western blotting were performed to determine the expression levels of type Ⅱ collagen and aggrecan in different Miyazaki grades of nucleus pulposus tissue. The nucleus pulposus cells were isolated by trypsin and collagenase Ⅱ and cultured to observe cells morphology under light microscope. The activity ratio of nucleus pulposus cells was determined by trypan blue staining and the growth curve of nuclear cells was drawn by CCK-8 kit. Results: The nucleus pulposus cells density in grade Ⅱ was higher than that in grade Ⅲ and Ⅳ, followed by that in grade Ⅴ. The expression of type Ⅱ collagen and aggrecan was the highest in grade Ⅱ, followed by that in grade Ⅲ, Ⅳ and grade Ⅴ, with significant differences between every two groups (P<0.05). In addition, the cells in grade Ⅱ were triangular and short spindle in shape, with pretty well refractive index. While the cells in grade Ⅲ had long processes, vague outline and pseudopodium. For the grade Ⅳ and Ⅴ, these cells had many different shapes, longer processes, blurred outlines and poor refractive index. The cell activity ratio was (94.8±2.8)% in grade Ⅱ group, (84.3±2.6)% in grade Ⅲ group, (75.1±4.8)% in grade Ⅳ group and (66.1±3.3)% in grade Ⅴ group, with significant differences between every two groups(P<0.05). The cell growth rate in grade Ⅱ of passage 1 cells were markedly faster than that of grade Ⅲ(P<0.05), and that of grade Ⅲ was faster than that of grade Ⅳ(P<0.05), so was that of grade Ⅳ than grade Ⅴ(P<0.05). Conclusions: The Miyazaki grading system could reflect biological characteristics of nucleus pulposus cells and the degree of disc degeneration well and provide an important reference for clinical treatment and diagnosis.