Biological Characteristics Of Nucleus Pulposus Cells In Cervical Intervertebral Discs Based On Miyazaki Grades

Part I Separation and culture of human cervical nucleus pulposus cells by enzymatic digestionObjective:To isolated and cultured nucleus pulposus cells from human cervical intervertebral disc and to observed the changes of biological characteristics of nucleus pulposus cells and provided an important experimental basis for subsequent research.Method:Nucleus pulposus tissue collected from 8 patients with cervical spondylotic myelopathy in our hospital and single cell suspension was obtained after isolated by trypsin and type Ⅱ collagenase,than placed them in cell culture incubator for cell culture and identification.Morphological changes,growth rateand cell attachment time and confluenced time of nucleus pulposus cellswere observed under microscope during culture Result:The nucleus pulposus cells were round after isolating.After 5-7 days,the cells began to adhered and proliferate,and the edge of the remaining tissue fragments the appeared cell cluster.After 10-14 days,the cells were triangular and short-stem in shape and the cells proliferated faster.4 weeks later,the cell confluence was about 85%,but the cell outline was not clearand the shape was not full.As to passage 1 cells,the cell proliferation was not obvious after 2 days culture,and the cell proliferation was accelerated at 3-7 days and entered into logarithmic growth phase.After 10 days,the cell proliferation slowed down and entered into the plateau phase,at which time the number of nucleus pulposus cells reached a peak.Subsequently,cell proliferation and apoptosis were unbalancedand the number of cells began to decline.The cell attachment time and 85%confluenced time of the newly isolated cells were 7.1±2.9 days and 27.4±3.8 days,respectively,and the times of which were significantly shortened in the passage 1 cells,which were 0.8±0.1days and 9.1±2.3 days respectively.The cell attachment time and85%confluenced time were 0.7±0.2 days and 7.4±1.8 days in passage 2,respectively.However,the cell attachment time was 1.7±0.9 days and the 85%confluenced time was 13.2±2.5 days,all that began to rise(P<0.05).Conclusion:The proliferation of nucleus pulposus cells in vitro was slow,the culture period was too much longerand the cell morphology changes with the number of passages.Therefore,it is difficult to obtain nucleus pulposus cells with better activity and higher purity,but it plays a key role in the selection of seed cells for tissue engineering intervertebral discs.Part Ⅱ Biological characteristics of nucleus pulposus cells in cervical intervertebral discs based on Miyazaki gradesObjective:To explore the differences in biological characteristics of cervical nucleus pulposus cells with different Miyzaki grades and assessed the consistency of Miyazaki grade and disc degeneration degree about cervical intervertebral disc.Methods:The nucleus pulposus was collected form 36 patients undergone ACCF or ACDF in our hospital from December 2017 to July 2018 and divided into 4 groups according to Miyazaki grade,grade Ⅱ(n=6),grade Ⅲ(n=10),grade Ⅳ(n=14)and grade V(n=16).There mean age were 43.2±5.1 year and 17 male and 19 female patients included.Tissue sections staining with toluidine blue and immunohistochemical staining were used to observe the density,distribution and protein expression of nucleus pulposus.Western blotting was performed to determine the expression levels of type Ⅱ collagen and aggrecan in nucleus pulposus tissue with different grades.The nucleus pulposus cells were isolated by trypsin and collagenase Ⅱ and cultured to observe cells morphology under light microscope.In addition,adhering time and 90%confluenced time were recorded during cell culture.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 higher Miyazaki grade,the lower density of nucleus pulposus cells and the expression levels of type Ⅱ collagen and aggrecan.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(94.1±4.6)%in grade Ⅱ group,(84.5±6.2)%in grade Ⅲ group,(77.5±5.8)%in grade IV group and(66.4±4.9)%in grade V group and there were significant differences between every two groups(P<0.05).The PO cell adhering time was 108.4±19.2h in grade Ⅱ,165.6±26.4 h in grade Ⅲ,218.4±31.2 h in gradeⅣand 249.6±40.8h in grade V,but 90%confluenced time was 21.4±4.4 days in grade 11,26.5±2.3 days in grade Ⅲ,30.2±2.9 days in grade Ⅳ and 33.6±3.8 days in gradeⅤ,there were significant differences between every two groups(P<0.05).During P1-P2 of each group,the adherence time and 90%confluenced time were significantly shortened compared with the P0 generation,but extended in P3 generation(P<0.05).There were significant differences between thees groups(P<0.05).The cell growth rate in grade Ⅱ of passage 2 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).Conclusion:The Miyazaki grading system could reflect biological characteristics of nucleus pulposus cells well and provide an important reference for clinical treatment and diagnosis.