Comparison Of Biological Characteristics Of Nucleus Pulposus Cells In Intervertebral Discs With Different Grade Of Pfirrmann Grading System

Intervertebral discs play a significant role in the support, durability, and flexibility of the spine. Disc degeneration is a relatively common problem. There is a known strong association between disc degeneration and disc-related pain. As the population to age, the incidence and prevalence of pain related to disc degeneration will undoubtedly increase. This situation causes heavy economic burden on the society. A thorough comprehension of disc degeneration is Fundamental to an appropriate treatment strategy. The culture of the disc cells is the premise to research disc degeneration on the cell level and a source of cells for intervertebral disc engineering. However, there is few article about the culture of human cervical nucleus pulposus cells. So the part one of this article aims to successfully establish a system of the culture of cervical nucleus pulposus cells. Magnetic resonance imaging (MRI) is the most important method for the clinical assessment of intervertebral disc pathology. The signal characteristics of the disc in T2-weighted MRIs reflect changes caused by aging or degeneration. Pfirrmann and his colleagues develop a grading system for lumbar disc degeneration based on MRI signal intensity, disc structure, distinction between nucleus and anulus, and disc height. This grading system is simple and comprehensive, with intra- and inter-observer reliability sufficient to discriminate between the different grades, therefore provides a standardized and reliable assessment method of disc degeneration for research and clinical purposes. the part two of this article aims to compare biological characteristics of nucelus pulposus cells in different grade of this system.Part one---Cultivation and identification of human cervical nucleus pulposus cellsObjective: To establish the system for culturing the human cervical nucleus pulposus cells and to identify their phenotypes. Methods The human cervical nucleus pulposus cells were isolated by collagenase digestion method and cultured in monolayer by culture solutions of DF12 + 20%FBS. Morphologic changes and growth of the cells were detected by microscope. The cell cycle and apoptosis rate were detected by Flow cytometric. The morphological structure and the phenotype were identified with the toluidine blue staining and immunocytochemistry means. Result The apoptosis rate of primary cells was 6.1±1.4%. The Cells at S phase were 7.3±0.5%. The cells were polygonal or short wedged morphology. The second passage cells grew more fast than that of the primary cells. The cells displayed intense toluidine blue metachromasia. The cells expressed collagen typeⅡ, but only a few elliptic gaint cells expressed CK8. Conclusion The human cervical nucleus pulposus cells were isolated and cultured in monolayer successfully. A few cells of the nucleus pulposus still maintain notochord cells phenotype in adult specimens.Part two --- Comparison of biological characteristics of nucleus pulposus cells in intervertebral discs with different grade of Pfirrmann grading systemObjective: To compare the biological characteristics of nucleus pulposus cells in intervertebral discs with different grade of Pfirrmann grading system. Method :Human nucleus pulposus cells were isolated by collagenase digestion method and cultured.they were grouped based on the Pfirrmann grading system. Group A represents grade II. Group B represents grade III. Group C represents grade IV. Cell vitality assay were examined with Trypan blue staining. Morphologic changes of the cells were observed by an inverted microscope. Cell ultra-structure morphological changes were examined using transmission electron microscopy. Cell growth curve was determined by cell counting . Proteoglycans expressions were examined by Alican blue chromatometry. These indexes were compared among 3 groups above。Results: Group A has the highest cell density and its cell vitality is 91.6±4.3%. Cell vitality of Group B is 83.5±6.7%. Group C has the lowest cell density and its cell vitality is 74.8±5.9%. There were statistical significances ( P < 0.05). The cells from group A were short spindle or polygonal shaped. They had clear outlines and rich cytoplasm with a lot of rough endoplasmic reticulum and mitochondria. The cells from group C had long processes and vague outlines. Much vacuoles located in their cytoplasm. Additionally, the entosis phenomenon had been observed. Under the same cellular density and culture conditions , the growth rate of passage 2 cells from the three groups were statistically significant respectively. The content of glycosaminoglycan of group A, B and C were 408.23±29.25mg/L,273.05±52.44mg/L,91.73±38.06 mg/L, respectively. There were significant differences among these groups (P﹤0.05). Conclusion:Pfirrmann classification of disc degeneration can be very good reaction in degenerative degree of nucleus pulposus cells. It is a simple and effective disc degeneration grading system.