Trypsin plus type II collagenase digestion for isolation of nucleus pulposus cells: the optimal glucose concentration in complete medium

doi:10.3969/j.issn.2095-4344.2016.20.002

Abstract

Abstract:

BACKGROUND: Intervertebral disc degeneration is the pathological basis of degenerative spinal diseases. Studies on the influential factors of intervertebral disc degeneration contribute to the prevention and treatment of degenerative spinal disease.
OBJECTIVE: To observe the growth and proliferation of nucleus pulposus cells isolated by trypsin plus type II collagenase digestion in complete medium with different glucose concentrations, exploring the optimal glucose concentration for growth of nucleus pulposus cells.
METHODS: Nucleus pulposus cells isolated and cultured by trypsin plus type II collagenase digestion method were observed under an inverted microscope, and the cell number was counted. Morphology of nucleus pulposus cells was observed after hematoxylin-eosin staining and toluidine blue staining. Collagen type II immunoreactivity was detected by immunohistochemical staining combined with immunofluorescent staining. Nucleus pulposus cells were incubated in complete medium containing various glucose concentrations (0, 6.25, 12.5, 17.5, and 25 mmol/L) for 24 hours, and then cell proliferation and apoptosis were determined.
RESULTS AND CONCLUSION: The stained nucleus pulposus cells showed polygonal and short spindle, with one or two nuclei. Cellular pseudopod appeared gradually and then became slim with increased passage numbers. The isolated and cultured nucleus pulposus cells positively expressed collagen type II and aggrecan Proliferative activity of nucleus pulposus cells cultured in medium with 17.5 mmol/L glucose was significantly higher than that in medium with 0 and 25 mmol/L glucose (P < 0.05 or P < 0.01). There was no significant difference in cell apoptosis between these groups except for 0 mmol/L glucose (P < 0.05). These results confirm that a large number of nucleus pulposus cells can be harvested by trypsin plus type II collagenase digestion and the optimal glucose concentration is 17.5 mmol/L.

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: Subject Headings, Tissue Engineering, Intervertebral Disk, Apoptosis, Glucose

Zhang Cun-xin, Ma Jin-feng, Wang De-chun. Trypsin plus type II collagenase digestion for isolation of nucleus pulposus cells: the optimal glucose concentration in complete medium[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(20): 2899-2906.

Figures/Tables 1

2.1 髓核细胞体外分离培养形态学观察及鉴定结果胰蛋白酶联合Ⅱ型胶原酶消化分离髓核组织后，可收获较多髓核细胞。光学显微镜下观察发现髓核细胞从网状的纤维组织中释放出来。原代髓核细胞接种后倒置显微镜下观察可见细胞呈球形，大小不等，悬浮在培养液中，三四天后可见有细胞贴壁。细胞贴壁后呈圆形并且变大，伸出伪足后呈三角形、多角形或短梭形，细胞核较大，有时可见双核，核仁明显，胞浆中可见分泌颗粒，细胞折光性好。培养约1周后，髓核细胞完全贴壁，细胞伸出的伪足相互连接成片。培养约15 d可见大部分细胞汇合，细胞周围可见基质样物质沉淀。培养约3周时，原代髓核细胞汇合可达85%以上。苏木精-伊红染色后，可见髓核细胞呈多角形、短梭形等不规则形状，胞核位于细胞中央或偏向一侧胞壁，染成蓝色，胞浆染成粉红色，越靠近胞核，胞浆着色越深。细胞免疫组化染色后可见胞质中Ⅱ型胶原蛋白染成黄褐色，越靠近细胞核着色越深。免疫荧光染色可见Ⅱ型胶原蛋白呈绿色荧光，大部分位于胞质中，且靠近胞核处荧光强度越强，胞核呈蓝色荧光。见图1。 2.2 髓核细胞生长曲线变化通过对1-5代髓核细胞计数观察发现，细胞呈“S”型曲线生长，见图2。传代后第1天，细胞生长缓慢，从第2-4天细胞生长迅速，第五天细胞生长进入停滞期，以后细胞生长更为缓慢，几乎不再增殖。且随着细胞传代次数的增加，细胞的生长活性逐渐降低。经观察发现，第1-3代细胞生长活性较强，细胞形态相对维持不变。第4代髓核细胞开始发生退变，属于过渡阶段。第5代之后，细胞生长增殖能力显著减退，细胞形态发生变化，由多角形、短梭形逐渐变成长梭形。 2.3 葡萄糖浓度筛选结果与17.5 mmol/L组相比， 25 mmol/L组细胞增殖受抑制(P < 0.05)；6.25， 12.5 mmol/L组细胞增殖比率与对照组(17.5 mmol/L)相比差异无显著性意义；0 mmol/L组细胞增殖受抑制显著(P < 0.01)，见图3。与对照组(17.5 mmol/L)比较，当培养液中葡萄糖浓度为6.25，12.5，25 mmol/L时，细胞凋亡差异无显著性意义(P > 0.05)，当培养液中葡萄糖浓度0 mmol/L时，细胞凋亡率明显增加(P < 0.05)。见图4。"

References

[1] Tsuji T, Watanabe K, Hosogane N, et al. Risk factors of radiological adjacent disc degeneration with lumbar interbody fusion for degenerative spondylolisthesis. J Orthop Sci. 2015.

[2] Ma JF, Zang LN, Xi YM, et al. MiR-125a Rs12976445 Polymorphism is Associated with the Apoptosis Status of Nucleus Pulposus Cells and the Risk of Intervertebral Disc Degeneration. Cell Physiol Biochem. 2016;38(1): 295-305.

[3] Battie MC, Videman T. Lumbar disc degeneration: epidemiology and genetics. J Bone Joint Surg Am. 2006;88 Suppl 2:3-9.

[4] Watanabe T, Sakai D, Yamamoto Y, et al. Human nucleus pulposus cells significantly enhanced biological properties in a coculture system with direct cell-to-cell contact with autologous mesenchymal stem cells. J Orthop Res. 2010;28(5):623-630.

[5] Erwin WM, Islam D, Inman RD, et al. Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration. Arthritis Res Ther. 2011;13(6):R215.

[6] Gebhard H, Bowles R, Dyke J, et al. Total disc replacement using a tissue-engineered intervertebral disc in vivo: new animal model and initial results. Evid Based Spine Care J. 2010;1(2):62-66.

[7] 李树文,武海军,银和平,等.Ⅱ型胶原酶消化结合组织块贴壁法分离培养兔髓核细胞[J].中国组织工程研究, 2013, 17(39):6861-6866.

[8] 李全修,陈伯华,刘勇,等.构建兔髓核细胞诱导人骨髓间充质干细胞向类髓核细胞分化的体外模型[J].中国组织工程研究与临床康复,2009,13(45):8961-8965.

[9] 于占革,杨威,温莹,等.兔不同节段椎间盘髓核细胞培养特性的比较[J].中国脊柱脊髓杂志,2010,20(8):689-693.

[10] Kong JG, Park JB, Lee D, et al. Effect of high glucose on stress-induced senescence of nucleus pulposus cells of adult rats. Asian Spine J. 2015;9(2):155-161.

[11] Neidlinger-Wilke C, Wurtz K, Urban JP, et al. Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure. Eur Spine J. 2006;15 Suppl 3:S372-S378.

[12] Zheng CJ, Chen J. Disc degeneration implies low back pain. Theor Biol Med Model. 2015;12:24.

[13] Sambrook PN, Macgregor AJ, Spector TD. Genetic influences on cervical and lumbar disc degeneration: a magnetic resonance imaging study in twins. Arthritis Rheum. 1999;42(2):366-372.

[14] Wang F, Cai F, Shi R, et al. Aging and age related stresses: a senescence mechanism of intervertebral disc degeneration. Osteoarthritis Cartilage. 2016; 24(3):398-408.

[15] Bali T, Kumar MN. Relative Contribution of Upper and Lower Lumbar Spinal Segments to Flexion/Extension: comparison between Normal Spines and Spines with Disc Disease in Asian Patients. Asian Spine J. 2015; 9(5):770-775.

[16] Ding F, Shao ZW, Xiong LM. Cell death in intervertebral disc degeneration. Apoptosis. 2013; 18(7):777-785.

[17] Xu TT, Liao F, Jin HT, et al. Research advance on intervertebral disc degeneration and cell death. Zhongguo Gu Shang. 2015;28(7):673-678.

[18] Yu X, Li Z, Shen J, et al. MicroRNA-10b promotes nucleus pulposus cell proliferation through RhoC-Akt pathway by targeting HOXD10 in intervetebral disc degeneration. PLoS One. 2013;8(12):e83080.

[19] Shi L, Teng H, Zhu M, et al. Paeoniflorin inhibits nucleus pulposus cell apoptosis by regulating the expression of Bcl-2 family proteins and caspase-9 in a rabbit model of intervertebral disc degeneration. Exp Ther Med. 2015;10(1):257-262.

[20] Woiciechowsky C, Abbushi A, Zenclussen ML, et al. Regeneration of nucleus pulposus tissue in an ovine intervertebral disc degeneration model by cell-free resorbable polymer scaffolds. J Tissue Eng Regen Med. 2014;8(10):811-820.

[21] 王新光,叶伟,郭汉明,等.白细胞介素-1β诱导人椎间盘髓核细胞凋亡[J].中国医药导报,2011,8(21):20-24.

[22] 丁树伟,徐学振.椎间盘组织工程种子细胞的选取、鉴定及培养难点[J].中国医药指南,2013,11(11):470-472.

[23] Mern DS, Thome C. Identification and characterization of human nucleus pulposus cell specific serotypes of adeno-associated virus for gene therapeutic approaches of intervertebral disc disorders. BMC Musculoskelet Disord. 2015;16:341.

[24] Kadow T, Sowa G, Vo N, et al. Molecular basis of intervertebral disc degeneration and herniations: what are the important translational questions? Clin Orthop Relat Res. 2015;473(6):1903-1912.

[25] 肖剑,赵剑,潘欣,等.兔腰椎间盘髓核细胞的培养及形态观察[J].中国矫形外科杂志,2001,8(8):65-66.

[26] 张传志,周跃,李长青,等.兔髓核细胞体外培养和重组人转化生长因子-β1对其代谢影响的研究[J].中国脊柱脊髓杂志,2006,16(4):280-283.

[27] Gu W, Zhu Q, Gao X, et al. Simulation of the progression of intervertebral disc degeneration due to decreased nutritional supply. Spine (Phila Pa 1976). 2014;39(24):E1411-E1417.

[28] Chen JW, Ni BB, Zheng XF, et al. Hypoxia facilitates the survival of nucleus pulposus cells in serum deprivation by down-regulating excessive autophagy through restricting ROS generation. Int J Biochem Cell Biol. 2015;59:1-10.

[29] Kong JG, Park JB, Lee D, et al. Effect of high glucose on stress-induced senescence of nucleus pulposus cells of adult rats. Asian Spine J. 2015;9(2):155-161.

[30] Park EY, Park JB. Dose- and time-dependent effect of high glucose concentration on viability of notochordal cells and expression of matrix degrading and fibrotic enzymes. Int Orthop. 2013;37(6): 1179-1186.

[31] Naqvi SM, Buckley CT. Extracellular matrix production by nucleus pulposus and bone marrow stem cells in response to altered oxygen and glucose microenvironments. J Anat. 2015;227(6):757-766.

[32] Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35(4):495-516.

[33] Zhang X, Chen Y, Jenkins LW, et al. Bench-to-bedside review: Apoptosis/programmed cell death triggered by traumatic brain injury. Crit Care. 2005;9(1):66-75.