ISOLATION AND IDENTIFICATION OF RAT INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS AT DIFFERENT SEGMENTS AND COMPARATIVE STUDY ON BIOLOGICAL CHARACTERISTICS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHUHouyi ,  WANGYuntao , WANGFeng , SHIRui , SONGPeng , KANGXingui

Department of Spinal Surgery, Zhongda Hospital, Southeast University, Nanjing Jiangsu, 210009, P. R. China;

Corresponding?author：

WANGYuntao, Email: wangyttod@seu.edu.cn

Keywords：

Intervertebral disc; Nucleus pulposus cells; Cell viability; Cell property; Caudal vertebrae; Lumbar vertebrae; Rat

DOI：

10.7507/1002-1892.20150280

Video：

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Objective To isolate nucleus pulposus cells (NPCs) from the caudal and lumbar intervertebral disc of rat, and to identify the morphology and to compare the characteristics. Methods The whole spine was separated from 8-week-old Sprague Dawley rats under the sterile conditions. NPCs of different segments (lumbar group: L_1,2-L₆, S₁; caudal group: C_1,2-C_17,18) were cultured by adherent cultivation approach. Cellular morphologic change was noted by HE staining and continuous observation under inverted phase contrast microscope. Besides, the aggrecan and collagen type Ⅱexpression were examined by toluidine blue and immunocytochemistry staining respectively. The total protein contents, senescence level, and the cell viability of passage 1-5 (P1-5) were detected. The growth curves of the P1 cells in lumbar and caudal groups were determined by cell counting kit 8. Results The NPCs were isolated and identified successfully. The adherence time of the primary cells (the cell fusion reached 90%) in lumbar group was significantly longer than that in caudal group in primary generation (P<0.05). HE staining showed that cytoplasm was pink with the blue nucleus. Lumbar disc NPCs were spindle. The larger caudal disc NPCs were polygonal or irregular. Toluidine blue staining showed that the proteoglycan was stained as blue. In the cytoplasm of cells, collagen type Ⅱwas stained as brown surround the blue-black nucleus. The cell viability had no significant difference between lumbar and caudal groups and between different passages in the same group (P>0.05). The caudal disc NPCs reached their logarithmic growth phase after 3 days of culture, while the cells in lumbar segments did after 4-5 days of culture. The cell proliferation in caudal segments was more than that in lumbar segments at 3-9 days (P<0.05). The difference in the total protein contents was not significant between cells at P1-5 in 2 groups (P>0.05), and the caudal disc NPCs had higher protein contents than lumbar disc NPCs (P<0.05). There was no significant difference in cell senescence rate between cells at P1, P2, and P3 in 2 groups (P>0.05), but significant difference was shown in senescence rate between 2 groups in cells at P4 and P5 (P<0.05). Conclusion Caudal disc NPCs have a better status, which is more suitable for experiment as a seed cell than the lumbar disc NPCs in the same generation.

Citation： ZHUHouyi, WANGYuntao, WANGFeng, SHIRui, SONGPeng, KANGXingui. ISOLATION AND IDENTIFICATION OF RAT INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS AT DIFFERENT SEGMENTS AND COMPARATIVE STUDY ON BIOLOGICAL CHARACTERISTICS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(10): 1295-1300. doi: 10.7507/1002-1892.20150280 Copy

1.	Wang YT, Wu XT, Chen H, et al. Endoscopy-assisted posterior lumbar interbody fusion in a single segment. J Clin Neurosci, 2014, 21(2):287-292.
2.	Le Maitre CL, Binch AL, Thorpe AA, et al. Degeneration of the intervertebral disc with new approaches for treating low back pain. J Neurosurg Sci, 2015, 59(1):47-61.
3.	Wang YT, Wu XT, Wang F. Regeneration potential and mechanism of bone marrow mesenchymal stem cell transplantation for treating intervertebral disc degeneration. J Orthop Sci, 2010, 15(6):707-719.
4.	Wang SZ, Rui YF, Lu J, et al. Cell and molecular biology of intervertebral disc degeneration:current understanding and implications for potential therapeutic strategies. Cell Prolif, 2014, 47(5):381-390.
5.	王鋒, 王運濤, 吳小濤. NPCs修復椎間盤退行性變的研究進展. 中國修復重建外科雜志, 2009, 23(7):864-867.
6.	Wang F, Wu XT, Zhuang SY, et al. Ex vivo observation of human nucleus pulposus chondrocytes isolated from degenerated intervertebral discs. Asian Spine J, 2011, 5(2):73-81.
7.	Mern DS, Beierfu? A, Thomé C, et al. Enhancing human nucleus pulposus cells for biological treatment approaches of degenerative intervertebral disc diseases:a systematic review. J Tissue Eng Regen Med, 2014, 8(12):925-936.
8.	張濤, 阮狄克, 王德利, 等. 組織工程髓核種子細胞的研究進展. 中國脊柱脊髓雜志, 2012, 22(2):175-178.
9.	Jiang L, Yuan F, Yin X, et al. Responses and adaptations of intervertebral disc cells to microenvironmental stress:a possible central role of autophagy in the adaptive mechanism. Connect Tissue Res, 2014, 55(5-6):311-321.
10.	Yang L, Zhu L, Dong W, et al. Reactive oxygen species-mediated mitochondrial dysfunction plays a critical role in high glucose-induced nucleus pulposus cell injury. Int Orthop, 2014, 38(1):205-206.
11.	Rodrigues-Pinto R, Richardson SM, Hoyland JA. An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration. Eur Spine J, 2014, 23(9):1803-1814.
12.	王運濤, 吳小濤, 王鋒, 等. 髓核中p16~(INK4a)的表達及其對椎間盤退變的影響. 中國修復重建外科雜志, 2014, 28(12):1508-1512.
13.	Galbusera F, van Rijsbergen M, Ito K, et al. Ageing and degenerative changes of the intervertebral disc and their impact on spinal flexibility. Eur Spine J, 2014, 23 Suppl 3:S324-332.
14.	王海, 熊承杰, 黃博, 等. 椎間盤組織工程學種子細胞來源的研究進展. 中國脊柱脊髓雜志, 2012, 22(1):77-81.
15.	于占革, 楊威, 溫瑩, 等. 兔不同節段椎間盤NPCs培養特性的比較. 中國脊柱脊髓雜志, 2010, 20(8):689-693.
16.	趙丹慧, 吳成愛, 王娜, 等. 實驗動物椎間盤特征在選擇椎間盤退變模型中的意義. 中國脊柱脊髓雜志, 2012, 22(9):854-856.
17.	Kim JH, Deasy BM, Seo HY, et al. Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro. Spine (Phila Pa 1976), 2009, 34(23):2486-2493.
18.	Arkesteijn IT, Smolders LA, Spillekom S, et al. Effect of co-culturing canine notochordal, nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration. Arthritis Res Ther, 2015, 17(1):60.
19.	Gantenbein B, Calandriello E, Wuertz-Kozak K, et al. Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia. BMC Musculoskelet Disord, 2014, 15:422.
20.	馬凱歌, 邵增務, 王佰川, 等. 脊索細胞培養基促進BMSCs增殖分化的研究. 中國修復重建外科雜志, 2012, 26(5):601-606.
21.	Yurube T, Hirata H, Kakutani K, et al. Notochordal cell disappearance and modes of apoptotic cell death in a rat tail static compression-induced disc degeneration model. Arthritis Res Ther, 2014, 16(1):R31.
22.	Omlor GW, Nerlich AG, Tirlapur UK, et al. Loss of notochordal cell phenotype in 3D-cell cultures:implications for disc physiology and disc repair. Arch Oothop Trauma Surg, 2014, 134(12):1673-1681.

1. Wang YT, Wu XT, Chen H, et al. Endoscopy-assisted posterior lumbar interbody fusion in a single segment. J Clin Neurosci, 2014, 21(2):287-292.
2. Le Maitre CL, Binch AL, Thorpe AA, et al. Degeneration of the intervertebral disc with new approaches for treating low back pain. J Neurosurg Sci, 2015, 59(1):47-61.
3. Wang YT, Wu XT, Wang F. Regeneration potential and mechanism of bone marrow mesenchymal stem cell transplantation for treating intervertebral disc degeneration. J Orthop Sci, 2010, 15(6):707-719.
4. Wang SZ, Rui YF, Lu J, et al. Cell and molecular biology of intervertebral disc degeneration:current understanding and implications for potential therapeutic strategies. Cell Prolif, 2014, 47(5):381-390.
5. 王鋒, 王運濤, 吳小濤. NPCs修復椎間盤退行性變的研究進展. 中國修復重建外科雜志, 2009, 23(7):864-867.
6. Wang F, Wu XT, Zhuang SY, et al. Ex vivo observation of human nucleus pulposus chondrocytes isolated from degenerated intervertebral discs. Asian Spine J, 2011, 5(2):73-81.
7. Mern DS, Beierfu? A, Thomé C, et al. Enhancing human nucleus pulposus cells for biological treatment approaches of degenerative intervertebral disc diseases:a systematic review. J Tissue Eng Regen Med, 2014, 8(12):925-936.
8. 張濤, 阮狄克, 王德利, 等. 組織工程髓核種子細胞的研究進展. 中國脊柱脊髓雜志, 2012, 22(2):175-178.
9. Jiang L, Yuan F, Yin X, et al. Responses and adaptations of intervertebral disc cells to microenvironmental stress:a possible central role of autophagy in the adaptive mechanism. Connect Tissue Res, 2014, 55(5-6):311-321.
10. Yang L, Zhu L, Dong W, et al. Reactive oxygen species-mediated mitochondrial dysfunction plays a critical role in high glucose-induced nucleus pulposus cell injury. Int Orthop, 2014, 38(1):205-206.
11. Rodrigues-Pinto R, Richardson SM, Hoyland JA. An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration. Eur Spine J, 2014, 23(9):1803-1814.
12. 王運濤, 吳小濤, 王鋒, 等. 髓核中p16~(INK4a)的表達及其對椎間盤退變的影響. 中國修復重建外科雜志, 2014, 28(12):1508-1512.
13. Galbusera F, van Rijsbergen M, Ito K, et al. Ageing and degenerative changes of the intervertebral disc and their impact on spinal flexibility. Eur Spine J, 2014, 23 Suppl 3:S324-332.
14. 王海, 熊承杰, 黃博, 等. 椎間盤組織工程學種子細胞來源的研究進展. 中國脊柱脊髓雜志, 2012, 22(1):77-81.
15. 于占革, 楊威, 溫瑩, 等. 兔不同節段椎間盤NPCs培養特性的比較. 中國脊柱脊髓雜志, 2010, 20(8):689-693.
16. 趙丹慧, 吳成愛, 王娜, 等. 實驗動物椎間盤特征在選擇椎間盤退變模型中的意義. 中國脊柱脊髓雜志, 2012, 22(9):854-856.
17. Kim JH, Deasy BM, Seo HY, et al. Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro. Spine (Phila Pa 1976), 2009, 34(23):2486-2493.
18. Arkesteijn IT, Smolders LA, Spillekom S, et al. Effect of co-culturing canine notochordal, nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration. Arthritis Res Ther, 2015, 17(1):60.
19. Gantenbein B, Calandriello E, Wuertz-Kozak K, et al. Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia. BMC Musculoskelet Disord, 2014, 15:422.
20. 馬凱歌, 邵增務, 王佰川, 等. 脊索細胞培養基促進BMSCs增殖分化的研究. 中國修復重建外科雜志, 2012, 26(5):601-606.
21. Yurube T, Hirata H, Kakutani K, et al. Notochordal cell disappearance and modes of apoptotic cell death in a rat tail static compression-induced disc degeneration model. Arthritis Res Ther, 2014, 16(1):R31.
22. Omlor GW, Nerlich AG, Tirlapur UK, et al. Loss of notochordal cell phenotype in 3D-cell cultures:implications for disc physiology and disc repair. Arch Oothop Trauma Surg, 2014, 134(12):1673-1681.

Chinese Journal of Reparative and Reconstructive Surgery

ISOLATION AND IDENTIFICATION OF RAT INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS AT DIFFERENT SEGMENTS AND COMPARATIVE STUDY ON BIOLOGICAL CHARACTERISTICS

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