Changes of the nucleus pulposus after in vitro culture of rabbit spinal motion segments

doi:10.3969/j.issn.2095-4344.2015.51.008

Abstract

Abstract:

BACKGROUND: In vitro organ culture model of the intervertebral disc as a contact between in vivo tests and cell culture can maintain cell activity in a native matrix, thus retaining the important cell-cell and cell-matrix interactions.
OBJECTIVE: To observe the changes in the nucleus pulposus during in vitro culture of rabbit spinal motion segments.
METHODS: A total of 50 spinal motion segments were harvested from 13 New Zealand white rabbits under aseptic conditions. These specimens were maintained for organ culture with hyperosmotic media (410 mOsm/kg), then 10 discs were observed by hematoxy-eosin staining and immunohistochemistry of type II collagen as well as proteoglycan content and viability of nucleus pulposus cells were determined before and at 3, 7, 14, 21 days after culture, respectively.
RESULTS AND CONCLUSION: Hematoxylin-eosin staining showed the structure of intervertebral disc tissue remained intact after 14 days of culture, but severely degenerated at 21 days of culture. The intensity value of type II collagen immunohistochemical staining in the nucleus pulposus had no significant changes within 14 days (P > 0.05), but the staining became shallow at 21 days, which was significantly different from that at 3, 7, 14 days
(P < 0.05). PAS/AB staining of proteoglycan of the nucleus pulposus showed no decrease in tinting strength within 7 days, but the strength became weakened slightly at 14 days and further weakened at 21 days. The intensity value of fluorescence staining of nucleus pulposus cells decreased at 21 days and the viability of nucleus pulposus cells decreased significantly as compared with that at 3, 7, 14 days (P < 0. 05). These findings indicate that the degeneration of the nucleus pulposus of rabbit spinal motion segment showed no significant changes at 14 days of culture, but became remarkable at 21 days of culture, indicating that the spinal motion segement can be used as an in vitro experimental model to study the biomechanical changes of the intervertebral disc within 14 days of culture.

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

CLC Number:

R318

Feng Min-shan, Zhan Jia-wen, Zhu Li-guo, Zhang Ping, Wang Yuan, Bai Jian-qi. Changes of the nucleus pulposus after in vitro culture of rabbit spinal motion segments [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(51): 8241-8246.

Figures/Tables 4

References

[1] 杨松波,高春华,庞晓东,等.椎间盘退变模型的研究进展[J].脊柱外科杂志,2012,10 (5): 315-317.
[2] Risbud MV, Izzo MW, Adams CS, et al.An organ culture system for the study of the nucleus pulposus: description of the system and evaluation of the cells. Spine (Phila Pa 1976), 2003;28(24): 2652-2658; discussion 2658-2659.
[3] Richardson SM, Walker RV, Parker S, et al.Intervertebral disc cell-mediated mesenchymal stem cell differentiation.Stem Cells.2006;24(3): 707-716.
[4] 牛朋彦,熊伟,李锋,等.渗透压负荷对兔椎间盘器官培养模型的影响[J].中国脊柱脊髓杂志,2009,19 (10): 729-734.
[5] Korecki CL, Costi JJ,Iatridis JC.Needle puncture injury affects intervertebral disc mechanics and biology in an organ culture model. Spine (Phila Pa 1976).2008;33(3): 235-241.
[6] Boden SD, McCowin PR, Davis DO, et al.Abnormal magnetic-resonance scans of the cervical spine in asymptomatic subjects. A prospective investigation. J Bone Joint Surg Am.1990; 72(8): 1178-1184.
[7] Chan SC, Gantenbein-Ritter B, Leung VY, et al. Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture. Spine J.2010;10(6): 486-496.
[8] Rannou F, Lee TS, Zhou RH, et al.Intervertebral disc degeneration: the role of the mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload. Am J Pathol.2004; 164(3): 915-924.
[9] Jim B, Steffen T, Moir J, et al.Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential. Eur Spine J.2011;20(8): 1244-1254.
[10] 张聪明,刘强,李钢,等.大鼠椎间盘器官整体培养条件下髓核组织的变化[J].中国医疗前沿,2010,5(6): 19-20.
[11] Gantenbein B, Grunhagen T, Lee CR, et al.An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs. Spine (Phila Pa 1976).2006;31(23): 2665-2673.
[12] Gruber HE,Hanley EN, Jr.Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation.BMC Musculoskelet Disord.2000;1: 1.
[13] Zhang Y, Phillips FM, Thonar EJ, et al.Cell therapy using articular chondrocytes overexpressing BMP-7 or BMP-10 in a rabbit disc organ culture model. Spine (Phila Pa 1976).2008; 33(8): 831-838.
[14] Ariga K,Yonenobu K,Nakase T, et al.Mechanical stress-induced apoptosis of endplate chondrocytes in organ-cultured mouse intervertebral discs: an ex vivo study. Spine (Phila Pa 1976),2003. 28(14): 1528-1533.
[15] Kim KW, Lim TH, Kim JG, et al.The origin of chondrocytes in the nucleus pulposus and histologic findings associated with the transition of a notochordal nucleus pulposus to a fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs. Spine (Phila Pa 1976).2003;28(10): 982-990.
[16] Gawri R, Mwale F, Ouellet J, et al.Development of an organ culture system for long-term survival of the intact human intervertebral disc[J]. Spine (Phila Pa 1976),2011. 36(22): 1835-1842.
[17] 李钢,范顺武,赵凤东,等.大鼠椎间盘器官整体培养条件下髓核组织的变化[J].中国修复重建外科杂志,2010,5 (4): 385-390.
[18] Haschtmann D, Stoyanov J V, Ettinger L, et al.Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system. Spine (Phila Pa 1976).2006;31(25): 2918-2925.
[19] Ohshima H, Urban J P,Bergel DH.Effect of static load on matrix synthesis rates in the intervertebral disc measured in vitro by a new perfusion technique. J Orthop Res.1995;13(1): 22-29.
[20] Hutton WC, Ganey TM, Elmer WA, et al.Does long-term compressive loading on the intervertebral disc cause degeneration?. Spine (Phila Pa 1976).2000;25(23): 2993-3004.
[21] Paul CP, Zuiderbaan HA, Zandieh Doulabi B,et al.Simulated-physiological loading conditions preserve biological and mechanical properties of caprine lumbar intervertebral discs in ex vivo culture. PLoS One.2012;7(3): e33147.
[22] Oshima H, Ishihara H, Urban J P, et al.The use of coccygeal discs to study intervertebral disc metabolism. J Orthop Res. 1993; 11(3): 332-338.
[23] Lee CR, Iatridis JC, Poveda L, et al.In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies[J]. Spine (Phila Pa 1976).2006;31(5): 515-522.
[24] Korecki CL, MacLean JJ,Iatridis JC.Characterization of an in vitro intervertebral disc organ culture system. Eur Spine J. 2007; 16(7): 1029-1037.
[25] Hutton WC, Elmer WA, Boden SD, et al.The effect of hydrostatic pressure on intervertebral disc metabolism. Spine (Phila Pa 1976).1999;24(15): 1507-1015.
[26] Chiba K, Andersson GB, Masuda K, et al.A new culture system to study the metabolism of the intervertebral disc in vitro. Spine (Phila Pa 1976).1998;23(17): 1821-1827; discussion 1828.
[27] Lim TH, Ramakrishnan PS, Kurriger GL, et al.Rat spinal motion segment in organ culture: a cell viability study. Spine (Phila Pa 1976).2006;31(12): 1291-1297; discussion 1298.
[28] Seol D, Choe H, Ramakrishnan P S, et al.Organ culture stability of the intervertebral disc: rat versus rabbit. J Orthop Res.2013; 31(6): 838-46.
[29] Urban J P,Maroudas A.Swelling of the intervertebral disc in vitro. Connect Tissue Res.1981;9(1): 1-10.
[30] 徐宏光,章平治,宋俊兴,等.退变大鼠椎间盘器官培养模型的建立及其意义[J].中国骨与关节外科,2012,5(3): 233-237,197.
[31] 牛朋彦,熊伟,李锋,等.软骨终板通透性对体外培养大鼠髓核细胞生物学特性的影响[J].中国脊柱脊髓杂志,2011,21(7): 597-602.
[32] 董妙珠,肖萍,叶于薇,等.PAS、AB染色法在软骨蛋白黏多糖检测中的运用[J].上海预防医学杂志,2004,16(9): 419-420.
[33] 徐无忌,李悦,原超.六味地黄丸含药血清对椎间盘Ⅰ型和Ⅱ型胶原表达的影响[J]. 中国组织工程研究,2013,17 (26): 4857-4864.
[34] 徐宏光,章平治,宋俊兴,等.循环机械压力诱导下兔椎间盘退变器官模型的建立及意义[J]. 中国骨与关节外科,2014,7(1): 45-51.
[35] Poot M, Zhang YZ, Kramer JA, et al.Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem.1996;44(12): 1363-1372.
[36] Isenberg JS,Klaunig JE.Role of the mitochondrial membrane permeability transition (MPT) in rotenone-induced apoptosis in liver cells. Toxicol Sci,2000. 53(2): 340-351.
[37] Haschtmann D, Stoyanov J V,Ferguson S J.Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole-organ intervertebral disc model. J Orthop Res.2006;24(10): 1957-1966.
[38] Hutton WC, Murakami H, Li J, et al.The effect of blocking a nutritional pathway to the intervertebral disc in the dog mode. J Spinal Disord Tech.2004;17(1): 53-63.