Local effects of adipose-derived mesenchymal stem cells injected into the degenerative intervertebral discs

doi:10.3969/j.issn.2095-4344.2017.01.014

Abstract

Abstract:

BACKGROUND: Domestic studies have shown that adipose-derived mesenchymal stem cells can differentiate into osteoblasts, osteoblasts and other types of bone cells under certain conditions, which are used to repair damaged bone or cartilage tissue.
OBJECTIVE: To observe the effect of adipose-derived mesenchymal stem cells on rabbit intervertebral disk degeneration.
METHODS: Thirty-six adult New Zealand rabbits were randomized into normal (no treatment), control and experimental groups. Rabbit models of intervertebral disk degeneration were made in the latter two groups using nucleus pulposus aspiration method. Fourteen days after modeling, model rabbits were given injection of BrdU-labeled allogeneic adipose-derived mesenchymal stem cell suspension (50 μL, 1×10⁹/L) in the experimental group or same volume of normal saline in the control group. Thereafter, MRI, hematoxylin-eosin staining and immunohistochemical staining were performed at 2, 4, 8 weeks after injection.
RESULTS AND CONCLUSION: MRI findings showed: unchanged disc height and clear and uniform T2-weighted intervertebral disk signals in the normal group; narrowed intervertebral space and weakened T2-weighted signals in the control group that occurred at 4 weeks and gradually developed with time; and increased disc height and T2-weighted signals in the experimental group. Intervertebral disk degeneration shown by hematoxylin-eosin staining occurred at 2 weeks after injection and aggravated in the control group, but in the experimental group, the degeneration was alleviated significantly compared with the control group. Additionally, the transplanted BrdU-labeled adipose-derived mesenchymal stem cells survived and proliferated in the rabbit intervertebral discs. To conclude, adipose-derived mesenchymal stem cells contribute to the recovery from intervertebral disk degeneration.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cell Transplantation, Intervertebral Disk Degeneration, Tissue Engineering

CLC Number:

R394.2

Shen Xiang, Xu Hong-guang, Ma Ming-ming, Zhang Qian, Xiao Liang, Suo Qi-feng. Local effects of adipose-derived mesenchymal stem cells injected into the degenerative intervertebral discs[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 77-81.

Figures/Tables 1

2.1 实验动物数量分析 36只新西兰大白兔均进入结果分析，无脱失。 2.2 细胞形态学观察及表面标记物检测结果从兔脂肪组织中分离出的脂肪间充质干细胞形状大多呈现圆形或类圆形，体积较小，孵育时干细胞24 h即可见有细胞贴壁，第3-5天细胞贴壁生长，呈集落样分布逐渐形成多个细胞克隆，细胞形态呈现为短梭形、三角形等，细胞逐渐融合；第5-7天，脂肪间充质干细胞形态均一，呈现为呈长梭形或纤维状；培养至第10天，脂肪间充质干细胞铺满大部分培养瓶底面，细胞形态呈栅栏状或漩涡状排列，融合达90%以上。正常传代后，细胞贴壁生长迅速，以均匀分布的长梭形细胞平铺生长(图1)。流式细胞仪检测结果显示，脂肪间充质干细胞表面表达标记物CD29(约81.3%)，但不表达CD45(仅有11.1%)。 2.3 兔椎间盘放射学检测结果 MRI检查显示，正常组各时间点椎间盘高度没有不变，T2加权相上椎间盘信号清晰、均一。对照组、实验组移植后第4周开始椎间隙变窄，T2加权相信号减弱；随着时间推移，对照组椎间盘间隙逐渐狭窄，同时椎间盘的T2加权相信号也越来越低，与正常组、实验组移植第4，8周MRI检测结果相比差异有显著性意义(P < 0.05)；移植后第4周，实验组椎间隙高度和T2加权相信号逐渐增强，与正常组椎间盘MRI检测结果比较差异无显著性意义(P > 0.05)；实验组组内不同时间点间MRI检测结果比较差异无显著性意义(P > 0.05)，对照组组内各时间MRI检测结果比较差异有显著性意义(P < 0.05)，见表1，图2。结果提示脂肪间充质干细胞对兔椎间盘退变模型有修复作用。 2.4 兔椎间盘组织学观察结果与正常组比较，对照组移植后2周椎间盘退变，椎间盘髓核有缩小趋势，髓核与纤维环之间距离增大，交界区出现连续性中性形态；而在第4周时，髓核组织不完整，且呈簇状出现空泡样变性；第8周，出现分界不清，外层纤维环进入髓核中央，同时髓核组织内细胞数量明显减少。与对照组相比，实验组椎间盘组织退变进程明显减缓，移植第4，8周时，髓核细胞数量减少、形态变化等均较轻，脂肪间充质干细胞对椎间保护有显著的保护作用，呈现中央髓核细胞比较完整，分界清晰，细胞呈团状生长，见图3。 2.5 移植BrdU标记脂肪间充质干细胞示踪检测结果移植后2，4，8周，BrdU标记的阳性细胞数量逐渐减少，并且从椎间盘中央区域向组织外周迁移，见图4，说明脂肪间充质干细胞可在兔椎间盘退变组织中正常存在及增殖。"

References

[1] Urban JP,Roberts S.Degeneration of the intervertebral disc. Arthritis Resther.2003;5:120-130.

[2] Waddell G.Low back disability.A syndrome of Western civilization.Neurosurg Clin N Am.1991;2(4):719-738.

[3] 冯传汉,张铁良.临床骨科学[M].2版.北京:人民卫生出版社, 2004:1925-1934.

[4] Gruber HE,Hanley EN Jr.Ultrastructure of the human intervertebral disc during agingand degeneration: comparison of surgical and controlspecimens. Spine. 2002; 27:798-805.

[5] Loma K,Riihimaki H,Luukkonen R,et al.Lowback pain in relation to lumbar discdegene-ration.Spine.2000;25:487-492.

[6] Luoma K,Vehmas T,Raininko R,et al.Lumbosacral transitional ver tebra: relation to disc degeneration and low back pain. Spine.2004;29:200-205.

[7] 吴在德,吴肇汉.外科学[M].6版.北京:人民卫生出版社,2003: 872-875.

[8] Mochida J,Nishimura K,Nomura T,et al.The im portance of preserving disc structure in surgical approaches to lumbar disc herniation.Spine.1996;21:1556-1564.

[9] Gregory CA,Gunn WG,Peister A,et al.An Alizarin red-based assay of mineralization byadherent cells in culture: comparison with cetylpyridiniurn chloride extraction.Anal Biochem.2004;329(1):77-84.

[10] Hayashi O,Katsube Y,HiroseM,et al.Comparison of osteogenic ability of rat mesenchyal stem cells from bone marrow,periosteum,and adipose tissue.Caleif Tissue Int. 2008;82(3):238-247.

[11] Planat-Benard V,Silvestre JS,Cousin B,et al.Plasticity of human adipose lineage cells toward endothelial cells: Physiological and therapeutic perspectives.Cireulation. 2004;109(5):656-663.

[12] Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from human adipose tissue: implications for cell-based therapies.Tissue Eng.2001;7(2):211-228.

[13] Kern S,Eichler H,Stoeve J,Kluter H,et al.Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood ,or aipose tissue.Stem Cells.2006;24: 1294-1301.

[14] Izadpanah R,Trygg C,Patel B,et al.Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue.Cell Biochem.2006;99:1285-1297.

[15] Rodriguez AM,Elabd C, Deltel F, et al. Adipocyte differentiation of multipotent cells established from human adipose tissue.Biochem Biophys Res Commun. 2004;315: 255-263.

[16] Casteilla L,Planat-Benard V,Cousin B,et al.Plasticity of adipose tissue:a promising therapeutic avenue in the treatment of cardiovascular and blood diseases?Aich Mal Coeur Vaiss.2005;98(9):922-926.

[17] Oedayrajsingh-Vanna MJ,Van HS,KniPPenberg M,et al.Adipose tissue-derived mesenchymal stem cell yield and growth charaeteristics are affected by the tissue-harvesting procedure.Cytotherapy.2006;8(2):166-177.

[18] Hombach-Klonisch S,Panigrahi S,Rashedi I,et al.Adult stem cells and their trans differentiation potential-perspeetives and therapeutic applications.J Mol Med (Berl).2008;86(12): 1301-1314.

[19] 中华人民共和国科学技术部.关于善待实验动物的指导性意见. 2006-09-30.

[20] 陆东风,吴昊,黄璟,等.新生SD大鼠心脏干细胞的体外分离培养与鉴定[J].南方医科大学学报,2006,26(11):97-100.

[21] Strauer BE,Brehm M,Zeus T,et al.Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans.Circulation. 2002; 106(15):1913-1918

[22] Makino S,Fukuda K,Miyoshi S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro.J Clin Invest. 1999;103(5):697-705.

[23] Kadivar M,Khatami S,Mortazavi Y,et al.In vitro cardiomyo genic potential of human umbilical vein derived mesenchymal stem cells.Biochem Biophys Res Commun.2006;340(2): 639-647.

[24] Cheng F,Zou P,Yang H,et al.Induced differentiation of human cord blood mesenchymal stem progenitor cells into cardiomyocyte-like cells in vitro.Huazhong Univ Sci Technolog Med Sci.2003;23(2):154.

[25] 罗凯,单根法,钟兹,等.脐血间充质干细胞肌源件诱导分化的研究[J].上海第二医科大学学报,2005,25(2):134-137．

[26] Huang JL,Yang SX.Differentiation of mesenchymal stem cells derived from human umbilical cord into cardiomyocytes. J Clin Rehabil Tissue Eng Res. 2008;12(47):9367-9370.

[27] 冯应君,杨汉东,汤天军.脐带血干细胞诱导分化为心肌样细胞电生理特性的实验研究[J].实用医学杂志,2007,23(5):613-616.

[28] Gang EJ,Jeong JA,Hong SH,et al.Skeletal myogenic differentiation 0f mesenchymal stem cells isolated from human umbilical cord blood.Stem Cells.2004;22:617-624．

[29] Siepe M,Akhyari P,Lichtenberg A,et al.Stem cells used for cardiovascular tissue engineering. Eur J Cardiothorac Surg. 2008;34:242-254.

[30] Zuk PA,Zhu M,Ashjian P,et al.Human adipose tissue is a source of multipotent stem ceils. Mol Biol Cell.2002; 13: 4279-4295．

[31] Sakai D,Mochida J,Iwashina T,et al.Differentiation of mesenchymal stem cells transplanted to a rabbit degenerative disc model model:potential and limitations for stem cell therapy in disc regeneration. Spine. 2005;30(21): 2379-2387.