Treatment of osteoarthritis using colony-forming cells in stromal vascular fraction of adipose tissue

doi:10.3969/j.issn.2095-4344.2015.14.004

Abstract

Abstract:

BACKGROUND: Stromal vascular fraction digested from adipose tissue is a mixture of a variety of cells, and clone-like cell components may have the real ability to maintain the stemness of mesenchymal stem cells

OBJECTIVE: To separate, passage and culture colony-forming cells in the stromal vascular fraction of adipose tissue, and to test its ability to repair the knee cartilage in animal models.

METHODS: Stromal vascular fraction isolated from fresh adipose tissue was cultured to collect clone-like adherent cells that were subject to digestion and passage until the third generation. A rabbit model of osteoarthritis was established surgically, and the third generation of colony-forming cells were transplantated into the articular cavity of model rabbits. Model rabbits with no cell transplantation served as controls. Cartilage regeneration in the knee joint of model rabbits were observed and compared.

RESULTS AND CONCLUSION: At 3 days after transplantation of stromal vascular fraction cells, clone-like cell colonies appeared in adherent cells; at 5 days, the amount of clone-like cell colonies and surrounding single cells were both increased. At the passage 3, the clone-like cell colonies were also visible, which were fusiform in shape. Gross observation and MRI examination showed that these colony-forming cells could promote cartilage repair in the joint cavity, increase secretion of synovial fluid, and help to control inflammation and narrow ulceration area. Experimental findings are beneficial for optimizing the isolation and culture of adipose-derived mesenchymal stem cells and provide a new way for the clinical treatment of osteoarthritis.

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

全文链接：

Key words: Adipose Tissue, Mesenchymal Stem Cell Transplantation, Osteoarthritis

CLC Number:

R394.2

Lv Pin-lei, Su Yue-han, Cao Yun, Wang Zheng. Treatment of osteoarthritis using colony-forming cells in stromal vascular fraction of adipose tissue[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(14): 2149-2154.

Figures/Tables 2

2.1 兔脂肪血管基质部分中原代克隆形成细胞的形态分离新鲜兔脂肪组织获得血管基质部分，接种后24 h内细胞贴壁。培养3 d时，增殖的贴壁细胞中出现克隆样生长的细胞集落，见图1A，2A。集落内部细胞形态较小，呈多边形；集落边缘细胞形态较长，呈梭形。培养5 d时，非克隆样生长的单个细胞数量也已较多；细胞集落较3 d时形态更大、更密实，见图1B，2B。当集落中细胞数大于100或集落间发生接触现象时，立即挑取克隆进行消化传代。 2.2 兔脂肪血管基质部分中克隆形成细胞的增殖情况将兔脂肪组织血管基质部分中克隆形成细胞挑选出来传代培养至第3代，细胞增殖能力旺盛，多呈梭形，但仍存在大量细胞呈集落形态分布，见图3。 2.3 实验动物数量分析共8只成年雄性新西兰大白兔，模型组3只，实验组3只，正常组2只。模型组和实验组通过改良Hulth法进行手术造模，手术过程中以及手术成功后均未见异常现象发生。 2.4 细胞移植前后膝关节大体观察结果正常组关节软骨外观呈浅蓝白色，半透明；关节面光滑平整，光泽明亮，无裂纹及软化灶；关节边缘规则齐整，无骨赘形成，见图4A。通过手术将兔后肢膝关节部位前后交叉韧带及内侧副韧带切断，完整切除内侧半月板，造成膝关节运动不稳定。术后驱赶12周后，模型兔膝关节内滑膜大部分呈结节样增生，滑液量少混浊，表面有重度裂纹及溃疡形成，多达骨质，软骨下骨暴露，有大量骨赘形成，见图4B。关节腔内细胞移植4周后，模型兔膝关节面较移植前平滑，滑液量增加，溃疡面缩小，见图5。 2.5 MRI检测骨关节炎模型兔的膝关节修复情况通过MRI成像系统可以清楚地看到，正常组兔的膝关节中存在半月板，而模型组和实验组兔中均不存在半月板；模型组兔关节面软骨有明显的磨损迹象，软骨较薄，胫骨头后侧有明显骨赘形成；实验组兔移植细胞治疗后，关节面软骨较模型组增厚，且较光滑，见图6。"

References

[1] Lawrence RC, Felson DT, Helmick CG, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum. 2008;58(1):26-35.
[2] Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage. 2002;10(6):432-463.
[3] Lee CR, Grodzinsky AJ, Hsu HP, et al. Effects of harvest and selected cartilage repair procedures on the physical and biochemical properties of articular cartilage in the canine knee. J Orthop Res. 2000;18(5):790-799.
[4] 张荣耀,毕晓娟,马艳,等.全骨髓法培养C57小鼠骨髓间充质干细胞的生物学特性[J].中国组织工程研究,2014,18(1):45-50.
[5] 宋丽娟,毕晓娟,马艳,等.小鼠脂肪源干细胞的生物学特性及增殖特点[J].新疆医科大学学报,2013,36(4):479-483.
[6] 邢昕,吕蓉,李素萍,刘忠,等.体外分离培养人源性脂肪间充质干细胞及其鉴定[J].中国输血杂志,2013,26(8):708-712.
[7] 侯晓琳,郁卫东,崔梅花,等.小鼠脂肪间充质干细胞的分离培养及肠道归巢[J].中国组织工程研究,2015,19(6):854-860.
[8] 吕品雷,苏约翰,崔大祥,等.荧光磁性纳米粒子标记三维培养系统中的脂肪间充质干细胞[J].中国组织工程研究,2015,19(1):44-48.
[9] Kassem M, Kristiansen M, Abdallah BM. Mesenchymal stem cells: cell biology and potential use in therapy. Basic Clin Pharmacol Toxicol. 2004;95(5):209-214.
[10] 吴清法,王立生,吴祖泽.间充质干细胞的来源及临床应用[J].军事医学科学院院刊, 2002,26(3):213-217.
[11] 文铁,刘玲,吕国华,等.年龄对人骨髓间充质干细胞体外生长与衰老的影响[J].中国组织工程研究与临床康复,2009,13(32): 6243-6248.
[12] Strem BM, Hicok KC, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med. 2005;54(3):132-141.
[13] Taha MF, Hedayati V. Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells. Tissue Cell. 2010;42(4):211-216.
[14] Kang BJ, Ryu HH, Park SS, et al. Comparing the osteogenic potential of canine mesenchymal stem cells derived from adipose tissues, bone marrow, umbilical cord blood, and Wharton's jelly for treating bone defects. J Vet Sci. 2012;13(3): 299-310.
[15] 苏约翰,卫超,吕品雷,等.利用细胞外基质大规模扩增临床级人脂肪间充质干细胞[J].中国组织工程研究,2014,18(10):1521- 1531.
[16] 翟吉良,翁习生,邱贵兴.骨关节炎动物模型的建立及选择[J].中国矫形外科杂志, 2007,15(11):843-845.
[17] 金刚.骨关节炎动物模型研究进展[J].中国民族民间医药, 2014, 23(13):24,26.
[18] 刘献祥,李西海,周江涛.改良Hulth造模法复制膝骨性关节炎的实验研究[J].中国中西医结合杂志, 2005, 25(12):1104-1108.
[19] 詹楚宁,付拴虎,程俊,等.骨关节炎实验动物模型研究进展[J]. 大众科技, 2013,15(12):121-123.
[20] Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science. 1998;279(5356):1528-1530.
[21] Dennis JE, Merriam A, Awadallah A, et al. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res. 1999;14(5):700-709.
[22] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143-147.
[23] Smith JR, Pochampally R, Perry A, et al. Isolation of a highly clonogenic and multipotential subfraction of adult stem cells from bone marrow stroma. Stem Cells. 2004;22(5):823-831.
[24] Deng J, Petersen BE, Steindler DA, et al. Mesenchymal stem cells spontaneously express neural proteins in culture and are neurogenic after transplantation. Stem Cells. 2006;24(4): 1054-1064.
[25] Engler AJ, Sen S, Sweeney HL, et al. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677-689.
[26] Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science. 19994;284 (5417):1168-1170.
[27] 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.
[28] Okura H, Saga A, Fumimoto Y, et al. Transplantation of human adipose tissue-derived multilineage progenitor cells reduces serum cholesterol in hyperlipidemic Watanabe rabbits. Tissue Eng Part C Methods. 2011;17(2):145-154.
[29] Banas A, Teratani T, Yamamoto Y, et al. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology. 2007;46(1):219-228.
[30] Williams AR, Hare JM. Mesenchymal stem cells: biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease. Circ Res. 2011;109(8): 923-940.
[31] Chung DJ, Hayashi K, Toupadakis CA, et al. Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia. Res Vet Sci. 2012;92(1):66-75.
[32] Spencer ND, Chun R, Vidal MA, et al. In vitro expansion and differentiation of fresh and revitalized adult canine bone marrow-derived and adipose tissue-derived stromal cells. Vet J. 2012;191(2):231-239.
[33] Reich CM, Raabe O, Wenisch S, et al. Isolation, culture and chondrogenic differentiation of canine adipose tissue- and bone marrow-derived mesenchymal stem cells--a comparative study. Vet Res Commun. 2012;36(2):139-148.
[34] Kang BJ, Lee SH, Kweon OK, et al. Differentiation of canine adipose tissue-derived mesenchymal stem cells towards endothelial progenitor cells. Am J Vet Res. 2014;75(7): 685-691.
[35] Wang QZ,Zhou LL,Guo Y,et al.Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis.Neural Regen Res. 2013;8 (35): 3353-3358.
[36] Prockop DJ. Repair of tissues by adult stem/progenitor cells (MSCs): controversies, myths, and changing paradigms. Mol Ther. 2009;17(6):939-946.
[37] 韩爽,卢世璧,刘强,等.自体脂肪间充质干细胞复合人脐带Wharton胶支架修复兔膝关节软骨缺损[J].中国组织工程研究, 2012,16(19):3496-3501.
[38] Yang YF,Ma T,Gong K,et al.Adipose-derived mesenchymal stem cell transplantation promotes adult neurogenesis in the brains of Alzheimer’s disease mice.Neural Regen Res. 2014; 9(8): 798-805.
[39] Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006;24(5):1294-1301.
[40] Izadpanah R, Trygg C, Patel B, et al. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem. 2006;99(5):1285-1297.