In vitro isolation and culture of chondrocytes from human osteoarthritis patients

doi:10.3969/j.issn.2095-4344.1791

Abstract

Abstract:

BACKGROUND: Numerous studies addressing cells in osteoarthritis still focus on the in vitro culture of articular chondrocytes in animal models. Degeneration in the animal models is not completely consistent with that in human osteoarthritis. How to construct an in vitro cell model using chondrocytes from human osteoarthritis and to study changing trend of its characteristic proteins is the key to simulating the cartilage degeneration in human osteoarthritis in vivo.
OBJECTIVE: To investigate the in vitro isolation and culture of human chondrocytes from osteoarthritis patients, to observe the morphological characteristics of human osteoarthritis chondrocytes from primary to third generation, and to study the changes in biological characteristics of chondrocyte-related proteins in different generation.
METHODS: The study protocol was in line with the ethic requirements of Chinese PLA General Hospital with the approval No. S2017-23-7. Six cases undergoing arthroplasty for severe osteoarthritis (2 males and 4 females, age 62-72 years old with a mean age of (68.3±3.39) years) were enrolled. The chondrocytes from abandoned cartilage tissue in these patients were isolated and cultured by one-step enzymatic digestion (type II collagenase), and then subcultured, so as to construct an in vitro chondrocyte culture system for osteoarthritis. The morphology of cells at different generations was observed by inverted phase contrast microscope. Hematoxylin-eosin staining, toluidine blue staining and type II collagen immunofluorescence staining were used for cell identification. Western blot was used to detect the expressions of Col2a, Aggrecan and matrix metalloproteinase-13 in each generation of chondrocytes at the fusion rate of 70%.
RESULTS AND CONCLUSION: After digestion using type II collagenase, the cells scattered around the tissue mass could be observed at about 1 week of culture, and these cells could be subcultured for further study after about 3 weeks. Morphological observation, hematoxylin-eosin staining, toluidine blue staining and type II collagen immunofluorescence staining proved that human chondrocytes were successfully cultured. The relative expression of Col2a and Aggrecan in chondrocytes at the third generation was significantly decreased as compared with that in primary cells (P < 0.01), and the expression gradually decreased with the subculture times. Matrix metalloproteinase-13 expression gradually increased with the increase of subculture times (P < 0.01). In conclusion, chondrocytes can be successfully isolated from the osteoarthritis specimens by one-step digestion of type II collagenase and then subcultured. The dedifferentiation of osteoarthritis chondrocytes in vitro increases with the increase of subculture times and the expression of functional proteins decreases as a whole. The chondrocytes within three generations present with cartilage degeneration in osteoarthritis and may be the best choice for experimental studies.

Key words: osteoarthritis, joint replacement, human chondrocytes, one-step enzymatic digestion, type II collagenase, type II collagen, aggrecan, National Natural Science Foundation of China

CLC Number:

Wei Yu, Wei Min. In vitro isolation and culture of chondrocytes from human osteoarthritis patients[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4056-4061.

Figures/Tables 2

2.1 软骨细胞形态倒置相差显微镜观察，培养1周左右可见组织块周围爬出散在的细胞，多为短梭形、三角形，少数为多角形，瓶底可见散在分布的单个细胞；2周左右可见组织块周围多量的细胞成簇生长，为梭形或多角形，见图1A；3周左右瓶底细胞已经连接成片，融合率为70%左右，部分细胞伸出伪足，形态欠规则，似成纤维细胞样。经传代后细胞增殖速度明显加快，24 h内细胞全部贴壁，两端伸出伪足，1周左右可长满瓶底。随着传代次数的增多，细胞形态似逐渐去分化为成纤维细胞，两端伸出数量不等的伪足，细胞形态愈发不规则，且生长速度开始下降，3代后上述特征逐渐表达明显，见图1B-D。 2.2 软骨细胞的不同染色观察结果苏木精-伊红染色可见第1代软骨细胞基本为三角形或多角形，细胞核呈蓝紫色，卵圆形或圆形，细胞质呈粉红色，随着传代次数的增加，至第3代软骨细胞时形态逐渐变为长梭形。甲苯胺蓝染色可见细胞核为深蓝色，胞浆着色稍浅呈浅蓝色，其内可见少量蓝紫色异染颗粒，传代至第3代时多为长梭形细胞，胞浆染色变浅，形态似成纤维细胞，见图2。 2.3 Ⅱ型胶原的免疫荧光染色观察结果 Ⅱ型胶原免疫荧光显示第1代细胞胞浆及胞膜出现清晰绿色荧光，细胞核区域在不同波段荧光激发下可见DAPI(蓝色)复染，表明培养的细胞表达特征性Ⅱ型胶原，并主要分布于胞浆和细胞膜上，这些都是软骨细胞的特征，见图3。 2.4 Western blot检测软骨细胞Col2a、MMP-13及Aggrecan的蛋白表达分别对原代和第1-3代人关节炎软骨细胞进行Western blot检测，扫描测定灰度值，并进行分析，见图4。随着软骨细胞培养代数的增加，Col2a与Aggrecan的表达在原代最强(1.029±0.042，0.397±0.025)，之后逐渐下降，至第3代分别为0.463±0.020，0.154±0.147，差异有显著性意义(P < 0.05)，其中Cola2的表达各代差异均有显著性意义(P < 0.01)。MMP-13的蛋白表达量随着培养代数逐渐升高，原代和第1-3代分别为0.387±0.004，0.418±0.027，0.663±0.013，0.790±0.031，差异均有显著性意义(P < 0.01)，见表1。"

References

[1] Roberts BC, Thewlis D, Solomon LB, et al. Systematic mapping of the subchondral bone 3D microarchitecture in the human tibial plateau: Variations with joint alignment. J Orthop Res. 2017;35(9):1927-1941.

[2] Appleton CT. Osteoarthritis year in review 2017: biology. Osteoarthritis Cartilage. 2018;26(3):296-303.

[3] 谢平金,余翔,柴生颋,等.川芎嗪干预早期膝骨关节炎大鼠软骨Ⅱ型胶原纤维α1基因与血管内皮生长因子mRNA及miR20b的表达[J].中国组织工程研究, 2018,22(12):1846-1851.

[4] 欧阳家耀,张海严,方航,等.小鼠软骨前体细胞ATDC5中沉默骨膜蛋白基因能延缓软骨退变[J].中华实验外科杂志, 2018, 35(1):15-18.

[5] 刘明东,盛天金,王万宗.胰蛋白酶及Ⅱ型胶原酶消化获取关节软骨细胞[J].中国组织工程研究,2010, 14(46):8551-8554.

[6] Altman R, Asch E, Bloch D, et al. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum. 1986;29(8):1039-1049.

[7] 邓林峡,余慕雪,潘思年,等.比较单独消化法及分步消化法培养新生大鼠原代软骨细胞的生物学特性[J].中国组织工程研究, 2018,22(25):4047-4052.

[8] 杨物鹏,许建中.软骨细胞培养及其调控[J].中国矫形外科杂志, 2000,7(8):800-803.

[9] Lu Y, Dhanaraj S, Wang Z, et al. Minced cartilage without cell culture serves as an effective intraoperative cell source for cartilage repair. J Orthop Res. 2006;24(6):1261-1270.

[10] 唐新,郑果,黄强,等.人骨关节炎软骨细胞的体外培养及细胞形态学分析[J].实用骨科杂志,2015,21(7):614-618.

[11] Filip A, Pinzano A, Bianchi A, et al. Expression of the semicarbazide-sensitive amine oxidase in articular cartilage: its role in terminal differentiation of chondrocytes in rat and human. Osteoarthritis Cartilage. 2016;24(7):1223-1234.

[12] Manning WK, Bonner WM Jr. Isolation and culture of chondrocytes from human adult articular cartilage. Arthritis Rheum. 1967;10(3):235-239.

[13] Green WT Jr. Behavior of articular chondrocytes in cell culture. Clin Orthop Relat Res. 1971;75:248-260.

[14] Klagsbrun M. Large-scale preparation of chondrocytes. Methods Enzymol. 1979;58:560-564.

[15] 童迅,赵海恩,张栋,等.人关节软骨细胞的体外分离、培养与鉴定[J].现代生物医学进展,2012,12(16):3040-3044.

[16] Darling EM, Pritchett PE, Evans BA, et al. Mechanical properties and gene expression of chondrocytes on micropatterned substrates following dedifferentiation in monolayer. Cell Mol Bioeng. 2009;2(3):395-404.

[17] 童迅,贠喆,张栋,等.人正常及骨关节炎软骨细胞体外培养的对照研究[J].现代生物医学进展, 2013,13(24):4648-4653.

[18] 胡炯,李笑颜,邓廉夫,等.人正常软骨细胞和骨性关节炎软骨细胞体外培养对照研究[J].中国中医骨伤科杂志, 2010,18(10):8-12.

[19] Kokubo M, Sato M, Yamato M, et al. Characterization of layered chondrocyte sheets created in a co-culture system with synoviocytes in a hypoxic environment. J Tissue Eng Regen Med. 2017;11(10):2885-2894.

[20] Qi WN, Scully SP. Extracellular collagen regulates expression of transforming growth factor-beta1 gene. J Orthop Res. 2000;18(6):928-932.

[21] Yik JH, Hu Z, Kumari R, et al. Cyclin-dependent kinase 9 inhibition protects cartilage from the catabolic effects of proinflammatory cytokines. Arthritis Rheumatol. 2014;66(6):1537-1546.

[22] Shiomi T, Lemaître V, D'Armiento J, et al. Matrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs in non-neoplastic diseases. Pathol Int. 2010;60(7):477-496.

[23] Wang M, Sampson ER, Jin H, et al. MMP13 is a critical target gene during the progression of osteoarthritis. Arthritis Res Ther. 2013;15(1):R5.

[24] Ruan G, Xu J, Wang K, et al. Associations between knee structural measures, circulating inflammatory factors and MMP13 in patients with knee osteoarthritis. Osteoarthritis Cartilage. 2018;26(8):1063-1069.

[25] Li H, Wang D, Yuan Y, et al. New insights on the MMP-13 regulatory network in the pathogenesis of early osteoarthritis. Arthritis Res Ther. 2017;19(1):248.