Intra-articular migration of transplanted bone marrow mesenchymal stem cells in rats with articular cartilage injury

doi:10.3969/j.issn.2095-4344.0514

Abstract

Abstract:

BACKGROUND: The application of mesenchymal stem cells (MSCs) in the treatment of cartilage damage has become a hot spot of research. Further studies on the distribution of MSCs in the body after injection and on the underlying mechanism of action are needed.
OBJECTIVE: To observe the migration of bone marrow mesenchymal stem cells (BMSCs) after injection into the region of osteochondral defect.
METHODS: Thirty Sprague-Dawley rats were randomized into two groups (n=15 per group). In the control group, the femoral tochlear was exposed but an osteochondral defect was not made; and after the suture, PKH26-labeled BMSCs were directly injected into the articular cavity of rats. In the experimental group, a cartilage defect of 1 mm in diameter and 1 mm in depth was made in the rat femoral trochlea, and 5×106 PKH26-labeled BMSCs were injected into the defect after operation. At 1, 3 and 7 days after injection, the femoral condyle was taken to make frozen sections followed by DAPI staining. The distribution of BMSCs was observed under laser scanning confocal microscope.
RESULTS AND CONCLUSION: In the control group, PKH26-labeled BMSCs were not transferred to the subchondral bone. In the experimental group, BMSCs were detected in the subchondral bone area at 1, 3 days after injection of PKH26-BMSCs in the bone cartilage defect area, and the BMSCs were also found in the bone marrow cavity at 7 days after injection. In conclusion, BMSCs in the articular cavity cannot migrate into the subchondral bone and bone marrow cavity unless the cartilage of the femoral condyle is damaged.

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: Osteochondrosis, Bone Marrow, Mesenchymal Stem Cell Transplantation, Cell Migration Assays, Tissue Engineering

CLC Number:

R394.2

Sun Bai-chuan, Wang Shou-feng, Liu Xue-jian, Zhang Kai-hong, Chen Peng, Huang Shao-dai, Lu Chang-feng, Wang Chong, Yu Wen, Wang Yu, Zhang Zeng-zeng, Zhou Cheng-fu, Peng Jiang. Intra-articular migration of transplanted bone marrow mesenchymal stem cells in rats with articular cartilage injury[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2699-2704.

Figures/Tables 1

2.1 细胞形态原代培养至第2天，倒置显微镜下观察，大部分细胞从骨片周围爬出，细胞呈长梭形；第3天时，细胞爬出的数量增多；第4天时，细胞数量明显增多，细胞呈长梭形围绕骨片生长，部分细胞集落间有融合现象，当细胞密度达50%时，1︰1传代。第1-3代骨髓间充质干细胞均呈长梭形，细胞形态无明显差异(图2)。 2.2 流式细胞鉴定结果流式细胞仪检测细胞表面抗原，高表达CD29和CD90，低表达CD34和CD45(图3)。 2.3 成脂、成骨、成软骨诱导分化能力成脂诱导10 d后，大部分细胞内出现圆形脂滴，细胞由长梭形变成圆形或多边形，油红O染色后脂滴呈红色(图4A)；成骨诱导3周后，细胞呈结层状、结节状生长，失去原始细胞结构，茜素红染色呈红色(图4B)；成软骨诱导4周后，细胞由扁平的细胞团块逐渐变成球形，并且软骨球不断增大、变圆，软骨球经石蜡切片阿利新蓝染色后软骨基质呈蓝色(图4C)，甲苯胺蓝染色后软骨细胞呈紫红色(图4D)。 2.4 荧光显微镜观察骨髓间充质干细胞标记情况 PKH26染色的骨髓间充质干细胞标记率达98%，染料均匀分布在细胞膜上，在倒置荧光显微镜下呈红色荧光，细胞轮廓清晰，胞膜完整。继续培养24 h后，细胞贴壁生长，形态良好，荧光显微镜下呈红色。细胞传代后荧光显微镜下仍呈红色荧光(图5)。 2.5 激光共聚焦显微镜下观察PKH26标记的骨髓间充质干细胞在体内的迁移情况关节腔内注射PKH26标记的骨髓间充质干细胞，第1，3，7天行股骨髁冰冻切片，DAPI染色，在激光共聚焦荧光显微镜下观察。假手术组：图6A为DAPI染色结果，细胞发出蓝色荧光，图6B为PKH26标记的骨髓间充质干细胞发出红色荧光，图6C为明场下的大鼠股骨髁冰冻切片，图6D为重叠图像。结果显示：正常大鼠关节腔注射PKH26标记的骨髓间充质干细胞，未见明显红色荧光细胞向软骨及软骨下骨区迁移。骨软骨缺损组：图7A为DAPI染色结果，细胞发出蓝色荧光，图7B为PKH26标记的骨髓间充质干细胞发出红色荧光，图7C为明场下的大鼠股骨髁冰冻切片，图7D为重叠图像。注射细胞后第1天，骨髓间充质干细胞在骨软骨缺损区；注射细胞后第3天，红色荧光细胞在缺损旁软骨及软骨下骨区；注射细胞后第7天，在骨髓腔内可见大量的PKH26标记的骨髓间充质干细胞。"

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