Autologous bone marrow mesenchymal stem cell transplantation for the treatment of early femoral head necrosis

doi:10.3969/j.issn.2095-4344.0927

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) as ideal seed cells have been worldwide confirmed to improve the microcirculation in the necrotic area of the femoral head.
OBJECTIVE: To observe the efficacy of autologous BMSCs transplantation in the treatment of early necrosis of the femoral head.
METHODS: (1) In vitro experiment. Osteoblasts were co-cultured with BMSCs using Transwell system as experimental group, osteoblasts cultured normally as blank group, and osteoblasts cultured in serum-free medium as control group. MTT was used to detect cell viability at 3 days of culture, flow cytometry used to detect cell apoptosis, and ELISA used to detect levels of vascular endothelial growth factor and bone morphogenetic protein 2 in the culture medium. (2) In vivo experiment. Thirty New Zealand white rabbits were randomized into three groups: blank group, control group (core decompression plus normal saline injection), and experimental group (core decompression plus autologous BMSCs injection). Femoral head samples from white rabbits were taken for hematoxylin-eosin and immunohistochemical staining at 7 days after cell transplantation.
RESULTS AND CONCLUSION: (1) Compared with the control and blank groups, the experimental group showed increased osteoblast proliferation, decreased osteoblast apoptosis, and enhanced levels of vascular endothelial growth factor and bone morphogenetic protein 2 in the culture medium (all P < 0.05). (2) In the control group, broken trabecular bone in the femoral head, evident adipocyte necrosis and a large number of empty bone lacunae were detected. In the experimental group, the number of osteoblasts and capillaries was increased as compared with the control group. The expression levels of vascular endothelial growth factor and bone morphogenetic protein 2 in the necrotic area of the femoral head were significantly increased in the experimental group compared with the control group (P < 0.05). To conclude, BMSCs transplantation can improve the microcirculation in the necrotic area of the femoral head by increasing the expression of vascular endothelial growth factor and bone morphogenetic protein 2.

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: Bone Marrow, Mesenchymal Stem Cell Transplantation, Vascular Endothelial Growth Factors, Bone Morphogenetic Proteins, Femur Head Necrosis, Tissue Engineering

CLC Number:

R394.2

Chen Xiang, Wang Wei-zhuo, Xue Jian-li, Xia Li-feng, Li Tao, Qiu Ling, Song Huan-jin. Autologous bone marrow mesenchymal stem cell transplantation for the treatment of early femoral head necrosis[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 4007-4013.

Figures/Tables 1

2.1 成骨细胞活力、凋亡及血管内皮生长因子、骨形态发生蛋白2水平空白组成骨细胞吸光度值为0.273 2± 0.030 8，对照组成骨细胞吸光度值为0.174 6±0.045 6，实验组成骨细胞吸光度值为0.263 6±0.052 6。对照组与实验组吸光度值差异有显著性意义(P < 0.05)，提示成骨细胞与BMSCs共培养时，可以促使成骨细胞活力增强(图1A)。空白组成骨细胞总凋亡率为(5.6±2.1)%，对照组总凋亡率为(16.3±2.8)%，实验组总凋亡率为(8.8±3.6)%。各组间比较差异有显著性意义(P < 0.05)，提示成骨细胞与BMSCs共培养时，可减少成骨细胞凋亡(图1B)。空白组、对照组、实验组血管内皮生长因子水平分别为(266.4±10.2)，(322.1±10.3)，(391.7±18) ng/L；空白组、对照组、实验组骨形态发生蛋白2水平分别为(14.95± 2.26)，(11.80±3.17)，(17.58±2.01) μg/L。组间比较行方差分析：①血管内皮生长因子组间比较F=668.86，P=0.000，进一步行Dunnett T检验，每两组间比较P=0.000，提示每两组的血管内皮生长因子水平差异有显著性意义；②骨形态发生蛋白2组间比较F=473.847，P=0.000，进一步行Dunnett T检验，每两组间比较P=0.000，提示每两组的骨形态发生蛋白2水平差异有显著性意义(图1C，D)。 2.2 体内实验结果造模4周后动物双后肢活动减少；治疗后观察手术动物一般情况良好，伤口无红肿、无渗液；治疗后3 d患肢无主动活动，1周后患肢开始自主活动，期间无动物死亡。 2.2.1 MRI检测造模效果行兔股骨头冠状位(OA)及矢状位(OC)MRI检查，MRI结果T2加权抑脂序列可见：空白组兔股骨头区信号均一，未有异常信号表现(图2A，B)；实验组可见兔股骨头区呈现不均匀的低信号，关节腔内可见少量关节积液，实验组MRI检查符合早期股骨头缺血性坏死期表现，提示造模成功(图2C，D)。 2.2.2 苏木精-伊红染色结果获取标本时，按原手术切口逐层切开，各组动物切口周围组织纤维化粘连，无流脓、无溢液，取兔股骨头区域制备切片行苏木精-伊红染色(图3)。空白组股骨头骨小梁排列规律，骨小梁饱满，细胞核较大位于中央，骨小梁中罕见空骨凹陷，骨髓细胞增殖活跃，骨小梁边缘可见成骨细胞依附。对照组股骨头骨小梁内成骨细胞坏死，有大量空骨陷窝，骨髓脂肪细胞增大，主要分布在髓芯减压孔道区；实验组股骨头骨小梁结构较紊乱，成骨细胞增多，新生血管较多，骨髓脂肪细胞增多，但较对照组脂肪细胞增大；骨小梁中仍有骨细胞，空骨陷窝较空白组宽大，细胞核周可见空晕，但空骨陷窝较对照组明显减少；骨小梁周围成骨细胞较空白组减少，较对照组增多。每组每只动物标本随机选取3张切片，每张切片随机选取5个视野进行计数。空白组、对照组、实验组血管数分别为(8.4±1.3)条，(3.5±1.2)条，(6.2±1.5)条，空骨陷窝率分别为(13.9±7.5)%，(58.3±7.3)%，(37.4±6.6)%。各组血管数、空骨陷窝率比较，差异有显著性意义(P < 0.05)。 2.2.3 免疫组化染色结果血管内皮生长因子染色阳性细胞在镜下呈棕黄色，其主要分布于血管内皮细胞胞质和细胞膜内。空白组血管内皮生长因子阳性表达(图4A)。对照组血管减少，出现血栓，血管内皮生长因子呈阴性表达(图4B)；实验组新生血管增多，血管内皮生长因子呈弱阳性，提示骨髓间充质干细胞移植可使兔股骨头区血管内皮生长因子表达增加(图4C)。空白组、对照组、实验组血管内皮生长因子免疫组化染色吸光度值分别为0.285 9±0.066 0，0.187 2±0.037 5，0.250 0±0.058 5，组间比较差异有显著性意义(P < 0.05)。骨形态发生蛋白2阳性染色表达在成骨细胞胞质和细胞膜内，成骨细胞主要存在于骨小梁周围边缘。空白组可见骨小梁周围成骨细胞胞质骨形态发生蛋白2阳性表达(图5A)。对照组成骨细胞罕见，骨小梁周围成骨细胞胞质骨形态发生蛋白2阴性表达(图5B)。实验组骨小梁成骨细胞增多，细胞胞质骨形态发生蛋白2阳性表达(图5C)，提示骨髓间充质干细胞移植可使兔股骨头坏死区骨形态发生蛋白2表达增加。空白组、对照组、实验组骨形态发生蛋白2免疫组化染色吸光度值分别为0.238 9±0.033 5，0.132 0±0.037 3，0.213 1±0.051 3，组间比较差异有显著性意义(P < 0.05)。"

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