Bone marrow mesenchymal stem cells co-cultured with allogenic bone in the articular cavity

doi:10.3969/j.issn.2095-4344.2016.19.003

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells and allogenic bones are commonly used as seed cells and scaffolds, respectively, for constructing tissue-engineered cartilage through in vitro co-culture. The loose body of the knee joint can survive in the articular cavity for a long time, and maintain certain characteristics of cartilage tissues. Therefore, the articular cavity can provide a good environment for the growth and development of chondrocytes.
OBJECTIVE: To investigate the effect of bone marrow mesenchymal stem cells co-cultured with allogenic bone in the articular cavity.
METHODS: Bone marrow mesenchymal stem cells were isolated from five newborn New Zealand white rabbits. One adult New Zealand rabbit was enrolled to prepare allogenic bone for co-culture with bone marrow mesenchymal stem cells. Afterwards, bone marrow mesenchymal stem cells and allogenic bone composites were cultured in the articular cavity (intracavitary culture group) or in vitro as in vitro culture group, respectively; the normal cartilage tissues grew in the articular cavity as control group. Cells were observed by hematoxylin-eosin staining and type II collagen immunohistochemistry staining at 4, 8 and12 weeks of culture.
RESULTS AND CONCLUSION: At 12 weeks culture, hematoxylin-eosin staining showed: in the control group, chondrocytes arranged tightly and directionally with red stained cytoplasm and cartilage matrix as well as blue nuclei; in the intracavitary culture group, the scaffold was mostly absorbed and chondrocytes grew into the scaffold in a certain direction with smaller shape, while cytoplasm and cartilage matrix were red stained, blue nuclei appeared; in the in vitro culture group, abundant chondrocytes proliferated in a disordered arrangement. Immunohistochemistry staining showed: the absorbance (A) values in the intracavitary culture group showed a continuous increase, but no obvious change was in the other two groups. Moreover, at 4, 8 and 12 weeks of culture, A values in the control group and intracavitary culture group were significantly higher than that in the in vitro culture group (P < 0.05); at 4 and 8 weeks, A value in the intracavitary culture group was significantly lower than that in the control group (P < 0.05), but at 12 weeks, there was no significant difference in A value between the two groups (P > 0.05). These results suggest that the tissue-engineered cartilage can be constructed by bone marrow mesenchymal stem cells co-cultured with allogenic bone under in vitro and in vivo environment, especially in the articular cavity.

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

CLC Number:

R394.2

Gao Feng-guang, Zhou Bai-sui, Mu Hai-bo. Bone marrow mesenchymal stem cells co-cultured with allogenic bone in the articular cavity[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(19): 2756-2762.

Figures/Tables 2

2.1 实验动物数量分析关节腔内观察实验纳入15只成年新西兰白兔，全部进入结果分析，无脱失。 2.2 兔骨髓间充质干细胞培养结果对兔骨髓间充质干细胞进行原代培养，培养后1 d后，出现少量细胞贴壁生长。接种3 d，贴壁生长细胞数量增加。经传代培养，细胞呈旋涡状或螺旋状，传至第3代未出现明显的改变(见图1)。 2.3 兔骨髓间充质干细胞成骨诱导结果对第3代骨髓间充质干细胞进行软骨定向诱导之后，连续培养2周，可观察到细胞周围出现基质分泌(见图2)。 2.4 同种异体骨形态观察制备得到的同种异体骨支架材料触之具有一定的轻度和韧性，可剪裁为不同的大小(见图3)。经电镜扫描，材料呈疏松多孔状结构(见图4)。 2.5 术后动物情况观察对新西兰白兔进行同种异体骨-骨髓间充质干细胞复合物膝关节腔内植入之后，未出现明显的排异反应。术后切口干燥，未出现渗出、红肿，动物活动正常，未出现明显的关节活动受限。术后动物均正常饮食，未出现动物死亡。 2.6 苏木精-伊红染色结果培养12周后进行苏木精-伊红染色和观察，可见：①正常对照组：细胞质和软骨基质出现红染，细胞核出现蓝染，软骨细胞按照一定的方向呈紧密状有序排列。②腔内培养组：细胞质和软骨基质出现红染，细胞核出现蓝染，支架材料基本吸收。软骨细胞长入支架之中，细胞按照一定应力方向排列，且形态变小。③体外培养组：软骨细胞出现大量增殖，但呈无序状排列。 2.7 免疫组织化学染色结果经免疫组织化学染色和观察，正常对照软骨细胞外基质中可发现大量棕黄色颗粒，经Ⅱ型胶原染色呈现出强阳性。腔内培养组经Ⅱ型胶原染色呈现出阳性，胞浆和基质均为棕黄色，且随着时间的不断推移，棕黄色颗粒数量越来越多。4周体外培养组可观察到出现少量Ⅱ型胶原表达的情况；8周时胞周存在棕黄色颗粒；12周经免疫组织化学反应Ⅱ型胶原呈强阳性，软骨细胞胞浆及胞外基质可观察到少量的黄色颗粒。经计数可得，随着培养时间的推移，腔内培养组的A值呈现出不断上升的情况，体外培养组和正常对照组均未出现明显的改变。且培养4，8，12周，正常对照组和腔内培养组的A值均显著高于体外培养组(P < 0.05)。培养4，8周，腔内培养组的A值均显著低于正常对照组(P < 0.05)。但培养12周，两组之间A值经比较差异无显著性意义(P > 0.05)。具体结果见表1及图5。"

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