Chondrogenic co-culture of allogenic decalcified bone matrix and bone marrow mesenchymal stem cells in the joint cavity: comparison of cartilage traits in the same joint cavity

doi:10.3969/j.issn.2095-4344.2014.08.004

Abstract

Abstract:

BACKGROUND: Loose bodies in the knee are found to survive for a long term and maintain certain histophysiological properties of cartilage tissue. Therefore, a bold hypothesis is proposed that the joint cavity may be a preferred environment for chondrocyte growth and development, supporting the concept of “intracavitary culture and intracavitary transplantation”.
OBJECTIVE: To observe the trait difference of chondrogenic culture with allogenic decalcified bone matrix and bone marrow mesenchymal stem cells in the joint cavity or in vitro versus cartilage in the same cavity.
METHODS: There were three groups in this experiment: in in vitro culture group, bone marrow mesenchymal stem cells from newborn rabbits undergoing chondrogenic culture were co-cultured with decalcified bone matrix from adult rabbits in vitro; in intracavitary culture group, bone marrow mesenchymal stem cells from newborn rabbits undergoing chondrogenic culture were co-cultured with decalcified bone matrix from adult rabbits in the joint cavity; normal cartilage in the same cavity served as control group.
RESULTS AND CONCLUSION: (1) After 12 weeks of culture, in the in vitro culture group, hematoxylin-eosin staining showed a small amount of chondrocytes proliferated, with blue-stained nuclei; toluidine blue staining showed chondrocytes arranged disorderly, surrounded by a small amount of matrix; Masson staining showed a small positive area and irregular cell arrangement; type II collagen immunohistochemistry staining showed a few of yellow particles in the cytoplasm and extracellular matrix. (2) After 12 weeks of culture, in the intracavitary culture group, hematoxylin-eosin staining showed proliferation of chondrocytes with blue-stained nuclei; toluidine blue staining showed cluster-shaped arrangement of chondrocytes surrounded by the matrix with lacuna formation; Masson staining showed there were many positive cells with blue-stained matrix that arranged in a certain stress direction; immunohistochemical identification of type II collagen was positive, and brown-yellow stained particles could be discerned in the extracellular matrix. These findings indicate that tissue-engineered cartilage can be generated by co-culture of allogenic decalcified bone matrix and bone marrow mesenchymal stem cells in the joint cavity or in vitro, and the cartilage cultured in the joint cavity is more close to normal cartilage than that cultured in vitro.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: biocompatible materials, tissue engineering, cartilage, stem cells

CLC Number:

R318

Xu Bin, Zhou Liang, Wang Ying-ming, Qian San-xiang. Chondrogenic co-culture of allogenic decalcified bone matrix and bone marrow mesenchymal stem cells in the joint cavity: comparison of cartilage traits in the same joint cavity[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(8): 1165-1171.

Figures/Tables 3

2.1 骨髓间充质干细胞的分离培养及诱导体外培养：原代培养24 h后少量圆形细胞贴壁，多数细胞仍呈悬浮状态。接种3 d后贴壁细胞增多，其中少数细胞伸出伪足变成纺锤样、长梭形，细胞核较大，呈圆形或椭圆形。4-6 d可见细胞集落形成，以后集落数目渐增多，10 d左右可达约90%融合。增殖传代的细胞贴壁迅速，约3 h大部分细胞贴壁，细胞呈长梭形均匀生长，形态比原代培养的细胞更均匀，细胞生长旺盛、增殖迅速，胞核明显，核仁清晰，核浆比例大，4-6 d即能达到90%融合，细胞形态均匀，平行排列。呈螺旋状或漩涡状，传至第3代时无明显变化(图1A)。成软骨诱导：取第3代骨髓间充质干细胞，加入软骨定向诱导液诱导培养14 d后可见细胞周围有基质分泌 (图1B)。 2.2 同种异体脱钙骨的形态观察脱钙骨支架材料维持其原有形态并有一定韧性和强度，可按要求修剪成一定形状，呈蜂窝状多孔的三维立体结构，表面孔隙与深层孔隙相通，表面纤维有少部分断裂(图1C,D)。 2.3 组织标本观察新西兰白兔施行膝关节腔内复合物植入后无明显排异反应，手术切口干燥，无红肿、渗出，膝关节活动无明显受限。取材时见关节液清亮，关节腔无明显粘连、滑膜增生、关节游离体或骨赘形成。饲养过程中未出现动物死亡。大体形态观察：体外培养组，术后4周时，标本表面凹凸不平明显，内部空隙较多，随着时间延长，至术后12周时，标本表面凹凸不平变平坦，内部空隙逐渐减少，但内部尚有部分空隙(图1E)。腔内培养组，术后4周时，标本表面凹凸不平，内部空隙较多，随着时间延长，标本表面逐渐形成透明软骨样物质，内部孔隙空隙逐渐减少，至12周时，形成类似正常软骨样外观(图1F)。同腔正常软骨表面光滑，呈半透明状(图1G)。"

石蜡切片苏木精-伊红染色：体外培养组，术后4周时见细胞核蓝染，软骨细胞少量，无软骨陷窝形成，8周时软骨细胞增多；12周时软骨细胞已大量增殖(图2A)。但各时期软骨细胞排列无序。腔内培养组，术后4周时标本支架内软骨细胞数量明显较多，软骨陷窝形成，陷窝周围基质深染；术后8周时标本支架内软骨细胞数量多，细胞周围分泌着色均一的玻璃样基质，且渗入支架结构内部，脱钙骨支架有部分被吸收，12周时细胞核蓝染，细胞质及软骨基质红染，支架基本被吸收，软骨细胞充分渗透进入支架内，形态变小，呈一定应力方向排列(图2B)。同腔正常软骨细胞核蓝染，细胞质及软骨基质红染，软骨细胞排列致密，按一定方向排列有序(图2C)。石蜡切片甲苯胺蓝染色：体外培养组，术后4周时软骨细胞少量形成，排列紊乱，周围少量基质包绕；8周时软骨细胞增殖，排列无序；12周时细胞较多，排列无序，细胞周围基质包绕(图2D)。腔内培养组各时间取材标本甲苯胺蓝染色均可见软骨细胞分泌的酸性玻璃样基质被染成红色或紫红色，随时间发展染色增多，支架等结构被染成蓝色(图2E)。同腔正常软骨细胞排列致密，按一定方向排列有序(图2F)。石蜡切片Masson染色：体外培养组，术后4周时少量细胞外基质包绕；8周时细胞外基质增多，软骨细胞排列无序；至12周时软骨细胞多且排列紊乱，基质蓝染(图2G)。腔内培养组各时间取材标本染色均可见胶原染色阳性，后期较早期染色更明显，至12周时软骨基质蓝染，软骨细胞多，按一定应力方向排列(图2H)。同腔正常软骨软骨基质蓝染，软骨细胞排列致密，按一定方向排列有序(图2I)。石蜡切片Ⅱ型胶原免疫组织化学(SP法)染色：体外培养组，术后4周时可见少量的Ⅱ型胶原表达；8周时胞周可见棕黄色反应物颗粒, 周边的少量零散细胞多呈阴性反应；12周时Ⅱ型胶原免疫组织化学反应呈强阳性，软骨细胞胞浆及胞外基质少量黄色颗粒(图2J)。腔内培养组，Ⅱ型胶原免疫组织化学染色阳性，胞浆和基质呈棕黄色，且随时间后延，细胞外基质中棕黄色颗粒越多，表现也越明显(图2K)。同腔正常软骨细胞外基质中出现大量棕黄色颗粒，Ⅱ型胶原染色强阳性(图2L)。每组免疫组织化学切片均随机选取10个不重叠视野，利用荧光显微镜及摄像系统×200倍视野分组别摄片。再将图片传输录入捷达801D形态学分析系统软件中，通过软件分析计算出平均吸光度(A)值、积分吸光度等。吸光度值越大，则光线被吸收程度就越大，免疫复合物的颜色就越深，溶质含量就越高；反之吸光度值越小则颜色越浅。"

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