Cartilage construction in nude mice with microencapsulated stem cells derived from human umbilical cord Wharton’s jelly

doi:10.3969/j.issn.2095-4344.2014.08.006

Abstract

Abstract:

BACKGROUND: Cartilage extracellular matrix with a large number of signaling molecule proteins and factors is likely to be an ideal material for tissue engineering cartilage.
OBJECTIVE: To investigate the possibility of calcium alginate and cartilage extracellular matrix combined with microencapsulated stem cells derived from human umbilical cord Wharton’s jelly to construct ectopic tissue-engineered cartilage in nude mice.
METHODS: Microfilament suspension of the cartilage extracellular matrix was prepared. Human stem cells derived from Wharton’s jelly of the umbilical cord were inoculated in to calcium alginate and cartilage extracellular matrix gel microspheres as experimental group. Stem cells derived from human umbilical cord Wharton’s jelly were incubated in simple alginate gel microspheres as control group. After in vitro culture, the microspheres were implanted into the dorsal subcutaneous tissue of nude mice. Samples were taken after 4 weeks, respectively, for gross and histological observation.
RESULTS AND CONCLUSION: The stem cells exhibited parallel-chondrocyte morphology in microspheres, which grew and proliferated quite well during in vitro culture. A new parallel-cartilaginous tissue was found in the subcutaneous tissue 4 weeks after surgery in the experimental group, and the tissue was positive for hematoxylin-eosin, safranine O, toluidine blue and collagen II. A large number of parallel-chondrocytes and cartilage lacuna-like structures were observed under a microscope with no obvious inflammatory reaction around the microspheres. The control group showed the partial degradation of microspheres, surrounded by only a small number of inflammatory cells and lymphocytes. Calcium alginate and cartilage extracellular matrix microspheres have a rather good histocompatibility which can be used to construct parallel-cartilaginous tissues by implanting stem cell-microspheric compound into the subcutaneous tissue of nude mice.

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: umbilical cord, mesenchymal stem cells, alginates, cartilage, extracellular matrix, tissue engineering

CLC Number:

R318

Yang Jian-hua, Liu Shu-yun, Zhao Peng, Lu Shi-bi, Zhang Li, Huang Jing-xiang, Zhao Bin,Xu Wen-jing, Guo Quan-yi. Cartilage construction in nude mice with microencapsulated stem cells derived from human umbilical cord Wharton’s jelly[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(8): 1179-1184.

Figures/Tables 1

2.1 软骨细胞外基质的成分表征显微镜下软骨外基质悬液成微丝状，扫描电镜显示悬液微丝为三维螺旋胶原结构(图1A)。阿尔辛蓝染色和Ⅱ型胶原染色均阳性(图1B，C)。Hochest33258染色荧光显微镜观察示无细胞及核物质残留，表明软骨脱细胞完全且保留了软骨细胞外基质的大部分成分(图1D)。 2.2 人脐带Wharton胶间充质干细胞体外培养形态学观察组织块接种24 h后细胞开始从组织块中爬出，分散贴壁生长，72 h后，贴壁细胞增多，生长活跃，细胞呈不规则星形或多角形，可见多处有圆形、哑铃形细胞分裂像，呈聚集性生长，可见细胞增殖较快(图2A)。原代细胞培养15 d左右爬满培养瓶，传代后生长速度明显加快。传到第3代时，可见细胞生长状态良好，多呈多角形和梭形，分散均匀生长增殖活跃(图2B)。 2.3 微球化干细胞的形态学观察干细胞接种到微球，实验组倒置显微镜下示细胞分布均匀，生长活跃(图2C)。环境扫描电镜示混合凝胶材料呈三维网状多孔结构(图2D)，包裹内的细胞生长状态良好，像软骨细胞样呈圆形或椭圆形，分泌外基质，周围形成软骨陷窝样结构。 2.4 微球体内移植后的大体和组织学观察裸鼠饲养4周过程中无死亡，伤口愈合良好，没有感染迹象，包埋凝胶微球材料位于皮下，形成皮丘，位置较固定，过量麻醉处死取材，4周时，外观乳白色类软骨样(图3A)，略缩小有少量降解，质较硬，基本保持球形，周围被透明筋膜组织包裹，与周围组织有粘连且少量血管浸润。标本行苏木精-伊红染色，可见微球周围没有/轻微炎症反应，细胞显示圆形，周围基质着色明显，分泌大量基质，分化为类软骨样形态(图3B)；甲苯胺蓝染色阳性，细胞周围有明显的蓝色(图3C)。番红O染色阳性，细胞周围有明显的红色包裹细胞，可见细胞分泌了丰富的聚糖类的软骨细胞外基质物质(图3D)；Ⅱ型胶原免疫组化染色阳性，显示细胞核深棕色，周围基质呈棕黄色，可见干细胞在凝胶中可合成、分泌Ⅱ型胶原等基质(图3E)，材料上形成了明显的软骨陷窝样结构。对照组显示微球部分降解，周围仅有少量炎症细胞及淋巴细胞。"

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