Chondrogenesis of adipose tissue-derived stem cells induced by auricular chondrocytes from microtia in vivo

doi:10.3969/j.issn.2095-4344.2017.21.006

Abstract

Abstract:

BACKGROUND: Due to quantity and quality deficiencies, chondrocytes from microtia are difficult to act as seed cells to construct an ear cartilage scaffold with the normal human auricle size.
OBJECTIVE: To test the hypothesis that auricular chondrocytes from microtia can promote chondrogenic differentiation and chondrogenesis of human adipose tissue-derived stem cells (ADSCs) at non-chondrogensis site in vivo, which is the preparatory work for preparation of human tissue-engineered auricle cartilage scaffold.
METHODS: Human ADSCs at passage 3 and auricular chondrocytes at passage 2 were mixed at a ratio of 7:3 and 5.0×10¹⁰/L mixed cells were suspended in 0.2 mL of 30% Pluronic F-127, and then the mixture was injected subcutaneously into Balb/c nude mice as experimental group. Auricular chondrocytes or ADSCs at the concentration of 5.0×10¹⁰/L were mixed with 0.2 mL of Pluronic F-127 and injected respectively as positive and negative control groups. 1.5×10¹⁰/L auricular chondrocytes were mixed and injected as low-concentration chondrocyte control group. All specimens were collected at the 8th week post-injection. Newborn tissues in nude mice were taken out for morphological examination, wet weight measurement, determination of glycosaminoglycans, histological and immunohistochemical staining.
RESULTS AND CONCLUSION: The wet weight of specimens in the experimental group was over 80% of that in the positive control group, and the wet weight of specimens in the low-concentration chondrocyte control group was less than 30% of that in the positive control group. The average wet weight and glycosaminoglycan content were significantly higher in the experimental and positive control group than in the negative control and low-concentration chondrocyte control groups (P < 0.05). In all the groups except for the negative control group, mature cartilage lacunas could be observed by histological staining and collagen type II could be detected for expression by immunohistochemistry to different extents. In the low concentration chondrocyte control group, cartilage lacunas were incompact and inhomogeneous, and the extracellular matrix was slightly stained. In the negative control group, mature cartilage lacunae and collagen type II could not be detective. To conclude, auricular chondrocytes from microtia can promote chondrogenic differentiation and chondrogenesis of ADSCs at the non-chondrogenesis site in vivo.

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: Chondrocytes, Fats, Stem Cells, Tissue Engineering

CLC Number:

R394.2

Cai Zhen, Jiang Hai-yue. Chondrogenesis of adipose tissue-derived stem cells induced by auricular chondrocytes from microtia in vivo[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3312-3319.

Figures/Tables 2

2.1 实验动物数量分析纳入裸鼠数量24只，分4组，全部进入结果分析，无脱失。 2.2 新生组织大体观察体内植入8周后，4组细胞- Pluronic F-127复合物均形成了较成熟的组织，新生组织周围有一层透明纤维膜包裹，纤维膜表面可见血管分支走行。共移植组与残耳软骨细胞组、低浓度残耳软骨细胞对照组注射部位均有白色半透明状类似软骨组织形成，共移植组与残耳软骨细胞组标本组织外观和弹性与正常软骨极为相似，外有一层透明包膜，未见有明显的血管；低浓度残耳软骨细胞对照组大部分标本表面光泽度及弹性差，脂肪干细胞组生成质软的纤维样组织，色淡黄色。各组标本大小不等，低浓度残耳软骨细胞对照组标本明显小于共移植组、残耳软骨细胞组和脂肪干细胞组(见图1)。 2.3 新生组织平均湿质量与糖胺多糖含量比较平均湿质量：共移植组的平均湿质量达到残耳软骨细胞组的80%以上；低浓度残耳软骨细胞对照组平均湿质量低于残耳软骨细胞组的30%，见图2。糖胺多糖平均含量：统计学分析结果表明，共移植组与残耳软骨细胞组的糖胺多糖含量差异无显著性意义(P=0.086 5 > 0.05)，但脂肪干细胞组和低浓度残耳软骨细胞对照组的糖胺多糖含量均显著低于共移植组和残耳软骨细胞组(P < 0.01)，见图3。 2.4 新生组织组织学检查 2.4.1 苏木精-伊红染色共移植组、残耳软骨细胞组与低浓度残耳软骨细胞对照组组织内均有成熟的软骨陷窝形成，但陷窝大小不规则，细胞浓度与细胞排列不均匀；脂肪干细胞组为纤维样组织，未见软骨陷窝形成。低浓度残耳软骨细胞对照组软骨陷窝数量较共移植组和单纯残耳软骨细胞组少，排列松散，胞外基质着色较共移植组和单纯残耳软骨细胞组淡，见图4。 2.4.2 Safranin O染色共移植组、单纯残耳软骨细胞组与低浓度残耳软骨细胞对照组组织内胞外基质均染成红色，可见大量软骨陷窝形成；单纯脂肪来源的干细胞组基质染色阴性，未见软骨陷窝形成；低浓度残耳软骨细胞对照组可见胞外基质红染较共移植组和单纯残耳软骨细胞组着色浅且不均匀，且软骨陷窝排列松散，陷窝数量较共移植组与单纯残耳软骨细胞组少，见图5。 2.4.3 甲苯胺蓝染色共移植组、单纯残耳软骨细胞组组织内均可见大量染成深蓝色的胞外基质，其间可见大量成熟的软骨陷窝；单纯脂肪来源的干细胞组基质染色阴性，未见软骨陷窝形成；低浓度残耳软骨细胞对照组可见胞外基质蓝染不均匀，且软骨陷窝排列松散，陷窝数量较共移植组与单纯残耳软骨细胞组少，见图6。 2.4.4 Masson三色染色共移植组、单纯残耳软骨细胞组与低浓度残耳软骨细胞对照组软骨胞外基质有大量染成绿色的胶原存在；单纯脂肪来源的干细胞组可见大量绿色胶原纤维存在，未见软骨陷窝；低浓度残耳软骨细胞对照组胞外基质中绿色胶原含量较共移植组与单纯残耳软骨细胞组明显减少，且软骨陷窝排列松散不均匀，见图7。 2.4.5 Verhoeff铁苏木精染色共移植组、单纯残耳软骨细胞组与低浓度残耳软骨细胞对照组软骨基质有染成蓝黑色的弹性纤维存在，证实有弹性软骨存在；单纯脂肪来源的干细胞组未见蓝黑色弹力纤维，可见红染的胶原纤维存在；低浓度残耳软骨细胞对照组基质中蓝黑色弹性数量较共移植组与单纯残耳软骨细胞组明显减少，见图8。 2.4.6 Ⅱ型胶原免疫组化染色共移植组、残耳软骨细胞组与低浓度残耳软骨细胞对照组均可见到在成熟软骨陷窝周围有棕黄色沉淀，即Ⅱ型胶原表达；单纯脂肪来源的干细胞组未见Ⅱ型胶原表达；低浓度残耳软骨细胞对照组与共移植组和单纯残耳软骨细胞组相比，胞外基质着色浅不均匀，见图9。"

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