“自组装”工程化软骨在裸鼠体内的生物学改建

Biological redevelopment of self-assembled tissue-engineered cartilage in nude mice

目的应用“自组装”培养技术联合生长分化因子5（GDF-5）体外构建工程化软骨，比较裸鼠体内植入前后软骨相关生物学特性的差异。方法密度梯度离心法分离培养成人骨髓间充质干细胞（hMSCs）。将第3代hMSCs用含GDF-5的软骨诱导液定向诱导培养，诱导3周后重悬细胞，以5×10^6个／ml的细胞终质量浓度接种于2％琼脂糖包被的24孔板，行自组装培养，体外培养6周后部分标本植入裸鼠皮下，再经体内6周后取材。通过大体观察、组织学、免疫组织化学、逆转录．聚合酶链反应（RT-PCR）、生物化学和生物力学检测等方法对体内植入前后标本的软骨相关生物学特性进行比较。结果体外培养6周时预分化的hMSCs“自组装”形成了软骨样组织，但质地较软，弹性较差。体内植入6周后，“自组装”工程化软骨能维持良好的软骨外观。体外标本的糖胺聚糖（GAG）含量为（6．32±0．47）ug/mg，弹性模量为（5．40±0．64）MPa，分别仅是体内标本的69．8％和26．1％。组织学染色显示体内标本的异染基质及Ⅱ型胶原显色程度均明显强于体外标本，RT-PCR结果也反映出这一变化。结论应用“自组装”技术联合GDF-5构建的工程化软骨在裸鼠体内皮下环境中能维持良好的软骨特性，并且体内环境能促进其生物学改建及进一步发育成熟。

Objective To construct engineered cartilage in vitro by the technique of self-assembly, combined with GDF-5 and to compare cartilage-related bio-charaeteristics of engineered cartilage before and after/n vivo implantation. Methods hMSCs were isolated by Fieoll density-gradient centrifugation. hMSCs at passage 3 were induced with chondrogenic medium containing 100 ug/L GDF-5 for 3 weeks. Three weeks later, the cells were suspended and then inoculated into each well of 2% agarose-coated 24-well plates at a density of 5 × 10^6/ml. After culture for 6 weeks in vivo, some constructs were implanted subcutaneously into nude mice and harvested at 6th week post-implantation. Gross observation, glycosaminoglyean （GAG） quantification, biomeehanical test, histology, immunohistochemistry and reverse transcription-polymerase chain reaction （RT-PCR） were used to compare cartilage-related bio-eharacteristics of engineered cartilage before and after in vivo implantation. Results At 6th week in vitroafter culture, the self-assembled cartilage-like constructs were generated from pre-differentiated hMSCs, with a weak intension. At 6th week post-implantation, self-assembled engineered constructs still maintained their cartilage-like appearance, the GAG content （ 6. 32 ± 0.47 ） ug/mg and compressive modulus （ 5.40 ± 0. 64） MPa of in vitro specimens, respectively, reached 69.8% and 26. 1% of in vivo specimens. Histologically, the aggreean and collagen type II staining of in vivo specimens was stronger than that of in vitro specimens, which was consistent with the results of RT-PCR. Conclusion The self-assembled engineered cartilage can maintain fine cartilage characteristics in subcutaneous environment. Moreover,in vivo implantation may promote biological redevelopment and further maturation of engineered cartilage.