载姜黄素电纺膜包裹模式维持干细胞再生软骨体内稳定性的实验研究

Curcumin-loaded nanofilm stabilize in vivo chondrogenesis of stem cell-engineered cartilage using an encapsulation model

目的探索载姜黄素电纺膜包裹模式用于维持干细胞再生软骨体内稳定性的可行性。方法将具有抗血管化作用的姜黄素与聚L-丙交酯-己内酯(PLCL)混合,并采用静电纺丝技术制备载姜黄素PLCL电纺膜,以单纯PLCL电纺膜作为对照组,行大体观及扫描电镜观察其表面形貌。随后在体外与人脐静脉血管内皮细胞共培养6 h,以探究载姜黄素PLCL电纺膜抗血管生成能力。另外,取兔骨髓间充质干细胞(BMSC)接种于明胶多孔支架,经体外成软骨诱导培养3周后,行大体及组织学检测以验证再生软骨组织。最后,将载姜黄素PLCL电纺膜紧密封装BMSC再生软骨组织,并植入裸鼠皮下培养6周后,行大体观察、Micro-CT及组织学检测,观察BMSC再生软骨的骨化及稳定软骨表型情况。结果有别于白色的PLCL电纺膜,载姜黄素PLCL电纺膜呈黄色大体观,但两者具有相似的纳米纤维样结构。体外血管形成实验表明,相较于PLCL电纺膜,载姜黄素PLCL电纺膜具有显著的抗血管生成能力。体外成软骨诱导实验表明,BMSC-明胶支架呈软骨样大体观,HE染色显示软骨陷窝结构及软骨特异性细胞外基质分泌,番红O染色证实有蛋白聚糖基质产生。裸鼠体内实验表明,PLCL组出现明显的血管入侵,而载姜黄素PLCL组无肉眼可见的血管入侵。Micro-CT图像显示PLCL组出现大量骨小梁组织,而载姜黄素PLCL组仅出现极少的骨小梁结构。HE及番红-固绿染色进一步证实PLCL组具有大量典型的骨小梁结构和典型骨特异性细胞外基质,而载姜黄素PLCL组为典型的陷窝结构及软骨特异性细胞外基质。结论载姜黄素电纺膜包裹模式可以有效维持干细胞再生软骨的体内稳定性。

Objective To explore the feasibility of a curcumin-loaded nanofilm using an encapsulation model to stabilize in vivo chondrogenesis of stem cell-engineered cartilage.Methods Curcumin(CUR)with anti-angiogenic activities was loaded into poly(L-lactide-co-caprolactone)(PLCL)and underwent electrospun process to produce a CUR/PLCL nanofilm,and a PLCL nanofilm was identically prepared as a control group.The morphologies of PLCL and CUR/PLCL nanofilms were observed using SLR camera and scanning electron microscope.The anti-angiogenic activity of CUR/PLCL nanofilm was evaluated through the in vitro co-culture with human umbilical vein endothelial cells for 6 hours.In addition,rabbit-derived bone marrow stem cells(BMSCs)were seeded into a porous gelatin scaffold and underwent chondrogenic culture for 3 weeks.The BMSC engineered cartilage was evaluated using SLR camera and histological staining.Finally,the BMSC engineered cartilage was embedded using the CUR/PLCL nanofilm and implanted into nude mice subcutaneously for 6 weeks.The achieved samples were evaluated using SLR camera,micro-CT,and histological staining examinations,aiming to distinguish chondrogenesis and ossification conditions.Results In contrast to the white PLCL nanofilm,the CUR/PLCL nanofilm showed yellow color,whereas both nanofilms exhibited similar nano-scaled structure.The in vitro tube formation assay suggested that the CUR/PLCL nanofilm possessed enhanced anti-angiogenic activity than PLCL nanofilm.A BMSC engineered cartilage tissue was successfully achieved via in vitro chondrogenic culture of the BMSC-gelatin construct for 3 weeks,as evidenced by cartilage-like appearance in photograph,typical lacunar structure and cartilage-specific extracellular matrix formation in HE staining,and positive stained glycosaminoglycan in safranin-O staining.After subcutaneously implanted into nude mice for 6 weeks,the gross images indicated that the PLCL group was obvious vascularized whereas the CUR/PLCL group was absent from vascular invasion.The micro-CT images suggested that the PLCL group exhibited abundant bone trabecular structure but not in the CUR/PLCL group.The HE and saf-O/FG staining further confirmed that the PLCL group showed typical bone trabecular structure and bone-specific matrix,and the CUR/PLCL group displayed typical mature lacuna and cartilage-specific extracellular matrix.Conclusion CUR/PCL nanofilm could stabilize in vivo chondrogenesis of stem cell-engineered cartilage using an encapsulation model.