可载药的磁性聚己内酯/明胶微球支架的制备及其体外成骨性能的研究

doi:10.13591/j.cnki.kqyx.2022.04.001

摘要/Abstract

摘要： 目的制备出可载药的磁性颗粒支架,并研究其体外成骨性能。方法使用复乳法制备聚己内酯(polycaprolactone,PCL)/明胶微球(PCL/gel microsphere,PGM)支架,通过相差显微镜、场发射电子显微镜(field emission scanning electron microscope,FESEM)观测形貌;使用热重分析(thermal gravity analysis,TGA)和体外降解实验检测PGM成分占比与降解质量变化趋势;通过CCK-8(cell counting kit-8)筛选合适的油酸四氧化三铁纳米颗粒(nano oil-acid coated Fe₃O₄ particles,OA@Fe₃O₄)包埋浓度并制备Fe-PGM;在该磁性微球中载入人源重组骨形态发生蛋白2(recombinant human bone morphogenetic protein-2,rhBMP-2),并使用倒置荧光显微镜(inverted fluorescence microscope,IFM)观察PGM、Fe-PGM、Fe-PGM+BMP-2三组的大鼠骨髓间充质干细胞(rat bone mesenchymal stem cell,rBMSC)的粘附。成骨诱导第7、14天后,通过实时荧光定量逆转录聚合酶链式反应(quantitative real-time reverse transcription polymerase chain reaction,qRT-PCR)检测PGM、Fe-PGM、Fe-PGM+BMP-2三组微球表面rBMSC成骨相关基因的表达水平。结果相差显微镜及FESEM图片显示PGM由多孔状PCL支架与内部明胶水凝胶构成;热重分析及体外降解提示,明胶与PCL的质量比约为50%,体外降解质量损失曲线平稳;CCK-8结果显示铁含量为0.4%的Fe-PGM在各时间点与PGM相比未表现细胞增殖抑制(P＜0.05);IFM观察到PGM、Fe-PGM、Fe-PGM+BMP-2均有良好的细胞粘附,并且BMP-2的加入使得Fe-PGM+BMP-2有更好的细胞铺展;qRT-PCR结果提示,OA@Fe₃O₄的加入使Fe-PGM及Fe-PGM+BMP-2组rBMSC的成骨相关基因表达水平较PGM高,BMP-2则进一步增大了Fe-PGM+BMP-2与Fe-PGM之间的差距(P＜0.05)。结论磁性PGM颗粒支架具有良好的细胞学特性,并且其载药功能可赋予其更好的粘附与成骨性能。

关键词: 颗粒支架, 油酸四氧化三铁纳米颗粒, 载药, 骨组织工程

Abstract: Objective To prepare drug-loaded magnetic granular scaffolds and investigate their osteogenesis behavior in vitro. Methods The polycaprolactone (PCL)/gelatin microsphere (PGM) scaffolds were constructed via a double-emulsion method. The phase contrast microscope and field emission scanning electron microscope (FESEM) were applied for morphology observation. Thermal gravity analysis (TGA) and in vitro degradation test were used to figure out the proportion of ingredients and the trend of mass loss. Cell counting kit-8 (CCK-8) assay was conducted to optimize the concentration of nano oil-acid coated Fe₃O₄ particles (OA@Fe₃O₄), and the final Fe-PGM were prepared which were subsequently loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2). The adhesion of rat bone mesenchymal stem cells (rBMSC) on PGM, Fe-PGM and Fe-PGM+BMP-2 was evaluated by inverted fluorescent microscope (IFM). Osteogenesis-related gene expression of rBMSC on PGM, Fe-PGM and Fe-PGM+BMP-2 was detected by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) 7 and 14 days after osteogenic induction. Results Photos of phase contrast microscope and FESEM demonstrated that PGM was composed of porous PCL skeleton and inner gelatin hydrogel. TGA and in vitro degradation test indicated that PCL and gelation was about 50% (wt.) of PGM respectively. Curve of mass loss in vitro was smooth. Results of CCK-8 showed that no inhibition on the proliferation of rBMSC was found in 0.4% Fe-PGM group compared to that in PGM group (P＜0.05). Acceptable cell adhesion was observed via IFM in PGM, Fe-PGM and Fe-PGM+BMP-2 group. Additionally, involvement of BMP-2 bettered cell sprawl in Fe-PGM+BMP-2 group. qRT-PCR analysis indicated a higher expression level of osteogenesis-related gene in Fe-PGM and Fe-PGM+BMP-2 group than that in PGM group owing to OA@Fe₃O₄, while application of BMP-2 further broadened the gap between Fe-PGM and Fe-PGM+BMP-2 group (P＜0.05). Conclusion The magnetic PGM scaffold has favorable cytological characteristics, and its drug-loading property could further improve adhesion and osteogenic differentiation of rBMSC on it.

Key words: granular scaffold, nano oil acid coated Fe₃O₄ particles, drug-loading, bone tissue engineering

中图分类号:

R783.1

周昉, 刘俊, 胡姝颖, 史凡, 严佳, 章非敏. 可载药的磁性聚己内酯/明胶微球支架的制备及其体外成骨性能的研究[J]. 口腔医学, 2022, 42(4): 289-295.

ZHOU Fang, LIU Jun, HU Shuying, SHI Fan, YAN Jia, ZHANG Feimin. Study on drug-loaded magnetic polycaprolactone/gelatin microsphere scaffolds and their osteogenesis behavior in vitro[J]. Stomatology, 2022, 42(4): 289-295.

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