介孔生物活性玻璃/聚己内酯短纤维骨组织工程支架体外抑炎功能研究

In vitro anti⁃inflammatory effects of mesoporous bioactive glasses/polycaprolactone fibrous bone tissue engineering scaffolds

目的利用介孔生物活性玻璃纳米颗粒(mesoporous bioactive glass nanoparticles,MBGN)增强聚己内酯(poly⁃capro⁃lactone,PCL)静电纺丝纳米纤维制备3D骨组织工程支架,并探究该支架材料的体外抑炎功能。方法通过静电纺丝获得PCL静电纺丝膜,溶胶-凝胶法制得MBGN。经过分散、均质化、筛选、冻干、明胶热自组装、羧甲基壳聚糖表面修饰等步骤,制备得到MBGN增强PCL静电纺丝纳米纤维3D骨组织工程(bone tissue engineering,BTE)支架PCL@MBGN。扫描电镜、能谱仪等检测支架表征。CCK⁃8法和活死细胞染色法检测支架的生物相容性。采用脂多糖(lipopolysaccharide,LPS)刺激RAW264.7细胞12 h,诱导巨噬细胞向M1型极化后用含不同MBGN添加量的PCL@MBGN支架浸提液刺激细胞48 h,流式细胞术检测M1、M2型巨噬细胞特征性表面标记分子CD86、CD206表达水平,并使用qRT⁃PCR法检测炎性相关细胞因子肿瘤坏死因子⁃α(tumor necrosis factor alpha,TNF⁃α)、白细胞介素1β(interleukin⁃1 beta,IL⁃1β)、白细胞介素4(interleukin⁃4,IL⁃4)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOs)的转录水平。结果随着MBGN添加量增加,扫描电镜下支架表面球状微粒增加,但没有显著影响支架的宏观形貌。PCL@MBGN在体外不仅拥有良好的生物相容性,而且可抑制LPS诱导的促炎型M1巨噬细胞极化,CD86表达降低(P<0.05),在一定程度上促进抑炎型M2巨噬细胞极化,CD206表达增高(P<0.05);抑制IL⁃1β、TNF⁃α、iNOs等促炎细胞因子的基因转录(P<0.05),同时还促进抑炎细胞因子IL⁃4表达(P<0.05),发挥体外免疫调节作用。结论本研究成功制备了具有3D结构的纳米纤维支架PCL@MBGN,MBGN的加入没有显著影响支架的形貌且支架无细胞毒性,具有抑炎作用。PCL@MBGN在炎症环境下的骨缺损修复中具有很大的应用前景。

Objective To synthesize mesoporous bioactive glass nanoparticles(MBGN)⁃enhanced poly⁃caprolactone(PCL)electrospun nanofiber 3D bone tissue engineering(BTE)scaffolds and to investigate the in vitro anti⁃inflammatory function of this scaf⁃fold.Methods PCL membranes were obtained by electrospinning;MBGN was synthesized by sol⁃gel method,and the MBGN⁃enhanced PCL electrospun nanofiber 3D bone tissue engineering scaffold PCL@MBGN was synthesized by dispersion,homogenization,filtering,freeze⁃drying,thermal self⁃assembly of gelatin,and surface modification with carboxymethyl chitosan,etc.The scaffolds were characterized by scanning electron microscopy and energy dispersive spectrometer.CCK⁃8 assay and live/dead staining assay were used to detect the biocompatibility of the scaffolds.Lipopolysaccharide(LPS)was used to stimulate RAW264.7 cells for 12 h to induce polar⁃ization toward M1 type,and then PCL@MBGN extracts with different MBGN additions were used to stimulate cells for 48 h.The ex⁃pression level characteristic surface markers of M1 and M2 macrophages,CD86 and CD206,were detected by flow cytometry.qRT⁃PCR was used to detect the transcriptional levels of inflammation⁃related cytokines such as tumor necrosis factor alpha(TNF⁃α),inter⁃leukin⁃1 beta(IL⁃1β),interleukin⁃4(IL⁃4),inducible nitric oxide synthase(iNOS),and other cytokines.Results With the increase of MBGN addition,the spherical particles on the fiber surface increased under SEM,but did not significantly affect the macroscopic morphology of the scaffolds.PCL@MBGN not only possessed good in vitro biocompatibility,but also inhibited LPS⁃induced polarization of pro⁃inflammatory M1 macrophages(P<0.01).To some extent,the scaffolds promoted the polarization of anti⁃inflammatory M2 macrophages(P<0.01),inhibited the transcription of pro⁃inflammatory cytokines,including IL⁃1β,TNF⁃αand iNOs(P<0.05),and promo⁃ted the transcription of anti⁃inflammatory cytokine IL⁃4(P<0.05),exerting an in vitro anti⁃inflammatory effect.Conclusion In this study,a nanofibrous scaffold PCL@MBGN with 3D structure was successfully prepared.The addition of MBGN did not significantly affect the morphology of the scaffold.The scaffolds were non⁃cytotox⁃ic,had anti⁃inflammatory effects.The results suggest that PCL@MBGN has great potential for application in bone defect repair in in⁃flammatory environments.