目的 探讨钛表面微米坑复合纳米管(micropitanotube topography,MNT)形貌对骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMMSC)内质网应激(endoplasmic reticulum stress,ERS)的激活及其与成骨分化的联系,以期为生物材料表面形貌设计提供依据.方法 纯钛试件分为4组:对照组(仅抛光处理)、毒胡萝卜素组(thapsigargin,TG,细胞接种于抛光钛表面,0.1 μmol/L TG处理9h)、MNT5组和MNT20组(抛光钛经酸蚀形成微米坑后,分别用5和20V阳极氧化处理制备纳米管).扫描电镜观察试件表面形貌,接种BMMSC并成骨诱导7、14和21 d后,分别行碱性磷酸酶(alkaline phosphatase,ALP)、胶原和矿化结节染色及半定量分析,透射电镜观察接种12h后BMMSC内质网形态的变化.荧光实时定量PCR检测ERS相关基因[免疫球蛋白重链结合蛋白质(immunoglobulin heavy chain binding protein,BiP)、蛋白激酶受体样内质网激酶(protein kinase receptorlike endoplasmic reticulum kinase,PERK)、转录激活因子4(activating transcription factor 4,ATF4)]的表达.结果 成骨诱导后TG和MNT20组ALP分泌、胶原合成和矿化结节形成均显著高于对照组(P＜0.05).相比对照组,TG、MNT5和MNT20组BMMSC内质网腔不同程度扩张、膨胀;与对照组相比,TG组BiP、PERK、ATF4表达量(分别为1.87±0.10、2.24±0.35、1.85±0.14),MNT5组BiP、ATF4表达量(分别为1.27±0.09、1.25 ±0.04)和MNT20组BiP、PERK、ATF4表达量(分别为1.44±0.09、2.40±0.60、1.48±0.05)显著增加,差异均有统计学意义(P＜0.05).结论 钛表面微米坑复合纳米管形貌可不同程度激活BMMSC ERS,而激活的ERS可能促进了钛表面形貌诱导的成骨分化.%Objective To explore the activation of endoplasmic reticulum stress (ERS) in bone marrow mesenchymal stem cell (BMMSC) and its effect on osteogenic differentiation induced by micropitanotube topography (MNT),so as to provide guidance for the topography design of biomaterials.Methods Four sample groups were fabricated:polishing control group (polished titanium,PT,no treatment),thapsigargin treatment (TG,0.1 μmol/L TG treated for 9 h),MNT5 and MNT20 (anodized at 5 V and 20 V after acid etching).Scanning electron microscope (SEM) was used to observe the topography of Ti samples.The alkaline phosphatase (ALP) production,collagen secretion and extracellular matrix (ECM) mineralization of BMMSC (osteogenic induced for 7,14 and 21 d) on Ti samples were detected to evaluate the osteogenic differentiation.After 12 h incubation,the shape and size of ER was examined using a transmission electron microscope (TEM),and ERS-related genes including immunoglobulin heavy chain binding protein (BiP),protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4) were detected by quantitative real-time PCR (qRT-PCR).Results After 7,14and 21 d of induction,the ALP production,collagen secretion and ECM mineralization in TG and MNT20 all significantly increased compared to PT (P＜0.05).The cells grown on TG,MNT5 and MNT20 surfaces displayed gross distortions of the ER.Compared to PT,BiP,PERK,ATF4 mRNA expression in TG was respectively 1.87 ± 0.10,2.24± 0.35,1.85 ± 0.14;BiP,ATF4 mRNA expression in MNT5 were respectively 1.27±0.09,1.25±0.04;BiP,PERK,ATF4 mRNA expression in MNT20 were respectively 1.44±0.09,2.40± 0.60,1.48±0.05 (P＜0.05).Conclusions MNT triggered different degree of ERS,and the activated ERS may promote MNT-induced osteogenic differentiation.
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