磁刺激下Fe_(3)O_(4)@ZIF-8纳米颗粒影响骨髓间充质干细胞的成骨分化

Fe3O4@ZIF-8 nanoparticles affect osteogenic differentiation of bone marrow mesenchymal stem cells under magnetic stimulation

背景:骨髓间充质干细胞在组织工程和骨再生中具有重要作用,然而如何促进骨髓间充质干细胞的成骨分化仍然是一个挑战。目的:探讨磁刺激下Fe_(3)O_(4)@ZIF-8纳米颗粒对骨髓间充质干细胞成骨分化的影响。方法:采用水热法合成沸石咪唑酯骨架(ZIF-8),采用一锅法合成具有磁性的Fe_(3)O_(4)@ZIF-8纳米颗粒(材料制备中分别加入2.5,5,10,20μg的Fe_(3)O_(4)),通过扫描电镜、X射线光电子能谱、X射线衍射、振动样品磁强计检测等对Fe_(3)O_(4)@ZIF-8纳米颗粒进行表征,筛选出合适的材料进行后续实验。提取4周龄SD大鼠骨髓间充质干细胞,分别与不同质量浓度(25,50,75,100,125μg/mL)的Fe_(3)O_(4)@ZIF-8纳米颗粒溶液共培养,CCK-8法检测细胞增殖,筛选出最佳的材料溶液质量浓度;筛选出材料溶液质量浓度后,施加磁刺激(磁场强度分别为0,50,100,150 MT),CCK-8法检测细胞增殖,筛选出最佳的磁场强度与Fe_(3)O_(4)@ZIF-8纳米颗粒,用于骨髓间充质干细胞诱导分化实验。将SD大鼠骨髓间充质干细胞分别与ZIF-8、Fe_(3)O_(4)@ZIF-8、Fe_(3)O_(4)@ZIF-8(磁场干预)纳米颗粒溶液共培养,以单独培养的细胞为空白对照,成脂诱导后进行油红O染色,成骨诱导后进行碱性磷酸酶、茜素红染色与Runx2蛋白质量浓度检测。结果与结论:(1)扫描电镜下可见Fe_(3)O_(4)@ZIF-8纳米颗粒呈现十二面体结构,随着材料中Fe_(3)O_(4)含量的增加,纳米颗粒的粒径增大,选择粒径约250 nm(该粒径下的纳米颗粒功能性及生物安全性较稳定)的Fe_(3)O_(4)@ZIF-8纳米颗粒(材料制备中分别加入5,10μg的Fe_(3)O_(4))进行后续实验。(2)CCK-8检测结果显示,在100MT磁场作用下,50μg/mL的Fe_(3)O_(4)@ZIF-8纳米颗粒(材料制备中加入10μg的Fe_(3)O_(4))能够显著促进骨髓间充质干细胞的增殖,选择该条件下的纳米颗粒进行骨髓间充质干细胞成骨诱导分化实验。(3)成骨诱导后,Fe_(3)O_(4)@ZIF-8(磁场干预)组骨髓间充质干细胞的碱性磷酸酶活性、细胞外基质矿化程度与Runx2蛋白质量浓度均高于其他3组(P<0.05);成脂诱导后,Fe_(3)O_(4)@ZIF-8(磁场干预)组骨髓间充质干细胞的脂滴形成少于其他3组(P<0.05)。(4)结果表明,在特定的磁场条件下Fe_(3)O_(4)@ZIF-8纳米颗粒可以促进骨髓间充质干细胞的成骨分化。

BACKGROUND:Bone marrow mesenchymal stem cells play a pivotal role in tissue engineering and bone regeneration.However,promoting the osteogenic differentiation of bone marrow mesenchymal stem cells poses a significant challenge.OBJECTIVE:To examine the influence of Fe_(3)O_(4)@ZIF-8 nanoparticles on the osteogenic differentiation of bone marrow mesenchymal stem cells under magnetic stimulation.METHODS:Zeolite imidazolate skeleton(ZIF-8)was synthesized by hydrothermal method,and magnetic Fe_(3)O_(4)@ZIF-8 nanoparticles were synthesized by one-pot method(2.5,5,10,and 20µg Fe_(3)O_(4) were added to the preparation materials,respectively).The Fe_(3)O_(4)@ZIF-8 nanoparticles were characterized by scanning electron microscopy,X-ray photoelectron spectroscopy,X-ray diffraction,and vibration sample magnetometer detection,and suitable materials were selected for subsequent experiments.Bone marrow mesenchymal stem cells of 4-week-old SD rats were extracted and co-cultured with Fe_(3)O_(4)@ZIF-8 nanoparticle solution with different mass concentrations(25,50,75,100,and 125μg/mL),respectively.Cell proliferation was detected by CCK-8 assay,and the optimal material solution mass concentration was selected.After the mass concentration of the material solution was screened,magnetic stimulation was applied(magnetic field intensity was 0,50,100,and 150 MT,respectively).Cell proliferation was detected by CCK-8 assay,and the best magnetic field intensity and Fe_(3)O_(4)@ZIF-8 nanoparticles were selected for the experiment of induced differentiation of bone marrow mesenchymal stem cells.SD rat bone marrow mesenchymal stem cells were co-cultured with ZIF-8,Fe_(3)O_(4)@ZIF-8,and Fe_(3)O_(4)@ZIF-8(magnetic field intervention)nanoparticle solution,respectively.The single cultured cells were used as blank controls.Lipid induction was followed by oil red O staining.After osteogenesis induction,alkaline phosphatase,alizarin red staining and Runx2 protein concentration were detected.RESULTS AND CONCLUSION:(1)Under scanning electron microscopy,Fe_(3)O_(4)@ZIF-8 nanoparticles showed a dodecahedral structure.With the increase of Fe_(3)O_(4) content in the material,the particle size of the nanoparticles increased.Fe_(3)O_(4)@ZIF-8 nanoparticles(5 and 10µg Fe_(3)O_(4) was added to the material preparation)with a particle size of about 250 nm(stable functional and biosafety of nanoparticles at this particle size)were selected.(2)The results of CCK-8 assay showed that 50μg/mL Fe_(3)O_(4)@ZIF-8 nanoparticles(with 10µg Fe_(3)O_(4) added to the preparation of the material)could significantly promote the proliferation of bone marrow mesenchymal stem cells under a 100 MT magnetic field.The nanoparticles under this condition were selected for the osteogenic induction differentiation experiment of bone marrow mesenchymal stem cells.(3)After osteogenic induction,the alkaline phosphatase activity,extracellular matrix mineralization degree,and Runx2 protein mass concentration of bone marrow mesenchymal stem cells in Fe_(3)O_(4)@ZIF-8(magnetic field intervention)group were higher than those in other three groups(P<0.05).After adipogenic induction,the lipid droplet formation of bone marrow mesenchymal stem cells in Fe_(3)O_(4)@ZIF-8(magnetic field intervention)group was lower than that in the other three groups(P<0.05).(4)The results show that Fe_(3)O_(4)@ZIF-8 nanoparticles can promote osteogenic differentiation of bone marrow mesenchymal stem cells under specific magnetic field conditions.