三维多孔支架表面成型二氧化钛纳米管阵列体外促进人牙髓干细胞粘附及成骨分化

Titanium dioxide nanotube arrays formed on 3D porous scaffold facilitated adhesion and osteogenic differentiation of human dental pulp stem cells in vitro

目的本实验计划将TiO_(2)纳米管阵列复合至二氧化锆(ZrO_(2))三维多孔支架表面,并探究对人牙髓干细胞(hDPSCs)早期粘附和增殖的影响。方法模板法制作ZrO_(2)三维多孔支架,质量分数5%、15%、30%TiO_(2)浆料涂层后500℃处理2 h, 10 mol/L的NaOH 110℃浸泡12、24、36 h,测定支架压缩强度及孔隙率,SEM观察表面形态,能谱仪分析表面元素类型和含量。选择出表面形态最优样品组为实验组,未成型纳米管样品为对照组,LRS测定表面物质的晶型并接种hDPSCs, SEM、鬼笔环肽染色观察细胞粘附。CCK-8检测细胞增殖,ALP检测成骨分化。结果 15%TiO_(2)涂层及NaOH处理24 h使ZrO_(2)支架表面具备了TiO_(2)(金红石)纳米管阵列,这类支架压缩强度为1.76 MPa,孔隙率为79%。细胞实验结果显示,实验组比对照组获得了更好的细胞丝状伪足和支架延伸,ALP表达显著增高,但CCK-8结果无差异。结论本实验在ZrO_(2)三维多孔支架表面成功构建成型TiO_(2)纳米管阵列,略微增加了支架压缩强度并可以促进hDPSCs的早期粘附和成骨分化,但对细胞增殖无明显影响。

Objective To fabricate titanium dioxide(TiO_(2)) nanotube arrays on the surface of 3 D porous zirconium dioxide(ZrO_(2))scaffolds and detect their influence on adhesion and proliferation of human dental pulp stem cells(hDPSCs). Methods ZrO_(2) porous scaffolds made by replication technique were coated with TiO_(2) slurry of 5%, 15%, 30% mass fractions respectively. After being heated for 2 h at 500 ℃, samples were respectively immersed in 10 mol/L NaOH for 12, 24, 36 h at 110℃. Compressive strength and porosity of scaffolds were characterized, and SEM scanned their surface, while EDS examined their element types and proportions. Confirmed crystal structures of, the experimental group(formed with best TiO_(2) nanotube arrays)and the control group(without TiO_(2) nanotube arrays) were determined by LRS and they were seeded with hDPSCs. Cells’ adhesion was visualized by SEM and phalloidin, while their osteogenic differentiation was determined by ALP and proliferation was determined by CCK-8. Results Confirmed as rutile, TiO_(2) nanotube arrays were formed on the surface of samples treated with 15% TiO_(2) slurry and NaOH for 24 h. The compressive strength was 1.76 MPa, and the porosity was 79%. Compared with the control group, cells from the experimental group had more pseudopodia, extended better after being seeded for 6 h and produced significantly higher ALP, while results of CCK-8 showed no statistical difference. Conclusion This experiment successfully forms TiO_(2) nanotube arrays on the surface of 3 D porous ZrO_(2) scaffolds. The TiO_(2) nanotube arrays mildly strengthen scaffolds and facilitate adhesion and osteogenic differentiation of human dental pulp stem cells in vitro, but have no influence on proliferation.