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黏附及成骨分化

›› 2021, Vol. 41 ›› Issue (6): 486-492.

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黏附及成骨分化

于泓川,何懋典,刘鹃,李军霞,帅逸,臧圣奇,金磊

南京医科大学金陵临床医学院（东部战区总医院）

收稿日期:2021-01-14 修回日期:2021-03-05 出版日期:2021-06-28 发布日期:2021-06-25
通讯作者: 金磊 E-mail:ljin@nju.edu.cn
基金资助:
基于新型仿生材料的口腔炎性组织来源间充质干细胞免疫调节及组织再生机制的研究;国家自然科学基金;江苏省青年基金;江苏省青年基金

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

Received:2021-01-14 Revised:2021-03-05 Online:2021-06-28 Published:2021-06-25

摘要/Abstract

摘要： 目的　本实验计划将TiO 纳米管阵列复合至二氧化锆（ZrO）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三维多孔支架，质量分数5%、15%、30%TiO浆料涂层后500 ℃处理 h，10 mol/L的NaOH 110 ℃浸泡1、4、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涂层及NaOH处理4 h使ZrO支架表面具备了TiO（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三维多孔支架表面成功构建成型TiO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。

关键词: 钛, 纳米管, 骨组织工程, 多孔支架, 牙髓干细胞

Abstract: Objective　 To fabricate titanium dioxide (TiO2) nanotube arrays on the surface of 3D porous zirconium dioxide（ZrO2）scaffolds and detect their influence on adhesion and proliferation of human dental pulp stem cells (hDPSCs). Methods　 ZrO2 porous scaffolds made by replication technique were coated with TiO2 slurry of 5%, 15%, 30% mass fractions respectively. After being heated for 2h at 500℃, samples were respectively immersed in 10mol/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 which by LRS, the experimental group（formed with best TiO2 nanotube arrays）and the control group (without TiO2 nanotube arrays) 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, TiO2 nanotube arrays were formed on the surface of the samples treated with 15% TiO2 slurry and NaOH for 24 hours. 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 6h and produced significantly higher ALP, while results of CCK-8 showed no statistical difference. Conclusions　 This experiment successfully forms TiO2 nanotube arrays on the surface of 3D porous ZrO2 scaffolds. The TiO2 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.

Key words: titanium, nanotubes, bone-tissue-engineering, scaffold, dental pulp stem cells

中图分类号:

R783.1

于泓川何懋典刘鹃李军霞帅逸臧圣奇金磊. 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黏附及成骨分化[J]. 口腔医学, 2021, 41(6): 486-492.

参考文献 22

[1]	Perez JR, Kouroupis D, Li DJ, et al.Tissue engineering and cell-based therapies for fractures and bone defects[J]. Front Bioeng Biotechnol, 2018, 6: 105.
[2]	Su EP, Justin DF, Pratt CR, et al.Effects of titanium nanotubes on the osseointegration,cell differentiation,mineralisation and antibacterial properties of orthopaedic implant surfaces[J].Bone Joint J, 2018, 100-B(1 Supple A):9-16
[3]	Oh S, Brammer KS, Li YS, et al.Stem cell fate dictated solely by altered nanotube dimension[J].Proc Natl Acad Sci USA, 2009, 106(7):2130-2135
[4]	Sugimoto K, Tsuchiya S, Omori M, et al.Proteomic analysis of bone proteins adsorbed onto the surface of titanium dioxide[J]. Biochem Biophys Rep, 2016, 7: 316-322.
[5]	Bjursten LM, Rasmusson L, Oh S, et al.Titanium dioxide nanotubes enhance bone bonding in vivo[J].J Biomed Mater Res A, 2010, 92(3):1218-1224
[6]	Bose S, Vahabzadeh S, Bandyopadhyay A.Bone tissue engineering using 3D printing[J].Mater Today, 2013, 16(12):496-504
[7]	熊美萍, 贾高智, 袁广银.全降解镁合金组织工程支架孔隙特征与性能关系研究[J].口腔医学, 2020, 40(11):971-975
[8]	Zhu YL, Zhu RQ, Ma J, et al.In vitro cell proliferation evaluation of porous nano-zirconia scaffolds with different porosity for bone tissue engineering[J].Biomed Mater, 2015, 10(5):055009-
[9]	Xuan K, Li B, Guo H, et al.Deciduous autologous tooth stem cells regenerate dental pulp after implantation into injured teeth[J].Sci Transl Med, 2018, 10(455):e-a
[10]	Zhu L, Ma J, Mu R, et al.Bone morphogenetic protein 7 promotes odontogenic differentiation of dental pulp stem cells in vitro[J]. Life Sci, 2018, 202: 175-181.
[11]	韩璧瑶, 穆睿, 朱磊, 等.胰岛素样生长因子-基因转染对人牙髓干细胞体外矿化性能的影响[J].医学研究生学报, 2020, 33(9):919-925
[12]	朱克荣, 陈强, 邓昱, 等.低温拉曼光谱研究二氧化钛纳米晶的相变[J].光散射学报, 2008, 20(4):329-332
[13]	杨婷, 陶硕, 张旗.消退素对氧化应激下人牙髓干细胞生物学性能的影响[J].口腔医学, 2020, 40(12):1061-1066
[14]	Koons GL, Diba MN, Mikos AG.Materials design for bone-tissue engineering[J].Nat Rev Mater, 2020, 5(8):584-603
[15]	Schemitsch EH.Size matters: Defining critical in bone defect size![J].J Orthop Trauma, 2017, 31(Suppl 5):S20-S22
[16]	Sarraf M, Nasiri-Tabrizi B, Yeong CH, et al.Mixed oxide nanotubes in nanomedicine: A dead-end or a bridge to the future?[J].Ceram Int, 2021, 47(3):2917-2948
[17]	Carter DR, Hayes WC.Bone compressive strength: The influence of density and strain rate[J].Science, 1976, 194(4270):1174-1176
[18]	Jones RE, Salhotra A, Robertson KS, et al.Skeletal stem cell-schwann cell circuitry in mandibular repair[J].Cell Rep, 2019, 28(11):2757-2766
[19]	Lyons JG, Plantz MA, Hsu WK, et al.Nanostructured biomaterials for bone regeneration[J]. Front Bioeng Biotechnol, 2020, 8: 922.
[20]	Ghanian MH, Farzaneh Z, Barzin J, et al.Nanotopographical control of human embryonic stem cell differentiation into definitive endoderm[J].J Biomed Mater Res A, 2015, 103(11):3539-3553
[21]	Li YR, Wang S, Dong YJ, et al.Effect of size and crystalline phase of TiO2 nanotubes on cell behaviors: A high throughput study using gradient TiO2 nanotubes[J].Bioact Mater, 2020, 5(4):1062-1070
[22]	Lin D, Chai YJ, Ma YF, et al.Rapid initiation of guided bone regeneration driven by spatiotemporal delivery of IL-8 and BMP-2 from hierarchical MBG-based scaffold[J]. Biomaterials, 2019, 196: 122-137.