钛表面氧化锌与纤连蛋白复合修饰应用于抗种植体周炎的研究

doi:10.15541/jim20210125

无机材料学报 ›› 2021, Vol. 36 ›› Issue (12): 1316-1322.DOI: 10.15541/jim20210125

所属专题：【生物材料】骨骼与齿类组织修复

钛表面氧化锌与纤连蛋白复合修饰应用于抗种植体周炎的研究

张大卫¹(), 朱立远², 卢红亮², 王佐林¹()

1.同济大学口腔医学院同济大学附属口腔医院, 上海牙组织修复与再生工程技术研究中心, 上海 200072
2.复旦大学微电子学院, 上海智能电子与系统研究院, 专用集成电路和系统国家重点实验室, 上海 200433

收稿日期:2021-03-03 修回日期:2021-04-07 出版日期:2021-12-20 网络出版日期:2021-04-30
通讯作者: 王佐林, 教授. E-mail: zuolin@tongji.edu.cn
作者简介:张大卫(1994-), 男, 硕士研究生. E-mail: david_zhang@tongji.edu.cn
基金资助:
国家重点研发计划项目(2018YFE0202200);上海市自然科学基金(18ZR1405000)

Titanium Modified with ZnO Nanofilm and Fibronectin: Preventing Peri-implantitis and Biocompatibility

ZHANG Dawei¹(), ZHU Liyuan², LU Hongliang², WANG Zuolin¹()

1. Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Department of Implantology, School & Hospital of Stomatology, Tongji University, Shanghai 200072, China
2. State Key Laboratory of ASIC and System, Shanghai Institute of Intelligent Electronics & Systems, School of Microelectronics, Fudan University, Shanghai 200433, China

Received:2021-03-03 Revised:2021-04-07 Published:2021-12-20 Online:2021-04-30
Contact: WANG Zuolin, professor. E-mail: zuolin@tongji.edu.cn
About author:ZHANG Dawei(1994-), male, Master candidate. E-mail: david_zhang@tongji.edu.cn
Supported by:
National Key R&D Program of China(2018YFE0202200);Natural Science Foundation of Shanghai(18ZR1405000)

摘要/Abstract

摘要：

种植体周炎会导致种植体周牙龈附着丧失、骨组织吸收, 甚至种植体松动、脱落。本研究采用原子层沉积技术联合硅烷化修饰在钛表面制备氧化锌纳米薄膜和纤连蛋白复合涂层。选取种植体周炎相关致病菌伴放线聚集杆菌和牙龈卟啉单胞菌用作该复合涂层抗菌效果的体外验证。同时, 检测牙龈成纤维细胞在钛、氧化锌修饰钛及纤连蛋白与氧化锌复合修饰钛表面黏附、增殖及其相关基因的表达。在大鼠上颌第一磨牙位点通过注射细菌法建立种植体周炎模型, 并比较不同材料的钛钉周围骨破坏范围和牙龈组织中的炎症程度。实验结果显示, 该复合涂层对伴放线聚集杆菌和牙龈卟啉单胞菌的24 h抗菌率分别为80.9%和75.7%; 牙龈成纤维细胞在复合修饰钛片表面展现出良好的黏附、增殖状态; 在大鼠种植体周炎模型中, Micro-CT结果显示, 纯钛钉周骨组织吸收为(1.14±0.71) mm, 而经复合修饰的钛钉周围骨组织吸收仅为(0.37±0.28) mm。石蜡切片观察和qPCR定量检测结果显示, 纯钛钉周牙龈及牙槽骨组织的炎症反应较重, 而经复合修饰的钛钉周围组织炎症反应明显减轻。因此, 该复合修饰可能在牙种植体防御细菌入侵、增强牙龈附着和降低牙龈炎症反应方面发挥作用。

关键词: 钛, 氧化锌, 纤连蛋白, 种植体周炎

Abstract:

Peri-implantitis can lead to peri-implant attachment loss, bone absorption, and even implant loose or lost. In this study, the atomic layer deposition technique and silanization were applied to prepare a composite zinc oxide (ZnO) /fibronectin (Fn) coating on the surface of titanium (Ti). Peri-implantitis related bacteria, including Aggregatibacter actinomycetemcomitans (A. a) and Porphyromonas gingivalis (P. g), were chosen to evaluate the antibacterial effect of the composite coating. Spreading, proliferation and associated gene expression of human gingival fibroblasts on Ti, Ti/ZnO, and Ti/ZnO/Fn were evaluated. In vivo peri-implantitis model was established on SD rat maxilla through the infusion of mixed bacterial suspension. The peri-implant bone resorption and inflammatory infiltration in gingiva were investigated. The results showed that antibacterial efficacies of the composite coating against A. a and P. g for 24 h were up to 80.9% and 75.7%, respectively. Fibroblasts on the surface of Ti/ZnO/Fn showed adhesion and proliferation in vitro. In the model of peri-implantitis, Micro-CT showed that the amount of peri-implant bone absorption of Ti was (1.14±0.71) mm, while the amount of Ti/ZnO/Fn was only (0.37±0.28) mm. Moreover, the observation of paraffin section and qPCR revealed that inflammation level of gingiva and alveolar bone around Ti was lower in Ti/ZnO/Fn than in other groups. In conclusion, the composite coating for dental implants might be effective on defensing bacterial invasion, enhancing gingiva attachment and preventing inflammation response of gingiva.

Key words: titanium, zinc oxide, fibronectin, peri-implantitis

中图分类号:

TQ174

张大卫, 朱立远, 卢红亮, 王佐林. 钛表面氧化锌与纤连蛋白复合修饰应用于抗种植体周炎的研究[J]. 无机材料学报, 2021, 36(12): 1316-1322.

ZHANG Dawei, ZHU Liyuan, LU Hongliang, WANG Zuolin. Titanium Modified with ZnO Nanofilm and Fibronectin: Preventing Peri-implantitis and Biocompatibility[J]. Journal of Inorganic Materials, 2021, 36(12): 1316-1322.

图/表 10

图1 (a) Ti/ZnO和Ti/ZnO/Fn的制备过程示意图, (b, e) Ti、(c,f) Ti/ZnO和(d,g) Ti/ZnO/Fn的SEM(b~d)低倍照片及 (e~g) 相应的高倍照片

Fig. 1 (a) Schematic diagram of fabrication progress of Ti/ZnO and Ti/ZnO/Fn, and SEM images of (b, e) Ti, (c, f) Ti/ZnO and (d, g) Ti/ZnO/Fn at low (b-d) and high (e-g) magnification ALD: Atomic layer deposition

图2 (a)Ti、Ti/ZnO和Ti/ZnO/Fn的XPS全谱, (b)Ti、Ti/ZnO、Ti/ZnO/APTES和Ti/ZnO/Fn的N1s窄谱, 以及(c)Ti/ZnO/APTES和Ti/ZnO/Fn的FT-IR光谱图

Fig. 2 (a) XPS spectra of Ti, Ti/ZnO, and Ti/ZnO/Fn, (b) high-resolution spectra of N1s peak of Ti, Ti/ZnO, Ti/ZnO/APTES, and Ti/ZnO/Fn, and (c) FT-IR spectra of Ti/ZnO/APTES and Ti/ZnO/Fn Ti/ZnO/APTES: Ti disc with ZnO nanofilm modified with APTES

图3 复合材料的24 h抗A. a和P. g 效果及其机理

Fig. 3 Antibacterial effects and mechanisms of Ti, Ti/ZnO, and Ti/ZnO/Fn against A. a and P. g for 24 h (a) Typical bacteria colonies of A. a and P. g on Ti, Ti/ZnO, and Ti/ZnO/Fn (left) with average corresponding antibacterial rates (right); (b) Fluorecent images of dead bacteria (red) and total bacteria (green) on surfaces of Ti, Ti/ZnO, and Ti/ZnO/Fn after incubation (left) and their dead/total ratio (right); (c) Cumulative zinc ion release curves of Ti/ZnO and Ti/ZnO/Fn A. a: Aggregatibacter actinomycetemcomitans; P. g: Porphyro-monas gingivalis. The error bars indicate standard deviations. *P < 0.05, **P < 0.01, and ***P < 0.005 (n=3, One-way ANOVA)

图4 复合材料的细胞相容性

Fig. 4 Cytocompatibility of Ti/ZnO/Fn on human gingival fibroblasts (a) Fluorescent images of hGFs cultured on Ti, Ti/ZnO and Ti/ZnO/Fn for 6 and 12 h with vinculin staining (red) and nuclei staining (blue); (b) SEM images of hGFs attachment on Ti, Ti/ZnO and Ti/ZnO/Fn for 6 and 12 h; (c) Quantitative measurement of spreading area of hGFs on Ti, Ti/ZnO and Ti/ZnO/Fn after 6 and 12 h culture; (d) Cell viabilities cultured on Ti, Ti/ZnO and Ti/ZnO/Fn after 1, 3 and 7 d hGFs: human gingival fibroblasts. Error bars indicate standard deviations. **P < 0.01, and ***P < 0.005 (n=3, One-way ANOVA)

图5 hGFs在Ti、Ti/ZnO和Ti/ZnO/Fn表面培养3 d后(a) Col1、(b)Col3和(c)ITGB1a基因的相对表达量

Fig. 5 Quantification of the gene expression of (a) Col1, (b) Col3 and (c) ITGB1a in hGFs on Ti, Ti/ZnO and Ti/ZnO/Fn for 3 d by qPCR Data are normalized against reference gene expression of glyceraldehyde- 3-phosphate dehydrogenase and standardized with Ct (cycle threshold) of Ti. Error bars indicate standard deviations. **P < 0.01 and ***P < 0.005 (n=3, One-way ANOVA)

图6 Ti、Ti/ZnO和Ti/ZnO/Fn制成的钛钉植入大鼠牙槽骨4 w后(a)Micro-CT矢状面截图, (b)钛钉周组织石蜡切片H＆E染色照片, 和(c)钛钉周牙龈组织中IL1β、Arg1、TNFα、IL4、TGFβ1基因的相对表达量

Fig. 6 (a) Sagittal pictures of Micro-CT, (b) H&E-stained tissues around screws, and (c) quantification of the gene expression of IL1β, Arg1, TNFα, IL4, and TGFβ1 by qPCR in gingiva tissues around Ti, Ti/ZnO and Ti/ZnO/Fn screws after being inserted into maxillae of rats for 4 w Data are normalized against reference gene expression of glyceraldehyde-3-phosphate dehydrogenase and standardized with Ct of control. Control: Ti screws infused with bacteria culture medium; Ti, Ti/ZnO and Ti/ZnO/Fn: Ti, Ti/ZnO and Ti/ZnO/Fn screws infused with mixed bacterial suspension. Error bars indicate standard deviations. *P < 0.05, **P < 0.01, and ***P < 0.005 (n=3, One-way ANOVA)

表S1 人牙龈成纤维细胞相关基因正反引物序列

Table S1 Specific forward and reverse primer sequences of detected genes of HGFs

Gene	Forward primer sequence (5′-3′)	Reverse primer sequence (3′-5′)
GAPDH	GCACCGTCAAGGCTGAGAAC	TGGTGAAGACGCCAGTGGA
Col1	TCTAGACATGTTCAGCTTTGTGGAC	TCTGTACGCAGGTGATTGGTG
Col3	GCAAATTCACCTACACAGTTCTGGA	CTTGATCAGGACCACCAATGTCATA
ITGB1α	TGTGTCAGACCTGCCTTGGTG	AGGAACATTCCTGTGTGCATGTG

表S2 大鼠炎症相关基因正反引物序列

Table S2 Specific forward and reverse primer sequences of detected genes of rat

Gene	Forward primer sequence (5′-3′)	Reverse primer sequence (3′-5′)
GAPDH	CCCCAATGTATCCGTTGTG	CTCAGTGTAGCCCAGGATGC
IL1β	GACCTGTTCTTTGAGGCTGACA	CTCATCTGGACAGCCCAAGTC
Arg1	AGCAGAGACCCAGAAGAATG	TTTCCTTTCAGTTCCTTCAG
TNFα	GACAAGGCTGCCCCGACTAT	GGGAGACTCCTCCCAGGTACA
IL4	TCGCTTGCCTTGGTGGTC	TGTGATGTTGCTCAGCTCCTC
TGFβ1	AGGACCTGGGTTGGAAGTGG	AGTTGGCATGGTAGCCCTTG

表S3 Ti、Ti/ZnO和Ti/ZnO/Fn的表面元素含量

Table S3 Elemental chemical composition of Ti, Ti/ZnO and Ti/ZnO/Fn

Sample	C1s/%	O1s/%	N1s/%	Si2p3/%	Ti2p3/%	Zn2p3/%
Ti	57.69	31.07	1.92	0.02	9.26	0.05
Ti/ZnO	38.94	37.22	2.26	0.08	1.07	20.44
Ti/ZnO/Fn	63.91	21.2	11.88	2.27	0.13	0.6

图S1 使用荧光探针标记的在材料表面培养24 h细菌胞内ROS及其含量

Fig. S1 Reactive oxygen species (ROS) detected by fluorescence probes in bacteria cultured on materials for 24 h (a) Fluorescent images of ROS in A. a and P. g cultured on Ti/ZnO and Ti/ZnO/Fn; (b) Quantitative measurement of area of ROS in A. a and P. g Error bars indicate standard deviations: *P < 0.05 (n=3, Student’s t test)

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钛表面氧化锌与纤连蛋白复合修饰应用于抗种植体周炎的研究

Titanium Modified with ZnO Nanofilm and Fibronectin: Preventing Peri-implantitis and Biocompatibility

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