口腔疾病防治 ›› 2022, Vol. 30 ›› Issue (6): 398-405.DOI: 10.12016/j.issn.2096-1456.2022.06.003

• 基础研究 • 上一篇    下一篇

钛表面不同硅烷偶联c(RGDfK)环肽的表征及生物相容性分析

周齐悦1(), 洪高英1, 吴桐1, 陈晨2, 谢海峰1()   

  1. 1.南京医科大学附属口腔医院修复科 江苏省口腔疾病研究重点实验室 江苏省口腔医学转化医学工程研究中心,江苏 南京(210029)
    2.南京医科大学附属口腔医院牙体牙髓科 江苏省口腔疾病研究重点实验室 江苏省口腔医学转化医学工程研究中心,江苏 南京(210029)
  • 收稿日期:2021-09-01 修回日期:2021-11-11 出版日期:2022-06-20 发布日期:2022-04-15
  • 通讯作者: 谢海峰
  • 作者简介:周齐悦,硕士研究生,Email: 2531960376@qq.com
  • 基金资助:
    国家自然科学基金项目(81970927);江苏省卫生健康委科学技术基金项目(M2020066);江苏高校优势学科建设工程资助项目(2018-87)

Characterization and biocompatibility analysis of different silanes coupling c(RGDfK) cyclic peptide on titanium surfaces

ZHOU Qiyue1(), HONG Gaoying1, WU Tong1, CHEN Chen2, XIE Haifeng1()   

  1. 1. Department of Prosthodontics, Affiliated Stomatology Hospital of Nanjing Medical University, Jiangsu Province Key Laboratory of Oral Diseases, Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing 210029, China
    2. Department of Endodontics, Affiliated Stomatology Hospital of Nanjing Medical University, Jiangsu Province Key Laboratory of Oral Diseases, Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing 210029, China
  • Received:2021-09-01 Revised:2021-11-11 Online:2022-06-20 Published:2022-04-15
  • Contact: XIE Haifeng
  • Supported by:
    grants from National Natural Science Foundation of China(81970927);Medical Research Projects of the Health Department of Jiangsu Province(M2020066);Priority Academic Program Development of Jiangsu Higher Education Institutions(2018-87)

摘要:

目的 探讨用四种不同硅烷将c(RGDfK)[(cyclo(Arg-Gly-Asp-d-Phe-Lys)]环肽固定于钛表面的效率及生物相容性。方法 钛表面经碱热处理(OH组),分别用3-氨丙基三乙氧基硅烷(3-aminopropyltriethoxysilane,APTES)(OHAP组)、3-氯丙基三乙氧基硅烷(3-chloropropyltriethoxysilane,CPTES)(OHCP组)、3-巯基丙基三甲氧基硅烷(3-mercaptopropyltriethoxysilane,MPTS)(OHMPT组)、3-异丁烯酰氧丙基三甲氧基硅烷(γ-methacryloxypropyltrimethoxysilane,γ-MPS)(OHMPS组)四种硅烷固定c(RGDfK)环肽,构建钛-硅烷-c(RGDfK)环肽涂层,钛片表面未处理组为空白对照组(NT组)。利用扫描电镜、接触角计观察各组涂层表面形貌及润湿性变化,通过X射线光电子能谱分析钛表面元素组成,4,6-二氨基-2-苯基吲哚(4,6-diamino-2-phenylindole,DAPI)与鬼笔环肽荧光染色后使用激光共聚焦显微镜观察材料表面小鼠前成骨细胞MC3T3-E1黏附情况,细胞计数(cell counting kit-8,CCK-8)试验和碱性磷酸酶(alkaline phosphatase,ALP)活性测定评价材料表面MC3T3-E1细胞增殖及成骨分化情况。结果 扫描电镜观察可见碱热处理后钛表面形成海绵状三维立体网状结构,硅烷-c(RGDfK)环肽涂层附着其上,各组润湿性较未处理钛片均有较大提高,OHMPS组Si/Ti、酰胺-N/Ti元素比最高;OHAP组细胞黏附形态最佳;OHAP组、OHMPT组、OHMPS组细胞增殖及ALP活性均显著高于对照组(P <0.05);OHCP组细胞增殖活性及ALP活性与对照组无统计学差异。结论 MPTS、CPTES、γ-MPS三种硅烷均能够作为偶联剂将c(RGDfK)环肽结合于钛表面,促进MC3T3-E1细胞黏附、增殖和分化,其中γ-MPS偶联c(RGDfK)环肽的效果最好,而MPTS、CPTES、γ-MPS偶联c(RGDfK)环肽后具有相似的生物学性能。

关键词: 钛种植体, c(RGDfK), 硅烷偶联剂, 碱热处理, 表面处理, MC3T3-E1细胞, 细胞黏附, 成骨分化, 碱性磷酸酶

Abstract:

Objective To compare the efficiency and biocompatibility of four different silanes on immobilizing c(RGDfK) peptide on titanium surface. Methods After alkali-heat treatment (group OH), the titanium surface was treated with 3-aminopropyltriethoxysilane (APTES) (group OHAP), 3-chloropropyltriethoxysilane (CPTES) (group OHCP) (3-mercaptopropyltrimethoxysilane (MPTS) (group OHMPT) and 3-isobutyryloxy propyltrimethoxysilane(γ- MPS) (group OHMPS) to immobilize the c(RGDfK) cyclic peptide and constructa titanium-silane-c(RGDfK) coating. The NT group was the blank control group. The surface morphology and wettability of the coatings were detected using scanning electron microscopy and contact angle measurement. The elemental composition of the titanium surface was analyzed using X-ray photoelectron spectroscopy. After fluorescent staining with 4’,6-diamino-2-phenylindole (DAPI) and phalloidin, the adhesion of mouse preosteoblast MC3T3-E1 cells on the surface of the materials was observed using laser confocal microscopy. Cell counting kit-8 (CCK-8) and alkaline phosphatase (ALP) activity assays were used to evaluate the proliferation and osteogenic differentiation of MC3T3-E1 cells on the surface of the materials, respectively. Results Scanning electron microscope observation showed a spongy-like 3-dimensional network formed on the titanium surface after alkali-heat treatment with silane-c(RGDfK) coating adhesion. The wettability of each group was greatly improved compared to the untreated titanium surface. The element ratios of Si/Ti and amide-N/Ti in the OHMPS group were the highest. The OHAP group exhibited the best cell adhesion effect. The cell proliferation and ALP activity of the OHAP, OHMPT, and OHMPS groups were significantly higher than the control group (P <0.05); there was no statistical difference between the OHCP group and the control group. Conclusion MPTS, CPTES and γ-MPS covalently immobilized cyclic peptide c(RGDfK) on the titanium surface, which promoted adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells. Theγ-MPS conjugated C (RGDfK)cyclic peptide exhibited the best effect. MPTS, CPTES and γ-MPS coupled with c(RGDfK) cyclic peptides had similar biological properties.

Key words: titanium implants, c(RGDfK), silane coupling agent, alkali-heat treatment, surface treatment, MC3T3-E1, cell adhesion, osteogenic differentiation, ALP

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