郝港桐,陈俊英,李莉,张鼎,翁亚军.钛表面PAMAM-BVLD/TPS修饰层构建及TPS浓度对生物相容性的影响[J].表面技术,2023,52(2):343-351.
HAO Gang-tong,CHEN Jun-ying,LI Li,ZHANG Ding,WENG Ya-jun.Construction of PAMAM-BVLD/TPS Modification Layer on Titanium Surface and Effect of TPS Concentration on Biocompatibility[J].Surface Technology,2023,52(2):343-351 |
| 钛表面PAMAM-BVLD/TPS修饰层构建及TPS浓度对生物相容性的影响 |
| Construction of PAMAM-BVLD/TPS Modification Layer on Titanium Surface and Effect of TPS Concentration on Biocompatibility |
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| DOI：10.16490/j.cnki.issn.1001-3660.2023.02.032 |
| 中文关键词: 表面改性 比伐卢定 促内皮 抗凝 细胞相容性 |
| 英文关键词:surface modification Bivalirudin endothelium-promoting anticoagulantion cytocompatibility |
| 基金项目:国家自然科学基金(31870955) |
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| Author | Institution |
| HAO Gang-tong | Key Laboratory of Advanced Materials Technology of Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China |
| CHEN Jun-ying | Key Laboratory of Advanced Materials Technology of Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China |
| LI Li | Key Laboratory of Advanced Materials Technology of Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China |
| ZHANG Ding | Key Laboratory of Advanced Materials Technology of Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China |
| WENG Ya-jun | Key Laboratory of Advanced Materials Technology of Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China |
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| 中文摘要: |
| 目的 在钛表面构建BVLD/TPS修饰层，并研究TPS浓度对其表面生物相容性的影响，从而筛选出最优TPS浓度。方法 将树枝状分子(PAMAM)接到钛表面，借助其端部氨基基团，将抗凝分子BVLD、促内皮功能分子TPSLEQRTVYAK肽(TPS)引入，构建出PAMAM-BVLD/TPS修饰层，重点研究TPS浓度对构建层生物相容性的影响。采用扫描电镜(SEM)、免疫荧光染色、傅里叶变换红外吸收光谱仪(FT-IR)、水接触角测量等对修饰层形貌、结构、表面亲疏水性等进行表征;使用酸性橙、Micro-BCA对Ti表面修饰层的活性基团进行测试。采用血小板粘附与激活试验、活化部分凝血活酶时间(APTT)评价血液组分与修饰层之间的相互作用。采用半体内循环试验评价修饰层与全血的相互作用。结果 成功构建修饰层，修饰层表面血小板粘附数量和激活程度均明显降低，其中TPS浓度为3 mol/L时，激活程度较低。APTT时间显著延长，其中修饰层APTT为37 s左右，相比对照组延长10 s左右。细胞相容性评价结果表明，与Ti表面相比，修饰层表面ECs粘附数量和增殖活性均增加，其中TPS浓度为3、5 mol/L时，内皮细胞生长情况最好，半体内循环试验结果表明，修饰层能够显著抑制血栓的形成。结论 在钛表面成功构建了BVLD/TPS修饰层，并初步探索出TPS的最优浓度，即当TPS浓度为3 mol/L时，其血液相容性以及细胞学评价良好，可以用于钛表面生物修饰层的研发与应用。 |
| 英文摘要: |
| At present, stent implantation is the main way for clinical treatment of atherosclerosis. However, after implantation, restenosis, thrombosis, inflammation and other problems may lead to treatment failure because the surface biocompatibility of implant materials does not meet the clinical requirements. Surface modification is one of the main methods to improve the biocompatibility of materials. At present, since the functional molecules modified by single function molecule can no longer meet the requirements of implant materials, the modification of multiple biomolecules on the surface of scaffolds has become a new hot spot. Therefore, the work aims to select anticoagulant molecule BVLD (Bivalirudin) and the endothelial- promoting molecule TPS (TPSLEQRTVYAK peptide) to construct BVLD/TPS modification layer on titanium surface and study the effect of TPS concentration on the biocompatibility of titanium surface, so as to obtain the optimal TPS concentration. For covalent construction of PAMAM-BVLD/TPS modification layer, titanium as a cardiovascular metal implant material, was firstly polished and then activated by NaOH to create a large number of hydroxyl reactive functional groups for electrostatic binding of positively charged PAMAM. Subsequently, according to the amino and amidation reaction of PAMAM terminal, BVLD and TPS were covalent immobilized onto surface, the PAMAM-BVLD/TPS modification layer was thus formed. Scanning electron microscopy (SEM) was used to observe the changes of the surface morphology of titanium. It was found that the surface roughness of titanium grafted with BVLD and TPS decreased. In immunofluorescence staining, the surface of the polypeptide grafted with FITC fluorescein gave off a large area of fluorescence. In FT-IR results, the successful graft of PAMAM could only be preliminarily judged. However, the successful graft of polypeptide could not be judged because the functional groups of the grafted molecules were similar. The acid orange experiment could further help to prove the successful graft of PAMAM. According to the Micro BCA experiment, the density of phenolic hydroxyl groups on the surface of the material was detected. The content of phenolic hydroxyl groups on the surface of the grafted peptide samples was significantly different from that of the contrast samples due to the unique phenolic hydroxyl group of polypeptide, proving the successful graft of the polypeptide. In the measurement of water contact Angle (WCA), the polypeptide molecule had better hydrophilicity than the other molecules, and with the increase of TPS concentration, the hydrophilicity was further improved. These experiments proved the successful construction of the BVLD/TPS modification layer. The effect of TPS concentration on the biocompatibility of the modification layer was studied in detail. In the platelet adhesion and activation experiment, the platelet activation degree on the titanium plane surface of the grafted polypeptide decreased, and the activation degree was the lowest when the TPS concentration was 3 mol/L. In the APTT experiment, the partial activation time of thrombin grafted with BVLD sample was still significantly longer than control sample, and the TPS concentration had no significant effect on the time. The APTT time of the modification layer was about 37 s, which was about 10 s longer than that of the control group. The experiment results of endothelial cell adhesion and proliferation disclosed that, compared with the control group, the number and activity of the endothelial cells adhered to the modification layer surface grafted by polypeptide increased, and the growth of the endothelial cells was excellent when the TPS concentration was 3 and 5 mol/L. Based on the above results, it was proved that the biocompatibility of modified titanium surface was optimal at the TPS concentration of 3 mol/L. Half body experiment showed that the modification layer could significantly inhibit thrombosis. Obviously, the co-immobilization of BVLD and TPS onto biomaterials surface, is expected to be used for the cardiovascular interventional materials with functionalization. BVLD/TPS modification layer is successfully constructed on titanium surface, and the optimal concentration of TPS is preliminarily explored, that is, when the concentration of TPS is 3 mol/L, its blood compatibility and cytological evaluation are good, which can be used for the development and application of biological modification layer on titanium surface. |
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