纯钛表面接枝二磷酸腺苷、纤维蛋白原对提高血小板活性的研究
发布时间：2018-11-04 17:28
【摘要】：目的:口服抗血小板药物广泛应用于心血管疾病患者的治疗和预防。越来越多的缺牙心血管疾病患者为提高生活质量选择种植修复治疗。种植成功的关键是形成骨结合。血小板在骨结合过程中具有重要意义。抗血小板药物通过不同途径抑制血小板活性,不利于骨结合的形成。本研究的目的是在钛表面接枝功能性促进血小板活化的物质二磷酸腺苷(Adenosine Diphosphate,ADP)、纤维蛋白原(Fibrinogen,FIB),构建一个特异性促进血小板粘附、聚集活化的表面,以期这种表面修饰促进长期口服抗血小板药物患者的创口凝血、提高种植体-骨结合,为开发针对长期服用抗血小板药物治疗患者专用口腔种植体提供实验依据。方法:通过聚多巴胺涂层修饰,在钛表面接枝功能性分子ADP、FIB。实验分为四组:钛组(Ti)、ADP组(Ti+DOP+ADP)、FIB组(Ti+DOP+FIB)、ADP与FIB混合接枝组(Ti+DOP+ADP+FIB)。对改性后的材料表面进行表征分析,通过X射线光电子能谱分析(XPS)分析材料表面的元素种类;通过静态接触角检测材料表面的亲水性,验证材料是否成功接枝。采集服用抗血小板药物(阿司匹林或氯吡格雷)治疗一年以上且系统疾病稳定,可以安全行口腔种植治疗的患者血液作为病例组(n=3),健康志愿者血液作为对照组(n=3),分别制备血小板悬浮液。血小板粘附实验由场发射扫描电镜(FE-SEM)定性检测材料表面血小板的黏附量以及形貌;乳酸脱氢酶(LDH)实验定量测定血小板的黏附量。血小板激活实验通过免疫荧光显微镜观察材料表面吸附的血小板膜表面CD41、CD62的表达情况。培养人骨髓间充质干细胞(hMSCs),通过CCK8实验检测细胞增殖情况,判断材料表面对人骨髓间充质干细胞是否具有毒副作用。结果:XPS结果中ADP组表面P元素出现,FIB组S元素出现,以及ADP与FIB混合接枝组P、S元素的出现,表明钛表面有功能性分子ADP、FIB的成功接枝。测定材料静态接触角结果表明改性后的材料表面亲水性提高,接触角的变化也进一步验证材料的成功接枝。场发射扫描电镜实验结果显示:相比于钛组,ADP组、FIB组、ADP与FIB混合接枝组表面可见大量成簇的血小板样结构粘附。LDH乳酸脱氢酶定量实验结果显示:相比于钛组,ADP组、FIB组、ADP与FIB混合接枝组乳酸脱氢酶含量均有提高,其中ADP与FIB混合接枝组可以显著提高血小板的聚集(P0.05)。免疫荧光显微镜观察材料表面血小板激活情况,血小板膜表面CD41、CD62表达情况与血小板粘附实验结果一致。hMSCs细胞增殖实验显示四组材料在1,3,5天无统计学差异(P0.05)。结论:通过聚多巴胺涂层修饰钛表面,接枝二磷酸腺苷、纤维蛋白原,可以成功构建出一个特异性促进血小板粘附、聚集活化的功能性表面。改性后的钛表面,尤其是ADP与FIB混合接枝改性的钛功能化表面可以促进抗血小板组和健康组血小板的黏附、聚集与活化,并对人骨髓间充质干细胞无毒性作用。因此可作为种植体表面修饰的方法之一,应用于长期服用抗血小板药物治疗且牙缺失需要种植修复的患者,提高种植体的骨整合能力,从而有望提高这类患者的种植修复效果(已申请新型实用专利)。
[Abstract]:Objective: Oral antiplatelet drugs are widely used in the treatment and prevention of cardiovascular diseases. More and more patients with missing tooth and cardiovascular diseases are choosing planting and repairing treatment for improving the quality of life. The key to the success of planting is the formation of bone-binding. platelets play an important role in osseointegration. Anti-platelet drugs inhibit platelet activity through different ways, which is not conducive to the formation of bone binding. The purpose of this study is to construct a surface that specifically promotes platelet adhesion, aggregation and activation by grafting functionality on the surface of titanium to promote platelet-activated substance bisphosphate (ADP), fibrinogen (FIB), so that the surface modification promotes wound coagulation of a long-term oral anti-platelet drug patient and improves the implant-bone combination and provides an experimental basis for the development of a special oral implant for the long-term administration of anti-platelet drugs. Methods: Poly (dopamine) coating was modified to graft functional molecules ADP, FIB on the surface of titanium. The experiment was divided into four groups: titanium group (Ti), ADP group (Ti + DOP + ADP), FIB group (Ti + DOP + FIB), ADP and FIB mixed grafting group (Ti + DOP + ADP + FIB). the surface of the modified material is characterized and analyzed, the element type of the surface of the material is analyzed by the Xray photoelectron spectroscopy (XPS), the hydrophilicity of the surface of the material is detected by the static contact angle, and whether the material is successfully grafted is verified. Platelet suspensions were prepared as case group (n = 3) and healthy volunteer blood as control group (n = 3). Platelet adhesion test was qualitatively detected by field emission scanning electron microscope (FE-SEM). The adhesion amount of platelets was measured quantitatively by LDH assay. Platelet activation assay was used to observe the expression of CD41 and CD62 on the surface of platelet membrane adsorbed on the surface of the material by immunofluorescence microscopy. Cultured human bone marrow mesenchymal stem cells (hMSCs) detected cell proliferation through CCK8 experiment, and judged whether the material surface had toxic and side effects on human bone marrow mesenchymal stem cells. Results: The surface P element of ADP group appeared in the XPS results, the S element of FIB group appeared, and the P and S elements of ADP and FIB mixed grafting group showed that the titanium surface had functional molecular ADP, FIB was grafted successfully. The static contact angle of the measured material indicates that the surface hydrophilicity of the modified material is improved, and the change of the contact angle further verifies the successful grafting of the material. The results of field emission scanning electron microscopy showed that there was a large number of clusters of platelet-like structures adhered to the surface of the mixed grafting group of ADP and FIB in the group of titanium, ADP, FIB, ADP and FIB. The results showed that the content of lactic dehydrogenase in the mixed graft group of ADP and FIB was increased compared with the group of titanium, ADP, FIB, ADP and FIB, among which ADP and FIB mixed grafting group could significantly improve platelet aggregation (P0.05). The expression of CD41 and CD62 on the surface of platelet membrane was consistent with that of platelet adhesion experiment. There was no significant difference in the proliferation of hMSCs in 1, 3 and 5 days (P0.05). Conclusion: The functional surface of platelet adhesion and aggregation activation can be successfully constructed by modifying the surface of titanium, grafting diphosphate and fibrinogen by polydopamine coating. The modified titanium surface, especially ADP and FIB, can promote the adhesion, aggregation and activation of platelet aggregation in anti-platelet group and healthy group, and has no toxicity to human bone marrow mesenchymal stem cells. Therefore, one of the methods which can be used as the surface modification of the implant can be applied to patients who have long-term administration of anti-platelet drugs and require planting and repair, so that the osseointegration ability of the implant is improved, and the planting and repairing effect of the implant is expected to be improved (new practical patents have been applied).
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.6
本文编号：2310625
[Abstract]:Objective: Oral antiplatelet drugs are widely used in the treatment and prevention of cardiovascular diseases. More and more patients with missing tooth and cardiovascular diseases are choosing planting and repairing treatment for improving the quality of life. The key to the success of planting is the formation of bone-binding. platelets play an important role in osseointegration. Anti-platelet drugs inhibit platelet activity through different ways, which is not conducive to the formation of bone binding. The purpose of this study is to construct a surface that specifically promotes platelet adhesion, aggregation and activation by grafting functionality on the surface of titanium to promote platelet-activated substance bisphosphate (ADP), fibrinogen (FIB), so that the surface modification promotes wound coagulation of a long-term oral anti-platelet drug patient and improves the implant-bone combination and provides an experimental basis for the development of a special oral implant for the long-term administration of anti-platelet drugs. Methods: Poly (dopamine) coating was modified to graft functional molecules ADP, FIB on the surface of titanium. The experiment was divided into four groups: titanium group (Ti), ADP group (Ti + DOP + ADP), FIB group (Ti + DOP + FIB), ADP and FIB mixed grafting group (Ti + DOP + ADP + FIB). the surface of the modified material is characterized and analyzed, the element type of the surface of the material is analyzed by the Xray photoelectron spectroscopy (XPS), the hydrophilicity of the surface of the material is detected by the static contact angle, and whether the material is successfully grafted is verified. Platelet suspensions were prepared as case group (n = 3) and healthy volunteer blood as control group (n = 3). Platelet adhesion test was qualitatively detected by field emission scanning electron microscope (FE-SEM). The adhesion amount of platelets was measured quantitatively by LDH assay. Platelet activation assay was used to observe the expression of CD41 and CD62 on the surface of platelet membrane adsorbed on the surface of the material by immunofluorescence microscopy. Cultured human bone marrow mesenchymal stem cells (hMSCs) detected cell proliferation through CCK8 experiment, and judged whether the material surface had toxic and side effects on human bone marrow mesenchymal stem cells. Results: The surface P element of ADP group appeared in the XPS results, the S element of FIB group appeared, and the P and S elements of ADP and FIB mixed grafting group showed that the titanium surface had functional molecular ADP, FIB was grafted successfully. The static contact angle of the measured material indicates that the surface hydrophilicity of the modified material is improved, and the change of the contact angle further verifies the successful grafting of the material. The results of field emission scanning electron microscopy showed that there was a large number of clusters of platelet-like structures adhered to the surface of the mixed grafting group of ADP and FIB in the group of titanium, ADP, FIB, ADP and FIB. The results showed that the content of lactic dehydrogenase in the mixed graft group of ADP and FIB was increased compared with the group of titanium, ADP, FIB, ADP and FIB, among which ADP and FIB mixed grafting group could significantly improve platelet aggregation (P0.05). The expression of CD41 and CD62 on the surface of platelet membrane was consistent with that of platelet adhesion experiment. There was no significant difference in the proliferation of hMSCs in 1, 3 and 5 days (P0.05). Conclusion: The functional surface of platelet adhesion and aggregation activation can be successfully constructed by modifying the surface of titanium, grafting diphosphate and fibrinogen by polydopamine coating. The modified titanium surface, especially ADP and FIB, can promote the adhesion, aggregation and activation of platelet aggregation in anti-platelet group and healthy group, and has no toxicity to human bone marrow mesenchymal stem cells. Therefore, one of the methods which can be used as the surface modification of the implant can be applied to patients who have long-term administration of anti-platelet drugs and require planting and repair, so that the osseointegration ability of the implant is improved, and the planting and repairing effect of the implant is expected to be improved (new practical patents have been applied).
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.6
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