純鈦表面接枝二磷酸腺苷、纖維蛋白原對(duì)提高血小板活性的研究
發(fā)布時(shí)間：2018-11-04 17:28
【摘要】：目的:口服抗血小板藥物廣泛應(yīng)用于心血管疾病患者的治療和預(yù)防。越來越多的缺牙心血管疾病患者為提高生活質(zhì)量選擇種植修復(fù)治療。種植成功的關(guān)鍵是形成骨結(jié)合。血小板在骨結(jié)合過程中具有重要意義�？寡“逅幬锿ㄟ^不同途徑抑制血小板活性,不利于骨結(jié)合的形成。本研究的目的是在鈦表面接枝功能性促進(jìn)血小板活化的物質(zhì)二磷酸腺苷(Adenosine Diphosphate,ADP)、纖維蛋白原(Fibrinogen,FIB),構(gòu)建一個(gè)特異性促進(jìn)血小板粘附、聚集活化的表面,以期這種表面修飾促進(jìn)長(zhǎng)期口服抗血小板藥物患者的創(chuàng)口凝血、提高種植體-骨結(jié)合,為開發(fā)針對(duì)長(zhǎng)期服用抗血小板藥物治療患者專用口腔種植體提供實(shí)驗(yàn)依據(jù)。方法:通過聚多巴胺涂層修飾,在鈦表面接枝功能性分子ADP、FIB。實(shí)驗(yàn)分為四組:鈦組(Ti)、ADP組(Ti+DOP+ADP)、FIB組(Ti+DOP+FIB)、ADP與FIB混合接枝組(Ti+DOP+ADP+FIB)。對(duì)改性后的材料表面進(jìn)行表征分析,通過X射線光電子能譜分析(XPS)分析材料表面的元素種類;通過靜態(tài)接觸角檢測(cè)材料表面的親水性,驗(yàn)證材料是否成功接枝。采集服用抗血小板藥物(阿司匹林或氯吡格雷)治療一年以上且系統(tǒng)疾病穩(wěn)定,可以安全行口腔種植治療的患者血液作為病例組(n=3),健康志愿者血液作為對(duì)照組(n=3),分別制備血小板懸浮液。血小板粘附實(shí)驗(yàn)由場(chǎng)發(fā)射掃描電鏡(FE-SEM)定性檢測(cè)材料表面血小板的黏附量以及形貌;乳酸脫氫酶(LDH)實(shí)驗(yàn)定量測(cè)定血小板的黏附量。血小板激活實(shí)驗(yàn)通過免疫熒光顯微鏡觀察材料表面吸附的血小板膜表面CD41、CD62的表達(dá)情況。培養(yǎng)人骨髓間充質(zhì)干細(xì)胞(hMSCs),通過CCK8實(shí)驗(yàn)檢測(cè)細(xì)胞增殖情況,判斷材料表面對(duì)人骨髓間充質(zhì)干細(xì)胞是否具有毒副作用。結(jié)果:XPS結(jié)果中ADP組表面P元素出現(xiàn),FIB組S元素出現(xiàn),以及ADP與FIB混合接枝組P、S元素的出現(xiàn),表明鈦表面有功能性分子ADP、FIB的成功接枝。測(cè)定材料靜態(tài)接觸角結(jié)果表明改性后的材料表面親水性提高,接觸角的變化也進(jìn)一步驗(yàn)證材料的成功接枝。場(chǎng)發(fā)射掃描電鏡實(shí)驗(yàn)結(jié)果顯示:相比于鈦組,ADP組、FIB組、ADP與FIB混合接枝組表面可見大量成簇的血小板樣結(jié)構(gòu)粘附。LDH乳酸脫氫酶定量實(shí)驗(yàn)結(jié)果顯示:相比于鈦組,ADP組、FIB組、ADP與FIB混合接枝組乳酸脫氫酶含量均有提高,其中ADP與FIB混合接枝組可以顯著提高血小板的聚集(P0.05)。免疫熒光顯微鏡觀察材料表面血小板激活情況,血小板膜表面CD41、CD62表達(dá)情況與血小板粘附實(shí)驗(yàn)結(jié)果一致。hMSCs細(xì)胞增殖實(shí)驗(yàn)顯示四組材料在1,3,5天無統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:通過聚多巴胺涂層修飾鈦表面,接枝二磷酸腺苷、纖維蛋白原,可以成功構(gòu)建出一個(gè)特異性促進(jìn)血小板粘附、聚集活化的功能性表面。改性后的鈦表面,尤其是ADP與FIB混合接枝改性的鈦功能化表面可以促進(jìn)抗血小板組和健康組血小板的黏附、聚集與活化,并對(duì)人骨髓間充質(zhì)干細(xì)胞無毒性作用。因此可作為種植體表面修飾的方法之一,應(yīng)用于長(zhǎng)期服用抗血小板藥物治療且牙缺失需要種植修復(fù)的患者,提高種植體的骨整合能力,從而有望提高這類患者的種植修復(fù)效果(已申請(qǐng)新型實(shí)用專利)。
[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).
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R783.6
本文編號(hào)：2310624
[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).
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R783.6
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