本文選題：生物活性玻璃 + 創(chuàng)面修復(fù)　； 參考：《華南理工大學(xué)》2014年博士論文

【摘要】：通過研制新型的皮膚軟組織創(chuàng)面修復(fù)材料，幫助患者修復(fù)缺損或難愈的皮膚創(chuàng)面組織，更好地恢復(fù)人體皮膚軟組織功能是醫(yī)學(xué)界和生物醫(yī)學(xué)材料學(xué)界多年來一直在探索并試圖解決的重要問題。生物活性玻璃45S5是采用高溫熔融法制備的一種無機(jī)活性材料，其化學(xué)組成為(質(zhì)量份比)：45%SiO2、4.5%Na2O、24.5%CaO和6%P2O5，該生物活性玻璃作為傳統(tǒng)具有代表性的一類無機(jī)活性材料在皮膚軟組織損傷修復(fù)領(lǐng)域已經(jīng)有了一定的應(yīng)用和相關(guān)研究報(bào)道，但是一些新型生物活性玻璃如溶膠－凝膠生物活性玻璃(Sol-gelbioactive glass, SGBG)和納米生物活性玻璃（Nanoscale bioactive glass, NBG）等在這方面的研究比較少，亦少見文獻(xiàn)報(bào)道。事實(shí)上，這些新型生物活性材料較傳統(tǒng)生物活性玻璃45S5有著一些更優(yōu)良的性能，如材料的化學(xué)均勻性可達(dá)分子級(jí)別，并具有納米介孔結(jié)構(gòu)、巨大的比表面積以及較高的化學(xué)活性和吸附特性等。 本論文圍繞溶膠-凝膠生物活性玻璃調(diào)控糖尿病難愈創(chuàng)面評(píng)價(jià)體系的建立及其修復(fù)機(jī)理進(jìn)行研究，以鏈脲佐菌素STZ誘導(dǎo)的糖尿病大鼠全層皮膚缺損模型來模擬糖尿病難愈創(chuàng)面的修復(fù)過程，通過透明膜描記法評(píng)估創(chuàng)面愈合率，并分別通過HE染色、免疫組化染色、透射電鏡等進(jìn)一步觀察分析成纖維細(xì)胞、上皮細(xì)胞、血管內(nèi)皮細(xì)胞增殖，以及相關(guān)生長(zhǎng)因子（VEGF和FGF-2）的表達(dá)，與此同時(shí)，通過MTT法測(cè)定觀察生物活性玻璃對(duì)人成纖維細(xì)胞增殖，系統(tǒng)研究并證實(shí)了溶膠-凝膠生物活性玻璃對(duì)糖尿病難愈創(chuàng)面的修復(fù)作用。具體研究工作如下： 首先，通過溶膠-凝膠制備工藝獲得溶膠-凝膠生物活性玻璃SGBG（摩爾組成：60%SiO2、36%C和4%P2O5），并采用溶膠-凝膠共沉淀法、結(jié)合冷凍干燥技術(shù)制備了顆粒尺寸在納米級(jí)的生物活性玻璃NBG（摩爾組成：60%SiO2、36%CaO和4%P2O5），研究了加入分散劑聚乙二醇（PEG-10000）對(duì)生物活性玻璃顆粒的分散性能、微觀形貌和生物活性的影響。結(jié)果表明：沒有加入PEG制得的NBG顆粒呈現(xiàn)不規(guī)則形態(tài)，粒徑小于50nm，加有PEG的NBG顆粒形狀趨于規(guī)則的球形，分散性大大提高，顆粒粒徑在40～100nm，而且加入PEG的濃度越高，制備的NBG顆粒粒徑越小。通過比較NBG與SGBG在模擬人體體液（Simulated bodyfluid, SBF）中的表面礦化研究，發(fā)現(xiàn)NBG比SGBG有更高的生物活性。 其次，通過體外細(xì)胞培養(yǎng)，MTT法評(píng)價(jià)45S5、SGBG和NBG三種生物活性玻璃材料的細(xì)胞增殖活性，研究不同濃度、不同作用時(shí)間下生物活性玻璃對(duì)人成纖維細(xì)胞體外生長(zhǎng)增殖的影響。通過酶解法從人包皮中成功提取成纖維細(xì)胞，，并通過免疫蛋白組化鑒定其為成纖維細(xì)胞，其中HMB45呈陰性，角蛋白呈陰性，S100呈陰性，波形蛋白呈陽性，均符合成纖維細(xì)胞的特點(diǎn)。結(jié)果，三種生物活性玻璃浸提液均無明顯細(xì)胞毒性，且在合適的濃度下有利于成纖維細(xì)胞的增殖，但三種生物活性玻璃的作用不同，45S5在細(xì)胞培養(yǎng)早期有利于成纖維細(xì)胞的增殖，而NBG則在后期對(duì)細(xì)胞的作用更為顯著。 在糖尿病難愈創(chuàng)面動(dòng)物模型建立方面，本研究旨在探討生物活性玻璃對(duì)一般皮膚創(chuàng)面和糖尿病皮膚創(chuàng)面的修復(fù)作用。以SD大鼠及鏈脲佐菌素誘導(dǎo)的糖尿病SD大鼠為動(dòng)物模型，將制備的生物活性玻璃膏劑應(yīng)用于大鼠背部全層皮膚缺損創(chuàng)面，并進(jìn)行相應(yīng)的修復(fù)評(píng)價(jià)和機(jī)理探討。實(shí)驗(yàn)中根據(jù)生物活性玻璃的種類將創(chuàng)面分為四組，即熔融生物活性玻璃45S5組（45S5）、溶膠-凝膠生物活性玻璃58S組（SGBG-58S）、納米生物活性玻璃58S組（NBG-58S）及凡士林對(duì)照組。和SGBG-58S相比，NBG-58S顆粒是由相對(duì)分散的納米顆粒構(gòu)成。通過對(duì)創(chuàng)面愈合率及創(chuàng)面愈合時(shí)間的統(tǒng)計(jì)計(jì)算發(fā)現(xiàn)，生物活性玻璃組能促進(jìn)創(chuàng)面的快速愈合，且SGBG-58S和NBG-58S這兩種生物活性玻璃的愈合效果更佳。組織學(xué)分析表明生物活性玻璃能促進(jìn)成纖維細(xì)胞的增殖和肉芽組織的快速形成，并且，免疫組化分析提示與創(chuàng)面修復(fù)有關(guān)的兩種生長(zhǎng)因子VEGF和FGF2在生物活性玻璃的刺激下有高效的表達(dá)。通過透射電鏡觀察顯示，生物活性玻璃組處理的創(chuàng)面中成纖維細(xì)胞更為成熟，含有更多的粗面內(nèi)質(zhì)網(wǎng)及其他細(xì)胞器，并能在術(shù)后7天就已經(jīng)形成了成熟的血管，SGBG-58S和NBG-58S效果比45S5更為明顯。綜上所述，生物活性玻璃，尤其是SGBG-58S和NBG-58S能促進(jìn)正常和糖尿病皮膚創(chuàng)面的快速愈合，在創(chuàng)面修復(fù)領(lǐng)域中具有廣泛的應(yīng)用前景。 此外，研究首次探索性地將45S5生物活性玻璃復(fù)合云南白藥膏劑應(yīng)用于上述建立的評(píng)價(jià)體系，為開發(fā)治療糖尿病創(chuàng)面外用藥物提供參考和新的思路。
[Abstract]:By developing a new type of skin soft tissue repair material, it is an important problem that the medical and biomedical materials academics have been exploring and trying to solve for many years to help the patients to repair the defect or difficult skin tissue and to better restore the soft tissue function of human skin. The active glass 45S5 is prepared by high temperature melting method. A kind of inorganic active material, whose chemical composition is (mass ratio):45%SiO2,4.5%Na2O, 24.5%CaO and 6%P2O5. The bioactive glass has been used as a representative kind of inorganic active material in the field of skin soft tissue damage repair. The sol-gel bioactive glass (Sol-gelbioactive glass, SGBG) and nanoscale bioactive glass (Nanoscale bioactive glass, NBG) are rare and rarely reported in this field. In fact, these new bioactive materials have some better properties than the traditional bioactive glass 45S5, such as the chemistry of materials. Homogeneity can reach molecular level, and has nano mesoporous structure, large specific surface area, high chemical activity and adsorption characteristics.
In this paper, the establishment and repair mechanism of the evaluation system of diabetic refractory wound by sol-gel bioactive glass were established and the repair process of diabetic skin defect was simulated with streptozotocin STZ induced diabetic rat full layer skin defect model, and the healing rate of wound healing was evaluated through the permeable membrane tracing method. HE staining, immunohistochemical staining and transmission electron microscopy were used to further observe the proliferation of fibroblasts, epithelial cells, vascular endothelial cells, and the expression of related growth factors (VEGF and FGF-2). At the same time, the proliferation of bioactive glass to human fibroblast cells was observed by MTT method, and the sol-gel biological activity was systematically studied and confirmed. The repairing effect of sex glass on diabetic refractory wounds is as follows:
First, sol-gel bioactive glass SGBG (molar composition: 60%SiO2,36%C and 4%P2O5) was obtained by sol-gel preparation process, and a sol gel co precipitation method was used to prepare bioactive glass NBG (mole composition: 60%SiO2,36%CaO and 4%P2O5) of particle size at nanoscale (60%SiO2,36%CaO and 4%P2O5), and the dispersant was studied. The effect of polyethylene glycol (PEG-10000) on the dispersibility, micromorphology and biological activity of bioactive glass particles shows that the NBG particles without PEG are irregular in shape, the particle size is less than 50nm, the shape of NBG particles with PEG tends to a regular shape, and the dispersion property is greatly improved, the particle size is 40 to 100nm, and the particle size is added. The higher the concentration of PEG, the smaller the particle size of the prepared NBG particles. By comparing the surface mineralization of NBG and SGBG in the simulated body fluid (Simulated BodyFluid, SBF), it is found that NBG has a higher biological activity than SGBG.
Secondly, the cell proliferation activity of three bioactive glass materials of 45S5, SGBG and NBG was evaluated by MTT method in vitro, and the effects of different concentration and time on the growth and proliferation of human fibroblasts in vitro were studied. The fibroblasts were successfully extracted from human foreskin by enzymatic hydrolysis, and the immunoglobulin was obtained through the immunoglobulin. HMB45 was negative, keratin was negative, S100 was negative, vimentin was positive, and all of the three bioactive glass extracts had no obvious cytotoxicity, and it was beneficial to the proliferation of fibroblasts at the appropriate concentration, but three kinds of bioactive glass. 45S5 is beneficial to fibroblast proliferation in the early stage of cell culture, while NBG plays a more significant role in the later stage.
The purpose of this study was to explore the effect of bioactive glass on the general skin wounds and diabetic skin wounds in the establishment of the diabetic skin wound animal model. The diabetic SD rats induced by SD rats and streptozotocin were used as animal models, and the bioactive glass plaster was applied to the full skin defect of the back of the rat. In the experiment, the wounds were divided into four groups according to the species of bioactive glass, namely, the fused bioactive glass 45S5 group (45S5), the sol-gel bioactive glass 58S group (SGBG-58S), the nano bioactive glass 58S group (NBG-58S) and the Vaseline control group. Compared with SGBG-58S, NBG-58S particles were compared. It is composed of relatively dispersed nanoparticles. By statistical calculation of wound healing rate and wound healing time, the bioactive glass group can promote the rapid healing of the wound, and the healing effect of the two bioactive glasses, SGBG-58S and NBG-58S, is better. The tissue analysis table Ming Sheng active glass can promote the proliferation of fibroblasts. And the rapid formation of granulation tissue, and the immunohistochemical analysis suggested that two growth factors VEGF and FGF2 related to the wound repair were highly expressed under the stimulation of bioactive glass. Through transmission electron microscopy, it was found that the fibroblasts were more mature in the wound surface of the bioactive glass group and contain more rough endoplasmic reticulum and more rough endoplasmic reticulum. Other organelles have formed mature blood vessels at 7 days after the operation. The effect of SGBG-58S and NBG-58S is more obvious than that of 45S5. To sum up, bioactive glass, especially SGBG-58S and NBG-58S, can promote the rapid healing of normal and diabetic skin wounds, and have extensive application prospects in the area of wound repair collar.
In addition, it is the first time to apply the 45S5 bioactive glass compound of Yunnan white ointment to the evaluation system established above, and provide a reference and new idea for the development of the treatment of external use drugs for diabetic wounds.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R587.1;R318.08

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