溶胶—凝胶生物活性玻璃对糖尿病难愈创面修复机理研究

发布时间：2018-05-18 02:41

本文选题：生物活性玻璃 + 创面修复　；参考：《华南理工大学》2014年博士论文

【摘要】：通过研制新型的皮肤软组织创面修复材料，帮助患者修复缺损或难愈的皮肤创面组织，更好地恢复人体皮肤软组织功能是医学界和生物医学材料学界多年来一直在探索并试图解决的重要问题。生物活性玻璃45S5是采用高温熔融法制备的一种无机活性材料，其化学组成为(质量份比)：45%SiO2、4.5%Na2O、24.5%CaO和6%P2O5，该生物活性玻璃作为传统具有代表性的一类无机活性材料在皮肤软组织损伤修复领域已经有了一定的应用和相关研究报道，但是一些新型生物活性玻璃如溶胶－凝胶生物活性玻璃(Sol-gelbioactive glass, SGBG)和纳米生物活性玻璃（Nanoscale bioactive glass, NBG）等在这方面的研究比较少，亦少见文献报道。事实上，这些新型生物活性材料较传统生物活性玻璃45S5有着一些更优良的性能，如材料的化学均匀性可达分子级别，并具有纳米介孔结构、巨大的比表面积以及较高的化学活性和吸附特性等。本论文围绕溶胶-凝胶生物活性玻璃调控糖尿病难愈创面评价体系的建立及其修复机理进行研究，以链脲佐菌素STZ诱导的糖尿病大鼠全层皮肤缺损模型来模拟糖尿病难愈创面的修复过程，通过透明膜描记法评估创面愈合率，并分别通过HE染色、免疫组化染色、透射电镜等进一步观察分析成纤维细胞、上皮细胞、血管内皮细胞增殖，以及相关生长因子（VEGF和FGF-2）的表达，与此同时，通过MTT法测定观察生物活性玻璃对人成纤维细胞增殖，系统研究并证实了溶胶-凝胶生物活性玻璃对糖尿病难愈创面的修复作用。具体研究工作如下：首先，通过溶胶-凝胶制备工艺获得溶胶-凝胶生物活性玻璃SGBG（摩尔组成：60%SiO2、36%C和4%P2O5），并采用溶胶-凝胶共沉淀法、结合冷冻干燥技术制备了颗粒尺寸在纳米级的生物活性玻璃NBG（摩尔组成：60%SiO2、36%CaO和4%P2O5），研究了加入分散剂聚乙二醇（PEG-10000）对生物活性玻璃颗粒的分散性能、微观形貌和生物活性的影响。结果表明：没有加入PEG制得的NBG颗粒呈现不规则形态，粒径小于50nm，加有PEG的NBG颗粒形状趋于规则的球形，分散性大大提高，颗粒粒径在40～100nm，而且加入PEG的浓度越高，制备的NBG颗粒粒径越小。通过比较NBG与SGBG在模拟人体体液（Simulated bodyfluid, SBF）中的表面矿化研究，发现NBG比SGBG有更高的生物活性。其次，通过体外细胞培养，MTT法评价45S5、SGBG和NBG三种生物活性玻璃材料的细胞增殖活性，研究不同浓度、不同作用时间下生物活性玻璃对人成纤维细胞体外生长增殖的影响。通过酶解法从人包皮中成功提取成纤维细胞，，并通过免疫蛋白组化鉴定其为成纤维细胞，其中HMB45呈阴性，角蛋白呈阴性，S100呈阴性，波形蛋白呈阳性，均符合成纤维细胞的特点。结果，三种生物活性玻璃浸提液均无明显细胞毒性，且在合适的浓度下有利于成纤维细胞的增殖，但三种生物活性玻璃的作用不同，45S5在细胞培养早期有利于成纤维细胞的增殖，而NBG则在后期对细胞的作用更为显著。在糖尿病难愈创面动物模型建立方面，本研究旨在探讨生物活性玻璃对一般皮肤创面和糖尿病皮肤创面的修复作用。以SD大鼠及链脲佐菌素诱导的糖尿病SD大鼠为动物模型，将制备的生物活性玻璃膏剂应用于大鼠背部全层皮肤缺损创面，并进行相应的修复评价和机理探讨。实验中根据生物活性玻璃的种类将创面分为四组，即熔融生物活性玻璃45S5组（45S5）、溶胶-凝胶生物活性玻璃58S组（SGBG-58S）、纳米生物活性玻璃58S组（NBG-58S）及凡士林对照组。和SGBG-58S相比，NBG-58S颗粒是由相对分散的纳米颗粒构成。通过对创面愈合率及创面愈合时间的统计计算发现，生物活性玻璃组能促进创面的快速愈合，且SGBG-58S和NBG-58S这两种生物活性玻璃的愈合效果更佳。组织学分析表明生物活性玻璃能促进成纤维细胞的增殖和肉芽组织的快速形成，并且，免疫组化分析提示与创面修复有关的两种生长因子VEGF和FGF2在生物活性玻璃的刺激下有高效的表达。通过透射电镜观察显示，生物活性玻璃组处理的创面中成纤维细胞更为成熟，含有更多的粗面内质网及其他细胞器，并能在术后7天就已经形成了成熟的血管，SGBG-58S和NBG-58S效果比45S5更为明显。综上所述，生物活性玻璃，尤其是SGBG-58S和NBG-58S能促进正常和糖尿病皮肤创面的快速愈合，在创面修复领域中具有广泛的应用前景。此外，研究首次探索性地将45S5生物活性玻璃复合云南白药膏剂应用于上述建立的评价体系，为开发治疗糖尿病创面外用药物提供参考和新的思路。
[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.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.1;R318.08

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