含铜硼酸盐生物玻璃支架与纤维对骨缺损与皮肤缺损修复作用的研究
发布时间：2019-06-03 21:02
【摘要】：第一部分含铜硼酸盐生物玻璃支架对骨缺损修复作用的研究目的:利用合成生物材料缓释促成骨和促成血管无机金属离子(如铜离子)为骨组织再生提供了一个新的思路,也成为了近年来骨组织工程领域研究的热点之一。本实验通过制备不同铜含量的硼酸盐生物玻璃(BG-Cu)支架,研究其在模拟体液(SBF)中的离子释放和羟基磷灰石(HA)转化情况;探索BG-Cu支架体外细胞毒性以及是否增强骨髓间充质干细胞(BMSCs)的成骨和成血管分化能力;尝试通过植入BG-Cu支架促进骨缺损处的成骨和成血管能力,以达到修复大尺寸骨缺损的目的。方法:通过有机泡沫法制备含不同铜浓度(0、0.5、1.0和3.0 wt.%)的BG支架,并对支架进行孔隙率、场发射环境扫描电镜(SEM)、X射线粉末多晶衍射仪(XRD)、力学强度等检测表征其理化性能。通过ICP-AES法测定支架中铜离子的释放曲线。在体外,将BMSCs接种于支架,通过SEM、CCK-8、ALP活性和q RT-PCR等方法研究支架的细胞相容性以及支架对细胞的粘附、增殖和分化的影响。体内将支架植入SD大鼠颅骨缺损区,并通过序列荧光标记、Microfil灌注、Micro-CT、组织学检测等方法对其成骨和成血管的效果进行评价。结果:不同铜含量掺杂之后,BG支架的表面形貌、孔隙率和力学强度没有发生明显改变。在SBF中,BG-Cu支架释放铜离子并发生HA转化。体外实验中,BG-Cu支架对细胞没有明显毒性作用,BMSCs在支架上能够良好粘附及增殖。相比于BG支架,BG-Cu可以显著促进BMSCs的ALP活性以及成骨(RUNX2、BMP-2 and OPN)/成血管(VEGF和b FGF)相关基因的表达。在缺损区植入BG-3Cu支架,结果发现新生骨组织和血管明显高于植入BG支架和空白对照。结论:1.通过有机泡沫法能够得到与人体松质骨微观结构和力学强度相似的Cu-BG支架,具有良好的体外生物活性;在SBF中,BG-Cu支架释放具有成血管效应的铜离子并发生HA转化。2.与BG支架相比,BG-Cu支架可以促进细胞的粘附、增殖以及成骨/成血管分化。3.BG-3Cu支架在体内有效促进缺损区的骨组织和血管的再生和修复。第二部分含铜硼酸盐生物玻璃纤维对皮肤缺损修复作用的研究目的:血管生成(Angiogenesis)是组织创面修复与再生中一个极其重要的过程。铜元素在血管生成的过程中扮演关键角色。本实验通过制备含铜硼酸盐生物玻璃(Cu-BG)纤维,研究其在模拟体液(SBF)中的离子释放和HA转化情况;探索Cu-BG纤维浸提液对细胞的毒性、对人脐静脉内皮细胞(HUVECs)的迁移、成管和VEGF分泌能力以及对成纤维细胞的成血管相关基因表达情况的影响;尝试通过全层皮肤缺损处植入Cu-BG纤维,研究其对皮肤再生和修复作用的相关机制。方法:采用喷吹法制备含不同铜浓度(0、0.5、1.0和3.0 wt.%)的BG纤维,并对其进行相关理化性能表征。通过ICP-AES法测定纤维中铜离子的释放曲线。在体外制备纤维的浸提液,并通过CCK-8研究不同BG纤维浸提液对HUVECs和成纤维细胞的毒性作用。通过transwell、成管活力和ELISA检测不同BG纤维浸提液对HUVECs的迁移、成管和VEGF分泌能力的影响。通过q RT-PCR检测不同BG纤维浸提液对成纤维细胞成血管相关基因表达情况。体内将纤维植入SD大鼠全层皮肤缺损区,并通过大体照片、Microfil灌注(Micro-CT扫描)及组织学检测等方法对成血管和促进皮肤创面愈合的效果进行评价。结果:制备的玻璃纤维的外观和质感如棉絮。在不同铜含量掺杂之后,BG纤维的表面形貌无明显差异,纤维的直径约为0.4到1.2μm,平均为0.85μm。在SBF中,Cu-BG纤维释放铜离子并发生HA转化。体外实验中,纤维的浸提液对HUVECs和成纤维细胞没有明显毒性作用,Cu-BG纤维浸提液可以显著提高HUVECs迁移、成管和VEGF分泌能力,促进成纤维细胞成血管相关基因 (VEGF、b FGF和PDGF)表达。体内将纤维植入SD大鼠全层皮肤缺损区,证实3Cu-BG纤维显著促进血管新生和加快皮肤创面愈合。结论:1.通过喷吹法能制备外观和质感如棉絮的BG纤维,并且具有良好的体外生物活性;在SBF中,Cu-BG纤维释放具有成血管效应的铜离子并发生HA转化。2.纤维的浸提液对HUVECs和成纤维细胞没有明显毒性作用,此外与BG纤维浸提液相比,Cu-BG纤维浸提液可以提高HUVECs迁移、成管和VEGF分泌能力,促进成纤维细胞成血管相关基因表达。3.3Cu-BG纤维在体内有效促进血管新生和加快皮肤创面愈合。
[Abstract]:The purpose of this study is to provide a new way for the regeneration of bone defect with the slow release of synthetic biomaterial and the formation of inorganic metal ions (such as copper ions). And has become one of the hot spots in the field of bone tissue engineering in recent years. The ion release and the conversion of hydroxyapatite (HA) in the simulated body fluid (SBF) were studied by the preparation of a borate bioglass (BG-Cu) stent with different copper content. To explore the in vitro cytotoxicity of BG-Cu stent and to enhance the osteogenic and vascular differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and to promote the osteogenesis and vascular ability of the bone defect by implanting the BG-Cu stent in order to achieve the purpose of repairing large-size bone defects. Methods: Different copper concentrations (0, 0.5, 1.0 and 3.0 wt.) were prepared by organic foam method. The physical and chemical properties of the BG stent were characterized by porosity, field emission, scanning electron microscopy (SEM), X-ray powder polycrystalline diffractometer (XRD) and mechanical strength. The release profile of copper ions in the stent was determined by ICP-AES. In vitro, BMSCs were seeded on the scaffold, and the cell compatibility of the scaffold and the effects of the stent on the cell adhesion, proliferation and differentiation were studied by means of SEM, CCK-8, ALP activity and q RT-PCR. In vivo, the stent was implanted into the skull defect area of SD rat, and the effects of bone and blood vessel were evaluated by sequential fluorescent labeling, Microfil perfusion, Micro-CT, and histological examination. Results: After different copper content, the surface morphology, porosity and mechanical strength of BG stent did not change significantly. In the SBF, the BG-Cu stent releases copper ions and undergoes HA conversion. In vitro, the BG-Cu stent has no obvious toxic effect on the cells, and the BMSCs can adhere well and proliferate on the scaffold. BG-Cu significantly promoted the ALP activity of BMSCs as well as the expression of bone formation (RUNX2, BMP-2 and OPN)/ vascular (VEGF and b FGF)-related genes as compared to the BG stent. The BG-3Cu stent was implanted in the defect area, and the results showed that the new bone tissue and the blood vessel were significantly higher than that of the implanted BG stent and the blank control. Conclusion:1. The Cu-BG stent with similar microstructure and mechanical strength of the human cancellous bone can be obtained by the organic foam method, and has good in-vitro biological activity; in the SBF, the BG-Cu stent releases the copper ions with the blood vessel effect and the HA conversion occurs. The BG-3Cu stent can promote the regeneration and repair of bone tissue and blood vessels in the defect area in vivo. The second part of the study on the repair of skin defect with the biological glass fiber of copper-containing borate: Angiogenesis is a very important process in the repair and regeneration of tissue wound. The copper element plays a key role in the process of angiogenesis. In this experiment, the ion release and HA conversion in the simulated body fluid (SBF) were studied by the preparation of a copper-containing borate bioglass (Cu-BG) fiber, and the toxicity of the Cu-BG fiber extract to the cells was explored, and the migration of human umbilical vein endothelial cells (HUVECs) was studied. The effect of the ability of the tube-forming and VEGF secretion and the expression of the fibroblast-related genes in the fibroblasts was studied. The mechanism of the regeneration and repair of the skin was studied by implanting Cu-BG in the skin defect of the whole layer. Methods: The concentration of different copper (0, 0.5, 1.0 and 3.0 wt.) was prepared by a blowing method. %) BG fibers and related physical and chemical properties. The release curve of copper ions in the fiber was determined by ICP-AES. The extract of fibers was prepared in vitro and the toxicity of different BG fiber extracts to HUVECs and fibroblasts was studied by CCK-8. The effects of different BG fiber extracts on the migration of HUVECs, tube-forming and VEGF secretion were measured by transwell, tube-forming activity and ELISA. The expression of vascular-related genes in fibroblasts was detected by q-RT-PCR. In vivo, the fibers were implanted into the whole-layer skin defect area of SD rats, and the effects of blood vessel and skin wound healing were evaluated by general photo, Microfil perfusion (Micro-CT scan) and histological examination. As a result, the appearance and texture of the prepared glass fiber are such as cotton wool. After doping with different copper content, the surface morphology of the BG fiber was not significantly different, the diameter of the fiber was about 0.4 to 1.2. m u.m, and the average was 0.85. m u.m. In the SBF, the Cu-BG fiber releases the copper ions and the HA conversion occurs. In in vitro experiments, the extract of the fiber did not have a significant toxic effect on the HUVECs and the fibroblasts, and the Cu-BG fiber extract can remarkably improve the migration of the HUVECs, the growth of the tube and the VEGF, and promote the expression of the fibroblast-related genes (VEGF, b FGF and PDGF). In vivo, the fiber was implanted into the whole-layer skin defect area of SD rats, and it was confirmed that the 3Cu-BG fiber significantly promoted the angiogenesis and accelerated the healing of the skin wound. Conclusion:1. In the SBF, the Cu-BG fiber releases copper ions with a blood vessel effect and has an HA conversion. The extract of the fiber did not have a significant toxic effect on the HUVECs and fibroblasts, and in addition to the BG fiber extract, the Cu-BG fiber extract can improve the secretion of the HUVECs, the tube-forming and the VEGF, 3.3Cu-BG fiber is effective in promoting angiogenesis and accelerating skin wound healing in vivo.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08;R68
[Abstract]:The purpose of this study is to provide a new way for the regeneration of bone defect with the slow release of synthetic biomaterial and the formation of inorganic metal ions (such as copper ions). And has become one of the hot spots in the field of bone tissue engineering in recent years. The ion release and the conversion of hydroxyapatite (HA) in the simulated body fluid (SBF) were studied by the preparation of a borate bioglass (BG-Cu) stent with different copper content. To explore the in vitro cytotoxicity of BG-Cu stent and to enhance the osteogenic and vascular differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and to promote the osteogenesis and vascular ability of the bone defect by implanting the BG-Cu stent in order to achieve the purpose of repairing large-size bone defects. Methods: Different copper concentrations (0, 0.5, 1.0 and 3.0 wt.) were prepared by organic foam method. The physical and chemical properties of the BG stent were characterized by porosity, field emission, scanning electron microscopy (SEM), X-ray powder polycrystalline diffractometer (XRD) and mechanical strength. The release profile of copper ions in the stent was determined by ICP-AES. In vitro, BMSCs were seeded on the scaffold, and the cell compatibility of the scaffold and the effects of the stent on the cell adhesion, proliferation and differentiation were studied by means of SEM, CCK-8, ALP activity and q RT-PCR. In vivo, the stent was implanted into the skull defect area of SD rat, and the effects of bone and blood vessel were evaluated by sequential fluorescent labeling, Microfil perfusion, Micro-CT, and histological examination. Results: After different copper content, the surface morphology, porosity and mechanical strength of BG stent did not change significantly. In the SBF, the BG-Cu stent releases copper ions and undergoes HA conversion. In vitro, the BG-Cu stent has no obvious toxic effect on the cells, and the BMSCs can adhere well and proliferate on the scaffold. BG-Cu significantly promoted the ALP activity of BMSCs as well as the expression of bone formation (RUNX2, BMP-2 and OPN)/ vascular (VEGF and b FGF)-related genes as compared to the BG stent. The BG-3Cu stent was implanted in the defect area, and the results showed that the new bone tissue and the blood vessel were significantly higher than that of the implanted BG stent and the blank control. Conclusion:1. The Cu-BG stent with similar microstructure and mechanical strength of the human cancellous bone can be obtained by the organic foam method, and has good in-vitro biological activity; in the SBF, the BG-Cu stent releases the copper ions with the blood vessel effect and the HA conversion occurs. The BG-3Cu stent can promote the regeneration and repair of bone tissue and blood vessels in the defect area in vivo. The second part of the study on the repair of skin defect with the biological glass fiber of copper-containing borate: Angiogenesis is a very important process in the repair and regeneration of tissue wound. The copper element plays a key role in the process of angiogenesis. In this experiment, the ion release and HA conversion in the simulated body fluid (SBF) were studied by the preparation of a copper-containing borate bioglass (Cu-BG) fiber, and the toxicity of the Cu-BG fiber extract to the cells was explored, and the migration of human umbilical vein endothelial cells (HUVECs) was studied. The effect of the ability of the tube-forming and VEGF secretion and the expression of the fibroblast-related genes in the fibroblasts was studied. The mechanism of the regeneration and repair of the skin was studied by implanting Cu-BG in the skin defect of the whole layer. Methods: The concentration of different copper (0, 0.5, 1.0 and 3.0 wt.) was prepared by a blowing method. %) BG fibers and related physical and chemical properties. The release curve of copper ions in the fiber was determined by ICP-AES. The extract of fibers was prepared in vitro and the toxicity of different BG fiber extracts to HUVECs and fibroblasts was studied by CCK-8. The effects of different BG fiber extracts on the migration of HUVECs, tube-forming and VEGF secretion were measured by transwell, tube-forming activity and ELISA. The expression of vascular-related genes in fibroblasts was detected by q-RT-PCR. In vivo, the fibers were implanted into the whole-layer skin defect area of SD rats, and the effects of blood vessel and skin wound healing were evaluated by general photo, Microfil perfusion (Micro-CT scan) and histological examination. As a result, the appearance and texture of the prepared glass fiber are such as cotton wool. After doping with different copper content, the surface morphology of the BG fiber was not significantly different, the diameter of the fiber was about 0.4 to 1.2. m u.m, and the average was 0.85. m u.m. In the SBF, the Cu-BG fiber releases the copper ions and the HA conversion occurs. In in vitro experiments, the extract of the fiber did not have a significant toxic effect on the HUVECs and the fibroblasts, and the Cu-BG fiber extract can remarkably improve the migration of the HUVECs, the growth of the tube and the VEGF, and promote the expression of the fibroblast-related genes (VEGF, b FGF and PDGF). In vivo, the fiber was implanted into the whole-layer skin defect area of SD rats, and it was confirmed that the 3Cu-BG fiber significantly promoted the angiogenesis and accelerated the healing of the skin wound. Conclusion:1. In the SBF, the Cu-BG fiber releases copper ions with a blood vessel effect and has an HA conversion. The extract of the fiber did not have a significant toxic effect on the HUVECs and fibroblasts, and in addition to the BG fiber extract, the Cu-BG fiber extract can improve the secretion of the HUVECs, the tube-forming and the VEGF, 3.3Cu-BG fiber is effective in promoting angiogenesis and accelerating skin wound healing in vivo.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08;R68
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