含銅硼酸鹽生物玻璃支架與纖維對骨缺損與皮膚缺損修復作用的研究
發(fā)布時間：2019-06-03 21:02
【摘要】：第一部分含銅硼酸鹽生物玻璃支架對骨缺損修復作用的研究目的:利用合成生物材料緩釋促成骨和促成血管無機金屬離子(如銅離子)為骨組織再生提供了一個新的思路,也成為了近年來骨組織工程領域研究的熱點之一。本實驗通過制備不同銅含量的硼酸鹽生物玻璃(BG-Cu)支架,研究其在模擬體液(SBF)中的離子釋放和羥基磷灰石(HA)轉化情況;探索BG-Cu支架體外細胞毒性以及是否增強骨髓間充質干細胞(BMSCs)的成骨和成血管分化能力;嘗試通過植入BG-Cu支架促進骨缺損處的成骨和成血管能力,以達到修復大尺寸骨缺損的目的。方法:通過有機泡沫法制備含不同銅濃度(0、0.5、1.0和3.0 wt.%)的BG支架,并對支架進行孔隙率、場發(fā)射環(huán)境掃描電鏡(SEM)、X射線粉末多晶衍射儀(XRD)、力學強度等檢測表征其理化性能。通過ICP-AES法測定支架中銅離子的釋放曲線。在體外,將BMSCs接種于支架,通過SEM、CCK-8、ALP活性和q RT-PCR等方法研究支架的細胞相容性以及支架對細胞的粘附、增殖和分化的影響。體內(nèi)將支架植入SD大鼠顱骨缺損區(qū),并通過序列熒光標記、Microfil灌注、Micro-CT、組織學檢測等方法對其成骨和成血管的效果進行評價。結果:不同銅含量摻雜之后,BG支架的表面形貌、孔隙率和力學強度沒有發(fā)生明顯改變。在SBF中,BG-Cu支架釋放銅離子并發(fā)生HA轉化。體外實驗中,BG-Cu支架對細胞沒有明顯毒性作用,BMSCs在支架上能夠良好粘附及增殖。相比于BG支架,BG-Cu可以顯著促進BMSCs的ALP活性以及成骨(RUNX2、BMP-2 and OPN)/成血管(VEGF和b FGF)相關基因的表達。在缺損區(qū)植入BG-3Cu支架,結果發(fā)現(xiàn)新生骨組織和血管明顯高于植入BG支架和空白對照。結論:1.通過有機泡沫法能夠得到與人體松質骨微觀結構和力學強度相似的Cu-BG支架,具有良好的體外生物活性;在SBF中,BG-Cu支架釋放具有成血管效應的銅離子并發(fā)生HA轉化。2.與BG支架相比,BG-Cu支架可以促進細胞的粘附、增殖以及成骨/成血管分化。3.BG-3Cu支架在體內(nèi)有效促進缺損區(qū)的骨組織和血管的再生和修復。第二部分含銅硼酸鹽生物玻璃纖維對皮膚缺損修復作用的研究目的:血管生成(Angiogenesis)是組織創(chuàng)面修復與再生中一個極其重要的過程。銅元素在血管生成的過程中扮演關鍵角色。本實驗通過制備含銅硼酸鹽生物玻璃(Cu-BG)纖維,研究其在模擬體液(SBF)中的離子釋放和HA轉化情況;探索Cu-BG纖維浸提液對細胞的毒性、對人臍靜脈內(nèi)皮細胞(HUVECs)的遷移、成管和VEGF分泌能力以及對成纖維細胞的成血管相關基因表達情況的影響;嘗試通過全層皮膚缺損處植入Cu-BG纖維,研究其對皮膚再生和修復作用的相關機制。方法:采用噴吹法制備含不同銅濃度(0、0.5、1.0和3.0 wt.%)的BG纖維,并對其進行相關理化性能表征。通過ICP-AES法測定纖維中銅離子的釋放曲線。在體外制備纖維的浸提液,并通過CCK-8研究不同BG纖維浸提液對HUVECs和成纖維細胞的毒性作用。通過transwell、成管活力和ELISA檢測不同BG纖維浸提液對HUVECs的遷移、成管和VEGF分泌能力的影響。通過q RT-PCR檢測不同BG纖維浸提液對成纖維細胞成血管相關基因表達情況。體內(nèi)將纖維植入SD大鼠全層皮膚缺損區(qū),并通過大體照片、Microfil灌注(Micro-CT掃描)及組織學檢測等方法對成血管和促進皮膚創(chuàng)面愈合的效果進行評價。結果:制備的玻璃纖維的外觀和質感如棉絮。在不同銅含量摻雜之后,BG纖維的表面形貌無明顯差異,纖維的直徑約為0.4到1.2μm,平均為0.85μm。在SBF中,Cu-BG纖維釋放銅離子并發(fā)生HA轉化。體外實驗中,纖維的浸提液對HUVECs和成纖維細胞沒有明顯毒性作用,Cu-BG纖維浸提液可以顯著提高HUVECs遷移、成管和VEGF分泌能力,促進成纖維細胞成血管相關基因 (VEGF、b FGF和PDGF)表達。體內(nèi)將纖維植入SD大鼠全層皮膚缺損區(qū),證實3Cu-BG纖維顯著促進血管新生和加快皮膚創(chuàng)面愈合。結論:1.通過噴吹法能制備外觀和質感如棉絮的BG纖維,并且具有良好的體外生物活性;在SBF中,Cu-BG纖維釋放具有成血管效應的銅離子并發(fā)生HA轉化。2.纖維的浸提液對HUVECs和成纖維細胞沒有明顯毒性作用,此外與BG纖維浸提液相比,Cu-BG纖維浸提液可以提高HUVECs遷移、成管和VEGF分泌能力,促進成纖維細胞成血管相關基因表達。3.3Cu-BG纖維在體內(nèi)有效促進血管新生和加快皮膚創(chuàng)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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R318.08;R68
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