含銅硼酸鹽生物玻璃支架與纖維對(duì)骨缺損與皮膚缺損修復(fù)作用的研究
發(fā)布時(shí)間：2019-06-03 21:02
【摘要】：第一部分含銅硼酸鹽生物玻璃支架對(duì)骨缺損修復(fù)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(jī)金屬離子(如銅離子)為骨組織再生提供了一個(gè)新的思路,也成為了近年來骨組織工程領(lǐng)域研究的熱點(diǎn)之一。本實(shí)驗(yàn)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(zhuǎn)化情況;探索BG-Cu支架體外細(xì)胞毒性以及是否增強(qiáng)骨髓間充質(zhì)干細(xì)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(jìn)骨缺損處的成骨和成血管能力,以達(dá)到修復(fù)大尺寸骨缺損的目的。方法:通過有機(jī)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(duì)支架進(jìn)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(xué)強(qiáng)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(xì)胞相容性以及支架對(duì)細(xì)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(biāo)記、Microfil灌注、Micro-CT、組織學(xué)檢測等方法對(duì)其成骨和成血管的效果進(jìn)行評(píng)價(jià)。結(jié)果:不同銅含量摻雜之后,BG支架的表面形貌、孔隙率和力學(xué)強(qiáng)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(zhuǎn)化。體外實(shí)驗(yàn)中,BG-Cu支架對(duì)細(xì)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(jìn)BMSCs的ALP活性以及成骨(RUNX2、BMP-2 and OPN)/成血管(VEGF和b FGF)相關(guān)基因的表達(dá)。在缺損區(qū)植入BG-3Cu支架,結(jié)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(duì)照。結(jié)論:1.通過有機(jī)泡沫法能夠得到與人體松質(zhì)骨微觀結(jié)構(gòu)和力學(xué)強(qiáng)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(yīng)的銅離子并發(fā)生HA轉(zhuǎn)化。2.與BG支架相比,BG-Cu支架可以促進(jìn)細(xì)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(jìn)缺損區(qū)的骨組織和血管的再生和修復(fù)。第二部分含銅硼酸鹽生物玻璃纖維對(duì)皮膚缺損修復(fù)作用的研究目的:血管生成(Angiogenesis)是組織創(chuàng)面修復(fù)與再生中一個(gè)極其重要的過程。銅元素在血管生成的過程中扮演關(guān)鍵角色。本實(shí)驗(yàn)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(zhuǎn)化情況;探索Cu-BG纖維浸提液對(duì)細(xì)胞的毒性、對(duì)人臍靜脈內(nèi)皮細(xì)胞(HUVECs)的遷移、成管和VEGF分泌能力以及對(duì)成纖維細(xì)胞的成血管相關(guān)基因表達(dá)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(duì)皮膚再生和修復(fù)作用的相關(guān)機(jī)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(duì)其進(jìn)行相關(guān)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(duì)HUVECs和成纖維細(xì)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(duì)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(duì)成纖維細(xì)胞成血管相關(guān)基因表達(dá)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(xué)檢測等方法對(duì)成血管和促進(jìn)皮膚創(chuàng)面愈合的效果進(jìn)行評(píng)價(jià)。結(jié)果:制備的玻璃纖維的外觀和質(zhì)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(zhuǎn)化。體外實(shí)驗(yàn)中,纖維的浸提液對(duì)HUVECs和成纖維細(xì)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(jìn)成纖維細(xì)胞成血管相關(guān)基因 (VEGF、b FGF和PDGF)表達(dá)。體內(nèi)將纖維植入SD大鼠全層皮膚缺損區(qū),證實(shí)3Cu-BG纖維顯著促進(jìn)血管新生和加快皮膚創(chuàng)面愈合。結(jié)論:1.通過噴吹法能制備外觀和質(zhì)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(yīng)的銅離子并發(fā)生HA轉(zhuǎn)化。2.纖維的浸提液對(duì)HUVECs和成纖維細(xì)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(jìn)成纖維細(xì)胞成血管相關(guān)基因表達(dá)。3.3Cu-BG纖維在體內(nèi)有效促進(jìn)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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R318.08;R68
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