本文選題：骨再生 + 血管化��； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：近年來,用于骨缺損修復(fù)的人工替代材料的研究取得了很大的進(jìn)展,大量新型的修復(fù)材料已在臨床上取得了廣泛的應(yīng)用。然而不同的材料由于其理化性能、機(jī)械性能以及生物應(yīng)用性能方面的不同,修復(fù)效果差異很大。生物材料在植入體內(nèi)后,宿主的免疫應(yīng)答反應(yīng)是影響其最終應(yīng)用效果的最重要的因素之一。在早期的研究中,組織修復(fù)的研究策略傾向于抑制宿主的免疫應(yīng)答來提高移植的成功率。而目前普遍接受的觀點認(rèn)為,宿主的免疫應(yīng)答能夠?qū)M織再生修復(fù)起到積極的免疫調(diào)控作用,從而促進(jìn)缺損修復(fù)以及組織再生。單核細(xì)胞是宿主免疫應(yīng)答調(diào)控中的重要細(xì)胞,同時也是骨改建以及骨缺損修復(fù)進(jìn)程中不可或缺的關(guān)鍵細(xì)胞之一,單核細(xì)胞與植入體內(nèi)的生物材料之間的相互作用也是影響生物材料移植成功的關(guān)鍵因素之一。仿生纖維內(nèi)硅化膠原支架(SCS),以膠原纖維內(nèi)部無定形水合二氧化硅有序沉積為特征,是一種具有良好理化性能和機(jī)械性能的生物材料。在前期的研究中,這種支架材料,在體外實驗中表現(xiàn)出了良好的促進(jìn)骨再生修復(fù)的潛力。然而其在體內(nèi)應(yīng)用于骨缺損的修復(fù)效果尚不明確,同時宿主免疫系統(tǒng)對其產(chǎn)生的反應(yīng)目前仍未得到相關(guān)的評估。在本研究中,我們對仿生纖維內(nèi)硅化膠原支架材料在骨缺損修復(fù)中的應(yīng)用進(jìn)行了全面的生物學(xué)評估,驗證了其在動物模型上修復(fù)骨缺損的效果,并對其與宿主的免疫調(diào)控之間的相互作用進(jìn)行了相關(guān)的探索,驗證了該支架材料與單核細(xì)胞之間的作用,以及通過調(diào)控單核細(xì)胞來促進(jìn)組織血管化、種子細(xì)胞募集,以及促進(jìn)骨再生的作用。1.研究思路在第一部分實驗中,我們采用膠原纖維模板誘導(dǎo)納米液相礦物質(zhì)前體(無定形硅酸)纖維內(nèi)定向沉積的技術(shù),構(gòu)建出了仿生纖維內(nèi)硅化膠原支架,并使用顯微CT(Micro-CT)技術(shù)和透射電子顯微鏡(TEM)技術(shù)對所構(gòu)建出的纖維內(nèi)硅化膠原支架進(jìn)行了形貌觀察;隨后對其在體液環(huán)境中的硅酸的緩釋水平進(jìn)行了測定。通過小鼠體內(nèi)異位植入模型對仿生纖維內(nèi)硅化膠原材料的生物相容性進(jìn)行了全面的評價:通過淋巴細(xì)胞二次刺激增殖實驗評價硅化膠原的免疫原性;采用流式細(xì)胞技術(shù)(Flow Cytometry)評價異位植入的材料對循環(huán)淋巴細(xì)胞水平以及活性的影響;通過ELISA實驗觀察異位植入的材料對循環(huán)炎癥因子水平的影響;通過植入部位組織學(xué)切片的HE染色來觀察原位炎癥細(xì)胞的浸潤情況,為其進(jìn)一步用于骨缺損修復(fù)提供了實驗基礎(chǔ)。在第二部分實驗中,我們應(yīng)用纖維內(nèi)硅化膠原支架修復(fù)了小鼠的顱骨缺損,并對其修復(fù)效果進(jìn)行了評估。在植入后即刻、1個月、3個月時采用Micro-CT技術(shù)對骨缺損的修復(fù)情況、新骨形成量以及骨密度進(jìn)行了測定;采用貫序熒光標(biāo)記技術(shù)對修復(fù)后3個月時的骨改建活性進(jìn)行了評估;使用修復(fù)后3個月時的組織標(biāo)本制作硬組織切片,采用Van Gieson染色(VG staining)和von Kossa銀染(VK sliver staining)技術(shù)對切片分別進(jìn)行染色,在組織學(xué)水平觀察原位骨再生的情況;在修復(fù)后3個月時,采用血管造影技術(shù)對缺損原位的血管再生水平進(jìn)行評估;在修復(fù)后1個月時進(jìn)行組織學(xué)切片,通過免疫熒光技術(shù)(Immunofluorescence)、免疫組織化學(xué)技術(shù)(Immunohistochemistry)對缺損部位相關(guān)的單核巨噬細(xì)胞,以及骨修復(fù)過程中的相關(guān)細(xì)胞因子表達(dá)水平進(jìn)行評估,并采用TRAP染色(tartrate-resistant acid phosphatase staining)對骨改建活性進(jìn)行評估。在第三部分實驗中,我們通過體外實驗進(jìn)一步對纖維內(nèi)硅化膠原支架材料對單核細(xì)胞的調(diào)控作用進(jìn)行了深入研究。通過細(xì)胞增殖實驗、凋亡實驗、細(xì)胞內(nèi)活性氧水平測定實驗來檢測支架材料對單核細(xì)胞增殖、凋亡、活性氧水平的影響;采用TRAP細(xì)胞染色技術(shù)來檢測支架材料對單核細(xì)胞分化的影響;通過細(xì)胞免疫熒光染色技術(shù)、q RT-PCR技術(shù)和Western Blot技術(shù)檢測支架材料對單核細(xì)胞相關(guān)細(xì)胞因子分泌水平的影響;通過Transwell細(xì)胞遷移實驗檢測支架材料與單核細(xì)胞相互作用后對骨髓間充質(zhì)干細(xì)胞(BMSCs)和血管內(nèi)皮前體細(xì)胞(EPCs)遷移的影響;通過Matrigel血管形成實驗檢測對EPCs成血管能力的影響;通過添加中和抗體來檢驗支架材料調(diào)控單核細(xì)胞影響細(xì)胞遷移與血管生成的關(guān)鍵細(xì)胞因子。在第四部分實驗中,我們采用仿生硅化膠原支架材料修復(fù)SD大鼠的股骨缺損,進(jìn)一步對支架材料通過影響單核細(xì)胞調(diào)控骨再生的信號通路進(jìn)行了研究。通過顯微CT技術(shù)、免疫組織化學(xué)技術(shù)以及Van Geison染色技術(shù)對支架材料修復(fù)后1個月時,缺損部位的成骨水平,以及血管生成水平進(jìn)行了評估;采用免疫熒光雙標(biāo)技術(shù)對缺損原位的單核細(xì)胞以及相關(guān)細(xì)胞因子的表達(dá)水平進(jìn)行評估;并進(jìn)一步在體外實驗中,采用Western Blot技術(shù)對單核細(xì)胞相關(guān)的分子信號通路進(jìn)行了檢測;采用Western Blot技術(shù)、TRAP細(xì)胞染色技術(shù)以及Transwell細(xì)胞遷移實驗、Matrigel血管形成實驗驗證相關(guān)信號通路對單核細(xì)胞分化和分泌的影響;最后通過體內(nèi)應(yīng)用通路抑制劑,來觀察相關(guān)信號通路阻斷后,股骨缺損修復(fù)水平的改變。2.實驗結(jié)果第一部分仿生纖維內(nèi)硅化膠原支架的制備與表征1)透射電鏡觀察下,仿生纖維內(nèi)硅化膠原支架材料的纖維內(nèi)部間隙中可見無定形的二氧化硅有序沉積,從而形成明顯的帶狀結(jié)構(gòu)。Micro-CT結(jié)果可見仿生纖維內(nèi)硅化膠原支架為具有多孔隙特征的三維網(wǎng)狀纖維支架結(jié)構(gòu)。在模擬體液環(huán)境中,材料能夠穩(wěn)定的緩釋硅酸,在1-10天時緩釋速度較快,10-30天時緩釋速度趨于平穩(wěn),平均每100mg硅化膠原在10m L PBS中暴露30天后,溶液中的硅酸濃度能達(dá)到約1.2mmol/L的水平。2)仿生硅化膠原支架材料的生物相容性評價結(jié)果顯示:材料具有較低的免疫原性,對淋巴細(xì)胞的二次刺激并沒有引起明顯的增殖反應(yīng)(p0.05);同時,材料的異位植入對循環(huán)淋巴細(xì)胞的數(shù)量、活性沒有影響(p0.05),循環(huán)中的炎癥因子濃度也維持在正常水平(p0.05);原位組織學(xué)切片的HE染色結(jié)果顯示,硅化膠原支架在植入后7天、14天發(fā)生了明顯的吸收,材料周圍無明顯炎癥細(xì)胞浸潤,證明了仿生硅化膠原支架材料具有良好的生物相容性,可以進(jìn)一步安全地應(yīng)用于體內(nèi)骨缺損修復(fù)。第二部分仿生纖維內(nèi)硅化膠原支架修復(fù)小鼠顱骨缺損的體內(nèi)實驗研究3)Micro-CT結(jié)果顯示仿生纖維內(nèi)硅化膠原支架材料能夠明顯的提升小鼠顱骨缺損的修復(fù)水平(p0.05)。和對照組相比,在術(shù)后三個月時,能夠觀察到形成了更多的新骨,同時其修復(fù)后的骨組織的改建活動更加活躍。4)與對照組相比,在術(shù)后三個月硅化膠原支架材料修復(fù)后的顱骨缺損區(qū)域形成了更多的血管組織,其血管長度、厚度、連接性均明顯高于對照組(p0.05)。5)在術(shù)后一個月時,硅化膠原支架材料修復(fù)的缺損區(qū)域出現(xiàn)了更多的CD31+Endomucin+雙陽性的血管,并且有更多的PDGF-BB表達(dá);缺損區(qū)域內(nèi)TRAP陽性的單核細(xì)胞數(shù)量明顯增多,同時單核細(xì)胞表達(dá)更多的趨化因子SDF-1以及轉(zhuǎn)化生長因子TGF-β1;在硅化膠原支架材料修復(fù)后,缺損區(qū)域有更多的骨髓間充質(zhì)干細(xì)胞標(biāo)志物Nestin以及血管內(nèi)皮生長因子VEGF的表達(dá),提示募集到了更多的修復(fù)種子細(xì)胞(p均0.05)。第三部分仿生纖維內(nèi)硅化膠原支架調(diào)控單核細(xì)胞影響成骨成血管的實驗研究6)硅化膠原支架材料對單核細(xì)胞的增殖、凋亡和細(xì)胞內(nèi)活性氧水平均沒有影響(p0.05);材料自身所緩釋的浸提液對BMSCs和EPCs的遷移也沒有影響(p0.05)。7)硅化膠原支架材料能夠促進(jìn)單核細(xì)胞向TRAP陽性的單核細(xì)胞分化(p0.05),該類型的單核細(xì)胞能夠在轉(zhuǎn)錄水平和翻譯水平表達(dá)更多的相關(guān)細(xì)胞因子(SDF-1,TGF-β,VEGF,PDGF-BB)(p均0.05)。8)使用硅化膠原支架材料對單核細(xì)胞進(jìn)行刺激后的條件培養(yǎng)基能夠明顯的促進(jìn)BMSCs和EPCs的遷移(p0.05),及EPCs的成血管能力(p0.05);中和抗體實驗證明條件培養(yǎng)基中的SDF-1,TGF-β和PDGF-BB是促進(jìn)遷移的關(guān)鍵細(xì)胞因子,而TGF-β,VEGF,PDGF-BB則是促進(jìn)EPCs成血管的關(guān)鍵細(xì)胞因子。第四部分仿生纖維內(nèi)硅化膠原支架調(diào)控單核細(xì)胞的信號轉(zhuǎn)導(dǎo)機(jī)制研究9)硅化膠原支架材料對不同類型的骨缺損修復(fù)均有良好的應(yīng)用,在大鼠股骨部分缺損的動物模型中,材料能夠明顯的促進(jìn)血管與骨的再生(p0.05),缺損局部形成了更多的血管結(jié)構(gòu)和更多的小梁骨結(jié)構(gòu)(p0.05),表明其具有明顯的促進(jìn)愈合和骨重建的作用;免疫熒光雙標(biāo)的結(jié)果顯示,缺損區(qū)域有更多的CD31+Emcn+雙陽性的血管生成(p0.05),同時表達(dá)更多的TRAP+的單核細(xì)胞(p0.05),單核細(xì)胞的PDGF-BB分泌也明顯增多(p0.05)。10)在體外實驗中,硅化膠原支架材料刺激后,單核細(xì)胞的P38和ERK1/2被激活;使用通路抑制劑分別抑制P38和ERK1/2后,發(fā)現(xiàn)單核細(xì)胞向TRAP陽性細(xì)胞的分化和相關(guān)細(xì)胞因子(SDF-1,TGF-β,VEGF,PDGF-BB)的分泌水平明顯下降(p0.05),其與支架材料共培養(yǎng)后的條件培養(yǎng)基促進(jìn)細(xì)胞遷移和EPCs成血管的能力也明顯下降(p0.05);進(jìn)一步的體內(nèi)實驗研究表明,在P38抑制后,大鼠股骨缺損局部形成的CD31+Emcn+雙陽性的血管明顯減少(p0.05),TRAP陽性單核細(xì)胞的數(shù)目以及單核細(xì)胞表達(dá)PDGF-BB的水平也明顯降低(p均0.05),表明硅化膠原支架材料能夠通過激活P38信號通路來進(jìn)一步促進(jìn)單核細(xì)胞的分化和分泌能力,從而影響骨缺損修復(fù)的進(jìn)程。3.結(jié)論1)仿生纖維內(nèi)硅化膠原支架材料表現(xiàn)出了低免疫原性的特征,能夠使宿主的免疫炎癥反應(yīng)控制在較低的水平范圍內(nèi),并且不會引起局部組織的炎癥細(xì)胞浸潤以及循環(huán)炎癥細(xì)胞和炎癥因子的上升,具有良好的生物相容性,在生物應(yīng)用方面具有明顯的優(yōu)勢。2)纖維內(nèi)仿生硅化膠原支架能夠應(yīng)用于不同類型的骨缺損修復(fù)(小鼠顱骨缺損,大鼠股骨部分缺損),可以促進(jìn)缺損局部的血管再生和骨重建,具有良好的骨缺損修復(fù)效果。3)在骨缺損的修復(fù)早期,仿生纖維內(nèi)硅化膠原支架材料能夠通過對單核細(xì)胞P38信號通路的激活,促進(jìn)單核細(xì)胞向TRAP陽性單核細(xì)胞分化,分泌更多的相關(guān)細(xì)胞因子(SDF-1,TGF-β,VEGF,PDGF-BB);一方面能夠促進(jìn)局部的血管生成,尤其是形成更多的CD31+Emcn+雙陽性血管,更好的促進(jìn)成骨成血管的協(xié)同作用(Coupling),另一方面能夠募集更多的宿主種子細(xì)胞(BMSCs,EPCs)到缺損區(qū)域,從而進(jìn)一步促進(jìn)局部的血管生成和骨再生。綜上所述,仿生纖維內(nèi)硅化膠原支架材料具有良好的生物相容性,在動物骨缺損模型的修復(fù)中表現(xiàn)出了良好的修復(fù)效果,能夠明顯促進(jìn)缺損局部的血管化和骨生成;仿生纖維內(nèi)硅化膠原支架材料能夠通過對單核細(xì)胞的調(diào)控,在修復(fù)早期促進(jìn)單核細(xì)胞的分化和分泌功能,從而進(jìn)一步促進(jìn)局部的血管化和宿主種子細(xì)胞的募集,與傳統(tǒng)的骨修復(fù)材料需要額外加載種子細(xì)胞或者細(xì)胞因子相比,在應(yīng)用方面具有便捷性和明顯的優(yōu)勢,在骨組織的再生修復(fù)的應(yīng)用中具有明顯的轉(zhuǎn)化潛力和應(yīng)用前景。
[Abstract]:In recent years, great progress has been made in the research of artificial substitute materials for bone defect repair. A large number of new types of restorative materials have been widely used in clinical practice. However, different materials have great differences in repair results because of their physical and chemical properties, mechanical properties and biological application performance. Biological materials are implanted in the implant. The immune response of the host is one of the most important factors affecting the final application of the host. In the early study, the research strategy of tissue repair tends to inhibit the host's immune response to improve the success rate of the transplant. Monocyte is one of the most important cells in the regulation of host immune response, and it is also one of the key cells in the process of bone remodeling and bone defect repair. The interaction between mononuclear cells and biomaterials implanted in the body also affects biomaterials. One of the key factors for the success of transplanting is that the silicified collagen scaffold (SCS) in the bionic fiber is characterized by the orderly deposition of amorphous silica in the collagen fibers. It is a biological material with good physical and chemical properties and mechanical properties. In the earlier study, the scaffold showed a good effect on promoting bone re in vitro. However, the response of the host immune system to the repair of bone defects remains unclear, and the response of the host immune system to it has not yet been evaluated. In this study, we conducted a comprehensive biological assessment of the application of the silicified collagen scaffold materials in the biomimetic fibers in the repair of bone defects. The effect of the repair of bone defect on animal model and the interaction between it and the host immune regulation were explored, and the effect between the scaffold and mononuclear cells was verified, and the role of the mononuclear cells to promote tissue vascularization, seed cell recruitment, and the effect of promoting bone regeneration through the regulation of mononuclear cells, as well as the.1. research idea of promoting bone regeneration. In the first part of the experiment, we used collagen fibrous templates to induce the deposition of nanoscale precursor (amorphous silicic acid) fibers in the fibrous precursor (amorphous silicic acid) fibers. We constructed the silicified collagen scaffold in the bionic fiber, and used the microscopical CT (Micro-CT) and transmission electron microscopy (TEM) technology to build the silicified collagen scaffold in the fibers. The slow-release level of silicic acid in the body fluid environment was measured. The biocompatibility of the silicified collagen in the bionic fiber was evaluated by the model of ectopic implantation in mice. The immunogenicity of silicified collagen was evaluated by the lymphocyte two stimulation proliferation test, and the flow cytometry was used. The effects of ectopic implanted materials on circulating lymphocyte level and activity were evaluated by Flow Cytometry. The effects of ectopic implanted materials on circulating inflammatory factors were observed by ELISA experiments, and the infiltration of cells in situ was observed by HE staining in the tissue section of the implanted site to further repair bone defects. In the second experiment, we used fibrous silicified collagen scaffold to repair the defect of the skull in mice and evaluate its repair effect. The repair of bone defects, the new bone shape and bone density were measured by Micro-CT technique at the moment of 1 months and 3 months after implantation. Bone remodeling activity at 3 months after repair was evaluated by sequence fluorescence labeling technique. Tissue specimens were made with Van Gieson staining (VG staining) and von Kossa silver staining (VK sliver staining) technique to stain the slices respectively after 3 months of repair. The bone regeneration in situ was observed at the histological level. 3 months after repair, angiographic technique was used to evaluate the level of vascular regeneration in situ. Tissue sections were performed at 1 months after repair, immunofluorescence technique (Immunofluorescence), immunohistochemical technique (Immunohistochemistry) for mononuclear macrophages related to the defect site, and bone repair process. The expression level of related cytokines was evaluated and TRAP staining (tartrate-resistant acid phosphatase staining) was used to evaluate the bone remodeling activity. In the third part of the experiment, we further studied the regulation of the silicified collagen scaffold material on mononuclear cells through in vitro experiments. The effect of scaffold material on the proliferation, apoptosis and reactive oxygen level of monocyte was detected by the proliferation test, apoptosis experiment and intracellular reactive oxygen level test. The effect of scaffold material on the differentiation of monocyte was detected by TRAP cell staining technique, and cell immunofluorescence staining technique, Q RT-PCR technique and Western Blot technique detection Branch The effect of the scaffold on the secretory level of monocyte related cytokines, and the effect of the Transwell cell migration test on the migration of bone marrow mesenchymal stem cells (BMSCs) and vascular endothelial progenitor cells (EPCs) after the interaction between the scaffold and mononuclear cells, and the effect of the Matrigel angiogenesis test on the vascular ability of EPCs; In the fourth experiment, we used biomimetic silicified collagen scaffolds to repair the femur defect of SD rats in the fourth part of the experiment, and further on the signaling pathway of the scaffold through the influence of mononuclear cells to regulate bone regeneration. The level of osteogenesis and angiogenesis of the defect site were evaluated by microCT, immunohistochemistry and Van Geison staining technique at 1 months after the repair of the scaffold. The expression of mononuclear cells in situ and the expression of related cytokines were evaluated by double immunofluorescence technique. In vitro, Western Blot technique was used to detect the molecular signaling pathway related to monocyte; Western Blot, TRAP cell staining and Transwell cell migration experiments were used to test the effect of signal pathway on the differentiation and secretion of monocyte by Matrigel angiogenesis. Internal application of pathway inhibitors to observe the changes in the level of repair of femur defect after the blocking of related signal pathways.2. experimental results in part 1: preparation and characterization of silicified collagen scaffolds in bionic fibers 1) under transmission electron microscopy, amorphous silicon dioxide ordered sinks in the internal gaps of silicified collagen scaffolds in biomimetic fibers were observed. In the simulated body fluid environment, the material can stabilize the sustained release silicic acid in the simulated body fluid environment, the slow release rate is faster in 1-10 days, and the slow release rate tends to be stable at 10-30 days, and the average silica gel per 100mg is on average in the simulated body fluid environment. 30 days after exposure to 10m L PBS, the concentration of silicic acid in the solution reached about 1.2mmol/L level.2) the biocompatibility evaluation of the biomimetic silicified collagen scaffold showed that the material had a lower immunogenicity and no obvious proliferation response to the two stimulation of the lymphocyte (P0.05); at the same time, the ectopic implantation of the material was followed by The number of ring lymphocytes, activity did not affect (P0.05), the concentration of inflammatory factors in the circulation was also maintained at normal level (P0.05). In situ histological section HE staining results showed that the silicified collagen scaffold was absorbed on the 7 day after implantation, and there was no obvious infiltration of inflammatory cells around the material, which proved that the biomimetic silicified collagen scaffold was proved. The material has good biocompatibility and can be further used in the repair of bone defect in vivo. In the body of second bionic fibers, silicified collagen scaffold for repairing the skull defect of mice in vivo 3. Micro-CT results showed that the biomimetic fibrous silicified collagen scaffold material could significantly improve the repair level of the skull defect in mice (p0.0 5. Compared with the control group, more new bone was observed at three months after the operation, and the remodeling activity of the bone tissue was more active after the repair. Compared with the control group, more vascular tissue was formed in the area of the skull defect after three months after the repair of the silicified collagen scaffold material, and the length, thickness and connectivity of the blood vessel were more than that of the control group. It was significantly higher than the control group (P0.05).5) at one month after the operation, more CD31+Endomucin+ double positive blood vessels were found in the defect area of the silicified collagen scaffold, and more PDGF-BB was expressed, and the number of TRAP positive monocytes in the defect region was significantly increased, while more chemokine SDF-1 was expressed by mononuclear cells. And transforming growth factor TGF- beta 1; after the repair of silicified collagen scaffold, more bone marrow mesenchymal stem cell marker Nestin and vascular endothelial growth factor VEGF were expressed in the defect region, suggesting that more repair seed cells were raised (P all 0.05). The influence of the third part of the imitated fibrous silicified collagen scaffold on mononuclear cells Experimental study of osteogenic vascularization 6) silicified collagen scaffold material has no effect on the proliferation, apoptosis and intracellular reactive oxygen water (P0.05) of mononuclear cells (P0.05), and the release of the material itself has no effect on the migration of BMSCs and EPCs (P0.05).7) silicified collagen scaffold materials can promote monocyte to TRAP positive mononuclear cells P0.05, the type of mononuclear cells can express more related cytokines (SDF-1, TGF- beta, VEGF, PDGF-BB) (P 0.05).8 at transcriptional and translation levels (P 0.05).8) using silicified collagen scaffolds to stimulate mononuclear cells to stimulate the migration of BMSCs and EPCs (P0.05), and EPCs vascular capacity (P) 0.05) 0.05) the neutralization antibody test showed that SDF-1, TGF- beta and PDGF-BB in the conditioned medium were the key cytokines to promote migration, while TGF- beta, VEGF, PDGF-BB were the key cytokines to promote EPCs angiogenesis. The fourth part of the biomimetic fibrous silicified collagen scaffold regulates the signal transduction mechanism of mononuclear cells (9) silicified collagen scaffold material Different types of bone defect repair have good application. In the animal model of the partial defect of the femur, the material can obviously promote the regeneration of blood vessel and bone (P0.05). The defect forms more vascular structure and more trabecular bone structure (P0.05), indicating that it has a significant effect on promoting healing and bone reconstruction; The results of double labeling showed that there were more CD31+Emcn+ double positive angiogenesis (P0.05), more TRAP+ mononuclear cells (P0.05), and more PDGF-BB secretion of mononuclear cells (P0.05).10). In vitro, after the silicified collagen scaffold was stimulated, the P38 and ERK1/2 of mononuclear cells were activated; the use of the pathway was inhibited. After inhibition of P38 and ERK1/2, the secretion of mononuclear cells to TRAP positive cells and related cytokines (SDF-1, TGF- beta, VEGF, PDGF-BB) decreased significantly (P0.05). The ability to promote cell migration and EPCs angiogenesis after co culture with scaffolds was also significantly decreased (P0.05); further in vivo The results showed that after P38 inhibition, the CD31+Emcn+ double positive blood vessels of the rat femur defect were significantly reduced (P0.05), the number of TRAP positive mononuclear cells and the level of PDGF-BB in monocyte decreased significantly (P 0.05), indicating that the silicified collagen scaffold could further promote the single nucleus by activating the P38 signaling pathway. The differentiation and secretion capacity of cells, which affects the process of bone defect repair,.3. conclusion 1) 1) the silicified collagen scaffold in the bionic fiber shows the characteristics of low immunogenicity, which can control the host's immune response to a lower level, and do not cause inflammatory cell infiltration in the local tissue and circulate inflammatory cells. With the increase of inflammatory factors, it has good biocompatibility and has obvious advantages in biological applications..2) bionic silicon in fibers.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R318.08;R68
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本文編號：2101039