仿生纤维内硅化胶原支架材料调控单核细胞促骨原位再生的研究

发布时间：2018-07-05 17:11

  本文选题：骨再生 + 血管化　； 参考：《第四军医大学》2017年博士论文

【摘要】：近年来,用于骨缺损修复的人工替代材料的研究取得了很大的进展,大量新型的修复材料已在临床上取得了广泛的应用。然而不同的材料由于其理化性能、机械性能以及生物应用性能方面的不同,修复效果差异很大。生物材料在植入体内后,宿主的免疫应答反应是影响其最终应用效果的最重要的因素之一。在早期的研究中,组织修复的研究策略倾向于抑制宿主的免疫应答来提高移植的成功率。而目前普遍接受的观点认为,宿主的免疫应答能够对组织再生修复起到积极的免疫调控作用,从而促进缺损修复以及组织再生。单核细胞是宿主免疫应答调控中的重要细胞,同时也是骨改建以及骨缺损修复进程中不可或缺的关键细胞之一,单核细胞与植入体内的生物材料之间的相互作用也是影响生物材料移植成功的关键因素之一。仿生纤维内硅化胶原支架(SCS),以胶原纤维内部无定形水合二氧化硅有序沉积为特征,是一种具有良好理化性能和机械性能的生物材料。在前期的研究中,这种支架材料,在体外实验中表现出了良好的促进骨再生修复的潜力。然而其在体内应用于骨缺损的修复效果尚不明确,同时宿主免疫系统对其产生的反应目前仍未得到相关的评估。在本研究中,我们对仿生纤维内硅化胶原支架材料在骨缺损修复中的应用进行了全面的生物学评估,验证了其在动物模型上修复骨缺损的效果,并对其与宿主的免疫调控之间的相互作用进行了相关的探索,验证了该支架材料与单核细胞之间的作用,以及通过调控单核细胞来促进组织血管化、种子细胞募集,以及促进骨再生的作用。1.研究思路在第一部分实验中,我们采用胶原纤维模板诱导纳米液相矿物质前体(无定形硅酸)纤维内定向沉积的技术,构建出了仿生纤维内硅化胶原支架,并使用显微CT(Micro-CT)技术和透射电子显微镜(TEM)技术对所构建出的纤维内硅化胶原支架进行了形貌观察;随后对其在体液环境中的硅酸的缓释水平进行了测定。通过小鼠体内异位植入模型对仿生纤维内硅化胶原材料的生物相容性进行了全面的评价:通过淋巴细胞二次刺激增殖实验评价硅化胶原的免疫原性;采用流式细胞技术(Flow Cytometry)评价异位植入的材料对循环淋巴细胞水平以及活性的影响;通过ELISA实验观察异位植入的材料对循环炎症因子水平的影响;通过植入部位组织学切片的HE染色来观察原位炎症细胞的浸润情况,为其进一步用于骨缺损修复提供了实验基础。在第二部分实验中,我们应用纤维内硅化胶原支架修复了小鼠的颅骨缺损,并对其修复效果进行了评估。在植入后即刻、1个月、3个月时采用Micro-CT技术对骨缺损的修复情况、新骨形成量以及骨密度进行了测定;采用贯序荧光标记技术对修复后3个月时的骨改建活性进行了评估;使用修复后3个月时的组织标本制作硬组织切片,采用Van Gieson染色(VG staining)和von Kossa银染(VK sliver staining)技术对切片分别进行染色,在组织学水平观察原位骨再生的情况;在修复后3个月时,采用血管造影技术对缺损原位的血管再生水平进行评估;在修复后1个月时进行组织学切片,通过免疫荧光技术(Immunofluorescence)、免疫组织化学技术(Immunohistochemistry)对缺损部位相关的单核巨噬细胞,以及骨修复过程中的相关细胞因子表达水平进行评估,并采用TRAP染色(tartrate-resistant acid phosphatase staining)对骨改建活性进行评估。在第三部分实验中,我们通过体外实验进一步对纤维内硅化胶原支架材料对单核细胞的调控作用进行了深入研究。通过细胞增殖实验、凋亡实验、细胞内活性氧水平测定实验来检测支架材料对单核细胞增殖、凋亡、活性氧水平的影响;采用TRAP细胞染色技术来检测支架材料对单核细胞分化的影响;通过细胞免疫荧光染色技术、q RT-PCR技术和Western Blot技术检测支架材料对单核细胞相关细胞因子分泌水平的影响;通过Transwell细胞迁移实验检测支架材料与单核细胞相互作用后对骨髓间充质干细胞(BMSCs)和血管内皮前体细胞(EPCs)迁移的影响;通过Matrigel血管形成实验检测对EPCs成血管能力的影响;通过添加中和抗体来检验支架材料调控单核细胞影响细胞迁移与血管生成的关键细胞因子。在第四部分实验中,我们采用仿生硅化胶原支架材料修复SD大鼠的股骨缺损,进一步对支架材料通过影响单核细胞调控骨再生的信号通路进行了研究。通过显微CT技术、免疫组织化学技术以及Van Geison染色技术对支架材料修复后1个月时,缺损部位的成骨水平,以及血管生成水平进行了评估;采用免疫荧光双标技术对缺损原位的单核细胞以及相关细胞因子的表达水平进行评估;并进一步在体外实验中,采用Western Blot技术对单核细胞相关的分子信号通路进行了检测;采用Western Blot技术、TRAP细胞染色技术以及Transwell细胞迁移实验、Matrigel血管形成实验验证相关信号通路对单核细胞分化和分泌的影响;最后通过体内应用通路抑制剂,来观察相关信号通路阻断后,股骨缺损修复水平的改变。2.实验结果第一部分仿生纤维内硅化胶原支架的制备与表征1)透射电镜观察下,仿生纤维内硅化胶原支架材料的纤维内部间隙中可见无定形的二氧化硅有序沉积,从而形成明显的带状结构。Micro-CT结果可见仿生纤维内硅化胶原支架为具有多孔隙特征的三维网状纤维支架结构。在模拟体液环境中,材料能够稳定的缓释硅酸,在1-10天时缓释速度较快,10-30天时缓释速度趋于平稳,平均每100mg硅化胶原在10m L PBS中暴露30天后,溶液中的硅酸浓度能达到约1.2mmol/L的水平。2)仿生硅化胶原支架材料的生物相容性评价结果显示:材料具有较低的免疫原性,对淋巴细胞的二次刺激并没有引起明显的增殖反应(p0.05);同时,材料的异位植入对循环淋巴细胞的数量、活性没有影响(p0.05),循环中的炎症因子浓度也维持在正常水平(p0.05);原位组织学切片的HE染色结果显示,硅化胶原支架在植入后7天、14天发生了明显的吸收,材料周围无明显炎症细胞浸润,证明了仿生硅化胶原支架材料具有良好的生物相容性,可以进一步安全地应用于体内骨缺损修复。第二部分仿生纤维内硅化胶原支架修复小鼠颅骨缺损的体内实验研究3)Micro-CT结果显示仿生纤维内硅化胶原支架材料能够明显的提升小鼠颅骨缺损的修复水平(p0.05)。和对照组相比,在术后三个月时,能够观察到形成了更多的新骨,同时其修复后的骨组织的改建活动更加活跃。4)与对照组相比,在术后三个月硅化胶原支架材料修复后的颅骨缺损区域形成了更多的血管组织,其血管长度、厚度、连接性均明显高于对照组(p0.05)。5)在术后一个月时,硅化胶原支架材料修复的缺损区域出现了更多的CD31+Endomucin+双阳性的血管,并且有更多的PDGF-BB表达;缺损区域内TRAP阳性的单核细胞数量明显增多,同时单核细胞表达更多的趋化因子SDF-1以及转化生长因子TGF-β1;在硅化胶原支架材料修复后,缺损区域有更多的骨髓间充质干细胞标志物Nestin以及血管内皮生长因子VEGF的表达,提示募集到了更多的修复种子细胞(p均0.05)。第三部分仿生纤维内硅化胶原支架调控单核细胞影响成骨成血管的实验研究6)硅化胶原支架材料对单核细胞的增殖、凋亡和细胞内活性氧水平均没有影响(p0.05);材料自身所缓释的浸提液对BMSCs和EPCs的迁移也没有影响(p0.05)。7)硅化胶原支架材料能够促进单核细胞向TRAP阳性的单核细胞分化(p0.05),该类型的单核细胞能够在转录水平和翻译水平表达更多的相关细胞因子(SDF-1,TGF-β,VEGF,PDGF-BB)(p均0.05)。8)使用硅化胶原支架材料对单核细胞进行刺激后的条件培养基能够明显的促进BMSCs和EPCs的迁移(p0.05),及EPCs的成血管能力(p0.05);中和抗体实验证明条件培养基中的SDF-1,TGF-β和PDGF-BB是促进迁移的关键细胞因子,而TGF-β,VEGF,PDGF-BB则是促进EPCs成血管的关键细胞因子。第四部分仿生纤维内硅化胶原支架调控单核细胞的信号转导机制研究9)硅化胶原支架材料对不同类型的骨缺损修复均有良好的应用,在大鼠股骨部分缺损的动物模型中,材料能够明显的促进血管与骨的再生(p0.05),缺损局部形成了更多的血管结构和更多的小梁骨结构(p0.05),表明其具有明显的促进愈合和骨重建的作用;免疫荧光双标的结果显示,缺损区域有更多的CD31+Emcn+双阳性的血管生成(p0.05),同时表达更多的TRAP+的单核细胞(p0.05),单核细胞的PDGF-BB分泌也明显增多(p0.05)。10)在体外实验中,硅化胶原支架材料刺激后,单核细胞的P38和ERK1/2被激活;使用通路抑制剂分别抑制P38和ERK1/2后,发现单核细胞向TRAP阳性细胞的分化和相关细胞因子(SDF-1,TGF-β,VEGF,PDGF-BB)的分泌水平明显下降(p0.05),其与支架材料共培养后的条件培养基促进细胞迁移和EPCs成血管的能力也明显下降(p0.05);进一步的体内实验研究表明,在P38抑制后,大鼠股骨缺损局部形成的CD31+Emcn+双阳性的血管明显减少(p0.05),TRAP阳性单核细胞的数目以及单核细胞表达PDGF-BB的水平也明显降低(p均0.05),表明硅化胶原支架材料能够通过激活P38信号通路来进一步促进单核细胞的分化和分泌能力,从而影响骨缺损修复的进程。3.结论1)仿生纤维内硅化胶原支架材料表现出了低免疫原性的特征,能够使宿主的免疫炎症反应控制在较低的水平范围内,并且不会引起局部组织的炎症细胞浸润以及循环炎症细胞和炎症因子的上升,具有良好的生物相容性,在生物应用方面具有明显的优势。2)纤维内仿生硅化胶原支架能够应用于不同类型的骨缺损修复(小鼠颅骨缺损,大鼠股骨部分缺损),可以促进缺损局部的血管再生和骨重建,具有良好的骨缺损修复效果。3)在骨缺损的修复早期,仿生纤维内硅化胶原支架材料能够通过对单核细胞P38信号通路的激活,促进单核细胞向TRAP阳性单核细胞分化,分泌更多的相关细胞因子(SDF-1,TGF-β,VEGF,PDGF-BB);一方面能够促进局部的血管生成,尤其是形成更多的CD31+Emcn+双阳性血管,更好的促进成骨成血管的协同作用(Coupling),另一方面能够募集更多的宿主种子细胞(BMSCs,EPCs)到缺损区域,从而进一步促进局部的血管生成和骨再生。综上所述,仿生纤维内硅化胶原支架材料具有良好的生物相容性,在动物骨缺损模型的修复中表现出了良好的修复效果,能够明显促进缺损局部的血管化和骨生成;仿生纤维内硅化胶原支架材料能够通过对单核细胞的调控,在修复早期促进单核细胞的分化和分泌功能,从而进一步促进局部的血管化和宿主种子细胞的募集,与传统的骨修复材料需要额外加载种子细胞或者细胞因子相比,在应用方面具有便捷性和明显的优势,在骨组织的再生修复的应用中具有明显的转化潜力和应用前景。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R318.08;R68
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本文编号：2101039