碳纳米材料治疗开放骨折后感染性骨缺损及伤口疤痕的研究

发布时间：2018-05-30 23:15

本文选题：开放性骨折 + 感染性骨缺损　；参考：《第二军医大学》2017年博士论文

【摘要】：随着我国工业及交通的高速发展,交通事故、高处坠落等高能量损伤导致的开放性骨折发病率逐年升高,而开放性骨折后易发生骨感染。针对感染性骨缺损的一系列病理特点,学者们主要将其归纳为以下几个方面1)细菌生物膜形成;2)抗生素在局部难以形成有效浓度;3)局部血运破坏;4)骨质再生困难。所以开发具有抗菌及成骨双重效能的植骨材料对于治疗开放性骨折导致的感染性骨缺损十分重要。另一个影响开放性骨折患者预后的并发症是伤口疤痕。高能量损伤导致的开放性骨折中,局部软组织受损严重,伤口不规则;即使开放性骨折患者的骨组织能在较长的治疗周期内愈合,但是如果遗留明显的疤痕组织,会对患者日常生活造成困扰,限制关节的活动度,降低开放性骨折患者术后的满意度。防止瘢痕形成的关键包括以下几个方面:抑制成纤维细胞过度增殖,抑制胶原的过度沉积,促进伤口部位的纤维组织有序生长。然而现阶段的治疗手段在减少增生性瘢痕形成的效果上尚不尽人意。根据开放性骨折感染及骨缺损的治疗需求,碳纳米材料中的石墨烯作为植骨材料的优越性体现在以下方面:1、改良支架的理化性能,满足骨填充需要;2、生物相容性及其促骨形成性能优良;3、对干细胞增殖和成骨分化具有积极影响;4、材料本身具备一定的抗菌性能;5、具备特殊的结构特点可作为理想的药物载体。另一方面,碳纳米管作为碳纳米材料的另一种存在形式,因其独特的结构和丰富的表面可修饰性,已经广泛运用于生物医学领域。研究已发现有序排列的碳纳米管可诱导多种细胞取向性生长生长。表明取向性的碳纳米管(ACNTs)作为组织工程支架,可控制细胞生长的位置和方向。本研究的目的旨在利用碳纳米材料独特的理化性质,制备成骨性能良好且本身具备一定抗菌性能的三维复合支架,并搭载抗生素,用于开放性骨折后感染及骨缺的治疗;再者,利用碳纳米材料优良的可塑性,制备取向性的碳纳米管薄膜,通过实验研究明确其抑制瘢痕形成的效应,并探究其机理,为碳纳米材料在组织工程中的进一步应用提供新的思路。我们通过自组装的方法,以抗坏血酸为还原剂,合成还原型氧化石墨烯/纳米羟基磷灰石复合支架(RGO-nHA),通过SEM,AFM,TEM观察其微观表征;通过TGA,FTIR等手段表征其理化性质;通过药物体外释放实验测定复合支架缓释万古霉素的性能;选用L929、MC3T3细胞,评估支架对其单克隆形成和凋亡的影响;选用BMSCs细胞,通过细胞计数、荧光染色、SEM及CLSM等方法评估支架对细胞粘附增殖,细胞形态的影响;通过茜素红染色,ALP活性检测等手段评估载药支架的成骨性能;通过平板菌落计数,荧光染色等手段评估支架抑制细菌增殖及生物膜形成的情况;通过液体培养基和固体培养基抑菌圈实验评估支架的抗菌性能及其持久性;通过体外溶血实验,评估支架的血液相容性,并通过SEM观察红细胞形态变化;通过将复合支架植入小鼠皮下,观察其在体内吸收的情况,评估其对小鼠关键脏器的影响;建立新西兰大白兔感染性骨缺损模型,将复合支架分组植入新西兰大白兔模型局部病灶内,通过影像学检测、血生化、以及组织学检测评估感染控制和骨再生情况。另外,我们通过化学气相沉积法制备有序性的碳纳米管阵列,制备成ACNTs薄膜,通过SEM,TEM,SAXRD等方法观察其微观形貌并评估其有序性;通过细胞增殖实验、EdU染色及细胞凋亡实验评估其对细胞增殖的影响及其细胞安全性;通过荧光染色和SEM观察ACNTs诱导细胞骨架重配并定向生长的情况;通过基因芯片分析,PCR、Western-blot等方法研究其抑制胶原沉积、抑制细胞增殖、诱导细胞定向生长的机制;运用新西兰大白兔增生性瘢痕模型,通过大体观察、组织学分析等方法验证ACNTs抑制增生性瘢痕形成的效果。结果表明,所制备的RGO-nHA复合支架具备相互连通的三维多孔结构,掺入nHA后成骨性能也得到增强,可促进细胞长入、促进骨质再生;在此复合支架上负载万古霉素之后,由于与石墨烯的π-π键合,药物可以在初始阶段实现较快速的释放,然后进行缓慢的药物释放,该药物缓释特点与石墨烯的固有的抗菌活性相结合之后可确保快速治疗感染并提供对细菌的持久抑制作用。体内实验也证明该载药系统可有效治疗感染性骨缺损。再者,我们通过化学气相沉积法成功合成了ACNTs,结果表明ACNTs可有效抑制成纤维细胞的过度增殖,引导细胞的取向性生长,抑制胶原沉积,而且体外实验结果证实其无细胞细胞毒性。机制分析表明,ACNTs主要通过改变细胞增殖,细胞骨架、细胞运动性以及胶原分泌相关基因表达而起作用。最后,我们采用兔耳疤痕模型对其活体效应进行了评估,表明ACNTs可有效抑制增生性疤痕的形成。以上研究利用石墨烯、碳纳米管两种碳纳米材料独特的理化及生物特性,创新性地将其引入开放性骨折导致的感染性骨缺损及伤口疤痕的治疗中,通过系列体外及动物活体实验证明碳纳米材料可作为治疗感染性骨缺损和伤口疤痕的理想组织工程支架,这为利用新型的碳纳米材料解决临床难题提供了新的思路。
[Abstract]:With the rapid development of industry and transportation in China, the incidence of open fracture caused by high energy damage, such as traffic accidents and high falling, is increasing year by year, and bone infection occurs easily after open fracture. For a series of pathological features of infectious bone defect, scholars mainly classify it as the following 1) bacterial biofilm formation; 2) It is difficult to form an effective concentration locally; 3) local blood transport damage; 4) bone regeneration is difficult. Therefore, it is important to develop bone grafting materials with dual efficacy of antibacterial and osteogenesis for the treatment of open fracture caused by infectious bone defects. Another complication that affects the prognosis of open fracture is scar scar. High energy damage Guide In the open fracture, the local soft tissue is badly damaged and the wound is irregular. Even if the bone tissue of the open fracture patient is healed within a long period of treatment, if a clear scar tissue is left over, the patient's daily life will be plagued, the activity of the joint is restricted, and the satisfaction of the open fracture patients can be reduced. The key to scar formation includes the following aspects: inhibiting hyperproliferation of fibroblasts, inhibiting excessive deposition of collagen, and promoting the orderly growth of fibrous tissue in the wound site. However, treatment at the present stage is not satisfactory in reducing the effect of hypertrophic scar formation. The advantages of graphene as bone grafting material in carbon nanomaterials are shown in the following aspects: 1, the physical and chemical properties of the modified scaffold meet the needs of bone filling; 2, biocompatibility and bone formation performance are excellent; 3, it has a positive effect on stem cell proliferation and osteogenesis differentiation; 4, the material itself has certain antibacterial properties; 5, with a special knot. The structure characteristics can be used as an ideal drug carrier. On the other hand, as another form of carbon nanomaterials, carbon nanotubes have been widely used in biomedical field because of their unique structure and rich surface modifier. The study has found that ordered arrays of carbon nanotubes can induce a variety of cell oriented growth and growth. The purpose of this study is to make use of the unique physical and chemical properties of carbon nanomaterials to prepare a three-dimensional composite scaffold with good bone properties and have certain antibacterial properties, and to take antibiotics for open fracture infection and bone deficiency, as the purpose of this study is to use the unique physicochemical properties of carbon nanomaterials. Furthermore, using the excellent plasticity of carbon nanomaterials to prepare the oriented carbon nanotube films, the effect of inhibiting the formation of cicatrars is determined by experimental study, and the mechanism is explored to provide a new idea for the further application of carbon nanomaterials in tissue engineering. The synthetic reductive graphene oxide / nano hydroxyapatite composite scaffold (RGO-nHA) was used to observe its microscopic characterization through SEM, AFM, and TEM. The physicochemical properties of the composite scaffold were characterized by TGA, FTIR and other means. The properties of the sustained release vancomycin were measured by the drug release experiment in vitro, and L929, MC3T3 cells were selected to evaluate the formation and withering of the scaffolds. The effect of BMSCs cells, cell count, fluorescent staining, SEM and CLSM were used to evaluate the effect of scaffolds on cell adhesion and proliferation, cell morphology, and the osteogenesis of the stent was evaluated by alizarin red staining, ALP activity detection and so on. The formation of biomembrane; the antibacterial performance and persistence of the scaffold were evaluated through the liquid culture and the solid medium bacteriostasis test. The blood compatibility of the scaffolds was evaluated through the in vitro hemolysis test, and the changes of the erythrocyte morphology were observed by SEM. The absorption of the composite scaffold was observed by implanting the composite scaffold into the mice, and the evaluation of its absorption in the body was observed. The influence on the key organs of the mice was established, and a New Zealand white rabbit model of infectious bone defect was established, and the composite scaffold was grouped into the local lesion of New Zealand white rabbit model. The infection control and bone regeneration were evaluated by imaging examination, blood biochemistry, and histological examination. Furthermore, we prepared the order by chemical vapor deposition. ACNTs films were prepared by carbon nanotube arrays. Their micromorphology was observed by SEM, TEM, SAXRD and so on. The effects on cell proliferation and cell safety were evaluated by cell proliferation experiments, EdU staining and apoptosis experiments, and cytoskeleton redistribution and directional growth were induced by fluorescence staining and SEM observation of ACNTs. The mechanism of inhibiting collagen deposition, inhibiting cell proliferation and inducing cell directional growth were studied by gene chip analysis, PCR, Western-blot and other methods. The effect of ACNTs inhibition on hyperplastic scar formation was verified by general observation and histological analysis. The RGO-nHA composite scaffold has a three-dimensional porous structure which is connected with each other. After adding nHA, the osteogenic performance is enhanced, which promotes the growth of the cells and promotes bone regeneration. After loading vancomycin on the composite scaffold, the drug can be released quickly at the initial stage due to the pion bond with graphene, and then a slow drug can be carried out. After the release of the drug, the drug release characteristics combined with the inherent antibacterial activity of graphene can ensure rapid treatment of infection and provide persistent inhibitory effect on bacteria. In vivo experiments also proved that the drug delivery system can effectively treat infectious bone defects. Furthermore, we successfully synthesized ACNTs by chemical vapor deposition, and the results show that ACNTs It can effectively inhibit the excessive proliferation of fibroblasts, guide the orientation growth of the cells, inhibit the deposition of collagen, and in vitro experimental results confirm the cytotoxicity of the cells. The mechanism analysis shows that ACNTs plays a role mainly by changing the cell proliferation, cytoskeleton, cell motility and gene expression related to the secretion of colloid. Finally, we take it. The rabbit ear scar model was used to evaluate its living effect, which showed that ACNTs could effectively inhibit the formation of hyperplastic scar. The above study uses the unique physicochemical and biological characteristics of graphene and carbon nanotube two carbon nanomaterials, and innovatively introduces them to the treatment of infected bone defects and wound scars caused by open fracture. A series of in vitro and animal living experiments have proved that carbon nanomaterials can be used as an ideal tissue engineering scaffold for the treatment of infected bone defects and wound scars. This provides a new way of thinking for the use of new carbon nanomaterials to solve clinical problems.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R683;TB383.1

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