發(fā)布時(shí)間：2018-05-30 23:15

  本文選題：開放性骨折 + 感染性骨缺損��； 參考：《第二軍醫(yī)大學(xué)》2017年博士論文

【摘要】：隨著我國(guó)工業(yè)及交通的高速發(fā)展,交通事故、高處墜落等高能量損傷導(dǎo)致的開放性骨折發(fā)病率逐年升高,而開放性骨折后易發(fā)生骨感染。針對(duì)感染性骨缺損的一系列病理特點(diǎn),學(xué)者們主要將其歸納為以下幾個(gè)方面1)細(xì)菌生物膜形成;2)抗生素在局部難以形成有效濃度;3)局部血運(yùn)破壞;4)骨質(zhì)再生困難。所以開發(fā)具有抗菌及成骨雙重效能的植骨材料對(duì)于治療開放性骨折導(dǎo)致的感染性骨缺損十分重要。另一個(gè)影響開放性骨折患者預(yù)后的并發(fā)癥是傷口疤痕。高能量損傷導(dǎo)致的開放性骨折中,局部軟組織受損嚴(yán)重,傷口不規(guī)則;即使開放性骨折患者的骨組織能在較長(zhǎng)的治療周期內(nèi)愈合,但是如果遺留明顯的疤痕組織,會(huì)對(duì)患者日常生活造成困擾,限制關(guān)節(jié)的活動(dòng)度,降低開放性骨折患者術(shù)后的滿意度。防止瘢痕形成的關(guān)鍵包括以下幾個(gè)方面:抑制成纖維細(xì)胞過度增殖,抑制膠原的過度沉積,促進(jìn)傷口部位的纖維組織有序生長(zhǎng)。然而現(xiàn)階段的治療手段在減少增生性瘢痕形成的效果上尚不盡人意。根據(jù)開放性骨折感染及骨缺損的治療需求,碳納米材料中的石墨烯作為植骨材料的優(yōu)越性體現(xiàn)在以下方面:1、改良支架的理化性能,滿足骨填充需要;2、生物相容性及其促骨形成性能優(yōu)良;3、對(duì)干細(xì)胞增殖和成骨分化具有積極影響;4、材料本身具備一定的抗菌性能;5、具備特殊的結(jié)構(gòu)特點(diǎn)可作為理想的藥物載體。另一方面,碳納米管作為碳納米材料的另一種存在形式,因其獨(dú)特的結(jié)構(gòu)和豐富的表面可修飾性,已經(jīng)廣泛運(yùn)用于生物醫(yī)學(xué)領(lǐng)域。研究已發(fā)現(xiàn)有序排列的碳納米管可誘導(dǎo)多種細(xì)胞取向性生長(zhǎng)生長(zhǎng)。表明取向性的碳納米管(ACNTs)作為組織工程支架,可控制細(xì)胞生長(zhǎng)的位置和方向。本研究的目的旨在利用碳納米材料獨(dú)特的理化性質(zhì),制備成骨性能良好且本身具備一定抗菌性能的三維復(fù)合支架,并搭載抗生素,用于開放性骨折后感染及骨缺的治療;再者,利用碳納米材料優(yōu)良的可塑性,制備取向性的碳納米管薄膜,通過實(shí)驗(yàn)研究明確其抑制瘢痕形成的效應(yīng),并探究其機(jī)理,為碳納米材料在組織工程中的進(jìn)一步應(yīng)用提供新的思路。我們通過自組裝的方法,以抗壞血酸為還原劑,合成還原型氧化石墨烯/納米羥基磷灰石復(fù)合支架(RGO-nHA),通過SEM,AFM,TEM觀察其微觀表征;通過TGA,FTIR等手段表征其理化性質(zhì);通過藥物體外釋放實(shí)驗(yàn)測(cè)定復(fù)合支架緩釋萬古霉素的性能;選用L929、MC3T3細(xì)胞,評(píng)估支架對(duì)其單克隆形成和凋亡的影響;選用BMSCs細(xì)胞,通過細(xì)胞計(jì)數(shù)、熒光染色、SEM及CLSM等方法評(píng)估支架對(duì)細(xì)胞粘附增殖,細(xì)胞形態(tài)的影響;通過茜素紅染色,ALP活性檢測(cè)等手段評(píng)估載藥支架的成骨性能;通過平板菌落計(jì)數(shù),熒光染色等手段評(píng)估支架抑制細(xì)菌增殖及生物膜形成的情況;通過液體培養(yǎng)基和固體培養(yǎng)基抑菌圈實(shí)驗(yàn)評(píng)估支架的抗菌性能及其持久性;通過體外溶血實(shí)驗(yàn),評(píng)估支架的血液相容性,并通過SEM觀察紅細(xì)胞形態(tài)變化;通過將復(fù)合支架植入小鼠皮下,觀察其在體內(nèi)吸收的情況,評(píng)估其對(duì)小鼠關(guān)鍵臟器的影響;建立新西蘭大白兔感染性骨缺損模型,將復(fù)合支架分組植入新西蘭大白兔模型局部病灶內(nèi),通過影像學(xué)檢測(cè)、血生化、以及組織學(xué)檢測(cè)評(píng)估感染控制和骨再生情況。另外,我們通過化學(xué)氣相沉積法制備有序性的碳納米管陣列,制備成ACNTs薄膜,通過SEM,TEM,SAXRD等方法觀察其微觀形貌并評(píng)估其有序性;通過細(xì)胞增殖實(shí)驗(yàn)、EdU染色及細(xì)胞凋亡實(shí)驗(yàn)評(píng)估其對(duì)細(xì)胞增殖的影響及其細(xì)胞安全性;通過熒光染色和SEM觀察ACNTs誘導(dǎo)細(xì)胞骨架重配并定向生長(zhǎng)的情況;通過基因芯片分析,PCR、Western-blot等方法研究其抑制膠原沉積、抑制細(xì)胞增殖、誘導(dǎo)細(xì)胞定向生長(zhǎng)的機(jī)制;運(yùn)用新西蘭大白兔增生性瘢痕模型,通過大體觀察、組織學(xué)分析等方法驗(yàn)證ACNTs抑制增生性瘢痕形成的效果。結(jié)果表明,所制備的RGO-nHA復(fù)合支架具備相互連通的三維多孔結(jié)構(gòu),摻入nHA后成骨性能也得到增強(qiáng),可促進(jìn)細(xì)胞長(zhǎng)入、促進(jìn)骨質(zhì)再生;在此復(fù)合支架上負(fù)載萬古霉素之后,由于與石墨烯的π-π鍵合,藥物可以在初始階段實(shí)現(xiàn)較快速的釋放,然后進(jìn)行緩慢的藥物釋放,該藥物緩釋特點(diǎn)與石墨烯的固有的抗菌活性相結(jié)合之后可確�？焖僦委煾腥静⑻峁⿲�(duì)細(xì)菌的持久抑制作用。體內(nèi)實(shí)驗(yàn)也證明該載藥系統(tǒng)可有效治療感染性骨缺損。再者,我們通過化學(xué)氣相沉積法成功合成了ACNTs,結(jié)果表明ACNTs可有效抑制成纖維細(xì)胞的過度增殖,引導(dǎo)細(xì)胞的取向性生長(zhǎng),抑制膠原沉積,而且體外實(shí)驗(yàn)結(jié)果證實(shí)其無細(xì)胞細(xì)胞毒性。機(jī)制分析表明,ACNTs主要通過改變細(xì)胞增殖,細(xì)胞骨架、細(xì)胞運(yùn)動(dòng)性以及膠原分泌相關(guān)基因表達(dá)而起作用。最后,我們采用兔耳疤痕模型對(duì)其活體效應(yīng)進(jìn)行了評(píng)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、碳納米管兩種碳納米材料獨(dú)特的理化及生物特性,創(chuàng)新性地將其引入開放性骨折導(dǎo)致的感染性骨缺損及傷口疤痕的治療中,通過系列體外及動(dòng)物活體實(shí)驗(yàn)證明碳納米材料可作為治療感染性骨缺損和傷口疤痕的理想組織工程支架,這為利用新型的碳納米材料解決臨床難題提供了新的思路。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R683;TB383.1

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