本文選題：利福平 + 外消旋聚乳酸　； 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：骨關(guān)節(jié)結(jié)核(Osteoarticular TB)是最常見肺外結(jié)核之一,其發(fā)病率近年來逐年增高,但治療效果不甚理想。研究表明實施病灶清除術(shù)后遺留的骨缺損能否有效重建,在手術(shù)治療骨關(guān)節(jié)結(jié)核中發(fā)揮著至關(guān)重要作用,決定著手術(shù)治療骨關(guān)節(jié)結(jié)核成敗與否�，F(xiàn)有骨缺損修復(fù)材料如自體骨、同種異體骨、鈦網(wǎng)(titanium mesh)、聚甲基丙烯酸甲酯(Polymethylmethacrylate, PMMA)骨水泥等均存在各自不足,制約了手術(shù)治療骨關(guān)節(jié)結(jié)核發(fā)展。因此,開發(fā)理想的骨結(jié)核缺損修復(fù)材料迫在眉睫。 外消旋聚乳酸(Poly-DL Lactic Acid, PDLLA)和納米羥基磷灰石(nano-hydroxyapatite, nHA)是最具潛力的兩類骨修復(fù)材料和藥物轉(zhuǎn)運系統(tǒng)(drug delivery system, DDS)載體材料,被廣泛應(yīng)用于生物醫(yī)學(xué)多個領(lǐng)域。利福平(Rifampin,RFP)為一線抗結(jié)核藥物。本課題中,我們在轉(zhuǎn)化醫(yī)學(xué)理念指導(dǎo)下,針對結(jié)核性骨缺損特點,利用溶劑揮發(fā)法成功設(shè)計并制備出載RFP多孔復(fù)合物(RFP/PDLLA/nHA復(fù)合物),并對其相關(guān)性能進行了重點研究,探索了其在結(jié)核性骨缺損治療領(lǐng)域的臨床應(yīng)用潛能。本論文研究內(nèi)容主要包括以下五個方面： 1.多孔RFP/PDLLA/nHA復(fù)合物制備及表征。以RFP、PDLLA、nHA為原料,利用溶劑揮發(fā)法制備出海綿狀疏松多孔RFP/PDLLA/nHA復(fù)合修復(fù)材料。以孔隙率(porosity)、載藥量(drug loading,DL)、包封率(Entrapment efficiency, EE)、成型情況等綜合判斷優(yōu)化制備工藝,篩選出最佳原料配比(wt.%)為RFP:PDLLA: nHA=2:10:1.此比例下,孔隙率為83.35+1.50%,RFP包封率高達76.77+0.82%,總載藥量高達12.82±0.14%。X射線衍射(X-ray differaction, XRD)和傅里葉變換紅外光譜(Fourier transform infrared spectroscope, FTIR)證實制備前后未發(fā)生明顯物象改變,未引入新雜質(zhì)。 2.多孔RFP/PDLLA/nHA復(fù)合物降解、藥物釋放特性研究。實驗發(fā)現(xiàn)該復(fù)合物具備優(yōu)良降解性能和藥物緩釋特性,降解過程中可見明顯孔壁結(jié)構(gòu)塌陷,大量降解小孔形成。復(fù)合物體內(nèi)外降解失重率(weight loss ratio, WLR)均明顯快于純PDLLA材料；其在體外存在短暫藥物爆釋現(xiàn)象,隨后可維持穩(wěn)定釋放12w以上；其體外累積釋藥分數(shù)(cumulative release percentage, CRP)與失重率高度相關(guān),存在明顯線性關(guān)系：Y=11.63+0.9549*X (R2=0.9836, SD=1.17, n=12)。 3.多孔RFP/PDLLA/nHA復(fù)合物生物安全性研究。細胞安全性實驗證實其對MC3T3-E1細胞無明顯細胞毒性；其與多孔PDLLA/nHA材料均具備良好細胞適應(yīng)性。通過將復(fù)合物植入昆明小鼠體內(nèi)初步探討其組織相容性,結(jié)果提示該復(fù)合物具備良好組織相容性；小鼠手術(shù)切口愈合可,生存狀態(tài)良好,病理學(xué)觀察表明其并未誘發(fā)嚴重急性炎癥及排斥反應(yīng)。材料周圍IL-6、TNF-a表達含量早期輕度升高,但存在迅速下降,向組織修復(fù)期快速轉(zhuǎn)變趨勢。 4.多孔RFP/PDLLA/nHA復(fù)合物骨誘導(dǎo)性探討。采用將材料與MC3T3-E1細胞誘導(dǎo)分化培養(yǎng)的方式,初步探討其體外骨誘導(dǎo)性。實驗發(fā)現(xiàn)多孔RFP/PDLLA/nHA組中MC3T3-E1細胞堿性磷酸酶(alkaline phosphatase, ALP)、骨鈣素(osteocalcin,OCN)以及Ⅰ型膠原(Collagen-I, COL-I)表達量顯著高于同期多孔RFP/PDLLA材料。結(jié)果表明多孔RFP/PDLLA/nHA復(fù)合物能顯著加快誘導(dǎo)MC3T3-E1細胞向成熟成骨細胞(osteoblast, OB)分化,具備優(yōu)良的體外骨誘導(dǎo)性。 5.多孔RFP/PDLLA/nHA復(fù)合物抗結(jié)核性能研究。采用材料與結(jié)核桿菌(Mycobacterium tuberculosis, M. tuberculosis)共培養(yǎng)的方式,結(jié)合MicroMGITTM熒光判讀儀、抗酸染色、金胺“O”染色(auramine O fluorescent dyes),流式細胞儀(flow cytometry, FCM)多種手段探討了多孔RFP/PDLLA/nHA及PDLLA/nHA兩種復(fù)合材料體外抗結(jié)核性能,結(jié)果表明前者具備優(yōu)良抗結(jié)核性能,通過藥物釋放能有效殺滅M. tuberculosis;而后者則可與M. tuberculosis友好相處。實驗同時發(fā)現(xiàn)FCM用于生物醫(yī)用材料功能化、安全性評價操作簡便、準確率高,值得推廣。 上述結(jié)果表明多孔RFP/PDLLA/nHA復(fù)合物作為結(jié)核性骨缺損修復(fù)材料具備一定臨床應(yīng)用潛能；但其在活體內(nèi)作用效能有待深入探討,材料特性對產(chǎn)物性能影響的具體機制亦有待進一步研究。本論文成功實施,為治療骨關(guān)節(jié)結(jié)核提供了新選擇,亦為骨科其他類似疾病研究提供了新設(shè)計靈感。
[Abstract]:Bone and joint tuberculosis (Osteoarticular TB) is one of the most common extrapulmonary tuberculosis. Its incidence is increasing year by year, but the effect of the treatment is not very satisfactory. The study shows whether the reconstruction of bone defects left by the debridement and the important role in the surgical treatment of bone and joint tuberculosis determines the surgical treatment of bone and joint tuberculosis. The existing bone defect repair materials such as autogenous bone, allograft bone, titanium mesh (titanium mesh) and polymethyl methacrylate (Polymethylmethacrylate, PMMA) bone cement have their own shortcomings, which restrict the development of the surgical treatment of bone and joint tuberculosis. Therefore, it is urgent to develop an ideal repair material for bone tuberculosis defects.
Poly-DL Lactic Acid (PDLLA) and nano hydroxyapatite (nano-hydroxyapatite, nHA) are the most potential two types of bone repair materials and drug transport systems (drug delivery system, DDS), which are widely used in many biomedical domain. Rifampin (Rifampin, RFP) is the first line of anti tuberculosis drugs. Under the guidance of the concept of translational medicine, we successfully designed and prepared the RFP porous complex (RFP/PDLLA/nHA complex) by solvent evaporation in the light of the characteristics of tuberculous bone defect, and focused on its related properties, and explored its clinical potential in the field of tuberculous bone defect treatment. The main content of this paper is the research content of this paper. Includes the following five aspects:
The preparation and characterization of 1. porous RFP/PDLLA/nHA complex. Using RFP, PDLLA and nHA as raw materials, the porous porous RFP/PDLLA/nHA composite restorations were prepared by solvent evaporation method. The optimized preparation process was synthesized with porosity (porosity), drug loading (drug loading, DL), encapsulation rate (Entrapment efficiency, EE), forming conditions and so on. The optimum raw material ratio (wt.%) is RFP:PDLLA: nHA=2:10:1., the porosity is 83.35+1.50%, the encapsulation efficiency of RFP is as high as 76.77+0.82%, and the total loading amount is up to 12.82 + 0.14%.X ray diffraction (X-ray differaction, XRD) and Fu Liye transform infrared spectroscopy (Fourier transform). Like change, not the introduction of new impurities.
Study on the degradation and drug release characteristics of 2. porous RFP/PDLLA/nHA complex. It is found that the compound has excellent degradation properties and drug release characteristics. The pore wall structure collapses and a large number of micropores are formed during the degradation process. The degradation weight loss rate (weight loss ratio, WLR) both inside and outside the compound object is obviously faster than the pure PDLLA material; There is a transient release of drug release in vitro, which can subsequently maintain stable release of more than 12W, and the cumulative release fraction (cumulative release percentage, CRP) in vitro is highly correlated with the weight loss rate, and there is a significant linear relationship: Y=11.63+0.9549*X (R2=0.9836, SD=1.17, n=12).
Study on biological safety of 3. porous RFP/PDLLA/nHA complex. Cell safety test showed that it had no obvious cytotoxicity to MC3T3-E1 cells, and had good cellular adaptability with porous PDLLA/nHA materials. The composite was implanted in Kunming mice and Its Histocompatibility was preliminarily discussed. The results suggest that the complex has good tissue phase. The survival condition of the mice was good. The pathological observation showed that it did not induce severe acute inflammation and rejection. The expression level of IL-6 and TNF-a around the material was slightly elevated in the early stage, but it declined rapidly and changed rapidly to the period of tissue repair.
4. bone inducibility of porous RFP/PDLLA/nHA complex. The bone inducibility in vitro was preliminarily explored by inducing differentiation and culture of material and MC3T3-E1 cells. The experiment found MC3T3-E1 cell alkaline phosphatase (alkaline phosphatase, ALP), osteocalcin (osteocalcin, OCN) and type I collagen (Collagen-I, COL-I) in the porous RFP/PDLLA/nHA group. The expression was significantly higher than that of the porous RFP/PDLLA material at the same time. The results showed that the porous RFP/PDLLA/nHA complex could significantly accelerate the differentiation of MC3T3-E1 cells to mature osteoblasts (osteoblast, OB), and have excellent in vitro bone inducibility.
5. the study of anti tuberculosis performance of 5. porous RFP/PDLLA/nHA complex. Using the co culture of materials and Mycobacterium tuberculosis (Mycobacterium tuberculosis, M. tuberculosis), combined with MicroMGITTM fluorescent reading instrument, acid stain, gold amine "O" staining (auramine O fluorescent dyes), and many means of flow cytometry. The anti tuberculosis performance of two kinds of composite materials of hole RFP/PDLLA/nHA and PDLLA/nHA in vitro shows that the former has excellent anti tuberculosis performance and can effectively kill M. tuberculosis through drug release, and the latter can be friendly to M. tuberculosis. The experiment also found that FCM is used for the functionalization of biomedical materials, and the safety evaluation is simple and accurate. High rate, worthy of promotion.
These results show that the porous RFP/PDLLA/nHA complex has the potential of clinical application as a repairing material for tuberculosis bone defect, but the effect of the compound in vivo needs further study. The specific mechanism of the effect of material properties to the performance of the product needs further study. The successful implementation of this paper provides a new choice for the treatment of bone and joint tuberculosis. Choose, provides a new design inspiration for Department of orthopedics and other similar diseases research.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R529.2

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