载利福平多孔复合物作为结核性骨缺损修复材料的研究

发布时间：2018-06-15 12:05

本文选题：利福平 + 外消旋聚乳酸　；参考：《华中科技大学》2014年博士论文

【摘要】：骨关节结核(Osteoarticular TB)是最常见肺外结核之一,其发病率近年来逐年增高,但治疗效果不甚理想。研究表明实施病灶清除术后遗留的骨缺损能否有效重建,在手术治疗骨关节结核中发挥着至关重要作用,决定着手术治疗骨关节结核成败与否。现有骨缺损修复材料如自体骨、同种异体骨、钛网(titanium mesh)、聚甲基丙烯酸甲酯(Polymethylmethacrylate, PMMA)骨水泥等均存在各自不足,制约了手术治疗骨关节结核发展。因此,开发理想的骨结核缺损修复材料迫在眉睫。外消旋聚乳酸(Poly-DL Lactic Acid, PDLLA)和纳米羟基磷灰石(nano-hydroxyapatite, nHA)是最具潜力的两类骨修复材料和药物转运系统(drug delivery system, DDS)载体材料,被广泛应用于生物医学多个领域。利福平(Rifampin,RFP)为一线抗结核药物。本课题中,我们在转化医学理念指导下,针对结核性骨缺损特点,利用溶剂挥发法成功设计并制备出载RFP多孔复合物(RFP/PDLLA/nHA复合物),并对其相关性能进行了重点研究,探索了其在结核性骨缺损治疗领域的临床应用潜能。本论文研究内容主要包括以下五个方面： 1.多孔RFP/PDLLA/nHA复合物制备及表征。以RFP、PDLLA、nHA为原料,利用溶剂挥发法制备出海绵状疏松多孔RFP/PDLLA/nHA复合修复材料。以孔隙率(porosity)、载药量(drug loading,DL)、包封率(Entrapment efficiency, EE)、成型情况等综合判断优化制备工艺,筛选出最佳原料配比(wt.%)为RFP:PDLLA: nHA=2:10:1.此比例下,孔隙率为83.35+1.50%,RFP包封率高达76.77+0.82%,总载药量高达12.82±0.14%。X射线衍射(X-ray differaction, XRD)和傅里叶变换红外光谱(Fourier transform infrared spectroscope, FTIR)证实制备前后未发生明显物象改变,未引入新杂质。 2.多孔RFP/PDLLA/nHA复合物降解、药物释放特性研究。实验发现该复合物具备优良降解性能和药物缓释特性,降解过程中可见明显孔壁结构塌陷,大量降解小孔形成。复合物体内外降解失重率(weight loss ratio, WLR)均明显快于纯PDLLA材料；其在体外存在短暂药物爆释现象,随后可维持稳定释放12w以上；其体外累积释药分数(cumulative release percentage, CRP)与失重率高度相关,存在明显线性关系：Y=11.63+0.9549*X (R2=0.9836, SD=1.17, n=12)。 3.多孔RFP/PDLLA/nHA复合物生物安全性研究。细胞安全性实验证实其对MC3T3-E1细胞无明显细胞毒性；其与多孔PDLLA/nHA材料均具备良好细胞适应性。通过将复合物植入昆明小鼠体内初步探讨其组织相容性,结果提示该复合物具备良好组织相容性；小鼠手术切口愈合可,生存状态良好,病理学观察表明其并未诱发严重急性炎症及排斥反应。材料周围IL-6、TNF-a表达含量早期轻度升高,但存在迅速下降,向组织修复期快速转变趋势。 4.多孔RFP/PDLLA/nHA复合物骨诱导性探讨。采用将材料与MC3T3-E1细胞诱导分化培养的方式,初步探讨其体外骨诱导性。实验发现多孔RFP/PDLLA/nHA组中MC3T3-E1细胞碱性磷酸酶(alkaline phosphatase, ALP)、骨钙素(osteocalcin,OCN)以及Ⅰ型胶原(Collagen-I, COL-I)表达量显著高于同期多孔RFP/PDLLA材料。结果表明多孔RFP/PDLLA/nHA复合物能显著加快诱导MC3T3-E1细胞向成熟成骨细胞(osteoblast, OB)分化,具备优良的体外骨诱导性。 5.多孔RFP/PDLLA/nHA复合物抗结核性能研究。采用材料与结核杆菌(Mycobacterium tuberculosis, M. tuberculosis)共培养的方式,结合MicroMGITTM荧光判读仪、抗酸染色、金胺“O”染色(auramine O fluorescent dyes),流式细胞仪(flow cytometry, FCM)多种手段探讨了多孔RFP/PDLLA/nHA及PDLLA/nHA两种复合材料体外抗结核性能,结果表明前者具备优良抗结核性能,通过药物释放能有效杀灭M. tuberculosis;而后者则可与M. tuberculosis友好相处。实验同时发现FCM用于生物医用材料功能化、安全性评价操作简便、准确率高,值得推广。上述结果表明多孔RFP/PDLLA/nHA复合物作为结核性骨缺损修复材料具备一定临床应用潜能；但其在活体内作用效能有待深入探讨,材料特性对产物性能影响的具体机制亦有待进一步研究。本论文成功实施,为治疗骨关节结核提供了新选择,亦为骨科其他类似疾病研究提供了新设计灵感。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R529.2

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