生物活性骨组织工程支架的研究及介孔羟基磷灰石的初步探讨

发布时间：2018-06-19 04:13

本文选题：纳米羟基磷灰石 + 聚丙交酯-乙交酯　；参考：《吉林大学》2012年博士论文

【摘要】：近来，工程学和材料学的众多进展，推动了骨组织工程学的发展。支架材料由天然材料向人工合成高分子材料发展，由单一支架材料向复合材料及表面修饰材料发展。天然骨基质是一种无机/有机结合的复合材料，包括天然形成的聚合物（胶原蛋白）和生物矿物质（磷灰石）。并且天然骨基质中羟基磷灰石以小于100nm的晶体与胶原纤维紧密结合构成纳米羟基磷灰石/胶原复合材料。因此，构建无机/有机结合的纳米复合材料能够模拟天然骨基质，能更好的促进骨组织的修复。由此，制备了纳米复合材料模拟天然骨基质，一方面本研究利用冷冻干燥法制备了不同n-HA含量的n-HA/PLGA多孔复合材料支架，并从孔隙率，力学强度，扫描电镜，接触角对其进行了表征，并探讨了不同的n-HA对多孔复合材料支架结构的影响，及对成骨细胞增殖的影响。另一方面PLGA与n-HA的复合，在一定程度上可以提高PLGA的生物活性和骨引导、诱导能力，但仍然不能满足修复大面积骨缺损的需要，本研究利用冷冻干燥法制备了多孔复合材料支架，并引入了具有生物活性的廉价易获得小分子化合物，二甲基砜（MSM）。探讨了MSM含量的不同对多孔复合材料支架结构功能和生物学行为的影响。本研究还合成了介孔纳米羟基磷灰石，并初步探讨了表面活性剂比率的不同对其结构和介孔结构对蛋白担载性能的影响。为组织工程支架在临床的应用提供实验依据，并开发具有生物活性的廉价的组织工程支架材料。第一部分HA/PLGA多孔复合材料支架冷冻干燥法制备及表征和细胞增殖的研究。课题组前期工作中了，研究了不同的冷冻速率对多孔支架材料的影响。结果显示4℃冻存制备的多孔支架，具有更大的孔径，更高的孔隙率。本研究选用4℃作为冻存温度，探讨n-HA含量的不同对多孔复合材料支架结构性能和细胞增殖的影响。利用冷冻干燥法制备了不同n-HA含量的n-HA/PLGA多孔复合材料支架，n-HA含量分别为0wt.%、5wt.%、10wt.%、20wt.%和40wt.%。利用扫描电镜观察多孔支架材料的表面形貌，乙醇溶液置换法测试复合材料的孔隙率，电子式万能试验机测试复合材料的力学强度。n-HA与PLGA铺膜接触角测试仪测量接触角。小鼠成骨前体细胞（MC3T3-E1）种植到多孔支架上，MTT法检测不同材料在3d、7d和14d对成骨细胞增殖的影响。结果显示：支架孔隙分布均匀，且孔隙保持连通。当n-HA含量为10wt.%和20wt.%时，显示了较好孔隙率，润湿性和较高的力学强度，能有效的促进成骨细胞的增殖。第二部分生物活性MSM/HA/PLGA多孔复合材料支架的制备及表征。前期工作中发现n-HA/PLGA复合材料，在一定程度上虽然可以提高PLGA的生物活性和骨引导、诱导能力，，但仍然不能满足修复大面积骨缺损的需要。因此，本研究利用冷冻干燥法引入了具有生物活性并且廉价易获得小分子化合物二甲基砜（MSM），探讨了MSM含量的不同对多孔复合材料的影响。利用扫描电镜观察多孔支架材料的表面形貌，乙醇溶液置换法测试复合材料的孔隙率，MSM、n-HA与PLGA铺膜接触角测试仪测量接触角。电子式万能试验机测试复合材料的力学强度。利用电感耦合等离子光谱发生仪测试了多孔支架中MSM的实际含量和累积释放量。结果显示：制备的支架具有均匀的孔隙分布，及良好的贯通性。MSM含量为0.1%时孔隙率和力学强度较高，冷冻干燥法能有效的将MSM引入组织工程支架，未造成MSM的明显丢失，并能延长MSM的释放时间。第三部分MSM/HA/PLGA多孔复合材料支架的细胞增殖、黏附和成骨研究。前期工作中，制备了MSM/HA/PLGA多孔复合材料支架，本研究探讨多孔复合材料支架对成骨细胞增殖、黏附和骨修复的影响。利用MTT法，DAPI染色和碱性磷酸酶活性测定，观察多孔支架材料对成骨细胞增殖和黏附渗透的影响，将支架材料植入兔桡骨缺损部位，术后4w和12w采用X光片检测骨形成量，以评价材料的骨修复能力。结果显示：MSM含量为0.1%时显示了较好的成骨增殖和黏附能力，能有效的促进骨修复。第四部分介孔纳米羟基磷灰石的制备表征及蛋白担载的研究。揭示表面活性剂的比率介孔n-HA结构的影响，以及介孔结构对HA吸附和释放牛血清白蛋白（BSA）的影响。利用湿沉淀法，用CTAB作为模板在室温下成功合成了介孔n-HA，用X射线衍射仪对合成的n-HA相位和晶体结构进行了表征。用傅里叶红外变换光谱对合成纳米粒子的化学结构基团进行测定。用场发射扫描电镜进行表面形貌分析。用透射电镜观察纳米粒子的尺寸和形状。用多角度光散射仪测试粒径。用氮气吸附测试合成粒子的孔隙结构参数。用BSA测试样品对蛋白担载释放性能的影响。结果显示：合成的样品均为纯相纳米低结晶度的介孔材料，当CTAB比率为0.5%时，显示了良好的介孔结构，较大的蛋白吸附和较长的释放时间。
[Abstract]:Recent advances in engineering and material science have promoted the development of bone tissue engineering. Scaffolds have developed from natural materials to synthetic polymers, from single scaffolds to composite materials and surface modified materials. Natural bone matrix is an inorganic / organic composite, including naturally formed polymers. Collagen and biological minerals (apatite). And hydroxyapatite in the natural bone matrix, with a compact combination of less than 100nm crystals and collagen fibers, constitutes a nano hydroxyapatite / collagen composite. Therefore, the construction of inorganic / organic composite nanocomposites can simulate the natural bone matrix and can better promote the repair of bone tissue.
As a result, nano composites were prepared to simulate natural bone matrix. On the one hand, the porous n-HA/PLGA composite scaffolds with different n-HA content were prepared by freeze-drying method and characterized by porosity, mechanical strength, scanning electron microscope and contact angle, and the influence of different n-HA on the structure of porous composite scaffold was discussed. And the effect on osteoblast proliferation. On the other hand, the combination of PLGA and n-HA, to some extent, can improve the biological activity of PLGA, bone guidance and induction, but still can not meet the needs of repairing large area bone defects. A small molecular compound, two methyl sulfone (MSM) was easily obtained. The effects of different MSM content on the structure and biological behavior of the porous composite scaffold were investigated. The mesoporous nano hydroxyapatite was also synthesized in this study. The effects of the different surface active agent ratio on the structure and the pore structure of the mesoporous structure on the performance of the protein loading were also preliminarily discussed. To provide experimental evidence for clinical application of tissue engineering scaffolds, and develop cheap bioengineering scaffolds with bioactivity.
The first part of the study on the preparation, characterization and cell proliferation of HA/PLGA porous composite scaffolds. In the previous work, the effects of different freezing rates on porous scaffolds were studied. The results showed that the porous scaffolds prepared at 4 degrees centigrade had larger pore diameter and higher porosity. This study was selected at 4 degrees C for this study. The effects of different n-HA content on the structural properties and cell proliferation of porous composite scaffolds were investigated. The n-HA/PLGA porous composite scaffolds with different n-HA content were prepared by freeze-drying method. The n-HA content was 0wt.%, 5wt.%, 10wt.%, 20wt.% and 40wt.%., respectively, to observe the surface morphology of porous scaffold materials by scanning electron microscope. The porosity of the composite was measured by the ethanol solution replacement method. The mechanical strength of the composite material was measured by the electronic universal testing machine (.N-HA) and the contact angle tester of PLGA membrane. The mouse osteogenic precursor cells (MC3T3-E1) were planted on the porous scaffold. The effects of different materials on the proliferation of osteoblasts were detected by different materials in 3D, 7d and 14d. The pore distribution is uniform and the pores remain connected. When the content of n-HA is 10wt.% and 20wt.%, it shows good porosity, wettability and high mechanical strength, which can effectively promote the proliferation of osteoblasts.
The preparation and characterization of the second bioactive MSM/HA/PLGA porous composite scaffold. In the previous work, it was found that n-HA/PLGA composites, to some extent, could improve the biological activity of PLGA, bone guidance and induction, but still could not meet the needs of repairing large area bone defects. Therefore, this study uses the freeze drying method. Two methyl sulfone (MSM), which has biological activity and cheap and easy to obtain small molecular compound (MSM), has been studied. The effect of different MSM content on porous composite material is investigated. The surface morphology of porous scaffold is observed by scanning electron microscope, the pore rate of composite material is measured by the method of ethanol solution replacement, and the measurement connection of MSM, n-HA and PLGA membrane contact angle tester The mechanical strength of the composite was tested by an electronic universal testing machine. The actual content and cumulative release of MSM in the porous scaffold were measured by an inductively coupled plasma spectrometer. The results showed that the prepared scaffolds had a uniform pore distribution and a good porosity and high mechanical strength when the good penetrating.MSM content was 0.1%. Freeze drying can effectively introduce MSM into tissue engineering scaffolds without causing significant loss of MSM and prolonging the release time of MSM.
The third part of the cell proliferation, adhesion and osteogenesis of MSM/HA/PLGA porous composite scaffold. In the early work, the MSM/HA/PLGA porous composite scaffold was prepared. The effects of porous composite scaffold on osteoblast proliferation, adhesion and bone repair were investigated. The MTT method, DAPI staining and alkaline phosphatase activity were used to observe the multi porous composite scaffold. The effect of scaffold material on osteoblast proliferation and adhesion permeability, the scaffold material was implanted into the rabbit radius defect site. 4W and 12W were used to measure the bone shape to evaluate the bone repair ability after 4W and 12W. The results showed that when the content of MSM was 0.1%, the bone proliferation and adhesion ability was better, and the bone repair could be promoted effectively.
The fourth part of the preparation of mesoporous nano hydroxyapatite and the study of protein loading. The effect of the ratio of mesoporous n-HA on the ratio of surfactant and the effect of mesoporous structure on HA adsorption and release of bovine serum albumin (BSA). The mesoporous n-HA was successfully synthesized with CTAB as a template at room temperature and was diffracted by X ray diffraction. The phase and crystal structure of the synthesized n-HA were characterized. The chemical structural groups of the synthesized nanoparticles were measured by Fourier transform infrared spectroscopy. The surface morphology was analyzed by field emission scanning electron microscopy. The size and shape of the nanoparticles were observed by transmission electron microscopy. The particle size was measured by the multi angle light scattering instrument. The nitrogen gas was used to measure the particle size. The pore structure parameters of the particles were tested. The effect of samples on the release performance of protein loaded with BSA showed that the synthesized samples were mesoporous materials with low crystallinity in pure phase. When the ratio of CTAB was 0.5%, the good mesoporous structure, large protein adsorption and longer release time were shown.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08

【参考文献】