基于聚富马酸丙二醇酯弹性材料的制备及其对睑板全层缺损的修复研究

发布时间：2018-06-01 19:32

本文选题：聚富马酸丙二醇酯 + 增韧改性　；参考：《浙江大学》2017年博士论文

【摘要】：第一部分聚富马酸丙二醇酯/甲基丙烯酸羟乙酯弹性材料优化制备与性能研究目的利用甲基丙烯酸羟乙酯作为共聚单体对聚富马酸丙二醇酯进行增韧改性,探索制备比传统PPF韧性更好的材料的可行性,获得制备弹性材料的甲基丙烯酸羟乙酯共聚单体最优比例,在此基础上调节HEMA共聚单体比例对PPF交联密度、亲水性及力学新能进行精确调控,以期构建出在结构和力学性能上对天然睑板组织有更高相似性的材料,为睑板替代物的研制提供一个新的途径。方法首先对兔睑板组织微观形貌、力学性能进行分析,在此基础上,采用具有良好生物相容性的PPF、HEMA进行共聚,通过对HEMA共聚单体比例的调整,优化共聚物各项参数,制备基于PPF的弹性材料。傅里叶转换红外线光谱分析共聚物化学键及化学组成,差扫描量热仪表征共聚物热性能,扫描电镜和透射电镜检测共聚物表面和断面微观结构,静态水接触角对共聚物的亲疏水性进行分析比较,万能测试机检测共聚物压缩及拉伸力学强度,分析评价PPF/HEMA共聚材料作为睑板替代材料的结构与性能仿生可行性。结果兔睑板平均杨氏模量为5.67±1.53 MPa。交联固化后的PPF-HEMA共聚物能够在3400 cm-1和1740cm-1处看到较强的羟基和羟基的伸缩振动峰,对比不同投料比的样品中羟基和羰基振动峰的相对强度,随着样品中投料的HEMA量增加,羟基相对羰基含量增加。PPF-HEMA0.5、PPF-HEMA1、PPF-HEMA2的水接触角随着HEMA单体投料比的增加而降低;溶胀率分别为16%、20%和30%左右。溶胀前,除PPF-HEMA0.5样品(14.3 ±2.6 MPa)以外,其他实验组和对照组压缩模量均在19 MPa左右。拉伸模式下,PPF-HEMA 0.5样品在溶胀前断裂伸长率就能达到20%左右,PPF-HEMA 1和PPF-HEMA 2样品的伸长率可以由溶胀前的不到10%提高到溶胀后的30～40%左右。在湿态循环加载条件下,各组分PPF-HEMA经过50次循环,样品形貌保持完整,应力-应变曲线重合较好,应力值分别为原始的90.1%,82.5%,and 85.9%,反应材料具有较好的抗疲劳性。结论本部分研究选用HEMA为共聚单体,以1:0.5、1:1和1:2三种质量置比投料,与PPF共聚制备了韧性材料。PPF-HEMA交联体系中,通过增加HEMA共聚单体的比例改变固化后材料的化学组成,并在相当程度上提高材料的溶胀性能,制备了一种具有良好的韧性和抗疲劳性的弹性材料,为模拟天然睑板替代材料的研究和应用奠定了实验基础。第二部分聚富马酸丙二醇酯/甲基丙烯酸羟乙酯组织工程多孔支架的制备及其理化性能和生物相容性研究目的利用生物相容性良好的明胶微粒作为致孔剂制备具有适度孔径、高孔隙率和连通性好的多孔支架,在微观层次探讨PPF-HEMA共聚材料的化学组分、孔隙结构对材料降解性能等理化性能和细胞黏附、增殖等生物活性的影响,进一步在二维培养条件下验证该组织工程PPF-HEMA多孔支架对睑板缺损修复修复研究中所涉及的目标细胞的适用性。方法采用明胶粒子致孔法制备不同孔径、不同共聚比例的多孔PPF-HEMA支架,扫描电镜观察支架的形貌和结构并测定支架孔径,排乙醇体积法测多孔支架孔隙率,根据1,2,3,6,9和12周支架的失重率分析体外降解性能。浸提液法用HDFs细胞考察材料的细胞毒性,并采用PrestoBlne试剂盒对人结膜基质细胞和人结膜上皮细胞两种不同目标细胞在支架上的黏附与增殖能力进行检测,免疫荧光染色特异性标记物观察支架上细胞形态,以评价组织工程PPF-HEMA多孔支架的生物相容性。结果明胶粒子致孔法180-280μm明胶微粒所制备的PPF-HEMA多孔支架具有规整的外形,而280-450μm、480-600μm明胶微粒致所制备的PPF-HEMA多孔支架因孔隙较大,280-450μm孔径支架外形较为粗糙不规,480-60μm孔径支架甚至出现开裂。180-280μm明胶模板所制备的 PPF-HEMA 0.5、PPF-HEMA 1 和 PPF-HEMA 2多孔支架孔隙率分别在88.9±2.3%,82.3±1.6%和84.6±3.1%,与Image J软件分析商业化的ADM孔隙率为91.5±3.2%相比无明显差异。扫描电镜可见多孔支架具有规整的外形,良好的孔径分布和连通性。体外PBS孵育12周内,PPF-HEMA2组的支架质量损失在第9周为10.9±1.3%,到12周质量损失也仅达11.4±0.7%;而PPF-HEMA0.5和PPF-HEMA1到12周质量损失分别达到15%和20%。MTT法检测不同PPF-HEMA浸提液之间以及对比空白对照组,各时间点细胞增殖情况无明显差异(P0.05)。在PPF-HEMA材料体系中,HEMA共聚单体的加入通过改变共聚体的亲水性而调控支架的细胞相容性在PPF-HEMA材料体系中,PPF-HEMA 1能支持一定的CjECs增殖,但PPF-HEMA 0.5和PPF-HEMA 2多孔支架虽有一定细胞黏附,但并不有效地支持目标细胞CjSCs生长。结论利用180-280μm明胶微粒致孔剂法可制备出具有适度孔径、高孔隙率和连通性好的PPF-HEMA多孔支架,支架在体外降解12周质量损失小于20%,具有良好的稳定性,通过改变PPF-HEMA投料比可以通过调节共聚体的交联密度可调控PPF-HEMA多孔支架体外降解行为。PPF-HEMA 1能支持一定的CjECs增殖,但PPF-HEMA0.5PPF-HEMA2基本不能支持细胞增殖。第三部分聚富马酸丙二醇酯/甲基丙烯酸羟乙酯多孔支架材料的体内生物学效应及眼睑重建效果研究目的分析评价组织工程PPF-HEMA多孔支架的体内生物相容性,同时观测其原位缺损重建修复效果,为睑板替代材料的构建提供新的思路和合成方法,为眼睑重建用组织工程睑板替代材料的临床应用奠定前期实验基础。方法构建兔全层睑板缺损动物模型,以去人脱细胞真皮基质作为对照,植入PPF-HEMA多孔支架材料。通过观察眼睑缺损修复效果、组织切片HE染色和Masson染色观察炎症反应和材料-组织反应情况,评价PPF-HEMA多孔支架材料的可操作性和在体生物相容性。结果成功构建兔全层睑板缺损动物模型,术后8周ADM组和PPF-HEMA 2组眼睑缺损完全修复,未见任何成角畸形,PPF-HEMA 1组可见轻度成角畸形,对比阴性对照组可见明显眼睑成角畸形及疤痕形成。组织学切片HE染色可见术后1周各组炎症反应明显,同一时间实验组与对照组炎症反应评分无统计学差异,随植入时间增加各组炎症反应均明显减轻,术后8周与术后1周炎症反应评分有统计学差异。组织学切片 Masson 染色可见PPF-HEMA多孔支架材料孔隙内新生结缔组织长入,支架周围胶原纤维束包裹,ADM组、PPF-HEMA 1和PPF-HEMA 2组Image J 测量其厚度分别为 28.0 ± 9.3 μm,65.2 ± 16.8 μm 和 63.5 ± 17.3 μm。结论组织工程PPF-HEMA多孔支架具有良好的体内生物相容性,在兔全层睑板缺损动物模型中PPF-HEMA 2组表现出更好的修复效果,较低的炎症反应以及较慢的降解速率,相比于ADM其炎症反应无明显差异,但长期稳定性更好。本研究结果为仿天然睑板结构与性能高分子材料的研究奠定了扎实的实验基础,为临床上眼表疾病的治疗提供了一条新的途径。
[Abstract]:The optimization of the preparation and properties of polyfumaric acid propanediol ester / hydroxyethyl methacrylate elastic material. Aim to toughen the polyfumaric acid propanediol ester with hydroxyethyl methacrylate as a copolymerized monomer, explore the feasibility of preparing the material better than the traditional PPF toughness, and obtain the methyl propylene for the preparation of the elastic material. The optimal proportion of hydroxyethyl ester copolymers is based on the ratio of HEMA copolymers to PPF crosslinking density, hydrophilicity and new mechanical energy, in order to construct a material with higher similarity in structure and mechanical properties to natural eyelid tissue, and to provide a new way for the development of eyelid substitutions. The microstructure and mechanical properties of the rabbit eyelid tissue were analyzed. On this basis, PPF and HEMA with good biocompatibility were used to copolymerized. By adjusting the proportion of HEMA copolymers, the parameters of the copolymers were optimized and the elastic materials based on PPF were prepared. The chemical bond and chemical composition of the copolymers were analyzed by Fourier transform infrared spectroscopy. The thermal performance of the copolymer was detected by differential scanning calorimeter. The microstructure of the surface and section of the copolymer was detected by scanning electron microscope and transmission electron microscope. The static water contact angle was used to analyze the hydrophobicity of the copolymer. The universal testing machine was used to detect the compressive strength of the copolymer and the tensile strength of the copolymer. The PPF/HEMA copolymers were analyzed and evaluated as the junctions of the replacement material of the eyelid plate. The results show that the average young's modulus of the rabbit's eyelid is 5.67 + 1.53 MPa. after curing, and the PPF-HEMA copolymer can see the strong hydroxyl and hydroxyl expansion vibration peaks at 3400 cm-1 and 1740cm-1. The relative intensity of the hydroxyl and carbonyl vibration peaks in the samples with different feeding ratios is compared with the HEMA quantity of the samples in the sample. With the increase of the content of hydroxyl group, the relative carbonyl content of.PPF-HEMA0.5, PPF-HEMA1, PPF-HEMA2 decreased with the increase of the ratio of HEMA monomers, and the swelling rate was 16%, 20% and 30% respectively. Before the swelling, the compression modulus of the other experimental and control groups was around 19 MPa except the PPF-HEMA0.5 sample (14.3 + 2.6 MPa). Under tensile mode, PPF-HE The elongation at break of MA 0.5 can reach about 20% before swelling, and the elongation of PPF-HEMA 1 and PPF-HEMA 2 can be increased from less than 10% before swelling to about 30 to 40% after swelling. Under the condition of wet cyclic loading, each component PPF-HEMA passes through 50 cycles, the sample morphology is intact, stress strain curves reclose well, stress The value is 90.1%, 82.5%, and 85.9%, respectively, and the reaction material has good fatigue resistance. Conclusion this part of the study selected HEMA as a copolymerized monomer, 1:0.5,1:1 and 1:2 three kinds of mass ratio of material, and PPF copolymerization with PPF to prepare the.PPF-HEMA crosslinking system of ductile material and change the chemical properties of the solidified material by increasing the proportion of HEMA copolymers. An elastic material with good toughness and fatigue resistance was prepared to improve the swelling properties of the material to a considerable extent. The experimental foundation for the research and application of the natural replacement material for the natural eyelid plate was established. The preparation and theory of the second part polyfumaric acid propanediol ester / hydroxyethyl methacrylate porous scaffold Studies on chemical properties and biocompatibility with good biocompatible gelatin particles as porosities to prepare porous scaffolds with moderate pore size, high porosity and good connectivity. The chemical composition of PPF-HEMA copolymers, pore structure on material degradation properties, cell adhesion, proliferation and so on are discussed at the micro level. The effect of substance activity was further verified under two dimensional culture. The applicability of the tissue engineering PPF-HEMA porous scaffold to the target cells in the repair and repair of the eyelid defect was verified. Method the porous PPF-HEMA scaffold with different pore size, different copolymerization ratio was prepared by the gelatin particle hole method, and the morphology and junction of the scaffold were observed by scanning electron microscope. The pore size of the scaffold was measured and the porosity of the porous scaffold was measured by the method of ethanol volume. The degradation performance in vitro was analyzed according to the weight loss rate of the 1,2,3,6,9 and 12 weeks scaffold. The cytotoxicity of the material was examined by HDFs cells in the leaching solution, and the PrestoBlne Kit was used to treat the two different target cells of human conjunctival stromal cells and human nodal epithelial cells on the scaffold. The cell morphology on the scaffold was observed by the specific markers of immunofluorescence staining to evaluate the biocompatibility of the porous scaffolds of the tissue engineering PPF-HEMA. Results the PPF-HEMA porous scaffolds prepared by gelatin particles 180-280 um m gelatin particles have a regular shape, and 280-450 m, 480-600 mu m gelatin micro The porous PPF-HEMA scaffolds prepared by the particles are relatively rough, and the 280-450 - m diameter of the scaffold is rough and irregular. The 480-60 - M aperture bracket even appears PPF-HEMA 0.5 prepared by the cracking.180-280 - M gelatin template. The porosity of the porous scaffold for PPF-HEMA 1 and PPF-HEMA 2 is 88.9 + 2.3%, 82.3 + 1.6% and 84.6 +, respectively, and Image J software The porosity of the commercialized ADM was 91.5 + 3.2%. The scanning electron microscope showed that the porous scaffold had a regular shape, a good pore size distribution and connectivity. In the 12 week incubation of PBS, the mass loss of the PPF-HEMA2 group was 10.9 + 1.3%, and the mass loss was only 11.4 + 0.7% in the 12 week, while PPF-HEMA0.5 and PPF-HEMA1 to 1. The 2 weeks of mass loss reached 15% and 20%.MTT method to detect different PPF-HEMA extracts and contrast blank control group. There was no significant difference in cell proliferation at all time points (P0.05). In the PPF-HEMA material system, HEMA copolymers were added by changing the hydrophilicity of the copolymer to regulate the cell compatibility of the scaffold in the PPF-HEMA material body In the system, PPF-HEMA 1 can support a certain number of CjECs proliferation, but the porous scaffolds of PPF-HEMA 0.5 and PPF-HEMA 2 have certain cell adhesion, but they do not effectively support the CjSCs growth of the target cells. Conclusion the porous scaffold with moderate pore size, high pore rate and good connectivity can be prepared by using 180-280 micron gelatin microparticles to produce porous scaffolds with good pore size, high porosity and connectivity. The mass loss of 12 weeks in vitro is less than 20% and has good stability. By changing the ratio of PPF-HEMA feeding ratio, the degradation behavior of PPF-HEMA porous scaffold can be regulated by adjusting the crosslinking density of the copolymer,.PPF-HEMA 1 can support a certain CjECs proliferation, but the PPF-HEMA0.5PPF-HEMA2 base can not support cell proliferation. Third part of the poly (fumarate) The biological effects of propanediol / hydroxyethyl methacrylate porous scaffold materials and the effect of eyelid reconstruction are studied to evaluate the biocompatibility of tissue engineered PPF-HEMA porous scaffolds in vivo, and to observe the effect of reconstruction and repair of the defects in situ for the construction of palpebral replacement materials. Reconstruction of eyelid reconstruction with tissue engineering palpebral replacement material in the clinical application of the earlier experimental basis. Method to construct a rabbit full layer blepharo defect animal model, to remove the human acellular dermal matrix as the control, implanted PPF-HEMA porous scaffold material. By observing the effect of eyelid defect repair, HE staining and Masson staining of the tissue section were used to observe the inflammatory reaction. The maneuverability and biocompatibility of PPF-HEMA porous scaffold were evaluated with material tissue reaction. Results the rabbit model of full layer blepharo defect was successfully constructed. 8 weeks after operation, the eyelid defect was completely repaired in group ADM and PPF-HEMA 2, no angular deformity was found, and mild angle deformity in group PPF-HEMA 1 could be seen, compared with negative control group. HE staining showed that there was no significant difference in the inflammatory response between the experimental group and the control group at the same time 1 weeks after the operation. The inflammatory response in each group was significantly reduced with the time of implantation, and there was a statistically significant difference between the 8 weeks after the operation and the 1 weeks after the operation. Masson staining showed that the new connective tissue in the porous scaffold of PPF-HEMA was long into the pores, and the collagen fiber bundles were wrapped around the scaffold. The thickness of ADM group, PPF-HEMA 1 and PPF-HEMA 2 groups was 28 + 9.3 mu m, 65.2 + 16.8 micron and 63.5 + 17.3 micron M., respectively. In the animal model of full layer eyelid defect in rabbits, the PPF-HEMA 2 groups showed better repair effect, lower inflammatory response and slower degradation rate, and had no significant difference compared with ADM, but the long-term stability was better. The results of this study lay the foundation for the study of natural eyelid structure and properties of polymer materials A solid experimental foundation has provided a new way for the treatment of ocular surface diseases.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R318.08

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