聚氨酯形状记忆过程中相分离微纳结构与回复应变对细胞形态的调控研究

发布时间：2018-05-20 13:53

本文选题：聚氨酯 + 形状记忆过程　；参考：《重庆大学》2016年博士论文

【摘要】：形状记忆聚氨酯(SMPU)具有良好的生物相容性、力学性能和形状记忆性能,在生物医学领域备受关注。SMPU软段与硬段因热力学不相容而形成的相分离微纳结构是其具有形状记忆效应(SME)的结构基础,而赋形、固定和形状回复则是其体现SME的必需过程。赋形和回复这两个动态过程可改变相分离微纳结构;回复过程还可产生回复应变。为了更深入地理解SME对SMPU生物相容性的影响机制,指导SMPU的结构设计和加工,需深入认识SMPU形状记忆过程中相分离微纳结构的变化及其与回复应变对细胞行为的调控。但迄今为止,尚缺乏相关研究。本文采用广角X射线衍射(WAXD)、小角X射线散射(SAXS)、傅里叶全反射红外光谱(ATR-FTIR)、差示扫描量热(DSC)、原子力显微镜(AFM)、扫描电镜(SEM)等技术考察了拉伸赋形与形状回复过程对SMPU薄膜相分离微纳结构的影响及影响机制,并以成骨细胞为模型细胞考察了微纳结构与回复应变对细胞形态的调控作用。主要研究内容和结论如下:(1)拉伸赋形对SMPU薄膜微纳结构的影响本文采用软段为聚乳酸-聚乙二醇三嵌段共聚物(PDLLA-PEG-PDLLA),硬段为六亚甲基二异氰酸酯(HDI)和哌嗪(PPZ)的嵌段SMPU作为模型材料,以聚(DL-乳酸)(PDLLA)为对照材料,采用溶液浇注法制备SMPU和PDLLA薄膜,考察拉伸赋形(拉伸率为0%,50%,100%,200%)对SMPU相分离微纳结构的影响,为深入理解赋形对细胞形态的调控及调控机理奠定基础。(1)未赋形SMPU薄膜具有明显的相分离结构,硬段逐级聚集形成硬段微区、独立“岛”结构和纳米纤维,在软段相中形成无序的硬段相化学“图案”;而对照材料PDLLA薄膜表面均一、平滑,仅出现因微弱结晶而导致的相分离现象。(2)拉伸赋形使光滑的PDLLA表面出现了垂直于拉伸方向排列的纳米突起。而在SMPU表面,拉伸赋形导致硬段微区与“岛”结构的长轴垂直于拉伸方向并且沿拉伸方向聚集,最终导致纳米纤维平行于拉伸方向,表明拉伸赋形可以改变SMPU的表面微纳结构并提高纳米纤维的有序性,提示对SMPU拉伸赋形可望成为一种制备有序化学“图案”的新方法。(2)拉伸赋形所致相分离微纳结构对细胞粘附与蛋白吸附的影响分别以成骨细胞和纤连蛋白(Fn)为模型细胞和模型蛋白,采用免疫荧光染色技术考察拉伸诱导的微纳结构对细胞形态与蛋白吸附的影响。结果显示:细胞长轴与Fn组装的蛋白纤维在SMPU薄膜表面均平行于拉伸方向,而在PDLLA薄膜表面则均垂直于拉伸方向,表明拉伸诱导的表面微纳结构可以调控细胞取向与蛋白组装,提示赋形处理可影响SMPU材料-细胞相互作用。同时,该结果进一步证实,拉伸赋形可成为一种制备有序化学“图案”以调控细胞行为的新方法。(3)回复介质对薄膜形状回复与微纳结构的调控回复过程是SMPU体现形状变化的过程,在医学应用中通常是在体液环境中发生。考察体液回复环境对SMPU相分离微纳结构和形状回复的影响及其机制,可为全面、深入地理解SMPU生物相容性提供理论基础,也可指导SMPU医学应用方案的制定。因此,本研究将拉伸赋形SMPU薄膜两端固定以保持应变恒定,将其置于32°C空气或细胞培养液DMEM中静置24h;随后,去除薄膜两端固定装置并分别升高空气或DMEM的温度使薄膜自由回复。通过考察介质环境对薄膜形状回复和表面微纳结构的影响,探讨体液环境影响SMPU形状记忆过程的相关机理。“受限”处理旨在使薄膜经历薄膜-细胞复合物在形状回复之前的处理过程(详见(4)),以便更准确地理解材料回复对细胞行为的调控机制。(1)定量检测了回复力和回复率的动态变化,发现薄膜在前20 min回复较快,在2 h内方可完成回复。“受限”处理对拉伸赋形薄膜的表面微纳结构无显著影响,但会因为应力松弛而降低薄膜形状回复力和回复率。薄膜在DMEM中的形状回复力和回复率明显低于空气中,DSC分析表明其机制与水分子增塑SMPU链段而导致应力松弛增强有关。上述结果表明,介质环境对形状回复性能有明显影响。(2)在空气中回复时,薄膜表面的硬段组装未完全回复到无序状态,多数纳米纤维与拉伸方向呈40°夹角排列,这与应力松弛导致的薄膜形状未完全回复有关;而在DMEM中回复时,薄膜表面硬段聚集增强,出现明显的纤维状和点状硬段微区,且纳米纤维与拉伸方向间的夹角也多高于40°,这可能与水分子增塑引起的不完全回复以及水分子与软段形成的氢键作用有关。上述结果表明,回复介质明显影响SMPU薄膜的表面微纳结构,这为后续深入理解回复过程对细胞行为的影响奠定了基础。(4)薄膜形状回复应变与微纳结构对细胞形态的调控回复应变可能对细胞施加持续力学刺激。因此,考察回复应变对细胞行为的调控作用,可为SMPU生物相容性的评价提供一条基于生物力学的新思路。为考察回复应变和微纳结构对细胞形态的调控作用,本研究将成骨细胞接种于拉伸赋形SMPU薄膜表面,于32°C下培养24h后去除薄膜两端固定装置,并将薄膜-细胞复合物转入37°C细胞培养箱中让薄膜自由回复。利用活细胞工作站追踪细胞对薄膜形状回复的响应,并用免疫荧光染色技术观察薄膜回复后成骨细胞形态随培养时间的变化,利用CCK-8技术初步考察薄膜形状回复对成骨细胞后期(1-7d)增殖行为的影响。(1)SMPU薄膜回复的总应变随拉伸率增加而增大,拉伸率为50%、100%和200%时其回复总应变(%)分别为17.55±3.34、27.87±2.93和34.80±2.60。(2)活细胞工作站和免疫荧光染色结果显示:在薄膜回复初期(≤4h),SMPU回复应变使成骨细胞趋于垂直于拉伸方向排列,且细胞取向变化与拉伸率有关,表明SMPU回复应变作为一种力刺激可以调控成骨细胞形态。(3)SMPU薄膜形状回复24h后,细胞取向与薄膜表面纳米纤维取向一致,表明随着细胞培养时间的延长,回复应变对细胞取向的调控作用会逐渐被基底微纳结构所取代。(4)CCK-8检测结果显示,薄膜形状回复后1-7天内,回复薄膜表面的细胞数明显高于未回复薄膜表面。该结果表明,回复应变可以作为一种力学刺激调控细胞形态并促进细胞增殖,SMPU可望用作一种新型的动态细胞培养基底。综上所述,本论文在系统考察赋形、回复和回复介质对SMPU相分离微纳结构调控的基础上,揭示了SMPU形状记忆过程中其微纳结构与回复应变对细胞形态的调控及相关机理,为深入理解SMPU生物相容性提供了理论基础,相关研究结果对SMPU材料的设计与加工具有明显的指导意义。同时,本论文也初步验证了SMPU在有序图案化加工技术和动态细胞培养系统中的潜在应用价值。
[Abstract]:Shape memory polyurethane (SMPU) has good biocompatibility, mechanical properties and shape memory properties. In the biomedical field, the phase separation micro nano structure formed by the thermodynamic incompatibility between the.SMPU soft segment and the hard segment is the structural basis of the shape memory effect (SME), while the shape, the fixed and the shape recovery are the embodiment of the SME. The two dynamic processes can change the phase separation micro nano structure, and the recovery process can also produce the response strain. In order to understand the mechanism of the effect of SME on the biocompatibility of SMPU, and to guide the structure design and processing of SMPU, it is necessary to understand the changes of the phase separation microstructure in the memory process of the SMPU shape and its relationship. Back strain regulation on cell behavior. But so far, there is a lack of relevant research. In this paper, wide angle X ray diffraction (WAXD), small angle X ray scattering (SAXS), Fourier total reflection infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), atomic force microscopy (AFM), scanning electron microscopy (SEM) and other techniques have been used to investigate the process of shape and shape recovery. The influence and influence mechanism of SMPU thin film phase separation micro nano structure and the effect of osteoblast on the regulation of cell morphology by micro nano structure and response strain. The main research contents and conclusions are as follows: (1) the effect of stretching on the microstructure of SMPU thin films in this paper is the soft segment of polylactic acid polyethylene glycol block copolymerization The hard segment (PDLLA-PEG-PDLLA), the block SMPU of six methylene diisocyanate (HDI) and piperazine (PPZ) was used as model material, and SMPU and PDLLA thin films were prepared by solution pouring method with poly (DL- lactic acid) (PDLLA) as the control material. The effect of tensile morphology (tensile rate of 0%, 50%, 100%, 200%) on the microstructure of SMPU phase separation was investigated. The shape has laid the foundation for the regulation and regulation mechanism of cell morphology. (1) the unshaped SMPU film has a distinct phase separation structure, hard segments gather to form hard segments, independent "island" structure and nanofibers, forming a disordered hard phase chemical "pattern" in the soft segment, while the surface of the control material PDLLA film is homogeneous and smooth. The phenomenon of phase separation caused by weak crystallization. (2) stretching and forming the smooth surface of the PDLLA surface appears to be a nano projection perpendicular to the direction of tension. On the SMPU surface, the stretch forming causes the hard segment and the long axis of the "island" structure to be perpendicular to the tensile direction and gather along the tensile direction, eventually leading to the nanofibers parallel to the direction of tension. It is indicated that stretching can change the surface microstructure of SMPU and improve the order of nanofibers. It is suggested that the shape of SMPU can be a new method to prepare the ordered chemical "pattern". (2) the effects of the phase separation microstructures on the cell adhesion and protein adsorption are osteoblasts and fibronectin (Fn), respectively. The effects of tensile induced micro nanostructures on cell morphology and protein adsorption were investigated by immunofluorescence staining. The results showed that the protein fibers assembled by the long axis of the cell and Fn were parallel to the direction of tension on the surface of the SMPU film, while the surface of the PDLLA film was perpendicular to the tensile direction, indicating that the tensile induction was induced. Surface microstructures can regulate cell orientation and protein assembly, suggesting that shaped treatment can affect the interaction between SMPU materials and cells. At the same time, the result further confirms that stretching can be a new method to prepare ordered chemical "patterns" to regulate cell behavior. (3) the control of the shape recovery and microstructure of the membrane by the recovery medium. The process of recovery is the process of shape change in SMPU, which usually occurs in the body fluid environment in medical applications. The effect of the body fluid recovery environment on the SMPU phase separation and the shape recovery and its mechanism can provide a theoretical basis for comprehensive and in-depth understanding of the biocompatibility of SMPU, and also guide the formulation of the SMPU medical application. Therefore, the tension - shaped SMPU film is fixed at both ends to keep the strain constant, and 24h is placed in the 32 degree C air or cell culture solution DMEM. Then, the film is removed and the temperature of the air or DMEM is elevated to the free recovery of the film. The film shape recovery and the surface micronano structure are investigated by the investigation of the medium environment. The influence of the body fluid environment on the SMPU shape memory process. "Limited" treatment aims to make the film undergo the process of the membrane cell complex before the shape recovery (4), in order to more accurately understand the regulation mechanism of the material response to the cell behavior. (1) the quantitative detection of the dynamic changes in the recovery and recovery rate It is found that the film has a fast recovery in the first 20 min and can complete the recovery in 2 h. "Limited" treatment has no significant effect on the surface microstructure of the stretched thin film, but it will reduce the shape recovery force and recovery rate because of the stress relaxation. The shape recovery force and recovery rate of the thin film in DMEM are obviously lower than that in the air, and the DSC analysis shows that the mechanism of the film is lower than that in the air. The results show that the medium environment has an obvious influence on the shape recovery performance. (2) the hard segment assembly of the surface of the film is not completely recovered to the disorder state, and most nanofibers are arranged in a 40 degree angle in the direction of tensile, which is caused by the stress relaxation in the air. (2) in the air, the hard segment assembly of the film surface is not completely recovered to the disordered state. The film shape is not fully recovered, but when the DMEM is recovered, the hard segment aggregation of the film surface is enhanced, and the fiber and spot hard segments appear obviously, and the angle between the nanofibers and the tensile direction is more than 40 degrees. This may be related to the incomplete recovery caused by the plasticization of water molecules and the hydrogen bond formed by the water molecules and the soft segments. The results show that the recovery medium obviously affects the surface micro nano structure of SMPU film, which lays the foundation for further understanding the effect of the recovery process on the cell behavior. (4) the response strain of the film shape recovery and the response strain of the microstructures to the cell morphology may be stimulated by the continued mechanical stimulation of the cells. Therefore, the response strain to the cells is investigated. The regulation of behavior can provide a new idea based on biomechanics for the evaluation of biocompatibility of SMPU. In order to investigate the regulation of response strain and micro nano structure on the cell morphology, the osteoblasts were inoculated on the surface of the stretched SMPU thin film. After the culture of 24h under 32 degree C, the two ends fixtures were removed and thin film thin films were removed. The cell complex was transferred into the 37 degree C cell culture box to make the film free recovery. The response of the cells to the film shape recovery was traced by the live cell workstation. The changes of the morphology of the osteoblasts were observed with the immunofluorescence staining technique, and the CCK-8 technique was used to examine the shape recovery of the film to the later stage of osteoblast (1-7d). The effect of proliferation behavior. (1) the total strain of SMPU film was increased with the increase of tensile rate, and the total strain was 50%, 100% and 200% (%) were 17.55 + 3.34,27.87 + 2.93 and 34.80 + 2.60. (2), respectively, and the results of immunofluorescence staining showed that at the early stage of the film recovery (less than 4h), the SMPU response strain made osteoblasts. The change of the cell orientation was related to the tensile rate in the vertical direction. It showed that the SMPU response strain could regulate the morphology of osteoblasts as a kind of force stimulation. (3) after the shape of SMPU film returned to 24h, the orientation of cell orientation was consistent with the orientation of nanofiber on the surface of the film. The control effect is gradually replaced by the basal microstructure. (4) the results of CCK-8 detection show that the number of cells on the surface of the film is significantly higher than that of the unrecovered membrane within 1-7 days after the shape of the film is restored. The results show that the response strain can be used as a mechanical stimulus to regulate cell morphology and promote cell proliferation, and SMPU is expected to be used as a new kind of new type of cell. On the basis of the systematic investigation of the regulation of the SMPU phase separation structure, this paper reveals the regulation and mechanism of the micro nano structure and the response strain on the cell morphology in the process of SMPU shape memory, and provides a theoretical basis for understanding the biocompatibility of SMPU in depth. The relevant research results have obvious guiding significance for the design and processing of SMPU materials. At the same time, this paper also preliminarily verifies the potential application value of SMPU in the ordered patterned processing technology and the dynamic cell culture system.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08

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