聚乳酸纳米纤维支架的构建与改性及其纳米力学性能研究
发布时间：2018-07-18 13:22
【摘要】：组织工程支架在再生医学中发挥了举足轻重的作用，其目的是修复和重建缺失或功能损伤的组织器官。支架可以为组织再生提供三维模板和人工细胞外基质环境。而通过模拟天然细胞外基质的结构(如细胞外基质组分中胶原的纳米纤维结构)以及优良的表面性能来构建支架，是组织工程非常重要的研究领域。 本研究通过热致相分离法制得了具有高孔隙率和高比表面积的三维聚乳酸(PLLA)纳米纤维支架。对热致相分离过程早期即PLLA纳米纤维成型过程中的结构演变以及纳米尺度的性能变化规律进行了研究。利用扫描电子显微镜、X射线衍射、红外光谱、差示扫描量热法、X射线光电子能谱、接触角测试、原子力显微镜以及力谱测试等对其形貌、相转变、结晶行为、表面化学结构、表面性能、亲疏水性能、高分子链的构象、表面粘附力以及弹性模量的变化规律进行了研究。通过引入柠檬酸(CA)和氧等离子体处理来调节支架的表面性质，并研究了其对细胞行为的影响。 热致相分离法主要包括以下四个步骤：PLLA的溶解、冷冻凝胶化、溶剂置换和真空冷冻干燥。热致相分离过程是由温度决定的，首先形成的是由纳米颗粒缩合而成的无定形沉积物，随后PLLA成核结晶。在冷冻凝胶化不充分的情况下，如冷冻1min，温度由60°C降至15°C的情况下形成的是α-型晶体，构成片状网络结构；而冷冻凝胶化足够时间、温度降至0°C及以下时，形成较无序的α′-型晶体且温度越低含量越高，形态为纳米纤维结构并逐渐均一化。最终的PLLA纳米纤维的直径分布比较均匀，尺寸在150-350nm范围内，这与天然胶原纤维的尺寸相近。随着冷冻凝胶化时间的递增，PLLA样品的结晶度逐渐增大，，且高分子链的结构发生了重排，排列变得更加紧密、有序。PLLA纳米纤维成型过程中，结构的重排导致非极性的甲基在表面富集，使得接触角增大、表面疏水性增强。 通过在生理盐水和磷酸盐缓冲液中进行的体外水解降解实验以及细胞生物学实验对具有不同凝胶化时间的PLLA支架的生物降解性能和生物相容性进行表征。PLLA纳米纤维成型过程中的形貌、结晶行为、化学结构、表面性能以及高分子链构象的变化和其亲疏水性、降解性能以及生物相容性具有直接的关系。PLLA的结晶以及疏水性的增强不利于PLLA支架的水解降解，但是PLLA纳米纤维支架更适于细胞的黏附和增殖。 利用AFM、TEM等对PLLA纳米纤维的成型机理以及微观结构做了进一步的研究，并通过AFM力谱测试、纳米压痕、纳米刻蚀等功能对PLLA纳米纤维成型过程中以及形变前后的纳米力学性能(粘附力、弹性模量等)的变化规律进行了分析。PLLA溶液通过液-液相分离形成富-高分子相和富-溶剂相，随后在富-高分子相中成核结晶形成纳米颗粒状晶体，颗粒状晶体有序排列形成了尺寸较小的纤维单元(70-100nm)，最后较小的纤维再横向自组装形成完整的PLLA纳米纤维。PLLA的表面结构(如高分子链的构象以及排列方式)等的变化和其纳米力学性能有直接的关系。PLLA纳米纤维由于高分子链排列更紧密、更有序，因而具有更高的表面粘附力以及弹性模量。利用纳米刻蚀技术在单根纳米纤维表面进行划痕，产生的形变由挤压应力和剪切应力共同作用，分别使PLLA纳米纤维的高分子链紧密排列和取向有序排列。形变后的表面粘附力和弹性模量都有明显的增大。 为了改善PLLA纳米纤维支架的表面亲疏水性，进一步提高其细胞亲和性，在热致相分离法制备三维PLLA纳米纤维支架的基础上，通过引入CA和氧等离子体处理两种方法对PLLA支架进行表面改性。并对改性前后PLLA支架的形貌、物理化学结构、表面性能的变化进行表征，并通过细胞生物学评价(细胞培养与种植、细胞黏附与增殖、细胞活性、成骨诱导、基因表达等)对表面改性后的PLLA支架的细胞相容性及其对干细胞原位诱导的影响进行了研究。表面改性后，都引入了强极性的 COOH基团，使得PLLA支架的接触角大幅度降低，改善了其亲水性，使其处于最适于细胞培养的范围内(40-80°)。表面改性提高了PLLA支架的细胞亲和性，促进了mBMSCs细胞的黏附、铺展，且有大量细胞触角和纳米纤维相结合。细胞的增殖速率和活性也有明显的提高。在成骨诱导实验中，从碱性磷酸酶染色和茜素红染色观察到改性前后的PLLA支架上的细胞都分泌了碱性磷酸酶和钙结晶，而改性后的支架上细胞的碱性磷酸酶活性有显著的提高。通过RT-PCR基因表达实验对主要的成骨标识物(ALP、COL、OCN和Cbfa-1/Runx-2)在分子水平上的表达进行检测，并证明改性后的PLLA支架更能促进这些成骨基因的表达。由此可以得出：所有的PLLA支架都适于mBMSCs细胞的成骨诱导，且在表面改性后的PLLA支架上成骨诱导效果更好。
[Abstract]:Tissue engineering scaffolds play a pivotal role in regenerative medicine with the aim of repairing and reconstructing tissue organs that are missing or functionally damaged . The scaffold can provide three - dimensional templates and an artificial extracellular matrix environment for tissue regeneration . The scaffold can be constructed by simulating the structure of natural extracellular matrix , such as the nanofiber structure of collagen in extracellular matrix components , and excellent surface properties , and is a very important field of research in tissue engineering .
The changes of morphology , phase transition , crystal behavior , surface chemical structure , surface properties , hydrophobic properties , conformation of polymer chain , surface adhesion and elastic modulus were studied by scanning electron microscope , X - ray diffraction , infrared spectrum , differential scanning calorimetry , X - ray photoelectron spectroscopy , contact angle test , atomic force microscope and force spectrum test . The effects of citric acid ( CA ) and oxygen plasma treatment on the surface properties of scaffolds were studied .
The thermal - induced phase separation method mainly comprises the following four steps : dissolving , freezing gelation , solvent replacement and vacuum freeze drying in the following four steps : firstly , forming an amorphous deposit formed by condensation of nano particles , forming amorphous deposits formed by condensation of nano particles , and then nucleating and crystallizing .
The results showed that the crystallinity of the polymer chains increased gradually with the increasing of the gelation time , and the arrangement became more compact and orderly . During the formation of the polymer chains , the rearrangement of the structure led to the accumulation of the nonpolar methyl groups on the surface , so that the contact angle increased and the surface hydrophobicity was enhanced .
The biodegradation performance and biocompatibility were characterized by in vitro hydrolytic degradation experiment and cell biology experiment in physiological saline and phosphate buffer . The morphology , crystallization behavior , chemical structure , surface property and polymer chain conformation were directly related to the changes of morphology , crystallization behavior , chemical structure , surface property and polymer chain conformation .
In this paper , the mechanism of nano - fiber formation and its microstructure were studied by AFM , TEM , etc . The changes of mechanical properties ( adhesion , elastic modulus , etc . ) were studied by AFM , nano - indentation and nano - etching .
涓轰簡鏀瑰杽PLLA绾崇背绾ょ淮鏀灦鐨勮〃闈翰鐤忔按鎬
本文编号：2132029
[Abstract]:Tissue engineering scaffolds play a pivotal role in regenerative medicine with the aim of repairing and reconstructing tissue organs that are missing or functionally damaged . The scaffold can provide three - dimensional templates and an artificial extracellular matrix environment for tissue regeneration . The scaffold can be constructed by simulating the structure of natural extracellular matrix , such as the nanofiber structure of collagen in extracellular matrix components , and excellent surface properties , and is a very important field of research in tissue engineering .
The changes of morphology , phase transition , crystal behavior , surface chemical structure , surface properties , hydrophobic properties , conformation of polymer chain , surface adhesion and elastic modulus were studied by scanning electron microscope , X - ray diffraction , infrared spectrum , differential scanning calorimetry , X - ray photoelectron spectroscopy , contact angle test , atomic force microscope and force spectrum test . The effects of citric acid ( CA ) and oxygen plasma treatment on the surface properties of scaffolds were studied .
The thermal - induced phase separation method mainly comprises the following four steps : dissolving , freezing gelation , solvent replacement and vacuum freeze drying in the following four steps : firstly , forming an amorphous deposit formed by condensation of nano particles , forming amorphous deposits formed by condensation of nano particles , and then nucleating and crystallizing .
The results showed that the crystallinity of the polymer chains increased gradually with the increasing of the gelation time , and the arrangement became more compact and orderly . During the formation of the polymer chains , the rearrangement of the structure led to the accumulation of the nonpolar methyl groups on the surface , so that the contact angle increased and the surface hydrophobicity was enhanced .
The biodegradation performance and biocompatibility were characterized by in vitro hydrolytic degradation experiment and cell biology experiment in physiological saline and phosphate buffer . The morphology , crystallization behavior , chemical structure , surface property and polymer chain conformation were directly related to the changes of morphology , crystallization behavior , chemical structure , surface property and polymer chain conformation .
In this paper , the mechanism of nano - fiber formation and its microstructure were studied by AFM , TEM , etc . The changes of mechanical properties ( adhesion , elastic modulus , etc . ) were studied by AFM , nano - indentation and nano - etching .
涓轰簡鏀瑰杽PLLA绾崇背绾ょ淮鏀灦鐨勮〃闈翰鐤忔按鎬
本文编号：2132029
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