医用功能性可降解聚氨酯复合体系构建的研究

发布时间：2018-07-25 11:23

【摘要】：软骨组织无血管、无神经、无淋巴，在关节腔内仅靠滑液来获取营养，其代谢主要以无氧酵解为主，决定了其有限的自身修复能力。然而，现有的修复治疗技术无法实现从生物学环境及力学环境来进行构建，以适于软骨的再生修复，从而使得临床上关节软骨的修复至今难以取得突破性的进展。根据生物学环境及力学环境来重构是未来软骨再生修复的重要研究方向，而如何实现以可降解材料为基础构建软骨再生的生物学环境及力学环境是未来软骨修复材料研究的重点与热点。本研究从上述角度出发，合成了一种新型的功能性医用可降解聚氨酯（PU），在此基础上，通过改变合成PU的软硬段比例构建了不同模量的PU材料，然后采用相转变-粒子沥滤法制备了不同孔结构特性的PU多孔支架，以满足骨和软骨再生对力学环境要求的不同。同时，通过表面改性的方法构建了适于软骨及骨再生的PU表面微环境以及通过复合的方法构建了适于软骨及骨再生的功能性微球/PU复合支架,以此来满足骨和软骨再生对生物学环境要求的不同，期望应用于一体化关节软骨组织工程。在构建不同弹性模量的PU材料上，以赖氨酸二异氰酸乙酯为硬段，平均分子量为2000的聚己内酯二醇为软段，具有药理活性的异山梨醇为扩链剂，采用不同的软硬段比例合成了不同模量的PU。利用FTIR、1H-NMR、GPC、XRD、DSC对合成的聚合物进行了表征。FTIR、1H-NMR结果表明合成的聚合物的结构是典型PU的结构；GPC测试结果显示聚合物的数均分子量超过5万，分布指数在1.6~2.0，分子量分布较窄；DSC分析结果显示软段比例较大合成得到的聚合物在42℃存在着结晶熔融峰，而硬段比例较大合成得到的聚合物，没有结晶熔融峰；而XRD结果证明合成的聚合物存在部分结晶，但是结晶不是很完整。通过材料的拉伸力学试验，结果证明合成得到的PU材料具有很好的弹性，其断裂伸长率超过700%；同时其酶解性能也表明，相比较传统的PLGA材料的降解性能，合成得到的PU具有更慢的降解速度，且降解后溶液呈弱碱性，表现出更加理想的降解特性。该PU材料，可以满足软骨组织工程需要承受一定的负荷的要求，且解决工程支架植入到体内与自体组织相连不紧密，而在界面上产生剪切力造成植入体与自体相分离的问题，同时不至于因为材料降解而产生酸性积累，导致无菌性炎症的发生。潜在应用于骨及软骨组织工程。在构建适于软骨及骨再生的表面微环境上，对PU进行了一系列的表面改性。首先利用1,3-丙二胺与PU链上的酯基基团发生胺解反应，在PU的表面形成游离的胺基，然后利用产生的胺基，一是通过与Ⅰ型胶原在EDC/NHS的作用下进行化学反应，使材料的表面接枝上胶原，构建骨再生的微环境，有利于骨细胞的增殖和分化；二是将游离的胺基酸化，使材料表面带正电荷，再通过静电作用力，在材料表面进行层层自组装硫酸软骨素和Ⅰ型胶原，构建软骨再生的微环境。RBITC-Col、QCM、XPS和AFM测试结果证明胶原和硫酸软骨素成功地吸附在PU的表面，使材料的表面变得更平整，形成比较均一的纳米级形貌结构，这样的一个表面纳米结构应该有利于细胞的粘附，促进细胞的增殖和分化。在不同孔结构特性的PU支架制备上，采用相转变-粒子沥滤法，通过改变良溶剂和不良溶剂的比例以及造孔剂的比例等来控制PU支架的孔径、分布、孔之间的连通性以及支架的力学性能等。结果表明添加了不良溶剂和造孔剂制备得到的PU三维支架是由大小不同的孔构成，孔与孔之间连通性较好，大孔孔径可达几百微米，孔隙率超过75%，可以满足细胞在支架上生长、增殖的需要；同时，支架的抗压性能较好，具有较好的形变回复能力，通过加入不良溶剂得到的三维支架在压缩过程中不会发生崩塌的现象。当PU溶液的浓度为14.5%，良溶剂和不良溶剂的比例为2:1，造孔剂与PU的质量比为5:1时，且在37℃进行干燥除去溶剂制备得到的PU三维多孔支架的性能较好。此时得到的支架的孔隙率为84.2%，孔径大于100μm所占的比例为87.5%，且孔之间的连通性较好；压缩应变为20%时的抗压强度为0.31MPa，满足软骨组织工程的力学性能要求。在构建适于软骨及骨再生的功能性微球/PU复合支架上，分别采用乳化法制备了明胶/肝素微球和双乳化溶剂挥发法制备了内部具有多孔结构的PLGA/氧氟沙星载药微球。明胶浓度，乳化剂浓度，水油比对明胶/肝素微球的粒径和分布有较大的影响。通过明胶微球包裹肝素，为明胶微球吸附bFGF提供活性位点，尽量保持bFGF的活性，构建软骨再生的缓释结构体系。而在PLGA/氧氟沙星载药微球制备中，在内水相中加入介孔二氧化硅、透明质酸、多聚赖氨酸对微球的粒径、分布及载药效率和释放都有影响。内水相中添加剂的物理吸附作用和静电吸引作用可以改善高亲水性药物在内水相中的留存量，并提高药物的包封率，但静电作用也可能会影响表面活性剂的乳化效果，破坏乳液的稳定性，造成较低的包封率。内水相中添加剂的亲水性的增加改善了高分子材料整体的亲水性，提高了亲水性药物在微球表面的吸附率，造成初期爆释较高。通过PLGA微球包裹氧氟沙星，构建骨再生的缓释结构体系。通过制得的功能性微球与PU三维多孔支架相复合，考察微球在PU支架中的分布，结果表明微球较均匀地分布在支架的孔壁和孔的里面，说明了这一功能性PU复合体系构建的可行性。通过PU材料的生物相容性及PU材料对滑膜干细胞分化为软骨细胞实验，，对PU材料的生物学性能进行了评价。结果表明不管是PU材料还是PU自组装胶原/硫酸软骨素材料，两者都没有毒性或者毒性很小，且支持细胞的生长和增殖。相比较单纯的PU材料，在PU材料表面组装上胶原和硫酸软骨素更有利于滑膜干细胞的生长及向软骨细胞分化。未来，单一的生物材料在复杂组织的再生中将难以起主导作用。把各种信号因子复合在材料主体结构上，以实现材料体系的多功能、多效用，将成为组织工程材料构建的发展趋势。本研究从力学环境和生物学环境角度构建了功能性PU复合支架材料体系，为一体化软骨组织工程的开发应用奠定了基础，为未来多功能复合支架材料的研究提供了一定的参考依据。
[Abstract]:The cartilage tissue has no blood vessel, no nerve, no lymph, and is only dependent on the synovial fluid to obtain nutrition in the articular cavity. Its metabolism is mainly based on anaerobic fermentation, which determines its limited self repair ability. However, the existing repair and treatment technology can not be constructed from the biological environment and the mechanical environment to fit for the regeneration of cartilage. It is difficult to make a breakthrough in the repair of articular cartilage in clinical. Reconstruction is an important research direction in the future of cartilage regeneration based on the biological environment and mechanical environment. How to build the biological environment and mechanical environment on the basis of biodegradable materials to build cartilage regeneration is the key point in the study of cartilage repair materials in the future. And hot spots.
In this study, a new functional medical degradable polyurethane (PU) was synthesized. On this basis, different moduli of PU materials were constructed by changing the proportion of soft and hard segments of synthetic PU. Then phase transition particle leaching method was used to prepare PU porous scaffolds with different pore structure properties to meet the regenerative force of bone and cartilage. At the same time, the PU surface microenvironment suitable for cartilage and bone regeneration is constructed by surface modification, and a functional microsphere /PU composite scaffold suitable for cartilage and bone regeneration is constructed by compound method, in order to meet the different biological environment requirements for bone and cartilage regeneration, and be expected to be applied to integration. Articular cartilage tissue engineering.
On the PU materials with different modulus of elasticity, the lysine two isocyanate was used as the hard segment and the average molecular weight was 2000. The pharmacologically active isosorbide was used as the chain extender. The PU. of different moduli was synthesized by different soft and hard segments, and the synthesized polymers were made by FTIR, 1H-NMR, GPC, XRD, DSC. The results of.FTIR, 1H-NMR show that the structure of the synthesized polymer is the structure of the typical PU; the results of GPC test show that the average molecular weight of the polymer is more than 50 thousand, the distribution index is 1.6~2.0, the distribution of the molecular weight is narrow, and the result of DSC analysis shows that the polymer obtained by the larger proportion of the soft segments has the crystallization melting peak at 42, and the ratio of the hard segment. The polymer obtained by the larger synthesis did not crystallize the melting peak, and the XRD results showed that the synthesized polymer was partially crystallized, but the crystallization was not very complete. Through the tensile mechanical test of the material, the results showed that the synthesized PU material had good elasticity, its elongation at break was over 700%, and its enzymatic properties also showed that compared with the results of its enzymatic hydrolysis, Compared with the degradation performance of the traditional PLGA material, the synthesized PU has a slower degradation rate, and the solution is weak alkaline after degradation, showing a more ideal degradation characteristic. The PU material can meet the requirement of the cartilage tissue engineering to bear a certain load, and the solution of the scaffold is not closely connected with the autograft in the body. The shear force on the interface causes the separation of the implant from the autologous phase, and it does not produce acid accumulation because of the degradation of the material, which leads to the occurrence of aseptic inflammation. It is potentially used in bone and cartilage tissue engineering.
In the construction of a surface microenvironment suitable for cartilage and bone regeneration, a series of surface modification has been carried out on PU. First, the amine solution of 1,3- propyl two amine and ester group on PU chain is used to form a free amino group on the surface of PU, and then the produced amine group is used, one is by chemical reaction with type I collagen under the action of EDC/NHS. The surface of the material is grafted with collagen, and the microenvironment of bone regeneration is constructed, which is beneficial to the proliferation and differentiation of the bone cells. Two the free amino group is acidified, the surface of the material is positively charged, and the electrostatic force is used to build the self assembled chondroitin sulfate and type I gluin on the surface of the material, and the microenvironment.RBITC-Col, QCM, XPS of the cartilage regeneration is constructed. The results of the AFM test showed that collagen and chondroitin sulfate were successfully adsorbed on the surface of PU, making the surface of the material more smooth and forming a relatively uniform nanoscale structure. Such a surface nanostructure should be beneficial to cell adhesion, promote cell proliferation and differentiation.
In the preparation of PU stents with different pore structure characteristics, the phase transition particle leaching method was used to control the pore size, distribution, connectivity between the holes and the mechanical properties of the scaffolds by changing the proportion of good solvent and bad solvent and the proportion of pore forming agent to control the mechanical properties of the scaffolds. The results showed that the PU obtained by the bad solvent and pore making agent was added to the PU. Three dimensional scaffolds are composed of different sizes of holes, with good connectivity between holes and holes, a large pore diameter of up to a few hundred microns and a porosity of more than 75%, which can meet the needs of cell growth and proliferation on the scaffold. At the same time, the compression performance of the scaffold is good, and the three-dimensional scaffold obtained by adding bad solvent is pressed. When the concentration of the PU solution is 14.5%, the proportion of the good solvent and the bad solvent is 2:1, the mass ratio of the pore forming agent to PU is 5:1, and the performance of the PU three-dimensional porous scaffold obtained by removing the solvent at 37 C is better. The porosity of the support frame is 84.2%, the pore size is more than 100 mu m. The compressive strength is 0.31MPa when the compressive strain is 20%, which meets the requirements of mechanical properties of cartilage tissue engineering.
In the construction of a functional microsphere /PU composite scaffold suitable for cartilage and bone regeneration, gelatin / heparin microspheres and double emulsified solvent evaporation method were prepared by emulsification. The concentration of gelatin, the concentration of emulsifier, and the ratio of water to oil to gelatin / heparin microspheres were larger than those of the gelatin / heparin microspheres. By wrapping the heparin with gelatin microspheres, the active site was provided for the gelatin microsphere to adsorb bFGF, and the activity of bFGF was maintained as far as possible to construct the sustained release structure of cartilage regeneration. In the preparation of PLGA/ ofloxacin microspheres, mesoporous silica, hyaluronic acid and polylysine were added to the internal aqueous phase, and the particle size, distribution and drug loading of polylysine were added to the microspheres. The physical adsorption and electrostatic attraction of additives in the internal water phase can improve the retention of high hydrophilic drugs in the internal water phase and increase the encapsulation efficiency of the drugs, but the electrostatic effect may also affect the emulsifying effect of the surfactant, destroy the stability of the emulsion, and cause the lower encapsulation efficiency. The hydrophilicity of the additive improves the hydrophilicity of the polymer as a whole, improves the adsorption rate of the hydrophilic drug on the surface of the microspheres, and causes a high initial detonation. Through the encapsulation of ofloxacin by PLGA microspheres, the sustained release structure of bone regeneration is constructed. The functional microspheres are combined with the PU three-dimensional porous scaffold. The distribution of microspheres in the PU scaffold shows that the microspheres are distributed evenly in the pores and holes of the scaffolds, indicating the feasibility of this functional PU complex system.
The biological properties of PU materials were evaluated by the biocompatibility of PU materials and the differentiation of synovial stem cells into chondrocytes by PU materials. The results showed that both PU and PU self assembled collagen / chondroitin sulfate have no toxicity or small toxicity, and support cell growth and proliferation. Collagen and chondroitin sulfate assembled on the surface of PU are more conducive to the growth and differentiation of synovial stem cells into chondrocytes.
In the future, a single biological material will be difficult to play a leading role in the regeneration of complex tissues. Combining various signal factors on the material body structure to realize the multi-function and utility of the material system will become the development trend of the construction of tissue engineering materials. This study has constructed the functional PU complex from the angle of mechanical and biological environment. The scaffold material system has laid a foundation for the development and application of the integrated cartilage tissue engineering, and provides some reference for the future research of multi-functional composite scaffold materials.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08;TQ323.8

【参考文献】