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Preparation And Evaluation Of Heparinized Chitosan/Soy Protein Composites As Anticoagulation Materials

Posted on:2012-12-05Degree:DoctorType:Dissertation
Country:ChinaCandidate:X M WangFull Text:PDF
GTID:1114330344951749Subject:Physiology
Abstract/Summary:PDF Full Text Request
Anticoagulant material, an important part of biological materials, which has been widely used as medical device and tissue engeering materials contacted with blood, such as artificial heart valve, hemodialysis system, extracorporeal circulation system, blood vessels embolic agents, heart pacemakers, artificial vascular, stents, interventional catheter, surgical line. The materials which directly contact with blood should have not only histocompatibility without inflammation, but also have good blood compatibility such as antithrombotic, without clotting phenomenon. In addition, it is required that the materials'mechanical properties such as elasticity and ductility are similar with human tissue and favorable fatiguedurability. Therefore, it is importantly to develop biomedical anticoagulant materials which have strong antithrombotic ability, good biocompatibility and mechanical properties without injecting anticoagulants as a substitutes.Heparin is the most commonly clinlic anticoagulation drug. The mostly delivery method is intravenous injection. However, it caused some side-effects such as dosage, bleeding, thrombocytopenia, allergies. Heparinization is one of the most effective ways that reduce the side effects which direct injection of heparin caused. And heparinized materials are the uppermost anticogulant materials.In this work, acetic acid was used as the co-solvent and two kinds of natural polymers, chitosan and soy protein, were used as main raw materials to prepare chitosan/soy protein composites with different composition and structure. The heparinized biomaterials were prepared with the physical or chemical interaction between heparin molecules and protein molecules, as well as chitosan molecules. The distribution and binding degree of heparin ware controlled by different heparinization technologies. The biological characteristics of the chitosan/soy protein composites including blood compability and histocompatibility were systematically evaluated by in vitro testing and in vivo animal experiments. Meanwhile, the factors affected on the properties and function of the new heparinized biomaterials were revealed. The main contents are as following: (1) Preparation and characterization of chitosan/soy protein composite membrane materials:A series of chitosan/soy protein composite membrane was prepared by blending and casting using chitosan and soy protein isolate (SPI) as the raw materials and acetic acid aqueous solution as co-solvent. The effects of SPI content on the structure and properties of the final chitosan/soy protein composite membranes were investigated by scanning electron microscope (SEM), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), water contact angle, tensile testing, in vitro cell culture and in vivo implantation experiments. The results revealed that the crystallinity decreased and the hydrophilicity of the composite membranes increased with the increase of SPI content. It was found that the mechanical properties of chitosan/soy protein composite membranes were excellent when SPI content was 10%. SPI had obvious promoted cell proliferation and improved cytocompatibility, tissue compatibility and biodegradability of the membranes. Therefore, it could regulate the microstructure, mechanical properties and biological properties of the materials by adjusting SPI content.(2) Preparation, characterization and hemocompability evaluation of surface-heparinized chitosan/soy protein composite membranes:Using EDC as cross-linker, heparin was grafted on to the surface of chitosan/soy protein composite membranes for hepariniztion. The structure and properties of the surface-heparinized chitosan/soy protein composite membranes were investigated by assay with toluidine blue, attenuated total reflection-fourier transforms infrared spectrometry, water contact angle test and tensile testing; The cytocompability and hemocompability of surface-heparinzed chitosan/soy protein composite membranes in vitro cell culture and anticoagulation experiments. The results revealed that the hydrophilicity of chitosan/soy protein composite membranes were remarkably improved by suface-grafting of heparin, but the mechanical properties decreased and the content of immobiled-heparin was 0.59~1.12μg/cm2. Moreover, the heparinized membranes could improve of growth of L929 cells and showed good cytocompability. Surface-heparinization could effectively reduce platelet adhesion, extend the plasma recalcification time (surface-heparinization groups 338 s~352 s, non-heparinization groups 112 s~201 s), curb thrombosis and reduce the hemolysis rate. It indicated that the surface-heparinized membranes have good blood compatibility.(3) Preparation, characterization and hemocompability evaluation of heparinized chitosan/soy protein composite membranes:A series of heparinized chitosan/soy protein composite membranes were prepared by blending, with or without cross-linking. The structure and properties of the heparinized chitosan/soy protein composite membranes were investigated by assay with toluidine blue, Attenuated total reflection-Fourier transforms infrared spectrometry, water contact angle test and tensile testing; The cytocompability and hemocompability of the heparinized chitosan/soy protein composite membranes were evaluated by in vitro cell culture and anticoagulation experiments. The results revealed that the amount of immobilized-heparin of the cross-linking heparinized chitosan/soy protein composite membranes was higher than that on the non-crosslinking heparinized and surface-heparinized chitosan/soy protein composite membranes (crosslinking heparinized groups 1.24~2.47μg/cm, non-crosslinking heparinized groups 0.67-1.29μg/cm2, surface-heparinization groups 0.59~1.12μg/cm2). The cross-linking heparinized membranes were more hydrophilic than the non-crosslinking heparinized membranes, and showed the mechanical properties than he non-crosslinking heparinized and surface-heparinized membranes. Moreover, the cross-linking heparinized membranes could improve the proliferation of L929 cells, and showed good cytocompability. Cross-linking heparinization can effectively reduce platelet adhesion, extended the plasma recalcification time (crosslinking heparinized groups 903 s~1179 s, non-crosslinking heparinized groups 292 s~306 s, surface-heparinization groups 338 s~352 s, non-heparinization groups 112 s~201 s), curb thrombosis and reduce the hemolysis rate. The cross-linking heparinized membranes showed better blood compatibility than the non-crosslinking heparinized membranes and surface-heparinized membranes.(4) The chitosan/soy protein sponges were fabricated in order to enhance the biodegradability of chitosan-based composites. A series of chitosan/soy protein isolate sponges were prepared by freeze-drying process. The effects of soy protein content on the structure and properties of the sponges were investigated by FT-IR, XRD and SEM. The biocompatibility and biodegradability of chitosan/soy protein sponges were evaluated by in vitro cell culture and in vivo implantation experiments. The results showed that the chitosan/soy protein sponges exhibited uniform three-dimensional porous structure, and the pore structure of the chitosan/SPI sponges can keep well with an increase in soy protein content. The in vitro cell culture process showed that the incorporation of soy protein isolate in the composite sponges could provide nutrients for cell growth, which was beneficial to the cell's growth and spread on the sponges, as well as to the enhancement of cell viability during cell culture. The sponges implanted into rats showed good biocompatibility and biodegradability. The biodegradation rate of the chitosan/soy protein sponges was higher than the corresponding membranes, which is due to the incorporation of SPI, the three-dimensional porous structure. Therefore, chitosan/soy protein sponges may be used as cell scaffolds with good biodegradability and biodegradability.(5) Preparation and characterization of chitosan-coated cellulose/soy protein composite membrane with improved physical properties and hemocompability:A series of cellulose/soy protein membranes was prepared using NaOH/urea solution as co-solvent, then chitosan (50000Da) was coated on the surface of cellulose/soy protein membranes. The original cellulose/soy protein composite membrane and chitosan-coated cellulose/soy protein composite membrane were characterized by FT-IR, XRD, SEM, water contact angle testing, and tensile testing. Chitosan-coated cellulose/SPI membranes had smoother surface microstructure and enhanced mechanical properties as compared with the corresponding cellulose/SPI membranes. The cytocompatibility and hemocompatibility of cellulose/SPI membranes and coated cellulose/SPI membranes were evaluated by cell culture, MTT assay, in vitro platelet adhesion testing, plasma recalcification time measurement, and hemolysis assay. The higher cell adherence and improved cytocompatibility of chitosan-coated cellulose/SPI membranes were mainly ascribed to the coated chitosan and the altered surface microstructure of cellulose/SPI membranes. Chitosan-coated cellulose/SPI membranes also showed lower platelet adhesion, longer PRT, and a lower hemolysis rate, all resulting from the good hemocompatibility of chitosan and the smoother membrane surface after chitosan coating. Undoubtedly, surface coating with chitosan improved the microstructure, mechanical properties, cytocompatibility, and hemocompatibility, thus widening the possible range of applications of cellulose/soy protein-based biomaterials.In conclusion, several series of chitosan/soy protein membranes, heparinized chitosan/SPI membranes and chitosan/soy protein composite sponges were successfully prepared in this work. The structure and properties of the membranes and sponges were characterized by methods of polymer chemistry and physics. The cytocompatibility and tissue compatibility of the biocomposites were evaluated by in vitro cell culture and in vivo animal experiments. And the hemocompatibility was evaluated by anticoagulation experiments. Thereby it obtained the chitosan/soy protein composite biomaterials that had good cytocompatibility, tissue compatibility and good hemocompatibility, and provided the theory basis and the experimental data for potential applications.
Keywords/Search Tags:Chitosan, Soy protein, Composite, Haparinize, Biocompatibility, Hemocompatibility
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