Preparation,Modification And Biocompatibility Evaluation Of Regenerated Cellulose/SPI Composite Beads

As the most abundant renewable natural polymer on the earth, cellulose has received increasing attention all over the world. With good biodegradability, biocompatibility, and nontoxicity to cells and tissue, cellulose has been widely utilized in the biomaterial field. Because of these unique properties, cellulose has been converted into films, fibers, sponges, and spherical particles or beads. Cellulose-based composite beads were synthesized by using cellulose as the matrix, which had potential applications in the field of chromatography fillers, protein immobilization, cell carriers and drug carrier/release systems due to its biodegradability and biocompatibility. Therefore, it has important significance to explore novel preparation of cellulose-based beads and broaden their applications. Over the last decades, various methods which based on dropping and dispersion techniques for the preparation of cellulose beads have been reported. The high-voltage electrostatic technique is an improved way based on dropping method. It can be used to prepare beads with uniform and controllable size, and is a relatively environmentally friendly, safe, convenient and continuous method. It is widely used to prepare beads in the field of biomedical field. Therefore, it is a simple and easy way for the preparation of cellulose-based beads using high-voltage electrostatic technique.Soy protein isolate (SPI) is an abundant and attractive protein with many biological activities. SPI had potential applications in biomedical fields because of its good biodegradability and biocompatibility. Meanwhile, with a good processing performance, SPI and its derivant can be made into various materials such as films, gels, fibers and sponges. As a biomaterial, SPI may have great potential for applications in the areas of the drug release, wound dressings and scaffold materials. In our previous work, SPI modified cellulose films and sponges showed improved mechanical properties, cytocompatibility, biological safety and biodegradability.Cellulose and SPI were used as the main raw materials in this thesis. Firstly, a high-voltage electrostatic technique was used to prepare a series of regenerated cellulose/SPI beads (RCSB). Secondly, their physical and chemical properties, cytotoxicity and cytocompatibility of the resultant RCSB were systematically investigated. Thirdly, to improve the shortcomings of RCSB, the beads were chemically modified by reaction with heparin on the surface of RCSB. The physical properties, cytotoxicity and cytocompatibility of the resultant heparinized RCSB were also investigated. Finaly, the interactions between bone marrow mesenchymal stem cells (BMSCs) and heparinized RCSB were investigated. We expect to provide a theoretical and experimental support for the future application of cellulose-based beads in biomedical field. The main contents of this thesis include the following several aspects:(1) Construction and characterization of regenerated cellulose/SPI composite beads using high-voltage electrostatic technique:Cellulose and SPI were used as the main raw materials, then dissolved in NaOH/urea aqueous system at low temperature. Regenerated cellulose/SPI beads (RCSB) with diameter ranging from micro to millimeter were fabricated using an environmentally friendly high-voltage electrostatic technique. The structure and physical properties of RCSB were characterized by optical microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), solid-state 13C NMR, water contact angle measurement, and thermogravimetric analysis (TGA). The resultant composite beads were round with nano-sized pores on the surface. The size of the dried beads ranged from 300 to 1500 mm and could be controlled by changing fabrication parameters such as the voltage used and the cellulose/SPI ratio. The FT-IR, XRD,13C NMR, and TGA results revealed that strong hydrogen bond interactions formed between cellulose and SPI in the beads during the regeneration process, and that the composite beads had good thermal stability, they could be sterilized at 121 ? as biomaterials. The surface hydrophilicity/hydrophobicity of the beads could be altered by adjusting the SPI content in the beads. This work provided a facile, environmentally friendly, and controllable method for the construction of cellulose/SPI-based beads.(2) The cytocompatibility and hemocompatibility evaluation of regenerated cellulose/SPI composite beads:The cytocompability and hemocompability of the above RCSB were evaluated by MTT and in vitro cell culture experiment, hemolysis ratio testing assay, red blood cells incubation experiment, and anticoagulation experiments. The results showed that the RCSB had noncytotoxicity and the L929 cells adhered and grew well on the surface of the beads. In some cases, cells layered on bead surfaces and exhibited good proliferation, especially on the composite beads which the SPI content was 10% and 30%. Moreover, most of the cells adhered to the surface of the composite beads showed shuttle-shaped. RCSB exhibited non-anticoagulation and exhibited no toxicity to red blood cells.(3) Preparation, characterization, cytocompatibility and hemocompability evaluation of surface-heparinized regenerated cellulose/SPI composite beads (HRCSB):Heparin was grafted on to the surface of RCSB for heparinization by using EDC as crosslinker. The structure and properties of the HRCSB were characterized by toluidine blue assay, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and water contact angle measurement; The cytocompability and hemocompability of HRCSB were also evaluated through in vitro cell culture experiment, hemolysis ratio testing assay, and anticoagulation experiments. The results showed that the hydrophilicity of RCSB were improved by surface-grafting of heparin.The surface content of heparin on HRCSB increased with the increase of WSPI. The heparinized beads had noncytotoxicity and could improve the adhesion and expansion of L929 cells on the surface. Surface-heparinization of RCSB could reduce the hemolysis rate, effectively reduce platelet adhesion and activation, obviously extend the plasma recalcification time (PRT), activated partial thromboplastin (APTT), Thrombin time (TT) and curb thrombosis. The results showed that the HRCSB exhibited good cytocompatibility and hemocompability.(4) Evaluation of the interactions and osteoinductive effect of BMSCs on HRCSB:The BMSCs were isolated and expanded by the whole bone marrow adherent culture method. The BMSCs were directly co-cultured with HRCSB, proliferation, adhesion and osteoinductive effect were detected through MTT, SEM, fluorescence microscopy, and ostoblasts associated markers as alkaline phosphatase (AKP), and calcium. The results showed that the BMSCs adhered and grew well on the surface of the beads with SPI content more than 30%. The HRCSB had noncytotoxicity to BMSCs when the SPI content was 30% and 50%. The BMSCs adhered and grew well on the surface of the beads with SPI content more than 30%. The composition of SPI in HRCSB can promote cell proliferation and the differentiation of BMSCs into osteoblasts. With the SPI content was 30% and 50%, the HRCSB were able to promote differentiation of BMSCs into osteoblast and mineralization in a certain extent. Therefore, HRCSB fabricated in this work may have potential applications in bone tissue engineering as cell carriers.To sum up, a series of regenerated cellulose/SPI composite beads and surface-heparinized regenerated cellulose/SPI composite beads were prepared successfully in this thesis. The structure and physical properties of the beads were characterized through polymer physical and chemical methods. The biocompatibility and hemocompatibility of the beads was systematically evaluated by MTT, in vitro cell culture experiments and anticoagulation experiments. The interactions and osteoinductive effect of surface-heparinized beads on the differentiation of BMSCs into osteoblasts were also investigated. The results indicated that the composite beads exhibited good biocompatibility and hemocompatibility. In one word, present thesis indicated that these new regenerated cellulose/SPI composite beads and surface-heparinized beads fabricated by high-voltage electrostatic technique showed potential applications as biomaterials, especially as cell carriers in the tissue engineering field, providing a theoretical significance and experimental support for the future application.