Construction And Application Of Hydroxypropyl Chitosan/Soy Protein Composites In Tissue Engineering

Chitosan is the alkaline polysaccharide existing in nature,and has been widely used in various tissue engineering fields due to its excellent properties such as biocompatibility,biodegradation,antibacteria,non-toxic and non-immune reaction.However,chitosan is insoluble in water and general organic solvents,and soluble in dilute acids,thus its application in biomedical field is greatly limited.Hydroxypropyl chitosan(HPCS)is an important functional derivative of chitosan,which is obtained by etherification of chitosan and propylene oxide under alkali conditions.It not only has good biocompatibility and biodegradation,but also improves the polarity and solubility of chitosan.HPCS has been used in tissue engineering,drug,fruit and vegetable freshkeeping agent,cosmetics,sewage treatment and disease treatment,due to its prosperities like moisture retention,antibacteria,adsorption,gel-forming,emulsification and surface activity.Therefore,according to the excellent properties of HPCS,it is of great theoretical significance and scientific value to expand the application scope and potential of new biomaterials based on HPCS in the biomedical field.Soy protein isolate(SPI)is a kind of natural macromolecule with abundant sources,low cost,edible and versatility.As a biodegradable green raw material,SPI has good solubility,emulsification,foaming,film-forming and hydration,and has been widely used in packaging,foam,biodegradable plastics,food development,fiber products and other fields.In addition,SPI has good biodegradability and biocompatibility,which not only have a variety of beneficial effects on the body,especially on bone health,cancer prevention,insulin sensitivity,weight control,inflammation,immune cell activity and mineral absorption,but also can promote cell proliferation and tissue regeneration.Further,as a plant protein,SPI can be mixed with other materials to prepare a variety of biological materials,including membranes,sponges,gels,microspheres and fibers.Thus,the development of environmentally friendly protein materials based on SPI and the in-depth exploration of its potential application in the biomedical field have important theoretical value and application value.In this thesis,a series of HPCS/SPI composite films(HCSF),composite hydrogels(HCSH)and composite sponges(HCSS)were developed via casting method,chemical crosslinking method and freeze-drying method and their structure,physicochemical and biological properties were comprehensively evaluated.According to the good biocompatibility of HCSF,its potential as wound dressing was evaluated by the fullthickness skin defect model in SD rats.According to the coagulation effect of HCSH,the hemostatic model of New Zealand rabbit liver was used to evaluate its feasibility as hemostatic material.According to the physicochemical and biological properties of HCSS and its influence on the proliferation and growth of RSC96,the optimum proportion of HCSS was selected for the construction of new nerve conduit(HCSSC).The HCSSC was further combined with the bone marrow mesenchymal stem cells(BMSCs)and Schwann cells derived from bone marrow mesenchymal stem cells(dBMSCs),and the repair effect was evaluated by the 10 mm sciatic nerve defect experiment in SD rats.The main content of this work includes the following aspects:(1)Construction of the HPCS/SPI composite films and their application as wound dressing: A series of HPCS/SPI composite films were developed using HPCS and SPI as main raw materials and epichlorohydrin as crosslinking agent via the process of blending,crosslinking and drying.The structure and physical properties of HCSF were tested by Fourier infrared spectrum,X-ray diffraction,scanning electron microscopy,water absorption ratio and mechanical testing.The cytocompatibility were evaluated by MTT,live/dead cell assay and direct cell contact assay.Blood compatibility were evaluated by hemolysis rate testing and plasma recalcification time.The feasibility of using it as wound dressing was evaluated by skin defect repair experiment.The results showed that SPI can regulate the water absorption and tensile strength of the HCSF.In addition,the HCSF has good cytocompatibility and blood compatibility,and can accelerate the speed of wound healing,showing potential application as wound dressing.(2)Construction of the HPCS/SPI composite hydrogels and their application as hemostatic material: A series of HPCS/SPI composite hydrogels were developed using HPCS and SPI as main raw materials and epichlorohydrin as crosslinking agent via the process of blending,crosslinking and gel-forming.The structure and physical properties of HCSH were characterized by Fourier infrared spectrum,X-ray diffraction,scanning electron microscopy,water absorption ratio,swelling ratio,swelling behavior of the pH sensitive,mechanical testing and rheological properties testing.The cytocompatibility and in vitro coagulation effects were evaluated by MTT,hemolysis rate testing and routine coagulation testing.The New Zealand rabbit liver hemostatic model was used to explore the potential of HCSH as a hemostatic material.The results showed that SPI could regulate the pore size,mechanical properties and gelation time of HCSH.The swelling property of HCSH can be controlled by the change of pH value.It can be used as hemostatic material because of its good cytocompatibility and good coagulation ability.(3)Construction and biocompatibility evaluation of HPCS/SPI composite sponges: A series of HPCS/SPI composite sponges were developed using HPCS and SPI as main raw materials and epichlorohydrin as crosslinking agent via the process of blending,crosslinking and freeze-drying.The structure and physical properties of HCSS were tested by Fourier infrared spectrum,X-ray diffraction,scanning electron microscopy,water absorption ratio,water retention ratio and mechanical testing.The cytocompatibility and blood compatibility were evaluated by direct cell contact test,hemolysis rate testing and erythrocyte contact testing.The related mechanism of HCSS on proliferation and growth of RSC96 were evaluated by CFSE proliferation experiment,cell cycle experiment,ROS testing and qPCR experiment.The results showed that the HCSS had the interpenetrating pore structure,good mechanical properties,good cytocompatibility and blood compatibility,as well as has the ability to promote the proliferation and growth of RSC96.(4)Construction of nerve guide conduits from HPCS/SPI composite sponges and its application for peripheral nerve repair: According to the third part of this work,the HCSSC with 50% SPI content were fabricated by the process of blending,crosslinking,mold filling and freeze-drying.The BMSCs and dBMSCs were injected into the HCSSC to evaluate the nerve repair effect.The structure of HCSSC was observed by scanning electron microscope.The phenotype and function of dBMSCs were identified by AM staining,immunofluorescence staining,qPCR and western-blot.The biocompatibility of HCSSC was evaluated by direct cell contact assay.The repair effect of HCSSC loaded with BMSCs and dBMSCs were comprehensively evaluate by sciatic nerve functional index,electrophysiology,H&E staining,immunofluorescence staining and gastrocnemius muscle testing.The results showed that the HCSSC had the porous structure,which was conducive to cell adhesion and growth.The extracted BMSCs had high purity,and dBMSCs had the phenotype and function of Schwann cells.The results of animal experiments verified that dBMSCs-loaded HCSSC has a good repair effect on the damaged nerve,and has potential application prospect in the field of peripheral nerve tissue engineering.To sum up,in this work,the two natural macromolecular materials of HPCS and SPI were taken as the research objects,and their excellent properties were explored in depth.A series of HPCS/SPI composite films,composite hydrogels and composite sponges with different forms were successfully prepared.The structures and physicochemical properties of these three scaffolds were characterized by chemical and physical methods.The cytocompatibility and blood compatibility were systematically evaluated by cell culture experiments and blood experiments.According to comprehensive consideration of structure,water absorption and mechanical properties of three scaffolds,we respectively apply them to the full-thickness skin defect,liver bleeding experiment and peripheral nerve defect study.Through the corresponding index system of evaluation,the results indicated that the HCSF,HCSH and HCSS has potential application prospects on wound dressings,hemostatic materials and nerve conduits,and provide experimental support for expanding the application of hydroxypropyl chitosan-based biomaterials in the field of biomedicine,as well as theoretical basis for the development and design of new tissue engineering biomaterials.