Construction Of Chitin Based Hydrogel And Its Application In Tissue Engineering

Chitin,widely existed in shells of crustacean and insect,is the second mostpolysaccharide after cellulose in nature.The new materials prepared by chitin has advantages of high biological safety,environmental protection and pollution-free.However,due to the extensive intramolecular and intermolecular hydrogen bonds,its melting temperature is even higher than thermal decomposition temperature and the chitin processing is still difficult,it is still a challenge to fabricate the functional chitin-based materials.The purpose of this thesis is to use green and environmentally friendly alkaline/urea aqueous system to dissolve chitin to prepare regenerated chitin hydrogel membrane materials.Based on this,the bioactive chitin composite hydrogel membrane materials are designed and constructed.And the functions of chitin hydrogel membrane are also studied as tissue engineering materials in nerve repair and periodontal repair.We hope it can also replace the related clinical biomedical materialsInnovations in this thesis include:(1)The partially deacetylated chitin was dissolved in alkaline/urea aqueous to incorporate the conductive poly(3,4-ethylenedioxothiophene)nanoparticles for preparing the chitin/PEDOT composite hydrogel membrane.The chitin-based nerve conduit for promoting peripheral nerve regeneration was obtained through modifying with cell adhesive peptide.(2)Zinc oxide was first dissolved in alkaline/urea aqueous.Then the chitin was dissolved in the mixed solution,and chitin-Zn O composite hydrogel membrane with zinc oxide nanoparticles uniformly dispersed can be obtained after regeneration.The as prepared hydrogel membrane can be used as bone guided barrier membrane for periodontal repair.(3)The chitin hydrogel membrane coated with polydopamine and polyzwitterionic can be obtained through rapid deposition method catalyzed by copper sulfate/hydrogen peroxide.After spun coating,chitin Janus hydrogel membrane was obtained and the as prepared membrane exhibited antibacterial performance,cell proliferation promotion activity,indicating potential in bone tissue regeneration.The main contents and conclusions in this thesis include the following three parts.Chitin incorporated with PEDOT nanoparticles composite hydrogel membrane(Ch T-PEDOT)was prepared by dissolved partially deacetylated chitin(DA80%)and PEDOT NPs in alkaline/urea aqueous and regenerated in ethanol.The mechanical property of chitin composite hydrogels were enhanced with the amount of PEDOT NPs increased.Meanwhile,the cell adhesive peptide CRGD can be modified on hydrogel membrane surface due to the amino group exposure by partially deacetylated chitin.In vitro cell experiments showed that proper amount of PEDOT nanoparticles in hydrogel membrane can improve cell viability.Importantly,the expression of nerve cell-related proteins were increased under the electrical stimulation,and the CRGD peptide modified on hydrogel membrane surface can significantly enhance cell adhesion.When curled into hollow nerve guidance conduit and then implanted on 10 mm sciatic nerve defect in rats,the membrane can efficiently promoted the connection of repaired nerve after 20 weeks operation.What's more,the recoverability of gastrocnemius muscle in hydrogel membrane group was close to autograft group.The thickness of axon myelin sheath for regenerated nerve in the composite hydrogel membrane group was significantly higher than that in chitin group,and close to the autograft group.Immunohistochemistry and immunofluorescence analysis showed that the composite material was more beneficial to the microvascular formation,Schwann cell adhesion and proliferation in injury nerve part,leading to the nerve regeneration.The conventional chitin incorporated with Zn O NPs hybrid materials were usuallyprepared by physical mixing,which resulted to the poor mechanical property and finally unable to be used as tissue engineering materials.Since zinc oxide can be dissolved in alkaline/urea aqueous solution in the form of[Zn(OH)₄]^2-,chitin incorporated with zinc oxide composite hydrogel film(Ch T-Zn O)can be obtained by directly dissolving chitin in alkaline/urea solution contained zinc hydroxide.In the process of chitin dissolution,a small amount of quaternary ammonium salt is added into chitin solution to enhance the solubility of chitin molecular chain to avoid the rapid gelation of the complex solution.In vitro experiment,we found that the composite hydrogel membrane can sustainably and stably release Zn²⁺,which had excellent antibacterial effect on Gram-negative P.gingivalis and Gram-positive S.aureus,as well as facilitating bone formation of mesenchymal stem cells(MSCs).As a barrier membrane implanted into rats with periodontal bone defect,we found that the composite hydrogel membrane had a certain promoting effect on alveolar bone regeneration compared with pure chitin group after 8 weeks operation.Pure chitin hydrogel membrane always has low antibacterial activity due to the large amount of acetylamino rather than amino groups.Herein,the chitin hydrogel membrane coated with polydopamine(Ch T-PDA)was prepared by rapid deposition of dopamine and zwitterionic monomer in 1 hour with copper sulfate/hydrogen peroxide as catalyst.Because the Cu²⁺can be stabilized into hydrogel membrane by polydopamine and chitin,the composite membrane can slowly and sustainably release low concentrations of Cu²⁺,which finally kill Gram-negative E coli and Gram-positive S aureus.In vitro cell experiments showed that the released Cu²⁺from composite hydrogel membrane can improve the proliferation of MC3T3-E1.Additionally,zwitterionic sulfobetaine methacrylate monomers can also be polymerized while dopamine deposition was occurred,and finally formed composite membrane which equipped with anti-protein adsorption as well as anti-bacterial adhesion.The chitin Janus composite hydrogel membrane can be obtained by coating a gelatin solution containing hydroxyapatite on one side of the membrane,which is expected to be effective in bone tissue repair.