Synthesis Of Injectable Chitin-based Hydrogel Forming In-situ And Application In Three-dimensional Cell Culture

The injectable hydrogels forming in-situ can encapsulate the active therapeutic molecules and/or cells homogeneously,and be injected into the body by minimally invasive surgery to fill any shape of defects.Due to their three-dimensional（3D）hydrophilic network similar to the natural extracellular matrix,the injectable hydrogels have received increasing attention in the biomedical field such as drug delivery,cell carrier and tissue repair.Chitin,an important natural nitrogen-containing polysaccharide,is biocompatible and biodegradable.In recent years,chitin and its derivatives have been constructed into a series of new injectable hydrogels for biomedical applications.Thermosensitive carboxymethyl chitin（CMCH）and hydroxypropyl chitin（HPCH）have been successfully synthesized homogeneously through the“green”solvent of alkali（Na OH,KOH or Li OH）/urea aqueous systems in our group.In this study,the phenolic hydroxyl groups were introduced into the CMCH chain to increase the hydrogel strength by enzymatic crosslinking.The Diels-Alder or Schiff base reaction was introduced into the thermosensitive HPCH hydrogel system to improve the strength of hydrogel by the dually（physically and chemically）crosslinking.The biocompatibility and application in 3D cell culture were investigated.In chapter 1,a brief overview of the research progress on chitin-based hydrogels and injectable hydrogels in the biomedical field.In addition,the features and applications of 3D cell culture have been briefly presented.In chapter 2,an enzymatically crosslinkable tyramine-modified carboxymethyl chitin（CMCH-Tyr）polymer was synthesized by incorporating the phenol groups to the water-soluble CMCH chain,resulted in a new injectable CMCH-Tyr hydrogel formed in-situ in the presence of horseradish peroxidase（HPR）and H₂O₂ under physiological conditions.The CMCH-Tyr hydrogel showed much better mechanical properties than those of the thermosensitive in-situ forming physical-crosslinking CMCH hydrogel.The gelation time,strength and biodegradation rate of the CMCH-Tyr hydrogels can be adjusted by varying the concentrations of HPR and H₂O₂ in the certain range.In vitro cytotoxicity assays and in vivo in-situ injection study showed non-toxicity,favorable gel formation,and good tissue biocompatibility of the enzyme-catalyzed CMCH-Tyr hydrogel.Thus,the biodegradable and biocompatible CMCH-Tyr hydrogels may hold great potential for 3D cell culture and tissue engineering.In chapter 3,based on the thermosensitive HPCH polymer,a novel thermosensitive and dully crosslinkable furyl-modified hydroxypropyl chitin（FGE-HPCH）polymer was synthesized by incorporating the furan ring.An injectable and dually crosslinked hydrogel was prepared in-situ by integration of the thermosensitive physical crosslinking and Diels-Alder click reaction in the presence of crosslinker maleimide-terminated PEG（MAL-PEG-MAL）.The dually crosslinked chitin-besed hydrogel showed much higher mechanical strength and slower biodegradation rate in contrast with the solely physically crosslinked thermosensitive HPCH hydrogel.The dually crosslinked hydrogel could be degraded by lysozyme.In addition,the strength and degradation rate of the FGE-HPCH hydrogel could be controlled by adjusting the molar substitution degrees of furyl and the molar ratio of furyl to maleinide of the crosslinker MAL-PEG-MAL.3D cell culture in the hydrogel displayed sustainable proliferation and good ability of self-assembling to form multicellular spheroids.In vivo investigation of the thermosensitive chitin-based dually crosslinked hydrogel showed favorable injectability,in-situ thermogelation and good biocompatibility.Thus the injectable,biodegradable and biocompatible dually crosslinked chitin-based hydrogel holds great potential for being applied in 3D cell culture and tissue repair.In chapter 4,injectable and dually crosslinked hydrogel containing collagen （HPCH-PEGDCHO-Col）was developed based on the thermosensitive HPCH polymer by introduction of collagen and Schiff base reaction in the presence of aldehyde-terminated PEG（PEGDCHO）.This not only effectively increased the hydrogel strength,but also promoted the adhesion of cells to the hydrogel,the cell proliferation and the size increase of the multicellular spheroids.The HPCH-PEGDCHO-Col hydrogel also had the ability of self-healing due to the dynamic acylhydrazone bond（-CH=N-）.Meanwhile,the hydrogel maintained good stability under physiological conditions and could be degraded by lysozyme,having no significant cytotoxicity.The strength and degradation rate of the hydrogel could be controlled by adjusting the molar ratio of aldehyde groups to amino groups.In vivo assay of the HPCH-PEGDCHO-Col hydrogel displayed excellent injectability,favorable in-situ gelation,good biodegradability and biocompatibility.Therefore,the injectable and dually crosslinked HPCH-PEGDCHO-Col hydrogel is expected to be a good scaffold material for tumor model and cartilage tissue repair.In chapter 5,the thermosensitive HPCH hydrogel was investigated as a scaffold for3D tumor cells culture.Tumor（HepG2,HeLa and C6）cells grew well in the 3D scaffold of HPCH hydrogel and could form multicellular spheroids.This 3D tumor cell growth was very similar to the real tumor tissue in vivo.Further,Hep G2 cells after 3D cell culture in HPCH hydrogel were found to experience dedifferentiation and the upregulation of stemness,displaying biological characteristics of stemness,such as the ability of forming multicellular spheroids,migration,drug resistance and subcutaneous tumor formation.Thus the thermosensitive HPCH hydrogel holds good potential to be applied in the fields of long-term stem cell culture,tumor drug development and tumor models.