In-situ Forming Injectable Modified Chitin Hydrogels

"Chemistry of Natural Resources", the theme of the 249th National Meeting & Exposition of the American Chemical Society in 2015, addresses topics important to human life in the 21st century. As the requirement of sustainable development for modern industrial and economy and the increasing shortage of fossil resources such as coal and oil, the development and utilization of natural biomass has become especially important. As the most abundant natural polysaccharide after cellulose, chitin possesses non-toxicity, biodegradability and good compatibility, thus holding great potential in bio medical applications. This work focused on the construction of thermo-sensitive chitin derivative hydrogels through etherification of chitin in a "green" solvent, NaOH/urea aqueous solution, by freezing/thawing method, and the correlation between structure and properties of these hydrogels. Meanwhile, the potential application of the modified chitin hydrogels in biomedical fields was evaluated by cell and animal experiments.The primary content and conclusions of this work are described in the following five parts.In Chapter 1, the progress and applications in regenerative medicine of chitin-based hydrogels and injectable hydrogels were reviewed.In Chapter 2, novel carboxymethyl chitins (CMCHs) with controlled degree of substitution (DS) and high degree of acetylation (DA) were synthesized homogeneously in aqueous NaOH/urea solution through etherification with sodium monochloroacetate, which possess less de-N-acetylation and less de-polymerization than the traditional heterogeneous reactions. The structure of the obtained CMCHs was precisely determined by proton NMR in acidic deuterated aqueous solution for the first time. Different and broad DS of CMCHs can be facilely and accurately adjusted in the range of 0.035 to 0.74. The CMCH-4 with lower DS of carboxymethylation exhibited dual thermo- and pH-sensitive properties and formed hydrogels at body temperature, which were not toxic and displayed promising future for biomedical applications.In Chapter 3, thermo-sensitive and injectable carboxymethyl chitin (CMCH) hydrogels were developed for three-dimensional (3D) cell culture. The CMCH solutions exhibited low viscosity at low temperatures and rapid thermo-responsive sol-to-gel phase transition behavior at 370℃. The gelation time of the obtained CMCH solutions could be easily tuned by varying temperature and the degree of carboxymethylation, which facilitates the encapsulation of cell/drug for injectable hydrogel. Moreover, the CMCH hydrogels in PBS buffer remained stable and continuous porous structure and could be degraded in the presence of lysozyme or hyaluronidase. Cytotoxicity test demonstrated that the CMCH hydrogels are non-cytotoxic, and HeLa cells proliferated sustainably and self-assembled to 3D multicellular spheroids with high cell activity on the surface of CMCH-14 hydrogeL The in-situ forming CMCH hydrogel encapsulated with COS-7 cells significantly promoted cell survival and proliferatioa In addition, CMCH hydrogels showed promising in-situ gel formation and tissue biocompatibility in preliminary in vivo mouse study. Therefore, CMCH hydrogel would be a promising candidate as injectable scaffold for 3D cell culture and soft tissue engineering.In Chapter 4, novel hydroxypropyl chitins (HPCH) with thermo-sensitivity were synthesized homogeneously in aqueous NaOH/urea solution through etherification of chitin with propylene epoxide. The HPCH solutions underwent reversible thermo-sensitive sol-gel transition The thermo-sensitivity and gelation behavior of HPCH solutions was studied systematically using dynamic light scattering and rheological test. The influence of the degree of N-acetyl, the degree of hydroxypropyl substitution, polymer concentration and the ionic strength in solution on the gelation behavior was also investigated. The gelation mechanism is discussed and mainly owned to the balance between the hydrophobic N-acetyl groups and the introduced hydrophilic hydroxypropyl groups of the modified chitin.In Chapter 5, the properties of the hydroxypropyl chitin (HPCH) hydrogels were studied for three-dimensional (3D) cell culture and cartilage regeneration in vivo. The HPCH solutions exhibited low viscosity at low temperatures and rapidly gelled at 370℃, and the transition was thermo-reversible. The mechanical strength and 3D connected porouse structure of HPCH hydrogels could be easily tuned by the degree of hydroxypropylation and polymer concentration The HPCH hydrogels remained stable in PBS buffer and could be degraded by lysozyme in vitro. Due to the strong hydrophilicity of HPCH hydrogels, cells cultured on the surface of hydrogel and within the hydrogel self-assembled to multi-cellular spheroids with high proliferation rate. HPCH solution gelled rapidly in situ after subcutaneous injection in mice. The forming hydrogel could be dedraded in vivo accompanied by acute inflammation which subsided after 4 weeks. Cartilage-like tissue was generated after subcutaneous injection of HPCH solution encapsulated withchondrocytesc into nude mice for 8 and 12 weeks. The tissue exhibited typical cartilage structure with strong deposition of extracellular matrix (ECM), proteoglycan and collagen II, indicating that HPCH hydrogel was a promising candidate scaffold for cartilage repair.A series of thermo-sensitive chitin derivative hydrogels were synthesized homogeneously by etherification of chitin in the NaOH/urea solvent system. These hydrogels exhibited promising potential in cell delivery,3D cell culture in vitro and cartilage tissue engineering fields. This work is producing significant scientific and technological accomplishments for design and development of new modified chitin in biomedical materials.