Preparation And Osteogenic Properties Of Ultrastrong Chitin Nanocomposite Hydrogel

Chitin hydrogel has good biocompatibility,osteogenic activity and antibacterial and anti-inflammatory properties for potential application as artificial periosteal,but it generally has defects of weak mechanical strength and toughness.Chitin whiskers（CHWs）,a needle rod-like nanoscale crystallites,not only retain the biological activities of chitin but also possess outstanding strength and modulus.Importantly,CHWs and chitin matrix are composed of the same component,which could be speculated that a good interfacial bond between the whiskers and matrix will be achieved.However,the solvents used to dissolve the chitin matrix are commonly strongly alkaline;the protonation of CHWs is severely affected under such an alkaline circumstance,so that the electrostatic repulsion between the whiskers is weakened and the agglomerates of the CHWs are easily formed.As a result,it is difficult to obtain the potential reinforcement effect of the whiskers.At the same time,chitin has certain limitations to promote vascularization.In addition,the anisotropic characteristics of natural periosteum can affect its strength,toughness and cell behavior.Based on the above considerations and analysis,the main purpose of this study is to construct a kind of nanocomposite hydrogel of chitin with excellent mechanical strength,osteogenic activity and vascular ability.For this purpose,the following research works were carried out:1.Maleation chitin whiskers（m CHWs）with lots of-COOH groups were designed,which can stably exist and homogeneously disperse in the alkaline chitin matrix due to their relatively strong electronegativities.In addition,CHWs were also prepared by acid hydrolysis of chitin as a comparative analysis.Then,the CHWs and m CHWs were utilized as reinforcing fillers to enhance the mechanical strength of the chitin hydrogel.As expected,the CHWs played an extremely limited role in reinforcing the chitin hydrogel.Conversely,due to good dispersibility and strong interfacial interaction,the m CHWs exhibited a significantly superior enhancement effect on the chitin hydrogel.Moreover,the tensile strength and modulus of the as-prepared m CHW/chitin composite hydrogels were strongly dependent on the content of m CHWs,and the highest values of 5.8±0.2 and 5.2±0.6 MPa were obtained with 3 wt%m CHWs content.Importantly,both CHWs and m CHWs can facilitate the adhesion,proliferation,and differentiation of mouse embryo osteoblast precursor（MC3T3-E1）cells.Moreover,the m CHWs also play these promoting effects in a content-dependent manner.Interestingly,CHWs and m CHWs mainly contain-NH₂ and-COOH groups,respectively,which will lead to the different surface protein adsorption and calcium-phosphate mineralization.But there are very small differences in cell adhesion,proliferation,and differentiation between the corresponding 3%CHW/chitin and 3%m CHW/chitin composite hydrogels.2.To better simulate the structure and performance of natural periosteum,the 100%PC-m CHW/C composite hydrogel was constructed by combining the methods of physical and chemical double cross-linking and pre-stretching and drying,which showed a highly oriented structure.In addition,the tensile properties of the PC-m CHW/C composite hydrogel were affected by the degree of pre-tensile strain.When the pre-tensile strain is 100%,the corresponding100%PC-m CHW/C composite hydrogel exhibits the highest tensile strength（19.7±2.4 MPa）and the maximum young’s modulus（66.7±1.9 MPa）.On this basis,the deferoxamine（DFO）was further successfully introduced on the surface of the 100%PC-m CHW/C composite hydrogel by the aid of polydopamine（PDA）layer.The presence of PDA and DFO has a slight effect on the initial orientation morphology of the surface of 100%PC-m CHW/C-PDA/DFO composite hydrogel,but the internal ordered structure can maintain well.The tensile strength and elongation at break of the 100%PC-m CHW/C-PDA/DFO composite hydrogel are 17.6±1.4 MPa and47.6±3.6%,respectively.The introduction of PDA and DFO slightly weakened the mechanical strength of 100%PC-m CHW/C-PDA/DFO composite hydrogel,while improved its toughness slightly.In addition,DFO can continuously and effectively release from composite hydrogel,which is expected to promote the osteogenic differentiation and angiogenesis of cells.3.Based on the above research,rat bone marrow mesenchymal stem（BMSCs）and human umbilical vein endothelial（HUVECs）cells were selected to explore the cytocompatibility,osteogenic activity and vascular ability of 100%PC-m CHW/C-PDA/DFO composite hydrogel,respectively.The results show that the anisotropic structure,PDA layer and DFO have a significant promotion effect on cell adhesion,proliferation,osteogenic differentiation and angiogenic activity.Particularly,the DFO can effectively synergistically improve the osteogenesis and vacularization ability of 100%PC-m CHW/C-PDA/DFO composite hydrogel.