Fabrication Of Chitosan-Silica Nanostructured Hybrid Materials And Cell Compatibility Investigation

Organic-inorganic hybrid materials are specific nanocomposites,in which the organic and inorganic components can interact at the molecular level.The organic and inorganic components distributed homogenously and there are no significant phase separating.Comparing with the conventional composites,the interaction between organic and inorganic components of hybrid materials is greatly enhanced,and the mechanical properties are significantly improved.The natural polymer derivative chitosan attracted researchers’interests due to its good biocompatibility,biodegradability,and advantages of easy chemical modification.Bone defect repairing requires scaffold materials with improved mechanical properties,controllable 3D structure and good biological performance.Inorganic materials such as bioceramics and bioactive glass are brittle,easily fractured and thus induce secondary injury,which cannot be applied in the defect that would experience cyclic load.While the organic polymers exhibit poor bioactivity.The performance of organic/inorganic composites is improved compared with inorganics or polymers,however,the weak interaction between the organic components and inorganic components in conventional composites making them possess low mechanical strength,which is mainly depended on the continuous polymer phases.Therefore,conventional materials do not fully meet the requirements of scaffold materials for tissue engineering.Chitosan-silica hybrids（CSHs）have been reported recently and suggested they have good cellular compatibility,bioactivity and promoting wound repair,which could be potentially applied in regenerative therapy.However,chitosan-silica hybrids prepared by previous works possess poor mechanical properties,which may due to the complex mechanism of chemical reactions in forming CSHs.In addition,the previous works could not fabricate CSHs with controllable three-dimensional structure.Since the defects of each patient are different in shapes,it is important that scaffolds could be produced customized.In the recent years,3D printing technology provided effective method to construct scaffolds with controlled three-dimensional structure.The essential issue of this study is to fabricate chitosan-silica scaffolds with improved mechanical properties,controllable 3D structure with porosity and pore connectivity.Firstly in this study,chitosan-silica hybrids were prepared by a facile sol-gel method.The hybrids were characterized by IR,²⁹Si-NMR,transmission electron microscopy,thermal analysis,water contact angle measurement,compression tests and in vitro mineralization.The biological properties are evaluated by co-culturing with mouse mesenchymal stem cells（mBMSCs）in vitro.Porous CSH scaffolds were prepared by 3D printing technique.The mechanical properties and in vitro mineralization of the scaffolds were characterized.The mouse bone morrow mesenchymal stem cells were cultured in vitro to evaluate the cell compatibility of the scaffolds.Furthermore,chitosan-silica porous scaffolds were modified by covalent bonding with gelatin by using silane coupling agent.The effects of the surface modification on the chemical and biological properties of the scaffolds are also studied.（1）The effects of different acidic conditions on the structure and properties of chitosan-silica hybrid materials（CSHs）synthesized by sol-gel method were studied.The chitosan-silica nanocomposite materials with uniform and uniform appearance of organic and inorganic components at nanometer scale can be successfully prepared by sol-gel method catalyzed by weak acid（acetic acid,pH=4）.The silica components in CSHs tend to aggregate in strong acid conditions and produce phase separation,and the degree of separation increases with the increase in the acidity of the reaction system.The compressive strength of CSH50 is42.6±3.3MPa,the elastic modulus is 312±27MPa,and the strain at fracture is 41.7±2.1%,which are much higher than CSH50-HCl-pH4 and CSH50-HCl-pH 2.8 which prepared under strong acid conditions.The mechanism of the synthesis of chitosan-silica type I hybrid materials by sol-gel method is discussed.Chitosan-silica Hybrid materials with different organic/inorganic components were prepared by sol-gel method using weak acid（acetic acid,pH=4）as catalyst,which shows high mechanical strength and good toughness.The results of mouse mesenchymal stem cells（mBMSCs）co-cultured on the hybrids were evaluated by CLSM.After 24 h incubation,the adhesion of m BMSCs on CSH60,CSH70 and chitosan was good,while the adhesion and proliferation of cells on CSH50wasrelatively inferior.Comparing with the control group,there was no significant difference between CSH50 and control group,indicating that CSHs were not cytotoxic.（2）3D printing technology had utilized in fabricating CSHs scaffolds,the hybrid gels with mediated rheological properties were firstly prepared by sol-gel method.A series of CSH scaffolds were fabricated by extrusion 3D printing.In addition,CSH-FD scaffolds with dual level of porous structure were fabricated by combining 3D printing technic and freeze-drying process.The microstructure,mechanical properties and in vitro mineralization of the scaffolds were studied.The biological properties of the scaffolds were also studied.The results showed that the porosities of CSH60 and CSH60-FD scaffolds were 46.5±3.3%and 88.5±4.1%,respectively,and the pores were connected in three dimensional.CSH scaffolds had good mechanical properties.The mechanical strength of CSH60 scaffolds was the highest,with a compressive strength of 21.5±3.0MPa and a fracture strain at 42.5±3.2%.The mechanical strength of CSH-FD scaffolds was lower and totally flexible.In vitro cell culturing experiments showed that m BMSCs spread and proliferated well on CSH-FD scaffolds.And the osteogenic differentiation of CSH60-FD was stronger than CSH50 and CSH70.（3）The surface of CSH scaffolds was modificated by grafting gelatin onto the silica network usingγ-glycidoxypropyltrimethoxysilane（GPTMS）as coupling agent.CSH-Gel scaffolds were fabricated by 3D printing,and the effects of surface modification on the chemical structure and bioactivity of the scaffolds were studied.The results reveal that the modification promoted the adhesion and proliferation of cells on the scaffolds.