Preparation And Properties Of Composite Liquid Crystalline Hydrogels For 3D Bioprinting

3D bioprinting can form complex structures and shapes,providing a broad prospect for the construction of vascular tissue engineering scaffolds.However,the poor mechanical properties of bioinks limit the development of 3D printing,so the preparation of high-performance bioinks is the key to the progress of 3D bioprinting.The structure and properties of hydrogels are similar to those of extracellular matrix,and they are commonly used substrates for the preparation of bioinks.Hydrogels for3 D bioprinting should possess desirable printability with biocompatibility and mechanical properties.Studies have shown that natural blood vessel walls are liquid crystalline.In addition,liquid crystallinity also allows the hydrogel to maintain lower viscosity at higher concentrations,reducing excessive print shear damage.In view of this,gelatin(Gel),quaternary ammonium chitosan(HACC),and sodium cromolyn(DSCG)were selected as substrates to prepare two groups of hydrogel materials for bioprinting.Gelatin and chitosan are both natural hydrogel materials widely used in tissue engineering with excellent biocompatibility.Sodium cromoglycate is a lyotropic liquid crystal,and the carboxylate group of sodium cromoglycate can be ionically cross-linked with quaternary ammonium ions.In order to obtain better mechanical properties and stability of the printed structure,the gelatin was further grafted with methacrylic anhydride to prepare an ion-crosslinking/photocrosslinking double network hydrogel.The specific research contents are as follows:(1)Preparation and characterization of Gel/HACC/DSCG hydrogels: GelatinHACC and DSCG composite hydrogels were prepared by using the ionic interaction of HACC and DSCG.The results of spectroscopic analysis confirmed the interaction of quaternary ammonium ions and carboxylate ions of DSCG and HACC.The liquid crystal structure and phase transition temperature of Gel/HACC/DSCG hydrogels were determined by wide-angle X-scattering(WAXS),differential scanning calorimetry(DSC)and polarized light microscopy(POM).Cell culture experiments confirmed that Gel/HACC/DSCG gel has good cytocompatibility and has a certain induction effect on cell oriented growth.Rheological tests show that Gel/HACC/DSCG hydrogels have shear-thinning properties,and can achieve rapid recovery of viscosity after removing shear force,indicating good printability and cell compatibility.The swelling ratio and degradation tests showed that the Gel/HACC/DSCG hydrogels degraded faster and had higher swelling ratios,which meant that the materials might deform and degrade prematurely during long-term cell culture.(2)Preparation and characterization of Gel MA/HACC-DSCG hydrogels.In order to improve the stability of hydrogels,Gel MA was prepared by grafting gelatin with methacrylic anhydride.Gel MA/HACC-DSCG hydrogel was obtained by swelling in saturated solution.Infrared analysis of the composite hydrogel material confirmed the coexistence of photocrosslinking and ionic crosslinking of Gel MA and HACC/DSCG double network structure.The liquid crystal structure and phase transition temperature of Gel/HACC/DSCG hydrogels were determined by WAXS,DSC and POM images.The mechanical properties of Gel MA/HACC-DSCG hydrogel are better than Gel/HACC/DSCG hydrogel by mechanical test.Rheological tests show that the uncrosslinked Gel MA/HACC has a lower overall viscosity and is suitable for a wider range of printing conditions.The swelling experiments and degradation experiments showed that the double crosslinking of Gel MA/HACC-DSCG slowed down the degradation rate of the hydrogel and reduced the swelling ratio,which means that Gel MA/HACC-DSCG has better fidelity and dimensional stability.(3)Study on the printability of Gel/HACC/DSCG and Gel MA/HACC-DSCG materials: In the Gel/HACC/DSCG system,the Gel/HACC/DSCG4% hydrogel showed better mechanical properties and stability.Gel/HACC/DSCG4% hydrogel has shear-thinning properties and can quickly recover viscosity after shearing.In the Gel MA/HACC-DSCG system,Gel MA/HACC3%-DSCG,Gel MA/HACC4%-DSCG and Gel MA/HACC5%-DSCG were selected to study the effect of 3D printing parameters on the structure,so as to explore the optimized printing conditions.Scanning electron microscope(SEM)images of the printed hydrogel filaments show the surface and core structures.Since the effect of printing shear force on the hydrogel is attenuated in the radial direction,the outer layer of the hydrogel filaments is subjected to strong shearing action,the structure is dense and the pore size is small,the core layer is weakly sheared,and the structure is loose and the pore size is large.Hollow cylindrical structures with a certain height were printed with Gel MA/HACC3%-DSCG,Gel MA/HACC4%-DSCG,and Gel MA/HACC5%-DSCG,showing the potential to construct complex stereostructures.(4)Gel MA/HACC3%-DSCG,Gel MA/HACC4%-DSCG and Gel MA/HACC5%-DSCG hydrogels with better mechanical properties were selected to test their hemocompatibility and histocompatibility.The results showed that the three hydrogels had low hemolysis rates,did not cause platelet aggregation,and had good blood compatibility.The results of histocompatibility experiments showed that higher HACC content could cause more severe inflammatory response,and materials with lower HACC content had better histocompatibility.(5)Statistical analysis was performed on the staining results of live/dead cells printed with cells,and the results showed that the survival rate of Gel/HACC/DSCG4%was 55.22%.This is because although Gel/HACC/DSCG4% has the property of shear thinning,the viscosity during printing is still large,and the shear force during the printing process causes damage to the cells.Since Gel MA/HACC-DSCGs were printed using less viscous uncrosslinked Gel MA/HACC solutions,all three components showed high cell viability above 70%.