Self-healing Gelatin Colloidal Gels As Bio-inks For 3D Bioprinting

3D bioprinting is based on additive manufacturing technology,which uses 3D printer to construct engineered cellularized constructs using cells,biomaterials and biological factors,and to achieve tissue mimics with biological functionality similar to human tissue/organs.It has great potential for applications in tissue engineering,regenerative medicine,drug screening and pathological model.As one of the representative methods of 3D bioprinting,extrusion printing is generally faced with some typical problems,such as the lack of ink types,cell shear damage,difficulty in the transformation of printing structure and function. Therefore,the research and development of new ink materials is imperative.Colloidal gel is a new type of gel composed of discontinuous particles dispersed in a continuous phase of solvent,which typically showed shear thinning properties and unique viscoelastic properties.By introducing reversible colloidal particles,colloidal gels are endowed with self-healing properties,making it important in the field of injectable medical filling materials and 3D bio-printable inks.However,upon being used as injectable carrier for cells,the shear force produced in this process will cause damage to the embedded cells,which limits its application in biological printing and injectable cell-loaded scaffolds.In order to solve this problem,gelatin-based colloidal gels were used as extrusion-based bioprinting ink to realize the printing and shaping of cell-loaded colloidal gels.Hereby a microfluidic technique was proposed to encapsulate cells in alginate microgels in order to protect the cells from the damage by shear stress.The microgels generation based on microfluidic,rheological properties and printability of gelatin gel,and the activity and functionality of cells under microgels protection were systematically characterized and studied.Firstly,we generated the alginate microgels with high throughput and remained cell viability.We compared the equilibrium dissociation constant between Ca²⁺ions and EDTA versus NTA molecules in different pH using Isothermal titration calorimetry（ITC）.The results showed that Ca-NTA was easier to dissociate Ca²⁺ions and gelation was faster than Ca-EDTA in the same pH,additionally,its dissociation environment is nearly neutral.Moreover,to determine the effects of different flow rates and acid concentrations on the generation of microgels we used microfluidic generated microgels and observe the sphericity of microgels,the results showed that the generation process was stable.Using Ca-NTA as the calcium source encapsulate NIH-3T3 cells in alginate microgels,the results showed that the size of microgels was uniform,the cells encapsulation obeyed Poisson distribution with 21%single cell encapsulation.To study cytotoxicity for difference calcium complexes as crosslinker and cells functionality we cocultured NIH-3T3 with calcium complexes and cultured encapsulated mesenchymal stem cells（MSCs）with osteogenic medium.The results indicated that Ca-NTA was biocompatible and cells encapsulated in microgels with high cell viability.We further demonstrated the functionality of MSCs in alginate microgels prepared using Ca-NTA as evidenced by the osteogenesis of encapsulated MSCs upon inductive culture.It provided cornerstone for cells that encapsulated in bio ink based colloidal gels with high activity.Secondly,we study the rheological performance and 3D printability about gelatin-based colloidal gels.To determine the rheological parameters of gelatin-based colloidal gels we used rheological tests.The results showed that the storage modulus of gelatin-based colloidal gels increased with the mass volume fraction and it also demonstrated behavior of shear thinning and self-healing.To establish the relationship between printability with the storage modulus we used the mechanical testing machine to determine the printability.The results showed that the printed line was uniform and continuous when the storage modulus is 2 kPa.To determine scaffold from macro and micro we used ultra-depth microscope and scanning electron microscope respectively.The results showed that the material with a storage modulus of 2 kPa showed a more uniform printed line and larger pores.Finally,we further investigated feasibility of constructing 3D tissue mimics based on microgels protected bio-ink.Additionally,we also determined the activity and the function after construction.To evaluate the shear damage of cells that the fluorescent dyes and CCK-8cell viability assay were used.Results showed that membrane damage of NH-3T3 cells in 2kPa and further the nuclear damage in 10 kPa.To detect the effect of calcium crosslink on shear resistance that 5-aminofluorescein-labeled alginate microgels were used.The results showed that the microgels crosslinked by 100 mM Ca2+maintain completeness in 10 kPa.To demonstrate the cell activity and biofunction we cultured MSCs in vitro and in vivo.It indicated the microgels not only protected cells activity but also the biofunction of osteogenic in vitro.The rat femoral defect model suggested the cells encapsulated in microgels promoted the bone regeneration and repaired in vivo.In summary,this work developed a shear-thinning and self-healing colloidal gels as bio inks for 3D construction of cellularized tissue mimics.By preparation of protective microcarriers loaded with microfluidics has effectively solved the problem of cell damage caused by shear force in bioprinting.Based on this shear thinning and self-healing colloidal gels,3D printing of tissue engineering scaffolds with high cell viability and stable cell function were demonstrated.This study opens up new ideas for the preparation of self-healing colloidal gel as bioprinting inks,and provides a theoretical basis and experimental support for the application of biological 3D printing in tissue engineering and regenerative medicine.