Study On The Charging Performance Of Cell-laden Alginate Bio-ink And Its 3D Bioprinting

Three dimensional bioprinting?3D bioprinting?has received extensive attention in tissue engineering and regenerative medicine because of the ability to print organ or tissue repair with complex structures,precise dimensions and high biological activity.However,the existing methods of 3D bioprinting generally use mechanical force,thermal stress and laser as a driving force,cause great damage to cells.Since cells can grow and divide under DC electric field with high voltage,the use of a DC electric field as a driving force is expected to reduce cells damage during 3D printing.Alginate is a kind of polyanion materials and the calcium alginate?A-Ca?gel microspheres formed by cross-linking with Ca²⁺have a small amount of negative electricity.By increasing the charge of A-Ca gel microspheres,it is expected to move,arrange and pile up under the electric field according to the established route to complete 3D printing.Chondroitin sulfate?CS?is a glycosaminoglycan that plays an important role in maintaining the normal physiological functions of cells and the relative stability of cellular environment.Sulfate groups on CS molecules are more easily to be ionized than carboxyl groups of alginate to form sulfate ion(-SO^3-).Therefore,according to the previous work of the research group,this dissertation provide a basis for the development of bio-inks driven by electric field via the investigation of the influencing factors and rules of the charge of A-Ca gel microspheres.Meanwhile,via the addition of CS in alginate bio-ink to enhance the protection of bio-ink to cell during printing process and through the employment of gelatin substrates containing Ca²⁺to solve the problem that alginate bio-ink is difficult to be printed into a certain shape.Moreover,via the employment of chick chorioallantoic membranes?CAM?to seek a new method to evaluate the vascularization of porous scaffolds achieved by 3D bioprinting.In this study,sodium alginate?SA?was employed as the basic material of bio-ink and A-Ca gel microspheres were printed by extrusion-high voltage electrostatic method.After the observation and calculation by inverted microscope,it was found that the SA concentration,needle inner diameter,voltage size,feed rate,needle tip height and cross-linking time all had influence on the size and the balling performance of A-Ca gel microspheres.At a certain concentration in the range from 50 to 100 mM,the concentration of CaCl₂ solution had no effect on the size and the balling performance of A-Ca gel microspheres.The charge of the gel microspheres was measured by conductometric titration and Faraday cup-coulometer.It was found that the A-Ca gel microspheres contained a small amount of negative charge.The charge was positive correlation with the amount of ionizable carboxyl group in A-Ca gel microspheres.Different concentrations of CS and SA were physically blended to prepare chondroitin sulfate/sodium alginate?CS/SA?bio-ink.These bio-ink were printed by extrusion-high voltage electrostatic method to get chondroitin sulfate/calcium alginate?CS/A-Ca?gel microspheres.Inverted microscope,scanning electron microscope?SEM?,fourier transform infrared?FTIR?and Faraday cup-coulometer were employed to evaluate the size,surface morphology,structure and charge amount of CS/A-Ca gel microspheres.Via the employment of physiological saline and protein solutions with different molecular weights to study the degradation and permeability of CS/A-Ca gel microspheres.Inverted microscopy and SEM images showed that the diameter and pores of CS/A-Ca gel microspheres increased when CS was added.FTIR showed that CS and SA interacted with Ca²⁺and formed a gel.The results of Faraday cup-coulometer demonstrated that with the increasing of CS concentration,the charge amount of the CS/A-Ca gel microspheres can be limitedly increased.The results of degradation and permeability indicated that the addition of CS can promote the degradation of the gel microspheres and increase their permeability to promote substance in and out.Finally,the CS concentration of 0.33 wt%was chose to be the optimal concentration due to the large charge,small size,good balling and permeability.Moreover,a moderate rate of the CS/A-Ca gel microspheres was achieved in this concentration.The CS/SA bio-ink with optimal CS concentration was mixed with human umbilical vein endothelial cells?HUVECs?to prepare CS/A-Ca gel microspheres containing HUVECs by 3D bioprinting.Faraday cup-coulometer was used to measure the charge of CS/A-Ca/HUVECs gel microspheres.Alamar Blue assay,laser confocal microscope and real-time quantitative PCR detection system?qPCR?were used to evaluate the effect of CS/A-Ca/HUVECs gel microspheres on the internal cell growth.The charge test results showed that the charge of CS/A-Ca gel microspheres containing HUVECs was further increased.The results of Alamar Blue assay,laser confocal microscopy and qPCR showed that cells growth and proliferation were normal in CS/A-Ca/HUVECs gel microspheres.Meanwhile the addition of CS can promote HUVECs growth and related gene expression of angiogenesis which can increase the likelihood of blood vessel formation.TheCS/A-Cabio-inkcontainingHUVECswasprintedthrougha laboratory-customized 3D printer.CS/A-Ca bio-ink was cross-linked through a gelatin substrate containing CaCl₂ to construct a“9-grid”scaffold?CS/A-Ca/HUVECs-S?.The effect of feed rate,gelatin concentration and CaCl₂ concentration on the formability of CS/A-Ca/HUVECs-S during the printing process was studied.CS/A-Ca/HUVECs-S immersed in physiological saline is to study its degradability.Alamar Blue assay,laser confocal microscopy and qPCR were used to evaluate the cell growth of CS/A-Ca/HUVECs-S.The results of CS/A-Ca/HUVECs-S formability showed that the optimal feeding speed was 0.003 m L/s,the concentration of CaCl₂ was 200 mM and the gelatin concentration was 20 wt%during the printing.Under these parameters,the structure of“9-grid”scaffold was complete and clear.Alamar Blue assay,laser confocal microscopy and qPCR results were consistent with the CS/A-Ca/HUVECs gel microspheres,showed that the addition of CS was more beneficial to the expression of gene associated with the growth and angiogenesis of HUVECS in CS/A-Ca/HUVECs-S?It increased the possibility of blood vessel formation.ex vivo chick chorioallantoic membrane?CAM?model with abundant blood vessels was developed.Porous hydroxyapatite ceramic?HA?and calcium phosphate cement with segmental porous structure?CPCs?were implanted into CAM to investigate the vascularization.Stereomicroscope and scanning electron microscope?SEM?were employed to observe the angiogenesis on the surface of CPCs.The vascular density,diameters and numbers were quantified by Image-Pro Plus and Nano Measurer.Furthermore,undecalcified histological staining was conducted to observe the inner angiogenesis of porous HA.Microscopic observations and quantitative results showed that the porous structure can promote the growth of blood vessels.Hematoxylin-eosin?H&E?staining showed that the blood vessels grew into the HA through the pore structure.These results indicated that three-dimensional porous structure would promote the angiogenesis.The present CAM model would provide a convenient,efficient and low-cost approach to evaluate the vascularization of porous scaffolds.This study discussed the influencing parameters and rules of the balling performance and the charge of A-Ca gel microspheres made by extrusion-high voltage electrostatic method.The increasing charge amount and bioactivity of A-Ca gel microspheres were achieved by the addition of CS.The problem that alginate bio-ink was not easy to shape was solved by the employment of gelatin substrate containing Ca²⁺.Meanwhile,a new method was provided to evaluate the vascularization of 3D printing scaffolds rapidly and simply by using CAM model.Moreover,a scientific basis was provided for the group who want to study on the 3D bioprinting by electric field driven.