| Objects:(1)To improve existing 3D bioprinting processes and biomaterials and promote 3D printing efficiency and printing quality;(2)To explore the effect of solvent PBS ionic strength(or concentration)of Alg-Gel bioink on printability and shape maintenance of printed constructs;(3)To explore the effect of solvent PBS ionic strength(or concentration)of Alg-Gel bioink on the biological behaviors of epidermal stem cells;(4)To explore the effect of regulation of the ionic strength of solvent PBS and the concentration of gelatin in Alg-Gel bioink on the maintenance of sternness and differentiation of epidermal stem cells.Materials and methods:(1)Selecting sodium alginate samples with uniform molecular weight and less impurity content by detecting the molecular weight distribution of different raw material samples;Selecting suitable printing temperature and printing nozzles by detecting the viability of embedded epidermal stem cells in bioprinted constructs under different printing temperatures and printing nozzles.(2)Analysis of printability of Alg-Gel bioinks with different solvent by detecting of viscosity,shear stress,storage modulus and lose modulus;Analysis of surface morphology and shape maintenance of Alg-Gel bioinks with different solvent by observation of bioprinted constructs during culture in vitro.(3)Live-Dead staining was used to detect viability fluctuation of epidermal stem cells in bioprinted constructs during culture in vitro;Ki-67 staining was used to detect proliferation of epidermal stem cell;Immunofluorescence staining and RT-PCR was used to detect the expression of keratin proteins K5,K14,K8,and K18 of epidermal stem cells;Observation of cell aggregation in the bioprinted constructs was performed using microscope.(4)Using the improved 3D printing process to test the printability of Alg-Gel bioinks with different solvent ionic strength and gelatin concentration;Measuring and comparing the extension rate of printed constructs;Using Live/Dead staining to detect the fluctuation of cell viability in bioprinted constructs during culture in vitro;RT-PCR was used to quantitatively analyze the expression of K5,K14,K8,K18,K10,and E-cad of epidermal stem cells in constructs printed with different bioinks;Microscope was used to evaluate cell aggregation in the printed constructs.The cell proliferation and differentiation was analysed in combination to determine the cell behaviors.Results:(1)The molecular weight distribution of sodium alginate sample C is more concentrated,and its actual printed tissue contains less impurities.Printing nozzle with an diameter of≥ 340 μ m perform less shear stress on cells and less influence on the cell viability after printing,while printing nozzle with a diameter of<340 μm perform significantly increased shear stress on cells and cell viability after printing was significantly reduced.Cell viability was extremely low at a temperature of 4℃ in printing syringes and increased when temperature of the printing syringes rose between 10℃ and 20℃.Cell viability was significantly increased when the temperature of the printing syringes rose above 20℃.Different platform temperatures had little effect on cell viability as soon as the temperatures of print syringes were the same.(2)From bioink B-l to B-4,with the increase of solvent ionic strength,the storage modulus(G’)of Alg-Gel bioink increases significantly,and the loss modulus(G")decreases significantly;Viscosity(V)and shear stress were also decreased significantly.Surface of B-1 was flat and surface cells were expanded;Surface of B-2 showed a smooth gutter-like shape with cell extension and branching;The surface of B-3 and B-4 became loose and granular,and the cells on the surface did not expand or aggregate into cell clusters and were covered with bioink.During the in vitro culture,the bioprinted constructs of B-1 and B-2 maintained the printed morphology.Cells in the printed constructs were clearly visible.The bioprinted constructs of B-3 and B-4 were significantly swollen and degraded and B-4 was more serious.The printed surface of B-3 and B-4 is villous when viewed under the microscope and the cell shadow is blurred and there was a phenomenon of printed material gathering and scattering.(3)The printing process of 3D printing had little effect on cell viability,but with the prolonged culture time in vitro,the cell wiability slowly declined.During in vitro culture,cell proliferation decreases significantly,and the proliferation activity of B-1 falls below 50%while downward trend of B-2 was slower.The proliferation and aggregation of cells in the B-2 bioprinted constructs was more obvious,and the cell gradually became blurred and part of cell clusters formed irregularly distorted glandular-like structure between the 14th and the 28th day of culture.In the B-2 bioprinted constructs,K8 and K18 expression was significantly increased while K5 and K14 were continued to express.(4)The contemporary regulation of the solute gelatin concentration and the PBS ionic strength of the solvent of Alg-Gel bioink would significantly impact on its printability.Bioink with low gelatin concentration and medium or high ionic strength could not be printed successfully.Bioink with low concentration gelatin and bioink with medium gelatin concentration and high ionic strength showed higher expansion rate.Other bioinks could be printed successfully and their extension rates were similar.Changing the solute and solvent had no effect on the cell viability in the bioprinted constructs,however,mainly affecting cell aggregation and differentiation:bioink with high gelatin concentration and medium ionic strength significantly promoted cell proliferation,aggregation and differentiation into sweat glands while maintaining sternness.Cell proliferation was low in high ionic strength bioinks,but cell sternness could be maintained.In bioink with high concentration of gelatin and low ionic strength,cell proliferation is low and stemness was not maintained,however differentiation into epidermal keratinocytes could be promoted.Conclusions:(1)Based on the previous 3D printing process,the printing nozzle(340 μm)was optimized,the printing temperature(20 ℃)was increased,and the sodium alginate material sample C which was more suitable for 3D bioprinting was screened.They helped to improve the 3D printing efficiency and quality.(2)A method of regulating the physical properties of Alg-Gel bioink by changing the solvent PBS concentration(or ionic strength)was initially established.The higher the PBS concentration(or ionic strength)of the solvent,the lower the viscosity and stiffness of the Alg-Gel bioink,which would be prone to the shape destruction of the printed constructs.According to the results,Alg-Gel bioink B-2(solvent 0.5×PBS,ionic strength 0.082M)not only had good printability,but also print the constructs with low swelling ratio and degradation rate after long term culture in vitro.(3)The influence of Alg-Gel bioink solvent on the biological behavior of epidermal stem cells was explored.By excluding the influence of the type and concentration of biomaterials in bioink and the type of solvent,simply changing the ionic strength of the bioink solvent,it was confirmed that the printed constructs with the Alg-Gel bioink B-2 had significant boost to the biological behavior of epidermal stem cells.Compared with other bioinks,B-2 significantly increased the proliferation of epidermal stem cells,and promoted differentiation into sweat gland cells while maintaining sternness and forms a cell-aggregated glandular-like structure in printed constructs.(4)High gelatin concentration(5%)could promote the differentiation of epidermal stem cells into epidermal keratinocytes.Highe ionic strength(0.156M)could maintain the sternness of epidermal stem cells.Medium gelatin concentration and ionic strength(3%and 0.082M)could promotes differentiation into sweat gland cells while maintaining sternness of them.This findings provided a sufficient theoretical basis for the construction of 3D printed skin substitute model containing sweat gland. |
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