The Study Of Construction Of Multifunctional 3D Printing Hydrogel System And Repairing Spinal Cord Injury

【Objective】The purpose of this study is to construct a 3D printed multifunctional hydrogel scaffold system loaded with BMSCs and BDNF.We plan to analyze its spatial structure and drug releasing properties,and to evaluate its safety in animal experiments by cytotoxicity experiments and functional determination,providing a theoretical basis for the future application of hydrogel scaffolds in animals to repair spinal cord injury.【Methods】1.Preparation and characterization of the 3D printed hydrogel scaffoldThe printing ink was composed of 10% GelMA hydrogel,BMSCs and LAP cross-linking agent.The 3D bioprinter was used to print a hydrogel scaffold containing BMSCs in a certain shape.After printing for 24 hours,the scaffold and cell morphology were observed under a scanning electron microscope.Sustained-releasing test: the initial concentration of BDNF in each scaffold is 0.5 μg,PBS is added and exchanged once a day,and the PBS solution at different time points were collected separately.After the collection of all samples is finished,the ELISA kit was used to detect the release of BDNF at each time point.2.Biocompatibility analysis of hydrogel scaffold systemAfter the the hydrogel scaffold was finished for 24 hours,the samples of the 2D culture medium group and the 3D printed scaffold group were pretreated.Live/Dead Cell Staining Kit was used to detect the cell survival rate of each group after printing,the cell morphology was observed and photographed with a laser confocal microscope.In the same way,the CCK8 kit was used to test the proliferation of the cells 1 day,4 days,7 days,and 14 days after printing.3.Functional determination of 3D printed hydrogel systemSeven days after the preparation of hydrogel scaffold,1000 μLTrizol was added to the 2D and 3D cultured cell samples.After the cells were fully lysed,30 μl of RNA-free water was added to dissolve the RNA,and 1 μl of total RNA was measured for OD260.Then,we added RNase inhibitor(50U/μl),primers(50p M/μl),and RNAto take a reverse transcription in a 1.5ml centrifuge tube.Under certain conditions,PCR amplification and q PCR were used to detect the rxpression of CD29,CD44,CD73,and CD166 mRNA.【Result】1.BMSC and GelMA hydrogel were configured as bioprinting inks,and the hydrogel scaffolds were printed successfully by 3D bioprinting technology.Through SEM observation,the porous and three-dimensional network structures can be seen clearly,and BMSCs were evenly distributed to grow on each part of the scaffold.In the 3D printed hydrogel scaffold system,the release dose of BDNF was the largest on the first day,the sustained release capacity was stabilized within a week,and the biological active dose of BDNF was still maintained at 2-4 weeks after printed.2.The result of Live/Dead Cell Staining showed that BMSCs survived well in2 D medium and 3D hydrogel scaffolds 24 h after printed.It can be clearly seen in a high magnification field that the cells in the 2D culture medium were distributed in a long spindle shape,and the cells were arranged closely,some of them contacted each other.The living cells in the 3D scaffold were arranged in an orderly manner with the scaffold structure,and dispersed evenly inside or on the surface of the scaffold.The cell density at 3D scaffolds in each field of view is a bit lower than 2D,but after each field of view is superimposed layer by layer,the total number of images after synthesised is binger than 2D group.In the cytotoxicity experiment,the cells in the2 D group increased at day4 compared with the 1st day,but the cell viability decreased on the 7th and 14 th day.While in the 3D scaffold,the cells showed a continuous growing trend.On the whole,BMSCs had higher survival rates in the experimental group than in the control group on day 1,4,7,and 14,respectively.The difference between the two groups was statistically significant(P <0.05),proving that 3D bioprinting hydrogel scaffolds had lower toxicity,higher safety and stronger cell proliferation than 2D culture medium.3.2D and 3D cultured BMSCs expressed a certain amount of CD29,CD44,CD73,and CD166 on the cellsurface.Among them,the expression of CD29 in the 3D hydrogel scaffold group was less than that in the 2D group,and the difference was statistically significant(P <0.05).There was no significant difference between thegroups in the expression of CD44,CD73,and CD166(P > 0.05).【Conclusion】1.In this experiment,a multifunctional 3D bioprinted hydrogel scaffold system with excellent properties such as porous,sustained release,and uniform cell distribution was successfully prepared.The scaffold system had good biocompatibility,and it was safe for cell proliferation.2.3D printed hydrogel scaffolds can reduce the migration of bone marrow mesenchymal stem cells by reducing the expression of CD29,so that cells could gather inside the scaffold better and promote axonal growth effectively.3.Cytokines released in the hydrogel scaffold system continuously,changing the location and function of the BMSCs,increasing the number of cells differentiated toward neurons,and improving the efficiency of axon regeneration,thereby it could repair spinal cord injury by multiple mechanisms in the future.