Construction Of SDF-1α-loaded Microcavity Hydrogels And Experimental Study On Their Recruitment Of ATDC5 Cells For Cartilage Differentiation

Objective:In this study,the researcher constructed a hydrogel scaffold loaded with SDF-1αgelatin microspheres based on the principles of tissue engineering.By controlling the dissolution of gelatin microspheres to release SDF-1αand form a microporous structure,the researcher discussed the recruitment ability of SDF-1αreleased by the scaffolds to ATDC5 cells and the cartilage differentiation effect of the microporous hydrogel culture system of ATDC5 cells.This study is expected to provide an experimental basis for the future use of this system in repairing cartilage defects.Methods:1.Preparation and degradation observation of gelatin microspheresGelatin microspheres with particle size of 80-120μm were prepared by double emulsification.After modified by Rhodamine B,these gelatin microspheres were observed to degrade in PBS solution and alginate hydrogel respectively.The stability of the microspheres was enhanced by cross-linking with Genipin,and the optimal microsphere cross-linking time was determined using the same observation method.2.Construction and cell recruitment of SDF-1α-loaded microporous hydrogelsMicroporous hydrogels were prepared respectively at the ratios of 0.1g:1m L,0.2g:1m L,0.3g:1m L and 0.4g:1m L microspheres sodium alginate,while microspheres were used to load500ng/m L,1000ng/m L and 2000ng/m L SDF-1αsolution.The concentration of SDF-1αin different groups of microporous hydrogels culture solution was detected by ELISA method to determine the optimal scaffold preparation parameters.The recruitment ability of SDF-1αsolution in different concentration and microporous hydrogel scaffolds to ATDC5 cells was determined by Transwell assay.3.Construction of microporous hydrogel culture system for ATDC5 cells and its chondrogenic differentiationGelatin microspheres were loaded with 1000ng/m L SDF-1αsolution,and two groups of hydrogel particles were prepared in the ratio of 0.3g microspheres:1m L ATDC5 cell alginate mixture with a cell density of 6×10~6/m L.In addition,two groups of hydrogel particles with the same parameters were prepared by taking blank cross-linked gelatin microspheres according to the same method.The above four groups of hydrogel particles were transferred to well plates,and cartilage differentiation was induced by using chondrogenic induction solution as the medium,in which exogenous TGF-β3 was added to two of the four groups of induction solution.Corresponding samples were collected periodically during the culture process.The level of SDF-1αconcentration in the culture system was detected by the ELSIA method.The cell proliferation activity in the culture system was detected by the CCK-8 method.And the expression of cartilage-related characteristic genes and proteins was detected according to Q-PCR and Western Blot.Results:1.Well-phenotyped gelatin microspheres were successfully prepared by double emulsification method.Through electron microscopic observation,it can be seen that porous structures was formed on the surface of microspheres,which was favorable for water absorption and cytokine loading.The degradation rate of the microspheres could be controlled by controlling the cross-linking time of Genipin,and the degradation time of the microspheres cross-linked for 30 minutes in alginate hydrogel was about 5-6 days.2.SDF-1α-loaded microporous hydrogel scaffolds were successfully constructed.The dissolution of gelatin microspheres was used to form pore structures inside the hydrogel and release SDF-1αcontinuously.By ELSIA experiment,it was determined that the optimal ratio of gelatin microspheres to alginate was 0.3g:1m L,and the optimal concentration of SDF-1αsupported by microspheres was 1000ng/m L.Transwell experiment showed that the recruitment effect of SDF-1αon ATDC5 cells was the strongest when the concentration of SDF-1αin culture medium was 10ng/m L,and the hydrogel scaffolds could promote the migration of ATCD5 cells.3.The cell microporous hydrogel culture system loaded with SDF-1αwas successfully constructed.The results showed that the cell proliferation activity of SDF-1αloaded microporous hydrogel and TGF-β3 co-culture group was the highest.This culture system was able to maintain the concentration of SDF-1αin the culture medium while releasing more SDF-1αafter all microspheres dissolved.The expression level of cartilage marker gene in the co-culture of SDF-1αloaded microsphere hydrogel and TGF-β3 was higher than that in the control group,and the WB results also showed that COLⅡwas higher in the two-factor co-culture group.The WB results also showed a higher expression of COLⅡ.It was confirmed that SDF-1αmicroporous hydrogel culture system could promote chondrogenic differentiation of ATDC5cells under the synergistic effect of TGF-β3.Conclusions:In this study,the microporous hydrogel culture system loaded with SDF-1αcan release SDF-1αstably,which has a promotion effect on the migration of ATCD5 cells,and its synergistic effect with TGF-β3 can effectively promote the chondrogenic differentiation of ATDC5 cells.The SDF-1α-loaded microporous hydrogel may provide a new method for cartilage tissue engineering as a new material for cartilage defect repair,and future studies can further optimize the system to provide a more ideal culture environment for chondrogenic differentiation of seed cells.