Experimental Study On Injectable Porous Chitosan Microsphonges As Cell Carriers For Engineering Tissue

Objective In this study,a porous chitosan microsponges with uniform and controllable size,shape memory and injectable properties was prepared by a microfluidic emulsion method,and evaluated the potential of this new kind scaffold material as a cell carrier for tissue engineering.Methods Chitosan microsponges were prepared by microfluidic emulsion combined with further freezing and in situ thawing processes.Chitosan microsponges with different concentrations were prepared,and chitosan microsponges with different diameters were prepared by changing the tube diameter and flow rate.The surface morphology and internal porous structure of microsponges were observed by optical microscope,scanning electron microscope and micro-CT;the porosity,swelling rate and degradation rate of chitosan microsponges were measured.The shape memory ability of chitosan microsponges was determined by compression experiments.Through antibacterial experiments,the antibacterial ability of chitosan microsponges was measured.Rat bone marrow mesenchymal stem cells were isolated and cultured,and the expression rates of surface marker antigens CD29,CD45,CD90 and CD11b/c of r BMSCs were detected by flow cytometry.The cytotoxicity and proliferation promoting ability of chitosan microsphonges were detected by Cell Counting Kit-8（CCK-8）.The effect of chitosan microsponges on cell viability was detected by Live/Dead experiment.Chitosan microspsponges and r BMSCs were co-cultured,and the cell adhesion rate was determined;after the cells were co-cultured with chitosan microsponges for 1 d,4 d and 7 d,the morphology and growth state of the cells were observed by scanning electron microscope and confocal laser scanning microscope respectively.Results Chitosan microsponges with porous,uniform size,injectable and shape memory were successfully prepared by a self-made microfluidic device combined with freezing and in situ thawing.The diameter of the chitosan microsponges can be controlled by changing the tude diameter and flow rate.Scanning electron microscopy and Micro-CT showed that the the surface and interior of chitosan microsponges presented porous network structure.Compression experiments show that chitosan microsponges have good shape-memory property.The chitosan microsponges have a relatively high porosity,about 84%.And the swelling ratio is as high as about 3800%.The chitosan microsponges were found to undergo a relatively slow degradation process in vitro.The surface marker antigens CD29 and CD90 of r BMSCs were highly expressed by flow cytometry,and the expression of CD11b/c and CD45 was low,which proved that the cell source was reliable.The results of CCK-8 showed that chitosan microsponges were non-toxic and could promote cell proliferation.The Live/Dead assay indicated that the cell viability was good,and no dead cells with red staining were observed.Laser scanning confocal microscopy observed that r BMSCs grew well on the surface of the microspheres,stretched along the surface of the chitosan microsponges,and the cells bridged nearby microsponges together.Conclusion Chitosan microsponges have a highly porous structure and good sharp-memory property.In vitro cell co-culture experiments showed that it has high biocompatibility and strong support for the adhesion and proliferation of r BMSCs.Chitosan microsponges are expected to be have great potential as efficient cell carriers for tissue engineering,especially as injectable scaffolds for the repair of irregular tissue defects.