Repair Of Acute Liver Injury In SD Rats By 3D Porous Chitosan Microspheres Combined With Adipose Stem Cells

Objective1.Simple 3D porous chitosan microspheres with stable pore size and porosity were prepared in vitro.Then soybean protein was coated on the surface of pure chitosan microspheres to compare the pore size,porosity and cell compatibility of the two microspheres with adipose stem cells.2.Microspheres with good compatibility with adipose-derived mesenchymal stem cells?ADSCs?were selected for general scanning electron microscopy?SEM?and CCK-8proliferation experiments to observe the proliferation ability of ADSCs on 3D porous chitosan microspheres.3.Adipose-derived mesenchymal stem cells?ADSCs?and porous chitosan microspheres were transplanted into SD rats to repair acute liver injury.Method1.3D porous chitosan microspheres and coated soybean protein were prepared by Beijing University of Science and Technology.Two kinds of finished microspheres were placed on the carrier platform.The diameter,pore size and porosity of the microspheres were observed by scanning electron microscopy after spraying gold.After sterilization with ⁶⁰GO,the 3-D porous chitosan microspheres were mixed with50 x 10⁴ adipose mesenchymal stem cells in a centrifugal tube.After 2 hours,the microspheres were cultured in a incubator at 37?in a 6-well plate.At the 1st,4th and7th day,the microspheres were removed for live/dead staining and the cell expansion was observed under confocal microscopy.2.Adipose tissue of 48h SD suckling rats was isolated,cultured and subcultured by type II collagenase magnetic bead centrifugation.Immunophenotype of adipose mesenchymal stem cells was detected and identified by flow cytometry,and the potential of inducing adipogenesis,osteogenesis and chondrogenesis was also identified.3-D porous chitosan microspheres combined with adipose mesenchymal stem cells survived 1,4 and 7 days later./After fixed,dehydrated,dried and sprayed gold,adipose mesenchymal stem cells with fixed cell number were inoculated into 96-well plate and cultured continuously in the extract of 3-D porous chitosan microspheres.CCK-8 kit was added and the growth curve was measured by enzyme labeling.3.Acute rat liver injury model was established by intraperitoneal injection of 10%CCl4-olive oil?10ml/kg?.3D porous chitosan microspheres combined with adipose-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells were transplanted to the liver surface respectively.Rat venous blood was collected at different time points for automatic biochemical detection of liver function in different groups?n=3 rats?,and then rat liver was harvested.The histological changes of liver in different groups?n=3 rats?were observed by HE method.Adipose-derived mesenchymal stem cells?ADSCs?were labeled with PKH-26 kit and combined with porous chitosan microspheres.Simple cells and microunits were transplanted into rat liver surface.Rat liver was frozen at different time points and stained with DAPI to observe the survival rate of ADSCs with the prolongation of treatment time.Result1.Under confocal microscopy after staining,we observed that the 3-D porous chitosan microspheres coated with soybean protein were slightly higher than the pure chitosan microspheres on the first day,and expanded a lot on the fourth and seventh days than the pure chitosan microspheres.With the prolongation of time,adipose mesenchymal stem cells migrated and grew slowly into the microspheres.After the fourth and seventh days of culture,the cell activity was significantly higher than that on the first day.Significance?P<0.05?.2.Adipose-derived mesenchymal stem cells cultured with type II collagenase grew adherently and fused gradually.After stable passage,the cell morphology and growth rate did not change significantly.Adipose-derived mesenchymal stem cells expressed CD29,CD90?99.9%,97.6%?and did not express CD11b,CD45?4.2%,1.8%?by flow cytometry.After 24 hours,a large number of cells adhered to the microspheres.Over time,a large number of adipose mesenchymal stem cells proliferated and slowly migrated to the microspheres.After 4 and 7 days of culture,the number of cells was significantly higher than that on the first day?P<0.05?The morphology of adipose-derived mesenchymal stem cells on the microspheres remained unchanged,and the adhesion of adipose-derived mesenchymal stem cells was good.The proliferation of adipose-derived mesenchymal stem cells in 96-well plate was observed by CCK-8,which proved that 3D porous chitosan microspheres had no obvious cytotoxicity.3.Liver function test and HE staining showed that the rat model of acute liver injury was successfully constructed.The results showed that the liver function of the rats recovered to near normal level on the third day of treatment?P>0.05?,and there was significant difference between the acute liver injury group and the blank control group?P<0.05?.HE staining showed that transplantation of adipose-derived mesenchymal stem cells-soybean protein+3D porous chitosan microspheres 1,2 and 3 days after acute liver injury was superior to intrahepatic injection of adipose-derived mesenchymal stem cells?P<0.05?.The treatment effect of the two groups was better than that of the control group?P<0.05?.On the 7th day,the liver function of all groups was similar to that of the blank control group.The results showed that the liver tissue damage and necrosis were significantly improved in all groups.The fluorescence intensity of PKH-26 was observed under fluorescence microscope after 1 day,1 week and 2 weeks.It was found that the number of adipose-derived mesenchymal stem cells decreased with the prolongation of time,and there were still residual adipose-derived mesenchymal stem cells at 2 weeks.Conclusion1.3D porous chitosan microspheres coated with soybean protein have better compatibility than simple microspheres,and adipose stem cells have stronger proliferation ability on soybean protein coated microspheres than simple microspheres.2.Adipose-derived mesenchymal stem cells?ADSCs?are abundant in resources,easy to obtain and do not involve ethical issues.Adipose-derived mesenchymal stem cells?ADSCs?have good proliferation and adhesion ability on 3D porous chitosan microspheres coated with soybean protein,which indicates that 3D porous chitosan microspheres coated with soybean protein are good microcarriers for stem cells in liver tissue engineering research.3.3D porous chitosan microspheres combined with adipose-derived stem cells and simple stem cell transplantation can improve the liver function of rats with acute liver injury.The therapeutic effect of microspheres combined with adipose-derived stem cells is better than that of intrahepatic injection of adipose-derived stem cells alone.It shows that 3D porous chitosan microspheres are good microcarriers in liver tissue engineering.