Genipin Crosslinking Improved The Biological Performance Of Rat Kidney Decellularized Matrices

[Objective] Pure acellular scaffolds have some shortcomings,such as brittleness enhancement,poor formability and easy degradation.It is often necessary to improve the biological properties of scaffolds through cross-linking reaction.The main purpose of this study is to prepare acellular whole kidney scaffolds crosslinked with genipin by immersion cross-linking method,so as to improve the degradation time,anti-inflammatory and biomechanical properties of acellular renal scaffolds,in order to closer to clinical needs.[Methods] Eighty healthy SD rats weighing about 250 g were randomly divided into normal group,cross-linked group,glutaraldehyde cross-linked group and genipin cross-linked group.Rat kidneys,renal arteries and veins were isolated and connected with peristaltic pump.The kidneys obtained from rat kidneys after PBS perfusion and blood removal were taken as normal group.Rat kidneys were successively infused with heparinized PBS solution,1% Triton X-100,1% sodium dodecyl sulfate(SDS)and deionized water to complete the preparation of rat kidney acellular biological scaffolds.In the glutaraldehyde-crosslinked group,1 500 ml of 0.625% glutaraldehyde(GA)solution was continuously infused for 12 hours;in the genipin-crosslinked group,chemical cross-linking was performed at 37 ? for 24 hours in 0.5% genipin solution;in the kidney acellular scaffold without glutaraldehyde and genipin cross-linking was used as the uncrosslinking group.HE,Masson,immunofluorescence staining microscopy and electron microscopy were used to observe the histomorphology and ultrastructure of the scaffolds;mechanical tensile test was used to detect and compare the mechanical properties of the scaffolds;subcutaneous embedding test was used to detect the anti-inflammatory and anti-degradation ability of the scaffolds;cell scaffolds co-culture test was used to detect the effects of different cross-linkers on cell growth and proliferation.[Results] After crosslinking with genipin,HE and Masson staining showed that collagen fibers arranged more tightly and orderly,fibers gathered in glomeruli,and the fluorescence staining of collagen I and collagen IV was enhanced.Scanning electron microscopy showed that the fibers of the cross-linked acellular scaffold did not break continuously,the three-dimensional space structure was honeycomb-like,and the typical glomerular structure was more clear;Mechanical properties test showed that the elastic modulus of the kidney in the cross-linked group was significantly higher than that in the uncrossed group.Subcutaneous embedding experiments showed that the number of inflammatory cells in the genipin cross-linked group was significantly lower than that in the non-cross-linked group at 3 and 7 days after transplantation,and the glomerular outline of the scaffold was still clearly visible and not degraded at 14 days after transplantation.Inflammatory cells further infiltrated and degraded in the non-cross-linked group and the normal group,and the boundary between the scaffold and the host tissue was blurred.The co-culture experiment of cell scaffolds showed that vascular endothelial cells in each group could adhere to the surface of the scaffolds and grow well after crosslinking with genipin.[Conclusions] Genipin crossinking rat renal decellularized scaffold is simple and feasible,and has good biocompatibility,anti-inflammatory and anti-degradation properties,which makes the three-dimensional spatial structure of the renal decellularized scaffold more full and three-dimensional,greatly improves the biological properties of the scaffolds,and lays a better foundation for the later cell implantation and organ regeneration.