Genipin Cross-linked Decellularized Scaffold Induces Regeneration Of Rat Defected Kidney

[Objective] The regenerative capacity of the kidneys is almost zero.Some scholars have tried to use decellularized kidney biological scaffolds to mediate the regeneration of injured kidneys,and some progress has been made.However,the simple decellularized biological scaffold has disadvantages such as easy collapse,deformation and rapid degradation.After the kidney scaffold is implanted into partially nephrectomized rats,if it degrades too quickly,it cannot provide a good growth environment for the migration and regeneration of tissues or cells.To solve this problem,cross-linking agents are usually needed to improve the performance of scaffolds.This study intends to improve the performance of decellularized kidney biological scaffolds by Genipin cross-linking method,which is beneficial to the regeneration of tissues and cells,and can be used for kidney damage Regeneration and repair provide better conditions.[Methods] Ninety SD rats,weighing about 230 g,were completely randomly divided into the normal group,the uncrosslinked scaffold group and the genipin crosslinked scaffold group.Blood vessels were ligatedfrom rats,kidneys were isolated,and PBS was removed by abdominal aorta perfusion to serve as the normal group;isolated kidneys were perfused with heparin solution,1% TritonX-100,1% sodium dodecyl sulfate(SDS),Deionized water was used to obtain the kidney biological scaffold after decellularization as an uncrosslinked scaffold group.The prepared decellularized biological scaffold was immersed in a 0.5%Genipin solution in a 37 ℃ incubator for 12 hours to obtain Genipin crosslinked scaffold group.The morphological characteristics of the kidneys of the three groups were observed with HE,Masson,and immunofluorescence staining respectively;the vascular structure of the three groups of kidneys was observed by vascular corrosion molds,and the retention of the vascular structure of the kidneys in the stent group was analyzed;type I ollagen enzymatic degradation,the three groups of kidneys were tested for anti-degradation ability.Cells were co-cultured with scaffolds.CCK8,Transwell migration,and immunofluorescence experiments were used to detect the effects of scaffolds on cell survival and growth.The left lower kidney of rats was excised to prepare prepared scaffolds.The injury resection site was made up to observe the effect of scaffold implantation in the body after cross-linking on the regeneration ability of injured kidney.[Results] After the decellularization process of normal rat kidneys,a simple decellularized kidney biological scaffold was obtained.The resultsof HE,Masson staining and immunostaining staining showed that the biological scaffold had almost completely removed the nucleus components in the tissue,and retained the extracellular matrix components.The collagen fibers of kidney bioscaffolds cross-linked by genipin are neatly and densely arranged,similar to the normal group,and the outline of the glomeruli and the fibrous structure gathered in the glomerular region can be clearly seen.It can also be seen from the fluorescence results that the cellular components are completely removed,and the specific fluorescent staining of type Ⅰ collagen and type Ⅳcollagen is enhanced.Renal vascular corrosion casting results show that the complete vascular structure remains in the decellularized kidney biological scaffold,and the scaffold after cross-linking with genipin also retains the similar complete vascular structure in the kidney of the normal group,but does not damage to vascular structures in the scaffold.Type Ⅰcollagen enzymatic degradation experiments showed that the anti-degradation ability of decellularized kidney biological scaffolds was significantly improved after genipin cross-linking.Vascular endothelial cells were co-cultured with kidney scaffolds in each group.The results showed that the kidney scaffolds in each group had cell adhesion growth.CCK8 results showed that renal scaffold was not cytotoxic to vascular endothelial cells after cross-linking with genipin.Cell compatibility can promote cell proliferation to a certain extent.The results of in vivoimplantation of biological scaffolds showed that after genipin cross-linked decellularized kidney scaffolds,it was seen that the uncross-linked decellularized scaffolds quickly degraded and the tissue was blurred.After genipin cross-linked renal scaffold complemented the defect site,it was apparent that cells moved into the scaffold and the glomerular structure was clearly visible.[Conclusion] Genipin cross-linked rat kidney scaffolds complementing the kidney defect in the rat can enhance the scaffold-induced kidney regeneration and repair ability.