The Study On Preparation Method Of Decellularized Scaffold Of Tissue-engineered Kidney With Anticoagulation And Induced Recellularization Function

Kidney transplantation is the most effective treatment for end-stage renal disease(ESRD).Its clinical application has been severely constrained by the shortage of renal donors.Therefore,researchers are committed to constructing the not only anatomical structures like but also biologically active artificial kidneys for alternative treatment of ESRD.In recent years,with the continuous improvement of medical research level and tissue engineering technology of regenerative medicine,more and more human tissues and organs have been constructed in vitro,and some have been used in clinical practice,bringing new hope for the construction of artificial kidneys.The regeneration strategy of tissue engineered organs is to cultivate seed cells by expansion in vitro,colonize them in a biological scaffold with three-dimensional structure,induce cell proliferation and directed differentiation by reconstructing tissue regeneration microenvironment,and gradually construct functional units and play a joint role to achieve organ regeneration.However,due to the complex structure of the kidney and the important functions of filtering metabolic waste,regulating body fluid balance and endocrine in the body,no teams have yet constructed a tissue engineered kidney that can achieve in vivo transplantation and truly play an alternative function as well.Based on the current research level,in order to realize the physiological structure and functional reconstruction of engineered kidneys,the following key issues need to be solved:(1)Preparation of biocompatibility scaffold materials with complete blood and urinary tract structure as well as growth factors;(2)Determining the development mechanism of the kidney and the differentiation system of various renal functional cells;(3)Remodeling the physiological microenvironment that satisfies the organ culture conditions in vitro;(4)Resolving the thrombosis and survival problem in vivo after the implantation of the engineered kidney.The above are the key issues in the process of engineering kidney construction and only on this basis can we further explore the interaction mechanism among various functional units of the kidney and finally achieve the reconstruction of the whole organ in vitro.In this study,the following work was done from the preparation,anticoagulation modification and recellularization of tissue engineered kidney scaffold.Firstly,we optimized the conditions of renal decellularization techniques.On the basis of previous studies,the various renal decellularized extracellular matrix scaffold preparation processes have been summarized and analyzed,and the reagents and methods causing potential damage to the extracellular matrix(ECM)have been eliminated.Triton X-100 and Sodium Dodecyl Sulfate(SDS)were used to decellularize kidneys.Then,after optimization of perfusion conditions and preliminary experiments,1% TritonX-100,0.5% SDS,0.5% SDS+ 1% TritonX-100 three experimental groups were designed.The above three groups of reagents were used to perfuse the renal artery to Sprague Dawley rats at a flow rate of 2 mL/min.The kidneys of the rat were perfused,and it was found that the use of SDS alone effectively removed the kidney cell components within 12 hours,resulting in a completely transparent extracellular matrix scaffold.By using SDS alone with reducing flow rate we could obtain the completely transparent kidney decellularized extracellular matrix within 12 hours as low as 0.4 mL/min.Next,this experiment attempted to apply a high hydrostatic pressure and repeated freeze-thaw pretreatment method to perfuse the rat kidney.Although it can cause the cell membrane to rupture before perfusion,it cannot further shorten the chemical reagent elution time.On the contrary,because the pressure destroys the natural structure of the kidney,the cell debris cannot smoothly flow out from the lumen with the eluent.Even if the eluate time is extended,a clear decellularized extracellular matrix also cannot be obtained.Therefore,a perfusion decellularization method by using a single reagent SDS,a low concentration,and a low flow rate was finally determined.Secondly,we successfully modified anticoagulant ability of renal decellularized extracellular matrix scaffolds.In this study,a short peptide composed of ten amino acids capable of specifically binding collagen(CQDSETRTFY),collagen binding peptide(CBP)and heparin,was conjugated to form a CBP-Heparin compound,then CBP-Heparin binds to the collagen portion of the decellularized kidney ECM to achieve heparinizing the scaffold after perfusion.It was confirmed by toluidine blue staining,heparin quantification and heparin release test that CBP-Heparin compound can be stably bound to the decellularized extracellular matrix.This study then verified the anticoagulant ability of CBP-Heparin-modified renal decellularized extracellular matrix scaffold.The renal decellularized extracellular matrix scaffold modified with Heparin and CBP-Heparin as well as PBS were divided into three groups for blood perfusion experiments and stained by H&E.Scanning electron microscopy and platelet marker CD61 immunofluorescence staining showed that the number of platelets and red blood cells in the renal decellularized extracellular matrix of the CBP-Heparin group was significantly reduced,and it could effectively prevent thrombosis.Thirdly,we effectively improved the recellularization level of renal decellularized extracellular matrix scaffold.In this study,the human umbilical venous endothelial cells(HUVECs)expanded in vitro were perfused into the Heparin,CBP-Heparin-modified renal decellularized extracellular matrix scaffold via the renal artery and were cultured for 7 days in a microenvironment suitable for cell growth.The scaffolds were taken at 1 day,3 days,and 7 days,respectively.By Immunofluorescence staining of Tubulin and endothelial cells marker CD31,it was demonstrated that the CBP-Heparin-modified renal decellularized extracellular matrix scaffold is more conducive to the adhesion,migration and growth of endothelial cells,which promotes its reendothelialization and creates the conditional basis for vascular and functional reconstruction of tissue engineered kidney.In summary,our study optimizes the process and method of preparation of renal decellularized scaffolds and uses CBP-Heparin to perform anticoagulant modification of the whole decellularized kidneys,effectively improving the level of recellularization of the scaffolds.This study provides good experimental and technical support to realize the regeneration of tissue engineered kidney in the future.