The Application Of Collagen-binding SDF And Functional Collagen Scaffoldsfor Spinal Cord Injury Repair And Myocardial Regeneration

BackgroundSpinal cord injury and myocardial infarction are the worldwide medical problems.There is no effective treatment in clinic,and stem cells can promote spinal cord injury repair and myocardial regeneration.Neural stem cells(NSCs)have the ability to differentiate into neurons,oligodendrocytes and astrocytes,which are the ideal cells for spinal cord injury repair.Cardiac stem cells and endothelial progenitor cells are the ideal cells for myocardial regeneration.However,stem cells transplantation for the treatment of spinal cord injury and myocardial infarction is difficult to be used in clinical application and transformation due to the limitations of cell source,immunogenicity and injury microenvironment.Many studies suggested that there were endogenous neural stem cells in the adult spinal cord,and endogenous cardiac stem cells and endothelial progenitor cells,etc.in the heart.After spinal cord injury and myocardial infarction,endogenous stem cells can migrate to the injured site under the guidance of SDF.SDF is a kind of chemokine and its receptor CXCR4 expresses on the surfaces of neural stem cells,cardiac stem cells,endothelial progenitor cells and mesenchymal stem cells,which is the main signal molecule mediating endogenous stem cells homing after tissue injury.Therefore,it is possible to promote spinal cord injury repair and myocardial regeneration by localized delivery of SDF at the injured site to recruit endogenous stem cells,which can avoid the restriction of exogenous stem cell transplantation in clinical application.However,native SDF(NAT-SDF)diffuses and degrades rapidly in vivo,reduces its therapeutic efficacy and results in some side effects,and the injured microenvironment is not conducive to the survival and differentiation of stem cells.In this study,we developed a recombinant SDF(CBD-SDF),which could specifically bind to collagen and achieve controlled release.Moreover,we prepared the collagen scaffolds to guide neural and myocardial regeneration.CBD-SDF-modified collagen scaffolds could not only enhance the recruitment of endogenous stem cells by controlling release of CBD-SDF,but also provide an instructive microenvironment and mechanical support for neural and myocardial regeneration.In addition,in order to promote the repair of spinal cord injury,we also used a clinical drug,VPA,inducing the neuronal differentiation of endogenous neural stem cells in vivo.The use of collagen scaffold to controlled release of CBD-SDF and recruit endogenous neural stem cells could simultaneously solve the problems of stem cell source,signal molecule delivery and regenerative microenvironment in the process of nerve and heart regeneration,which was more suitale for clinical application and transformation.Contents and methods1.Preparation of recombinant SDF contains the collagen-binding domainThe native human SDF(NAT-SDF)gene was amplified from human fibroblasts mRNA by polymerase chain reaction.CBD-SDF was constructed by incorporating the collagen-binding domain(CBD)into the C-terminus of NAT-SDF.NAT-SDF or CBD-SDF gene was inserted into the pET28a vector,and then transformed into the BL21 strain(DE3)of Escherichia coli for protein expression.Proteins were purified by a Nickel affinity chromatography column and identified by tricine SDS-PAGE and western blotting.ELISA was used to evaluate the binding and controlled release ability of CBD-SDF with collagen.2.CBD-SDF-modified collagen scaffold combined with VPA administration for spinal cord injury repairWe isolated and cultured the NSCs,the expression of nestin and CXCR4 on the surface of NSCs were detected by immunostaining method.Transwell system was used to study the migration and adhesion of NSCs mediated by CBD-SDF.Subsequently,we prepared a functional collagen scaffold for spinal cord repair by tethering the CBD-SDF on the linearly ordered collagen scaffold(LOCS).In the rat complete spinal cord injury model,we studied the function of functional collagen scaffold combined with VPA(i.p)in the recruitment of endogenous neural stem cells,neurons regeneration,synapses formation and glial scar formation and so on.Finally,the BBB scoring system was used to evaluate the locomotion recovery in rats during the 8 weeks post-surgery.3.CBD-SDF and collagen membrane for myocardial regeneration(1)Transwell system was used to study the migration of mesenchymal stem cells and c-kit positive(c-kit~+)stem cells mediated by CBD-SDF.In the rat acute myocardial infarction model,CBD-SDF was injected into the ischemic area,and western blot was used to detect the binding ability of CBD-SDF and VEGF expression.At 3 days post-surgery,immunofluorescence staining was used to detect the c-kit~+endogenous stem cells.Masson's trichrome-staining was used to analyze the scar size and the thicknessof the left ventricle(LV)wall.An anti-von Willebrand Factor(vWF)antibody was used to evaluate the density of the capillary vessels.(2)We prepared a 3-dimensional collagen membrane,and then analyzed its microstructure and mechanical ability by scanning electron microscope and mechanical measurement system,respectively.The functional cardiac patch was developed by tethering CBD-SDF on the collagen membrane,and ELISA was used to analyze the controlled release of CBD-SDF from collagen.The functional cardiac patch was implanted into a rat myocardial defect model.At 3 days post-surgery,immunofluorescence staining was used to detect the c-kit~+and CD31~+endogenous stem cells.At one month post-surgery,anti-von Willebrand Factor(vWF)antibody and anti-?-sarcomeric actin(?-SMA)antibody were used to evaluate the regeneration of the capillary vessels and myocardium,respectively.Result1.CBD-SDF could bind to collagen and achieve controlled releaseWe successfully constructed the recombinant expression vectors of NAT-SDF and CBD-SDF,and obtained stably expressed E.coli cell lines.The purified CBD-SDF could specifically bind to collagen and achieve controlled release.2.CBD-SDF modified LOCS combined with VPA administration could promote spinal cord injury repairNestin and CXCR4 were co-expressed on NSCs.Both of CBD-SDF and NAT-SDF could promote the migration of NSCs,and there was no significant difference between them,indicating that the incorporation of CBD had not affected the bioactivity of SDF.VPA could promote the neural differentiation of NSCs but inhibit their astrocytic differentiation.In the rat complete spinal cord injury model,CBD-SDF-modified LOCS combined with VPA administration could simultaneously promote the recruitment and neuronal differentiation of endogenous NSCs,promote neural regeneration and synapses formation,inhibit scar formation,and finally improve locomotor function.3.CBD-SDF and the functional cardiac patch could promote angiogenesis and myocardial regeneration(1)In the rat acute myocardial infarction model,CBD-SDF could bind to the endogenous collagen and achieve controlled release,enhance the recruitment of c-kit~+endogenous stem cells and promote the expression of VEGF in the ischemic area.Finally,CBD-SDF could reduce scar size,increase the wall thickness of left ventricular,promote angiogenesis and improve cardiac function.(2)We prepared the 3D collagen membrane for myocardial regeneration.The collagen membrane had good pore size,which was favorable for cell adhesion and growth.Moreover,the collagen membrane had good mechanical properties,which could provide mechanical support for myocardial regeneration.A functional cardiac patch was constructed by tethering CBD-SDF on the collagen membrane,which could achieve controlled release from the collagen membrane.In the rat myocardial defect model,the functional cardiac patch could effectively enhance the recruitment of c-kit~+and CD31~+endogenous stem cells,promote angiogenesis and myocardial regeneration.ConclusionTo sum up,in this study,we developed a collagen-binding SDF,which could bind to collagen and achieve controlled release.The combination of collagen scaffold and CBD-SDF could achieve controlled release of SDF and enhance the recruitment of endogenous neural stem cells,which could simultaneously solve the problems of stem cell source,signal molecules delivery and regenerative microenvironment in the process of nerve and heart regeneration.