| Background:Spinal cord injury (SCI) is a trauma of the central nervous system with high disabilityrate. Tissue engineering approaches have great potential for treating SCI. The classicalstrategy is to use a biomimetic scaffold for cell-loading, in vitro proliferation, andconsequent in vivo implantation. The scaffold provides a suitable microenvironment for cellpenetration and adequate mechanical strength to support cell adhesion and proliferation.Various polymeric materials such as poly (lactic-co-glycolic acid)(PLGA), collagen,and chitosan have been used as tissue-engineered scaffolds for SCI repair; however, thesescaffolds fail to mimic the complex three-dimensional (3D) structure of the spinal cord,Thus they are undesirable for successful spinal cord regeneration. Recently, bio-derivedacellular extracellular matrices have been used for tissue engineering applications. Cells areremoved from the materials (decellularization), leaving behind the extracellular matrix(ECM) and its3D structure. These bio-derived acellular scaffolds provide the frameworkfor cell adhesion, migration, proliferation, and differentiation; and they subsequently induceangiogenesis. They can be further implanted with minimal rejection and completelyreplaced with parasitifer. These biomaterials are of great interest and have been successfullyused in the skin, bladder, small intestine, heart valves, blood vessels, skeletal muscle, andperipheral nerve.In the previous study, the research group attempts to decellularize rat spinal cord,hoping to remove cells and myelin components within the spinal cord, while retainingcollagen, fibronectin, laminin and other ECM as well as its3D spatial structure, to constructa new biological scaffold that conform to spinal cord physiological needs and its anatomicalstructure. But the established decellularizing method is not sound, it can easily destroy the 3D structure of the spinal cord ECM. And there are also residual cells in the scaffold treatedby this method. Scaffolds constructed with this method have deficiencies such as poorbiomechanical intensity, weak anti-enzymolysis capacity, and poor structural stability. Thesubject plans to optimize the preparing method of acellular spinal cord scaffolds on basis ofprevious work, so that to prepare rat acellular spinal cord scaffolds that have beenthoroughly decellularized while retaining integrate ECM structure. Genipin (GP) chemicalcrosslinking processing is applied to modify rat acellular spinal cord scaffolds, so that toenhanceits biomechanical properties, anti-enzymolysis capacity and structural stability. Thebiocompatibility of modified and optimized rat acellular spinal cord scaffolds and its impactto rat bone marrow mesenchymal stem cells (BMSCs) will be evaluated. We are in hope ofproviding a new ideal biological scaffold for SCI repairing tissue engineering research.Objectives:1. To modify preparing methods of rat acellular spinal cord scaffolds, that canthoroughly remove cells, myelin sheath and other immunogenic components within thespinal cord, while retaining the integrity of the natural3D structure of spinal cord ECM aswell.2. To establish an in vitro isolating and culturing method for rat BMSCs withcomparatively higher purity, and study its multipotential differentiation inducingtowards osteoblasts, adipocytes and neural cells, to provide ideal seed cells for SCIrepairing.3. To evaluate the influence of GP-crosslinking to biomechanical properties,anti-enzymolysis capacity, structural stability and other physical and chemical properties aswell as biocompatibility of rat acellular spinal cord scaffolds, and to provide a new idealscaffold for SCI repairing tissue engineering research.Methods:1. Acellular spinal cord scaffolds were prepared by the improved and previousmethods respectively, and a comparative study was performed on their general morphology,microstructure, excellent rate, DNA residue, porosity, water content, enzymolysis rate andother characteristics using visual inspection, light microscopy, scanning electronmicroscopy, DNA detection kit, agarose gel electrophoresis and other techniques.2. Rat BMSCs were isolated and cultured in vitro by density gradient centrifugation combining with differential adhesion method, using the MTT method to test cellproliferation viability, and flow cytometry to identify expression of cell surface marker.After differentiation of the cells towards osteoblasts, adipocytes and neural cells wereinduced, alizarin red staining, alkaline phosphatase staining, oil red O staining and NSE,GFAP immunofluorescence staining were performed respectively, to detect themultipotential differentiation of the rat BMSCs.3. Rat acellular spinal cord scaffolds were crosslinked by GP and glutaraldehyde (GA)respectively, and then a comparative study was performed on their general morphology,microstructure, porosity, water content, enzymolysis rate, structural stability andbiomechanical properties, using visual inspection, light microscopy, scanning electronmicroscopy, biomechanical testing instrument and other techniques.4. Rat BMSCs were seeded on uncrosslinked, GP-crosslinked, and GA-crosslinked ratacellular spinal cord scaffolds respectively, then they were cultured in vitro, the threegroups were comparatively studied on their cytotoxicity, cell adhesion capacity andbiological behaviors of the cell such as growth and migration on the scaffolds, by MTTassay, light microscopy, electron microscopy and other techniques.5. Normal spinal cord, uncrosslinked, GP-crosslinked and GA-crosslinked rat acellularspinal cord scaffolds were implanted subcutaneously into allogeneic rats, and their internalinflammatory response, absorption process and immunogenicity were comparativelystudied by HE staining, CD4and CD8immunohistochemistry staining.Results:1. Rat acellular spinal cord scaffolds prepared by modified method were decellularizedmore thoroughly, and a more complete ECM3D structure was retained. And their excellentrate was65%, DNA residue of35.7±18.5ng/mg dry weight, which are better than thosewith the ones prepared by unmodified method; while the pore size range was10~100μm,porosity rate of85.9±6.3%, water content of284.1±12.7%, and their in vitro enzymolysisrate was fast, all of which were almost as the same with those of unmodified method.2. Rat BMSCs isolated and cultured by density gradient centrifugation combining withdifferential adhesion method possessed of typical fibroblast-like spindle-shapedmorphology, a purity of about98%, vigorous proliferation, expressing CD44, CD90molecules without expressing CD34and CD45molecules, and having multipotential differentiation inducing towards osteoblasts, adipocytes and neural cells.3. Comparing with uncrosslinked scaffolds, ultimate tensile strength and elasticmodulus of GP-crosslinked rat acellular spinal cord scaffolds increased from0.112±0.071MPa,0.564±0.113MPa to0.193±0.064MPa,1.541±0.082MPa respectively, and theanti-enzymolysis capacity and stability in PBS were significantly enhanced, while generalmorphology, microstructure, porosity rate, and pore size were maintained, but the watercontent decreased slightly to229.7±12.5%, above results were basically the same withGA-crosslinked group.4. GP-crosslinked and uncrosslinked scaffolds were with comparatively strong celladhesion ability, cell relative growth rate of nearly100%, cytotoxicity rating of0to1, nosignificant cytotoxicity, rat BMSCs can grow, migrate, and secrete ECM normally on thetwo groups of scaffolds, which is superior to GA-crosslinked group.5. GP-crosslinked scaffolds implanted into allogenic rats, only caused mild foreignbody reaction, and the in vivo absorption time was prolonged significantly comparing withuncrosslinked groups, immunogenicity was extremely weak, almost the same with that ofGA-crosslinked scaffolds, and significantly better than the uncrosslinked groups.Conclusions:1. This study successfully improved the preparation method of rat acellular spinal cordscaffolds; the modified method can thoroughly remove cells and myelin components whilecomparatively retaining intact natural3D ECM structure. And the prepared rat acellularspinal cord scaffolds were with natural3D network pore structure and good porosity rateand water content2. Rat BMSCs with high purity that were successfully isolated and cultured usingdensity gradient centrifugation combining with differential adhesion method, possess ofmultipotential differentiation inducing towards osteoblasts, adipocytes and neural cells, andcan be used as ideal seed cells to repair SCI.3. GP-crosslinked rat acellular spinal cord scaffolds, whose biomechanical properties,anti-enzymolysis capacity and structural stability had been significantly enhanced, possessof similar general morphology, natural3D structure, good porosity, and water content tonormal spinal cord. GP shares the same effect with the traditional chemical crosslinkingagent GA. 4. GP-crosslinked rat acellular spinal cord scaffolds possessed of good cellularcompatibility and tissue compatibility, extremely low immunogenicity, and showed goodbiocompatibility, which is significantly better than that of crosslinked with GA, while theirin vivo absorption time was significantly prolonged comparing with uncrosslinked ones. Ithas provided SCI repair tissue engineering with a new ideal biological scaffold. |
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