Experimental Study Of Degradable Tissue Engineering Small-diameter Vessel

【Objective】:To construct the degradable small-diameter vessel and determine its biomechanical properties in vitro and degradation properties in vivo.CD₃₄⁺ cells,which isolated from bone marrow of carine,were cultured and expansion.Then evaluate their biological peoperties and seed them to the artificial scaffold.The result may be useful for futher research of tissue engineering small-diameter vessel.【Methods】:Biodegradable polydioxanone(PDS) suture has the excellent properties of strength and elasticity,which was selected to prefabricate to a mesh tube as the scaffold.The biomechanical properties of PDS and scaffold were measured by elongation test.Constructed the multilayering scaffold with PDS(polydioxanone,PDS) fibers woven as middle elasticity layer and coved with SIS(small intestinal submucosa,SIS), the inner layer was coated with collagen sponge which was added with 10%CS (ChondroitinSulfate,CS).Compare its biomechanical properties such as burst strength,rupture intensity and compliance with canine carotid artery.The scaffolds were implanted in the dorsal muscle of dogs for periods of 2,4,8,12,24 weeks.Then histological and TEM observation were performed to study the change of the tested materials in the process of degradation.CD₃₄⁺ cells were isolated from bone marrow of carine by an magnetic activated cell sorting(MACS) and cultured in DMEM with VEGF in it.Differentiation of CD₃₄⁺ cells into endothelial cells were examed by CD₃₁ and vWF immunofluorescence and innmunohistochemistry stain.Endothelial cell function were assesed through low-density lipoprotein(Ac- LDL) -DiL incorpration.Endothelial cells were seeded onto the surface of small-diameter vessel grafts and observed by scanning electron microscopy.【Results】:(1)PDS monofilament fiber has prior biomechanical properties including flexibility and elasticity,which is favored to be knitted.Then a macro-porous tubular mesh was prepared by the specific micro-calibre knitting.(2)The burst strength of the scaffold is(43.5±8.3)kPa,and the rupture intensity (19.10±1.56)N,the strain ratio(42.88±3.16)%,the radial compliance(5.96±0.87) %/100mmHg.(3)In the process of degradation,SIS and Coll-CS layer preserve the basic shape in 2-4 weeks,and the pores structure of Coll-CS sponge started to break and amalgamate.In 12 weeks,the collagen sponge had been completely absorbed and placed with neonatal collagen fiber,the degradation of PDS material accelerated and it had been completely absorbed in 24 weeks.There was neither hyperplasia or necrosis nor other rejection reaction after the implanted.(4) Detected by the flow cytometry,the average number of the CD₃₄⁺ cells isolated by magnetic activated cell sorting(MACS) was 79.27±5.85%.Observed with inverted phase contrast microscope,the CD₃₄⁺ cells isolated from bone marrow were round shape.After the cultivation of 24h,the cells pasted wall were appearanced and the cytocolony were emerged at 1 week.Then the cells reach to logarithm period and reach to peak period with appearance of pavementing stones at 2 weeks.(5) The immunofluorescence and innmunohistochemistry stain were positive for CD₃₁ and vWF.Meanwell,the endothelial cell function were proved by low-density lipoprotein(Ac-LDL) -DiL absorption.(6) The scanning electron microscopy showed that large amount of endothelial cells growing on the surface of small-diameter vessel grafts.The endothelial cells had expanded apperrance and some grow into the honeycomb holes of collagen sponge.【Conclusion】:(1) Biodegradable polydioxanone(PDS) suture has the excellent properties of strength and elasticity,which is favored to be knitted.(2) The scaffold having a satisfactory biomechanical properties that meet the mechanical requirements during the implantation in vivo as a tissue engineered blood vessel scaffold.(3) The biomimetic small-diameter artificial scaffold has appropriate degradation time and has little rejection reaction of tissue.(4) CD₃₄⁺ cells can be isolated from bone marrow by magnetic activated cell sorting and were able to be induced into endothelial cells with VEGF in vitro.(5) The small-diameter vessel scaffold made ideal endothelialization with endothelial cells.