Objective To observe whether Cyclo-RGDfK (Arg-Gly-Asp-D-Phe-Lys) could enhance the adhesion of myofibroblast to decellularized scaffolds and upregulate the expression of Integrin αVβ3 gene. Methods Myofibroblast from the rat thoracic aorta was acquired by primary cell culture. The expression of Vimentin and α-smooth muscle actin(α-SMA) has been detected by immunoflurescent labeling. Decellularized valves have been randomly divided into three groups (each n=7). Group A (blank control): valves do not receive any pretreatment; Group B: valves reacted with linking agent NEthylN(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) for 36 hours before being seeded; Experimental group: Cyclo-RGD peptide has been covalently immobilized onto the surface of scaffolds by linking agent EDC. The fifth generation of myofibroblast has been planted on the scaffolds of each group. The adhesion of myofibroblast to the scaffolds was evaluated by HE staining and electron scanning microscope. The expression of Integrin αVβ3 was quantified by halfquantitative reverse transcriptionpolymerase china reaction (RT-PCR). Results We can see that myofibroblast has exhibited b positive staining for Vimentin and α-SMA. Besides, it has been shown that the expression of Integrin αVβ3 was much higher in the experimental group than that of the group A and group B(Plt;0.05). There was no statistically difference in group A and group B (P=0.900). Conclusion RGD pretreatment does enhance the adhesive efficiency of seeding cells to the scaffolds and this effect may be related to the upregulation of Integrin αVβ3.
【Abstract】 Objective To design a novel small-cal iber vascular graft using a decellularized allogeneic vascularscaffold pre-loaded with bFGF. Methods The decellularized canine common carotid were obtained by a detergent-enzymatic procedure, then the scaffolds were covalently l inked with heparin and pre-loaded with bFGF, the amount of binding bFGF and releasing curve were assayed by ELISA. Canine BMSCs expanded in vitro were seed on the scaffolds to observe the effects of binding bFGF on prol iferation. Both bFGF pre-loaded and non-pre-loaded decellularized grafts were implanted in canines as carotid artery interposition for 8 weeks, the patency was examined by digital subtraction angiography and histological method. Results Histology and electron microscopic examination of the decellularized scaffolds showed that cellular components were removed completely and that the extracellular matrix structure remained intact. The amount of binding bFGF positively related to the concentration of bFGF. There was a significant difference in the amount of binding bFGF between two different scaffoldsthroughout all bFGF concentrations(P lt; 0.05), and up to 100 ng/mL, the local and sustained release of bFGF from the heparin treated scaffolds were assayed up to 20 days. Additionally, MTT test showed the bFGF-preloaded scaffolds significantly enhanced the prol iferation of seeded BMSCs in vitro compared with non-bFGF-preloaded scaffolds at 3 days after seeding and thereafter(P lt; 0.01). Furthermore, in vivo canine experiments revealed that all 8 bFGF-pre-loaded scaffolds remained patent after 8 weeks of implantation, and host cell l ined the lumen and populated the wall. Only 1 non-bFGF-pre-loaded scaffold was patent, and the other 7 grafts were occluded because of thrombsus formation. Conclusion This study provides a new strategy to develop a small diameter vascular graft with excellent biocompatibil ity and high patency rate.
Objective To compare the effect of fabricating decellularized scaffold of homograft bioprosthetic tube valved (HBTV) with two kinds of cell detergents and to provide a homograft bioprosthetic scaffold for fabrication of tissueengineering heart valve (TEHV). Methods The active cells in the HBTV, which conserved by liquid nitrogen, were decellularized by low osmotic pressure of Tris buffer, in which containing sodium dodecylsulphate (SDS) and deoxycholic acid (DOA) respectively. The leaflets or aortic wall was fixed with fixative and stained with hematoxylin and eosin, collagen fibers or elastic fibers for observation and photographs by light microscope or by scanning electron microscope (SEM) after decellularized. Results When the leaflets of HBTV were incubated togetherwith 0.03% SDS or 0.5% DOA of Tris buffer respectively for 48 hours, the activeendothelial cells (ECs) in the leaflets were not only decellularized completely, but also reserved the collagen fibers or elastic fibers integrally, which is two of the main components of extracellular matrix (ECM). A part of fibroblast inthe center leaflets was reserved. The morphologic structure of leaflets after decellularized was not significantly different from that before decellularized. The concentration of SDS was increased to 0.1% when decellularized the cells of aortic wall, but DOA was still kept 0.5%. Conclusion The better decellularizedscaffold of HBTV obtained was disposed by 0.03%-0.1% SDS or 0.5% DOA, which wasadvantageous to adhesiveness and amplification of implantation cells on the decellularized scaffold of HBTV in order that HBV reendothelialized or for the TEHVfabricated in vitro.