| None of the currently available heart valve replacement devices posses any regenerative or growth potential. Accordingly the reconstruction of viable heart valves, with ability to grow, repair and remodel, without immunological response and the need for anticoagulation, is a major goal. A variety of different approaches all aim to generate a starter matrix resembling the native valve in design and function as much as possible and in parallel allowing repopulation with autologous cells. The use of biologic scaffolds derived from decellularized mammalian tissues is commonplace. Such scaffolds are composed of extracellular matrix (ECM) and have been successfully used in a variety of tissue engineering and regenerative medicine applications.Preservation of the complex composition and three-dimensional ultrastructure of the ECM is highly desirable but it is recognized that all methods of decellularization result in disruption of the architecture and potential loss of surface structure and composition. The most effective agents for decellularization of each tissue and organ will depend upon many factors, including the tissue’s cellularity, density, lipid content, and thickness. Minimization of these undesirable effects rather than complete avoidance is the objective of decellularization.Objective of this study was to investigate the impact of decellularization protocol with new detergent-Octyl-glucopyranoside (OGP) on extracellular matrix integrity of porcine pericardium (PP) compared with other traditional detergents-sodium dodecylsulfate (SDS) and sodium deoxycholate in combination with Triton X-100(SDT). The main study contents are as follows:Efficiency of decellularization of OGP using different concentrations (0.5%(w/v),1%(w/v),2%(w/v) and5%(w/v)) was investigated to optimize appropriate decellularized concentration of OGP. Haematoxylin and eosin (HE) and Weigert stain were used to assess effect of OGP decellularization method. There were a great number of cells remaining in the native PP on the histological section of HE staining. The collagen fibers of section were wave-like and compact, whereas the elastin fibers of section were clear and continuous as shown by Weigert staining. While in PP processed by0.5%(vv/v) OGP, cell debrises were still observed by HE stain. Compared native PP, cells of PP were completely removed when the concentration of OGP was1%(w/v), and collagen fibers of PP treated by2%(w/v) OGP and5%(w/v) OGP were sparse and deranged, especially for5%(w/v) OGP decellularization. Collagen fibers of PP treated by1%(w/v) OGP were kept wavelike and the elastin fibers of section were clear and continuous. It was obvious that collagen fibers and elastin fibers of PP treated by1%(w/v) OGP were preserved. The effect of different detergents was comparative studied on cell extraction of PP. For native PP. there were a great number of cells remaining in the PP on the histological section of HE staining and Movat pentachrome stain. The collagen fibers of section were wavelike and compact, whereas the elastin fibers of section were clear and continuous as shown by Weigert staining and Movat pentachrome stain. In the OGP treatment group, no cells remained in PP and the collagen fibers of section were kept wavelike. and the elastin fibers of section were clear and continuous. In the SDS treatment group, no cells remained in PP, but the collagen fibers of section were broken and deranged. The elastin fibers in the PP were malalignment. In the SDT treatment group, HE and Movat pentachrome stained sections of the tissues revealed an incomplete cell removal from the matrix. Black-blue stained nuclei were still found in the tissues. Weigert stain and Movat pentachrome stain showed elastic fiber distorted partly in comparison with native tissue. The results can be also demonstrated by the determination of DNA content and the morphology and structural observed by scanning electron microscopy (SEM). Biochemical compositions and biomechanical properties were well preserved as shown by biochemical assays and uniaxial tensile test except that glycosaminoglycans markedly decreased. Moreover, the results obtained in the MTT study and haemolysis test further indicated that the cytotoxicity and haemolysis ratio of decelluiarized PP by OGP was significantly lower than that by SDS and SDT.In conclusion, this work demonstrated the feasibility to use environmentally-friendly and nontoxic OGP as a novel decellularization reagent in the decellularization of biological tissues. The results suggest that the OGP method is superior to either the SDS method or the SDT method, as it achieves not only the complete decellularization but also the preservation of both the mechanical function and the matrix structure of the tissues, it might be a suitable approach to construct tissue engineering heart valve scaffold. |
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