Oliver Bloch, Wilhelm Erdbrügger, Wolfgang Völker, Alexander Schenk, Steffen Posner, Wolfgang Konertz, Pascal M. Dohmen
Med Sci Monit 2012; 18(12): BR487-492
Available online: 2012-12-01
Background: Only limited information is available regarding the influence of decellularization on the extracellular matrix in heart valves. Within the extracellular matrix proteoglycans (PG) play a central role in the structural organization and physical functioning of valves and in their capability of settling with endothelial and interstitial cells partially myofibroblasts. We have therefore estimated the effects of decellularization using deoxycholic acid on the structure of the extracellular matrix and PG´s in porcine aortic valves.
Material/Methods: Cupromeronic blue was used, alone or in combination with OsO4/thio-carbo-hydrazide/OsO4 for electron microscopic visualization. For PG and glycosaminoglycan (GAG) investigation a papain digestion was employed in combination with photometric determination using dimethylmethylene blue.
Results: The results indicate that deoxycholic acid affects the compartmentation of the PG-associated interstitial network not significantly. Compared to controls the PG-rich network was preserved even after deoxycholic acid treatment for 48 h. In parallel to electron microscopy immune assays (ELISA) showed smooth muscle cell -actin to be reduced to 0.96%±0.71 and total soluble protein to 6.68%±2.0 (n=3) of untreated controls. Protein loss corresponded well with the observations in electron micrographs of rupture and efflux of cell content. Further signs of lysis were irregular cell contours and loss of the basement membrane.
Conclusions: Efficient cell-lysis without disintegration or loss of integrity of the interstitial PG network can be achieved by treatment of aortic valves with deoxycholic acid for 48h. This protocol might also be suitable for clinical use to optimize conditions for growth and autologous remodelling of valves.
Keywords: Tissue Engineering, Xenograft, Proteoglycans - analysis, Glycosaminoglycans - analysis