Development of Bioartificial Myocardium using Collagen Scaffold Functionalized with RGD Peptides
Olivier Schussler1, Walid Al Chare1, Mariana Louis-Tisserand3, Robert Michelot1, Malcolm Wood2, Didier Heude1, Alain Carpentier1, JuanCarlos Chachques1, Dan Salomon2, Yves Lecarpentier4; 1Laboratory of Biosurgery University Paris V Pompidou Hospital, Paris, France; 2The Scripps Research Institut MEM 241, La jolla, CA; 3INSERM U689 Paris VII University, Paris, France; 4Dpt explorations CardioRespiratoires, Le Kremlin Bicêtre, France
Comment on this Abstract
Objective: Collagen matrix (CM) seeded with neonatal cardiomyocytes represents the unique scaffold in which contractile activity has been demonstrated in vitro. However, contractility is variable and terminal cell differentiation has been achieved only in the presence of the tumor extract: Matrigel™ that also compromises nutriment diffusion. In addition, angiogenesis in collagen scaffolds remains very poor. We hypothesized that improving cell matrix interactions by coupling adhesive peptides containing Arg-Gly-Asp (RGD) which interact with integrin adhesion molecules on endothelial cells and cardiomyocytes would enhance cell survival, differentiation and angiogenesis without requiring Matrigel.
Methods: Mouse endothelial cells (MS1) (n=7) or neonatal rat cardiomyocytes (n=16) were cultured in RGD~CM. Controls were cellurized CM-RGD+/- cultivated in the presence +/- of soluble RGD(sRGD)(n=11). Angiogenesis was evaluated quantitatively and qualitatively by electron microscopy. Parameters of contractility (spontaneous or under electrostimulation) during contraction and relaxation phases were mesuared by using a force length microtransducer under isotonic or isometric conditions. Cell number was evaluated by Flow Cytometry and apoptosis by in situ labeling with annexin-V-FITC and confocal examination.
Results: By EM, vascular profiles 8.0+/-1.2 per mm2 were present only in RGD+CM (p<0.001). In addition these profiles were ramified in 45% of cases. sRGD prevents angiogenesis. Contractile activity was present in 80% of RGD+ vs 50% of RGD- constructs. All isotonic and isometric mechanical parameters, spontaneous and electro-stimulated contraction and relaxation, were improved in RGD+ constructs (each p<0.01). Stimulation threshold was decreased in RGD+ (<3V/cm) and was in the range of that required for papillary muscle. At optimal electrostimulation frequency (0.17 Hz), RGD+ had a nearly 3-fold increase in both maximum extent of shortening (31+/-3 vs 9+/-4μm, p<0.05) and maximum shortening velocity (633+/-180 vs 256+/-55 μm/s, p<0.05). As in the myocardium, matrix contraction and relaxation appear to be an active mechanism. By classical and confocal microscopy, cardiomyocytes changed their morphology in RGD+ scaffolds with reorganization of contractile apparatus and development of cross-striation.
Conclusion: By promoting angiogenesis and contractile activity, the use of collagen matrix modified with RGD adhesive peptides may be an important step for the development of engineered artificial myocardium.
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