RHEOLOGY AND MECHANICAL STABILITY OF A CARDIAC EXTRACELLULAR MATRIX AND BACTERIAL NANOCELLULOSE SCAFFOLD

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ABSTRACT Introduction: The regenerative limitations of the myocardium represent a major challenge for cardiovascular reconstructive surgery due to the inability of conventional therapies to fully restore myocardial architecture and function. Strategies based on decellularized extracellular matrix (dECM) associated with biomimetic three -dimensional biomaterials may provide mechanical and bioactive support, promoting cell adhesion, organization, and maturation for myocardial regeneration. Objective: To obtain cardiac dECM with preserved structural and biochemical properties and to evaluate the str uctural, rheological, and biological properties of a hybrid three -dimensional composite composed of bacterial nanocellulose, dECM biogel, and cardiomyocytes for cardiovascular tissue engineering applications. Materials and Methods: Canine hearts obtained f rom natural deaths at the Veterinary Hospital of the University of Marília were subjected to decellularization using 1% sodium dodecyl sulfate (SDS) under continuous agitation (100 rpm), followed by serial washing steps with buffered solution. The study was approved by the Animal Ethics Committee of the University of Marília (protocol 22/2024). Decellularization efficacy was assessed through macroscopic analysis, light microscopy, scanning electron microscopy, residual genomic DNA quantification, and immuno histochemistry. The obtained matrix was frozen, lyophilized, and pulverized for biogel production. Viscoelastic characterization was performed using rotational oscillatory shear rheometry. Amplitude sweep tests defined the linear viscoelastic region, while frequency sweep tests determined the storage modulus (G′) and loss modulus (G″). Creep -recovery assays evaluated viscoelastic resilience and permanent deformation, whereas steady-state flow curves analyzed hysteresis area and yield stress. The hybrid composite consisted of bacterial nanocellulose as the mechanical support, dECM biogel as the bioactive component, and in vitro cultured cardiomyocytes. Recellularization was evaluated regarding cell adhesion, viability, and cellular organization. Results: The 1% SDS protocol promoted complete decellularization after 30 days, demonstrating superiority over the 0.5% SDS plus Triton X-100 protocol, evidenced by the absence of cellular nuclei, residual DNA levels below 10 ng/mg dry weight, and preservation of the t hree-dimensional fibrillar architecture. Immunohistochemistry confirmed preservation of collagen types I and IV, fibronectin, laminin, and elastin. Rheological analysis demonstrated predominantly elastic behavior (G′ > G″), with storage modulus values rang ing from 36 -47 kPa for pure nanocellulose and increasing to 42 -54 kPa for the hybrid composite, compatible with the stiffness range of native myocardium. The material exhibited high viscoelastic resilience, low permanent deformation after cyclic loading, pseudoplastic shear-thinning behavior, low thixotropy, and yield stress compatible with bioprinting applications. Recellularization demonstrated efficient cardiomyocyte adhesion, maintenance of viability, and progressive cell proliferation guided by the fib rillar topography, without structural collapse. Conclusion: The proposed decellularization protocol efficiently produced a structurally preserved and biochemically intact cardiac dECM. The three -dimensional hybrid composite demonstrated biomimetic mechanic al behavior, structural stability, and effective support for cardiomyocyte adhesion and proliferation, highlighting its potential as an experimental platform for myocardial reconstruction and cardiovascular tissue engineering applications.