Gelatin and carbon-based nanotubes scaffold for cardiac tissue engineering: A preliminary study

Objectives: A realistic goal for cardiac muscle engineering is the design of a scaffold able to mimic both the tissue-specific architecture and mechanical properties as well as the main physiological functions. The aim of this study was the set up of gelatin and carbon nanotubes (CNT) scaffolds for cardiac tissue engineering applications. Materials and methods: Gelatin-based scaffolds (Sc) and CNT in percentage of 0.3% and 0.9% (n = 12), cross-linked with genepin 0.2%, were prepared. H9c2 cell line was cultured for 10 days in DMEM, supplemented with 10% of FBS (C10%, n = 10). Myoblast differentiation was induced by adding 1% FBS (C1%, n = 6), while cardiac phenotype by 10 nM all trans-retinoic acid (CRA, n = 6). Cell viability, citotoxicity, phenotype differentiation and immunohistochemical assay were performed. After 10 days cells and Sc were collected in tri-reagent and RNA was extracted for RealTime PCR analysis. In order to evaluate cardiac phenotype, the natriuretic peptide (NP) and endothelin (ET) system were studied. To confirm cellular interaction by gap junction formation, connexin (CX)-43 was measured. Results: Immunohystochemistry study revealed that C1% showed the presence of elongated myotubes, typical of skeletal phenotype and dissimilar from myoblast of control condition. CRA was induced to cardiac phenotype showing round and multinucleated nuclei. Data were also confirmed by a significantly increased expression of NP system in CRA with respect to C10% and C1% except for NP receptor-C that significantly decreased in CRA. Furthermore, CRA revealed an increased of both ET-A and ET-B receptors in parallel with a decreased ET-1 expression with respect to C10% and C1%. In CNT Sc cell viability was similar both at 0.3% than and 0.9% and resulted decreased at 3 days probably for adapting at the Sc. NP and ET system expression decreased in CNT0.3% and CNT0.9% with respect to C10% as well as CX43 mRNA (p < 0.01), mainly due to a lacking of complete differentiation in cardiac phenotype during these few days. Conclusions: In this study the addition of retinoic acid during serum reduction favours a cardiac phenotype at the expense of skeletal muscle trans-differentiation, confirmed by NP and ET system expression. Moreover, the lacking of a complete differentiation of cells on CNT-Sc highlights the need of more day of culture to realize this process. Nevertheless further analysis on novel biomaterials to enhance cell growth/proliferation and to support the damaged heart will be needed to bring heart tissue engineering into clinical application, these results are a useful starting point to develop new Sc-based biomaterials.