2D and 3D Electrospun Silk Fibroin Gelatin Coatings to Improve Scaffold Performances in Cardiovascular Applications

3D scaffolds and 2D matrices fabricated by electrospinnig show morphology similar to that of native ECM, however their mechanical and biological properties are often inadequate, particularly in applications in contact with blood, e.g. in blood vessel substitutes. Biocoatings can improve the performance of these substrates, in particular cross-linked gelatin is among the most used substances.

In this work, a gelatin coating was applied to electrospun silk fibroin (ESF) mats and tubes intended for the regeneration of cardiovascular tissues. The crosslinking reaction used is based on a Michael-type addition in water that promotes the formation of covalent bonds between gelatin amino groups and β-carbons of N-N’-methylene bis-acrylamide (MBA)[1].

Interestingly, when the reacting mixture is applied to a substrate containing primary or even secondary amino groups, these groups can participate in the reaction, being incorporated into the gelatin coating, thus increasing the coating stability on the surface.

ESF mats and tubes, obtained as described in [2] were coated with gelatin MBA-crosslinked in situ by loading or dipping the ESF samples with the crosslinking solution, by use of static or dynamic home-made systems. SEM analysis on coated samples showed a homogeneous coating with gelatin penetrating the whole thickness of the SF matrix {»120 µm for mats and » 212 µm for tubes), with an increase of thickness of about 40% in wet conditions. Water uptake tests indicated for coated samples a faster and higher swelling (1600% after 14 days) than not coated ones (500%), due to the presence of gelatin.

Tensile mechanical tests showed higher values of ultimate stress and elastic modulus for silk fibroin samples (s_b=2.4, E=1.82 MPa) compared to gelatin-coated ones (s_b=1.2, E=0.58 MPa), with not significant differences in the ultimate deformation (»150%).

Indirect cytocompatibility tests, performed by culturing L929 cells in the presence of eluates obtained by immersing coated and uncoated samples up to 7 days in culture medium, demonstrated a cell viability higher than the control. In direct contact tests using L929 cells, a good cytocompatibility was demonstrated by both coated and uncoated ESF samples, with a cell viability increasing with the culture time (up to 7 days) and a flattened and stretched morphology at SEM.

Primary human umbilical vein endothelial cell (HUVEC), obtained by enzymatic digestion (Cittadella Hospital, PD,I) were seeded onto ESF and ESF-coated samples and cultured under standard tissue culture conditions. Cell adhesion on the matrices was analysed by OM after fixing with formalin and staining with toluidine blue. After 7 days from seeding, cell proliferation was evaluated by a protein assay (BCA Protein Assay kit) and the results indicated a significantly higher (p<0.05) cell growth on gelatin-coated ESF samples.

Overall, these results point out that the described gelatin coating allows producing a structure with adequate mechanical properties for cardio-vascular applications and biological characteristics even better than those of silk fibroin.

References

[1]Contessi Negrini, N., Tarsini, P., Tanzi, M.C., Farè, S., Chemically crosslinked gelatin hydrogels as scaffolding materials for adipose tissue engineering (2019) J. Appl. Polym. Sci. 136 (8), 47104

[2] Marelli B, Alessandrino A, Fare S, Freddi G, Mantovani D, Tanzi MC. Compliant electrospun silk fibroin tubes for small vessel bypass grafting. Acta Biomater. (2010) 6:4019–26.