Oriented micro-pores to promote scaffold vascularization.

A high level of vascularisation at injured tissue is the cornerstone for a stable blood clot formation, cellular colonization and subsequent tissue regeneration. Compelling evidence suggests scaffold architecture as relevant factor for maximizing vascularisation, under the rationale of “contact guidance” concept: an implanted scaffold may promote micro-vessel development in certain directions, driving endothelial cell orientation, and thus efficiently promote vascularisation of the whole construct. Here, we propose the application of an innovative technique to produce film-like scaffold with highly oriented porosity. Chitosan scaffolds were produced by electrodeposition by optimizing different process parameters to obtain a controlled porosity. After fabrication, they were cross-linked with genipin, we performed a complete physico-chemical and biological characterization and, using a murine model of subcutaneous implantation, we evaluated animal immune response to scaffold, its integration with surrounding tissues and related vascularisation at 3 and 6 weeks. Random and oriented porous scaffold were compared. Morphological characterization was performed by optical microscopy and SEM, showing scaffolds with an oriented porosity (about 500 μm) and a random microporosity due to H2 bubble production; on the other hand, random porous structure revealed an heterogeneous porosity. In water, both scaffolds showed analogous swelling degree. At 24h after MS1 murine endothelial cell seeding, SEM images showed cells adhering the surfaces of scaffolds following pore shape; at 72h, extracellular matrix deposition was also observed. After subcutis implantation, histological analysis indicated no inflammatory response, while lymphocyte proliferation assay confirmed absence of chitosan-specific immune response. At 3 weeks, Masson’s trichrome staining showed higher degree of tissue ingrowths and vascularization with oriented scaffold; nevertheless, this difference was negligible at 6 weeks, as well as vessel maturation degree (CD31 and α-SMA immunostainings). Our data suggest a putative role of micropattern oriented porosity in enhancing early vascularization of scaffold, thus promoting scaffold integration and new tissue regeneration.