Introduction: The full recovery of injured tissue requires stable blood clot formation with high neovascularization degree[1]. Fine-tunable, micropatterned porous scaffold, acting as core template for clot stability and cell homing, can drive endothelial cell tube formation via “contact guidance”[2]-[4], promoting neovascularization within the entire construct. However, the knowledge about micropatterning effect on angiogenesis is still scanty. Via chitosan (CH) electrodeposition (ECD)[5],[6], we tested hierarchical micropatterned scaffolds for neovascularization. Each had an array of open, 500 μm-wide pores, with the same orientation but differently spaced (700 or 900 μm). The substrate with oriented pores could favor endothelial cell migration to form the microvascular network, more than in presence of a random porosity.
Materials and Methods: CH-ECD on aluminum grids was tuned, for micropatterning, by 500 μm-wide holes in the grids[5],[6], at intervals of 700 or 900 μm. After crosslinking (1% (w/w) genipin), morphology was analyzed by SEM; swelling by percentage of water uptake (WU) and tensile tests by Dynamic Mechanical Analyzer. Mouse endothelial cells (MS1) viability was tested by MTT assays at 1-3-7 days after cell seeding, and with 1 week-eluates. At 7 days, cell density within scaffold was calculated by fluorescence analysis of cross-sections (30-60-90 μm-deep)[7]. The most promising micropatterned scaffold was compared to the random pore one, via subcutaneous implantation in wild-type mice, for 3 and 6 weeks. Masson’s trichrome histological staining was used to detect inflammatory reaction and tissue ingrowth; immunofluorescence (anti-CD31, anti-α-SMA) allowed vessels’ semi-quantitative analysis[8] (means’ comparison: t-test, p<0.05).
Results and Discussion: Random pores ranged between 10 and 500 µm of diameter, while micropatterned oriented pores were of ≈ 450 µm of diameter, regular and open, differently spaced according to grids dimensions (Fig.1).
All scaffolds displayed a marked and fast WU increase in 10 min, till a plateau, and an elastic tensile stress/strain behavior, affected by crosslinking. The effect of patterning was negligible. The high cytocompatibility at MTT assays was confirmed by SEM and fluorescence images, demonstrating, at 7 days, cells adhering, in depth, within scaffolds, although at a lesser extent with random and 900 μm-spaced pore scaffolds. In vivo test, using 700 μm-spaced pore scaffolds, showed tissue ingrowth without inflammation. At 6 weeks, the density of vessels, around and inside scaffolds, was higher for the micropatterned porosity over the random one (Fig.2).
Conclusions: Our findings identified a promising micropattern of porosity to enhance angiogenesis, supporting the importance of scaffold design to maximize the vascular network. The perspectives will explore the effect on neovascularization of pore size, enlarging the ECD-micropatterning application to fields facing highly oriented tissue regeneration.
LA and LDN thank MIUR for economic support through FIRB Futuro in Ricerca (Surface-associated selective transfection — SAST, RBFR08XH0H); AC was partially supported by PRIN 2010–2011 (PRIN 20102ZLNJ5_006), financed by the Ministry of Education, University and Research (M.I.U.R.), Rome, Italy.
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