5. References

prompt alterations in endothelial cells [20]. Herein we found that the ECM deposited by the papillary subset was rich in collagen type I, which might explain the early development of vessel-like structures in our bilayer constructs generated from pre-selected fibroblasts. In vitro, collagen I interacts with the specific a1b1 and a2b1 integrins from the endothelial cells, which induce precapillary cord organization and, consequently, capillary-like structures formation [40]. Interestingly, the ECM produced by the reticular subset was rich in laminin. This protein is known to trigger the migration of endothelial cells through the activation of a3b1 integrin in these cells [41,42] to expand the vessel-like structures formed [43].

Therefore, the laminin-rich ECM might have prompted the migration of the endothelial cell, initially seeded in the papillary layer, to the reticular part beneath along the culture, while also having a role in the stabilization of the formed structures [44], at latter culture times in both dermal layers.

Therefore, our results seem to further support the existence of an intricate signaling network responsible for the modulation of the vascular network within the different dermal layers that is highly dependent on the environment generated by the respective fibroblasts subpopulation.

Overall, we provide evidence that pre-selection of papillary and reticular fibroblasts is relevant in promoting the in vitro prevascularization of skin TE constructs. Notwithstanding, while the optimization of the culture conditions to allow the conservation of fibroblast subpopulation’s phenotypes has not been achieved, their use in the construction of skin constructs for tissue engineering applications is hampered.

Anyhow, vascularized in vitro skin models built from pre-selected fibroblasts subsets offer advantages in terms of representation of tissue functionality being therefore a valuable study platform.

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