Numerous advances have been made in the field of skin tissue engineering in recent years to overcome skin injuries, burns and pathologies. Most of skin substitutes are exogenous matrices requiring the contribution of both host fibroblasts and endothelial cells and, therefore, a long time to become functional after implantation. In view of this, here we investigate the potential of a specific pre-vascularized dermis (PVD) obtained by seeding freshly isolated fibroblasts onto gelatin microbeads and adding, at a certain culture time, endothelial cells (HUVECs) [1]. More in detail, we implanted our engineered skin on the back of nu/nu mice in a full thickness skin defect model which, respect to the subcutaneous pocket we previously experienced, is more functional for both general and wound healing applications.
At different timepoints (3, 7, 14, 21 and 42 days) we retrieved our skin biohybrids and analysed them by histology and immunofluorescence. Animal studies were performed following the guidelines of EU (2010/63/EU).
Our main objective is to study the behaviour and the degree of integration of our skin substitute in the host organism. First of all, an appreciable integration of a pre-vascularized substitute with host tissue results in a fast anastomosis between the two vascular networks. However, since integration is a complex process there are other aspects which have to be considered. For example, a major limitation of skin substitutes in clinical application is to mimic the physiological sensitivity of the host skin.
Regarding vascularization, we looked for the expression of the lectins Griffonia Semplicifolia and Ulex Europaeus Agglutinin I (UEA I) to mark murine and human vessels, respectively. We noticed, starting from day 14
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onwards, the onset of numerous anastomosis between the two vascular networks (human and murine). Afterwards, from the innervation standpoint, we looked at the expression of Neurofilament-M, PGP9.5, NGF and BDNF along with the corresponding receptors TrkA and TrkB. Interestingly, we noticed a partial reinnervation of our skin substitutes already 42 days after implantation through the appreciable expression of PGP9.5, a promising result in comparison with other skin substitutes where the reinnervation is observed not earlier than after 8 weeks of implantation [2]. Therefore, since neurotrophins guide fibroblasts differentiation into myofibroblasts, also increasing skin tensile strength, we analysed the variation of the Young Modulus of our samples via indentation test. Moreover, to better frame the expression of our markers we performed a quantitative analysis through PCR.
In conclusion, our skin substitute, when implanted in a full thickness skin defect model, shows an earlier vascularization and innervation time compared to other substitutes described in the literature leading us to investigate the connection between vascularization and innervation during tissue development and maturation
