Angiogenesis and innervation during the integration of engineered skin substitutes

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 Abstract 21 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

Orexin-A Prevents Lipopolysaccharide-Induced Neuroinflammation at the Level of the Intestinal Barrier

In states of intestinal dysbiosis, a perturbation of the normal microbiome composition, the intestinal epithelial barrier (IEB) permeability is increased as a result of the disruption of the epithelial tight junction protein network, in which occludin is mostly affected. The loss of IEB integrity promotes endotoxemia, that is, bacterial lipopolysaccharide (LPS) translocation from the intestinal lumen to the circulatory system. This condition induces an enhancement of pro-inflammatory cytokines, which leads to neuroinflammation through the gut-brain axis. Orexin-A (OX-A), a neuropeptide implicated in many physiological functions and produced mainly in the brain lateral hypothalamic area, is expressed also in several peripheral tissues. Orexin-producing neurons have been found in the myenteric plexus to project to orexin receptor 1 (OX-1R)-expressing enterocytes of the intestinal villi. In the present study we investigated the protective role of OX-A against LPS-induced increase of IEB permeability and microglia activation in both an in vivo and in vitro model of the gut-brain axis. By exploiting biochemical, immunocytochemical, immunohistochemical, and functional approaches, we demonstrate that OX-A preserves the IEB and occludin expression, thus preventing endotoxemia and subsequent neuroinflammation

Anatomical templates for tissue (re)generation and beyond

Induced pluripotent stem cells (iPSCs) represent a valuable alternative to stem cells (SCs) in regenerative medicine overcoming their ethical limitations, like embryo disruption. Takahashi and Yamanaka in 2006 reprogrammed, for the first time, mouse fibroblasts into iPSCs through the retroviral delivery of four reprogramming factors: Oct3/4, Sox2, c-Myc and Klf4 (SKOM). Since then, several studies started reporting the derivation of iPSC lines from animals other than rodents for translational and veterinary medicine. Here, we review the potential of using these cells for further intriguing applications, such as "cellular agriculture". IPSCs, indeed, can be a source of in vitro, skeletal muscle tissue, namely "cultured meat", a product that improves animal welfare and encourages the consumption of healthier meat along with environment preservation. Also, we report the potential of using iPSCs, obtained from endangered species, for therapeutic treatments for captive animals and for assisted reproductive technologies as well. This review offers a unique opportunity to explore the whole spectrum of iPSC applications from regenerative translational and veterinary medicine to the production of artificial meat and the preservation of currently endangered species. This article is protected by copyright. All rights reserved

Induced Pluripotent Stem Cells as Vasculature Forming Entities

Tissue engineering (TE) pursues the ambitious goal to heal damaged tissues. One of the most successful TE approaches relies on the use of scaffolds specifically designed and fabricated to promote tissue growth. During regeneration the guidance of biological events may be essential to sustain vasculature neoformation inside the engineered scaffold. In this context, one of the most effective strategies includes the incorporation of vasculature forming cells, namely endothelial cells (EC), into engineered constructs. However, the most common EC sources currently available, intended as primary cells, are affected by several limitations that make them inappropriate to personalized medicine. Human induced Pluripotent Stem Cells (hiPSC), since the time of their discovery, represent an unprecedented opportunity for regenerative medicine applications. Unfortunately, human induced Pluripotent Stem Cells-Endothelial Cells (hiPSC-ECs) still display significant safety issues. In this work, we reviewed the most effective protocols to induce pluripotency, to generate cells displaying the endothelial phenotype and to perform an efficient and safe cell selection. We also provide noteworthy examples of both in vitro and in vivo applications of hiPSC-ECs in order to highlight their ability to form functional blood vessels. In conclusion, we propose hiPSC-ECs as the preferred source of endothelial cells currently available in the field of personalized regenerative medicine

Induced Pluripotent Stem Cells as Vasculature Forming Entities

Tissue engineering (TE) pursues the ambitious goal to heal damaged tissues. One of the most successful TE approaches relies on the use of scaolds specifically designed and fabricated to promote tissue growth. During regeneration the guidance of biological events may be essential to sustain vasculature neoformation inside the engineered scaold. In this context, one of the most eective strategies includes the incorporation of vasculature forming cells, namely endothelial cells (EC), into engineered constructs. However, the most common EC sources currently available, intended as primary cells, are aected by several limitations that make them inappropriate to personalized medicine. Human induced Pluripotent Stem Cells (hiPSC), since the time of their discovery, represent an unprecedented opportunity for regenerative medicine applications. Unfortunately, human induced Pluripotent Stem Cells-Endothelial Cells (hiPSC-ECs) still display significant safety issues. In this work, we reviewed the most eective protocols to induce pluripotency, to generate cells displaying the endothelial phenotype and to perform an ecient and safe cell selection. We also provide noteworthy examples of both in vitro and in vivo applications of hiPSC-ECs in order to highlight their ability to form functional blood vessels. In conclusion, we propose hiPSC-ECs as the preferred source of endothelial cells currently available in the field of personalized regenerative medicine

Orexin-A Prevents Lipopolysaccharide-Induced Neuroinflammation at the Level of the Intestinal Barrier

In states of intestinal dysbiosis, a perturbation of the normal microbiome composition, the intestinal epithelial barrier (IEB) permeability is increased as a result of the disruption of the epithelial tight junction protein network, in which occludin is mostly affected. The loss of IEB integrity promotes endotoxemia, that is, bacterial lipopolysaccharide (LPS) translocation from the intestinal lumen to the circulatory system. This condition induces an enhancement of pro-inflammatory cytokines, which leads to neuroinflammation through the gut-brain axis. Orexin-A (OX-A), a neuropeptide implicated in many physiological functions and produced mainly in the brain lateral hypothalamic area, is expressed also in several peripheral tissues. Orexin-producing neurons have been found in the myenteric plexus to project to orexin receptor 1 (OX-1R)-expressing enterocytes of the intestinal villi. In the present study we investigated the protective role of OX-A against LPS-induced increase of IEB permeability and microglia activation in both an in vivo and in vitro model of the gut-brain axis. By exploiting biochemical, immunocytochemical, immunohistochemical, and functional approaches, we demonstrate that OX-A preserves the IEB and occludin expression, thus preventing endotoxemia and subsequent neuroinflammation