Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery

Critical lower limb ischemia is a severe disease. A common approach is infrainguinal bypass. Synthetic vascular prosthesis, are good conduits in high-flow low-resistance conditions but have difficulty in their performance as small diameter vessel grafts. A new approach is the use of native decellularized vascular tissues. Cell-free vessels are expected to have improved biocompatibility when compared to synthetic and are optimal natural 3D matrix templates for driving stem cell growth and tissue assembly in vivo. Decellularization of tissues represent a promising field for regenerative medicine, with the aim to develop a methodology to obtain small-diameter allografts to be used as a natural scaffold suited for in vivo cell growth and pseudo-tissue assembly, eliminating failure caused from immune response activation. Material and methods. Umbilical cord-derived mesenchymal cells isolated from human umbilical cord tissue were expanded in advanced DMEM. Immunofluorescence and molecular characterization revealed a stem cell profile. A non-enzymatic protocol, that associate hypotonic shock and low-concentration ionic detergent, was used to decellularize vessel segments. Cells were seeded cell-free scaffolds using a compound of fibrin and thrombin and incubated in DMEM, after 4 days of static culture they were placed for 2 weeks in a flow-bioreactor, mimicking the cardiovascular pulsatile flow. After dynamic culture, samples were processed for histological, biochemical and ultrastructural analysis. Discussion. Histology showed that the dynamic culture cells initiate to penetrate the extracellular matrix scaffold and to produce components of the ECM, as collagen fibres. Sirius Red staining showed layers of immature collagen type III and ultrastructural analysis revealed 30 nm thick collagen fibres, presumably corresponding to the immature collagen. These data confirm the ability of cord-derived cells to adhere and penetrate a natural decellularized tissue and to start to assembly into new tissue. This achievement makes natural 3D matrix templates prospectively valuable candidates for clinical bypass procedures

Catchability and survival of the resources during fishing operation

The present Ph.D. thesis aims to test and evaluate by-catch reduction devices (BRDs) that minimize the retention of undersized fish and do not penalize revenues of the fishing industry. Considering that a fraction of fish that escape from fishing gear or that are rejected at the sea probably does not survive (unaccounted mortality), it is a major concern for sustainable fisheries management, as unaccounted mortality may lead to biased stock assessment since they will tend to underestimate fishing mortality and overestimate stock size. In this context, in the present Ph.D. thesis, the escape survival (i.e. survival of the fish escaped through the trawl net codend) of the Mullus barbatus Linnaeus 1758 and the discard survival (survival of fish rejected at the sea after being hauled on deck) of Trachurus trachurus were evaluated for the first time in the central Mediterranean Sea. In conclusion, the use of underwater lights in Mediterranean trawl fisheries should be carefully regulated through ad hoc measures that are currently lacking, to minimize the potential impacts of artificial light on some already overexploited stocks. Even if further works should be carried out in the future to test BRDs performances in different areas and seasons, the T90 50 mm codend and the Grid-T45 40 mm seem promising tools to reduce the catch of undersized individuals and contribute to mitigating the current overfishing of Parapenaeus longirostris and Merluccius merluccius. The escape survival of M. barbatus was high and thanks to an improved methodology the bias in the sampling was minimized. However, for improved stock assessment of M. barbatus, the experiment should be repeated to provide accurate escape mortality estimates. While the discard survival of T. trachurus was very low and according to the landing obligation (Reg. EU 1380/2013) all the juveniles of the species should be landed.