*4.6. Vascular Tissue*

Vascular tissue is related to the transport of nutrients, oxygen, CO2, hormones, and blood cells through the body. Essentially, it comprises arteries, arterioles, and veins, while only arteries are the common target for decellularization approaches. Arteries are elastic blood vessels that carry oxygenated blood from the heart to the whole body. The main arteries are composed of three tissue layers, from inside out: intima, media, and adventitia. The tunica intima's ECM contains mainly laminin and collagen type IV, while the medium layer is principally composed of collagen type III, elastin, glycoproteins, and GAGs. In contrast, the ECM of the outermost tunica consists primarily of collagen type I and elastin, but it also contains proteoglycans such as biglycan and decorin, as well

as thrombospondin-2 [114]. Although arteries have a very complex structure, it is very important to maintain the ECM components in the interest of keeping the ECM properties intact, such as elasticity and resistance.

Arterial diseases such as pulmonary arterial hypertension, restenosis, and peripheral arterial diseases are now targeted with experimental and promising stem cell therapies [115–118]. On the other hand, peripheral and coronary artery bypasses are clinical procedures often based on artery replacement by autologous graft transplantation. The use of natural or synthetic biopolymers as grafting materials is a clinically feasible option [119,120], but decellularized artery grafts are gaining increased research attention as artery bypass grafting materials due to their proper molecular and mechanical properties and their reduced immunogenicity [121,122].

Before the artery decellularization technique starts, there are different steps that are recommended for better results. Some protocols sugges<sup>t</sup> to lyse blood cells by washing the arteries in distilled water while shaking [120], while others include three freeze–thaw cycles with EDTA [50]. As in many other tissues, decellularization is mainly based on the activity of detergents such as SDS, EDTA, SDC, CHAPS, Triton X-100, or DOC [27,50,56,120,123,124], used individually or in combination [50]. Some protocols report trypsin and hypo/hypertonic solutions used together with these detergents [27,50,56], while the use of enzymatic DNA and RNA removal as one of the final steps is also recommended [27,56]. All of these chemical methods report quite good results, both in vitro and in vivo, implying the use of detergents as a valuable approach to vascular decellularization, while improvements related to avoiding immunogenicity and cytotoxicity are required [121].

Recently, arterial decellularization mediated by supercritical and pressurized CO2 has been described. It briefly consists of a high-pressure syringe pump that delivers liquid CO2 and ethanol or limonene (as cosolvents) through a preheated extraction vessel. Samples are treated with scCO2 and endonucleases to remove residual cosolvent and DNA. This approach yields a nearly intact decellularized tissue free of cells, lipids, and nucleic acids, proposing an alternative to traditional decellularization methods. Nevertheless, further in vitro and in vivo analyses need to be completed [125].
