*4.5. Cardiovascular Tissue*

The heart is a muscular organ whose function is mainly the pumping of blood through the circulatory system. The ECM of the heart is quite specific and is composed predominantly of collagens (types I, II, and III), fibrillin, hyaluronan, laminin, fibronectin, and proteoglycans [34,103,104]. Due to its composition, it shows grea<sup>t</sup> strength, flexibility, and durability [105].

Cardiovascular diseases are the target of multiple regenerative medicine approaches. Stem cell-related approaches have been tested as promising therapies for myocardial infarction, chronic ischemic myocardial dysfunction, and nonischemic dilated cardiomyopathy, among others. Moreover, regenerative and repair strategies have also been investigated as alternatives to heart transplant procedures. On the other hand, research e fforts are directed toward defining regenerative or reparative approaches as a substitute to heart valve replacement procedures, a clinically required procedure in certain patients of valvular heart disease (VHD). In all of these cardiovascular regenerative research contexts, decellularized cardiac tissue is now thoroughly assayed, sometimes as a biocompatible and cytokine-carrying implantable sca ffold, and in other cases, as a recellularized carrier of therapeutic cells [106,107]. Indeed, whole tissue decellularization, followed by recellularization, is a strategy that many labs are working on [108].

In comparison to other tissues, which are hard to decellularize, current methods yield decellularized heart tissue that retains a grea<sup>t</sup> number of its original properties, including its elasticity. The most frequent method to achieve heart decellularization is the use of specific detergents combined with other decellularizing agents. Detergents such as SDS, sodium deoxycholate, PEG, or Triton X-100 work very well as decellularization agents in valves, tissue pieces, and in a whole heart decellularization context. These detergents are often used together [76], but they have also been individually compared and optimized. For example, for whole porcine heart decellularization, Ferng et al. suggested 3% SDS as the optimal detergent, especially when perfused at a pressure between 90 and 120 mmHg, while discouraging the use of CHAPS or OGP due to their inability to successfully decellularize the tissue. Physical and enzymatic methods are often used before or after the detergent-based decellularization step. For example, osmotic shock before decellularization induces loosening of the ECM, while pretreatment with trypsin–EDTA also improves further decellularization steps [30,109,110]. After the detergent-based decellularization step(s), and in order to achieve more porosity in the sca ffold, hearts are often freeze-dried [109–111], and sucrose may be added to the freezing media in order to avoid damaging or denaturizing biomolecules in the heart sca ffold [111]. Focused on heart valve decellularization, postdetergent treatment, enzymes at low concentrations have been used in order to remove the waste of nucleic acids [112], a treatment that does not a ffect collagen valves or elastane and is compatible with further cell seeding strategies [30].

Some authors claim the most common detergents used as decellularizing agents for hearts may cause ECM denaturation and further loss of mechanical properties. That is why some physical and enzymatic treatments have been assayed with no detergent step for heart decellularization. For example, Seo et al. based their study on the supercritical fluid technology using the scCO2–EtOH cosolvent system to decellularize hearts, showing that the supercritical carbon dioxide method maintains higher GAG and collagen levels in the remaining decellularized sca ffolds [113]. For heart valves, in terms of enzymatic methods, an accutase solution, followed by nuclease treatment, has been reported [112], while other proteolytic and collagenolytic enzymes combined with nucleases are also able to e ffectively remove nearly all nucleic acids [112]. Other methods have been assayed, but seem more controversial in terms of the achieved nucleic acid removal, such as the use of a combination of proteases and chelating agents, e.g., trypsin–ethylenediaminetetraacetic acid [112].
