*4.7. Dermal Tissue*

The skin's main function is to protect, but it is also in charge of thermoregulation and perception. Dermal tissue is complex, with different layers, such as the epidermis, dermis, and subdermis, each with different compositions, properties, and functions. The epidermis is the outermost layer, and its main function is protection. Behind it, the dermis softens stress and strain, while also provides a sense of touch, elasticity, and heat. The inner layer is the subdermis and it is in charge of insulation. Dermal tissue, as many tissues, is composed of different collagens (85% of the dermal tissue's ECM), matricellular proteins, elastin, fibrillin, and also other fiber-forming proteins, such as vitronectin and fibronectin, which are necessary for wound healing. The ECM of dermal tissue is also formed by proteoglycans and GAGs, with functions related to hydration and osmosis balance. Examples of these are hyaluronic acid, decorin, and versican [126].

Dermal conditions such as cutaneous burns and scars require regenerative medicine approaches to restore dermal function. These therapeutic targets comprise stem cell transplants, growth factors, and tissue engineering. When scars, burns, and wounds are not able to heal on their own, they require replacement of the dermal barrier. It is very common to find skin equivalent and reconstruction research in the literature using primary human fibroblasts and keratinocytes, regularly supplemented with a collagen type I matrix or ascorbate [127,128]. In terms of decellularized approaches, acellular dermal matrices (ADMs) are widely used in clinical regenerative medicine approaches because of their biological and structural organization. ADMs can be of both animal and human origin, and they have a lot of different applications, such as the regeneration of skin tissue in burn, wound, and scar reconstructions, among others.

The majority of commercial ADMs are based on patented or proprietary decellularization protocols. Among the experimental research examples, the first step is always the mechanical isolation of the dermal layer, which is obtained by individual or combined chemical, physical, and biological methods supplemented with agitation. Dermal tissue can be incubated in hypotonic buffer for cell lysing [129] before the dermal decellularization step, which is usually carried out with detergents such as Triton X-100, DOC, N-lauroylsarcosinate (NLS), or SDS, and often in combination with trypsin, BSA, EDTA, and/or dispase [25,76,124,129–132]. Detergents can be also combined with acids and bases for the hydrolytic degradation of residual nucleic acids or even hair, but they can damage the ECM [109]. Some protocols describe the use of a further step in endonucleases treatment to remove residual genetic material [129,132].

It is worth noting that there are some other methods that have been reported that are detergent-free, such as osmotic shock and latrunculin B treatment, but they are not mainstream in the dermal tissue decellularization field [132].

#### *4.8. Tissues Related to Respiratory System*

Taking oxygen and expelling carbon dioxide is a function of the respiratory system, which is formed by multiple organs. Some of them, such as the trachea, lungs, and even diaphragm, have been tested as raw materials for decellularization purposes [133–135].

The trachea is related to essential physiological functions, such as airway protection, phonation, and breathing. It is composed of hyaline cartilage, fibrous tissue, respiratory epithelium, and smooth muscle, with cartilage being the most prominent [134]. Trachea damage requiring replacement surgery can be a result of trauma, neoplastic diseases, or congenital stenosis. Tracheal reconstruction in these cases is still a grea<sup>t</sup> challenge, while currently, autologous tissue and cell transplantation, with or without additional grafting material, seems the best solution [136,137]. In this sense, decellularized allograft and xenograft trachea is a material that has been tested in in vivo settings. While hyaline cartilage is part of this tissue, its decellularization protocols are slightly different to the ones described for cartilage, being tissue freeze-drying, followed by detergent treatment, the most common [138]. Some protocols add DNases into the detergent treatment step to ensure the elimination of the nuclear material. Commonly, mild reagents are used with several cycles of repeated treatment. According to the reports, this procedure yields mild disruption of the mucosa layer, with preservation of the majority of the remaining tissue structure [139].

The function of the lungs is related to gas exchange from the environment to the bloodstream. The structure of the bronchi in the lungs is similar to the trachea, while air circulates from the bronchi to the bronchioles on its way to alveolar airspace, where gas exchange occurs. The regenerative capacity of the lungs is low, and a broad spectrum of severe lung diseases, such as obstructive diseases, fibrosis, and sarcoidosis, may require a lung transplantation procedure [140]. In this context, decellularized lung tissue has been tested as an experimental alternative to transplantation. Perfusion of detergents such as SDS and Triton X-100 is a technique useful to decellularize mouse and rat cadaveric lung tissue, thereby preserving the vascular and airway structures of the tissue [141–143]. Due to the complexity of lung tissue and the need for instant functionality of used implants, a lung's dECM requires recellularization with epithelial and endothelial cells via cell infusion and bioreactors for ex vivo generation, maturation, and maintenance of the so-called bioartificial lungs. In vivo implantation of these organs in rats yields anastomosis, but long-term success is still to be achieved [143,144].

#### *4.9. Tissues Related to Gastrointestinal Tract*

The gastrointestinal tract is a complex microenvironment with different parts and multiple related organs. It is in charge of digesting food to extract energy and nutrients and to absorb them. Damage to the gastrointestinal tract or the related organs can be caused by stress, injury, or diseases that affect one or several tissues of the gastrointestinal tract, for example, trauma, surgeries, neoplasia, cancers, fibrosis, inflammatory bowel disease, esophagectomy, and congenital or acquired defects [145]. The

complex anatomy of the gastrointestinal tract makes the use of bioengineered sca ffolds a di fficult task, requiring the use of multilayered structures and the seed of di fferent types of cells, depending on the tissue anatomy [146]. The esophagus and intestines have been the main target of decellularization in gastrointestinal tissue, along with the related organs such as the liver and pancreas, and decellularization has often been complemented with the seeding of functional cells [147]. Research of the esophagus and pancreas is still in the initial stages of development [148]; therefore, here, we focus on intestine and liver tissue decellularization.

The intestine has a complex cellular and matrix architecture with multiple gradients, and it is therefore di fficult to replicate using simple sca ffolds. The three-dimensional architecture of the intestine is maintained by the ECM, composed of an intricate network of fibrous structural proteins (proteoglycans and glycoproteins), along with fibronectin, laminin isoforms, collagens, and heparin sulfate proteoglycans (HSPGs). Furthermore, multiple cell phenotypes are present in the intestine, including stem cells, pericryptal myofibroblasts, fibroblasts, endothelial cells, pericytes, immune cells, neural cells, and smooth muscle cells [149]. The intestinal stem cell niche is a well-known dynamic environment located at the base of crypts and embedded within a specific ECM in which the intestinal stem cells (ISCs) reside and control proliferation, di fferentiation, and tissue homeostasis. That is why multiple research e fforts have been conducted to bioengineer the intestinal stem cell niche, including the use of decellularized tissue [150]. For intestine decellularization, a combination of chemical and enzymatic solutions (perfusion of sodium deoxycholate, use of DNase, immersion in a hypotonic solution, etc.) is used to remove cells in this tissue, while maintaining the 3D structure [151,152]. Recent studies have shown that the integrin e ffector protein focal adhesion kinase (FAK) is essential for intestinal regeneration, and thus the preservation of FAK in decellularized tissue sca ffolds is essential for regenerative purposes [153].

Hydrogels derived from decellularized intestine are useful for generating endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and small intestine organoids [154]. Moreover, decellularized intestine sca ffolds have been used for regenerative purposes in many other tissues, due to their intrinsic ability to induce site-specific in vivo cellular repopulation and regeneration without the need for an in vitro recellularization step. For example, decellularized intestine has been used for regeneration purposes in vascular [155], cardiac [156], dura mater [157], abdominal wall [158], bladder [159], bowel [160], corneal [161], esophagus [162], tendon [163], ligament, cartilage (meniscus) [164], dermal [165], and bone tissues [166].

In recent decades, several methods have been implemented for liver decellularization, aiming to preserve the liver's major ECM components such as laminin, elastin, fibronectin, collagen types I and IV, and sulfated glycosaminoglycan (sGAG) [167]. The most promising of such methods is the perfusion decellularization method, in which detergents (SDS and/or Triton X-100), hyperosmotic (NaCl), and enzymatic (DNase) solutions are injected intravascularly [168]. Additionally, liver fragments can be immersed into the detergent solutions under mechanical agitation, and the previously described detergent–enzymatic method can be combined with high G-force oscillation treatment to reduce processing times. In such decellularized livers, the structural properties and the protein composition of the ECM are maintained, while they show good biocompatibility and neovascularization in vivo [168,169].
