*4.3. Adipose Tissue*

White adipose tissue is defined as a connective tissue that stores energy in the form of lipids (triglycerides), insulates the body, and provides cushioning and support for subcutaneous tissues and internal organs. It is composed of clusters of fat-storing cells (adipocytes) surrounded by a reticular fiber network and interspersed small blood vessels. The key ECM proteins of adipose tissue are collagen type I, collagen type IV, and laminin. Collagen with laminin provides anchoring sites and barrier functions for adipocytes. Collagen types IV and VII and laminin are major components of the basement membrane [84].

Reconstruction of soft tissue defects is needed after certain tumor resections, external injury, or due to congenital malformations, and presents a major challenge in plastic and reconstructive surgery. At present, the main complications related to adipose tissue reconstruction include capsular contracture, necrosis and donor site morbidity, and immune rejection, and therefore, new clinical approaches are required to improve the success rate. The subcutaneous adipose tissue discarded from surgical operations represents an abundant and easy-to-collect human tissue source, processable by dECM biomaterial [85]. In this sense, allograft and xenograft dECM biomimetic sca ffolds have proved to be e ffective tools for promoting tissue repair and regeneration in numerous preclinical and clinical studies [86–88].

The optimal adipose tissue decellularization includes the extraction of lipids (delipidation), followed by the extraction of cells and cell components, thereby maintaining key proteins and the 3D structure. Human and porcine are the most common sources of adipose tissue extraction, and there are two di fferent kinds of initial adipose tissue samples useful for decellularization purposes. Such samples can be solid tissue derived from resection surgery, usually performed in the abdominal area, which has to be cut into small pieces for decellularization. On the other hand, liposuction-derived samples are gel-like tissues that require homogenization and centrifugation as the initial step for separation of the lipid phase. After initial processing, decellularization can be achieved using detergent-based or detergent-free protocols [89].

A detergent-free method for adipose tissue decellularization was described by Flynn et al. in 2010, in which the dECM was produced with a combination of multiple physical and chemical strategies, such as freeze–thaw cycles in hypotonic bu ffer to loosen the ECM, isopropanol to remove lipids, and enzymatic digestion with trypsin–EDTA, DNase, RNase, and lipase to remove cells and lipids. The resulting dECM conserves the collagen architecture and provides a microenvironment for the di fferentiation of human adipose stem cells [90,91]. A further published similar protocol demonstrated, by immunohistochemical staining, that laminin and collagen type IV remain abundant in the decellularized matrix. In vitro and in vivo models with microporous foams and hydrogel sca ffolds with cells both demonstrate strong support for adipogenesis and induce an angiogenic response and formation of new adipose tissue [55,92]. dECM sca ffolds generated by similar detergent-free methods, with a combination of isopropanol, trypsin, EDTA, and DNase–RNAse in gel-like liposuction-derived samples, served as support for human adipose-derived stem cells and adipose regeneration [93].

In adipose tissue, the use of detergents for decellularization seems to increase the risk of matrix protein denaturalization and degradation. Wang et al. reported a method with multiple sequential physical and chemical steps, including a polar solvent extraction and Triton X-100 treatment, which resulted in the maintenance of collagens but the absence of laminin in the final dECM [94]. Similar results were obtained when SDS was used during the decellularization process [95]. Note that although laminin was absent in Triton-X-100-treated samples, in vivo studies have confirmed that the dECM undergoes vascularization and adipose tissue regeneration at Day 30 of implantation, which is consistent with other reports on the adipose tissue-derived matrix.

#### *4.4. Skeletal Muscle and Tendons*

Muscles are connective tissues formed by contractile fibers. Skeletal muscles are responsible for voluntary movement and homeostasis, and they are attached to bones by collagen fibrillar structures

called tendons. Skeletal muscle is divided into several innervated and vascularized subtypes. Given the complex structure of skeletal muscle, it is difficult to pinpoint the exact distribution and composition of the ECM. Collagen is the most common component, as it contains collagen types I, II, III, IV, V, VI, XI, XII, XIV, XV, and XVIII. GAGs are ubiquitous in the ECM, while interactions between proteins and glycans are particularly important to regulate protein distribution. Moreover, ECM glycoproteins and cell membrane–protein interactions transmit the mechanical force in the muscle and are active during muscle injury regeneration [96].

Skeletal muscle loss is often the result of a traumatic injury. In this sense, reconstruction surgery may be required to recover functionality [97]. The first option is always an autologous transfer from nearby tissue, but this implicates a partial loss of functionality or volume. Among other approaches, the use of the muscle's decellularized ECM is a promising treatment, due to several reasons. Decellularized muscle xenografts are feasible, as a muscle's ECM is similar among different species, thereby minimizing the risk of the immune response [98,99]. Moreover, the dECM from skeletal muscle shows good integration in vivo, promoting vascularization, remodulation, and differentiation.

The initial skeletal muscle decellularization protocols included physical methods such as freeze–thawing or proteases, which have been further proven too aggressive for proper muscle tissue decellularization. At present, and aiming to preserve the matrix content and tissue structure, some less aggressive detergents, salt solutions, and nucleus-specific enzymes are the preferred decellularization methods. Most of the protocols use weak acids or detergents, such as sodium deoxycholate or Triton X-100 and SDS, respectively, followed by DNase treatment—all of them at low concentrations and exposition times and with multiple repeated cycles [100,101]. In some cases, trypsin is used in low concentrations and for short times, ensuring it does not damage in excess the protein structure.

Tendon tissue is a highly fibroelastic structure that connects muscles to bones. A tendon's ECM is mainly formed of collagen type I, elastin, and proteoglycans, and it provides mechanical and elastic capabilities. Collagen, in particular, constitutes up to 80% of the dry mass of tendons. These proteins are organized by creating fibers, fascicles, and, finally, the tendon itself. Other than that, there is a huge network of proteoglycans and other elastic macromolecules [49].

Tendons have a natural healing capacity, but they can be damaged if the injury goes beyond this healing capacity. Damage to tendons can be the result of severe trauma or the result of continuously repeated injuries during recovery processes [47]. When using a material to repair possible damage, the main properties required are mechanical and elastic capacities. In this context, dECM derivate materials show good regenerative properties, particularly the ones created with decellularized tendons, as they are assimilated easily and promote new tissue formation.

In order to decellularize tendons, detergents are the most commonly used reagents. Triton X-100 and SDS have been tested and compared independently as tendon decellularization methods, with and without a previous freeze–thawing cycle [102]. Triton X-100 treatment shows less efficiency in cell lysis, with no significant cell removal, and induces damage to the tendon structure. On the other hand, SDS is more effective as a tendon decellularization agent, with less damage to the ECM components and collagen structure [102]. Tri-n-butyl phosphate (TnBP) detergent has also been used for tendon decellularization purposes, with improved results compared to previous methods. Specifically, TnBP treatment results in a significant decrease in cell density, without disruption of the collagen matrix, even when used in relatively high concentrations [47].
