*4.2. Cartilage Tissue*

Cartilage in adult animals is a connective, smooth, and resilient tissue. Hyaline cartilage is avascular, and it is present in the stress points of skeletal tissue, such as bone heads, where it provides flexibility and prevents abrasion and damage [77]. It is also present in the rib cage, nose, larynx, and trachea, while its extracellular matrix is composed of collagens, mainly collagen type II, glycosaminoglycans (GAGs), and laminin [78]. GAGs are closely related to cartilage mechanical properties and help the tissue to cope with sudden external forces [24]. Elastic cartilage is a supportive structure for tissues such as the outer ear and epiglottis, and histologically, it is similar to hyaline cartilage, but with much more elastic fibers [21]. On the other hand, fibrocartilage is the only cartilage with collagen type I in its structure, because it is a mixture of fibrous tissue and cartilaginous tissue, with unique toughness and elasticity properties, and is present in intervertebral discs and menisci, among others.

Hyaline cartilage is the most commonly targeted cartilage in regenerative treatment—in particular, the one covering the end of bones in articulations. Damage to articular cartilage is usually related to trauma or pathology, and can cause pain, osteoarthritis, or even loss of functionality [78]. Surgical interventions attempt to solve these conditions, but at present, they are largely temporary solutions, pushing research toward searching for new regenerative approaches. The lack of vascularization greatly limits the number of nutrients and oxygen that can reach the inner parts of the tissue. This condition makes cartilage regrowth particularly challenging [79]. Therefore, tackling articular cartilage regeneration is a challenging goal. The literature is extensive in terms of approaches aiming to regenerate articular cartilage tissue, using di fferent kinds of implantable sca ffolds [80,81]. In this sense, cartilage dECM derivatives have been used for coating other implantable materials or as a 3D-printed structure for knee and meniscal regeneration, among others.

The basics for articular cartilage decellularization are the same for the di fferent types of cartilages, and in general, decellularization aims to keep in the matrix as many GAGs as possible. Of note, as the di fferent cartilages have di fferent permeability properties, it is paramount to set up proper conditions for optimum decellularization in each case.

Tissue snap freezing or freeze–thawing is a common pretreatment in many decellularization protocols [24,58]. Similarly, pretreatment with hypotonic and hypertonic solutions is also a popular method, used to induce apoptosis by osmotic pressure [11]. These methods do not decellularize the structure by itself, but they help further decellularization methods to work better and to reach the inner parts of the tissue. It has been proved that snap freezing does not a ffect the matrix component, and it has no significant negative impact on the structure [82]. Conversely, initial tissue homogenization, suggested in some articles, leads to the significant loss of GAGs and structural proteins [83].

Further to physical pretreatments, decellularization has been assayed with enzymatic or chemical detergent methods. The enzyme trypsin–EDTA is the most common decellularization approach reported [83]. Trypsin–EDTA breaks both the proteins that hold the cell in place inside the matrix, as well as the cell membrane proteins. Based on protease activity, the main setback of using this technique is the degradation of the proteins of potential interest in the final dECM. In order to prevent this, the exposure time to trypsin–EDTA needs to be highly controlled, and it is usually limited to 6–24 h [51].

Regarding the detergents used as decellularization reagents, Triton X-100 and SDS are the most commonly used ones. Both cause some extent of damage to the structure of the matrix, but the mechanical integrity of the matrix maintains at an acceptable level. Note that both incur damage to GAG content and integrity, being Triton X-100 the worse in this sense [26,45].

An additional issue to be taken into account regarding cartilage tissue is the need for enzymatic treatment with nuclease activity to prevent waste nucleic acid material from sticking to the matrix [11]. In elastic cartilage, the nuclease time may be necessarily longer to ensure decellularization. In articular cartilage, on the other hand, being a less dense structure, the risk of having DNA material stick to the protein matrix is lower.

The digestion of GAGs using chondroitinase ABC (ChABC) has been reported as a cartilage decellularization method. It is not a commonly used technique; although it facilitates the removal of native chondrocytes, it reduces the mechanical properties of the tissue as well [11].
