# Decellularized Scaffolds for Skin Repair and Regeneration
## 3 Decellularization Methods
### Main Decellularizing Agents  
#### 3.2.1 Detergents  
Detergents are the most common decellularizing agents used to process skin, SIS, mesothelium, bladder, and AM, regardless of the tissue origin. SDS is the strongest detergent used for tissue decellularization. It is ionic and disrupts all interactions between biomacromolecules. This agent is the most reported in decellularization protocols, and numerous teams have used it to process porcine SIS, porcine bladder wall, human amnion, human skin, rat flap, or fibroblast cell sheets [68; 80; 81; 89; 92; 95]. Patents often mention its use for the production of commercial acellular matrices used in clinic [83; 84; 86]. SDS treatments are commonly performed by immersion in a 0.5% w/v solution (range of concentrations reported in literature: 0.1-1%) and last for hours. Thick and complex tissues containing a dense conjunctive layer or a multilayered epithelium, such as mesothelium, skin, and bladder wall are processed from 12 to 24 h. In contrast, thinner or less complex structure treatments last from 0.5 h for fibroblast sheets to 4-12 h for SIS. All decellularization studies that include SDS in concentrations >0.1% in their protocols, have reported a total removal of cells and cellular debris from the native tissue and low levels of remaining DNA. Some combinations with nucleases have been reported on amnion and bladder wall for SDS concentrations lower than 0.1% [68; 92]. Although SDS is an excellent agent for decellularization, it has been frequently reported to damage ECM. Loss of global ECM density as well as GAG content and damage to the collagen network have particularly been observed in the matrix produced by fibroblast sheets [89] or in complex tissues such as human skin [96] and porcine SIS [81]. The increase in SDS concentration from 0.05% to 0.5% on fibroblast sheets or in exposure time from 6 to 12 h on SIS has been directly correlated with the amplification of ECM alterations. Scanning electron microscopy studies also suggest rearrangements of ECM fibers and variations in scaffold porosity after SDS treatment, even if no common and systematic effects can be generalized. The exposure of porcine SIS to the combination of 1% SDS for 12 h and 1% Triton X-100 for 0.5 h leads to complete cell removal but exerts similar adverse effects on ECM quality, coupled with a low cell development and metabolic activity after hemangioma stem cell (HEMSC) seeding [75]. Different SDS/Triton X-100 combinations have also been tested on rat fascicocutaneous flaps, leading to high DNA content removal after perfusion of 1% SDS for 24 or 72 h, followed by a 0.5-h 1% Triton X-100 treatment [95]. Modifications of the 2D structure of the ECM have been noted for the 72-h SDS perfusion using histological staining, but no other investigation has characterized the effects on ECM in detail. The switch of the 72-h SDS treatment from perfusion to immersion was associated with a decrease in DNA removal. Basement membrane preservation after exposure to SDS is essential for epithelialization of the scaffold. The preservation of collagen IV, collagen VI, and laminin has been confirmed by immunostaining in porcine bladder wall (Tris HCl 10 mM overnight, 0.1% SDS for 24 h) [92] and softly decellularized human skin (1 M NaCl 24 h, 0.5% SDS 1 h) [97], but not in porcine SIS (0.05% trypsin 12 h, 0.5% SDS 4 h). Trypsin was shown to be compatible with basement membrane preservation at a higher concentration [81]. In human AM softly decellularized by a 24-h immersion in 0.03% SDS coupled with DNAse I and RNAse I treatments, maintenance of the basement membrane components but damage to the mesenchymal matrix architecture have been observed [68]. However, the mechanical properties were not significantly altered, and the final scaffold exhibited characteristics, e.g., failure strain, failure stress, an elastin phase slope, and a collagen phase slope similar to native tissue.