# Decellularized Scaffolds for Skin Repair and Regeneration
## 3 Decellularization Methods
### Main Decellularizing Agents
#### 3.2.1 Detergents
DNA in dermis and fat compartments was quantified under 50 ng/mg of dry flap (10-fold reduction compared to native tissue). The general structure (2D and 3D) of the ECM and GAG content were preserved, but no evaluation of the basement membrane was described. Vascular endothelial GF was easily detectable by immunohistochemistry in fat tissue, but very low staining appeared in dermis. The growth of human umbilical vein endothelial cells and adipose stromal cells was shown between days 1 and 7 post-seeding. Sodium deoxycholate (SD) is another ionic detergent applied as a decellularizing agent. It is commonly used at concentrations ranging from 2% to 4% and disrupts interactions between biomacromolecules. Its action, however, is frequently associated with DNA precipitation in spite of the loss of DNA-histone interactions, as has been confirmed [100]. The incubation of tissues with nucleases is thus automatically applied after exposure to SD, lasting for a few hours at concentrations from 5 to 10 kU/mL. The capacity of SD to decellularize tissue alone has been reported in a study dedicated to the whole decellularization of rat intestine [101]. All cells and cell debris were removed with perfusion of a 4% SD solution for 4 h (connection to the superior mesenteric artery), followed by a DNAse I treatment for 3 h, and ECM density was preserved. In contrast, an incomplete decellularization of porcine SIS was observed after a 12 h immersion in 4% SD and a DNAse I treatment. Some cell nuclei were still visible in the conjunctive layer, associated with a level of remaining DNA that was 40% higher than that detected in tissue treated with SDS (40-45 versus 30 ng/mg dry tissue, respectively) [82]. However, other tissues that are compatible with perfusion and rich in ECM, such as aorta vein or skeletal muscle [102; 103], can be totally decellularized using similar SD treatments. A possible explanation for the incomplete cell removal in porcine SIS could be the limited SD penetration into the tissue during immersion compared to perfusion. As perfusion cannot be applied to all tissues, especially to skin that lacks an accessible vascular tree, immersion strategies have been developed by combining SD with other agents to aid in cell removal. In porcine skin where the multilayered epithelium is strongly anchored to the basement membrane, an exposure of the tissue to 1 M NaCl and 0.5% Triton X-100 for 24 h prior to a 4%, 24-h SD and DNAse I treatments, successfully removed cells from both the epidermis and dermis compartments [74]. In rat, a fascicutaneous flap decellularization protocol using 4% SD and DNAse I was also reported, but further characterization of the acellular matrix is needed to characterize its properties [90]. SD-based decellularization approaches have been reported to be excellent for ECM preservation when used on tissues such as aorta, muscle, and lungs. This probably explains the effort invested in the development of protocols dedicated to skin-repair acellular matrix production despite problems with the perfusion-to-immersion switch. In porcine SIS, comparison of SD treatment to a double SDS/Triton X-100 exposure indicated a better quality of the final ECM matrix, even if some cells were still detected in the tissue. The 3D structure of the matrix was very close to that of the native tissue architecture, whereas other detergents increased porosity and modified fiber organization. Mechanical properties (failure strain, elastic modulus) were also similar to control tissue, and metabolic activity of HEMSCs at 96 h post-seeding was 10 times that of the activity of cells grown on the SDS/Triton X-100 matrix [75]. As previously described, a combination with NaCl and Triton X-100 on porcine skin is associated with basement membrane and ECM density preservation and a very satisfying biocompatibility in vivo [74]. Sodium lauroylsarcominate (NLS, ionic) is a detergent that is less commonly reported for skin-repair matrix production. In mouse skin, a 1% NLS treatment for 24 h leads to complete removal of cells [87].