# Decellularized Scaffolds for Skin Repair and Regeneration
## 3 Decellularization Methods
### Main Decellularizing Agents
#### 3.2.1 Detergents
This study is the only one to report decellularization of human AM using SDS, and the soft treatment applied could be an interesting approach to preserving ECM mechanics and integrity. Finally, no other comparison of tissue biomechanics before and after decellularization has been reported. Even if ECM damage is often underlined after SDS treatments, biocompatibility of the resulting scaffolds has been demonstrated for several native tissues. In vitro, matrices derived from porcine skin [80], porcine SIS [82], human AM [68], and fish skin [98] do not exert cytotoxic or proliferation-limiting effects. Cytotoxic tests performed by direct or conditioned-medium contact, metabolic arrays, or visual observations by light microscopy several days after cell seeding have confirmed the compatibility of the acellular matrices with cell development. These studies used NIH 3T3 fibroblasts, primary fibroblasts, bone marrow mesenchymal stem cells (BMSCs), or human lung carcinoma cells. Implantation tests have also been reported for some acellular matrices obtained using SDS. Porcine SIS-derived matrix implanted subcutaneously in rat has shown a very satisfying colonization and establishment of host fibroblasts 4 weeks post-operatively. A classic immune response involving giant cells and infiltrated inflammatory cells has been described but started to decrease after 14 days of implantation [81]. Triton X-100 is a non-ionic detergent that can disrupt interactions between biomacromolecules more gently than SDS. It is very commonly used alone or with other agents to generate matrices for skin repair derived from porcine SIS [82; 99], porcine skin [68], porcine mesothelium [94], mouse skin [87], and rat flap [72]. Almost all protocols apply this detergent at the same concentration of 0.5% v/v, with a few using up to 1%. The ability of Triton X-100 to fully decellularize a native tissue has been evaluated with porcine SIS exposure to increasing concentrations of detergent from 0.5% to 2% [99]. Acellular scaffolds were obtained after 48 h of immersion in all conditions, but ECM damage, especially loss of density, was observed for concentrations higher than 1%. On mouse skin, successful decellularization using 0.5% Triton X-100 for 24 h also has been reported, associated with matrix alterations, especially loss of elastin and increased stiffness compared to native skin [87]. Limited colonization by primary fibroblasts and poor metabolic activity was also described. Surprisingly, during wound healing experiments in mouse, new blood vessel formation was observed in the grafted matrix, whereas a low dermis fibroblast infiltration occurred in accordance with in vitro experiments. Deleterious effects of 0.5% Triton X-100 on mouse skin may be the result of its limited thickness and cell abundance compared to SIS. To modulate the effects on ECM and favor cell development, combinations of Triton X-100 with other agents have been tested at reduced exposure times. As mentioned above, the combination of 0.1% SDS for 12 h with 1% Triton X-100 for 0.5 h failed to produce a satisfying porcine SIS acellular matrix [75] because the scaffold obtained was not compatible with cell growth in vitro. In contrast, co-exposure of porcine skin to 0.5% Triton X-100 and hypertonic 1 M NaCl, followed by 2% sodium deoxycholate and DNAse I treatments, appeared more promising [74]. Density and integrity of dermis ECM were preserved, especially collagen I and fibronectin networks, and the preservation of basement membrane was confirmed by the detection of collagen VII and laminin. Other observations revealed that, after a 6-month abdominal implantation in monkey, new blood vessels and dense colonization by host cells could be observed (global immune response not estimated). In rat, a dermis/adipose flap was successfully decellularized by perfusion using a combination of 1 M NaCl (epidermis removal and tonic shock), trypsin, and 1% Triton X-100 for 48 h [72].