# Decellularized Scaffolds for Skin Repair and Regeneration
## 3 Decellularization Methods
### Complementary Agents  
#### Enzymes  
Nucleases are the most common complementary agents included in decellularization protocols independently of the source of native tissue. Nucleic acids, especially DNA, are well known for their high immunogenic properties. To limit nucleic acid levels as much as possible, treatments with DNAse I, DNAse II, or benzonse are performed after exposure of the tissue to the main decellularizing agent(s) [68; 74; 75]. Applied concentrations, ranging from 50 U to 10 kV/mL, depend on the properties of the tissue (type, thickness, cell density) and the exposure to main decellularizing agent(s) (nature, duration, concentration, temperature). Regarding the choice between benzonse or DNAse, no clear rationale is available in the literature. Finally, for highly cellularized tissues, processing can also include RNAse treatments. As mentioned above, strict protease-based approaches are not sufficient to remove all cells from the tissues used for the production of skin-repair matrices. Accutase, a commercial mix of proteolytic and collagenolytic enzymes classically used for soft cell-detachment from two-dimensional (2D) culture supports, failed to decellularize human AM: the epithelial layer was totally removed after 1 h of incubation, but many mesenchymal cells were still detected [76]. Trypsin has also been tested on human AM combined with EDTA, allowing for complete elimination of cells in both epidermal and mesenchymal compartments and retention of basement membrane components. However, the ability of the matrix to drive sufficient primary fibroblast infiltration and proliferation after a 7-day in vitro culture has not been demonstrated. The same group investigated the effects of dispase, a bacterial protease targeting collagen IV, laminin, and fibronectin [77]. They achieved complete decellularization but observed ECM alterations and important losses in basement membrane structure, as had been already reported [78; 79]. Cell seeding was not assayed because of the low probability of epithelialization success using a basement membrane with such damage. Although still undocumented as an efficient decellularized agent, trypsin can be combined with physical, acid-, base-, or detergent-based protocols [80; 81; 82]. The action of trypsin is achieved by immersion of the tissue in an isotonic solution containing from 0.25% to 0.1% w/v of enzyme, separately from the other treatments of the protocol. This exposure is often combined with EDTA (0.1%) and preferentially applied between delipidation and principal decellularization steps. Tissue exposure to trypsin needs to be controlled; its ability to cleave a broad range of substrates means that deleterious effects on ECM structure can be exerted under overly long or too concentrated trypsin treatments. In porcine skin, different kinds of \"fracturations\" and density loss were observed in the ECM of the dermis compartment after an 18-h 0.25% trypsin exposure [80], whereas a denserand less porous ECM network was obtained for the same decellularization protocol excluding this preliminary step. Similar observations have been reported for human amnion with shorter exposure at the same concentration [76]. Surprisingly, none of the studied patents report strict trypsin incubation during the processing of human skin, human AM, and porcine SIS for acellular matrix production for skin repair [83; 84; 85]. Only one recent patent presenting a decellularization method for fish skin includes a trypsin step, but at a concentration lower than 0.001% [86]. Dispase, mentioned above as deleterious to human AM in isotonic solution, has surprisingly been described in two publications as a good complement for skin decellularization. On porcine skin, application of a soft dispase treatment (0.56 U/mL, 4 \({}^{\circ}\)C in isotonic solution), followed by SDS and soft trypsin exposures, resulted in good cell removal without any adverse effect on ECM post- 4- and 12-h treatments.