# Decellularized Scaffolds for Skin Repair and Regeneration
## 3 Decellularization Methods
### Main Decellularizing Agents
#### 3.2.3 Physical Approaches  
'Supercritical conditions' refers to combinations of particularly high temperature and high pressure so that a fluid reaches a physical state combining the properties of both liquid and gas. This physical state allows a good penetration of the fluid inside tissues, just like a gas, with a macromolecule solubilization capacity similar to that of a liquid. Supercritical CO\({}_{2}\) has been used initially to gently sterilize natural materials and tissues [107]. In recent years, supercritical CO\({}_{2}\) decellularization was developed on vascular tissues [108], and most recently, processing of human skin and porcine skin has been reported.  
On human skin, processing with supercritical CO\({}_{2}\) after a hypertonic 1 M NaCl exposure has produced results similar to those with SDS in terms of cellular content removal (DNA level post-treatment <30 ng/mg of dried ECM) [91]. Small fractures reducing global ECM density were detected, but the fibronectin network seems to be unaffected. A surprising 3D structure was observed on scanning electron microscopy, however, characterized by punctate globular structures in the ECM network. Proliferation and viability of primary bovine chondrocytes were documented, but fibroblasts growth assessment was not presented. That study additionally underlined the importanceof tissue saturation with the CO\({}_{2}\)-philic detergent LS-54 prior to CO\({}_{2}\) exposure to enhance removal of cellular components.  
A recent paper presented an approach combining supercritical CO\({}_{2}\) with NaOH (from 0.1 to 1 M, exact concentration unknown) applied on porcine skin [109]. Observations in the ECM network were similar to those previously reported with human skin, and promising results were obtained in a porcine wound healing model with the matrices seeded with autologous cells before grafting. This protocol is patented and used to produce ABCcolla(r) Collagen Matrix (ACRO Biomedical, Kaohsiung, Taiwan).  
Freeze-thawing is a special case that is difficult to categorize as a main or complementary agent because the complete elimination of cells using only extreme variations of temperature has not been fully demonstrated. With cell sheets, no complete decellularization has been achieved applying three cycles of freeze-thawing [89]. However, with human mesothelium, a combination of three freeze-thawing cycles with trypsin, DNAse I, and lipase VI exposures and isopropanol treatment to remove lipids has been reported as effective in removing all cells from the tissue. As trypsin and IPL cannot fully decellularize a tissue, the effect of freeze-thawing may be determinant in eliminating cells content [76]. Supplementary studies of the tissue after each step of the protocol are necessary to characterize the effects of each agent. Regardless, this study suggests that the combination of several complementary agents could be an interesting approach to investigate [76].  
Regarding hydrostatic pressure, no protocol has been reported that has used it to decellularize tissues to produce acellular matrices for skin repair.