# Decellularized Scaffolds for Skin Repair and Regeneration
## 3 Decellularization Methods
### Promising New Approaches  
Investigations are under way to find new main and complementary agents for tissue decellularization. A main issue when using acellular matrices for grafts in clinic is the immune response elicited by the exogenous tissue, sometimes leading to graft rejection. Galactose-\(\alpha\)-(1, 3)-galactose (\(\alpha\)-gal) is an oligosaccharide sequence in some sugar-linked proteins and lipids of non-primate cell membranes. This oligosaccharide is a strong immunogenic epitope in humans and is especially responsible for the rejection of pig-derived organs. A method has been specifically developed to eliminate this antigen from porcine skin [74]. A classic decellularization protocol combining hypotonic NaCl, Triton X-100, SD, and DNAse (already described above) has been completed by the addition of an \(\alpha\)-galactosidase incubation step, with no observed modification in the ECM structure compared to galactosidase-free treatment. After abdominal implantation in a monkey model, anti-\(\alpha\)-gal antibody levels were similar for matrices derived from galactosidase-treated and KO gal -/- pig tissues, whereas amounts for non-galactosidase-treated matrix were 16 and 25 times higher at 20 and 70 days post-surgery, respectively. Despite such promising results, no other similar studies have been published.  
In terms of main decellularizing agents, latrunculin B, a sea sponge toxin that inhibits actin polymerization [110], has recently been used to decellularize mouse skin [87]. Combined with 0.6 M NaCl, 1 M KI, dispase II, and benzonase exposures, latrunculin B treatment (50 nM in DMEM, 2 h) led to a complete cell removal, with a satisfying conservation of ECM density and quality (GAG and hydroxypolin levels, elastin distribution similar to native tissue). No changes in the biomechanics of the tissue were detected, in contrast to results with NLS or Triton X-100 treatments tested in parallel. In vivo, in a mouse wound healing model, infiltration of numerous host fibroblasts, formation of new blood vessels, and total re-epithelialization of the wound were observed.  
Many protocols have been published that propose techniques adapted for tissue decellularization (Table 2). Some of these methods are already patented to produce skin-repair matrices used in clinic. Although classic decellularizing agents are efficient at cell removal, adverse effects on the ECM are frequently reported. The main difficulty in the development of decellularization methods is thus to find the fine balance between sufficient removal of cell content and sufficiently limited ECM modification to maintain its properties driving the infiltration and the development of host cells.  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline  & \multicolumn{2}{c}{**Physical**} & \multicolumn{2}{c}{**Acids and Bases**} & \multicolumn{2}{c}{**Detergents**} \\ \hline
**Method** & Freeze & Supercritical & & & & & \\  & Thawing & CO2 & & & & & \\  & & & & & & & \\ \hline
**Common application** & & & & & & & \\  & (no-conserv) & (no-conserv) & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\ \hline \end{tabular}
\end{table}
Table 2: Summary of the different main decellularizing agents and their characteristics.