Beyond Decellularization: Remnant Mitochondrial DNA Can Act as Hidden Damage-Associated Molecular Pattern

Tissue decellularization aims to obtain bioscaffolds for regenerative applications by removing all cellular components while preserving the extracellular matrix (ECM) architecture. Although decellularization removes the majority of linear nuclear DNA (nDNA), residual amounts remain detectable. However, the fate of circular mitochondrial DNA (mtDNA) after decellularization has not yet been reported. Cell death or injury can cause the release of mtDNA, which is resistant to breakdown by exonucleases. Extracellular mtDNA acts as a damage-associated molecular pattern (DAMP) that can trigger immune responses. The aim of this study is to assess the presence of residual mtDNA in the liver, bile duct, and vascular scaffolds after decellularization and whether this causes inflammatory responses in macrophages. Decellularized tissues showed a marked reduction in total DNA content well below the threshold of 50 ng/mg tissue. However, in liver and vascular scaffolds, a relative increase in the mtDNA:nDNA ratio was detected in the remnant DNA fraction. Residual mtDNA in bioscaffolds acted as DAMPs causing macrophage activation, as shown by increased cell proliferation and cytokine production. Strategies to further reduce remnant mtDNA were tested. We found that treatment with the endonuclease enzyme HpaII was effective in degrading residual mtDNA. Importantly, mtDNA removal resulted in a significantly reduced macrophage activation. In conclusion, our study shows that mtDNA is relatively resistant to the decellularization procedure and can act as a DAMP in bioscaffolds. This underscores the importance of removing mtDNA from decellularized bioscaffolds to improve the immunocompatibility for biomedical applications.