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

Area of Science:

• Biomaterials Science
• Immunology
• Regenerative Medicine

Background:

• Tissue decellularization creates extracellular matrix (ECM) bioscaffolds for regenerative medicine.
• Decellularization effectively removes nuclear DNA (nDNA) but residual amounts remain.
• The fate and immunogenic potential of mitochondrial DNA (mtDNA) after decellularization are unknown.

Purpose of the Study:

• To assess residual mtDNA in decellularized liver, bile duct, and vascular scaffolds.
• To determine if residual mtDNA triggers inflammatory responses in macrophages.
• To evaluate strategies for reducing mtDNA in bioscaffolds.

Main Methods:

• Quantification of residual DNA (nDNA and mtDNA) in decellularized scaffolds.
• Assessment of macrophage activation (proliferation, cytokine production) upon exposure to scaffolds.
• Treatment of scaffolds with endonuclease HpaII to degrade mtDNA.

Main Results:

• Decellularized scaffolds showed reduced total DNA but a higher relative mtDNA:nDNA ratio.
• Residual mtDNA in scaffolds activated macrophages, increasing proliferation and cytokine release.
• HpaII treatment effectively degraded mtDNA and significantly reduced macrophage activation.

Conclusions:

• Mitochondrial DNA (mtDNA) is resistant to decellularization and acts as a damage-associated molecular pattern (DAMP).
• Residual mtDNA in bioscaffolds can elicit inflammatory responses, impacting immunocompatibility.
• Targeted removal of mtDNA, e.g., using HpaII, is crucial for enhancing the safety and efficacy of decellularized bioscaffolds in biomedical applications.