Antigen Removal Strategy in Laryngeal Tissue Engineering Mitigates Extracellular Matrix Collagen Degradation

Introduction: Preservation of the extracellular matrix (ECM) in addition to removal of immunogenic components is crucial for maintaining structural integrity and facilitating proper cellular integration in tissue engineered laryngeal constructs. The goal of this study was to evaluate the efficacy of an antigen removal (AR) technique in engineering a laryngeal vascularized composite allograft (LVCA).

Methods: This experimental study was conducted over one year. Mouse LVCAs were harvested and perfused through a bioreactor system using either a sodium dodecyl sulfate (SDS)-based (n= 9) or AR-based (n= 14) protocol. Untreated LVCAs were harvested to serve as the normal control (NC) group (n=8). The level of decellularization was assessed using Qubit dsDNA testing. H&E, picrosirius red, and alcian blue staining were used to assess ECM composition. QuPath software for digital pathology analysis was used to quantitatively assess collagen I (Col1) and collagen III (Col3) content based on intensity.

Results: Following perfusion, analysis revealed that both the SDS and AR groups exhibited significantly reduced levels of dsDNA compared to NC tissue (SDS vs NC, p=0.001; AR vs NC, p< 0.001). Col1 and Col3 intensity was significantly reduced in the muscle tissue following SDS treatment compared to the NC group (Col1, p < 0.001; Col3, p=0.003). No significant difference in Col1 and Col3 intensity were observed between the AR and NC groups. In the larynx cartilage, the intensity of Col1 was significantly increased in the AR group compared to NC (p=0.007), while it was decreased in the SDS group compared to NC (p=0.39). The intensity of Col3 was significantly reduced in cartilage following SDS treatment (p=0.007). No significant difference was seen between the AR and NC groups.

Conclusions: Perfusion-based engineering utilizing an AR protocol results in superior preservation of Col1 and Col3 in the laryngeal ECM compared to conventional decellularization methods. This improved retention of important ECM components may facilitate improved structural integrity and cellular integration of LVCAs.