QS4: Optimizing The Decellularization Of The Rodent Epigastric Free Flap: A Comparison Of Automated SDS-based Protocols

Abstract

Purpose: Raising flaps to cover complex wounds with exposed critical structures are lengthy operations that result in donor site morbidity. Tissue engineering research is developing with great promise to build replacement tissues without morbidity. Decellularization removes whole cells and cell debris, and is the initial step to create a scaffold with an intact vascular network. Benchmark measurement of the overall cellular level is quantification of the DNA content, where 50 ng/mg is classically considered as a threshold. Although perfusion decellularization and recellularization approaches have shown exceptional promise in whole organ engineering, there is minimal crossover into the microsurgical field. Sodium dodecyl sulfate (SDS)-based protocols are known to have deleterious effects on the ultrastructure and capillary network of the scaffolds, but remain the predominant choice for decellularization protocols. This study aims to optimize the SDS exposure protocol for automated decellularization by comparing different SDS perfusion times to gain better understanding of the balance between decellularization and scaffold preservation. Methods: A 3D-printed closed-system bioreactor capable of continuously perfusing fluid throughout the vasculature was used for decellularization of free flaps. 2x2 cm fasciocutaneous free flaps from the epigastric region of the rat were harvested, and the vascular pedicle was isolated as a single artery and vein. Three flaps were evaluated in each group. The artery (inflow) and vein (outflow) were cannulated to monitor preservation of the vasculature. 1% SDS solution was perfused in different durations (3, 5 and 10 days) based on several protocols found in the literature. Automated SDS perfusion was followed by 1 day of 1% Triton X-100 and 1 day of 1xPhosphate-buffered saline (PBS), all of which were at the perfusion pressure of 120 mmHg. Results: For vasculature analysis, continual perfusion into the artery and out of the vein within the bioreactor was assessed throughout the decellularization process. H&E, Masson’s trichrome, and Verhoeff-Van Gieson staining were performed to assess architecture and locale of residual nuclei. Residual DNA was quantified by the fluorescent marker PicoGreen. 5 days of 1% SDS solution had the least residual DNA content (1.309±0.807 ng/mg) followed by 10 days (12.684±14.184 ng/mg) and 3 days (82.387±71.595 ng/mg), p<0.001. The DNA content ratio of skin over subcutaneous tissues was consistent across all protocols with skin having twice as much residual DNA after each protocol. The vascular network was visualized for qualitative assessment with the perfusion of hardening cast to create contrast against soft tissue to visualize with microCT imaging. Conclusion: A decellularization protocol of 5 days of 1% SDS solution followed by 1 day of 1% Triton X-100 and 1 day of 1xPBS was the most successful to keep the residual DNA content at a minimum, while preserving the structural integrity of the tissues. The bioreactor is capable of running automated and repeatable protocols using several solutions and continuously perfusing fluid throughout the vasculature of a free flap. This compact and integrated system can decrease hands-on time and be used in the future for further recellularization of scaffolds to bioengineer soft tissue replacement without donor site morbidity.