Automated Decellularization of the Rodent Epigastric Free Flap: A Comparison of Sodium Dodecyl Sulfate–Based Protocols

Abstract

Introduction Free tissue transfer to cover complex wounds with exposed critical structures results in donor-site morbidity. Perfusion decellularization and recellularization of vascularized composite tissues is an active area of research to fabricate complex constructs without a donor site. Sodium dodecyl sulfate (SDS)-based protocols remain the predominant choice for decellularization despite the deleterious effects on tissue ultrastructure and capillary networks. We aimed to develop an automated decellularization process and compare different SDS perfusion times to optimize the protocol. Methods A three-dimensional-printed closed-system bioreactor capable of continuously perfusing fluid through the vasculature was used for decellularization. The artery and vein of rat epigastric fasciocutaneous free flaps were cannulated and connected to the bioreactor. Protocols had varying durations of 1% SDS solution (3, 5, and 10 days) followed by 1 day of 1% Triton X-100 and 1 day of 1x phosphate-buffered saline. The residual DNA was quantified. Microarchitecture of the constructs was assessed with histology, and the vascular network was visualized for qualitative assessment. Results The structural integrity and the microarchitecture of the extracellular matrix was preserved in the 3- and 5-day SDS perfusion groups; however, the subcutaneous tissue of the 10-day protocol lost its structure. Collagen and elastin structures of the pedicle vessels were not compromised by the decellularization process. Five-day SDS exposure group had the least residual DNA content (p < 0.001). Across all protocols, skin consistently had twice as much residual DNA over the subcutaneous tissues. Conclusion A compact and integrated bioreactor can automate decellularization of free flaps to bioengineer regenerative constructs for future use in reconstruction of complex defects. A decellularization protocol with 5 days of 1% SDS exposure was the most successful to keep the residual DNA content at a minimum while preserving the structural integrity of the tissues.

A Model to Study Wound Healing Over Exposed Avascular Structures in Rodents With a 3D-Printed Wound Frame

“…In addition, excessively large, and heavy chambers can restrict the movements of the animal and prevent housing of multiple animals in 1 cage, which further increases distress on the animal and overall cost of the experiment for longer durations of follow-up. 3D printing is an advancing modality, which is becoming more accessible in different aspects of research in regenerative medicine and reconstructive surgery 27,35 . The wound frame described in the current study offers benefits over previously described studies to allow for future reproducible and affordable experiments across various aspects of wound healing research (Fig.…”

“…3D printing is an advancing modality, which is becoming more accessible in different aspects of research in regenerative medicine and reconstructive surgery. 27,35 The wound frame described in the current study offers benefits over previously described studies to allow for future reproducible and affordable experiments across various aspects of wound healing research (Fig. 10).…”

“…Flap harvest was performed as described previously 26,27 . Briefly, the epigastric area was marked to design a 20 × 20 mm flap.…”

“…Flap harvest was performed as described previously. 26,27 Briefly, the epigastric area was marked to design a 20 Â 20 mm flap. A full-thickness incision, including the skin and subcutaneous tissues above the facia, was made and the superficial inferior epigastric vessels were exposed.…”

Optimized Decellularization of a Porcine Fasciocutaneaous Flap

“…The perfusion decellularization technique has been shown to be applicable to a number of different organs and tissues, including those used for reconstructive surgery [15,16]. A limited number of studies reported the decellularization of fasciocutaneous flaps from small [17][18][19][20] and large animals [21,22]. These studies have shown success in the removal of the cells using histological staining and/or the quantification of DNA content, and a few investigated the scaffold further for ECM characterization and biocompatibility [21].…”