A Quick and Reliable Method to Decellularize a Gracilis Flap A Crucial Step Toward Building a Muscle M. Diya Sabbagh, MD,* Si-Gyun Roh, MD,* Junting Liu, MD,* Mohamed Morsy, MD,*† Amjed Abu-Ghname, MD,* Chunfeng Zhao, MD,‡ and Brian T. Carlsen, MD* Introduction: Tissue loss as a consequence of congenital anomalies, trauma, ma- lignancy, or gangrene represents a major health care problem in the United States. Because younger individuals are disproportionately affected, the costs are magnified over time and the resultant individual and societal effects are tremendous. The currently available options to restore soft tissue defects are associated with donor site morbidities. Vascularized composite allotransplantation may provide form, function, and esthetics without a donor site; however, it comes with the significant risk associated with toxic immunosuppression (Biomaterials. 2015;61:246-256, Ann Plast Surg. 2015;75(1):112-116, Transplantation. 2009;88(2):203-210). Engineered tissues offer promise in finding viable alternatives to allograft and autologous tissues. In this study, we present our simple and quick method to decellularize a muscle without disrupting the vascular network integrity or the ex- tracellular matrix. Optimizing the decellularization process is a crucial step toward creating an “off-the-shelf ” flap that can be used for soft tissue reconstruction. Methods: The superficial gracilis muscle of 20 rats were harvested on their circulation and decellularized using perfusion with Krebs-Henseleit buffer and sodium dodecyl sulfate for 6 hours. These flaps were evaluated by gross morphology, histology, DNA quantification, integrity of the vascular network, scanning electron microscopy, and transmission electron microscopy. Results: All samples were decellularized successfully as determined by DNA content and histological analysis for cellular content. The vascular network was preserved in all samples. Conclusions: We present a quick, simple, and affordable method to decellularize a muscle flap through the vascular network. Our proposed method is efficient and can be completed in a significantly shorter time when compared with other methods. It is also safe and does not affect integrity of tissue, and this is essential for a reliable recellularization. Key Words: external fixator, free flaps, reconstruction (Ann Plast Surg 2019;83: 709–715) S oft tissue defects are common and typically result from accidents, cancer ablation, congenital defects, or reconstructive procedures. Despite the overwhelming need for medical and surgical interventions to improve outcomes after amputations and disabling tissue loss, the capacity to restore function remains limited. 1 Autologous tissue reconstruction in the form of local and free tissue transfer represents the predominant approach to dealing with such problems. This often fails to restore form, function, or esthetics. Furthermore, this approach comes with a significant cost to the patient, with surgical risk and donor site morbidity. Recently, the transfer of allogeneic tissue, such as hand and face transplantation, has become a clinical reality. Although vascularized composite allotransplantation may provide form, function, and esthetics without a donor site, it comes with the significant risk associated with toxic immunosuppressive medications. 2–4 Eliminating the need for immunosuppressive medications would revolutionize the fields of transplantation and reconstructive surgery and expand their applications. Tissue engineering provides us with the opportunity to create an autologous, bioartificial graft from patient derived cells. Such grafts would provide a great alternative to allogenic grafts because these grafts would overcome the classic drawbacks of tissue reconstruction: immu- nogenicity, quality, and quantity. 4 Despite the potential of tissue engi- neering to improve outcomes after tissue loss, the field has largely failed at the ultimate goal of tissue and organ replacement. Two important limitations have hampered progress and tempered enthusiasm for the field. The primary obstacle is the requirement for a blood supply to enable cells to survive within a 3-dimensional matrix. With rare ex- ceptions (ie, cartilage and cornea), cells will not grow and differentiate if they reside more than 200 μm from a blood capillary. 5–7 Conse- quently, tissue engineering in the conventional sense has been limited to very thin constructs whereby the cells obtain nutrients and eliminate waste by simple diffusion. The second obstacle is the requirement for the extracellular matrix (ECM) for maintenance of tissue function. The ECM is now recognized as a critical player in cell patterning, mi- gration, proliferation, differentiation, and function. Interestingly, these properties are tissue specific: ligands and growth factors within the ECM bind cell surface receptors and are critical for signaling and tro- phic stimulation. In addition, the ECM provides important spatial cues and facilitates mechanical signal transduction. 4,8 Tissue transfer represents an important tool in the armamentar- ium of the reconstructive surgeon. However, donor site morbidity and the limited availability of soft tissue that is suitable to be used to cover very large defects highlight the importance of finding a better alterna- tive. In pursuit of this goal, we have been working toward creating a vascularized composite tissue graft. We have previously demonstrated efficient and successful decellularization of a fasciocutaneous flap in a rat model with preservation of the vascular network and pedicle. 5 Although this approach holds promise, we were not able to achieve complete sterilization of this flap. Thus, we have directed our efforts to apply our model to a sterile flap. In our previous study, we suc- cessfully decellularized a normal epigastric flap from residual DNA content perspective. However, none of the decellularized flaps maintained physiologic vascular integrity. Recently, we conceived a novel approach to solve this problem and we were able to decellularize a gracilis flap without compromising the vascular net- work integrity or the ECM. Furthermore, we were able to achieve complete decellularization without exposing the tissue to excess amounts of detergents and, therefore, maintain the elasticity and in- tegrity of the ECM. In this study, we present our decellularization technique, which provided us with a model where the vascular Received March 21, 2019, and accepted for publication, after revision June 25, 2019. From the *Division of Plastic Surgery, Mayo Clinic, Rochester, MN; †Department of Orthopedic Surgery, Assiut University Hospital, Assiut, Egypt; and ‡Division of Orthopedic Research, Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN. Conflicts of interest and sources of funding: This study was supported by a generous grant from the Mayo Clinic Esam and Dalal Obaid Center for vascularized composite tissue allotransplantation. The authors declare no conflict of interest. Reprints: Brian T. Carlsen, MD, Division of Plastic Surgery, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail: Carlsen.Brian@Mayo.edu. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.annalsplasticsurgery.com). Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/19/8306–0709 DOI: 10.1097/SAP.0000000000002054 TRANSPLANTATION SURGERY AND RESEARCH Annals of Plastic Surgery • Volume 83, Number 6, December 2019 www.annalsplasticsurgery.com 709 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.