RSS-Feed abonnieren
DOI: 10.1055/s-0042-1760110
Automated Decellularization of the Rodent Epigastric Free Flap: A Comparison of Sodium Dodecyl Sulfate–Based Protocols
![](https://www.thieme-connect.de/media/jrm/202307/lookinside/thumbnails/10-1055-s-0042-1760110_22050158-1.jpg)
Abstract
Background 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.
Keywords
decellularization - vascularized tissue engineering - fasciocutaneous free flap - extracellular matrix - allograft* These authors contributed equally.
Publikationsverlauf
Eingereicht: 19. Mai 2022
Angenommen: 31. Oktober 2022
Artikel online veröffentlicht:
30. Dezember 2022
© 2022. Thieme. All rights reserved.
Thieme Medical Publishers, Inc.
333 Seventh Avenue, 18th Floor, New York, NY 10001, USA
-
References
- 1 Hallock GG. Complications of 100 consecutive local fasciocutaneous flaps. Plast Reconstr Surg 1991; 88 (02) 264-268
- 2 Buchanan PJ, Kung TA, Cederna PS. Evidence-based medicine: wound closure. Plast Reconstr Surg 2016; 138 (3, Suppl): 257S-270S
- 3 Mirzabeigi MN, Wilson AJ, Fischer JP. et al. Predicting and managing donor-site wound complications in abdominally based free flap breast reconstruction: improved outcomes with early reoperative closure. Plast Reconstr Surg 2015; 135 (01) 14-23
- 4 Hallock GG. Complications of the free-flap donor site from a community hospital perspective. J Reconstr Microsurg 1991; 7 (04) 331-334
- 5 Siemionow M, Ortak T, Izycki D. et al. Induction of tolerance in composite-tissue allografts. Transplantation 2002; 74 (09) 1211-1217
- 6 Kueckelhaus M, Fischer S, Seyda M. et al. Vascularized composite allotransplantation: current standards and novel approaches to prevent acute rejection and chronic allograft deterioration. Transpl Int 2016; 29 (06) 655-662
- 7 Ott HC, Clippinger B, Conrad C. et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med 2010; 16 (08) 927-933
- 8 Ott HC, Matthiesen TS, Goh SK. et al. Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Nat Med 2008; 14 (02) 213-221
- 9 Song JJ, Guyette JP, Gilpin SE, Gonzalez G, Vacanti JP, Ott HC. Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nat Med 2013; 19 (05) 646-651
- 10 Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials 2011; 32 (12) 3233-3243
- 11 Scarritt ME, Pashos NC, Bunnell BA. A review of cellularization strategies for tissue engineering of whole organs. Front Bioeng Biotechnol 2015; 3 (MAR): 43
- 12 Folkman J, Hochberg M. Self-regulation of growth in three dimensions. J Exp Med 1973; 138 (04) 745-753
- 13 Poornejad N, Buckmiller E, Schaumann L. et al. Re-epithelialization of whole porcine kidneys with renal epithelial cells. J Tissue Eng 2017; 8: 2041731417718809
- 14 Robertson MJ, Dries-Devlin JL, Kren SM, Burchfield JS, Taylor DA. Optimizing recellularization of whole decellularized heart extracellular matrix. PLoS One 2014; 9 (02) e90406
- 15 Lu TY, Lin B, Kim J. et al. Repopulation of decellularized mouse heart with human induced pluripotent stem cell-derived cardiovascular progenitor cells. Nat Commun 2013; 4: 2307
- 16 Montserrat N, Garreta E, Izpisua Belmonte JC. Regenerative strategies for kidney engineering. FEBS J 2016; 283 (18) 3303-3324
- 17 Gilpin SE, Ren X, Okamoto T. et al. Enhanced lung epithelial specification of human induced pluripotent stem cells on decellularized lung matrix. Ann Thorac Surg 2014; 98 (05) 1721-1729 , discussion 1729
- 18 Guyette JP, Gilpin SE, Charest JM, Tapias LF, Ren X, Ott HC. Perfusion decellularization of whole organs. Nat Protoc 2014; 9 (06) 1451-1468
- 19 Shandalov Y, Egozi D, Koffler J. et al. An engineered muscle flap for reconstruction of large soft tissue defects. Proc Natl Acad Sci U S A 2014; 111 (16) 6010-6015
- 20 Jank BJ, Goverman J, Guyette JP. et al. Creation of a bioengineered skin flap scaffold with a perfusable vascular pedicle. Tissue Eng Part A 2017; 23 (13-14): 696-707
- 21 Qu J, Van Hogezand RM, Zhao C, Kuo BJ, Carlsen BT. Decellularization of a fasciocutaneous flap for use as a perfusable scaffold. Ann Plast Surg 2015; 75 (01) 112-116
- 22 Zhang Q, Johnson JA, Dunne LW. et al. Decellularized skin/adipose tissue flap matrix for engineering vascularized composite soft tissue flaps. Acta Biomater 2016; 35: 166-184
- 23 Giatsidis G, Guyette JP, Ott HC, Orgill DP. Development of a large-volume human-derived adipose acellular allogenic flap by perfusion decellularization. Wound Repair Regen 2018; 26 (02) 245-250
- 24 Gilbert TW. Strategies for tissue and organ decellularization. J Cell Biochem 2012; 113 (07) 2217-2222
- 25 Maghsoudlou P, Georgiades F, Smith H. et al. Optimization of liver decellularization maintains extracellular matrix micro-architecture and composition predisposing to effective cell seeding. PLoS One 2016; 11 (05) e0155324
- 26 Price AP, Godin LM, Domek A. et al. Automated decellularization of intact, human-sized lungs for tissue engineering. Tissue Eng Part C Methods 2015; 21 (01) 94-103
- 27 Schilling BK, Lamberti KK, Snowden MJ. et al. Design and fabrication of an automatable, 3D printed perfusion device for tissue infusion and perfusion engineering. Tissue Eng Part A 2020; 26 (5-6): 253-264
- 28 Casal D, Pais D, Iria I. et al. A model of free tissue transfer: the rat epigastric free flap. J Vis Exp 2017; (119) 55281
- 29 Gerli MFM, Guyette JP, Evangelista-Leite D, Ghoshhajra BB, Ott HC. Perfusion decellularization of a human limb: a novel platform for composite tissue engineering and reconstructive surgery. PLoS One 2018; 13 (01) e0191497
- 30 Bae H, Puranik AS, Gauvin R. et al. Building vascular networks. Sci Transl Med 2012; 4 (160) 160ps23
- 31 Seetapun D, Ross JJ. Eliminating the organ transplant waiting list: the future with perfusion decellularized organs. Surgery 2017; 161 (06) 1474-1478
- 32 Kim IH, Ko IK, Atala A, Yoo JJ. Whole kidney engineering for clinical translation. Curr Opin Organ Transplant 2015; 20 (02) 165-170
- 33 Fu RH, Wang YC, Liu SP. et al. Decellularization and recellularization technologies in tissue engineering. Cell Transplant 2014; 23 (4-5): 621-630
- 34 Cui H, Chai Y, Yu Y. Progress in developing decellularized bioscaffolds for enhancing skin construction. J Biomed Mater Res A 2019; 107 (08) 1849-1859
- 35 Naik A, Griffin MF, Szarko M, Butler PE. Optimizing the decellularization process of human maxillofacial muscles for facial reconstruction using a detergent-only approach. J Tissue Eng Regen Med 2019; 13 (09) 1571-1580
- 36 Naik A, Griffin M, Szarko M, Butler PE. Optimizing the decellularization process of an upper limb skeletal muscle; implications for muscle tissue engineering. Artif Organs 2020; 44 (02) 178-183
- 37 Duisit J, Maistriaux L, Bertheuil N, Lellouch AG. Engineering vascularized composite tissues by perfusion decellularization/recellularization: review. Curr Transplant Rep 2021; 8 (02) 44-56
- 38 Bengur FB, Solari MG. Extracorporeal perfusion of vascularized composite tissues: the bridging role of an emerging technology in reconstructive microsurgery. Plast Reconstr Surg Glob Open 2022; 10 (10) e4578
- 39 Egro FM, Schilling BK, Fisher JD. et al. The future of microsurgery: vascularized composite allotransplantation and engineering vascularized tissue. J Hand Microsurg 2022; (e-pub ahead of print). DOI: 10.1055/s-0042-1757182.
- 40 Singer VL, Jones LJ, Yue ST, Haugland RP. Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal Biochem 1997; 249 (02) 228-238
- 41 Holden MJ, Haynes RJ, Rabb SA, Satija N, Yang K, Blasic Jr JR. Factors affecting quantification of total DNA by UV spectroscopy and PicoGreen fluorescence. J Agric Food Chem 2009; 57 (16) 7221-7226
- 42 Lupon E, Lellouch AG, Acun A. et al. Engineering vascularized composite allografts using natural scaffolds: a systematic review. Tissue Eng Part B Rev 2022; 28 (03) 677-693
- 43 Jank BJ, Xiong L, Moser PT. et al. Engineered composite tissue as a bioartificial limb graft. Biomaterials 2015; 61: 246-256
- 44 Duisit J, Maistriaux L, Taddeo A. et al. Bioengineering a human face graft: the matrix of identity. Ann Surg 2017; 266 (05) 754-764
- 45 Duisit J, Amiel H, Wüthrich T. et al. Perfusion-decellularization of human ear grafts enables ECM-based scaffolds for auricular vascularized composite tissue engineering. Acta Biomater 2018; 73: 339-354