Development of an efficient perfusion-based protocol for whole-organ decellularization of the ovine uterus as a human-sized model and in vivo application of the bioscaffolds
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The main purpose of this investigation was to determine an efficient whole-organ decellularization protocol of a human-sized uterus and evaluate the in vivo properties of the bioscaffold.
Twenty-four ovine uteri were included in this investigation and were decellularized by three different protocols (n 6). We performed histopathological and immunohistochemical evaluations, 4,6-diamidino-2-phenylindole (DAPI) staining, DNA quantification, MTT assay, scanning electron microscopy, biomechanical studies, and CT angiography to characterize the scaffolds. The optimized protocol was determined, and patches were grafted into the uterine horns of eight female Wistar rats. The grafts were extracted after 10 days; the opposite horns were harvested to be evaluated as controls.
Protocol III (perfusion with 0.25% and 0.5% SDS solution and preservation in 10% formalin) was determined as the optimized method with efficient removal of the cellular components while preserving the extracellular matrix. Also, the bioscaffolds demonstrated native-like biomechanical, structural, and vascular properties. Histological and immunohistochemical evaluations of the harvested grafts confirmed the biocompatibility and recellularization potential of bioscaffolds. Also, the grafts demonstrated higher positive reaction for CD31 and Ki67 markers compared with the control samples which indicated eminent angiogenesis properties and proliferative capacity of the implanted tissues.
This investigation introduces an optimized protocol for whole-organ decellularization of the human-sized uterus with native-like characteristics and a prominent potential for regeneration and angiogenesis which could be employed in in vitro and in vivo studies. To the best of our knowledge, this is the first study to report biomechanical properties and angiographic evaluations of a large animal uterine scaffold.
KeywordsUterus Infertility Tissue engineering Regeneration Bioscaffold
We would like to express our sincere gratitude to Dr. Torabi for providing the organs and Mr. Reza Esmaili and Mr. Nourbakhsh for their kind cooperation during this project.
This study was funded by Tehran University of Medical Sciences (grant number 96-03-30-36497).
Compliance with ethical standards
All the animal procedures were approved by The Animal Ethics Committee of the Tehran University of Medical Sciences, School of Medicine and Education Section of Basic Sciences and were performed in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals.
Conflict of interest
The authors declare that they have no conflict of interest.
- 2.Kisu I, Mihara M, Banno K, Umene K, Araki J, Hara H, et al. Risks for donors in uterus transplantation. Reprod Sci. 2013 Dec;20(12):1406–15. https://doi.org/10.1177/1933719113493517.
- 4.Brännström M, Johannesson L, Bokström H, Kvarnström N, Mölne J, Dahm-Kähler P, et al. Live birth after uterus transplantation. Lancet. 2015;385:607–16.Google Scholar
- 5.Mats Brännström. Uterus transplantation and beyond. J Mater Sci Mater Med (2017) 28:70DOI https://doi.org/10.1007/s10856-017-5872-0, 70.
- 6.Hellström M, Bandstein S, Brännström M. Uterine tissue engineering and the future of uterus transplantation. Ann Biomed Eng. 2017 Jul;45(7):1718–30 Epub 2016 Dec 19.Google Scholar
- 9.Santoso EG, Yoshida K, Hirota Y, Aizawa M, Yoshino O, Kishida A, et al. Application of detergents or high hydrostatic pressure as decellularization processes in uterine tissues and their subsequent effects on in vivo uterine regeneration in murine models. PLoS One. 2014;9(7):e103201.Google Scholar
- 10.Hiraoka T, Hirota Y, Saito-Fujita T, Matsuo M, Egashira M, Matsumoto L, et al. STAT3 accelerates uterine epithelial regeneration in a mouse model of decellularized uterine matrix transplantation. JCI insight. 2016;1(8).Google Scholar
- 13.Hellström M, Moreno-Moya JM, Bandstein S, Bom E, Akouri RR, Miyazaki K, et al. Bioengineered uterine tissue supports pregnancy in a rat model. Fertil Steril. 2016;106(2):487–96 e1.Google Scholar
- 15.Barakat O, Abbasi S, Rodriguez G, Rios J, Wood RP, Ozaki C, et al. Use of decellularized porcine liver for engineering humanized liver organ. J Surg Res. 2012;173(1):e11–25.Google Scholar
- 16.Hashemi J, Pasalar P, Soleimani M, Khorramirouz R, Fendereski K, Enderami SE, et al. Application of a novel bioreactor for in vivo engineering of pancreas tissue. J Cell Physiol. 2018;233(5):3805–16.Google Scholar
- 17.Kajbafzadeh AM, Khorramirouz R, Kameli SM, Fendereski K, Daryabari SS, Tavangar SM, et al. Three-year efficacy and patency follow-up of decellularized human internal mammary artery as a novel vascular graft in animal models. J Thorac Cardiovasc Surg. 2019;157(4):1494–502.Google Scholar
- 19.Kitahara H, Yagi H, Tajima K, Okamoto K, Yoshitake A, Aeba R, et al. Heterotopic transplantation of a decellularized and recellularized whole porcine heart. Interact Cardiovasc Thorac Surg. 2016;22(5):571–9.Google Scholar
- 21.Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med. 2008;14(2):213–21.Google Scholar
- 22.Myers KM, Elad D. Biomechanics of the human uterus. Wiley Interdiscip Rev Syst Biol Med. 2017;9(5).Google Scholar
- 23.Bhrany AD, Lien CJ, Beckstead BL, Futran ND, Muni NH, Giachelli CM, et al. Crosslinking of an oesophagus acellular matrix tissue scaffold. J Tissue Eng Regen Med. 2008;2(6):365–72.Google Scholar
- 25.Williams C, Liao J, Joyce EM, Wang B, Leach JB, Sacks MS, et al. Altered structural and mechanical properties in decellularized rabbit carotid arteries. Acta Biomater. 2009;5(4):993–1005.Google Scholar
- 27.Cebotari S, Tudorache I, Jaekel T, Hilfiker A, Dorfman S, Ternes W, et al. Detergent decellularization of heart valves for tissue engineering: toxicological effects of residual detergents on human endothelial cells. Artif Organs. 2010;34(3):206–10.Google Scholar