A Combination of Physical and Chemical Treatments Is More Effective in The Preparation of Acellular Uterine Scaffolds

Document Type : Original Article

Authors

1 Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

2 Department of Tissue Engineering, School of Medicine, Shahroud, University of Medical Sciences, Shahroud, Iran

3 Sexual Health and Fertility Research Center, University of Medical Sciences, Shahroud, Iran

Abstract

Objective:
Decellularized uterine scaffold, as a new achievement in tissue engineering, enables recellularization and
regeneration of uterine tissues and supports pregnancy in a fashion comparable to the intact uterus. The acellular
methods are methods preferred in many respects due to their similarity to normal tissue, so it is necessary to try to
introduce an acellularization protocol with minimum disadvantages and maximum advantages. Therefore, this study
aimed to compare different protocols to achieve the optimal uterus decellularization method for future in vitro and in
vivo bioengineering experiments.

Materials and Methods:
In this experimental study, rat uteri were decellularized by four different protocols (P) using
sodium dodecyl sulfate (SDS), with different doses and time incubations (P1 and P2), SDS/Triton-X100 sequentially
(P3), and a combination of physical (freeze/thaw) and chemical reagents (SDS/Triton X-100). The scaffolds were
examined by histopathological staining, DNA quantification, MTT assay, blood compatibility assay, FESEM, and
mechanical studies.

Results:
Histology assessment showed that only in P4, cell residues were completely removed. Masson’s trichrome
staining demonstrated that in P3, collagen fibers were decreased; however, no damage was observed in the collagen
bundles using other protocols. In indirect MTT assays, cell viabilities achieved by all used protocols were significantly
higher than the native samples. The percentage of red blood cell (RBC) hemolysis in the presence of prepared scaffolds
from all 4 protocols was less than 2%. The mechanical properties of none of the obtained scaffolds were significantly
different from the native sample except for P3.

Conclusion:
Uteri decellularized with a combination of physical and chemical treatments (P4) was the most favorable
treatment in our study with the complete removal of cell residue, preservation of the three-dimensional structure,
complete removal of detergents, and preservation of the mechanical property of the scaffolds.

Keywords

References

1. Johannesson L, Dahm-Kähler P, Eklind S, Brännström M. The future of human uterus transplantation. Women’s Health (Lond). 2014; 10(4): 455-467.
2. Jones BP, Saso S, Yazbek J, Thum MY, Quiroga I, Ghaem-Maghami S, et al. Uterine transplantation: scientific impact paper No. 65 April 2021. BJOG. 2021; 128(10): e51-e66.
3. Kuo CY, Baker H, Fries MH, Yoo JJ, Kim PCW, Fisher JP. Bioengineering strategies to treat female infertility. Tissue Eng Part B Rev. 2017; 23(3): 294-306.
4. Magalhaes RS, Williams JK, Yoo KW, Yoo JJ, Atala A. A tissueengineered uterus supports live births in rabbits. Nat Biotechnol. 2020; 38(11): 1280-1287.
5. Schutte SC, Taylor RN. A tissue-engineered human endometrial stroma that responds to cues for secretory differentiation, decidualization, and menstruation. Fertil Steril. 2012; 97(4): 997-1003.
6. House M, Sanchez CC, Rice WL, Socrate S, Kaplan DL. Cervical tissue engineering using silk scaffolds and human cervical cells. Tissue Eng Part A. 2010; 16(6): 2101-2112.
7. Campbell GR, Turnbull G, Xiang L, Haines M, Armstrong S, Rolfe BE, et al. The peritoneal cavity as a bioreactor for tissue engineering visceral organs: bladder, uterus, and vas deferens. J Tissue Eng Regen Med. 2008; 2(1): 50-60.8. Porzionato A, Stocco E, Barbon S, Grandi F, Macchi V, De Caro R. Tissue-engineered grafts from human decellularized extracellular
matrices: a systematic review and future perspectives. Int J Mol Sci. 2018; 19(12): 4117.
9. Ciampi O, Bonandrini B, Derosas M, Conti S, Rizzo P, Benedetti V, et al. Engineering the vasculature of decellularized rat kidney scaffolds using human induced pluripotent stem cell-derived endothelial cells. Sci Rep. 2019; 9(1): 8001.
10. 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): 213-221.
11. Negishi J, Funamoto S, Kimura T, Nam K, Higami T, Kishida A. Porcine radial artery decellularization by high hydrostatic pressure. J Tissue Eng Regen Med. 2015; 9(11): E144-E151.
12. Hashimoto Y, Funamoto S, Kimura T, Nam K, Fujisato T, Kishida A. The effect of decellularized bone/bone marrow produced by highhydrostatic pressurization on the osteogenic differentiation of mesenchymal stem cells. Biomaterials. 2011; 32(29): 7060-7067.
13. Cebotari S, Tudorache I, Ciubotaru A, Boethig D, Sarikouch S, Goerler A, et al. Use of fresh decellularized allografts for pulmonary valve replacement may reduce the reoperation rate in children and young adults: early report. Circulation. 2011;124 (11 Suppl): S115-S123.
14. Badylak SF. Decellularized allogeneic and xenogeneic tissue as a bioscaffold for regenerative medicine: factors that influence the host response. Ann Biomed Eng. 2014; 42(7): 1517-1527.
15. Daryabari SS, Kajbafzadeh AM, Fendereski K, Ghorbani F, Dehnavi M, Rostami M, et al. 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. J Assist Reprod Genet. 2019; 36(6): 1211-1223.
16. 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.
17. Keane TJ, Swinehart IT, Badylak SF. Methods of tissue decellularization used for the preparation of biologic scaffolds and in vivo relevance. Methods. 2015; 84: 25-34.
18. Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials. 2006; 27(19): 3675-3683.
19. Song M, Liu Y, Hui L. Preparation and characterization of acellular adipose tissue matrix using a combination of physical and chemical treatments. Mol Med Rep. 2018; 17(1): 138-146.
20. White LJ, Taylor AJ, Faulk DM, Keane TJ, Saldin LT, Reing JE, et al. The impact of detergents on the tissue decellularization process: a ToF-SIMS study. Acta Biomater. 2017; 50: 207-219.
21. Wolcott NS, Sit KK, Raimondi G, Hodges T, Shansky RM, Galea LAM, et al. Automated classification of estrous stage in rodents using deep learning. Sci Rep. 2022; 12: 17685.
22. Helmer KS, Cui Y, Chang L, Dewan A, Mercer DW. Effects of ketamine/xylazine on expression of tumor necrosis factor-alpha, inducible nitric oxide synthase, and cyclo-oxygenase-2 in rat gastric mucosa during endotoxemia. Shock. 2003; 20(1): 63-69.
23. Ichimura T, Kasai M, Imai K, Yamauchi M, Fukuda T, Yasui T, et al. A difficult to diagnose case of low‑grade endometrial stromal sarcoma with smooth muscle differentiation treated with laparoscopic surgery: a case report. Mol Clin Oncol. 2022; 16(4): 92.
24. Ozawa A, Sakaue M. New decolorization method produces more information from tissue sections stained with hematoxylin and eosin stain and masson-trichrome stain. Ann Anat. 2020; 227: 151431.
25. Mehdizadeh Kashi A, Tahemanesh K, Chaichian Sh, Joghataei MT, Moradi F, Tavangar SM, et al. How to prepare biological samples and live tissues for scanning electron microscopy (SEM). Galen Medical Journal. 2014; 3(2): 63-80.
26. Autian J. Biological model systems for the testing of the toxicity of biomaterials. In: Kronenthal RL, Oser Z, Martin E, editors. Polymer science and technology. Boston: Springer; 1975; 181-203.
27. Abdulghani S, Mitchell GR. Biomaterials for in situ tissue regeneration: a review. Biomolecules. 2019; 9(11): 750.
28. Campo H, Baptista PM, López-Pérez N, Faus A, Cervelló I, Simón C. De-and recellularization of the pig uterus: a bioengineering pilot study. Biol Reprod. 2017; 96(1): 34-45.
29. Gilpin A, Yang Y. Decellularization strategies for regenerative medicine: from processing techniques to applications. Biomed Res Int. 2017; 2017: 9831534.
30. Hellstrom M, Bandstein S, Brannstrom M. Uterine tissue engineering and the future of uterus transplantation. Ann Biomed Eng. 2017; 45(7): 1718-1730.
31. Zhang X, Chen X, Hong H, Hu R, Liu J, Liu C. Decellularized extracellular matrix scaffolds: recent trends and emerging strategies in tissue engineering. Bioact Mater. 2021; 10: 15-31.
32. Yao Q, Zheng YW, Lin HL, Lan QH, Huang ZW, Wang LF, et al. Exploiting crosslinked decellularized matrix to achieve uterus regeneration and construction. Artif Cells Nanomed Biotechnol. 2020; 48(1): 218-229.
33. Tiemann TT, Padma AM, Sehic E, Bäckdahl H, Oltean M, Song MJ, et al. Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation. Mol Hum Reprod. 2020; 26(3): 167-178.
34. Yu H, Chen Y, Kong H, He Q, Sun H, Bhugul PA, et al. The rat pancreatic body tail as a source of a novel extracellular matrix scaffold for endocrine pancreas bioengineering. J Biol Eng. 2018; 12: 6.
35. Faulk DM, Carruthers CA, Warner HJ, Kramer CR, Reing JE, Zhang L, et al. The effect of detergents on the basement membrane complex of a biologic scaffold material. Acta Biomater. 2014; 10(1): 183-193.
36. Momtahan N, Panahi T, Poornejad N, Stewart MG, Vance BR, Struk JA, et al. Using hemolysis as a novel method for assessment of cytotoxicity and blood compatibility of decellularized heart tissues. ASAIO J. 2016; 62(3): 340-348.
37. de la Maza A, Parra JL. Vesicle-micelle structural transitions of phospholipid bilayers and sodium dodecyl sulfate. Langmuir. 1995; 11(7): 2435-2441.
38. Hwang J, San BH, Turner NJ, White LJ, Faulk DM, Badylak SF, et al. Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide. Acta Biomater. 2017; 53: 268-278.
39. Andersen KK, Oliveira CL, Larsen KL, Poulsen FM, Callisen TH, Westh P, et al. The role of decorated SDS micelles in sub-CMC protein denaturation and association. J Mol Biol. 2009; 391(1): 207-226.
40. Nouri A, Hajian M, Monsefi M. Tissue engineering of mouse uterus using menstrual blood stem cells (MenSCs) and decellularized uterine scaffold. Stem Cell Res Ther. 2021; 12(1): 475.

Files

Cited by

Share

How to cite

Statistics