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Fabrication of testicular decellularized scaffolds using a hybrid SDS-supercritical CO₂ method: process optimization,
Mojtaba Rastgou-Maeini1,2, Ali Zeinolabedini Hezave3,4, Seyed Hadi Anjamrooz1
1Tissue Engineering Lab., Anatomy Department, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Background:
Infertility is a serious health issue that has social and economic impacts on families. The construction of a bioengineered testis as an alternative solution can enhance infertility treatment processes. The principles of tissue engineering are derived from the natural reconstruction and repair of tissues, where scaffolds, alongside cells and signaling molecules, play a key role. Therefore, the aim of this study is to prepare a decellularized testicular scaffold from mouse tissue based on a supercritical carbon dioxide protocol and to investigate its histological properties, cell viability, and compatibility.
Methods:
The whole rat testis tissue was decellularized using 1% SDS as pretreatment, Ethanol 70% as Co-solvent and Supercritical Carbon Dioxide. Optimal acellular removal was confirmed through DNA content analysis and staining with Hematoxylin-Eosin. The preservation of extracellular matrix structures was evaluated through glycosaminoglycans (GAGs) extraction and histological staining techniques, including periodic acid-Schiff, aldehyde fuchsin, Alcian blue, Masson's trichrome, and Scanning electron microscopy (SEM). Additionally, the cytotoxicity of the scaffold was assessed using the MTT assay.
Results:
The optimal protocol for decellularization was determined to involve a combination of 1% SDS for 8 h and Supercritical Carbon Dioxide at parameters of 200 bar pressure, 45 °C, and a duration of 5 h. Evidence provided by quantitative and qualitative assessments of DNA, GAGs, SEM, and histochemical analyses confirmed effective cellular removal while adequately preserving the extracellular matrix of the rat testis post-decellularization. Additionally, the biocompatibility of the prepared scaffold was established through MTT assay, indicating favorable biological compatibility.
Conclusion:
Despite the fact that the supercritical carbon dioxide protocol alone was unable to produce a decellularized scaffold with the desired standard characteristics, its combined use with 1% SDS not only reduced the decellularization time compared to other methods but also effectively preserved the extracellular matrix structures.
