Masoomikarimi Masoomeh, Salehi Majid, Noorbakhsh Farshid, Rajaei Samira
Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Department of Tissue Engineering, School of Medicine, Shahroud, University of Medical Sciences, Shahroud, Iran. Email:
Cell J. 2023 Jan 1;25(1):25-34. doi: 10.22074/cellj.2022.8396.
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.
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.
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.
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.
去细胞子宫支架作为组织工程学的一项新成果，能够使子宫组织重新细胞化和再生，并以与完整子宫相当的方式支持妊娠。去细胞方法在许多方面因其与正常组织的相似性而成为首选方法，因此有必要尝试引入一种缺点最少、优点最多的去细胞方案。因此，本研究旨在比较不同方案，以获得未来体外和体内生物工程实验的最佳子宫去细胞方法。
在本实验研究中，使用四种不同方案（P）对大鼠子宫进行去细胞处理，方案分别为：使用不同剂量和孵育时间的十二烷基硫酸钠（SDS）（P1和P2）；依次使用SDS/Triton-X100（P3）；以及物理（冻融）和化学试剂（SDS/Triton X-100）的组合（P4）。通过组织病理学染色、DNA定量、MTT分析、血液相容性分析、场发射扫描电子显微镜（FESEM）和力学研究对支架进行检测。
组织学评估表明，只有在P4方案中细胞残余物被完全去除。Masson三色染色显示，在P3方案中胶原纤维减少；然而，使用其他方案时未观察到胶原束受损。在间接MTT分析中，所有使用方案获得的细胞活力均显著高于天然样本。在所有4种方案制备的支架存在的情况下，红细胞（RBC）溶血百分比均小于2%。除P3外，所获得的支架的力学性能与天然样本均无显著差异。
在我们的研究中，采用物理和化学处理相结合的方法（P4）去细胞的子宫是最有利的处理方式，其能完全去除细胞残余物，保留三维结构，完全去除去污剂，并保留支架的力学性能。
