Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University Bratislava 811 08, Slovakia.
National Institute of Rheumatic Diseases, Piestany 921 12, Slovakia.
Exp Biol Med (Maywood). 2023 Jun;248(12):1034-1042. doi: 10.1177/15353702231162092. Epub 2023 Apr 18.
Recently, several scaffolds have been introduced for urethral tissue engineering. However, acellular human urethral scaffold harvested from deceased donors may provide significant advantages compared to synthetic, composite, or other biological scaffolds. This study aims to develop the protocol for decellularization of the human urethra that preserves substantial extracellular matrix (ECM) components, which are essential for subsequent recellularization mimicking the natural environment of the native ECM. A total of 12 human urethras were harvested from deceased donors. An equal part of every harvested urethra was used as a control sample for analyses. The protocol design was based on the enzyme-detergent-enzyme method. Trypsin and Triton X-100 were used to remove cells, followed by DNase treatment to remove DNA residues. Subsequently, the specimens were continually rinsed in deionized water for seven days. The efficiency of decellularization was determined by histochemistry, immunohistochemical staining, scanning electron microscopy (SEM), and DNA quantification. Histological analysis confirmed cell removal and preservation of urethral structure after decellularization. The preservation of collagen IV and fibronectin was confirmed by histologic examination and immunohistochemical staining. SEM confirmed the maintenance of the ultrastructural architecture of ECM and fibers. DNA content in decellularized urethra was significantly lower compared to the native sample ( < 0.001), and so the criteria for decellularized tissue were met. Cytotoxicity analysis data showed that the matrix-conditioned medium did not contain soluble toxins and had no significant inhibitory effect on cell proliferation, providing evidence that the decellularized samples are not toxic. This study demonstrates the feasibility of the enzyme-detergent-enzyme-based decellularization protocol for removing cellular components and maintaining urethral ECM and its ultrastructure. Moreover, obtained results provide solid ground for recellularization and urethral tissue engineering, which will follow.
最近，已经有几种支架被引入到尿道组织工程中。然而，与合成的、复合的或其他生物支架相比，从已故供体中获取的无细胞人尿道支架可能具有显著优势。本研究旨在开发一种脱细胞人尿道的方案，该方案可以保留大量的细胞外基质（ECM）成分，这些成分对于随后的再细胞化过程至关重要，因为它可以模拟天然 ECM 的环境。共采集了 12 个人的尿道，每个采集的尿道的等分部分被用作分析的对照样本。方案设计基于酶-去污剂-酶方法。使用胰蛋白酶和 Triton X-100 去除细胞，然后使用 DNAse 处理去除 DNA 残留。随后，将标本在去离子水中连续冲洗 7 天。通过组织化学、免疫组织化学染色、扫描电子显微镜（SEM）和 DNA 定量来确定脱细胞效率。组织学分析证实了脱细胞后细胞的去除和尿道结构的保留。组织学检查和免疫组织化学染色证实了胶原 IV 和纤维连接蛋白的保留。SEM 证实了 ECM 和纤维的超微结构的保持。与天然样本相比，脱细胞尿道中的 DNA 含量明显降低（<0.001），因此达到了脱细胞组织的标准。细胞毒性分析数据表明，基质条件培养基不含有可溶性毒素，对细胞增殖没有显著的抑制作用，这证明了脱细胞样本没有毒性。本研究证明了基于酶-去污剂-酶的脱细胞方案去除细胞成分和保持尿道 ECM 及其超微结构的可行性。此外，获得的结果为随后的再细胞化和尿道组织工程提供了坚实的基础。
