Department of Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo 05508-270, Brazil.
Department of Physics, State University of Maringá, Maringá 87020-900, Brazil.
Cells. 2023 Jul 15;12(14):1864. doi: 10.3390/cells12141864.
The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine. Recently, biomimetic ovarian tissue reconstruction was proposed to reestablish ovarian endocrine functions. Despite many decellularization methods proposed, there is no established protocol for whole ovaries by detergent perfusion that is able to preserve tissue macro and microstructure with higher efficiency. This generated biomaterial may have the potential to be applied for other purposes beyond reproduction and be translated to other areas in the tissue engineering field. Therefore, this study aimed to establish and standardize a protocol for porcine ovaries' decellularization based on detergent perfusion and ultrasonication to obtain functional whole-ovary scaffolds. For that, porcine ovaries ( = 5) were perfused with detergents (0.5% SDS and 1% Triton X-100) and submitted to an ultrasonication bath to produce acellular scaffolds. The decellularization efficiency was evaluated by DAPI staining and total genomic DNA quantification. ECM morphological evaluation was performed by histological, immunohistochemistry, and ultrastructural analyses. ECM physico-chemical composition was evaluated using FTIR and Raman spectroscopy. A cytocompatibility and cell adhesion assay using murine fibroblasts was performed. Results showed that the proposed method was able to remove cellular components efficiently. There was no significant ECM component loss in relation to native tissue, and the scaffolds were cytocompatible and allowed cell attachment. In conclusion, the proposed decellularization protocol produced whole-ovaries scaffolds with preserved ECM composition and great potential for application in tissue engineering.
脱细胞支架在人工组织重建中的应用是再生医学中一种具有巨大治疗潜力的方法。最近，仿生卵巢组织重建被提出以重建卵巢内分泌功能。尽管已经提出了许多脱细胞方法，但目前还没有一种能够以更高效率保留组织宏观和微观结构的去污剂灌注全卵巢脱细胞方法。这种生物材料可能具有超越生殖功能的潜在应用，并可以转化为组织工程领域的其他领域。因此，本研究旨在建立和标准化一种基于去污剂灌注和超声处理的猪卵巢脱细胞方法，以获得功能性全卵巢支架。为此，用去污剂（0.5%SDS 和 1%Triton X-100）灌注猪卵巢（=5），并进行超声处理以产生脱细胞支架。通过 DAPI 染色和总基因组 DNA 定量评估脱细胞效率。通过组织学、免疫组织化学和超微结构分析评估 ECM 形态。使用傅里叶变换红外光谱（FTIR）和拉曼光谱评估 ECM 理化组成。使用鼠成纤维细胞进行细胞相容性和细胞黏附试验。结果表明，所提出的方法能够有效地去除细胞成分。与天然组织相比，ECM 成分没有明显损失，支架具有细胞相容性并允许细胞附着。总之，所提出的脱细胞方案产生了具有保留 ECM 组成的全卵巢支架，具有在组织工程中应用的巨大潜力。
