Otolaryngology - Head and Neck Surgery, Biomedical Engineering, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9035, USA.
Tissue Eng Part A. 2009 Nov;15(11):3537-46. doi: 10.1089/ten.TEA.2009.0064.
This study investigated the potential of the decellularized human umbilical vein (HUV) as an allogeneic, acellular extracellular matrix (ECM) scaffold for engineering the vocal fold lamina propria in vitro. HUV specimens with Wharton's jelly on the abluminal surface were uniformly dissected from native umbilical cords using an automated procedure and subjected to a novel saline-based decellularization treatment for removal of potentially antigenic epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the resulting acellular constructs and cultured for 21 days. The structures of decellularized and fibroblast-repopulated HUV constructs and the attachment, proliferation, and infiltration of fibroblasts were examined with light microscopy and scanning electron microscopy. Changes in the relative densities of collagen in the constructs associated with decellularization and recellularization were quantified using digital image analysis. In addition, fibroblasts infiltrating the scaffolds were released by cell recovery and quantified by counting. Viscoelastic properties of the scaffolds were measured using a linear, simple-shear rheometer at phonatory frequencies. Results showed that an acellular ECM construct with an intact three-dimensional structure of Wharton's jelly was fabricated. Vocal fold fibroblasts readily attached on the abluminal surface of the construct with high viability, with significant cellular infiltration up to approximately 600 microm deep into the construct. A significant increase in collagen expression was observed with recellularization. The elastic modulus and dynamic viscosity of the fibroblast-repopulated scaffolds were comparable to those of the human vocal fold lamina propria. These findings supported the potential of the construct as a possible surgical allograft for vocal fold restoration and reconstruction.
本研究探讨了脱细胞人脐静脉（HUV）作为同种异体、无细胞细胞外基质（ECM）支架的潜力，用于体外工程化喉声带固有层。使用自动化程序从天然脐带中均匀分离出具有 Wharton 胶的 HUV 标本，并对其进行新型盐水基脱细胞处理，以去除潜在的抗原表位。从原代培养的人喉成纤维细胞接种到所得的无细胞构建体上，并培养 21 天。用光镜和扫描电子显微镜检查脱细胞和纤维细胞再填充的 HUV 构建体的结构以及纤维细胞的附着、增殖和渗透。使用数字图像分析定量测量与脱细胞和再细胞化相关的构建体中胶原的相对密度变化。此外，通过细胞回收释放渗透到支架中的成纤维细胞并通过计数进行量化。使用线性简单剪切流变仪在发音频率下测量支架的粘弹性特性。结果表明，制造出具有完整三维 Wharton 胶结构的无细胞 ECM 构建体。喉成纤维细胞在构建体的无细胞表面上容易附着，具有高活力，细胞渗透深度可达约 600 微米。再细胞化后观察到胶原表达显著增加。纤维细胞再填充支架的弹性模量和动态粘度与人类声带固有层相当。这些发现支持了该构建体作为可能的声带修复和重建手术同种异体移植物的潜力。
