Xu Chet C, Chan Roger W, Tirunagari Neeraj
Graduate Program in Biomedical Engineering, University of Texas Southwestern Medical Center, Dallas, TX 75390-9035, USA.
Tissue Eng. 2007 Mar;13(3):551-66. doi: 10.1089/ten.2006.0169.
A novel method for preparing an acellular xenogeneic extracellular matrix scaffold for tissue engineering was developed. Bovine vocal fold lamina propria specimens were treated with high-concentration sodium chloride, nucleic acid digestion, and ethanol dehydration for decellularization and removal of immunogenic foreign epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the acellular scaffolds and cultured for 21 days. The decellularized and the recellularized scaffolds were examined by light microscopy, fluorescent microscopy, and scanning electron microscopy. Collagen synthesis and release by fibroblasts were quantified by the Sircol assay, whereas the synthesis and release of hyaluronic acid, decorin, and fibronectin were assessed by enzyme-linked immunosorbent assays. Viscoelastic shear properties of the scaffolds were quantified by a simple-shear rheometer at frequencies of up to 250 Hz. Preliminary results showed that a biodegradable, acellular extracellular matrix scaffold with an intact basement membrane and 3-dimensional structure of the matrix proteins was engineered. Vocal fold fibroblasts readily attached to and infiltrated the scaffold with high viability and active protein synthesis, demonstrating the biocompatibility. The elastic shear modulus and dynamic viscosity of the acellular scaffold and the fibroblast-repopulated scaffold were comparable to those of the human vocal fold cover. These findings support the potential of the scaffold as a xenograft for vocal fold reconstruction and regeneration.
开发了一种用于组织工程的新型脱细胞异种细胞外基质支架制备方法。对牛声带固有层标本进行高浓度氯化钠处理、核酸消化和乙醇脱水，以实现脱细胞并去除免疫原性外来表位。将原代培养的人声带成纤维细胞接种到脱细胞支架上并培养21天。通过光学显微镜、荧光显微镜和扫描电子显微镜对脱细胞和再细胞化的支架进行检查。通过Sircol分析法对成纤维细胞的胶原蛋白合成和释放进行定量，而通过酶联免疫吸附测定法评估透明质酸、核心蛋白聚糖和纤连蛋白的合成和释放。通过简单剪切流变仪在高达250Hz的频率下对支架的粘弹性剪切特性进行定量。初步结果表明，构建了一种具有完整基底膜和基质蛋白三维结构的可生物降解脱细胞细胞外基质支架。声带成纤维细胞易于附着并以高活力和活跃的蛋白质合成能力浸润支架，证明了其生物相容性。脱细胞支架和再细胞化支架的弹性剪切模量和动态粘度与人声带覆盖层的相当。这些发现支持了该支架作为声带重建和再生异种移植物的潜力。
