Joint Department of Biomedical Engineering, North Carolina State and University of North Carolina-Chapel Hill, Raleigh, NC, United States of America.
Comparative Medicine Institute, North Carolina State University, Raleigh, NC, United States of America.
Biomed Mater. 2021 Nov 26;17(1). doi: 10.1088/1748-605X/ac361f.
Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g. DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the standardization, cost-effectiveness, and scale-up process. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization systems to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor's effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allow for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulationsuggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.
细胞外基质（ECM）是一种由生物活性分子组成的复杂结构，代表着局部组织微环境。脱细胞 ECM 生物材料利用这些生物分子应用于再生医学。一种潜在的治疗应用是使用特定于声带（VF）的 ECM 来修复损伤后的 VF。ECM 支架是通过脱细胞过程衍生而来的，该过程旨在去除不需要的免疫原性生物分子（例如 DNA），同时保留 ECM 的组成。脱细胞的有效性通常在最后通过量化 ECM 属性来评估，例如最终 dsDNA 含量。然而，ECM 制造的批间变异性仍然是标准化、成本效益和扩大规模过程的一个重大挑战。用于过程控制的工具数量有限，严重限制了揭示脱细胞过程参数与 ECM 属性之间的相关性。在这项研究中，我们开发了一种适用于经典批量方法和半连续脱细胞化系统的技术，以实时追踪两种喉部组织的脱细胞化过程。我们假设监测生物反应器的 260nm 处的流出物吸光度随时间的变化将提供组织中 DNA 释放的代表性图谱，从而允许进行过程优化。获得了喉部组织的 DNA 释放图谱，并成功地用于优化 VF 固有层-ECM（auVF-ECM）水凝胶的衍生。该水凝胶具有与常用生物材料相当的流变特性，可用于治疗 VF 损伤。此外，auVF-ECM 水凝胶在脂多糖刺激下促进了 THP-1 巨噬细胞中 CCR7 的下调，表明具有一些抗炎特性。结果表明，吸光度图谱是脱细胞化过程中 DNA 去除的良好代表，因此提供了优化未来方案的重要工具。
