Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts, USA.
Department of Biomedical Engineering, Bucknell University, Lewisburg, Pennsylvania, USA.
Tissue Eng Part C Methods. 2020 Jun;26(6):317-331. doi: 10.1089/ten.TEC.2020.0083. Epub 2020 Jun 9.
Horseradish peroxidase (HRP) has been investigated as a catalyst to crosslink tissue-engineered hydrogels because of its mild reaction conditions and ability to modulate the mechanical properties of the matrix. Here, we report the results of the first study investigating the use of HRP to crosslink fibrin scaffolds. We examined the effect of varying HRP and hydrogen peroxide (HO) incorporation strategies on the resulting crosslink density and structural properties of fibrin in a microthread scaffold format. Primary (1°) and secondary (2°) scaffold modification techniques were evaluated to crosslink fibrin microthread scaffolds. A primary scaffold modification technique was defined as incorporating crosslinking agents into the microthread precursor solutions during extrusion. A secondary scaffold modification technique was defined as incubating the microthreads in a postprocessing crosslinker bath. Fibrin microthreads were enzymatically crosslinked through primary, secondary, or a combination of both approaches. All fibrin microthread scaffolds crosslinked with HRP and HO via primary and/or secondary methods exhibited an increase in dityrosine crosslink density compared with uncrosslinked control microthreads, demonstrated by scaffold fluorescence. Fourier transform infrared spectroscopy indicated the formation of isodityrosine bonds in 1° HRP crosslinked microthreads. Characterization of tensile mechanical properties revealed that all HRP crosslinked microthreads were significantly stronger than control microthreads. Primary (1°) HRP crosslinked microthreads also demonstrated significantly slower degradation than control microthreads, suggesting that incorporating HRP and HO during extrusion yields scaffolds with increased resistance to proteolytic degradation. Finally, cells seeded on HRP crosslinked microthreads retained a high degree of viability, demonstrating that HRP crosslinking yields biocompatible scaffolds that are suitable for tissue engineering. The goal of this work was to facilitate the logical design of enzymatically crosslinked fibrin microthreads with tunable structural properties, enabling their application for engineered tissue constructs with varied mechanical and structural properties.
辣根过氧化物酶（HRP）已被研究作为交联组织工程水凝胶的催化剂，因为其具有温和的反应条件和调节基质机械性能的能力。在这里，我们报告了首次研究使用 HRP 交联纤维蛋白支架的结果。我们研究了改变 HRP 和过氧化氢（HO）掺入策略对纤维蛋白在微丝支架形式中的交联密度和结构特性的影响。评估了主要（1°）和次要（2°）支架修饰技术以交联纤维蛋白微丝支架。主要支架修饰技术定义为在挤出过程中将交联剂掺入微丝前体溶液中。次要支架修饰技术定义为在微丝后处理交联剂浴中孵育微丝。通过主要、次要或两者的组合方法对纤维蛋白微丝进行酶促交联。通过 HRP 和 HO 进行交联的所有纤维蛋白微丝支架（通过主要和/或次要方法）与未交联的对照微丝相比，显示出二酪氨酸交联密度增加，这通过支架荧光证实。傅里叶变换红外光谱表明在 1°HRP 交联的微丝中形成了同二酪氨酸键。拉伸力学性能的表征表明，所有 HRP 交联的微丝均明显强于对照微丝。1°HRP 交联的微丝也表现出比对照微丝明显更慢的降解，这表明在挤出过程中掺入 HRP 和 HO 可产生对蛋白水解降解具有更高抵抗力的支架。最后，接种在 HRP 交联微丝上的细胞保持了高度的活力，这表明 HRP 交联产生了生物相容性的支架，适用于具有不同机械和结构特性的组织工程。这项工作的目的是促进具有可调结构特性的酶交联纤维蛋白微丝的逻辑设计，使其能够应用于具有不同机械和结构特性的工程化组织构建体。
