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 (H2O2) incorporation strategies on the resulting crosslink density and structural properties of fibrin in a microthread scaffold format. Primary (1º) and secondary (2º) 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 post-processing crosslinker bath. Fibrin microthreads were enzymatically crosslinked through primary, secondary, or a combination of both approaches. All fibrin microthread scaffolds crosslinked with HRP and H2O2 via primary and/or secondary methods exhibited an increase in dityrosine crosslink density compared to uncrosslinked control microthreads, demonstrated by scaffold fluorescence. Fourier transform infrared spectroscopy indicated the formation of isodityrosine bonds in 1º HRP crosslinked microthreads. Characterization of tensile mechanical properties revealed that all HRP crosslinked microthreads were significantly stronger than control microthreads. Primary (1º) HRP crosslinked microthreads also demonstrated significantly slower degradation than control microthreads, suggesting that incorporating HRP and H2O2 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 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.

中文翻译：

---

辣根过氧化物酶催化的纤维蛋白微丝支架交联

辣根过氧化物酶（HRP）由于其温和的反应条件和调节基质机械性能的能力，已被研究用作交联组织工程水凝胶的催化剂。在这里，我们报告第一项研究结果的调查，该研究使用HRP交联血纤蛋白支架。我们检查了不同的HRP和过氧化氢（H2O2）掺入策略对所产生的微丝支架形式的纤维蛋白的交联密度和结构特性的影响。评价了主要（1º）和次要（2º）支架修饰技术以交联纤维蛋白微螺纹支架。主要的支架改性技术定义为在挤出过程中将交联剂掺入微丝前体溶液中。辅助支架改性技术定义为在后处理交联剂浴中孵育微丝。纤维蛋白微丝通过主要，次要或两种方法的组合酶促交联。与未交联的对照微丝相比，通过一级和/或二级方法与HRP和H2O2交联的所有血纤蛋白微丝支架均显示二硬脂酸交联密度的增加，这通过支架荧光显示。傅里叶变换红外光谱表明在1ºHRP交联的微丝中形成了异丁氨酸键。拉伸机械性能的表征表明，所有HRP交联的微螺纹均明显强于对照微螺纹。HRP交联的主（1º）微丝也显示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H2O2产生的支架对蛋白水解降解的抵抗力增强。最后，接种在HRP交联微线上的细胞保留了高度的活力，这表明HRP交联产生了适合组织工程的生物相容性支架。这项工作的目的是促进具有可调节结构特性的酶促交联纤维蛋白微线的逻辑设计，使其能够用于具有不同机械和结构特性的工程组织构造。接种在HRP交联微线上的细胞保留了高度的活力，表明HRP交联产生了适合组织工程的生物相容性支架。这项工作的目的是促进具有可调节结构特性的酶促交联纤维蛋白微线的逻辑设计，使其能够用于具有不同机械和结构特性的工程组织构造。接种在HRP交联微线上的细胞保留了高度的活力，表明HRP交联产生了适合组织工程的生物相容性支架。这项工作的目的是促进具有可调节结构特性的酶促交联纤维蛋白微线的逻辑设计，使其能够用于具有不同机械和结构特性的工程组织构造。