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 (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 H₂O₂ 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 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 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.

中文翻译：

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辣根过氧化物酶催化纤维蛋白微丝支架的交联。


辣根过氧化物酶（HRP）因其温和的反应条件和调节基质机械性能的能力而被研究作为交联组织工程水凝胶的催化剂。在这里，我们报告了第一项研究的结果，该研究调查了使用 HRP 交联纤维蛋白支架。我们研究了不同的 HRP 和过氧化氢 (H ₂ O ₂ ) 掺入策略对微线支架形式的纤维蛋白的交联密度和结构特性的影响。评估了初级（1°）和次级（2°）支架修饰技术以交联纤维蛋白微丝支架。主要的支架改性技术被定义为在挤出过程中将交联剂掺入微丝前体溶液中。二次支架修饰技术被定义为在后处理交联剂浴中孵育微线。纤维蛋白微丝通过初级、次级或两种方法的组合进行酶促交联。通过支架荧光证明，与未交联的对照微线相比，通过主要和/或次要方法与HRP和H ₂ O ₂交联的所有纤维蛋白微线支架表现出二酪氨酸交联密度的增加。傅里叶变换红外光谱表明在 1° HRP 交联微丝中形成了异二酪氨酸键。拉伸机械性能的表征表明，所有 HRP 交联的微线均明显强于对照微线。 初级（1°）HRP 交联微线还表现出比对照微线明显更慢的降解，这表明在挤出过程中掺入HRP 和H ₂ O ₂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，证明 HRP 交联产生适合组织工程的生物相容性支架。这项工作的目标是促进具有可调结构特性的酶交联纤维蛋白微线的逻辑设计，使其能够应用于具有不同机械和结构特性的工程组织结构。