Horseradish Peroxidase-Catalyzed Crosslinking of Fibrin Microthread Scaffolds

Abstract

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 (H 2 O 2 ) 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 H 2 O 2 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 H 2 O 2 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.

Engineering sulfated polysaccharides and silk fibroin based injectable IPN hydrogels with stiffening and growth factor presentation abilities for cartilage tissue engineering

“…Moreover, enzymatic crosslinking is highly tunable and cyto-compatible and therefore, has been used widely for many tissue engineering applications. 39,40…”

“…Moreover, enzymatic crosslinking is highly tunable and cyto-compatible and therefore, has been used widely for many tissue engineering applications. 39,40 Although the presence of CMC in scaffolds is known to provide resilience, it lacks the ability to bind to growth factors. Therefore, to enable growth factor binding ability and its longterm presentation to cells, CMC was subjected to chemical sulfation to obtain sulfated-CMC (s-CMC) (Fig.…”

“…The process of employing horseradish peroxidase and hydrogen peroxide-mediated enzymatic crosslinking has previously been utilized to introduce orthogonal crosslinking in polymers containing phenol groups to develop hydrogel systems for various tissue engineering applications. 40,54 Since phenol groups are present in silk fibroin and in t-CMC, the radicals generated due to the action of an enzyme are responsible for the emergence of tyrosine-tyrosine, tyramine-tyramine and tyrosine-tyramine crosslinks in between the polymeric chains giving rise to a crosslinked hydrogel network. 55 Firstly, FTIR spectroscopy was employed to verify the presence of functional groups of the modified polymers in the fabricated CMC/t-CMC/silk and s-CMC/t-CMC/silk IPN hydrogels.…”

Tissue engineering modalities in skeletal muscles: focus on angiogenesis and immunomodulation properties

“…Among several peroxidase types, horseradish peroxidase (HRP) isolated from Armoracia rusticana has been extensively employed in the fabrication of hydrogels. HRP can be isolated from renewable sources with affordable costs for extraction and purification processes [ 199 , 200 ]. However, the activity of HRP in the presence of hydrogen peroxide can yield reduced cell viability [ 201 ].…”

Optimizing the physical properties of collagen/hyaluronan hydrogels by inhibition of polyionic complexes formation at pH close to the collagen isoelectric point

“…This hypothesis is supported by the increase in denaturation temperature up to 59 1C in pure collagen hydrogel after photocrosslinking, indicating fibrils were stabilized possibly through new tyrosine/tyrosine covalent bonds. 27,28 The difference of collagen denaturation temperature between pure and collagen/THA hydrogels could be due to the formation of supplementary tyrosine/tyramine bounds.…”