Azide-functional extracellular matrix coatings as bioactive platform for bioconjugation.

: In recent years, the development and application of decellularized extracellular matrices (ECM) for the use as biomaterials has grown rapidly. These cell-derived matrices (CDMs) represent highly bioactive and biocompatible materials consisting of a complex assembly of biomolecules. Even though CDMs mimic the natural microenvironment of cells in vivo very closely, they still lack specifically addressable functional groups, which are often required to tailor biomaterial functionality by bioconjugation. To overcome this limitation, metabolic glycoengineering (MGE) emerged as a powerful tool to equip CDMs with chemical groups such as azides. These small chemical handles are known for their ability to undergo bioorthogonal click reactions, which represent a desirable reaction type for bioconjugation. However, ECM insolubility makes its processing very challenging. In this contribution, we isolated both unmodified ECM as well as azide-modified clickECM by osmotic lysis. In a first step, these matrices were concentrated to remove excessive water from the decellularization step. Next, the hydrogel-like ECM films were mechanically fragmentized resulting in easy to pipet suspensions with fragment sizes ranging from 7.6 - 31.3 µm (as indicated by the mean d90 and d10 values). The biomolecular composition was thereby not impaired as proven by immunohistochemistry. The suspensions were used for the reproducible generation of surface coatings, which proved to be homogenous in terms of ECM fragment sizes and coating thicknesses (mean coating thickness was found to be 33.2 ± 7.3 µm). Furthermore, they were stable against fluid-mechanical abrasion in a laminar flow cell. When primary human fibroblasts were cultured on the coated substrates, an increased bioactivity was observed. By conjugating the azides within the clickECM coatings with alkyne-coupled biotin molecules, a bioconjugation platform was obtained, where biotin-streptavidin interaction could be used. The applicability was demonstrated by equipping the bioactive clickECM coatings with horseradish peroxidase (HRP) as a model enzyme.