Azide-Functional Extracellular Matrix Coatings as a Bioactive Platform for Bioconjugation

Abstract

In recent years, the development and application of decellularized extracellular matrices (ECMs) for use as biomaterials have grown rapidly. These cellderived 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 a biomaterial functionality by bioconjugation. To overcome this limitation, metabolic glycoengineering has 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 the unmodified ECM and azide-modified clickECM by osmotic lysis. In a first step, these matrices were concentrated to remove excessive water from the decellularization step. Next, the hydrogellike ECM and clickECM films were mechanically fragmentized, resulting in easy to pipette suspensions with fragment sizes ranging from 7.62 to 31.29 μm (as indicated by the mean d 90 and d 10 values). The biomolecular composition was not impaired as proven by immunohistochemistry. The suspensions were used for the reproducible generation of surface coatings, which proved to be homogeneous in terms of ECM fragment sizes and coating thicknesses (the 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 the biotin−streptavidin interaction could be used. Its applicability was demonstrated by equipping the bioactive clickECM coatings with horseradish peroxidase as a model enzyme.

Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods

“…Fibroblastderived matrices (both, azide-modied as well as unmodied, from now on referred to as (click)ECM when both types of ECM are discussed) were generated and isolated as previously described. 17 In brief, cells were seeded in tissue culture polystyrene dishes (F 14.5 cm) at a density of 3.2 Â 10 6 cells per dish (20 000 cells per cm 2 ) on day 1 and were then cultured for another seven days in DMEM supplemented with 10% FCS, 1% P/S, and 50 mg mL À1 Na-L-ascorbate. On day 5, cells were treated with either 625 mL 50 mM GalNAc (resulting in the unmodied ECM, "ECM") or 625 mL 50 mM Ac 4 GalNAz per dish (resulting in the azide-modied "clickECM").…”

“…Aer isolation through osmotic lysis, the isolated (click)ECM was concentrated and homogenised as previously described. 17 In brief, concentration of isolated (click)ECM was achieved using sterile ultracentrifugation tubes with regenerated cellulose membranes (molecular weight cut-off (MWCO): 10 kDa). Aer centrifugation (4000 rpm for 90 minutes), concentrated (click)ECM was recovered and transferred into a microcentrifuge tube using a pipette.…”

“…This reaction leads to a covalent attachment via triazole rings and can therefore be used for bioorthogonal bioconjugation. [13][14][15][16][17] Using the same monosaccharide but adipose-derived stem cells (ASCs) instead of broblasts, Nellinger et al demonstrated that this method can be used to generate azide-modied tissue-specic ECM. 18 As an alternative to the above mentioned Ac 4 -GalNAz, Gutmann et al demonstrated the successful modication of NIH-3T3 broblasts-derived ECM using the glucosamine derivate 2-azidoacetylamino-2-deoxy-(1,3,4,6)tetra-O-acetyl-D-glucopyranoside (Ac 4 GlcNAz).…”

“…18 As an alternative to the above mentioned Ac 4 -GalNAz, Gutmann et al demonstrated the successful modication of NIH-3T3 broblasts-derived ECM using the glucosamine derivate 2-azidoacetylamino-2-deoxy-(1,3,4,6)tetra-O-acetyl-D-glucopyranoside (Ac 4 GlcNAz). 19,20 Despite the successful modication of ECM with azides via MGE and their demonstrated applicability as surface coatings or bioconjugation platforms, 10,17,19 it is not yet understood if MGE changes properties of azide-modied ECM (e.g. the overall protein content or the biomolecular composition) beyond the modication with azide groups.…”

“…Bioanalytical characterisation of these materials is distinctly challenging due to the insoluble and highly complex nature of cell-derived matrices. 17,21 Yet, qualitative as well as quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specic biomolecules is essential for the course of biomaterial research in order to eventually understand and predict the prospective functions and performance of the developed biomaterial.…”