The extracellular matrix (ECM) is a complex network of biomolecules which surrounds the cells in a human tissue. Primary cells are capable of producing an ECM in vitro which can be isolated after several days of culture. Because of its diverse functions and due to the high biological activity, the isolated ECM is a promising biomaterial for the use in tissue engineering and regenerative medicine The ECM is able to promote cell adhesion, proliferation, and differentiation in a tissue specific manner. However, the use of ECM as coatings or scaffolds is limited, e. g. due to minor mechanical stability. In our approach we used Metabolic Glyco Engineering (MGE) to introduce azide groups into the glycan structures of the ECM to create a variously deployable »clickECM«.
Histochemical and immunofluorescence analysis were performed in order to evaluate the incorporation of azides into this clickECM and to further characterize the biological composition. We could show that MGE can be used to introduce azide groups into the ECM of human cells. This clickECM consists of glycans, collagens, and non-collagenous proteins whereby the ratio of these biomolecules is the same in clickECM and unmodified ECM.
To investigate the accessibility of the incorporated azides, substrates were functionalized with activated alkynes and clickECM was covalently immobilized via a copper-free click reaction resulting in a significantly increased coating stability compared to a conventional, physisorbed ECM coating. The incorporated azides could also be accessed by bi-functionalized hydrogel crosslinking molecules to increase the density and the stability of the clickECM coating compared to a conventional coating with unmodified ECM. The covalent immobilization of the clickECM mediates an increased stability while preserving the high biological activity of the human ECM. Crosslinking of the clickECM with alkyne-functional crosslinking agents increases the coating density as well as the coating stability.
We propose that our clickECM is a promising technology to e. g. generate coatings with an increased stability with high biological complexity or to introduce selected (bio)molecules into the clickECM.