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clickECM - a new approach to covalently immobilize human ECM on artificial surfaces

: Keller, Silke; Ruff, Sybil Mara; Bach, Monika; Kluger, Petra

Bionanomaterials 17 (2016), Nr.s1, S.S59
ISSN: 2193-0651
ISSN: 2193-066X
German Society for Biomaterials (Annual Conference) <2016, Aachen>
Deutsche Gesellschaft für Biomaterialien (DGBM Jahrestagung) <2016, Aachen>
Fraunhofer IGB ()

The extracellular matrix (ECM) is a complex three-dimensional network of biomolecules that surround the cells within a human tissue. The ECM is tissue-specific and is considered the natural environment of the cells. Due to the high biological activity, various types of ECM are able to promote cell adhesion, proliferation and differentiation in a tissue specific manner [1]. Primary cells are capable of producing an ECM in vitro, which can be isolated after several days of culture [2]. Due to its diverse functions, the isolated ECM is a promising biomaterial for use in tissue engineering and regenerative medicine.
However, if this isolated ECM is used as a biomaterial for surface coatings, a major limitation is the lack of mechanical stability, since physisorption is the primary source of adhesion between the molecules [3].
To overcome this limitation our approach was to covalently immobilize the human ECM on artificial surfaces. Therefore, we performed metabolic oligosaccharide engineering (MOE) to introduce click groups into the glycan structures of the ECM. Here we demonstrate that this method is suitable to fabricate a “clickable” human ECM (clickECM) which can be covalently immobilized on click-functionalized artificial surfaces via copper-free click reaction [4] (Fig. 1).
We cultured primary human dermal fibroblasts over a period of six days to generate a human dermal ECM. By treating the cells with an azidemodified monosaccharide (Ac4GalNAz), we were able to introduce click functionalities into the glycans of the ECM. Histochemical and immunofluorescence analysis were performed to characterize the biological composition of the resulting clickECM in comparison with untreated ECM and human skin dermis. Then, artificial surfaces were functionalized with activated alkynes for the covalent immobilization of clickECM. Coating of unmodified ECM via physisorption served as a control. The stability of these coatings was compared by calculating the percentage area covered with ECM before and after an intensive washing step in detergent-supplemented buffer. The bioactive properties of these coatings were evaluated by quantifying the cell proliferation of HaCaT cells seeded on either clickECM-coated or uncoated glass substrates.
We could show that MOE can be used to introduce click groups into the ECM of human dermal fibroblasts. This clickECM is comparable to the unmodified ECM and to human skin dermis in terms of the distribution of glycans and typical ECM components. The clickECM can be covalently immobilized on alkyne-modified surfaces resulting in a significant increase in coating stability compared to a conventional ECM coating via physisorption (Fig. 2). Cell proliferation of HaCaT cells was significantly enhanced on the clickECM-coated surfaces compared to uncoated glass substrates. These results demonstrate that the covalent immobilization provides high stability, while also preserving the biological activity of the human ECM.
We therefore propose that clickECM is a promising biomaterial for the use in in tissue engineering and regenerative medicine e. g. as a tissue-specific coating material to mimic the natural environment of cells.