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Azide-bearing cell-derived extracellular matrices (“clickECMs”) have emerged as a highly exciting new class of biomaterials. They conserve substantial characteristics of the natural extracellular matrix (ECM) and offer simultaneously small abiotic functional groups that enable bioorthogonal bioconjugation reactions. Despite their attractiveness, investigation of their biomolecular composition is very challenging due to the insoluble and highly complex nature of cell-derived matrices (CDMs). Yet, thorough qualitative and quantitative analysis of the overall material composition, organisation, localisation, and distribution of typical ECM-specific biomolecules is essential for consistent advancement of CDMs and the understanding of the prospective functions of the developed biomaterial. In this study, we evaluated frequently used methods for the analysis of complex CDMs. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and (immune)histochemical staining methods in combination with several microscopic techniques were found to be highly eligible. Commercially available colorimetric protein assays turned out to deliver inaccurate information on CDMs. In contrast, we determined the nitrogen content of CDMs by elementary analysis and converted it into total protein content using conversion factors which were calculated from matching amino acid compositions. The amount of insoluble collagens was assessed based on the hydroxyproline content. The Sircol™ assay was identified as a suitable method to quantify soluble collagens while the Blyscan™ assay was found to be well-suited for the quantification of sulphated glycosaminoglycans (sGAGs). Eventually, we propose a series of suitable methods to reliably characterise the biomolecular composition of fibroblast-derived clickECM.
An advanced ‘clickECM’ that can be modified by the inverse-electron demand Diels-Alder reaction
(2022)
The extracellular matrix (ECM) represents the natural environment of cells in tissue and therefore is a promising biomaterial in a variety of applications. Depending on the purpose, it is necessary to equip the ECM with specific addressable functional groups for further modification with bioactive molecules, for controllable cross-linking and/or covalent binding to surfaces. Metabolic glycoengineering (MGE) enables the specific modification of the ECM with such functional groups without affecting the native structure of the ECM. In a previous approach (S. M. Ruff, S. Keller, D. E. Wieland, V. Wittmann, G. E. M. Tovar, M. Bach, P. J. Kluger, Acta Biomater. 2017, 52, 159–170), we demonstrated the modification of an ECM with azido groups, which can be addressed by bioorthogonal copper-catalyzed azide-alkyne cycloaddition (CuAAC). Here, we demonstrate the modification of an ECM with dienophiles (terminal alkenes, cyclopropene), which can be addressed by an inverse-electron-demand Diels-Alder (IEDDA) reaction. This reaction is cell friendly as there are no cytotoxic catalysts needed. We show the equipment of the ECM with a bioactive molecule (enzyme) and prove that the functional groups do not influence cellular behavior. Thus, this new material has great potential for use as a biomaterial, which can be individually modified in a wide range of applications.
Natural extracellular matrix (ECM) represents an ideal biomaterial for tissue engineering and regenerative medicine approaches. For further functionalization, there is a need for specific addressable functional groups within this biomaterial. Metabolic glycoengineering (MGE) provides a technique to incorporate modified monosaccharide derivatives into the ECM during their assembly, which was shown by us earlier for the production of a modified fibroblast-derived dermal ECM.
Gelatin is one of the most prominent biopolymers in biomedical material research and development. It is frequently used in hybrid hydrogels, which combine the advantageous properties of bio‐based and synthetic polymers. To prevent the biological component from leaching out of the hydrogel, the biomolecules can be equipped with azides. Those groups can be used to immobilize gelatin covalently in hydrogels by the highly selective and specific azide–alkyne cycloaddition. In this contribution, we functionalized gelatin with azides at its lysine residues by diazo transfer, which offers the great advantage of only minimal side‐chain extension. Approximately 84–90% of the amino groups are modified as shown by 1H‐NMR spectroscopy, 2,4,6‐trinitrobenzenesulfonic acid assay as well as Fourier‐transform infrared spectroscopy, rheology, and the determination of the isoelectric point. Furthermore, the azido‐functional gelatin is incorporated into hydrogels based on poly(ethylene glycol) diacrylate (PEG‐DA) at different concentrations (0.6, 3.0, and 5.5%). All hydrogels were classified as noncyctotoxic with significantly enhanced cell adhesion of human fibroblasts on their surfaces compared to pure PEG‐DA hydrogels. Thus, the new gelatin derivative is found to be a very promising building block for tailoring the bioactivity of materials.
Due to its availability and minimal invasive harvesting human adipose tissue-derived extracellular matrix (dECM) is often used as a biomaterial in various tissue engineering and healthcare applications. Next to dECM, cell-derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. So far both types of ECM were investigated extensively toward their application as (bio)material in tissue engineering and healthcare. However, a systematic characterization and comparison of soft tissue dECM and cdECM is still missing. In this study, we characterized dECM from human adipose tissue, as well as cdECM from human adipose-derived stem cells, toward their molecular composition, structural characteristics, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans compared with cdECMs. Structural characteristics revealed an immature state of the fibrous part of cdECM samples. By the identified differences, we aim to support researchers in the selection of a suitable ECM-based biomaterial for their specific application and the interpretation of obtained results.
In recent years, the development and application of decellularized extracellular matrices (ECMs) for use as biomaterials have 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 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 hydrogel-like 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 d90 and d10 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.