Cross-linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix (ECM) in tissue engineering. However, the quantification of modified groups in GM is still an unsolved issue, although this is the key factor for tailoring the physicochemical material properties. In this contribution, H-C-HSQC NMR spectra are used to gain detailed structural information on GMs and of 2-fold modified gelatin containing methacryloyl and acetyl groups (GMAs). Distinctive identification of methacrylate, methacrylamide, and acetyl groups present in GMs and GMAs revealed an overlap of methacrylamide and modified hydroxyproline signals in the H NMR spectrum. Considering this, we suggest a method to quantify methacrylate and methacrylamide groups in GMs precisely based on simpleH NMR spectroscopy with an internal standard. Quantification of acetylation in GMAs is also possible, yet, 2D NMR spectra are necessary. The described methods allow direct quantification of modified groups in gelatin derivatives, making them superior to other, indirect methods known so far.
Gelatin methacryloyl (acetyl) (GM(A)) is increasingly investigated for various applications in life sciences and medicine, for example, drug release or tissue engineering. Gelatin type A and type B are utilized for GAM(A) and GBM(A) preparation, but the impact of gelatin raw material on modification reaction and resulting polymer properties is rather unknown so far. Therefore, the degrees of modification (DMA) and physicochemical properties of five GAM(A) and GBM(A) derivatives are compared: The degrees of methacryloylation (0.32–0.98 mmol g−1) are indistinguishable for GAM(A) and GBM(A) as are the sol‐gel temperatures. Isoelectric points, solution viscosities, and hydrodynamic radii which are distinct for GA and GB, converge with increasing DMA. Interestingly, differences are measured for the storage moduli and equilibrium degrees of swelling of respective GA and GB derivative‐based hydrogels, in spite of their comparable DMA. This underlines the importance of GM(A) characterization beyond the modification degree.
Chemically cross-linkable gelatin methacryloyl (GM) derivatives are getting increasing attention regarding biomedical applications. Thus, thorough investigations are needed to achieve full understanding and control of the physico-chemical behavior of these promising biomaterials. We previously introduced gelatin methacryloyl acetyl (GMA) derivatives, which can be used to control physical network formation (solution viscosity, sol-gel transition) independently from chemical cross-linking by variation of the methacryloyl-to-acetyl ratio. It is known that temperature dependent physical network formation significantly influences the mechanical properties of chemically cross-linked GM hydrogels. We investigated the temperature sensitivity of GM derivatives with different degrees of modification (GM2, GM10), or similar degrees of modification but different methacryloyl contents (GM10, GM2A8). Rheological analysis showed that the low modified GM2 forms strong physical gels upon cooling while GM10 and GM2A8 form soft or no gels. Yet, compression testing revealed that all photo cross-linked GM(A) hydrogels were stronger if cooling was applied during hydrogel preparation. We suggest that the hydrophobic methacryloyl and acetyl residues disturb triple helix formation with increasing degree of modification, but additionally form hydrophobic structures, which facilitate chemical cross-linking.
Cellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.
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