Abstract
The present work reports on the development of photo-cross-linkable gelatins sufficiently versatile to overcome current biopolymer two-photon polymerization (2PP) processing limitations. To this end, both the primary amines as well as the carboxylic acids of gelatin type B were functionalized with photo-cross-linkable moieties (up to 1 mmol/g) resulting in superior and tunable mechanical properties (G′ from 5000 to 147000 Pa) enabling efficient 2PP processing. The materials were characterized in depth prior to and after photoinduced cross-linking using fully functionalized gelatin-methacrylamide (gel-MOD) as a benchmark to assess the effect of functionalization on the protein properties, cross-linking efficiency, and mechanical properties. In addition, preliminary experiments on hydrogel films indicated excellent in vitro biocompatibility (close to 100% viability) both in the presence of MC3T3 preosteoblasts and L929 fibroblasts. Moreover, 2PP processing of the novel derivative was superior in terms of applied laser power (≥40 vs ≥60 mW for gel-MOD at 100 mm/s) as well as post-production swelling (0–20% vs 75–100% for gel-MOD) compared to those of gel-MOD. The reported novel gelatin derivative (gel-MOD-AEMA) proves to be extremely suitable for direct laser writing as both superior mimicry of the applied computer-aided design (CAD) was obtained while maintaining the desired cellular interactivity of the biopolymer. It can be anticipated that the present work will also be applicable to alternative biopolymers mimicking the extracellular environment such as collagen, elastin, and glycosaminoglycans, thereby expanding current material-related processing limitations in the tissue engineering field.
Highlights
Gelatin and its derivatives are of specific interest in the field of biomaterials because they are characterized by high biocompatibility combined with excellent cell-interactive properties due to the presence of Arg-Gly-Asp (RGD) motifs in the gelatin backbone.[1−3] In addition, gelatin is derived from collagen, which is a major component of the natural extracellular matrix (ECM), rendering it an ideal ECM mimic.[4−7] it is a costeffective,[8,9] food and drug administration (FDA) approved,[10] bioresorbable polymer that can be degraded enzymatically.[5,6,9,11,12]
We anticipated that an increase in photoreactive functionalities using the gelatin carboxylic acids would positively affect the cross-link density of the resulting hydrogels
Starting from gel-MOD with a degree of substitution (DS) of 97% (0.37 mmol methacrylamides/g of gelatin), we targeted the partial modification of the carboxylic acid functionalities present in the glutamate and aspartate side chains
Summary
Gelatin and its derivatives are of specific interest in the field of biomaterials because they are characterized by high biocompatibility combined with excellent cell-interactive properties due to the presence of Arg-Gly-Asp (RGD) motifs in the gelatin backbone.[1−3] In addition, gelatin is derived from collagen, which is a major component of the natural extracellular matrix (ECM), rendering it an ideal ECM mimic.[4−7] it is a costeffective,[8,9] food and drug administration (FDA) approved,[10] bioresorbable polymer that can be degraded enzymatically.[5,6,9,11,12]To date, one of the most commonly applied hydrogel materials for biofabrication and tissue engineering purposes is methacrylamide-modified gelatin (gel-MOD) or gelatin-methacryloyl hydrogels (gel-MA), which can be obtained by functionalization of the primary amines of the (hydroxy)lysine and ornithine side groups present in gelatin with methacrylic anhydride.[4,13−22] (Figure 1A) As a result, a photo-cross-linkable derivative is obtained that is suitable for laser-based rapid prototyping techniques including two photon polymerization (2PP).[5,6,23−25] Important material limitations remain, in terms of mechanical and swelling properties after cross-linking. With the aim of overcoming the limitations described above to improve the two-photon polymerization potential of gelatin hydrogels, we report a novel photo-cross-linkable gelatin. In this respect, the two-photon polymerization potential refers to several aspects of 2PP including the minimally required spatiotemporal energy to obtain reproducible structures. The two-photon polymerization potential refers to the mimicry between the applied CAD and the final structure obtained This feature is determined both by postproduction swelling of the hydrogel during the development process as well as the mechanical properties of the material. By introducing additional cross-linkable functionalities, the hydrogels of this novel derivative will be characterized by a higher network density after cross-linking and outperform currently reported gelatin derivatives[32−35] (e.g., gel-MOD/gel-MA, gel-SH, ...) in 2PP potential
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