Abstract
There exists a clear clinical need for adipose tissue reconstruction strategies to repair soft tissue defects which outperform the currently available approaches. In this respect, additive manufacturing has shown to be a promising alternative for the development of larger constructs able to support adipose tissue engineering. In the present work, a thiol-ene photo-click crosslinkable gelatin hydrogel was developed which allowed extrusion-based additive manufacturing into porous scaffolds. To this end, norbornene-functionalized gelatin (Gel-NB) was combined with thiolated gelatin (Gel-SH). The application of a macromolecular gelatin-based thiolated crosslinker holds several advantages over conventional crosslinkers including cell-interactivity, less chance at phase separation between scaffold material and crosslinker and the formation of a more homogeneous network. Throughout the paper, these photo-click scaffolds were benchmarked to the conventional methacrylamide-modified gelatin (Gel-MA). The results indicated that stable scaffolds could be realized which were further characterized physico-chemically by performing swelling, mechanical and in vitro biodegradability assays. Furthermore, the seeded adipose tissue-derived stem cells (ASCs) remained viable (>90%) up to 14 days and were able to proliferate. In addition, the cells could be differentiated into the adipogenic lineage on the photo-click crosslinked scaffolds, thereby performing better than the cells supported by the frequently reported Gel-MA scaffolds. As a result, the developed photo-click crosslinked scaffolds can be considered a promising candidate towards adipose tissue engineering and a valuable alternative for the omnipresent Gel-MA. Statement of SignificanceThe field of adipose tissue engineering has emerged as a promising strategy to repair soft tissue defects. Herein, Gel-NB/Gel-SH gelatin-based hydrogel scaffolds were produced using extrusion-based additive manufacturing. Using a cell-interactive, thiolated gelatin crosslinker, a homogeneous network was formed and the risk of phase separation between norbornene-modified gelatin and macromolecular crosslinkers was reduced. UV-induced crosslinking of these materials is based on step growth polymerization which requires less free radicals to enable polymerization. Our results demonstrated the potential of the developed scaffolds, due to their favourable physico-chemical characteristics as well as their adipogenic differentiation potential when benchmarked to Gel-MA scaffolds. Hence, the hydrogels could be of great interest towards future development of adipose tissue constructs and tissue engineering in general.
Highlights
Gelatin is frequently used in various biomedical applications
The amine groups of RCPhC1 and gelatin were modified with photo-crosslinkable methacrylamide functionalities (RCPhC1-MA and Gel-MA, respectively) to avoid the use of potentially toxic crosslinkers such as glutaraldehyde and to reduce the long reaction times that are associated with 1-ethyl3-[3-dimethylaminopropyl]carbodiimide hydrochloride-N-hydroxysuccinimide (EDC-NHS) crosslinking (30 min versus 16 h, respectively).[16,17]
During the modification of RCPhC1-MA, 1 equivalent of methacrylic anhydride was added with respect to the primary amines of the lysine moieties resulting in a degree of substitution (DS) of 90% (0.60 mmol methacrylamide per g RCPhC1)
Summary
Gelatin is frequently used in various biomedical applications. gelatin is generally extracted from an animal source, which can result in issues with reproducibility as well as pathogen transmittance. The drawbacks include problems with product reproducibility due to batch to batch variations and the risk of pathogen transmittance including prions.[9,10] We hypothesize that a recombinant peptide based on human collagen type I could be an attractive alternative to animal-derived gelatin. Lithium(2,4,6-trimethylbenzoyl)phenylphosphinate (Li-TPO-L or LAP) was applied in the experiments as a photo-initiator (PI) instead of the more often used PI Irgacure 2959 (I-2959) The latter PI has a molar extinction coefficient of 4 MÀ1 cmÀ1 at 365 nm, while Li-TPO-L has a molar extinction coefficient between 200 and 300 MÀ1 cmÀ1 at 350–380 nm, which results in a superior reactivity compared to I-2959.18–20 The developed hydrogels were physico-chemically characterized by gel fraction determination, swelling experiments and mechanical tests as well as in vitro biological assays and cell encapsulation experiments. We hypothesized that RCPhC1-MA could be an attractive alternative to animalderived Gel-MA in terms of reproducibility, processing, mechanical strength, swelling properties and biocompatibility
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