Bioactive Limonene-Derived Oligomer in Gelatin Hydrogels: Impact of Cross-Linking Chemistry on Physicochemical Properties and Wound Healing Performance.

Abstract

Antioxidant photo-cross-linkable hydrogels have garnered significant interest for biomedical applications, but challenges such as additive thermal instability and interference with cross-linking remain. Here, we report the first systematic comparison of a limonene-derived oligomer (PLM) incorporated into two distinct photo-cross-linkable gelatin platforms, highlighting how cross-linking chemistry governs antioxidant performance and network integrity. PLM was incorporated at 5% and 10% (w/w, relative to gelatin), comparing the performance of gelatin-methacryloyl (GelMA) and gelatin-norbornene (GelNB) hydrogels in terms of cross-linking efficiency, antioxidant retention, release profile, and biocompatibility upon PLM incorporation. While PLM negatively affected the physicochemical properties of GelMA, its incorporation into GelNB did not show similar drawbacks. High-resolution magic angle spinning (HR-MAS) ¹H NMR spectra revealed a significant drop in double bond consumption (DC) for GelMA, from 71% to 46% in GelMA/PLM10. In contrast, GelNB demonstrated nearly complete DC (98%) even in GelNB/PLM10, indicating efficient cross-linking despite the presence of the antioxidant. Release profiles suggested a Fickian diffusion mechanism, with higher diffusivity values for GelNB, further highlighting differences in the matrix networks. Additionally, while PLM increased GelMA's antioxidant capacity from 6% to 19%, GelNB with PLM showed an impressive enhancement, reaching up to 90%. Both GelMA and GelNB hydrogels with 5% (w/w) PLM supported healthy cell morphology and viability above 85% over 7 days. <i>In-vivo</i>, GelNB/PLM5 demonstrated excellent biocompatibility over 18 days, with no significant inflammation compared to controls. Notably, this sample achieved the highest wound closure (94.90%) by day 18, outperforming the positive control (Sorbalgon). Overall, the results demonstrate that the thiol-ene cross-linking pathway enables superior preservation of antioxidant functionality and network integrity, positioning GelNB/PLM5 as a promising candidate for advanced wound-healing applications.