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

## TL;DR

This study compares two types of gelatin-based hydrogels with an antioxidant oligomer, showing that one type (GelNB) performs better in preserving antioxidant properties and healing wounds.

## Contribution

The first systematic comparison of limonene-derived oligomer effects on GelMA and GelNB hydrogels, revealing cross-linking chemistry's impact on antioxidant retention and wound healing.

## Key findings

- GelNB hydrogels with PLM showed nearly complete cross-linking and higher antioxidant capacity (up to 90%) compared to GelMA.
- GelNB/PLM5 achieved 94.90% wound closure in vivo, outperforming the control.
- GelNB/PLM5 demonstrated excellent biocompatibility with no significant inflammation over 18 days.

## 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. In-vivo, 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.