Immunomodulatory omega-3 injectable hydrogel for vital pulp therapy

Biomaterials that both seal the pulp during direct pulp capping (DPC) and actively control inflammation are unusual. Here, we engineered injectable gelatin methacryloyl (GelMA) hydrogels carrying omega-3 polyunsaturated fatty acids. Our null hypothesis states that GelMA and GelMA-omega-3 hydrogels do not differ in their physicochemical, mechanical, cytocompatibility, immunomodulatory, or in vivo effects.

Distinct omega-3 concentrations were added to GelMA, and the resulting hydrogel was photocrosslinked. Scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) were used to examine the hydrogels' microstructure and composition. Swelling, degradation, compression, and rheological tests were also performed. Dental pulp stem cell (DPSC) viability and mineralization capacity were assessed with or without lipopolysaccharide (LPS). LPS-challenged RAW 264.7 macrophages were exposed to hydrogel-conditioned media to measure TNF-α and IL-6 levels. In vivo implantation of the hydrogels was conducted to determine biocompatibility, macrophage polarization (iNOS/CD163), and TGF-β expression. Statistics used one-way ANOVA with Tukey's post-hoc test or two-way ANOVA with the Holm-Šídák test (p<0.05).

SEM revealed a more fibrous, heterogeneous network in the omega-3 hydrogels. Incorporating omega-3 reduced swelling and slowed mass loss compared to GelMA. Although a high omega-3 load decreased Young's modulus, the ultimate strength remained similar. FTIR confirmed omega-3 incorporation. Crosslinked hydrogels were solid-like, shear-thinning, thermally stable, and recovered post-shear. All formulations were cytocompatible. Without LPS, mineralization levels were similar across groups. With LPS, high omega-3 concentration decreased mineralization, while low concentration matched the control. All hydrogels suppressed LPS-induced TNF-α and IL-6; omega-3 further reduced IL-6 compared to GelMA. In vivo, omega-3 hydrogels increased early cell influx that resolved by day 28, enhanced M2 macrophage marker expression at 7 and 28 days, lowered the M1/M2 ratio over time, and elevated TGF-β expression early on with sustained levels.

Omega-3 hydrogels are biocompatible and actively modulate inflammation, supporting their potential as an immunomodulatory strategy for DPC.