Vanillin-based functionalization strategy to construct multifunctional microspheres for treating inflammation and regenerating intervertebral disc

Abstract

Intervertebral disc degeneration (IVDD) is one of the main causes of low back pain. Although local delivery strategies using biomaterial carriers have shown potential for IVDD treatment, it remains challenging for intervention against multiple adverse contributors by a single delivery platform. In the present work, we propose a new functionalization strategy using vanillin, a natural molecule with anti-inflammatory and antioxidant properties, to develop multifunctional gelatin methacrylate (GelMA) microspheres for local delivery of transforming growth factor β3 (TGFβ3) toward IVDD treatment. In vitro, functionalized microspheres not only improved the release kinetics of TGFβ3 but also effectively inhibited inflammatory responses and promoted the secretion of extracellular matrix (ECM) in lipopolysaccharide-induced nucleus pulposus (NP) cells. In vivo, functionalized platform plays roles in alleviating inflammation and oxidative stress, preserving the water content of NP and disc height, and maintaining intact structure and biomechanical functions, thereby promoting the regeneration of IVD. High-throughput sequencing suggests that inhibition of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling might be associated with their therapeutic effects. In summary, the vanillin-based functionalization strategy provides a novel and simple way for packaging multiple functions into a single delivery platform and holds promise for tissue regeneration beyond the IVD.

Keywords: Intervertebral disc degeneration; Microsphere; Regeneration; Transforming growth factor β3; Vanillin.

Graphical abstract

Scheme 1

Summary of fabricating vanillin functionalized microspheres for the IVD regeneration. (A) The detailed method and process of fabricating vanillin functionalized microspheres. (B) The mechanism for the IVD regeneration.

Fig. 1

Characterizations of microspheres. (A, B) Monodisperse GelMA microspheres of two sizes and corresponding particle size distributions (n = 50). (C) SEM images of GelMA microspheres (GM) and vanillin functionalized microspheres (V@GM). (D) FTIR spectra of GM and V@GM. (E) Frequency sweep rheological analysis of GM and V@GM.

Fig. 2

TGFβ3 controlled release property of functionalized microspheres. (A, B) In vitro release kinetics of TGFβ3 in GM and V@GM (n = 3). (C) Representative light photomicrographs of migrated NPCs cultured with microspheres. (D) Quantitative analysis of migrated cell density (n = 3). *, p < 0.05; ns, no significant difference.

Fig. 3

Anti-inflammatory effect of functionalized microspheres. (A) Quantifications of inflammation-related genes expression (Il1b, Il6, Ptgs2, and Tnfa) in NPCs cultured on microspheres by qRT-PCR after 3 days (n = 3). (B) COX2 immunofluorescence images after 5 days. *, p < 0.05; ns, no significant difference.

Fig. 4

The matrix metabolism of NPCs on different microspheres with LPS treatment. (A) Quantifications of matrix metabolism-related genes expression (Krt19, Acan, Col2a1, and Mmp3) in NPCs on microspheres by qRT-PCR after 3 days (n = 3). (B) COL II immunofluorescence images after 5 days. Quantifications of (C) GAG and (D) hydroxyproline of NPCs after 14 days (n = 3). *, p < 0.05; ns, no significant difference.

Fig. 5

Transcriptomic analysis. (A) Volcano plot of DEGs. Red dots indicated upregulated genes; Blue dots indicated downregulated genes. (B) Heatmap showed a series of DEGs related to inflammation and matrix metabolism in NPCs. (C) GO analysis showed the top 20 biological processes involved in DEGs. (D) KEGG enrichment bubble plot showed the top 20 pathways involved in DEGs. (E) GSEA showed that the “cytokine receptor activity” and “collagen catabolic process” were inhibited in the V-T@GM group. (F) Western blotting of AKT and p-AKT.

Fig. 6

Imaging evaluation of the animal experiment. (A) PA images of NP tissue in situ 7 days after injections of different microspheres. (B) X-ray images (right) and MRI images (left) of the rats' IVDs (marked by red wireframe) after 4 weeks and 8 weeks. Quantitative analyses of (C) PA signals (n = 3), (D) DHI% (n = 3), and (E) the optical density of IVDs (n = 3). *, p < 0.05; ns, no significant difference.

Fig. 7

Histological evaluation of the animal experiment. (A) Gross morphologies of the IVD at 4 weeks and 8 weeks. H&E staining (the first row), SO/FG staining (the second row), and immunohistochemistry staining of COL II (the third row) at (B) 4 weeks and (C) 8 weeks. Histological grade of sections in different groups at (D) 4 weeks and (E) 8 weeks (n = 3). *, p < 0.05; ns, no significant difference.

Fig. 8

Biomechanical evaluation of the animal experiment. (A) Stress-strain curves, (B) compression moduli, (C) stress-relaxation curves, and (D) t_1/2 (n = 3) of IVDs at 8 weeks.