Poster Presentation 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

Introduction
    Glycans are essential in protein folding, cellular signalling, inflammation, and cell survival, making them potential therapeutic targets. Sialylation, a terminal glycan modification, plays a key role in physiological and pathological processes. Both dogs and humans experience intervertebral disc (IVD) degeneration, presenting similarly in terms of clinical signs, diagnostics, and treatment. However, the impact of glycosylation profiles on canine IVD homeostasis has not been studied. The glycosylation inhibitor 3F-peracetyl-Neu5Ac (Neu5Ac-inhib), which inhibits sialyltransferase, was locally released by a hyaluronic acid (HA) hydrogel to determine its efficacy as a regenerative therapy for canine IVD degeneration. It hypothesized that it would effectively inhibit hyper-sialylation in this model, attenuating the degenerative response.

Methods
     Animal experiments were performed with ethical approval from the Research Ethics Committee of Tokai University Hospital, Japan. Ten female beagle dogs (12 months) were each subjected to five experimental conditions at eight lumbar spinal levels. Disc degeneration was induced by aspiration of nucleus pulposus tissue in four of the experimental conditions and allowed to progress for four weeks; the final group was approached but not invaded. Three experimental groups were given subsequent HA-hydrogel injections: one unloaded, one loaded with low-dose Neu5Ac-inhib, and one loaded with high-dose Neu5Ac-inhib; the final experimental group was given a saline injection. Prior to animal sacrifice, radiographic disc height index measurements  were obtained and IVDs were imaged on a Phillips Ingenia Ambition 3-Tesla MRI instrument, and sagittal T1, T2W and T2 maps were recorded on a monthly basis. IVDs were collected 12 weeks post-induction of degeneration and assessed by histological, glycomics, and proteomics analysis. Six samples per condition were selected for matrix-assisted laser desorption/ionisation–mass spectrometry imaging (MALDI-MSI) N-glycan analysis. Tissue sections were digested with PNGase-F enzyme before being coated in an α-cyano-4-hydroxycinnamic acid matrix by spray deposition using an HTX-TM-Sprayer™. Tissue sections were analysed using a Bruker scimaX^® instrument with a resolving power of 200.000 at m/z 933.31.

Results
     Disc height index significantly reduced in saline-treated discs but recovered in low and high dose Neu5Ac-inhib hydrogel treatment. MRI analysis demonstrated an improved Pfirrmann grade after Neu5Ac-inhib-loaded HA treatment. Histological analysis revealed a reduction in degenerative scoring in treatment groups compared to the saline injection. MALDI-MSI analysis revealed distinct and reproducible N-glycan expression for each experimental group. Inhibition of sialylation by Neu5Ac-inhib yielded a presented N-glycome that resembled untreated control tissues, including a reduction of presented sialylated N-glycan species and an increased presentation of tetra-antennary N-glycans compared with the unloaded hydrogel and saline treated controls (Figure 1). Furthermore, dose-dependent responses could be identified. This correlated with proteomic analysis which demonstrated conserved extracellular matriz regulation in Neu5Ac-inhib groups and a reduction in inflammatory signalling.

Discussion

     The N-glycome changes with canine IVD degeneration like that of humans compared to equivalent healthy IVD. This study successfully showcases the regenerative response to the sialylation inhibitor Neu5Ac-released by the HA-hydrogel in a preclinical canine model of IVD degeneration. Furthermore, radiological imaging highlights the impact of Neu5Ac-inhib on disc physiology, showing improved disc height in discs treated with Neu5Ac-inhib loaded HA.