Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

INTRODUCTION

Intervertebral disc (IVD) degeneration, often associated with low back pain, is characterized by the loss of viable cells and decreased extracellular matrix content in the nucleus pulposus (NP). Notochordal cells (NCs) and their secretory products demonstrate a significant capacity to regenerate the intervertebral disc and restore its biomechanical function. However, human NCs are scarcely available since they are lost early in life and it is difficult to maintain their phenotype in culture. The aim of this study was to investigate the potential of a decellularized NC matrix (dNCM)-based hydrogel to stimulate IVD regeneration and to function as an instructive biomaterial for cell-based therapies.

METHODS

First, the optimal formulation of dNCM crosslinked with polyethylene glycol (PEG) was determined based on gel physio-chemical and cell viability studies. Porcine NCs were encapsulated within the dNCM-PEG hydrogel and cultured for 14 days (n=6). Read-outs included cell viability, morphology, phenotype, and matrix-production capacity. Based on these results, the potential of the dNCM-PEG gel was investigated in an acute injury rat tail model (n=8). On day 7 and 28, IVDs were collected and evaluated for disc height index (DHI), degeneration score, matrix production, and cell phenotype. Additionally, we investigated whether NCM-PEG gel promoted in vitro differentiation of induced pluripotent stem cells (iPSCs)-derived mesendoderm progenitor cells (MEPCs) towards notochordal-like cells (NLCs) (n=1). Cell viability, morphology, and phenotype were assessed on days 3, 7, and 14. Finally, the regenerative capacity of dNCM-PEG hydrogel alone and in combination with MEPCs, was evaluated by injecting bovine NP explants after inducing degeneration and culturing in a simulated loading environment. On day 27, read-outs included MEPC survival, lactate release, glucose uptake, matrix production, and cell phenotype.

RESULTS

Porcine NCs showed not only high viability after 14 days of culture within the hydrogel, but also good maintenance of NC morphology (CAV1+ vacuolated cells), matrix production (ACAN, COLII), and phenotype (T, panKRT, FOXF1, PAX1). Preliminary in vivo results indicated improved IVD cell morphology and matrix production in rat IVDs on days 7 and 28 after injection with dNCM-PEG gel. MEPCs remained viable up to day 14 after encapsulation together with the expression of early (T, FOXA2) and mature NC markers (panKRT) indicating successful differentiation toward the notochordal-lineage. Additionally, MEPCs encapsulated in dNCM-PEG gel survived in dynamically loaded bovine NP explants for up to 27 days. Both dNCM-PEG hydrogel alone and together with MEPCs resulted in increased immunopositivity of panKRT and T compared to degenerated controls. However, the DNA content and Ki67 immunopositivity were only increased in dNCM-PEG+MEPCs. Biomaterial injection did not immediately restore NP explants biomechanically at 27 days follow up.

DISCUSSION

In conclusion, dNCM-PEG hydrogel stimulates IVD regeneration and functions as an instructive cell carrier that protects and promotes a healthy NC-phenotype. While immediate biomechanical regeneration did not occur after the hydrogel injection, a biological regeneration of NP cells may lead to long-term restoration and thereby contribute to alleviation discogenic pain.

This work was funded by Horizon 2020 (825925), the Dutch Arthritis Society (LLP22), and Science Foundation Ireland (SFI) (13/RC/2073_P2).