Development of gellan gum-based microparticles/hydrogel matrices for application in the intervertebral disc regeneration

Abstract

Low back pain is one of the most reported medical conditions associated to intervertebral disc (IVD) degeneration. Nucleus pulposus (NP) is often regarded as the structure where intervertebral disc degeneration begins. Gellan gum-based (GG) hydrogels for acellular and cellular tissue engineering strategies have been developed for finding applications as NP substitutes. The innovative strategy is based on the reinforcement of the hydrogel matrix with biocompatible and biodegradable GG microparticles (MPs), which are expected to improve the mechanical properties, while allowing to tailor its degradation rate. In this study, several GG MPs/hydrogels discs formulations were prepared by means of mixing high (HAGG 0.75% (w/v)) and low acyl (LAGG 2% (w/v)) GG aqueous solutions at different ratios, namely 75%:25% (v/v), 50%:50% (v/v), 25%:75% (v/v), respectively. The GG MPs size was measured using a stereo microscope and their dispersion within the hydrogel matrix was evaluated by means of staining the MPs with Toluidine Blue-O. The developed GG MPs/hydrogel discs were physico-chemically characterized by Fourier-transform infrared spectroscopy and 1H-nuclear magnetic resonance spectroscopy. The swelling behaviour and degradation rate were assessed by immersion in a phosphate buffer saline solution for the period of 14 days. The morphology and mechanical behaviour were investigated by scanning electron microscopy and dynamic mechanical analysis, respectively. The mechanical properties of the hydrogels discs were improved by mixing the gels with the MPs. In addition, the possible cytotoxicity of the leachables released by MPs/hydrogel discs was screened in vitro, using a mouse lung fibroblast cell line (L929 cells). In order to investigate the encapsulation efficacy of L929 cells into the GG MPs/hydrogel discs, cells were stained with DAPI blue/Texas Red-Phalloidin and observed by confocal microscopy, after 24, 48 and 72 hours of culturing. A cell viability assay was also performed using Calcein AM staining. The cell culture studies demonstrated that MPs/hydrogel discs are non-cytotoxic over L929 cells. It was also demonstrated that L929 cells can be successfully encapsulated into the GG MPs of different formulations, remaining viable after 72 hours of culturing. This study showed that GG hydrogel matrices reinforced with cell-loaded MPs could be a candidate strategy for NP regeneration.