Objectives: The intervertebral disc(IVD) central core is made by a gel-like tissue structure composed of more than 80% of water. This highly hydratated structure is named Nucleus Pulposus (NP). Proteoglycans (PGs) such as versican, biglycan, decorin, fibromodulin, luminican and especially aggrecan are the main constituents of the NP matrix as well as collagen type II. The important role of NP in IVD function relates with the withstanding of loads along spine absorbing shocks. The purpose of this work is focused on creating novel Gellan gum-based (GG) hydrogel formulations. GG microparticles (MPs) dispersed in a GG matrix are the novelty for finding application as NP substitute. The ongoing experiment comprises de GG functionalization through addition of methacrylated groups. Envisioning overcome some drawbacks present in normal GG the functionalization will allow, i.e., the photopolymerization in situ of the biomaterial.
Methods: High acyl (HA) and Low acyl (LA) Gellan Gum (GG) at different ratio (75%:25% (v/v); 50%:50% (v/v), 25%:75% (v/v)), HAGG 0.75% and LAGG 2%, were mixed in order to prepare solutions to be used as formulations of GG MPs/hydrogels matrix. The GG MP/hydrogel matrix formulations were characterized by dynamic mechanical analysis (DMA), swelling behaviour and degradation rate. The toxic effect of GG MPs/hydrogel discs leachables onto the cells was investigated in vitro using a mouse lung fibroblast-like cell (L929 cells) line. Live/Dead cell viability assay was also carried out in order to assess the encapsulation efficacy; meanwhile DAPI/Phalloidin staining was performed to evaluate cell morphology. The Methacrylated Gellan Gum (GG-MA) was prepared following the protocol described in Silva-Correia et al, 2010. Mechanism reactions occurred in presence of glycidyl methacrylate (GMA) by addition to a solution of LAGG at 2% (w/v). The reaction was running over 24hours at room temperature controlling the pH at 8.5 with sodium hydroxide 1 M (NaOH).
Results and Discussion: GG MPs size was measured using a stereo microscope by means of staining the MPs with Toluidine Blue-O. This method also allowed evaluating the MPs dispersion and matrix cohesion. From DMA analysis it was observed that the range of 50-500 mg/mL of incorporated MPs is the optimal concentration to reinforce GG matrices and mimic the natural NP. It was demonstrated the non–cytotoxic effect of MPs/hydrogels over L929 cells. In fact, L929 cells were successfully encapsulated in all formulations of GG MPs and remaining viable over 72 hours of culturing. The resulting product from methacrylation reaction was evaluated by nuclear magnetic resonance (NMR) to assess the reaction efficiency and consequently, the degree of substitution.
Conclusions: Methacrylated Gellan gum and GG MP/hydrogel matrix are promising strategies for finding application as suitable replacement for the degenerated NP.