The intervertebral disc central core is made by a gel-like tissue structure composed of more than 80% of water, Nucleus Pulposus (NP).
Proteoglycans such as versican and especially aggrecan are the main
constituents of the NP matrix as well as collagen type II. The purpose
of this work is 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 methacrylated groups addition. In order to optimize some properties of GG, the functionalization
will allow us to improve the water solubility and photopolymerization
in situ of the biomaterial. 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 performed 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 [1]. Mechanism
reactions occurred in presence of glycidyl methacrylate by addition to a
solution of LAGG at 2% (w/v). The reaction was running over 24 h at
room temperature controlling the pH at 8.5 with sodium hydroxide
1 M. GG MPs size was measured using a stereo microscope by 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. It was demonstrated
the non–cytotoxic effect of MPs/hydrogels over L929 cells. In fact,
L929 cells were successfully encapsulated in all GG formulations GG
MPs and remaining viable over 72 h of culturing. The resulting product
from methacrylation reaction was evaluated by nuclear magnetic resonance to assess the reaction efficiency and the degree of substitution.
Methacrylated Gellan gum and GG MP/hydrogel matrix are promising
hydrogels to be used in tissue engineering strategies for treatment of
the degenerated NP.
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