Synthesis and Characterization of Oxidized Polysaccharides for In Situ Forming Hydrogels

Abstract

Polysaccharides are widely used as building blocks of scaffolds and hydrogels in tissue engineering, which may require their chemical modification to permit crosslinking. The goal of this study was to generate a library of oxidized alginate (oALG) and oxidized hyaluronic acid (oHA) that can be used for in situ gelling hydrogels by covalent reaction between aldehyde groups of the oxidized polysaccharides (oPS) and amino groups of carboxymethyl chitosan (CMC) through imine bond formation. Here, we studied the effect of sodium periodate concentration and reaction time on aldehyde content, molecular weight of derivatives and cytotoxicity of oPS towards 3T3-L1 fibroblasts. It was found that the molecular weights of all oPs decreased with oxidation and that the degree of oxidation was generally higher in oHA than in oALG. Studies showed that only oPs with an oxidation degree above 25% were cytotoxic. Initial studies were also done on the crosslinking of oPs with CMC showing with rheometry that rather soft gels were formed from higher oxidized oPs possessing a moderate cytotoxicity. The results of this study indicate the potential of oALG and oHA for use as in situ gelling hydrogels or inks in bioprinting for application in tissue engineering and controlled release.

Keywords: alginate; cytotoxicity; fibroblasts; hyaluronic acid; hydrogels; in situ gelling; oxidation.

Figure 1

Sodium periodate-mediated oxidation of alginate and hyaluronic acid. The small letters denote the number of repeating units of β-d-mannuronate (m), α-l-guluronate (n; upper part), and hyaluronic acid (n; lower part); the letters o and r denote the number of repeating units for the units of the polysaccharide chain that were oxidized by NaIO₄, while the letters p and q denote the number of repeating units in the polymer chain, that were not oxidized.

Figure 2

ATR-FTIR spectra of native and oxidized derivatives of (A) alginate and (B) hyaluronic acid. The curves are vertically shifted for a better comparison.

Figure 3

Molecular weight distribution of native and oxidized derivatives of (A) alginate and (B) hyaluronic acid as determined by gel permeation chromatography (GPC).

Figure 4

(A) Fluorescence micrographs after staining with vital stain 5(6)-carboxyfluorescein diacetate (CFDA) and (B) viability studies by Q-blue assay of 3T3-L1 fibroblasts incubated in native and oxidized alginate solutions in phosphate buffered saline (PBS) with a concentration of 5 g L⁻¹ for 24 h; scale bar: 10 μm. Data represent mean ± SD values normalized to control, n = 10, * p ≤ 0.05; compared to native alginate (nALG).

Figure 5

(A) Fluorescence micrographs after staining with vital stain CFDA and (B) viability studies by Q-blue assay of 3T3-L1 fibroblasts incubated in native and oxidized hyaluronic acid solutions in PBS with a concentration of 5 g L⁻¹ for 24 h; scale bar: 10 μm. Data represent mean ± SD values normalized to control, n = 10, * p ≤ 0.05; compared to native hyaluronic acid (nHA).

Figure 6

Rheological properties of the hydrogels at 37 °C. Each curve corresponds to the average of three different samples. (A) Crosslinking process represented with storage modulus as a function of reaction time at a frequency of 1 Hz and 0.1% strain. (B) Dependence of the complex modulus (G*) on the strain amplitude at a frequency of 1 Hz of the already crosslinked hydrogels, measured after the crosslinking process. (C,D) Evolution of the storage (G’) and loss (G”) moduli, respectively, as a function of the frequency at 0.1% stain of the crosslinked hydrogels.

Figure 7

Viability studies by Q-blue assay of C3H10T1/2 mouse stem cells incubated in hydrogels, containing carboxymethyl chitosan (CMC) and oxidized hyaluronic acid or oxidized alginate for 24 and 72 h. Data represent mean ± SD values normalized to control, n = 4, * p ≤ 0.05.