Crosslinking Kinetics of Methylcellulose Aqueous Solution and Its Potential as a Scaffold for Tissue Engineering

Abstract

Thermosensitive, physically crosslinked injectable hydrogels are in the area of interests of various scientific fields. One of the representatives of this materials group is an aqueous solution of methylcellulose. At ambient conditions, methylcellulose (MC) is a sol while on heating up to 37 °C, MC undergoes physical crosslinking and transforms into a gel. Injectability at room temperature, and crosslinkability during subsequent heating to physiological temperature raises hopes, especially for tissue engineering applications. This research work aimed at studying crosslinking kinetics, thermal, viscoelastic, and biological properties of MC aqueous solution in a broad range of MC concentrations. It was evidenced by Differential Scanning Calorimetry (DSC) that crosslinking of MC is a reversible two-stage process, manifested by the appearance of two endothermic effects, related to the destruction of water cages around methoxy groups, followed by crosslinking via the formation of hydrophobic interactions between methoxy groups in the polymeric chains. The DSC results also allowed the determination of MC crosslinking kinetics. Complementary measurements of MC crosslinking kinetics performed by dynamic mechanical analysis (DMA) provided information on the final storage modulus, which was important from the perspective of tissue engineering applications. Cytotoxicity tests were performed using mouse fibroblasts and showed that MC at low concentration did not cause cytotoxicity. All these efforts allowed to assess MC hydrogel relevance for tissue engineering applications.

Keywords: DMA; DSC; cellular tests; crosslinking kinetics; methylcellulose; thermosensitive hydrogel.

Figure 1

DSC heating scans of MC solutions with various concentrations (indicated)—(a) heat flow normalized to the sample weight, and (b) heat flow normalized to the methylcellulose (MC) weight. Curves subtracted using polynomial. For comparison, the curves are shifted in y-axis.

Figure 2

DSC cooling curves of the MC solutions with various concentrations (indicated) normalized to the MC weight. Curves subtracted using polynomial. For comparison, the curves are shifted in the y-axis.

Figure 3

Deconvolution of the thermal effects registered during heating for MC concentrations—(a) 1.5 wt.% and (b) 9 wt.%.

Figure 4

The absolute value of the heat exchanged upon heating during the exothermic, the low-temperature (endo 1) and the high-temperature (endo 2) endothermic effects versus the MC content.

Figure 5

Phase diagram for various concentrations of MC constructed using the onset temperature.

Figure 6

The rates, k, of water cages formation (exo), their destruction (endo 1), and of crosslinking (endo 2), as a function of—(a) the MC content and (b) the temperature, T_onset.

Figure 7

G′ versus time at 37 °C for several MC concentrations—(a) enlarged view of the low G′ range, and (b) the whole G′ range.

Figure 8

dG′/dt versus time for several MC solutions—(a) enlarged view of low MC concentrations, and (b) the whole dG′/dt range.

Figure 9

Crosslinking rate, k, determined by DMA, versus the MC content.

Figure 10

PrestoBlue cell proliferation results for the MC hydrogel for several MC concentrations (after 3 days).

Figure 11

SEM of L929 cells on (a) tissue culture plastic (TCP) (control) and (b) hydrogel containing 1 wt.% of MC (50 µL volume).

Figure 12

Fluorescence microscopy of stained L929 cells on—(a) TCP (control) and (b) hydrogel containing 1 wt.% MC (50 µL volume).