A methylcellulose/agarose hydrogel as an innovative scaffold for tissue engineering

Abstract

In situ crosslinked materials are the main interests of both scientific and industrial research. Methylcellulose (MC) aqueous solution is one of the representatives that belongs to this family of thermosensitive materials. At room temperature, MC is a liquid whereupon during temperature increase up to 37 °C, it crosslinks physically and turns into a hydrogel. This feature makes it unique, especially for tissue engineering applications. However, the crosslinking rate of MC alone is relatively slow considering tissue engineering expectations. According to these expectations, the crosslinking should take place slowly enough to allow for complete injection and fill the injury avoiding clogging in the needle, and simultanously, it should be sufficiently fast to prevent it from relocation from the lesion. One of the methods to overcome this problem is MC blending with another substance that increases the crosslinking rate of MC. In these studies, we used agarose (AGR). These studies aim to investigate the effect of different AGR amounts on MC crosslinking kinetics, and thermal, viscoelastic, and biological properties. Differential Scanning Calorimetry (DSC) and dynamic mechanical analysis (DMA) measurements proved that AGR addition accelerates the beginning of MC crosslinking. This phenomenon resulted from AGR's greater affinity to water, which is crucial in this particular crosslinking part. In vitro tests, carried out using the L929 fibroblast line and mesenchymal stem cells (MSCs), confirmed that most of the hydrogel samples were non-cytotoxic in contact with extracts and directly with cells. Not only does this type of thermosensitive hydrogel system provide excellent mechanical and biological cues but also its stimuli-responsive character provides more novel functionalities for designing innovative scaffold/cell delivery systems for tissue engineering applications.

Fig. 1. DSC heating scans registered for selected compositions of MC without and with the addition of AGR plus example results of the peak deconvolution. Normalization to MC weight.

Fig. 2. Effect of agarose addition on the peak temperature and the peak heat of the deconvoluted peaks: (a) and (d) LT peak, (b) and (e) MT peak and (c) and (f) HT peak.

Fig. 3. Average G′ as a function of time for various concentrations of MC and MC/AGR.

Fig. 4. The time derivative of G′ for various MC and MC/AGR concentrations.

Fig. 5. Crosslinking rate (k) determined from DMA results, vs. the MC/AGR content.

Fig. 6. Final G′ for various MC and MC/AGR concentrations. Statistical significance **p < 0.001, ****p < 0.0001.

Fig. 7. The L929 (a) viability and (b) cell number, determined on MC/AGR hydrogels after 1 and 3 days. Statistical significance: *p < 0.05.

Fig. 8. Fibroblasts distribution in 3D cultures by an FM after 1 and 3 days. The slice views (the bottom of the well) and 3D views show various distributions in 3D culture depending on MC/AGR concentration.

Fig. 9. Representative pictures, rendered by maximum intensity projection (MIP) in orthogonal projection, of hBM-MSCs seeded and cultured for 5 days on the following hydrogel combinations: MC5/AGR1.5, MC3/AGR3, MC5/AGR3.5, and MC5/AGR5.

Fig. 10. Cell distribution in 3D cultures by an FM after 1, 3, and 5 days. The slice views and 3-D views show cell distributions in 3D culture depending on MC/AGR concentration.