(182af) Development of Injectable Hydrogels for Cartilage Tissue Engineering

Articular cartilage is an avascular tissue with limited self-healing capability. Damages to this tissue demand more efficient solutions than the ones currently available and tissue engineering-based approaches are promising in this scenario, combining appropriate choices of scaffolds, cells and bioactive agents. Among scaffold options, thermosensitive hydrogels present the advantage of being injectable, requiring minimally invasive surgical procedures. Two drugs well known to help reliving the pain associated to articular cartilage lesions are dexamethasone (DEX) and sodium diclofenac (SD). Another drug of interest in cartilage treatment is gallic acid (GA), which can crosslink the collagen fibers of the cartilage matrix, avoiding their degradation.

In this context, the objective of the present work was then to develop thermoresponsive hydrogels by combining methylcellulose (MC), carboxymethyl chitosan (C), xanthan gum (X) and carrageenan (CR), and to incorporate the above mentioned bioactive agents in the most promising formulations.

The results showed that glycerol had to be incorporated in the polymer mixtures to decrease the gelling temperature from formulations containing methylcellulose, xanthan gum and carboxymethyl chitosan from around 37 °C to approximately 28 °C, a temperature considered adequate for the intended purpose. A hydrogel consisting of methylcellulose and carrageenan showed a gelling temperature of 40 °C, which was decreased to only 37 °C with the addition of glycerol. The pH of the formulations was also analyzed, and values in the range of 6.9 to 7.4 were targeted. Since both formulations, when prepared in water, showed pH values above 7.9, phosphate buffered saline (PBS) was used alternatively to solubilize the components. The gelling temperature obtained for the formulation consisting of methylcellulose, xanthan gum and carboxymethyl chitosan was practically not affected with the use of PBS and, therefore this formulation was selected for the additional studies.

The drugs were added to the hydrogel at a concentration of 100 µmol/L for DEX and DS, and at 200 µmol/L for GA, and the proliferation of human mesenchymal stem cells (hMSC) in this matrix was then evaluated.

No significant decrease was noticed in MSC viability after 7 days of culture, indicating that the hydrogel is not cytotoxic to the cells. The analysis of DNA content during 28 days in the hydrogel showed that the cells had high mobility in the matrix due to swelling, since after 14 days the number of cells decreased about 3 times in comparison to the time immediately after inoculation. This fact could be positive in cartilage tissue engineering, as the cells could effectively move towards the cartilage defect, enhancing the regeneration of the affected area. The cells which remained trapped in the matrix were able to proliferate, as confirmed by increased DNA content in days 21 and 28 after inoculation, showing that the hydrogel could work as a cell reservoir for relatively long periods.

In conclusion, the hydrogel formulation containing MC, X and C loaded with the tested bioactive agents can be used in cartilage tissue engineering, as it presents adequate gelling temperature, is not cytotoxic and allows appropriate proliferation of the tested cells.

Acknowledgements: The authors acknowledge the support from CAPES-Brazil and CNPq-Brazil.