With material characteristics that can easily be tuned to mimic the natural tissue environment, hydrogels are interesting biomaterials for tissue engineering. Of particular interest are dynamic hydrogels that provide mechanical cues to encapsulated cells. As highly mechanosensitive cells, chondrocytes are known to respond to mechanical stimulation in native cartilage. In this research, we developed a thermoresponsive polymer by grafting chains of N-isopropylacrylamide (NIPAM) and 2-hydroxylethyl acrylate (HEA) onto a gelatin backbone, and methacrylating (Ma) the HEA groups. The resulting GelNHMa polymer was used to form hydrogels able to provide cyclic mechanical stimulation to encapsulated Articular Cartilage Progenitor Cells (ACPCs) by repeated shrinking and swelling of the material over time (Fig. 1).

 Materials and Methods

GelNHMa was synthesised by grafting chains of NIPAM and HEA onto a bromine-modified gelatin backbone via an atom transfer radical polymerization (ATRP) reaction. Subsequent methacrylation of the HEA moieties yielded the photocrosslinkable polymer GelNHMa. Hydrogels were formed by photo crosslinking on ice, and shrunken by incubation at 37 °C for 24 hours. Cell compatibility was determined by encapsulating ACPCs and monitoring the cell viability and metabolic activity. The shrinking kinetics at 37 °C and swelling kinetics at room temperature (RT) were determined by measuring the hydrogel diameter at regular intervals. A mechanical stimulation cycle was defined as 3 hour swelling at RT followed by 20 hour shrinking at 37 °C (Fig. 2A).

Results

The shrinking extent and gel stability were tuned by adjusting the NIPAM/HEA chain lengths and the degree of methacrylation, respectively, yielding a hydrogel that shrinks up to 65% in volume at 37°C. ACPCs encapsulated in these gels showed good viability in the material, and did not show signs of cellular stress upon repeated shrinking and swelling of the material. Different regimen of swelling-shrinking cycles were then tested for their influence on chondrocyte differentiation and extracellular matrix deposition. Over a period of 29 days in culture, ACPCs were shown to produce cartilaginous ECM, with presence of glycosaminoglycans (Fig. 2B) and collagens (Fig. 2C) detected.

Conclusion

In this study, we developed a thermo-adaptive hydrogel based on the polymer GelNHMa. The material showed good stability, temperature-based shrinkability and cycleability. Encapsulated ACPCs showed good viability and functionality by producing cartilaginous ECM. Current experiments focus on investigating mechanotransduction pathways that are activated upon cyclic stimulation.