Engineered extracellular microenvironment with in situ hydrazone-linked hydrogels for tissue engineering

Hydrogels are exceptional biomaterials for a wide range of pharmaceutical and biomedical applications. In fact, in situ formation of hydrogels is desired for minimally invasive techniques for tissue regenerative medicine.... [ view full abstract ]

Hydrogels are exceptional biomaterials for a wide range of pharmaceutical and biomedical applications. In fact, in situ formation of hydrogels is desired for minimally invasive techniques for tissue regenerative medicine. Hydrogels have been used as scaffolds, because of their tissue like properties, biocompatibility, high water content and high permeability for nutrients and metabolites. However, engineering hydrogels with tunable mechanical properties is crucial since the extracellular matrix surrounding cells, provides mechanical signals to regulate a variety of cell behaviors.

Here, in situ crosslinkable gelatin – gellan gum hydrogels were developed via hydrazone chemistry. Hydrazide modified gelatin was synthetized in order to form hydrogels through covalent interaction with the aldehydes present in the gellan gum backbone. Aldehydes in gellan gum were generated after the cleavage of vicinal diols with sodium periodate. The hydrogels were characterized mechanically and the stability was studied in cell culture medium. To evaluate cell biocompatibility, human fibroblasts were exposed with the different gelatins and hydrogel formulations.  Live/dead staining was used to visually assess cell viability and morphology of fibroblast encapsulated in hydrogels. In addition, elongation and proliferation were evaluated using confocal microscopy and optical projection tomography. Finally, the interaction of cardiomyocytes with the hydrogels was studied in 2D and 3D models.

Hydrazide modification of gelatin and oxidation of gellan gum facilitate the spontaneous covalent bonding between gelatin and gellan gum, aiding rapid gelation with and homogeneous crosslinking distribution. In addition, gelation time and mechanical properties can be controlled by varying the concentration of the components. During compression, the hydrogels showed elastic behavior, which is similar to real soft tissue. Hydrogels showed good cytocompatibility and provided a suitable microenviroment for cell culturing. In addition, human fibroblast showed elongated morphologies after 24h incubation.  Preliminary study with human induced pluripotent stem cell derived cardiomyocytes showed favorable cell response to the hydrogels since they are beating spontaneously in 2D and 3D in vitro studies

Thus, we can create tunable 3D microenvironments for the cell spreading and proliferation. Our work may provide insight into the design of biomaterials for cardiac cell culture as well as tissue regeneration.