|Ph.D Student||Maya Schnabel Lubovsky|
|Subject||Nano Scale Cell-Matrix Interactions through Amorphous|
Hydrogels Polymeric Networks
|Department||Department of Biomedical Engineering||Supervisors||Professor Seliktar Dror|
|Professor Emeritus Talmon Yeshayahu|
In the body, cells are confined by the extracellular matrix (ECM), a complex 3D array of proteins and polysaccharides, that encapsulates most stromal cells in the body. In this study we used a biosynthetic hydrogel matrix to study the nano-scale interactions associated with the interface between mesenchymal cells and their encapsulating milieu.
The Seliktar research group has developed a biomimetic material that can harness the bioactive properties of fibrinogen, but still retain control over physical features of the material, based on a synthetic polymer conjugate. In this study we conjugated fibrinogen to polyethylene glycol (PEG) polymer to create a biosynthetic precursor with tunable physicochemical properties, based on the ratio between the two constituents. A hydrogel matrix is formed from the biocompatible fibrinogen-polymer adducts by free-radical polymerization using light activation (photopolymerization). For cellular hydrogels, cells were entrapped within the biocompatible hydrogel matrix by light-activated free-radical polymerization from a pre-polymer solution containing dispersed fibroblasts. These materials display irreversible light-activated chemical cross-linking. The synthetic PEG polymer provides control over the matrix properties, whereas the protein provides the biofunctionality needed for the fibroblasts to survive after gelation, including cell adhesion and degradation motifs.
This work is focused specifically on providing the fundamental scientific understanding of cell/biomaterial interactions at the nano-scale. For that goal we used state-of-the-art cryogenic high- resolution scanning electron microscopy (cryo-HRSEM). Until now, investigations of cellular hydrated hydrogels using cryo-HRSEM have not been reported in the literature. The common drying techniques that are usually applied to hydrogels for preparing SEM specimens lead to substantial nano-structural artifacts. For the high-resolution imaging we have developed a modified specimen preparation methodology. The characterization of the fully hydrated cellular hydrogels was performed on freeze-fractured samples. We were able to obtain SEM micrographs with nanoscale resolution, showing intimate contact between cellular processes and the hydrogel ECM.
Contrast enhancement in the highly hydrated ECM was achieved using gadolinium and iodine-containing molecules, which labeled scaffold proteins. The staining process was developed and tested in combination with the state-of-the-art cryo-SEM methodology. This methodology allowed us to study the subcellular interactions occurring at the cell-hydrogel interface, while preserving the native structure of the hydrated polymer network.
Cryogenic transmission electron microscopy (cryo-TEM) was also used for material characterization, validation of the contrast-enhancement methodology, and backing the cryo-SEM observations.
Our findings underscore the need and value of sophisticated methodologies for properly visualizing cell-compatible hydrogels in tissue engineering.