Biodegradable biopolymers are widely used to create matrices for tissue engineering. Protein-based hydrogel materials are known to well mimic the natural cell environment, and thus provide optimal conditions for tissue regeneration. Matrices from regenerated fibroin (Fb) are water-soluble. To prevent solubility of Fb-based matrices and to improve their mechanical properties chemical cross-linking is used. However, it may not be effective enough because of rather low content of primary amino groups in the Fb structure. Several possible conformational states of Fb are known, namely water-soluble α-helices and insoluble β-folded structures. Creating conditions for the α → β conformational transition at the hydrogel formation from regenerated Fb allows to influence the polymer solubility.
The aim of the study was to prepare Fb-based cryostructures cross-linked with genipin and to study their structure, some physicochemical properties, as well as to evaluate their biocompatibility in an in vitro model.
To obtain porous cryostructures (matrices), Fb solutions (20 and 30 wt.%,) were incubated at -10°C for 30 min and freeze-dried for 18 h. The obtained porous matrices were cross-linked by incubation in a genipin solution (10 wt.%) in ethanol for 24 h. Using confocal laser microscopy, the matrices were shown to provide an open-pore system. The mean pore sizes were 149 ± 7 µm and 355 ± 14 µm for the Fb-20 (20 wt.%) and Fb-30 (30 wt.%) cryostructures, respectively. The absence of solubility was ensured both by Fb transformation into the β-conformational state and by chemical cross-linking with genipin in an alcohol medium. Cytotoxicity of fibroin cryostructures was studied by extract test using mouse fibroblasts L929 as model cells. The number of viable cells was determined by MTT-test. Some decrease in cell viability was observed at their incubation in undiluted extracts from the matrices. However, after incubation of the cells with the extracts no changes were found in cell morphology. After diluting the extracts, cell viability increased. To study cell behavior in the matrix, L929 cells and immortalized human mesenchymal stem cells (hTERT-MSC) were seeded in matrices and cultured for 7 days. Cell distribution and morphology in the cryostructures were analyzed by confocal microscopy after 7 days of cultivation. Both hTERT-MSC and L929 cells formed a dense cell monolayer on Fb-20 and Fb-30 cryostructures.