Cell Culture Biomaterials Research Articles

Hydrogels with tunable stress relaxation regulate stem cell fate and activity.

Natural extracellular matrices (ECMs) are viscoelastic and exhibit stress relaxation. However, hydrogels used as synthetic ECMs for three-dimensional (3D) culture are typically elastic. Here, we report a materials approach to tune the rate of stress relaxation of hydrogels for 3D culture, independently of the hydrogel’s initial elastic modulus, cell-adhesion-ligand density and degradation. We find that cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) are all enhanced in cells cultured in gels with faster relaxation. Strikingly, MSCs form a mineralized, collagen-1-rich matrix similar to bone in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa. We also show that the effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility and mechanical clustering of adhesion ligands. Our findings highlight stress relaxation as a key characteristic of cell-ECM interactions and as an important design parameter of biomaterials for cell culture.

Pluripotency maintenance of amniotic fluid-derived stem cells cultured on biomaterials.

The stem cell fates of pluripotency and differentiation are regulated by not only soluble biological cues but also insoluble biochemical cues (i.e., extracellular matrix (ECM)) and the physical cues of cell culture biomaterials (i.e., elasticity). We investigated the maintenance of pluripotency and the differentiation lineages of human amniotic fluid-derived stem cells (hAFSCs) cultured on poly(vinyl alcohol-co-itaconic acid) (PVA) hydrogels grafted with several types of ECM and corresponding oligopeptides in expansion medium. hAFSCs cultured on soft PVA hydrogels (12.2 kPa) that were grafted with oligopeptides derived from fibronectin and vitronectin showed high pluripotency, which was evaluated by Oct4, Sox2 and Nanog expression. The hAFSCs grown on soft PVA hydrogels (12.2 kPa) grafted with each oligopeptide showed higher pluripotency, as assessed by Oct4 and Nanog expression, than hAFSCs grown on stiff PVA hydrogels (25.3 kPa) grafted with the same oligopeptides and a much higher pluripotency than those grown on rigid tissue-culture polystyrene dishes. Soft biomaterials appeared to be adequate to maintain the pluripotency of hAFSCs. Surprisingly, hAFSCs that showed higher pluripotency on PVA hydrogels grafted with oligopeptides derived from fibronectin and vitronectin also expressed higher levels of early differentiation markers for three germ layers in expansion medium. This result suggests that hAFSCs are heterogeneous and that this population contains highly pluripotent stem cells and stem cells that can be easily differentiated.

Hyaluronic Acid-Based Hydrogels Crosslinked by Copper-Catalyzed Azide-Alkyne Cycloaddition with Tailorable Mechanical Properties

Biopolymers of the extracellular matrix are attractive starting materials for providing degradable and biocompatible biomaterials. In this study, hyaluronic acid-based hydrogels with tunable mechanical properties were prepared by the use of copper- catalyzed azide-alkyne cycloaddition (known as "click chemistry"). Alkyne-functionalized hyaluronic acid was crosslinked with linkers having two terminal azide functionalities, varying crosslinker density as well as the lengths and rigidity of the linker molecules. By variation of the crosslinker density and crosslinker type, hydrogels with elastic moduli in the range of 0.5-4 kPa were prepared. The washed materials contained a maximum of 6.8 mg copper per kg dry weight and the eluate of the gel crosslinked with diazidostilbene did not show toxic effects on L929 cells. The hyaluronic acid-based hydrogels have potential as biomaterials for cell culture or soft tissue regeneration applications.

Application of two morphologically different fibrillar and filamentous insulin amyloids as a biomaterial for cell culture surfaces

Synthetic biomaterials, such as polymers and self-assembling polypeptides have been developed for cell culture. The self-assembly of misfolded proteins, which is so-called ‘amyloid’, has also been demonstrated to work as a cell adhesive biomaterial. In this study, we demonstrated that two morphologically different insulin amyloids, fibrils and filaments, can be used as biomaterials for cell culture. We previously showed that the cytotoxicity of insulin filaments is markedly lower than that of fibrils. Both types of insulin amyloid-coated dishes showed higher cell adhesion and cell proliferation ability compared to non-coated dishes. Interestingly, insulin filaments showed higher cell adhesion and cell proliferation ability compared to insulin fibrils. These results strongly suggest that insulin amyloids, and insulin filaments in particular, can be used as a biomaterial for cell culture surfaces.

Supramolecular hydrogels inspired by collagen for tissue engineering

Supramolecular hydrogels are promising biomaterials for cell culture in 2-D and 3-D environments. Inspired by the chemical structure of collagen, which bears the repeating tripeptide of glycine-Xaa-4R-hydroxyproline (GXO; Xaa is any one of the natural amino acids), we designed and synthesized a small library of supramolecular hydrogelators (a total of 6). We found that four of the hydrogels were suitable for NIH 3T3 cell culture in the 2-D environments. Gel 2, the best hydrogel, has properties that are similar to those of collagen for 3T3 cell culture. These findings not only provide more supramolecular hydrogel candidates for tissue engineering, but also offer a new strategy for designing biomaterials that mimic nature.

Thermal behavior and mechanical properties of physically crosslinked PVA/Gelatin hydrogels

Poly (vinyl alcohol)/Gelatin hydrogels are under active investigation as potential vascular cell culture biomaterials, tissue models and vascular implants. The PVA/Gelatin hydrogels are physically crosslinked by the freeze-thaw technique, which is followed by a coagulation bath treatment. In this study, the thermal behavior of the gels was examined by differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis (DMTA). Rheological measurement and uniaxial tensile tests revealed key mechanical properties. The role of polymer fraction in relation to these mechanical properties is explored. Gelatin has no significant effect on the thermal behavior of PVA, which indicates that no substantial change occurs in the PVA crystallite due to the presence of gelatin. The glass transition temperature, melting temperature, degree of crystallinity, polymer fraction, storage modulus ( G ′ ) and ultimate strength of one freeze-thaw cycle (1FT) hydrogels are inferior to those of 3FT hydrogels. With coagulation, both 1FT and 3FT hydrogels shifted to a lower value of T g , melting temperature and polymer fraction are further increased and the degree of crystallinity is depressed. The mechanical properties of 1FT, but not 3FT, were strengthened with coagulation treatment. This study gives a detailed investigation of the microstructure formation of PVA/Gelatin hydrogel in each stage of physical treatments which helps us to explain the role of physical treatments in tuning their physical properties for biomechanical applications.