INTRODUCTION: There is a growing interest in biomimetic materials which are capable of recapitulating the native extra-cellular environment and drive/guide cell behaviour. Hyaluronan (HA) and fibrin (FB) are two natural extracellular components that each play a pivotal role in modulating cellular behaviour, such as proliferation, migration and differentiation. Cells are capable of sensing their surrounding mechanical environment and responding via a process known as mechanotranduction. This mechanosensitivity involves cytoskeletal deformation and propagation of mechanical signals throughout the cell which is known to affect cell differentiation. We aim to engineer composite hydrogels made of HA and FB that are characterised by different mechanical properties with the ultimate goal of guiding cell response.
METHODS: HA conjugated with tyramine (Ds 11%) was mixed with FB in mass ratios 1:1, 2:1 and 4:1 keeping a constant [FB] (2mg/ml) and varying [HA]. Rheological properties of the composites were evaluated with frequency sweep and stress ramp tests and compared with those of the individual components. Fluorescent confocal microscopy was used to evaluate fibrous fibrin network structure within the hydrogels. Human Mesenchymal Stromal Cells (hMSCs) were encapsulated in HA/FB hydrogels at 3*106 cells/ml and cultured up to 28 days in standard chondrogenic medium. Cell morphology and the organization of actin cytoskeletal structures were evaluated at 1 and 3 days post encapsulation by phalloidin staining and microscopy. RNA was purified from samples cultured for 28 days and gene expression analyses were performed with RT-qPCR.
RESULTS: 1:1 and 2:1 HA/FB composite hydrogels had up to 5-fold higher storage modulus than the sum of the pure components, indicating a synergistic effect of combining HA and FB. This effect disappeared in the condition 4:1 HA/FB whose mechanical properties approximated pure HA. Furthermore, the 1:1 and 2:1 HA/FB composites had similar storage modulus to each other while 4:1 had a higher storage modulus. Confocal microscopy showed fibrous space-spanning network in all composites materials. The morphology of hMSCs cultured in HA/FB hydrogels was different in the different composites. Within 3 days, cells in the 1:1 hydrogel had an elongated morphology with several branches. On the contrary, in the 4:1 condition, cells retained a more spherical phenotype. In all hydrogels, cells expressed typical chondrogenic markers COL2A1, ACAN and SOX9, with a higher expression in hydrogels with higher relative percentage of HA.
DISCUSSION & CONCLUSIONS: By modulating hyaluronan and fibrin relative ratios we were able to engineer composite hydrogels with different mechanical properties. For certain ratios, we observed mechanical reinforcement, possibly linked to a prestressed state of the fibrin network in the presence of higher amounts of hyaluronan. Cells encapsulated in these materials acquired different cytoskeletal conformation within 3 days suggesting that the different HA/FB composites influence early cell response and possibly guide cell differentiation. While all the investigated conditions supported chondrogenic differentiation of MSCs, we could observe a higher expression of chondrogenic markers correspondingly to higher HA percentage in composites materials. This aligns with the expected spherical morphology for chondrocytes which is observed at higher HA concentrations.