Introduction: Given the significance of hydrogels as cell-instructive materials, it is important to understand how their chemical and physical properties direct cell fate. Recently, we introduced visible light crosslinking of tyramine-modified hyaluronan (HA-Tyr) as an alternative to previously established horseradish peroxidase (HRP)/H2O2 mediated crosslinking. HA-Tyr provides a broad range of gel mechanics while maintaining bio-functionality (less than 10% HA modification)[1],[2]. We hypothesized, that given the different crosslinking chemistries and gelation mechanisms, mesenchymal stem cell (MSC) behavior on these HA-Tyr hydrogels might be differentially modulated.
Methods: HA-Tyr (6.5% Tyr-substitution) was synthesized as previously described[1]. HA-Tyr films of 7 kPa (3.5% w/v) were prepared enzymatic (HRP) (0.68 mM H2O2/1 Unit/ml HRP) or singlet-oxygen mediated (EO) (0.02% EosinY, visible light 60 sec), or these gelation mechanisms were combined by sequential crosslinking with 0.34 mM H2O2/1 Unit/ml HRP followed by 0.02% EosinY, 30 sec light (HRP/EO). These gels were in-situ modified with RGD (500 μM). Substrates were characterized with proton nuclear magnetic resonance (1H NMR), rheometry and atomic force microscopy (AFM). Juvenile bovine MSCs were plated onto gels (5.000/cm2), cultured for 4 hours and 8 hours, and then fixed and stained for analysis. For CD44 blocking studies, MSCs were incubated with anti-CD44 before plating. Phalloidin, focal adhesions and YAP (a transcriptional regulator) were visualized by immunofluorescence. Cell area and YAP nuclear localization were quantified using ImageJ[3], and focal adhesions quantified with FAAS[4]. The study was repeated three times and significance determined by ANOVA with Tukey`s post hoc test, p<0.05.
Results: 1H NMR analysis showed that low di-tyramine bonds formation was unaffected by the RGD functionalization. Rheometry and AFM demonstrated that both HRP and EO crosslinking mechanisms formed elastic hydrated polymer networks with little stress relaxation. MSC spreading area increased on HRP crosslinked substrates relative to EO matrices.
Monitoring cell spreading on EO substrates showed a plateau after 8 hours relative to HRP and HRP/EO substrates (4 hours). HRP crosslinking led to greater cell focal adhesion length and increased nuclear translocation of YAP compared to EO and HRP/EO gels.
Discussion: The crosslinking chemistry of soft HA-Tyr hydrogels has a substantial impact on MSC behavior. The presence of EO and visible light did not impair RGD functionality, as confirmed by similar MSC behavior on HRP/EO crosslinked substrates (Figure 1 + 2). CD44 blocking studies suggested that MCS behavior is mediated by the crosslinking and RGD rather than direct interactions with the HA (data not shown). Ongoing work is focused on identifying nano-topographical features of HA-Tyr gels and the mechanistic basis that may regulate the behavior and differentiation of MSCs.
Conclusion: These findings highlight the importance of considering the crosslinking chemistry of HA hydrogels as an additional cue that may regulate stem cell fate on these engineered cell-instructive materials.
European Society for Biomaterials (ESB) for the Racquel LeGeros Travel Award for Claudia Loebel; COST Action MP1005 NAMABIO
References:
[1] Loebel, C., et al., Biomacromolecules (2015) DOI: 10.1021/acs.biomac.5b00363
[2] Loebel, C., et al., Carbohydr Polym (2015) 115, 325
[3] Dupont, S., et al., Nature (2011) 474 (7350), 179
[4] Berginski, M. E., and Gomez, S. M., F1000Res (2013) 2, 68