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Biomaterials

Biomaterials - Poster Session E

Poster C3 - The Influence of Substrate Stiffness and Geometrical Constraints on the Hippo Pathway

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Mechanobiology investigates the impact of mechanical forces on biological systems, with substrate stiffness being a crucial factor influencing cell behavior. Bone marrow-derived mesenchymal stem cells (BD-MSCs) are multipotent cells capable of differentiating into various cell types and have been shown to differentiate and proliferate differently depending on substrate stiffness. The YAP/TAZ protein plays a primary role in this process but its role under varying stiffness and geometrical constraints is not fully understood. This study seeks to investigate BD-MSC proliferation on polyacrylamide hydrogel substrates with varying stiffnesses and geometries to examine the role of the Hippo signaling pathway, specifically YAP/TAZ activity. To fully understand the impact of nuclear YAP localization on cellular mechanical pathways, it is essential to understand the process of YAP translocation. This translocation occurs due to ECM cohesion factors which inactivate the Hippo pathway and result in the movement of YAP to the nucleus. One major influence on ECM modification is substrate stiffness. By incorporating geometry as a factor into hydrogels, stiffness gradients and boundaries are created, resulting in cell shapes that would not exist on non-patterned substrates. To achieve this, macro-topological shapes were introduced by utilizing a protein layer between the hydrogel and BD-MSCs. This allows cells to grow into predefined structures, exhibiting different mechanobiology that the stiffness would typically elucidate. Elongated cell shapes express pronounced nuclear YAP localization, while rounded shapes on substrates exhibit less, in correlation with ECM adhesion properties. Introducing specific cell shapes enhances the study by demonstrating how geometric constraints alter cellular pathways.

Materials and

Methods:  Polyacrylamide hydrogel substrates coated with fibronectin were employed to create a range of stiffness from 10 kPa to 100 kPa. In order to instill macro-topological patterns onto the hydrogel substrates, PDMS gel was made on top of spin coated silicon disks to create stamps of varying patterns. The stamps were then used to stamp patterns of fibronectin onto the surface of polyacrylamide gels. BD-MSCs were then passaged onto the hydrogel surfaces and permitted to grow then fixed and stained for actin, nucleus, and YAP protein to visualize the translocation of the protein with cell adhesion factors. Fluorescent microscopy will be used to assess YAP protein localization and activity, utilizing its co-activation properties with actin and the nucleus to visualize nuclear translocation. This method enabled the investigation of how different cell environments, induced by the substrate topography and stiffness, influence BD-MSC proliferation.

Results, Conclusions, and Discussions: Findings regarding the influence of specific shapes on proliferation are pending further fluorescent microscopy analysis for lab geometric samples. Preliminary expectations suggest that YAP nuclear translocation will be influenced both by substrate stiffness and cell shape highlighting the significance of the Hippo signaling pathway manipulation on cell proliferation. Elongated and spread-out cell shapes on non-patterned substrates exhibited more pronounced nuclear YAP localization, while more rounded shapes on non-patterned substrates showed less nuclear YAP. These findings suggest that both substrate stiffness is a critical factor in modulating YAP/TAZ signaling and, consequently, BD-MSC proliferation while shape influence has yet to be determined.
Initial findings confirm that BD-MSC proliferation is significantly influenced by substrate stiffness, extending previous observations to higher stiffness values. The observed increase in YAP nuclear translocation on stiffer substrates underscores the importance of the Hippo signaling pathway in mechanobiological regulation. Furthermore, the introduction of specific cell shapes through macro-topological patterns on substrates indicates that geometric constraints can modulate cellular mechanotransduction pathways and proliferation rates while the extent of which is pending observation. These insights could provide a more comprehensive understanding of how mechanical environments affect BD-MSC behavior, with potential implications for tissue engineering and regenerative medicine.
The modification of cell geometry and stiffness examined in this study highlights the nuanced response of BD-MSCs to mechanical cues, emphasizing the critical role of substrate environment in mechanical pathway dependent proteins such as YAP/TAZ. The enhanced YAP/TAZ signaling on stiffer substrates suggests a mechanotransductive pathway that could be targeted for therapeutic interventions. Future research should further explore the molecular mechanisms underlying these observations and consider the potential for manipulating substrate stiffness or alternate geometries in clinical applications to optimize stem cell therapies. Additionally, the findings on the influence of specific cell shapes suggest that geometric constraints further influence cellular behavior, highlighting the need to consider both mechanical forces and substrate geometry in mechanobiological studies. Understanding these dynamics is expected to provide deeper insights into BD-MSC behavior in various mechanical environments and inform further research in biomaterials engineering and the study of other cell types and tissues.

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