Cellular responses to culture substrates with programmable anisotropy
July 14-18, 2019
Physiologically relevant culture substrates are needed to accurately model cell and tissue function in vitro to characterize function in both healthy and altered (diseased) states. In addition to their use as model systems, exerting control over cellular function in a biochemical engineering process through cell-substrate interactions may reveal new ways to increase yield or efficiency. While knowledge of cellular responses to elastic substrates has advanced greatly, it was only recently recognized that cellular interactions with viscous components of networks alters mammalian cell spreading, migration, proliferation, and differentiation. Matrix studies have shown varying results in response to stress relaxation timescales however, indicating that multiple factors contribute to the cell's interpretation of its mechanical microenvironment. We hypothesize that there is an additional, critical design parameter that has not been considered: the length scales over which cells sense mechanical properties. This work seeks to investigate these questions using a new type of culture substrate based on cytocompatible liquid crystalline (LC) polymers. This work focuses on the design, synthesis, and characterization of new biomaterial substrates whose viscoelastic properties can be manipulated by controlling the liquid crystalline (LC) ordering within the material. These materials also have the ability to morph in shape in response to an external stimulus (e.g. light), which may be applied during in vitro culture to result in dynamic culture substrates. A unique feature is that order can be programmed from the molecular scale to the macroscale, which permits study of how cells interact with the substrates across different length scales. To enable these studies, liquid crystallinity must be maintained in a hydrated network, which is inherently challenging because swelling of polymers tends to increase the distance between LC molecules to weaken their ordering. This work prepares new LC networks using Click chemistry, which was selected for its efficiency under mild reaction conditions that can be used to incorporate more sensitive biological molecules. This work seeks to combine the dynamic properties of these LC materials with their low cytotoxicity, stability in a hydrated phase, and ability to be processed into scaffolds and gels for use as hydrated and responsive culture substrates. The goals are to first characterize the impact of composition on liquid crystalline ordering and culture substrate properties before quantifying the impacts of substrate anisotropy and mechanics, programmed at different length scales, on mesenchymal stem cell differentiation. To prepare the materials, alkyne-terminated liquid crystalline monomers (mesogens) and azide-terminated polyether chain extenders (PEO poly(ethylene oxide); PPO poly(propylene oxide)) were synthesized and purified by modifying established reactions. Chain extender molecular weight and composition were varied to afford control over water uptake and LC organization. For one-step LC network synthesis, chemically crosslinked networks were synthesized by polymerizing the mesogens and chain extenders with a tetraazide crosslinker. To enable cell encapsulation, a two-step network synthesis was used, where azide-terminated LC prepolymers were crosslinked in water using multifunctional strained alkyne. Scaffolds were also prepared to enable 3D studies by polymerizing the reactive mixture in the presence of sodium chloride (sieved to 500-600 μm) and extracting the salt once the reaction was complete. All LC networks were found to organize into the smectic phase. By varying the composition and molecular weight of the chain extender, the material’s elastic modulus and stability of the LC phase was tailored. The networks were found to display reversible shape changing, where the films extended in the LC phase and contracted in the isotropic phase. Composition was found to impact the ability of the network to change shape and the amount of strain generated. Additionally, stress relaxation experiments conducted in the hydrated state showed that networks that were isotropic were found to respond elastically, but LC networks displayed more viscous responses. Mesenchymal stem cells incubated with extractable materials displayed no differences in cellular toxicity compared to tissue culture controls. Cells were found to attach and proliferate on the hydrated LC networks, but attachment was found to be about 50% that of the tissue culture plastic. Adsorption of gelatin with fibronectin onto the networks successfully increased cell attachment. Cell spreading and differentiation (adipogenic vs. osteogenic) studies are ongoing at the time of abstract submission. Ultimately, this work lays the synthetic groundwork for a new synthetic platform for LC biomaterials that can be adapted to include biological molecules as well as investigates LC network utility as a dynamic culture substrate.
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Kelly A. Burke, Yongjian Wang, and Ranjan Srivastava, "Cellular responses to culture substrates with programmable anisotropy" in "Biochemical and Molecular Engineering XXI", Christina Chan, Michigan State University, USA Mattheos Koffas, RPI, USA Steffen Schaffer, Evonik Industries, Germany Rashmi Kshirsagar, Biogen, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/biochem_xxi/33