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    Biomechanics

    Defining the Macro- and Nanomechanics of Mechanically Tunable Biomaterials to Improve Control of Cell Behavior

    (D-139) Defining the Macro- and Nanomechanics of Mechanically Tunable Biomaterials to Improve Control of Cell Behavior

    Friday, October 13, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row D - Poster # 139
    Introduction::

    Biomaterials-based tissue engineering approaches have utilized silk fibroin (SF) from the Bombyx mori silkworm because of its biocompatibility and tunable mechanical properties. SF has been used to synthesize several classes of biomaterials including silk gels, sponges, catheters, and nanoparticles. Despite their excellent versatility and cytocompatibility, a known issue with silk materials is that identical cell types cultured within supposedly identical silk constructs and receiving the same biochemical cues from the scaffolds and the media can behave in disparate and unpredictable ways, indicating the cells are potentially receiving different mechanical cues from the scaffolds. In this preliminary study, we set out to understand if the macroscale mechanical properties of silk scaffolds, obtained through traditional mechanical testing, differ from the micro- and nanoscale mechanical properties of the scaffolds, which are the properties the cells are experiencing within their length scale. To evaluate and identify any differences, we utilized Atomic Force Microscopy (AFM) and used extracellular-matrix based gels as a control material, with less batch to batch variability. We hypothesized that the nanomechanical data obtained from AFM imaging would differ than the previously reported macroscale modulus values obtained through tensile and compression testing.   Our goal is to explain and predict the variability in cell behavior that occurs despite cells being grown in the same kind of material (gel and sponge). We hypothesized that observed differences in cell behavior in seemingly identical constructs are due to discrepancies between the macroscale and micro- and nanoscale mechanics of the materials.  



    Materials and Methods:: SF for gels and sponges was prepared from Bombyx mori cocoons using an established, widely standardized protocol2. For ECM-only gels, ECM was isolated from rat quadriceps muscle and prepared by mixing with diH2O at 30 mg/ml and gelling for 30 min at 37°C. ECM coated SF sponges were prepared by saturating SF sponges with 500 μL of 10 mg/ml ECM and allowing gelation for 30 min at 37°C. Mechanical testing of all samples was carried out within 24 hours of preparation. AFM imaging was done in ambient conditions using Bruker’s Dimension FastScan Bio SPM with a 64-bit Nanoscope V Controller utilized in combination with FastScan-B, ScanAsyst-Fluid, to analyze the SF gels, SF sponges and ECM-coated, and PeakForce-HiRs-F-B probes gels, to analyze the ECM gels.  8 to 50 measurements were taken per sample, and a minimum of n=3 samples were evaluated per material, timepoint, and condition. Young’s Modulus values were derived from the resulting force curves using AtomicJ. All gel materials were mechanically analyzed on a custom load frame in the Northwest Tissue Mechanics Lab. SF sponge compression testing was performed on an Instron UTS with a 10N load cell. Samples were preloaded to 0.05N and compressed to 30% strain at 1% strain/sec-1. Young’s modulus values were derived from the slope of the stress-strain curves in the region between 1 and 25% strain. Differences between the Young’s modulus values obtained from AFM and compression testing were evaluated using an unpaired two-tailed T-test in Prism, with significance set to p < 0.05. 

    Results, Conclusions, and Discussions::

    Results: In all SF gels, the Young’s moduli obtained from AFM were significantly higher than those reported from compression testing (p < 0.0001). Similarly, the Young’s moduli obtained through AFM for both the SF sponges and the ECM-coated SF sponges were significantly higher than those obtained through compression testing (p = 0.0227 and p < 0.0001, respectively). However, there was no significant difference in Young’s moduli obtained from ECM gels between AFM and compression testing (p = 0.1712), suggesting the ECM material obtained is more uniform, with less batch-to-batch variability, than the SF.  


    Conclusions: For SF gels, SF sponges, and ECM-coated SF sponges, AFM reported significantly higher Young’s modulus values when compared to the modulus values obtained from compression testing. In ECM gels, although the modulus values obtained through AFM testing were increased, there was no significant difference between the modulus values obtained from AFM and compression testing. The lack of significance is likely due to a small sample size.
    Discussion:     The results of this study highlight the differences that exist in the macromechanical and nanomechanical properties of the observed biomaterials. These findings suggest that traditional macroscale mechanical testing, such as compression testing, can not accurately assess the mechanical properties of biomaterials at the cellular level. To better understand how mechanical properties affect cell behavior, it is likely that the nanomechanical properties, assessed through modalities such as AFM, need to be tested. Understanding how nanomechanical properties can affect cellular behavior would allow for biomaterials with highly tailored mechanical gradients to be synthesized in hopes to steer cellular differentiation towards the regeneration of complex tissues. 



    Acknowledgements (Optional): :

    References (Optional): : We would like to thank the Surface Science Lab for assistance with obtaining and interpreting the nanomechanical measurements, and the Northwest Tissue Mechanics Lab for the use of their custom load frame. We acknowledge support from the Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grants #P20GM103408, P20GM109095, and 1C06RR020533. We also acknowledge support from The Biomolecular Research Center at Boise State, BSU-Biomolecular Research Center, RRID:SCR_019174, with funding from the National Science Foundation, Grants #0619793 and #0923535; the M. J. Murdock Charitable Trust; Lori and Duane Stueckle, and the Idaho State Board of Education.