Speaker
Description
INTRODUCTION
Biophysical parameters are integral to various pathophysiologies, such as fibrosis and cancer. Systematic investigations of such parameters and how they influence cell behaviour and disease progression are feasible in 3D in vitro models of disease.
In this project we used collagen I, the most abundant extracellular matrix protein, to engineer 3D constructs, both as soft hydrogels and dense models. Many techniques have been used to explore biomechanics but limitations surrounding methodology and different elastic moduli, makes direct comparison of measurements difficult.
We aimed to establish a reproducible technique to measure shear elastic modulus, as a measure of material stiffness, by oscillatory rheometry.
METHODS
3D Collagen I Scaffolds: Acellular soft and dense collagen type I gels, were manufactured with a starting concentration of 2 mg/ml. To fabricate hydrogels with 0.2% density a mix of collagen type I, 10xMEM and neutralising agent composed of 10 M NaOH was prepared and 1 M HEPES buffer. This mix was set for 15 mins at 37oC.
Dense scaffolds were created by compressing hydrogels using RAFT absorbers (RAFTTM) to expel liquid, for a final collagen density of 10%. All Scaffolds were maintained in RPMI 1640 with 10% FBS 1% P/S, in a humidified atmosphere at 37°C, 5% CO2 air.
Rheology: Amplitude sweep and frequency sweep testing was conducted on collagen type I scaffolds, using the Kinexus Pro+ Rheometer (Netzsch). 20 mm parallel plate geometry with a solvent trap was used, testing at 1Hz frequency and 37°C. Testing determines shear complex modulus (G*), which defines the elastic storage modulus (G’) and viscous loss modulus (G’’), and phase angle (θ). Testing parameters set by normal force (N) or gap size (h). Storage modulus (G’), phase angle (θ), LVE region and stress (σ) strain rate (ẏ) curves were analysed. Moduli were calculated by averaging the last 10 points in the LVE region.
RESULTS
Dense collagen scaffolds had a higher storage modulus, averaging between 578 Pa ±181 (S.D.). Whereas soft gels exhibited lower storage moduli around 33 Pa ± 4.7 (S.D.). Strain stress curves from amplitude sweep indicate a longer linear viscoelasticity region (LVER) in soft scaffolds, with phase angles between 7° and 9°. In comparison, dense gels had phase angles between 10° and 11°.
Frequency sweep testing indicates the collagen scaffolds behave as viscoelastic solids. Setting testing parameters by normal force or gap size showed greater stiffness in dense gels. However, overall storage moduli measurements were higher in all scaffolds when setting by normal force.
CONCLUSION
Collagen density influences the tissue’s mechanical properties as soft scaffolds exhibited greater viscoelasticity compared to dense constructs. These scaffolds can mimic physiologically relevant tissue stiffness, which can be used to investigate the interactions between key cell types and the biophysical microenvironment.
Testing by set normal force resulted in measurements being higher, although those experiments also had much lower gap size compared to experiments set by gap. The sensitivity of the construct’s biomechanics in response to stress is evident. Further understanding of this will guide our design for more biomimetic 3D disease models.
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