Concentration Independent Modulation of Local Micromechanics in a Fibrin Gel

Abstract

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues.

In situ calibration of position detection in an optical trap for active microrheology in viscous materials

“…One outstanding question in the field of mechanobiology is determining how the tissue mechanics impact cell fate, as many tissues exhibit nonlinear viscoelastic responses [1,11,12]. Optical trap based active microrheology is well suited and is increasingly used for biomechanical measurements [11][12][13][14][15][16]35].…”

“…The technique can accurately measure pN-scale forces at length scales from µm to nm [6,7]. In optical trap-based active microrheology, mechanical properties of the local microenvironment are determined by measuring the induced motion of a trapped particle due to the applied force [8][9][10][11][12][13][14]. Briefly, near infrared light is used to apply spring-like oscillations that drive embedded probes used as local mechanical sensors.…”

Micromechanics of cellularized biopolymer networks

“…Therefore, we expect the micromechanical properties of a cellularized collagen gel to be remodeled by embedded cells (28)(29)(30). To test this hypothesis, we seed strongly contracting mesenchymal cells (mouse fibroblast cells NIH 3T3 and human breast cancer cells MDA-MB-231) in the collagen matrix at low density and measure the micromechanical response around a single isolated cell.…”

Molecular interference of fibrin's divalent polymerization mechanism enables modulation of multiscale material properties

“…Local material response functions within hydrogels were measured by active microrheology (AMR) as previously described [29, 30]. Fibrin gels were polymerized as described in previous sections but with the addition of 2 μm microbeads added to the unpolymerized solution at a concentration of 1 mg/mL.…”