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.
Highlights
Hydrogels polymerized from natural, synthetic, or hybrid molecules are commonly used as extracellular matrix (ECM) for the study of cellECM interactions as well as for medically implantable biomaterials and potential scaffolds for tissue regeneration [1,2]
In gels prepared for active microrheology (AMR), 20 ml of a 20 mg/ml solution of 2 mm diameter silica beads and 50 ml fetal bovine serum (FBS) were mixed with the sterile-filtered fibrinogen solution for every 1 ml of gel. 1 ml of this final gel solution was added to 20 ml of polymerization-initiating thrombin (Sigma, 50 U/ml) previously aliquoted into a 35 mm glass bottom Petri dish (No 1.5 glass, Matek)
We found agreement between macro and microrheology of 2.5 mg/ml fibrin gels, but not for 5 mg/ml and 10 mg/ml fibrin, where microrheology reports a softer matrix than macrorheology (Table 1)
Summary
Hydrogels polymerized from natural, synthetic, or hybrid molecules are commonly used as ECMs for the study of cellECM interactions as well as for medically implantable biomaterials and potential scaffolds for tissue regeneration [1,2]. The design of a hydrogel that mimics the physiological microenvironment requires consideration of a multitude of factors including micromechanical properties [3,4,5], biocompatibility, ligand concentration [6], biotransport kinetics, and pore size [7,8,9,10]. Complex interactions between these factors contribute to the transduction of cellular signals, which in turn determines cell survival, proliferation, and phenotype. While phenotypic changes have been demonstrated in such systems [14], their relevance is debatable in the context of understanding basic physiology
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
