Mechanically Active and Tunable Extracellular Matrix Fibers

Mechanically Active and Tunable Extracellular Matrix Fibers Introduction Dry fiber mechanical testing method • Protein fibers are found in many contexts, such as in collagen fibers in the extracellular matrix (ECM) • Our goal is to understand components that contribute to fiber mechanical properties and mechanobiology • Ehlers-Danlos Syndrome (EDS) is a • Wet spun fibers are attached to a sacrificial frame connective tissue disorder associated • The frame is clamped, and its edges are melted away with mutations in collagen or collagen• The fiber is stretched and the force in the fiber and the fiber’s associated proteins, such as Aortic displacement are measured using an Instron microtester Carboxypeptidase-Like Protein (ACLP)1 2 ACLP improves fiber mechanical properties • ACLP mutations cause changes in collagen fibril nanoscale structure (A, arrows1) • We used wet-spun fibers of collagen and ACLP to model EDS • Wet-spun and pulled fibers can be used to evaluate how ligand binding to ECM fibers changes with fiber stretch Motivation • Tissue mechanics are altered in disease states (i.e. tumors) • Changes in fiber strain could cause changes in ligand binding, leading to alterations in tissue response to disease Method 1. Treat silicone membrane with aminosilanes and glutaraldehyde 2. Add labeled protein fibers in a grid pattern 3. Stretch membrane 4. Allow ligand to bind 5. Primary and secondary antibody binding 6. Image, then divide antibody image by fiber image Syringe pump • Collagen I (Col I) and Col I + ACLP fibers were spun and tested (A) to obtain stress-strain curves (B, representative curves) • Diameter (C) is measured by imaging the fibers • Modulus is the slope of the linear region of the stress-strain curve, toughness is the area under the curve, and tensile strength is the maximum stress attained • The smaller diameter of Col I + ACLP suggests ACLP may change the microscopic fiber structure Summary • Wet spinning can be used to generate many types of protein fibers • We have developed methods to test single protein fibers • ACLP improves the mechanical properties of collagen I fibers in a dry state • We have developed methods to determine whether ligands have strain dependent binding to a wide variety of fibers • Fibronectin remains strain-sensitive when incorporated into a fiber with another protein (silk), which presents opportunities to combine the mechanical properties of silk with the biological properties of fibronectin Future work Fibronectin is strain-sensitive in a silk-fibronectin fiber Mechanical testing A. Example images for each experimental condition. (i) shows images of the fiber labeled with a peptide that is sensitive to fibronectin strain. (ii) shows labeling of fibronectin or silk within the fiber. (iii) shows the peptide label divided by protein label. Wet Spinning • Wet spinning is used to form protein fibers • A protein solution is extruded out of a glass syringe into a bath of isopropyl alcohol by a syringe pump • In the alcohol bath, water diffuses out of the fiber and the fiber solidifies Determining the impact of fiber strain on ligand binding B. Results. Silkfibronectin fibers have strain-dependent binding to the peptide, while silk fibers do not. This shows fibronectin retains its strainsensitive properties when incorporated into silk fibers. • We have developed a method to test single ECM fibers under wet/hydrated conditions • We will use this system to determine how composition of ECM fibers impacts mechanical properties Strain-Sensitive binding • Using the platform presented here, we are identifying new strain-sensitive ligand-protein binding partners • We will use native tissue to confirm these interactions also occur in a native setting