The Mechanosensitivity of Type I Collagen to MMP Cleavage

Student: Robert Camp
Department: Mechanical & Industrial Engineering
Advisor: Jeffrey Ruberti

Abstract

In general, the goal will be to quantify collagen’s mechanosensitivity to enzymatic cleavage by MMPs. Data such as the time to cleavage as a function of mechanical load, enzyme bonding, and cleavage force kinetics will be recorded. Using this data, a spring constant curve for collagen will be developed. This understanding can be applied to other potential Mechanomes.

To study collagen’s mechanosensitivity to enzymatic attack, it will be necessary to develop a means to fix a single collagen monomer such that a measurable force may be applied to it. Firstly, the collagen will have to be prepared so that it can be “grabbed”. By using antibodies at either end of a procollagen molecule and covalent functionalization, we will attach a 1.0 μm diameter paramagnetic particle to one end and 2.0 μm diameter nonmagnetic polystyrene bead to the other. This will give two “handles” that can be used to manipulate the procollagen monomer.

I will construct a magnetic trap based on a design by Yan, Skoko, and Marko of the University of Illinois at Chicago.1 This device will be used to manipulate the procollagen. This design uses 3 1.0 mm micropipettes and a 200 μm diameter magnet mounted at the end of a tapered glass rod operating in a 500 μl well that is mounted to a inverted microscope. The first micropipette is the “bead-catching” pipette, has an inside diameter of 2 μm, and is mounted to a micromanipulator. The glass rod with the magnet is also attached to a micromanipulator. The second micropipette is the “loading” pipette and has an inner diameter of 20 μm and is mounted to a manual three-axis manipulator. The final micropipette is the “force-measuring” pipette. It has an inside tip diameter of 2 μm and it is mounted on the microscope at a fixed position.

Using this setup we can inject the double bead attached collagen to the test fixture using the loading pipette. Once in the well, the bead-catching pipette can be used to capture the nonmagnetic bead of the collagen pair using suction. The procollagen bead pair then can be transferred to the force-measuring pipette where it is also held in place with suction. Once it is attached, the pipette containing the magnet can be used to stretch the procollagen monomer and apply a load.

Dr. Ruberti estimates that the nominal force on a single collagen monomer varies widely in the body; this varies from roughly 3.5 pN in the cornea to upwards of 250 to 300 pN in an Achilles tendon. This near-field magnetic tweezer can possibly achieve the forces necessary to simulate these conditions.

In previous studies done by Sun, et al2 it has been shown by using optical tweezers that a collagen molecule can be fully stretched with a force of around 3 pN. To ensure that we will be stretching the monomer only axially, the stage containing the magnet will be translated to pull the monomer to maximum length while applying only nominal loads (less than 10 pN). Now that the procollagen monomer is held perpendicularly between the two pipettes, forces can now be applied in a uniform manner. Forces of 10, 20, 50, 100, 150, 200, 250, and 300 pN will be applied in successive experiments. While the procollagen monomer is being stretched, the MMP solution will be introduced. For each loading condition the cleavage time will be recorded. For each force loading, there will be at least four successful runs, for a total of at least 32 experimental data points.

It is expected that this experiment will demonstrate the relationship between mechanical load and cleavage time. If this experiment demonstrates the cleavage rate is affected by the mechanical load applied to the collagen, Dr. Jeffrey Ruberti’s hypothesis on collagen mechanical properties will be strongly supported. Even if the null hypothesis is shown, this experiment will still supply important data on MMP binding kinetics, MMP binding forces, and MMP cleavage rates on a single collagen molecule.