robert camp mechanical engineering extracellular matrix engineering research laboratory (emerl)...

Robert CampMechanical Engineering

Extracellular Matrix Engineering Research Laboratory(EMERL)

Advisor: Jeff Ruberti

AgendaWho are you?What is Collagen?Why is the mechanosensitivity of collagen

important? (alternately, Why did IGERT give you money?)

How are you going to test this?Where is all this cool work being done?Current StatusConclusions and Question/Answer

Who are you?Robert CampPhD Student, Mechanical EngineeringEducation

Rochester Institute of Technology BS Mechanical/Aerospace Engineering

University of New Hampshire MS Mechanical Engineering

Work ExperienceHigh Energy PhysicsInfrared Optomechanics

What is Collagen?

Principal load bearing and most abundant protein in vertebrate animals

Defined as (GLY-X-Y)n

3 left-handed helical alpha chains form one right-handed helix collagen monomer

Source: Wikipedia.org

Collagen Type I27 different types of collagen found in

natureCollagen Type I is the basis of researchOne of the fibril forming collagens

Type I, II, III, V, XIMonomer Size

300 nanometers long1.5 nanometers in diameter

Source: Wikipedia.org

Collagen Formation

Collagen is secreted from the cell as procollagen

Source: Kadler 1996

Collagen Formation

The N and C terminal propeptides are cleaved to allow self-assembly into fibrils

Entropy driven assembly, but the control of organization is unknown

Source: Kadler 1996

Collagen FormationCollagen fibrils can

grow under mechanical load in vivo

There is no engineering analog for this kind of material

Source: Kadler 1996

Member of the MechanomeMechanome- The constellation of all

molecules that are load responsive in natureKnown: Strain-stabilized against thermal

denaturationSuspected: Stabilized against enzymatic

cleavage by applied loads.This is the hypothesis that I am currently

attempting to disprove

Strain Stabilized CollagenSource:Amit Bhole, 2007

Why is this important?Osteoarthritis

$60 Billion effect on the economyCollagen no longer in tension

Fluid Sacs

Cleaved Collagen

Normal

DegradingCollagen in tension

Escaping Fluid Sacs

Why is this important?Basic scientific understanding of collagenous

matrix development, remodeling, and repairKnown: Our connective tissue self-optimizes

to carry applied loadsIs it the Fibroblast that is smart, or is it the

Collagen?

Fibroblast

Collagen

OR

Why is this important?Enhanced applied science

This mechanism could be exploited by tissue engineers to “sculpt” collagen scaffoldings with collagen monomers, cleavage enzymes, and a applied load

The scaffolding would be load optimized New class of self-optimizing, healable

materialsExploiting the concept of the load-stabilization

of a self-assembling molecule

The goalTo use a magnetic trap to apply a force to a

collagen monomer to see how it effects the enzymatic cleavage rate

How is this going to be done?Near field magnetic trap

Based on a design developed at the University of Illinois, Chicago to apply loads to single DNA Molecules DNA: 50X Longer then Collagen

Collagen

Force Measuring Pipette

Loading Pipette

Bead Catching Pipette

Permanent Magnet

Source: Yan, et al 2004

The magnetic trapUnlike tradition magnetic traps, this one

stretches the sample perpendicular to the image plane, instead of parallel with it

Internal Magnet

Traditional Magnetic Trap

Near Field Magnetic Trap

Not to Scale

The MaterialProcollagen will be

used so that we may exploit the propeptides for bead attachments

The procollagen will be isolated from cultured bovine corneal fibroblasts in the EMERL lab

PropeptidesPropeptides

Source: Wikipedia.org

The beads2 Beads must be covalently attached to the

procollagenBetween 1 and 3 micrometers in diameterOne plastic, one paramagnetic

Procollagen propeptide-antibody-functionalized beadProcollag

en

Paramagnetic Bead

Not Even Close to Scale

Antibody

Different Antibody

Procollagen + Paramagnetic Bead

Plastic Bead

Force Measuring Micropipette

Not to Scale

Source: Sun 2002

TAP

The experiment

Closer, But Still Not to Scale

Magnet Pipette

Once the bead is loaded, the magnet is brought into the cell to apply the load on the procollagen

Paramagnetic Bead

Antibodies removed for clarity

The experiment

Closer, But Still Not to Scale

Matrix metalloproteinase 8 (MMP-8) will be introduced into the cell

MMP is the enzyme that cleaves collagen

MMP-8

The experiment

Closer, But Still Not to Scale

Time to cleavage will be recorded

Four successful runs are required for each load

Applied loads1 pN for control10, 20, 50, 100, 150, 200,

250, 300 pN

The final resultsIf this experiment demonstrates the cleavage

rate is affected by the mechanical load applied to the collagen, our hypothesis on collagen mechanosensitivity 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.

So where is this being done?Extracellular Matrix Engineering Research

Laboratory (EMERL) in 234 EganThe lab is under the Department of

Mechanical Engineering, but our research is more within the auspices of Biomedical Engineering

Primarily research collagen

One of two Nikon T2000 microscopes in the lab

Current StatusWorking with Dr. Shashi Murthy, Chemical

Engineering Department, Northeastern University to determine the best method to attach the procollagen to the beads

Working on a method to calibrate the force measuring pipettes using microfluid flow

Artist Julian Voss-Andreae’s 11 foot tall sculpture of Collagen

“The most exciting phrase to hear in science, the one that heralds new discoveries, isn’t ‘eureka!’, but rather ‘hmmmm, that’s funny’.” Isaac Asimov