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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