Cartilage Injury Protocol
Dominik Haudenschild at SCORE, July 2008
· Harvest cartilage from bovine knee femoral condyles,
o preferably 9-12 month old animals, up to 20 months is OK but older and the cartilage is too thin, younger and the growth plate is not fused.
o 6mm biopsy punches on condyle, then osteotome to release discs
· Allow to ‘recover’ 24 hours in DMEM/Ascorbate/PenStrep/10% CS
· Treat with fresh media containing +/- 4mM Acrylamide monomer for 48 hours
· Injure on Instron at 250N (this is about 8MPa, causing injury but not crushing cartilage)
o Move head to contact
o Apply 250N in 0.1 seconds
o Hold at 250N for 0.5 seconds
o Move back to contact position in 0.1 seconds
· Stain for Necrosis 2-3 hours later with modified live/dead stain as follows
o Make 1mg/ml EtBr stock, dilute this in media at 40ul/10ml (=Dead Stain)
o Make 1:1000 dilution of Calcein-AM in media (=Live Stain)
o Stain cartilage in ‘dead’ stain for 30-45 minutes
o Cut explant in ½ through center of disc
o Stain in ‘live’ stain for 30 minutes
o Lay cut ends onto glass-bottom dishes and image live/dead on confocal scope
o Place explants in Z-fix for 24 hours, then 70% Ethanol until ready to paraffin embed and stain for caspase activation/parp cleavage
· Stain for Apoptosis 3 days later
o Use modified live/dead stain and paraffin embedding as described above
o Apoptosis is detectable after >6 hours, peaks day 2, and remains detectable to day 3.5
Experimental Design:
|
|
Control |
Acrylamide Treated |
||
|
Necrosis |
Apoptosis |
Necrosis |
Apoptosis |
|
|
Control |
3 explants |
3 explants |
3 |
3 |
|
Injury |
3 |
3 |
3 |
3 |
Tuesday Harvest tissue
Wednesday Start Acrylamide Treatment
Friday 12:pm Injure
Friday 3pm Live/dead for necrosis
Monday Live/dead for apoptosis
Instron Notes
Load cells: 1000lb = ~5,000N capacity
Load cells: 4000lb = ~20,000N
Want Load cell to be in range between 15% to 85% of capacity
· On switch = white switch on back of machine, turn on and allow computer to warm up.
· Directions: + is up, - is down
· On startup, zero is near middle of the 4inch range of motion
· Calibration light is blinking:
o Push calibration button
o Calibration menu
o Calibrate function
o Restore if it is already calibrated
o *Auto – autocalibrates
· Turn on Hydraulics
· High Activator button
o Can move up & down ΔΔ is fast, Δ is slow
· Position sample
o Loosen Torque bolt ½ turn
o Raise or lower with red knobs
o Lock into place with torque wrench
· Set Limits
o 1500N with action “unload” (load)
o 100mm is total travel, so set travel limit to + 15mm, -15mm
· Injury protocol is to find contact manually, then 250N in 100ms, then hold 500ms and return to contact
· Set up testing device
o HAND TIGHTEN NUT ON LOAD CELL to avoid damaging sensitive load cell
o Zero load
§ First, turn of load protection,
§ Calibrate Load Setup
§ Cal
§ Balance
· On Computer Program,
o Control mode = LOAD
o Collect data, enter a new file for each sample (8.3 naming convention of windows)
o Sample at 50Hz
o Don’t save .DAT file each time
o Return MMI Control
