Patella Plan

Ismail Olumegbon & Annina SaukkoJuly 14th, 2015

Preparation1)Take a patella out of freezer and melt it in the water bath (room temperature, 30 min)2)Saw the patella with the band saw3)Clean the band saw4)Mark the injury sites (diameter 12 mm) and control sites (8mm red)5)Saw the marks for CT with dremel to the bone (dark blue)6)Mark cross underneath the patella to the upper part to mark in which direction the patella is viewed7)take a picture and draw an image of the orientation and injuries of the sample

1)approx. time 1.5 hours

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OCT

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OCT

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OCT measurements procedure:

With the Catheter well connected, air bubble is driven-out using a syringeThe OCT is set to live viewUsing the OCT set-up, the Patella measurement site is kept flat, parallel and close to the catheter.A distance of 1mm is maintain throughout the measurementsA pull back scan is then performed along the line of interest For each pull back, start the pull back at the initial mark on the sample along the direction of the CatheterAt a fixed distance from the start of the pull back, check for alignment using the corresponding OCT slices. The above procedure is repeated for all 13 measurement lines

OCT

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eOCT measurements Timeline:Number of measurements sites: 4Number of OCT measurements lines per site: 2Number of control measurements:5Total number of measurement lines: (4 x 2) + 5 = 13There will be 3 scans per lineTotal number of OCT scans to be measured: 13 x 3 = 39 Approximate time for the OCT measurements is 2 hours

NIR

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NIR

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NIR measurements procedure:

Keep room as dark as possible and switch on the systems, and allow the NIR lamp to warm up for 15 minutes.Optimize the integration time and averaging to be used for the whole experiment.Using the setup, ensure perpendicular alignment of the probe with the region of interest to be measured.Keep the Patella surface covered with PBS soaked gauze. Only open the region of interest, which should be sprayed with PBS every 2 mins.Bring the NIR probe as close as possible to the measurement location, and take the absorbance measurement. Export to ASCII

NIR measurement sites

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eNIR measurements Number of measurements sites: 3, Number of Locations per site: 4Number of controls: 5Total number of measurement locations: 4×3+ 5 = 17Each location will be measured 3 timesTotal number of measurements: 3 x 17 = 51Approximate time of NIR measurements is 2 hours

Mold for Patella When paraffin is cooled enough the

patella the lid, with patella clued on in, is set to the paraffin

Cooling in fridge for 15 min Cooling in freezer for ~1 h After the mold loosens from the plastic

box, the paraffin is shaped so that there is as little paraffin as possible in the edges of the mold

Approx. time 2 hours

Melting of paraffin and other preparation for the mold is done while Ismail is working on NIR measurements

Patella is glued on the lid using epoxy Gauze is used to keep the patella moist 3 coats of kitchen film under and on top

of the patella Painter’s tape is used to ensure the

flatness outside the patella

CT

• 1) Add PBS (50 – 80ml depending on the sample):

• Native CT scan with 200 µm


• remove the PBS

• 2) Add Hexabrix:

• CT scan with 200 µm


• wait for 45 minutes



• remove the Hexabrix

• rinse the patella in PBS (750 ml) for 2 hours

*We still need to decide whether to use verity or dual energy CT

• 3) Add PBS:



• remove PBS

• 4) Add the mixture of Hexabrix and Bi203:



• wait for 45 minutes



• Rinse in PBS (750 ml) for 2 hours

• Approx. time 8 hours

DAY 1Time Task Who Approx. time

8:00 – 11:00 Preparation I 3 hours

11:00 -13:00 NIR I 2 hours

13:00 -15:00 Mold prep. A 2 hours

13:00 – 15:00 PBS degassing I 2 hours

15:00 – 17:00 OCT I 2 hours

17:00 – 01:00 CT A 8 hours

01:00 – 01:15 Checking the thickness of the cartilage

A 15 min

01:15 – 01:30 Finishing A 15 min

total ~ 18 hours I: 9 hours A: 10,5 hours

Cut and holes 2 holes:

depth 0.5 mm, diameter 1.0 mm

depth 1.0 mm, diameter 1.4 mm

Cut: width 3.6 mm

Approx. time 20 min

Chisel for the cut

Trypsin move the trypsin from freezer to the fridge

the day before dilute trypsin with PBS (no EDTA) to 1

mg/ml warm up the trypsin in incubator 15 min to

37.5 degrees screw the patella to the holder glue the tube on the patella with super

glue cover the rest of the patella with moist

(PBS) gauze dose trypsin (10 ml) in the tube move the system to the incubator for 90

min (37.5 degrees) unscrew the patella remove the trypsin carefully detach the tube Remove the glue • approx. time 2 hours

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Shocker

Mechanical damage procedure:

The region of interest on the Patella is kept perpendicular to the drop tower.The mass is gently lowered with a string through the drop tower until it touches the patella surface.The site of interest is re-align until it is parallel with the tip of the impact mass. The impact mass is then raised.

With the shocker gently fixed at the height of interest (63cm), the impact mass is lowered until it reaches this height.The shocker is then gently removed, allowing the impact mass to drop on the sample

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Surface Abrasion procedure:

The Instron is powered on. It is allowed to warm up. The cooler is then switched on.Calibration is done with the load cell in airUpon calibration, the Patella and holder assembly is tightly fixed to the base.The indenter is the manually lowered until the Emory paper (P60) is in contact with the flatly oriented location of interest.Using the Merlin software, a 5% strain is applied on the sample at 0.001mm/secUsing the MAX software, a 1-cycle rotation is then performed by the Instron to abrase the surface in 4sec.

t(s)0 4

DAY 2Time Task Who Appox. time

9:00 – 09:30 Cut & Holes I 30 min

9:30 – 10:15 Mech. inj. I 45 min

10:15 – 11:00 Abrasion I 1 hour

11:00 – 13:00 Trypsin A 2 hours

11:00 – 13:00 PBS Degassing I 2 hours

13:00 – 15:00 NIR I 2 hours

15:00 – 17:00 OCT I 2 hours

17:00 – 01:00 CT A 8 hours

01:00 – 01:15 Finishing the day

A 15 min

total ~ 17 hoursI: 8 hours 15 minsA: 12 hours

Patella Plan

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