-- aerdemir 2015-08-17 13:03:30 Tissue testing protocols are currently being evaluated, more details are available at /ProtocolEvaluation

Contents

Target Outcome
Prerequisites
1. Infrastructure
2. Previous Protocols
Tissue types
1. Primary tissues
2. Secondary tissues
Protocols
Mach-1 limitations and desired features
Testing protocol feasibility assessment and related Mach-1 parameter tuning
References

Target Outcome

Material behavior for all primary and secondary tissues necessary for required representative constitutive models.

Prerequisites

Infrastructure

For more details see Infrastructure/ExperimentationMechanics.

Previous Protocols

For more details see Specifications/Specimens.

For more details see Specifications/SpecimenPreparation.

For more details see Specifications/ExperimentationAnatomicalImaging

For more details see Specifications/ExperimentationJointMechanics

Tissue types

Primary tissues

Cartilage	1. Medial femoral condyle
	2. Lateral femoral condyle
	3. Medial tibial plateau
	4. Lateral tibial plateau
	5. Trochlear groove
	6. Patella
Meniscus	1. Medial meniscus
	2. Lateral meniscus
Ligaments	1. Anterior cruciate ligament
	2. Posterior cruciate ligament
	3. Lateral collateral ligament
	4. Medial collateral ligament
	5. Patellar ligament
Tendon	1. Quadriceps tendon

Secondary tissues

Medial Capsule
Lateral Capsule
Medial patellofemoral ligament
Transverse ligament

Protocols

Ligaments and tendon

Experiment Conditions

Multi-step tensile stress-relaxation test supported by video data to characterize viscoelastic behaviour of the samples.

Measurements

Force-displacement data (unfiltered @ 100 Hz unless downsampled)
Video data (RAW format, 640x480 @ 10 Hz unless downsampled)

Operating Procedures

Sample preparation

Once the ligaments and quadriceps tendon are separated for tissue testing sample preparation, the thicker ligaments (ACL, PCL, MCL and LCL) and tendon will be thinned for tensile testing. This is done using a cryostat (Available at Histochemistry Core at Biomedical Engineering, CCF).
The tissues will be thinned to be under 1 mm.
Once the thin segments are obtained, using a dummbell shaped punch, tensile testing samples will be prepared.
The punch is 10 mm by 2 mm between the flared sections used for clamping the tissue.
MCL is thin enough to be punched without any further thinning.
Punch specifications are provided in infrastructure page.
Samples will be taken from the mid-substance region of the ligaments and along the long axis of the fibers.
- Sample MCL

Thickness measurement

Once the samples are punched out, the thickness of the samples will be measured using the optical thickness measurement system.

Width measurement

Width of the sample can be measured optically (using camera) once the sample is placed in the testing setup.

Note: The dimensions will be recorded in a separate text file in the sample specific folder.

Test set up

The punched sample will be placed in serrated metal clamps for mounting in the tissue testing machine.
Punch details can be found on Infrastructure page.
Tissue adhesive will be used along with the metal clamps to prevent the test samples from slipping during the mechanical tests.
Markers (using a maker pen) will be placed on the sample for video strain measurement.
Calibration for the load cell of the mechanical testing system will be verified before each test.
Specimens will be kept immersed in a PBS bath and tested at room temperature.
Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).

Determination of reference length

For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
Place the complementary puzzle pieces of the clamp in machine and align the clamp heads (not overlapping or touching , align then side by side).

-- colbrunn 2015-10-27 16:12:05 can this be a picture or a diagram?

Set displacement to zero.
Set force to zero
Place sample in the testing system. .
Preload to 10 gf at a loading rate of 0.05 mm/s
Wait: 10 min.
Keep it in force control for 10 minutes at 10 gf.
Record displacement as reference length.
Unload

Testing

A ruler will be placed in the testing chamber to aid optical strain measurement.
Stress relaxation tests will be conducted on each sample.
The protocols with entire sequences are programmed in the system and can be repeated with adjustments based on sample dimensions.
The force/displacement data will be acquired at 100 Hz and the video data is acquired at 10Hz.
- Zero Load
- Find Contact: preload by 10 gf, 0.05 mm/s
- Wait: 600 sec.; 00:10:00.
- Move Relative: to load sample (by 1.5% nominal strain)
- Sinusodial: preconditioning with an amplitude of 1.5% strain for 10 cycles at 2 Hz.
- Move Relative: to unload sample (by 1.5% nominal strain). This will bring it back to initial length found with 10gf.
- Move Relative: ramp to 3% strain at 20%/s.
- Wait: 10 sec.; 00:00:10.
- Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
- Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
- Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
- Move Relative: ramp to 6% strain at 20%/s.
- Wait: 10 sec.; 00:00:10.
- Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
- Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
- Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
- Move Relative: ramp to 9% strain at 20%/s.
- Wait: 10 sec.; 00:00:10.
- Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
- Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
- Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
- Move Relative: to unload sample ( move 5 mm opposite to loading direction)

Note:

Make sure for each step of the test sequences the location of file is appropriately selected.

Data analysis

Once the test is complete, the acquired data is reviewed using the Mach1 Analysis software. The test should be repeated if necessary.
The data can be loaded and visualized in the analysis software, and if the rates and duration for which the rate is applied are as expected, the data will pass the check. Alternatively, the data can be run through the analysis python script to assess the data quality more thoroughly.

Sample removal and storage

Once the test is complete the sample will be carefully removed from the system, wrapped in saline soaked paper towel, placed in an appropriately named ziplock bag, and stored in the freezer in BioRobotics lab.
The sample naming convention can be found in data management wiki page.

Data storage

The collected data will be immediately transferred to Midas (local storage at Cleveland Clinic).

Cartilage

Experiment Conditions

Multi-step tensile stress-relaxation test supported by video data (for tensile tests) to characterize viscoelastic behaviour of the samples.

Measurements

Force-displacement data (unfiltered @ 100 Hz unless downsampled) Video data (RAW format, 640x480 @ 10 Hz unless downsampled)

Operating Procedures

Sample preparation

For details visit Specifications/SpecimenPreparation
Once the tibia and femur articular surfaces are exposed after dissection, rectangular strips of cartilage will be separated from the bone using a scalpel.
Using a 5 mm by 1 mm punch tensile samples will be obtained.
A 5 mm diameter punch will be used to obtain cylindrical, full thickness cartilage sample for confined and unconfined compression tests.
A vibratome will be used to obtain thin uniform thickness samples (settings will be optimized and reported).

Thickness measurement

Once the samples are punched out, the thickness of the samples will be measured using the optical thickness measurement system (OTMS).
The system can be found in room ND1-06A.
For details of development and validation of the OTMS, visit Specifications/ExperimentationTissueThickness

Width measurement

Tensile sample: Width of the sample can be measured optically (using camera) once the sample is placed in the testing setup.

Note: The dimensions will be recorded in a separate text file in the sample specific folder.

Test set up

Confined Compression test

It is recommended that the load cell be calibrated before each test.
The sample will be placed in a confined compression chamber and the whole assembly will be fixed in a saline bath and kept at room temperature during testing.
A stainless steel filter will be placed on the sample and an indentor will be used to compress the sample while it is enclosed in the compression chamber.
Stress relaxation tests will be conducted on each sample.

Unconfined compression test

Same sample will be used for both confined and unconfined compression tests.
The sample will be placed on a flat compression platform and the whole assembly will be fixed in a saline bath and kept at room temperature during testing.
Stress relaxation tests will be conducted on each sample using a flat indentor.
It is recommended that the experimenter wait for at least 30 mins between the two tests.

Tensile test

It is recommended that the load cell be calibrated before each test.
The punched sample will be placed in serrated metal clamps for mounting in the tissue testing machine.
Tissue adhesive will be used along with the metal clamps to prevent the test samples from slipping during the mechanical tests.
Markers (using marker pens) will placed on the sample for video strain measurement.
The load cell of the mechanical testing system will be calibrated before each test.
Specimens will be kept immersed in a saline bath and tested at room temperature.

Determination of reference length

Compression test : Not required
Tensile test
- For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
- Place the complementary puzzle pieces of the clamp in machine and aligh the clamp heads (not overlapping or touching , align then side by side).
- Set displacement to zero.
- Set force to zero
- Place sample in the testing system. .
- Preload to 10 gf at a loading rate of 0.05 mm/s
- Wait: 10 min.
- Record displacement as reference length.
- Unload

Testing

Compression test (confined and unconfined)

Stress relaxation tests will be conducted on each sample.
The protocols with entire sequences are programmed in the system and can be repeated with adjustments based on sample dimensions.
The force/displacement data will be acquired at 100 Hz.
Move Relative: to unload sample (from reference length by 2% nominal strain)
Zero Load
Find Contact: preload by 10 gf, 0.05 mm/s
Wait: 600 sec.; 00:10:00.
Move Relative: to load sample (by 2.5% nominal strain)
Sinusodial: preconditioning with an amplitude of 2.5% strain for 10 cycles at 2 Hz.
Move Relative: to unload sample (by 2.5% nominal strain)
Move Relative: ramp to 5% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: ramp to 10% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: ramp to 15% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: to unload sample (move 2 mm opposite to loading direction)

Tensile test

A ruler will be placed in the testing chamber to aid optical strain measurement.
Stress relaxation tests will be conducted on each sample.
The protocols with entire sequences are programmed in the system and can be repeated with adjustments based on sample dimensions.
The force/displacement data will be acquired at 100 Hz and the video data is acquired at 10Hz .
Recommended: before running any test with longer hold times, the test should be run once with 2-3 sec hold time to ensure the procedure is working as expected.
Move Relative: to unload sample (from reference length by 2% nominal strain)
Zero Load
Find Contact: preload by 10 gf, 0.05 mm/s
Wait: 600 sec.; 00:10:00.
Move Relative: to load sample (by 2.5% nominal strain)
Sinusodial: preconditioning with an amplitude of 2.5% strain for 10 cycles at 2 Hz.
Move Relative: to unload sample (by 2.5% nominal strain)
Move Relative: ramp to 5% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: ramp to 10% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: ramp to 15% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: to unload sample (move 2 mm opposite to loading direction)

Note: Make sure for each step of the test sequences the location of file is appropriately selected.

Data analysis (All tests)

Once the test is complete, the acquired data is reviewed using the Mach1 Analysis software. The test should be repeated if necessary.

Sample removal and storage (All tests)

Once the test is complete the sample will be carefully removed from the system, wrapped in saline soaked paper towel, placed in an appropriately named ziplock bag, and stored in the freezer inBioRobotics lab.

Data storage (All tests)

The collected data will be immediately transferred to Midas (local storage at Cleveland Clinic).

Menisci

Experiment Conditions

Multi-step tensile stress-relaxation test supported by video data (for tensile tests) to characterize viscoelastic behaviour of the samples.

Measurements

Force-displacement data (unfiltered @ 100 Hz unless downsampled) Video data (RAW format, 640x480 @ 10 Hz unless downsampled)

Operating Procedures

Sample preparation

Each meniscus will be divided into three sections radially.
The middle section will be selected so that it is just enough to accommodate the tensile punch (5 mm by 1 mm test length).
Thin strips will be obtained (depth wise) from the middle section and using a 5 mm by 1 mm punch tensile samples will be obtained.
5 mm diameter samples will be punched from the anterior or posterior sections of the menisci and used in compression tests.
A vibratome will be used to obtain thin uniform thickness samples (settings will be optimized and reported).

Thickness measurement

Once the samples are punched out, the thickness of the samples will be measured both optically and using a constant-pressure (~0.001 MPa) linear variable displacement transducer (LVDT) probe system available in ND –.

Width measurement

Tensile sample: Width of the sample can be measured optically (using camera) once the sample is placed in the testing setup.

Note: The dimensions will be recorded in a separate text file in the sample specific folder.

Test set up

Confined Compression test

It is recommended that the load cell be calibrated before each test.
The sample will be placed in a confined compression chamber and the whole assembly will be fixed in a saline bath and kept at room temperature during testing.
A stainless steel filter will be placed on the sample and an indentor will be used to compress the sample while it is enclosed in the compression chamber.
Stress relaxation tests will be conducted on each sample.

Unconfined compression test

Same sample will be used for both confined and unconfined compression tests.
The sample will be placed on a flat compression platform and the whole assembly will be fixed in a saline bath and kept at room temperature during testing.
Stress relaxation tests will be conducted on each sample using a flat indentor.

Tensile test

It is recommended that the load cell be calibrated before each test.
The punched sample will be placed in serrated metal clamps for mounting in the tissue testing machine.
Sand paper and tissue adhesive will be used along with the metal clamps to prevent the test samples from slipping during the mechanical tests.
Markers (india ink) will placed on the sample for video strain measurement. The ink can be found in testing accessories drawer.
The load cell of the mechanical testing system will be calibrated before each test.
Specimens will be kept immersed in a saline bath and tested at room temperature.

Determination of reference length

Compression test : None
Tensile test
- For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
- Place the complementary puzzle pieces of the clamp in machine and aligh the clamp heads (not overlapping or touching , align then side by side).
- Set displacement to zero.
- Set force to zero
- Place sample in the testing system. .
- Preload to 10 gf at a loading rate of 0.05 mm/s
- Wait: 10 min.
- Record displacement as reference length.
- Unload

Testing

Compression test (confined and unconfined)

Stress relaxation tests will be conducted on each sample.
The protocols with entire sequences are programmed in the system and can be repeated with adjustments based on sample dimensions.
The force/displacement data will be acquired at 100 Hz.
Move Relative: to unload sample (from reference length by 2% nominal strain)
Zero Load
Find Contact: preload by 10 gf, 0.05 mm/s
Wait: 600 sec.; 00:10:00.
Move Relative: to load sample (by 2.5% nominal strain)
Sinusodial: preconditioning with an amplitude of 2.5% strain for 10 cycles at 2 Hz.
Move Relative: to unload sample (by 2.5% nominal strain)
Move Relative: ramp to 5% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: ramp to 10% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: ramp to 15% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: to unload sample (move 2 mm opposite to loading direction)

Tensile test

A ruler will be placed in the testing chamber to aid optical strain measurement.
Stress relaxation tests will be conducted on each sample.
The protocols with entire sequences are programmed in the system and can be repeated with adjustments based on sample dimensions.
The force/displacement data will be acquired at 100 Hz and the video data is acquired at 10Hz .
Recommended: before running any test with longer hold times, the test should be run once with 2-3 sec hold time to ensure the procedure is working as expected.
Move Relative: to unload sample (from reference length by 2% nominal strain)
Zero Load
Find Contact: preload by 10 gf, 0.05 mm/s
Wait: 600 sec.; 00:10:00.
Move Relative: to load sample (by 2.5% nominal strain)
Sinusodial: preconditioning with an amplitude of 2.5% strain for 10 cycles at 2 Hz.
Move Relative: to unload sample (by 2.5% nominal strain)
Move Relative: ramp to 5% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: ramp to 10% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: ramp to 15% strain at 20%/s.
Wait: 10 sec.; 00:00:10.
Wait: 100 sec.; 00:01:40 (downsampling at a frequency of 1 Hz for all data).
Wait: 1000 sec.; 00:16:40 (downsampling at a frequency of 0.1 Hz for all data).
Wait: 690 sec.; 00:11:30 (downsampling at 0.01 Hz for all data).
Move Relative: to unload sample (move 2 mm opposite to loading direction)

Note: Make sure for each step of the test sequences the location of file is appropriately selected.

Data analysis (All tests)

Once the test is complete, the acquired data is reviewed using the Mach1 Analysis software. The test should be repeated if necessary.

Sample removal and storage (All tests)

Data storage (All tests)

The collected data will be immediately transferred to Midas (local storage at Cleveland Clinic).

Capsule

Conditions

Measurements

Force-deformation behaviour

Operating Procedure

Mach-1 limitations and desired features

System limitation: Acceleration/deceleration times too long and affect tissue relaxation behavior. Solution: Software update by Biomomentum Inc. to reduce accleration/deceleration times (version 4.3.1.9).

System test: Tests run using a foam sample (unconfined compression). 5% strain applied at 100%/s assuming 1mm, 2mm, 5mm and 12mm.

Mach-1-v-4.3.1.9-test.pdf

Testing protocol feasibility assessment and related Mach-1 parameter tuning

Alternative 1
mach1-capabilities-test.ods
Alternative 2
Preconditioning
Strain rate representation of primary tissues under various loading scenarios/ activities.
Trial tissue samples (non oks) tested at 20%/s stain rate
overview.odt
Evaluation of feasibility, repeatability and reproducibility of test protocols
/ProtocolEvaluation

References

1. Seitz, Andreas Martin, Fabio Galbusera, Carina Krais, Anita Ignatius, and Lutz Dürselen. “Stress-relaxation Response of Human Menisci Under Confined Compression Conditions.” Journal of the Mechanical Behavior of Biomedical Materials 26 (October 2013): 68–80. doi:10.1016/j.jmbbm.2013.05.027. http://www.sciencedirect.com/science/article/pii/S175161611300204X

2. Korhonen RK1, Laasanen MS, Töyräs J, Rieppo J, Hirvonen J, Helminen HJ, Jurvelin JS. "Comparison of the equilibrium response of articular cartilage in unconfined compression, confined compression and indentation." Journal of Biomechanics 2002 Jul;35(7):903-9. http://www.ncbi.nlm.nih.gov/pubmed/12052392

3. Shaokoon Cheng, Elizabeth C. Clarke, Lynne E. Bilston. "The effects of preconditioning strain on measured tissue properties." ournal of Biomechanics 42 (2009) 1360–1362. http://www.ncbi.nlm.nih.gov/pubmed/19394022

4. Mija Lee, William Hyman. "Modeling of failure mode in knee ligaments depending on the strain rate." BMC Musculoskelet Disord. 2002; 3: 3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC65677

5. Duenwald SE1, Vanderby R Jr, Lakes RS. "Stress relaxation and recovery in tendon and ligament: Experiment and modeling." Biorheology. 2010;47(1):1-14. doi: 10.3233/BIR-2010-0559. http://www.ncbi.nlm.nih.gov/pubmed/20448294

6. Subrata Pal. "Mechanical Properties of Biological Materials." Design of Artificial Human Joints & Organs 2014, pp 23-40. http://link.springer.com/chapter/10.1007%2F978-1-4614-6255-2_2