TableOfContents

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

Secondary tissues

1. Medial Capsule
2. Lateral Capsule
3. Medial patellofemoral 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.
• Samples will be taken from the mid-substance region of the ligaments and along the long axis of the fibers.
  • attachment:mcl-sample.png Sample MCL

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

• 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.
• 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 PBS bath and tested at room temperature.
• Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).
  • attachment:mach1.png

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 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

• 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 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)
• Move Relative: ramp to 3% strain at 100%/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 100%/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 100%/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.

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.

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

• 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 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 100%/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 100%/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 100%/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 100%/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 100%/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 100%/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 100%/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 100%/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 100%/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 100%/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 100%/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 100%/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).

Capsule

Conditions

• TBD

Measurements

• Force-deformation behaviour

Operating Procedure

• TBD

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

---

Mach-1 limitations and desired features

System limitation: Acceleration/deceleration times too long and affect tissue relaxation behavior. Solution: Software updatde 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.

attachment:Mach-1-v-4.3.1.9-test.pdf

---

Note The information from this point onward will be moved to Discussion page.

Data acquisition by Elvis Danso

Note:

• As part of his pre-doctoral work, Elvis Danso collected mechanical testing data for 30 tissue samples. The findings will be summarized here.
• Tissues tested:
  • Medial/ lateral, femoral and tibial cartilage (indentation and tensile test)
  • Patellar groove, cartilage (indentation and tensile test)
  • Patella (indentation and tensile test)
  • Medial/ lateral menisci (indentation and tensile test)
  • ACL, PCL, MCL, LCL, patellar ligament, quadriceps tendon(tensile test)
• ACL
  • attachment:acl.pdf

Pilot tissue mechanical tests

attachment:sample-list.xls

Sample 1

Sample: Cylindrical compression sample

Tissue: Cartilage

Location: Lateral tibial plateau

Dimensions (measured using LVDT): Thickness = 2.47 mm, Diameter= 5 mm.

Test: Unconfined compression

Data:

attachment:data.txt

Note: Pre-stress value is determined from 1. finding load when the load cell starts accumulating load (as close to zero as possible and as allowed by the load cell resolution) 2.and the area of cross section. For this sample the diameter was measured using a ruler (thickness measurement system is currently not calibrated for >3 mm, need gauges). This prestress value will be used for all the test samples for cartilage compression tests.

Sample 2

Sample: Dumbbell tensile sample

Tissue: Cartilage

Location: Lateral Femoral Condyle

Dimensions (measured using LVDT): Thickness = mm, Length= mm, width = mm

Test: Uniaxial tensile test

Data:

Sample 3

Sample: Dumbbell tensile sample

Tissue: MCL

Location: Mid-substance

Dimensions (measured using LVDT): Thickness = mm, Length= mm, width = mm

Test: Uniaxial tensile test

Data: