• Specifications
• ExperimentationTissueMechanics

For detailed tissue testing protocol evaluation and all previous attempts, check /ProtocolEvaluation

Protocol evaluation and repeatability assessment summary

Repeatability tests done on two rubber samples, Unconfined compression test (1000 preconditioning cycles, ramp before and after preconditioning, 15% max strain @ 20%/s, 30 min hold, 5mm diameter sample): oks00TR5-rubber-rubber1-01_04-repeatability.pdf Additional 4 repeatability tests done for rubber sample 1 from previous set,unconfined compression test oks00TR5-rubber-01_08-repeatability.pdf Rubber repeatability at lower strain rates oks00TR5-rubber2-01-04-repeatability.pdf Rubber sample 3, unconfined compression rubber3-01_04-repeatability.pdf Rubber sample 3, unconfined compression, 8 tests rubber3_01_08-repeatability.pdf

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
4. Transverse ligament

Protocols

Note: Maintain a readme file in each specimen data folder (both Mach1 and optical thickness measurement system) with all the relevant notes (dimensions, type of test, any other test specific information etc)

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 @ 2.5 KHz unless downsampled)
• Video data (RAW format, 640x480 @ 10 Hz unless downsampled)

Operating Procedures

Sample preparation

• Once the ligaments and quadriceps tendon are harvested , they need to be thinned in order to get 1mm thick (uniform thickness) tensile testing samples. The tissues are thinned using a cryostat. For details of ligament/tendon tissue testing sample preparation, go to Specifications/SpecimenPreparation

• Samples should 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, measure the thickness of the samples using the optical thickness measurement system (OTMS). For details on how to use the OTMS go to Specifications/ExperimentationTissueThickness

• Keep the sample immersed in saline for an hour before measuring the thickness. Make sure that the sample is always kept in saline when it is not being handled for thickness measurement, placing in clamps etc.

Note: Use appropriate file naming convention to name the image file (.jpg). This will automatically reflect in the result .xml and .png files.

Width measurement

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

Test set up

Tensile test

• For zeroing position.
  • Place the complementary puzzle pieces of the clamp in machine and align the clamp heads (not overlapping or touching , align them one behind the other).
  • Set displacement to zero.
  • Note: this sets the zero position of the system using which starting length of the specimen is determined.

• Preparing sample before placing in the testing system
  • Place the punched sample in serrated metal clamps for mounting in the tissue testing machine.
  • Clamp details can be found on Infrastructure page.
  • Use tissue adhesive along with the metal clamps to prevent the test samples from slipping during the mechanical tests.
  • Place markers (using a maker pen) on the sample for video strain measurement.
  • Calibrate the load cell before each test.
  • Keep the specimen immersed in saline bath throughout the testing and test at room temperature.
  • Place a ruler in the testing chamber to aid optical strain measurement. Make sure the ruler and sample are in the same plane when seen from the camera.
  • Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).

    

Testing

Note: The force/displacement data should be acquired at 2.5 kHz and the video data at 10Hz (video data obtained only for stress relaxation)

Tensile test

1. Determination of reference length

• Use force filter.

• For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
• Place sample (held by clamps) in the testing system.
• Set force to zero
• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the length in mm.
• Record displacement as reference length.
• Unload by 1mm

2. Preconditioning

• Do not use force filter.

• The protocol for tensile preconditioning is programmed in the system and can be run with adjustments based on sample dimensions. (t-1r-1000pc-1r-1u)
• Move Absolute: to bring sample to 300 microns from the reference initial position @ 0.005 mm/s
• Move Relative: 6 % nominal strain at 20%/s
• Move Relative: -6 % nominal strain at 20%/s
• Move Relative: to load sample (by 12.5% nominal strain)
• Sinusoidal: preconditioning with an amplitude of 2.5% strain for 1000 cycles at 2 Hz.
• Move Relative: to unload sample (by 12.5% nominal strain)
• Move Relative: 6 % nominal strain at 20%/s
• Move Relative: -6 % nominal strain at 20%/s
• Unload completely by 1mm.

3. Determination of reference length

• Use force filter.

• For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
• Place sample (held by clamps) in the testing system.
• Set force to zero
• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the length in mm.
• Record displacement as reference length.
• Unload by 2mm

4. Stress Relaxation

• Do not use force filter

• Protocol: t-3xrh-1u
• Move Absolute to reference length obtained from previous step - 300 microns.
• Move Relative: ramp to 2% strain at 20%/s for ligament and 2% strain for tendon at 20%/s(all strain and rate measurements based on new reference length).(add 300 microns)
• 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 4% strain at 20%/s for ligament and 4% strain for tendon 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 for ligament and 6% strain for tendon 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 3 mm opposite to loading direction)

Note:

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

• All data is stored in data.txt file in a main data folder. After the test is complete move this file to appropriate folder with the right specimen name. For naming convention go to Specifications/DataManagement

• Maintain a readme.txt file in the specimen master specimen folder to note down any relevant information for all the tests performed for that oks specimen.

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 carefully remove the sample from the system, wrap in saline soaked paper towel, place in an appropriately named ziplock bag, and store in the freezer in BioRobotics lab.

• The sample naming convention can be found in data management wiki page.

Data storage

• Transfer the collected data immediately 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 @ 2.5kHz 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, separate rectangular strips of cartilage from the bone using a scalpel.
• Using a 5 mm by 1 mm punch obtain the tensile sample (full thickness) (Along the fiber orientation in the superficial zone).
• Using a 5 mm diameter punch obtain cylindrical sample (full thickness) for confined and unconfined compression tests.
• Use the vibratome to obtain uniform thickness samples.

• For details go to, Specifications/SpecimenPreparation

Thickness measurement

• Once the samples are punched out, measure the thickness using the optical thickness measurement system (OTMS).
• The set up is located in room ND1-06A.

• For details of use, development and validation of the OTMS, visit Specifications/ExperimentationTissueThickness

• Keep the samples immersed in saline for an hour before measuring the thickness. Make sure that the sample is always kept in saline when it is not being handled for thickness measurement, placing in test set up etc.

Width measurement

• Width of the sample can be measured optically (for tensile samples using camera) once the sample is placed in the testing setup. For compression samples the diameter is assumed to be 5mm (from the punch dimensions)

Test set up

Confined Compression test

• Calibrate the load cell before each test.
• Place the sample in the confined compression test chamber and fix the whole assembly in a saline bath. All tests are done at room temperature.
• Place the stainless steel filter on the sample.
• Use the confined compression indentor to compress the sample while it is enclosed in the compression chamber.
• Test is done at room temperature.

Unconfined compression test

• Calibrate the load cell before the test.
• Use the same sample for both confined and unconfined compression tests.
• Place the sample on a flat compression platform and fix the whole assembly in a saline bath.
• Test is done at room temperature.
• Conduct stress relaxation test using a flat indentor.

Tensile test

• For zeroing position.
  • Calibrate the load cell before each test.
  • Place the complementary puzzle pieces of the clamp in machine and align the clamp heads (not overlapping or touching , align them side by side).
  • Set displacement to zero.
  • Note: this sets the zero position of the system using which starting length of the specimen is determined.

• Preparing sample before placing in the testing system
  • Place the punched sample in serrated metal clamps for mounting in the tissue testing machine.
  • Clamp details can be found on Infrastructure page.
  • Use tissue adhesive along with the metal clamps to prevent the test samples from slipping during the mechanical tests.
  • Place markers (using a maker pen) on the sample for video strain measurement.
  • Keep the specimen immersed in saline bath throughout the testing and test at room temperature.
  • Place a ruler in the testing chamber to aid optical strain measurement. Make sure the ruler and sample are in the same plane when seen from the camera.
  • Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).

Testing

Compression test (confined and unconfined)

1. Determination of reference thickness

• Thickness obtained from OTMS is used to calculate required strains and strain measurements.

• To find the initial position to begin testing use 'find contact' protocol.

• Use force filter.

• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the position.
• Unload by 1mm

2. Preconditioning

• Do not use force filter

• Use compression preconditioning protocol ().
• Move Absolute: to bring sample to 300 microns from the reference initial position @ 0.005 mm/s
• Move Relative: 15 % nominal strain at 20%/s
• Move Relative: -15 % nominal strain at 20%/s
• Move Relative: to load sample (by 12.5% nominal strain)
• Sinusoidal: preconditioning with an amplitude of 2.5% strain for 1000 cycles at 2 Hz.
• Move Relative: to unload sample (by 12.5% nominal strain)
• Move Relative: 15 % nominal strain at 20%/s
• Move Relative: -15 % nominal strain at 20%/s
• Unload completely by 1mm.

NOTE: Check the preconditioning data before proceeding with the stress relaxation test. If the preconditioning data is not satisfactory, try to assess the cause. Usually it is specimen non uniformity for compression samples or the test set up may have a lose component. If after these the reason is not certain, and test cannot be repeated the same day/ next day, freeze the sample until the issues can be resolved.

3. Determination of reference thickness

• Thickness obtained from OTMS is used to calculate required strains and strain measurements.

• Run 'find contact' protocol.

• Use force filter.

• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the position.
• Unload by 1mm

4. Stress Relaxation

• Do not use force filter

• Use protocol c_3xrh_1u
• Move Absolute: 300 microns off of the position obtained by above 'find contact' protocol.
• Move Relative: ramp to 5% strain (plus 300 microns) 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

1. Determination of reference length

• Use force filter.

• For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
• Place sample (held by clamps) in the testing system.
• Set force to zero
• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the length in mm.
• Record displacement as reference length.
• Unload by 2mm

2. Preconditioning

• Do not use force filter.

• The protocol for tensile preconditioning is programmed in the system and can be run with adjustments based on sample dimensions (preconditioning-t).
  • Move absolute to the displacement/ position/ length measured from 'initial length' protocol
  • Move Relative: to load sample (by 7.5% nominal strain)
  • Sinusodial: preconditioning with an amplitude of 7.5% strain for 1000 cycles at 2 Hz.
  • Move Relative: to unload sample (by 7.5% nominal strain). This will bring it back to initial length found with 10gf.
  • Move Relative: by 1mm to further unload sample to no load state

NOTE: Check the preconditioning data before proceeding with the stress relaxation test. If the preconditioning data is not satisfactory, try to assess the cause. Usually it is the grips for tensile samples or the test set up may have a lose component. If after these the reason is not certain, and test cannot be repeated the same day/ next day, freeze the sample until the issues can be resolved.

3. Determination of reference length

• Use force filter.

• For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
• Place sample (held by clamps) in the testing system.
• Set force to zero
• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the length in mm.
• Record displacement as reference length.
• Unload by 2mm

4. Stress Relaxation

• Do not use force filter

• Use protocol t_3xrh_1u
• Move Absolute to reference length obtained from previous step.
• Move Relative: ramp to 5% strain at 20%/s (all strain and rate measurements based on new reference length).
• 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 3 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, review the acquired data using the Mach1 Analysis software. For a more detailed analysis, use the python script developed for tusste mechanical testing data analysis. The test should be repeated if necessary.

Sample removal and storage (All tests)

Once the test is complete, remove the sample carefully from the system, wrap in saline soaked paper towel, place in an appropriately named ziplock bag, and store in the freezer in BioRobotics lab.

Data storage (All tests)

Transfer all collected data immediately 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 @ 2.5 kHz unless downsampled) Video data (RAW format, 640x480 @ 10 Hz unless downsampled)

Operating Procedures

Sample preparation

• Divide each meniscus into three sections radially.
• The middle section should be selected such that it is just enough to accommodate the tensile punch (5 mm by 1 mm test length).
• Obtain thin strips (depth wise) using the vibratome from the middle section and using a 5 mm by 1 mm punch obtain tensile samples.
• Punch 5 mm diameter sample from the anterior or posterior sections of the menisci and use in compression tests.
• Use vibratome to obtain thin uniform thickness samples.

• For details on sample preparation, go to Specifications/SamplePreparation

Thickness measurement

• Once the samples are punched out, measure the thickness using the optical thickness measurement system (OTMS).
• The set up is located in room ND1-06A.

• For details of use, development and validation of the OTMS, visit Specifications/ExperimentationTissueThickness

• Keep the samples immersed in saline for an hour before measuring the thickness. Make sure that the sample is always kept in saline when it is not being handled for thickness measurement, placing in test set up etc.

Width measurement

• Width of the sample can be measured optically (for tensile samples using camera) once the sample is placed in the testing setup. For compression samples the diameter is assumed to be 5mm (from the punch dimensions)

Test set up

Confined Compression test

• Calibrate the load cell before each test.
• Place the sample in the confined compression test chamber and fix the whole assembly in a saline bath. All tests are done at room temperature.
• Place the stainless steel filter on the sample.
• Use the confined compression indentor to compress the sample while it is enclosed in the compression chamber.

Unconfined compression test

• Calibrate the load cell before the test.
• Use the same sample for both confined and unconfined compression tests.
• Place the sample on a flat compression platform and fix the whole assembly in a saline bath.
• Test is done at room temperature.
• Conduct stress relaxation test using a flat indentor.

Tensile test

• For zeroing position.
  • Place the complementary puzzle pieces of the clamp in machine and align the clamp heads (not overlapping or touching , align them side by side).
  • Set displacement to zero.
  • Note: this sets the zero position of the system using which starting length of the specimen is determined.

• Preparing sample before placing in the testing system
  • Place the punched sample in serrated metal clamps for mounting in the tissue testing machine.
  • Clamp details can be found on Infrastructure page.
  • Use tissue adhesive along with the metal clamps to prevent the test samples from slipping during the mechanical tests.
  • Place markers (using a maker pen) on the sample for video strain measurement.
  • Calibrate the load cell before each test.
  • Keep the specimen immersed in PBS bath throughout the testing and test at room temperature.
  • Place a ruler in the testing chamber to aid optical strain measurement. Make sure the ruler and sample are in the same plane when seen from the camera.
  • Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).

Testing

Compression test (confined and unconfined)

1. Determination of reference thickness

• Thickness obtained from OTMS is used to calculate required strains and strain measurements.

• To find the initial position to begin testing use 'find contact' protocol.

• Use force filter.

• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the position.
• Unload by 1mm

2. Preconditioning

• Do not use force filter

• Use compression preconditioning protocol.
• Move Absolute: to bring sample to the reference initial position @ 0.005 mm/s
• Move Relative: to load sample (by 7.5% nominal strain)
• Sinusoidal: preconditioning with an amplitude of 7.5% strain for 1000 cycles at 2 Hz.
• Move Relative: to unload sample (by 7.5% nominal strain)
• Unload completely by 1mm.

3. Determination of reference thickness

• Thickness obtained from OTMS is used to calculate required strains and strain measurements.

• Run 'find contact' protocol.

• Use force filter.

• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the position.
• Unload by 1mm

4. Stress Relaxation

• Do not use force filter

• Move Absolute: to the position obtained by above 'find contact' protocol.
• 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

1. Determination of reference length

• Use force filter.

• For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
• Place sample (held by clamps) in the testing system.
• Set force to zero
• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the length in mm.
• Record displacement as reference length.
• Unload by 2mm

2. Preconditioning

• Do not use force filter.

• The protocol for tensile preconditioning is programmed in the system and can be run with adjustments based on sample dimensions.
  • Move absolute to the displacement/ position/ length measured from 'initial length' protocol
  • Move Relative: to load sample (by 7.5% nominal strain)
  • Sinusodial: preconditioning with an amplitude of 7.5% strain for 1000 cycles at 2 Hz.
  • Move Relative: to unload sample (by 7.5% nominal strain). This will bring it back to initial length found with 10gf.
  • Move Relative: by 1mm to further unload sample to no load state

3. Determination of reference length

• Use force filter.

• For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
• Place sample (held by clamps) in the testing system.
• Set force to zero
• Preload to 10 gf at a loading rate of 0.005 mm/s
• Wait: 1 min.
• Note the length in mm.
• Record displacement as reference length.
• Unload by 2mm

4. Stress Relaxation

• Do not use force filter

• Move Absolute to reference length obtained from previous step.
• Move Relative: ramp to 5% strain at 20%/s (all strain and rate measurements based on new reference length).
• 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 3 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, review the acquired data using the Mach1 Analysis software. For a more detailed analysis, use the python script developed for tusste mechanical testing data analysis. The test should be repeated if necessary.

Sample removal and storage (All tests)

Once the test is complete, remove the sample carefully from the system, wrap in saline soaked paper towel, place in an appropriately named ziplock bag, and store in the freezer in BioRobotics lab.

Data storage (All tests)

Transfer all collected data immediately to Midas (local storage at Cleveland Clinic).

---

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

Cartilage

• Biomechanics of articular cartilage and determination of material properties. XIN L. LU and VAN C. MOW. Med Sci Sports Exerc. 2008.

• A human knee joint model considering fluid pressure and fiber orientation in cartilages and menisci. K.B. Gu, L.P. Li. Med Eng Phs (2011).

• Nonlinear tensile properties of bovine articular cartilage and their variation with age and depth. Charlebois M1, McKee MD, Buschmann MD. J Biomech Eng. 2004.

• Compressive and tensile properties of articular cartilage in axial loading are modulated differently by osmotic environment. Korhonen RK1, Jurvelin JS. Med Eng Phys. 2010.

• Variations in the intrinsic mechanical properties of human articular cartilage with age, degeneration, and water content. Armstrong CG, Mow VC. J Bone Joint Surg Am. 1982.

• Heterogeneity of tibial plateau cartilage in response to a physiological compressive strain rate. Deneweth JM1, Newman KE, Sylvia SM, McLean SG, Arruda EM. J Orthop Res. 2013.

• Anatomic variation of depth-dependent mechanical properties in neonatal bovine articular cartilage. Silverberg JL1, Dillavou S, Bonassar L, Cohen I. J Orthop Res. 2013.

• Biomechanical considerations in the pathogenesis of osteoarthritis of the knee. Heijink A1, Gomoll AH, Madry H, Drobnič M, Filardo G, Espregueira-Mendes J, Van Dijk CN. Knee Surg Sports Traumatol Arthrosc. 2012.

• Comparison of the equilibrium response of articular cartilage in unconfined compression, confined compression and indentation. Korhonen RK1, Laasanen MS, Töyräs J, Rieppo J, Hirvonen J, Helminen HJ, Jurvelin JS. J Biomech. 2002.

• The 'instantaneous' compressive modulus of human articular cartilage in joints of the lower limb. Shepherd DE1, Seedhom BB. Rheumatology (Oxford). 1999.

• Mechanical testing of intra-articular tissues. Relating experiments to physiological function. Christopher D. Smitha, b, Spyros Masourosa, b, Adam M. Hilla, Andrew L. Wallacec, Andrew A. Amisb, c, Anthony M.J. Bulla Current Orthopaedics 2008.

• Macro-, micro- and ultrastructural investigation of how degeneration influences the response of cartilage to loading. Thambyah A1, Zhao JY, Bevill SL, Broom ND.J Biomech. 2016.

• Highly nonlinear stress-relaxation response of articular cartilage in indentation: Importance of collagen nonlinearity. Mäkelä JT1, Korhonen RK2. Osteoarthritis Cartilage. 2007.

• Estimation of mechanical properties of articular cartilage with MRI - dGEMRIC, T2 and T1 imaging in different species with variable stages of maturation. Nissi MJ1, Rieppo J, Töyräs J, Laasanen MS, Kiviranta I, Nieminen MT, Jurvelin JS.Osteoarthritis Cartilage. 2013.

• Noninvasive dualMRI-based strains vary by depth and region in human osteoarthritic articular cartilage. Griebel AJ1, Trippel SB, Neu CP. J Mech Behav Biomed Mater. 2016.

• Mechanical properties of normal and osteoarthritic human articular cartilage. Robinson DL1, Kersh ME, Walsh NC, Ackland DC, de Steiger RN, Pandy MG. Int J Clin Exp Pathol. 2015.

Meniscus

Ligaments

Tendons

Other

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

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