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

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 sample 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 @ 2KHz 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

• 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).

    

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 sample (held by clamps) in the testing system.
• Set force to zero
• Preload to 5 gf at a loading rate of 0.005 mm/s
• Wait: 2 min.
• Note the length in mm.
• Record displacement as reference length.
• Unload

• Use force filter.

Testing

• Stress relaxation
  • The protocol for tensile stress relaxation is programmed in the system and can be repeated with adjustments based on sample dimensions.
  • The force/displacement data will be acquired at 2.5 kHz and the video data is acquired at 10Hz.

Note: Do not filter force data.

• Move absolute to the displacement/ position/ length measured from 'initial length' protocol
• 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.
• 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

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).
• 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.

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).

Determination of reference length / thickness

• Compression test : Thickness obtained from OTMS is used to calculate required strains and strain measurements.
• Tensile test
  • For tensile tests the 'initial length' protocol (programmed in Mach1) is performed first to obtain the initial length of the sample.
  • At this point, the zero position should have been set before measuring sample length.
  • Place sample in the testing system.

  • NOTE: Use force filter.

  • Preload to 5 gf at a loading rate of 0.005 mm/s
  • Wait: 10 min.
  • Record displacement as reference length.
  • Unload

Testing

Compression test (confined and unconfined)

• Conduct stress relaxation tests on each sample.
• The protocols with stress relaxation sequences are programmed in the system and can be repeated with adjustments based on sample dimensions.

• Acquire the force/displacement data at 2.5Khz. Do not use force filter

• Move Absolute: to bring sample to the reference initial position @ 0.005 mm/s
• 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

• Conduct stress relaxation tests each sample.
• The protocols with tensile stress relaxation sequences are programmed in the system and can be repeated with adjustments based on sample dimensions.

• The force/displacement data will is acquired at 2.5 kHz and the video data is acquired at 10Hz. Do not use force filter

• Move Absolute: to bring sample to the reference initial length @ 0.005 mm/s
• 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, 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 @ 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)

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).

---

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

---