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

-- ["aerdemir"] DateTime(2014-09-23T12:08:01Z) What does patellar groove / femoral groove mean? Is the cartilage sample from femur?

Secondary tissues

1. Capsule

-- ["aerdemir"] DateTime(2014-09-23T12:08:01Z) Any other tissue?

Protocols

Ligaments

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)

-- ["aerdemir"] DateTime(2014-09-23T12:23:59Z) We may need to blend test settings and operating procedures together to have a coherent sequence of actions

Ligaments

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)

-- ["aerdemir"] DateTime(2014-09-23T12:23:59Z) We may need to blend test settings and operating procedures together to have a coherent sequence of actions

Operating Procedures

Sample preparation

• Once the ligaments are separated for tissue testing sample preparation, the thicker ligaments (ACL, PCL, MCL and LCL) will be thinned for tensile testing. This is done using a cryostat (Available at Histochemistry Core at Biomedical Engineering, CCF).
• The ligaments 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.

Preparation

• 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 'find contact' step 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 Position
• Zero Load
• Find Contact: preload by 10 gf, 0.05 mm/s
• Wait: 600 sec.; 00:10:00.
• Move Relative: to load sample (by 0.75% nominal strain)
• Sinusodial: preconditioning with an amplitude of 0.75% strain for 10 cycles at 2 Hz.
• Move Relative: to unload sample (by 0.75% 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 (by 10% nominal strain)

Notes:

• Make sure for each step of the test sequences the location of file is appropriately selected.
• It is recommended that once the test is complete, the acquired data is reviewed using the Mach1 Analysis software. The test should be repeated if necessary.
• 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
• The collected data will be immediately transferred to Midas.

-- ["aerdemir"] DateTime(2014-09-23T12:23:59Z) Let's get the description of ligament procedure cleaned up. In following, we will work on the rest of the document using ligament procedure as an example.

-- ["aerdemir"] DateTime(2014-09-23T12:23:59Z) Let's get the description of ligament procedure cleaned up. In following, we will work on the rest of the document using ligament procedure as an example.

Cartilage

Conditions

• Uniaxial tension
  • Preload: 10 gf, 0.05mm/s
  • Wait: 10 min (this is initial length)
  • upload tensile test sequence, adjust parameters based on intial length.
  • Preload: 10 gf,0.05 mm/s
  • Wait: 10 min
  • Preconditioning: amplitude: 2.5% strain, 10 cycles, 2Hz
  • Stress relaxation: 5%, 10%,and 15% strain at 100%/s.
  • Hold time after each strain application will be 30 min
  • Video data will be recorded at 10Hz.
  • Force/ displacement data acquisition frequency will be set to 100Hz.
• Confined compression
  • Preload: 10 gf, 0.05mm/s
  • Wait: 10 min
  • Preconditioning: amplitude: 2.5% strain, 10 cycles, 2Hz
  • Stress relaxation: 5%, 10%, and 15% strain at 100%/s
  • Hold time after each strain application will be 30 min
• Unconfined compression
  • Preload: 10 gf, 0.05mm/s
  • Wait: 10 min
  • Preconditioning: amplitude: 2.5% strain, 10 cycles, 2Hz
  • Stress relaxation: 5%, 10%, and 15% strain at 100%/s
  • Hold time after each strain application will be 30 min

Measurements

• Force-deformation behaviour
• Video data for strain measurement / Poisson's ratio calculation.

Operating Procedure

Note: A typical test day for all three tests from one cartilage location would include testing sample in unconfined compression, then testing a tensile sample followed by retesting the compression sample under confined compression.

• Tensile test
  • 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 vibratome will be used to obtain thin uniform thickness samples (settings will be optimized and reported).
  • Once the samples are punched out, the thickness of the samples will be measured.
  • Decision is still pending on whether the thickness will be measured optically or using a constant-pressure (~0.001 MPa) linear variable displacement transducer (LVDT) probe.
  • Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).
  • The sample will be placed in serrated tensile clamps (using tissue adhesive and sand paper) and the whole assembly will be fixed in a saline bath (in the testing system) and kept at room temperature during testing.
  • Markers (india ink) are placed on the sample for video strain measurement.
  • A ruler is also placed in the testing chamber to aid optical strain measurement.
  • Stress relaxation tests will be conducted on each sample.
• Confined compression
  • once the tibia and femur articular surfaces are exposed after dissection, rectangular strips of cartilage will separated from the bone using a scalpel.
  • A 5 mm diameter punch will be used to obtain cylindrical, full thickness cartilage samples.
  • A vibratome will be used to obtain uniform thickness samples (settings will be optimized and reported).
  • Once the samples are punched out and cut uniformly, the thickness of the samples will be measured.
  • Decision is still pending on whether the thickness will be measured optically or using a constant-pressure (~0.001 MPa) linear variable displacement transducer (LVDT) probe.
  • It is recommended that the load cell be calibrated before each test.
  • Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).
  • 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
  • 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.

Menisci

Conditions

• Uniaxial tension
  • Preload: 10 gf, 0.05mm/s
  • Wait: 10 min (this is initial length)
  • upload tensile test sequence, adjust parameters based on intial length.
  • Preload: 10 gf,0.05 mm/s
  • Wait: 10 min
  • Preconditioning: amplitude: 2.5% strain, 10 cycles, 2Hz
  • Stress relaxation: 5%, 10%,and 15% strain at 100%/s.
  • Hold time after each strain application will be 30 min
  • Video data will be recorded at 10Hz.
  • Force/ displacement data acquisition frequency will be set to 100Hz.

• Confined compression
  • Preload: 10 gf, 0.05mm/s
  • Wait: 10 min
  • Preconditioning: amplitude: 2.5% strain, 10 cycles, 2Hz
  • Stress relaxation: 5%, 10%, and 15% strain at 100%/s
  • Hold time after each strain application will be 30 min
• Unconfined compression
  • Preload: 10 gf, 0.05mm/s
  • Wait: 10 min
  • Preconditioning: amplitude: 2.5% strain, 10 cycles, 2Hz
  • Stress relaxation: 5%, 10%, and 15% strain at 100%/s
  • Hold time after each strain application will be 30 min

Measurements

• Force-deformation behaviour
• Video data for strain measurement / Poisson's ratio calculation.

Operating Procedure

• Tensile test
  • 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 tensile sample will be punched from the circumference.
  • Once the samples are punched out, the thickness of the samples will be measured.
  • Decision is still pending on whether the thickness will be measured optically or using a constant-pressure (~0.001 MPa) linear variable displacement transducer (LVDT) probe.
  • It is recommended that the load cell be calibrated before each test.
  • Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).
  • The sample will be placed in serrated tensile clamps (using tissue adhesive and sand paper) and the whole assembly will be fixed in a saline bath and kept at room temperature during testing.
  • Markers (india ink) are placed on the sample for video strain measurement.
  • A ruler is also placed in the testing chamber to aid optical strain measurement.
  • Stress relaxation tests will be conducted on each sample.
• Confined compression
  • 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 uniform thickness samples (settings will be optimized and reported).
  • Once the samples are punched out and cut uniformly, the thickness of the samples will be measured.
  • Decision is still pending on whether the thickness will be measured optically or using a constant-pressure (~0.001 MPa) linear variable displacement transducer (LVDT) probe.
  • It is recommended that the load cell be calibrated before each test.
  • Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).
  • 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
  • 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.

Tendons

Conditions

• Uniaxial tension
  • Preload: 10 gf, 0.05mm/s
  • Wait: 10 min (this is initial length)
  • upload tensile test sequence, adjust parameters based on initial length.
  • Preload: 10 gf, 0.05 mm/s
  • Wait: 10 min
  • Preconditioning: amplitude: 1.5% strain, 10 cycles, 2Hz.
  • Stress relaxation: 3%, 6%, and 9% strain at 100%/s
  • Hold time after each strain application will be 30 min
  • Video data will be recorded at 10Hz.
  • Force/ displacement data acquisition frequency will be set to 100Hz.

Measurements

• Force-deformation behaviour
• Video data for strain measurement / Poisson's ratio calculation.

Operating Procedure

• Once the tendon is separated for tissue testing sample preparation, it will be thinned for tensile test. This is done using a cryostat (Available at Histochemistry Core at Biomedical Engineering, CCF). The tendon 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 2mm between the flared sections used for clamping the tissue.
• Once the samples are punched out, the thickness of the samples will be measured.
• Decision is still pending on whether the thickness will be measured optically or using a constant-pressure (~0.001 MPa) linear variable displacement transducer (LVDT) probe.
• The 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.
• Tests will be conducted on MA056-V500c material testing machine (Biomomentum Inc, Laval, Québec, Canada).
• Specimens will be kept immersed in a saline bath and kept at room temperature during testing.
• Stress relaxation tests will be conducted on each sample.

Capsule

Conditions

Measurements

• Force-deformation behaviour

Operating Procedure

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

---

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: