NOTE There are 4 test machines so four groups will perform this experiment at one time.

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1 Laboratory 4: Confined Compression Testing of Articular Cartilage November 20/21, 2002 BIOEN 5201 Introduction to Biomechanics Instructor: Jeff Weiss TA: Carlos Bonifasi-Lista Objective: The objective of this laboratory is to determine the response of bovine articular cartilage under confined compression loading using incremental stress relaxation tests. Relating articular cartilage deformation to tissue health is invaluable to the study of arthritis and cartilage injury/healing. Articular cartilage is a biphasic material, i.e. it consists of two phases. In this case those phases are the solid matrix phase and an interstitial fluid phase, 20 and 80 percent, respectively. The flow of interstitial fluid during use of joints is an important mechanism for the mechanical response of cartilage as well as nutrition. The movement of the interstitial fluid is crucial to the lubrication of joints. NOTE There are 4 test machines so four groups will perform this experiment at one time. Equipment required (each test station): 1 mini materials test machine 1 PC with NI A/D card and NI motor controller card, NI Labview software gram load cell (Sensotec, limit-stop protected, waterproof) 1 confining disc and chamber 1 large sintered steel filter 2 small sintered steel filters Sample height-sizing acrylic block 1 set Allen wrenches Digital calipers 0.9% normal saline in squirt bottle Supplies required: CD-R for data backup Bovine patella Razor blade NOTE BE EXTREMELY CAREFUL WITH THE LOAD CELL! IT CAN BE DESTROYED EASILY VIA OVERLOADING. Load cell calibration enter the load cell calibration factor (Newtons per volt) in the Labview VI based on the calibration page provided with the load cell from the manufacturer (excitation is 2.5 volts) and the information provided by the TA.

2 Harvest of Test Sample If necessary, defrost the patella (Figure 1) by setting it in a deep tray under warm, running water in a ziploc bag, submerging the tissue as much as possible under the warm water bath. Allow the warm water to bathe the plastic bag containing the tissue while the setup continues. If part of it is not submerged in the water bath, be sure to rotate it periodically to allow it to defrost uniformly. Figure 1: Bovine patella. Once the tissue is thawed, use a trephine drill bit in connection with a drill press to core a plug from the cartilage (Figure 2). From the area of the cartilage that was cut around with the trephine bit, use a razor blade to remove this cylinder-shaped piece of cartilage separated on its sides, but still attached at the bottom, by cutting the attachment between the cartilage and the bone toward the bottom. Figure 2: Trephining burr. Place this extracted sample into the sample height-sizing block (acrylic block with several small collinear holes), and cut the top of the specimen off with a razor blade so that it is flush with the top surface of the block. Allow the sample to soak in saline solution for at least 5 minutes before testing begins. Measure the thickness of the sample using digital calipers. Setting up the Test Place the circular sintered steel filter in the bottom of tank. Set the confining chamber on top of the circular filter (Figure 3). Place the cylindrical sintered steel filter plug and cartilage sample in the center of the confinement chamber. Then place another cylindrical sintered steel filter on top of the cartilage sample. Figure 3: Tank, confining chamber and circular filter. This filter should extent at least 2-3 mm past the top of the confining chamber. The cartilage should rest between the two cylindrical sintered steel filters (Figure 4 note that this figure does not show the lower cylindrical filter). Bring the indenter block down until it is just above the tissue. Cartilage Indenter Filter Pour saline solution into the tank until the filters and cartilage are submerged in solution. Filter Confining Chamber Figure 4: Schematic of test configuration. Blue arrows indicate direction of fluid flow.

3 Performing the Incremental Stress Relaxation Test Adjust the load cell balance so that the output is 0 volts. Move the indenter down onto the top filter SLOWLY, IN SMALL INCREMENTS, until the load cell reads approximately 5 grams of load (0.05 N). Reset the encoder position to zero at this point. This will be the starting position for the stress relaxation testing. You will be applying increments of axial compression equivalent to 10% strain. A total of 5 increments will be applied, resulting in an axial strain of 50% at the end of the test. Based on your thickness measurement, determine the required actuator movements. BE CAREFUL YOU ARE DOING A COMPRESSION TEST AGAINST THE BOTTOM OF THE TEST MACHINE AND IF YOU ENTER TOO MUCH DISPLACEMENT YOU WILL DESTROY THE LOAD CELL. You need to make sure that you pick your times for each relaxation test so that the load is as close to equilibrated as possible. The load will equilibrate faster at lower strain levels (e.g. in less than 5 minutes at 10% strain) than at high strain levels (e.g., could take up to ½ hour at 50% strain). Use the custom VI designed by Carlos to apply the incremental strains and record the resulting stress relaxation behavior. Back up load-time and actuator displacement-time data onto CD-R. Cleanup Remove the sample from the acrylic tray and dispose of it in a small, red, autoclavable biomaterial waste bag. Seal the bag and place in the freezer Decant the saline solution from the acrylic chamber, rinse the chamber and tank with water, and then clean with alcohol or other available cleaning agent such as Orthozyme. Clean any instruments used for dissecting or handling tissue with Orthozyme diluted with water in a small dissection tray. Rinse these off with water and let them air dry on a chux. When dry, wipe them with rubbing alcohol. Wipe off the indenter block and freezer lid handle with rubbing alcohol. Wash the dissection tray with antibacterial soap and air dry. Wash counter tops with antibacterial soap and spray with Lysol disinfectant spray when dry.

4 When clean-up is finished, throw away all paper towels and gloves. Data analysis: The objective of the data analysis is to determine both the peak and equilibrium stresses and to determine the relaxation behavior of the cartilage as a function of applied axial compressive strain. Equilibrium stress-strain behavior: Investigators have determined that the finite-strain elastic response of articular cartilage under confined compression can be well-described by the following 1-D constitutive model (which is based on a 3D hyperelastic strain energy formulation): 2 2 e 1 λ 1 T = Hao e β λ 2β 1 2 λ ( 1) Here, H ao is the aggregate modulus of the solid phase and β controls the rate of increase of the slope of the stress-strain curve. Note since the cross-sectional area does not change, the axial Cauchy stress T e and the 1 st P-K stress Π e are equivalent for this test configuration. λ is the stretch ratio or 1-ε, where ε is compressive strain. Using the values for the stress after equilibration of each of the stress-relaxation tests (6 sets including [0,0]), determine the material coefficients H ao and β by performing a nonlinear least squares curve fit to the above equation in Sigmaplot (or equivalent software). You will need to define a user-defined function to do this ( statistics regression wizard select category user-defined ). Or, you can edit an existing function definition from, for instance, the exponential growth group, and then save it as your own function. Carlos can advise on this. Reduced relaxation curves: Create reduced relaxation curves (as per Lab 3) for all 5 incremental stress relaxation tests. Plot them similarly to Lab 3, all on the same graph, with the x-axis as log(time) and the y-axis displaying the value of the reduced relaxation function (which should start at a value of 1 at t=0). Discuss any differences between the relaxation curves at each strain level. Percent relaxation: Make an x-y plot showing the percent relaxation (y-axis) as a function of strain level (x-axis). Time to relax: Make an x-y plot showing the time-to relaxation (y-axis) as a function of strain level (x-axis). Why do you think that relaxation takes longer at higher strain levels? Please follow the formatting guidelines for the previous lab reports.

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