TableOfContents

Target Outcome

Prerequisites

Infrastructure

Previous Protocols

Protocols

Specimen Storage

  1. Specimen Storage
    1. Storage Location
      1. Specimens should be stored in the walk-in freezer on the third floor in room ND3-77B
      2. Serological testing sheets should be scanned and saved in the repository directory upon delivery
      3. A log should be filled out on the data sheet found in ND3-77B with:
        1. Specimen number
        2. PI name being Ahmet Erdemir
        3. Shelf where the leg was placed labeled accordingly
        4. Source: Human
      4. Serological testing sheets for each specimen should be accounted for and should remain in room ND3-77B so long as a specimen still remains in the freezer.
    2. Specimen Packaging
      1. Specimens can be directly placed in the freezer in their original packaging after being procured from the company
      2. Specimens should be packaged as followed after handling it:
        1. Wrap specimen in a blue chuck (found in cabinet in room ND1-04).
        2. Specimen chuck should be labeled with the specimen number, the side (right or left) and what it is (knee)
        3. The wrapped specimen should be placed in a red biohazard bag found in the same cabinet in ND1-04 as the chucks and labeled in the same fashion as the chuck.

-- ["Stemmer"] 7-9-14: Does the wrapped specimen go back in the freezer? Is there another option like a refrigerator or cooler if the researcher still needs to work on the specimen in the near future (like the next day)

Specimen Thawing

  1. Specimen thawing

    1. The specimen should be taken to the BioRobotics Cadaver Lab in room ND1-04 and thawed for 12-18 hours (potentially upto 24 hours) prior to specimen preparation at room temperature.

    2. The specimen should be unwrapped in order to thaw in less than 24 hours.
    3. For thawing periods greater than 24 hours (i.e. through the weekend) leave the specimen wrapped.

Specimen Dissection

  1. Specimen dissection:
    1. Mark proximal and distal regions for tissue removal
      1. Femur: 4.5 inches proximal, measured from the epicondylar axis
      2. Tibia: 4.5 inches distal, measured from the epicondylar axis
    2. Remove all tissue proximal to thigh mark to expose hip.
    3. Isolate the quadriceps tendon by removing tissue around it and keep it hydrated by wrapping it in saline soaked towel. Make sure to leave adequate amount of quadriceps tendon for clamping.
    4. Remove a four inch wide strip of tissue distal to tibia mark to expose tibia and fibula shafts. All the tissue on the tibia and fibula shafts should be removed. The tibia should be exposed as proximal as possible without damaging joint capsule to allow room for plugs (see below).
    5. The rest of the soft-tissue will remain throughout testing due to possible effects peripheral soft-tissue has on joint response as described in attachment:Peripheral_Soft-Tissue_Effects.pdf

Patella Registration and Optotrak Marker Alignment

  1. Collect data to associate Optrorak and registration marker coordinate systems for patella: This can be done in parallel with specimen dissection or before/after the whole experiment is done.
    1. Place an Optotrak marker on a piece of wood.
    2. Place patella base plug on the same piece of wood.
    3. Place patella Optotrak marker on patella base plug.
    4. Secure the wood (not needed but good).
    5. Make sure Optotrak system is calibrated and ready to roll.
    6. Record location of Optotrak markers (wood + patella base plug) (these are measured in global Optrotrak coordinate system)
    7. Replace patella Optotrak marker with patella registration marker assembly on patella base plug.
    8. Record location of Optotrak marker on wood and patella registration marker assembly divots with Optotrak probing device in the order illustrated below.
    9. Remove base plug from wood.
    10. Now, we can establish the transformation matrix between patella registration marker coordinate system and patella Optotrak marker coordinate system.

Optotrak Base Plug Placement on Bones

  1. Base plug placement

    1. The use of an optoelectronic camera system (Optotrak, Northern Digital Inc., Waterloo, Ontario, Canada) requires repeatable placement of the orthopedic research pin markers http://www.ndigital.com/lifesciences/spineresearchpins.php

    2. In order to ensure repeatable placement, three plastic (MRI compatible) fixtures were designed to be rigidly secured to the tibia, femur, and patella throughout experimentation.

    3. The fixtures and markers can be found in the labeled shelves in the BioRobotics Cadaver Lab.

    4. The fixtures are composed of:
      1. A threaded base plug attached to the bone through the use of three brass screws. Bone cement is used to ensure firm connection between the screws and bone. For experiments requiring MRI imaging, polymer based plugs should be used. If MRI imaging is not required, brass plugs are permissible.
      2. A second brass fixture (for motion tracking) can be securely screwed onto the plug through the used of two pins in a repeatable fashion. This brass fixture will hold the Optotrak markers for the tibia and femur (see figure below). For patella it will hold the Optotrak markers OR registration marker assembly.
        • attachment:optotrak_mounting_assembly.png
    5. The fixtures mounted on the bones with caution to the following issues:
      1. Three screws (#3) will secure each fixture to the bone.
      2. Holes should be drilled on the bone to secure the screws, using fixture as a template. Use drill bit diameter ~.088 in; tapping is not needed. Drill length should be short to ensure patella cartilage is not compromised.
      3. Before screwing in, use a k-wire in one hole to hold plug still when screwing in others.
      4. To ensure the screws securing the base plugs to the bone don't loosen.
        1. Make small batches of bone cement (one plug at a time), to avoid cement hardening (clumped up cement is not good; make a new batch). Bone cement should be mixed in the fume hood and masks should be worn.
        2. Dip the screw in the bone cement; wipe off excess cement from screw before inserting.
        3. Screw into the bone prior to hardening.
      5. If a screw shears off, continue as long as 2 screws are fully engaged; leave the sheared one in plug.
      6. If all screws shear then remove screws and try drilling bigger holes first. You may want to test with non-cemented screws first to see if they shear.
      7. Femur - ~4 inches from the epicondylar axis
        1. The fixture should be mounted on the medial side of the femur and the pin holes should be aligned parallel to the femoral shaft with the two screw holes oriented on the anterior side and the single screw hole oriented on the posterior side (brass base plug shown below)
          • attachment:femur_optotrak_mount.png
      8. Tibia - ~3 inches from the epicondylar axis
        1. The fixture should be mounted on the lateral side of the tibia and the pin holes should be aligned parallel to the tibial shaft with the two screw holes oriented on the anterior side and the single screw hole oriented on the posterior side (brass base plug shown below)
          • attachment:tibia_optotrak_mount.png
      9. Patella - on the center of the patella
        1. Expose the bone by cutting the skin on a vertical line and retract to the sides to make room for the plug.

        2. The fixture should be mounted on the anterior side of the patella tibia and the pin holes should be aligned such that the two screw holes are on the inferior side. -- ["aerdemir"] DateTime(2014-11-01T23:42:42Z) Need a figure to show optotrak mount alignment for patella.

Registration Marker Placement on Femur and Tibia

  1. Placement of registration markers
    1. Three spherical markers should be rigidly mounted to the tibia and the femur as close to joint line as possible without damaging ligamentous structures (~2 to 2.5 inches). The volume of placement is simply driven by the field of view constraints of anatomical imaging.
      1. Spherical markers are hollow plastic spheres (10 mm radius ) filled with water based gel and are attached to the bones using plastic screws (#8-32) for the tibia and femur for MRI registration. The screw axis and sphere centers should be concentric. If not, careful handling is necessary to prevent rotation of the markers after their installation on the bone.
        • attachment:spherical_marker.png
    2. Tissue may need to dissected to make room for the markers.
    3. Holes to place the registration markers should be drilled on the bone using drill bit size of 0.136 in. Holes should be tapped for #8-32 using a drill wire gage 29.
    4. Femur spheres should be mounted on the anterior or anteromedial, medial, lateral sides of the bone.
    5. Tibia spheres should be mounted on the medial, lateral, posterior sides of the bone.
      • attachment:knee_with_registration_markers.png
      • Patella registration markers is not needed at this point as their association to patella Optrotrak markers should be obtained as described above.

Optorak Marker Assembly on Bones

  1. Mounting of Optotrak markers:
    1. Place the femur Optotrak marker on femur base plug
    2. Place the tibia Optotrak marker on tibia base plug
      1. The tibia optotrak marker is a rigid assembly consisting of three normal Optotrak markers, allowing for a larger Optotrak measurement view attachment:Tibia_Optotrak_Marker.png
    3. Place the patella Optotrak marker on patella base plug

Acquisition of Anatomical Landmark Locations

  1. Anatomical landmark data collection:
    1. Move specimen to Optotrak measurement view.
    2. Make sure Optotrak system is previously calibrated and ready to roll
    3. Use the digitizing probe to record anatomical locations along with Optotrak marker position/orientation output, measured with respect to the global Optotrak coordinate system for each respective bone
    4. The following anatomical locations should be recorded:
      1. Femur landmarks
        1. F1. Lateral epicondyle of the femur (most lateral point)
        2. F2. Medial epicondyle of the femur (most medial point)
        3. F3-F6. 4 points around the epiphyseal line of the femur
      2. Tibia landmarks
        1. T1. Medial tibial plateau (most medial point)
        2. T2. Lateral tibial plateau (most lateral point)
        3. T3. Medial malleolus of the tibia (most medial point)
        4. T4. Lateral malleolus of the fibula (most lateral point)
      3. Patella landmarks
        1. P1. Most lateral point
        2. P2. Most medial point
        3. P3. Most superior point
        4. P4. Most inferior point
    5. Now, we can describe the anatomical points in their respective Optotrak body coordinate systems.

Acquisition of Registration Marker Locations

  1. Registration marker data collection
    1. Keep specimen within Optotrak measurement view.
    2. Use the digitizing probe to record registration marker locations along with Optotrak marker position/orientation output, measured with respect to the global Optotrak coordinate system for each respective bone
    3. Twelve points on each spherical marker should be digitized such that they are distributed evenly about the sphere surface
    4. The spheres should be digitized in the following order:
      1. Femur: anterior, medial, lateral markers
      2. Tibia: lateral, medial, posterior markers
    5. Now, we can describe registration markers in their respective Optotrak body coordinate systems.

Preparation of Specimen for Anatomical Imaging

  1. Prepare specimen for MRI:
    1. Remove Optotrak markers from their respective base plugs on femur, tibia, and patella
    2. Cut bones

      1. Use a hack saw found in labeled cabinet in the BioRobotics Cadaver Lab

      2. Clamp and cut femur ~7.5 inches proximal to the epicondylar axis. File the end of the bones to prevent sharp edges.
      3. Clamp and cut tibia and fibula ~7.5 inches distal to the epicondylar axis. File the end of the bones to prevent sharp edges.
      4. Secure tibia and fibula to each other using a zip tie and a spacer.
      5. The foot should be preserved for future testing (following storage procedures described above)
      6. Place patella registration marker assembly on patella
    3. Now, the specimen is ready to go through ["Specifications/ExperimentationAnatomicalImaging"]

Preparation of Specimen for Mechanical Joint Testing

  1. Prepare specimen for joint testing
    1. After specimen comes from MRI; the registration markers can be removed.
    2. Potting
      1. Preparation
        1. Safety
          1. Gloves, face shields, gowns, protective eye-wear and welders gloves should be used at all times by people handling hot liquid metal
        2. Melting woods metal

          1. The woods metal found in the hood of the BioRobotics Cadaver Lab should be melted.

          2. For quickly melting it, turn the temperature of the hotplate to 300 degrees until melted.
          3. Once melted:
            1. If the woods metal isn't already in the aluminum pitcher, CAREFULLY pour it into it and place the pitcher on the hotplate.
            2. Turn the temperature to 100 degrees.

        3. Two aluminum tubes (3 inches long, 2.5 inch OD) can be found in the labeled cabinet in the BioRobotics laboratory.

        4. Clay can be found in the BioRobotics laboratory in the first cabinet on the left side of the room. Approximately, a fist full should be taken.

      2. Potting the tibia end.
        1. Before potting, check whether Optotrak markers will fit on plugs with the pot.
        2. Roll the clay flat so that the aluminum tube can sit on it and be completely sealed.
        3. Place the clay on the the dissection table on the blue side of a chuck.
        4. If there are drill-bit holes in the tube, orient the tube on the clay so the hole side is not touching the clay
        5. Seal the bottom of the tube completely with clay and plug any holes with bits of clay
        6. Place tibia in the tube and hold the extended knee in place
          1. Verify the length from the bottom of the tube to the epicondylar axis as ~7.5 inches
          2. Verify the center of the epicondylar axis is inline, vertically, with the bottom of the tube, from both sagittal and anterior views.
        7. Use a beaker to fill the tube with water and refill the beaker.
        8. A foil funnel found in the hood should be held over the top of the tube with forceps by the person holding the knee.
        9. A different person should CAREFULLY ladle the HOT woods metal into the funnel filling the tube completely.
        10. Cold water from the beaker can be poured on the tube several times in order to quickly solidify the woods metal.
        11. Approximately 5 minutes are needed for it to completely harden.
        12. Remove the clay from the bottom of the tube.
        13. Flip the knee so that the bottom of the tibia tube is pointed up.
        14. Top the tube off by CAREFULLY ladling hot woods metal into it.
        15. Pour cold water on top of it again and wait for it to harden.
        16. Using a chisel remove the excess wood metal that hardens over edge of the pot.
        17. Secure the fibula to the tibia by passing a drill bit (or a wood screw) through both bones and leaving it there. Try to keep relative positioning of the tibia and fibula anatomical.
      3. Potting the femur end.
        1. Before potting, check whether Optotrak markers will fit on plugs with the pot.
        2. Roll the clay flat so that the second aluminum tube can sit on it and be completely sealed.
        3. Place the clay on the dissection table on the blue side of a chuck.
        4. If there are drill-bit holes in the tube, orient the tube on the clay so the hole side is not touching the clay
        5. Seal the bottom of the tube completely with clay and plug any holes with bits of clay.
        6. Place femur in the tube and hold the extended knee in place.
          1. Verify the length from the bottom of the tube to the epicondylar axis as ~7.5 inches
          2. Verify the center of both tubes are inline, vertically, from both sagittal and anterior views.
          3. Use a beaker to fill the tube with water and refill the beaker.
        7. A foil funnel found in the hood should be held over the top of the tube with forceps by the person holding the knee.
        8. A different person should CAREFULLY ladle the HOT woods metal into the funnel filling the tube completely.
        9. Cold water from the beaker can be poured on the tube several times in order to quickly solidify the woods metal.
        10. Approximately 5 minutes are needed to completely harden.
        11. Remove the clay from the bottom of the tube.
        12. Flip the knee so that the bottom of the femur tube is pointed up.
        13. Top the tube off by CAREFULLY ladling hot woods metal into it.
        14. Pour cold water on top of it again and wait for it to harden.
        15. Using a chisel remove the excess wood metal that hardens over edge of the pot.

-- ["Stemmer"] 7-9-14: An image of potting the bones could be helpful

  1. Secure potted specimen
    1. Two 3 inch long drill bits (~1/8 inches in diameter) will be driven in to each tube in order to prohibit motion between the bone and the pot.
    2. Holes in the tube should be utilized if possible.
    3. Each drill bit should be driven in so that it goes through both sides of the tube and through the bone.
    4. Attempts to drill through the part of the tube closest to the joint should be made.
    5. The two drill bits on each bone should NOT be oriented parallel to one another.
  2. Prepare extensor mechanism (for patellofemoral joint testing only)
    1. These activities may happen throughout the various specimen preparation stages listed above. They are provided in here to ensure their execution.
    2. Clamp quadriceps tendon (using liquid nitrogen)

      1. Different sized cable-pulling grips can be found in the labeled cabinet in the BioRobotics laboratory.

        1. Example figure below shows a (a) schematic of the clamp setup, (b) a close-up view of the clamp and tendon in the nitrogen freezing tube and (c) a potted knee with a clear view of the quadriceps tendon clamp in place.
      2. The tendon should be carefully isolated as close to the joint center as possible.
      3. The clamp should be positioned such that its loop will be slightly above the bottom plane of femur pot. The tendon may need to be cut to accommodate this positioning.
      4. Suture can be used to consolidate the quadriceps tendon into a uniform and dense tubular structure using the whipstitch technique.
      5. Suture at the end of the tendon should be threaded through and out of the pulling end of the clamp.
      6. The clamp should then be compressed so the tendon can be fully inserted into it by pulling on the suture.
      7. Once fully in the clamp, applying tension to the clamp should fully encapsulate the tendon.
      8. applying a manual load to the clamp should not cause the tendon to slip out
      9. If the tendon does slip out after applying a manual load, consider using a smaller diameter and/or longer clamp
      10. Tendon should be ready for freeze clamping during the experiment; when appropriate follow the steps below:
    3. Prepare quadriceps area for pressure sensor insertion (which can be conducted during the experimentation)
      1. Specific preparation for patellofemoral testing includes accommodating the pressure sensor. The sensor should be small enough to fit inside the patellofemoral joint but large enough to cover the contact surface.
      2. Please refer to ["Specifications/PressureCalibration"] for specifics about preparing the sensor for testing.
      3. A space beneath the quadriceps tendon will have to be opened adequately to accommodate the pressure sensor (left in the above picture) Without disrupting the surrounding capsular structure, the sensor insertion area should be cleaned up to ensure that the sensor does not catch to any tissue structure during insertion.
      4. The sensor can be inserted in one of the following ways depending on the knee size:
        1. Insertion method for large knees and for better securing of the sensor in place:
          1. Vertical incisions, one medial to and the other lateral, will be made proximal to the tibial insertion of the patellar tendon. Space these wide enough to "feed" the bottom of the sensor through these holes. Make sure the incisions to not compromise any mechanically important features of the joint.
          2. Eyelets (grommets) will need to be assembled after assessing approximate location of the pressure sensor relative to the specimen. Tie suture threads are tied to these eyelets so the sensor can be easily positioned (and pulled taut) through the vertical incisions during testing.
        2. Insertion method for small knees and for quick placement of sensor:
          1. This method does not use any eyelets or suture threads to pull the sensor through vertical incisions. Vertical incisions are therefore not necessary.
          2. Curve the sensor transversely to give it some stiffness along the insertion direction. Push the sensor through the joint as far as possible, feeding it in the joint space with your fingers. The sensor may be placed when the specimen is on the robot.
  3. Place Optotrak marker assembly on patella, femur, and tibia base plugs.
  4. Now, the specimen is ready to go through ["Specifications/ExperimentationJoint/Mechanics"]

Preparation of Specimen for Mechanical Tissue Testing

Note Photos should be taken at every step with appropriate labeling to facilitate cataloging.

Harvesting the tissues

Once joint testing is completed, all primary and secondary tissues will be harvested.

-- ["bonnert2"] What does "closely as possible" mean? Remove as much as possible? Or remove to be as close to the capsule as possible?

attachment:medial_meniscus.png attachment:lateral_meniscus.png

attachment:femur_cartilage.png attachment:tibia_cartilage.png

Tissue Preservation

If time constraints during tissue harvesting do not allow sample preparation (see below), the tissues should be properly preserved.

Test Sample Preparation

Cartilage

-- ["bonnert2"] - how many cartilage strips should there be? This wasn't clarified in the harvesting section... As many as possible?

  1. ~Thickest portion (determined by sight) of the load bearing region
  2. ~Thickest portion (determined by sight) of the non-load bearing region

Meniscus

-- ["Stemmer"] 7-9-14: The steps for using the vibratome are slightly repetitive. Maybe just refer the users to the previous section where the steps are initially described.

Ligaments and Tendon

Data Storage

All data relevant to specimen preparation, e.g., anatomical landmark locations, registration marker positions, etc. should be uploaded to the in-house data management system (http://cobicore.lerner.ccf.org/midas, accessible only within the Cleveland Clinic network) for prospective organization (Open Knee(s) Community). Following organization, a documented version of the data will be disseminated at Open Knee(s) project site (https://simtk.org/home/openknee).