Target Outcome
- Overall readiness of the specimen to start experimentation
- Readiness of the specimen for registration
- Readiness of the specimen for anatomical imaging
- Readiness of the specimen for joint mechanics testing
- Readiness of the specimen for tissue mechanics testing
Prerequisites
Infrastructure
- ["Infrastructure/ExperimentationMechanics"]
Previous Protocols
- ["Specifications/Specimens"].
Related Protocols
- ["Specifications/Registration"]
- ["Specifications/ExperimentationAnatomicalImaging"]
- ["Specifications/ExperimentationJointMechanics"]
- ["Specifications/ExperimentationTissueMechanics"]
Protocols
Specimen Storage
- Specimen Storage
- Storage Location
- Specimens should be stored in the walk-in freezer on the third floor in room ND3-77B
- Serological testing sheets should be scanned and saved in the repository directory upon delivery
- A log should be filled out on the data sheet found in ND3-77B with:
- Specimen number
- PI name being Ahmet Erdemir
- Shelf where the leg was placed labeled accordingly
- Source: Human
- 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.
- Specimen Packaging
- Specimens can be directly placed in the freezer in their original packaging after being procured from the company
- Specimens should be packaged as followed after handling it:
- Wrap specimen in a blue chuck (found in cabinet in room ND1-04).
- Specimen chuck should be labeled with the specimen number, the side (right or left) and what it is (knee)
- 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.
- Storage Location
-- ["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
- Specimen thawing
The specimen should be taken to the BioRobotics Cadaver Lab in room ND1-04 and thawed for 12-18 hours prior to specimen preparation at room temperature.
- The specimen should be unwrapped in order to thaw in less than 24 hours
- For thawing periods greater than 24 hours (i.e. through the weekend) leave the specimen wrapped
-- ["Stemmer"] 7-9-14: Does the specimen have to sit on an absorbent pad/tablecloth of some sort? If the specimen sits on something, does that material have to be changed periodically? How do you know when it is done thawing (12-18 hours seems like a large range)
Specimen Dissection
- Specimen dissection:
- Mark proximal and distal regions for tissue removal
- Femur: 5 inches proximal, measured from the epicondylar axis
- Tibia: 4 inches distal, measured from the epicondylar axis
- Remove all tissue proximal to thigh mark to expose hip.
- 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.
- 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).
- 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
- Mark proximal and distal regions for tissue removal
-- ["Stemmer"] 7-9-14: How wide is the four inch strip? Should the strip be horizontal or run parallel with the tibia and fibula?
Patella Registration and Optotrak Marker Alignment
- 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.
- Place an Optotrak marker on a piece of wood.
- Place patella base plug on the same piece of wood.
- Place patella Optotrak marker on patella base plug.
- Secure the wood (not needed but good).
- Make sure Optotrak system is calibrated and ready to roll.
- Record location of Optotrak markers (wood + patella base plug) (these are measured in global Optrotrak coordinate system)
- Replace patella Optotrak marker with patella registration marker assembly on patella base plug.
- Record location of Optotrak marker on wood and patella registration marker assembly divots with Optotrak probing device.
- attachment:digitizing_probe.jpg
- Remove base plug from wood.
- 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
- Base plug placement
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
- 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.
The fixtures and markers can be found in the labeled shelves in the BioRobotics Cadaver Lab.
- The fixtures are composed of:
- 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.
- 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
- The fixtures mounted on the bones with caution to the following issues:
Three screws will secure each fixture to the bone (-- ["aerdemir"] DateTime(2014-10-06T12:44:57Z) Size? Location?).
Holes should be drilled on the bone to secure the screws (-- ["aerdemir"] DateTime(2014-10-06T12:44:57Z) Size? Location?), using fixture as a template. Drill length should be short to ensure patella cartilage is not compromised.
- Before screwing in, use a k-wire in one hole to hold plug still when screwing in others.
- To ensure the screws securing the base plugs to the bone don't loosen.
- 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.
- Dip the screw in the bone cement; wipe off excess cement from screw before inserting.
- Screw into the bone prior to hardening.
- If a screw shears off, continue as long as 2 screws are fully engaged; leave the sheared one in plug.
- 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.
- Femur - ~4 inches from the epicondylar axis
- 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
- 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)
- Tibia - ~3 inches from the epicondylar axis
- 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
- 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)
- Patella - on the center of the patella
- Expose the bone by cutting the skin on a vertical line and retract to the sides to make room for the plug.
-- ["aerdemir"] DateTime(2014-10-06T12:44:57Z) What is the orientation of attachment? I.e., in which direction the two screw wholes are oriented?
Registration Marker Placement on Femur and Tibia
- Placement of registration markers
- 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.
Spherical markers are hollow plastic spheres (10 mm radius ) filled with water based gel and are attached to the bones using plastic screws (-- ["aerdemir"] DateTime(2014-10-06T12:52:35Z) Size?) 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
- Tissue may need to dissected to make room for the markers.
Holes to place the registration markers should be drilled on the bone using drill bit size of @SI@ Drill bit size? and tapped using tap size of -- ["aerdemir"] DateTime(2014-10-06T12:52:35Z) Tap size?
- Femur spheres should be mounted on the anterior, medial, lateral sides of the bone.
- 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.
- 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.
Optorak Marker Assembly on Bones
- Mounting of Optotrak markers:
- Place the femur Optotrak marker on femur base plug
- Place the tibia Optotrak marker on tibia base plug
- 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
- Place the patella Optotrak marker on patella base plug
Acquisition of Anatomical Landmark Locations
- Anatomical landmark data collection:
- Move specimen to Optotrak measurement view.
- Make sure Optotrak system is previously calibrated and ready to roll
- 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
- The following anatomical locations should be recorded:
- Femur landmarks
- F1. Lateral epicondyle of the femur (most lateral point)
- F2. Medial epicondyle of the femur (most medial point)
- F3-F6. 4 points around the epiphyseal line of the femur
- Tibia landmarks
- T1. Medial tibial plateau (most medial point)
- T2. Lateral tibial plateau (most lateral point)
- T3. Medial malleolus of the tibia (most medial point)
- T4. Lateral malleolus of the fibula (most lateral point)
- Patella landmarks
- P1. Most lateral point
- P2. Most medial point
- P3. Most superior point
- P4. Most inferior point
- Femur landmarks
- Now, we can describe the anatomical points in their respective Optotrak body coordinate systems.
Acquisition of Registration Marker Locations
- Registration marker data collection
- Keep specimen within Optotrak measurement view.
- 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
- Twelve points on each spherical marker should be digitized such that they are distributed evenly about the sphere surface
- The spheres should be digitized in the following order:
- Femur: anterior, medial, lateral markers
- Tibia: medial, lateral, posterior markers
- Now, we can describe registration markers in their respective Optotrak body coordinate systems.
Preparation of Specimen for Anatomical Imaging
- Prepare specimen for MRI:
- Remove Optotrak markers from their respective base plugs on femur, tibia, and patella
- Cut bones
Use a hack saw found in labeled cabinet in the BioRobotics Cadaver Lab
- Clamp and cut femur ~8 inches proximal to the epicondylar axis. File the end of the bones to prevent sharp edges.
- Clamp and cut tibia and fibula ~7 inches distal to the epicondylar axis. File the end of the bones to prevent sharp edges.
- Secure tibia and fibula to each other using a zip tie and a spacer.
- The foot should be preserved for future testing (following storage procedures described above)
- Place patella registration marker assembly on patella
- Now, the specimen is ready to go through ["Specifications/ExperimentationAnatomicalImaging"]
Preparation of Specimen for Mechanical Joint Testing
- Prepare specimen for joint testing
- After specimen comes from MRI; the registration markers can be removed.
- Potting
- Preparation
- Safety
- Gloves, face shields, gowns, protective eye-wear and welders gloves should be used at all times by people handling hot liquid metal
- Melting woods metal
The woods metal found in the hood of the BioRobotics Cadaver Lab should be melted.
- For quickly melting it, turn the temperature of the hotplate to 300 degrees until melted.
- Once melted:
- If the woods metal isn't already in the aluminum pitcher, CAREFULLY pour it into it and place the pitcher on the hotplate.
- Turn the temperature to 100 degrees.
Two aluminum tubes (3 inches long, 2.5 inch OD) can be found in the labeled cabinet in the BioRobotics laboratory.
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.
- Safety
- Potting the tibia end.
- Secure the fibular to the tibia by passing a drill bit through both bones and leaving it there. Try to keep relative positioning of the tibia and fibula anatomical.
- Before potting, check whether Optotrak markers will fit on plugs with the pot.
- Roll the clay flat so that the aluminum tube can sit on it and be completely sealed.
- Place the clay on the the dissection table on the blue side of a chuck.
- If there are drill-bit holes in the tube, orient the tube on the clay so the hole side is not touching the clay
- Seal the bottom of the tube completely with clay and plug any holes with bits of clay
- Place tibia in the tube and hold the extended knee in place
- Verify the length from the bottom of the tube to the epicondylar axis as ~7 inches
- Verify the center of the epicondylar axis is inline, vertically, with the bottom of the tube, from both sagittal and anterior views.
- Use a beaker to fill the tube with water and refill the beaker.
- A foil funnel found in the hood should be held over the top of the tube with forceps by the person holding the knee.
- A different person should CAREFULLY ladle the HOT woods metal into the funnel filling the tube completely.
- Cold water from the beaker can be poured on the tube several times in order to quickly solidify the woods metal.
- Approximately 5 minutes are needed for it to completely harden.
- Remove the clay from the bottom of the tube.
- Flip the knee so that the bottom of the tibia tube is pointed up.
- Top the tube off by CAREFULLY ladling hot woods metal into it.
- Pour cold water on top of it again and wait for it to harden.
- Using a chisel remove the excess wood metal that hardens over edge of the pot.
- Potting the femur end.
- Before potting, check whether Optotrak markers will fit on plugs with the pot.
- Roll the clay flat so that the second aluminum tube can sit on it and be completely sealed.
- Place the clay on the dissection table on the blue side of a chuck.
- If there are drill-bit holes in the tube, orient the tube on the clay so the hole side is not touching the clay
- Seal the bottom of the tube completely with clay and plug any holes with bits of clay.
- Place femur in the tube and hold the extended knee in place.
- Verify the length from the bottom of the tube to the epicondylar axis as ~8 inches
- Verify the center of both tubes are inline, vertically, from both sagittal and anterior views.
- Use a beaker to fill the tube with water and refill the beaker.
- A foil funnel found in the hood should be held over the top of the tube with forceps by the person holding the knee.
- A different person should CAREFULLY ladle the HOT woods metal into the funnel filling the tube completely.
- Cold water from the beaker can be poured on the tube several times in order to quickly solidify the woods metal.
- Approximately 5 minutes are needed to completely harden.
- Remove the clay from the bottom of the tube.
- Flip the knee so that the bottom of the femur tube is pointed up.
- Top the tube off by CAREFULLY ladling hot woods metal into it.
- Pour cold water on top of it again and wait for it to harden.
- Using a chisel remove the excess wood metal that hardens over edge of the pot.
- Preparation
-- ["Stemmer"] 7-9-14: An image of potting the bones could be helpful
- Secure potted specimen
- Two 3 inch drill bits will be driven in to each tube in order to prohibit motion between the bone and the pot.
- Holes in the tube should be utilized if possible.
- Each drill bit should be driven in so that it goes through both sides of the tube and through the bone.
- Attempts to drill through the part of the tube closest to the joint should be made.
- The two drill bits on each bone should NOT be oriented parallel to one another.
- Prepare extensor mechanism (for patellofemoral joint testing only)
- Clamp quadriceps tendon (using liquid nitrogen)
Different sized cable-pulling grips can be found in the labeled cabinet in the BioRobotics laboratory.
- 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.
- The tendon should be carefully isolated as close to the joint center as possible.
- Suture can be used to consolidate the quadriceps tendon into a uniform and dense tubular structure using the whipstitch technique.
- Suture at the end of the tendon should be threaded through and out of the pulling end of the clamp.
- The clamp should then be compressed so the tendon can be fully inserted into it by pulling on the suture.
- Once fully in the clamp, applying tension to the clamp should fully encapsulate the tendon.
- applying a manual load to the clamp should not cause the tendon to slip out
- If the tendon does slip out after applying a manual load, consider using a smaller diameter and/or longer clamp
- Clamp quadriceps tendon (using liquid nitrogen)
-- ["aerdemir"] DateTime(2014-10-06T13:20:54Z) I am not sure when the following freeze-clamping steps should be. After preparing the tendon-clamp assembly? Or, after preparing pressure sensor? Or at the time of patellofemoral joint testing?
- Place the tendon-clamp assembly in a tube with a hose clamp.
- Pour liquid nitrogen into the tube to ensure initial freezing of the tendon-clamp assembly. Use a chuck to protect the knee when pouring liquid nitrogen.
- Prepare quadriceps area for pressure sensor insertion (which can be conducted during the experimentation)
- Specific preparation for patellofemoral testing includes accommodating the pressure sensor. The sensorshould be small enough to fit inside the patellofemoral joint but large enough to cover the contact surface.
- Please refer to ["Specifications/PressureCalibration"] for specifics about preparing the sensor for testing.
- A space beneath the quadriceps tendon will have to be opened adequately to accommodate the pressure sensor (left in the above picture)
- 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.
- Be sure suture threads are tied to the eyelets so the sensor can be easily positioned (and pulled taut) during testing.
- Place Optotrak marker assembly on patella, femur, and tibia base plugs.
- 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.
- Starting with an incision axially, skin, fat and muscle tissues will be removed as closely as possible.
-- ["bonnert2"] What does "closely as possible" mean? Remove as much as possible? Or remove to be as close to the capsule as possible?
- Quadriceps tendon-patella-patellar ligament complex is exposed at this point and will be carefully separated from the joint.
- Once the joint space is exposed, lcl will be worked on and carefully harvested (by cutting closely at the insertion sites).
- The lateral meniscus will be exposed enough to be harvested intact.
- Next the mcl-medial meniscus complex will be harvested together as the meniscus is attached to the mcl and will be easier to separate once the combined structure is separated from the joint.
- This will expose the acl and pcl which can be easily harvested once all the surrounding tissues are removed.
- As soon as the tissues are removed, the labeler will label the tissue clearly identifying the type and anatomical directions of tibia and femur insertion site ends, i.e., depiction of tibial and femoral ends, anatomical orientation of sides, e.g., medial or lateral, anterior or posterior.
- The labeler will place the ligament in a prelabeled bag indicating the specimen #, ligament name, and orientation.
- Naming convention can be found in data management specifications.
- The labeler will place the meniscus in a prelabeled bag indicating the specimen #, meniscus side, and orientation.
- The labeler will place the tendon in a prelabeled bag indicating the specimen #, tendon name, and orientation.
attachment:medial_meniscus.png attachment:lateral_meniscus.png
- The tibia, femur, and patella with articular cartilage intact on all bone surfaces will be separated and labeled.
- Rectangular strips of cartilage will be removed using a scalpel blade as close to the bone surface as possible
attachment:femur_cartilage.png attachment:tibia_cartilage.png
- Cartilage strips should be placed in a prelabeled bags indicating the location (pictures will be taken at every step to later associate every strip and its location).
Tissue Preservation
If time constraints during tissue harvesting do not allow sample preparation (see below), the tissues should be properly preserved.
- Each tissue should be separately wrapped in saline soaked gauze and stored in labeled bags.
- Each tissue should be placed in a biohazard bag/ ziplock bag or test tube with a copy of the label described above.
- Each tissue should be kept in a freezer connected to a backup generator. A freezer is also available on the third floor of Department of Biomedical Engineering, Cleveland Clinic.
- When storing tissues in the freezer, the login sheet outside should be filled in assigning the the shelf location, specimen label and date.
Test Sample Preparation
Cartilage
- For confined compression
- 5 mm diameter full thickness cylindrical samples will be acquired (punch specifications can be found in the experimentation mechanics infrastructure page).
- For each cartilage strip two samples will be punched from:
-- ["bonnert2"] - how many cartilage strips should there be? This wasn't clarified in the harvesting section... As many as possible?
- ~Thickest portion (determined by sight) of the load bearing region
- ~Thickest portion (determined by sight) of the non-load bearing region
- To punch out samples, harvested cartilage strips will be placed on the plate of a hand press located in ND1-06A in the Department of Biomedical Engineering, Cleveland Clinic
- A rubber sheet will be placed underneath the sample.
- The punch will sit on top of the tissue over the region where the sample should be harvested.
- The press will be lowered completely until a cylindrical sample separates from the harvested tissue.
- If tissue testing will be performed at a later date, samples should be wrapped in saline soaked gauze individually and labeled accordingly: medial or lateral, load bearing or not, anterior or posterior, orientation
- The samples should then be stored in the freezer with the rest of the harvested tissue and samples, e.g.
- To obtain uniform thickness samples, samples will be shaved minimally using a vibratome (Leica VT1200S) immediately prior to testing.
- The vibratome is available in ND1-06A in the Department of Biomedical Engineering, Cleveland Clinic.
- Tissue adhesive should be applied to the articular side of the cartilage and placed on the plate.
- The plate should sit on the magnet of the vibratome.
- The blade should be fixed of the holder of the vibratome.
- The plate should be manually raised until the sample and blade are aligned and the top of the sample is in the same plane as the blade.
- Amplitude and speed settings should be adjusted [TBD]
- Slowly move the blade towards the sample.
- Remove 50 microns at a time until a flat surface is achieved (this assessment is done visually).
- A tissue debonder should be used to detach the sample from the plate so as to preserve the articulating surface.
- The vibratome is available in ND1-06A in the Department of Biomedical Engineering, Cleveland Clinic.
Meniscus
- For uniaxial tension
- Sample dimensions are 5 X 2 mm (800 micron thickness) dumbbells.
- Each meniscus will be divided into three regions (anterior, middle and posterior).
- The tensile sample will be obtained from the middle region.
- The 5 X 2 mm tensile punch should be oriented on the meniscus in the middle region and it should be divided so that the tensile punch sits completely on the middle block, circumferentially.
- To obtain uniform thickness samples, samples will be cut using a vibratome immediately prior to testing.
- The vibratome can be found in ND1-06A in the Department of Biomedical Engineering, Cleveland Clinic.
- All tools and accessories can be found in the top drawer below it.
- The circular plate and residual glue should be removed.
- Glue should be applied to the tibial side of the meniscus block and placed on the plate.
- The plate should sit on the magnet on the vibratome.
- The blade should be fixed on the holder of the vibratome.
- The plate should be manually raised until the sample and blade are aligned and the top of the sample is in the same plane as the blade
- Amplitude and speed settings should be adjusted [we will put in ball-park once we know]
- Slowly move the blade towards the sample.
- Remove 50 microns at a time until a flat surface is achieved.
- Once flat surface is achieved that can accommodate the tensile sample, 800 microns thick sample will be removed using vibratome.
Multiple samples can be obtained and should be labeled by layer -- ["aerdemir"] DateTime(2014-01-13T23:55:09Z) It may help to provide examples of sample labeling.
- A debonder will be used to remove the sample.
- Each layer should be placed on the plate on the press found in ND1-06A in the Department of Biomedical Engineering, Cleveland Clinic.
- A rubber sheet should be placed on top of the sample.
- The punch will sit on top of the rubber sheet over the region where the sample should be harvested.
- The press will be lowered completely until the dumbbell sample separates.
- For confined compression
- Sample dimensions are anticipated to be 5 mm diameter discs and full thickness.
- To obtain uniform thickness samples on the posterior and anterior blocks, samples will be cut using the vibratome immediately prior to testing.
- The vibratome can be found in ND1-06A in the Department of Biomedical Engineering, Cleveland Clinic.
- All tools and accessories can be found in the top drawer below it.
- The circular plate and glue should be removed.
- Glue should be applied to the tibial side of the meniscus block and placed on the plate.
- The plate should sit on the magnet on the vibratome.
- The blade should be fixed on the holder of the vibratome.
- The plate should be manually raised until the sample and blade are aligned and the top of the sample is in the same plane as the blade.
- Amplitude and speed settings should be adjusted [we will put in ball-park once we know]
- Slowly move the blade towards the sample .
- Remove 50 microns at a time until a flat surface is achieved.
- A debonder can be used to remove the remaining sample from the plate.
- Each block should be placed on the plate on the press found in ND1-06A in the Department of Biomedical Engineering, Cleveland Clinic.
- A rubber sheet should be placed on top of the sample
- The punch will sit on top of the rubber sheet over the region where the sample should be harvested.
- The press will be lowered completely until the 5 mm diameter disk separates.
-- ["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
- For uniaxial tension
- Sample dimensions will be 10 X 2 mm (~1 mm thickness) dumbbells
To cut the tissue to desired dimensions, a cryostat will be used (-- ["aerdemir"] DateTime(2014-01-14T00:06:54Z) Company, Model, etc.?). Reservation on a cryostat can be made at the Histochemistry Core in the Department of Biomedical Engineering at Cleveland Clinic, by contacting Eddie Uhl.
- Tissue should be kept frozen.
- Remove the gauze under cool water and place the tissue in dry ice.
- Measure the tissue thickness.
- If the ligament thickness is greater than 1 mm, the cryostat will be used to remove excess tissue. This is anticipated to be the case for quadriceps tendon, ACL and PCL.
- Eddie Uhl will use the cryostat to provide tissue sheets of 1 mm thickness.
- Once the sample is in the cryostat, 50 micron segments will be removed incrementally until 0.5-1 mm of tissue is removed.
- The sample will be flipped in the cryostat and another 0.5-1 mm of tissue will be removed.
- The previous two steps should be repeated until a tissue sheet of ~1 mm remains. Calipers are used to measure the thickness before each flip (3-4 measurements are taken and averaged).
- The tissue sheets will be punched in the longitudinal direction by placing them on the plate on the press found in ND1-06A in the Department of Biomedical Engineering, Cleveland Clinic.
- A rubber sheet will be placed on top of the sample.
- The 10 X 2 mm dumbbell punch will sit on top of the rubber sheet over the region where the sample should be harvested.
- The press will be lowered completely until the dumbbell sample separates.
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).
Notes for Organization
-- ["aerdemir"] DateTime(2014-03-14T19:49:36Z) Jason asks if we can check potential deformation Optotrak markes by assemblimg them the same way on a rigid jig before and after experiment?
-- ["aerdemir"] DateTime(2014-03-14T19:49:36Z) Snehal asks if we can do registration marker probing before and after mri to ensure they stay at the same place.
Specimen preparation steps include specimen initialization (to start experimentation) and are required before activities of registration, anatomical imaging, joint testing, and tissue testing. Steps involved are interleaved as listed below and provided in the following illustration.
-- ["aerdemir"] DateTime(2013-12-09T14:48:00Z) Jason, please remove the numbers in the figure so that we don't need to refer to a separate page.
-- ["hallorj"] DateTime(2013-12-09T22:02:57Z) Ahmet, I'm happy to remove the numbering but just in case, I kept this workflow consistent with main flowchart in the ["Specifications"] page.
- Specimen preparation: [#Initialization initialization]
- Result: prepared specimen for acquisition of specimen-specific anatomical features
- Output: coordinates of landmarks on anatomy, robot, and motion analysis system.
- See ["Specifications/Registration"] for the use of this output to establish and relate coordinate systems.
- Specimen preparation for [#Registration registration]
- Result: readiness of the specimen to acquire registration data (placement of registration markers)
- Output: coordinates on registration markers as measured by experimental digitization system
- See ["Specifications/Registration"] for the use of this output to establish and relate coordinate systems.
Specimen preparation for [#AnatomicalImaging anatomical imaging]
- Result: readiness of the specimen to conduct MRI
- ["Specifications/ExperimentationAnatomicalImaging"]
- Note that coordinates on registration markers as measured by MRI will be used for registration, see ["Specifications/Registration"].
Specimen preparation for [#JointTesting joint mechanics testing]
- Result: readiness of the specimen to conduct joint mechanics testing
- ["Specifications/ExperimentationJointMechanics"]
Specimen preparation for [#TissueTesting tissue mechanics testing]
- Result: readiness of the specimen to conduct tissue mechanics testing
- ["Specifications/ExperimentationTissueMechanics"]
-- ["aerdemir"] DateTime(2014-03-14T18:58:34Z) The following detailed workflow was generated by me, Jason, and Snehal to clarify sequence of operations for specimen preparation.
Specimen Storage
Storage Location
- Specimens should be stored in the walk-in freezer on the third floor in room ND3-77B
- A log should be filled out on the data sheet found in ND3-77B with:
- Specimen number
- PI name being Ahmet Erdemir
- Shelf where the leg was placed labeled accordingly
- Source: Human
- 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.
Specimen Packaging
- Specimens can be directly placed in the freezer in their original packaging after being procured from the company
- Specimens should be packaged as followed after handling it:
- Wrap specimen in a blue chuck (found in cabinet in room ND1-04).
- Specimen chuck should be labeled with the specimen number, the side (right or left) and what it is (knee)
- 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.
Preliminary Steps
- The specimens should be acquired based on ["Specifications/Specimens"].
- Full leg should be procured (foot to femoral head) and kept in the third floor freezer in room ND3-77B.
- All serological testing sheets should be accounted for upon arrival and a data log found on a shelf in room ND3-77B should be filled out describing what shelf each specimen was stored on.
- Serological testing sheets should be scanned and saved in the repository directory
The specimen should be taken to the BioRobotics Cadaver Lab in room ND1-04 and thawed for 12-18 hours prior to specimen preparation at room temperature.
Use a hack saw found in labeled cabinet in the BioRobotics Cadaver Lab and cut the femur 8 inches proximal to the epicondylar axis and the tibia 7 inches distal to the epicondylar axis. Cut the fibula as proximal to the joint line as possible without damaging ligamentous structures.
- The foot should be preserved for future testing. It should be wrapped in a chuck found on the bottom shelf of the cadaver supplies cabinet in the robot lab.
- The chuck should be labeled with the specimen number, the side (right or left) and what it is (foot). The wrapped foot should be placed in a red biohazard bag found in the same cabinet as the chucks and labeled in the same fashion as the chuck. The foot should be taken to the third floor freezer (ND3-77B) and placed a shelf. A log should be filled out on the data sheet found in ND3-77B with the PI name being Ahmet Erdemir and the shelf where the foot was placed labeled accordingly.
- Upon completion of preparation, the serological testing sheets for each specimen should be returned to ND3-77B so long as a specimen still remains in the freezer.
- Specimen demographics (e.g., age, height, weight, race etc) will be recorded in the simVITRO setup screen and stored in the setup configuration file.
Remove all soft tissue on the femur side proximal to 5 inches and distal to 9 inches from the epicondylar axis while preserving the entire quadriceps tendon -- ["aerdemir"] DateTime(2014-03-10T17:44:00Z) Tara, what does "proximal to 5 inches and distal to 9 inches from the epicondylar axis" mean?
- Wrap the quadriceps tendon in a paper towel soaked in saline.
Remove all soft tissue on the tibia side distal to 4 inches and proximal to 8 inches from the epicondylar axis. -- ["aerdemir"] DateTime(2014-03-10T17:44:00Z) Tara, what does "proximal to 4 inches and distal to 8 inches from the epicondylar axis" mean?
- 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
-- ["hallorj"] DateTime(2014-01-28T14:54:34Z) Tara, what "data log" should be filled out in the above description and where is this data log? At this point we should probably also create a repository directory for each specimen and catalog the specimen number and other relevant data in a readme file.
-- ["aerdemir"] DateTime(2014-03-10T17:44:00Z) Specimen related information should also be logged in at a specimen wiki page.
-- ["hallorj"] DateTime(2014-01-28T14:54:34Z) Do we need to decide how to prepare the quadriceps tendon? Related, are we going to be using the style of clamp from the previous tests?
Preparation for Registration
Mounting of Base Plugs
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
- In order to ensure repeatable placement, plastic (MRI compatible) fixtures were designed to be rigidly secured to the tibia and femur throughout experimentation.
The fixtures and markers can be found in the labeled shelves in the BioRobotics Cadaver Lab.
- The fixtures are composed of:
- 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.
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. -- ["aerdemir"] DateTime(2013-12-09T15:50:33Z) We need quantitative information documenting the boundaries of repatability.
attachment:optotrak_mounting_assembly.png
- The centers of the fixtures should be mounted on the:
- Tibia - ~5 inches from the epicondylar axis
- 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
- Femur - ~6 inches from the epicondylar axis
- 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
Patella - on the center of the patella -- ["aerdemir"] DateTime(2014-03-10T17:50:47Z) The alignment of patella base plug should be described to permit appropriate orientation of the patella registration marker assembly.
- Tibia - ~5 inches from the epicondylar axis
Mounting of Registration Markers
Markers need to be placed on femur, tibia, and patella to associate imaging and joint testing coordinate systems.
Tibia & Femur
- Three spherical markers should be rigidly mounted to the tibia and the femur as close to joint line as possible without damaging ligamentous structures (~50-60 mm)
- Tibia spheres should be mounted on the medial, lateral, posterior sides of the bone.
- Femur spheres should be mounted on the anterior, medial, lateral sides of the bone.
attachment:knee_with_registration_markers.png
-- ["aerdemir"] DateTime(2014-03-10T17:56:27Z) Jason, can you inform the team about more appropriate and detailed location of the tibia and femur marker sets? Also, we need a new picture.
- Spherical markers are hollow plastic spheres (10 mm radius ) filled with water based gel and are attached to the bones using plastic screws for the tibia and femur for MRI registration.
attachment:spherical_marker.png Anchor(Patella_MRI_marker)
Patella
MRI [:Specifications/Registration#Patella+Registration:patella registration] consists of a 3D printed attachment with three resin filled spheres. There are 12 divots on the attachment that can be used to register where the spheres are with respect to the attachment.
-- ["aerdemir"] DateTime(2013-12-09T15:50:33Z) What is the reproducibility of this registration?
The MRI registration attachment is mounted to the patella using the same [:Specifications/SpecimenPreparation#Marker+Mounting:fixture] used to mount the optoelectronic markers.
-- ["aerdemir"] DateTime(2014-03-10T18:06:19Z) The image should reflect new patella registration marker assemble.
attachment:patella_registration.png
-- ["aerdemir"] DateTime(2014-03-10T18:06:19Z) At this stage the specimen is appropriately dissected; femur, tibia, and patella have base plugs attached; femur and tibia have separate registration markers attached; and patella has the registration marker assembly attached on its base plug. In a sense, the specimen is ready for anatomical imaging.
SPECIMEN CAN GO TO ["Specifications/ExperimentationAnatomicalImaging"]
Anatomical Landmark Digitization
After the base plugs and Optotrak markers have been secured to the specimen (to femur, tibia, and patella), the anatomical landmarks can be digitized with respect to the markers. A digitizing probe should be used to take each point with respect to the bone's respective marker. This can be done when the specimen is mounted on the robot or placed inside Optorak camera sight, i.e. measurement volume. This activity can be done before or after anatomical imaging. Nonetheless, it is advised to do it when the specimen is mounted on the robot immediately before joint testing.
attachment:Digitizer.png
- The tibia coordinate system requires the digitization of the following points
- T1. Medial tibial plateau (most medial point)
- T2. Lateral tibial plateau (most lateral point)
- T3. Medial malleolus of the tibia (most medial point)
- T4. Lateral malleolus of the fibula (most lateral point)
- The femur coordinate system requires the digitization of the following points
- F1. Lateral epicondyle of the femur (most lateral point)
- F2. Medial epicondyle of the femur (most medial point)
- F3-F6. 4 points around the epiphyseal line of the femur
- The patella coordinate system requires the digitization of the following points
- P1. Most lateral point
- P2. Most medial point
- P3. Most superior point
- P4. Most inferior point
-- ["aerdemir"] DateTime(2014-03-10T18:30:04Z) At this point, we should have data to establish transformation matrices between tibia anatomical and Optotrak marker coordinate systems, femur anatomical and Optotrak marker coordinate systems, and patella anatomical and Optotrak marker coordinate systems.
Registration Marker Digitization
- Patella
The digitization of points and the registration of the patella marker coordinate system should be done prior to base plug assembly.
- The base plug should be rigidly secured to a piece of wood that is fixed in space.
- The patella Optotrak marker should be secured to the base plug and the position/orientation data should be recorded to identify where the sensor is in space.
- The patella registration marker attachment should be mounted on the plug and the 12 divots should be digitized using the digitizing probe.
attachment:digitizing_probe.png
- Tibia and Femur
- Twelve points on each spherical marker should be probed using the digitizing probe such that they are distributed evenly about the sphere surface
- The spheres should be digitized in the following order
- Tibia: medial, lateral, posterior markers
- Femur: anterior, medial, lateral markers
- This activity should be performed when the specimen is mounted on the robot.
-- ["aerdemir"] DateTime(2014-03-10T18:30:04Z) At this point, we should have data to establish transformation matrices between tibia Optotrak marker and registration coordinate systems, femur Optotrak marker and registration coordinate systems, and patella Optotrak marker and registration coordinate systems.
Preparation for Joint Testing
Potting the Specimen
Initial Steps
Remove the MRI markers from the specimen as they have already been digitized. -- ["aerdemir"] DateTime(2014-03-10T18:50:42Z) This is not necessary for femur and tibia.
- Gloves, face shields, gowns, protective eye-wear and welders gloves should be used at all times by people handling hot liquid metal
The woods metal found in the hood of the BioRobotics Cadaver Lab should be melted.
- For quickly melting it, turn the temperature of the hotplate to 300 degrees until melted.
- Once melted:
- If the woods metal isn't already in the aluminum pitcher, CAREFULLY pour it into it and place the pitcher on the hotplate.
- Turn the temperature to 100 degrees.
Two aluminum tubes (3 inches long, 2.5 inch OD) can be found in the labeled cabinet in the BioRobotics laboratory.
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.
Tibia
- Roll the clay flat so that the aluminum tube can sit on it and be completely sealed.
- Place the clay on the the dissection table on the blue side of a chuck.
- If there are drill-bit holes in the tube, orient the tube on the clay so the hole side is not touching the clay
- Seal the bottom of the tube completely with clay and plug any holes with bits of clay
- Place tibia in the tube and hold the extended knee in place
- Verify the length from the bottom of the tube to the epicondylar axis as ~7 inches
- Verify the center of the epicondylar axis is inline, vertically, with the bottom of the tube, from both sagittal and anterior views.
- Use a beaker to fill the tube with water and refill the beaker.
- A foil funnel found in the hood should be held over the top of the tube with forceps by the person holding the knee.
- A different person should CAREFULLY ladle the HOT woods metal into the funnel filling the tube completely.
- Cold water from the beaker can be poured on the tube several times in order to quickly solidify the woods metal.
- Approximately 5 minutes are needed for to completely harden.
- Remove the clay from the bottom of the tube.
- Flip the knee so that the bottom of the tibia tube is pointed up.
- Top the tube off by CAREFULLY ladling hot woods metal into it.
- Pour cold water on top of it again and wait for it to harden
Femur
- Roll the clay flat so that the second aluminum tube can sit on it and be completely sealed.
- Place the clay on the dissection table on the blue side of a chuck.
- If there are drill-bit holes in the tube, orient the tube on the clay so the hole side is not touching the clay
- Seal the bottom of the tube completely with clay and plug any holes with bits of clay.
- Place femur in the tube and hold the extended knee in place.
- Verify the length from the bottom of the tube to the epicondylar axis as ~8 inches
- Verify the center of both tubes are inline, vertically, from both sagittal and anterior views.
- Use a beaker to fill the tube with water and refill the beaker.
- A foil funnel found in the hood should be held over the top of the tube with forceps by the person holding the knee.
- A different person should CAREFULLY ladle the HOT woods metal into the funnel filling the tube completely.
- Cold water from the beaker can be poured on the tube several times in order to quickly solidify the woods metal.
- Approximately 5 minutes are needed to completely harden.
- Remove the clay from the bottom of the tube.
- Flip the knee so that the bottom of the femur tube is pointed up.
- Top the tube off by CAREFULLY ladling hot woods metal into it.
- Pour cold water on top of it again and wait for it to harden.
Drill Bits
- Two 3 inch drill bits will be driven in to each tube in order to prohibit motion between the bone and the pot.
- Holes in the tube should be utilized if possible.
- Each drill bit should be driven in so that it goes through both sides of the tube and through the bone.
- Attempts to drill through the part of the tube closest to the joint should be made.
- The two drill bits on each bone should NOT be oriented parallel to one another.
Clamping the Quadriceps Tendon
Different sized cable-pulling grips can be found in the labeled cabinet in the BioRobotics laboratory.
- The tendon should be carefully isolated as close to the joint center as possible.
- Suture can be used to consolidate the quadriceps tendon into a uniform and dense tubular structure
- Suture at the end of the tendon should be threaded into the clamp and pulled through so the full length of the tendon sits snugly in the clamp.
-- ["aerdemir"] DateTime(2014-03-10T18:50:42Z) Is this for freeze clamping? More details need to be provided in here.
-- ["hallorj"] DateTime(2013-12-09T22:02:57Z) One thing we need to describe is how to prepare the tendon/clamp for the linear actuator
Patellofemoral
- Specific preparation for patellofemoral testing includes accommodating the pressure sensor.
- Please refer to ["Specifications/PressureCalibration"] for specifics about preparing the sensor for testing.
- A space beneath the quadriceps tendon will have to be opened adequately to accommodate the pressure sensor (left in the above picture)
- 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.
- Be sure suture threads are tied to the eyelets so the sensor can be easily positioned (and pulled taut) during testing.
Preparation for Tissue Testing
Photos should be taken at every step with appropriate labeling to facilitate cataloging.
Harvesting the tissues
Once joint testing is completed, the surgeon will harvest each the following tissue types, which will be labelled and stored until preparation of tissue testing samples.
Refer to attachment:Specimen_sample_labeling.pdf as a labeling guide.
- Ligaments (MCL, LCL, ACL,PCL and patellar ligament) and quadricep tendon will be procured by cutting at each insertion site using a scalpel blade.
- As soon as the ligament is removed, the surgeon will give a second person (the labeler) the ligament clearly identifying the type of the tissue and anatomical directions of tibia and femur insertion site ends, i.e., depiction of tibial and femoral ends, anatomical orientation of sides, e.g., medial or lateral, anterior or posterior.
- The labeler will place the ligament in a prelabeled bag indicating the specimen #, ligament name, and orientation
-- ["aerdemir"] DateTime(2014-01-13T23:30:43Z) Provide naming convention in here.
- [Illustration to follow]
- Both lateral and medial menisci will be procured
attachment:medial_meniscus.png attachment:lateral_meniscus.png
- As soon as each meniscus in its entirety is removed, the surgeon will give the labeler the meniscus clearly identifying the side, medial or lateral and anatomical directions, superior or inferior and anterior or posterior
The labeler will place the meniscus in a prelabeled bag indicating the specimen #, meniscus side, and orientation -- ["aerdemir"] DateTime(2014-01-13T23:30:43Z) Provide naming convention in here.
- [Illustration to follow]
- The surgeon will remove the clamp from the quadriceps tendon and harvest it at the patella insertion site
- The labeler will place the tendon in a prelabeled bag indicating the specimen #, tendon name, and orientation
- [Illustration to follow]
- The labeler will place the tendon in a prelabeled bag indicating the specimen #, tendon name, and orientation
- The surgeon will separate the tibia, femur, and patella femur with articular cartilage intact on all bone surfaces
- Rectangular strips of cartilage will be removed using a scalpel blade as close to the bone surface as possible
attachment:femur_cartilage.png attachment:tibia_cartilage.png
- The surgeon will separate the tibia, femur, and patella femur with articular cartilage intact on all bone surfaces
- Rectangular strips of cartilage (from the entire articular surface) will be removed using a scalpel blade as close to the bone surface as possible
- Cartilage strips should be placed in a prelabeled bags indicating the location (pictures will be taken at every step to later associate every strip and its location).
- [Illustration to follow]
Tissue Preservation
If time constraints during tissue harvesting do not allow sample preparation (see below), the tissues should be properly preserved.
- Each tissue should be separately wrapped in saline soaked gauze and stored in labeled bags.
- Each tissue should be placed in a biohazard bag with a copy of the label described above.
- Each tissue should be kept in a freezer connected to a backup generator. A freezer is provided on the third floor of Department of Biomedical Engineering, Cleveland Clinic.
- When storing tissues in the freezer, the login sheet outside should be filled in assigning the the shelf location, specimen label and date.
Test Sample Preparation
Cartilage
- For confined compression
- 5 mm diameter full thickness cylindrical samples will be acquired (punch specifications can be found in the experimentation mechanics infrastructure page).
- For each cartilage strip two samples will be punched from:
- ~Thickest portion (determined by sight) of the load bearing region
- ~Thickest portion (determined by sight) of the non-load bearing region
- To punchout samples, harvested cartilage strips will be placed on the plate of a hand press located in NDB-10 in the Department of Biomedical Engineering, Cleveland Clinic
- A rubber sheet will be placed on top of the sample
- The punch will sit on top of the rubber sheet over the region where the sample should be harvested
- The press will be lowered completely until a cylindrical sample separates from the harvested tissue
- If tissue testing will be performed at a later date, samples should be wrapped in saline soaked gauze individually and labeled accordingly: medial or lateral, load baring or not, anterior or posterior, orientation
- The samples should then be stored in the freezer with the rest of the harvested tissue and samples, e.g.
- To obtain uniform thickness samples, samples will be shaved minimally using a vibratome (Leica VT1200S) immediately prior to testing.
- The vibratome is available in ND1-06A in the Department of Biomedical Engineering, Cleveland Clinic.
- All related tools and accessories can be found in the top drawer below.
- The circular plate and any residual glue should be removed.
- Tissue adhesive should be applied to the articular side of the cartilage and placed on the plate.
- The plate should sit on the magnet of the vibratome.
- The blade should be fixed of the holder of the vibratome.
- The plate should be manually raised until the sample and blade are aligned and the top of the sample is in the same plane as the blade.
- Amplitude and speed settings should be adjusted [we will put in ball-park once we know]
- Slowly move the blade towards the sample
- Remove 50 microns at a time until a flat surface is achieved (this assessment is done visually).
- A tissue debonder can be used to detach the sample from the plate so as to preserve the articulating surface.
Meniscus
- For uniaxial tension
- Sample dimensions are 5 X 2 mm (800 micron thickness) dumbbells.
- Each meniscus will be divided into three regions (anterior, middle and posterior).
- The tensile sample will be obtained from the middle region.
- The 5 X 2 mm tensile punch should be oriented on the meniscus in the middle region and it should be divided so that the tensile punch sits completely on the middle block, circumferentially.
- To obtain uniform thickness samples, samples will be cut using a vibratome immediately prior to testing.
- The vibratome can be found in NDB-12 in the Department of Biomedical Engineering, Cleveland Clinic.
- All tools and accessories can be found in the top drawer below it.
- The circular plate and residual glue should be removed.
- Glue should be applied to the tibial side of the meniscus block and placed on the plate.
- The plate should sit on the magnet on the vibratome.
- The blade should be fixed on the holder of the vibratome.
- The plate should be manually raised until the sample and blade are aligned and the top of the sample is in the same plane as the blade
- Amplitude and speed settings should be adjusted [we will put in ball-park once we know]
- Slowly move the blade towards the sample.
- Remove 50 microns at a time until a flat surface is achieved.
- Once flat surface is achieved that can accommodate the tensile sample, 800 microns thick sample will be removed using vibratome.
Multiple samples can be obtained and should be labeled by layer -- ["aerdemir"] DateTime(2014-01-13T23:55:09Z) It may help to provide examples of sample labeling.
- A debonder will be used to remove the sample.
- Each layer should be placed on the plate on the press found in NDB-14 in the Department of Biomedical Engineering, Cleveland Clinic.
- A rubber sheet should be placed on top of the sample.
- The punch will sit on top of the rubber sheet over the region where the sample should be harvested.
- The press will be lowered completely until the dumbbell sample separates.
- For confined compression
- Sample dimensions are anticipated to be 5 mm diameter discs and full thickness.
- To obtain uniform thickness samples on the posterior and anterior blocks, samples will be cut using the vibratome immediately prior to testing.
- The vibratome can be found in NDB-12 in the Department of Biomedical Engineering, Cleveland Clinic.
- All tools and accessories can be found in the top drawer below it.
- The circular plate and glue should be removed.
- Glue should be applied to the tibial side of the meniscus block and placed on the plate.
- The plate should sit on the magnet on the vibratome.
- The blade should be fixed on the holder of the vibratome.
- The plate should be manually raised until the sample and blade are aligned and the top of the sample is in the same plane as the blade.
- Amplitude and speed settings should be adjusted [we will put in ball-park once we know]
- Slowly move the blade towards the sample .
- Remove 50 microns at a time until a flat surface is achieved.
- A debonder can be used to remove the remaining sample from the plate.
- Each block should be placed on the plate on the press found in NDB-14 in the Department of Biomedical Engineering, Cleveland Clinic.
- A rubber sheet should be placed on top of the sample
- The punch will sit on top of the rubber sheet over the region where the sample should be harvested.
- The press will be lowered completely until the 5 mm diameter disk separates.
Ligaments and Tendon
- For uniaxial tension
- Sample dimensions will be 10 X 2 mm (~1 mm thickness) dumbbells
To cut the tissue to desired dimensions, a cryostat will be used (-- ["aerdemir"] DateTime(2014-01-14T00:06:54Z) Company, Model, etc.?). Reservation on a cryostat can be made at the Histochemistry Core in the Department of Biomedical Engineering at Cleveland Clinic, by contacting Eddie Uhl.
- Tissue should be kept frozen.
- Remove the gauze under cool water and place the tissue in dry ice.
- Measure the tissue thickness.
- If the ligament thickness is greater than 1 mm, the cryostat will be used to remove excess tissue. This is anticipated to be the case for quadriceps tendon, ACL and PCL.
- Eddie Uhl will use the cryostat to proved tissue sheets of 1 mm thickness.
- Once the sample is in the cryostat, 50 micron segments will be removed incrementally until 0.5-1 mm of tissue is removed.
- The sample will be flipped in the cryostat and another 0.5-1 mm of tissue will be removed.
- The previous two steps should be repeated until a tissue sheet of ~1 mm remains. Calipers are used to measure the thickness before each flip (3-4 measurements are taken and averaged).
- The tissue sheets will be punched in the longitudinal direction by placing them on the plate on the press found in NDB-14 in the Department of Biomedical Engineering, Cleveland Clinic.
- A rubber sheet will be placed on top of the sample.
- The 10 X 2 mm dumbbell punch will sit on top of the rubber sheet over the region where the sample should be harvested.
- The press will be lowered completely until the dumbbell sample separates.