The past two weeks at the MOI have been packed full of 12 hour days due to the influx of rat and canine models from the vet school. The vet school students practiced their necropsy techniques and sent over 8 rat models at the beginning of week 6 and 3 canine tails on week 7. With the arrival of these new rat models and canine tissues, I had to harvest all of the available tail IVDs, place them in the flexcell plates and vacuum system, culture them under load for 6 days each, and perform media changes and the end-of-culture procedure. The lab also had an MOI organized study day this week in which I was one of the first five people in the world to evaluate a successful ACL to ACL allograft!
I received the 8 rat tail models on Monday of Week 6 and immediately started the harvesting procedure. All of the harvesting procedures for IVDs have to be performed sterilely, so I had to lay sterile mats in the hood, gather all the necessary sterile equipment, and use sterile gloves. I used a scalpel to first remove the tails from the rats at the IVD closest to the beginning of the tail. Due to the thick skin that surrounds rat tails, I had to locate the disks by squeezing the tail and feeling for the vertebrae and slightly raised IVDs that lie between them. This is quite a difficult skill to acquire and it took me several harvests before I became comfortable locating the IVDs by feel. I then sliced through the middle of the IVD and placed all of the tails in a chlorhexidine solution. I disposed of all of the rat bodies in the biohazard bins and began the IVD extraction portion of the harvest procedure. To remove all of the IVDs, I had to first slice off all of the rough skin on the exterior of the tail. I made two cuts opposite each other and pulled each skin slice off the muscle that surrounds the vertebrae of the tail. Then, I began to remove all of the muscle and fat that surround the tail vertebrae and IVDs. I used a pair of rongeurs to shred this tissue off of the tail. I learned that you have to be really forceful with twisting and pulling this tissue off in order to remove the tissue. You cannot be cautious of damaging the tissue or else stripping the muscle could easily take you close to an hour to complete. After removing all of the surrounding tissue, I used a pair of bone cutters to cut out each of the IVDs. I left a small piece of vertebral bone (bone plate) on each side of the IVD so that I could trim it flat on the wet saw. These bone plates have to be very flat in order to allow the IVDs to stand upright in the flexcell plates. If the bone plates are not completely flat, an even load cannot be applied to the IVD and they have to used in the non-load plate. I used the wet saw and a specially designed IVD jig to make the cut and ensure that each of the bone plates were smooth and flat. After finishing the whole harvest process, I had removed and trimmed 48 discs from all of the tails. I placed these disks into 12 flexcell plates separated into two different experimental groups and a negative control group. Four of the plates would undergo a .50 MPa load in the vacuum system. Four non-loaded plates would serve as the negative control groups. The last 4 plates would undergo the .25 MPa load. All of the plates had half of their IVDs injured with an 18 gauge needle to simulate an annular tear commonly experienced by patients with IVD degeneration disease. The needle is used to pierce the outer annular fibers of the disc and allow for parts of the nucleus pulposus to leak out of the annulus fibrosus. The four plates in each group had two plates with IVDs from rats 1,2,3,4 and two plates with IVDs from rats 5,6,7,8 to ensure that IVDs from different rats were being exposed to each of the treatments. The rat tail IVD 6-day "Trucker" regimen that I developed during my first culture was used for all 12 plates. On Day 3, I performed a media change for biomarker analysis in assays at the end of my project. On Day 6, I completed the end-of-culture procedure by evaluating cell viability and viable cell density in all of the discs. To perform cell viability testing, I had to bisect all of the IVDs so that the actual IVD could be exposed to the calcein AM and ethidium homodimer stains. My results have been very consistent since my first rat tail IVD load model with the .50 MPa, injured IVDs being the least viable and the no-load, uninjured IVDs being the most viable. These results support our previous IVD load studies that have also supported the correlation between higher loads combined with injury and increased cell death.
When I received the 3 canine tail IVDs, I performed the exact same harvest process that I used on the rat tail IVDs. However, I used an immense pair of rongeurs and bone cutters and extracted 27 IVDs due to the fewer number present in canine tails in comparison to rat tails. These IVDs were placed into 5 flexcell plates and were split into two different experimental groups and a negative control group. Three of the plates would receive a .50 MPa load and one of these plates would also receive an IL-1B (inflammatory cytokine) treatment. The two negative control group plates were both no-load plates. One of the plates received the IL-1B treatment and the other was negative for the treatment. Each of the plates had 6 IVDs, two (one injured/one uninjured) from each of the 3 canine tails, except for the 5th plate. The 5th plate had the 3 extra IVDs from each of the canine tails and was used as the third .50 MPa load group. Day 3 of the protocol called for a media change and Day 6 called for the end-of-culture procedure just like in the rat tail load culture. My results were very similar to my previous canine load cultures. The IL-1B, injured, .50 MPa experimental group displayed the lowest viability and the uninjured negative control group without the IL-1B treatment showed the greatest viability. These are the results Dr. Stoker and I have come to expect with the IL-1B treatment. It seems to cause a great deal of cell death within the tissue, which supports our hypothesis that chronic inflammation plays a role in the degeneration of tissues from the knee joint to the spine.
Additionally, the lab also had an MOI organized study day this week. This was a new experience for me because the lab's study days are usually funded and lead by Arthrex surgeons and vets. Dr. Cook, my PI and the head veterinary orthopaedic surgeon, was the head surgeon for this study day, which allowed me to assist him in the procedure in ways I was not permitted to in the Arthrex studies. The study day's goal was to arthroscopically evaluate a fresh ACL to ACL allograft that was fixated with the suspensory technique (w/cortical buttons) 6 weeks prior in a canine model. Dr. Cook first manually felt for tibial translation in the knee and then gauged lameness as he watched the canine's gait. The canine was then put under anesthesia and the arthroscopic examination followed. An arthroscopic evaluation consists of scoping the knee joint with a small camera on the end of a probe to examine the condition of the tissue. Dr. Cook instructed us on how to perform an arthroscopy and evaluate a canine knee while he was inspecting the ACL allograft and the surrounding tissues. I learned that all of the healthy cartilage, ligaments and other tissue should look white, smooth, and shiny. As Dr. Cook moved in and out of the joint compartments he showed us the cartilage, menisci, and allograft and they all seemed to be pretty white, smooth and shiny. He was really happy with the outcomes and judged each allograft to be successful. He explained that he saw minimal fraying of the ACL, solid fixation and integration of the ACL allograft, and no signs of tissue damage or degeneration in the surrounding tissues. Dr. Cook, Dr. Stoker, Dr. Bozynski and I were the first people in the world to successfully fixate and examine a successful ACL to ACL allograft. It certainly looks like a promising method of ACL reconstruction and has the potential to completely revolutionize the method of reconstruction. I am very excited to have had the opportunity to be a part of such an exciting breakthrough! (Make sure to check out the arthroscopic images below)
When I received the 3 canine tail IVDs, I performed the exact same harvest process that I used on the rat tail IVDs. However, I used an immense pair of rongeurs and bone cutters and extracted 27 IVDs due to the fewer number present in canine tails in comparison to rat tails. These IVDs were placed into 5 flexcell plates and were split into two different experimental groups and a negative control group. Three of the plates would receive a .50 MPa load and one of these plates would also receive an IL-1B (inflammatory cytokine) treatment. The two negative control group plates were both no-load plates. One of the plates received the IL-1B treatment and the other was negative for the treatment. Each of the plates had 6 IVDs, two (one injured/one uninjured) from each of the 3 canine tails, except for the 5th plate. The 5th plate had the 3 extra IVDs from each of the canine tails and was used as the third .50 MPa load group. Day 3 of the protocol called for a media change and Day 6 called for the end-of-culture procedure just like in the rat tail load culture. My results were very similar to my previous canine load cultures. The IL-1B, injured, .50 MPa experimental group displayed the lowest viability and the uninjured negative control group without the IL-1B treatment showed the greatest viability. These are the results Dr. Stoker and I have come to expect with the IL-1B treatment. It seems to cause a great deal of cell death within the tissue, which supports our hypothesis that chronic inflammation plays a role in the degeneration of tissues from the knee joint to the spine.
Additionally, the lab also had an MOI organized study day this week. This was a new experience for me because the lab's study days are usually funded and lead by Arthrex surgeons and vets. Dr. Cook, my PI and the head veterinary orthopaedic surgeon, was the head surgeon for this study day, which allowed me to assist him in the procedure in ways I was not permitted to in the Arthrex studies. The study day's goal was to arthroscopically evaluate a fresh ACL to ACL allograft that was fixated with the suspensory technique (w/cortical buttons) 6 weeks prior in a canine model. Dr. Cook first manually felt for tibial translation in the knee and then gauged lameness as he watched the canine's gait. The canine was then put under anesthesia and the arthroscopic examination followed. An arthroscopic evaluation consists of scoping the knee joint with a small camera on the end of a probe to examine the condition of the tissue. Dr. Cook instructed us on how to perform an arthroscopy and evaluate a canine knee while he was inspecting the ACL allograft and the surrounding tissues. I learned that all of the healthy cartilage, ligaments and other tissue should look white, smooth, and shiny. As Dr. Cook moved in and out of the joint compartments he showed us the cartilage, menisci, and allograft and they all seemed to be pretty white, smooth and shiny. He was really happy with the outcomes and judged each allograft to be successful. He explained that he saw minimal fraying of the ACL, solid fixation and integration of the ACL allograft, and no signs of tissue damage or degeneration in the surrounding tissues. Dr. Cook, Dr. Stoker, Dr. Bozynski and I were the first people in the world to successfully fixate and examine a successful ACL to ACL allograft. It certainly looks like a promising method of ACL reconstruction and has the potential to completely revolutionize the method of reconstruction. I am very excited to have had the opportunity to be a part of such an exciting breakthrough! (Make sure to check out the arthroscopic images below)
The ACL to ACL allograft before fixation. A suspensory fixation method was used and the cortical buttons are pictured on either side of the graft. (6 weeks ago/Week after my arrival)
Arthroscopic images of the canine knee joint as it underwent the ACL Reconstruction with the ACL to ACL allograft. (6 weeks ago)
An X-ray taken immediately post-op that shows the cortical buttons used for the ACL to ACL allograft reconstruction. The staples that were used over the sutures to close the wound can also be seen. (6 weeks ago)
Arthroscopic images of the knee joint during the 6-week post-op evaluation.
(Bottom two pictures are of the articular cartilage)
(Top center picture is the ACL to ACL allograft)
(Top left is an x-ray that shows the cortical buttons used in fixation of the graft)
(Top right is an image of the femoral condyle. The ligament in the center of the joint is the ACL to ACL allograft)
A closer look at the blades of the bone-cutters
The IVDs extracted from the 3 canine tails (9 per tail)
A closer look at the canine tail IVDs in DMEM/F12 media
My plate layout for media collection. All treatment groups (Rat number, injured/uninjured, load amount) are included.
The twelve flexcell plates used for the rat tail IVDs. The 2 24-well plates on the right were used for cell viability.
Bisection of the rat tail IVDs before cell viability. The IVD on the left is what the discs look like after culture. You can see the exposed nucleus pulposus and annulus fibrosus on the bisected disk on the right.
A portion of one of the canine tails during the IVD extraction process. The exposed bone protruding from the tail muscle and fat is the vertebral column and a single IVD. The muscle and fat lying around the tail is the tissue I stripped off to access the vertebral column.
The IVD after it is cut from the vertebral column and stripped of the muscle and fat.
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