As mentioned in my previous blog, I have been awaiting ligament, menisci, and cartilage tissue from a canine or human knee joint for (interleukin) IL-1B tissue culture. IL-1B is an inflammatory cytokine that has been proven to increase rates of tissue degeneration and osteoarthritis development in the Thompson Lab. Dr. Stoker wants me to experiment with different types of knee tissues in a co-culture with varying levels of this cytokine to determine its effects on the entire knee joint. This co-culture uses an insert permeable to the media to separate the two tissue samples from physical contact, while allowing them to share the same media. This creates an extremely accurate model for knee tissues in their native environment due to their exposure to the same synovial fluid in the joint. This model would then be treated with the IL-1B and cultured for 21 days. During these 21 days, the media would be collected every three days for biomarker evaluation at the end of the study. This biomarker evaluation is performed by a plate reader that identifies different proteins within an assay that can indicate tissue degeneration and osteoarthritis severity. These proteins can include proteolytic enzymes such as aggrecanase, which break down the proteoglycans in the cartilage matrix. The presence of this protein is a positive indicator of osteoarthritis development. After the culture and media changes are complete, the remaining tissue would undergo cell viability and viable cell density testing. This includes cutting flat slices from the tissues on the wet saw and staining them with calcein AM and ethidium homodimer for imaging under the fluorescent microscope. The image displays the cell viability by fluorescing red or green. Viable cell density is performed with a cell counting program, which determines how many cells are in the tissue in a given area. Finally, the tissue is sent to Dr. Bozynski, the lab pathologist, for histopathological assessment. All of these assessments would provide data for the condition of the tissue after the IL-1B treatment and would allow us to determine the effects of IL-1B on the entire knee joint. Unfortunately, I still have not received these tissues and have been told by Dr. Stoker that these tissues are sometimes difficult to come by depending on the time of year. Euthanization of canine models for tissue harvest is unpredictable due to the lab's dependence on animal shelters and the veterinary school. Furthermore, in order to obtain human tissue, the patient must give consent for the harvest of the tissue during an ACL reconstruction or total knee replacement, which is also quite a rare situation. Dr. Stoker and I have decided that in the meantime, I should begin my independent project with rat tail intervertebral disks. This project will be part of the intervertebral disk degeneration disease load study that just received grant approval.
Intervertebral disks (IVDs) are fibrocartilaginous joints that lie between the vertebrae and allow for shock absorption and mobility in the spine. They are made up of the nucleus pulposus, a soft gel-like substance made of proteoglycans and water, and the annulus fibrosus, the tough outer layer of the disk composed of interwoven ligament fibers. The nucleus pulposus lies in the middle of the disk and is what allows for the shock absorbing characteristic of IVDs. The annulus fibers prevent the nucleus pulposus from herniating out of the disk by sealing it and evenly distributing pressure. Intervertebral disk degeneration disease occurs when these disks collapse and cease to function. The degeneration of these disks begins with an annular tear, which happens as a result of the natural aging process of the spine or an injury. When these tears heal, they are replaced by scar tissue, which is much weaker than the annulus fibers. Over time or with repeated injury, the annulus tears and heals with scar tissue, gradually weakening the disks. As these disks are injured and weakened, the nucleus pulposus loses a significant amount of its water content. This water loss prevents the nucleus pulposus from serving as a cushion and the IVD collapses as the vertebrae on the top and bottom move closer together. This results in improper alignment of the vertebrae, which puts excessive pressure on the facet joints causing wear and the formation of bone spurs. These bone spurs can then grow into the spinal canal and pinch the spinal cord and surrounding nerves. This process is known as spinal stenosis. As the disks collapse, nerve roots can be pinched and the nucleus pulposus can also herniate out of the disk irritating nerves and causing increased inflammation and pain. Symptoms for degenerative disk disease can include tingling, numbness, and extreme pain in the lower back, hips, and legs.
Due to the lack of available tissues for the knee joint IL-1B tissue culture, Dr. Stoker assigned me to the rat tail IVD portion of the intervertebral disk degeneration disease load study. The rat tail IVDs are readily available from the university vet school and they can handle relatively high levels of compression without dying. They also share a very similar structure to human IVDs. This makes them a perfect model for this study. The study aims to identify the different types of loads that are harmful to IVDs as well as any biomarkers associated with IVD degeneration disease. According to Dr. Stoker, it has been concluded in several papers that truck drivers have a significantly higher risk of IVD degeneration disease due to many factors. I had to sort through several journals on NCIB and Pubmed to find similar articles with the same conclusions. I then presented them to Dr. Stoker for approval. As a result, the first experiment I performed included culturing rat tail IVDs under a load that simulates driving in a truck seat for an extended period of time. However, prior to beginning this experiment, I had to obtain the rat models from the vet school and harvest the IVDs from the tail. Harvesting the IVDs proved to be fairly simple and required a pair of hemostats and bone cutting forceps. The tissue surrounding the vertebral column in the rat tail had to be removed with the hemostats. The IVDs were then harvested with the bone cutting forceps by cutting off each of the vertebrae surrounding the disks. The rat tail IVDs were placed in a DMEM/F12 media and stored in the incubator for tissue culture. Before applying the compression to the IVDs, three different experimental groups were created. Each plate received two injured and two non-injured IVDs. This injury consisted of piercing the annular wall with a needle simulating an annular tear. One of the plates received a .25 MPa load and the other received a .50 MPa load. The third plate was the no-load control group. The lab uses the Flexcell system and culture plates to apply a load to tissues. The Flexwell plates are six-well plates with rubber membranes on the bottom and a plastic insert that screws on to the top. These plates are placed in a vice that is attached to the Flexcell vacuum system, which actually applies the load. The vacuum system forces the rubber membrane up against the plastic insert and compresses the rat tail IVD. I had to program this vacuum system to compress at a frequency of 1 Hz to simulate the vibrations and pulsating compressions experienced while sitting and driving in a truck. Additionally, the vacuum had to be programmed to apply the load for five hours with a one hour break before another six hours. A twelve-hour break followed to simulate sleep. These regimens were carefully planned out to follow the legal driving limits that commercial truck drivers have to follow while on the road. Overall, this system models the loads and vibrations that the spinal column experiences during a commercial truck driver's day. The rat tail IVDs will be left in the Flexcell system for another six days and will receive a media change at the three-day mark. This media will be used in an assay to identify any biomarkers associated with IVD degeneration disease in a plate reader at the end of the study. The IVDs will also undergo cell viability and viable cell density testing at the end of the study to determine the type of load which caused the most damage to the tissues. Once the rat tail IVD "Trucker" model is completed, I will select another demographic of patients proven to have a suspiciously high rate of IVD degeneration disease. I will then have to reprogram the Flexcell system to simulate the loads the spinal column experiences on a daily basis according to that demographic's lifestyle. I am thinking about using a professional football player model for the next experiment, which would consist of large compressions to the IVDs at staggered intervals instead of the continuous pulse used in the trucker model. This study will provide data on the loads that are most damaging to the IVDs and if there are any biomarkers that can be used for diagnosis during the pre-IVD degeneration disease stages.
Another live canine study day was held during this week and the graduate students and I were instructed to meet the PIs at the veterinary school. The study currently going on involves 12 mixed breed hounds that are being treated for hip dysplasia symptoms. Arthrex, an orthopaedic medical device company, is funding the study and wants the lab to evaluate a new hip arthroplasty prosthetic. These 12 canines were radiographed during last week's study day to evaluate hip dysplasia severity. This week, they underwent a GAITrite evaluation, which involves having each of the dogs walk and run on a 25-foot mat studded with pressure sensors. Next week, they will undergo a hip arthroplasty with the arthrex prosthetic and will be reevaluated on the GAITrite once fully recovered. The data shows how much weight the canine is putting on each limb during their walk/run and how even and balanced their gate is. From this data, the pre-op and post-op lameness can be compared and sent back to Arthrex who can use it for further refinement of the prosthetic. My role in the GAITrite evaluations was to walk the canines up and down the mat while making sure their gait was uninterrupted. This proved to be extremely difficult because many of the dogs are scared to walk on the mat and either refuse to step on it or hop awkwardly and interrupt their natural gaits. The canines have to perform a perfect walk from the beginning of the mat to the end for the data to be valid, so the uncooperative ones had to be repeatedly tested until they finally gave in and stopped resisting. The whole team was drenched in sweat and exhausted by the end of this test. In addition, I have continued to attend all of the graduate classes and journal clubs that Dr. Stoker and Dr. Cook teach. In the last journal club, we discussed biomarker traces that can be found in serum and urine and the different assays used to identify them. We also discussed how they may be able to be used as an indicator for osteoarthritis allowing for diagnosis before the degeneration is radiographically detectable. In the graduate level classes, we have started our own tissue digests and cultures and have started discussing western blots and how they can be used in our studies due to the arrival of a new plate reader that can read western and dot blots. I look forward to continuing my rat tail IVD "Trucker" load study and participating in the full hip arthroplasty surgery day next week.
Intervertebral disks (IVDs) are fibrocartilaginous joints that lie between the vertebrae and allow for shock absorption and mobility in the spine. They are made up of the nucleus pulposus, a soft gel-like substance made of proteoglycans and water, and the annulus fibrosus, the tough outer layer of the disk composed of interwoven ligament fibers. The nucleus pulposus lies in the middle of the disk and is what allows for the shock absorbing characteristic of IVDs. The annulus fibers prevent the nucleus pulposus from herniating out of the disk by sealing it and evenly distributing pressure. Intervertebral disk degeneration disease occurs when these disks collapse and cease to function. The degeneration of these disks begins with an annular tear, which happens as a result of the natural aging process of the spine or an injury. When these tears heal, they are replaced by scar tissue, which is much weaker than the annulus fibers. Over time or with repeated injury, the annulus tears and heals with scar tissue, gradually weakening the disks. As these disks are injured and weakened, the nucleus pulposus loses a significant amount of its water content. This water loss prevents the nucleus pulposus from serving as a cushion and the IVD collapses as the vertebrae on the top and bottom move closer together. This results in improper alignment of the vertebrae, which puts excessive pressure on the facet joints causing wear and the formation of bone spurs. These bone spurs can then grow into the spinal canal and pinch the spinal cord and surrounding nerves. This process is known as spinal stenosis. As the disks collapse, nerve roots can be pinched and the nucleus pulposus can also herniate out of the disk irritating nerves and causing increased inflammation and pain. Symptoms for degenerative disk disease can include tingling, numbness, and extreme pain in the lower back, hips, and legs.
Due to the lack of available tissues for the knee joint IL-1B tissue culture, Dr. Stoker assigned me to the rat tail IVD portion of the intervertebral disk degeneration disease load study. The rat tail IVDs are readily available from the university vet school and they can handle relatively high levels of compression without dying. They also share a very similar structure to human IVDs. This makes them a perfect model for this study. The study aims to identify the different types of loads that are harmful to IVDs as well as any biomarkers associated with IVD degeneration disease. According to Dr. Stoker, it has been concluded in several papers that truck drivers have a significantly higher risk of IVD degeneration disease due to many factors. I had to sort through several journals on NCIB and Pubmed to find similar articles with the same conclusions. I then presented them to Dr. Stoker for approval. As a result, the first experiment I performed included culturing rat tail IVDs under a load that simulates driving in a truck seat for an extended period of time. However, prior to beginning this experiment, I had to obtain the rat models from the vet school and harvest the IVDs from the tail. Harvesting the IVDs proved to be fairly simple and required a pair of hemostats and bone cutting forceps. The tissue surrounding the vertebral column in the rat tail had to be removed with the hemostats. The IVDs were then harvested with the bone cutting forceps by cutting off each of the vertebrae surrounding the disks. The rat tail IVDs were placed in a DMEM/F12 media and stored in the incubator for tissue culture. Before applying the compression to the IVDs, three different experimental groups were created. Each plate received two injured and two non-injured IVDs. This injury consisted of piercing the annular wall with a needle simulating an annular tear. One of the plates received a .25 MPa load and the other received a .50 MPa load. The third plate was the no-load control group. The lab uses the Flexcell system and culture plates to apply a load to tissues. The Flexwell plates are six-well plates with rubber membranes on the bottom and a plastic insert that screws on to the top. These plates are placed in a vice that is attached to the Flexcell vacuum system, which actually applies the load. The vacuum system forces the rubber membrane up against the plastic insert and compresses the rat tail IVD. I had to program this vacuum system to compress at a frequency of 1 Hz to simulate the vibrations and pulsating compressions experienced while sitting and driving in a truck. Additionally, the vacuum had to be programmed to apply the load for five hours with a one hour break before another six hours. A twelve-hour break followed to simulate sleep. These regimens were carefully planned out to follow the legal driving limits that commercial truck drivers have to follow while on the road. Overall, this system models the loads and vibrations that the spinal column experiences during a commercial truck driver's day. The rat tail IVDs will be left in the Flexcell system for another six days and will receive a media change at the three-day mark. This media will be used in an assay to identify any biomarkers associated with IVD degeneration disease in a plate reader at the end of the study. The IVDs will also undergo cell viability and viable cell density testing at the end of the study to determine the type of load which caused the most damage to the tissues. Once the rat tail IVD "Trucker" model is completed, I will select another demographic of patients proven to have a suspiciously high rate of IVD degeneration disease. I will then have to reprogram the Flexcell system to simulate the loads the spinal column experiences on a daily basis according to that demographic's lifestyle. I am thinking about using a professional football player model for the next experiment, which would consist of large compressions to the IVDs at staggered intervals instead of the continuous pulse used in the trucker model. This study will provide data on the loads that are most damaging to the IVDs and if there are any biomarkers that can be used for diagnosis during the pre-IVD degeneration disease stages.
Another live canine study day was held during this week and the graduate students and I were instructed to meet the PIs at the veterinary school. The study currently going on involves 12 mixed breed hounds that are being treated for hip dysplasia symptoms. Arthrex, an orthopaedic medical device company, is funding the study and wants the lab to evaluate a new hip arthroplasty prosthetic. These 12 canines were radiographed during last week's study day to evaluate hip dysplasia severity. This week, they underwent a GAITrite evaluation, which involves having each of the dogs walk and run on a 25-foot mat studded with pressure sensors. Next week, they will undergo a hip arthroplasty with the arthrex prosthetic and will be reevaluated on the GAITrite once fully recovered. The data shows how much weight the canine is putting on each limb during their walk/run and how even and balanced their gate is. From this data, the pre-op and post-op lameness can be compared and sent back to Arthrex who can use it for further refinement of the prosthetic. My role in the GAITrite evaluations was to walk the canines up and down the mat while making sure their gait was uninterrupted. This proved to be extremely difficult because many of the dogs are scared to walk on the mat and either refuse to step on it or hop awkwardly and interrupt their natural gaits. The canines have to perform a perfect walk from the beginning of the mat to the end for the data to be valid, so the uncooperative ones had to be repeatedly tested until they finally gave in and stopped resisting. The whole team was drenched in sweat and exhausted by the end of this test. In addition, I have continued to attend all of the graduate classes and journal clubs that Dr. Stoker and Dr. Cook teach. In the last journal club, we discussed biomarker traces that can be found in serum and urine and the different assays used to identify them. We also discussed how they may be able to be used as an indicator for osteoarthritis allowing for diagnosis before the degeneration is radiographically detectable. In the graduate level classes, we have started our own tissue digests and cultures and have started discussing western blots and how they can be used in our studies due to the arrival of a new plate reader that can read western and dot blots. I look forward to continuing my rat tail IVD "Trucker" load study and participating in the full hip arthroplasty surgery day next week.
The Flexcell Program displaying the .5MPa compression load on the rat tail IVDs. You can see that the load is pulsated to simulate the vibrations and jostling of the spinal column during commercial truck driving.
The Flexcell plates in the vice and inside of the incubator. The Flexcell vacuum attaches to the plates through two tubes on the bottom of the vice. The non-load control group is below the vice.
An idea of how the GAITrite evaluations are performed.
Comments
Post a Comment