My fourth week at the Missouri Orthopaedic Institute has been the busiest and most action-packed of them all! Surgery days, end-of-culture days, harvest days, and an immense number of media changes made for a truly unforgettable week.
Although I have yet to receive any knee joint tissues, I have been working extensively with my rat tail IVD "Trucker" model that I mentioned in my previous blog. After harvest, I cultured the IVDs under a load that simulated the compression and vibration that the spinal column experiences while driving in an 18-wheeler. The IVDs were cultured in Flexcell plates and vices that connect to a vacuum system, which forces a rubber membrane to compress the IVD against a plastic insert. I developed and programmed a 6-day regimen that followed legal commercial driving limits and allowed for a 1 Hz/.25/.50 MPa pulsating load in order to accurately simulate the vibrations and jostling that occurs while driving. All three of the plates were filled with a DMEM/F12 media and placed in the incubator for culture. The two .25/.50 MPa load plates were attached to the vacuum system to begin compression. The no-load control plate was also placed in the incubator for the 6-day experimental period. I performed a media change on the third day and collected the media for biomarker analysis at the end of the study. Day 6 was an end-of-culture day and I collected media once again and completed cell viability and viable cell density testing. This involved staining and imaging with the fluorescent microscope and using a cell counting program to determine the overall viability of the IVDs. I have gone over how these evaluations are executed and analyzed in my previous blog posts. Through the viable cell assessments, I concluded that a higher compression load results in a much lower viable cell density. Images of the IVDs that underwent a .50 MPa load and the needle-simulated annular tear injury displayed complete death of annulus fibrosis and nucleus pulposus cells. The .25 MPa injured load showed lower viable cell density than the uninjured load, but still had greater viability than the .50 MPa experimental group. The no-load control group had no abnormal cell death and were very viable. Overall, this experiment went exactly as planned and will serve as a protocol for other IVD load studies. I trimmed the IVDs and placed them in a papain buffer overnight for tissue digest. In addition, the "trucker" regimen I developed and programmed will be used by the lab to test other tissues and further experiment with the commercial trucker IVD disc degeneration disease issue. After completing this rat tail IVD "trucker" load model, I notified the veterinary school regarding my need for more rat tails to harvest the IVDs and continue with different load models.
The lab also received canine cervical, lumbar, and tail section samples from the veterinary medical diagnostic lab (VMDL) for several of the IVD studies that are currently being done. The canine tail section was given to me in order to develop an imaging protocol for tail IVDs in the Thompson Lab. I was also told to prepare some of the IVDs for my next load study. Dr. Stoker was impressed by my imaging techniques for the rat tail IVDs on the fluorescent microscope and instructed me to write out a protocol for both rat and canine tail IVDs for the undergraduate and graduate students to follow. I was to experiment with keeping and removing the vertebral bone surface on the IVD to determine which provided for a higher quality image. Due to my previous experiments with the rat tail IVDs, I knew that keeping the bone on the sides of the IVD was the best way to image them because it allowed them to lay flat on the microscope slide. This allowed for a clearer picture due to the microscope's ability to focus on the entire disk. I also determined that bisecting the IVD instead of trying to cut off both sides of the bone was the most effective way to keep the annulus fibrosus and nucleus pulposus intact for imaging. However, I did have to experiment with imaging techniques for the canine tail IVDs due to my inexperience in working with them. First, I had to sterilely clean the canine tail section in order to reduce the risk of contamination in the plate. This involved skinning and washing the tail with a chlorhexidine antiseptic in order to remove hair and any excess blood while killing any present bacteria. I then used a pair of large hemostats and bone cutting forceps to remove the surrounding muscle and tissue from the vertebral column in the tail. I cut out each of the IVDs by slicing the vertebrae on either side of the disc. The next step involved using the wet saw to accurately slice off the majority of the bone leaving only a thin layer on either side of the IVD. These thin bone plates allow for the IVD to stand up in the Flexcell plate and receive even amounts of pressure throughout the tissue. If not trimmed, the thick vertebral bone pieces block any diffusion of the media into or out of the IVD and lead to tissue death during culture. After trimming each of the IVDs on this wet saw, I transferred them from the 6-well Flexcell plate into two 24-well plates with 1 ml of PBS solution. One plate would be used for the canine tail IVD load study that I would begin in the next few days and the other plate was for the imaging protocol project. I took the IVDs that were being used for the imaging protocol project and trimmed one bone plate off of half of the IVDs and off both sides for the other half. This would allow me to determine if having the bone side face down on the microscope slide produced a better image than just tissue against the slide. I predicted that having the bone side face down would allow for the canine IVD to lay flat on the microscope slide allowing for more efficient focusing like I had seen with the rat tail IVDs. After staining and imaging both the bone-off and bone-on samples, I determined that the bone-on canine IVDs were much too tall and unstable to sit flat on the microscope slide due to the added height from the bone plates. The bone-off IVDs were much easier to squeeze flat between the microscope slides allowing for a clearer picture. I reported to Dr. Stoker and let him know what I thought about imaging the bone-off and bone-on IVDs. He agreed with me after I showed him my images and told me that the protocol would be adjusted to include my findings. Rat tail IVDs were to be imaged with both bone plates on and had to be bisected before placed on the slide. Canine tail IVDs were to be imaged with both bone plates removed and the annulus fibrosus and nucleus pulposus alone on the microscope slides.
The other group of canine tail IVDs was used to continue my tail IVD load study. I had already harvested and trimmed the IVDs from the tail beforehand, so I immediately placed them into two 6-well Flexcell plates with 5ml of DMEM/F12 media. One plate would receive a .50MPa load and the other would be the no-load control plate. I also treated half of the IVDs in both plates with IL-1B, an inflammatory cytokine, to induce inflammation and determine its effects on the viability of an IVD under load. I had to design and program another 6-day regimen that applied a heavier load to these canine tail IVDs due to their larger diameter and height in comparison to the rat tail IVDs. The load was stepped up to .50 MPa for all of the IVDs, which required approximately 3lbs of force to be exerted by the vacuum system. The samples will be kept in the incubator until day 3 when they will receive a media change for biomarker analysis on the plate reader. Day 6 will be another end-of-culture day and I will have to perform cell viability and viable cell density testing as well as a tissue digest.
In addition to all of the canine and rat tail IVD load models that I was working on this week, the lab also had three surgery study days. Arthrex, a leader in the production of surgical supplies and joint prosthetics, funded a study in which the Thompson Lab for Regenerative Orthopaedics was to evaluate a new method of canine hip arthroplasty. The previous two study days involving the hip radiographs and GAITrite evaluations were all pre-assessments for these surgery study days. 11 of our research dogs diagnosed with moderate to severe hip dysplasia through the hip radiographs were selected to receive the total hip arthroplasties. The surgery days were to run from 5:30am to 6:30pm on Tuesday, Wednesday, and Thursday. Dr. Cook, the director and head orthopaedic surgeon at MOI, wanted to get through four on the first and second day, and the remaining 3 on the final day. On Tuesday morning at around 5:00am, I walked down to the veterinary medical diagnostic lab (VMDL) to prepare the canine models and the operating room for surgery. I had bleached the entire OR the day before, so I only had to organize the surgical equipment and sterile gowns for the surgeons who would be arriving later in the morning. I then went to retrieve the first canine from her room in the VMDL and transported her to the prep room. In the prep room, the veterinary technician inserted an IV and injected a light anesthetic followed by propofol to completely put the canine to sleep. I then assisted with intubating the canine with an endotracheal tube, which was then attached to a veterinary anesthesia machine. The machine continuously pumps a mix of oxygen and isoflurane into the canine's lungs, while assisting with carbon dioxide removal. The machine also allows the anesthesia assistant to manual respirate for the canine through a squeeze bag if the canine stops breathing on its own. I then inserted an esophageal stethoscope to monitor the canine's heart and respiration rate throughout the prep and surgery periods. I completed the surgery prep procedure by shaving the appropriate limb, cleaning it with an antiseptic scrub, and wrapping and hanging the limb from a leg support. I then proceeded to do the anesthesia checks, which involved measuring oxygen and isoflurane levels from the anesthesia machine, recording heart and respiration rate through the stethoscope, checking capillary refill time through the gingiva to monitor blood pressure, and evaluating the palpebral reflex and jaw tone to determine anesthetic depth. For all eleven of the surgeries throughout the three days, I was assigned to preparing the canine for surgery as well as being the anesthesia assistant in the OR. Being the anesthesia assistant in the OR was extremely exciting, however it required a great deal of focus to constantly monitor the canine's vitals during the three-hour surgery. My other jobs included post-operative care, resterilizing the OR and the instruments needed by the surgeons, and safe transport of the canines after sedation and surgery. Overall, these canines were constantly monitored by the students and attending surgeons and veterinarians and received a full hip replacement that will hopefully help them regain a normal gait and a healthy lifestyle. Their femoral head and acetabulum were removed by an oscillating saw and replaced with titanium prosthetics. So far, the canines have been doing very well and are already walking and exercising daily. Hip radiographs were performed immediately subsequent to surgery and GAITrite evaluations will be redone in the next few days to compare pre-op and post-op lameness.
I am also continuing to attend all journal clubs and graduate classes instructed by Dr. Cook and Dr. Stoker. I am learning a great deal about western blots through these lectures and have even attempted a few with the graduate students. I look forward to another exciting week in the Thompson Lab for Regenerative Orthopaedics!
Although I have yet to receive any knee joint tissues, I have been working extensively with my rat tail IVD "Trucker" model that I mentioned in my previous blog. After harvest, I cultured the IVDs under a load that simulated the compression and vibration that the spinal column experiences while driving in an 18-wheeler. The IVDs were cultured in Flexcell plates and vices that connect to a vacuum system, which forces a rubber membrane to compress the IVD against a plastic insert. I developed and programmed a 6-day regimen that followed legal commercial driving limits and allowed for a 1 Hz/.25/.50 MPa pulsating load in order to accurately simulate the vibrations and jostling that occurs while driving. All three of the plates were filled with a DMEM/F12 media and placed in the incubator for culture. The two .25/.50 MPa load plates were attached to the vacuum system to begin compression. The no-load control plate was also placed in the incubator for the 6-day experimental period. I performed a media change on the third day and collected the media for biomarker analysis at the end of the study. Day 6 was an end-of-culture day and I collected media once again and completed cell viability and viable cell density testing. This involved staining and imaging with the fluorescent microscope and using a cell counting program to determine the overall viability of the IVDs. I have gone over how these evaluations are executed and analyzed in my previous blog posts. Through the viable cell assessments, I concluded that a higher compression load results in a much lower viable cell density. Images of the IVDs that underwent a .50 MPa load and the needle-simulated annular tear injury displayed complete death of annulus fibrosis and nucleus pulposus cells. The .25 MPa injured load showed lower viable cell density than the uninjured load, but still had greater viability than the .50 MPa experimental group. The no-load control group had no abnormal cell death and were very viable. Overall, this experiment went exactly as planned and will serve as a protocol for other IVD load studies. I trimmed the IVDs and placed them in a papain buffer overnight for tissue digest. In addition, the "trucker" regimen I developed and programmed will be used by the lab to test other tissues and further experiment with the commercial trucker IVD disc degeneration disease issue. After completing this rat tail IVD "trucker" load model, I notified the veterinary school regarding my need for more rat tails to harvest the IVDs and continue with different load models.
The lab also received canine cervical, lumbar, and tail section samples from the veterinary medical diagnostic lab (VMDL) for several of the IVD studies that are currently being done. The canine tail section was given to me in order to develop an imaging protocol for tail IVDs in the Thompson Lab. I was also told to prepare some of the IVDs for my next load study. Dr. Stoker was impressed by my imaging techniques for the rat tail IVDs on the fluorescent microscope and instructed me to write out a protocol for both rat and canine tail IVDs for the undergraduate and graduate students to follow. I was to experiment with keeping and removing the vertebral bone surface on the IVD to determine which provided for a higher quality image. Due to my previous experiments with the rat tail IVDs, I knew that keeping the bone on the sides of the IVD was the best way to image them because it allowed them to lay flat on the microscope slide. This allowed for a clearer picture due to the microscope's ability to focus on the entire disk. I also determined that bisecting the IVD instead of trying to cut off both sides of the bone was the most effective way to keep the annulus fibrosus and nucleus pulposus intact for imaging. However, I did have to experiment with imaging techniques for the canine tail IVDs due to my inexperience in working with them. First, I had to sterilely clean the canine tail section in order to reduce the risk of contamination in the plate. This involved skinning and washing the tail with a chlorhexidine antiseptic in order to remove hair and any excess blood while killing any present bacteria. I then used a pair of large hemostats and bone cutting forceps to remove the surrounding muscle and tissue from the vertebral column in the tail. I cut out each of the IVDs by slicing the vertebrae on either side of the disc. The next step involved using the wet saw to accurately slice off the majority of the bone leaving only a thin layer on either side of the IVD. These thin bone plates allow for the IVD to stand up in the Flexcell plate and receive even amounts of pressure throughout the tissue. If not trimmed, the thick vertebral bone pieces block any diffusion of the media into or out of the IVD and lead to tissue death during culture. After trimming each of the IVDs on this wet saw, I transferred them from the 6-well Flexcell plate into two 24-well plates with 1 ml of PBS solution. One plate would be used for the canine tail IVD load study that I would begin in the next few days and the other plate was for the imaging protocol project. I took the IVDs that were being used for the imaging protocol project and trimmed one bone plate off of half of the IVDs and off both sides for the other half. This would allow me to determine if having the bone side face down on the microscope slide produced a better image than just tissue against the slide. I predicted that having the bone side face down would allow for the canine IVD to lay flat on the microscope slide allowing for more efficient focusing like I had seen with the rat tail IVDs. After staining and imaging both the bone-off and bone-on samples, I determined that the bone-on canine IVDs were much too tall and unstable to sit flat on the microscope slide due to the added height from the bone plates. The bone-off IVDs were much easier to squeeze flat between the microscope slides allowing for a clearer picture. I reported to Dr. Stoker and let him know what I thought about imaging the bone-off and bone-on IVDs. He agreed with me after I showed him my images and told me that the protocol would be adjusted to include my findings. Rat tail IVDs were to be imaged with both bone plates on and had to be bisected before placed on the slide. Canine tail IVDs were to be imaged with both bone plates removed and the annulus fibrosus and nucleus pulposus alone on the microscope slides.
The other group of canine tail IVDs was used to continue my tail IVD load study. I had already harvested and trimmed the IVDs from the tail beforehand, so I immediately placed them into two 6-well Flexcell plates with 5ml of DMEM/F12 media. One plate would receive a .50MPa load and the other would be the no-load control plate. I also treated half of the IVDs in both plates with IL-1B, an inflammatory cytokine, to induce inflammation and determine its effects on the viability of an IVD under load. I had to design and program another 6-day regimen that applied a heavier load to these canine tail IVDs due to their larger diameter and height in comparison to the rat tail IVDs. The load was stepped up to .50 MPa for all of the IVDs, which required approximately 3lbs of force to be exerted by the vacuum system. The samples will be kept in the incubator until day 3 when they will receive a media change for biomarker analysis on the plate reader. Day 6 will be another end-of-culture day and I will have to perform cell viability and viable cell density testing as well as a tissue digest.
In addition to all of the canine and rat tail IVD load models that I was working on this week, the lab also had three surgery study days. Arthrex, a leader in the production of surgical supplies and joint prosthetics, funded a study in which the Thompson Lab for Regenerative Orthopaedics was to evaluate a new method of canine hip arthroplasty. The previous two study days involving the hip radiographs and GAITrite evaluations were all pre-assessments for these surgery study days. 11 of our research dogs diagnosed with moderate to severe hip dysplasia through the hip radiographs were selected to receive the total hip arthroplasties. The surgery days were to run from 5:30am to 6:30pm on Tuesday, Wednesday, and Thursday. Dr. Cook, the director and head orthopaedic surgeon at MOI, wanted to get through four on the first and second day, and the remaining 3 on the final day. On Tuesday morning at around 5:00am, I walked down to the veterinary medical diagnostic lab (VMDL) to prepare the canine models and the operating room for surgery. I had bleached the entire OR the day before, so I only had to organize the surgical equipment and sterile gowns for the surgeons who would be arriving later in the morning. I then went to retrieve the first canine from her room in the VMDL and transported her to the prep room. In the prep room, the veterinary technician inserted an IV and injected a light anesthetic followed by propofol to completely put the canine to sleep. I then assisted with intubating the canine with an endotracheal tube, which was then attached to a veterinary anesthesia machine. The machine continuously pumps a mix of oxygen and isoflurane into the canine's lungs, while assisting with carbon dioxide removal. The machine also allows the anesthesia assistant to manual respirate for the canine through a squeeze bag if the canine stops breathing on its own. I then inserted an esophageal stethoscope to monitor the canine's heart and respiration rate throughout the prep and surgery periods. I completed the surgery prep procedure by shaving the appropriate limb, cleaning it with an antiseptic scrub, and wrapping and hanging the limb from a leg support. I then proceeded to do the anesthesia checks, which involved measuring oxygen and isoflurane levels from the anesthesia machine, recording heart and respiration rate through the stethoscope, checking capillary refill time through the gingiva to monitor blood pressure, and evaluating the palpebral reflex and jaw tone to determine anesthetic depth. For all eleven of the surgeries throughout the three days, I was assigned to preparing the canine for surgery as well as being the anesthesia assistant in the OR. Being the anesthesia assistant in the OR was extremely exciting, however it required a great deal of focus to constantly monitor the canine's vitals during the three-hour surgery. My other jobs included post-operative care, resterilizing the OR and the instruments needed by the surgeons, and safe transport of the canines after sedation and surgery. Overall, these canines were constantly monitored by the students and attending surgeons and veterinarians and received a full hip replacement that will hopefully help them regain a normal gait and a healthy lifestyle. Their femoral head and acetabulum were removed by an oscillating saw and replaced with titanium prosthetics. So far, the canines have been doing very well and are already walking and exercising daily. Hip radiographs were performed immediately subsequent to surgery and GAITrite evaluations will be redone in the next few days to compare pre-op and post-op lameness.
I am also continuing to attend all journal clubs and graduate classes instructed by Dr. Cook and Dr. Stoker. I am learning a great deal about western blots through these lectures and have even attempted a few with the graduate students. I look forward to another exciting week in the Thompson Lab for Regenerative Orthopaedics!
Hip radiograph after the total hip arthroplasty. Femoral head and acetabulum titanium prosthetic on right hip joint.
Picture of my point of view while monitoring the canine under anesthesia.
No-load control plate for the canine tail IVD load study.
.50 MPa loaded plate attached to the vacuum system. (Canine tail IVD load study)
Our first attempt at a western blot in one of the graduate student classes.
My new regiment for the canine tail IVD load study. (3lbs of force for .50 MPa compression load)
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