After landing in the small, but lively city of Columbia, Missouri on a runway much too narrow and short for comfort, I was greeted by the enormous healthcare complex that makes up the University of Missouri Hospital. I had never seen such a vast collection of specialty hospitals and was surprised to find the Truman VA Hospital, Fischel Cancer Center, and Orthopaedic Institute all within walking distance of the main University Hospital. Each of the hospitals was built only a few years ago and all of them boast a very modern architecture. It was truly a remarkable sight to see and I grew more and more excited as we passed the glass front doors of each building. We arrived at my dorm on Mizzou's campus, which is only a few blocks south of the University Hospital. My daily commute would be a two-minute walk to the front doors of the Orthopaedic Institute. I could not have imagined a more convenient location to live.
I entered the Missouri Orthopaedic Insititute the next morning and rode the elevator all the way up to the fourth floor where the Thompson Laboratory for Regenerative Orthopaedics is located. In the lobby, I met both of my PIs, Dr. Cook and Dr. Stoker, who gave me a tour of the Thompson Lab. The expansive lab consists of small subbranches, which include a bioengineering lab, a tissue culture room, a histology lab, and the administrative offices. I was told that I would be spending most of my time in the tissue culture room, which houses several laminar flow hoods, incubators, and enormous freezers used to store and safely work with a variety of tissues. Shortly after, I was introduced to the lab pathologist, Dr. Bozynski, who showed me around the histology lab and introduced me to the five graduate students in the lab. Two of them were new to the lab and the others had been there for nearly five years, which made becoming accustomed to the lab very easy for me. I always had someone to approach with questions and had several other students to interact with throughout the initial training. So far, I have been treated identically to these graduate students and have been assigned the same responsibilities and introductory projects. This has been fantastic and has given me the opportunity to show off my knowledge and lab skills to the PIs as they assign me projects usually given to graduate students. Dr. Bozynski then introduced me to the veterinary team that works in the lab. They were all very welcoming and told me a little about the large animal models that are used in the lab. This list includes canine, lapine, caprine, ovine, bovine, and equine models and several other smaller animal models. Dr. Stoker then gathered the group of graduate students and I and drove us down to the veterinary school where the lab used to be located. They had recently moved into the Missouri Orthopaedic Institute and had not yet finished moving all of the tissue samples from the freezers in the veterinary school. We were instructed to carry in the lab's industrial grade coolers and transport the remaining tissue samples from the freezers into the lab's van without allowing any of the tissues to thaw. I opened one of the freezers to begin my task and was taken aback when I found the freezers stocked full of detached canine limbs. However, I started grabbing and loading the coolers as quickly as possible to prevent any unnecessary warming of the tissues and quickly grew comfortable handling the frozen canine limbs. We loaded the coolers back into the van and sped back to the lab. Once we returned to the lab, we unloaded approximately fifty limbs back into the immense -80 degrees Celsius freezer labeled "K9". Dr. Stoker than introduced me to Sebastian, the Ph.D. student who I would be working closely with for the rest of my time in the lab. Sebastian is a Fulbright Scholar from Colombia and has spent the last few months working on culturing human and canine ACL cells under load to determine the effects of different physical therapy methods after ACL reconstruction. Sebastian and I talked for about an hour about ACL reconstruction and the work he was currently doing with ACL tissue. During this conversation with Sebastian, I realized that I was going to fit in extremely well in the lab due to everyone's shared interests in orthopaedics and ACL reconstruction.
Over the next two weeks, I spent my time learning about all of the current studies the lab was performing, learning the ins and outs of working safely with human and canine tissue, and beginning my independent project. Before I touched a pipette, Dr. Stoker made sure that I understood that the main goal behind all of the studies completed at MOI was to discover better surgical and therapeutic techniques for traumatic orthopaedic injuries in order to reduce recovery time and the risk of osteoarthritis. Due to my previous experience with a traumatic knee injury that required a full year of rehabilitation and increased my risk of osteoarthritis significantly, MOI's goal has a personal touch and inspires me to work diligently in the lab every day. The main studies going on in the lab include a cell viability study on tissues stored using a specific preservation system, an intervertebral disc degeneration study, and an ACL cell and tissue culture study. Dr. Stoker wanted me to experience each of the studies so that I could observe the different laboratory techniques used in MOI. I thought that this would be a valuable learning experience because I would have to decide which techniques to use for my independent study within the next few days. The cell viability study was being performed by the two senior graduate students. The study involves imaging slices of human articular cartilage from osteochondral grafts preserved in an MOI produced media. The cartilage samples are harvested from either the femoral condyle, tibial plateau, menisci, or trochlear groove. They are then sliced into flat pieces on a low-speed wet saw. The pieces are then placed into a PBS solution and stained with both a live and dead cell stain. The live cell stain, calcein AM, is a cell-permeant dye that is metabolized by living cells and fluoresces green under a fluorescent microscope. The dead cell stain, ethidium homodimer, is a cell-impermeant stain that only enters cells with holes in the plasma membrane. Once inside these dead or dying cells, the stain binds to the DNA and fluoresces red under the fluorescent microscope. The tissue samples are then imaged under the fluorescent microscope under two different wavelengths that activate each of the stains. The images are superimposed on each other and the living and dead cells can be observed to determine overall cell viability in the tissue sample. A cell counting program is then used to determine viable cell density in the sample. All of this data will be used in a Missouri Osteochondral Preservation System (MOPS) viability study. Due to the need for immediate processing of these samples as soon as they are delivered from the operating room, I have been added to the "on-call" list, which keeps a record of which graduate student is responsible for the cell viability tests that day. I had to undergo several hours of training with Dr. Stoker on how to properly work with human tissues and a surgical scalpel. In addition, I was taught how to remove thin slices of cartilage from these samples for chondrocyte culture in a separate study. Next, I observed the intervertebral disk degeneration study being performed by a veterinary team member and an orthopaedic spine surgeon from MOI. The study uses canine spine and tail IVDs as well as human IVDs to serve as models for disk degeneration disease. The main function of IVDs is to serve as a shock absorber for the spine. They are located in between vertebrae and are made up of the nucleus pulposus and annulus fibrosus. Degenerative disk disease often results in the herniation of the disk as the nucleus pulposus pushes out of the annulus fibrosus. This study is being performed to determine if overweight, diabetic patients have an increased risk of degenerative disk disease due to high blood glucose levels and abnormal insulin-transferrin-selenium levels. The IVD is removed from the spine or tail with a low-speed wet saw, and the nucleus pulposus and annulus fibrosus are harvested and cultured in different types of media in a 24 well plate. These different types of media have varied glucose and ITS levels to determine what kind of effects these substances have on the IVD. While observing this study, I was instructed to change and collect the media every other day and harvest the canine tail IVDs on the wet saw upon their arrival. These media changes often overlap with the weekend, so I had to be in the lab on Saturday and Sunday for two media changes last week. I only live a short walk away and thoroughly enjoyed being in the lab and perfecting my pipetting skills over the weekend. The media is transferred to a collection plate and will be experimented with in ELISA assays in the near future. The ELISA assays will be used to identify different biomarkers associated with disk degeneration disease with the help of a plate reader machine. These media transfers and ELISA assays require an enormous amount of pipetting, and I have AP Bio to thank for preparing me so well.
The final study I observed was Sebastian's human and canine ACL culture experiment. As previously mentioned, he is performing monolayer cell culture and tissue culture with ACL samples. While the cells and tissues are being cultured, they are placed under different types and amounts of load. This is performed with a Flexcell system, which uses special 6 well plates to place compression and tension loads on samples. This study aims to determine the benefits or disadvantages of a more rigorous physical therapy protocol that places more load on an ACL reconstruction graft before complete healing, but results in quicker muscle strengthening. This is the study that I will actively participate in the most, so I made sure to take notes on Sebastian's culturing protocols and Flexcell procedures. I learned very quickly that sterility in the culture room is of utmost importance due to the considerable risk of contamination in your cultures. To become and remain sterile in the culture room, you must wear shoe covers, sterile surgical gloves, and douse your arms and hands in alcohol every minute or so. It is quite a tedious process, but it is vitally important to successful tissue and cell cultures. Sebastian also taught me how to identify different ACL cells and observe the differentiation of ligamentocytes and fibroblasts in his cultures. After, I worked with Sebastian to develop a procedure for my independent project. Due to my analysis of numerous ACL reconstruction articles in Dr. Peretz's spring EXP class, I knew that many scientists believe that inflammation after ACL tear and reconstruction is a direct cause of osteoarthritis development in the affected knee and was fascinated by this idea. I mentioned this to Sebastian and he told me about how they often culture their cartilage samples in IL-1B, an inflammatory cytokine, to determine if inflammation has any effect on the tissues. He told me that testing ACL cell and tissue cultures with IL-1B or other inflammatory cytokines could be an interesting addition to the study. Additionally, he taught me all about co-cultures, which he also believes could be useful in my study. A co-culture would allow me to place two different samples in the same well in order to share the same media as they do in the native knee joint. Currently, I am awaiting either canine or human articular cartilage, menisci, or ligament samples in order to begin this project. These samples are sometimes difficult to come by because a canine model from the vet school or animal shelter has to be euthanized in order to harvest these tissue samples. The other option would require patient consent to harvest some of this tissue during surgery. Both of these cases are quite rare and until then, I will continue to participate in Sebastian's ACL cell culture, the cell viability tests, the IVD studies, and the occasional chondrocyte culture.
Throughout the two weeks I have been in the lab, I have also traveled to the vet school to help with studies involving the live canine models. This involves transporting the dogs through the hallways and into and out of the radiograph or examination rooms. I participated in a hip dysplasia radiograph study day most recently, and I was responsible for making sure the correct canine was removed from their room and transported safely to and from the radiograph room after sedation. I was also responsible for keeping track of which canines received the sedation reversal drug and the exact time they became sternal. I monitored the canines during their sternal recumbency period and brought them back to their rooms as soon as they regained complete consciousness and neuromuscular control. Additionally, over the last two weeks, I have sat in on all of the graduate student classes and journal clubs taught by Dr. Stoker and Dr. Cook. Currently, the classes are mainly focused on cell and tissue culture, and they have helped me tremendously while working in the tissue culture room and with planning my independent project. The journal clubs are focused on discussing any orthopaedic literature that Dr. Stoker and Dr. Cook find interesting and relatable to the studies being performed in the lab. To my surprise, I have been able to follow the discussions about the literature and must thank Dr. Peretz for teaching me how to read these types of articles and emphasizing how important comprehending literature is in the spring EXP class. Overall, I have had an extremely eventful first two weeks in the lab and I look forward to obtaining the human or canine knee joint tissue samples in order to begin my IL-1B ACL culture study.
I have included some pictures taken during my first two weeks below. (WARNING: Some of these pictures are quite graphic due to the nature of the studies being performed in the lab.)
A view of the histology section of the lab.
The fluorescent microscope used in the cell viability imaging and viable cell density calculations.
I entered the Missouri Orthopaedic Insititute the next morning and rode the elevator all the way up to the fourth floor where the Thompson Laboratory for Regenerative Orthopaedics is located. In the lobby, I met both of my PIs, Dr. Cook and Dr. Stoker, who gave me a tour of the Thompson Lab. The expansive lab consists of small subbranches, which include a bioengineering lab, a tissue culture room, a histology lab, and the administrative offices. I was told that I would be spending most of my time in the tissue culture room, which houses several laminar flow hoods, incubators, and enormous freezers used to store and safely work with a variety of tissues. Shortly after, I was introduced to the lab pathologist, Dr. Bozynski, who showed me around the histology lab and introduced me to the five graduate students in the lab. Two of them were new to the lab and the others had been there for nearly five years, which made becoming accustomed to the lab very easy for me. I always had someone to approach with questions and had several other students to interact with throughout the initial training. So far, I have been treated identically to these graduate students and have been assigned the same responsibilities and introductory projects. This has been fantastic and has given me the opportunity to show off my knowledge and lab skills to the PIs as they assign me projects usually given to graduate students. Dr. Bozynski then introduced me to the veterinary team that works in the lab. They were all very welcoming and told me a little about the large animal models that are used in the lab. This list includes canine, lapine, caprine, ovine, bovine, and equine models and several other smaller animal models. Dr. Stoker then gathered the group of graduate students and I and drove us down to the veterinary school where the lab used to be located. They had recently moved into the Missouri Orthopaedic Institute and had not yet finished moving all of the tissue samples from the freezers in the veterinary school. We were instructed to carry in the lab's industrial grade coolers and transport the remaining tissue samples from the freezers into the lab's van without allowing any of the tissues to thaw. I opened one of the freezers to begin my task and was taken aback when I found the freezers stocked full of detached canine limbs. However, I started grabbing and loading the coolers as quickly as possible to prevent any unnecessary warming of the tissues and quickly grew comfortable handling the frozen canine limbs. We loaded the coolers back into the van and sped back to the lab. Once we returned to the lab, we unloaded approximately fifty limbs back into the immense -80 degrees Celsius freezer labeled "K9". Dr. Stoker than introduced me to Sebastian, the Ph.D. student who I would be working closely with for the rest of my time in the lab. Sebastian is a Fulbright Scholar from Colombia and has spent the last few months working on culturing human and canine ACL cells under load to determine the effects of different physical therapy methods after ACL reconstruction. Sebastian and I talked for about an hour about ACL reconstruction and the work he was currently doing with ACL tissue. During this conversation with Sebastian, I realized that I was going to fit in extremely well in the lab due to everyone's shared interests in orthopaedics and ACL reconstruction.
Over the next two weeks, I spent my time learning about all of the current studies the lab was performing, learning the ins and outs of working safely with human and canine tissue, and beginning my independent project. Before I touched a pipette, Dr. Stoker made sure that I understood that the main goal behind all of the studies completed at MOI was to discover better surgical and therapeutic techniques for traumatic orthopaedic injuries in order to reduce recovery time and the risk of osteoarthritis. Due to my previous experience with a traumatic knee injury that required a full year of rehabilitation and increased my risk of osteoarthritis significantly, MOI's goal has a personal touch and inspires me to work diligently in the lab every day. The main studies going on in the lab include a cell viability study on tissues stored using a specific preservation system, an intervertebral disc degeneration study, and an ACL cell and tissue culture study. Dr. Stoker wanted me to experience each of the studies so that I could observe the different laboratory techniques used in MOI. I thought that this would be a valuable learning experience because I would have to decide which techniques to use for my independent study within the next few days. The cell viability study was being performed by the two senior graduate students. The study involves imaging slices of human articular cartilage from osteochondral grafts preserved in an MOI produced media. The cartilage samples are harvested from either the femoral condyle, tibial plateau, menisci, or trochlear groove. They are then sliced into flat pieces on a low-speed wet saw. The pieces are then placed into a PBS solution and stained with both a live and dead cell stain. The live cell stain, calcein AM, is a cell-permeant dye that is metabolized by living cells and fluoresces green under a fluorescent microscope. The dead cell stain, ethidium homodimer, is a cell-impermeant stain that only enters cells with holes in the plasma membrane. Once inside these dead or dying cells, the stain binds to the DNA and fluoresces red under the fluorescent microscope. The tissue samples are then imaged under the fluorescent microscope under two different wavelengths that activate each of the stains. The images are superimposed on each other and the living and dead cells can be observed to determine overall cell viability in the tissue sample. A cell counting program is then used to determine viable cell density in the sample. All of this data will be used in a Missouri Osteochondral Preservation System (MOPS) viability study. Due to the need for immediate processing of these samples as soon as they are delivered from the operating room, I have been added to the "on-call" list, which keeps a record of which graduate student is responsible for the cell viability tests that day. I had to undergo several hours of training with Dr. Stoker on how to properly work with human tissues and a surgical scalpel. In addition, I was taught how to remove thin slices of cartilage from these samples for chondrocyte culture in a separate study. Next, I observed the intervertebral disk degeneration study being performed by a veterinary team member and an orthopaedic spine surgeon from MOI. The study uses canine spine and tail IVDs as well as human IVDs to serve as models for disk degeneration disease. The main function of IVDs is to serve as a shock absorber for the spine. They are located in between vertebrae and are made up of the nucleus pulposus and annulus fibrosus. Degenerative disk disease often results in the herniation of the disk as the nucleus pulposus pushes out of the annulus fibrosus. This study is being performed to determine if overweight, diabetic patients have an increased risk of degenerative disk disease due to high blood glucose levels and abnormal insulin-transferrin-selenium levels. The IVD is removed from the spine or tail with a low-speed wet saw, and the nucleus pulposus and annulus fibrosus are harvested and cultured in different types of media in a 24 well plate. These different types of media have varied glucose and ITS levels to determine what kind of effects these substances have on the IVD. While observing this study, I was instructed to change and collect the media every other day and harvest the canine tail IVDs on the wet saw upon their arrival. These media changes often overlap with the weekend, so I had to be in the lab on Saturday and Sunday for two media changes last week. I only live a short walk away and thoroughly enjoyed being in the lab and perfecting my pipetting skills over the weekend. The media is transferred to a collection plate and will be experimented with in ELISA assays in the near future. The ELISA assays will be used to identify different biomarkers associated with disk degeneration disease with the help of a plate reader machine. These media transfers and ELISA assays require an enormous amount of pipetting, and I have AP Bio to thank for preparing me so well.
The final study I observed was Sebastian's human and canine ACL culture experiment. As previously mentioned, he is performing monolayer cell culture and tissue culture with ACL samples. While the cells and tissues are being cultured, they are placed under different types and amounts of load. This is performed with a Flexcell system, which uses special 6 well plates to place compression and tension loads on samples. This study aims to determine the benefits or disadvantages of a more rigorous physical therapy protocol that places more load on an ACL reconstruction graft before complete healing, but results in quicker muscle strengthening. This is the study that I will actively participate in the most, so I made sure to take notes on Sebastian's culturing protocols and Flexcell procedures. I learned very quickly that sterility in the culture room is of utmost importance due to the considerable risk of contamination in your cultures. To become and remain sterile in the culture room, you must wear shoe covers, sterile surgical gloves, and douse your arms and hands in alcohol every minute or so. It is quite a tedious process, but it is vitally important to successful tissue and cell cultures. Sebastian also taught me how to identify different ACL cells and observe the differentiation of ligamentocytes and fibroblasts in his cultures. After, I worked with Sebastian to develop a procedure for my independent project. Due to my analysis of numerous ACL reconstruction articles in Dr. Peretz's spring EXP class, I knew that many scientists believe that inflammation after ACL tear and reconstruction is a direct cause of osteoarthritis development in the affected knee and was fascinated by this idea. I mentioned this to Sebastian and he told me about how they often culture their cartilage samples in IL-1B, an inflammatory cytokine, to determine if inflammation has any effect on the tissues. He told me that testing ACL cell and tissue cultures with IL-1B or other inflammatory cytokines could be an interesting addition to the study. Additionally, he taught me all about co-cultures, which he also believes could be useful in my study. A co-culture would allow me to place two different samples in the same well in order to share the same media as they do in the native knee joint. Currently, I am awaiting either canine or human articular cartilage, menisci, or ligament samples in order to begin this project. These samples are sometimes difficult to come by because a canine model from the vet school or animal shelter has to be euthanized in order to harvest these tissue samples. The other option would require patient consent to harvest some of this tissue during surgery. Both of these cases are quite rare and until then, I will continue to participate in Sebastian's ACL cell culture, the cell viability tests, the IVD studies, and the occasional chondrocyte culture.
Throughout the two weeks I have been in the lab, I have also traveled to the vet school to help with studies involving the live canine models. This involves transporting the dogs through the hallways and into and out of the radiograph or examination rooms. I participated in a hip dysplasia radiograph study day most recently, and I was responsible for making sure the correct canine was removed from their room and transported safely to and from the radiograph room after sedation. I was also responsible for keeping track of which canines received the sedation reversal drug and the exact time they became sternal. I monitored the canines during their sternal recumbency period and brought them back to their rooms as soon as they regained complete consciousness and neuromuscular control. Additionally, over the last two weeks, I have sat in on all of the graduate student classes and journal clubs taught by Dr. Stoker and Dr. Cook. Currently, the classes are mainly focused on cell and tissue culture, and they have helped me tremendously while working in the tissue culture room and with planning my independent project. The journal clubs are focused on discussing any orthopaedic literature that Dr. Stoker and Dr. Cook find interesting and relatable to the studies being performed in the lab. To my surprise, I have been able to follow the discussions about the literature and must thank Dr. Peretz for teaching me how to read these types of articles and emphasizing how important comprehending literature is in the spring EXP class. Overall, I have had an extremely eventful first two weeks in the lab and I look forward to obtaining the human or canine knee joint tissue samples in order to begin my IL-1B ACL culture study.
I have included some pictures taken during my first two weeks below. (WARNING: Some of these pictures are quite graphic due to the nature of the studies being performed in the lab.)
The massive -80 degrees Celsius freezers that house the tissue samples. You can see that there is one labeled "HU" for human and "K9" for canine.
The many different kinds of media that are used for cell and tissue culture. Many of these contain different amounts of glucose and ITS for the IVD diabetic model study. For my independent study, I will have to make my own media with various kinds of inflammatory cytokines.
A photo from the media changes I performed over the weekend. The collection plates are pictured on top of the blue sterile mats. It is very important to maintain a sterile environment when changing the media to prevent any sort of contamination.
The 24-well plates used to culture different tissues. The pink liquid is the MOI media that the tissues are cultured in.
The spinal surgeon cuts away the outer human IVD tissue in order to reveal and harvest the nucleus pulposus and annulus fibrosus for culture.
Canine knee joint after dissection of the hind limb. You can see the ACL and PCL in the femorotibial compartment and the menisci on the tibial plateau.
Incubator used to store the tissues and cells during culture. It has an airtight seal and continuously pumps carbon dioxide into the chamber. The carbon dioxide serves as a buffer for the tissue samples in the media.
A human articular cartilage graft that I imaged with the fluorescent microscope after staining with calcein AM and ethidium homodimer. You can see that this graft was very viable and the patient that received it should have a very low risk of graft failure. (Green=live cells/Red=dead cells)
A photo of one of the graduate students removing articular cartilage from a human tibial plateau for cell viability testing.
One of the laminar flow hoods in the culture room used for cell and tissue culture and media changes.
Wow. Sounds like you landed in exactly the right lab. I'm so glad your first 2 weeks went so well!
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