Over the past two weeks, I finished processing, loading, and evaluating my final canine tail and rat tail IVD samples that I received from the vet school. Then, I used my collection of IVD load study media plates and tissue digest plates to run several different types of fluorescence and absorbance-based assays. The quantitative data gathered from the plate reader was then run through a T-Test to identify the statistically significant values. I also participated in my last study day at the Vet Med Diagnostic Lab.
The vet school delivered three rat tails and three canine tails at the beginning of week 8 after their biweekly necropsy. These were my 8th set of canine/rat tail IVD samples and I breezed through the harvest and loading procedures due to my mastery of the protocols. I harvested 6 IVDs from each of the rat tails and 9 IVDs from each of the canine tails with the rongeurs and bone cutters. All of the muscle and fat surrounding the vertebral column was stripped and the IVDs were exposed and cut out. The IVDs were trimmed on the wet saw to remove the excess vertebral bone and create level bone plates. This allowed for upright placement of the IVDs in the flexcell plates during the tissue culture step. All 45 of the IVDs were placed into 4-well flexcell plates and separated into the different load/injury/cytokine treatment groups. The canine tail IVD plates consisted of two .50 MPa loaded plates, two .25 MPa loaded plates, and two no-load negative control plates. The 7th plate contained only 3 IVDs and was placed into the .25 MPa group to increase our sample size for the .25 MPa load treatment for the IVD load study Orthopaedic Research Society (ORS) Abstract. Half of the IVDs in each plate were injured with an 18 gauge needle to simulate an annular tear. One plate in each of the load and no-load treatment groups received the 10 ng/ml Il-1B (inflammatory cytokine) treatment. The 18 rat tail IVDs were placed into identical treatment groups, but I had to adjust the concentration of the Il-1B to account for the tissue size. I diluted the 10ng/ml stock solution to 1ng/ml and aliquoted it into separate falcon tubes for later use. The rat tail IVD load plates were organized into two .50 MPa plates, one .25 MPa plate, and one no-load negative control plate. Each plate contained 4 IVDs except for the 5th plate, which housed only two. I used this plate as an additional .25 MPa load plate. All of the plates had half of their discs injured with the same 18 gauge needle. Half of the discs also received the 1ng/ml IL-1B treatment. I made sure that an equal number of injured and uninjured disks were receiving the IL-1B treatment to ensure that we tested the effects of the cytokine treatment on both treatment groups. The 12 Flexcell plates were fixed in the vice and attached to the Flexcell vacuum system inside of the incubator. Instead of programming the same "Trucker" regimen that I had developed to use on the previous six load groups, I reinstated the 10 min interval load regimen that had been used by the lab before my arrival. Dr. Stoker wanted me to compare the two different regimens by observing the viability of the tissues and their biomarker levels at the end of the study. This information will be used to supplement my canine and rat tail IVD load study ORS abstract for the February conference. The Flexcell plates were cultured under load in the incubator for 6 days. On day 3, I collected the media for the biomarker analysis step and performed a media change. On day 6, I performed the final media change and the cell viability testing/viable cell density count as part of the end-of-culture procedure. I bisected all of the IVDs to expose the nucleus pulposus and annulus fibrosus to the ethidium homodimer (dead-cell stain) and calcein am (live-cell stain). The discs were then imaged under the fluorescent microscope and viable cells were counted with a cell-counting computer program. I have gone into the fine details of this staining and counting process in my previous blogs. The next step involved measuring each of the IVD's diameter to determine the exact load in MPa that was applied to the disc. This load differs depending on the diameter of the IVD and due to the significant variation in diameter in mammal tail IVDs, this was a necessary step. An equation is used to solve for this load in MPa based on the diameter and force (lbs) that was applied by the Flexcell vacuum as part of the regimen. I then trimmed off all of the bone plates on the discs with a scalpel and placed them into a deep well with a papain solution buffer to digest the tissues. The images and cell count are very important components of this study due to their importance in determining the viability of the discs in the different treatment groups. However, Dr. Stoker has set up an assay protocol for cultured tissue samples in order to obtain quantitative data that can be run through a T-Test with the lab's statistician. To prepare for this assay protocol, all of the media sample and tissue digest plates had to be collected.
After gathering all of my deep well plates, I used my excel sheet that I had been keeping up to date with tissue weights, media plate layouts, and tissue diameters to organize the plates. I wound up with 4 rat tail IVD media plates and 4 tissue digest plates as well as 4 canine tail IVD media plates and tissue digest plates. I then created a sample list for all 16 of these deep well plates by naming all of them by rat/canine number, load, injury, and cytokine. After naming all of the samples I made one enormous sample list with all of the samples I had collected during my 9 weeks in the lab. I had collected nearly 1500 media and tissue digest samples by my 9th week and had to run assays on all of these samples by the end of my 10th week. I made sure to ask the vet school to hold any new rat tail and canine tails until I had completely finished all of my assays. I then began the actual assay protocol and opened up the binder stacked full of the procedures to find my first assay procedure. The protocol calls for the media and tissue GAG (glycosaminoglycan) assays to be performed first. GAG is a polysaccharide that is a direct cartilage-degradation product. In IVD degeneration disease, a depletion in the levels of GAGs and proteoglycans is observed. This decrease can cause loss of IVD function and pain associated with the disease. Both assays are dimethyl methylene blue (DMMB) assays, however the tissue GAG assay calls for the tissue digest solution whereas the media GAG assay requires the media collected during the media changes. This assay is an absorbance-based assay and measures the amount of GAG in each sample based upon the intensity of light the plate reader measures as a beam passes through the sample solution. The plate reader is a very expensive and extremely complex machine that saves, organizes, and exports all of this quantitative data for statistical analysis. The media and tissue GAG assays require the mastery of serial dilutions and multi-chamber pipetting to create an acceptable standard curve and accurate readings. It took me several attempts until I completely understood how to lay out the assay plates, perform the standard curve serial dilutions, and calculate each dilution of the stock solutions. I was able to effortlessly move through these two assays after a few days. My next challenged involved the PGE2 assay, which the protocol called for next. The PGE2 assay is a fluorescence-based enzyme-linked immunosorbent assay (ELISA). PGE2 is one of the most important catabolic factors involved in the development of osteoarthritis. Dr. Stoker recognized that in previous load studies, compression of tissues resulted in a significant increase in PGE2 levels. He figured that it would be very interesting to see if my "trucker" regimen had similar results. This assay calls for a solution of PGE2 bound to a fluorescein fluorophore. This solution is added to your media plate samples with a separate solution of monoclonal antibodies to PGE2. These antibodies bind to PGE2 in a competitive manner. A low amount of the analyte (PGE2 in the media sample) results in more antibodies bound to the fluorophore probe solution added to the samples. This results in a high signal measured by the plate reader. A high amount of analyte results in fewer antibody molecules bound to the fluorophore causing a low signal during the read-out. Therefore, the signal is inversely proportionate to the concentration of PGE2 in the media plate samples and standards. The PGE2 assay is certainly one of the more confusing assays I performed and it involved numerous washes to rid the plate of any reagents not bound by the antibodies. This allowed the plate to read only the fluorescence of the fluorophore probes that were bound to the analyte. The next assay on the procedure was the MMP activity assay, which I ran with several other samples from different studies. MMPs are proteinases involved with the degradation of the extracellular matrix. When these MMPs are active in chondrocytes, cartilage degrades and osteoarthritis begins to develop. The MMP assay is a fluorescence-based assay used to detect if certain MMPs that act on chondrocytes are active in media samples. This assay also includes a number of serial dilutions to create the standards and other less complex dilutions used to aliquot stock solutions. The assay uses a fluorescence resonance energy transfer peptide as an MMP activity indicator. This peptide is not fluorescent when intact, however when it is cleaved by active MMPs, it regains its fluorescence. The plate reader can measure the fluorescent signal strength and will then report this data in an excel sheet that is exported and used for statistical analysis. The assay protocol listed the nitric oxide assay after MMP activity to give you a small break. The nitric oxide assay is the simplest of all the assays the lab runs and requires only 15 minutes to run. Increased nitric oxide levels are found in joints that are severely osteoarthritic, which has lead many to believe that it is involved in the pathogenesis of the disease. It is known to mediate the degradative effects of inflammatory cytokines (such as the IL-1B used in my cytokine treatment) and tumor necrosis factor alpha. Nitrite in the media plate samples reacts with a fluorescent probe known as DAN reagent. NaOH is then added to each of the wells to enhance the fluorescence to a frequency that the plate reader can measure. The final assay that I ran is known to be difficult and require the most caution of all of the assays. The cytokine assay uses color-coded magnetic beads coated with analyte-specific antibodies (cytokine-specific antibodies) to measure the amount of specific cytokines in the media plate samples. The assay uses a Luminex machine which uses two different frequency lasers to identify a bead and the analyte bound to it and determine the magnitude of the signal. This assay requires serial dilutions for the standards, the addition of the beads, and then several washes. The addition of beads and the washing procedure are what make the assay quite difficult to follow and very easy to ruin. When adding and washing the beads, a magnet must be added to the bottom of the plate so as to not let the beads mix with the sample before incubation or get washed out of the plate into the sink. The magnets get taken on and off the plate at several steps throughout the procedure and if you forget to take it off or add it on a second too early, the entire assay must be restarted due to the chance of incorrect binding of the beads or a loss of the beads entirely during the rinse procedure with the wash buffer. Several of the PhD students have made mistakes with this assay in the past and were forced to face the wrath of Dr. Stoker. Due to its nearly 1000 dollar cost to run per plate, I can understand Dr. Stoker's anger, and therefore was extremely cautious while running this assay. Fortunately, when I ran the plate in the Luminex machine, it immediately started recognizing beads and the corresponding cytokine. We test for cytokines such as IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, MCP-1, MIP-1alpha, and TNF-alpha. The majority of these are inflammatory cytokines, which can result in the chronic inflammation that is observed in OA and IVD degeneration disease. After finishing the assay protocol, I lined up my sample list with the data that was exported by the plate reader and performed a T-Test with Dr. Stoker. He uses a computer program that uses the data line-up to calculate all of the statistically significant differences in the assay measurement data. The program is pretty complex, but it does most of the calculations for you and returns a sheet that compares each of the treatment groups against each other. For instance, if you want the program to compare load groups, it will compile a list of all of the loaded and non-loaded samples. Then, it will highlight all of the significant differences in the assay measurements between the treatment groups. For my 16 plates of canine/rat tail IVD samples, the results were almost exactly what I expected. The .50 load, injured, IL-1B samples had the highest measurements of GAG, MMP Activity, NO, and Cytokines, and the lowest reading for PGE2 (Inverse relationship). The no-load, non-injured, negative samples had the lowest expression of GAG, MMP Activity, NO, and Cytokines and the highest measurement of PGE2. The .25 load group fell just in between the two extremes just as expected. The injury and IL-1B treatments in the .25 load group seemed to increase the measurements in all of the assays except for PGE2. For one of the canine sample, the no-load groups had the highest measurements of all of the analytes and the lowest score for PGE2. These were the exact opposite results compared to the other 8 canine tail models. It did not take me long to recognize that the canine expressing the strange measurements was one of the canine models I processed from the Humane Society Shelter. The shelter called the lab on a Sunday, and I volunteered to come in right away and process the canine upon arrival. However, when it came to culture the tail IVDs, I was short on Flexcell inserts because Dr. Stoker had sent them to the autoclave room before the weekend. I called Dr. Stoker and he told me that we would have to try to preserve them in media outside of the culture room for the night. I did as I was told, but when I arrived in the morning one of the three plates was contaminated due to the non-sterile environment. I still carried through with the culturing procedure, but made sure to mark which samples were contaminated. Sure enough, the contaminated samples all wound up in loaded plates due to the small number of IVDs obtained and were non-viable by the time I cultured them under load in the Flexcell machine. Dr. Stoker and I determined that the contaminated loaded samples were completely dead by the day 3 and day 6 media changes and were producing none of the biomarkers measured for in the assay protocol. I went back into the cell viability images and confirmed that the discs were non-viable at the end of culture. This explained why the non-loaded, non-contaminated, samples had higher measurements of the biomarkers at the end of culture. They were still somewhat viable and had continued to produce biomarkers throughout the entire 6 day culture period. Overall, my canine and rat tail IVD load study was very successful and supported my hypothesis fully.
My last study day at the VMDL consisted of the extraction of canine bone marrow aspirate concentrate (BMAC) and the separation of platelet-rich plasma from canine blood. Dr. Cook performed the BMAC harvest from the left humerus with a 15 gauge BMA needle. He performed the procedure on three canines and was done in about half an hour. The veterinary team then performed a blood draw from the jugular vein for the PRP separation. The vets let me insert and draw the blood after they had sedated the canines and instructed me on the procedure. We drew around 40 ml of blood from each canine. The blood was then run through an Arthrex centrifuge that spins and separates the blood into red blood cells, platelet-poor plasma, and platelet-rich plasma. The PRP was separated into a syringe and was brought back to the lab with the BMAC for use on a tendon study. The RBC and PPP left in the separation bag were disposed of in the biohazard bin at the lab. Furthermore, I have continued to sit in on all of the graduate level topics classes and attend the weekly lab journal club. Next week will be my 10th and last week in at the Thompson Laboratory for Regenerative Orthopaedics. I look forward to Dr. Peretz's visit on Tuesday of next week and can't wait to show her all the exciting studies and surgical procedures that are being performed here at the Missouri Orthopaedic Institute.
The equation used to find the actual load (MPa) applied to each of the discs based on their diameter and the force (lbs) that the Flexcell vacuum applied to each of the plates.
After gathering all of my deep well plates, I used my excel sheet that I had been keeping up to date with tissue weights, media plate layouts, and tissue diameters to organize the plates. I wound up with 4 rat tail IVD media plates and 4 tissue digest plates as well as 4 canine tail IVD media plates and tissue digest plates. I then created a sample list for all 16 of these deep well plates by naming all of them by rat/canine number, load, injury, and cytokine. After naming all of the samples I made one enormous sample list with all of the samples I had collected during my 9 weeks in the lab. I had collected nearly 1500 media and tissue digest samples by my 9th week and had to run assays on all of these samples by the end of my 10th week. I made sure to ask the vet school to hold any new rat tail and canine tails until I had completely finished all of my assays. I then began the actual assay protocol and opened up the binder stacked full of the procedures to find my first assay procedure. The protocol calls for the media and tissue GAG (glycosaminoglycan) assays to be performed first. GAG is a polysaccharide that is a direct cartilage-degradation product. In IVD degeneration disease, a depletion in the levels of GAGs and proteoglycans is observed. This decrease can cause loss of IVD function and pain associated with the disease. Both assays are dimethyl methylene blue (DMMB) assays, however the tissue GAG assay calls for the tissue digest solution whereas the media GAG assay requires the media collected during the media changes. This assay is an absorbance-based assay and measures the amount of GAG in each sample based upon the intensity of light the plate reader measures as a beam passes through the sample solution. The plate reader is a very expensive and extremely complex machine that saves, organizes, and exports all of this quantitative data for statistical analysis. The media and tissue GAG assays require the mastery of serial dilutions and multi-chamber pipetting to create an acceptable standard curve and accurate readings. It took me several attempts until I completely understood how to lay out the assay plates, perform the standard curve serial dilutions, and calculate each dilution of the stock solutions. I was able to effortlessly move through these two assays after a few days. My next challenged involved the PGE2 assay, which the protocol called for next. The PGE2 assay is a fluorescence-based enzyme-linked immunosorbent assay (ELISA). PGE2 is one of the most important catabolic factors involved in the development of osteoarthritis. Dr. Stoker recognized that in previous load studies, compression of tissues resulted in a significant increase in PGE2 levels. He figured that it would be very interesting to see if my "trucker" regimen had similar results. This assay calls for a solution of PGE2 bound to a fluorescein fluorophore. This solution is added to your media plate samples with a separate solution of monoclonal antibodies to PGE2. These antibodies bind to PGE2 in a competitive manner. A low amount of the analyte (PGE2 in the media sample) results in more antibodies bound to the fluorophore probe solution added to the samples. This results in a high signal measured by the plate reader. A high amount of analyte results in fewer antibody molecules bound to the fluorophore causing a low signal during the read-out. Therefore, the signal is inversely proportionate to the concentration of PGE2 in the media plate samples and standards. The PGE2 assay is certainly one of the more confusing assays I performed and it involved numerous washes to rid the plate of any reagents not bound by the antibodies. This allowed the plate to read only the fluorescence of the fluorophore probes that were bound to the analyte. The next assay on the procedure was the MMP activity assay, which I ran with several other samples from different studies. MMPs are proteinases involved with the degradation of the extracellular matrix. When these MMPs are active in chondrocytes, cartilage degrades and osteoarthritis begins to develop. The MMP assay is a fluorescence-based assay used to detect if certain MMPs that act on chondrocytes are active in media samples. This assay also includes a number of serial dilutions to create the standards and other less complex dilutions used to aliquot stock solutions. The assay uses a fluorescence resonance energy transfer peptide as an MMP activity indicator. This peptide is not fluorescent when intact, however when it is cleaved by active MMPs, it regains its fluorescence. The plate reader can measure the fluorescent signal strength and will then report this data in an excel sheet that is exported and used for statistical analysis. The assay protocol listed the nitric oxide assay after MMP activity to give you a small break. The nitric oxide assay is the simplest of all the assays the lab runs and requires only 15 minutes to run. Increased nitric oxide levels are found in joints that are severely osteoarthritic, which has lead many to believe that it is involved in the pathogenesis of the disease. It is known to mediate the degradative effects of inflammatory cytokines (such as the IL-1B used in my cytokine treatment) and tumor necrosis factor alpha. Nitrite in the media plate samples reacts with a fluorescent probe known as DAN reagent. NaOH is then added to each of the wells to enhance the fluorescence to a frequency that the plate reader can measure. The final assay that I ran is known to be difficult and require the most caution of all of the assays. The cytokine assay uses color-coded magnetic beads coated with analyte-specific antibodies (cytokine-specific antibodies) to measure the amount of specific cytokines in the media plate samples. The assay uses a Luminex machine which uses two different frequency lasers to identify a bead and the analyte bound to it and determine the magnitude of the signal. This assay requires serial dilutions for the standards, the addition of the beads, and then several washes. The addition of beads and the washing procedure are what make the assay quite difficult to follow and very easy to ruin. When adding and washing the beads, a magnet must be added to the bottom of the plate so as to not let the beads mix with the sample before incubation or get washed out of the plate into the sink. The magnets get taken on and off the plate at several steps throughout the procedure and if you forget to take it off or add it on a second too early, the entire assay must be restarted due to the chance of incorrect binding of the beads or a loss of the beads entirely during the rinse procedure with the wash buffer. Several of the PhD students have made mistakes with this assay in the past and were forced to face the wrath of Dr. Stoker. Due to its nearly 1000 dollar cost to run per plate, I can understand Dr. Stoker's anger, and therefore was extremely cautious while running this assay. Fortunately, when I ran the plate in the Luminex machine, it immediately started recognizing beads and the corresponding cytokine. We test for cytokines such as IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, MCP-1, MIP-1alpha, and TNF-alpha. The majority of these are inflammatory cytokines, which can result in the chronic inflammation that is observed in OA and IVD degeneration disease. After finishing the assay protocol, I lined up my sample list with the data that was exported by the plate reader and performed a T-Test with Dr. Stoker. He uses a computer program that uses the data line-up to calculate all of the statistically significant differences in the assay measurement data. The program is pretty complex, but it does most of the calculations for you and returns a sheet that compares each of the treatment groups against each other. For instance, if you want the program to compare load groups, it will compile a list of all of the loaded and non-loaded samples. Then, it will highlight all of the significant differences in the assay measurements between the treatment groups. For my 16 plates of canine/rat tail IVD samples, the results were almost exactly what I expected. The .50 load, injured, IL-1B samples had the highest measurements of GAG, MMP Activity, NO, and Cytokines, and the lowest reading for PGE2 (Inverse relationship). The no-load, non-injured, negative samples had the lowest expression of GAG, MMP Activity, NO, and Cytokines and the highest measurement of PGE2. The .25 load group fell just in between the two extremes just as expected. The injury and IL-1B treatments in the .25 load group seemed to increase the measurements in all of the assays except for PGE2. For one of the canine sample, the no-load groups had the highest measurements of all of the analytes and the lowest score for PGE2. These were the exact opposite results compared to the other 8 canine tail models. It did not take me long to recognize that the canine expressing the strange measurements was one of the canine models I processed from the Humane Society Shelter. The shelter called the lab on a Sunday, and I volunteered to come in right away and process the canine upon arrival. However, when it came to culture the tail IVDs, I was short on Flexcell inserts because Dr. Stoker had sent them to the autoclave room before the weekend. I called Dr. Stoker and he told me that we would have to try to preserve them in media outside of the culture room for the night. I did as I was told, but when I arrived in the morning one of the three plates was contaminated due to the non-sterile environment. I still carried through with the culturing procedure, but made sure to mark which samples were contaminated. Sure enough, the contaminated samples all wound up in loaded plates due to the small number of IVDs obtained and were non-viable by the time I cultured them under load in the Flexcell machine. Dr. Stoker and I determined that the contaminated loaded samples were completely dead by the day 3 and day 6 media changes and were producing none of the biomarkers measured for in the assay protocol. I went back into the cell viability images and confirmed that the discs were non-viable at the end of culture. This explained why the non-loaded, non-contaminated, samples had higher measurements of the biomarkers at the end of culture. They were still somewhat viable and had continued to produce biomarkers throughout the entire 6 day culture period. Overall, my canine and rat tail IVD load study was very successful and supported my hypothesis fully.
My last study day at the VMDL consisted of the extraction of canine bone marrow aspirate concentrate (BMAC) and the separation of platelet-rich plasma from canine blood. Dr. Cook performed the BMAC harvest from the left humerus with a 15 gauge BMA needle. He performed the procedure on three canines and was done in about half an hour. The veterinary team then performed a blood draw from the jugular vein for the PRP separation. The vets let me insert and draw the blood after they had sedated the canines and instructed me on the procedure. We drew around 40 ml of blood from each canine. The blood was then run through an Arthrex centrifuge that spins and separates the blood into red blood cells, platelet-poor plasma, and platelet-rich plasma. The PRP was separated into a syringe and was brought back to the lab with the BMAC for use on a tendon study. The RBC and PPP left in the separation bag were disposed of in the biohazard bin at the lab. Furthermore, I have continued to sit in on all of the graduate level topics classes and attend the weekly lab journal club. Next week will be my 10th and last week in at the Thompson Laboratory for Regenerative Orthopaedics. I look forward to Dr. Peretz's visit on Tuesday of next week and can't wait to show her all the exciting studies and surgical procedures that are being performed here at the Missouri Orthopaedic Institute.
A canine tail IVD after bisection. The annulus fibrosus surrounds the darker, jelly-like nucleus pulposus in the center of the IVD. This disc would then be stained for cell viability imaging.
Bone plates shaved off a canine IVD prior to tissue digest.
Canine IVDs with bone plates trimmed before tissue digest. These discs have already gone through the cell viability imaging and viable cell density count.
Fluorescent microscope image of a rat tail IVD after staining. (Green=living cells/Red=deade cells) The finer dots make up the annulus fibrosus and the blobs of green on the right side of the image are the nucleus pulposus pushed off to the side on the miscroscope slide.
An image of the fluorescence-based assay plates
A small group of the assays run on the media and tissue digest samples.
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