Week 5: Manav Surti

 This week, I was able to shadow Dr. Sabrina Strickland in the OR, as she worked on an osteochondral allograft case. This patient presented with many different conditions pertaining to their knee, needed two osteochondral allografts, two osteotomies, and several graft implants. It was very cool to see a patellar OCA, as the patella is a tissue that is confined by the medial and lateral patellofemoral ligaments, the patellar tendon, and the quadriceps tendon, as well as others. The cartilage that articulates with the patellofemoral groove is thus, on the underside of the patella. Defects on the underside of the patella are then, as you can imagine, really hard to treat because they require for the patella to be turned while still being confined by the connective tissue. Dr. Strickland performed a patellar eversion, being able to turn the patella to expose defective cartilage while the patella is still connected to the ligaments and tendons. 

    After this surgery, I collected both the donor patellar and hemicondylar tissue from her to continue mitochondrial testing on them. This run through of the experiment was much easier to conduct, as I had built up some technique from my previous run of this experiment a few weeks ago. I also conducted a dose response of my TMRM, polarized mitochondria, dye. I did this because I was unable to see the TMRM signal on the deconvolution microscope I am using for my project. This time I was very happy to see polarized mitochondria signal within the donor tissue. For context, again, I believe that the surgical perturbations we do to extract cartilage plugs from cadaveric donors is not harsh enough to kill the embedded chondrocytes but still harsh enough to depolarize their mitochondria, setting up long-term consequences for patients that are receiving OCA surgeries. 

    This time, I also took images (outlined below) to better showcase my protocol that I have developed for plug extraction from cadaveric human donor tissue. I have not had the chance to transfer my microscope images as of yet, but I hope to include some pilot results of mitochondrial staining in an upcoming blog post!

Fig 1a. Cadaveric donor hemicondyle (left) obtained for cytogenic and metabolic testing after surgical needs were met by Dr. Sabrina Strickland. 


Fig 1b. Arthrex brand OATS kit is plunged into the articular surface of the donor tissue. An autoclaved blue sheet piece is placed between the concave collared pin that is used to extrude the plug. This was done to prevent extensive metal on cartilage damage. OATS blade is hammered into the surface til a maximal depth of 10mm. 

Fig 1c. OATS blade is twisted to break the subchondral osseus component, and is cleanly taken out of the tissue. This leaves a 6mm hole from where a 6mm cartilage plug was taken, simulating surgical extrusion of a cartilage plug for placement within a patient. 


Fig 1d. For this experimental run, I took two 6mm osteochondral plugs. These plugs were extruded into a petri dish with phosphate buffered saline (PBS). As seen, mature human cartilage is typically on the order of 1-1.5mm thick. 


Fig 1e. Using a freshly autoclaved blade, the osseus and chondral components are separated, leaving behind a 6mm diameter and 1.5mm thick cartilage plug. 



Fig 1f. Each cartilage plug is bisected (using bovine synovial fluid on the blade to preserve chondrocytes). One hemicylinder is set aside for mitochondrial staining with Mitotracker Green and Tetramethyl Rhodamine Methyl-Ester, Perchlorate. The other hemicylinder is set aside for viability staining with Calcein AM and Ethidium Homodimer-1.

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