Week 6: Manav Surti

    This week I observed Dr. Sabrina Strickland in the OR again, as she had another osteochondral allograft (OCA). This OCA was again, a patellar graft. This was really interesting to see again, following last week, since the patellar articular cartilage is on the underside of the structure. This patient had a very large focal defect in their patellar cartilage that was causing severe cartilage on bone sliding on the trochlear groove. To address this, Dr. Strickland took an orthotopic region of a donor cadaveric patella to replace the focal defect. The defect spanned 20mm, so Dr. Strickland had to take most of the donor patella for the patient's surgery; this left me with very little cartilaginous tissue to work with when the donor tissue was transferred to me. 

    I once again went through my experimental protocol on this donor patella. This was really difficult to do since most of the cartilage plug was taken. Nonetheless, I tried to take the best samples I could from the patella. These were taken using the protocol I wrote about in my last blog post, week 5. This time, the main limiter was the lack of tissue to work with; all of my staining and imaging seemed to work well! This gives me more hope for the next, and final time, that Dr. Gomoll and Dr. Strickland will be able to give me donor OCA tissue while I'm at immersion. 

    



Fig 1. CalceinAM and Ethidium Homodimer staining on healthy, donor articular cartilage. 

    This image (fig1) characterizes live chondrocytes in green and dead chondrocytes in red. I was quite harsh when taking the cartilage plug out of this patella. I was not trying to simulate surgical environments in this experiment, but the way in which I had to handle this patella in order to get a decent chunk of cartilage may be similar to the manner in which a surgeon handles donor tissue under the pressure of surgery. Regardless, most of the chondrocytes located deep within the tissue remain alive, while there is quite widespread death at the surface. 


Fig 2. MitoTracker Green FM and Tetramethyl Rhodamine, Methyl Ester, Perchlorate staining on healthy, donor articular cartilage. 

    This image (fig2) characterizes mitochondral polarization. The mitochondria within the chondrocytes should be polarized, maintaining a highly negative concentration of ions on the inner side of the organelles. This is integral to a functional electron transport chain and generation of ATP. When disrupted, the chondrocyte may still be detected as alive, but it could be very dysfunctional, and on its way to programmed cell death. The red dye in this image stains ONLY the polarized (functional) mitochondria, while the green dye stains all mitochondria, regardless of polarity. Thus, the green signal is essentially a positional marker of where chondrocytes are within the cartilage matrix, while a lack of a red signal overlapping a green signal is representative of a mitochondria that has lost its polarity. As seen in the image, a lot of the mitochondria toward the surface of the articular cartilage are depolarized (as seen by a lack of TMRM red signal). 

    This could be immensely useful to clinicians who have long evaluated solely chondrocyte viability in OCA tissues, and not functionality. Next (and last) iteration of this experiment, I hope to combine all of the techniques and protocols I've been able to design in this short period of time, and get many quantifiable images of mitochondrial depolarization in healthy, donor articular cartilage. 

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