Week 4: Manav Surti
This week was a research heavy week, as I spent a few days in clinic but most of the days prepping to begin my research project. On Monday and Thursday, I was in the clinic observing Dr. Gomoll and I was able to see some post-operational patients who had undergone osteochondral allograft (OCA) surgery, as well as learn some new terms such as Lateral Extra-Articular Tenodesis (LET) which can tighten a patient's iliotibial (IT) band, allowing their ACL graft to be more stable, preventing rupture.
On Tuesday and Wednesday I prepped for, and carried out, my first experimental run-through for my project. To provide some background, osteochondral allografts have been used for a very long time to treat the so-called, "middle ground," of cartilage defects. This is to say, techniques such as arthroplasty or osteotomy are often used for widespread cartilage damage and in cases where it is not worth preserving the native articular cartilage. Techniques like osteochondral autograft transfer (OAT) and autologous chondrocyte implantation (ACI) are useful for mechanical and biological recapitulation of the native cartilage structure, but are often not as useful for deep and focal cartilage defects that have the potential to carry damage into the surrounding articular cartilage. Even more concerning are the mechanical differences seen between load bearing and non-load bearing regions of articular cartilage, with non-load bearing cartilage having both macro- and microscopic differences, making OAT a somewhat outdated technique. An ideal whole-tissue graft would incorporate the native collagen type 2 hyaline cartilage ECM and the underlying osseus component while also being orthotopic for accurate load-bearing. Fresh tissue of such nature can be obtained from cadaveric donors who have healthy articular cartilage [1], [2]. It is important to note that chondrocyte viability is of the upmost importance to upkeep in cadaveric donor tissue, as chondrocytes will continue to integrate native, hyaline cartilage into a patient defect site. Early evaluation of chondrocyte viability used histologic measures and gross analysis, but the advent of confocal microscopy and real-time fluorescent dyes have made direct evaluation of chondrocyte viability less demanding. Recent evaluations of donor tissue, and also graft retrievals from patients after they've failed, have revealed that, if kept in ideal conditions, chondrocytes do survive, proliferate, and persist for years within the patient.
This is where my project comes in; viability and functionality are two different matters. Although chondrocytes could be surviving at the time of implantation, their functional components, namely, mitochondria (MT), could be dysfunctional. To test this, many research groups and clinicians have conducted colorimetric metabolic assays that test whether the cells are producing any type of metabolic component. This is an inherently flawed metric, since MT can stay dysfunctional for hours prior to carrying out action that can be detected on a colorimetric metabolic assay. Thus, it is vital to test real time MT dysfunction and depolarization. In the Bonassar lab, we have previously characterized MT depolarization in real time, in a model of posttraumatic osteoarthritis (PTOA). Thus, the tools used to visualize real-time (milliseconds to seconds) depolarization of MT should be easily transferrable to human donor tissue samples (or so I thought).
Getting wetlab access, as well as starting up in a wetlab space I am not used to has definitely had some obstacles. I was able to access the Dr. Tony Chen's wetlab space within the HSS research institute, and he and his postdoc Vince Sise have provided incredible help in getting me started with this project here. Vince has been instrumental in answering my endless questions, and Dr. Chen has been instrumental in ideating with me to help these ideas come to fruition. I have thus far been able to use the Arthrex OATS kit to take a 6mm plug of human, donor cartilage, and fluorescently stain it with dyes that record MT polarization. Thus, a lack of TMRM, my red dye that signifies MT polarization, can indicate that the MT within the donor tissue are actually depolarized prior to implantation. The Bonassar lab has also previously found that peracute MT depolarization precedes and may even be the cause of apoptosis, and subsequent inflammation and ECM catabolism that perpetuates biological OA and downstream mechanical symptoms. Thus, if we are implanting tissue with already depolarized MT into patients, we could be setting them up to get biological variants of OA that could lead to painful mechanical symptoms later in life. This has been a difficult experiment to run here, with my dyes potentially going through too many thaw cycles on their journey here, rendering them useless. Next week, I plan to try a dose response of my dyes to see if I simply didn't add enough to see them on the microscope this week, or if the dyes truly are shot. I also have a Calcein/EthD kit to crosscheck fluorescence viability as a positive control.
References
[1] V. S. Nikolaou and P. V. Giannoudis, “History of osteochondral allograft transplantation,” Injury, vol. 48, no. 7, pp. 1283–1286, Jul. 2017, doi: 10.1016/j.injury.2017.05.005.
[2] S. Sl, G. J, B. K, C. J, S. J, and B. W, “Fresh osteochondral allograft transplantation for the knee: current concepts,” The Journal of the American Academy of Orthopaedic Surgeons, vol. 22, no. 2, Feb. 2014, doi: 10.5435/JAAOS-22-02-121
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