My name is Kuntul Chaudhary, and I'm currently a PGY-3 resident in the Department of Physical Medicine and Rehabilitation at the University of Pittsburgh Medical Center. I am thrilled to share our research on the mechanistic studies of platelet-rich plasma, and I'm very fortunate to have this research sponsored by the Foundation for PM&R, Scott F. Nadler-Passore Musculoskeletal Research Grant. In preparing for this talk, I wanted to learn more about Dr. Nadler. In reading about him online and learning from my attendings, I found he was cherished by colleagues, friends, and patients alike. He was a pioneer in the field of musculoskeletal research, authoring 76 articles and case reports and 31 chapters, books, and monographs. However, true to the nature of a physician-scientist, Dr. Nadler excelled in patient care in his clinical practice, listed 15 times as a best doctor by various regional and national publications. As I prepare for my own career as a physician-scientist, I will always hold Dr. Nadler's memory close as his namesake grant served as a launching point to my own scientific endeavors. Thank you, Dr. Nadler. And with that, I have no other disclosures. As one of the great humorists of American literature, Mark Twain writes, age is an issue of mind over matter. If you don't mind, it doesn't matter. Unfortunately, there's a lot of matter to have an issue about, particularly when one's knees go from looking like this to this. Of the age-related impairments, osteoarthritis, as you saw in that last x-ray, is a degenerative joint disease and one of the most common causes of disability worldwide. In joints, two adjacent bones, such as a femur and a tibia, are covered by a specialized layer of articular cartilage and encased in a synovial capsule. The articular cartilage itself is composed of water, approximately 70%, with the remainder being composed of organic extracellular matrix components, mainly type II collagen, aggrecan, and other proteoglycans. There are several risk factors leading to the development of osteoarthritis. The predominant joint-level risk factors include joint injury, repetitive use for occupation or leisure, and joint malalignment. Person-level risk factors include female sex, joint biomechanics, genetic factors, adiposity, and of course, age. The pathophysiology of how a joint degenerates is still debated in literature. However, a generally accepted common pathway begins with disruption of the articular cartilage, which might be as small as a surface fibrillation, that then evolves into deep fissures associated with exfoliation of cartilage fragments, ultimately leading to delamination and exposure of the underlying bone. This leads to the common diagnostic findings, including alterations in the state of bone mineralization, the formation of bone cysts, and the appearance of osteophytes. This progressive degeneration leads to loss of joint function and pain, which significantly hinders the ability of individuals to live independently and maintain an acceptable quality of life. Shockingly enough, one of the estimates of symptomatic OA is up to 15% in individuals aged 56 to 84 years worldwide. This is certainly a significant public health concern. Unfortunately, as the lifespan of the average individual increases, along with person-level risk factors such as obesity and a sedentary lifestyle, the prevalence and incidence of osteoarthritis is expected to increase. As seen in this data by the National Health Interview Survey, the rate of osteoarthritis is expected to grow, almost doubling from 2000 to 2040. This will inevitably lead to the need for more invasive procedures such as joint arthroplasties or total knee replacements. However, surgery is not without risk and can lead to complications such as periprosthetic joint infection, aseptic loosening, and osteolysis due to wear, which can all have catastrophic consequences. So this begs the question, how can we restore the form and function of a degenerative joint in an effort to delay or eliminate more invasive procedures? This is where regenerative medicine plays a role, seeking to restore form and function through biological processes. Regenerative rehabilitation is a fairly broad field, including tissue transplantation, tissue engineering, gene therapy, and stimulation of the endogenous repair systems through injection of cellular materials such as blood. Blood itself has always been intriguing throughout history. Since the ancient times, drinking or transfusing blood of young individuals has been thought to be a potent rejuvenation method, which has been utilized widely in many cultures from Pliny the Elder's recounting of spectators rushing to drink the blood of fallen gladiators to Aztec blood rituals, and even in relatively recent literature. Throughout history, several have sought the fountain of youth in the blood of the young and the healthy. In modern times, Dr. Thomas Rando from Stanford University and others have studied the supposed fountain of youth through heterochronic parabiosis, in which a young animal is surgically sutured to an old animal such that they develop a single shared circulatory system which allows for the study of circulating factors contributing to the rejuvenation of the older animal and vice versa. Previous research in our own lab conducted by my co-PI, Dr. Amrita Sahu, has shown that heterochronic transfusion of young serum on aged muscle stem cells reveals a younger phenotype, as evidenced by the increased expression of myo-D, a marker of myogenesis. Additionally, functional output of old mice injected with young serum increased, as indicated by the increased specific force of the tibialis anterior muscle. This implicates that heterochronic transfusion can not only restore form, but also function. Unfortunately, and fortunately, we don't practice heterochronic parabiosis in common clinical practice. However, through the use of platelet-rich plasma, an autologous plasma mixture highly concentrated in platelets and derived from the centrifugation of whole blood, researchers may have found a way to capitalize on the innate regenerative capability of the human body, which is particularly robust in young individuals. Circulating in our bloodstream, we have several growth factors derived from platelets that work to repair damage incurred. The theory behind PRP is that if we concentrate this healing potential and provide a regenerative stimulus, we will be able to promote chondrogenesis and proliferation to revert a degenerative joint to its premorbid state. However, PRP remains a hot topic in many circles, as clinical and preclinical research is equivocal. In current literature, there is a gap in the study of patient characteristics such as gender, activity level, and of course, age. This had us asking a simple question, is all PRP the same? And more importantly, does age attenuate the effects of PRP? To compare the ability of PRP from young and old donors to promote a healthy chondrogenic cell profile, we sought to answer the question, what would happen if PRP from a young or old individual was injected into old and osteoarthritic knees? We hypothesized that young but not old PRP would help restore cartilage structure and function. We started by establishing baseline characteristics of healthy young and old chondrocytes. We plated cadaveric young or old non-arthritic human chondrocytes from knee joints onto polycarbonate trans wells and then evaluated the nuclear morphology using cell analysis software and the chondrogenic profile through immunofluorescence staining for two markers of chondrogenicity, type 2 collagen and SOX9. For our experimental group, we plated cadaveric osteoarthritic human chondrocytes onto trans wells and then treated these cells with PRP derived from young or old donors for four days in vitro. We then evaluated the nuclear morphology and chondrogenic profile. Nuclear morphology is of interest given that morphological aberrations have been associated with cell differentiation, development, and disease, thereby serving as a surrogate to cellular health. We first compared the nuclear morphology of our young and old chondrocytes using the cell profiler cell imaging analysis software. The features of the morphology were split into various components. We then did a similar process to compare the nuclear morphology of our untreated osteoarthritic chondrocytes or the osteoarthritic chondrocytes treated with either young or old PRP. We looked at the top 10 features contributing to the nuclear morphology in these two groups and found three features in common. We chose to further compare nuclear extent, roughly defined as nuclear size, with increased nuclear extent correlating to poorer cell health. As expected, the osteoarthritic cells had increased nuclear extent as seen in the light blue bars on the right side of the graph. However, the nuclear morphology of osteoarthritic chondrocytes treated with PRP from young donors, represented by the green bars and circles, demonstrated characteristics of young and healthy chondrocytes as evidenced by the decreased nuclear extent. This benefit, unfortunately, was lost with age as evidenced by minimal change in nuclear extent in osteoarthritic chondrocytes treated with PRP from old donors, represented here in the red. We also compared chondrogenicity in young and old chondrocytes, which was quantified through the immunofluorescent staining of type 2 collagen and SOX9. Young cells showed an increased type 2 collagen, which is represented here in the green, and increased SOX9 expression, represented here in the red. We then compared the immunofluorescence of type 2 collagen and SOX9 in osteoarthritic cells and our experimental groups treated with either young PRP or old PRP. As expected, the osteoarthritic chondrocytes displayed decreased type 2 collagen and SOX9 expression when compared to their young counterparts. Interestingly, PRP from young donors induced a more youthful phenotype in the osteoarthritic cells, as evidenced by increased type 2 collagen and SOX9 expression. However, this benefit was significantly blunted when the cells were cultured with PRP from old donors. To test the physiological relevance of our in vitro studies, we next conducted a set of in vivo experiments. Aged male mice received PRP injections using 6 of the 12 PRP samples utilized in the in vitro studies, which included 3 young and 3 old PRP samples. We utilized a total of 20 mice in these experiments. Animals were randomized to either experimental and control groups. To mitigate the effects of between-animal variability, we used a within-subject design. In the experimental group, each mouse received identical injections of one of the 3 young PRP samples into the right knee and an injection of one of the old PRP samples into the left knee. 4 biological replicates were analyzed in each sample, bringing the total number of experimental mice to 12. In the control group, each mouse received a saline injection into the right knee, while the left knee remained untreated. 8 mice were used in the control group. For PRP administration in the mice, we developed an intra-articular knee injection protocol using a lateral injection approach to mimic the standard of care in humans. Each mouse knee was held in a slightly flexed position, and a 31-gauge needle was then introduced anterolaterally beneath the patellar ligament and into the intra-articular space. A total of 10 microliters of either saline or PRP was injected into the knee joint. The knee was then cycled through flexion and extension 3 times, and the mouse was returned to its cage. Prior to the start of the experimental studies, two independent investigators confirmed the intra-articular localization of the injection using tripen blue dye injections into 12 mice. As demonstrated here, the novel injection approach was successful, as the dye localized only to the intra-articular space, which is demonstrated by the blue coloring which you see here in the dissected mouse joint. Fourteen days after the injections, mice were euthanized. Knee joints were harvested, fixed, and decalcified prior to paraffin embedding. Paraffin-fixed knee joints were subsequently sectioned and stained with safranin O fast-green hematoxylin to evaluate for cartilage integrity and synovial tissue health. We evaluated the cartilage integrity using the Osteoarthritis Research Society International score, which is typically shortened to ORSI, a histopathologic evaluation of osteoarthritic cartilage, which relies on a grading and staging component, with a higher grade indicating a more aggressive biologic progression and a higher stage indicating a wider disease extent. An independent investigator was blinded to the images obtained and completed the ORSI scoring to maintain rigor. Consistent with the in vitro data, the in vivo experiments revealed that young PRP improved cartilage integrity as evidenced by improved ORSI scores. In contrast, PRP derived from old donors demonstrated increased cartilage surface disruption and numerous empty chondrocyte lacunes. Additionally, we took a more quantitative histopathologic measure of cartilage integrity in addition to the qualitative measure of ORSI scores by assessing cartilage roughness. Cartilage roughness is measured by assessing the deviation of the cartilage surface from a fitted kerf. We found that young PRP decreased cartilage surface roughness, which is notable as cartilage roughness is known to increase with age and disease state, as noted by prior experiments completed in our lab by Dr. Ijeoma. Finally, we assessed synovial thickness of each knee to assess for synovial inflammation using ImageJ software. The synovium of the mice knee joints treated with PRP derived from age individuals also demonstrated a more inflammatory profile as evidenced by increased density of resident cells and increased synovial membrane thickness. Interestingly, comparing the synovium from mice who received young PRP or saline injections, we don't see a significant difference, which may indicate that old PRP provokes an inflammatory response. Putting this all together, young PRP had a rejuvenative effect on chondrocytes as compared to old PRP. With our experiments, we saw that ORSI scores indicating osteoarthritic progression and extent decreased after a single young PRP injection. With our linear mixed effect model, we found that the synovial thickness and ORSI scores were statistically correlated, and therefore we see that knees injected with young PRP are clustered toward the bottom left as indicated by the red dots, indicating lower ORSI scores and less synovium thickness. However, looking at the other side of the coin, the old PRP injections did not improve cartilage integrity in aged mice and in fact induced an inflammatory profile in the synovium. Putting them together, old PRP paradoxically worsened outcomes in aged mice. Through our experiments, we found that young but not old PRP induced restorative properties in osteoarthritic chondrocytes, which is definitely an exciting launching point for our future studies. Additionally, in vivo studies revealed that injection of young PRP induced a chondroprotective effect in aged mice. Although PRP is a popular OA treatment employed in the clinic, we found that the efficacy of PRP is attenuated with age and may paradoxically provoke an inflammatory response. In the future, we plan to repeat our in vivo experiments in the upcoming weeks with female PRP to evaluate for any sex differences, as all of our current experiments have used male PRP. Additionally, given the increasing prevalence of PRP use in the clinic, we are interested in assessing the potential for long-term regenerative effects in PRP in osteoarthritic joints. Finally, in the upcoming weeks, we will be delving into the mechanistic studies of PRP with a focus on extracellular vesicles, as we hope to translate this research from the bench to the bedside through future development of testing platforms in which clinicians can evaluate the efficacy of PRP prior to injection and strategies to augment the efficacy of PRP injections to bioengineered circulating factors that mimic youthful PRP. Thank you so much for listening. I have a few acknowledgments. Big thank you to my mentor, Dr. Fabricio Ambrosio, for encouraging my scientific endeavors and providing valuable guidance along the way. Another big thank you to our team, who has been instrumental in these collaborative efforts to complete these experiments, with special thanks to Drs. Ijeoma and Sahu, and our fantastic lab manager, Ms. Shinde. I would also like to thank the Foundation for PM&R for funding the study through the Scott F. Nadler-Pasor Musculoskeletal Research Grant, and finally to the RMSTP program for the support and guidance to put me on track to this exciting research. Thank you again for listening, and please feel free to reach out to me with any questions.

platelet-rich plasma

osteoarthritis

chondrogenesis

cartilage cells

inflammation

regenerative effects