ASSESSING THE IMPACT OF INCREASING VITAMIN C CONCENTRATIONS ON HIERARCHICAL COLLAGEN FIBER FORMATION BY MENISCAL FIBROCHONDROCYTES

Menisci are semi-lunar wedge-shaped discs that aid in load distribution and transfer in the knee. Menisci are capable of distributing loads due to a complex collagen organization dominated by circumferentially-aligned collagen fibers. With age, menisci have increased susceptibility to injury and degeneration, where collagen fibers become torn and no longer functional. Additionally, there is reduced healing with age, ultimately reducing the ability of the meniscus to distribute load in the knee, leading to the progression of osteoarthritis (OA) and an increased need for total knee replacements. OA is the most common joint condition, affecting over 32.5 million US adults and is characterized by inflammation and the progressive deterioration of articular cartilage. There is a growing need for a treatment that will drive collagen fiber regeneration to heal the meniscus and prevent the formation of OA. Vitamin C or ascorbic acid is a natural antioxidant that has been shown to stimulate collagen synthesis in dermal fibroblasts and thus could be a tool to help the meniscus heal better, in turn reducing OA progression. The objective of this thesis is to investigate whether vitamin C can accelerate and improve collagen fiber maturation by meniscal fibrochondrocytes isolated from neonatal bovine and aged humans. We hypothesize that increased concentrations of vitamin C will accelerate and improve collagen accumulation and fiber formation, regardless of cellular age or species, resulting in significantly stronger tissues. To do this, we first cultured neonatal bovine meniscal fibrochondrocytes in a previously developed culture device that guides cells to develop hierarchical fibers and dosed them with 200 µM, 400 µM, and 800 µM vitamin C over 6 weeks. We found that 400 µM and 800 µM vitamin C produced a significant increase in collagen accumulation early in culture compared to 200 µM control cultures, resulting in an accelerated increase in tissue tensile properties. However, we found no significant differences in fiber organization, composition, or mechanics between 400 µM and 800 µM, suggesting a threshold in vitamin C effect on neonatal bovine cells (Chapter 2). We then performed preliminary studies evaluating the effect of vitamin C on aged human meniscal fibrochondrocytes to see whether vitamin C had similar effects on aged human cells as it does on neonatal bovine. Interestingly, 400 and 800 µM vitamin C enhanced hierarchical fiber formation, increased collagen accumulation by 4 weeks, and accelerated improvements in tensile properties, in a dose-dependent fashion; however, by 6 weeks 200 µM cultures had similar improvements (Chapter 3). These findings suggest that vitamin C does increase the production of collagen in both neonatal bovine and aged human meniscal fibrochondrocytes, which shows promise as a therapeutic method for driving healing in aging menisci, however further optimization of dose concentration and duration is needed.