No abstract

Objective: Metabolic dysregulation has recently been identified as a key feature of osteoarthritis. Mechanical overloading has been postulated as a primary cause of this metabolic response. Current methods of real-time metabolic activity analysis in cartilage are limited and challenging. However, optical redox imaging leverages the autofluorescence of co-enzymes NAD(P)H and FAD to provide dye-free real-time analysis of metabolic activity. This technique has not yet been applied to cartilage. This study aimed to assess the effects of a compressive load on cartilage using optical redox imaging. Method: Cartilage samples were excised from porcine femoral condyles. To validate this imaging modality in cartilage, glycolysis was inhibited via 2-deoxy-D-glucose (2DG) and oxidative phosphorylation was inhibited by rotenone. Optical redox images were collected pre-and post-inhibition. To assess the effects of mechanical loading, samples were subjected to a compressive load and imaged for approximately 30 min. Load and strain parameters were determined using high-speed camera images in Matlab. A range of loading magnitudes and rates were applied across samples. Results: 2DG and rotenone demonstrated the expected inhibitory effects on fluorescence intensity in the channels corresponding to NAD(P)H and FAD, respectively. Mechanical loading induced an increase in NAD(P)H channel fluorescence which subsided by 30 min post-loading. Magnitude of loading parameters had mixed effects on metabolites. Conclusions: Optical redox imaging provides an opportunity to assess real-time metabolic activity in cartilage. This approach revealed a metabolic response to a single load and can be used to provide insight into the role of metabolism in mechanically-mediated cartilage degradation.

From the two metabolic processes in healthy cartilage, glycolysis has been associated with proliferation and oxidative phosphorylation (oxphos) with matrix synthesis. Recently, metabolic dysregulation was significantly correlated with cartilage degradation and osteoarthritis progression. While these findings suggest maturation predisposes cartilage to metabolic instability with consequences for tissue maintenance, these links have not been shown. Therefore, this study sought to address three hypotheses (a) chondrocytes exhibit differential metabolic activity between immaturity (0–4 months), adolescence (5–18 months), and maturity (>18 months); (b) perturbation of metabolic activity has consequences on expression of genes pertinent to cartilage tissue maintenance; and (c) severity of cartilage damage is positively correlated with glycolysis and oxphos activity as well as optical redox ratio in postadolescent cartilage. Porcine femoral cartilage samples from pigs (3 days to 6 years) underwent optical redox ratio imaging, which measures autofluorescence of NAD(P)H and FAD. Gene expression analysis and histological scoring was conducted for comparison against imaging metrics. NAD(P)H and FAD autofluorescence both demonstrated increasing intensity with age, while optical redox ratio was lowest in adolescent samples compared to immature or mature samples. Inhibition of glycolysis suppressed expression of Col2, Col1, ADAMTS4, and ADAMTS5, while oxphos inhibition had no effect. FAD fluorescence and optical redox ratio were positively correlated with histological degeneration. This study demonstrates maturation‐ and degeneration‐dependent metabolic activity in cartilage and explores the consequences of this differential activity on gene expression. This study aids our basic understanding of cartilage biology and highlights opportunity for potential diagnostic applications.

Cartilage loading is important in both structural and biological contexts, with overloading known to cause osteoarthritis. Cellular metabolism, which can be evaluated through the relative measures of glycolysis and oxidative phosphorylation, is important in disease processes across tissues. Details of structural damage coupled with cellular metabolism in cartilage have not been evaluated. Therefore, the aim of this study was to characterize the time- and location-dependent metabolic response to traumatic impact loading in articular cartilage. Cartilage samples from porcine femoral condyles underwent a single traumatic injury that created cracks in most samples. Before and up to 30 minutes after loading, samples underwent optical metabolic imaging (OMI). OMI measures the fluorescent intensity of byproducts of the two metabolic pathways, FAD for oxidative phosphorylation and NAD(P)H for glycolysis, as well as the redox ratio between them. Images were taken at varied distances from the center of the impact. Shortly after impact, fluorescence intensity in both channels decreased, while redox ratio was unchanged. The most dramatic metabolic response was measured closest to the impact center, with suppressed fluorescence in both channels relative to baseline. Redox ratio varied nonlinearly as a function of distance from the impact. Finally, both lower and higher magnitude loading reduced FAD fluorescence, whereas reduced NAD(P)H fluorescence was associated only with low strain loads and high contact pressure loads, respectively. In conclusion, this study performed novel analysis of metabolic activity following cartilage damage and demonstrated time-, distance-, and load-dependent response to traumatic impact loading.