Maturation‐ and degeneration‐dependent articular cartilage metabolism via optical redox ratio imaging

Abstract

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.

Inhibition of B-cell lymphoma 2 family proteins alters optical redox ratio, mitochondrial polarization, and cell energetics independent of cell state

“…No prior studies have directly related the ORR to cellular senescence. However, previous ORR imaging of young and old mouse articular cartilage supports this result as both NAD(P)H and FAD intensities increased with cartilage age 67 and senescence is also known to increase with age. 68 Interestingly, the increased energetic state with ABT-263 treatment alone is not seen when ABT-263 is used in combination with mTORC 1/2 inhibition (TAK-228).…”

Formalin fixation and paraffin embedding interfere with preservation of optical metabolic assessments based on endogenous NAD(P)H and FAD two photon excited fluorescence

“…Two-photon excited fluorescence (TPEF) can provide important insights regarding cellular metabolic function and dysfunction [6][7][8][9][10]. NAD(P)H (reduced nicotinamide adenine dinucleotide (phosphate)) and FAD (flavin adenine dinucleotide) are two naturally fluorescent co-enzymes involved in several metabolic pathways, such as glycolysis, oxidative phosphorylation, fatty acid synthesis, and oxidation and have been used to determine the oxido-reductive state of cells through the use of their TPEF intensity [8,9,[11][12][13]. Since the fluorescence characteristics of NADH and NADPH are very similar, we use NAD(P)H to refer to potential contributions from NADH and/or NADPH [10].…”