Autofluorescence Lifetime Reports Cartilage Damage in Osteoarthritis

Abstract

species are independent of the efficiency of excitation and detection of the emission, they can be more robust than fluorescence intensity measurements, particularly in complex scattering samples such as biological tissue 12. The application of fluorescence lifetime readouts to cartilage autofluorescence was reported in 2004 13 and subsequent work on ex vivo engineered cartilage tissue has shown the potential for autofluorescence lifetime (AFL) measurements to provide label-free readouts of cartilage degradation following specific treatments with collagenase, chondroitinase-ABC and ribose 14. We have previously reported that both the degradation of collagen and the removal of aggrecans from natural ex vivo cartilage are associated with a decrease in AFL of cartilage tissue following treatment with bacterial collagenase, human MMP-1 and, trypsin; and also aggrecanase induction by retinoic acid treatment of live cartilage 15. Collagen is a major contributor of cartilage autofluorescence through its intermolecular crosslinks 16,17. As the gaps between collagen fibrils are largely filled by aggrecans in cartilage tissue 4,5 , removal of aggrecan is likely to influence the chemical microenvironment of collagen crosslinks. This may explain why proteolytic removal of aggrecan from cartilage can reduce the AFL of cartilage collagen 15. Here we extend our previous work to evaluate the potential of AFL measurements to provide label-free readout of cartilage damage with a view to the development of a clinical instrument for the non-invasive monitoring of cartilage status. Specifically, this study focused on (1) how chemically-induced aggrecan depletion affects the AFL of intact murine, porcine and human cartilage; (2) whether AFL measurements can report local enzymatically-induced damage in the articular cartilage surface; (3) determining if AFL measurements can be used to detect natural erosion in human tibial plateau cartilage. Methods Materials. Bovine trypsin and retinoic acid were purchased from Sigma-Aldrich (Dorset, UK). Bacterial collagenase type 2 was purchased from Worthington Biochemical Corp (Lakewood, NJ, USA). Recombinant MMP-1 was expressed in E.coli, purified and activated as previously reported 18,19 .

Real-time multispectral fluorescence lifetime imaging using Single Photon Avalanche Diode arrays

“…2) reveal a well-demarcated region that is coincident with the area of collagen digestion, yielding shorter lifetime relative to non-digested areas. These results are in close agreement with previous studies of cartilage digestion 46,47,53 . The decrease in autofluorescence lifetime is more prominent in channels 1 and 2, i.e.…”

“…Specifically, spectral measurements show a ~10 nm shift towards longer wavelengths in the fluorescence emission of digested cartilage relative to non-digested cartilage (see Fig. 5), which is in agreement with previous studies 17,47 but directly contradicts another 53 .…”

“…To demonstrate our implementation in clinically relevant biological tissue, we measured the autofluorescence lifetime and spectral signatures of porcine articular cartilage. Measurements were realized before and after treatment of the articular surface with bacterial collagenase, to induce endogenous contrast derived from the localized digestion of collagen and consequent alteration of its fluorescence characteristics, in an effort to mimic natural degradation occurring in osteoarthritis 47,53 . The photon integration time for each autofluorescence acquisition was set to ~11 ms. For this temporal window, we measured a negligible contribution from background light originating from the LED (see Supplementary Fig.…”

“…The average power at the sample plane was kept below 25 μW at all times during measurements, which is below the maximum permissible exposure (MPE) for skin and eye. At a probe-to-target distance of 3 mm, this corresponds to a power density of less than 5 mW/cm 2 , which is similar or less than that used for excitation in previous in vivo or ex vivo investigations, using similar excitation strategies [36][37][38]47 . In none of these studies alterations in the biological characteristics of the sample as a result of laser excitation were reported.…”

NLRP3 Inflammasome Pharmacological Inhibitors in Glycyrrhiza for NLRP3-Driven Diseases Treatment: Extinguishing the Fire of Inflammation

“…Osteoarthritis (OA) is the most common arthritis in the elderly and is characterized by subchondral bone hyperplasia and articular cartilage degeneration, leading to the loss of joint function. 22 , 23 NLRP3 inflammasome is a potentially novel therapeutic target for the management of OA. 24 , 25 In vitro, Lico A suppresses NLRP3 inflammasome activation and GSDMD expression in primary mouse OA chondrocytes via nuclear factor erythroid-2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/NF-κB axis ( Figure 3 ), indicating that Lico A attenuates LPS-induced chondrocyte pyroptosis.…”