Spatial and longitudinal monitoring of cartilage and intervertebral disc extracellular matrix formation using proteoglycan building blocks and click chemistry: specificity, quantification, and detection upon subcutaneous implantation.

Abstract

BACKGROUND/OBJECTIVE: The development of regenerative therapies for osteoarthritis and intervertebral disc degeneration requires non-destructive methods to spatiotemporally monitor extracellular matrix (ECM) synthesis. This study aimed to establish a method for the use of bioorthogonal click chemistry for longitudinal and spatially resolved tracking of newly synthesized proteoglycans in cartilaginous tissues, including tools to assess specificity, enable quantification and for future application for noninvasive real-time tracking of ECM production by imaging. METHODS: Bovine articular cartilage explants, human nucleus pulposus tissue, and human chondrocyte cultures were metabolically labeled using azide-modified N-acetylgalactosamine (Ac4GalNAz) incorporation with copper-catalyzed and copper-free click chemistry. GAG-specific incorporation was verified by enzymatic digestion with chondroitinase ABC(ChABC) and hyaluronidase (HAse). Sequential labeling with spectrally distinct fluorophores enabled temporal tracking of matrix deposition. Volumetric quantification was performed using ImageJ on confocal microscopy Z-stacks. Near-infrared (NIR) dyes were employed for in vitro and subcutaneous imaging, with photoacoustic imaging used to enhance spatial resolution. RESULTS: Ac4GalNAz demonstrated negligible cytotoxicity at 50 μM in cartilage explants. In tissue explants, metabolic labeling showed high specificity for chondroitin sulfate (CS)-containing proteoglycans, confirmed by ChABC digestion. In contrast, 2D chondrocyte cultures showed ChABC-resistant labeling, suggesting incorporation into membrane-associated glycoproteins. Sequential copper-free labeling distinguished temporally distinct matrix synthesis within the same sample. Using NIR-labels after incorporation, explants were detectable both in vitro and following subcutaneous implantation, with only a moderate loss of signal intensity. Photoacoustic imaging appeared to provide higher spatial resolution, as shown by visualization of implant contours. CONCLUSIONS: Bioorthogonal metabolic labeling enables non-destructive, spatially resolved, and quantitative monitoring of proteoglycan synthesis in cartilaginous tissues. Labeling specificity is tissue-context dependent, requiring enzymatic validation for each model system. Integration with NIR fluorescence and photoacoustic imaging provides a foundation for future in vivo applications. STATEMENT OF SIGNIFICANCE: Monitoring newly synthesized extracellular matrix (ECM) in cartilaginous tissues is a critical challenge in regenerative medicine for osteoarthritis and intervertebral disc degeneration. Building on prior work demonstrating metabolic labeling feasibility in cartilage, this study substantially advances the field by establishing quantitative, spatiotemporally resolved proteoglycan tracking across multiple clinically relevant model systems, including nucleus pulposus tissue. We introduce volumetric 3D quantification of newly synthesized ECM from confocal z-stacks, sequential temporal labeling to distinguish matrix deposition at different timepoints, and near-infrared and photoacoustic imaging enabling subcutaneous detection. Critically, we identify model-dependent labeling specificity, highlighting the need for validation in each model. Together, these advances provide a comprehensive framework for non-destructive monitoring of ECM regeneration with direct translational potential.