Academic Journal
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.
| Τίτλος: | 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. |
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| Συγγραφείς: | Dilek Y; Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands., Rios JL; Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands., O'Riordan C; FUJIFILM VisualSonics Inc., Amsterdam, the Netherlands., Bruggink V; Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University, Jena, Germany; JeNaCell GmbH-An Evonik Company, Jena, Germany., Warmink K; Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands., Muenzebrock KA; Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands., Chen H; Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands., Fenu M; Department of Otorhinolaryngology, Head and Neck Surgery, Erasmus MC, University Medical Center Rotterdam, the Netherlands., Bloemhoff N; Department of Otorhinolaryngology, Head and Neck Surgery, Erasmus MC, University Medical Center Rotterdam, the Netherlands., Durongbhan P; Department of Biomedical Engineering, The University of Melbourne, Parkville, Australia., van Osch GJVM; Department of Otorhinolaryngology, Head and Neck Surgery, Erasmus MC, University Medical Center Rotterdam, the Netherlands; Department of Orthopedics and Sports Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands; Department of Biomechanical Engineering, Faculty of Mechanical Engineering, Delft University of Technology, Delft, the Netherlands., Stok KS; Department of Biomedical Engineering, The University of Melbourne, Parkville, Australia., Garcia JP; Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands; Department of Bioprocess Engineering, Wageningen University & Research, Wageningen, the Netherlands., Creemers LB; Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands; Department Anaesthesiology, Institute of Mental Health and Neuroscience, Division Neuroscience, University Maastricht, the Netherlands. Electronic address: laura.creemers@maastrichtuniversity.nl. |
| Πηγή: | Acta biomaterialia [Acta Biomater] 2026 Sep; Vol. 222, pp. 274-288. Date of Electronic Publication: 2026 Jul 29. |
| Τύπος έκδοσης: | Journal Article |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Elsevier Country of Publication: England NLM ID: 101233144 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1878-7568 (Electronic) Linking ISSN: 17427061 NLM ISO Abbreviation: Acta Biomater Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Kidlington, Oxford, UK : Elsevier, c2004- |
| Ιατρικοί όροι (MeSH): | Extracellular Matrix*/metabolism , Proteoglycans*/chemistry , Proteoglycans*/metabolism , Intervertebral Disc*/metabolism , Cartilage, Articular*/metabolism , Cartilage*/metabolism , Click Chemistry*, Chondrocytes/metabolism ; Chondrocytes/cytology ; Animals ; Cattle ; Humans |
| Περίληψη: | 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 (Ac4 GalNAz) 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. (Copyright © 2026 The Authors. Published by Elsevier Inc. All rights reserved.) |
| Competing Interests: | Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Caroline O'Riordan is an employee of FUJIFILM Corporation. All other authors have no conflicts of interest pertaining to the study presented. |
| Contributed Indexing: | Keywords: Bioorthogonal chemistry; Cartilage regeneration; Metabolic labeling; Photoacoustic imaging; Proteoglycan synthesis |
| Substance Nomenclature: | 0 (Proteoglycans) |
| Entry Date(s): | Date Created: 20260729 Date Completed: 20260916 Latest Revision: 20260916 |
| Update Code: | 20260917 |
| DOI: | 10.1016/j.actbio.2026.07.050 |
| PMID: | 42526826 |
| Βάση Δεδομένων: | MEDLINE |
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