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Lookup NU author(s): Abby Brumwell, Simran Raheja, Dr WILLIAM CAWLEY, Sarah Orr, Caitlin ToddORCiD, Professor David Deehan, Dr Nichola Conlon, Professor Matthias TrostORCiD, Dr Sarah RiceORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 2025 The Author(s). Objective: Human primary articular chondrocytes (hPACs) are routinely isolated from articular cartilage for pre-clinical OA research. Collagenase digest of tissue is an essential step, yet the impact of lengthy enzymatic incubation on the hPAC phenotype is unclear. We aimed to delineate this through proteomic analysis. Design: hPACs were isolated from human knee cartilage (n = 4) from patients undergoing total knee replacement. Collagenase treatment was performed with or without prior fixation of the tissue. Proteomes were quantified using LC-MS/MS. The Proteomic Ruler was employed to estimate protein copy numbers and cell protein masses. Significant differences in protein intensities were determined using paired t-testing and Benjamini-Hochberg correction. Proteomic data were integrated with existing transcriptomes (GSE217871) of hPACs and ground cartilage (ex vivo) RNA. Results: Following collagenase treatment, we identified 498 differentially expressed proteins (DEP) in the unfixed cells. We observed depletion of FOXO signaling and enrichment of ribosomal RNA processing, indicative of increased cell cycle progression. This was supported by depletion of cell cycle inhibitors including CDKN1C. Transcriptomic analysis identified 3937 differentially expressed genes (DEG), and a 53 % overlap in DEP and DEG. Propidium iodide staining did not identify significant differences in cell cycle between fixed and unfixed hPACs. Conclusions: We identified shifts in the proteome and transcriptome of hPACs following collagenase digest, supporting the use of tissue fixation before extracting nucleic acids for analysis where possible. Despite widespread expression changes, hPACs largely retain their chondrocyte phenotype. These datasets and analyses will serve as a valuable resource for the OA and cartilage research community.
Author(s): Brumwell A, Raheja S, Cawley W, Birkett-Jones E, Orr SE, Todd C, Deehan DJ, Conlon NJ, Trost M, Rice SJ
Publication type: Article
Publication status: Published
Journal: Osteoarthritis and Cartilage Open
Year: 2025
Volume: 7
Issue: 4
Print publication date: 01/12/2025
Online publication date: 23/08/2025
Acceptance date: 13/08/2025
Date deposited: 06/10/2025
ISSN (print): 1522-9653
ISSN (electronic): 2665-9131
Publisher: Elsevier Ltd
URL: https://doi.org/10.1016/j.ocarto.2025.100664
DOI: 10.1016/j.ocarto.2025.100664
Data Access Statement: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [https://www.ebi.ac.uk/pride/] with the dataset identifier PXD064948. Reviewers can access the data through the following details: Project accession: PXD064948; Token: BarcyaysuXdS. All other data and analysis code is available from the corresponding authors upon request.
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