Dataset Information

Impact of collagenase treatment on the proteome and transcriptome of human articular chondrocytes

ABSTRACT: Objective Human primary articular chondrocytes (hPACs) are routinely isolated from articular cartilage and for pre-clinical OA research. Collagenase digest of tissue is an essential step, yet the impact of enzymatic incubation on the phenotype of hPACs is unclear. We aim to delineate this through proteomic and transcriptomic analysis. Design hPACs were isolated from human knee cartilage (N=4) with and without prior fixation. Proteomes were quantified using LC-MS/MS. The Proteome 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 tissue. Results Collagenase treatment resulted in a 10% increase in protein mass/cell (P=0.06). Seven percent of the proteome (498 proteins) significantly changed (Padj. = 4.5x10-4 - 0.049). Pathway analysis revealed depletion of FOXO signaling terms (FDR = 0.049 – 0.024), and enrichment of ribosomal RNA processing terms (FDR = 0.025 – 2.9x10-11). Transcriptomic anlaysis revealed 3937 differentially expressed genes (P.adj<0.05), of which 97% of differentially-expressed proteins overlapped (R2 =0.83). Propidium iodide staining followed by flow-assisted cell sorting (FACS) (N=3) did not identify significant differences in cell cycle between fixed and unfixed chondrocytes (all P>0.05). Conclusions We identified shifts in the proteome and transcriptome of hPACs following collagenase digest, supporting use of tissue fixation before extracting nucleic acids for analysis. Despite widespread expression changes, hPACs retain their chondrocyte phenotype. These datasets and analyses will serve as a valuable resource for the OA research community.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Articular Chondrocyte

DISEASE(S): Osteoarthritis

SUBMITTER: Matthias Trost

LAB HEAD: Matthias Trost

PROVIDER: PXD064948 | Pride | 2025-10-20

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	chondro_raw_data_zipped.zip	Other
	report-first-pass.parquet	Other
	report-first-pass.pr_matrix.tsv	Tabular
	report-first-pass.stats.tsv	Tabular
	report-first-pass.tsv	Tabular

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Publications

A proteomics investigation of primary human articular chondrocyte isolation.

Brumwell Abby A Raheja Simran S Cawley William W Birkett-Jones Emily E Orr Sarah E SE Todd Caitlin C Deehan David J DJ Conlon Nichola J NJ Trost Matthias M Rice Sarah J SJ

Osteoarthritis and cartilage open 20250823 4

<h4>Objective</h4>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.<h4>Design</h4>hPACs were isolated from human knee cartilage (n = 4) from patients undergoing total knee replacement. Collagenase treatment was performed with or without p ...[more]

PMID: 41069381

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Project description:Osteoarthritis (OA) of the hand is a common disease resulting in pain and impaired function. The pathogenesis of hand OA (HOA) is elusive and models to study it have not been described so far. Culture of chondrocytes is a model to study the development of cartilage degeneration, which is a hallmark of OA and well established in OA of the knee and hip. In the current study we investigated the feasibility human chondrocyte culture derived from proximal interphalangeal (PIP) finger joints of dissecting room cadavers. Index and middle fingers without signs of osteoarthritis were obtained from 30 cadavers using two different protocols. Hyaline cartilage from both articulating surfaces of the proximal interphalangeal (PIP) joint was harvested and digested in collagenase. Cultured chondrocytes were monitored for contamination, viability, and expression of chondrocyte specific genes. Chondrocytes derived from knee joints of the cadavers were cultured under identical conditions. Gene expression comparing chondrocytes from PIP and knee joints was carried out using Affymetrix GeneChip Human 2.0 ST arrays. The resulting differentially expressed genes were validated by real-time PCR and immunohistochemistry.Chondrocytes harvested up to 101 hours after death of the donors were viable. mRNA expression of collagen 2A1, aggrecan and Sox9 was significantly higher in chondrocytes as compared to cultured fibroblasts. Comparison of gene expression by chondrocytes from PIP and knee joints yielded 528 differentially expressed genes. Chondrocytes from the same joint region had a higher grade of similarity than chondrocytes of the same individual. These results were validated using real-time PCR and immunohistochemistry.We demonstrate for the first time a reliable method for culture of chondrocytes derived from PIP joints. PIP chondrocytes show a specific gene expression pattern and could be used as tool to study cartilage degeneration in HOA. Three samples of cultured chondrocytes from knee and proximal interphalangeal finger joints were compared. Gene expression of the four most differentially regulated genes was confirmed by real-time PCR in 10 independent samples.