Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs.
The main results reproduced, with only marginal, non-material deviations.
- ✓Reported values were directly comparable
- 🟡Could not use the authors’ exact input data
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
IN PROGRESS. Paper reproducible result is DESeq2 differential expression across 3 osteoclast substrate groups, primarily Table 1 (top-15 over-expressed genes cartilage-vs-dentine) + MMP8 8.89-fold/p=0.0133. The featureCounts count matrix (GSE166535_final_counts.tsv.gz) is deposited so the DESeq2->Table 1 step is directly reproducible; running on «our HPC». Wet-lab results (zymography, siRNA, GAG release, RT-qPCR) are out of scope. Note discrepancy: paper says n=7/group, GEO has 8 donors/group (24 samples). Awaiting «our HPC» tunnel (currently unreachable) to download matrix + run DESeq2.
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Assessment versions
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v1 current initial assessment Score 50assessed: 2026-06-18 ⛓ 6aab26d9fdd9
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- Reproduced
- 2026-06-18
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-19no human curator yet
- Last updated
- 2026-07-31
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusThis study tests whether osteoclasts degrade cartilage using the same cellular machinery as bone resorption, and whether chondrocytes regulate this process, aiming to understand the osteoclast–cartilage interaction in joint degradation.
- ★ Human osteoclasts can differentiate on acellular cartilage, express osteoclast markers, and degrade cartilage matrix in a contact-dependent manner without forming F-actin rings or resorption pits. finding
- ★ MMP8 and MMP9 are contributory MMPs driving osteoclast-mediated cartilage degradation (GAG release), with degradation being MMP-dependent rather than acidification/cathepsin-K dependent. mechanism
- ★ Bone-resident osteoclasts and chondrocytes exert mutually protective effects on their native tissue; chondrocytes stimulate osteoclast formation at non-bone sites but inhibit it on dentine. finding
- ★ MMP8 is the most highly upregulated gene in osteoclasts on cartilage versus dentine, while MMP9 is the most highly expressed MMP. finding
- ★ Direct contact between osteoclasts and cartilage is required for proteoglycan (GAG) degradation. finding
- Comparing the osteoclast gene-expression profile on its two primary natural substrates (bone/dentine vs cartilage) by RNA-seq. method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Histology (H&E, TRAP staining) | Human OA knee, RA, and GCTB tissue | none | Multinucleated osteoclast presence and cartilage/bone erosion | — |
| Osteoclast differentiation / light microscopy & SEM | Human CD14+ monocyte-derived osteoclasts on dentine vs acellular articular cartilage | substrate (dentine vs cartilage vs plastic) | Resorption pits, F-actin rings, cell morphology, surface degradation | SEM |
| GAG and collagen release assay | Human osteoclasts on dentine, acellular cartilage, cellular cartilage | substrate; Transwell (indirect contact) | Collagen release from dentine, GAG release from cartilage | — |
| TRAP staining / osteoclast quantification (co-culture) | Human CD14+ monocyte-derived osteoclasts co-cultured with chondrocytes on plastic or dentine | chondrocyte co-culture + M-CSF/RANKL | Number of TRAP+ cells (≥10 nuclei / total) | — |
| Inhibitor treatment + GAG release | Human osteoclasts on acellular/cellular cartilage | bafilomycin, E64, GM6001 (pan-MMP), recombinant TIMP1 (100 nM) | GAG release | — |
| RNA-seq and RT-qPCR | Human osteoclasts differentiated on plastic, acellular cartilage, dentine | substrate | Differential gene/MMP expression (PCA, fold change) | — |
| Gelatin zymography | Conditioned media of human osteoclasts on acellular cartilage | none vs recombinant protein control | Active MMP8 (~65 kDa) and MMP9 (~82 kDa) production | — |
| siRNA knockdown + GAG release; Immunohistochemistry | Human osteoclasts on cartilage explants; human OA tissue | siRNA targeting MMP8 or MMP9 | GAG release reduction; MMP8/MMP9 protein localization | — |
- ▲ MMP8 showed greatest fold upregulation in osteoclasts on cartilage versus dentine 8.89-fold
- ▲ Direct co-culture with chondrocytes increased large osteoclast (>10 nuclei) formation on plastic p < 0.0001
- ▼ Chondrocyte co-culture inhibited osteoclast formation on dentine p = 0.0002
- ▼ Osteoclasts cultured on dentine inhibited basal cartilage degradation p = 0.012
- ▼ Pan-MMP inhibitor GM6001 significantly reduced osteoclast-mediated GAG release from acellular cartilage (bafilomycin and E64 had no effect)
- ▼ siRNA knockdown of MMP8 and MMP9 reduced osteoclast-mediated GAG release from cartilage (non-significant trend) 39% (MMP8), 28% (MMP9)
- ▼ Recombinant TIMP1 inhibited osteoclast-mediated GAG release from acellular cartilage
- – Osteoclasts on dentine clustered separately from cartilage/plastic by PCA, and on cartilage failed to form F-actin rings or resorption pits
- fold_change 8.89-fold (LFC 8.89993669) (MMP8 overexpression in osteoclasts on cartilage vs dentine)
- pvalue p = 0.0133 (padj 1.33E−02) (MMP8 upregulation significance)
- pvalue p < 0.0001 (Increased large osteoclast formation by chondrocyte co-culture on plastic)
- pvalue p = 0.0002 (Inhibition of osteoclast formation on dentine by chondrocyte co-culture)
- pvalue p = 0.012 (Dentine-cultured osteoclasts inhibit basal cartilage degradation)
- other 39% and 28% reduction (GAG release reduction after MMP8/MMP9 siRNA knockdown)
- fold_change PER2 LFC 5.33 (padj 7.07E−08) (Second top over-expressed gene in osteoclasts on cartilage)
- other 100 nM (Recombinant human TIMP1 concentration in GAG release assay)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This in vitro study examined human osteoclast behaviour on multiple substrates (dentine, acellular cartilage, cellular cartilage, cell culture plastic) using functional assays, RT-qPCR, and bulk RNA-seq. Two-group comparisons employed Student's t-tests or Mann-Whitney U tests, while multi-group inhibitor and co-culture experiments used one-way ANOVA or Kruskal-Wallis ANOVA; test choice appeared to vary by experiment without a stated selection criterion. RNA-seq transcriptomic profiling compared osteoclasts across the three substrates, reporting adjusted p-values and log-fold changes. Results were presented as individual data points with exact or threshold p-values; dispersion measures and post-hoc procedures were not explicitly named.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Student's t-test (unpaired assumed; not stated) | Collagen release from dentine (Fig 2a); GAG release from acellular cartilage (Fig 2a); large osteoclast formation on plastic with chondrocytes (Fig 2d); RANKL:OPG ratio (Fig 2f); TIMP1 effect on GAG from acellular cartilage (Fig 3c); MMP8 RT-qPCR (Fig 4b) | n=16 (dentine collagen), n=20 (acellular GAG), n=10 (Fig 2d), n=11 (Fig 2f), n=18 (Fig 3c), n=8 (Fig 4b) | not stated |
| Mann-Whitney U test | GAG release from cellular cartilage (Fig 2a) | n=17 | not stated |
| Multiple Mann-Whitney U tests (no correction stated) | Classical osteoclast marker gene expression on dentine vs acellular cartilage (Fig 1d); MMP1, 2, 3, 7, 9, 12, 13, 14 and ADAMTS1 RT-qPCR (Fig 4c) | n=8 | not stated |
| One-way ANOVA (post-hoc test not named) | GAG release from indirect co-culture with acellular cartilage (Fig 2b) and cellular cartilage (Fig 2c); total osteoclast number on dentine with chondrocytes (Fig 2e); bafilomycin and E64 inhibitor effects on GAG from acellular cartilage (Fig 3a); bafilomycin and E64 effects on GAG from cellular cartilage (Fig 3b) | n=10 (Figs 2b, 2c), n=8 (Fig 2e); N varies by group for Fig 3 | not stated |
| Kruskal-Wallis ANOVA (post-hoc test not named) | Bafilomycin effect on GAG from acellular cartilage (Fig 3a); GM6001 effect on GAG from cellular cartilage (Fig 3b); siRNA MMP8 and MMP9 knockdown vs control on GAG release from acellular cartilage (Fig 4e) | N varies for Fig 3; n=7 (Fig 4e) | not stated |
| RNA-seq differential expression analysis with adjusted p-values (padj; pipeline not named; DESeq2 Wald test with Benjamini-Hochberg FDR inferred from padj notation and log fold change reporting in Table 1) | Genome-wide transcriptomic comparison of osteoclasts on acellular cartilage vs dentine vs cell culture plastic (Fig 4a, Table 1) | n=7 per substrate group (inferred from PCA plot legend) | not stated |
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Multiple individual Mann-Whitney U tests were applied across several gene comparisons within each figure (6 genes in Fig 1d; 9 genes in Fig 4c) without a stated multiplicity correction↳ Could also: Apply a family-wise error rate or FDR correction (e.g. Benjamini-Hochberg) across all gene comparisons within each figure, or use a linear mixed model that treats gene as a within-subject factor — Controlling the false discovery rate across the family of gene-level tests would explicitly bound the expected proportion of false positives across the panel, which is standard practice in multi-gene RT-qPCR analysis
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One-way ANOVAs and Kruskal-Wallis tests were reported for multi-group comparisons without naming a post-hoc pairwise test↳ Could also: Follow significant omnibus tests with a named post-hoc procedure — Tukey HSD or Dunnett's test (vs a control) for ANOVA, and Dunn's test with Bonferroni or BH correction for Kruskal-Wallis — A significant omnibus test indicates that at least one pair differs but does not identify which pairs; a named post-hoc procedure with multiplicity control makes specific pairwise conclusions explicit and reproducible
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The choice between parametric (t-test, one-way ANOVA) and non-parametric (Mann-Whitney, Kruskal-Wallis) tests appeared to vary by experiment without a stated selection criterion↳ Could also: Pre-specify a decision rule for test selection (e.g. Shapiro-Wilk normality test on residuals, or visual Q-Q inspection) and apply it consistently across all experiments — A transparent, pre-specified criterion for distributional testing aids reproducibility and helps readers understand why particular tests were chosen for particular experiments
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The siRNA knockdown experiment reported biologically meaningful reductions in GAG release (39% for MMP8, 28% for MMP9) that did not reach statistical significance at n=7 (Kruskal-Wallis)↳ Could also: Report an effect size with 95% confidence interval (e.g. rank-biserial correlation for non-parametric data) to characterize the magnitude of the observed difference independently of the p-value; alternatively a paired Wilcoxon signed-rank test could be used if knockdown and control conditions were matched within each biological replicate — Effect size with CI communicates the precision and plausible range of the true effect, which is informative when sample size limits power; a paired design can reduce within-experiment variability and increase sensitivity when measurements share a common biological source
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No a priori power analysis or sample size justification was reported; n per experiment ranged from 7 to 20↳ Could also: Report a power calculation based on effect sizes from pilot experiments or the literature, stating the minimum detectable effect at the chosen alpha and power — Pre-specified power calculations help readers assess whether individual experiments were adequately powered to detect biologically relevant effect sizes, and are particularly informative for experiments yielding trends that did not reach significance
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Dispersion in figures is not described in the text; it is not stated whether error bars represent SD, SEM, or another measure↳ Could also: Explicitly label the dispersion measure in each figure legend and state it in the Methods; for small n (7–20), SD or 95% CI is often recommended over SEM because SEM shrinks with n and can visually understate variability — SD describes sample-level variability, SEM describes the precision of the mean estimate, and 95% CI conveys inferential range; stating which was used — and overlaying individual data points — allows readers to correctly interpret the spread and assess distributional assumptions
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-34811438
Paper: Larrouture et al. 2021, Sci Rep 11:22841. "Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs." DOI 10.1038/s41598-021-02246-7 · PMCID PMC8608887.
Code: https://github.com/cgat-developers/cgat-flow (CGAT toolkit; ruffus-based NGS pipelines — the framework named in Methods. Authors used "a computational pipeline calling scripts from the CGAT toolkit"; co-author A.P. Cribbs is a cgat-developers maintainer). Applying this framework + DESeq2 to the paper's own deposited data is a valid reproduction (BRIEF P16).
Data: GEO GSE166535 (SRA SRP305654 / BioProject PRJNA701237). Human
osteoclasts cultured 10 days on three substrates: plastic ("no-sub"), dentine
("dent"), and OA cartilage ("cart"). Illumina NextSeq 500, paired-end.
Series-level processed file: GSE166535_final_counts.tsv.gz (featureCounts matrix).
Reported pipeline (Methods)
- Alignment: STAR → human genome GRCh38.
- Quantification: featureCounts v1.4.6 (subread package), uniquely-mapped reads only.
- QC: "at least 14 million aligned reads per sample".
- Differential expression: DESeq2, three groups (plastic / dentine / cartilage).
- Orchestration: CGAT toolkit pipeline.
IN SCOPE (pipeline-derived → attempt to reproduce)
| # | Result | Paper location | Reproduction route |
|---|---|---|---|
| C1 | Table 1 — top-15 over-expressed genes in osteoclasts on cartilage vs dentine, with LFC + p-adj | Table 1 | DESeq2 on GSE166535_final_counts.tsv (deposited), contrast cart vs dent |
| C2 | MMP8 greatest upregulation: 8.89-fold, p = 0.0133 (cartilage vs dentine) | Results text + Table 1 row 1 | same DESeq2 run |
| C3 | PCA plot separating the three substrate groups | Fig 4 (PCA panel) | DESeq2/vst + prcomp on count matrix |
| C4 (stretch) | Full pipeline from raw FASTQ: STAR→GRCh38 + featureCounts → counts matching deposited matrix | Methods | Download SRP305654 (24 runs) on «infra», run STAR+featureCounts on «our HPC», compare to deposited final_counts.tsv |
The most faithful + feasible target is C1/C2: the count matrix is deposited, so the DESeq2 step that produces Table 1 can be reproduced directly. C4 (re-deriving counts from FASTQ) is the full-pipeline stretch goal.
Audit flag to test
Table 1 column "LFC" = 8.89993669 for MMP8 while the text says "8.89-fold". DESeq2 reports log2FoldChange. If DESeq2 log2FC(MMP8) ≈ 8.9, then "8.89-fold" in the text is a mislabel (a log2FC of 8.9 = ~477-fold). If instead the table's LFC column is linear fold (= 2^log2FC), DESeq2 log2FC(MMP8) should be ≈ 3.15. Reproducing the DESeq2 output resolves which, and whether 8.89 is the log2FC or the linear fold change. Record this in AUDIT.md.
OUT OF SCOPE (wet-lab / manual — not attempted)
- RT-qPCR validation of MMP expression (Fig 4).
- Gelatin zymography (Fig 4).
- siRNA knockdown of MMP8/MMP9 and GAG-release assays from cartilage explants (39% / 28% reduction) — wet-lab functional assays, no pipeline.
- Histology / immunostaining, resorption pit assays.
Known discrepancy to verify
Paper Methods state n = 7 biological replicates per group; GEO GSE166535 lists n = 8 donors per group (24 samples: BA, BB, BF, BH, BI, BJ, BK, BE). Confirm N actually present in the count-matrix columns and reconcile (possibly one donor/sample excluded from the published DE analysis).
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