Single-cell RNA sequencing reveals extensive fibrotic remodeling and pathogenic chondrocyte subpopulations in developmental dysplasia of the hip

Introduction

Developmental dysplasia of the hip (DDH) is one of the most common pediatric skeletal disorders, affecting approximately 0.56–3.8% of newborns worldwide (1,2). DDH encompasses a spectrum of anatomical abnormalities involving the femoral head and acetabulum, including instability, subluxation, and complete dislocation (3). If left unrecognized or inadequately treated, DDH disrupts hip joint biomechanics and markedly increases the risk of early-onset osteoarthritis (OA), leading to long-term functional disability (4).

The etiology of DDH remains incompletely understood. It is generally regarded as a multifactorial condition, influenced by both environmental and genetic factors. Reported risk factors include female sex, breech presentation, primiparity, oligohydramnios, and macrosomia (5). Several genes—such as GDF5 (6), ASPN (7), COL1A1 (8), and DKK1 (9)—have been implicated in DDH susceptibility; however, findings across studies remain inconsistent, underscoring the complexity of DDH pathogenesis. Notably, little is known about the cellular composition of acetabular cartilage in DDH, the molecular signatures of specific chondrocyte populations, or the extent to which cellular heterogeneity contributes to cartilage degeneration and disease progression.

Single-cell RNA sequencing (scRNA-seq) has recently transformed the characterization of cartilage biology by enabling the identification of previously unrecognized chondrocyte subpopulations and disease-associated transcriptional programs. This technology has revealed substantial chondrocyte heterogeneity in disorders such as OA (10), Kashin-Beck disease (KBD) (11), and intervertebral disc degeneration (IVDD) (12), offering new mechanistic insights into tissue remodeling and degeneration. However, to date, no single-cell transcriptomic studies have systematically investigated human DDH cartilage, leaving a critical gap in understanding the cellular landscape and pathological remodeling associated with this pediatric condition.

In this study, we performed scRNA-seq on acetabular cartilage obtained from children with DDH and age-matched controls to comprehensively characterize chondrocyte heterogeneity in DDH. By integrating differential gene expression, pathway enrichment, trajectory analysis, and cell-cell communication profiling, we constructed the first single-cell atlas of DDH cartilage. Our findings identify distinct pathogenic chondrocyte subpopulations and widespread fibrotic remodeling, providing new insights into DDH pathogenesis and highlighting potential translational targets for preserving joint integrity in affected children.

Methods

Ethical approval and informed consent

This study was approved by the Institutional Ethics Committee of the Children’s Hospital of Fudan University [Approval No. (2022) 205]. Written informed consent was obtained from the legal guardians of all pediatric participants. This study was conducted in accordance with the approved study protocol and the Declaration of Helsinki and its subsequent amendments.

Sample acquisition

Acetabular cartilage samples from children diagnosed with DDH were collected intraoperatively during corrective hip surgery, following parental consent. Control cartilage was obtained from pediatric patients without DDH who underwent lower-limb amputation due to traumatic injury. Macroscopically intact acetabular cartilage distant from the injury site was collected during surgery, minimizing the potential influence of acute trauma-related tissue responses. Discarded acetabular cartilage tissue was harvested with guardian permission. All specimens were immediately processed for downstream scRNA-seq and histological validation. Clinical information for the DDH and control donors, including age, sex, diagnosis, and sample site, is summarized in Table S1.

Representative radiographic materials of the DDH cases are shown in Figures S1-S3.

ScRNA-seq and Seurat processing

Raw sequencing data were processed using Seurat (v4.2) in R. Cells with fewer than 200 detected genes, cells within the top 1% of total gene counts, or exhibiting >10% mitochondrial gene expression were excluded. To correct potential batch effects between samples, data from different donors were integrated using the Harmony algorithm. After integration, principal component analysis (PCA) was performed, and the top principal components were used for downstream analysis. Graph-based clustering was conducted using the FindNeighbors and FindClusters functions in Seurat with a clustering resolution of 0.5. Cluster visualization was generated using t-distributed stochastic neighbor embedding (t-SNE). t-SNE was used for visualization as it provided clear separation of the identified chondrocyte populations in this dataset. Additional quality control steps were applied to minimize potential doublets and ambient RNA contamination. Filtering of cells with extremely high gene counts and low-quality transcript profiles helped reduce the likelihood of doublets and background RNA signals.

Cell-type identification and marker gene analysis

Marker genes for each cell cluster were identified using Seurat’s FindAllMarkers function with the Wilcoxon rank-sum test. Only genes expressed in ≥10% of cells within a cluster and displaying significant differential expression were retained. Cell identities were manually annotated based on canonical marker gene expression and prior published literature. Heatmaps, dot plots, and violin plots were generated using the DoHeatmap, DotPlot, and VlnPlot functions to visualize marker expression patterns.

Enrichment analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the clusterProfiler R package. The top 100 marker genes per cluster were used for enrichment analysis. Pathways with an adjusted P value <0.05 were considered significantly enriched. Results were visualized using built-in R functions.

Differentially expressed gene (DEG) analysis

DEGs between DDH and control groups within each chondrocyte subpopulation were identified using Seurat FindMarkers with the Wilcoxon likelihood-ratio test and default parameters. Criteria for DEG significance included expression in ≥10% of cells within a cluster and an average log fold-change >0.25. Given the limited number of donors in this pediatric cohort, results were interpreted with caution, and key transcriptional patterns were examined across individual samples to minimize potential donor-specific bias.

Pseudotime trajectory analysis

Chondrocyte developmental trajectories were reconstructed using the Monocle2 package to infer differentiation dynamics among chondrocyte populations. Genes were filtered based on the following criteria: expression in >10 cells, mean expression >0.1, and q value <0.01. Pseudotime ordering was performed to characterize cellular differentiation trajectories and lineage relationships among chondrocyte subsets.

Cell-cell communication analysis

Intercellular communication networks were inferred using the CellChat R package based on curated ligand-receptor interaction databases. Communication probabilities and signaling strengths were quantified using aggregateNet, while computeNetSimilarity assessed structural similarities among signaling pathways. Outgoing and incoming signaling roles were visualized using netAnalysis_signalingRole_heatmap, and aggregated ligand–receptor interactions were plotted using netVisual_aggregate.

RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from chondrocytes using the TRIzol reagent. Reverse transcription was performed using FastKing gDNA Dispelling RT SuperMix (TIANGEN, Beijing, China; KR118). RT-qPCR assays were conducted on an Applied Biosystems QuantStudio 3 system using Ultra-Rapid HotStart PCR Master Mix (Yeasen Biotechnology, Shanghai, China; 10157ES08). Gene expression was normalized to β-actin, and relative expression levels were calculated using the 2^−ΔΔCt method.

Clinical information for samples used in qPCR validation is summarized in Table S2.

Hematoxylin-eosin (H&E) staining

DDH and control cartilage samples were fixed in formaldehyde at 4 °C for 24 hours and decalcified in 15% ethylenediaminetetraacetic acid (EDTA). Paraffin-embedded specimens were sectioned at 4 µm, deparaffinized, and stained with hematoxylin for 10 minutes and eosin for 3 minutes. Images were captured using a Leica DM750 microscope equipped with a Leica ICC50 HD camera.

Safranin O-fast green staining

Cartilage tissues were fixed in 4% paraformaldehyde and decalcified in 15% EDTA for 4–5 weeks. After dehydration in graded ethanol and xylene, 4-µm paraffin sections were stained with 0.2% Safranin O for 5 minutes and 2% fast green for 15 seconds, followed by a brief rinse in 1% acetic acid. Stained sections were imaged using a Leica DM750 microscope.

Immunohistochemistry (IHC)

Sections were deparaffinized, rehydrated, and subjected to antigen retrieval using 10 mM citrate buffer (pH 6.0) at 60 °C overnight. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 15 minutes, followed by blocking with 1% sheep serum for 1 hour. Sections were incubated overnight at 4 °C with primary antibodies: anti-COL1A1 (Proteintech, Wuhan, China; 14695-1-AP, 1:1,000) and anti-COL3A1 (Abcam, Cambridge, UK; A11691, 1:500). After incubation with HRP-conjugated secondary antibodies and hematoxylin counterstaining, slides were visualized using a Leica DM750 microscope equipped with an ICC50 HD camera.

Statistical analysis

Statistical analyses were performed using R software. Differential expression analysis was conducted using the Wilcoxon rank-sum test in Seurat. For RT-qPCR validation, data are presented as mean ± standard deviation (SD), and comparisons between groups were performed using Student’s t-test. A P value <0.05 was considered statistically significant.

Results

Identification of nine transcriptionally distinct chondrocyte subpopulations in DDH cartilage

ScRNA-seq of acetabular cartilage from three children with DDH and two controls yielded 10,550 high-quality chondrocytes, all expressing canonical cartilage markers, including COL2A1, ACAN, and SOX9, confirming their chondrocyte identity despite transcriptional heterogeneity Unsupervised clustering analysis revealed nine transcriptionally distinct chondrocyte subpopulations, four of which (C1–C4) represent previously uncharacterized cell states in pediatric cartilage (Figure 1A,1B).

Figure 1 Identification of chondrocyte subsets and transcriptional signatures in human DDH cartilage. (A) t-SNE visualization of single-cell transcriptomes showing nine chondrocyte subsets identified in human acetabular cartilage. (B) t-SNE plots colored by sample origin, distinguishing DDH and control cells. (C) Heatmap showing scaled expression of representative marker genes defining each chondrocyte subset. (D) Proportional distribution of the nine chondrocyte populations in DDH and control samples. (E) Dot plot illustrating the expression patterns of canonical marker genes across the identified chondrocyte clusters. DDH, developmental dysplasia of the hip; t-SNE, t-distributed stochastic neighbor embedding.

The C1 subset expressed CD55 and PRG4, suggesting a lubricating or anti-inflammatory phenotype, whereas C2 was enriched for CYTL1 and CNMD, genes associated with cartilage maintenance. C3 displayed elevated expression of MMP2 and HTRA1, consistent with matrix-remodeling activity. C4, a subset detected predominantly in DDH samples, expressed SPP1, TUBB2A, and TUBB2B, indicating a potential DDH-specific chondrocyte phenotype. Marker genes and corresponding literature used for chondrocyte subset annotation are summarized in Table S3.

Among previously reported populations, regulatory chondrocytes (RegCs) exhibited homeostatic markers (CHI3L1, CFH, and PLA2G2A), dividing chondrocytes (DivCs) represented proliferative chondrocytes (TOP2A, CENPF, and CDK1), fibrotic chondrocyte 1 (FC1) showed fibrosis-related markers (ASPN and POSTN), FC2 expressed fibrocartilage-associated collagens (COL1A1 and COL1A2). Despite the upregulation of fibrosis-associated genes, these subsets retained expression of core chondrocyte markers, supporting their classification as fibrocartilage-like chondrocyte populations rather than contaminating fibroblasts. Hypertrophic chondrocytes (HTCs) demonstrated hypertrophic markers (IBSP, COL10A1, and PTH1R) (Figure 1C).

Comparative analysis of cell-type proportions showed that C1, FC1, and DivC were expanded in DDH cartilage, whereas C2, RegC, and HTC were more abundant in controls, indicating a shift toward proliferative and fibrotic phenotypes in DDH (Figure 1D). Expression patterns of representative marker genes across clusters are shown in Figure 1E. Cells from different donors were broadly distributed across the identified clusters, indicating that the clustering structure was not driven by donor-specific batch effects.

GO and KEGG analyses reveal enrichment of extracellular matrix (ECM) remodeling, ossification, and adhesion pathways in DDH-associated subsets

GO analysis demonstrated that C1 cells were enriched in insulin-like growth factor (IGF) signaling, with prominent expression of IGFBP5 and BMP2, whereas RegC, FC1, and FC2 were enriched in ECM organization and ossification, consistent with their putative roles in cartilage remodeling (Figure 2A). FC1 and FC2 displayed strong enrichment of matrix-remodeling genes, with MMP2 predominating in FC1 and MMP13 in FC2, suggesting enhanced cartilage degradation in DDH.

Figure 2 GO and KEGG pathway enrichment across chondrocyte subsets. (A,B) Bubble plot depicting the top 10 enriched GO biological process terms within seven major chondrocyte subsets, based on the top 100 highly variable genes identified in each subset. (C) KEGG pathway enrichment analysis showing significantly upregulated pathways in DDH-associated chondrocyte subsets compared with controls, based on DEGs identified using the FindMarkers algorithm. DEG, differentially expressed gene; DivC, dividing chondrocyte; FC, fibrotic chondrocyte; GO, Gene Ontology; HTC, hypertrophic chondrocyte; KEGG, Kyoto Encyclopedia of Genes and Genomes; RegC, regulatory chondrocyte.

KEGG pathway analysis further revealed activation of ECM-receptor interaction and focal adhesion pathways across multiple DDH-associated subsets, including C1, C3, FC1, FC2, and HTC (Figure 2B). RegC uniquely exhibited enrichment in glycolysis/gluconeogenesis pathways, suggesting a metabolic shift in response to an altered cartilage microenvironment. In addition, apoptosis-related pathways were significantly enriched across several DDH subpopulations, implicating programmed cell death in DDH pathogenesis. Gene set enrichment analysis further supported enrichment of extracellular matrix organization and hemostasis-related pathways (Figure 2C).

DDH cartilage exhibits a fibrotic and degenerative transcriptional program

Gene expression comparisons between DDH and control samples revealed marked transcriptional remodeling across multiple chondrocyte subpopulations. While C2 and HTC subsets showed largely similar expression patterns between groups, other clusters demonstrated substantial disease-associated differences.

In DDH cartilage, FC1 and FC2 subsets exhibited strong induction of fibrosis-related genes, including IGFBP4, THY1, and POSTN, as well as matrix-remodeling enzymes such as MMP14 and MMP13. Collagen genes COL1A1 and COL1A2 were also significantly elevated in these subsets (Figure 3A). These fibrotic markers were minimally expressed in control samples, indicating that fibrocartilage-like phenotypes are specifically activated in DDH cartilage.

Figure 3 Differential gene expression between DDH and control chondrocytes. (A) Dot plot showing the expression of top marker genes across chondrocyte subsets in DDH versus control samples, based on aggregated FindAllMarkers analysis. (B) Violin plots illustrating differential expression of representative fibrosis-, proliferation-, and cartilage matrix-related genes between DDH and control groups. DDH, developmental dysplasia of the hip.

The DivC subset showed selective upregulation of proliferative genes (CENPF, STMN1, CDK1, and TOP2A) in DDH, suggesting aberrant chondrocyte proliferation (Figure 3B). In contrast, key structural cartilage genes—COL2A1, ACAN, CHAD, and CNMD—were consistently downregulated in several DDH subpopulations, reflecting impaired ECM integrity.

These transcriptional findings were further supported by molecular and histological validation. RT-qPCR demonstrated decreased expression of COL2A1 and CYTL1, accompanied by increased expression of MMP13, COL3A1, and COL1A1 in DDH tissues (Figure 4A). Safranin O-fast green and H&E staining revealed reduced proteoglycan content, disrupted ECM organization, and decreased chondrocyte density. Immunohistochemistry further confirmed elevated deposition of COL1A1 and COL3A1 in DDH cartilage (Figure 4B), and Safranin O-fast green and H&E staining demonstrated reduced proteoglycan content and disrupted cartilage architecture (Figure 4C).

Figure 4 Molecular and histological validation of ECM alterations in DDH. (A) RT-qPCR analysis of COL2A1, CYTL1, MMP13, COL3A1, and COL1A1 expression in DDH and control acetabular tissues. (B,C) Representative images of IHC staining for COL1A1 and COL3A1, together with Safranin O-fast green and H&E staining, demonstrated ECM disorganization and altered collagen deposition in DDH cartilage. *, P<0.05; **, P<0.01; ***, P<0.001. C, control; DDH, developmental dysplasia of the hip; ECM, extracellular matrix; H&E, hematoxylin-eosin; IHC, immunohistochemistry; RT-qPCR, real-time quantitative polymerase chain reaction.

Trajectory and cell-cell communication analyses reveal progressive differentiation toward fibrotic states in DDH

Pseudotime trajectory analysis positioned C2 and HTC subsets at the earliest developmental stages, consistent with their roles in cartilage homeostasis and matrix maturation (Figure 5A-5E).

Figure 5 Pseudotime trajectory analysis of chondrocyte differentiation states. (A) Pseudotime trajectory illustrating the distribution of nine chondrocyte subsets along the inferred developmental path. (B) Visualization of pseudotime states assigned to individual chondrocytes. (C) Distribution of control and DDH cells along the trajectory. (D) Pseudotime progression highlighting developmental timing. (E) Expression dynamics of representative chondrocyte marker genes along pseudotime. C, control; DDH, developmental dysplasia of the hip; DivC, dividing chondrocyte; FC, fibrotic chondrocyte; HTC, hypertrophic chondrocyte; RegC, regulatory chondrocyte.

In contrast, DDH chondrocytes were preferentially distributed at later pseudotime states corresponding to C1, DivC, FC1, and FC2, indicating a progression toward proliferative and fibrotic phenotypes (Figure 5D). Along the trajectory, expression of cartilage matrix genes (COL2A1 and ACAN) gradually decreased, whereas fibrosis- and degradation-associated markers (MMP13) increased, revealing a shift toward degenerative remodeling (Figure 5E).

CellChat analysis further showed altered outgoing and incoming signaling patterns among chondrocyte subsets (Figure 6A). Representative ligand-receptor interactions supporting the highlighted pathways, including collagen- and TGF-β-related signaling, are shown in Figure 6B. Representative ligand-receptor pairs supporting these signaling pathways included interactions such as COL1A1-ITGA1 and TGFB1-TGFBR1.

Figure 6 Cell-cell communication networks altered in DDH cartilage. (A) Heatmap showing outgoing and incoming signaling patterns among chondrocyte subsets. (B) Dot plot showing representative ligand–receptor interactions supporting the highlighted signaling pathways. DDH, developmental dysplasia of the hip; DivC, dividing chondrocyte; FC, fibrotic chondrocyte; HTC, hypertrophic chondrocyte; RegC, regulatory chondrocyte.

Discussion

In this study, we constructed the first single-cell transcriptomic atlas of acetabular cartilage from children with DDH. Nine transcriptionally distinct chondrocyte populations were identified, including four previously unrecognized subsets (C1–C4). Compared with age-matched controls, DDH cartilage exhibited extensive alterations in cellular composition, ECM remodeling, and intercellular communication. Collectively, these findings uncover fundamental changes in chondrocyte states in cartilage obtained from dislocated hips in DDH. Importantly, these molecular alterations likely represent secondary adaptations to abnormal mechanical loading and joint instability associated with hip dislocation, rather than intrinsic defects of cartilage itself.

Cartilage obtained from dislocated hips demonstrated marked fibrotic remodeling and loss of chondrogenic identity.

One of the most prominent findings of this study is the widespread activation of fibrotic and matrix-degrading programs in DDH cartilage. FC1 and FC2 subsets in DDH exhibited strong induction of COL1A1, COL1A2, POSTN, MMP13, and MMP14, accompanied by reduced expression of cartilage-specific ECM genes such as COL2A1, ACAN, CHAD, and CNMD. These signatures were validated by qPCR, immunohistochemistry, and histological staining, which consistently demonstrated increased collagen I/III deposition and decreased proteoglycan content (13,14).

Similar fibrotic remodeling has been described in end-stage OA, where collagen I replacement and upregulation of matrix proteases mark irreversible degeneration (15,16). Our data suggest that DDH cartilage undergoes fibrotic remodeling much earlier in life, potentially driven by abnormal mechanical loading resulting from acetabular malformation (17,18). Such an early fibrotic transition may, at least in part, predispose DDH patients to the well-recognized risk of premature OA.

Disruption of key chondrogenic regulators may contribute to impaired cartilage maturation

Multiple genes critical for cartilage development were downregulated across DDH populations, including CYTL1, CHAD, S100A1 (15), and CNMD (16). CYTL1 promotes early chondrogenesis and protects against cartilage degradation, and its deficiency accelerates OA progression in mouse models (19,20). Similarly, CHAD contributes to collagen fibril organization and early skeletal development (21,22). In addition, S100A1 and CNMD play important roles in maintaining chondrocyte differentiation and cartilage homeostasis (23,24). The coordinated downregulation of these genes suggests a failure to maintain normal chondrocyte differentiation and ECM assembly, thereby contributing to the structural instability of DDH cartilage (25).

Single-cell trajectory analysis indicates a shift toward late, degenerative chondrocyte states

Pseudotime reconstruction positioned C2 and HTC cells at early developmental stages, whereas DDH chondrocytes were predominantly located in more advanced pseudotime states enriched for fibrotic and proliferative phenotypes (C1, FC1, FC2, and DivC). Along this trajectory, we observed progressive loss of cartilage matrix genes and increased expression of degradative enzymes such as MMP13. These findings are consistent with a scenario in which abnormal joint mechanics and altered loading conditions associated with hip dislocation drive chondrocytes toward degenerative and fibrosis-prone states, rather than maintaining stable articular cartilage identity (26,27).

Altered intercellular communication highlights collagen and TGF-β signaling as key drivers of DDH pathology

CellChat analysis revealed strengthened collagen signaling networks in nearly all DDH subsets, consistent with the widespread fibrosis observed histologically. In addition, the TGF-β pathway—known to promote ECM synthesis, fibrosis, and chondrocyte hypertrophy—was predominantly active in the C3 population. Dysregulated TGF-β signaling has been implicated in both OA and skeletal dysplasia (28-30), and our findings suggest that aberrant activation of this pathway may contribute to pathological ECM remodeling in DDH.

IGF axis dysregulation may underlie impaired cartilage growth in DDH

We observed altered expression of IGF-binding proteins (IGFBP1, IGFBP3, IGFBP5, and IGFBP6) within the C1 subset. Because IGFBPs modulate IGF availability and receptor signaling, disruptions in this axis could potentially impair normal cartilage growth and repair. Prior studies reporting reduced circulating IGFBP5 levels in infants with DDH further support this hypothesis and suggest that impaired IGF signaling may have diagnostic or therapeutic relevance. However, further mechanistic studies will be required to clarify the precise functional role of IGF signaling dysregulation in DDH (31,32).

Clinical implications

Together, our findings suggest that cartilage obtained from dislocated hips in DDH undergoes substantial molecular remodeling characterized by fibrosis, ECM disorganization, and altered chondrocyte differentiation. These transcriptional changes likely represent secondary responses to abnormal joint mechanics and loading conditions rather than primary intrinsic cartilage defects.

Understanding these secondary molecular adaptations may help explain why prolonged hip dislocation predisposes patients to early degenerative joint changes and may provide insights into potential strategies for preserving cartilage integrity once mechanical alignment of the hip is restored.

Limitations

There are several limitations in this study. First, the sample size and number of donors were limited due to the difficulty of obtaining pediatric acetabular cartilage samples. Although consistent transcriptional patterns were observed across DDH samples, donor-level variability cannot be completely excluded and should be addressed in future studies with larger cohorts.

Second, only cartilage tissue was analyzed; integration with synovium, ligament, or subchondral bone may reveal additional pathological interactions.

Third, functional experiments to validate the roles of candidate markers such as IGFBP5 or MMP13 were beyond the scope of this study. Future investigations using in vitro and in vivo models will be necessary to establish causal mechanisms.

Another limitation relates to the source of control cartilage, which was obtained from trauma-related surgical cases. Although macroscopically intact acetabular cartilage distant from the injury site was collected, potential stress- or inflammation-related transcriptional effects cannot be completely excluded. However, the primary molecular changes observed in this study mainly involved ECM remodeling rather than acute inflammatory signatures.

Conclusions

In summary, this study provides a comprehensive single-cell atlas of cartilage obtained from dislocated hips in DDH and reveals profound alterations in chondrocyte composition, ECM remodeling, and signaling pathways.

These findings offer new insights into the molecular adaptations associated with abnormal joint mechanics in DDH and may help inform future studies aimed at preserving cartilage health following correction of hip instability.

Acknowledgments

None.

Footnote

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-891/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-891/prf

Funding: This research was funded by the National Natural Science Foundation of China (No. 82272441).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-1-891/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Institutional Ethics Committee of the Children’s Hospital of Fudan University [Approval No. (2022) 205]. Written informed consent was obtained from the legal guardians of all pediatric participants. This study was conducted in accordance with the approved study protocol and the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Schoenecker PL, Clohisy JC, Millis MB, et al. Surgical management of the problematic hip in adolescent and young adult patients. J Am Acad Orthop Surg 2011;19:275-86. [Crossref] [PubMed]
2. Westacott DJ, Butler D, Shears E, et al. Universal versus selective ultrasound screening for developmental dysplasia of the hip: a single-centre retrospective cohort study. J Pediatr Orthop B 2018;27:387-90. [Crossref] [PubMed]
3. Jawadi AH, Wakeel A, Tamimi W, et al. Association analysis between four vitamin D receptor gene polymorphisms and developmental dysplasia of the hip. J Genet 2018;97:925-30.
4. Wyles CC, Heidenreich MJ, Jeng J, et al. The John Charnley Award: Redefining the Natural History of Osteoarthritis in Patients With Hip Dysplasia and Impingement. Clin Orthop Relat Res 2017;475:336-50. [Crossref] [PubMed]
5. Harsanyi S, Zamborsky R, Krajciova L, et al. Developmental Dysplasia of the Hip: A Review of Etiopathogenesis, Risk Factors, and Genetic Aspects. Medicina (Kaunas) 2020;56:153. [Crossref] [PubMed]
6. Hatzikotoulas K, Roposch ADDH Case Control Consortium, et al. Genome-wide association study of developmental dysplasia of the hip identifies an association with GDF5. Commun Biol 2018;1:56. [Crossref] [PubMed]
7. Sekimoto T, Ishii M, Emi M, et al. Copy number loss in the region of the ASPN gene in patients with acetabular dysplasia: ASPN CNV in acetabular dysplasia. Bone Joint Res 2017;6:439-45. [Crossref] [PubMed]
8. Zhao L, Tian W, Pan H, et al. Variations of the COL1A1 gene promoter and the relation to developmental dysplasia of the hip. Genet Test Mol Biomarkers 2013;17:840-3. [Crossref] [PubMed]
9. Liu S, Tian W, Wang J, et al. Two single-nucleotide polymorphisms in the DKK1 gene are associated with developmental dysplasia of the hip in the Chinese Han female population. Genet Test Mol Biomarkers 2014;18:557-61. [Crossref] [PubMed]
10. Liu Y, Da W, Xu MJ, et al. Single-cell transcriptomics reveals novel chondrocyte and osteoblast subtypes and their role in knee osteoarthritis pathogenesis. Signal Transduct Target Ther 2025;10:40. [Crossref] [PubMed]
11. Wang X, Ning Y, Zhang P, et al. Comparison of the major cell populations among osteoarthritis, Kashin-Beck disease and healthy chondrocytes by single-cell RNA-seq analysis. Cell Death Dis 2021;12:551. [Crossref] [PubMed]
12. Zhang Y, Han S, Kong M, et al. Single-cell RNA-seq analysis identifies unique chondrocyte subsets and reveals involvement of ferroptosis in human intervertebral disc degeneration. Osteoarthritis Cartilage 2021;29:1324-34. [Crossref] [PubMed]
13. Eyre DR, Weis MA, Wu JJ. Articular cartilage collagen: an irreplaceable framework? Eur Cell Mater 2006;12:57-63. [Crossref] [PubMed]
14. Han L, Grodzinsky AJ, Ortiz C. Nanomechanics of the Cartilage Extracellular Matrix. Annu Rev Mater Res 2011;41:133-68. [Crossref] [PubMed]
15. Benito MJ, Veale DJ, FitzGerald O, et al. Synovial tissue inflammation in early and late osteoarthritis. Ann Rheum Dis 2005;64:1263-7. [Crossref] [PubMed]
16. Appleton CTG, Pitelka V, Henry J, et al. TGF-β/SMAD2 signaling drives OA fibrosis. Arthritis Rheum 2007;56:418-29.
17. Andriacchi TP, Mündermann A. The role of ambulatory mechanics in the initiation and progression of knee osteoarthritis. Curr Opin Rheumatol 2006;18:514-8. [Crossref] [PubMed]
18. Li Y, Wei X, Zhou J, et al. Mechanical overload induces chondrocyte fibrosis via TGF-β signaling. J Orthop Res 2013;31:1745-51.
19. Kim JS, Ryoo ZY, Chun JS. Cytokine-like 1 (Cytl1) regulates the chondrogenesis of mesenchymal cells. J Biol Chem 2007;282:29359-67. [Crossref] [PubMed]
20. Jeon J, Oh H, Lee G, et al. Cytokine-like 1 knock-out mice (Cytl1-/-) show normal cartilage and bone development but exhibit augmented osteoarthritic cartilage destruction. J Biol Chem 2011;286:27206-13. [Crossref] [PubMed]
21. Larsson T, Sommarin Y, Paulsson M, et al. Cartilage matrix proteins. A basic 36-kDa protein with a restricted distribution to cartilage and bone. J Biol Chem 1991;266:20428-33.
22. Batista MA, Nia HT, Önnerfjord P, et al. Nanomechanical phenotype of chondroadherin-null murine articular cartilage. Matrix Biol 2014;38:84-90. [Crossref] [PubMed]
23. Diaz-Romero J, Nesic D. S100A1 and S100B: Calcium Sensors at the Cross-Roads of Multiple Chondrogenic Pathways. J Cell Physiol 2017;232:1979-87. [Crossref] [PubMed]
24. Zhu S, Qiu H, Bennett S, et al. Chondromodulin-1 in health, osteoarthritis, cancer, and heart disease. Cell Mol Life Sci 2019;76:4493-502. [Crossref] [PubMed]
25. Aigner T, Sstöve J. Collagens—major regulators in cartilage matrix assembly. Curr Opin Rheumatol 2003;15:657-63.
26. Hwang HS, Kim HA. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis. Int J Mol Sci 2015;16:26035-54. [Crossref] [PubMed]
27. Wei YS, Li DH, Liu WL, et al. Altered Chondrocyte Apoptosis Status in Developmental Hip Dysplasia in Rabbits. Balkan Med J 2016;33:639-44. [Crossref] [PubMed]
28. van der Kraan PM, van den Berg WB. TGF-β and osteoarthritis progression. Nat Rev Rheumatol 2012;8:649-56.
29. Blaney Davidson EN, Remst DFG, Vitters EL, et al. TGF-β induces ECM production and fibrosis in chondrocytes. Arthritis Rheum 2009;60:2589-99.
30. Long F. TGF-β family signaling in skeletal development. Cold Spring Harb Perspect Biol 2012;4:a009183.
31. Lupu F, Terwilliger JD, Lee K, et al. Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol 2001;229:141-62. [Crossref] [PubMed]
32. Demirel E, Şenocak E, Şenocak GNC, et al. Investigation of Igf-1, Igf-Bp3 and Igf-Bp5 levels in umbilical cord blood of infants with developmental dysplasia of the hip. Turk J Med Sci 2023;53:659-65. [Crossref] [PubMed]