A whole-genome transcriptome analysis of articular chondrocytes in secondary osteoarthritis of the hip

<div><p>Objective</p><p>To date, exhaustive gene expression analyses of chondrocytes in hip osteoarthritis (OA) have yielded specific gene expression patterns. No study has reported on the exhaustive transcriptome of secondary hip OA based on acetabular dysplasia in a Japanese population, while previous reports have focused on primary or idiopathic hip OA in Caucasian populations. This study aims to search for specific gene expression patterns of secondary hip OA chondrocytes by transcriptome analysis.</p><p>Design</p><p>Human articular cartilage was obtained from femoral heads following hemiarthroplasty for femoral neck fracture (N = 8; non-OA) and total hip arthroplasty for secondary hip OA (N = 12). Total RNA was extracted from the articular cartilage and submitted for microarray analysis. The obtained data were used to perform gene expression analysis, GO enrichment analysis and pathway analysis and were compared with data from primary hip OA in Caucasian populations in the literature.</p><p>Results</p><p>We identified 888 upregulated (fold change: FC ≥ 2) and 732 downregulated (FC ≤ 0.5) genes in hip OA versus non-OA chondrocytes, respectively. Only 10% of upregulated genes were common between the secondary and primary OA. The newly found genes prominently overexpressed in the secondary hip OA chondrocytes were <i>DPT</i>, <i>IGFBP7</i>, and <i>KLF2</i>. Pathway analysis revealed extracellular matrix (ECM)-receptor interaction as an OA-related pathway, which was similar to previous reports in primary hip OA.</p><p>Conclusions</p><p>This is the first study to report the genome-wide transcriptome of secondary hip OA chondrocytes and demonstrates new potential OA-related genes. Gene expression patterns were different between secondary and primary hip OA, although the results of pathway and functional analysis were similar.</p></div

Compared gene profiles.

<p>Venn diagram demonstrating the overlap of the differentially expressed genes between secondary and primary hip OA. Overlapping portion of the three circles indicates the genes significantly expressed in both studies in common (36 up-regulated genes and 56 down-regulated genes). In the inner circles on the left side, the area not overlapped with the primary OA’s circle indicate differentially expressed genes in secondary (316 up-regulated genes and 103 down-regulated genes).</p

Functional enrichment analysis using a gene set enrichment analysis (GSEA).

<p>Functional enrichment analysis using a gene set enrichment analysis (GSEA).</p

Femoral head of #NOF (fracture of neck of femur: non-OA) and OA.

<p>(A) A femoral head of #NOF. (B) A femoral head of a typical secondary OA. The articular cartilage pieces were obtained from the middle to deep layer of the non-weight bearing, macroscopically intact area from #NOF femoral head (white arrow heads), and the surface layer (black arrow heads) surrounding the weight-bearing, eburnated area (*) from OA femoral heads, respectively.</p

The upregulated pathways in OA chondrocytes.

<p>The upregulated pathways in OA chondrocytes.</p

The demographic data of the cartilage samples of non-OA and OA.

<p>The demographic data of the cartilage samples of non-OA and OA.</p

qRT-PCR replication study.

<p>The figures show relative gene expression of 12 prominently overexpressed gene in OA chondrocytes detected by the microarray analysis. <i>ASPN</i> (A), <i>COL1A2</i> (B), <i>COL2A1</i> (C), <i>COL3A2</i> (D), <i>COL5A1</i> (E), <i>KLF2</i> (F), <i>MXRA5</i> (G), <i>OGN</i> (H), <i>PCOLCE</i> (I), <i>SPARC</i> (J), <i>TGFBI</i> (K), and <i>TNFAIP6</i> (L). The data were shown as average ± standard error of mean (SEM) (**: <i>p</i> < 0.01).</p

Representative differentially expressed genes in OA and NOF cartilage.

<p>Representative differentially expressed genes in OA and NOF cartilage.</p

Histological images of fracture healing.

<p>(A) Alcian blue-stained sections. Scale bar = 500 μm. <i>DAP12</i>^{<i>–/–</i>} mice showed more residual cartilaginous tissue and delayed woven bone formation (arrow) at day 10. Substantially impaired cortical shell formation on the callus surface was observed in <i>DAP12</i>^{<i>–/–</i>} mice at day 14 (arrowhead). <i>DAP12</i>^{<i>–/–</i>} mice showed residual cartilage matrix in the peripheral callus, less original cortex absorption and delayed bone remodeling at day 28. (B) Quantitative histomorphometric analysis of the fractured callus in B6 and <i>DAP12</i>^{<i>–/–</i>} mice. There was no significant difference in callus volume between <i>DAP12</i>^{<i>–/–</i>} and B6 mice. Hematoma area was significantly larger in <i>DAP12</i>^{<i>–/–</i>} mice than in B6 mice at day 7. The newly formed bone area was significantly smaller in <i>DAP12</i>^{<i>–/–</i>} mice than in B6 mice at days 7 and 10. Cartilage area was significantly larger in <i>DAP12</i>^{<i>–/–</i>} mice than in B6 mice at day 10. *<i>p</i><0.05, **<i>p</i><0.01.</p

Micro-CT image analysis of fracture callus at day 14 in B6 and <i>DAP12</i>^{<i>–/–</i>} mice.

<p>(A) Representative axial images of fracture callus in B6 and <i>DAP12</i>^{<i>–/–</i>} mice. Dense trabecular structure and deteriorated cortical shell formation on the surface in <i>DAP12</i>^{<i>–/–</i>} mice compared with B6 mice. (B) Structural parameter analysis of fracture callus of B6 and <i>DAP12</i>^{<i>–/–</i>} mice. Trabecular thickness was significantly larger in <i>DAP12</i>^{<i>–/–</i>} mice than in B6 mice, and the percentage of new cortical shell area was significantly lower in <i>DAP12</i>^{<i>–/–</i>} mice. There were no significant differences in TV, BV/TV, Tb.N or Tb.Sp. *<i>p</i><0.05.</p