Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis

Abstract

Objectives: Osteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA.

Methods: Fluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations.

Results: Articular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone.

Conclusion: Our findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA.

Keywords: arthritis; chondrocytes; experimental; fibroblasts; osteoarthritis.

Figure 1

Pdgfrα-lineage progenitors, not articular chondrocytes, clonally expand to form osteophytes. (a) GFP expression (green) by cells in periosteum and synovium of the knee of a 15-week-old mouse carrying the Pdgfrα-H2BGFP transgene (n=2). (b) Adult Col2-CreER;Tom mice induced with tamoxifen at 2 weeks of age (n=8, 2 experiments, 7–8 weeks old). Note Tom-labelled cells (red) in articular and growth plate cartilage of the knee. (c) Pdgfrα-H2BGFP;Col2-CreER;Tom mice induced with tamoxifen at 2 weeks of age and analysed 2 weeks after DMM (n=4, plus n=3 Col2-CreER;Tom only). Note Pdgfrα-expressing cells (green) in osteophyte that are negative for Tom (red). (d) Tom expression (red) in Col2-CreER;Tom mice induced with tamoxifen at 2 weeks of age and analysed 8 weeks after DMM (n=6). (e–g) Pdgfrα-CreER;Confetti mice were induced with tamoxifen starting at 11 to 12 weeks of age, followed by DMM surgery and analysis 2 weeks later (n=4). (e) CFP (blue), YFP (yellow) and RFP (red) expression in contralateral knee serving as internal control. Arrows indicate labelled cells along periosteal surface. (f) CFP (blue), YFP (yellow) and RFP (red) expression in osteophyte of destabilised knee. Arrows indicate monochromatic chondrocyte clusters within periosteum, arrowheads indicate distinct monochromatic clusters of chondrocyte-like and fibroblast-like cells in overlying synovium. (g) Percentage of cells in osteophytes labelled with each of the fluorescent proteins, and total percentage of cells labelled (mean±95%CI, n=4). Fluorescence microscopy images in (a,b,d) show nuclear counterstain in blue. Dashed white lines in (c,d,f) indicate boundary between osteophyte and edge of tibia. Brightfield images of near-adjacent sections stained with Safranin O and Fast Green are shown on the left in (c–f). Scale bars in all panels indicate 100 μm. A, articular cartilage; CFP, cyan fluorescent protein; DMM, destabilisation of the medial meniscus; G, Growth plate; PS, periosteum and synovium junction; RFP, red fluorescent protein; S, synovium; YFP, yellow fluorescent protein.

Figure 2

Perivascular and Grem1-expressing skeletal stem cells do not contribute to osteophyte formation. (a–c) Nes-CreER;Tom mice, some also carrying Nes-GFP, were induced with tamoxifen neonatally. (a) Nes-traced cells (red) and Nes-GFP+ cells (green) in knee from 13-week-old unoperated mouse (n=3, 2 experiments, 6–13 weeks old). (b) Nes-traced cells (red, n=6) and Nes-GFP+ cells (green, n=3) in knee 2 weeks post-DMM. Arrows indicate labelled cells around blood vessels in synovium. (c) Nes-traced cells (red) in knee 8 weeks post-DMM (n=3). Arrows indicate labelled cells associated with bone marrow vasculature within osteophyte. (d,e) LepR-Cre;Tom mice underwent DMM surgery at 12 weeks and were analysed 8 weeks later. (d) LepR-traced cells (red) in uninjured contralateral knee serving as internal control (n=3). Arrows indicate labelled cells in synovium and periosteum. (e) LepR-traced cells (red) in destabilised knee (n=4). Arrows indicate labelled cells in synovium. (f) Grem1-CreER;Tom mice were induced with tamoxifen at 7 weeks of age and left unoperated (n=2) or analysed 2 weeks after DMM (n=3). Arrow indicates osteophyte. Fluorescence microscopy images in all panels show nuclear counterstain in blue. Dashed white lines in (b,c,e) indicate boundary between osteophyte and edge of tibia. Brightfield images of near-adjacent sections stained with Safranin O and Fast Green are shown on the left in (a–e). Scale bars in all panels indicate 100 μm. A, articular cartilage; DMM, destabilisation of the medial meniscus; G, growth plate; PS, periosteum and synovium junction; S, synovium.

Figure 3

Gdf5-lineage cells are a subset of Pdgfrα-expressing cells in the adult knee, (a) Tom+ Gdf5-lineage cells (red) in 14-week-old Gdf5-Cre;Tom mouse knee. Nuclear counterstain is shown in blue. Scale bar indicates 100 μm. (b) Knee of 11-week-old Pdgfrα-H2BGFP;Gdf5-Cre;Tom mouse showing Tom (red; Gdf5-lineage cells) and GFP expression (green; Pdgfrα-expressing cells) (n=3). Scale bar indicates 100 μm. (c–h) Freshly isolated cells from knees of Pdgfrα-H2BGFP;Gdf5-Cre;Tom mice (7–10 weeks old) were analysed by flow cytometry. See online supplementary figure 1 for gating strategies and FMO controls. (c) Representative flow plot showing Tom and GFP expression by single viable cells (n=9, 4 experiments). (d) Percentage of single viable cells that expressed one or both fluorescent labels (mean±95%CI, n=9, 4 experiments). (e–h) Phenotypic analysis detecting a range of mesenchymal and fibroblast (Gp38, CD90, CD73, CD51 and CD105), haematopoietic (CD45), endothelial (CD31) or erythrocyte (Ter-119) markers. (e) Representative flow plots showing expression of Tom and the indicated markers within single viable GFP+ cells (n=4–5 for each marker, 4 experiments). (f–h) Percentage of single viable cells that express the indicated markers within (f) GFP+Tom+ (Gdf5-lineage cells), (g) GFP+Tom-(other Pdgfrα-expressing cells), and (h) GFP-Tom-cell populations (mean±95%CI, n=4–5 for each marker, 4 experiments). A, articular cartilage; G, growth plate; PS, periosteum and synovium junction; S, synovium.

Figure 4

Joint-resident SSCs within the Gdf5-lineage form osteophytes, (a–c) Adult Gdf5-Cre;Tom mice underwent surgery at 9 weeks to induce DMM in one knee, with contralateral knee sham-operated, and BrdU administered from surgery until end of experiment 2 weeks later. (a) Tom+ Gdf5-lineage cells (red) and BrdU-labelled cells (green) in sham-operated knee (n=4). Arrows indicate Tom+ cells along the periosteal surface with incorporated proliferation label. (b) Tom+ Gdf5-lineage cells (red) and BrdU-labelled cells (green) in destabilised knee (n=4). Note Tom+ cells with incorporated proliferation label throughout the osteophyte. Co-staining for Tom (red) with Col2 (green) to reveal cartilage matrix surrounding Tom+ cells is shown on the far right (image from different mouse). (c) Percentage of cells in osteophytes that are Tom+ Gdf5-lineage cells, and percentage of cells in osteophytes that have incorporated the BrdU proliferation label (mean±95%CI, n=4). (d,e) Gdf5-Cre;Tom mice underwent DMM surgery at 9–14 weeks and were analysed 8 weeks after DMM. (d) Tom+ Gdf5-lineage cells (red) in mature osteophyte (n=7). Arrows indicate Tom+ cells lining endosteal surfaces and arrowheads indicate Tom+ osteocytes embedded within the bone of the osteophyte. Enlarged image on right shows Tom+ chondrocytes in the cartilage cap. (e) Percentage of cells in osteophytes that are Tom+ Gdf5-lineage cells (mean±95%CI, n=7), divided into the capping region and osteocytes within bone. Fluorescence microscopy images in all panels show nuclear counterstain in blue. Dashed white lines in (b,d) indicate boundary between osteophyte and edge of tibia. Brightfield images of near-adjacent sections stained with Safranin O and Fast Green are shown on the left. Boxed regions indicate areas shown at higher magnification on the right. Scale bars in all panels indicate 100 μm. A, articular cartilage; BrdU, bromodeoxyuridine; DMM, destabilisation of the medial meniscus; G, growth plate; PS, periosteum and synovium junction; S, synovium.

Figure 5

Sox9-expressing progenitors give rise to hybrid cells in the early osteophyte. (a,b,e) Sox9-CreER;Tom mice were induced with tamoxifen at 7 weeks of age. (a) Tom+ Sox9-traced cells (red) in articular cartilage (A), growth plate (G), and scattered within periosteum (P) of knee from 9-week-old uninjured mouse (n=3). Boxed region on left indicates area shown at higher magnification on the right (different tissue sections are shown). (b) Tom+ Sox9-traced cells (red) in osteophyte (outlined with dashed white line on far right) at 2 weeks post-DMM (n=3). Brightfield image of near-adjacent section stained with Safranin O and Fast Green is shown on the left. Boxed region is shown at higher magnification on the far right. (c,d) Double fluorescence in situ hybridisation in wild-type mouse knees at 1 week (c) or 2 weeks post-DMM (d) for indicated mRNA targets. Note co-expression of Col2a1 (red) with Col1a1, Ocn, Spp1 or Col10a1 (green) in the early osteophyte, and absence of Col1a1 in the tibial growth plate (G). Merged and individual channel images of the boxed osteophytes are shown to the right. n=3 for each probe combination. (e) Co-detection of Tom with Col2a1 and Col1a1 mRNA in osteophyte of Sox9-CreER;Tom mouse (n=3). Individual and merged channel images are shown. Note Tom+ Sox9-traced cells (magenta) coexpressing Col2a1 (green) and Col1a1 (red) in outlined area. Fluorescence microscopy images in all panels show nuclear counterstain in blue. Scale bars indicate 100 μm in (a,b) and 200 μm in (c–e). A, articular cartilage; DMM, destabilisation of the medial meniscus; G, growth plate; P, periosteum.

Figure 6

Contribution of Prg4-expressing progenitors to osteophytes. (a,b) Prg4-CreER;mTmG mice were induced with tamoxifen at 7 weeks of age, followed by surgery to induce DMM in one knee, with contralateral knee sham-operated. Prg4-traced cells were detected with anti-GFP antibody (green), and Col1a1 or Col2a1 mRNA expression by fluorescence in situ hybridisation (red). (a) GFP+ Prg4-traced cells at articular surface and in synovial lining in control sham-operated knee (n=3). Note membrane localisation of GFP was observed, indicating successful mTmG conversion. (b) GFP+ Prg4-traced cells at 2 weeks post-DMM. Note expansion in synovium but minimal contribution to hybrid cells that express Col1a1 and Col2a1 in the early osteophyte (n=3). Boxes indicate magnified images to the right, shown as merged and individual channel images. Arrowheads indicate rare Prg4-traced cells expressing Col2a1. (c–h) Prg4-CreER;Tom mice were induced with tamoxifen at 8 weeks of age. (c) Tom+ Prg4-traced cells (red) in synovial lining and superficial zone of articular cartilage in 10-week-old uninjured mouse (n=7, 3 experiments). Green: Col2 immunostaining (n=3). (d) Tom+ Prg4-traced cells (red) in osteophyte at 2 weeks post-DMM (n=8, 2 experiments). Note Tom+ cells in Col2+ (green) cartilage matrix and overlying synovial tissue. (e) Percentage of cells that expressed Tom at 2 weeks post-DMM in Col2+ cartilage matrix or Col2− tissue of the osteophyte (mean±95% CI, n=8, 2 experiments). (f) Tom+ Prg4-traced cells (red) in Col10+ (green) hypertrophic cartilage of osteophyte 2 weeks post-DMM, indicated by arrows (n=4). (g) Tom+ Prg4-traced cells (red) in osteophyte at 8 weeks post-DMM (n=7, 2 experiments). Green: Col2 immunostaining. (h) Percentage of cells that expressed Tom at 8 weeks post-DMM in Col2+ cartilage matrix or Col2-tissue of the osteophyte cap, or among osteocytes in the osteophyte bone (mean±95% CI, n=7, 2 experiments). Fluorescence microscopy images show nuclear counterstain in blue. Brightfield images of Safranin-O-stained near-adjacent sections are shown on the left in (d,g). Dashed white lines indicate boundary between osteophyte and edge of tibia in (d,f,g). Scale bars indicate 200 μm in (a,b) and 100 μm in (c,d,f,g). A, articular cartilage; DMM, destabilisation of the medial meniscus; G, growth plate; PS, periosteum and synovium junction; S, synovium.

Figure 7

Proposed model of osteophyte formation in OA. Our data show that Pdgfrα+ Gdf5-lineage progenitors, which in the normal joint are present at the junction of periosteum and synovium near the articular cartilage, are activated in OA to form both the cartilage and bone of the osteophyte. They include Prg4-expressing progenitors (orange) residing in synovial lining and Sox9-expressing progenitors (green) in the underlying periosteum. During the early stage of osteophyte formation, Sox9-expressing progenitors in periosteum give rise to hybrid skeletal cells that form a transient cartilage template which is remodelled to bone. Progeny of Prg4-expressing progenitors are recruited to the forming osteophyte and supply chondrocytes to the cartilage, but they make negligible contributions to osteoblast-lineage cells that form the bone. A, Articular cartilage; M, meniscus; OA, osteoarthritis.