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First published online 3 February 2004
doi: 10.1242/jcs.00912

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The surface of articular cartilage contains a progenitor cell population

Gary P. Dowthwaite¹, Joanna C. Bishop¹, Samantha N. Redman¹, Ilyas M. Khan¹, Paul Rooney², Darrell J. R. Evans^1,*, Laura Haughton¹, Zubeyde Bayram³, Sam Boyer⁴, Brian Thomson⁴, Michael S. Wolfe⁵ and Charles W. Archer^1,

¹ Cardiff School of Biosciences and Cardiff Institute of Tissue Engineering and Repair, Cardiff University, PO Box 911, Museum Avenue, Cardiff CF10 3US, UK
² Tissue Services, National Blood Service, Langley Lane, Sheffield S5 7JN, UK
³ Department of Histology and Embryology, Akdeniz University, 070 Campus Antalya, Turkey
⁴ Smith and Nephew Group Research Centre, York Science Park, Heslington, York YO10 5DF, UK
⁵ Centre for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA

View larger version (64K): [in a new window]   Fig. 1. Frozen sections (A,B) and isolated chondrocytes (C-F) from 7-day bovine articular cartilage immunolabelled for 5 (A,C,E) and ß1 (B,D,F) integrin subunits. 5 and ß1 integrin subunits are localised throughout the depth of the articular cartilage (A,B) although not every chondrocyte is labelled. Isolated chondrocytes from surface zone immediately after sequential pronase/collagenase isolation labelled with antibody to alpha 5 (C) and ß1 (D) subunits. Labelling for 5 (E) and ß1 (F) is also present 72 hours after differential adhesion to fibronectin. Fibronectin-EDA was localised in frozen tissue sections to the surface 2-3 cell layers (G). Integrin 5 and ß1 subunit expression was assessed by flow cytometry after sequential pronase/collagenase digestion (4 hours) and 72 hours after differential adhesion assay to fibronectin (H). At 4 hours and 72 hours, there was no difference in integrin subunit expression between surface zone chondrocytes (P>0.05), although during this time period the overall expression of 5 and ß1 subunits was significantly decreased (P<0.01). At 4 hours, middle zone chondrocytes had a higher expression of ß5 subunits relative to ß1 subunits (P<0.01), although there was no difference in expression after 72 hours (P>0.05).

View larger version (33K): [in a new window]   Fig. 2. Chondrocytes were subjected to differential adhesion to fibronectin as described in the Materials and Methods, and colonies consisting of more than 32 cells were counted at 6 and 10 days and the results expressed as colony forming efficiency as described in the Materials and Methods. (A,B) Colonies initially appeared at 6 days in samples derived from the surface zone subjected to differential adhesion to fibronectin for 20 minutes and the CFE of this cohort had increased by 10 days. Using 4 cells as being indicative of a colony the same trend was apparent, with surface zone cells subjected to differential adhesion to fibronectin for 20 minutes showing enhanced CFE at both 6 and 10 days (C,D). Colony size of surface (S), middle (M) and deep (D) zone chondrocytes was also assessed at 6 (E) and 10 (F) days. Cells were plated and the number of cells per colony was counted at 6 and 10 days. Surface zone chondrocytes formed bigger colonies when plated onto fibronectin for 20 minutes (SFN 20) at both 6 (E) and 10 (F) days than any other sample. There was no difference in colony size within any other cohort at either time point. In addition, the colony size of surface zone cells plated on fibronectin for 20 minutes was increased at 10 days compared with 6 days and there was no increase within any of the other cohorts. *P<0.01 compared with 6 days; **P<0.01 compared with all other cohorts at the same time point; ***P<0.001 compared with all other cohorts at the same time point. Abbreviations as in Table 1.

View larger version (17K): [in a new window]   Fig. 3. Seven-day bovine articular cartilage labelled with antibody to Notch 1 (A) and counterstained with propidium iodide. Chondrocytes within the uppermost 2-3 cell layers of the surface zone (arrows) label strongly for Notch 1. Chondrocytes were labelled with anti-notch 1 antibody and subjected to single-channel FACS analysis immediately after isolation (B). 86% of surface zone cells label positively for N1 compared with 10% and 34% from middle and deep zone, respectively. (*P<0.001 compared with middle and deep.) Chondrocytes were selected immunomagnetically and subjected to differential adhesion and initial adhesion and CFE assessed. Notch 1-selected surface zone cells (SFN N1) were more adherent than N1-selected middle (MFN N1) and deep zone (DFN N1) cells and unselected cells (SFN 20, MFN 20, DFN 20) (C). In addition, the CFE of surface zone cells selected for N1 was greater than notch-positive middle and deep zone cells and unselected cells (D). *P<0.001 compared with middle and deep, **P<0.001 compared with N1 selected and unselected middle and deep cells, ***P<0.01 compared with unselected surface zone cells, ****P<0.01 compared with selected and unselected middle and deep zone cells. Abbreviations as in Table 1.

View larger version (44K): [in a new window]   Fig. 4. Treatment with DAPT did not affect the adhesion of surface and deep zone chondrocytes to fibronectin (A) but abolished the CFE of surface zone cells at 6 and 10 (B) days. Indeed, the CFE of DAPT-incubated cells was not different from that of deep zone cells at either time point. Transfection with NICD rescued this abolition of CFE (C). NICD transfection did not increase CFE in cells not treated with DAPT (P>0.05). Cartilage explants were removed from 7-day bovine articular cartilage and cultured in the presence (D) or absence (E) of 50 nM DAPT for 7 days as described in Materials and Methods. Note that in the presence of DAPT, an acellular weakly stained band is present beneath the surface zone (arrows). These images represent a selection from 3 separate experiments each containing 6 explants per treatment. Note that the image in D is the other half of the explant from that shown in E. Using a graduated grid, the number of cells 0-100 and 101-200 µm from the articular surface was counted and the region 101-200 µm from the articular surface was shown to contain fewer cells in treated samples relative to controls (F). Explants were treated with DAPT for 7 days with the addition of BrdU on days 4, 5 and 6. Localisation of BrdU in controls (G) reveals cell proliferation, whereas there was no BrdU localisation in DAPT-treated samples (H). ns, P>0.05 compared with surf control; *P<0.01 compared with DAPT treated; **P<0.05 compared with all other cohorts, DMSO; 0.1%, dimethyl sulfoxide, Mock; no plasmid, PBS; fibronectin only.

View larger version (57K): [in a new window]   Fig. 5. lacZ-infected chondrocytes were injected into the wing bud of stage 22 chick embryos and incubated for stage 36 (10 days). ß-galactosidase-positive cells were present 24 hours after injection in the humerus (A,B, arrow), i.e. in the proximal region corresponding to the site of injection. After 10 days' incubation, ß-galactosidase activity was present in several tissues, including perimysium (C), tendon (D), bone (E, arrow) and articular fibrocartilage (F, arrow). Using anti-lacZ and bovine-specific collagen type I antibody, bovine cells and collagen were co-localised in articular fibrocartilage (G, arrow), tendon (H, arrow), perimysium (I, arrow) and bone (J, arrow). Samples from animals injected with deep zone cells contained few lacZ-positive cells and when present were not identifiable in any organised tissue (K, arrow).

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