Structure of pericellular matrix around agarose-embedded chondrocytes M.A. DiMicco, Ph.D., J.D. Kisiday, Ph.D., H. Gong, M.D., Ph.D., A.J. Grodzinsky, Sc.D.  Osteoarthritis and Cartilage  Volume 15, Issue 10, Pages 1207-1216 (October 2007) DOI: 10.1016/j.joca.2007.03.023 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Cell (open circles, left axis) and GAG (squares, right axis) content of chondrocyte agarose constructs increased with time in culture. n=4 gels from separate joints per timepoint. Osteoarthritis and Cartilage 2007 15, 1207-1216DOI: (10.1016/j.joca.2007.03.023) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Matrix deposition around representative agarose-embedded chondrocytes. Proteoglycan deposition increased with culture duration, evaluated by toluidine blue staining and TEM images (inset). Dense granules in the TEM images indicate proteoglycans that have been precipitated by treatment with ruthenium hexamine trichloride before uranyl acetate staining, and the extent of the electron-dense region agrees well with the toluidine blue staining. Bar=10μm. Osteoarthritis and Cartilage 2007 15, 1207-1216DOI: (10.1016/j.joca.2007.03.023) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Confocal microscopy images depicting the spatial distribution of collagen type VI around agarose-embedded chondrocytes after (A) 7, (B) 14, (C) 21, and (D) 28 days of culture. Bar=10μm. Osteoarthritis and Cartilage 2007 15, 1207-1216DOI: (10.1016/j.joca.2007.03.023) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 (A) Log plot of gene expression for matrix constituents collagen type II (col2), aggrecan (agg), biglycan (bgn), fibromodulin (fmod), fibronectin (fnect), collagen type VI (col6), and decorin (dcn), normalized to 18S content of each sample. Open bars represent the expression in agarose-embedded chondrocytes after 2 days in culture; closed bars are the expression levels in chondrocytes from freshly isolated calf patellofemoral groove cartilage (mean±s.e.m., n=5 joints). (B) Collagen type VI expression by agarose-embedded chondrocytes was relatively high after 2 days in culture compared with chondrocytes from cartilage (dashed line), and this expression decreased by day 7 and was relatively stable thereafter (mean±s.e.m., n=3–7 per time point; *P<0.05 vs day 2). Osteoarthritis and Cartilage 2007 15, 1207-1216DOI: (10.1016/j.joca.2007.03.023) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Quick-freeze/deep-etch image of chondrocyte pericellular matrix. (A) Day 7, agarose-embedded chondrocyte. Between the cell membrane (cm) and further-removed matrix (frm) is an area of radially oriented fibers (small arrows) connecting the cell membrane to a dense fibrous ring (large arrows) surrounding the cell at a distance corresponding to the extent of collagen type VI deposition observed by confocal microscopy (Fig. 3). (B) Matrix surrounding a chondrocyte within cartilage, illustrating the juxtaposition of the cell membrane (cm) with the PCM, composed of longer tangentially oriented fibers (large arrows) interspersed with smaller, radially oriented fibers (small arrows). The intracellular space is labeled “ic”. Bar=1μm. Osteoarthritis and Cartilage 2007 15, 1207-1216DOI: (10.1016/j.joca.2007.03.023) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions