S. Varghese, Ph. D. , P. Theprungsirikul, B. S. , S. Sahani, B. S. , N

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Glucosamine modulates chondrocyte proliferation, matrix synthesis, and gene expression S. Varghese, Ph.D., P. Theprungsirikul, B.S., S. Sahani, B.S., N. Hwang, B.S., K.J. Yarema, Ph.D., J.H. Elisseeff, Ph.D. Osteoarthritis and Cartilage Volume 15, Issue 1, Pages 59-68 (January 2007) DOI: 10.1016/j.joca.2006.06.008 Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 (A) Cell morphology of chondrocytes exposed to GlcN after 3 days of culture. (B) Cells exposed to GlcN proliferated and achieved comparable confluency to control cultures when they were exposed to GlcN after 6 days of initial plating. Photographs were taken after a total of 11 days of culture. Osteoarthritis and Cartilage 2007 15, 59-68DOI: (10.1016/j.joca.2006.06.008) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 GlcN tolerance of chondrocytes depends upon when GlcN is added to culture. Proliferation of cells after 11 days in monolayer at various experimental conditions. (B) Snapshots of monolayer cultures obtained via optical microscope at the time of GlcN exposure. Osteoarthritis and Cartilage 2007 15, 59-68DOI: (10.1016/j.joca.2006.06.008) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 (A) Growth kinetics of chondrocytes in culture medium containing different concentrations of GlcN. Chondrocytes were plated at a cell density of 5000cells/cm2, and exposed to GlcN immediately. Initial population doubling for the first 3 days after plating the cells. Cells were harvested after 24, 48, and 72h and counted using a coulter counter. (B) Population doubling times. (C) Cell proliferation for 5 days after plating the cells determined by WST-1 assay. Higher absorbance indicates more cells. Osteoarthritis and Cartilage 2007 15, 59-68DOI: (10.1016/j.joca.2006.06.008) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Real-time PCR of monolayer cultured chondrocytes indicates a dose-dependent up-regulation of (A) cartilage specific markers collagen type II and aggrecan mRNA and (B) TGF-β1 mRNA with GlcN treatment. Osteoarthritis and Cartilage 2007 15, 59-68DOI: (10.1016/j.joca.2006.06.008) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Biochemical analysis of DNA (cell proliferation), GAG, and collagen (n=6) for 3D constructs cultured with 0, 2, and 15mM GlcN. (a) Cell proliferation of chondrocytes encapsulated within hydrogel (b) GAG, and (c) collagen content for encapsulated chondrocytes normalized by DNA content (w/w; n=6). *P<0.05 and **P<0.007. Osteoarthritis and Cartilage 2007 15, 59-68DOI: (10.1016/j.joca.2006.06.008) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 (A) Safranin-O and, (B) collagen type II staining (nuclei of the cells were stained with DAPI (blue)) for 3D hydrogels constructs cultured in the indicated concentrations of GlcN. Osteoarthritis and Cartilage 2007 15, 59-68DOI: (10.1016/j.joca.2006.06.008) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 (A) Tunnel staining for apoptotic cells after incubated in the presence of varying GlcN concentrations. (B) Percentage of living cells after 6 weeks culture in presence of varying GlcN concentrations. Osteoarthritis and Cartilage 2007 15, 59-68DOI: (10.1016/j.joca.2006.06.008) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions