Characterization of proteoglycan production and processing by chondrocytes and BMSCs in tissue engineered constructs J.T. Connelly, Ph.D., C.G. Wilson,

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Characterization of proteoglycan production and processing by chondrocytes and BMSCs in tissue engineered constructs J.T. Connelly, Ph.D., C.G. Wilson, Ph.D., M.E. Levenston, Ph.D. Osteoarthritis and Cartilage Volume 16, Issue 9, Pages 1092-1100 (September 2008) DOI: 10.1016/j.joca.2008.01.004 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Safranin-O and immunofluorescence images of day 32 constructs. (A) Safranin-O; (B) aggrecan-G1 globular domain, green; (C) aggrecan-G3 globular domain, green; (D) versican hyaluronan binding domain, green; (E) type II collagen, green; (F) non-immune control. DNA is blue in all images. Scale bar=100μm. Osteoarthritis and Cartilage 2008 16, 1092-1100DOI: (10.1016/j.joca.2008.01.004) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Immunofluorescence and western blot analysis of the pericellular matrix. (A) Co-staining for collagen VI, red, and decorin, green; (B) co-staining for collagen VI, red, and biglycan, green; (C) fibronectin, green. DNA is blue in all images. Western blots of construct extracts for (D) collagen VI, (E) decorin, (F) biglycan, and (G) fibronectin. Scale bar=100μm. Osteoarthritis and Cartilage 2008 16, 1092-1100DOI: (10.1016/j.joca.2008.01.004) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Influence of aggrecanase and MMP inhibitors on ECM production. (A) WST-1 analysis of mitochondrial activity following 32 days of treatment with the aggrecanase (Agg Inh) or MMP(MMP Inh) inhibitors N=4/group. (B) Cumulative sGAG released to the media, N=6/group, *P<0.05 with BMSC only group. (C) Total sGAG accumulation within the construct at day 32, N=6/group, *P<0.05 with BMSC only group. (D) Immunofluroescence images of collagen II accumulation at day 32, green, and DNA, blue. Scale bar=100μm. Osteoarthritis and Cartilage 2008 16, 1092-1100DOI: (10.1016/j.joca.2008.01.004) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Western blot analysis of aggrecan processing. (A) Detection of aggrecan-G1 domain in BMSC construct extracts and day 32 media samples from control, aggrecanase (Agg Inh) or MMP (MMP Inh) inhibitors treatments. (B) Detection of the m-calpain generated neoepitope (DLS) in construct extracts. (C) Detection of aggrecan-G1 in native articular cartilage. Osteoarthritis and Cartilage 2008 16, 1092-1100DOI: (10.1016/j.joca.2008.01.004) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Construct mechanics. The dynamic compressive moduli of day 0 and day 32 constructs measured at 0.1Hz and 1.0Hz. *P<0.05 with BMSC only group, N=6/group. Osteoarthritis and Cartilage 2008 16, 1092-1100DOI: (10.1016/j.joca.2008.01.004) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions