Biochemical and functional modulation of the cartilage collagen network by IGF1, TGFβ2 and FGF2 Y.M. Jenniskens, M.Sc., W. Koevoet, B.Sc., A.C.W. de.

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Biochemical and functional modulation of the cartilage collagen network by IGF1, TGFβ2 and FGF2 Y.M. Jenniskens, M.Sc., W. Koevoet, B.Sc., A.C.W. de Bart, B.Sc., H. Weinans, Ph.D., H. Jahr, Ph.D., J.A.N. Verhaar, M.D., Ph.D., J. DeGroot, Ph.D., G.J.V.M. van Osch, Ph.D. Osteoarthritis and Cartilage Volume 14, Issue 11, Pages 1136-1146 (November 2006) DOI: 10.1016/j.joca.2006.04.002 Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Effect of growth factors on proteoglycan deposition in three-dimensional chondrocyte cultures. Proteoglycan deposition is shown after (A) 11 days of culture (n=6) and (B) 20 days of culture (n=12). *Indicates P<0.01 and # indicates P<0.05 compared to the control condition without growth factors, which is set at 100% (SD=8.2% at day 11 and SD=6.8% at day 20). Data are presented as mean %±SD. Osteoarthritis and Cartilage 2006 14, 1136-1146DOI: (10.1016/j.joca.2006.04.002) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Effect of growth factors on collagen deposition in three-dimensional chondrocyte cultures. Collagen deposition is shown after (A) 11 days of culture (n=6) and (B) 20 days of culture (n=12). *Indicates P<0.01 and # indicates P<0.05 compared to the control condition without growth factors, which is set at 100% (SD=2.5% at day 11 and SD=16.1% at day 20). Data are presented as mean %±SD. Osteoarthritis and Cartilage 2006 14, 1136-1146DOI: (10.1016/j.joca.2006.04.002) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 Effect of growth factors on collagen cross-linking. HP cross-links in newly formed collagen are shown after 20 days of culture (n=12). *Indicates P<0.01. Data are presented as mean±SD. Osteoarthritis and Cartilage 2006 14, 1136-1146DOI: (10.1016/j.joca.2006.04.002) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Immunohistochemical analysis of produced matrix. Collagen type II deposition in (A) cells cultured in the presence of IGF1; (B) TGFβ2 ; (C) FGF2 ; (D) cells cultured without growth factors; (E) collagen type I is mainly only present in large cell clusters, not in matrix of single cells; (F) is the isotype control. Magnification is 100×, with a 400× inset. Positive cells are indicated arrows, arrowheads indicate negative cells. Osteoarthritis and Cartilage 2006 14, 1136-1146DOI: (10.1016/j.joca.2006.04.002) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Gene expression analysis after 11 and 20 days of culture as n-fold change compared to the untreated control. (A) collagen type II, (B) aggrecan, (C) LOX, and (D) TLH expression. *Indicates P<0.01 compared to control. Data are presented as boxplots with median, 25th and 75th quartile for the gene expression with the control condition without growth factors set at 1. Osteoarthritis and Cartilage 2006 14, 1136-1146DOI: (10.1016/j.joca.2006.04.002) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 Overall MMP activity and MMP-1 expression. (A) Overall MMP activity was measured in the culture medium after 20 days of culture together with (B) the gene expression of the specific collagenase MMP-1 after 11 and 20 days of culture. *Indicates P<0.01 and # indicates P<0.05 compared to the control condition. Data are presented as mean±SD for the overall MMP activity and as boxplots with median, 25th and 75th quartile for the gene expression with the control condition without growth factors set at 1. Osteoarthritis and Cartilage 2006 14, 1136-1146DOI: (10.1016/j.joca.2006.04.002) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 7 Relation between mechanical properties and matrix constituents after 35 days. Biochemical and biomechanical properties of tested alginate disks after 35 days of culture as percentage compared to the control with (A) the equilibrium modulus of the newly formed matrix (control is 100±26%), (B) the secant modulus of the newly formed matrix (control is 100±23%), (C) proteoglycan deposition (control is 100±11%), (D) collagen deposition (control is 100±12%) and (E) HP cross-links per collagen molecule (control is 100±10%). *Indicates P<0.01 and # indicates P<0.05 compared to control. Data are presented as mean±SD. Osteoarthritis and Cartilage 2006 14, 1136-1146DOI: (10.1016/j.joca.2006.04.002) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions