Growth characterization of neo porcine cartilage pellets and their use in an interactive culture model  Carsten Lübke, Ph.D., Jochen Ringe, M.Sc., Veit Krenn, M.D., Gabriele Fernahl, Stine Pelz, M.Sc., Rüdiger Kreusch-Brinker, M.D., Michael Sittinger, Ph.D., Manrico Paulitschke, M.D.  Osteoarthritis and Cartilage  Volume 13, Issue 6, Pages 478-487 (June 2005) DOI: 10.1016/j.joca.2004.01.009 Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 (A) Illustration of morphometric pellet analysis. Pellet thickness was measured at five distinct pellet locations (shown by lines). Cell density and cell distribution in Alcian blue stained pellets were calculated by counting cells in 10 squares of a defined area (0.0025mm2). (B) Low power micrograph of 2-week-old pellet cultures. Scale indicates centimeter. Osteoarthritis and Cartilage 2005 13, 478-487DOI: (10.1016/j.joca.2004.01.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Thickness of pellets from 11 chondrocyte cultures. Pellets show a logarithmic growth over time. The thickness of each pellet was measured at five different locations of the pellet. Values are the mean±SD. Differences in pellet thickness are significant by t test (*P<0.05; **P<0.005; ***P<0.0005) compared to day 3. Osteoarthritis and Cartilage 2005 13, 478-487DOI: (10.1016/j.joca.2004.01.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 (A) Representative development in pellet thickness of one cell batch (n=1). Pellets show a logarithmic growth over time. Thickness of each pellet was measured at five different points on the pellet. (B) Cell density (♦) is defined as cells per square (10 squares per pellet) and relative cell density (▪) as the product of pellet thickness by cell number per square. Cell density declines but the relative cell density remains nearly unchanged. (C) Representative example of Ki67 expression in a pellet on day 17. Arrows represent Ki67 positive cells. No cluster formation could be observed. Scale bar=50μm. Values are the mean±SD. Differences in pellet thickness are significant by t test (*P<0.0005) compared to day 5. Differences in pellet cell density are significant by t test (*P<0.05; **P<0.005) compared to day 8. Osteoarthritis and Cartilage 2005 13, 478-487DOI: (10.1016/j.joca.2004.01.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Pictures of type II and type I collagen antibody labeling in chondrocyte pellets on days 8 and 19, respectively. Positive immunostaining of type II collagen is indicated by a red/brown color. Type I collagen labeled pellets did not show any immunoreactivity. Scale bar=50μm. Osteoarthritis and Cartilage 2005 13, 478-487DOI: (10.1016/j.joca.2004.01.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Histological and immunohistochemical analysis of co-cultures. (A–C) Alcian blue staining of cartilage proteoglycans and nuclear fast red counterstaining of pellet cryosections of (A) a 4-week-old chondrocyte pellet, (B) a 4-week-old chondrocyte pellet co-cultured for 2 weeks with IA RA synovial fibroblasts (HSE) and (C) a pellet co-cultured with normal, NI, synovial fibroblasts (K4IM). (D–F) Type II collagen and HLA-ABC antibody labeling in cryosections of (D, E) a 4-week-old chondrocyte pellet co-cultured for 2 weeks with K4IM cells, and (F) a pellet co-cultured with HSE cells. Positive immunostaining of type II collagen is indicated by a red/brown color. HLA-ABC positive cells are red dotted. Arrowheads indicate synovial cells. Scale bar=50μm. Osteoarthritis and Cartilage 2005 13, 478-487DOI: (10.1016/j.joca.2004.01.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 HPLC and semi-quantitative real-time PCR gene expression analysis of chondrogenic marker genes expressed by porcine pellet cultures. Cartilage matrix formation was assessed by protein expression of collagen fiber inherent hydroxyproline and by mRNA expression of typical ECM marker genes type I collagen, type II collagen and aggrecan. Hydroxyproline expression was calculated using 3,4-dehydroxyproline as an internal standard, whereas the expression of marker genes was calculated as the percentage of the expression of the housekeeping gene GAPDH. Osteoarthritis and Cartilage 2005 13, 478-487DOI: (10.1016/j.joca.2004.01.009) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions