Tissue engineering of stratified articular cartilage from chondrocyte subpopulations T.J. Klein, M.S., B.L. Schumacher, B.S., T.A. Schmidt, M.S., K.W.

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Tissue engineering of stratified articular cartilage from chondrocyte subpopulations T.J. Klein, M.S., B.L. Schumacher, B.S., T.A. Schmidt, M.S., K.W. Li, Ph.D., M.S. Voegtline, Ph.D., K. Masuda, M.D., E.J.-M.A. Thonar, Ph.D., R.L. Sah, M.D., Sc.D. Osteoarthritis and Cartilage Volume 11, Issue 8, Pages 595-602 (August 2003) DOI: 10.1016/S1063-4584(03)00090-6

Fig. 1 Schematic of methods used to engineer cartilage tissue using cells isolated separately from superficial and middle zones of articular cartilage. Homogeneous constructs were generated from chondrocytes of the superficial (S) or middle (M) zones. Stratified constructs (S/M) were formed by sequentially seeding different chondrocyte subpopulations, with time allowed between seedings for the initial layer to coalesce. Superficial (white) and middle (gray) zone chondrocytes and the tissues derived from them are shaded differently to emphasize their location in culture and in the engineered tissue. Osteoarthritis and Cartilage 2003 11, 595-602DOI: (10.1016/S1063-4584(03)00090-6)

Fig. 2 Structural, compositional, and functional properties of three types of cartilage constructs formed from superficial and/or middle zone chondrocytes after 1 and 2 weeks in culture. Wet weight (WW), DNA, GAG, and collagen (Col) were all normalized to the cross-sectional area of the construct. (F) The equilibrium modulus, HA0, was measured in confined compression. Data are expressed as mean±s.e.m. (P<0.001 (↔), P<0.01 (•), P<0.05 (♦)). Osteoarthritis and Cartilage 2003 11, 595-602DOI: (10.1016/S1063-4584(03)00090-6)

Fig. 3 Localization of matrix and secreted molecules in S, S/M, and M cartilage constructs cultured for 2 weeks. Some tissue sections (A, E, H) were stained with Alcian Blue to detect glycosaminoglycan. Other sections were immunostained for (B, F, I) type II collagen or (D, G, J) SZP, and counterstained with Methyl Green. Positive immunoreactivity is indicated by a purple color. Counterstaining results in a blue–green color. For clarity, higher power images (D1, G1, J1) of the top and (D2, G2, J2) of the middle of each construct are shown. To show specificity of immunostaining, high power images (C1, C2) of a section treated with non-specific control IgG are shown. Bar, 100μm. Osteoarthritis and Cartilage 2003 11, 595-602DOI: (10.1016/S1063-4584(03)00090-6)

Fig. 4 Depth-dependent SZP expression in each construct type after 2 weeks of culture. (A) Cells were counted in 0.2mm bins, with cells between 0.4mm from the top surface and 0.2mm from the bottom surface combined (…) to allow for variations in construct thickness. (B) SZP measured in spent medium from days 12 to 14 of construct culture by indirect ELISA, normalized to construct area and culture duration (P<0.001 (↔)). Osteoarthritis and Cartilage 2003 11, 595-602DOI: (10.1016/S1063-4584(03)00090-6)