R. Mhanna, E. Öztürk, P. Schlink, M. Zenobi-Wong

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Probing the microenvironmental conditions for induction of superficial zone protein expression R. Mhanna, E. Öztürk, P. Schlink, M. Zenobi-Wong Osteoarthritis and Cartilage Volume 21, Issue 12, Pages 1924-1932 (December 2013) DOI: 10.1016/j.joca.2013.08.017 Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Chondrocytes were harvested from cartilage tissue and serially passaged by culturing on tissue culture plastic for 4 days from passage 0 to passage 4. Analysis of Col2 mRNA levels (A) and SZP (B) showed a decrease in expression with passaging. Gene expression was normalized to expression in cartilage tissue (n = 3). Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10.1016/j.joca.2013.08.017) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Passage 3 chondrocytes were cultured on tissue culture plastic (TCP) for 4 days or encapsulated in alginate beads (Beads) for the same period to determine if expression of SZP and Col2 could be restored. qRT-PCR mRNA levels for (A) Col2 and (B) SZP on TCP and within alginate beads normalized to TCP gene expression (n = 5) and (C) immunostaining of SZP in cells cultured on TCP and in alginate beads, scale bar 50 μm. Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10.1016/j.joca.2013.08.017) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 The effect of oxygen tension on SZP and Col2 mRNA levels was quantified by qRT-PCR. (A) mRNA levels of SZP and (B) Col2 for cells seeded on TCP or encapsulated in alginate beads in normoxic or hypoxic conditions normalized to expression in TCP normoxic conditions (n = 3). (C) Immunostaining of SZP and Col2 in cartilage tissue and alginate beads under normoxic and hypoxic conditions. The curved dashed line marks the edge of the alginate bead and the dashed box is shown at a higher magnification in the insert (scale bar 50 μm for cartilage samples and 200 μm for alginate samples). (D) Schematic representing cartilage tissue and its different layers as well as the gradients of oxygen and tensile strain and the corresponding SZP and Col2 synthesis by cells of the different layers. Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10.1016/j.joca.2013.08.017) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Design for application of 3D cyclic strain. (A) STREX device. (B) New chamber designed for 3D loading. Metal tubes were inserted in the holes connecting the chamber to the machine to minimize non-uniform deformations around the holes. (C) Strain measured within a hydrogel in response to 10% compressive/tensile strain set by the instrument. (D) Glass beads embedded in an alginate gel (scale bar 200 μm). (E) Schematic representation of the chamber: The gel was injected in the blue area, letters (a, b and c) mark areas where images of glass beads were taken. Three images were taken 1 mm away from the small round pillar marked with an arrow (a), three images at the same level closer to the wall (b) and three images were taken in the center middle (c) to cover the whole gel in one plane. To assess variation in the z-axis, images were taken at two different depths resulting in a total of 18 images per gel. In each image four measurements of displacement were performed. (F) The homogeneity was assessed by measuring the displacement before/after compression/tension of 10–30 μm glass beads. Displacement was found to have no significant difference between the various locations (n = 3, P > 0.05). Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10.1016/j.joca.2013.08.017) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 The effect of mechanical strain on SZP and Col2 mRNA levels was measured by qRT-PCR. (A) SZP mRNA levels and (C) Col2 mRNA levels for cells seeded on collagen coated PDMS in response to 2 h strain applied 4 days post-seeding or applied every day for 2 h throughout the culture period (total of four times). (B) SZP mRNA levels and (D) Col2 mRNA levels for cells in response to the same strain regime when encapsulated in alginate gels (n = 4). Non-strained samples were used as reference sample and RPL13 was used as a housekeeping gene. Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10.1016/j.joca.2013.08.017) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10. 1016/j. joca Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10. 1016/j. joca Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10. 1016/j. joca Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10. 1016/j. joca Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2013 21, 1924-1932DOI: (10. 1016/j. joca Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions