Engineering of Rat Articular Cartilage on Porous Sponges: Effects of TGF-β 1 and Microgravity Bioreactor Culture

Figures & data

TABLE 1 The experimental groups

	Control	Group 1	Group 2
Culture Type	2D-Static	3D-Static	3D-Bioreactor
Seeding Technique	Static	Static	Dynamic
TGF β1 (5 ng/ml)	− / +	− / +	− / +

Abbreviations: 2D: two-dimensional culture; 3D: three-dimensional sponge culture.

Figure 1 Schematic presentation of the experimental procedures.

TABLE 2 Effect of seeding technique on cell loading capacity of PLGA sponge

Cell Number	Static Seeding	Dynamic Seeding
Introduced^[1]	50.000 cells/sponge	50.000 cells/sponge
Unattached^[2]	2.11 ± 0.82 × 10^4 cells	0.82 ± 0.47 × 10^4 cells
% Seeded^[3]	%57.9 *	%83.7*

^[1]Initially seeded chondrocytes with > 90% viability (by trypan blue dye exclusion test).

^[2]Unattached chondrocytes counted at Day 3 by trypan blue test (from the well where sponge was initially placed, and from the exchanged medium).

^[3]Calculated from the formula; [(Introduced-Unattached)/ Introduced] × 100; Mean ± SD; N = 3; statistical significance: P < 0.05 (*,*).

Figure 2 Representative SEM images of the macroporous PLGA sponges seeded with rat articular chondrocytes retrieved from (A) static, (B) bioreactor cultures after 28 days with TGF-β 1-induction. Infiltration of chondrocytes into sponge pores (C) and outer surface (D) can be visualized in high magnification images of B. Note the decreasing sponge pore size in bioreactor culture (B) compared to the static culture (A). Scale bars, A and B: 100 μ m; C and D: 10 μ m.

Figure 3 Change in the number of chondrocytes cultured on 35 mm-tissue culture dishes (2D-static), and inside sponges (3D-static and 3D-dynamic) under (A) standard, (B) TGF-β 1-induced culture conditions. Cell numbers at Day 0 were: ∼ 25.000 cells/dish and ∼ 50.000 cells/sponge. Absorbance values are presented as normalized means± SD from three independent experiments. (Continued)

Figure 4 Phase-contrast micrographs demonstrating the dark blue formazan crystals formed from MTT inside sponge pores after 14 days of dynamic culture: in (A) standard, (B) TGF-β 1-containing medium; scale bars: 200 μ m. Note the round-shaped chondrocytes (arrows) at the vicinity of the formazan crystals.

Figure 5 Light microscopy images demonstrating proteoglycan stainings. Alcian blue (A, B) and Safranin-O (C, D) stainings of rat articular chondrocytes on PLGA sponge under static (A and C) and bioreactor (B and D) culture conditions with TGF-β 1 at Day 28; scale bars: 200 μ m. Note the strong stainings of the samples retrieved from bioreactor cultures confirming the chondrogenic state.

Figure 6 Light microscopy images demonstrating immunohistochemical stainings. Anti-rat Collagen type II (A, B) and Anti-rat Aggrecan (C, D) stainings of rat articular chondrocytes on PLGA sponge under static (A and C) and bioreactor (B and D) culture conditions with TGF-β 1 at Day 28; scale bars: 200 μ m.