3D dynamic culture of rabbit articular chondrocytes encapsulated in alginate gel beads using spinner flasks for cartilage tissue regeneration

Abstract

Cell-based therapy using chondrocytes for cartilage repair suffers from chondrocyte dedifferentiation. In the present study, the effects of an integrated three-dimensional and dynamic culture on rabbit articular chondrocytes were investigated. Cells (passages 1 and 4) were encapsulated in alginate gel beads and cultured in spinner flasks in chondrogenic and chondrocyte growth media. Subcutaneous implantation of the cell-laden beads was performed to evaluate the ectopic chondrogenesis. It was found that cells remained viable after 35 days in the three-dimensional dynamic culture. Passage 1 cells demonstrated a proliferative growth in both media. Passage 4 cells showed a gradual reduction in DNA content in growth medium, which was attenuated in chondrogenic medium. Deposition of glycosaminoglycans (GAG) was found in all cultures. While passage 1 cells generally produced higher amounts of GAG than passage 4 cells, GAG/DNA became similar on day 35 for both cells in growth media. Interestingly, GAG/DNA in growth medium was greater than that in chondrogenic medium for both cells. Based on GAG quantification and gene expression analysis, encapsulated passage 1 cells cultured in growth medium displayed the best ectopic chondrogenesis. Taken together, the three-dimensional and dynamic culture for chondrocytes holds great potential in cartilage regeneration.

Figure 1

The setup of 3D dynamic culture. Morphology of P1 (a) and P4 (b) rACs cultured in 2D was observed under phase contrast microscope. (c) Gross view of cell-laden alginate beads; (d) SEM image of the interior microstructure of alginate beads laden with P1 rACs in chondrogenic medium for 28 d; (e) schematic illustration of suspension culture of cell-laden alginate beads in a spinner flask; (f) gross view of a spinner flask; (g) cell-laden alginate beads in a spinner flask.

Figure 2

Live/dead staining of rACs in alginate beads. (a) P1 rACs on day 0; (b) P1 rACs in chondrogenic medium for 35 d (Ind-P1rAC); (c) P1 rACs in growth medium for 35 d (Con-P1rAC); (d) P4 rACs on day 0; (e) P4 rACs in chondrogenic medium for 35 d (Ind-P4rAC); (f) P4 rACs in growth medium for 35 d (Con-P4rAC). Fluorescent images were taken at 4x, 10x, and 20x magnifications. Green indicates live cells and red indicates dead cells.

Figure 3

Growth and GAG production of rACs in alginate beads. (a) MTT assay; (b) DNA content; (c) GAG content; (d) GAG/DNA. Captions were defined as in Figure 2. Asterisk indicates P < 0.05.

Figure 4

Histological analysis of rACs in alginate beads. (a) P1 rACs on day 0; (b) Ind-P1rAC on day 35; (c) Con-P1rAC on day 35; (d) P4 rACs on day 0; (e) Ind-P4rAC on day 35; (f) Con-P4rAC on day 35. Captions were defined as in Figure 2.

Figure 5

Gene expression of rACs in alginate beads. (a) Collagen type I; (b) collagen type II; and (c) collagen type X. Captions were defined as in Figure 2. Asterisk indicates P < 0.05.

Figure 6

Cell growth and GAG production in implants. (a) MTT assay; (b) DNA content; (c) GAG/bead; (d) GAG/DNA. Asterisk indicates P < 0.05.

Figure 7

Gene expression of collagen type I (a), collagen type II (b), and collagen type X (c) of implants. Asterisk indicates P < 0.05.