Quantification of Changes in Morphology, Mechanotransduction, and Gene Expression in Bovine Articular Chondrocytes in Response to 2-Dimensional Culture Indicates the Existence of a Novel Phenotype

Abstract

Objective: Matrix-induced autologous chondrocyte implantation (ACI) offers a potential solution for cartilage repair but is currently hindered by loss of the chondrocyte differentiated phenotype. To further our understanding of the mechanism of dedifferentiation, changes in the phenotype in relation to mechanotransduction were recorded in response to monolayer culture.

Methods: Bovine cartilage explants were excised and chondrocytes cultured for 9 days (P1), 14 days (P2), and 21 (P3) days. Changes in morphology and regulatory volume increase (RVI; a mechanotransduction response) were determined by the expression of key genes by RT-PCR and confocal microscopy, respectively.

Results: A loss of a differentiated phenotype was observed in P1 with a reduction in sphericity and an overall increase in cell volume from 474.7 ± 32.1 µm(3) to 725.2 ± 35.6 µm(3). Furthermore, the effect of 2-dimensional (2-D) culture-induced dedifferentiation on mechanotransduction was investigated, whereby RVI and Gd(3+)-sensitive REV5901-induced calcium rise were only observed in 2-D cultured chondrocytes. A significant up-regulation of types I and II collagens and Sox9 was observed in P1 chondrocytes and no further significant change in type I collagen but a return to baseline levels of type II collagen and Sox9 upon further culture.

Conclusion: These data indicated the presence of an intermediate, mesodifferentiated phenotype and highlight the importance of mechanotransduction as a marker of the chondrocytic cell type.

Keywords: REV5901; calcium; collagen; dedifferentiation; phenotype; regulatory volume increase.

Figure 1.

Growth curve of bovine articular chondrocyte upon in vitro culture. Chondrocytes were subcultured upon 80% confluence, counted, and reseeded at the same density. A growth curve was plotted of log total cell number versus time and a linear regression used to calculate growth rate and doubling time. There was a steady increase in cell number only when cultured at LD. Data were pooled from 23 individual experiments (N = 12). Data shown as mean ± SEM. R² values for regression analysis are shown.

Figure 2.

Cellular dimensions and sphericity of chondrocytes as quantified using the Imaris Isosurface Surpass utility (Bitplane). A series of chondrocyte images were acquired using confocal microscopy were reassembled in Imaris (Bitplane), and isosurface “objects” were created in Imaris (Bitplane) where the depth (a), width (b), and length (c) of each object were determined. There was a significant 2-fold increase and decrease in chondrocyte length and depth, respectively. Moreover, there was a significant decrease in sphericity upon 9 days of culture, indicating a switch in morphology from as early as the first passage. Data pooled from 16 individual experiments (N = 4, n = 119). Data shown as mean ± SEM. Student t test values are shown and deemed nonsignificant (N/S) when P ≥ 0.05.

Figure 3.

The expression profiling of chondrocytes before and throughout culture. Changes in the expression of key genes compared to amounts in freshly isolated chondrocytes (P0) were recorded upon confluence after 9 days in culture (P1), 14 days in culture (P2), and 21 days in culture (P3). The level of expression of Sox9 declined upon culture despite the transient increase in P1. Both the expression levels of types II and I collagen increased during 2-D expansion, but the ratio of type II collagen to type I collagen expression decreased significantly during the first few days of culture. Transient increase in Sox9 and type II collagen during P1 suggested that the loss of phenotype might be a multistep process. There was however no change in NKCC1 expression for the duration of the culture period. Experiment repeated 6 times (N = 6). Data shown as mean ± SEM. Student t test values are shown and deemed nonsignificant (N/S) when P ≥ 0.05.

Figure 4.

The capacity of freshly isolated and 2-D expanded chondrocytes to exhibit RVI in response to hyperosmotic conditions. Freshly isolated (A), P1 (B), P2 (C), and P3 (D) chondrocytes were loaded with 5 µM calcein-AM and imaged using CLSM before and up to 20 minutes following a 42% hyperosmotic challenge. Freshly isolated chondrocytes did not exhibit RVI, whereas P1, P2, and P3 2-D cultured chondrocytes exhibited volume regulation (N = 4, n = 119). Data shown as mean ± SEM. Student t test values are shown and deemed nonsignificant (N/S) when P ≥ 0.05. R² values for regression analysis are shown.

Figure 5.

The effect of REV5901 loading on freshly isolated and 2-D cultured chondrocytes. (A) Freshly isolated and 2-D cultured chondrocytes were incubated with 3 µM Fluo-4 AM (Invitrogen) in BPS for 30 minutes prior to loading of 50 µM REV5901, whereby there was a sustained rise in [Ca²⁺]_i with freshly isolated chondrocytes exhibiting higher sensitivity to REV5901 than 2-D expanded chondrocytes. In the presence of 2 mM EGTA, there was a drop in maximum [Ca²⁺]_i rise from 37.97% ± 5.67% to 27.86% ± 3.17% and from 14.20% ± 1.70% to 2.01% ± 0.72% in freshly isolated and 2-D cultured chondrocytes, respectively. These data indicated a reduction in store contribution to REV5901-mediated [Ca²⁺]_i rise upon 2-D culture. (B) The dependency of freshly isolated and 2-D cultured chondrocytes on individual calcium channels was investigated by loading chondrocytes with 3 µM Fluo-4 AM (Invitrogen) in appropriate inhibitory conditions prior to loading 50 µM REV5901 and measurement of [Ca²⁺]_i changes. The inhibition factor in the presence of ruthenium red did not change upon culture, whereas inhibitions of sodium-sensitive calcium channels and stretch-sensitive calcium channels were increased and reduced, respectively. These data indicated a change in channel activity in response to 2-D culture. Experiment repeated 4 times (N = 6). Data shown as mean ± SEM. Student t test values are shown and deemed nonsignificant (N/S) when P ≥ 0.05.