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. 2012 Aug;60(8):576-87.
doi: 10.1369/0022155412449018. Epub 2012 May 19.

Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes

Drew W Taylor  1 , Nazish Ahmed, Anthony J Hayes, Peter Ferguson, Allan E Gross, Bruce Caterson, Rita A Kandel
Affiliations

Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes

Drew W Taylor et al. J Histochem Cytochem. 2012 Aug.

Abstract

To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.

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Figures

Figure 1.
Figure 1.
Glycosaminoglycan (GAG), collagen, and DNA contents of tissues formed by primary bovine P0 and human passaged P2 grown alone or in co-culture. GAG and collagen contents expressed relative to DNA in tissues formed up to 4 weeks of culture (n=9, three individual patients). The results from all experiments were pooled and expressed as mean ± SEM. Significance was assigned at p<0.05. (A) †bP0 at 2 weeks was significantly different from hP2 and hP2bP0 samples from the same time point (week 2); ‡bP0 at 4 weeks was significantly different from bP0 from the first week and hP2 from the same time point (week 4); and £hP2bP0 was significantly different from hP2bP0 from weeks 1 and 2 as well as hP2 from the same time point. (B) *bP0 at week 2 was significantly different from hP2 and hP2bP0 from the same time point (week 2); **bP0 at week 3 was significantly different from hP2 from the same time point (week 3); ***bP0 at week 3 was significantly different from hP2 from the same time point (week 4) and bP0 from weeks 1 and 2; ¥hP2bP0 at week 3 was significantly different from hP2bP0 at week 1; and ¥hP2bP0 at week 4 was significantly different from hP2 at same time point (week 4) and hP2bP0 from weeks 1 and 2. (C) CbP0 at 3 weeks was significantly different from hP2 samples from the same time point and bP0 samples from 1 and 2 weeks; €bP0 at 4 weeks was significantly different from hP2 samples at the same time point and bP0 samples from weeks 1 and 2; and €hP2bP0 at 4 weeks was significantly different from hP2 samples at the same time point and hP2bP0 samples from weeks 1 and 2.
Figure 2.
Figure 2.
Proteoglycan analysis of the in vitro formed tissues. Photomicrographs of histological sections of the different tissues over time and stained with either Alcian blue or antibodies reactive with specific proteoglycans (aggrecan, biglycan, and decorin). Insets: Photomicrographs of histological sections taken after 4 weeks of culture of the different tissues and stained with antibodies reactive to either biglycan or decorin (width of inset is equal to 30 µm). Every 10 units on the scale bar represents 100 μm.
Figure 3.
Figure 3.
Sulfated keratan and chondroitin distribution in the in vitro formed tissues. Photomicrographs of histological sections of the different tissues over time and stained with antibodies reactive with specific chondroitins (chondroitin-0-sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate) and keratan sulfate. Every 10 units on the scale bar represents 100 μm.
Figure 4.
Figure 4.
Type I and II collagens in the in vitro formed tissue. Photomicrographs of histological sections of the tissues formed from bP0, hP2, and hP2bP0 cultures over time and stained with antibodies reactive with type I or II collagen. Inserts: (–), type I collagen staining on bovine native cartilage as a negative control; (+), type I collagen staining on bovine native tendon as a positive control. Every 10 units on the scale bar represents 100 μm.
Figure 5.
Figure 5.
Transmission electron microscopy (TEM) of tissue formed by hP2bP0 co-culture. Transmission electron micrographs at 1 week (A), 2 weeks (B), 3 weeks (C), and 4 weeks (D) of culture. Four-week tissue showed a lack of direct cell-cell contact despite close proximity of two adjacent cells (E) and the presence of collagen fibers (F). Bars A-C = 1 µm; Bar D = 2 µm; Bar E = 0.5 µm; Bar F = 0.2 µm.
Figure 6.
Figure 6.
Gene expression over time. Samples were harvested at various time points from 24 hr (h) to 4 weeks (wk). Data are from one representative sample, repeated in triplicate, and are expressed as mean ± SEM on a logarithmic scale relative to human passaged cells grown alone (hP2). This experiment was repeated three times.
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