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. 2001 Aug;60(8):781–790. doi: 10.1136/ard.60.8.781

Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks

K Almqvist 1, L Wang 1, J Wang 1, D Baeten 1, M Cornelissen 1, R Verdonk 1, E Veys 1, G Verbruggen 1
PMCID: PMC1753804  PMID: 11454643

Abstract

OBJECTIVE—To produce tissue engineered cartilage by human articular chondrocytes in vitro for further use in in vivo manipulations for the treatment of cartilage defects.
METHODS—Human articular chondrocytes were cultured in 0.5%, 1.0%, and 2.0% of alginate for up to four weeks. The optimal concentration of an alginate matrix for cell replication and for aggrecan synthesis by chondrocytes was determined. DNA content in the different culture conditions was measured after two and four weeks. Aggrecan synthesis rates and accumulation in the surrounding extracellular matrix were assessed by [35S]sulphate incorporation after the same periods of culture. To follow the outgrowth of chondrocytes from the alginate beads, chondrocytes were cultured for four weeks in 0.5 or 1.0% alginate surrounded by 0.25 or 0.5% fibrin gel. DNA content of each culture was measured after different culture periods. Finally, human chondrocytes in 1.0% alginate beads were embedded in 0.5% fibrin gel for eight weeks. Immunohistochemical analysis for aggrecan, type I and II collagen was performed weekly.
RESULTS—At two weeks the DNA content in each culture significantly increased in 0.5 and 1.0% alginate cultures in comparison with baseline values. This increase continued until week 4 at the three alginate concentrations. Aggrecan synthesis at two weeks was highest in 0.5 and 1.0% alginate cell cultures. At four weeks aggrecan synthesis rates decreased independently of the alginate concentrations. Aggrecan mainly accumulated in the interterritorial matrix. Proliferation of chondrocytes in alginate and outgrowth of these cells in the surrounding fibrin gel were evident throughout the culture period. The accumulation of aggrecan and type II collagen around the cells, in alginate as well as in fibrin gel, gradually increased over the culture period. Type I collagen appeared after six weeks in alginate and in the surrounding fibrin.
CONCLUSION—Human chondrocytes proliferate in this culture system, show an outgrowth into the surrounding fibrin, and synthesise a cartilage-like matrix for up to eight weeks.



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Figure 1  .

Figure 1  

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DNA content in each culture from donor 1 (A), 2 (B), and 3 (C) was investigated at day 1, and at two and four weeks. Data are presented as mean values of three cultures; standard deviations and p values are given.

Figure 2  .

Figure 2  

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Total aggrecan synthesis rates per culture from donor 1 (A), 2 (B) and 3 (C) at two and four weeks in three different concentrations of alginate are given, with the relative amounts of the newly synthesised aggrecans in cell associated matrix (CAM), interterritorial matrix (ITM), and medium related to the total aggrecan synthesis (percentage value in column). Data are presented as mean values of three cultures. p values (0.5% v 1.0%, 1.0% v 2.0%, and 0.5% v 2.0%) at two and four weeks are given. The p values for aggrecan synthesis rates between two and four weeks are 0.27, 0.02, and 0.0003 for donor 1, 0.0004, 0.0006, and 0.006 for donor 2, and 0.31, 0.10, and 0.04 for donor 3 in 0.5%, 1.0%, and 2.0% alginate cultures, respectively.

Figure 3  .

Figure 3  

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Total aggrecan synthesis rates for each cell from donor 1 (A), 2 (B), and 3 (C) at two and four weeks in three different concentrations of alginate are given. The relative amounts of the newly synthesised aggrecans in cell associated matrix (CAM), interterritorial matrix (ITM), and medium related to the total aggrecan synthesis rates for each cell are identical to those in fig 2. Data are presented as mean values of three cultures. p Values (0.5% v 1.0%, 1.0% v 2.0%, and 0.5% v 2.0%) at two and four weeks are given. The p values for aggrecan synthesis rates between two and four weeks are <0.05 in all alginate concentrations for the three donors.

Figure 4  .

Figure 4  

Figure 4  

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Immunohistochemistry of chondrocytes cultured in alginate surrounded by fibrin gel. Staining of aggrecan, type II collagen, and type I collagen in alginate and fibrin at three days (A), two weeks (B), four weeks (C), and eight weeks (D). Magnification ×100.    

Figure 5  .

Figure 5  

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Paraffin section of chondrocytes cultured in alginate beads for two weeks and stained for aggrecan. A well defined cell associated matrix with newly synthesised aggrecans can be seen, as well as a slighter staining of the intercellular matrix. Magnification ×100.

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