ARTICLE |
Correspondence to: Susan Chubinskaya, Dept. of Biochemistry, Rush-PresbyterianSt Luke's Medical Center, 1653 W. Congress Parkway, Chicago, IL 60612. E-mail: schubins@rush.edu
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Summary |
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Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of 1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The
1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover. (J Histochem Cytocem 49:12111219, 2001)
Key Words: human articular chondrocytes, alginate beads, in situ hybridization, type II collagen, aggrecan, MMP-3, MMP-8
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Introduction |
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When chondrocytes are released from their native matrix and placed in culture, they dedifferentiate unless the culture conditions are carefully controlled. For example, in monolayer culture if not at high density, the chondrocytes become fibroblastic in appearance and no longer synthesize cartilage-specific matrix components, e.g., Type II collagen and aggrecan (
When bovine and human articular chondrocytes are suspended in alginate beads, these cells maintain their phenotype over long periods of time (
The purpose of this study was to extend the parameters of the alginate bead culture system to include analyses of mRNA expression by in situ hybridization (ISH) in human chondrocytes. A modification of the procedure as previously reported (1 (II) procollagen IIB, aggrecan core protein, and for MMP-3 and -8 (prostromelysin and procollagenase 2) were compared in chondrocytes of newborn and adult human cartilage both in freshly fixed tissue and in separated cells cultured in alginate beads. This study demonstrates that the genes for aggrecan core protein, MMP-3, and MMP-8 are continuously expressed in human chondrocytes cultured in alginate beads for 8 months. The expression of the Type II collagen (COL2A1) gene is detectable for the first 2 months of culture but by 3 months mRNA is no longer detectable, which is consistent with its absence in adult knee articular cartilage (
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Materials and Methods |
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Reagents
Chemicals, either reagent or molecular biology grade, were purchased from Sigma Chemical (St Louis, MO) unless otherwise noted. Pronase was purchased from Calbiochem (La Jolla, CA), collagenase P (Clostridium histolyticum) from Boehringer Mannheim (Indianapolis, IN), and fetal bovine serum (FBS) from Hyclone (Logan, UT). Low-viscosity alginate (Keltone, LV) was a gift from Kelco (Chicago, IL). Dulbecco's modified Eagle's medium/Ham's F12 medium (DMEM/ F12) was obtained in a 1/1 powder from Gibco (Grand Island, NY). All electrophoresis-grade chemicals were from Bio-Rad (Hercules, CA).
Tissue Acquisition
Full-thickness articular cartilage slices were dissected from the load-bearing regions of the tali of talocrural joints and femoral condyles of tibiofemoral joints from 10 adult human donors within 24 hr of death. Epiphyseal cartilage with diaphyseal growth plates was obtained from three newborn human donors less than 1 year old. The adult articular cartilages and epiphyseal cartilages were obtained through the Regional Organ Bank of Illinois. For the adult cartilage, only normal joints with no detectable fibrillations or osteophytes were selected.
Preparation of Chondrocytes and Alginate Beads
Chondrocytes were prepared for culture in alginate beads as previously described (
Histological Processing of Cartilage Tissue
Epiphyseal cartilage from three newborns and full-thickness articular cartilage from eight adult donors (3 x 3 mm2 pieces) were removed from the joints without subchondral bone or calcified cartilage and processed for ISH or immunohistochemistry (IHC) using RNase-free solutions as previously described (
Processing of Alginate Beads for ISH
The beads were processed as previously described (
Oligonucleotide Probes
Probes for human aggrecan core protein [25-mer antisense oligonucleotide probe complementary to bp 736760 (5'-ACAGAGGGACACGTCATATCGCTGT-3')] and collagen Type IIB [23-mer antisense oligonucleotide probe complementary to bp 7496 (5'-GCCAGGATGTCCGGCAACCAGGA-3')] (-thiol-35S]-dCTP using terminal deoxynucleotidyl transferase (Gibco) and hybridized under conditions of highest stringency corrected for each probe.
In Situ Hybridization
Cartilage processing under RNAase-free conditions followed the procedure of
Immunohistochemistry
Cartilage samples from two additional adult donors were obtained and processed for immunohistochemistry and immunoblotting after culture of the chondrocytes in alginate beads under conditions that were identical to those described for the ISH technique. Mouse anti-human Type II collagen antibody (a generous gift from Dr. T. Glant; Rush Medical College, Chicago, IL) was used as the primary antibody for indirect immunostaining. Deparaffinized sections of beads were washed for 5 min in PBS0.2% Tween-20, followed by incubation with chondroitinase ABC (5 U/ml) in 0.1 M sodium acetate, 0.1 M Tris, pH 7.3, for 30 min at 37C. The sections were washed in PBS0.2% Tween-20 before incubation with 80% methanol:20% of a 3% H2O2 for 20 min to block endogenous peroxidase activity. Nonspecific antibody binding sites were blocked by incubation with normal mouse sera [1:2000 dilution in PBS0.2% Tween-20 with 3% bovine serum albumin (BSA)]. The sections were then incubated for 1 hr in primary antibody (anti-human Type II collagen) diluted 1:1500 in PBS0.2% Tween-203% BSA. Control sections did not receive primary antibody; otherwise, they were treated identically. The sections were washed and incubated for 1 hr with horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce Chemicals; Rockford, IL) diluted 1:500 in PBS0.2% Tween-203% BSA. At the end of the incubation, the sections were rinsed in PBS0.2% Tween-20 three times for 5 min each and antibody binding was visualized using one tablet of 3,3'-diaminobenzidine tetrahydrochloride and one tablet of urea hydrogen peroxide. The sections were dehydrated through alcohols and xylene and coverslips were mounted with Acrytol.
Western Blotting
The identity of Type II collagen chains in the conditioned media, cell-associated matrix, and further-removed matrix was performed using immunoblotting. The cultures were stopped at 7 and 14 days. The media were harvested every 2 days and pooled for each 7-day period. The beads were dissolved in 55 mM Na-citrate, 0.15 M NaCl, 30 mM Na2EDTA, pH 6.8, following the procedure of
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Results |
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The histological appearance of human chondrocytes cultured in alginate beads and stained with Safranin O and fast green is shown in Fig 1. In the absence of Ca+2, the alginate beads depolymerize. To maintain the structural integrity of the beads and the entrapped chondrocytes, CaCl2 was included during fixation, followed by inclusion of BaCl2 for washing to permanently crosslink the alginate. After these modified fixation steps, beads were processed for histology and the sections were stained with Safranin O and fast green (Fig 1). Both the newly formed cartilage matrix and the alginate stained with Safranin O, indicating the presence of sulfate groups. However, the alginate stained more intensely than the matrix so that the chondrocytes with newly formed matrix could be distinguished from the alginate (Fig 1: white arrow indicates the newly formed matrix; black arrow indicates the alginate). The intensity of staining in the alginate was not due to deposition of sulfated glycosaminoglycans in the further-removed matrix alone because beads formed in the absence of cells and processed for histological staining with Safranin O were comparatively intensely stained (data not shown). The histological appearance of alginate beads could be also visualized with cresyl violet (Fig 3A, Fig 3C, Fig 3E, Fig 5A, and Fig 5C). This stain is used for ISH to counterstain the matrix of the tissue or the beads. With cresyl violet the alginate was less intensely stained and the newly formed matrix was more intensely stained (Fig 3C: open arrow indicates intensely stained newly formed matrix; closed arrows indicate less intensely stained alginate matrix).
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A second major modification made in the fixation step for ISH was the exclusion of CPC from the fixative. The presence of CPC prevented the identification of mRNA in chondrocytes cultured in alginate beads (data not shown).
The comparison between mRNA expression of aggrecan core protein, 1 (II) procollagen IIB, MMP-3, and MMP-8 in the chondrocytes cultured for 7 days in alginate beads and that in fresh tissue is summarized in Table 1. In the cartilage processed for ISH without culture, mRNA expression for aggrecan, MMP-3, and MMP-8 was detectable in the chondrocytes from both adult and young donors. Type II collagen message was detectable in the chondrocytes of the young donors but not in those of the adult in the intact knee tissue (Fig 2). Messages for all four genes were detectable in both newborn and adult chondrocytes cultured in alginate beads for 7 days (Table 1) and up to 2 months (Fig 3 and Fig 4 for Type II collagen expression). Whereas mRNA expression for aggrecan, MMP-3, and MMP-8 was detectable for an additional 6 months of chondrocyte culture in alginate (as an example, Fig 5 illustrates MMP-8 expression), message for Type II collagen became undetectable by 3 months in culture.
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To determine whether Type II collagen mRNA was translated into protein and deposited in the cell-associated and further-removed matrix compartments, after 7 and 14 days of culture the beads were dissolved and separated into two fractions as described in Materials and Methods. Western blotting (Fig 6) confirmed that after 7 days in culture the chondrocytes were synthesizing Type II collagen. At that time point, Type II collagen was present in the cell-associated matrix and further-removed matrix. No Type II collagen was detectable in the media. By 14 days in culture, Type II collagen was present in the media as well. Because the antibody recognizes both Types I and II collagen, Type I collagen standard was used as a negative control on the Western blots. The appearance of 2 (I) chain specific for collagen Type I would be indicative of phenotypic changes during chondrocyte cultures. As shown on the Western blot (Fig 6,
2 (I) chain marked by arrow), the
2 (I) chain that is present in the Type I collagen standard was absent from the matrix formed by the chondrocytes cultured in alginate beads.
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Immunohistochemical staining of the beads with the same Type II collagen antibody used for Western blotting showed that chondrocytes with their cell-associated matrix were intensely stained at 14 days of culture (Fig 7). The further-removed matrix was stained but not as heavily as the cell-associated matrix.
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Discussion |
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A method described for immunohistochemical evaluation of chondrocytes cultured in alginate gel (
Applying the modifications described above, we were able to detect messenger RNA in chondrocytes cultured in alginate beads through 8 months of culture. The presence of mRNA for MMP-3 and MMP-8 in the chondrocytes of uncultured newborn and adult human cartilage is consistent with earlier studies (
The gene for human aggrecan core protein is continuously expressed in both developing and adult cartilage (
The gene for Type II collagen (COL2A1) is expressed in young, growing cartilage (
Chondrocytes within adult cartilage have formed a network of surrounding collagen fibers in the territorial matrix. In the alginate beads, the chondrocytes, which have been stripped of their native matrix during processing, re-establish the matrix in the cell-associated and further-removed matrix compartments, similar to those previously reported for bovine chondrocytes ( or stimulated by transforming growth factor-ß or osteogenic protein-1. Moreover, our recent work (unpublished data) indicates that not only normal articular chondrocytes but also osteoarthritic chondrocytes can be studied in an alginate bead system.
In this study we have shown for the first time that the alginate bead system can be analyzed for gene expression by ISH and that chondrocytes over prolonged periods of culture (up to 8 months) continue to maintain their phenotype by expressing the cartilage-specific genes.
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Acknowledgments |
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Supported by NIH grant 2-P50-AR-39239, by a DAAD Fellowship (KH, Germany) and by the Deutsche Forschungsgemeinschaft (DFG Schu 1267/1-1; MS, Germany).
Received for publication November 15, 2000; accepted May 30, 2001.
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