Formation of nodular structures resembling mature articular cartilage in long–term primary cultures of human fetal epiphyseal chondrocytes on a hydrogel substrate

Abstract

Objective. To establish long-term cultures of human fetal epiphyseal chondrocytes under conditions that allow the preservation of a cartilage-specific phenotype.Methods. Chondrocytes isolated from 20-24-week human fetal epiphyseal cartilage were cultured for up to 180 days on plastic dishes previously coated with the hydrogel, poly-(2-hydroxyethyl methacrylate). Morphologic, ultrastructural, and biochemical characteristics of the cultures were examined at various intervals, and the expression of genes encoding types I, 11, and IX collagen and aggrecan core protein was determined by Northern hybridizations of total cellular RNA with human-specific complementary DNAs.Results. Human fetal epiphyseal chondrocytes cultured for 180 days under conditions that prevented their attachment to the underlying substratum formed nodular structures with morphologic and structural characteristics resembling mature articular cartilage. The cells in the center of the nodules remained spherical and were surrounded by an abundant cartilaginous extracellular matrix, as evidenced by histochemical and ultrastructural examinations. The cells in the periphery of the nodules acquired a discoid morphology and were surrounded by a sparse extracellular matrix. Biosynthetic studies demonstrated the maintenance of a Cartilagespecific phenotype throughout the 180 days of culture, Submitted for publication August 31, 1993; accepted in revised form March 20, 1994. with the production of aggrecan and types 11, IX, and XI collagens but not type I collagen. Northern hybridizations showed high levels of messenger RNAs (mRNAs) for aggrecan core protein, type I1 procollagen, and type IX collagen, but type I procollagen mRNA was not detectable even at 180 days of culture.

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis

“…Bovine fetal chondrocytes were isolated from epiphyseal cartilage (knee joint) obtained from late-secondtrimester fetuses (Animal Technologies, Tyler, TX). The chondrocytes were isolated according to the method of Reginato et al (36). Briefly, to remove adherent fibrous tissues, the cartilage was incubated in Hanks' medium containing trypsin and bacterial collagenase (2 mg/ml each) for 1 hour at 37°C.…”

“…HFCs from 3 separate specimens were isolated as described above. For each experiment, one-half of the cells were plated in 60-mm petri dishes that had been precoated with 0.9 ml of a 10% solution of poly-(2-hydroxyethyl methacrylate) (poly-HEMA; Polysciences, Malvern, PA) as previously described (36). Chondrocytes cultured under these conditions retain their differentiated phenotype for at least 180 days in culture (36).…”

“…For each experiment, one-half of the cells were plated in 60-mm petri dishes that had been precoated with 0.9 ml of a 10% solution of poly-(2-hydroxyethyl methacrylate) (poly-HEMA; Polysciences, Malvern, PA) as previously described (36). Chondrocytes cultured under these conditions retain their differentiated phenotype for at least 180 days in culture (36). In order to induce dedifferentiation, the remaining one-half of the chondrocytes from each sample were plated into 162-cm 2 tissue culture flasks at a density of 30,000/cm 2 .…”

“…The chondrocyte phenotype can be reexpressed in these cells by culturing them in suspension, in agarose, with alginate beads, or on a hydrogel substrate (17,19,25,28,(30)(31)(32)(33)(34)(35)(36). These changes in the biosynthetic profile of dedifferentiated chondrocytes resemble some of the phenotypic changes displayed by OA chondrocytes, and the matrix they produce is similar to that synthesized by chondroprogenitor cells (7)(8)(9)(10)(11)(12)(13)17,(37)(38)(39)(40).…”

“…For Northern blot analyses, 100-200 ng poly(Aϩ) RNA from the differentiated and dedifferentiated HFCs was electrophoresed on 1% agarose-formaldehyde gels and transferred to Hybond-Nϩ or XL membranes (Amersham Pharmacia Biotech, Piscataway, NJ) as described previously (36). The blots were hybridized in 5ϫ saline-sodium citrate (SSC), 2ϫ Denhardt's solution, 50% formamide, 0.1-0.5% sodium dodecyl sulfate (SDS), and 0.1 mg/ml denatured salmon sperm Northern analysis of poly(Aϩ) mRNA isolated from differentiated and dedifferentiated HFCs.…”