Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Abstract

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS(+3)) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.

Figure 1

Effect of 2D (monolayer) or 3D (porous collagen sponge) conditions on phenotype of bovine articular chondrocytes cultured for 1, 2, and 4 weeks. (a) Total sulfated glycosaminoglycan content (s-GAG) was measured by DMB assay and normalized to DNA content in each dish or sponge (n = 5). (b) Ratio of collagen type II (COL II) to collagen type I (COL I) was calculated from levels of each RNA as measured by quantitative, competitive RT-PCR assays and normalized to glyceraldehyde 3-phosphate dehydrogenase (G3PDH). (c) Ratio of aggrecan (AGG) to collagen type I (COL I) was calculated from levels of each RNA as measured by quantitative, competitive RT-PCR and normalized to G3PDH.

Figure 2

Photomicrographs of bovine articular chondrocytes cultured in collagen sponges (Toluidine blue stain). Matrix that contains sulfated glycosaminoglycans appears pink/purple and cell nuclei are blue. (a) Collagen sponge was precultured for 1 week in medium containing FBS. Scale bar is 50 μm. (b) Collagen sponge was cultured in FBS for an additional week. (c) After preculture, collagen sponge was cultured in media containing 2% Nutridoma for 7 days. (d) After preculture, collagen sponge was cultured in media containing ITS⁺³ for 7 days. Arrows indicate pycnotic cells.

Figure 3

Effect of culture media on expression levels of cartilage signature genes in bovine articular chondrocytes in collagen sponges. Media contained 5% fetal bovine serum (FBS), 2% Nutridoma (Nut) or 1% ITS⁺³. Gene expression levels of collagen type II (COL) and aggrecan (AGG) were measured by competitive RT-PCR and normalized to G3PDH.