Elsevier

Matrix Biology

Volume 23, Issue 4, July 2004, Pages 231-241
Matrix Biology

Fibroblast growth factor-2 in serum-free medium is a potent mitogen and reduces dedifferentiation of human ear chondrocytes in monolayer culture

https://doi.org/10.1016/j.matbio.2004.06.004Get rights and content

Abstract

The loss of the differentiated phenotype (dedifferentiation) during the expansion culture of donor chondrocytes remains a large problem in cartilage tissue engineering. Dedifferentiated chondrocytes produce other matrix components and therefore the tissue produced will be of less suitable quality. Previously, the addition of fibroblast growth factor-2 (FGF2) to a serum-containing medium (SCM) during expansion culture was shown to have positive effects on the phenotype of articular chondrocytes. In the present study, we focused on a more defined, serum-free medium (SFM), to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Adult human ear chondrocytes were expanded in serum-free medium supplemented with 100 ng/ml FGF2. Expansion culture in a conventional serum-containing medium (10% FCS) served as control. The cell yield during expansion culture in serum-free medium with FGF2 was significantly higher compared to serum-containing medium. In addition, chondrocytes expanded in the serum-free medium with FGF2 expressed a more differentiated phenotype at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I and aggrecan to versican. Also, a higher gene expression of Sox9 was found. Next, suspension in alginate and subsequent culture in vitro or subcutaneous implantation in nude mice was used to evaluate the capacity of the chondrocytes, expanded in either medium, to re-express the differentiated phenotype (redifferentiation) and to form cartilage. The observed beneficial effects of the serum-free medium with FGF2 on the chondrocyte phenotype at the end of monolayer culture were sustained on both transcriptional and extracellular level throughout both redifferentiation methods.

Introduction

Cartilage has limited intrinsic repair mechanisms. Therefore, cartilage defects beyond a certain magnitude will not heal spontaneously and can lead to significant joint morbidity Howel, 1986, Simon, 1999. During the last 15 years, new techniques, grouped under the name of tissue engineering, are being developed to treat cartilage defects using culture-expanded donor cells. Classically, 2D monolayer culture on a plastic surface with serum-supplemented medium is used for expansion culture. A serious side effect of monolayer expansion culture is loss of chondrocyte phenotype (dedifferentiation) Abbott and Holtzer, 1966, Holtzer et al., 1960, Von der Mark, 1986. During monolayer culture, chondrocytes spread onto the culture flask and expression gradually shifts from collagen type II and aggrecan to collagen type I and versican, molecules characteristic for a more fibroblast-like phenotype (Von der Mark, 1986). In an attempt to improve the quality of tissue-engineered cartilage by culture-expanded chondrocytes, various expansion media supplemented with growth factors were recently developed De Haart et al., 1999, Jakob et al., 2001, Mandl et al., 2002, Martin et al., 1999, Martin et al., 2001a, Pei et al., 2002, Quatela et al., 1993, Seko et al., 1995, Van Osch et al., 2002. The most widely used growth factor for this purpose is fibroblast growth factor-2 (FGF2). Previous studies showed that addition of FGF2 to the conventional serum-containing medium (SCM) increased both cell yield during monolayer expansion culture and the capacity of expanded chondrocytes to re-express cartilage specific molecules during subsequent 3D culture in vitro Martin et al., 1999, Martin et al., 2001a. Controversially, it was shown in these studies that FGF2 increased dedifferentiation during monolayer culture Jakob et al., 2001, Martin et al., 1999.

Most studies with growth factors were performed in the presence of serum. Addition of growth factors to serum-supplemented media could influence the actions of the growth factors due to possible interacting factors in the serum (Van Susante et al., 2000). Also, large variations in bioactivity can exist between different serum batches, making the reproducibility of results difficult (Yaeger et al., 1997). In a previous study, we were able to multiply immature ear chondrocytes in a defined serum-free medium (SFM) with FGF2 and thereby enhancing the quality of the matrix generated lateron in 3D tissue formation, compared to chondrocytes multiplied in medium with 10% serum (Mandl et al., 2002). However, in that study, we used high cell densities in monolayer (40,000 cells/cm2), which limited cell multiplication. The aim of the present study is to evaluate and further elucidate the effects of serum-free medium with FGF2 on cell expansion and redifferentiation capacity of human adult chondrocytes. Therefore, adult human ear chondrocytes were expanded in monolayer culture in either defined serum-free medium with 100 ng/ml FGF2 (SFM+FGF2) or conventional SCM without additional growth factors. Next, the ability of expanded chondrocytes to re-express the differentiated phenotype (redifferentiation) and to form cartilage was assessed via suspension in an alginate gel. The alginate-cell suspension was either cultured in vitro or implanted subcutaneously in nude mice after seeding into a biomaterial. Quantitative RT-PCR technique was used in combination with biochemical assays and histology to analyze the effects of our experimental medium with FGF2 on cellular phenotype at different stages of the process and the quality of extracellular matrix produced.

Section snippets

Monolayer expansion culture

On average, four times more proliferation was observed with serum-free medium with FGF2 (SFM+FGF2) compared to SCM (Fig. 1). Highest cell numbers were found with 7500 cells/cm2 in SFM+FGF2, were at least seven doublings had occurred after three passages. In all conditions, Q-RT-PCR data obtained from chondrocytes at the end of monolayer culture showed a very low expression of aggrecan and collagen type II, which is typical for chondrocyte dedifferentiation (Fig. 2a). The degree of

Discussion

Reducing chondrocyte dedifferentiation during monolayer culture offers a major challenge in cartilage tissue engineering. In the present study, we found that expansion of chondrocytes in a defined serum-free medium with a high concentration of FGF2 reduced dedifferentiation and subsequently favored chondrogenic expression during in vitro or in vivo redifferentiation methods. Alongside, total cell yield after three passages in monolayer was significantly enhanced when SFM+FGF2 had been used

Chondrocyte expansion

Pieces of ear cartilage from three adults (respectively 18, 43 and 46 years old), obtained as leftover material from patients undergoing reconstructive surgery of the nose using ear cartilage, was used. The perichondrium was removed and cartilage was digested for 1.5 h with pronase (Sigma, St. Louis, MO) followed by overnight digestion with collagenase B (Boehringer Mannheim, Germany). Chondrocytes were seeded in monolayer using two different densities: 7500 cells/cm2 and 15,000 cells/cm2.

Acknowledgements

The authors would like to thank Dr. G. Nolst-Trenite for providing the ear cartilage. Corrina de Ridder and Cindy Bolder are much appreciated for operating and nursing the nude mice. This work was funded by the Dutch Ministry of Economic Affairs in a grant of Senter (no. BTS00021). The monoclonal antibodies, II-II6B3 and M38, were obtained from the Developmental Studies Hybridoma Bank (DSHB) maintained by the Department of Pharmacology and Molecular Sciences, John Hopkins University School of

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