Chondrocytes Expressing Intracellular Collagen Type II Enter the Cell Cycle and Co-Express Collagen Type I in Monolayer Culture Adel Tekari, 1,2 Reto Luginbuehl, 3 Willy Hofstetter, 1 Rainer J. Egli 1,3 1 Group for Bone Biology and Orthopaedic Research, Department Clinical Research, University of Bern, Bern, Switzerland, 2 Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland, 3 RMS Foundation, Bettlach, Switzerland Received 25 March 2014; accepted 17 June 2014 Published online 16 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22690 ABSTRACT: For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re- introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells’ potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1503–1511, 2014. Keywords: chondrocyte; collagen type II; flow cytometry; bromodeoxyuridine; monolayer culture For more than two centuries, it is known that traumatic defects of the articular cartilage do not heal spontaneously. 1 Therapeutic interventions for the treatment of these defects aim to delay or prevent progression to osteoarthritis. More than twenty years ago, a promising cell-based repair strategy known as autologous chondrocyte transplantation (ACT) was introduced in clinics. 2,3 During ACT, chondrocytes are extracted from their native environment of cartilage tissue and grown in monolayer cultures for amplifica- tion in order to obtain the number of cells needed for therapy. 3 During the adaptation to environmental changes from cartilage tissue to the tissue culture plate, the chondrocytes begin to proliferate and ac- quire a fibroblast-like phenotype with decreased ex- pression of collagen type II (COL2) and increased expression of collagen type I (COL1). 4,5 When these cells are subsequently transferred into an environment favoring chondrogenesis, such as in vivo into a carti- lage defect during ACT or in vitro into a pellet culture systems that mimic the events of chondrogenesis and cartilage formation, 4 they have typically lost the capacity to form hyaline cartilage but rather produce a fibrocartilaginous tissue. 6,7 The synthesis of COL2 is characteristic of cells of the chondrocyte lineage, and COL2 is the main constituent of the cartilage matrix. COL2 is composed of three alpha-1 chains forming a triple helix struc- ture, and during posttranslational processing, proline residues are hydroxylated by the proline-4-hydroxy- lase enzyme, using ascorbic acid as an essential co- factor. 8 The procollagen is then exported to the extracellular domain, the non-helical C- and N-termi- nal ends are cleaved, and through polymerization, collagen fibrils and fibers are formed. In addition to being a major structural component of cartilage tissue, extracellular COL2 has been shown to act in a chondroinstructive manner on chondrocyte lineage cells through interaction with integrin receptors on the cell surface. 9,10 The importance of COL2 in carti- lage and chondrocytes is evident, and as such monitor- ing the expression of COL2 is a critical parameter to assess chondrocyte lineage cells. The purpose of this study was to characterize with a cellular resolution the phenotypic adaptations that articular chondrocytes undergo in monolayer culture after harvesting from cartilage tissue, with particular focus on the expres- sion of COL2 and COL1 proteins and to further understand why these cells lose the potential to form cartilage tissue when transferred into a microenviron- ment that supports chondrogenesis. MATERIALS AND METHODS Expansion of Primary Chondrocytes Bovine articular cartilage was harvested from the humeral heads of 2-year-old animals obtained within 24 h after death. Chondrocytes were isolated by sequential enzymatic digestion of the cartilage with 2.5 mg/ml pronase (Sigma–Aldrich, Buchs, Switzerland) for 1h and 450 mg/ml collagenase P (Roche Diagnostics, Rotkreuz, Switzerland) for 4h at 37˚C. The released cells were expanded in proliferation medium (DMEM/ Ham’s F12 [Gibco, Life Technologies, Zug, Switzerland]) containing 10% fetal bovine serum (FBS, Sigma–Aldrich) and penicillin/streptomycin (P/S, 100 units/ml and 100 mg/ml, re- spectively, Gibco). Near confluence, the cells were released with Trypsin/EDTA (Gibco) and re-plated until reaching passage 6 (P6). Twenty-four hours after isolation and then at Conflict of interest: None. Grant sponsor: RMS Foundation; Grant number: E10-0007. Correspondence to: Rainer J. Egli (T: þ41-32-644-1339; F: þ41-32-644-1176; E-mail: rainer.egli@rms-foundation.ch) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2014 1503