Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture

• Published in: Journal of orthopaedic research Vol. 32; no. 11; pp. 1503 - 1511
• Main Authors: Tekari, Adel, Luginbuehl, Reto, Hofstetter, Willy, Egli, Rainer J.
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.11.2014
• Subjects: Animals; Ascorbic Acid - chemistry; bromodeoxyuridine; Bromodeoxyuridine - chemistry; Cartilage, Articular - cytology; Cattle; Cell Culture Techniques; Cell Lineage; Cell Proliferation; Cells, Cultured; chondrocyte; Chondrocytes - cytology; Chondrogenesis; Collagen Type I - biosynthesis; collagen type II; Collagen Type II - biosynthesis; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Expression Regulation; Humans; monolayer culture; monolayer culture; flow cytometry; chondrocyte; bromodeoxyuridine; collagen type II
• ISSN: 0736-0266; 1554-527X
• 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. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1503–1511, 2014.