title: Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.
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.
authors.affiliation: Group for Bone Biology and Orthopaedic Research, Department Clinical Research, University of Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
authors.forename: Adel
authors.initials: A
authors.lastname: Tekari
authors.forename: Reto
authors.initials: R
authors.lastname: Luginbuehl
authors.forename: Willy
authors.initials: W
authors.lastname: Hofstetter
authors.forename: Rainer J
authors.initials: RJ
authors.lastname: Egli
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry; D001973:Bromodeoxyuridine / Q000737:chemistry; D002358:Cartilage, Articular / Q000166:cytology; D002417:Cattle; D018929:Cell Culture Techniques; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology*; D020219:Chondrogenesis; D024042:Collagen Type I / Q000096:biosynthesis*; D024043:Collagen Type II / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans
chemical_list: D024042:Collagen Type I; D024043:Collagen Type II; D001973:Bromodeoxyuridine; D001205:Ascorbic Acid
keywords: bromodeoxyuridine; chondrocyte; collagen type II; flow cytometry; monolayer culture