Probing the microenvironmental conditions for induction of superficial zone protein expression

• Published in: Osteoarthritis and cartilage Vol. 21; no. 12; pp. 1924 - 1932
• Main Authors: Mhanna, R, Öztürk, E, Schlink, P, Zenobi-Wong, M
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-12-2013
• Subjects: Animals; Cartilage, Articular - cytology; Cartilage, Articular - metabolism; Cattle; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Cellular Microenvironment - genetics; Cellular Microenvironment - physiology; Chondrocytes - metabolism; Chondrocytes dedifferentiation; Collagen Type II - genetics; Collagen Type II - metabolism; Hypoxia - genetics; Hypoxia - metabolism; Immunohistochemistry; Mechanical strain; Microenvironment; Oxygen tension; Proteoglycans - genetics; Proteoglycans - metabolism; Real-Time Polymerase Chain Reaction; Rheumatology; RNA, Messenger - analysis; Stress, Mechanical; Superficial zone protein; Tissue Engineering - methods; Chondrocytes dedifferentiation; Microenvironment; Oxygen tension; Superficial zone protein; Mechanical strain
• ISSN: 1063-4584; 1522-9653
• DOI: 10.1016/j.joca.2013.08.017

Summary Objective To determine the in vitro conditions which promote expression of superficial zone protein (SZP). Methods Chondrocytes from 6-month-old calves were expanded in monolayer culture and the expression of SZP in alginate bead and monolayer culture was quantified with quantitative real time-polymerase chain reaction (qRT-PCR) and immunostaining. The effect of oxygen tension on SZP expression was determined by qRT-PRC analysis of cells cultured in two dimension (2D) and three dimension (3D) under hypoxic (1% pO2 ) or normoxic (21% pO2 ) conditions. Finally, to examine the effect of cyclic tensile strain on expression of SZP in 2D and 3D cultures, chondrocytes encapsulated in alginate beams or seeded on type I collagen coated polydimethylsiloxane (PDMS) chambers were subjected to 5% strain at 1 Hz, 2 h/day for 4 days or 2 h at the fourth day of culture and mRNA levels were quantified. Results Bovine chondrocytes in monolayer showed a drastic decrease in SZP expression, similar in trend to the commonly reported downregulation of type II collagen (Col2) . Chondrocytes embedded in alginate beads for 4 days re-expressed SZP but not Col2. SZP expression was higher under normoxic conditions whereas Col2 was upregulated only in alginate beads under hypoxic conditions. Cyclic mechanical strain showed a tendency to upregulate mRNA levels of SZP. Conclusions A microenvironment encompassing a soft encapsulation material and 21% oxygen is sufficient for fibroblastic chondrocytes to re-express SZP . These results serve as a guideline for the design of stratified engineered articular cartilage and suggest that microenvironmental cues (oxygen tension level) strongly influence the pattern of SZP expression in vivo.