title: Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study.
abstract: OBJECTIVE
  Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas.
  
  
  METHODS
  Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA.
  
  
  RESULTS
  Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region.
  
  
  CONCLUSIONS
  These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of mechanosensitivity of cartilage and mechanical regulation of chondrocyte gene expression.
authors.affiliation: Orthopaedic Research Laboratories, Department of Orthopaedics, Brown Medical School/Rhode Island Hospital, Providence, RI 02903, USA. xu_yang@brown.edu
authors.forename: Xu
authors.initials: X
authors.lastname: Yang
authors.forename: Peter S
authors.initials: PS
authors.lastname: Vezeridis
authors.forename: Brian
authors.initials: B
authors.lastname: Nicholas
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Crisco
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Moore
authors.forename: Qian
authors.initials: Q
authors.lastname: Chen