Investigator: R. Lane Smith, PhD Project Staff: Dennis R. Carter, PhD; Gary S. Beaupré, PhD; Scott A. Yerby, PhD; George Sims, MD; John Zauner, MD; Stuart B. Goodman, MD, PhD; and David J. Schurman, MD Project Category: Arthritis - 2000 This study addressed mechanisms by which mechanical loads influence rehabilitation of joint function through tissue repair and regeneration. The goal was to develop fundamental information regarding cartilage cell biology that would be applicable to interventional techniques for restoration of joint function in arthritis. The experiments used cultured adult bovine articular cartilage chondrocytes to: (1) determine whether intermittent hydrostatic pressure will stimulate expression and assembly of extracellular matrix macromolecules and cell-surface proteins; (2) determine whether intermittent hydrostatic pressure will modulate expression of genes encoding cartilage matrix proteoglycan core protein in high density cell monolayers and in explant culture; (3) determine whether intermittent hydrostatic pressure will modulate expression of genes encoding the type II collagen in high density cell monolayers and in explant cultures. With respect to Objective 1, the effects of the intermittent hydrostatic pressure on matrix synthesis by immunohistochemical analysis of aggrecan and type II collagen showed increased deposition into the cell-associated matrix. Objectives 2 and 3 were both completed after the development of specific reverse transcription-polymerase chain reaction (RT-PCR) assays. The experimental approach included development of gene-specific primers for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of articular chondrocyte messenger RNA levels. The rationale in doing this work was that metabolic shift up or shift down in cells is accompanied by changes in the mRNA levels either by an increase in synthesis or by a change in stability. The experiments demonstrated that each of the primer sets yielded specific products that were quantified within each reaction. Analysis of the effects of intermittent hydrostatic pressure on chondrocyte mRNA levels when applied at times of 2, 4, 8, 12, and 24 hours confirmed that type II collagen signal was increased relative to unloaded cultures (p<0.05, Tukey Multiple Comparison GLM) whereas type I collagen levels were unchanged. The results of these experiments provided the basis for altering the pattern of loading such that the cells were exposed to the intermittent hydrostatic pressure on an interval basis. A second series of experiments tested the mRNA levels in cells treated with intermittent hydrostatic pressure for four hours per day for four days. The results showed that specific loading regimens could be effective in stimulating a sustained increase in the genetic expression of aggrecan and type II collagen mRNA. Type I collagen remained at very low levels in the adult chondrocytes and was not changed within the limits of detection using the RT-PCR protocols. The intracellular fibrillar matrix protein as represented by beta-actin mRNA signal was unchanged by the treatment with intermittent hydrostatic pressure. Publications and Presentations: A total of 12 presentations at national and international meetings were completed during the funding period. A representative publication is: Smith RL, Lin J, Trindade MCD, Shida J, Kajiyama G, Vu T, Hoffman AR, van der Meulen MCH, Goodman SB, Schurman DJ, Carter DR: Time-Dependent Effects of Intermittent Hydrostatic Pressure on Articular Chondrocyte Type II Collagen and Aggrecan mRNA Expression. J Rehabil Res Dev, 37(2):153-161, 2000. Funding Source: VA RR&D Merit Review |
