[Cytokine-induced nitric oxide production of joint cartilage cells in continuous passive movement. Anti-inflammatory effect of continuous passive movement on chondrocytes: in vitro study].
OBJECTIVE
The temporomandibular joint is a place of motion where release of proinflammatory cytokines like interleukin-1 beta (IL-1 beta) induces cartilage destruction via production of nitric oxide (NO). The purpose of this study was to evaluate the effects of continuous passive motion in the form of cyclic stretch on the synthesis of inducible nitric oxide synthase (iNOS).


METHODS
Articular chondrocytes were harvested from rabbit cartilage slices and cultured in F12 medium supplemented with 10% fetal calf serum. Subsequently, cells (10(5)/ml per well) were transferred to Flexercell plates, grown to 90% confluency, and subjected to one of the following regimens: (1) no mechanical strain (10 Hz, 20% elongation rate); (2) rhIL-1 beta (1 ng/ml); (3) mechanical strain; (4) mechanical strain and rhIL-1 beta. The cells were exposed to cyclic stretch for 24 h. Thereafter, cells were trypsinized and centrifuged on microscope slides in a cytospin centrifuge. The presence of iNOS was determined by indirect immunoperoxidase staining using polyclonal rabbit anti-iNOS as primary antibodies, and goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) as secondary antibodies. Diaminobenzidine was used as substrate for HRP. Total NO production was spectrophotometrically measured in the supernatants of cultures using Greiss reaction. All experiments were done in triplicates and the statistical significance was analyzed by Student's t-test.


RESULTS
Cells with treatment 1 and 3 did not exhibit presence of iNOS. IL-1 beta-treated cells in group 2 exhibited intense peroxidase staining. Cells in group 4 exposed to IL-1 and mechanical stress showed staining with considerably less intensity. Control cells treated with normal rabbit IgG as a primary antibody did not exhibit peroxidase staining. The data were further confirmed by measurement of statistically significant differences of NO levels in supernatants assessed in the four different treatment groups.


CONCLUSIONS
Our findings suggest that continuous passive motion exerts anti-inflammatory effects on chondrocytes, downregulating the quantity of iNOS-positive cells. Since NO acts as an intracellular, transcellular, and cytotoxic molecule, it has a basic importance for proper post-traumatic and postoperative TMJ function as well as the course and therapy of inflammatory TMJ diseases.
Gassner
R
R
Klinische Abteilung für MKG-Chirurgie, Universität Innsbruck. Robert.Gassner@uibk.ac.at
Buckley
M J
MJ
Piesco
N
N
Evans
C
C
Agarwal
S
S
D000818:Animals; D019902:Chondrocytes / Q000276:immunology* / Q000473:pathology; D016207:Cytokines / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D007375:Interleukin-1 / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010812:Physical Stimulation; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D013704:Temporomandibular Joint / Q000276:immunology* / Q000473:pathology
D016207:Cytokines; D007375:Interleukin-1; D011994:Recombinant Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase