Effect of hyaluronic acid (MW 500-730 kDa) on proteoglycan and nitric oxide production in human osteoarthritic chondrocyte cultures exposed to hydrostatic pressure.
OBJECTIVE
This study investigated the in vitro effects of hyaluronic acid (HA) of molecular weight (MW) 500-730 kDa on human articular chondrocytes cultivated for 48 h in the presence of interleukin-1beta (IL-1beta) with and without hydrostatic cyclical pressure.


METHODS
The effects of 10 and 100 microg/ml HA with and without IL-1beta were assessed in the culture medium of cells exposed to pressurization cycles in the form of sinusoidal waves (minimum pressure 1MPa, maximum pressure 5MPa) at a frequency of 0.25Hz for 3h, by the immunoenzymatic method on microplates for the quantitative measurement of human proteoglycans (PG) and by the Griess method for nitrites (NO). Morphological analyses were performed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).


RESULTS
The presence of IL-1beta determines a significant decrease in PG and a significant increase in NO concentrations measured in the culture medium. When the cells are cultured in the presence of IL-1beta and HA at the two concentrations, a statistically significant restoration of PG and a decrease in NO levels are observed. Under pressurization conditions, we observed that the PG concentration in the medium of cells presented a very significant increase in all the conditions used in the study, except for IL-1beta alone. NO production decreased very significantly in the presence of IL-1beta+HA 10 and IL-1beta+HA 100. The results of metabolic evaluation are confirmed by morphological findings obtained by TEM and SEM.


CONCLUSIONS
These in vitro studies confirm both the protective role of HA (MW 500-730 kDa), which counteracts the IL-1beta-induced effects, and the importance of pressure on chondrocyte metabolism and morphology.
Fioravanti
A
A
Rheumatology Unit, Department of Clinical Medicine and Immunological Sciences, University of Siena, Le Scotte Policlinico, Siena, Italy. fioravanti7@virgilio.it
Cantarini
L
L
Chellini
F
F
Manca
D
D
Paccagnini
E
E
Marcolongo
R
R
Collodel
G
G
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000368:Aged; D002358:Cartilage, Articular / Q000187:drug effects* / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology*; D006874:Hydrostatic Pressure; D007375:Interleukin-1 / Q000494:pharmacology; D008854:Microscopy, Electron / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008875:Middle Aged; D009569:Nitric Oxide / Q000096:biosynthesis*; D020370:Osteoarthritis, Knee / Q000378:metabolism*; D011509:Proteoglycans / Q000096:biosynthesis*
D000276:Adjuvants, Immunologic; D003470:Culture Media; D007375:Interleukin-1; D011509:Proteoglycans; D009569:Nitric Oxide; D006820:Hyaluronic Acid