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Antioxidant effect of hyaluronic acid in cultured embryonic cartilage: biochemical and morphological aspects
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Abstracts
A n t i o x i d a n t effect of h y a l u r o n i c acid in cultured e m b r y o n i c cartilage: b i o c h e m i c a l and m o r p h o l o g i c a l a s p e c t s BRUN, L. CARDARELLI A N D G. ABATANGELO Institute of Histology, University of Padua, Italy
D. DAGA-GORDINI, R. CORTIVO, P.
In order to test the scavenging effect of reactive oxygen species, we added hyaluronic acid (HA) to two free radical generating systems, activated neutrophils and Hypoxantine/Xantine Oxidase (20-60 ml) in 10 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and antibiotic. The neutrophils, added to chick sterna cultures, were activated with FMLP and Cytochalasin B for 3 h at 37°C. After incubation of the tissue in these two reactive oxygen species generating systems, the medium was removed and the cartilages, after washing with phosphate buffer solution (PBS), were incubated in fresh medium containing 35+S sulphate (10 Ci/ml). After 24 h the sternae were removed, washed and extracted with 4 ~ Gu-HC1 for 24 h at 4°C. Following extraction, the insoluble residue was dissolved in 1 M HC1 for 10 h at
110°C. The amount of radioactivity incorporated into Guanidinium extract and in the residue (HC1 digest) was measured and expressed as CPM/mg of dry tissue. Both Xantine Oxidase reaction and activated neutrophils decreased proteoglycan synthesis. On the other hand, HA was able to reverse this inhibition, especially in Xantine Oxidase system. From morphological observations using electron microscopy it has been possible to observe many ultrastructural changes of chondrocytes and extracellular matrix. Significative alterations were observed at the level of nuclear chromatin as well as at the level of Golgi areas and Rough Endoplasmic Reticulum (RER) which appeared enlarged with accumulating material in the cavities.
Morphological e v a l u a t i o n of the activity of hyaluronic acid on synovial m e m b r a n e and articular cartilage E. GOVONI*, L. FRIZZIERO'~, A. PERBELLINI:~ AND I. PASQUALI RONCHETTI§
*Milan University, Milan, tMaggiore Hospital, Bologna, $Fidia S.p.A., Abano Terme §Modena University, Modena, Italy To evaluate the efficacy of intra-articular treatment with hyaluronic acid with molecular weight between 500 and 730 kDa (HA), in 40 patients suffering from osteoarthritis of the knee in the inflammatory phase an open, uncontrolled clinical trial (intra-articular injection of HA at a dose of 20 mg[2 ml per week for 5 weeks) was carried out, together with: (i) an in vivo blind evaluation at the microscopicsubmacroscopic level by arthroscopy and microarthroscopy (performed at the admission and on day 180) of the grading and the extent of articular cartilage damage and the grading of the inflammatory alterations of the synovial membrane (using a scale of 0-100); (ii) an ex vivo analysis performed under blind conditions of the biopsy samples at the microscopic-submicroscopic level by light and electron microscopy.
The histological assessment criteria of efficacy were represented for the articular cartilage by the thickness of the amorphous superficial layer, the Mankin scale, and the Ultrastructural Chondrocyte Test System; for the synovial membrane by the thickness of the synovial lining, the number of inflammatory mononuclear cells, and the area of fibrosis. By arthroscopy, in 32.5~o of patients a reduction in the degree and/or extension of articular cartilage damage was found; 32 out of 40 patients showed a significant decrease in the synovitis grading. At histology, in articular cartilage, in the patients with 2nd degree lesions the rebuilding of the superficial amorphous layer, a reduced Mankin score and an improvement in chondrocyte vitality indices were found. The reduction of inflammatory features was a constant finding in synovial membrane.
Abstracts
A n t i o x i d a n t effect of h y a l u r o n i c acid in cultured e m b r y o n i c cartilage: b i o c h e m i c a l and m o r p h o l o g i c a l a s p e c t s BRUN, L. CARDARELLI A N D G. ABATANGELO Institute of Histology, University of Padua, Italy
D. DAGA-GORDINI, R. CORTIVO, P.
In order to test the scavenging effect of reactive oxygen species, we added hyaluronic acid (HA) to two free radical generating systems, activated neutrophils and Hypoxantine/Xantine Oxidase (20-60 ml) in 10 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and antibiotic. The neutrophils, added to chick sterna cultures, were activated with FMLP and Cytochalasin B for 3 h at 37°C. After incubation of the tissue in these two reactive oxygen species generating systems, the medium was removed and the cartilages, after washing with phosphate buffer solution (PBS), were incubated in fresh medium containing 35+S sulphate (10 Ci/ml). After 24 h the sternae were removed, washed and extracted with 4 ~ Gu-HC1 for 24 h at 4°C. Following extraction, the insoluble residue was dissolved in 1 M HC1 for 10 h at
110°C. The amount of radioactivity incorporated into Guanidinium extract and in the residue (HC1 digest) was measured and expressed as CPM/mg of dry tissue. Both Xantine Oxidase reaction and activated neutrophils decreased proteoglycan synthesis. On the other hand, HA was able to reverse this inhibition, especially in Xantine Oxidase system. From morphological observations using electron microscopy it has been possible to observe many ultrastructural changes of chondrocytes and extracellular matrix. Significative alterations were observed at the level of nuclear chromatin as well as at the level of Golgi areas and Rough Endoplasmic Reticulum (RER) which appeared enlarged with accumulating material in the cavities.
Morphological e v a l u a t i o n of the activity of hyaluronic acid on synovial m e m b r a n e and articular cartilage E. GOVONI*, L. FRIZZIERO'~, A. PERBELLINI:~ AND I. PASQUALI RONCHETTI§
*Milan University, Milan, tMaggiore Hospital, Bologna, $Fidia S.p.A., Abano Terme §Modena University, Modena, Italy To evaluate the efficacy of intra-articular treatment with hyaluronic acid with molecular weight between 500 and 730 kDa (HA), in 40 patients suffering from osteoarthritis of the knee in the inflammatory phase an open, uncontrolled clinical trial (intra-articular injection of HA at a dose of 20 mg[2 ml per week for 5 weeks) was carried out, together with: (i) an in vivo blind evaluation at the microscopicsubmacroscopic level by arthroscopy and microarthroscopy (performed at the admission and on day 180) of the grading and the extent of articular cartilage damage and the grading of the inflammatory alterations of the synovial membrane (using a scale of 0-100); (ii) an ex vivo analysis performed under blind conditions of the biopsy samples at the microscopic-submicroscopic level by light and electron microscopy.
The histological assessment criteria of efficacy were represented for the articular cartilage by the thickness of the amorphous superficial layer, the Mankin scale, and the Ultrastructural Chondrocyte Test System; for the synovial membrane by the thickness of the synovial lining, the number of inflammatory mononuclear cells, and the area of fibrosis. By arthroscopy, in 32.5~o of patients a reduction in the degree and/or extension of articular cartilage damage was found; 32 out of 40 patients showed a significant decrease in the synovitis grading. At histology, in articular cartilage, in the patients with 2nd degree lesions the rebuilding of the superficial amorphous layer, a reduced Mankin score and an improvement in chondrocyte vitality indices were found. The reduction of inflammatory features was a constant finding in synovial membrane.