Sensitivity of chondrocytes of growing cartilage to reactive oxygen species

Biochimica et Biophysica Acta 1425 (1998) 103^111

Sensitivity of chondrocytes of growing cartilage to reactive oxygen species Emanuela Fragonas a , Piero Pollesello b , Vladimir Mlina¨rik c , Renato To¡anin d , Cristina Grando a , Cristiana Godeas a , Franco Vittur a; * a

Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, L. Giorgieri 1, I-34127 Trieste, Italy b Orion Corporation, Orion Pharma, Reseach and Development, Drug Design Unit, NMR-Laboratory, P.O. Box 65, FIN-02101 Espoo, Finland c Magnetic Resonance Unit, Derer Hospital, Limbova¨ 5, Sk-83305 Bratislava, Slovakia d Poly-Bio¨s Research Center, Area di Ricerca, Padriciano 99, I-34012 Trieste, Italy Received 5 February 1998; revised 4 May 1998; accepted 19 May 1998

Abstract Vascular invasion of calcified cartilage, during endochondral ossification, is initiated and sustained by invasive cells (endothelial cells and macrophages) which degrade the tissue by releasing lytic enzymes. Concurrently, reactive oxygen species (ROS) are also released by these cells and we hypothesize that ROS also contribute to the degradation of the tissue. As a preliminary approach to this problem, the antioxidant activities and the effect of ROS on hypertrophic cartilage and chondrocytes (HCs) were investigated. Compared to resting or articular chondrocytes, HCs exhibited higher catalase but lower SOD specific activities and lower PHGPx concentration, thus revealing a defence activity specific against H2 O2 . Moreover, dose-dependent depletion of ATP occurred after few minutes of exposure to ROS, and a long-term treatment (16 h incubation with ROS) promoted the release of LDH activity and a significant variation of the poly- to monounsaturated fatty acid ratio. Finally, the incubation of HCs with low ROS doses induced the release of sedimentable alkaline phosphatase activity (matrix vesicles). How the obtained results fit the in vivo occurring events is discussed. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Reactive oxygen species; Hypertrophic chondrocyte; Growing cartilage; Matrix vesicle; NMR

1. Introduction Endochondral ossi¢cation is a multistep process Abbreviations: ROS, reactive oxygen species; SOD, superoxide dismutase; PHGPx, phospholipid hydroperoxide glutathione peroxidase; LDH, lactate dehydrogenase; HCs, RCs and ACs, hypertrophic, resting and articular chondrocytes; MUFA and PUFA, mono and polyunsaturated fatty acid chains; HX, hypoxanthine; XO, xanthine oxidase * Corresponding author. Fax: +39 (40) 676-3691; E-mail: [email protected]

during which chondrocytes of growth cartilage proliferate, become hypertrophic and generate the socalled `matrix vesicles' [1]. These extracellular bodies are the sites where the ¢rst crystals of hydroxyapatite can be detected. Thereafter the calci¢cation extends to the whole cartilage matrix [2,3], the calci¢ed tissue is invaded by capillaries, is degraded, and ¢nally substituted by newly laid-down bone [4]. Neovascularization is a fundamental step of endochondral ossi¢cation, but its mechanism and control are not yet fully understood. Capillary invasion is

0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 0 5 5 - 5

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initiated and sustained by terminal capillary cells. Anderson and Parker [5] proposed that the invasive cells are endothelial cells and accompanying macrophages. Schenk et al. [6,7] showed that, in the calci¢ed cartilage, transversal septa between chondrocytes are degraded by endothelial and perivascular cells while chondroclasts resorb mineralized longitudinal septa by secretion of proteases and collagenases. In£ammatory cells [8] release angiogenic stimulators, such as growth factors and cytokines. Endothelial cells, macrophages and chondroclasts, when activated, also release reactive oxygen species (ROS) [9]. It is known that ROS are active on various components of the cartilage matrix and their e¡ects on hyaluronic acid [10], proteoglycans and collagen [11,12] have been described. In addition, ROS may in£uence articular chondrocyte growth [13], promote damage of their membranes [14] and depress their metabolic pathways for energy production [15,16]. In the arthritic disorders of the joints, it was demonstrated that ROS can induce both matrix degradation and chondrocyte damage [17]. Our hypothesis is that similar phenomena can occur also during the neovascularization process in growth cartilage where ROS may participate in the demolition of the cartilaginous sca¡olding of the bone. As far as we know, the hypothesis that ROS generated by cells of the macrophagic lineage might participate in the process of endochondral bone formation has not yet been taken into consideration, and no information is available about the sensitivity of the growth plate cartilage to these active species. In particular, no data demonstrate a direct e¡ect of ROS on hypertrophic chondrocytes, the cells of growth plate directly exposed to the agents favoring the piercing by the vascular tips. Therefore, the evaluation of these e¡ects and of the defence mechanisms operating in growth plate chondrocytes against ROS can be particularly useful. In our study, di¡erent parameters were considered as follows: (1) the antioxidant activities of the cells were evaluated from the speci¢c activities of superoxide dismutase (SOD) and catalase and from the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPx); (2) the cell damage induced by ROS was evaluated from the percentage of the LDH activity released; (3) the blockade of the energetic metabolism was monitored

by the decrease of intracellular ATP; and (4) the tissue damage was demonstrated by following the variation in the saturation degree of fatty acid residues. Finally, the in£uence of ROS on matrix vesicle production was also tested. Articular and resting cartilages and chondrocytes were taken as controls, because the former are subjected to the action of ROS in vivo only when a pathological status develops, while the resting cannot be exposed to the action of exogenous ROS as a consequence of their position in the growth plate. 2. Materials and methods 2.1. Tissue dissection, cell isolation and culture conditions Growth cartilage was obtained from the scapulae of 6-month-old pigs (150^200 kg). Scapulae were accurately freed of perichondrium and periosteum (in the zone of bone^cartilage junction); the cartilaginous portion was cleaved and residues of mineralized cartilage were accurately scraped o¡. Resting and hypertrophic regions were selected and dissected as previously reported by Vittur et al. [18]. Tissue preparation, enzymatic treatment for isolation of resting and hypertrophic chondrocytes, and the conditions for monolayer cultures were as described by Pollesello et al. [19]. Only 7^10-day-old primary cultures were employed in order to avoid chondrocyte dedi¡erentiation. Suspension cultures were used in the experiments for matrix vesicles induction; in this case, chondrocytes were cultured 7^10 days in alginate gels as described by Grandolfo et al. [20]. To avoid contamination of the hypertrophic chondrocyte preparations by erythrocytes, the cell suspensions were fractionated by centrifugation on discontinuous Percoll density gradients (d = 1.01 and 1.08; 800Ug, 10 min), and the puri¢ed HCs were collected at the interface between the two solutions. For some control experiments, aliquots of articular cartilage were obtained from the proximal head of the humerus of the same animals, chondrocytes were isolated from this tissue by the same procedure reported for the RCs [19] and cultured in monolayers or in alginate for 10 days.

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2.2. Reactive oxygen species (ROS) ROS …O3 2 † were generated by the hypoxanthine (HX)-xanthine oxidase (XO) reaction [21] using 0.003^0.12 U ml31 of XO and 4 mM HX (Sigma, Milan, Italy) in Dulbecco's minimal essential medium (DMEM; Seromed, Berlin, Germany). 2.3. Quantitation of the ROS-induced chondrocyte damage The e¡ect of ROS on chondrocytes was evaluated in two di¡erent ways: namely the intracellular concentration of ATP was taken as an indicator of the short-term damage, whereas the long-term e¡ect was estimated by determining the free vs total lactate dehydrogenase activity ratio after treatment of 1-weekold monolayer cell cultures with increasing amounts of ROS for 16 h. 2.4.

31

P-NMR ex vivo experiments

The intracellular concentration of ATP was measured by 31 P-NMR spectroscopy on samples of ¢nely sliced cartilage as described earlier [22]. In such experiments, the exposure time of the tissue to ROS was 15^60 min. 31 P-NMR experiments were performed at 37³C on a Bruker AM 300 WB spectrometer equipped with a broad band probe. The tissue slices were placed in 10 mm (o.d.) NMR tubes. The tubes were then ¢lled with DMEM (with or without the ROS generating system) and spun during acquisition (10 Hz). The spectral width was 8.9 kHz, the number of data points 8K, the £ip angle 62³, and the repetition time 0.5 s. In order to increase the S/N ratio, an exponential multiplication of the time-domain data was performed prior to Fourier transformation. Chemical shifts are referred to phosphocreatine at pH 7.4 (0 ppm). Isolated RCs and HCs were also used in this type of experiments immediately after their isolation or after 7 days of culture in alginate gels. The obtained results were overimposable with those obtained by using minced tissue. 2.5. ROS e¡ect on cartilage lipids In order to evaluate the damage caused by ROS to

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the lipidic components of the tissue, the dissected samples were exposed to ROS (generated by 4 mM hypoxanthine and 0.12 U ml31 xanthine oxidase) at 37³C for 16 h in Dulbecco's medium. Thereafter, lipids were extracted by the following procedure. Samples were frozen and ground in liquid nitrogen in a ceramic mortar. After thawing, lipids were extracted with chloroform/methanol 2:1 (10 ml g31 wet tissue) at 4³C, in the dark and under a nitrogen atmosphere in order to prevent oxidation of the sample. The organic phase was washed as described by Folch et al. [23]. The solvent was exchanged to CDCl3 99.95% (Aldrich) and the samples were stored at 318³C until they were analyzed. 1 H-NMR and 13 C-NMR experiments were performed at 27³C on a Bruker ARX 400 spectrometer equipped with an inverse detection broadband probe, using 5 mm NMR tubes. Chemical shifts are referred to tetramethylsilane (TMS). 1 H-NMR spectra were acquired using a 60³ £ip angle, 256 scans, a repetition time of 4 s, spectral width of 5600 Hz, and data size of 16K points. Proton decoupled 13 C-NMR spectra were acquired using a spectral width of 8 kHz, 32K data points, a 45³ £ip angle, and a repetition time of 7 s. Fourier transformation of 1600 transients was performed after zero-¢lling of the time^domain data to 64K points. 2.6. Determination of enzymatic activities Lactate dehydrogenase (LDH) activity was measured according to Vassault [24]. Alkaline phosphatase activity was assayed according to Stagni et al. [25]. Catalase and SOD activities were measured on chondrocytes immediately after their isolation. The catalase activity was evaluated by the spectrophotometric method of Johansson et al. [26] while the superoxide dismutase activity was monitored according to Matsumoto et al. [27] using acetylated ferricytochrome c [28]. DNA was measured by the method of Labarca and Paigen [29]. 2.7. Evidence of phospholipid hydroperoxide glutathione peroxidase (PHGPx) The identi¢cation of PHGPx in RCs and HCs was obtained by electrophoresis and Western blot on cell

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protein lysates according to the technique of Roveri et al. [30]. The anti-PHGPx rabbit polyclonal antibody employed was a¤nity puri¢ed using a column where recombinant PHGPxcys46 was covalently linked [31]. PHGPx isolated from pig heart and rat testis mitochondrial membranes were used as in [32]. 2.8. Matrix vesicle production by hypertrophic chondrocytes Alginate culture of chondrocytes is an in vitro technique suitable for gaining and maintaining the hypertrophic phenotype of chondrocytes, for maintaining their normal intracellular calcium concentration, constant alkaline phosphatase speci¢c activity, and producing matrix vesicles [20]. Hypertrophic chondrocytes, cultured in alginate gels for 7^10 days, were then stimulated by ROS produced at 0.006 U ml31 xanthine oxidase for 16 h at 37³C. Media were then collected and stored. The alginate gels were then solubilized with citrate (50 mM citrate, 100 mM NaCl, 10 mM glucose, pH 7.4) and the cells were recovered from the resulting suspension by centrifugation at 800Ug for 5 min [20]. Matrix vesicles were then isolated from the ¢nal supernatant by di¡erential centrifugation [33] and their total alkaline phosphatase activity was measured and normalized to the total LDH of the combined media and ¢nal supernatants. The results were compared to those of control, untreated, cultures. 3. Results 3.1. Disproportionating enzymes of chondrocytes Table 1 shows the speci¢c activities of catalase and Table 1 Disproportionating activities of chondrocytes Chondrocytes from

Catalase (U mg31 DNA)

SOD (U mg31 DNA)

Hypertrophic cartilage Resting cartilage Articular cartilage

190.0 þ 68.5 (5) 75.8 þ 10.0 (5) 36.7 þ 5.43 (4)

33.17 þ 3.69 (9) 98.83 þ 9.57 (11) 61.17 þ 3.25 (13)

At least four di¡erent cell preparations were analyzed for their enzymatic activities immediately after isolation. Each value represents the mean of the indicated number of experiments þ S.D.

Fig. 1. Identi¢cation of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the hypertrophic (Hy) and resting (R) chondrocytes. Five to 10 Wl of the lysate (0.6 ml) derived from 2U106 cells, was subjected to polyacrylamide electrophoresis, blotting and immunostaining for PHGPx as described by Roveri et al. [30]. Reference proteins were PHBPx from pig heart (H) and rat testis (T).

SOD in chondrocytes isolated from hypertrophic cartilage and, for comparison, those of resting and articular cells. HCs are endowed with the highest catalase activity, but show the lowest SOD activity. The presence of PHGPx in HCs and RCs is demonstrated by the immunoblotting experiment presented in Fig. 1. In this semi-quantitative experiment, the relative dimensions of the decorated bands indicate that the enzyme is present in RCs at a higher concentration than that of the hypertrophic cells. 3.2. Damage induced by ROS on chondrocytes 3.2.1. Short-term treatment The ¢rst e¡ect of ROS on HCs was detected by 31 P-NMR spectroscopy (Fig. 2). The concentration of nucleotide triphosphate (mostly ATP) falls after 30 min of treatment with ROS, in a dose-dependent manner. When 0.12 U ml31 of xanthine oxidase are used for ROS production, the intracellular level of ATP falls rapidly under the detection limit. Similar results were obtained with resting and articular cartilages and also on freshly isolated or cultured chondrocytes (not shown). 3.2.2. Long-term treatment Fig. 3 shows the e¡ect of ROS generated at di¡erent concentrations of xanthine oxidase on the di¡erent chondrocyte types. Articular chondrocytes are the most sensitive to the ROS as they present a max-

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imum release of LDH activity at 0.02 U ml31 of xanthine oxidase a concentration ine¡ective on resting and hypertrophic chondrocytes. Six-fold more xanthine oxidase is needed to induce a signi¢cant release of LDH by resting and hypertrophic cells. The data reported in Table 2 show that, at 0.12 U ml31 of XO, ROS promote the damage of about 88% of articular chondrocytes, but of only 46% of resting and 25% of ossifying cells.

Fig. 3. Cytotoxic e¡ects of increasing ROS concentrations on cultured chondrocytes. The indicated concentrations of XO were used to generate increasing ROS concentrations. Con£uent cultures were exposed to ROS for 16 h at 37³C in DMEM. The cell damage was evaluated from the released LDH activity as percent of the total activity of the culture. The results were then normalized to the maximum release obtained and plotted. Bars indicate S.D.

3.3. E¡ect of ROS on cartilage lipids Fig. 4 shows the NMR spectra obtained for crude lipid extracts of resting and hypertrophic cartilage. In Fig. 4A, the 1 H-NMR spectrum of a lipid extract of cartilage is shown. Peaks assigned to double bonds, Table 2 Sensitivity of chondrocytes to ROS Fig. 2. 31 P-NMR spectra on untreated and ROS-treated hypertrophic cartilage. Spectra were collected for 20^30 min at the end of the treatment period. (a) Untreated cartilage. (b) ROStreated tissue at 0.12 U ml31 XO, 4 mM HX, 37³C, 1 h in DMEM. (c) ROS-treated tissue at 0.12 U ml31 XO, 4 mM HX, 37³C, 16 h in DMEM. Pst is the peak of 1 mM phosphate reference solution, pH 7.4. sealed in a glass capillary and inserted in the NMR tube containing the tissue suspension. Pi indicates the peak of inorganic phosphate in the NMR tube, NTPK;L;Q corresponds to the signals of triphosphorylated intracellular nucleotides (mostly ATP); PME indicates the phosphomonoesters. The decrease of ATP is indicated by the disappearance of the NTPK;L;Q signals.

Chondrocytes from

Free LDH activity (% of total activity of the culture)

Hypertrophic cartilage Resting cartilage Articular cartilage

24.7 þ 02.9 (8) 46.2 þ 15.3 (5) 87.6 þ 10.8 (7)

Con£uent monolayer cultures (7^10 days of culture) of the different chondrocyte types were exposed for 16 h at 37³C to the ROS generated at 0.12 U ml31 of XO and 4 mM HX in DMEM. The extent of cell damage was estimated from free LDH activity as percent of total LDH activity of the culture. Data are the mean þ S.D. of the indicated number of experiments on di¡erent cell preparations.

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E. Fragonas et al. / Biochimica et Biophysica Acta 1425 (1998) 103^111 Fig. 4. NMR spectra obtained for crude lipid extracts of resting and hypertrophic cartilage. (A) 1 H-NMR spectrum of a lipid extract from resting cartilage. The spectrum is divided into four regions. Signals attributed to protons belonging to double bonds (a) to di- and tri-acylated glycerols and to the phosphocholine and phosphoethanolamine parts of phospholipids (b) to methylene groups (c), and to terminal methyl groups of fatty acid chains (d). In the region (c), a peak is assigned to protons belonging to methylene groups located between two non-conjugated unsaturations of a fatty acid chain (e). In proximity of (d), signals from the methyl groups of cholesterol are shown (f,g). In particular, the peak of methyl-18 of cholesterol is well resolved (g). The major methylene peak in region (c) is truncated at 30% of its actual intensity. Integrals of the peaks were used to calculate the lipid composition. (B) Expansion of the 13 C-NMR spectra of crude lipid extracts from resting and hypertrophic cartilage before and after the treatment with ROS (as described in Section 2). The spectral region from 129.2 to 131 ppm, characteristic of the olephinic carbons belonging to mono- or di-unsaturated fatty acid chains is shown. 1, Hypertrophic, after treatment with ROS (0.12 U ml31 , 16 h, 37³C); and 2, its control; 3, ROS-treated (as above) resting cartilage; and 4, its control. Peaks are assigned to di¡erent fatty acid chains as follows: 18:1 (h), 18:2 (i), 16:1 (j), 14:1 (k), 18:3 (l) and 20:4 (m).

6

methyl- and methylene groups of the fatty acyl chains, to some of the phospholipid polar heads (phosphocholine and phosphoethanol-amine), to the di- and tri-acylated glycerol and to some of the methyl groups of cholesterol are marked. Fig. 4B shows the typical 13 C-NMR spectra of the lipid extracts from resting and ossifying cartilage before and after the treatment with ROS. The spectral region characteristic for the olephinic carbons is shown. Comparison with the spectral characteristics of the original compounds and with literature data [34] made it possible to identify the spectral pattern of the fatty acid

chains and the peaks characteristic for carbons 5 and 6 of cholesterol. Moreover, carbons belonging to non-conjugated double bonds on fatty acid chains could be also detected, being the most represented fatty acid chains 18:1, 18:2, 16:1 and 14:1. In order to obtain more information on the quantitative lipid composition of the samples, both the 1 H- and 13 C-NMR spectra were integrated as described elsewhere [34]. The cholesterol to total lipid molar ratio, unsaturation ratio, average chain length of the fatty acid chains and PUFA to MUFA ratio

Table 3 The lipid composition of control and ROS-treated hypertrophic and resting cartilages Cartilage

Cholesterol (a)

Mean chain length of fatty acids

Unsaturation (a)

PUFA/MUFA

Resting Resting, treated Hypertrophic Hypertrophic, treated

0.080 þ 0.004 0.090 þ 0.002 0.070 þ 0.003 0.080 þ 0.003

17.10 þ 0.12 16.87 þ 0.09 17.09 þ 0.12 16.77 þ 0.11

0.62 þ 0.01 0.59 þ 0.01 0.65 þ 0.01* 0.55 þ 0.02*

0.17 þ 0.01 0.09 þ 0.02 0.21 þ 0.02** 0.08 þ 0.02**

The values of cholesterol and unsaturation (a) are the cholesterol to total fatty acid molar ratio and the average number of unsaturations per fatty acid chain, respectively. Chain length is the mean number of carbon atoms constituting the fatty acid chains. PUFA/ MUFA is the molar ratio of the poly- to the mono-unsaturated fatty acids. The values are expressed as means þ S.E (n = 3); Samples derive from three di¡erent tissue preparations. *P 6 0.07, control vs. treated samples of the hypertrophic tissue. **P 6 0.025, control vs. treated samples of the hypertrophic tissue.

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Table 4 Release of matrix vesicles as estimated from the values of sedimentable alkaline phosphatase activity released from ROS-treated hypertrophic chondrocytes Alkaline phosphatase/LDH

Control

ROS-treated

Control/treated

18.85 þ 3.5

26.14 þ 3.0

0.72 þ 0.05

Hypertrophic chondrocytes, maintained for 7^10 days in suspension cultures in alginate gels, were treated for 16 h at 37³C in DMEM with 6 mU ml31 XO and 4 mM HX. The gels were then dissolved and matrix vesicles puri¢ed by di¡erential centrifugation as described in Section 2. The released LDH, as percentage of total activity of the cultures, was the same for both treated and untreated cultures. Data are reported as mean þ S.D. (n = 4) for the di¡erent cell preparations.

were calculated for resting and hypertrophic cartilages in control conditions as well as after treatment with ROS (see Table 3). The lipid composition of resting and hypertrophic cartilages in control conditions are similar. After treatment with ROS, the PUFA to MUFA ratio and the average unsaturation ratio decrease in both resting and hypertrophic tissues. However, such decrease is statistically signi¢cant only for hypertrophic cartilage. 3.4. Matrix vesicle production The ability of ROS in inducing hypertrophic chondrocytes to produce alkaline phosphatase-enriched matrix vesicles was also tested (Table 4). Cultured chondrocytes were treated with ROS produced at 0.006 U ml31 xanthine oxidase. The concentration of ROS was maintained low to avoid the disruption of the plasma membrane and a consequent artefactual liberation of alkaline phosphatase-containing debris. The membrane integrity was proved by the amount of the free LDH activity which was the same in treated and reference cultures. An increase of 30% in the ratio of the total sedimentable alkaline phosphatase activity to the free LDH activity of the culture after treatment of cultures with ROS indicates a signi¢cant increase in the production of alkaline phosphatase-containing bodies. 4. Discussion A ¢rst basic information can be drawn from our results: the di¡erentiated chondrocyte presents di¡erent phenotypes both in the growth plate and in the articular tissue. Moreover, in the growth plate, the

phenotype changes with the degree of di¡erentiation. A di¡erence among the di¡erent `chondrocytes' is represented by their di¡erent expression of dismutating activities. SOD and catalase activity in chondrocytes have been studied in the past with contrasting results by Davis et al. [35], Matsumoto et al. [27] and by Deahl et al. [36]. Our results show a high SOD speci¢c activity in resting chondrocytes, while a higher catalase speci¢c activity is expressed by the hypertrophic cells. While the distribution pattern of SOD con¢rms the results of Matsumoto et al. [27], that of catalase is just the opposite of that which was found by these authors. This contradiction may be ascribed to the di¡erent sources of cartilage; in fact, in their work, Matsumoto and coworkers studied chicken cartilage, which presents a vessel distribution at the bone/cartilage boundary di¡erent from that occurring in pig scapula. It is of relevance that Deahl et al. [36] detected catalase activity only at the level of the plasma membrane of hypertrophic chondrocytes. Even if their results are only qualitative, the authors suggest that this localization of the enzyme is related to the presence of H2 O2 and hydroperoxides in the extracellular matrix. In this study, we have demonstrated that catalase is present in all chondrocyte types, but also that it is expressed at most in hypertrophic cells. This fact suggests that HCs have a better protection against extracellular H2 O2 . Support of this hypothesis is given by the data of Teixeira et al. [37] which show that, at least in vitro, chondrocytes release antioxidant enzymes as a protection against hexogenous ROS. PHGPx (phospholipid hydroperoxide glutathione peroxidase) is a seleno-enzyme, invested of the role of protecting cell membranes against a peroxidative damage; our data show its presence in cartilage. Despite the fact that immunoblotting can give only a

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semi-quantitative evaluation of the amount of the enzyme present in the cells, RCs seem to express a higher amount of enzyme with respect to the hypertrophic cells and, therefore, they can potentially be more resistant to lipoperoxidation. It is worth mentioning that the ROS generated by the system hypoxanthine-xanthine oxidase is the peroxide radical …O3 2 † which could rapidly dismutate, by action of SOD, producing molecular oxygen and hydrogen peroxide. In its turn H2 O2 can also produce hydroxyl radicals (OHW ), through the Fenton reaction with Fe2‡ . As a consequence, it is highly probable that di¡erent ROS are acting in our experimental systems at the same time. The enzyme pattern of HCs shows that these cells are programmed to resist to the action of H2 O2 better than other chondrocyte types. However, it seems more prone to damage induced by O3 2 and more sensitive to lipoperoxidation. In fact, the results on the release of LDH activity after long-term treatment with ROS (Fig. 3 and Table 2) clearly show that the hypertrophic cells are more resistant than the other chondrocyte types. Moreover, lipoperoxidation is enhanced in HCs after treatment with ROS, as demonstrated by the signi¢cant decrease of the PUFA/ MUFA ratio and the average unsaturation ratio (Table 3). On the contrary, these two parameters are not signi¢cantly changed in the case of resting cells. This fact may be due to a di¡erent content in fatty acid residues [38], but it may be also due to the high PHGPx activity present in RCs exerting a lipid protection. Another point of interest is the observation that the degree of unsaturation of fatty acids from hypertrophic cartilage is signi¢cantly reduced after exposure of the tissue to the ROS. A similar decrease has been observed by Wuthier [38] by comparing the unsaturation level of fatty acids of hypertrophic cells and that of matrix vesicles. Ex vivo 31 P-NMR spectroscopy experiments were performed to follow, as a function of time, the decrease of intracellular ATP promoted by incubation with ROS. Apparently, there is no di¡erence among the various chondrocyte types and the fact that the concentration of ATP falls after a very short treatment with ROS is consistent with the observation of Baker et al. [15] that H2 O2 causes the oxidative inactivation of the 3-phosphoglyceraldehyde dehydro-

genase. The chondrocytes being mostly glycolytic [22], this enzyme inactivation by ROS can almost completely abolish ATP production. The fact that low ROS doses can stimulate matrix vesicle production is also of particular relevance. This phenomenon appears to be an active process of membrane budding instead of a simple loss of membrane debris as indicated by the release of LDH activity by the treated samples identical to that of controls. In addition, as it was recently demonstrated that low doses of ROS can induce cell proliferation [39], an artefactually higher matrix vesicle production by an increased number of cells in the treated cultures may be suspected. This seems not to be the case for two reasons: the fact that the total DNA of the ROS-treated cultures is unchanged with respect to that of the untreated one; and that the treatment time (16 h) is shorter than the duplication time (48 h in our experimental conditions) of the cells. In conclusion, the following information has been collected: HCs of growth plate have a unique pattern of antioxidant enzymatic activities and they are particularly protected against damage induced by H2 O2 . High ROS concentration induces a rapid disappearance of intracellular ATP at ¢rst and, thereafter, membrane lipid peroxidation and membrane disruption with the loss of cellular components. On the other hand, low doses of ROS induce an increase in matrix vesicle production. The information so far obtained in vitro appears to ¢t the events occurring in vivo during endochondral ossi¢cation. At the bone^cartilage interface the hypertrophic chondrocytes are destroyed. This may occur for various reasons, one of which can be the in£uence of high doses of ROS (probably as H2 O2 ) released by the active-resorbing cells of the vascular tips. The hypertrophic cells at more than one cell layer distance from the advancing tip would be exposed to lower doses of ROS and thus stimulated to produce matrix vesicles. Obviously, a direct demonstration of the involvement of ROS in these processes is still to be obtained in vivo. An evaluation of the molecular status (molecular weight, aggregation ability, ¢bril formation, etc.) of the matrix components, a detailed study on the lipid components of HCs and the identi¢cation of ROS in living tissue may contribute to the understanding of the true role of ROS

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in endochondral ossi¢cation. Such studies are in progress. Acknowledgements This work was supported by grants of the Ministry of University and Scienti¢c and Technological Research, and of the University of Trieste, Italy. Thanks are due to Profs. Ursini and Roveri for the anti-PHGPx antibody. The assistance of Miss. A. Giovannini is highly appreciated.

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