Cell Tissue Res (2004) 315:393–406DOI 10.1007/s00441-003-0830-1

R E G U L A R A R T I C L E

Ant�nio R. Pombinho · Vincent Laiz� ·Duarte M. Molha · Sandra M. P. Marques ·M. Leonor Cancela

Development of two bone-derived cell lines from the marine teleostSparus aurata ; evidence for extracellular matrix mineralizationand cell-type-specific expression of matrix Gla protein and osteocalcinReceived: 28 May 2003 / Accepted: 27 October 2003 / Published online: 5 February 2004� Springer-Verlag 2004

Abstract A growing interest in the understanding of theontogeny and mineralization of fish skeleton has emergedfrom the recent implementation of fish as a vertebratemodel, particularly for skeletal development. Whereasseveral in vivo studies dealing with the regulation of boneformation in fish have been published, in vitro studieshave been hampered because of a complete lack of fish-bone-derived cell systems. We describe here the devel-opment and the characterization of two new cell lines,designated VSa13 and VSa16, derived from the vertebraof the gilthead sea bream. Both cell types exhibit aspindle-like phenotype and slow growth when cultured inLeibovitz’s L-15 medium and a polygonal phenotype andrapid growth in Dulbecco’s modified Eagle medium(D-MEM). Scanning electron microscopy and von Kossastaining have revealed that the VSa13 and VSa16 cellscan only mineralize their extracellular matrix whencultured in D-MEM under mineralizing conditions, form-ing calcium-phosphate crystals similar to hydroxyapatite.We have also demonstrated the involvement of alkalinephosphatase, a marker of bone formation in vivo, and Glaproteins (osteocalcin and matrix Gla protein, MGP) in theprocess of mineralization. Finally, we have shown thatVSa13 and VSa16 cell lines express osteocalcin and MGPin a mutually exclusive manner. Thus, both cell lines arecapable of mineralizing in vitro and of expressing genes

found in chondrocyte and osteoblast cell lineages, em-phasizing the suitability of these new cell lines asvaluable tools for analyzing the expression and regulationof cartilage- and bone-specific genes.

Keywords Fish-bone-derived cell culturemineralization · Matrix Gla protein · Osteocalcin ·Gilthead sea bream, Sparus aurata (Teleostei)

Introduction

The gilthead sea bream Sparus aurata has recentlybecome one of the most important fish species foraquaculture in the Mediterranean basin and in southernPortugal. The presence of high levels of skeletal abnor-malities in farm-grown fishes is a major concern becauseof the decreased survival rate of abnormal fish and thesubsequent negative commercial implications. In an effortto understand bone and cartilage metabolism and themineralization process in this commercially importantmarine fish, several studies have recently been publisheddealing with the ontogeny and mineralization of theskeleton in this species (Faustino and Power 1998, 1999;Gavaia et al. 2000). In parallel, efforts have been madetoward the cloning of cDNAs and genes corresponding toproteins involved in fish bone and cartilage formation.Both osteocalcin (bone Gla protein, BGP) and matrix Glaprotein (MGP), two vitamin-K-dependent proteins rou-tinely used as markers for bone and cartilage in mam-malian systems, have been purified, their cDNAs andgenes cloned, and specific antibodies developed (Cancelaet al. 1995; Pinto et al. 2001, 2003; Simes et al. 2003). Inaddition, a number of hormones and their receptors,which are known to affect calcium homeostasis and/orbone metabolism in higher vertebrates, have been clonedin this species. These include the parathyroid hormone-related protein (PTH-rP; Flanagan et al. 2000), andestrogen (Socorro et al. 2000), prolactin (Santos et al.2001), thyroid hormone (Nowell et al. 2001), and calci-

A.R. Pombinho and V. Laiz� contributed equally to this workThis work was partially funded with grants from the PortugueseScience and Technology Foundation PRAXIS/BIA/11159/98,POCTI/34668/Fis/2000 and POCTI/BCI/48748/2002. V.L.,S.M.P.M and A.P. were the recipients of a postdoctoral fellowship(BPD/1607/2000 and BPD/9403/2002) and a CCMAR/Universityof Algarve fellowship, respectively

A. R. Pombinho · V. Laiz� · D. M. Molha · S. M. P. Marques ·M. L. Cancela ())CCMAR, Universidade do Algarve,Campus de Gambelas, 8005-139 Faro, Portugale-mail: lcancela@ualg.ptTel.: +351-289-800971Fax: +351-289-818353

um-sensing (Flanagan et al. 2002) receptors. However,studies to characterize the gene promoter and the regu-lation of gene expression have been hampered by the lackof a suitable well-characterized in vitro cell system that isderived from any marine teleost and that has been shownto be capable of expressing bone- or cartilage-specificmarkers and of undergoing mineralization in vitro.Indeed, although a number of fish cell lines from avariety of species are available, the large majority havebeen poorly characterized in terms of gene expression,since they were originally developed almost exclusivelyfor studies of virology and toxicology (Segner 1998).Furthermore, and to the best of our knowledge, none isderived from bone or cartilage.

In this report, we describe, for the first time, thedevelopment and initial characterization of two cell linesderived from vertebrae of S. aurata. Both are capable ofmineralizing in vitro, but they express osteocalcin andmatrix Gla protein in a mutually exclusive manner andtherefore represent a valuable tool for further studies ofthe expression of genes involved in tissue mineralization.

Materials and methods

Materials

Tissue culture media (Leibovitz’s medium, L-15, and Dulbecco’smodified Eagle medium, D-MEM), fetal bovine serum (FBS),Hanks’ balanced salts (HBS), antibiotics (penicillin and strepto-mycin), antimycotics (fungizone), trypsin-EDTA solution, and L-glutamine were purchased from Invitrogen (Barcelona, Spain).Tissue culture plates were purchased from Sarstedt (Rio de Mouro,Portugal). All other reagents were purchased from Sigma-Aldrich(Sintra, Portugal), unless otherwise stated.

Fish

Juvenile gilthead sea bream (S. aurata), obtained from naturalspawned eggs, were bred at 16–20�C in 100-l seawater tanks with a12 h light-dark photoperiod, aeration of 100 ml/min, and renewalflow of 1 tank/day. Artemia and/or rotifers were used to feed thefish up to 50 days after hatching, and then artificial food (Sorgal,Porto, Portugal) was given.

Cell culture

Preparation and maintenance

Cell cultures of mixed phenotype were obtained from vertebrae ofS. aurata by adapting previously established procedures (Robeyand Termine 1985). Briefly, vertebrae were collected with sterileinstruments from young healthy specimens of about 15 cm inlength, cleaned from adherent tissues, reduced manually to smallfragments (around 8 mm3), and digested in two steps (30 min and90 min) with 0.125% collagenase in HBS at 37�C. Fragments werewashed three times with Leibovitz’s L-15 medium supplementedwith 1% penicillin-streptomycin and 1% fungizone to eliminate anysurface bacteria and fungus and then placed in 100-mm tissueculture dishes and cultured in the same medium enriched with 10%fetal bovine serum (FBS) at 22�C in a humidified atmosphere. Cellswere allowed to migrate from fragments and attach to the plate forapproximately 2 weeks and were then collected with 1� trypsin-EDTA solution and placed into new plates with fresh medium. Cell

cultures were subsequently routinely sub-cultured (1:2) at earlyconfluence by trypsinization and cells up to passage 30 were usedfor experiments.

Phenotype analysis

Cells routinely grown in L-15 were temporarily subcultured in D-MEM supplemented with 10% FBS, 1% penicillin-streptomycin,1% fungizone, and 2 mM L-glutamine, and incubated at 33�C in a10% CO2 humidified atmosphere. The cell phenotype was observedunder an Axiovert 25 inverted light microscope (Zeiss, G�ttingen,Germany) equipped with phase-contrast and linked to a C-3030digital camera (Olympus, Hamburg, Germany).

Measurement of cellular proliferation

Cells were seeded in 6-well plates at 5�104 cells/well and culturedin L-15 or D-MEM supplemented with 1%, 5%, or 10% FBS. Atappropriate times, cells were washed twice with phosphate-bufferedsaline (PBS; pH 7.4), frozen at �80�C in 0.5 ml/well distilled water,detached by using a cell scraper, and collected in a 1.5-mlmicrocentrifuge tube. The protein content of each tube wasmeasured by using the Coomassie Plus Protein Assay Reagent(Pierce, Rockford, USA) and bovine serum albumin (BSA) as thestandard.

DNA transfection

VSa cells were seeded in 6-well plates at approximately 105 cells/well, cultured in D-MEM supplemented with 10% FBS for 24–48 hand then transfected with 0.5–1.5 �g mammalian expression vectorpEGFPN1 (Clontech, Madrid, Spain) containing the enhancedgreen fluorescent protein (EGFP) by using either FuGENE 6(Roche, Amadora, Portugal) or the calcium phosphate co-precip-itation method as previously described (Pfitzner et al. 1995).Production of EGFP in living VSa cells was investigated 48 h aftertransfection by using a DM LB epifluorescence microscope (Leica,Lisbon, Portugal). The transfection efficiency for each cell line andcondition was calculated from 12 reciprocal micrographs (fromthree independent experiments) taken by using a filter suitable forfluorescein iso-thiocyanate or bright field optics.

Extracellular matrix mineralization and nodule detection

Cells were seeded in 6-well plates at 105 cells/well and cultured inL-15 or D-MEM supplemented with 10% FBS. To induce miner-alization of the extracellular matrix (ECM), confluent culturesreceived medium supplemented with 50 �g/ml L-ascorbic acid(vitamin C), 10 mM b-glycerophosphate, and 4 mM CaCl2 in orderto give, according to the medium composition supplied by themanufacturer, final phosphate concentrations of 11.8 and 10.9 mMand final calcium concentrations of 5.3 and 5.8 mM, in L-15 andD-MEM, respectively. This medium (L-15/CaGPC or D-MEM/CaGPC) was changed twice a week until cells were fixed, andmineral was revealed by von Kossa staining. For this, cells werewashed three times with PBS at room temperature, fixed with 10%formaldehyde (in PBS) for 1 h at 4�C, washed three times withdistilled water, and then incubated with 5% silver nitrate for 30 minunder ultraviolet light. Formation of mineralized nodules in theECM was observed under an inverted light microscope. Relativelevels of ECM mineralization in cultures treated with levamisole(10 �M or 1 mM) or sodium-warfarin (5 �g/ml or 500 �g/ml) weredetermined by von Kossa staining and densitometric methods withQuantity 1 software (Bio-Rad, Amadora, Portugal).

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Scanning electron microscopy

Confluent VSa13 cells were grown for 3 weeks in D-MEM undermineralizing conditions on 13-mm tissue culture coverslips, fixedin 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2)for 3 h at 4�C, and washed twice in 0.1 M sodium cacodylate for15 min at the same temperature. Samples were dehydrated at roomtemperature in a graded ethanol series (30%, 50%, 75%, 95%, and100%; 5 min each), critical-point dried, coated with carbon, andscanned in a JSM 6301F scanning electron microscope (JEOL,Peabody, USA) at 15 kV.

X-ray diffraction

Confluent VSa13 cells were grown for 3 weeks in D-MEM undermineralizing or normal conditions and prepared according topreviously established procedures (Stanford et al. 1995). Cells werewashed twice with PBS, extracted in distilled water with a cellscraper, pelleted by centrifugation (1,500 g for 10 min), and washedthree times in 70% ethanol. Pellets were freeze-dried in an Alpha1–4 Christ apparatus (Braun Biotech International, Melsungen,Germany) and stored desiccated. Vertebrae samples were preparedaccording to the procedure described by Cancela et al. (1995).Briefly, clean vertebrae were manually fragmented into smallpieces, washed in distilled water and then in 100% acetone, anddried at room temperature. Fragments were frozen in liquidnitrogen, ground to a coarse powder, and subsequently passedthrough a 1-mm sieve. The resulting powder was washed with10 volumes 6 M guanidine-HCl (3�3 h), 10 volumes distilled water(3�3 h), and 10 volumes of 100% acetone (3�3 h), and finally driedat room temperature. Samples were analyzed by X-ray diffractionin an X’Pert diffractometer (Philips, Eindhoven, Netherlands) withCu Ka irradiation being filtered by nickel.

Measurement of alkaline phosphatase activity

Confluent VSa13 and VSa16 cells were grown for 3 weeks in D-MEM under mineralizing or normal conditions. At appropriatetimes, cells were washed twice with PBS and extracted in 1%Triton X-100 with a cell scraper. Alkaline phosphatase (ALPase)activity and protein concentrations were determined from cellsuspension by using commercially available kits (procedure No.104 from Sigma, and the BCA protein assay kit from Pierce).

Measurement of calcium and phosphate deposition

Confluent VSa13 cells were grown for 3 weeks in D-MEM undermineralizing or normal conditions. At appropriate times, cells werewashed twice with PBS and extracted in 0.1 N HCl with a cellscraper. The cell suspension was incubated for 4 h at 4�C withgentle shaking then centrifuged (13,000 g for 5 min). Calcium andphosphate concentrations were determined in the supernatant byusing commercially available kits (procedures No. 587 and 360,respectively, from Sigma).

Alcian blue staining

Confluent VSa13 and VSa16 cells were grown for 3 weeks in D-MEM under mineralizing or normal conditions then stained byAlcian blue. Cells were washed twice with PBS, fixed for 1 h with10% formaldehyde (in PBS), washed twice with 0.1 N HCl, andthen incubated for 5 h at room temperature with 0.5% (w/v) Alcianblue 8GX (in 0.1 N HCl). Unbound dye was removed by extensivewashes with distilled water. Coloration was observed under aninverted light microscope and quantified, after solubilization in 1%SDS, at 480 nm.

RNA preparation and Northern blot analysis

Total RNA was extracted from confluent cultures grown in L-15 orD-MEM as described by Chomczynski and Sacchi (1987). Aliquotsof total RNA (10 mg) were fractionated on 1% agarose-formalde-hyde gels and transferred to a Nytran SuPer Charge nylonmembrane (Schleicher & Schuell, Dassel, Germany) by capillaryblotting with 10� standard saline citrate buffer (SSC;1�SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.0). DNAprobes for S. aurata actin (255-bp fragment corresponding toposition 796–1050 in the coding region, GenBank accessionNo. X89920) or Argyrosomus regius MGP (672-bp fragmentcorresponding to position 233–904 in the cDNA, GenBank acces-sion No. AF334473) were radiolabeled with [a-32P]dCTP (3000 Ci/ml; Amersham Biosciences, Carnaxide, Portugal) by using therandom priming Rediprime II kit and purified of unincorporatednucleotides by using MicroSpin G-50 columns (Amersham Bio-sciences). All hybridizations were performed overnight at 42�C inULTRAhyb buffer (Ambion, Austin, USA). Blots were washed(2�5 min) in low stringency solution (2�SSC, 0.1% SDS) and then(2�15 min) in high stringency solution (0.1�SSC, 0.1% SDS) at42�C, and were subsequently exposed to an imaging screen for 6 h.The screen was analyzed with the Bio-Rad Molecular Imagersystem GS-505. Relative levels of mRNA were determined bydensitometric methods with Quantity 1 software and normalized bycomparison with b-actin.

Reverse transcription/polymerase chain reaction techniqueand Southern blot analysis

Total RNA (2.5 �g) was treated with RNase-free DNase I for 1 h(37�C) and reverse-transcribed by Moloney-murine leukaemia virus(MMLV) reverse transcriptase (Invitrogen) according to manufac-turer’s instructions. BGP cDNA was then amplified by thepolymerase chain reaction (PCR) in a GeneAmp 2400 thermalcycler (Perkin Elmer, Boston, USA) with Taq DNA polymerase(Promega, Madison, USA) and two specific primers, viz., SBG8Fand SBG11R (Pinto et al. 2001). PCRs were performed as follows:2 min at 94�C, 20 cycles of amplification (each cycle: 30 s at 94�C,30 s at 50�C, 1 min at 72�C), and a final elongation step of 10 minat 72�C. PCR products were size-separated by electrophoresis on a2% agarose gel, transferred onto a Nytran SuPer Charge nylonmembrane, and hybridized overnight with cDNA probes for S.aurata BGP and b-actin at 42�C in ULTRAhyb buffer. Probelabeling, purification, blot washing, exposition, screen analysis, andsignal intensity determination were performed as for Northern blotanalysis.

Results

Preparation of cell cultures

Dividing cell cultures derived from S. aurata vertebraexplants were obtained as described above and cultured at22�C in L-15 supplemented with 10% FBS and a 1%antibiotic/fungizone mix. After 1 week under theseconditions, cells migrating from tissue fragments andadhering to the surface of dishes were collected andsubcultured. Two cultures (VSa13 and VSa16) indepen-dently prepared from different fish specimens werefurther analyzed. After a few passages, both cell typesshowed a spindle-like phenotype (Fig. 1). At confluence,cell growth appeared to be contact-inhibited, and all cellsexhibited an apparent homogeneous morphology that wasmaintained after more than 35 passages. Cells survivedwell under these conditions of culture, although they grew

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slowly (doubling time: approximately 4 days; Fig. 2).After an initial phase of slow growth, only VSa13 showeda significant increase in growth rate (Fig. 2). Cells wereunable to sustain any type of consistent growth in L-15supplemented with 5% or less FBS (results not shown).After 10–15 passages, cells were transferred to D-MEMmedium supplemented with 10% FBS and cultured at33�C. This induced a clear change in phenotype, withcells showing a homogenous polygonal morphology(Fig. 1) and greatly improved their survival and growthrates (Fig. 2). Both cell types doubled in approximately36 h when cultured with 10% FBS and became contact-inhibited upon reaching confluence. VSa13 were largerthan VSa16 and therefore fewer cells were present in aconfluent well containing this cell type. Both cell typeswere able to survive in D-MEM supplemented with 5%FBS, but only VSa16 cells were able to continue dividingafter the initial divisions (Fig. 2). After five passages inD-MEM, cells were transferred back to the originalculture conditions (L-15 with 10% FBS at 22�C) andexhibited, after two passages, a spindle-like phenotypesimilar to the original one (Fig. 1), without any detectableloss in cell viability.

Mineralization of the ECM

To determine the ability of VSa13 and VSa16 cells tomineralize the ECM, confluent cultures were placed inmedium containing supplements required for mineraldeposition in mammalian cultures: b-glycerophosphate as

Fig. 1a–f Phase-contrast micrographs of S. aurata VSa13 (a–c)and VSa16 (d–f) cells cultured in different growth media. a, d Cellscultured in L-15 for 10–15 passages. b, e Cells cultured in L-15then transferred to D-MEM for five additional passages. c, f Cells

from b and e transferred to L-15 and cultured in this medium forfive additional passages. Cells were grown until confluence inmedium supplemented with 10% FBS prior to each passage. Bar100 �m

Fig. 2 Growth performances of VSa13 and VSa16 cells cultured inL-15 supplemented with 10% FBS (circles) or D-MEM supple-mented with 1% (diamonds), 5% (squares) or 10% (triangles) FBS.Cells were collected starting on the second day of plating and every2 days thereafter for the next 12 days of culture. Results areexpressed as microgram protein per well versus time of collectionafter initial seeding. Results are representative of three independentexperiments

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a source of inorganic phosphate and vitamin C as acollagen synthesis cofactor (Franceschi et al. 1994).Calcium chloride as a calcium source was also provided,since our previous experiments on S. aurata calcifiedtissue-derived cell cultures revealed that the ECM couldonly mineralize in vitro in a consistent manner after theaddition of at least 2 mM CaCl2 to the culture medium.The quantity of calcium added was later optimized to4 mM in bone-derived cell cultures (unpublished).

As negative controls for mineralization, companioncultures were left untreated and non-seeded wells re-ceived medium with supplements. To detect deposition ofmineral, 3-week-treated cultures were stained by the vonKossa method and observed under an inverted lightmicroscope (Fig. 3). Cells cultured in L-15 supplementedwith calcium, b-glycerophosphate and vitamin C(CaGPC), or L-15 alone exhibited similar phenotypes,and no mineral deposition could be detected (results notshown). In contrast, many silver-stained mineral noduleswere detected in cultures grown in D-MEM/CaGPC(Fig. 3). The nodules were of various sizes and wereessentially found at sites where cells were more con-densed. No nodules were detected in untreated cultures(Fig. 3) or in non-seeded wells (results not shown).Scanning electron-microscopic analysis of 3-week-treatedVSa13 cultures confirmed the presence of mineral nod-ules embedded in the ECM (Fig. 4).

Measurement of ALPase activity

ALPase activity was determined from extracts of VSa13and VSa16 cultures grown in D-MEM under control ormineralizing conditions throughout 4 weeks of experi-ment. ALPase activity strongly increased between 1 and

2 weeks in VSa16 cultures undergoing mineralization(over 18-fold) and continued to increase for the next2 weeks (Fig. 5) reaching, by 4 weeks of treatment, levels52-fold higher than those observed after 1 week oftreatment. This increase in ALPase activity was alsoobserved in VSa13 cells (5-fold after 2 weeks and 62-foldafter 4 weeks), but activity remained at the most (at4 weeks of treatment) 50% of that observed in VSa16cells (Fig. 5).

Nature of mineral deposition

Calcium and inorganic phosphate fractions associatedwith VSa13 mineralized ECM were quantified over a 4-week culture period. Deposition of both calcium andinorganic phosphate increased progressively (Fig. 6), witha molar calcium/inorganic phosphate ratio of 1.42€0.18(mean € SD), which is close to the theoretical ratio of1.67 for hydroxyapatite (HA) crystals, Ca10(PO4)6(OH)2,the major inorganic component of mammalian and fishmature bones.

To analyze further the nature of the mineral deposited,VSa13 cells were cultured under control or mineralizingconditions and collected, after 3 weeks, together with theECM. Samples were then analyzed by X-ray diffraction,and the results obtained with the cells were comparedwith the spectra obtained with highly crystalline syntheticHA and with the mineral extracted from S. auratavertebrae (Fig. 7). The diffractogram obtained with thecells was consistent with those obtained from both HAsynthetic crystals and mineral extracts of S. auratavertebrae, indicating the bio-apatitic nature of the mineraldeposited in the ECM of VSa13 cells. Similar results were

Fig. 3a–d Phase-contrast mi-crographs of von Kossa-stainedVSa13 (a, b) and VSa16 (c, d)cells cultured in D-MEM for3 weeks under control (a, c) ormineralizing conditions (b, d).White arrows indicate silver-stained mineral nodules. Bar100 �m

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Fig. 5 Alkaline phosphatase (ALPase) activity in VSa13 andVSa16 cells cultured in D-MEM for 4 weeks under mineralizingconditions. Data are from a representative assay (of at least threeidentical experiments) and are expressed as the net increase(control values are subtracted)

Fig. 6 Time-course of calcium (Ca) and phosphate (Pi) depositionin VSa13 cells (passage 30) grown in D-MEM/CaGPC (ND notdetected/below sensitivity of the assay). Data are from a represen-tative experiment (of at least three identical experiments) and areexpressed as the net increase (control values are subtracted)

Fig. 4a–d Scanning electronmicrographs of VSa13 cellscultured in D-MEM for 3 weeksunder mineralizing conditions.White arrows indicate mineralnodules. Bars 100 �m (a),20 �m (b), 5 �m (c), 1 �m (d)

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obtained with extracts from mineralizing VSa16 cells(results not shown).

Effect of levamisole and warfarin treatmenton ECM calcification

VSa cells were grown under normal and mineralizingconditions for 3 weeks and treated twice a week with twodifferent concentrations (verified in preliminary experi-ments not to be toxic to the cells) of either levamisole, astereospecific inhibitor of ALPase, or sodium warfarin, aninhibitor of Gla-containing protein g-carboxylation. ECMmineralization of both cell lines was inhibited by levami-sole treatments (Fig. 8), but VSa13 cells were found to bemore sensitive than VSa16 cells to high concentrations(93% versus 57% of inhibition, as compared with controlvalues). On the contrary, sodium-warfarin treatmentsincreased the mineralization of both VSa13 and VSa16ECM (Fig. 8), and again, VSa13 cells were found to bemore sensitive than VSa16 cells (150% versus 80% ofinduction, as compared with control values). Together,these results demonstrated the cell- and matrix-protein-dependent process of VSa13 and VSa16 ECM calcifica-tion and, in particular, the involvement of ALPase andGla proteins in this process.

Expression of BGP and MGP

To determine the relationship between ECM mineraliza-tion and BGP and MGP gene expression, VSa cells were

cultured for 4 weeks in L-15, L-15/CaGPC, D-MEM, orD-MEM/CaGPC, and the total RNA was extracted andprocessed for either RT-PCR coupled to Southern (BGP)or Northern (MGP) analyses. Results indicated that bothcell types had no detectable levels of BGP mRNA whencultured in L-15 or L-15/CaGPC (results not shown). Incontrast, a strong signal for BGP was observed in VSa16cells cultured in D-MEM/CaGPC and undergoing miner-alization, whereas no signal was detected in controlVSa16 cells cultured in D-MEM (Fig. 9). This signal wascomparable in size to the BGP transcript amplified fromS. aurata vertebrae, which was used as a positive control.No such induction of BGP gene expression was observedin VSa13 cells cultured in D-MEM/CaGPC (Fig. 9).

Fig. 8 Effect of levamisole and sodium warfarin on ECM miner-alization of VSa13 and VSa16 cells (Cont untreated cultures).Percentages of inhibition or induction of ECM mineralization arecalculated from control values and are indicated within each bar

Fig. 9 BGP gene expression in VSa13 and VSa16 cells cultured inD-MEM. Confluent cultures were grown for 4 weeks undermineralizing conditions (M4) or left untreated (C4). C0 representsuntreated samples at the start of the experiment. Total RNA(2.5 �g) was reverse-transcribed, amplified by PCR with BGP (S.aurata BGP)-specific primers, Southern hybridized with a 32P-labeled SaBGP probe, and exposed to an imaging screen. S. auratab-actin (ACT) was amplified by PCR from the same RT reactions.RNA extracted from S. aurata vertebra (Vert.) was used as apositive control

Fig. 7 X-ray diffractograms. Top Diffraction pattern of a highlycrystalline synthetic hydroxyapatite. Middle Diffraction pattern ofbone-derived apatite from S. aurata. Bottom Diffraction pattern ofmineralized VSa13 cultures. This result was obtained by subtract-ing the non-specific signal resulting from the biological material(obtained with control VSa13/D-MEM cultures) from the totalsignal obtained from VSa13/D-MEM/CaGPC

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In contrast, MGP mRNA was detected in VSa13 cellscultured either in L-15 or in D-MEM (Fig. 10). No MGPsignal was ever detected in VSa16 cells. In comparisonwith control conditions, MGP gene expression in VSa13cells cultured in L-15/CaGPC was strongly induced(66%) by 4 weeks of treatment, whereas it was stronglyinhibited (95%) in cells cultured in D-MEM/CaGPC.Together, these results indicated that (1) BGP and MGPgene expression patterns were cell-type- and ECM-status-dependent, (2) BGP gene expression was low andspecifically induced in VSa16 cells undergoing mineral-

ization, and (3) MGP gene expression was strong andspecifically reduced in VSa13 cells undergoing mineral-ization (D-MEM/CaGPC medium, Fig. 10) and wasinduced in VSa13 cells treated with mineralizing supple-ments but not undergoing mineralization (L-15/CaGPCmedium, Fig. 10). Finally, basal levels of MGP geneexpression were higher in cells cultured in D-MEM(Fig. 10).

Alcian blue staining of ECM acid mucopolysaccharides

Chondrocytic properties of VSa cell lines were investi-gated by measuring the accumulation of acid mucopoly-saccharides within the ECM of cells grown undermineralizing conditions. After 3 weeks of culture, bothVSa13 and VSa16 mineralized ECM exhibited positiveAlcian blue staining (no staining was detected in culturesgrown under control conditions), but a more intensecoloration was observed in VSa13 mineralized culture(Fig. 11). The quantification of the dye bound to thematrix showed that VSa13 cells produced 3.4 times moreacid mucopolysaccharides when mineralizing than didVSa16 cells, suggesting a chondrocyte origin for VSa13cells.

DNA transfection

To investigate further the suitability of VSa cells for thestudy of molecular pathways in bone and cartilageformation in vitro, the transfectability of both cell lineswas evaluated by measuring the synthesis of the EGFPafter transfection of pEGFPN1 by two different methods.The lipid-based FuGENE 6 reagent achieved significanttransfection efficiencies in VSa13 and VSa16 cell lines(30% and 12%, respectively) whereas no EGFP produc-tion was detected following transfection with the calciumphosphate method (Fig. 12). Interestingly, when usingFuGENE 6, VSa13 cells were 2.5 times more trans-fectable than VSa16 cells, independently of any toxicityattributable to the reagent. The same difference wasobserved when transfecting VSa cells with pCMVLacZ(Clontech) and measuring b-galactosidase synthesis (re-sults not shown).

Discussion

The purpose of this work has been to develop, for the firsttime, in vitro model systems derived from marine teleostbone suitable for analyzing bone- and cartilage-specificgene expression and regulation and their involvement inECM mineralization. In the present work, two bone-derived cell lines from the gilthead sea bream S. auratacapable of mineralizing their ECM have been generated.They are homogeneous, well adapted to growth instandard media supplemented with mammalian serum,and have been maintained for more than 80 passages

Fig. 10 MGP gene expression in VSa13 and VSa16 cells culturedin L-15 (top A) or D-MEM (middle B). Confluent cultures weregrown for 4 weeks under mineralizing conditions (M4) or leftuntreated (C4). C0 represents untreated samples at the start of theexperiment. Total RNA (15 �g RNA/lane) was size-fractionated,transferred onto a membrane, and Northern hybridized with a 32P-labeled MGP (Argyrosomus regius MGP) or ACT (S. aurata b-actin) probe. The membrane was exposed to an imaging screen andrelative expression (bottom C) was determined by densitometricmethods (ND not detected)

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exhibiting the same stable characteristics (our unpub-lished results).

Development of bone-derived cell lines from teleost fish

VSa13 and VSa16 cultures were initiated at 22�C in L-15,a medium well suited for supporting cell growth in non-CO2-equilibrated environments and widely used to culturefish cells (Bols et al. 1994; Chi et al. 1999; Fernandez etal. 1993; Ganassin et al. 1999; Saito et al. 2000; Tong etal. 1997). In this medium, both cell types exhibited a

spindle-like phenotype and a slow growth rate and wereunable to mineralize their ECM. In contrast, when cellswere grown at 33�C in D-MEM, a medium well suited forsupporting growth of a broad spectrum of mammalian celllines, including bone-derived cells (Cooper et al. 1998;Solomon et al. 2000), and also previously used tomaintain some fish cell lines (Bejar et al. 1997; Lehaneet al. 1999), both cell types exhibited a polygonalphenotype and a rapid growth rate and were able tomineralize their ECM. The two cell types were able toadapt to readily available standard media without specialrequirements, such as the addition of NaCl, a supplementneeded to sustain the growth of some marine fish cells(Clem et al. 1961; Law et al. 1978; Li et al. 1985).Changing the culture medium brought a change inphenotype, growth performance, and mineralizing abilityof these cells, indicating the presence of factors in D-MEM necessary for cell differentiation and its capabilityfor sustaining in vitro mineralization in fish cells aspreviously seen for mammalian cells. This medium-related change in cell phenotype was reversible withoutany detectable increase in cell mortality, thus demon-strating, (1) the complete reversibility of medium-depen-dent phenotype changes and (2) the ability of calcifiedtissue-derived cells from S. aurata to differentiate ac-cording to culture conditions and factors present in culturemedium. Major differences in pH, in the buffering systemand in carbohydrate, amino acid, and vitamin levels existbetween L-15 and D-MEM media (Table 1); thesedifferences are presumably involved in the phenotypicchanges observed. However, we did not try to identify thefactor(s) responsible, since our primary interest was tofind a commercially available medium that would supportfish-bone-derived cell growth and mineralization.

Fig. 11a–d Phase-contrast mi-crographs of Alcian blue-stained VSa13 (a, b) and VSa16(c, d) cells cultured in D-MEMfor 3 weeks under control (a, c)or mineralizing conditions (b,d). Inset: Quantification of thedye bound to the mineralizedextracellular matrix of VSa13and VSa16 cells. Bar 100 �m

Fig. 12 Efficiency of DNA transfection into VSa13 and VSa16cells. Cells were transfected with the mammalian expression vectorpEGFPN1 by using the FuGENE 6 reagent or the calciumphosphate coprecipitation method. Results are given as means €standard deviation (N.D. not detected)

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In vitro mineralization of S. aurata bone-derived cell lines

Results obtained from von Kossa staining and scanningelectron-microscopic analysis demonstrated the ability ofVSa cells to mineralize their ECM under specific condi-tions, in agreement with a number of studies performed inrat, mouse, and human osteoblast-like cell lines (Costaand Fernandes 2000; Fournier and Price 1991; Keller-mann et al. 1990; Otsuka et al. 1999; Stanford et al.1995). As previously determined for mammalian bone-derived cells, VSa13 and VSa16 cell lines require thepresence of both vitamin C and an external source ofinorganic phosphate to mineralize their ECM. In mam-malian systems, vitamin C has been shown to benecessary for the in vitro differentiation of a variety ofcell types, including adipocytes, myoblasts, chondrocytes,odontoblasts, and osteoblasts, in relation to the knownaction of vitamin C on collagen matrix synthesis(Franceschi et al. 1994). Vitamin-C-treated cultures areknown to form multilayered structures that contain anabundant, highly ordered, collagenous matrix and toexhibit increased synthesis of collagenous and non-collagenous proteins, and an increase in ALPase activity(Denis et al. 1994; Franceschi et al. 1994; Spindler et al.1989). If a source of organic phosphate, such as b-glycerophosphate, is present, a discrete zone of HA-containing mineral is formed within collagen fibrils(Steitz et al. 2001). Our results indicate that, in fish, asin higher vertebrates, vitamin C is required to increasecollagen matrix formation, possibly by stimulating thehydroxylation, secretion, and processing of type I procol-lagen components, as previously shown in mammalianmodel systems (Franceschi et al. 1994). These results arealso in agreement with data from in vivo studies that haveshown that, in fish, an absence of vitamin C in the dietresults in a higher incidence of fish skeletal abnormalitiesduring development and a corresponding increase in fishmortality (Lim and Lovell 1978; Madsen and Dalsgaard1999).

If additional calcium is not supplied to the culturemedium of VSa13 and VSa16 cells, ECM mineralizationdoes not occur. Indeed, no deposition of mineral can bedetected by von Kossa staining when VSa cells arecultured in D-MEM supplemented only with b-glycero-phosphate and vitamin C, even after extended periods ofincubation (up to 6 weeks; results not shown), indicatingthat the quantity of calcium initially present in medium(1.8 mM) is not sufficient to promote the formation ofcalcium-phosphate crystals in these cells. This result is in

contrast to findings described for bone-derived mam-malian cells (Mori et al. 1998), which typically do notrequire the addition of extra calcium to induce mineral-ization, in particular when cultured in D-MEM. Together,these results indicate that although levels of calciuminitially present in D-MEM are sufficient to supportmineralization in mammalian bone cells, this is not thecase for S. aurata-derived bone cells, which requirehigher extracellular calcium concentrations to sustainmineralization. These unexpected results may be corre-lated with in vivo data indicating that marine fish such asS. aurata, whose extracellular environment is rich incalcium salts (natural calcium levels in sea water are 400–420 mg/l), can sustain relatively high fluctuations ofcirculating plasma calcium concentrations without theinduction of any apparent physiological distress (Guer-reiro et al. 2002). Therefore, circulating calcium concen-trations required as a stimulus for initiating ECM min-eralization may be higher in marine fish than in mam-mals, in which calcium homeostasis is tightly regulated(the normal range for the total serum calcium is 2.1–2.5 mM), and in which relatively small fluctuations areknown to have drastic physiological effects and may evenimpair survival (Mundy 1990). Nodule formation in bothcell types is an active process, since no nodules have beendetected in untreated cultures or in non-seeded wellsreceiving the same supplemented medium (results notshown), indicating that mineral deposition is cell-depen-dent and not the result of any calcium/phosphate precip-itation, and the direct consequence of the addition of themineralization supplements. To demonstrate further thatnodule formation observed in cultures of VSa cells isspecific for both the cell type and the culture conditions, anon-related cell line derived from S. aurata caudal fin wascultured under the same conditions. No nodules weredetected after von Kossa staining of treated cultures(unpublished). Finally, we can conclude that, in contrastto D-MEM, L-15 does not provide conditions for ECMmineralization.

Involvement of bone-specific ALPase and Gla proteinsin ECM mineralization of VSa cell cultures

As previously determined for mammalian bone-derivedcells, mineralization of VSa13 and VSa16 ECM involvesthe bone-specific ALPase. We have observed a strongincrease in ALPase activity during ECM mineralization inboth cell lines (most probably through an increase in gene

Table 1 Comparison of L-15and D-MEM growth media

L-15 D-MEM

pH 7.6 7.3Buffering system Phosphate/free base amino acids/galactose CarbonatesCarbohydrates No glucose/galactose/high pyruvate High glucose/low pyruvateAmino acids Rich mediuma Poor mediuma

Vitamins Poor mediumb Rich mediumb

a L-15 contains 3.7 times more amino acids (in total) than in D-MEMb L-15 contains 4 times less vitamins than in D-MEM

402

expression, as reported in mammalian cells; Kondo et al.1997), concomitant with an increase in calcium andinorganic phosphate deposition. Our findings are in goodagreement with the proposed role of ALPase, viz., toprovide bone-derived cells with inorganic phosphate bydephosphorylating exogenous b-glycerophosphate. Inor-ganic phosphate is then associated with calcium to formHA crystals and build up the mineralized matrix (Bellowset al. 1992). The involvement of the bone-specificALPase in ECM mineralization was confirmed in VSacell lines by the strong reduction of nodule formationobserved when levamisole, a known inhibitor of ALPaseactivity (Vanbelle 1976), was added to the culturemedium of VSa cells. Gla proteins and in particularosteocalcin and matrix Gla protein have been unequivo-cally linked with tissue mineralization through mousegenetics (Ducy et al. 1996; Luo et al. 1997). Both arenegative regulators of mineralization and available dataindicate that BGP function is linked to the specificinhibition of crystal maturation (Boskey et al. 1998),whereas MGP seems to exert a more generalized functionto prevent tissue calcification in cartilage (Yagami et al.1999) and in the vascular system (Bostrom and Demer2000). We have observed an increase in ECM mineral-ization when warfarin, a known inhibitor of g-carboxyl-ation (Berkner 2000) is added to the culture medium ofVSa cells, suggesting the involvement of Gla-containingproteins (most probably MGP in VSa13 and osteocalcinin VSa16 cells) in mechanisms controlling nodule for-mation. The inhibitory/regulatory role of Gla proteins intissue calcification (MGP) and in crystal growth (osteo-calcin) through their calcium- and HA-binding propertieshas been clearly demonstrated in vitro (Hunter et al. 1996;Nishimoto and Price 1985; Roy and Nishimoto 2002).Therefore, the effect of warfarin on the ECM mineral-ization of our fish cell lines was expected. The presenceof MGP and osteocalcin transcripts in VSa13 and VSa16cells, respectively, and the regulation of their expressionaccording to ECM mineralization status (see below) havefurther confirmed their involvement in the calcificationprocess in fish, as previously seen in mammals.

Nature of mineral crystals deposited in VSa ECM

The structure of the mineral deposited in VSa cell culturesis similar to that of calcium HA salts. Both bone and scaleof teleosts are principally made up of calcium HA saltsembedded in a matrix of type I collagen fibers (Bigi et al.2000; Lee and Glimcher 1991). To date, no fish-derivedcell line has ever been shown to be able to mineralize invitro and produce an hydroxyapatite-like matrix similar tothat observed in mammalian cells undergoing in vitromineralization (Kellermann et al. 1990). Our findingssupport the hypothesis that bone formation is an ancientprocess and provides further evidence for the importanceof our newly developed cell lines as a valid model systemfor investigating the regulation of mineralization events

and the changes that might have occurred throughoutevolution.

BGP/MGP gene expression in VSa16 and VSa13 cells

BGP transcripts were not detectable in VSa16 or inVSa13 cells grown under normal culture conditions, butits expression was up-regulated in VSa16 cells duringmineralization in D-MEM, whereas MGP mRNA wasnever detected in these cells, under any of the experi-mental conditions used. These results indicate that VSa16cells represent an early stage of osteoblast differentiation,as seen in both the in vitro and the in vivo model systemsof bone formation in mammals (Lian and Stein 1999;Marie 2001), in which pre-osteoblast-like cells can beinduced to mineralize and, at this stage of differentiation,begin to express the BGP gene. This conclusion is furthersupported by the increase in ALPase activity observedduring mineralization and the low production of acidmucopolysaccharides under the same conditions. Ourresults suggest that fish pre-osteoblasts respond to min-eralizing stimuli as mammalian cells do and indicate thatour newly developed in vitro cell system is adequate forfurther analyses of the expression of genes related toosteoblast differentiation and mineralization in this ma-rine fish model. Our data also confirm that BGP is amarker gene for mineralization events in fish, a resultpreviously suggested by our earlier in vivo studies (Pintoet al. 2001, 2003; Simes et al. 2003).

VSa13 cells strongly express the MGP gene undernormal culture conditions in both L-15 and D-MEM, butbasal expression is higher in D-MEM. Based on recentdata suggesting the direct regulation of Xenopus MGPgene expression by extracellular calcium concentrations(Concei�¼o et al. 2002), we hypothesize that the higherbasal levels of MGP seen in VSa13 cells results from thehigher calcium content in D-MEM (1.8 mM) than in L-15(1.3 mM). Furthermore, this effect is at least partiallymediated via a limited region of the proximal XenopusMGP gene promoter, which is known to interact withspecific protein nuclear factors, thus indicating that one ofthese factors may be a calcium-sensing protein (Con-cei�¼o et al. 2002). Our efforts to correlate this calcium-dependent modulation of MGP gene expression with theconcomitant expression of the recently cloned S. auratacalcium-sensing receptor have not been successful, sincethe presence of this mRNA cannot be detected in ourcells, either before or after calcium treatment (unpub-lished). This is in agreement with data on the in vitroregulation of human MGP gene expression by extracel-lular calcium (Pi et al. 1999). Pi et al. (1999) have shownthat, although a slight increase in MGP gene expression(two-fold) can be detected, no correlation can be estab-lished with the presence, in their model system, of mRNAfor the calcium-sensing receptor previously identified.Taken together, the available data suggest that theregulation of MGP gene expression may be modulatedby direct changes in extracellular calcium concentrations

403

working through a calcium-sensing-receptor-independentmechanism.

Our present data also indicate that MGP gene expres-sion is strongly down-regulated by the mineralizationstatus of the ECM in VSa13 bone-derived cells. Indeed,MGP gene expression decreases in D-MEM culturestreated over 3 weeks for mineralization and formingnodules, whereas no change is observed in similarlytreated cells but cultured in L-15, in which mineralizationis not detected. Human osteoblasts derived from boneexplants and human osteosarcoma-derived cell lines havepreviously been shown to express the MGP gene in vitro(Cancela and Price 1992; Fraser et al. 1988). In rodents,MGP expression has been shown to be modulated duringin vitro differentiation and subsequent mineralization(Barone et al. 1991), whereas in vivo, MGP has beendescribed as a gene expressed within skeletal bones andonly by chondrocytes, not by osteoblasts (Luo et al.1997). In situ hybridization data suggests that MGP isexpressed by early or hypertrophic chondrocytes but notby mature chondrocytes in mammals (Luo et al. 1995) orin fish (Simes et al. 2003). The results obtained in VSa13cells point to the possibility that these represent either aspontaneously transformed osteoblast-like cell that hasacquired the capability of expressing MGP (as seen inother mammalian transformed bone cell lines) or a cellline representing one stage of a chondrocyte lineagecapable of differentiation into a mineralizing cell type.The presence, after induction of ECM mineralization, oflower levels of ALPase activity and a much higherproduction of acid mucopolysaccharides in VSa13 than inVSa16 cells is in agreement with this second hypothesis.Our finding that VSa13 cells undergo mineralization ofthe ECM once allowed to remain confluent for severaldays is consistent with in vivo studies showing thatchondrocytes must progress, prior to mineralization, froma proliferative to a differentiated hypertrophic stageexhibiting matrix vesicles within their ECM (Anderson1995). The condensation occurring in confluent cellcultures therefore probably has a differentiation effecton VSa13 cells resulting in the expression of thehypertrophic chondrocyte phenotype.

In conclusion, we have described the establishment oftwo bone-derived cell lines suitable not only for boneformation studies and bone-/cartilage-specific gene ex-pression and regulation, but also for classical toxicolog-ical and virological studies. The development of cell linesfrom S. aurata, a marine teleost widely used in aquacul-ture and of important economic value in the Mediter-ranean and southern Atlantic coast of Europe shouldbroaden the usefulness of this vertebrate, which isincreasingly becoming a model system for studies onthe hormonal regulation of calcium homeostasis in marinefish (Guerreiro et al. 2002; Ingleton 2002). Furthermore,it should allow the convenient study of the influence oftranscription factors and other regulatory proteins on theexpression levels of fish genes in cartilage and boneformation.

Acknowledgements The authors are grateful to C. Azevedo fromICBAS, University of Porto, for the scanning electron micrographspresented in this paper and to B. J. Goodfellow from theDepartment of Chemistry, University of Aveiro, for the X-raydiffractograms. We also thank the staff of the Laboratory ofMicrobiology, University of Algarve, for technical help with thefluorescence microscopy.

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