Isolation and Characterization of Calcium-accumulating Matrix ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Val. 260, No. 29, Issue of December 15,pp. 15972-15979,1985 Printed in U.5.A.

Isolation and Characterization of Calcium-accumulating Matrix Vesicles from Chondrocytes of Chicken Epiphyseal GrowthPlate Cartilage in PrimaryCulture* (Received for publication, June 14,1985)

Roy E. Wuthier, Jia E. Chin, John E. Hale, Thomas C. Register, Laura V. Hale, and Yoshinori Ishikawa From the Department of Chemistry, University of South Carolina, Columbia, South Carolina 29208

Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primaryculture. Thechondrocytes maintain normal morphology and synthesize type I1 collagen throughout the cultureperiod. The culturederived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stageof culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphataserich MV production occurs as the cells attain confluency. Increasingtheinitial chondrocyte seeding densityproportionatelyincreases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferatingzone, indicating that MV formation is dependent on cellular differentiation. MV released by the culturedchondrocytes were compared in protein andphospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, andthe phospholipid profiles, suggest that significant modification of the plasma membrane occurs duringM V formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkalinephosphatase substrates. This culture system thus appears to be a useful model for isolating for native MV and characterizing factors required vesicle formationand mineralization. Matrix vesicles (MV’) are known to be associated with the earliest detectable mineralization in cartilage (1, 2), intramembranous bone (3, 4), mantle dentin (5, 6), and alveolar bone (7, 8). Yet despite numerous st.udies of calcification induced by MV i n uitro (9-13), there is as yet no concensus as to the mechanism by which the vesicles induce this important biologicalprocess. One of the fundamental problems

* This work was supported in part by Grant AM-18983 from the United States Public Health Service, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The abbreviations used are: MV, matrix vesicles; DMEM, Dulbecco’s modified Eagle*s medium; TES,N-tris [hydroxymethyl] methyl-2-amino-ethanesulfonicacid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CDMV, culture-derived matrix vesicles; SCL, synthetic cartilage lymph.

which has hindered progress in this area hasbeen the lack of suitable methods for MV isolation. While the original collagenase method (14) has been used in numerous studies on MV-induced calcification (9-11, 15), there hasbeen a continuing question whether the nonspecific proteases present in crude collagenase (16) alter the vesicle proteins and thereby affect the results obtained (12, 17). Other methods of vesicle isolation which do not involve protein digestion have been used (12-13). While these preparations have the ability to rapidly induce mineralization, they generally suffer from lack of homogeneity. Furthermore, there have been notable differences between MV preparations in the requirements for organic phosphate substrates and in the kinetics of the mineralization. One approach that offers promise for enabling the isolation of native-type MV is the use of cultured chondrocytes. There have now been two reports on the isolation ofMV from primary cultures of chondrocytes (18, 19); however, because of differences i s culture conditions, the mechanisms by which these culture-derived vesicles were formed, and whether the isolated MV were capable of inducing calcification are unknown. Although other cell culture studies have reported the presence of MV associated with the onset of mineralization (20,21), we report here for the firsttime the isolation of native MV from cultured chondrocytes which possess the ability to accumulate large amounts of Ca” and Pi in uitro and induce mineralization. In this paper we describe the isolation of MV produced by primary cultures of hypertrophic chondrocytes, the characterization of their constituent proteinsand phospholipids, some conditions which affect their elaboration by cells in culture, and initial results regarding their ability to accumulate mineral ions in uitro. Our studies indicate that release of alkaline phosphatase-rich MVby chondrocytes in culture is closely related to the attainmentof cell confluency and/or the state of differentiation which leads to re-expression of alkaline phosphatase synthesis. Furthermore, they show that thevesicles so produced differ significantly in protein,but notphospholipid composition, from those released by crude collagenase digestion. Our findings indicate that there may be differences between vesicles produced early and those produced later in the post-confluent period of culture with respect to their ability to accumulatepineralions. A preliminary report briefly describing some of these findings has been published (22). EXPERIMENTALPROCEDURES

Materials-Dulbecco’s Modified Eagle’s Medium (DMEM), with L-glutamine, sodium pyruvate, and glucose (1 g/liter), fetal bovine serum, and antibiotic-antimycotic were obtained from Gibco (Grand

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Formation of Matrix Vesicles in Culture Island, NY).Trypsin (type 111,2 x crystallized from bovine pancreas), hyaluronidase (type VI-S), alkaline phosphatase substrate @-nitrophenylphosphate), TES, and SDS-PAGE protein M, standards were obtained from Sigma. Collagenase (CLS 11) was purchased from Worthington, and [3ZP]orthophosphoric acid (carrier-free),[45Ca]cal(67 cium chloride (NEZ-013,1.6 Ci/mol), [methyl-1’,2’-3H]thymidine Ci/mmol), and Omnifluor were from New England Nuclear. Sterile culture flasks (75 and150 cm2) and 24-well dishes were obtained from Corning Glass Works (Corning, NY). All other chemicals used were of reagent grade and were supplied by Fisher. Cell Culture-Chondrocytes were isolated from the hypertrophic region of epiphyseal growth plate cartilage of the tibia of 8-10-weekold hybrid broiler-strain chickens (Columbia Farms, West Columbia, SC) aspreviously reported (22-24). Cell viability was determined by trypan blue exclusion and countingwith a hemocytometer. Chondrocytes were generally seeded a t a density of2.7 X IO4 cells/cm2 of culture surface area. The cultures were supplied with DMEM, fetal bovine serum (lo%, v/v), penicillin/streptomycin (10,000 units and 10 pg/ml, respectively; 2%, v/v) and antimycotic (Fungisone (25 pg/ ml); 1%, v/v), the total volume being 0.15-0.20 ml/cm2 of culture surface area. Cultures were maintained in an incubatora t 37 “C with a 95% air, 5% CO2 atmosphere. The culture medium was changed every 3 days for the duration of the experiments. PHIThymidine Incorporation-To measure the rate of cell division, [3H]thymidinewas added to the culturemedia at an activity of 40 nCi/ml. 3 dayslater themedium was removed; the cells were fixed, and DNA was precipitated and extracted (25). Samples of NaOHextracted DNA were added to a mixture of 3.2 g of Omnifluor/liter in toluene and 20% (v/v) Triton N-101 and counted in a Intertechnique SL-30 liquid scintillation spectrometer. Collagen Typing-Chondrocytes were grown on glass coverslips, rinsed three times with phosphate-buffered saline, and fixed with 70% ethanol for 5 min.The fixed cells were incubated with polyclonal rabbit anti-type I collagen (gift of Dr. Thomas Borg, University of SC) or monoclonal mouse anti-type I1 collagen (gift of Dr. Kristofen Rubin, Uppsala University, Sweden) for15 min at room temperature in a moist chamber. Following three rinses with phosphate-buffered saline the coverslips were incubated 15 min a t room temperature as above with fluorescein-conjugated goat anti-rabbit IgG and goat antimouse IgG (Cappell,WestChester, PA), respectively. After three additionalrinseswithphosphate-bufferedsaline, coverslips were mounted in 10% glycerol and observed under epifluorescence using a Nikon diaphot microscope. To further verify the type of collagen produced by the cultured cells, it was subjected to cyanogen bromide cleavage and compared with authentic type I1 collagen isolated from chicken sternal cartilage by SDS-PAGE (26). Isolation of Matrix Vesicles-At the end of each 3-day interval of culture, the DMEM was decanted and saved. The chondrocyte cultures were refed with fresh DMEM containing the aforementioned supplements. The decanted DMEM was then incubated with the highly purified hyaluronidase (1 NationalFormularyunit/ml of DMEM) for 5 min a t 37 “C to digest any proteoglycan produced by the chondrocytes that was released into the culture medium. At the end of the digestion, the supernatant was centrifuged at 13,000 X g for 20 min using a Beckman SW 28 rotor to remove any cells and cellular debris, and the resulting supernatant was subjected to a second centrifugation a t 100,000 X g for 60 min to sediment the MV. Electron Microscopy-MV pellets were prepared for transmission electron microscopy by washing with 67 mM phosphate buffer (pH 7.4), fixation in sodium cacodylate-buffered (pH 7.4) glutaraldehyde (2%, w/v) and post fixation with 1% oso4before being embedded in Spurrstandard medium (27). Sections were stained withuranyl acetateand lead nitrate and examined under aPhillipsEM-300 electron microscope. Incorporation of ~2P]0rthophosphateinto Cell and Matrix Vesicle Phospholipids-Chondrocyte cell cultures were initiated as outlined previously. From the 3rd day of culture forward, 10 pCi of [“PI orthophosphate/25 ml of DMEM was added at each successive feeding. Phospholipids were extracted from the isolated chondrocytesand MV as previously described (28). Briefly, samples (with added carrier phospholipids) were extracted with chloroform/methanol (2:1, v/v) twice according to theprocedure of Folch et al. (29). The phospholipid composition of the lipid fraction was analyzed by two-dimensional chromatography on Whatman SG-81silica gel-loaded paper (30).The radioactivity of the lipid-bearing areas from the chromatograms was measured by liquid scintillation counting. For the cell and plasma membrane fractions (to which no carrier lipid was added) the amount

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of phospholipid in each class was determined by Pi analysis of the lipid-bearing areas (31) after digestion with 70% perchloric acid. Isolation of Cellular Membrane Fractions-Chondrocytes were harvested from the culture dishes after removal of the DMEM. The cell layer was freed from the flask using a Teflon scraper, and the cells were sedimented a t 1,000 X g for 10 min. The cell pellet was then homogenized in TES/MgClp/sucrose (TMS) buffer (pH 7.4), 50 mM, 0.25mM, 10% w/v, respectively, using a ground glass homogenizer. The suspension was subjected to differential centrifugation, the microsomal pellet so obtained being resuspended in the TMSbuffer and layered on a discontinuous 10/30/40/50% (w/w) TES-buffered sucrose gradient (12). After centrifugation a t 100,000 X g for 60 min, the gradientwas fractionated, assayed for alkalinephosphatase activity, and pooled for further biochemical analyses. Electrophoresis of Membrane Proteins-Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of MV proteins was performed using 10% polyacrylamide slab gels (130 X 200 X 1.5 mm), essentially as described by O’Farrell (32). The gels were stained for protein either by the Coomassie Blue method of Weber and Osborn (33), or of Blakesley and Boezi (34), or with the silver method of Wray etal. (35). SDS-PAGE protein molecular mass standards were: carbonic anhydrase (29 kDa), ovalbumin (45kDa), bovine serum albumin (66 kDa), phosphorylase B (97.4 kDa), P-galactosidase (116 kDa), and myosin (205 kDa). In some cases, the Coomassie-Blue stained gels were scanned using a Zenith soft laser scanning densitometer. Ion Uptake by Matrix Vesicles-Uptake of 45Caby the culturederived MV was as described previously (13), except that the assay

FIG. 1. P h a s e - c o n t r a s pt h o t o m i c r o g r a p h of c u l t u r e d chicken epiphyseal growth plate hypertrophic chondrocytes. Cells were seeded at a density of 2.7 X IO4 cells/cm2 and grown 21 days in Dulbecco’s modified Eagle’s medium to which wasadded 10% (v/v) fetal bovine serum (see “Experimental Procedures”). The cells have attained confluency and have attained typical polygonal morphology. Some cells (see arrows) display refractile boundaries, sites of the beginning of nodular multilayer formation. (Magnification, X 140).

Formation of Matrix Vesicles in Culture

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Time in C u l t u r e (Days)

FIG.2. Effect of initial cell seeding density on the production of alkaline phosphatase-rich MV by hypertrophic chondrocytes in culture. MV were harvested by differential ultracentrifugation of the spent culture medium (see “Experimental Procedures”). The flask surface area was 75 cm’; the seeding densities tested were: 1.7 X 10’ (O), 3.3 X lo3 (A), 6.6 X lo3 (A),1.3 X lo4 (O), and 2.7 X lo4 (0)cells/cm2. Arrow heads mark the time a t which the cells became confluent. Note the markedly higher and more rapid release of alkaline phosphatase-richMV at thehigher seeding densities. pNPP, p-nitrophenylphosphate.

/J

Time in Culture (Days)

FIG. 3. Relationship between cumulative production of alkaline phosphatase-rich MV and the length oftime in culture by chondrocytes grown in DMEM + 10%fetal bovine serum in cell culture. Cells were seeded at 2.7 X 10 cells/cm’. Inset, rate of MV production after each successive cell feeding.Values presented are the mean f S.E. of seven successive cultures. Note the reproducible cyclicpattern in therate of MV production. The marked decline in rate of MV formation which occurred shortly after seeding (days 4-9) corresponds to thetime of cell attachment, spreading, and early rapid cell division. pNPP, p-nitrophenylphosphate.

was scaled down to accomodate the lesser yields of vesicle protein available from the cultures. Uptake was monitored by liquid scintillation counting of the filter-retained radioactivity (36). Alkaline Phosphatase Assays-Assays of AP activity were as previously described by Cyboron et al. (37), using p-nitrophenylphosphate as a substrate. RESULTS

Hypertrophic chrondrocytes released from chicken epiphyseal growth plate cartilage attached to the culture vessels

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Time in Culture IDays)

FIG.4. Relationship between cell division,cellular alkaline phosphatase activity, and release of alkaline phosphatase-rich MV into theculture medium. Cells were seeded at an initial density of 2.5 X lo4 cells/cm2 in 16-mm diameter wells.Cell division was determined by measuring incorporation of acid-precipitable [3H]thysee “Experimental Procedures.” Cellular AP activity (+) midine (e), is the total recovered from each culture well with a teflon scraper; MV-alkaline phosphatase (V)was measured in the spent culture medium as described under “Experimental Procedures.” Inset, rate of cell division (0),([3H]thymidine incorporation/dish/3-dayculture period); rate of cellular (0)and MV (V) alkaline phosphatase production (nanomoles of p-nitrophenylphosphate (PNPP) hydrolyzed per min/dish/3-day culture period, X lo-’).

within 24 h of plating, began to flatten outand undergo mitosis, and usually attained confluency between days 10 and 15 of culture (Fig. 1). Effects of Seeding Density-The initial cell-seeding density had a profound effect on MV production (Fig. 2), optimal seeding density being about 3 x lo4 cells/cm2 of growing area. At lower seeding densities, the time to confluency was considerably lengthened, and the rateof MV production was measurably reduced. When cultures were seeded at much higher densities (6 X lo4-1.5 X lo5cells/cm2)there was no significant increase in the release of alkaline phosphatase-rich MV into the culture medium (data not shown). Time Course of Matrix Vesicle Production-Fig. 3 shows the mean production of MV in seven successive primary cell cultures using a single lot of fetal bovine serum. (Individual lots of fetal bovine serum varied in their ability to support cell growth and MV production.) After a brief burst in output of alkaline phosphatase-rich vesicles immediately after seeding the cells, MV production was minimal for several days. As the cultures approached confluency, the rate of alkaline phosphatase-rich MV production increased, rapid formation ofMV normally occurring for about 10 days (Fig. 3, inset). Beyond this point, the rate of MV production often declined, although some lots of serum cultures maintained high rates of MV production for as long as 35-40 days. Relationship between Cell Division, Cellular Alkuline Phosphatase, and Matrix Vesicle Production-Studies showed that there was a parallel relationship between the cumulative


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FIG. 5. Transmission electron micrographs of MV harvested at different stages of cell culture. Cells were grown as described in the Legend to Fig. 3 and samples of spent medium were subjected to differential centrifugation to sediment the released MV after treatment of the medium with highly purified hyaluronidase to disaggregate proteoglycans (see "Experimental Procedures"). The MV pellets were washed with phosphate buffer, fixed in isosmotic glutaraldehyde and Os04, and processed for transmission electron microscopy as described under "Experimental Procedures." MV released by collagenase digestion during chondrocyte isolation (a)and CDMV harvested at day 2 ( b ) ,day 12 (c), and day 37 ( d ) of the culture period are shown. Note the decrease in size and density of most MV isolated on day 12 (c) when rates of MV-alkaline phosphatase production were low (Fig. 4), and the reappearance of large numbers of electron-dense MV late in the culture ( d ) when alkaline phosphatase levels were high. The alkaline phosphatase-rich CDMV ( b and d ) are similar in morphology to thoseisolated from fresh tissue (Bars equal 1.0 p n ; )

(a).

incorporation of ["]thymidine, cellular levels of alkaline phosphatase, and release of alkaline phosphatase-rich MV into the culture medium (Fig. 4). The levels of cellular and MV alkaline phosphatase were low for the first 12 days of culture, but during the subsequent 12-day period in which an almostconstantrate of ["]thymidine incorporation occurred, cellular alkaline phosphatase activity increased proconditions, gressively (Fig. 4, inset). Under these experimental release of alkaline phosphatase-rich MV was closely correlated with cellular alkaline phosphatase levels ( r = 0.85, n = 9). The peak rate of MV production occurred just before the time when the rate of [3H]thymidine incorporationbegan to decline. Transmission Electron Microscopy of Culture-derived Matrix Vesicles (CDMV)-Fig. 5 compares vesicles released by the collagenase method ( a )with those produced by the chondrocytes at days 2 ( b ) ,12 ( c ) , and 37 ( d ) in culture. Vesicles released into the medium by the cultured chondrocytes at different stages of the culture were generally similar in appearance to those seen in vivo or after release by collagenase digestion. The CDMV contained electron-dense amorphous material, a feature seen most frequently early (day 2) and late (day 37) in the culture period at a time when alkaline phosphatase levels were high. Midway in culture, before major production of alkaline phosphatase-rich MV reappeared (see Figs. 3 and 4),isolated CDMV were smaller and less electron-

dense (Fig. 5c) than those seen earlier or later in the culture period. Note that a number of the CDMV obtained on day 2 were distorted and containedmineral crystallites (Fig. 5b). SDS-PAGE Protein Profiles of Culture-derived Matrix Vesicles-The protein composition of sucrose gradient-purified CDMV and collagenase-released MV, and tissue-derived and culture-derived chondrocyte plasma membrane fractions were analyzed by SDS-PAGE stained with Coomassie Blue and analyzed using a soft laser scanning densitometer (Fig. 6). (Two other methods of visualization of proteins from the various vesicle fractions were also used silver staining (35) and autoradiography of I4C-amino acid-labeled proteins. These methods gave very similar protein profiles, data not shown.) Although the proteinprofiles of CDMV and collagenase-released MV had similarities (e.g. major bands at 37, 44, and 55 kDa, CDMV had larger amounts of high-molecular mass and smaller amounts of low-molecular mass proteins. The major protein bands of CDMV (Fig. 6a) were found at 67, 100, 73, 55, and 44 kDa (in that order) and lesser bands at 37, 34, 31, and 26 kDa; collagenase-released MV (Fig. 6d) had major bands at 48,33,35,55, and44 kDa (in that order) and lesser bands at 37, 50, 100, 57, and 73 kDa. Only minor differences existed between the protein patterns of cultured chondrocyte-derived plasma membrane fractions(Fig. 6c) and those obtainedfrom plasma membrane fractionsisolated from fresh tissue microsomes (Fig. 6e). Both plasmamembrane


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‘

a

29K

MW Stds L 67K

(33K

100,000 X g sedimentable pellet. The rate of production of 32P-labeled MV phospholipids increased exponentially between days 6 and 15 (Fig. 7, inset), leveled (days 15-21), and then began to decline (day 24). The phospholipid composition of the CDMV (Table I) was similar to thatpreviously observed for collagenase-released MV (28, 38) and that reported by Glaser and Conrad (18) for CDMV. There was, however, a noticeably lower level of phosphatidylcholine, higher sphingomyelin, and less evidence of phospholipase A-degraded lipids. The principal differences between the CDMV and the plasma membrane fraction were a decreased proportion of phosphatidylcholine (45% less), increased sphingomyelin (220%more), and increased phosphatidylserine (260% more), confirming that a major change in phospholipid composition of the plasma membrane accompanies the formation of MV (39). 45Caand 32PiMetabolism by Culture-derived Matrix Vesicles-Initial studies revealed that MV produced by preconfluent cultured cells were capable of accumulating small amounts of &Ca from SCL (Fig. 8, inset). No organic phosphate substrate was needed for this process; however, rate of uptake was slower than that observed with homogenizationderived MV-enriched microsomal fractions (13). Further studies with SCL-washed MV derived frompost-confluent cultures showed that these MV were also capable of accumulating large amounts of 45Caand 32Pifrom the SCL. However, here, addition of an alkaline phosphatase substrate (1 mM ATP, Fig. 7) or 2 mM AMP (data not shown) to the 45Caand 32Pilabeled SCL was needed for mineral ion accumulation. DISCUSSION

Chondrocytes isolated from the hypertrophic zone of the growth plate of 8-10-week-old broiler-strain chickens, grown in DMEM supplemented with 10% fetal bovine serum, synthesized type 11, not type Icollagen (data not shown), produced FIG. 6. Scan of CoomassieBlue-stainedSDS-PAGE electro- abundant matrix proteoglycans, and released phospholipid-, pherogram of proteins from MV and plasma membranefrac- protein-, and alkaline phosphatase-rich MV into the culture tions from cultured cells and fresh tissue. Electrophoresis was medium. Our studies indicate that production of alkaline as described under “Experimental Procedures.” Protein molecular phosphatase-rich MV is a function of cellular differentiation. weight standards (a and f ) ; CDMV (b);plasma membrane fraction Chondrocytes isolated from the proliferating and hyperfrom cultured cells (CDPM) (c); collagenase-released MV (CRMV) (d); plasma membrane fraction from fresh cartilage MV-enriched trophic regions of the growth plate differed significantly in microsomes (MVEM-PM) ( e ) . Note thatthe CDMV had larger their ability to produce alkaline phosphatase-rich MV; those amounts of higher-molecular mass bands (55-100 kDa) and smaller from the proliferating region released fewer MV, even after amounts of lower-molecular mass bands (31-44 kDa) than theCRMV extended culture, than those isolated from the hypertrophic or the CDPM fractions. region. These findings confirm and extend previous studies by Glaser and Conrad (18) who showed that embryonic chick fractions had major bands at 33, 37, and 44 kDa, and lesser chondrocytes released alkaline phosphatase-rich MV into the bands at 100, 67-73, 55, 48, and 31 kDa. This indicates that culture medium. These studies are fundamentally different, relatively little change in plasma membrane protein compo- however, from those of Golub et al. (19), who manipulated the sition occurs between cultured cells and native tissue chon- medium in which the freshly isolated chondrocytes were drocytes. Comparing the protein profiles of chondrocyte-de- placed so as toinduce release of vesicles. Production ofMV by hypertrophic chondrocytes varied rived plasma membrane fractions (Fig:6c) with that of the CDMV (Fig. 6b), there were again notable differences. While markedly depending on the stage of culture: immediately after qualitatively the two fractions had similar-sized proteins, the isolation from the tissue, the cells released large amounts of relative amounts of the 100, 73, 67, and 55 kDa bands were alkaline phosphatase-rich MV, but as they attached to the much greater, and the 44, 37, and 33 kDa bands were less culture flask, flattened and began to divide, alkaline phosphathan those seen in thechondrocyte-derived plasma membrane tase production rapidly declined to minimal levels. Production of alkaline phosphatase and release of alkaline phosphatasefractions. Phospholipid Composition of Cultured Chondrocytes and rich MV into the culture medium, which were closely correMatrix Vesicles-By using [32P]orthophosphatesupplied con- lated, began to increase only after the cells hadattained tinuously to the culture medium at defined specific activity, confluency. However, production of alkaline phosphatase-rich the composition and patternof phospholipid synthesis in the MV was not simply due to cellular degeneration. Fig. 4 (inset) cultured cells and CDMV was studied (Fig. 7). Labeling of shows that therate of MVformation increased steadily during phospholipids was detected in CDMV in the first medium an extended period when cell division occurred at a nearly change after administration of isotope (arrow), and paralleled constant rate. Further,it is importantto note that thegrowth the reappearance of alkalinephosphataseactivityin the plate chondrocytes did not exhibit classic contact inhibition,

in Culture

Formation Vesicles of Mutrix

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FIG. 7. Incorporation of [32P]orthophosphateinto MV phospholipids by cultured hypertrophic chondrocytes. For details, see "Experimental Procedures". Closed symbols, cumulative synthesis ofMV phospholipids, inset, rate of MV phospholipid synthesis a t successive stages of cell culture (open symbols). Sphingomyelin (A,A), phosphatidylcholine (H,O), phosphatidylethanolamine (+, 0),and phosphatidylserine (V,V) are shown. Note that 32Plabeling ofCDMV phospholipids closely paralleled the appearance of alkaline phosphatase activity (0)in the CDMV fraction. pNPP, p-nitrophenylphosphate.

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Time in Culture (Days) TABLEI Phospholipid composition of matrix vesicles, chondrocytes, and chondrocyte plasma membrane fractions SPH, sphingomyelin; PC, phosphatidylcholine; LPC, lysophosphatidylcholine; PE, phosphatidylethanolamine; LPE, lysophosphatidylethanolamine;PS, phosphatidylserine; PI, phosphatidylinositol; PA, phosphatidic acid; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol. Values are the mean f S.E. percentages of the total lipid P; the number of samples analyzed is given at right in parentheses. Fraction

I:

SPH

I

PC

LPC

LPE

PE PS

Matrix vesicles 31.1 f 2.0"**28.8 f 1.2' 3.0 f 0.5 21.3 f 0.6'b 0.8 f 0.lo This report 15.7 48.9 14.5 0.8 Glaser and Conrad (18 13.1 f 1.9 7.8 f 1.2 f 3.0 3.3 f 1.3 36.8 13.3 f 2.5 Wuthier (28) 20.9 f 1.5 14.9 k 2.4 33.8 f 2.4 Peress et al. (38) Chondrocytes This report Wuthier (28)

8.4 5.6 f 0.3

19.3 63.3 0.9 57.9 + 1.1 1.7 f 0.3 19.1 f 0.6

PG

PI

PA DPG

Samples

% lipid P

2.8 2 1.7 3.2 k 0.2 0.9 f 0.1 0.5 f 0.1 1.0 f 0.1 2.8 8.6 1.4 f 0.3 0.4 +. 0.4 +. 0.2 4.6 1.4 f 0.3 2.8 2 0.8 3.2 +- 0.4 f 0.8 2.6 It 0.9 8.3 2.9 f 0.9

1.0 2.1 5.2 f 1.1 3.3

I'

12.3

1

12.3

k 0.34.4 f 0.40.2 f

10.3 0.1 0.5 f 0.1 1.2 f 0.2

1

(6) (1) (17) (5) (1) (9)

Chondrocyte plasma membrane fraction 11.5 1.9 14.4 53.6 This report f 1.5 1.3 f 1.3 12.2 Wuthier et al. (12) 13.9 f 1.1 56.8 f 2.8 2.8 f 0.5 0.5 57.1 I18.6 7.6 Glaser and Conrad (18 I I I Mean value differs significantly from the matrix vesicle values reported earlier (28,38); p S 0.01. Mean value differs significantly from chondrocyte plasma membrane value reported earlier (12);p S 0.01, ~~

'

but in some areas began to form nodular multilayers (Fig. 1). Again note that cell numbers continue to increase after the attainment of confluency (Fig. 4). These findings, coupled with the finding that MV production was affected by initial cell-seeding density, suggest that maintenance of alkaline phosphatase phenotypic expression and release ofMV is dependent on tissue-derived factors and/or the effects of cellcell contact. Studies inprogress indicate that both serum and tissue-derived growth factors are essential for this type of phenotypic expression. A significant question here is whether the MV released by the cultured chondrocytes are analogous to those observed i n situ in thegrowth plate. From a variety of criteria this appears

to be so. First, the ultramicroscopic morphology of the CDMV is very similar to that seen in tissue MV, or in MV isolated by crude collagenase digestion (Fig. 5), (14, 28). Second, the CDMV show the same type of phospholipid compositional relationship to theplasma membrane of chondrocytes as that seen in MV isolated directly from the fresh tissue (28, 38). They possess the well-established enrichment insphingomyelin and phosphatidylserine, and depletion of phosphatidylcholine (Table 1)reported in several earlier studies (18, 28, 38). Third, the CDMV are enriched in alkaline phosphatase, a classic MV marker enzyme (14, 23). Fourth, they possess the ability to accumulate large amounts of 45Caand 32Piin vitro using the metastable SCL. Finally, they possess a distinctive

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.,.,

FIG. 8. Time course of 45Ca and 32Pi uptake from SCL by MV collected during thepost-confluent period of chondrocyte culture. 45Cauptake, 0,

0 , - ATP,

32Piuptake, 0,Q + ATP, 0,

0. The biphasic uptake curve.of mineral ions is typical of MVinduced mineralization (11, 13).Net ion accumulation by the CDMV at late stages of incubation had 45Ca/32Piuptake ratios of 1.59, values indicative of formation of 45Ca-deficienthydroxyapatite. Note that ATPmarkedly stimulated uptake of both mineral ions. Inset, time course of 45Cauptake from SCL by MV harvested early in the pre-confluent period of culture. Ca2+uptake, which occurred in the absence of added organic phosphatesubstrates tothe SCL, although limited and relatively slow, was nevertheless much more than seen with post-confluent CDMV in the absence of alkaline phosphatase substrates. ,

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2535

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Incubation Time (Hours)

and reproducible protein electrophoretic profile which, although related to thatof the chondrocyte plasma membrane, appears to be unique to MV. (This finding is evidence against the idea that MV are simply products of cellular degeneration.) These studies also help resolve a long-standing question regarding the intrinsic protein constituents of MV. Vesicles isolated by crude collagenase digestion are subject to degradation of the exposed surface proteins because of the nonspecific protease constituents present (16). On the other hand, MV fractions isolated by nonenzymatic methods which rely on homogenization and fractionation (12) are heterogeneous because of the presence of intracellular membrane components. The CDMV reported here overcome several important criticisms of these otherstudies. First, the CDMV are isolated by the mildest of procedures: differential centrifugation of the vesicle-containing culture medium. Second, because no proteolytic digestion' or homogenization is involved, the CDMV should be in a virtually natiue state. The fact that theCDMV are capable of accumulating large amounts of 45Caand 3zPi suggests that this is indeed true. Thus, it is significant that CDMV (Fig. 6b) differ significantly in protein composition from MV isolated by crude collagenase digestion (Fig. 6d) or from plasma membrane fractions obtainedby homogenization and sucrose gradient fractionation methods (Fig. 6, c and e ) . The presence of several characteristic intense higher-molecular mass (67, 73, and 100 kDa) protein bands which are nearly absent from collagenase-released MV indicates that some proteolytic degradation ofMV proteinsmust occur during crude collagenase digestion. Nevertheless, it should be pointed outthat MV isolated from different species may differ The hyaluronidase used is a highly purified endoglycosidase from bovine testes which was used to degrade the polysaccharide chains of the proteoglycans abundantly produced by the cultured chondrocytes and released into the culture medium.

in sensitivity to protease degradation. For example, MV derived from rat alveolar bone (39) and cartilage (39-40), and fetal bovine cartilage (40), by collagenase digestion contain significantly more high-molecular mass protein than the collagenase-released chicken MV reported here. The absence, or significantly lower levels of certain highmolecular mass, and the enrichment of several lower-molecular mass (e.g. 33,35,and 37 kDa) bands in plasma membrane fractions obtained from both cultured and nativechondrocytes indicates that modification of the protein,as well as the phospholipid constituents occurs in theplasma membrane at sites of MV formation. Since the protein profiles of the native (Fig. 6e) and cultured chondrocyte (Fig. 6c) plasma membrane fractions were very similar, it is unlikely that thedifferences seen between collagenase-released MV and CDMV resulted from changes in gene expression by the cultured cells. Comment needs to be made regarding the uptake of 45Ca and 32Piby the CDMV. The levels of mineral ions accumulated by the CDMV (10.0 pmol of 45Caand 6.3 pmol of 32Pi/mgof protein) were far higher than those observed by Glaser and Conrad (18), amounts that exceed the ability of transport systems to accumulate in theabsence of precipitate formation. These levels of uptake aresimilar to those previously observed with MV-enriched microsomes (13) in which it was established by x-ray diffraction that apatite formation had OCcurred. That apatite formation did occur during CDMV incubation is supported by the fact that these high levelsof 45Ca and 32Piuptake occurred at ratios consistent with apatite formation. Thus, the data indicate that the CDMVwere capable of inducing mineral formation within a period of about 14 h, when provided with appropriate alkaline phosphatase substrates. Furthermore, the finding that the ability of CDMV to accumulate mineral ions varies depending upon the state of culture from which they were produced may provide insight

Formation of Matrix Vesicles in Culture into the nature of MV-induced calcification. For example, it may explain some apparent discrepancies between data obtained by various groups relative to theinvolvement of organic phosphatesubstratesin MV-mediated calcification. Initial studies by Ali and Evans (9),by Hsu and Anderson (lo), and by Vaananen (15) indicated that ATP was stimulatory, if not obligatory for MV calcification. On the otherhand, ourearlier studies with nonenzymatically released MV (12, 13), and studies by Felix et al. (11) with collagenase-released MV, suggest that preformed labile mineral within MV is a key component for the mineralizing activity of such preparations. Here we find evidence for the production of both types of vesicles by the cultured cells: thosenot requiring organic phosphate substrates, isolated early after release of cells from the native tissue; and those dependent upon phosphate substrates, produced by cells during the post-confluent period of culture. It is evident that MV isolated early in the culture period contained mineral ions carried over by the freshly isolated cells. In fact, the early CDMV (Fig. 5b) show the presence of numerous MV containing mineralcrystallites. On the other hand, it is highly unlikely that the mineral seen in these early CDMV was present extracellularly at the time of cell isolation. This is based on the fact that, before seeding, the cell pellets weTe washed with pH 6 buffer to remove residual mineral. Thus, it is possible that different types of MV exist in cartilage tissue, and thatthey may be capable of inducing calcification by different mechanisms. REFERENCES 1. Anderson, H. C. (1969) J. Cell Biol. 41,59-72 2. Bonucci, E. (1970) 2. Zellforsch. Mikrosk. Anat. 103, 192-217 3. Bernard, G. W., and Pease, D. C. (1969) Am. J. Anat. 125, 271290 4. Bonucci, E. (1971) Clin. Orthop. Relat. Res. 78,108-139 5. Sisca, R. F., and Provensa, D. V. (1972) Calcif. Tissue Res. 9, 116 6. Eisenman, D. R., and Glick, P. L. (1972) J. Ultrastruct. Res. 41, 18-28 7. Sela, J., Bab, I., and Muhlrad, A. (1978) Metab. BoneDis & Relat. Res. 1,185-191 8. Bab, I., Muhlrad, A., and Sela, J. (1979) Cell Tissue Res. 202, 17 9. Ali, S. Y., and Evans, L. (1973) Biochem. J. 134, 647-650 10. Hsu, H. H. T., and Anderson, H. C. (1975) Proc. Natl. Acad. Sci. U. S. A. 75,3805-3808 11. Felix, R., Herrmann, W., and Fleisch, H. (1978)Biochem. J. 170, 681-691 12. Wuthier, R. E., Linder, R. E., Warner, G. P., Gore, S. T., and

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