23 1

ARTICULAR CARTILAGE VESICLES GENERATE

CRYSTALS IN VITRO CALCIUM PYROPHOSPHATE DIHYDRATE-LIKE

BETH A. DERFUS, JOHN W. RACHOW, NEIL S. MANDEL, ADELE L. BOSKEY, MICHAEL BUDAY, VLADIMIR M. KUSHNARYOV, and LAWRENCE M. RYAN

Objective. To identify the morphology of a mineral-forming fraction of adult porcine hyaline artic- ular cartilage digest and characterize the mineral it forms.

Methods. Electron microscopy, Fourier trans- form infrared (FTIR) spectroscopy, x-ray microanalysis, compensated polarized light microscopy, and biochemical studies including 14C-labeled UDPG pyrophosphohydro- lase radiometric assay.

Results. This fraction of articular cartilage digest contained membrane-limited vesicles resembling growth

From the Division of Rheumatology, Medical College of Wisconsin, Milwaukee, the National Veterans Administration Crys- tal Identification Center, Milwaukee, Wisconsin, and the Depart- ment of Biochemistry, Cornell University Medical College and the Hospital for Special Surgery, New York, New York.

Supported by a Biomedical Research Center grant from the Arthritis Foundation, by a VA Merit Review award, and by NIH grants DK-30579, HL-29879, and S-R37-DEO-4141. Dr. Mandel is a VA Research Career Scientist. Dr. Ryan's work is supported by a Research Career Development Award from NIAMS (5-K04-AR- 01 502).

Beth A. Derfus, MD: Rheumatology Fellow, Medical Col- lege of Wisconsin; John W. Rachow, PhD, MD: Assistant Professor of Medicine, Division of Rheumatology , University of Iowa College of Medicine, Iowa City; Neil S. Mandel, PhD: Professor of Medi- cine, Biochemistry, Biophysics, and Orthopedic Surgery, Medical College of Wisconsin, and Associate Chief of Staff for Research, Zablocki VA Medical Center; Adele L. Boskey, PhD: Professor of Biochemistry, Cornell University Medical College, and Director, Ultrastructural Biochemistry Laboratory, Hospital for Special Sur- gery; Michael Buday: Senior Research Technologist, Medical Col- lege of Wisconsin and Zablocki VA Medical Center; Vladimir M. Kushnaryov, MD, PhD: Professor of Microbiology, Medical College of Wisconsin; Lawrence M. Ryan, MD: Professor of Medicine and Director, Division of Rheumatology, Medical College of Wisconsin.

Address reprint requests to Beth A. Derfus, MD, Box 118, Milwaukee County Medical Complex, 8700 West Wisconsin Ave- nue, Milwaukee, WI 53226.

Submitted for publication March 15, 1991; accepted in revised form September 4, 1991.

plate cartilage matrix vesicles and formed mineral after only 24 hours in physiologic salt solution containing 1 mM ATP. The mineral contained inorganic pyrophos- phate, 95% of which derived from ATP, and phosphate, 93% of which derived from inorganic phosphate in the medium. The FTIR spectrum of this mineral closely resembled the spectrum of standard calcium pyrophos- phate dihydrate (CPPD) crystals. Compensated polar- ized light microscopy showed positively birefringent, rod-shaped crystals morphologically identical to CPPD. Ca:P ratios, defined by energy-dispersive microanalysis, were also consistent with CPPD.

Conclusion. The articular cartilage vesicle frac- tion of porcine hyaline cartilage is capable of generating mineral that strongly resembles CPPD.

Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate crystals occur in articular cartilage and synovial fluid of patients with diverse arthropathies (1-3). Both crystal types may initiate or amplify articular tissue destruction ( 4 3 , but the source of these crystals has not been precisely identi- fied. A sedimentable fraction of adult porcine articular cartilage digest which supports ATP-enhanced cal- cium deposition under physiologic ionic conditions has been demonstrated (6). We sought to characterize the morphology of this fraction by electron microscopy for comparison with a similar mineralizing fraction of epiphyseal growth plate matrix vesicles (7,8). We then characterized the mineral produced by this fraction, comparing it with crystals observed in human articular diseases. We report that a vesicle-containing fraction of articular cartilage digest produces both calcium phosphate and calcium pyrophosphate mineral in

Arthritis and Rheumatism, Vol. 35, No. 2 (February 1992)

232 DERFUS ET AL

vitro, the latter of which has morphologic, biochemi- cal, and spectral characteristics of CPPD.

MATERIALS AND METHODS Articular cartilage vesicle (ACV) isolation. Hyaline

articular cartilage was obtained from femoral condyles and patellae of freshly slaughtered pigs. Cartilage slices were washed with Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY), and incubated overnight in DMEM with 2% penicillin/streptomycin/Fungizone (PSF; Gibco). Cartilage slices were then sequentially treated with 0. I % (weightholume) testicular hyaluronidase, type IV-S (Sigma, St. Louis, MO), 0.5% (w/v) pancreatic trypsin (Worthington, Freehold, NJ), and 0.2% (w/v) soybean trypsin inhibitor, type I-S (Sigma), for 10 minutes each, at 37". Cartilage was then washed with Hanks' balanced salt solution without calcium or magnesium (HBSS; Gibco) and digested with 0.2% (w/v) collagenase, type I1 (Sigma) for 45 minutes and 0.05% (w/v) collagenase with 0.2% (w/v) lactal- bumin hydrolysate (Gibco) for 20 hours (6). All reagents contained 1% PSF.

The cartilage digest was centrifuged in a Beckman L8-80M ultracentrifuge (Beckman, Irvine, CA) at 300g for 10 minutes to remove intact cells, at 20,OOOg for 10 minutes to remove large cell fragments, at 50,OOOg for 60 minutes to remove residual organelles and nuclei, and at 200,OOOg for 40 minutes to pellet membrane-bound vesicles (6). The pellet from the final centrifugation which contained ACVs was washed twice with HBSS and centrifuged at 200,OOOg for 40 minutes both times. The ACVs were suspended in HBSS to a final concentration of 3-6 mg of protein/ml (9).

Electron microscopy. Unmineralized ACV suspen- sion was washed twice with HBSS and centrifuged at 200,OOOg for 40 minutes after each wash. The pellet was fixed and refrigerated at 4°C in 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.2-7.4. After 3 washes with veronal acetate buffer, pH 7.2, the specimen was postfixed with 1% osmium tetroxide in veronal acetate buffer, pH 7.2, and embedded in Polybed 812 (Polysciences, Warrington, PA). The blocks were sectioned using an Ultracut ultramicrotome (Beckman). Sections were placed on 200-mesh copper grids, stained with uranyl acetate and Reynold's lead citrate, and examined in a Philips 400 electron microscope (Philips Electronic Instruments, Mahwah, NJ).

ACV mineralization. ACV mineralization was as- sayed in a calcifying salt solution (CSS) containing 2.2 mM CaCI,, 1.6 mM KH,PO,, 1 mM MgCl,, 85 mM NaCl, 15 mM KCI, 10 mM NaHCO,, 50 mM N-tris(hydroxymethy1) methyl-2-aminoethanesulfonic acid (Sigma), and 1 mM ATP disodium salt (Sigma), pH 7.6 (7). Twenty-five microliters of ACV suspension was added to 500 pl of CSS in 1.5-ml polypropylene micro test tubes, vortexed, and incubated in a 37°C water bath. CSS was labeled with 1 pCi/ml of 4sCa (Amersham, Arlington Heights, IL). After various incuba- tion periods, samples were centrifuged at 10,OOOg for 10 minutes in a refrigerated Microfuge B (Beckman). The supernatant was removed by pipette. The remaining pellet was washed twice by resuspension in 500 pl of ice-cold CSS without ATP, and centrifuged after each wash at 10,OOOg for

10 minutes. ,'Ca contained in the mineral phase was solubi- lized with concentrated HCI and counted in a 5-ml Scin- tiverse I1 (Fisher, Itasca, IL) in a Minaxi Tri-Carb 4000 series scintillation counter (Packard, Downers Grove, IL). Results were expressed as the percentage of the total ,'Ca in CSS that precipitated.

Control ACV mineralization assays were conducted for each experiment using one of 3 methods: substitution of 25 pl of heat-inactivated (56°C for 30 minutes) ACVs for the active ACVs, incubation of active ACVs in CSS at 4"C, or incubation of active ACVs in CSS prepared without ATP.

Characterization of ACV mineral phosphate moieties derived from ATP. ATP could contribute inorganic phos- phate (P,), pyrophosphate (PP,), or unaltered ATP to the mineralized pellet. To determine the contribution of ATP to the formation of ACV mineral, mineralized pellets formed in CSS containing ~ ~ ~ P - l a b e l e d ATP of known specific activity were analyzed. Pellets were first dissolved in 1 ml0.1N HCI. Intact ATP was estimated as a percentage of the counts adsorbed to a 2% activated-charcoal slurry. 32P-labeled Pi and "P-labeled PP, do not adsorb to charcoal. Nonadsorbed counts were further characterized. 32P-labeled Pi derived from ~ ~ ~ P - l a b e l e d ATP was estimated as the counts precip- itable as acid phosphomolybdate, according to a modifica- tion of the method described by Sugino and Miyoshi (10). PP, derived from ATP was represented by the 32P-labeled PP, counts remaining in the supernatant of the acid phosphomo- lybdate precipitate.

The presence of 3ZP-labeled PP, in the phosphomo- lybdate supernatant was confirmed by its susceptibility to hydrolysis with yeast inorganic pyrophosphatase (PP,ase). Following incubation with 10 pl of a 0.1 unit/ml stock solution of bakers yeast PP,ase (Sigma) in HBSS containing 0.5 mM MgCI, and 0.4 mM MgSO, for 1 hour at 25"C, the Pi precipitation was then repeated. Additional phosphomolyb- date-precipitable counts after PP,ase treatment compared with those without PP,ase treatment were considered to be PP,-derived. Furthermore, chemical quantification of PP, in some pellets was performed by the radiometric UDFG pyrophosphohydrolase assay described by Cheung and Su- hadolnik (11). The ATP-derived moieties (Pi, PP,, or ATP) contained in the mineralized pellet were expressed as the percentage of the total pellet counts or as nanomoles per pellet. All determinations were performed in triplicate.

Characterization of ACV mineral phosphate derived from CSS. Some phosphate in mineral pellets may derive from Pi in the CSS, rather than from ATP. In experiments parallel to those just described, CSS was labeled with 32P-labeled Pi rather than with ~ ~ ~ P - l a b e l e d ATP. The final mineral pellet content of 32P-labeled Pi derived from CSS was characterized as described above.

Statistical analysis. Results of all quantitations are expressed as the mean ? SD. The Wilcoxon rank sum test was used to compare paired samples.

Fourier transform infrared analysis. After incubation periods of 2, 8, 16, and 24 hours, 10 samples of unlabeled, mineralizing ACV were taken from each incubation group, pooled, and centrifuged at 200,OOOg for 40 minutes. The mineral-containing pellets were then washed with 5 ml of ice-cold CSS, centrifuged at 200,OOOg for 20 minutes, frozen at -7O"C, and lyophilized. Lyophilized mineral was incor-

CPPD-LIKE CRYSTALS IN PORCINE CARTILAGE 233

Figure 1. Electron micrographs of articular cartilage vesicles. A, Multiple membrane-limited vesicles are seen amidst an amorphous background (original magnification x 28,000). B, Trilaminar membranclimited vesicles occasionally contain electron-dense inclusions. No intact collagen fibers are seen (original magnification x 60,000).

porated into a KBr pellet under vacuum at 12,000 psi pressure and analyzed on a Nicolet 520 FTIR spectrometer. Forty spectral scans were computer-averaged to produce each 4,000400 cm-' FTIR spectrum. Two different sets of control ACV samples were analyzed: ACV aliquots in CSS without ATP, incubated at 37"C, and ACV aliquots in CSS, incubated at 4°C.

A computerized interactive program was used to subtract absorption bands of nascent, nonmineralizing, con- trol ACVs from those of the mineral phase. Spectra were consistently subtracted to a flat baseline for nascent protein peaks. The resulting mineral spectra were then compared with standard FTIR library spectra for octacalcium phos- phate, hydroxyapatite, amorphous calcium phosphate, cal- cium orthophosphates, and dicalcium phosphate dihydrate (brushite), and monoclinic (m) and triclinic (t) CPPD from the National Veterans Administration Crystal Identification Center (Milwaukee, WI). Additional hydroxyapatite and amorphous calcium phosphate reference spectra were gen- erated from samples kindly provided by Dr. Richard Men- delsohn and Nancy Pleshko (Department of Chemistry, Rutgers University, Newark, NJ).

Energydispersive x-ray microanalysis. Mineral gener- ated by 72-hour and 12-day incubations of vesicles in CSS was washed twice and transferred to a 200-mesh, Formvar- coated copper grid. After 1 minute, residual CSS was removed with a filter paper wick, and the crystals were allowed to air dry on the grid. A control grid containing only air-dried CSS was also prepared. Individual grids were then examined in a Philips EM-400 HTG scanning transmission electron microscope (STEM) equipped with EDAX model 9100 x-ray detector.

Crystals were visualized first with transmission elec- tron microscopy. STEM images were then used for the energy-dispersive x-ray microanalysis. The EDAX system, joined with a DEC-11 computer, expressed the elemental composition of the crystal, corrected for atomic number and relative losses of specific x-ray radiation in the detector and sample. Ca:P ratios were calculated for each crystal ana- lyzed. Three crystals from 72-hour ACV mineral were ex- amined in a preliminary study. Five crystals from 12-day ACV mineral were then examined.

Compensated polarized light microscopy. Twice- washed ACV mineral from the 72-hour incubation was

234 DERFUS ET AL

examined using a compensated polarized light microscope (Leitz, Midland, Ontario, Canada). Control samples from similar incubations of CSS without ACVs were examined for comparison. The presence of PP, was confirmed by testing positively birefringent crystals for susceptibility to dissolu- tion with yeast inorganic PP,ase (10 pl of a 1 .O unit/ml stock solution of bakers yeast PP$se in HBSS containing 0.5 mM MgCl, and 0.4 mM MgSO, for 80 minutes at 25°C). Control samples were incubated in identical fluid without PP,ase. All readings were performed blindly by one of us (LMR).

50

-g 40 a -8 30 E a 20 3 d- 10 $

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0 RESULTS 2h 8h 18h 24h 48h Electron micrographs reveal trilaminar mem-

brane-bound vesicles. Electron micrographs of the initially isolated 200,000~ pellet of articular cartilage

Figure 3. Articular cartilage vesicle mineralization, as determined by the percentage of total "Ca precipitated at the times shown. Bars show the mean and SD (see Results for n values). digest revealed homogeneous vesicles measuring 57 +-

14 nm in greatest dimension (n = 50) (Figure 1). The trilaminar membrane limiting the vesicles measured 6.6 ? 0.63 nm (n = 10). These ACVs are round or oval. No bacteria, cellular organelles, or intact collagen fibrils were identified. Numerous vesicles contained electron-dense material.

ACVs mineralize in the presence of ATP. ATP- dependent mineral formation in the presence of ACVs previously described by Wortmann et al (6) was con- firmed using the 45Ca-labeled mineralization assay. Incubation of 25 pl of ACVs at 37°C for 24 and 48 hours in 500 pl of CSS showed that 6.2 5 1.2% of total 45Ca precipitated at 24 hours (n = 4) and 15.1 5 2.9% (n = 5) precipitated at 48 hours (Figure 2), which differed significantly (P < 0.05, by Wilcoxon rank sum

2or -

ACV ACV CSS ACV-HI + + +

CSS CSS/-ATP css Figure 2. Articular cartilage vesicle (ACV) mineralization, as de- termined by the percentage of total 45Ca precipitated at 24 and 48 hours of incubation. CSS = calcifying salt solution; CSSI-ATP = CSS without ATP; ACV-HI = articular cartilage vesicles-heat inactivated. Bars show the mean and SD (see Results for n values).

test). Figure 2 also demonstrates that ACVs incubated under similar conditions but without ATP precipitated only 0.4 f 0.1% (n = 3) and 0.1 f 0.02% (n = 3) of the total 45Ca at 24 and 48 hours, respectively (P < 0.01 for precipitation with versus without ATP). When ACVs were deleted from the incubation mixture, 45Ca pre- cipitation was 0.2 & 0.1% (n = 4) and 0.1 * 0.03% (n = 5) at 24 and 48 hours, respectively (P < 0.01 for precipitation with versus without ACVs). Heat- inactivated ACVs showed similarly low levels (0.23 f 0.03% [n = 31 and 0.3 2 0.12% [n = 31 45Ca precipi- tation at 24 and 48 hours, respectively) (P < 0.01 for precipitation with active versus inactive ACVs).

ACV mineral 45Ca incorporation increases pro- gressively during the first 48 hours. ACVs incubated in CSS at 37°C for 2 4 8 hours with 45Ca demonstrated a sequential, progressive increase in mineral formation (Figure 3). Variation in the percentage of 45Ca precip- itated by vesicle preparations from separate experi- ments (Figures 2 and 3) was due to variation in the concentration of protein and the enzyme activity of the preparations. In numerous extended incubations un- der identical conditions, 45Ca precipitation plateaued at 24-72 hours. There was no more 45Ca precipitated at 1 week than at 72 hours (data not shown).

ATP contributes PPi to ACV mineral. The phos- phate moiety in ACV mineral may be derived from either the 1 mM ATP or the 1.6 mM Pi in CSS. Phosphate from these sources may be incorporated into ACV mineral as PP, or Pi, or adsorbed to mineral as intact ATP. Selective trace labeling of CSS in mineralization assays with either ~ ~ ~ P - l a b e l e d ATP or "P-labeled Pi, demonstrated the contribution of each source to the ACV mineral phosphate moiety compo-

CPPD-LIKE CRYSTALS IN PORCINE CARTILAGE 235

" ATP PPi Pi Phosphate-containing moiety in pellet

Figure 4. Characterization of articular cartilage vesicle (ACV) min- eral phosphate moiety. Parallel ACV preparations were incubated for 40 hours in calcifying salt solution containing either 32P-labeled inorganic phosphate (Pi) or y3*P-labeled ATP. The final ACV mineral phosphate moiety was characterized as ATP, inorganic pyrophosphate (PR), or Pi by calculations from the starting specific activity of each tracer, the percentage of the total tracer that precipitated, and the percentage of each moiety found in the mineral pellet. Bars show the mean and SD (see Results for n values).

sition (Figure 4). Of the total ~ ~ ~ P - l a b e l e d ATP incor- porated into ACV mineral after 40 hours of incubation, the majority, 168.1 & 9.9 nmoles (n = 3), precipitated in the form of PP,; a portion, 29.6 2 9.7 nmoles (n = 3), was adsorbed ATP; and the smallest fraction, 8.9 * 1.6 nmoles (n = 3), precipitated as Pi. Of the total 32P-labeled Pi incorporated into ACV mineral, the majority, 121.4 & 1.7 nmoles (n = 3), remained Pi, and only a small amount, 9.4 -+ 1.3 nmoles (n = 3), did not precipitate in the assay method we used (modification of the method of Sugino and Miyoshi [lo]). By com- parison, the majority of PP, in ACV mineral was derived from the ATP in CSS, while most of the Pi in ACV mineral was derived from Pi in CSS.

ATP-derived PP, increases with time. Figure 5 demonstrates the phosphate moieties contained in ACV mineral over time. The percent of total y3'P- labeled ATP transformed to pelletable PP, increased with time from 28.9 5 13.9% (n = 3) at 20 hours to 65.9 f 7.7% (n = 3) at 65 hours. The percentage of the total trace label in the form of adsorbed ATP declined from 43.9 & 22.4% (n = 3) at 20 hours to 23.3 k 9.8% (n = 3) at 65 hours.

ACV Pi moiety does not increase with time. The percentage of ~ ~ ~ P - l a b e l e d ATP in the form of Pi did not rise above 7% for the 3 incubation periods studied (Figure 6). The contribution of 1.6 mM Pi in CSS to ACV mineral phosphate moieties as determined by

20h 40h 65h Figure 5. ATP-derived phosphate moiety characterization of ACV mineral over time. Contribution of ATP at 20,40, and 65 hours was determined by sequential activated-charcoal binding followed by PFWi separation of r3*P-labeled ATP labeled ACV mineral. Bars show the mean and SD (see Results for n values). See Figure 4 for definitions.

32P-labeled Pi precipitation is shown in Figure 6. The majority of 32P-labeled Pi precipitated in ACV mineral in the form of Pi within the first 20 hours of incubation.

Additional studies confirm the presence of PP,. To assure that the saturated phosphomolybdate soluble fraction from the P,/PP, separation assay was PP,, additional samples were briefly exposed to bakers yeast PPiase before PiPP, separation. This brief expo- sure of ACV mineral to PP,ase hydrolyzed 85% of the putative PP,, confirming its chemical identity.

Further confirmation of PP, in ACV mineral was obtained by measurement of PP, using the I4C- labeled UDPG pyrophosphohydrolase radiometric as-

rn C 3

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8 PPi pi

c. a,

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20h 40h 65h Figure 6. Pi-derived phosphate moiety characterization of ACV mineral over time. Contribution of Pi at 20, 40, and 65 hours was determined by sequential activated-charcoal adsorption of ATP followed by PPi/Pi separation of ACV mineral that had been tagged with 32P-Pi. Bars show the mean and SD, some of which were too small to be seen (see Results for n values). See Figure 4 for definitions.

236 DERFUS ET AL

say (1 1). After 116 hours of incubation, ACVs gener- ated 3.19 t 0.34 nmoles of PPi/hour (n = 10). This amount is similar to the 3.54 t 0.14 nmoles of PP,hour measured using Sugino and Miyoshi's method and calculated by multiplying the total number of nano- moles of each trace label incorporated into the mineral by its percentage as PP,.

FTIR spectroscopy reveals that the spectra for nascent ACVs are similar to previously published spec- tra for growth plate matrix vesicles. Nascent, nonmin- eralizing ACVs incubated in either CSS at 4°C or CSS without ATP at 37°C were lyophilized to provide control spectra for FTIR subtraction from ACV min- eral spectra. Figure 7 shows that the FTIR spectrum for these nascent ACVs demonstrates many absorp- tion bands corresponding to organic moieties (1,650 cm-' [C=O] amide I, 1,550 cm-' [N-H, C-N] amide 11, and 1,050 cm-' [P-0-C]) previously re- ported for chick epiphyseal chondrocyte-released ma- trix vesicles (12). These peaks confirm the presence of

A

I I I I I I I B

2000 1800 1600 1400 1200 1000 800 600 400 Wavenumber (cm-1)

Figure 7. Fourier transform infrared spectra for nascent nonminer- alizing vesicle (A), and after subtraction of one articular cartilage vesicle spectrum from another (B).

2 Houm

I 24 Houm

I I I I I I I I

Wavenumber (cml)

2000 1800 1600 1400 1200 1000 800 600 400

Figure 8. Fourier transform infrared (FTIR) spectra of time- dependent articular cartilage vesicle (ACV) mineral maturation. The multiple peaks at 1,037-1,147 cm-I, single prominent peak at 910 cm-I, and multiple peaks at 480-580 cm-I emerge by 24 hours, signifying organizing mineral with peak absorptions at 1 day, con- sistent with monoclinic CPPD. Nascent ACV spectra were FTIR subtracted from ACV mineral spectra at each time point.

protein and phosphate bonds and are consistent with the presence of phospholipid-containing membrane in this nascent ACV preparation. Absorption peaks at 1,046 cm-' (narrow) and 1,165 cm-' (broad) may be attributable to phospholipid membrane bonds, but the presence of some small amount of mineralization in the control preparation cannot be excluded.

Subtraction of the spectra of 2 nascent ACV

CPPD-LIKE CRYSTALS IN PORCINE CARTILAGE 237

HA

Wavenumber (cm-1)

-ACVrnlned --mcppD

Wavenumber (cm-1)

Figure 9. Articular cartilage vesicle (ACV) mineral Fourier transform infrared (FTIR) spectrum at 24 hours, showing that the greatest similarity is with the JTIR spectrum of monoclinic calcium pyrophosphate dihydrate (m-CPPD). ACP = amorphous calcium phosphate; HA = hydroxyapatite; brushite = dicalcium phosphate dihydrate.

samples prepared either without ATP or at 4°C did not reveal any additional spectral maxima, thus indicating the reproducibility of nascent ACVs which serve as the control (Figure 7B).

Kinetic analysis of ACV mineralization as moni- tored by FTIR spectroscopy showed progressive mineral maturation over the first 24 hours. The spectral evolu- tion of organized ACV mineral over time is demon- strated in Figure 8. Spectral maxima due to mineral were not identified in the first 8 hours, but they were seen at 16 hours with a distinct maxima at 910 cm-'. This peak increased in absorbance relative to neigh- boring peaks by 24 hours, which indicates increased mineralization. Broader peaks at 1,037-1,147 cm-' and 480-580 cm-' at 16 hours sharpened by 24 hours.

Protein-associated mineral may generate spec- tra with slight peak wavenumber deviations from the spectra of pure mineral, due to protein-mineral molec- ular bond contacts which perturb mineral bond con-

formation and motion. FTIR subtraction of organic moiety absorption peaks will not correct this devia- tion. Spectral standards for mineral in association with organic moieties are not well characterized. The pro- cess of FTIR subtraction of partially overlapping peaks may itself cause slight deviations in peak wave- numbers as well. The following observations were made with modest allowance of +I5 cm-' for such deviations.

ACV mineral FTIR spectrum at 24 hours most closely resembles the standard spectrum for monoclinic CPPD. Figure 9 shows the FTIR mineral spectra of amorphous calcium phosphate, hydroxyapatite, brushite, and m-CPPD for comparison to the 24-hour ACV mineral spectrum. The single peak at 906-910 cm-', which was seen at 16 and 24 hours, also occurs strongly in the m-CPPD standard spectrum. This peak is not present in either hydroxyapatite or amorphous calcium phosphate spectra. The multiple peaks at

238 DERFUS ET AL

1,037-1,147 cm-’ noted in the 24-hour ACV mineral DISCUSSION spectra are also seen in the CPPD standard, whereas hydroxyapatite and amorphous calcium phosphate each have single absorption maxima at 1,035 cm- ’ and 1,053 cm-’, respectively. The dicalcium phosphate dihydrate (brushite) spectrum contains 3 distinct peaks within this range, but lacks the single strong absorp- tion maxima at 900-930 cm-’. The morphology of the 480-580 cm- ’ multiple peaks more closely corre- sponds to the double absorption peaks of CPPD or brushite than the singlet of amorphous calcium phos- phate at 51 1 cm-’ or the doublet of hydroxyapatite at 602 and 562 cm-’.

Transmission electron microscopy and energy- dispersive x-ray microanalysis demonstrate ACV min- eral Ca:P ratios consistent with CPPD. Examination of 72-hour ACV mineral with transmission electron mi- croscopy revealed crystals of various sizes and shapes that were separate from or associated with background electron-dense material. No bacterial contamination was seen. Energy-dispersive x-ray microanalysis of individual crystals showed Ca:P ratios ranging from 0.64 to 1.07. No higher Ca:P ratios consistent with hydroxyapatite were noted. Control samples with air- dried CSS on the grids revealed neither calcium nor phosphorus precipitation. Sample staining was omit- ted in order to avoid interference of the stain with the detection of the x-ray emission from the sample crys- tal. Since samples were unstained, low contrast did not allow identification of vesicles associated with these crystals.

Washed, mature, ACV mineral generated by 12-day incubation showed more homogeneous crys- tals, with Ca:P ratios of 0.94 _t 0.11 (n = 5) , which is very close to the theoretical Ca:P ratio of 1.0 for CPPD. These crystals were located amidst amorphous background material.

The ACV mineral pellet contains positively bire- fringent crystals. Compensated polarized light micros- copy of twice-washed 72-hour ACV mineral pellets revealed a population of strongly positively birefrin- gent, rod-shaped crystals. Twinning and bundling of crystals were common. This appearance is identical to that of CPPD. No bacteria were seen under high power magnification. Control samples from a similar incuba- tion of CSS without ACVs did not show crystals. Exposure of ACV mineral to PP,ase eliminated virtu- ally all of these positively birefringent crystals, thereby confirming their PPi content.

The vesicles isolated from adult porcine hyaline articular cartilage resemble previously characterized growth plate matrix vesicles. Our ACV preparations are largely composed of vesicles, but remnants of collagen or other organic matrix components may affect their mineralizing capacity. Our ACVs have trilaminar membranes and are approximately 60 nm in diameter. Anderson’s early electron micrographs of decalcified murine growth plate matrix clearly demon- strated trilaminar membranelimited vesicles of vari- ous shapes and with diameters ranging from 30 nm to 1 pm (13). Recent examinations using slam-freezing and freeze-substitution techniques confirmed the pres- ence of trilaminar membranelimited vesicles in mu- rine and chick growth plate, and identified 3 popula- tions of vesicle sizes: 50-70 nm, 100-150 nm, and 2W250 nm (14,lS). Structurally similar vesicles have also been identified in human osteoarthritic femoral head cartilage by Ali and Griffiths (16) and by Ohira and Ishikawa (17).

The association of mineral with growth plate vesicles first demonstrated by Anderson (13) has been confirmed using unstained, high magnification images with reduced dark room exposure, showing intact vesicles surrounded by crystal rods (14). In osteo- arthritic cartilage, vesicles associated with calcium phosphate mineral have been identified (16) and “oval bodies” laden with electron-dense material resembling crystal-laden vesicles are seen in close proximity to large mineral deposits (17). Some previous ultrastruc- tural studies of crystal deposition in articular cartilage, may have failed to demonstrate the direct participation of matrix vesicles because, without specimen decalci- fication, these large mineral deposits obscure the un- derlying matrix.

The utilization of nucleoside triphosphates in articular cartilage vesicle mineralization bears similar- ity to growth plate vesicle mineralization as well. Nucleoside triphosphate participation in growth plate vesicle calcification has been well characterized (7,8,18). Although ATP is not essential for calcium deposition by growth plate vesicles, it enhances min- eralization ( 19). Articular cartilage vesicles mineral- ized very poorly in the absence of an organic phos- phate source, confirming previous observations by Wortmann et a1 (6). We have shown, however, that the phosphate moiety contribution of ATP in ACV miner- alization mirrors that of growth plate vesicle mineral- ization described by Hsu and Anderson (18). In both systems, ATP contributes the majority of PP, found in

CPPD-LIKE CRYSTALS IN PORCINE CARTILAGE 239

the mineral, while Pi from the initial calcifying media contributes most of the vesicle-generated mineral Pi.

The FTIR spectrum of mineral generated by ACVs incubated with ATP showed absorption maxima closely corresponding to those of m-CPPD. This con- trasts with the FTIR spectra of maturing growth plate vesicle mineral incubated without ATP, which corre- sponds to hydroxyapatite (12). The prominent peak near 910 cm-' found in our mineral spectrum and in the CPPD spectrum is also prominent in the spectrum of tetrasodium pyrophosphate, and most likely repre- sents the bond stretching and/or bond nodes in the P-0-P chemical group. ACV mineral contained numerous, positively birefringent, rod-shaped crystals morphologically identical to CPPD under compen- sated polarized light microscopy. These crystals dis- solve with yeast PP,ase treatment. Energy-dispersive x-ray microanalysis revealed a distinct population of crystals associated with amorphous background mate- rial, with Ca:P ratios consistent with CPPD and lower than that of basic calcium phosphates such as hy- droxyapatite. Sufficient crystal bulk was not available for definitive x-ray powder diffraction analysis.

Taken together, the FTIR spectrum, the ap- pearance under polarized light microscopy, the sus- ceptibility of this mineral to pyrophosphatase, and the consistent Ca:P ratios substantiate the presence of CPPD-like crystals in mineral generated by articular cartilage vesicles. This vesicle mineralizing system is unique in its rapid generation of crystals with promi- nent features of CPPD using articular cartilage- derived protein after incubation under physiologic ionic conditions.

Studies of the kinetics of type I collagen gel CPPD deposition have shown that monoclinic CPPD deposits preceded triclinic CPPD deposits by at least 7 weeks (20). PP, concentrations less than 0.025 mM appeared to be necessary for t-CPPD deposition, while m-CPPD deposition occurred at PP, concentrations of 0.05 mM. The m-CPPD generated at 24 hours by ACVs may be followed by the formation of t-CPPD by these vesicles as well. Investigations of ACV mineral generated over longer incubation periods with lower PP, concentrations are under way.

Potential sources of nucleoside triphosphates in articular cartilage matrix include leaking degenerating chondrocytes or, possibly, degenerating vesicles themselves. Substrate may also diffuse into cartilage from synovial fluid (21), from cells in the fluid, or from synovial membrane surrounding the fluid. We recog- nize that the ATP substrate concentration used in this

in vitro system may be higher than cartilage matrix concentrations available for mineralization in vivo. Further characterization of the minimal ATP require- ment for ACV mineralization is in progress.

CPPD and basic calcium phosphate crystals frequently appear together in the Milwaukee shoulder syndrome, in osteoarthritis, and in chondrocalcinosis (22,23). In fact, 75% of CPPD crystal-contahing joint fluids also contain basic calcium phosphate crystals (22). Matrix vesicles from growth plate cartilage gen- erate hydroxyapatite, one of these basic calcium phos- phates, in the absence of ATP. In the presence of ATP, we have shown that ACVs form PP,, which may inhibit further hydroxyapatite formation and promote the formation of CPPD. Thus, PP, concentrations may determine not only the morphology of a particular crystal (monoclinic versus triclinic), but also the type of crystal (hydroxyapatite versus CPPD) generated in mineralizing systems. These articular cartilage vesi- cles may also be capable of hydroxyapatite generation under conditions of limited nucleoside triphosphate availability or rapid PP, hydrolysis.

In summary, the data presented characterize an articular cartilage vesicle fraction similar to previously described epiphyseal growth plate vesicles in electron microscopic morphology and utilization of ATP, and utilization of ATP. The FTIR spectrum of the ACV mineral is significantly different from epiphyseal growth plate vesicle mineral described previously. ACV mineral generated in the presence of ATP shows spectral peaks which correspond closely with those of CPPD. The Ca:P ratio of this mineral by x-ray mi- croanalysis is consistent with that of CPPD. Finally, the appearance of many, PP,ase-sensitive, positively birefringent rods, by polarizing light microscopy of ACV mineral, further underscores its similarity to CPPD. Articular cartilage vesicles and the mineral they form may provide further insight into potential pathogenic mechanisms for calcium pyrophosphate dihydrate crystal deposition diseases.

ACKNOWLEDGMENTS We thank Indira KUNP, Laureen Daft, Mary Facul-

jak, and Kim Kokoshka for their excellent technical and artistic assistance in the preparation of the manuscript. We also thank Dr. Richard Mendelsohn and Nancy Pleshko for advice in the interpretation of the FTIR spectra.

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