JOURNAL OF BONE AND MINERAL RESEARCH Volume 6, Number 5, 1991 Mary Ann Liebert, Inc., Publishers

Structural Studies of the Mineral Phase of Calcifying Cartilage C. REY,' K. BESHAH,2 R. GRIFFIN,' and M.J. GLIMCHER3

ABSTRACT The calcified cartilage of the epiphyseal growth plate of young calves has been studied by x-ray diffraction, Fourier transform infrared spectroscopy, magic angle 31Pnuclear magnetic resonance spectroscopy, and chemical composition. The powdered tissue was separated by density centrifugation as a function of mineral content and thus qualitatively of the age of the calcium-phosphorus mineral phase. The individual density centrifugation fractions were examined separately. X-ray diffraction of the samples, especially of the lowest density fractions, revealed very poorly crystalline apatite. Fourier transform infrared spectroscopy and 31P nuclear magnetic resonance spectroscopy revealed the presence of significant amounts of nonapatitic phosphate ions. The concentration of such nonapatitic phosphates increases during the early stages of mineralization but then decreases as the mineral content steadily rises until full mineralization is achieved. The total concentration of carbonate ions was found to be much lower in calcified cartilage than in bone from the same organ (scapula). The carbonate ions are located in both A sites (OH-) and B sites (PO:-), with a distribution similar to that found in bone mineral. However, discrepancies between infrared resolution factors of phosphate and carbonate bands are consistent with a heterogeneous distribution of carbonate ions in poorly organized domains of the solid phase of calcium phosphate. These initial studies permit one to characterize the calcium phosphate mineral phase as a very poorly crystalline, immature calcium phosphate apatite, rich in labile nonapatitic phosphate ions, with a low concentration of carbonate ions compared with bone mineral of the same animal, indeed from the bone of the same organ (scapula).

INTRODUCTION a great many studies of mineralization in the cartilage of epiphyseal growth plate,('-') only a very few have addressed the problem of the structural or chemical nature of the CaP mineral phase deposited in the extracellular organic r n a t ~ i x . ( ~The - ~ I CaP mineral phase in cartilage has for the most part been identified as poorly crystalline apatite because of the similarity of its composition, morphology, and x-ray and electron diffraction and optical characteristics to that of bone. As in the case of the bone mineral, there have been a number

A

LTHOUGH THERE HAVE BEEN

of suggestions that a precursor mineral phase occurs before the appearance of apatite crystals, and several calcium phosphate solid phases have been suggested that may play this role: amorphous calcium phosphate (ACP), brushite, and octacalcium phosphate (OCP).(',6)Most of the putative precursors were proposed on the basis of the morphologic size and habit of the crystals, their chemical composition, or the absence of crystalline structure, or by analogy with precipitates of calcium phosphate solids prepared in vitro under various conditions. Brushite was identified crystallographically in samples of embryonic bovine calcified cartilage(*)and chick bone,"") but as discussed else-

'Current address: Laboratoire de Physico-Chimie des Solides, Ecole Nationale Superieure de Chimie, Institut National Polytechnique de Toulouse, 3 1400 Toulouse, France. 'Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology, Building NW 14, Cambridge, MA 02139. 3Laboratory for the Study of Skeletal Disorders and Rehabilitation, Harvard Medical School, the Children's Hospital, Boston, MA 02115.

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516

where"" this brushite was probably formed during tissue preparation. In another group of experiments investigators have reported that the solid mineral phase consisted almost entirely of amorphous CaP.(3,12) In the present study we isolated the calcified cartilage of bovine epiphyseal growth plate from the scapulae of young calves(13) and examined the density centrifugation fractions as well as unfractionated samples by x-ray diffraction. Although x-ray and electron diffraction give valuable information of the long-range order of the solid CaP mineral phase, they are unable to resolve fine structural details of the local atomic arrangement of the mineral ions. Therefore, in addition to x-ray diffraction, our investigations of the structural characteristics of the mineral phase of calcifying cartilage included methods to examine both the long-range and short-range organization of the mineral ions: magic angle spinning sample 31P nuclear magnetic resonance spectroscopy (31P-NMR), and resolution-enhanced, Fourier transform infrared spectroscopy (FTIR). Moreover, to obtain a clearer picture of the potential temporal changes that occur in the mineral phase with time and maturation, samples of calcified epiphyseal growth plate cartilage of increasing mineral content were separated by gradient density centrifugation('0) and the individual fractions also studied by the same techniques.

MATERIALS AND METHODS Calcifying cartilage was prepared from the epiphyseal growth plate of scapulae of 2-month-old calves as described,'") except that one sample was prepared by first removing and discarding the most heavily calcified tissues and using only the next moderately calcified tissue. Analyses of the samples of calcified cartilage revealed that they contained essentially all type I1 collagen; no type I was detected using type I and type I1 collagen antib0dies.1~~) In addition, one further group of samples was prepared by dissecting and discarding all but the very lightly mineralized cartilage situated in the most proximal region of the cartilage. The latter two preparations concentrated the amount of the youngest CaP mineral phase in such samples. The tissue samples were lyophilized as soon as possible after dissection and ground in liquid nitrogen. Particles under 10 pm were separated by dry sieving at room temperature and a major portion of the samples submitted to the gradient density separation procedure in nonaqueous solvents.(1o)The lowest density fractions (1.4-1.5 to 1.6-1.7), which were found to contain only a few percent of the CaP mineral phase by weight, were treated with hydrazine(14'at room temperature to remove the majority of the organic matrix, thus increasing their mineral content to a level that permitted us to obtain clear data by the physical techniques (x-ray diffraction, "P-NMR, and FTIR) used. This was carried out as follows. Samples of calcified cartilage (50 mg to 3 g) were suspended in anhydrous hydrazine (530 ml) for 24 h at room temperature. The solid residue was separated by centrifugation, resuspended in hydrazine (2-

10 ml) for an additional hour, and centrifuged twice. The solid material was washed with 5-25 ml of anhydrous alcohol three times and allowed to dry under vacuum. Examination of the samples of calcified cartilage and of in vitro precipitated samples of ACP, small crystals of poorly crystalline apatite, OCP, brushite, and whole embryonic and postnatal bone, as well as density centrifugation samples of bone, showed that none of these major calcium phosphate solid phases were significantly modified by the hydrazine procedure, as deduced by examining the samples before and after treatment by x-ray diffraction and FTIR (Rey et al., unpublished results). However, a few very minimal alterations in the nonapatitic FTIR bands were noted after hydrazine extraction. The concentrations of the nonapatitic phosphate ions resulting from room temperature hydrazine extraction were much too small to permit quantitative calculations of the possible changes in their concentration. After room temperature hydrazine treatment approximately 30-m% of the organic matrix remained in the cartilage particles as calculated from amino acid analyses. The calcium concentrations of the samples were determined by atomic absorption spectroscopy~ls~ and phosphorus by colorimetry(16)after the organic matrix was removed by ignition or oxidation in a mixture of perchloric and nitric acids. Because of the small amount of sample available, chemical analysis for carbonate content'") was performed on only a few selected samples (Table 1). The carbonate content of other samples, including those separated by density fractionation, was estimated from resolution-enhanced FTIR spectra by comparing the intensity of the v2 carbonate band (871 cm-I) to that of vI phosphate (560 cm-l) band on deconvoluted spectra. The estimated error of this method is - 10%. X-ray diffraction patterns were obtained on a classic Debye-Scherrer camera, which allowed detection of the characteristic reflection of OCP (d = 18.7A). The magic angle "P-NMR experiments were performed on a homemade spectrometer working at 119 MHz in 24 x g field strength at controlled spinning speed of 2 KHz."' 19) The IR spectra were recorded from KBr pellets (1-2 mg sample in 300 mg KBr) on a Perkin-Elmer and an Analect FX 6260 FTIR spectrometer at 4 cm-Lnominal resolution. Resolution enhancement was obtained from constructor software using the self-deconvolution technique of The Perkin-Elmer deconvolution proKauppinen et al. gram involved two parameters: the bandwidth (BW) of IR bands and a sensitivity coefficient ( K ) . The Analect program required setting the bandwidth at half-height (BW/2), a sensitivity coefficient, and an apodization function. To facilitate a comparison between various spectra, the parameters of deconvolution were kept constant for all cartilage samples.(21-23) With the Perkin-Elmer instrument the best results were obtained with a BW of 25 cm-', K = 2 in the v3P04domain, and a BW of 18 cm-', K = 2.25 in the v,PO, domain. With the Analect instrument a Bessel apodization function, BW/2 = 18 cm-I, K = 2.1 in the v,PO, domain and BW/2 = 8 cm-l, K = 2.5 in the v,CO, domain, was used. To obtain data relating to the relative crystallinity of the samples, the resolution factors of v,PO, (560 and 600 cm-I) and VzcO, (878 and 871 cm-') bands,

517

STRUCTURAL STUDIES OF MINERAL CALCIFYING CARTILAGE

TABLE1.

CHEMICAL COMPOSITION OF THE MINERAL OF CALCIFIED CARTILAGE OF THE OF THE SCAPULAE OF 2-MONrH-OLD CALVES

EPIPHYSEAL GROWTH PLATE

~

COY' Ca(%) Wholea Wholeb Fractions 1.4-1.5 Deproteinized 1.5-1.6 Deproteinized 1.6-1.7 Deproteinized 1.7-1.8 1.8-1.9 Above 1.9

P(%)

(%)

co,=/p (atoms%)

Ca/P (molar ratios)

Ca/(P + CO?) (molar ratios)

0.74 1.75

0.65 1.26

0.021 0.029

8.3 6.0

0.88 1.08

0.81 1.02

0.71 18.7 3.4 20.5 10.4 20.8 14.3 16.7 20.4

0.56 11.8 2.22 12.3 6.2 12.0 8.2 9.3 11.2

0.017 0.57 0.032d 0.28 0.130d 0.39 0.158 0.195 0.470d

7.9 NDc 3.8 ND 5.4 NDC 5.0 6.5 10.9

0.98 1.23 1.19 1.28 1.30 1.34 1.35 1.37 1.41

0.91 1.16 1.14 1.23 1.24 1.28 1.29 1.29 1.27

aWhole cartilage (sample B, Fig. 2). bWhole cartilage (sample A, Fig. 2). CNot determined. dDetermined by chemical analysis. All other carbonate values were calculated from IR spectral band intensity ratios (see Materials and Methods).

which are related to the bandwidths since the band positions are nearly constant from sample to sample, were calculated as indicated in previous papers (Fig. l).(2'.24)

RESULTS Density fractionation and chemical composition of the samples The histograms representing the results of density centrifugation fractionation of two samples of calcified cartilage are shown in Fig. 2: one (Fig. 2A) dissected somewhat closer to the metaphyseal bony region (more calcified) than the other (Fig. 2B). In both samples the most prominent fraction has a density between 1.4 and 1.5 g/cm3, very close to the density (1.35) of uncalcified dry cartilage. The mineral content of the 1.4-1.5 fraction is very low, but it accounts for about 75-80% of the total mineral contained in sample A. In sample B a still sharper distribution was obtained: 90% or more of the mineral was found in the 1.4-1.5 fraction. Density fractionation of samples of whole calcified cartilage containing considerably more mineral were essentially similar to those already p~blished.''~) Chemical analyses of the whole cartilage tissue and of the density centrifugation fractions are shown in Table 1. Because many samples had very low contents of a CaP mineral phase, the values, particularly of phosphorus, may also include organic phosphate derived from phosphoproteins or phospholipids, for example, in addition to inor-

I I

700

600

500

cm400

FIG. 1. Resolution factor (RF) determination: RF aa'/aa".

=

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REY ET AL.

X-ray diffraction

A

%

-

80

- 40 n

W

8

U W

>

0

0

B

W

The x-ray diffraction patterns of whole calcified cartilage and of fractions of increasing density derived from them are shown in Fig. 3. The x-ray diffraction patterns of the lowest density centrifugation fractions are particularly poor. Only two reflections, corresponding to d spacings of 3.45 and 2.85 A, respectively, can be identified. These correspond to characteristic reflections of apatite and undoubtedly represent reflections generated by the very small amount of the very youngest and very poorly crystalline apatite present in the cartilage. When these same samples are treated with hydrazine at room temperature several other weak reflections of poorly crystalline apatite are also clearly observed. As the density and extent of mineral deposition in the fractions increase, the resolution and sharpness of the apatitic x-ray diffraction patterns increase.

Resolution-enhanced FTIR

%

80

Among the four vibrational modes of free PO:- and C0:- ions, we chose to study v, (900-1250 cm-') and the v4 (500-650 cm-') domains of PO,. It is in these domains where the most intense bands of the ions and of the v2 (800-900 cm-') domain of CO, occur. In addition, the sharp, medium-intensity band of the group is not obscured

40

A 1.3

1.4

1.5

1.6

1.7

1.8

1.9

DENSITY FIG. 2. Histogram of calcified cartilage of the epiphyseal growth plate of the scapulae of 2-month-old calves obtained by density centrifugation. (A) Samples collected closer to metaphysis (more calcified). (B) Samples collected farther from metaphysis (proximal portion of calcified epiphyseal growth plate, less calcified). The major fraction of the cartilage in both cases has a density of 1.41.5, representing a minimally calcified tissue containing very young calcium phosphate crystals.

B

C D

ganic orthophosphates. The Ca/(P + carbonate) ratios of the mineral phase of calcified cartilage, especially in the heaviest density centrifugation fractions or hydrazinetreated samples, in which the contributions from the organic constituents of the cells and extracellular matrix are very much smaller, are quite similar to those of young bone mineral, except for the notable exception that calcified cartilage is characterized by a low carbonate content compared to bone mineral, which typically exhibits a carbonate/P ratio of about 0.20.(251(carbonate/P ratio = 0.18 for scapula bone from same specimen.)

E FIG. 3. Examples of the x-ray diffraction patterns generated by samples of calcified cartilage from the epiphyseal growth plate of the scapulae of 2-month-old calves obtained by density centrifugation: (A) 1.4-1.5; (B) 1.4-1.5 deproteinized with hydrazine; ( C ) 1.5-1.6; (D) 1.7-1.8; (E) above 1.9.

STRUCTURAL STUDIES OF MINERAL CALCIFYING CARTILAGE

by the organic matrix or by the phosphate absorption bands (Fig. 4).Iz1) In the v,P04 domain the spectra of whole samples of calcifying cartilage and of density centrifugation fractions are characterized by intense bands due to phosphate ions in an apatitic environment at 600,575, and 560 cm-I. The low wavenumber portion of this domain shows broad absorptions at about 540 and 530 cm-I. These bands have been detected in bone and enamel mineral and assigned to apatitic and nonapatitic HPO, ions.'22) The broad 530 cm-I band is particularly intense in the cartilage mineral. This band has been found in both brushite and OCP but not in HP0,-containing apatites. On the high-wavenumber portion of this domain there is a clearly resolved band at about 610 cm-I. This band is also observed as a broad shoulder in the spectral patterns obtained from bone and enamel mineral and has been assigned to a phosphate ion in a labile, nonapatitic location. The intensity of this band decreases a great deal during maturation of the mineral phase in bone, enamel, and synthetic in vitro synthesized apatites.(22tIn addition a shoulder at 620 cm-* is particularly apparent in undeproteinized samples. This band may be associated with bands at 585 and 530 cm-l. All these bands are slightly altered by extraction with hydrazine at room temperature. Since these bands are also present in synthetic preparations of apatites, however, we have assigned them to those phosphate groups that are very labile. The intensities of the bands are much weaker in the very youngest crystals of bone and enamel and in freshly pre-

519

cipitated synthetic apatites and cannot be detected in the later stages of maturation of the mineral phase. Slight changes in the v,PO, spectra are noted in fractions of increasing density, especially in the calculated values of P0,RF. A larger RF indicates that the peak has a smaller bandwidth. This in turn provides information on the crystallinity of the solid sample.'26)Contrary to maturing bone mineral, the resolution of the bands reaches a maximum at intermediate levels of density (Table 2). The lowest density fractions show broad, poorly resolved IR bands, which progressively become even more poorly resolved, as can be seen in the samples of whole cartilage obtained farther proximally from the metaphyseal bone (fraction B, Fig. 2). As the mineral content of the density fractions increases until fraction 1.7-1.8 is reached, the bands appear narrower and better resolved. However, in density fractions greater than 1.7-1.8 the bandwidth again increases, accompanied by small variations in the intensities of the bands. Treatment with hydrazine alters the spectra only very slightly. The relative amount of the nonapatitic phosphate groups reaches a maximum in the 1.6-1.7 fractions. In addition, there are slight changes in the positions of the bands in several fractions, which may explain the broadening of the phosphate bands and the concomitant change in the resolution factor in samples of whole calcified cartilage compared to each of the density fractions. The spectrum of whole samples consists of a superimposition of very close, unresolved peaks that cannot be resolved by deconvolution. This results in an increase in bandwidth with an

W

0

z

a m a

0 v) m

a

1100

900

600

cm-

'

550

FIG. 4. Examples of resolution-enhanced FTIR spectra generated by samples of calcified cartilage from the epiphyseal growth plate of the scapulae of 2-month-old calves obtained by density centrifugation: (1) 1.4-1.5, deproteinized; (2) 1.6-1.7, deproteinized; (3) above 1.9, not deproteinized; (a) v,PO, domain; (b) v,PO, domain; (c) v,CO, domain.

REY ET AL.

520

TABLE2. CALCIFIED CARTILAGE OF THE EPIPHYSEAL GROWTH PLATEOF THE SCAPULAE OF 2-MONTH-OLDCALVESa

Resolution factors Samples Whole cartilageb Whole cartilagec Fractions

Intensity ratios of carbonate bands

C0,RF

P04RF

878 cm-l/871 cm-'

866 cm-'/871 cm-I

0.08

0.23 0.23

0.72 0.81

0.61 0.83

0.30 0.33 0.35 0.37 0.35 0.32

0.84 0.80 0.81 0.82 0.81 0.77

0.80 0.86 0.80 0.70 0.65 0.65

0.06

1.4- 1.5 1.5-1.6 1.6-1.7 1.7-1.8 1.8-1.9

0.07 0.07 0.06 0.07 0.09

Above 1.9

0.08

aFT-IR crystallinity and intensity parameters related to v,CO, and v,PO, bands. bWhole cartilage (sample B, Fig. 2). CWhole cartilage (sample A, Fig. 2).

apparent band intensity higher than the individual density fractions, which are much more homogeneous with respect to the mineral components. The v,PO, domain appears much more complex than the v4P04domain. The spectra of hydrazine-treated whole calcified cartilage are poorly resolved even after deconvolution. This is also true of the lowest density fractions. This is probably related to the poor crystallinity of the CaP phase in both these samples, since the whole cartilage samples consist principally of the lowest density fractions. However, as the density of the fractions increases, resolution of the peaks becomes progressively better except for the highest density, most heavily mineralized fraction. The main peaks in the domain of these wavenumbers are identical to those found in enamel and bone mineral (Fig. 4). Although precise assignments are difficult to make owing to the complexity of the spectra, comparison with synthetic calcium phosphates permits the identification of several of them.(23' The main, maximum band, at -1030 crn-', exists in stoichiometric apatites and may be assigned t o PO, groups in an apatitic environment. This has been shown to be the dominant band in mature old bone and enamel. The 1020 cm-I band is found in nonstoichiometric, HP0.- and C0,-containing apatites and OCP and may be considered characteristics of nonstoichiometric apatiticlike phosphates. Two main resolved peaks exist in the domain of high wavenumbers at 1115 and 1125 cm-I. Both these bands are seen in the spectra of OCP and freshly precipitated apatites, but only the 11 15 cm- band is found in mature bone mineral. Because these peaks are absent in well-crystallized apatites, they probably represent nonapatitic phosphate ions. The 1125 cm-' peak has been shown to be associated with the 620 cm-l band occurring in the v,P04 domain. Both disappear during the maturation of synthetic apatites. The shoulder at 1140 cm-I is assigned to HP0.-containing apatites. (') Although in this domain there is a strong similarity in the spectra of OCP and calcified cartilage, especially in the 1.6-1.7 density fraction

(Fig. 5), there are several important differences. For example, the band positions of OCP and calcified cartilage are not identical. Moreover, the moderate intensity of the 910 cm-l band, which is generally considered characteristic of OCP, does not appear at all in calcified cartilage, as one would expect considering the overall well-resolved spectra of the two samples. Finally, the 620 cm-l band in the v,P04 domain, which is absent from the spectra of OCP and the 610 cm-l band and is characteristic of OCP, has a much higher intensity in calcified cartilage than one would expect if the organization of the atoms and ions in OCP were alone contributing to this portion of the spectra of the calcified cartilage. As noted earlier, these differences suggest that the atomic arrangements occurring in a poorly crystalline mineral cannot be totally identified with those of highly three-dimensionally organized crystalline solid phases, even if there are some analogies between the Very small variations in the intensities of the bands are noticed in the density-fractionated samples. For example, the relative intensities of the 1020 and 1125 cm-' peaks increase as a function of the increased density of the fractions. The variations reach a maximum in the 1.6-1.7 and the 1.7-1.8 fraction and then decrease in the density fractions progressively greater than 1.7-1.8. These variations indicate that structural changes occur with time during the progressive increase in the amount of mineral phase deposited in the calcified cartilage and with changes in the organic matrix that also occur with maturation of the mineral phase. The v,CO, bands in calcified cartilage are of low intensity with only fair to poor resolution. Nevertheless, three components may be distinguished in a similar way to those in bone and enamel mineral. The well-resolved bands at 878 and 871 cm-l have been assigned to type A and type B carbonate apatites, respectively, corresponding to the location of the carbonate ions either in monovalent (OH-) or trivalent (PO:-) anionic sites in the apatitic lattice.(z1)The

521

STRUCTURAL STUDIES OF MINERAL CALCIFYING CARTILAGE

similar to those of bone mineral, although there is more dispersion. Finally, the relative intensity of the 866 cm-I band is higher in the lowest density fractions than in the highest density fractions. The resolution of these bands has often been used to assess the crystallinity of calcium phosphate solids. Comparing the degree of crystallinity calculated from the IR spectra of carbonate and phosphate of bone mineral reveals definite differences between the mineral of cartilage and the mineral of bovine scapular and chicken bone(z21and of synthetic apatites (Tables 1 and 2). Based on the IR phosphate bands, the mineral in calcified cartilage is more crystalline than that of bone and of synthetic apatites having the same C0,RF. A similar result has been observed in the youngest crystals of 1150

1050

950

31Pnuclear magnetic resonance spectroscopy w

0

z a m

U

0 v) m

a

960

900

600

040

Because of the large sample size needed for magic angle 31P-NMR,several batches were pooled and the study performed on a reduced scale in the density centrifugation fractions. The magic angle spinning 31P-NMRspectra of calcified cartilage are similar to those of bone mineral (Fig. 6). The main, broad phosphorus peak does not have a fine structure despite the fact that FTIR demonstrates the presence of several different types of phosphate ions. The inability to detect and resolve the spectra of the different phosphate ions by "P-NMR is due to the shielding of phosphorus nuclei by the oxygen atoms of the phosphate groups. Several side bands are present on each side of the main phosphate peak, the positions and relative intensities of which have been shown to depend on the nature of the phosphate ion. I18.191 The side bands observed in calcified cartilage are similar to those of young bone mineral and have been interpreted as due to the presence of HPOj- in a noncrystalline, brushite-like environment.(lsl Faint variations in the intensity of these bands are observed in the density fractions and the maximum content of the brushite-like environment peaks in the 1.5-1.6 density fraction.

550

cm-I FIG. 5. Comparison of FTIR deconvoluted spectra of fraction 1.6- 1.7 and octacalcium phosphate (Analect spectrometer): (A) v,PO, domain; (B) enlargement of undeconvoluted spectra showing the absence of 910 cm-' band; C) v,PO, domain.

DISCUSSION

The presence of type 11 collagen as the major species of collagen in samples of whole calcified cartilage and in the density fractions, and the absence of type I collagen, establish that the samples of calcified cartilage examined in these experiments are free of bone and are derived from the cartilage of the epiphyseal growth plate. The samples faint shoulder at 866 cm-I corresponds to a labile carbo- of whole calcified cartilage included only the very youngest nate location that has been shown to gradually disappear crystals, based on the fact that they consisted almost comduring maturation of the mineral as the crystals age.(zL.z4' pletely of the 1.4-1.5 density fraction and had a CaP minAlthough it has been shown that a broad HPO, band may eral content of only 2.8% by weight. This mineral content occur in the same domain (Fig. 5B), its presence does not is very much lower than even the least calcified fraction of introduce a significant distortion of the narrow carbonate bone obtained from embryonic chicks.(18.29-311 The 1.4-1.5 band. The intensity factor ratios and the resolution factors density fraction obtained by density centrifugation had a in the calcified cartilage could not be accurately deter- mineral content slightly less than that of the whole sample mined because of the low intensities of the VzcO, bands of calcified cartilage. Both the samples of whole calcified and the poor resolution of the deconvoluted spectra. The cartilage and the 1.4-1.5 density fraction obtained from it values for the carbonate bands in calcified cartilage are represent as early a stage (probably the earliest stage) of

522

REY ET AL. Overall, then, these data allow us to characterize the first detectable solid phase of CaP deposited in calcified cartilage as a very immature, poorly crystalline CaP apatite, rich in labile, nonapatitic phosphate ions, with a very low concentration of carbonate ions in the earliest deposited mineral compared with both the bone from the scapula of the same animal and with the very youngest bone (chick embryo) thus far studied.'2') Although the maturation process of the mineral phase of calcified cartilage in general appears to proceed as it does in bone r n i n e ~ a l , ( ~ ~ - ~ ~ ) as evidenced by x-ray diffraction and chemical composition, there are distinct and important differences, particularly in the concentration of atoms and ions and in the temporal changes that occur in the mineral phase, both chemically and structurally.

Crystallinity

A

The variations in the width of the IR bands are related to small distortions in the environment of specific ions. Such distortions may arise both near or on the surface of crystals and in the interior of crystals, due to heterogeneities of composition, strain, ionic substitutions or vacancies in the lattice, and so on. The variations of the width of IR spectral bands is conveniently measured in spectra with low or average resolution by the resolution factors of neighboring bands with nearly the same intensity and quasi-invariant position-like bands in v4P04and v2C03domains. However, information from broadening of the IR bands can be related only t o the most adjacent ions. The discrepancy between CO,RF and P04RF, observed in the density fractions of calcifying cartilage, suggests that there are two domains of different crystallinity that coexist in L the mineral phase of calcified cartilage: a carbonate-con+5 0 -5 -10 taining domain that is poorly organized with respect to the apatitic domains, and carbonate-free domains with a more kHz organized atomic organization and higher degree of crysFIG. 6. 31P-NMR spectra of density centrifugation frac- tallinity. The changes in the IR crystallinity parameters intions of calcified cartilage of 2-month-old calf scapulae: dicate that the crystallinity of the carbonated domains do (A) 1.4-1.5; (B) 1.5-1.6; (C) 1.6-1.7; (D) above 1.7. not substantially change during the progressive mineralization of the tissue. On the contrary, a slight increase in the crystallinity is observed in the carbonate-free domains as biologic calcification in vertebrate skeletal tissues that has mineralization progresses. This may be due to crystal been examined by x-ray diffraction, FTIR spectroscopy, growth or a better atomic organization of the previously and magic angle spinning 3'P-NMR. The x-ray diffraction deposited and/or the newly formed crystals. This seems a characteristics, the chemical composition, and the struc- more likely explanation since there appears to be little crystural assessment of the calcified cartilage samples are for tal growth with increasing mineralization.(3z) Since these the most part entirely consistent with the conclusion that phenomena occur when the carbonate content of the minthe crystals of calcified cartilage in the particular samples eral phase decreases, it appears consistent with the putative of whole cartilage and of the 1.4-1.5 density fraction rep- relationship between crystallinity and the concentration of resent very young newly deposited mineral. carbonate ions in the special case of the mineral phase of Like the earliest stage of calcification in bone, the very calcified cartilage. However, there are unquestionably earliest detectable mineral phase in both whole calcified other factors that may also control the crystallinity of the cartilage and in the 1.4-1.5 density fraction has the x-ray mineral phase of calcified cartilage, such as its interaction diffraction characteristics of poorly crystalline apa- with glycosamin~glycans,(~~~~~) Mg2+,(35.36) and P20:-.(37) tite. (29-31) Similarly, the absence of nonapatitic, crystalline phases in the calcified cartilage and the presence of disNonapatitic phosphate ions crete, nonapatitic CaP ionic clusters detected by 31P-NMR, possibly in a DCPD (brushite) configuration, are also simiThe relatively large concentration of nonapatitic phoslar to the findings we obtained in very young bone using phate ions is one of the main characteristics of the mineral similar techniques. ( 1 8 . z 2 ~ 1 9 ) phase of calcified cartilage. The concentration of the phos~~

I

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and that old bone, which has a relatively slow rate of remodeling and turnover of its mineral phase, contains more carbonate ion than young bone, which remodels at a much faster rate than old bone.(39)The change in the concentration of carbonate ions in bone mineral is inversely related to the changes in the concentration of nonapatitic phosphate ions. The same inverse relationship occurs in cartilage, suggesting that the introduction of carbonate ions into the cartilage mineral is related to a secondary change in the mineral phase leading to an increase in the apatitic characteristics of the solid phase at the expense of nonapatitic phosphates. On this basis the relatively low carbonate ion content in the mineral of calcified cartilage compared to bone may simply be attributed to the fact that the mineral in calcified cartilage is less mature than it is in bone. Although in general density-fractionated samples have densities proportional to the average of their constituent mineral, the proportionality factor may differ between animals of different ages or between different types of tiss u e ~ . ' ~This ~ ) is because the average age of crystals in a sample of any specific density depends strongly on the rates at which they form, develop, and mature. Despite the difficulty in comparing the age of mineral crystals from different tissues on the basis of physicochemical parameters, it is clear that maturation of apatite crystals is always associated with a decrease in the amount of nonapatitic phosphate ions.(z3)If we choose this parameter as a criterion of the maturity of crystals, it is possible to obtain at least some qualitative idea about the maturity of the crystals in different samples. With this criterion our data indicate that the youngest sample of cartilage contains crystals that are less mature than the youngest samples of bone crystals. ( 2 3 2 4 ) On the other hand, the crystals in samples of calcified cartilage of moderate density appear to contain more nonapatitic phosphate groups than those of the very highest or very lowest densities. This likewise may be a reflection of the rate of mineralization and/or maturation and suggests that there may have been a faster rate of crystal deposition in the tissue of intermediate density and/or a slower rate of crystal maturation. In summary, the CaP mineral phase in calcified cartilage can be identified as a very poorly crystalline, carbonated apatite, rich in nonapatitic phosphate ions, having a relative low content of carbonate ions. At all stages of calcification, including the earliest phase of calcification thus far studied in either bone or cartilage by the techniques described in this and earlier paper^,('^.^^-^') the only crystalline solid detected by x-ray diffraction was apatite. No evidence for a nonapatitic solid phase was found; even the very earliest solid CaP mineral phase deposited showed x-ray reflections of apatite: there was no evidence that the very earliest solid phase of CaP was amorphous. During Carbonate content the progressive development and maturation of the minThe second important characteristic specific to the min- eral phase the crystallinity of the mineral phase increases. eral phase of calcified cartilage is its low carbonate con- However, in contrast to bone mineral, the concentration tent. The changes accompanying maturation of solid of nonapatitic, labile phosphate ions also increases with have been clearly time. In the final stages of mineralization the characterisphases of CaP in biologic identified. It is well established, for instance, that the car- tics of the mineral phase in cartilage tend to approximate bonate content of bone mineral increases with maturity more closely those of bone mineral.

phate groups is particularly high in the fractions of medium density and appears to be directly related to the rapid initial progression of mineralization after the first deposition of the CaP solid phase. It is interesting that the maximum concentration of brushite-like, nonapatitic phosphate groups detected by 3'P-NMR occurs in a lower density fraction (1.5-1.6) than the maximum concentration of total nonapatitic phosphate ions detected by FTIR (1.6-1.7 and 1.7-1.8). The maximum crystallinity also occurs in samples of 1.7-1.8. In density fractions greater than 1.71.8 the amount of nonapatitic phosphate ions decreases slowly as more of the phosphate ions become apatitic in the mature mineral phase. Despite the analogies noted between the phosphate ions found in brushite and in OCP and those found in the relatively more poorly crystalline younger mineral phase of calcified cartilage, one cannot conclude from the present data that brushite and/or OCP are the precursors of the apatite solid phase in calcified cartilage since no crystal structure corresponding either to brushite or OCP was detected by x-ray diffraction, even in the very earliest stages of mineralization (1% or less of brushite and of OCP in the fully calcified cartilage can be detected), or in density fractions containing the highest concentrations of nonapatitic phosphate ions. Rather, it is more likely that the nonapatitic phosphate groups observed by 3'P-NMR and FTIR are constituents of a poorly crystalline apatite p h a ~ e , ( ' ~ . and ~ ~ .not ~ ' ) a distinct CaP solid phase different from apatite. One of the most surprising results is that the increase in crystallinity accompanying the progressively increased calcification of the cartilage tissue that occurs with time occurs simultaneously with a progressive increase in the concentration of nonapatitic phosphate groups. In bone there is a steady decrease in nonapatitic phosphate with increasing maturity and crystallinity. (18.22.23) The resolution factor of the v4P04bands is related to local distortions in the environment of the phosphate ions in apatitic domains. There are indications from partial dissolution experiments of poorly crystalline apatite, for example, ( 2 2 . 2 3 ) that there is an inhomogeneous distribution of nonapatitic phosphate ions in the solid, which may not perturb the majority of the constituents in apatite organization of the crystal lattice. Besides, we note the similarities between the IR spectra of OCP and some of the density fraction samples: OCP contains apatitic domains and the most intense bands of OCP in v,PO, domain are only slightly shifted with respect to those of apatite. Thus it is possible that the presence of an OCP-like environment may also not significantly alter the P0,RF index.

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ACKNOWLEDGMENTS This work was supported in part by grants from the National Institutes of Health (AR 34078 and AR 34081), the National Science Foundation (PCM-7901181), the Peabody Foundation, Inc., and an Institutional Grant from the Orthopaedic Research Education Foundation funded by Bristol-Myers/Squibb/ZimmerCorporation.

bone mineral and its synthetic analogues. Calcif Tissue Res 13~73-82. 18. Roufosse AH, Aue WP, Roberts JE, Glimcher MJ, Griffin RG 1984 Investigations of the mineral phase of bone by solid state phosphorus-3 1 magic angle sample spinning nuclear magnetic resonance. Biochemistry 23:6115-6120. 19. Aue WP, Roufosse AJ, Glimcher MJ, Griffin RG 1984 Solid state phosphorus-3 1 nuclear magnetic resonance study of

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Address reprint requests to: Dr. Melvin Glimcher The Children’s Hospital 300 Longwood Ave. Boston, MA 02115 Received in original form June 13, 1990; in revised form December I I , 1990; accepted December 13, 1990.