Calcif. Tiss. Res. 18, 155 160 (1975) 9 by Springer-Verlag 1975

Amorphous Calcium Precipitations in Human Aortic Valve K o o k M. K i m a n d B e n j a m i n F. T r u m p Departments of Pathology Veterans Administration Hospital and University of Maryland School of Medicine Baltimore, Maryland Received January 24, accepted March 24, 1975 Spheroidal, granular, and fibrillar particles were observed in human aortic valve calcifications. Spheroids were similar to amorphous calcium phosphate shown previously in synthetic preparations and in bone. The frequent coexistence of needle shaped hydroxyapatite crystals with granular and fibrillar particles suggests that the latter are also amorphous calcium phosphate and may be precursors of hydroxyapatite. All three types of particles had a tendency to form laminated and spherular secondary structures and failed to give a crystalline pattern by electron diffraction. Key words: Amorphous calcium phosphate - - Aortic valve - - Aging.

Introduction Calcification of the h u m a n aortic valve occurs regularly, increases gradually with age (Sell a n d Scully, 1965), a n d characteristically occurs along a zone of lipid a c c u m u l a t i o n i n dense fibrous tissue (Kim a n d H u a n g , 1971). A c c u m u l a t e d lipids i n the valves are d e g r a d a t i o n products of senescent a n d degenerate fibroblasts a n d these deposits appear to be the site of m i n e r a l i z a t i o n (Kim et al., 1974). The gradual n a t u r e of the calcification process i n v a l v u l a r connective tissue allowed us to observe amorphous calcium phosphate a n d its spatial relation to hyd r o x y a p a t i t e crystals. This appears to be the first visualization of amorphous calcium phosphate in non-skeletal tissue of m a n . F u r t h e r m o r e , more t h a n one morphologic t y p e of amorphous calcium phosphate m a y exist i n the aortic valve.

Materials and Methods Aortic valves were obtained from autopsies and surgical resections as previously described (Kim and Huang, 1971 ; Trump et al., 1973; Kim et al., 1974). The tissues were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) postfixed in osmium tetroxide, and embedded in Epon 812. Thin sections were examined either unstained or stained with lead citrate and/or uranyl acetate. Selected area electron diffraction was carried out oil unstained 1,000 A thick sections using a JEOL 100 B electron microscope.

Results Precipitations i n aortic valves, as a rule, occur i n spherular aggregates i n a n d a r o u n d cellular d e g r a d a t i o n products. The latter are p r e d o m i n a n t l y m e m b r a n o u s a n d t h e y appear to be derived from lysosomal debris (Kim et al., 1974). Three types of u n i t structures were n o t e d a m o n g various electron dense precipitations in the valves : spheroids, fine g r a n u l a r a n d fine fibrillar particles. For reprints: Dr. B.F. Trump, Department of Pathology, University of Maryland School of Medicine, 31 South Greene Street, Baltimore, Maryland 21201, U.S.A.

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Spheroids were the most c o m m o n type. T h e y measured 50-200 A in diameter, usually showed " h o l l o w " centers, and had a t e n d e n c y to form larger aggregations measuring up to several t h o u s a n d A in diameter. Needle shaped structures resembling crystals 1 were often observed on the periphery of these aggregates, or at times intermixed with spheroids (Figs. 1-4). W h e n the number of spheroids was smaller t h e y usually showed laminated circular and/or radial arrangements. I n some cases clusters of spheroids without hollow centers were found along the edge of aggregates, giving an irregular scalloped margin (Fig. 3). Finely granular particles were also present in large quantity. These formed either irregular solid clusters or circular and laminated secondary structures (Fig. 5). Crystal-shaped particles with a radial arrangement were frequently present on the periphery of or within the aggregates of these granules (Figs. 5-6). Fibrillar particles also formed spherular or circular aggregates with a radial arrangement of the fibrils (Fig. 7). Transformation of these fibrillar structures into needle-shaped crystals was also observed (Fig. 8); aggregates of spheroids were frequently seen in the center of fibrillar aggregates. Selected area electron diffraction of crystal-shaped particles yielded a diffraction pattern of calcium h y d r o x y a p a t i t e (Table 1; Fig. 9A), while spheroidal, granular and fibrillar particles did not show a crystalline p a t t e r n (Fig. 9 B).

Discussion

Spheroids seen in this s t u d y are similar to the particles observed b y Weber (1967) in a s t u d y of amorphous calcium phosphate. Molnar (1959) reported similar structures in frozen-dried developing mouse bone. The appearance of needleshaped h y d r o x y a p a t i t e crystals in apposition to the spheroidal aggregates is consistent with the t y p e of conversion described b y several workers who studied amorphous calcium phosphate in cell free systems (Eanes et al., t973; BreSevid and Ffirdi-Milhofer, 1972; West, 1971). The chemical nature of the finely granular and fibrillar precipitations is not certain; however, the appearance of needle-shaped crystals in apposition to granular particles, and the a p p a r e n t morphological transitions from fibrillar

Fig. 1. An aggregate of spheroids within an electron dense vesicle. Needle shaped particles are seen within the spheroidal aggregate and along the vesicle wall. Stained with uranyl acetate and lead citrate. Enlargement of the rectangular area is shown in Fig. 2, X 54600 Fig. 2. A part of aggregate of spheroids shown in Fig. 1. Spheroids have hollow centers and are approximately 120 A in diameter. Unstained, • 80000 Fig. 3. A half of a floral aggregate of amorphous particles with electron dense central core. Periphery of the aggregate is scalloped by spheroids without ceutral core, and giving an appearance of "budding". Unstained, • 71400 Fig. 4. A rounded aggregate of spheroids of approximately 85 A. in diameter. Spheroids form laminated and radially arranged secondary structures. Unstained, x 71400 1 Although it has been shown that plate-like crystals of hydroxyapatite can also occur (Bocciarelli, 1970), stereo-views of the crystals in this study at a magnification of X 50000 or higher revealed only the needle pattern.

Amorphous Calcium Phosphate in Aortic Valve

2

157

4

structures to needle-shaped hydroxyapatite crystals, suggest that these two types of particles m a y also represent amorphous precursors of hydroxyapatite. The reasons for spherular and laminated secondary structure formation of these amorphous precipitations are not clear. I n part, it appears to be a function of the organic matrix, i.e. cellular degradation products.

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5

6

7

8

Fig. 5. An aggregate of finely granular particles surrounded by radially arranged needle shaped crystals. Needles are also present among the amorphous granular particles. Stained with uranyl acetate and lead citrate, • 80190 Fig. 6. Circularly arranged granular particles with a central core. Needle shaped particles coexistent with these amorphous particles appear to develop from the latter, • 57420 Fig. 7. A spherular aggregate of thin fibrillar particles. Stained with uranyl acetate and lead citrate, • 57420 Fig. 8. Needle shaped crystals are present in the background of fibrillar particles. Unstained, • 57420

Amorphous Calcium Phosphate in Aortic Valve

A

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Fig. 9. (A) Electron diffraction of needle shaped crystals shows the pattern of calcium hydroxyapatite (left). (B) Diffraction pattern of spheroidal particles consists of a few hazy rings (right). Granular and fibrillar particles show diffraction patterns that are similar to the spheroidal particles Table 1. Lattice spacings of crystalline deposits in aortic v~lve compared with ASTM standard measurementsa given for hydroxyapatite hkl

dA aortic valve

dh OHAP

111 002 210 211 202 301 212 310 203 213 004, 441 501, 204

3.88 3.43 3.04 2.81 2.63 2.49 2.29 2.25 2.O0 1.76

3.88 3.44 3.08 2.81 2.63 2.52 2.29 2.26 2.00 1.75

1.70

1.72

1.56

1.58

a X-ray powder data file, ref. card 9-432. Philadelphia: American Society for Testing and Materials.

I t is concluded t h a t , in h u m a n aortic valve, the i n i t i a l phase separation of calcium occurs as a m o r p h o u s calcium phosphate followed b y t r a n s f o r m a t i o n into h y d r o x y a p a t i t e , a n d t h a t there could be more t h a n one morphological t y p e of amorphous precursors of calcium h y d r o x y a p a t i t e .

Aclcnowledgement.This is contribution ~r 165 from the Cellular Pathobiology Laboratory. This study was supported by the American Cancer Society, Maryland Division, Inc., and the Veterans Administration :Research Fund.

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Reterences 1. Bocciarelli, D. S. : Morphology of crystallites in bone. Calcif. Tiss. Res. 5 , 2 6 1 ~ 6 9 (1970) 2. Bre6evi6, L., Fiirdi-Milhofer, H. : Precipitation of calcium phosphates from electrolyte solutions. II. The formation and transformation of the precipitates. Calcif. Tiss. Res. 10, 82-90 (1972) 3. Eanes, E.D., Termine, J.D., Nylen, M.U.: An electron microscopic study of the formation of amorphous calcium phosphate and its transformatior~ to crystalline apatite. Calcif. Tiss. ges. 12, 143-158 (1973) 4. Kim, K.M., I-Iuang, S-N. : Ultrastructural study of calcification of human aortic valve. Lab. Invest. 25, 357-366 (1971) 5. Kim, K.M., Valigorsky, J.M., Mergner, W . J . , Jones, R.T., Pendergrass, R.E., Trump, B. F. : Aging changes of human aortic valve in relation to dystrophic ealcification. Human Path. (in press) 6. Molnar, Z. : Development of the parietal bone of young mice. I. Crystals of bone mineral in frozen dried preparation. J. Ultrastruct. Res. 3, 3 9 4 5 (1959) 7. Sell, S., Seully, R . E . : Aging changes in the aortic and mitral valves. I-Iistologic and histochemical studies, with observations on the pathogenesis of calcific aortic stenosis and calcification of the mitral annulus. Amer. J. Path. 46, 345-365 (1965) 8. Trump, B.F., Valigorsky, J.M., Dees, J . H . , Mergner, W . J . , Kim, K.M., Jones, R.T., Pendergrass, R.E., Garbus, J., Cowley, 1%A. : Cellular changes in human disease. Human Path. 4, 89-109 (1973) 9. Weber, J.C., Eanes, E.D., Gerdes, R . J . : Electron microscope study of non-crystalline ealeium phosphate. Arch. Bioehem. Biophys. 129, 723 724 (1967) 10. West, V.C.: Observations on phase transformation of a precipitated calcium phosphate. Calcif. Tiss. Res. 7, 212 219 (1971)

Amorphous calcium precipitations in human aortic valve.

Spheroidal, granular, and fibrillar particles were observed in human aortic valve calcifications. Spheroids were similar to amorphous calcium phosphat...
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