Calcif. Tiss. Res. 19, 85--90 (1975) 9 by Springer-Verlag 1975

Original Papers Mineralization of Invertebrate Cartilage R. G. E i l b e r g a n d D. A. Z u c k e r b e r g Fairleigh Dickinson University, School of Dentistry, Hackensaek, N.J. P. Person Veterans Administration Hospital, Brooklyn, New York and the Marine Biological Laboratory, Woods ttole, Mass. Received January 10, accepted April 9, 1975 Although invertebrate cartilage tissues do not mineralize in nature, it is now reported for the first time that when excised gill cartilage tissue from Limulus (horse shoe crab) is placed in an appropriate incubation medium metastable to hydroxyapatite, mineralization will occur. The mineralization is temperature dependent, and takes place at 37 ~ but not at 20 ~ Incubations in media metastable to calcite have not produced mineralization. Histologic examination of mineralized tissues showed mineral deposits predominantly within cells, and to ~ lesser extent in the matrix. X-ray diffraction of the deposited mineral revealed a typical biological hydroxyapatite pattern. Key words: Cartilage - - Mineralization - - Invertebrates - - Connective tissues.

Introduction I n v e r t e b r a t e cartilages c o r r e s p o n d in m a n y w a y s t o v e r t e b r a t e c a r t i l a g e : t h e i n v e r t e b r a t e tissues are h i g h l y h y d r a t e d , rich in acid g l y c o s a m i n o g l y c a n s a n d possess v a r y i n g a m o u n t s of collagen (Person a n d P h i l p o t t , 1969a, b; P h i l p o t t a n d Person, 1970). I n s o f a r as is known, i n v e r t e b r a t e cartilage tissues do n o t s y n t h e s i z e a d i s t i n c t m i n e r a l phase in t h e course of t h e i r n a t u r a l h i s t o r y , as do m a n y v e r t e b r a t e cartilages (Schaffer, 1930; P e r s o n a n d P h i l p o t t , 1969a). The failure of i n v e r t e b r a t e cartilages t o mineralize in vivo is of considerable i n t e r e s t , because o t h e r tissues do mineralize in animals w i t h n o n - m i n e r a l i z i n g cartilages. F o r e x a m p l e , in g a s t r o p o d molluscs, t h e o d o n t o p h o r e cartilages do n o t mineralize, while a shell of calcium c a r b o n a t e is f o r m e d b y t h e m a n t l e tissues in these s a m e animals. The genetic basis for cartilage m i n e r a l i z a t i o n is t h e r e f o r e u n d o u b t e d l y p r e s e n t in m a n y i n v e r t e b r a t e animals, b u t r e p r e s s e r or o t h e r influences m a y somehow h i n d e r expression of t h e genetic p o t e n t i a l (Person a n d P h i l p o t t , 1969 a). I n t h e a b o v e perspective, t h e p r e s e n t studies were p e r f o r m e d to l e a r n w h e t h e r o1" n o t t h e endoskeletal b r a n c h i a l or gill cartilages of Limulus polyphemus (horseshoe crab) could be i n d u c e d t o mineralize in vitro.

Materials and Methods Limulus polyphemus 50-75 mm and 125-150 mm in body length were obtained from the Marine Biological Laboratory, Woods Hole, Mass., and maintained in an Instant Ocean For reprints: Dr. P. Person, Veterans Administration Hospital, 800 Poly Place, Brooklyn, N.Y. 11209, USA.

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Aquarium System (100 gallon capacity) at 17 ~ They were fed twice weekly with pieces of fresh clam, and were vigorous and active when selected for use. Animals were sacrificed by exsanguination, following which the branchial cartilages were dissected and trimmed free of adherent tissues, as described previously (Person and Philpott, 1969a). I n some experiments, tissue slices 1/2-2 m m thick were immediately cut freehand a n d placed in incubating media. I n other experiments cubes or cylinders of tissue were used. The dissected cartilage bars were sometimes wrapped in aluminum foil and placed in cracked ice for periods up to 3 hours prior to use, without any a p p a r e n t detrimental effects on ability to mineralize. Based upon earlier work of Bachra (1963), incubating media were metastable to hydroxyapatite (Eilberg, Gould a n d Sobel, 1965) or to calcite (Eilberg, Levy and Iovino, 1972). The solutions were prepared as follows:

a) Hydroxyapatite Medium. To a 11 volumetric flask was added 100 ml of a solution containing NaHCOa (220 mM/1), KCI (50 mM/l) and NaC1 (702 mM/l). Next, 50 ml of a solution containing Na 2 HPO 4 (25.1 raM/l) and NaH2PO 4 (6.45 raM/1 as NaH2PO4.H20 ) was added. Following this, about 200 ml of distilled water was added. CO 2 was then bubbled through the solution for 1 min. Next, 11 ml of a CaCl 2 solution (250 mM/1 as CaCI2.2H~O ) was added, and the volume made to mark with distilled water. Nitrogen was bubbled through the resulting solution until a pH of 7.30 was reached. b) Calcite Medium. To a 11 volumetric flask was added 300 ml of a solution containing NaHCO3 (220 mM/1); MgCI 2 (0.20 mM/1) and NaCI (460 mM/1). C03 was then bubbled through the solution for 1 min. Next, 12 ml of a CaC12 solution (250 mM/1 as CaC12.2H20 ) weer added, and the volume made to mark with distilled H20. The resulting solution usually had a p H of 7.20. If not, N 2 was bubbled through until the p H reached the desired value. 45Ca was added to b o t h incubating solutions in order to follow the course of mineral formation, H20 was double distilled, and all glassware was acid washed. Tissues were weighed wet, placed in sealed 50~ml Erlenmeyer flasks filled to the brim with the solution metastable to hydroxyapatite or to calcite, and incubated for periods from 1-7 days at 20 ~ or 37 ~. A t the end of the incubation period, tissues were removed from the mineralizing solutions for chemical analysis. They were rinsed thoroughly with distilled water and ashed in a muffle furnace a t 550 ~ The ash was redissolved in 0.1 N HC1, a n aliquot was placed in scintillation fluid and analyzed for 4~Ca b y standard scintillation techniques. The total Ca content was then calculated. I n some instances ash weights were also determined. For X-ray diffraction, samples were air dried and Debye-Scherrer diffraction p a t t e r n s were made by Dr. Albert Hirschman, D e p a r t m e n t of Anatomy, Downstate Medical Center, Brooklyn, New York. For histologic study of haematoxylimeosin stained material, samples were removed from incubating media, placed in 10% buffered formalin and processed by standard methods. The Von-Kossa technique for localization of calcium was also employed.

Results T a b l e 1 s h o w s t h e r e s u l t s of 3 e x p e r i m e n t s i n w h i c h L i m u l u s gill c a r t i l a g e w a s i n c u b a t e d a t 37 ~ f o r v a r y i n g t i m e p e r i o d s i n s o l u t i o n s m e t a s t a b l e t o h y d r o xyapatite and to calcite, respectively. The data show that there was significant Ca u p t a k e b y t i s s u e s i n c u b a t e d i n t h e h y d r o x y a p a t i t e - m e t a s t a b l e solution, but not by tissues incubated in the calcite-metastable solution. Table 2 shows the r e s u l t s of a n e x p e r i m e n t i n w h i c h g r a v i m e t r i c d e t e r m i n a t i o n s o n a s h e d s a m p l e s w e r e also c a r r i e d o u t . C o r r e s p o n d i n g q u a n t i t i e s of m i n e r a l a s h w e r e f o u n d i n t h e m i n e r a l i z e d s p e c i m e n s . I t c a n b e s e e n f r o m T a b l e s 1 a n d 2 t h a t t h e r e w a s cons i d e r a b l e v a r i a t i o n i n C a u p t a k e i n e a c h of t h e e x p e r i m e n t s . T h i s v a r i a b i l i t y r e f l e c t s t h e f a c t t h a t t h e size a n d s u r f a c e a r e a of t i s s u e s p e c i m e n s w e r e n o t i d e n t i c a l i n e a c h e x p e r i m e n t , as d e s c r i b e d i n Materials and Methods. I n f u t u r e e x -

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Table 1. Calcium uptake by Limulus gill cartilage in solutions metastable to hydroxyapatite or to calcite Length of incubation period at 37 ~

Calcium uptake by 45Ca technique (mg Ca/gm wet cartilage) Sol. metastable to hydroxyapatite

Sol. metastable to calcite

Experiment 1:1 day 3 days 7 days

0.56 2.06 8.99

0.22 0.19 0.19

Experiment 2 : 1 day 3 days 7 days

9.33 26.30 37.70

0.18 a a

Experiment 3 : 1 day 3 days 7 days

3.17 15.87 17.59

0.75 0.58 0.36

a No analyses performed because of inadequate sample size.

Table 2. Comparison of calcium uptake by Limulus gill cartilage as determined by isotopic and gravimetric techniques Length of incubation period at 37 ~

Ca uptake by 45Ca technique (rag Ca/g cartilage)

Ca uptake by gravimetric technique (rag ash/g cartilage)

1 day 3 days 7 days

2.87 12.31 17.82

10.35 33.33 49.51

p e r i m e n t s which are p l a n n e d to d e t e r m i n e t h e kinetics of mineralization, we will use tissue slices of c o n s t a n t surface a r e a a n d volume. W h e n tissues were i n c u b a t e d in t h e h y d r o x y a p a t i t e - m e t a s t a b l e m e d i u m a t 20 ~ t h e r e was no u p t a k e of tsCa. Von K o s s a s t a i n showed significant deposits of Ca salts in t h e tissues from h y d r o x y a p a t i t e - m e t a s t a b l e incubations, b u t n o t from t h e c a l c i t e - m e t a s t a b l e s y s t e m s as m a y be seen in Figs. 1 a n d 2. I n specimens i n c u b a t e d for 7 days, t h e localization of t h e Von K o s s a deposits is p r i m a r i l y w i t h i n cells, with r e l a t i v e l y fewer deposits d e t e c t a b l e in t h e m a t r i x . X - r a y diffraction of powders m a d e from a i r - d r i e d tissue slices between 1 - 2 m m thick, gave a t y p i c a l p a t t e r n for biological h y d r o x y a p a t i t e in those sections i n c u b a t e d in t h e h y d r o x y a p a t i t e - m e t a s t a b l e solutions a t 37 ~ (Fig. 3B). However, o n l y diffuse p a t t e r n s of organic m a t e r i a l were o b t a i n e d from tissues i n c u b a t e d in c a l c i t e - m e t a s t a b l e solutions, or in h y d r o x y a p a t i t e - m e t a s t a b l e solutions a t 20 ~ (Fig. 3A). A c o m p a r i s o n diffraction p a t t e r n from a k n o w n s a m p l e of biological h y d r o x y a p a t i t e is sho~la in Fig. 3C.

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:Fig. 1. Appearance of non-mineralized L i m u l u s cartilage tissue incubated in ealcite-metastable medium. A similar appearance is also found in non-incubated control tissues t a k e n from animals freshly removed from the sea tank. Von-Kossa stain with eosin counterstain, • 200 Fig. 2. Appearance of mineralized L i m u l u s gill cartilage tissue following 7 days incubation in hydroxyapatite-metastable medium. Note the dark mineral granules dispersed primarily throughout the cells. The granule content of the matrix is much lower t h a n t h a t of cells. Von-Kossa stain with eosin counterstain, • 200

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Fig. 3. (A) X-ray diffraction pattern of non-mineralized cartilage incubated in calcite-metastable solution, or of non-incubated control tissues. (B) X-ray diffraction pattern of mineralized cartilage incubated for 7 days in hydroxyapatite-metastable medium, washed in distilled water and air-dried (compare with (C) below). (C) X-ray diffraction pattern of a known biological hydroxyapatite (courtesy of Dr. A. Hirschman)

Discussion

The present experiments establish that Limulus gill cartilages, which do not mineralize in vivo, possess the ability to undergo mineralogization with hydroxyapatite in vitro. The results also show that temperature is an important variable in this mineralization process. At 20 ~ which is within the temperature range in which the animal lives in the ocean, mineralization does not occur, while at 37 ~ which is well above the temperature range in which the animal lives, mineralization does occur. Of added interest is the fact that with a few notable exceptions, most mineralizing invertebrate organisms above the Porifera (sponges) form the calcium carbonate mineral phases, calcite or aragonite, or both. I t is therefore somewhat unexpected that Limulus gill cartilage should form an hydroxyapatite phase, which is characteristic of vertebrate cartilage mineralization. Of relevance to the above, may be the observation that in one invertebrate organism, the common shrimp found in waters of the Gulf Stream off the southern coast of the United States, hydroxyapatite is found in the tips of the claws of the legs of these animals (Trautz, 1961; and Trautz, personal communication). I n view of the fact that such waters are at relatively high temperatures, the possibility of a correlation between elevated temperatures and mineralization of invertebrate tissues by hydroxyapatite should and will, receive closer scrutiny.

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A f e a t u r e of t h e gill cartilage m i n e r a l i z a t i o n is t h a t for t h e 7 d a y i n c u b a t i o n i n t e r v a l s t u d i e d histologically, m i n e r a l u p t a k e a p p e a r s to be p r i m a r i l y intracellular, w i t h lesser m a t r i x i n v o l v e m e n t . A t i m e sequence s t u d y of t h e sites of mineral u p t a k e is p r e s e n t l y in progress in this l a b o r a t o r y . W e h a v e also f o u n d t h a t o t h e r i n v e r t e b r a t e cartilages will also mineralize h y d r o x y a p a t i t e , including t h e cranial cartilage of Loligo (squid), a n d t h e o d o n t o p h o r e cartilage of Busycon (marine snail), I n s u m m a r y , t h e p r e s e n t e x p e r i m e n t s establish for t h e first t i m e t h a t inverteb r a t e endoskeletal cartilage tissues have t h e c a p a c i t y to form h y d r o x y a p a t i t e . This a p p e a r s a unique t y p e of cartilage which could be used to s t u d y b o t h genetic a n d e n v i r o n m e n t a l factors associated with repression a n d i n d u c t i o n of mineralization. I n a v e r y real sense, this is an e x p e r i m e n t a l s y s t e m which has been prep a r e d for us b y n a t u r e .

References Bachra, B.: Precipitation of calcium carbonates and phosphates from metastable solutions. Ann. N.Y. Acad. Sci. 109, 251 255 (1963) Eilberg, R.G., Gould, D., Sobel, A.E.: Nucleating substances in saliva. Nature 207, 481 483 (1965) Eilberg, R.G., Levy, M., Iovino, E.: Mineralization in vitro of invertebrate shell proteins. Program and Abstracts, 50th General Session, Int. Assn, Dent. Res., Abstr. 812, March (1972) Person, P., Philpott, D.E.: The nature and significance of invertebrate cartilages. Biol. Revs. Cambridge Phil. Soc. 44, 1-16 (1969a) Person, P., Philpott, D.E.: The biology of cartilage I. Invertebrate cartilages: Limulus gill cartilage. J. Morphol. 128, 67 93, (1969b) Philpott, D.E., Person, P,: The biology of cartilage IX. Invertebrate cartilages: Squid head cartilage. J. Morphol. 131,417-430 (1970) Schaffer, J.: Die Stiitzgewebe. In: Handbuch dcr mikroskopischcnAnatomie des Menschen Von MSllendorf, W. ed.) p. 1 390. Zw. Bd., Zw. T., Berlin: Springer 1930 Trautz, O.: Crystalline organization of dental mineral. In: Structural and chemical organization of teeth, vol. I I (Miles, A.E.W. ed.), p. 201-246. New York: Academic Press 1967

Mineralization of invertebrate cartilage.

Although invertebrate cartilage tissues do not mineralize in nature, it is now reported for the first time that when excised gill cartilage tissue fro...
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