Journal of Dental Research

Journal of Dental Research http://jdr.sagepub.com/

Immunocytochemical Localization of Osteocalcin in Developing Rat Teeth I. Gorter de Vries, E. Quartier, P. Boute, E. Wisse and D. Coomans J DENT RES 1987 66: 784 DOI: 10.1177/00220345870660031601 The online version of this article can be found at: http://jdr.sagepub.com/content/66/3/784

Published by: http://www.sagepublications.com

On behalf of: International and American Associations for Dental Research

Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/66/3/784.refs.html

Downloaded from jdr.sagepub.com by guest on July 24, 2011 For personal use only. No other uses without permission.

Immunocytochemical Localization of Osteocalcin in Developing Rat Teeth§ I. GORTER DE VRIES, E. QUARTIER, P. BOUTE, E. WISSE1, and D. COOMANS2 Institute of Dentistry and 'Laboratory for Cell Biology and Histology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium

Osteocalcin was purified by gel chromatography from a crude extract obtained after decalcification of rat incisors. The apparent molecular weight, as determined by 5-15% SDS-polyacrylamide gel electrophoresis, was 18,000, and amino acid analysis revealed 60 y-carboxyglutamic acid residues per 1000. Antisera against osteocalcin, raised in rabbits, reacted specifically with osteocalcin when investigated by immuno-electroblotting of dentin crude extract. 4-pim cryosections of formaldehyde-fixed tooth germs showed positive immunocytochemical staining for osteocalcin in dentin and odontoblasts. The staining of the mantle dentin at the coronal sides of the tooth germs was more intense than that of the adjacent circumpulpal dentin, while the odontoblasts involved in the formation of mantle dentin showed stronger immunoreactivitv than did odontoblasts involved in circumpulpal dentin formation. This marked difference was not observed on the root sides of the tooth germs. In 1h-pm cryosections, osteocalcin immunoreactivity was found evenly distributed throughout the entire cell body, with the exception of the Golgi region, which was less intensely stained, while the nucleus and the cell process were negative. The positive staining reaction with anti-osteocalcin antiserum was found in dentin from the very onset of its formation in the fetus. In conclusion, our results demonstrate the presence of osteocalcin in odontoblasts and dentin. Its immunocytochemical localization may be compatible with a distinct role in early dentinogenesis.

J Dent Res 66(3):784-790, March, 1987

Introduction. Osteocalcin or bone Gla protein (BGP) is a major protein of bone (Hauschka et al., 1975; Price et al., 1976) and dentin (Linde et al., 1980), constituting from 5 to 10% of the noncollagenous proteins. In most species, it contains three y-carboxyglutamic acid (Gla) residues per molecule of approximately 50 amino acids. Gla is formed by the carboxylation of glutamic acid, a reaction which is dependent on vitamin K. The Gla residues bind calcium ions and are responsible for the high affinity of osteocalcin for hydroxyapatite (Price et al., 1976; Poser and Price, 1979; Hauschka and Carr, 1982). The abundance of osteocalcin among the non-collagenous proteins of bone and dentin, and the strong correlation of its serum concentration with the level of bone turnover (Price et al., 1980b) indicate an important function of osteocalcin in the metabolism of mineralized tissues. Osteocalcin in rat dentin consists of four closely related proteins (Linde et al., 1982). It was found to be synthesized in organ culture of dentin-odontoblast fragments (Dimuzio et al., 1983). Odontoblasts and dentin of rat molar tooth germs in vivo (Bronckers et al., 1985) and cultured in vitro (Finkelman and Butler, 1985) stain positive after incubation with antidentin osteocalcin antiserum. In this study, we have further investigated the immunocytochemical localization of osteocalcin, especially in incisor tooth germs, where root-side dentin is found in addition to crown-side dentin. We were also interested in the developmental appearance of osteocalcin in odonReceived for publication April 16, 1986 Accepted for publication October 17, 1986 §This investigation was supported by the Belgian "Fonds voor Geneeskundig Wetenschappelijk Onderzoek", grant number 3.0038.81. 2To whom correspondence and reprint requests should be addressed

784

toblasts and dentin during fetal life, since it has been reported that newly formed rat bone is devoid of the protein (Price et al., 1980a; Price et al., 1981).

Materials and methods. Purification of osteocalcin. -Incisors were excised from male

Wistar rats (weighing 200 g each), cleaned of adhering tissue, and stored frozen. All manipulations were carried out at 4TC. The incisors were broken and extracted with a solution containing 4 mol/L guanidine-HC1, 50 mmol/L Na-acetate, 10 mmol/L EDTA, and several protease inhibitors (Gorter de Vries et al., 1986) at pH 6.5. The tooth fragments were further extracted with 0.25 mol/L EDTA, 20 mmol/L Tris at pH 7.4, also containing protease inhibitors, resulting in a crude dentin extract. Dentin phosphoprotein was precipitated from the desalted extract with CaCl2. Osteocalcin was purified from the supernatant as described (Butler et al., 1981) and eluted as a single peak in repeated chromatography over a Sephadex G50 fine column (Pharmacia, Uppsala, Sweden) in 100 mmol/ L NH4HCO3 (Price et al., 1976). Characterization of osteocalcin.-The peak fractions of the Sephadex G-50 column were pooled and further analyzed by DEAE-cellulose chromatography, by amino acid analysis, and by polyacrylamide gel electrophoresis. The material was chromatographed at 4TC on a I x 6 cm column of DEAE-cellulose (DE-52, Whatman, Maidstone, England) with 50 mmol/L TrisHCl at pH 8.0. Elution was performed with a linear gradient of 0-0.6 mol/L NaCI over a total volume of 500 mL. For amino acid analysis, samples were hydrolyzed in 6 mol/L HC1 at 1 10C for 22 hours or, for the Gla determinations, in 2 mol/ L KOH at 1 0IC for 22 hours (Hauschka, 1977), since Gla decarboxylates to form glutamic acid on acid hydrolysis. Amino acid analysis was performed with a Beckman type 119 CL amino acid analyzer. Glutamic acid was used as internal reference for calculation of the final amino acid composition from the data obtained from the alkaline and acid hydrolysates, since Gla is converted quantitatively into Glu on acid hydrolysis (Lian et al., 1979). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) was carried out in 5-15% or 20% gels (Laemmli, 1970). Gels were stained with Coomassie Brilliant Blue or with Silver Stain (Biorad Laboratories, Utrecht, The Netherlands) and scanned with a Vernon Photometer. Preparation and characterization of antisera.-White New Zealand rabbits were immunized with 1 mg of osteocalcin, prepared as described, dissolved in 0.5 mL phosphate-buffered saline (PBS), and emulsified with 0.5 mL Freund's complete adjuvant (Difco, Detroit, MI) through multiple intracutaneous injections on the back and two intramuscular injections in the hind legs. After 80 days, booster injections were given subcutaneously with 100 pLg osteocalcin dissolved in 0.5 mL PBS and emulsified with an equal volume of Freund's incomplete adjuvant. Animals were bled eight days later from the central ear vein. Sera were tested for the presence of anti-osteocalcin antibodies by enzyme-linked immunosorbent assay (ELISA) on microtiter plates (Dynatech, Greiner, Nurtingen, West Ger-

Downloaded from jdr.sagepub.com by guest on July 24, 2011 For personal use only. No other uses without permission.

Vol. 66 No. 3

OSTEOCALCIN IN DEVELOPING RAT TEETH

785

0:0.4 (1~~~~~~~~~~~~~~~~~~~~.

E 0.8 E

z

EN 3UI OUE l

00r Go