Calcified Tissue Research

Calcif. Tiss. Res. 24, 223-229 (1977)

9 by Springer-Verlag 1977

Demonstration of Enamel Matrix Proteins on Root-Analogue Surfaces of Rabbit Permanent Incisor Teeth Steven E. Schonfeld and Harold C. Slavkin Laboratory for Developmental Biology, School of Dentistry, University of Southern California, Los Angeles, California 90007, USA

Summary. The continuously erupting rabbit incisor tooth is normally thought of as having an enamel covered " c r o w n " on its labial surface and a cementum covered "root" on its lingual surface. We have examined both surfaces of continuously erupting rabbit incisor teeth taken from near term embryos by a variety of means, including transmission and scanning electron microscopy, biochemical fractionation, and immunohistochemistry. In all cases, we could detect no qualitative difference in the early extracellular matrices taken from the labial and lingual surfaces of the teeth. Both matrices were shown to be composed of dentin and enamel, although the thickness and geometry of the enamel matrix on the lingual surface was somewhat different from that on the labial surface. Key

words: Enamel-cementum-morphology Immunocytochemistry - - Biochemistry

labial surface is covered with enamel and serves the function of the crown, whereas, the lingual surface is generally considered to be covered with cementum, and serves to anchor the tooth in the alveolus, a function normally ascribed to the root (Kronfeld, 1938; Selvig, 1963, 1967; Anderson and Stern, 1966; Shibata and Stern, 1967, 1968). One report, based on light microscopy, described the occurrence of enamel on the lingual surfaces of the most coronal portions of unerupted permanent rabbit incisor teeth (Hirschfeld, Weinreb and Michaeli, 1973). We report the results o f several experiments designed to determine if enamel proteins are, in fact, found on the lingual surface of the continuously erupting rabbit incisor tooth. This communication confirms and extends the earlier observations by a variety of criteria.

--

Materials and Methods

Introduction The permanent mandibular and anterior maxillary incisors of the rabbit are examples of continuously erupting teeth (Hirschfeld, Weinreb and Michaeli, 1973). A necessary consequence of continuous eruption is the loss of the functional dichotomy between the crown and the root which is normally found on teeth of finite eruption. A morphological adaptation to continuous eruption which is found in the rabbit incisor tooth is the occurrence of root and crown "analogues", which serve the mechanical functions normally performed by anatomically distinct crowns and roots. In the case of the continuously erupting rabbit incisor tooth (and in lagomorph and rodent incisors in general), the

Embryonic New Zealand white rabbits were obtained by Caeserian section from does during the 26th day of gestation. The does were sacrificed by injection of air into the medial ear vein. Embryos were placed on ice immediately following delivery. Maxillary anterior and mandibular permanent incisor teeth were dissected from the embryos and placed into a calcium and magnesium free phosphate buffered saline solution (CMF-PBS), pH 7.2, at 4 ~ C. Embryonic molar teeth were harvested in a similar manner. The tooth organs used for electron microscopy were not decalcified. Transmission Electron Microscopy. Embryonic 26-day incisor teeth were removed from the CMF-PBS, and fixed by the method of Hay and Revel (1969). The teeth were subsequently embedded in Epon 812, oriented in such a way that sections of both the labial and lingual sides of the same tooth could be obtained for comparisons. Sections were cut at approximately 1 /tm, stained with toluidine blue, and examined at the light microscopic level to verify ~Hentation. Thin sections were cut to silver or gold interference colors and stained with uranyl acetate and lead citrate. Scanning Electron Microscopy. The embryonic teeth were washed

Send offprint requests to S. E. Schonfeld at the above address

with fresh cold CMF-PBS and sonicated with a Biosonik sonicator

224

S.E. Schonfeld and H.C. Slavkin: Enamel Proteins on Root Analogue Surfaces of Rabbit Incisors

equipped with the standard 2.5 inch horn at 30% power to remove adherant cells and odontoblast cell processes, thus exposing the topography of the labial enamel surface and the lingual "rootanalogue" surface. The resulting aceUular matrices were again washed in CMF-PBS and sonicated a second time. After washing with CMF-PBS a third time, the sonicated extracellular tooth matrices were fixed as described (Hay and Revel, 1969). Following dehydration through graded alcohol solutions, the incisors were placed in a mixture of alcohol and acetone in a ratio of 1 : 1, and then in 100% acetone. The specimens were critical-point dried with CO s replacement to preserve the ultrastructural architecture of the matrix surfaces (Boyde and Wood, 1969). The teeth were coated with a conductive alloy prior to examination in the scanning electron microsope.

Extraction and Characterization of Aeellular Matrix Proteins by Polyaerylamide Gel Eleetrophoresis (PAGE). Embryonic New Zealand white rabbit incisor teeth were obtained as described. The teeth were cut into labial and lingual sections so that the electrophoretic mobilities of constituent proteins from both sides of the tooth could be compared. The incisor matrix pieces were demineralized prior to PAGE by treatment with 5% trichloroacetic acid (TCA) solution at 4 ~ C for 30 min. The TCA was removed with two washes of ice-cold methanol, and the matrices were extracted with 6 M urea in a 100 mM Tris-borate buffer at pH 8.6. A ratio of 7 ~tg of demineraliized matrix per #1 of Tris-borate buffer was used for the chemical extractions at 60 ~ C for 90 min (Guenther et al., 1977). The urea solutions were deionized immediately prior to use by passing them over a BioRad AG 50 l-X8 ion exchanged column. Chemically extracted extraceUular matrix proteins were analyzed using the method of Guenther et al. (1977). This method employs 7.5% acrylamide gels with a I :37.5 ratio of acylamide to bisacrylamide. The gels were 6 M in urea. Gels were cast in BioRad precision gel tubes (5 mm x 125 mm) and overlaid with 0.10 ml of 100% n-butanol. Electrode buffers for this system consisted of 100 mM Tris-borate, pH 8.6. The gels were routinely prerun at 200 V for 1 h using a voltage regulated power supply. Urea extracts of the matrix fractions (enamel containing labial portion and lingual root-analogue portion) were assayed for protein content (Lowry et al., 1951). Samples were reduced with 400 mM 2-mercaptoethanol and then incubated at 60 ~ C for 30 min in an atmosphere of 100% nitrogen immediately prior to electrophoresis. Sixty micrograms of protein solution was applied t o each gel. The gels were run for 5 h at a constant voltage of 200 V. After completing the electrophoresis, the gels were fixed and stained in a solution of 0.25% Coomassie Brilliant Blue R-250, 9.2% glacial acetic acid, and 40% methanol for 1 h at room temperature. The gels were then destained in a solution of 7.5% glacial acetic acid and 25% methanol prior to photography.

Preparation of Antibody. An alloimmune serum was produced in New Zealand white rabbits against embryonic New Zealand white rabbit molars of 26 days' gestation (Schonfeld, 1975, 1976; Schonfeld, Trump and Slavkin, 1977). At this stage of development, the molar matrices consisted only of enamel and dentin: Since the roots had not yet begun to form, there was no cementum present on these teeth. The embryonic molar matrices were sonicated as described, and washed several times with CMF-PBS, rinsed with deionized water, and lyophilized. The lyophilized tooth matrices were then pulverized in a liquid nitrogen mill. Powdered matrix was suspended in complete Freund's adjuvant at a concentration of 2 mg of matrix per ml of adjuvant. Young adult New Zealand white rabbits were then immunized by 6 consecutive weekly injections of the mixture. Control serum was

prepared by administering the adjuvant (without matrix) to another group of rabbits according to the same protocol. At the conclusion of the immunization program, the animals were bled, and serum was prepared. Immunoglobulins were then prepared from the serum by ammonium sulfate precipitation (Campbell et al., 1970). The concentration of immunoglobulin was brought to 5 mg/ml in a bicarbonate buffered saline solution, pH 7.2.

Immunofluorescence Microseopy. Indirect immunofluorescence microscopy was performed on the continuously erupting incisor teeth from embryonic (26-day) New Zealand white rabbits (Schonfeld, 1975, 1976; Schonfeld, Trump and Slavkin, 1977). Incisors were obtained and sonicated as described and were mounted on cryostat chucks in frozen saline. The teeth were oriented such that the long axis was parallel to the microtome knife, so that sections of 7 pm could be obtained which included both the labial and lingual surfaces. The sections were treated with either control or anti-matrix serum, each at a concentration of 0.67 mg/ml. After 10 min at 37 ~ C, the sera were washed off and the sections further treated with a fluorescein isothiocyanate (FITC) conjugated goat antiserum to rabbit 7S immunoglobulins (Hyland Laboratories, Costa Mesa, Ca.). The slides were washed again, and cover slips were mounted with a mixture of glycerol and buffered saline (Goldman, 1968). Observations and photomicrography were performed with a Zeiss fluorescence microscope equipped with an HBO 200 W/4 mercury lamp and bright field condenser. All photographs were taken at identical magnifications and exposure times with Kodak High Speed Ektachrome (Daylight) film. Prints from the color slides were made by a commercial photographic laboratory.

Results A p r e l i m i n a r y i n v e s t i g a t i o n w a s p r e s e n t e d in w h i c h t h e identification of a material which has the morphological c h a r a c t e r i s t i c s o f e n a m e l m a t r i x w a s o b s e r v e d o n t h e l a b i a l a n d lingual s u r f a c e s o f e m b r y o n i c m o u s e and rabbit incisor teeth using scanning electron micros c o p y ( S l a v k i n a n d B o y d e , 1974). H i r s c h f e l d a n d cow o r k e r s ( 1 9 7 3 ) h a v e also r e p o r t e d t h a t t h e c o r o n a l portions of unerupted rabbit incisors are covered with enamel. These observations were confirmed and extend e d in o u r p r e s e n t i n v e s t i g a t i o n s . Scanning electron micrographs of the entire labial a n d lingual s u r f a c e s o f 2 6 - d a y e m b r y o n i c r a b b i t incisors, f r o m w h i c h t h e cells h a v e b e e n r e m o v e d b y d i s s e c t i o n a n d s o n i c a t i o n , are s h o w n in F i g u r e s 1 A a n d 1C. T h e a r e a s w i t h i n t h e b o x e s w e r e s u b s e q u e n t l y p h o t o g r a p h e d at h i g h e r m a g n i f i c a t i o n (Figs. 1B a n d 1D). T h e s e h i g h e r m a g n i f i c a t i o n p h o t o m i c r o g r a p h s o f t h e l a b i a l a n d lingual ( r o o t - a n a l o g u e ) s u r f a c e s b o t h d e m o n s t r a t e d t h e c h a r a c t e r i s t i c h o n e y c o m b - l i k e appearance of mineralizing enamel matrix resulting from t h e T o m e s ' p r o c e s s pits. A l t h o u g h b o t h t h e l a b i a l a n d lingual surfaces contained the ultrastructural pattern characteristic of developing enamel, the matrix on the l i n g u a l s u r f a c e o f t h e t o o t h d e m o n s t r a t e d a signifi-

S.E. Schonfeldand H.C. Slavkin:Enamel Proteins on Root AnalogueSurfaces of Rabbit Incisors

225

Fig. 1. (A) Scanning electron micrograph of the labial surface of an acellular 26-day embryonic rabbit incisor x 50. (B) Area on panel A within box, showingthe honeycombedappearance characteristic of the mineralizingenamel matrix x 2000. (C) Scanningelectronmicrographof the lingual surface of an acellular 26-day embryonicrabbit incisor x50. (D) Area of panel (C) within box at 2000x magnification.Although this surface will eventually be coveredwith cementum, note that the early matrix (prior to actual cementum deposition) displays the honeycombed structures seen in panel (B). (Figurereduced to 75%)

cantly reduced amount of the enamel-like matrix material. It was observed that the area within each hexagon of enamel matrix (the Tomes' process pits) was greater on the lingual surface than on the labial surface, resulting in a reduced amount of the enamellike material. The height, or thickness, of the honeycomb-like structures on both labial and lingual surfaces was also determined by observing cross sections of acellular matrices which had been cut in half using scanning electron microscopy. Representative measurements of the thickness of the enamel-like material on the labial surface was 18/an, whereas the height on the root analogue surface was only 8.5/2m. It should be stressed that these measurements were made at a point on the tooth which was the same distance from the incisal tip on both the labial and lingual surfaces, resulting in measurements of matrix at the same developmental stage.

Areas which correspond to the rectangles shown in the low magnification scanning photomicrographs of Figure 1 were subsequently studied by transmission electron microscopy. Representative transmission electron micrographs (Fig. 2) demonstrated that the dentin matrices were calcified and contained collagen fiber bundles. The enamel matrix of the labial side (Fig. 2a) was characterized by the presence of crystals oriented orthogonal to the enamel-dentin jucntion, which is the usual appearance of developing enamel (Slavkin, et al., 1976). The lingual surface of the incisor tooth (Fig. 2b) demonstrated a morphology comparable to that observed for the labial side of the tooth. The major difference between the labial and lingual surfaces was a reduced thickness of the enamel-like matrix on the lingual surface. No evidence o f collagen fibers was seen in this matrix.

226

S.E. Schonfeld and H.C. Slavkin: Enamel Proteins on Root Analogue Surfaces of Rabbit Incisors

Fig. 2. (a) Transmission electron micrograph showing the labial enamel matrix and a portion of the dentin matrix. Note that the collagen fibers which can be seen in the dentin are absent in the enamel matrix, which is oriented in parallel columns at right angles to the dentinenamel junction x27,000. (b) Transmission electron micrograph showing the lingual matrix. Note the similarity between the right-hand portion of this matrix and the right-hand portion of the labial matrix x 27,000. (Reduced to 75%)

Fig. 3. Urea acrylamide gel patterns of proteins extracted from labial (a) and lingual (b) segments of the 26-day embryonic rabbit incisor. Note that the gel patterns appear qualitatively identical. Arrows indicate the 3 enamel protein bands which were identified by Guenther et al., 1977 The embryonic incisor tooth organs which were used in this study were cut into segments to c o m p a r e the electrophoretic mobilities of proteins extracted from the labial and lingual sides of the tooth. C o m p a r i s o n o f the electrophoretic profiles of labial and lingual protein extracts shows that there is no qualitative difference, indicating that these two sides of the tooth are c o m p o s e d of identical proteins at this stage o f development. Three o f the proteins which can be resolved in this system have been identified as enamel proteins (Guenther et al., 1977). W e observed that the bands corresponding to these three proteins stained m u c h less heavily on lingual gels as c o m p a r e d with labial gels. This is consistent with our earlier observations that

there is less enamel-like material on the lingual surface c o m p a r e d with the labial surface. In order to further investigate the similarity between these proteins which seem t o be shared between the labial and lingual matrices, indirect immunofluorescence m i c r o s c o p y was performed using an antiserum which was m a d e against intact molar matrices known to contain enamel. It should be noted that the intact molar matrices which were used as the immunogen to which the antiserum was raised contained no cementum at the stage o f development at which they were used. Therefore, any antibodies which were m a d e could only be directed against dentin or enamel extracellular matrix proteins.

S.E. Schonfeld and H.C. Slavkin: Enamel Proteins on Root Analogue Surfaces of Rabbit Incisors Embryonic rabbit incisors which were cut in longitudinal sections so as to contain both labial and lingual surfaces were treated with the rabbit anti-molar and FITC-conjugated goat anti-rabbit sera. Examination of these sections in the fluorescence microscope showed binding of the antibody to both the labial

227

and lingual surfaces of the teeth, but no binding to the dentin (Fig. 4a). Indirect immunofluorescence microscopy of comparable tooth sections treated with the control serum and the F1TC-conjugate did not show binding of the antibody, indicating that the antibody interaction with the labial and lingual surfaces was

Fig. 4. Indirect immunofluorescence photomicrographs showing the binding of an antibody directed against embryonic rabbit molar enamel to both the labial (enamel-bearing; upper portion of section) and lingual (root-analogue, lower portion of section) surfaces of an embryonic rabbit incisor (A). Immunofluorescencemicrograph of a similar tooth treated with control serum is shown in (B). See text for details

228

S.E. Schonfeld and H.C. Slavkin: Enamel Proteins on Root Analogue Surfaces of Rabbit Incisors

specific (Fig. 4b). This observation provides strong evidence that the same proteins are found on the labial and lingual surfaces of these teeth. The specificity of the antiserum has been confirmed by a modified radioimmunoelectrophoretic assay (Schonfeld, 1976; Schonfeld, Trump and Slavkin, 1977). These experiments showed that the antiserum was specific for two of the three protein bands which were identified as enamel specific in the urea-PAGE system employed, and that there were no antibodies present which were directed against dentin proteins.

Discussion The presence of enamel matrix protein on the lingual (root analogue) surface of the continuously erupting permanent rabbit incisors has been conclusively demonstrated by electron microscopic, immunohistochemical, and biochemical criteria. The basis for this demonstration is that we could find no qualitative difference between the enamel bearing labial surface and the lingual root analogue surface by any of these criteria. The localization of enamel matrix proteins on the lingual surface may represent a morphological curiosity peculiar to these specialized teeth. Alternatively, the presence of enamel proteins on root surfaces may be reflective of a more generalized phenomenon. In this regard, it is interesting to note that the innermost layer of acellular cementum (amorphous aceUular cementum) has been characterized by the absence of collagen fibers (Selvig, 1967; Listgarten, 1975). The absence of collagenous fibers in this layer is remarkable when one considers that t h e remainder of accelular and cellular cementum contains collagen. Could the amorphous acellular cementum actually be a layer (albeit very thin) of partially mineralized enamel matrix proteins ? In the case of the teeth studied in our experiments, the epithelial cells which were located adjacent to the lingual surface of the developing rabbit incisors were equivalent to cells of Hertwig's epithelial root sheath (Selvig, 1967). The observations of Lester (1969) indicated a high level of cytodifferentiation within epithelial root sheath cells. These ceils contain a well developed Golgi apparatus, consistent with the ability of these cells to synthesize and secrete extracellular matrix macromolecules (Lester, 1969). The findings presented in our studies indicated that the enamel matrix proteins were present on the lingual surface. This material was derived from the cells which were equivalent to the root sheath cells, which is a further indication of the functional differentiation of this cell population. Consistent with the idea that the ceils of Hertwig's

epithelial root sheath secrete a thin enemel matrix which contributes to amorphous acellular cementum are the observations of Paynter and Pudy (1958), who described a metachromatic "basemSnt membrane" as being the initial extracellular matrix of acellular cementum. They further noted that the "layer of amorphous cementum next to dentin first appeared as a basement membrane on the pulpal surface of the epithelial root diaphragm. It was continuous across the diaphragm and along the side of the root, becoming metachromatic at the level on the root where the epithelial root sheath disintegrated" (Paynter and Pudy, 1958). Shibata and Stern (1967) also described an amorphous, orthochromatic, PAS and alcian blue positive basement membrane adjacent to the outer layer of dentin on lingual surfaces of the rat incisor. They postulate that this material forms the innermost layer of acellular cementum. The existence of enamel matrix proteins in an incompletely mineralized form in the area generally described as amorphous cementum is consistent with our findings of enamel proteins on the root analogue surfaces of rabbit incisor teeth. Our observations are not in conflict with the observations of Lester, Paynter and Pudy, and Shibata and Stern. Further studies to localize enamel matrix proteins on more conventional root surfaces are in progress in our laboratories.

Acknowledgements. This research was supported by Grants DE00094 and DE-02858 from the National Institutes of Health, and by a grant from the California Dental Association. We gratefully acknowledge the technical assistance of Pablo Bringas, Sheryl Osborne, and Jack Worall.

References Anderson, G.S., Stern, I.B.: The proliferation and migration of the attachment epithelium on the cemental surfaceof the rat incisor. Periodontics 4, 115-123 (1966) Boyde, A., Wood, C.: Preparation of animal tissues for surfacescanning electron microscopy.J. Microsc. 90, 221-249 (1969) Campbell, D.H., Garvey, J.S., Cremer, N.E., Sussdorf, D.H.: In: Methods in immunology, p. 189-191. New York: W.A. Benjamin 1970 Goldman, M.: In: Fluorescent antibody methods, p. 149. New York: Academic Press 1968 Guenther, H.L., Croissant, R.C., Schonfeld, S.E., Slavkin, H.C.: Identification of four extracellular-matrix enamel proteins during embryonic-rabbittooth-organ development. Biochem. J. 163, 591-603 (1977). Hay, E.D., Revel, J.-P.: Fine structure of the developing avian cornea. In: Monographs in developmentalbiology (Wolski, A., Chen, P.S., eds.), p. 130. Basel: S. Karger 1969 Hirschfeld, Z., Weinreb, M. M., Michaeli, Y.: Incisors of the rabbit: morphology, histology, and development. J. Dent. Res. 52, 377-384 (1973) Kronfetd, R.: The biologyof cementum, J. Amer. Dent, Assoc. 25, 1451-146I (1938)

S.E. Schonfeld and H.C. Slavkin: Enamel Proteins on Root Analogue Surfaces of Rabbit Incisors Lester, K.S.: The unusual nature of root formation in molar teeth of the laboratory rat. J. Ultrast. Res. 28, 481-506 (1969) Listgarten, M.A.: AfibriUar dental cementum in the rat and hamster. J. Periodont. Res. 10, 158-167 (1975) Lowry, O.H., Roberts, N.R., Leiner, K.Y., Wu, M.L., Farr, A.L.: The quantitative histochemistry of brain. 1. Chemical methods. J. Biol. Chem. 207, 1-49 (1954) Paynter, K.J., Pudy, G.: A study of the structure, chemical nature, and development of cementum in the rat. Anat. Rec. 131, 233246 (1958) Schonfeld, S.E.: Demonstration of an alloimmune response to embryonic enamel matrix proteins. J. Dent. Res. 54, C 72-C77 (1975) Schonfeld, S.E.: Enamel proteins: distribution and autoimmune potential of a sequestered antigen. (Ph.D. dissertation.) Los Angeles: University Of Southern California 1976 Schonfeld, S.E., Trump, G.N., Slavkin, H.C.: Immunogenicity of two naturally occurring solid-phase enamel proteins. Proc. Soc. Exp. Biol. Med. 115, 111-114 (1977)

229

Selvig, K.A.: Electron microscopy of Hertwig's epithelial sheath and of early dentin and cementum formation in the mouse incisor. Acta odont. Stand. 21. 175-186 (1963) Selvig, K.A.: In: Studies on the genesis, composition, and fine structure of cementum. Bergen: Universitetsforlaget 1967 Shibata, F., Stern, I.B.: Hertwig's sheath in the rat incisor. I. Histologic study. J. Periodont. Res. 2, 227-237 (1967) Shibata, F., Stern, I.B.: Hertwig's sheath in the rat incisor. II. Autoradiographic study. J. Periodont. Res. 3, 227-237 (1967) Slavkin, H.C., Boyde, A.: Cementum: an epithelial secretory product? J. Dent. Res. 53, 157 (abstract) (1974) Slavkin, H.C., Mino, W., Bringas, P.: The biosynthesis and secretion of precursor enamel protein by ameloblasts as visualized by autoradiography after tryptophan administration. Anat. Rec. 185, 289-312 (1976)

Received May 25 / Revised August 4, August 12 / Accepted August 15, 1977

Demonstration of enamel matrix proteins on root-analogue surfaces of rabbit permanent incisor teeth.

Calcified Tissue Research Calcif. Tiss. Res. 24, 223-229 (1977) 9 by Springer-Verlag 1977 Demonstration of Enamel Matrix Proteins on Root-Analogue...
2MB Sizes 0 Downloads 0 Views