Archs oral Bid.
Vol. 35,
No. 8, pp. 603613,
1990
0003-9969/90 $3.00 + 0.00
Copyright0 1990PergamonPressplc
Printedin Great Britain.All rightsresewed
ULTRASTRUCTURAL CHA:RACTERIZATION RAT MOLAR
AND IMMUNOCYTOCHEMICAL OF CULTURED CELLS FROM STELLATE RETICULUM
G. E. WISE, V. L. RUDICK,A. M. BRUN-ZINKERNAGEL and W. FAN Department of Anatomy and Cell Biology, Texas College of Osteopathic Medicine, 3500 Camp Bowie Blvd, Fort Worth, TX 76107-2690, U.S.A.
(Accepted 26 February 1990) Summary-By scanning electron microscopy, the cultured cells were. stellate and had numerous filipodiacharacteristics of stellate reticulum cells in ho. By transmission electron microscopy, they contained bundles of intermediate filaments, numerous mitochondria, large electron-dense granules and desmosomes-all fealtures of the stellate reticulum in uiuo. Moreover, the stellate reticulum was the only region of the enamel organ in uiuo that contained large, electron-dense granules. By immunocytochemistry, the cultured cells contained cytokeratins, confirming their epithelial nature, and stellate reticulum cells in uivo and in uitrodid not have an EGF receptor. Thus, these combined ultrastructural and immunocytochemical findings suggest that the cell culture was of stellate reticulum. Key words: stellate reticulum, dental follicle, immunocytochemistry,
INTRODUCIION During the secretory phase of amelogenesis, the enamel organ is a highly complex, stratified epithelium consisting of columnar ameloblasts, a flattened stratum intermedium and the stellate reticulum. Ckcasionally there is a remnant of outer enamel epithelium at the cervical loop but by electron microscopy the few cells of this epithelium appear similar to those of the stellate reticu.lum (G. E. Wise and W. Fan, unpublished). Although the role of the ameloblast in enamel production is. well known (see Weinstock and Leblond, 1971; Warshawsky and Vugman, 1977; reviewed by Frank, 1979), the function of the remainder of the enamel organ remains obscure. We have shown (Wise and Fa.n, 1989) that the stratum intermedium contains alkaline phosphatase during this phase but the stellate reticulum does not. It has been suggested that the stellate reticulum is involved in ion transport (Kallenbach, 1966; Reith, 1970), but there is no experimental confirmation of this. For example, Munhoz and Leblond (1974) injected 4sCa intravenously into rats and found no label over the enamel organ. Moreover, there appears to be no proton pump in the stellate reticulum, but there is one in ameloblasts and the stratum intermedium (Sasaki et al., 1988). Cahill et al. (1988) have speculated that the enamel organ might serve as a ‘clock’ that regulates tooth eruption. Transforming growth factor beta has been demonstrated in the stellate reticulum of 13-day mouse embryos (Heine et al., 1987). Because this factor stimulates growth and proliferation of bone
Abbreuiutions: EGF, epithelial growth factor; PBS, phosphate-buffered saline.
ultrastructure.
cells (Robey et al., 1987; Centrella, McCarthy and Canalis, 1987), it could be that stellate reticulum synthesizes and/or releases factors that regulate tooth eruption. Thus, we have now attempted to isolate and culture the stellate reticulum, because having a pure population of stellate reticulum cells should allow their function to be characterized. We also describe the ultrastructure of normal stellate reticulum and compare it with that of our cultured cells. MATERIALS
AND METHODS
Isolation and culture of stellate reticulum ceils Stellate reticulum was taken from 7-lo-day-old Sprague-Dawley rats killed by cervical dislocation. Two to three rats were used for each isolation, although enough cells for culture may be obtained from one animal. The follicles and enamel organs were surgically removed from first molars and rinsed in Hank’s balanced saline solution containing 10% fetal bovine serum. These were then washed twice in Hank’s containing 1% trypsin from porcine pancreas (Sigma Chemical Co., St Louis, MO, U.S.A.) before incubation for 2 h at 4°C in the same solution. After trypsinization, the follicles were placed in Hank’s containing 20% fetal bovine serum, and the enamel organ was separated under a dissecting microscope using both trans- and epiillumination. The dissected tissue was cultured overnight in 6-well cluster dishes in growth medium containing 50% fetal bovine serum at 37°C in a humidified 95% .air/5% CO1 incubator. The growth medium consisted of Dulbecco’s modified Eagle’s/F12 mixture with Lglutamine and 15 mM HEPES, pH 7.3, supplemented 603
G. E WBE et al.
604
with the following hormones: insulin, 5 pg/ml; transferrin, 5 pg/ml; tri-iodothyronine, 3.25 pg/ml; prostaglandin E 1, hydrocortisone, 25 ng/ml; 20 ng/ml. After 24 h, the serum concentration was reduced to 10%. The medium was replaced approximately every 48-72 h. Electron microscopy
The cultured cells (5-25 days in culture) were fixed at room temperature in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h, followed by rinses in the buffer. They were then postfixed in aqueous 2% 0~0, for 1 h and dehydrated with ethanol. The dehydrated cells were placed in ethanol/Epon solutions of 1: 1 and 1: 2 for 30 min each and then left overnight in 100% Epon. They were then placed in fresh Epon and polymerized for 24 h at 70°C. Thinsections of the blocks were cut on a Porter-Blum MTZB ultramicrotome, stained with uranyl acetate and lead citrate and examined with an Hitachi H-600 electron microscope. Enamel organs and attached follicles were also fixed and dehydrated as described above. For scanning electron microscopy, the cultured cells were also fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h, dehydrated in graded solutions of ethanol, critical-point dried with CO*, mounted on stubs, coated with gold and examined with an ETEC Autoscan scanning electron microscope. Immunocytochemical localization of cytokeratins
Cells were grown on glass coverslips for one week after isolation before being rinsed in PBS, pH 7.3, and then fixed in freshly prepared 4% paraformaldehyde for 10 min at room temperature. The coverslips were then washed twice with PBS and the cells permeabilized for 12 min using 0.2% Triton X-100 in PBS. This was followed by 4 rinses of PBS over a 5-min period. The cells were then exposed to monoclonal anti-cytokeratin 8.13 (Sigma Immuno Chemicals), diluted 1:25 in PBS containing 3% bovine serum albumin, and incubated in a moist chamber for 60 min at room temperature. After 3 washes in PBS containing 1% Triton X-100, fluorochromelabelled IgG diluted 1: 100 in PBS/3% bovine serum albumin was applied and the cells incubated for 30min. The coverslips were washed in 3 changes of PBS and mounted in Mowiol containing 2.5% triethylenediamine (PolySciences, Warrington, PA, U.S.A.). In some cases, mouse ascites fluid (1:25) or anti-ovalbumin (1: 25) replaced anti-cytokeratin. Slides were viewed with a Zeiss Photomicroscope III equipped with epi-fluorescence, and photographed using ASA 1600 film.
Immunocytochemical localization of EGF receptor
For the in vivo localization of EGF receptor, the dental follicles and attached enamel organs of Id-day-old rats were removed and fixed in 10% neutral buffered formalin for 3 h at 4°C. The tissues were dehydrated in a graded series of ethanols and embedded in glycol methacrylate from the JB-4 Embedding Kit (Polysciences, Warrington, PA, U.S.A.) according to the method of Mostafa Meyer and Latorraco (1982) as modified by Wise and Fan (1989). Sections (1.6 pm) were cut with a Porter-Blum MTZB ultramicrotome and placed on slides. The slides were rinsed in 0.05 M tris buffer, pH 7.6, at room temperature and incubated in 3% H,O, for 10 min to eliminate endogenous peroxidase activity. Non-specific background staining was reduced by incubation in goat IgG (diluted 1: 5) for 60min. The slides were rinsed in tris buffer and incubated with a commercial monoclonal mouse anti-EGF receptor (clone 29.1, Mouse IgGl, Sigma Chemical Co.) at a 1: 100 dilution for 24 h at 4°C. They were then rinsed in buffer and incubated in peroxidase-conjugated goat antibody to mouse IgG (1: 100) for 60 min at room temperature. To detect the peroxidase, the slides were rinsed and incubated in 0.06% diaminobenzidine tetrahydrochloride/O.Ol% H,O, in tris for 10min at room temperature. The slides were then rinsed in water stained with haematoxylin and mounted in Permount. Controls included the following: (1) incubation with mouse ascites fluid (same source as specific antibody) instead of anti-EGF receptor, (2) omission of anti-EGF receptor, (3) use of mouse non-immune serum instead of anti-EGF receptor, and (4) pretreatment of sections with EGF before applying anti-EGF receptor. The same procedure was used for detecting antiEGF receptor as for the immunocytochemical localization of keratins except that anti-EGF receptor was used instead of anti-cytokeratin. Dilutions of 1:25, 1: 50 and 1: 100 were used. Histochemical localization of alkaline phosphatase The methods of Symons (1955) and Burstone (1958) were used to detect alkaline phosphatase, with a-naphthyl phosphate disodium salt as the substrate and Fast Blue B salt as the azo dye in 2% sodium barbital buffer (PH 9.2). The enamel organs and dental follicles from the first molars of 8-day-old rats were fixed in neutral buffered formalin and embedded in JB-4, as described earlier, and sections of this material were incubated with the above substrate and dye for 10 min at 37°C. Cultured cells derived from the enamel organs were also fixed on coverslips in
Plate 1 Fig. 1. Low-power electron micrograph of enamel organ and follicle of a first molar from a 7-day-old rat. Ameloblasts (A), stratum intermedium (SI), stellate reticulum (SR) and dental follicle (DF) are seen. Granules (arrows) are present in cells of the stellate reticulum and in the fibroblasts of the dental follicle. Note also the fenestrated capillary (C) in the dental follicle adjacent to the stellate reticulum. x 5300
Culture of stellate reticulum cells
Plate 1
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Culture of stellate reticulum cells neutral buffered formalin and incubated in the same medium to detect alkaline phosphatase. In some instances, the cultured cells were permeabilized, after fixation, with 0.2% Triton X-100. Controls for both the sections and cultured cells did not use the substrate in the medium. After incubation, the cells or sections were rinsed 3 times in distilled water and counterstained with Gill haematoxylin (Sigma Chemical Co.). RESULTS
In preparations of normal enamel organs, the stellate reticulum was continuous with the cells of the stratum inter-medium but separated from the dental follicle by a prominent basal lamina (Figs 1 and 2). There was an extensive, fenestrated capillary bed adjacent to the stellate reticulum (Figs 1 and 2); each capillary was surrounded by a basal lamina (Fig. 2). The cells of the stellate reticulum were indeed stellate and had numerous filipodia (Figs 1 and 2). They contained numerous mitochondria, extensive arrays of intermediate filaments often arranged in bundles, and were attached to each other by numerous desmosomes (Figs l-3). Gap junctions, some as long as 4.0 pm, were found by both thin-section electron microscopy and freeze-fracture electron microscopy (Fig. 4 and 4a). Most characteristic was the presence of electron-dense granules, which were not membrane-bound, with a mean diameter of approx. 0.6 pm, in the cells of the stellate reticulum and in the fibroblasts of the dental follicle (Fig. 1). The granules were not seen in the ameloblasts or in the stratum intermedium. The cells in culture reach confluency in 2 weeks and their morphology was similiar to that of the stellate reticulum in oiuo. In some instances, these cells were maintained through five passages of culture. They were stellate with numerous filipodia (Fig. 5). They also had intermediate filaments (often in bundles), numerous mitochondria, electrondense granules and desmosomes (Figs 68). The cultured cells were heavily stained for cytokeratins (Figs 9 and 10) and their fluorescence pattern suggested that they had intracellular filamentous network. The control preparations were not stained (Fig. 11). Immunoperoxidase localization of the EGF receptor in uiuo did not stain the stellate reticulum (Fig. 12) or any other region Iof the enamel organ. The stellate reticulum in vitro had an occasional cell faintly staining for EGF receptor, but in most instances, the cells did not stain.
607
The stratum intermedium of Sday-old rat first molars was the only part of the enamel organ to stain for alkaline phosphatase in uiuo (Fig. 13). The cultured cells did not stain for alkaline phosphatase (Fig. 14), neither did their controls. DISCUSSION
Our findings suggest that the cells we isolated from the enamel organ of 7-IO-day-old rats were from the stellate reticulum. Their morphology was similar to that of stellate reticulum cells in uiuo. Ultrastructurally, the similarities were even more striking. The presence in the cultured cells of intermediate filaments (often in bundles), numerous mitochondria and frequent desmosomal attachments confirmed their epithelial origin, as well as having a parallel with the appearances of intact stellate reticulum. Significantly, the cultured cells also contained electron-dense granules, such as were found in uiuo only in the stellate reticulum and fibroblasts of the dental follicle, not in ameloblasts and cells of the stratum intermedium. Contamination of our cultures by fibroblasts from the dental follicle was unlikely because the cultured cells stained with a monoclonal antibody to cytokeratins, an epithelial characteristic. The cultured &day rat molar cells were not derived from the stratum intermedium because its cells do not contain dense granules and because the cultured cells did not stain for alkaline phosphatase, unlike the stratum inter-medium of the same age in uiuo. The only other possible source of the cultured epithelial cells would be the ameloblasts, but this is most unlikely because the cultured cells contained numerous intermediate filaments and dense granules: ameloblasts have a terminal web, composed primarily of microfilaments, whereas the stellate reticulum contains an abundant array of intermediate filaments. From postpartum day 1 until eruption the only layer of the first molar enamel organ that contains dense granules is the stellate reticulum (Wise et al., 1988; Wise and Fan, 1989); granules were frequent in our cultured cells. Also, our cultured cells were stellate (see Figs 5 and 16): if they were derived from columnar ameloblasts, they would have had to undergo an extensive change in shape as well as acquiring the capacity to synthesize dense granules and numerous intermediate filaments. As mentioned in the Introduction, Cahill et al. (1988) have suggested that the stellate reticulum functions as a biological clock to regulate tooth
Plate 2 Fig. 2. Electron micrograph of stellate reticulum showing bundles of intermediate filaments (arrows) and numerous mitochondria within the cells. A prominent basal lamina (arrowheads) separates the stellate reticulum from the adjacent fenestrated capillary (C) of the dental follicle. x 15,500 Fig. 3. High-power electron micrograph of a desmosome in the stellate reticulum. Intermediate filaments (tonofilaments) are associated with the desmosome but also are seen in other regions of the cell. x 124,100 Fig. 4. Electron micrograph of an extensive gap junction (arrows) between two stellate reticulum cells. x 84,300 (a) Insert shows freeze-fracture view of a gap junction from the stellate reticulum. First molar, 9 days. x 102,800
G. E Wtsn et al.
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eruption. Although the dental follicle is necessary to tooth eruption (Cahill and Marks, 1980, 1982), the site(s) of regulation of eruption is unknown. Heine et al. (1987) found transforming growth factor beta in the stellate reticulum (see Introduction), and it is tempting to speculate that the stellate reticulum might function to elaborate growth factors that initiate and regulate tooth eruption. However, the absence of expression of EGF receptor in viva and in vitro in the enamel organ suggests that if EGF causes premature eruption it is located elsewhere. In the mouse embryo, radioactive EGF binds to the dental follicle (Partanen and Thesleff, 1987). It also binds to
the dental follicle of erupting mouse incisors and human premolars (Thesleff, Partanen and Rihtniemi, 1987). Thus, while EGF receptors may be localized in the dental follicle, the EGF itself may be synthesized by the stellate reticulum. In fact, the morphology of the stellate reticulum and dental follicle cells in oivo supports the speculation that the stellate reticulum might be secreting materials such as growth factors into the dental follicle. The layer of fenestrated capillaries in the dental follicle immediately adjacent to the stellate reticulum could be specialized for receiving secreted products, like the fenestrated capillaries in endocrine glands.
Plate 3 Fig. 5. Scanning electron micrograph of cultured cells. Note the epithelial appearance of the cells and the numerous fine cellular processes (tilipodia). x 450 Fig. 6. Low-power transmission electron micrograph of cultured cells. Note the bundles of intermediate filaments (arrowheads), desmosomes (arrows) and mitochondria (M). x 8700 Fig. 7. High-power electron micrograph of a cultured stellate reticulum cell showing an electron-dense granule (arrow). Intermediate filaments (IF) also are prominent. x 58,200 Plate 4 Fig. 8. Electron micrograph of cultured stellate reticulum cells showing a prominent desmosome (arrow) and numerous intermediate filaments (IF). x 62,500
Plate 5 Fig. 9. Phase-contrast
view of cultured stellate reticulum cells. x 800
Fig. 10. The same cells as seen in Fig. 9 after incubation with anti-cytokeratin and labelled with fluorescein IgG. The cells strongly fluoresce and their fluorescent pattern suggests that intermediate filaments have reacted with the anti-cytokeratin. x 800 Fig. 11. Cultured stellate reticulum cells after (control) incubation with mouse ascites fluid instead of anti-cytokeratin prior to labelling with fluorescein IgG; no fluorescence is seen. x 800 Plate 6 Fig. 12. Immunoperoxidase-stained section for EGF receptor within the intact enamel organ from the first molar of a Cday-old rat. Note the absence of staining for anti-EGF receptor in the entire enamel organ-ameloblasts (A), stratum intermedium @I) and stellate reticulum (SR). x 400 Fig. 13. Light micrograph of enamel organ and associated dental follicle from the first molar of an &day-old rat showing the staining of the stratum intermedium (arrows) for alkaline phosphatase. Note the absence of staining in the ameloblasts (A) and stellate reticulum (SR). x 365 Fig. 14. Cultured cells obtained from the first molar of an 8-day-old rat. Cells do not stain for alkaline phosphatase; note their stellate shape (arrows). x 200
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Culture of stellate reticulum cells thank Dr Lawrence Oakford for assistance in scamtin electron microscopy and MS Paula Ghena for typing the manuscript. This work was partially supported by an organized research grant from the Texas College of Osteopathic Medicine and, in part, by NIDR grant DE08088 to G.E.W. Acknowledgements-We
REFERENCES
Burstone M. S. (1958) Histochemical comparison ofnapthol AS-phosphates for the demonstration of phosphatase. J. natn. Cancer Inst. 20, 601615. Cahill D. R. and Marks S. C. Jr (1980) Tooth eruption: evidence for the central role of the dental follicle. J. oral Path. 9, 189-200. Cahill D. R. and Marks S. C. Jr (1982) Chronology and histology of exfoliation and eruption of mandibular premolars in dogs. J. Morph. 171, 213-218. Cahill D. R., Marks :S. C. Jr, Wise G. E. and Gorski J. P. (1988) A review and comparison of tooth eruption systems used in experimentation-a new proposal on tooth eruption. In: The Biological Mechanisms of Tooth Eruption and Root Resorption (Edited by Davidovitch Z.) vv. l-7. EBSCO Media. Birminaham. AL. Ceytrella M., McCarthy T: L. and Canalis E. (1987) Transforming growth factor beta is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cells cultures from fetal rat bone. J. biol. Chem. 262, 2869-2874.
Frank R. M. (1979) Tooth enamel: current state of the art. J. a&u. Res. 58(B), 684693. Heine V. I., Munoz E. F., Flanders K. C., Ellingsworth L. R., Lam H. Y. P., Thompson N. L., Roberts A. B. and Spom M. B. (1987) Role of transforming growth factor-B in the development Iofthe mouse embryo. J. Cell Biol. 105, 2861-2876. Kallenbach E. (1966) Electron microscopy of the papillary layer of rat incisor enamel organ during enamel maturation. J. Ultrastruct. Res. 14, 518-533. Mostafa Y. A., Meyer R. A. Jr and Latorraco R. (1982) A simple and rapid method for osteoclast identification using a histochemical method for acid phosphatase. Histochem. J. 14, 409-413.
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Munhoz C. 0. G. and Leblond C. P. (1974) Deposition of calcium phosphate into dentin and enamel as shown by radioautography of sections of incisor teeth following injection of “%a into rats. Calc. Tiss. Res. 15, 221-235. Partanen A.-M. and Thesleff I. (1987) Localization and quantitation of ‘*%epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages. Deul Biol. 120, 186-197. Reith E. J. (1970) The stages of amelogenesis as observed in molar teeth of young rats. J. Ulirastruct. Res. 30, 111-151.
Robey P. G., Young M. F., Flanders K. C., Roche N. S., Kondaiah P., Reddi A. H., Termine J. D., Spom M. B. and Roberts A. B. (1987) Osteoblasts synthesize and respond to TGF-beta in vitro. J. Cell Biol. 105,457463. Sasaki T., Tadokoro K., Yanagisawa T., Higashi S. and Garant P. R. (1988) H (-K)-ATPAse activity in the rat incisor enamel organ during enamel formation. Anat. Rec. 221, 823-833. Symons N. B. B. (1955) Alkaline phosphatase activity in developing teeth of the rat. J. Anat. 89, 238-241. _ Thesleff I.. Partanen A.-M. and Rihtniemi L. (1987) \ , Localization of epidermal growth factor receptors in mouse incisors and human premolars during eruption. Eur. J. Orthodont.
9, 24-32.
Warshawsky H. and Vugman I. (1977) A comparison of the protein synthetic activity of presecretory and secretory ameloblasts in rat incisors. Anat. Rec. 188, 143-171. Weinstock A. and Leblond C. P. (1971) Elaboration of the matrix glycoprotein of enamel by the secretory ameloblasts of the rat incisor as revealed by radioautography after galactose ‘H-iniection. J. Cell Biol. 51. 26-51. Wise G. E. and Fan W. (1989) Changes in the tartrateresistant acid phosphatase cell population in dental follicles and bony crypts of rat molars during tooth eruption. J. dent. Res. 68, 150-156. Wise G. E., Marks S. C. Jr, Cahill D. R. and Gorski J. P. (1988) Ultrastructural features of the dental follicle and enamel organ prior to and during tooth eruption. In: The Biological Mechanisms of Tooth Eruption and Root Resorption (Edited by Davidovitch Z.) pp. 243-249. EBSCO
Media, Birmingham, AL.
Vol. 35,
No. 8, pp. 603613,
1990
0003-9969/90 $3.00 + 0.00
Copyright0 1990PergamonPressplc
Printedin Great Britain.All rightsresewed
ULTRASTRUCTURAL CHA:RACTERIZATION RAT MOLAR
AND IMMUNOCYTOCHEMICAL OF CULTURED CELLS FROM STELLATE RETICULUM
G. E. WISE, V. L. RUDICK,A. M. BRUN-ZINKERNAGEL and W. FAN Department of Anatomy and Cell Biology, Texas College of Osteopathic Medicine, 3500 Camp Bowie Blvd, Fort Worth, TX 76107-2690, U.S.A.
(Accepted 26 February 1990) Summary-By scanning electron microscopy, the cultured cells were. stellate and had numerous filipodiacharacteristics of stellate reticulum cells in ho. By transmission electron microscopy, they contained bundles of intermediate filaments, numerous mitochondria, large electron-dense granules and desmosomes-all fealtures of the stellate reticulum in uiuo. Moreover, the stellate reticulum was the only region of the enamel organ in uiuo that contained large, electron-dense granules. By immunocytochemistry, the cultured cells contained cytokeratins, confirming their epithelial nature, and stellate reticulum cells in uivo and in uitrodid not have an EGF receptor. Thus, these combined ultrastructural and immunocytochemical findings suggest that the cell culture was of stellate reticulum. Key words: stellate reticulum, dental follicle, immunocytochemistry,
INTRODUCIION During the secretory phase of amelogenesis, the enamel organ is a highly complex, stratified epithelium consisting of columnar ameloblasts, a flattened stratum intermedium and the stellate reticulum. Ckcasionally there is a remnant of outer enamel epithelium at the cervical loop but by electron microscopy the few cells of this epithelium appear similar to those of the stellate reticu.lum (G. E. Wise and W. Fan, unpublished). Although the role of the ameloblast in enamel production is. well known (see Weinstock and Leblond, 1971; Warshawsky and Vugman, 1977; reviewed by Frank, 1979), the function of the remainder of the enamel organ remains obscure. We have shown (Wise and Fa.n, 1989) that the stratum intermedium contains alkaline phosphatase during this phase but the stellate reticulum does not. It has been suggested that the stellate reticulum is involved in ion transport (Kallenbach, 1966; Reith, 1970), but there is no experimental confirmation of this. For example, Munhoz and Leblond (1974) injected 4sCa intravenously into rats and found no label over the enamel organ. Moreover, there appears to be no proton pump in the stellate reticulum, but there is one in ameloblasts and the stratum intermedium (Sasaki et al., 1988). Cahill et al. (1988) have speculated that the enamel organ might serve as a ‘clock’ that regulates tooth eruption. Transforming growth factor beta has been demonstrated in the stellate reticulum of 13-day mouse embryos (Heine et al., 1987). Because this factor stimulates growth and proliferation of bone
Abbreuiutions: EGF, epithelial growth factor; PBS, phosphate-buffered saline.
ultrastructure.
cells (Robey et al., 1987; Centrella, McCarthy and Canalis, 1987), it could be that stellate reticulum synthesizes and/or releases factors that regulate tooth eruption. Thus, we have now attempted to isolate and culture the stellate reticulum, because having a pure population of stellate reticulum cells should allow their function to be characterized. We also describe the ultrastructure of normal stellate reticulum and compare it with that of our cultured cells. MATERIALS
AND METHODS
Isolation and culture of stellate reticulum ceils Stellate reticulum was taken from 7-lo-day-old Sprague-Dawley rats killed by cervical dislocation. Two to three rats were used for each isolation, although enough cells for culture may be obtained from one animal. The follicles and enamel organs were surgically removed from first molars and rinsed in Hank’s balanced saline solution containing 10% fetal bovine serum. These were then washed twice in Hank’s containing 1% trypsin from porcine pancreas (Sigma Chemical Co., St Louis, MO, U.S.A.) before incubation for 2 h at 4°C in the same solution. After trypsinization, the follicles were placed in Hank’s containing 20% fetal bovine serum, and the enamel organ was separated under a dissecting microscope using both trans- and epiillumination. The dissected tissue was cultured overnight in 6-well cluster dishes in growth medium containing 50% fetal bovine serum at 37°C in a humidified 95% .air/5% CO1 incubator. The growth medium consisted of Dulbecco’s modified Eagle’s/F12 mixture with Lglutamine and 15 mM HEPES, pH 7.3, supplemented 603
G. E WBE et al.
604
with the following hormones: insulin, 5 pg/ml; transferrin, 5 pg/ml; tri-iodothyronine, 3.25 pg/ml; prostaglandin E 1, hydrocortisone, 25 ng/ml; 20 ng/ml. After 24 h, the serum concentration was reduced to 10%. The medium was replaced approximately every 48-72 h. Electron microscopy
The cultured cells (5-25 days in culture) were fixed at room temperature in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h, followed by rinses in the buffer. They were then postfixed in aqueous 2% 0~0, for 1 h and dehydrated with ethanol. The dehydrated cells were placed in ethanol/Epon solutions of 1: 1 and 1: 2 for 30 min each and then left overnight in 100% Epon. They were then placed in fresh Epon and polymerized for 24 h at 70°C. Thinsections of the blocks were cut on a Porter-Blum MTZB ultramicrotome, stained with uranyl acetate and lead citrate and examined with an Hitachi H-600 electron microscope. Enamel organs and attached follicles were also fixed and dehydrated as described above. For scanning electron microscopy, the cultured cells were also fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h, dehydrated in graded solutions of ethanol, critical-point dried with CO*, mounted on stubs, coated with gold and examined with an ETEC Autoscan scanning electron microscope. Immunocytochemical localization of cytokeratins
Cells were grown on glass coverslips for one week after isolation before being rinsed in PBS, pH 7.3, and then fixed in freshly prepared 4% paraformaldehyde for 10 min at room temperature. The coverslips were then washed twice with PBS and the cells permeabilized for 12 min using 0.2% Triton X-100 in PBS. This was followed by 4 rinses of PBS over a 5-min period. The cells were then exposed to monoclonal anti-cytokeratin 8.13 (Sigma Immuno Chemicals), diluted 1:25 in PBS containing 3% bovine serum albumin, and incubated in a moist chamber for 60 min at room temperature. After 3 washes in PBS containing 1% Triton X-100, fluorochromelabelled IgG diluted 1: 100 in PBS/3% bovine serum albumin was applied and the cells incubated for 30min. The coverslips were washed in 3 changes of PBS and mounted in Mowiol containing 2.5% triethylenediamine (PolySciences, Warrington, PA, U.S.A.). In some cases, mouse ascites fluid (1:25) or anti-ovalbumin (1: 25) replaced anti-cytokeratin. Slides were viewed with a Zeiss Photomicroscope III equipped with epi-fluorescence, and photographed using ASA 1600 film.
Immunocytochemical localization of EGF receptor
For the in vivo localization of EGF receptor, the dental follicles and attached enamel organs of Id-day-old rats were removed and fixed in 10% neutral buffered formalin for 3 h at 4°C. The tissues were dehydrated in a graded series of ethanols and embedded in glycol methacrylate from the JB-4 Embedding Kit (Polysciences, Warrington, PA, U.S.A.) according to the method of Mostafa Meyer and Latorraco (1982) as modified by Wise and Fan (1989). Sections (1.6 pm) were cut with a Porter-Blum MTZB ultramicrotome and placed on slides. The slides were rinsed in 0.05 M tris buffer, pH 7.6, at room temperature and incubated in 3% H,O, for 10 min to eliminate endogenous peroxidase activity. Non-specific background staining was reduced by incubation in goat IgG (diluted 1: 5) for 60min. The slides were rinsed in tris buffer and incubated with a commercial monoclonal mouse anti-EGF receptor (clone 29.1, Mouse IgGl, Sigma Chemical Co.) at a 1: 100 dilution for 24 h at 4°C. They were then rinsed in buffer and incubated in peroxidase-conjugated goat antibody to mouse IgG (1: 100) for 60 min at room temperature. To detect the peroxidase, the slides were rinsed and incubated in 0.06% diaminobenzidine tetrahydrochloride/O.Ol% H,O, in tris for 10min at room temperature. The slides were then rinsed in water stained with haematoxylin and mounted in Permount. Controls included the following: (1) incubation with mouse ascites fluid (same source as specific antibody) instead of anti-EGF receptor, (2) omission of anti-EGF receptor, (3) use of mouse non-immune serum instead of anti-EGF receptor, and (4) pretreatment of sections with EGF before applying anti-EGF receptor. The same procedure was used for detecting antiEGF receptor as for the immunocytochemical localization of keratins except that anti-EGF receptor was used instead of anti-cytokeratin. Dilutions of 1:25, 1: 50 and 1: 100 were used. Histochemical localization of alkaline phosphatase The methods of Symons (1955) and Burstone (1958) were used to detect alkaline phosphatase, with a-naphthyl phosphate disodium salt as the substrate and Fast Blue B salt as the azo dye in 2% sodium barbital buffer (PH 9.2). The enamel organs and dental follicles from the first molars of 8-day-old rats were fixed in neutral buffered formalin and embedded in JB-4, as described earlier, and sections of this material were incubated with the above substrate and dye for 10 min at 37°C. Cultured cells derived from the enamel organs were also fixed on coverslips in
Plate 1 Fig. 1. Low-power electron micrograph of enamel organ and follicle of a first molar from a 7-day-old rat. Ameloblasts (A), stratum intermedium (SI), stellate reticulum (SR) and dental follicle (DF) are seen. Granules (arrows) are present in cells of the stellate reticulum and in the fibroblasts of the dental follicle. Note also the fenestrated capillary (C) in the dental follicle adjacent to the stellate reticulum. x 5300
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Culture of stellate reticulum cells neutral buffered formalin and incubated in the same medium to detect alkaline phosphatase. In some instances, the cultured cells were permeabilized, after fixation, with 0.2% Triton X-100. Controls for both the sections and cultured cells did not use the substrate in the medium. After incubation, the cells or sections were rinsed 3 times in distilled water and counterstained with Gill haematoxylin (Sigma Chemical Co.). RESULTS
In preparations of normal enamel organs, the stellate reticulum was continuous with the cells of the stratum inter-medium but separated from the dental follicle by a prominent basal lamina (Figs 1 and 2). There was an extensive, fenestrated capillary bed adjacent to the stellate reticulum (Figs 1 and 2); each capillary was surrounded by a basal lamina (Fig. 2). The cells of the stellate reticulum were indeed stellate and had numerous filipodia (Figs 1 and 2). They contained numerous mitochondria, extensive arrays of intermediate filaments often arranged in bundles, and were attached to each other by numerous desmosomes (Figs l-3). Gap junctions, some as long as 4.0 pm, were found by both thin-section electron microscopy and freeze-fracture electron microscopy (Fig. 4 and 4a). Most characteristic was the presence of electron-dense granules, which were not membrane-bound, with a mean diameter of approx. 0.6 pm, in the cells of the stellate reticulum and in the fibroblasts of the dental follicle (Fig. 1). The granules were not seen in the ameloblasts or in the stratum intermedium. The cells in culture reach confluency in 2 weeks and their morphology was similiar to that of the stellate reticulum in oiuo. In some instances, these cells were maintained through five passages of culture. They were stellate with numerous filipodia (Fig. 5). They also had intermediate filaments (often in bundles), numerous mitochondria, electrondense granules and desmosomes (Figs 68). The cultured cells were heavily stained for cytokeratins (Figs 9 and 10) and their fluorescence pattern suggested that they had intracellular filamentous network. The control preparations were not stained (Fig. 11). Immunoperoxidase localization of the EGF receptor in uiuo did not stain the stellate reticulum (Fig. 12) or any other region Iof the enamel organ. The stellate reticulum in vitro had an occasional cell faintly staining for EGF receptor, but in most instances, the cells did not stain.
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The stratum intermedium of Sday-old rat first molars was the only part of the enamel organ to stain for alkaline phosphatase in uiuo (Fig. 13). The cultured cells did not stain for alkaline phosphatase (Fig. 14), neither did their controls. DISCUSSION
Our findings suggest that the cells we isolated from the enamel organ of 7-IO-day-old rats were from the stellate reticulum. Their morphology was similar to that of stellate reticulum cells in uiuo. Ultrastructurally, the similarities were even more striking. The presence in the cultured cells of intermediate filaments (often in bundles), numerous mitochondria and frequent desmosomal attachments confirmed their epithelial origin, as well as having a parallel with the appearances of intact stellate reticulum. Significantly, the cultured cells also contained electron-dense granules, such as were found in uiuo only in the stellate reticulum and fibroblasts of the dental follicle, not in ameloblasts and cells of the stratum intermedium. Contamination of our cultures by fibroblasts from the dental follicle was unlikely because the cultured cells stained with a monoclonal antibody to cytokeratins, an epithelial characteristic. The cultured &day rat molar cells were not derived from the stratum intermedium because its cells do not contain dense granules and because the cultured cells did not stain for alkaline phosphatase, unlike the stratum inter-medium of the same age in uiuo. The only other possible source of the cultured epithelial cells would be the ameloblasts, but this is most unlikely because the cultured cells contained numerous intermediate filaments and dense granules: ameloblasts have a terminal web, composed primarily of microfilaments, whereas the stellate reticulum contains an abundant array of intermediate filaments. From postpartum day 1 until eruption the only layer of the first molar enamel organ that contains dense granules is the stellate reticulum (Wise et al., 1988; Wise and Fan, 1989); granules were frequent in our cultured cells. Also, our cultured cells were stellate (see Figs 5 and 16): if they were derived from columnar ameloblasts, they would have had to undergo an extensive change in shape as well as acquiring the capacity to synthesize dense granules and numerous intermediate filaments. As mentioned in the Introduction, Cahill et al. (1988) have suggested that the stellate reticulum functions as a biological clock to regulate tooth
Plate 2 Fig. 2. Electron micrograph of stellate reticulum showing bundles of intermediate filaments (arrows) and numerous mitochondria within the cells. A prominent basal lamina (arrowheads) separates the stellate reticulum from the adjacent fenestrated capillary (C) of the dental follicle. x 15,500 Fig. 3. High-power electron micrograph of a desmosome in the stellate reticulum. Intermediate filaments (tonofilaments) are associated with the desmosome but also are seen in other regions of the cell. x 124,100 Fig. 4. Electron micrograph of an extensive gap junction (arrows) between two stellate reticulum cells. x 84,300 (a) Insert shows freeze-fracture view of a gap junction from the stellate reticulum. First molar, 9 days. x 102,800
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eruption. Although the dental follicle is necessary to tooth eruption (Cahill and Marks, 1980, 1982), the site(s) of regulation of eruption is unknown. Heine et al. (1987) found transforming growth factor beta in the stellate reticulum (see Introduction), and it is tempting to speculate that the stellate reticulum might function to elaborate growth factors that initiate and regulate tooth eruption. However, the absence of expression of EGF receptor in viva and in vitro in the enamel organ suggests that if EGF causes premature eruption it is located elsewhere. In the mouse embryo, radioactive EGF binds to the dental follicle (Partanen and Thesleff, 1987). It also binds to
the dental follicle of erupting mouse incisors and human premolars (Thesleff, Partanen and Rihtniemi, 1987). Thus, while EGF receptors may be localized in the dental follicle, the EGF itself may be synthesized by the stellate reticulum. In fact, the morphology of the stellate reticulum and dental follicle cells in oivo supports the speculation that the stellate reticulum might be secreting materials such as growth factors into the dental follicle. The layer of fenestrated capillaries in the dental follicle immediately adjacent to the stellate reticulum could be specialized for receiving secreted products, like the fenestrated capillaries in endocrine glands.
Plate 3 Fig. 5. Scanning electron micrograph of cultured cells. Note the epithelial appearance of the cells and the numerous fine cellular processes (tilipodia). x 450 Fig. 6. Low-power transmission electron micrograph of cultured cells. Note the bundles of intermediate filaments (arrowheads), desmosomes (arrows) and mitochondria (M). x 8700 Fig. 7. High-power electron micrograph of a cultured stellate reticulum cell showing an electron-dense granule (arrow). Intermediate filaments (IF) also are prominent. x 58,200 Plate 4 Fig. 8. Electron micrograph of cultured stellate reticulum cells showing a prominent desmosome (arrow) and numerous intermediate filaments (IF). x 62,500
Plate 5 Fig. 9. Phase-contrast
view of cultured stellate reticulum cells. x 800
Fig. 10. The same cells as seen in Fig. 9 after incubation with anti-cytokeratin and labelled with fluorescein IgG. The cells strongly fluoresce and their fluorescent pattern suggests that intermediate filaments have reacted with the anti-cytokeratin. x 800 Fig. 11. Cultured stellate reticulum cells after (control) incubation with mouse ascites fluid instead of anti-cytokeratin prior to labelling with fluorescein IgG; no fluorescence is seen. x 800 Plate 6 Fig. 12. Immunoperoxidase-stained section for EGF receptor within the intact enamel organ from the first molar of a Cday-old rat. Note the absence of staining for anti-EGF receptor in the entire enamel organ-ameloblasts (A), stratum intermedium @I) and stellate reticulum (SR). x 400 Fig. 13. Light micrograph of enamel organ and associated dental follicle from the first molar of an &day-old rat showing the staining of the stratum intermedium (arrows) for alkaline phosphatase. Note the absence of staining in the ameloblasts (A) and stellate reticulum (SR). x 365 Fig. 14. Cultured cells obtained from the first molar of an 8-day-old rat. Cells do not stain for alkaline phosphatase; note their stellate shape (arrows). x 200
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Culture of stellate reticulum cells thank Dr Lawrence Oakford for assistance in scamtin electron microscopy and MS Paula Ghena for typing the manuscript. This work was partially supported by an organized research grant from the Texas College of Osteopathic Medicine and, in part, by NIDR grant DE08088 to G.E.W. Acknowledgements-We
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Warshawsky H. and Vugman I. (1977) A comparison of the protein synthetic activity of presecretory and secretory ameloblasts in rat incisors. Anat. Rec. 188, 143-171. Weinstock A. and Leblond C. P. (1971) Elaboration of the matrix glycoprotein of enamel by the secretory ameloblasts of the rat incisor as revealed by radioautography after galactose ‘H-iniection. J. Cell Biol. 51. 26-51. Wise G. E. and Fan W. (1989) Changes in the tartrateresistant acid phosphatase cell population in dental follicles and bony crypts of rat molars during tooth eruption. J. dent. Res. 68, 150-156. Wise G. E., Marks S. C. Jr, Cahill D. R. and Gorski J. P. (1988) Ultrastructural features of the dental follicle and enamel organ prior to and during tooth eruption. In: The Biological Mechanisms of Tooth Eruption and Root Resorption (Edited by Davidovitch Z.) pp. 243-249. EBSCO
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