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Patterns of Fluoro-Gold Entry into Rat Molar Enamel, Dentin, and Pulp
M.R. Byers1,2,* and
K.J. Yoon Lin2
1 Dept. of Anesthesiology, Box 356540 , and
2 Dept. of Endodontics, Univ. of Washington, Seattle, WA 98195-6540;
Correspondence: *corresponding author, Byersm{at}u.washington.edu
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ABSTRACT
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Permeabilities of enamel and dentin are not fully understood despite their importance for caries, restorative materials, and pulp-dentin-enamel interactions. We have found that Fluoro-Gold is useful for examining tooth permeability, and we designed studies to test the effects of aging, injury, neural function, and dentinal repair on its influx into vital rat teeth. We used fluorescence microscopy and immunocytochemistry to show that Fluoro-Gold rapidly penetrates enamel, the dentin-enamel junction, and outer dentinal acellular tubules, and then concentrates in odontoblasts, where it remains for weeks. As predicted, influx was greatest in immature teeth, and formation of reparative dentin impeded it. We expected that denervation would disrupt influx, because of neural regulation of dentinal fluid movement, but it did not. Damage to odontoblasts under injured dentin caused increased influx and efflux of Fluoro-Gold. Analysis of our data suggests that permeabilities of enamel and dentin to Fluoro-Gold are age-related, inter-dependent, and regulated by odontoblasts.
Key Words: odontoblasts • aging • innervation • injury • reparative dentin
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INTRODUCTION
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The bi-directional permeability of enamel to small molecules in vivo and in vitro has been demonstrated with radioactive tracers, water, or conductance (Bartelstone, 1951; Linden, 1968; ten Bosch et al., 2000). It is greatest in teeth with immature enamel, and it appears to require a partnership with dentin. In addition, electrical stimulation of tooth surfaces in vitro causes charged dyes to penetrate through enamel into dentin and pulp (Mumford, 1959). Fluoro-Gold is commonly used for retrograde neuronal tracing (Fried et al., 1989; Chang et al., 1990; Wessendorf, 1991; Qian and Naftel, 1994), but here we have analyzed its ability to penetrate through enamel into rat teeth in vivo. We have analyzed its rate and route of entry and the effects of age, type of tooth, injury, presence of reparative dentin, and denervation. Our specific hypotheses were: (1) that Fluoro-Gold influx would decrease during tooth maturation as the organic/aqueous proportion of enamel decreases and dentin thickens; (2) that less would be found in inflamed pulps due to vasodilation and increased pulpo-dentinal fluid outflow (Olgart, 1996; Pashley, 1996); (3) that formation of reparative dentin would block its movement; and (4) that sensory denervation would affect Fluoro-Gold labeling patterns because of neural regulation of pulpal and dentinal interstitial fluid flow (Vongsavan and Matthews, 1992; Berggreen and Heyeraas, 2000).
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MATERIALS & METHODS
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Animals and Anesthesia
These studies of 54 male Sprague-Dawley rats were approved according to the University of Washington animal welfare guidelines. Each rat received intraperitoneal Equithesin (4.25% chloral hydrate, 0.97% sodium pentobarbital, 0.35-0.45 mL/100 g) prior to Fluoro-Gold application, dental procedures, denervation surgery, and terminal fixation. The sample size per group (N) for each experiment represents the number of treated jaws.
Fluoro-Gold
Fluoro-Gold (2-hydroxy-4, 4'-diamidinostilbene; Fluorochrome Inc., Denver, CO, USA) is a carcinogen (molecular weight, 532 D) whose fluorescent component is hydroxystilbamidine (Wessendorf, 1991). Fluoro-Gold crystals were inserted into the first molar sulcus on its mesial side, where they dissolved within minutes. If hemorrhage or sulcular fluid efflux caused washout, additional crystals were added to produce a standard yellow bolus after the five-minute application period. Application controls received a three-day exposure to circulating Fluoro-Gold (N = 2) (intraperitoneal 0.5% solution, 15 mg/Kg; Merchenthaler, 1991), placement of crystals for 1-2 hrs on molar occlusal surfaces (N = 3), or no Fluoro-Gold (N = 10, negative controls).
Timing and Aging Studies
We used fluorescence or immunocytochemical detection of Fluoro-Gold after placement into the first molar sulcus of adult rats (2-5 months old) at 1-2 hrs (N = 5), 4 hrs (N = 2), 1 day (N = 2), 3-4 days (N = 18), 7-8 days (N = 5), or 13 days (N = 2). We compared those jaws with three-day labeling in juvenile rats (3-4 wks old, N = 8; or 6 wks, N = 6) and old rats (10-12 months, N = 11).
Injuries
(1) Flap or Scrape Injury
We made a gingival flap at each first molar in 6 rats, and then the right molars also received a scrape injury (N = 12) by means of a 1-2 Gracey curette to the exposed cervical enamel and dentin, as described earlier (Taylor et al., 1988), for comparison with the flap-only left molars (N = 12). Fluoro-Gold was then placed on the injured areas of all 4 teeth per rat and examined 3 days later.
(2) Reparative Dentin
We made Class V injuries to the mesial surfaces of maxillary first molars using a #2 round bur to drill through the free gingiva and halfway into dentin, followed by etching and air drying, as described (Taylor and Byers, 1990) at 11 days (N = 4) prior to Fluoro-Gold application, followed by 3 more days for tooth labeling.
(3) Pulp Exposure
We made small pulp exposures in both maxillary first molars of 5 rats using a #1/4 round bur and placed Fluoro-Gold onto the pulp (N = 10), and sealed it with Cavit (Premier, Norristown, PA, USA) for 3-8 days.
(4) Denervation
We cut the right inferior alveolar nerve from the lateral approach, as described previously (Berger et al., 1983) in 6 rats, with 3 others sham-operated by exposure of the lateral mandible without canal entry. The rats were re-anesthetized 2-3 days later for Fluoro-Gold placement in the right (denervated) and left (intact) first mandibular molar sulcus for subsequent three-day labeling.
Tissue Fixation and Preparation
Each rat was deeply anesthetized and perfusion-fixed for 10 min with 200 mL of 4% paraformaldehyde in 0.1 M phosphate buffer plus 0.2% picric acid, pH 7.4. Jaws were excised, post-fixed, decalcified in cold 4 N formic acid in 0.5 M sodium formate (pH 2.5-3), rinsed in 0.1 M phosphate buffer (pH 7.4), and equilibrated in 30% sucrose. Longitudinal, mesio-distal serial sections were cut at 40 µm on a freezing microtome and collected in phosphate-buffered saline.
Immunocytochemistry
Using a polyclonal antibody (AB153, 1:10,000 dilution; Chemicon, Temecula, CA, USA), we detected Fluoro-Gold patterns in every fourth section. Floating sections were incubated in primary antibody at 4°C for 3 days, reacted with biotinylated goat anti-rabbit IgG (1 µL/mL; Vector), followed by the avidin-biotin complex (ABC, Vector, Burlingame, CA, USA), detected with diaminobenzidine (0.75 mg/mL; Sigma, St. Louis, MO, USA) in 0.1 M Tris-buffered saline with 0.0125% hydrogen peroxide. Fully reacted sections were mounted on gelatin-coated slides, then counterstained, in some cases, with methylene blue. Immunospecificity was tested by omission of primary antibody.
Fluoro-Gold Passage Through Enamel
Maxillary first molars in two 10-month-old and two six-week-old rats were labeled for 2 hrs by sulcular Fluoro-Gold placement, followed by fixation with 4% formaldehyde, dehydration, and embedment in methacrylate resin (Microbed, Electron Microscopy Services, Fort Washington, PA, USA). Undecalcified sections were cut slowly at 40-µm thickness by means of a Leica-1600 microtome with diamond blade. Sections were examined by fluorescence microscopy (UV excitation, blue emission) and digital photography.
Analyses and Statistics
The reacted sections were coded and analyzed blind by both investigators using a Nikon FXA microscope. Each investigator measured the longitudinal extent of the labeled odontoblast layer along the mesial side of the first molar in two midline sections per jaw. The four values per jaw were then averaged, and means per group were compared by the Student-Newman-Keuls test. Each jaw was also assessed for two Fluoro-Gold patterns: (1) concentration in odontoblast layer or (2) diffuse spread into deep pulp. The occurrence of those patterns was then compared for juvenile, adult, old, and injury groups by non-parametric Kruskal-Wallis one-way ANOVA and the Dunn Test.
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RESULTS
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Patterns at 3 Days
There was strong Fluoro-Gold in odontoblasts and adjacent pulp cells of the mesial cervical pulp for all adult first molars (Figs. 1A, 1B ). It also spread around the sulcus to label the intermediate and distal pulp horns and usually the second molar as well. There was no Fluoro-Gold in radicular dentin or pulp, but intercuspal odontoblasts were often labeled, as would be expected by the yellowing of cervical and intercuspal enamel that was observed as Fluoro-Gold dissolved and flowed around the sulcus. There was also some labeling of the dead track tubules over reparative dentin at cusp tips. The adjacent gingiva and connective tissue had intense label. Fluoro-Gold in the sulcus of maxillary incisors (3 rats, 6 incisors) did not produce any labeling of pulp or dentin, despite excellent gingival label (Fig. 1C).
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Figure 1. Labeling in adult rat teeth. (A,B) Fluoro-Gold was placed in the sulcus (*) on the mesial side of the first molar. Three days later, it was found heavily labeling the odontoblasts (left arrow) near that site, and less intensely for cervical odontoblasts on other sides of the tooth (right arrow). There was no label of root pulp, but some intercuspal odontoblasts were labeled. (C) Similar Fluoro-Gold placement in rat incisor sulcus (*) did not produce any label of pulp (p) 3 days later. (D-G) Analysis of timing of Fluoro-Gold influx demonstrated labeled dentin plus a few odontoblasts (arrows) 1 hr after FG placement (D), many more odontoblasts at 4 hrs for cervical (E) and intercuspal (F) odontoblasts, with strong label of odontoblasts persisting at 13 days (G). Dentinal labeling was prominent at 1 and 4 hrs but weak or absent at 3-13 days, and was blocked by reparative dentin (RD, in Fig. 1E ). (H) Intraperitoneal injection of Fluoro-Gold produced weak label of all odontoblasts (arrow) in all teeth. (I) At high magnification, Fluoro-Gold can be seen outlining acellular tubules of outer dentin (OD) and within odontoblast processes (arrows) of inner dentin (ID) 4 hrs after application. (J,K) Fluorescence microscopy of undecalcified teeth shows Fluoro-Gold in enamel (En) and staining inner dentin (den) and pulp (P) at 2 hrs after placement in the mesial sulcus next to first molars. The endogenous fluorescence of unlabeled teeth (K) was much less. Scales: A, 0.5 mm; B-H, 0.2 mm; I, 0.05 mm.
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Timing of Entry
By 1 hr, Fluoro-Gold was in outer cervical dentin, with some initial labeling of inner dentin and odontoblasts (Fig. 1D). By 4 hrs, Fluoro-Gold was into cervical or intercuspal dentin, underlying odontoblasts, and dead track tubules overlying reparative dentin (Figs. 1E, 1F). The odontoblast labels at 1, 7-8, and 13 days were similar to that at 3 days (Figs. 1A, 1B, 1G), but Fluoro-Gold had cleared from dead tracks and dentinal tubules by 7-8 days. There was weak uniform labeling of odontoblasts 3 days after intraperitoneal placement (Fig. 1H). High magnification showed that Fluoro-Gold labeling of outer dentin was along tubule walls, while odontoblast processes became stained in middle and inner dentin (Fig. 1I). None of the immuno-controls (no primary antibody, no FG label) had FG staining (not shown). No pulpal Fluoro-Gold was found when crystals were just placed on molar cusp tips for 1-1.5 hrs, though dead tracks were well-labeled under those conditions (not shown).
Initial Route of Influx
The implanted Fluoro-Gold dissolved within minutes and spread around the first molar sulcus and into intercuspal grooves, and often continued on to the second molar. It stained enamel yellow within 10 min (Appendix Fig., L; www.dentalresearch.org). This staining was greater and spread further in young rats, and it was gone by 1 day. Sections of undecalcified teeth were examined by fluorescence microscopy at 2 hrs after application of Fluoro-Gold. The dye was especially evident in enamel, inner dentin, and pulp, and it was much brighter than endogenous fluorescence (Figs. 1J, 1K).
Age Effects
Labeling at 3 days in juvenile rats (3-4 wks old) was stronger and much more extensive than for two- to five-month-old adults (Figs. 1A, 2A, Table), although unerupted teeth were completely unlabeled (Fig. 2B). Old rats (10-12 months) had thick dentin and flat odontoblasts (Figs. 2C, 2D, 2E) compared with the thin dentin and columnar odontoblasts of young rats, but each group concentrated Fluoro-Gold in odontoblast cell bodies. Fluoro-Gold in all juvenile samples had spread along the sulcus to label both the first and second molars, while none of the old rats was labeled beyond the first molar.
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Figure 2. Comparison of immature and very old rat molars. (A,B) The Fluoro-Gold label of odontoblasts (arrows) was widespread and intense for first (M1) and second (M2) molars 3 days after placement on the mesial side of M1 in juvenile rats. The third molar (B) was not yet erupted and was unlabeled. (C) Year-old molars have much less odontoblast labeling (arrow) compared with immature teeth (AB), and Fluoro-Gold did not reach the posterior side of either the first or the second molar. (D,E) Differences in odontoblast height and labeling for young and old molars are shown in these higher-magnification views from Figs. 2A and 2C. Scale bars: A, C, 0.5 mm; B, D, E, 0.2 mm.
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Injury
Molars with the flap procedure had normal Fluoro-Gold distribution at 3 days (Fig. 3A, Table). However, under the scrape injuries, much more was found in dentin (Figs. 3B, 3c, 3D) than normal, and it also spread into central pulp (Table). A longer extent of the mesial odontoblast layer was labeled at the scrape injury sites (Table), although at a lower intensity than directly under the application site (Figs. 3B, 3C). The distal sides of those molars still had Fluoro-Gold located in odontoblasts, as in intact teeth (Fig. 3C). Root dentin and pulp also had label in most injured teeth. When we injured teeth 11 days prior to three-day Fluoro-Gold application, we found that the induced reparative dentin was an excellent barrier. The Fluoro-Gold collected in dead tracks outside the reparative dentin and was mostly blocked from entering pulp (Figs. 3E, 3F). When Fluoro-Gold was implanted into pulp, it extended all the way through dentin where odontoblasts were damaged, but failed to enter dentinal tubules if the odontoblast layer was intact (Fig. 3G). Thus, Fluoro-Gold patterns for scrape-injury, repaired, or implanted molars were each distinctive and differed from the labeling of odontoblasts of intact molars.
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Figure 3. FG patterns for different dental injuries. (A) A flap procedure (f) on the mesial side of the first molar without tooth injury gave patterns similar to those in normal teeth. (B) Scraping of exposed crown and root after a gingival flap procedure (f/s) gave strong labeling of coronal and root dentin, weak label of odontoblasts beneath the labeled dentin, and diffuse label throughout the mesial pulp. (C) Odontoblasts, dentin, and adjacent pulp on the distal side of the molar in panel B were labeled at the normal intensity. (D) Higher magnification of the injury site in Fig. 3B, showing variable intensities in the odontoblast layer. (E) Prior induction of reparative dentin (RD) blocked entry of most of the FG, which persisted in the dentinal tubules that were blocked by the reparative matrix. (F,G) When Fluoro-Gold was placed into molar pulps (*) through small cavities, it spread throughout the pulp and into dentin, where the odontoblast layer was broken, but was kept out of dentin by intact odontoblast layers (Od). Scale bars: A,B,C,E,F = 0.2 mm. D,G = 0.1 mm.
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Denervation
We transected the right inferior alveolar nerve 2-3 days prior to placement of Fluoro-Gold, as confirmed by neural labeling in adjacent sections (not shown), to determine whether neuro-regulation of pulpal blood flow and interstitial fluid pressure would affect Fluoro-Gold entry. In all 6 animals with first molar denervation, the patterns were similar to those of contralateral innervated control teeth and 3 sham-operated rats (not shown).
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DISCUSSION
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Fluoro-Gold (FG) is an excellent neuronal label in animal studies (Chang et al., 1990; Qian and Naftel, 1994). We show here that it is also useful for tracing permeabilities of enamel, dentin, and pulp. When Fluoro-Gold was placed in the rat molar sulcus, it dissolved rapidly and passed through enamel to label the cervical dentin-enamel junction, peritubular matrix in outer dentin, and odontoblast processes and cell bodies. It collected in the odontoblast cell bodies by 1 day and remained there at least 2 wks. Fluoro-Gold did not penetrate rat incisors at all, but placement was on the palatal side, where enamel is absent. This negative finding for incisors is consistent with results of earlier work showing the importance of a dentin-enamel partnership when fluid moves through enamel (Linden, 1968).
We confirmed our first hypothesis, that Fluoro-Gold penetration would decrease with age as enamel reduces its proportion of organic channels and dentin thickens. Our prediction that reparative dentin would impede its influx was also confirmed. However, two other hypotheses were not supported. We had expected reduced entry into inflamed pulps at the scrape-injury sites, because of increased dentinal fluid outflow and reduced dye penetration under those conditions (Olgart, 1996; Pashley, 1996; Vongsavan et al., 2000). In addition, sensory denervation did not affect Fluoro-Gold patterns, a surprising outcome given the neural regulation of pulpal blood flow and of interstitial fluid flow in teeth (Vongsavan and Matthews, 1992; Matthews and Vongsavan, 1994; Olgart, 1996; Berggreen and Heyeraas, 2000). Penetrations of dyes, isotopes, anesthetics, bacterial products, or restorative materials have been shown to be affected by dental fluid outflow and have been greater for extracted than for vital teeth (Edwall and Kindlova, 1971; Pashley et al., 1981; Potts et al., 1985; Bergenholtz et al., 1996; Vongsavan and Matthews, 1992; Vongsavan et al., 2000). In our study, Fluoro-Gold had excellent entry into vital tooth pulp that increased at sites of injury, despite the presumed increased fluid outflow, showing unusual properties compared with other kinds of tracers.
Enamel is often viewed as an inert tissue, but organic material and water occur between the prisms (Bartelstone, 1951; Linden, 1968; Pashley, 1996; Shellis, 1996; ten Bosch et al., 2000), and that is where Fluoro-Gold appeared to cross enamel (Fig. 1J). In extracted young human teeth, cervical enamel has more dye flow than the rest of the crown (Linden, 1968). Here, cervical enamel was the preferred pathway, but that would also be expected from the sulcular placement of Fluoro-Gold. Entry was much stronger in juvenile teeth, where the enamel interprismatic region is proportionally greater than in old teeth. The dentin was also much thinner in the young teeth, and odontoblast processes reach closer to the dentin-enamel junction (Byers and Sugaya, 1995), which might also favor influx.
Efflux of dentinal fluid has been well-studied at cut dentin (Turner et al., 1989; Vongsavan and Mattthews, 1992; Ciucchi et al., 1995; Pashley, 1996), but few have investigated materials penetrating inward through intact teeth or moving inward during fluid outflow at cut dentin. Fluoro-Gold represents a tool to accomplish this and to determine the timing of entry. The staining patterns after Fluoro-Gold was sealed into pulps also revealed some aspects of its outflow. The pulp-dentin border forms an effective barrier to the outflow of many agents (Thomas, 1985; Turner et al., 1989; Bishop, 1992; Pashley, 1996), including Fluoro-Gold (Fig. 3G), as long as the odontoblast layer was present. Where the odontoblasts were lost, the implanted FG moved out into dentin to fill the tubules entirely in a pattern that was completely different from influx patterns.
In conclusion, we have shown that Fluoro-Gold enters vital rat molars in patterns that reveal pathways of permeability of enamel and dentin, and that show reduced entry after tooth maturation or at reparative dentin. The increased entry in injured teeth and its entry into denervated teeth were both surprising, because they were accomplished despite fluid outflow that occurs at etched injured dentin (Pashley, 1996) and loss of neural regulation of that outflow. This appears to be the first demonstration of trans-dental passage and long-term odontoblastic retention of an orally applied label in intact vital teeth.
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ACKNOWLEDGMENTS
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We thank Drs. E.H. Chudler, G.W. Harrington, and Zongyang Sun for advice; and Paul Haun, Peggy O’Neill, Helen Youm, and Norma Anderson for technical help. This research is supported by NIH grants #DE05159 (MRB) and #K16DE00161 (KJYL). Preliminary publication was: Yoon KJ, Byers MR (2001). Pattern and penetration of a novel label for injured and uninjured vital tooth pulp tissues. J Endodon 27:224 (Am Assoc Endodontists). This paper is based in part on a thesis submitted by Dr. K.J. Yoon Lin to the graduate faculty of the University of Washington (2001) for a Master of Science degree.
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FOOTNOTES
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A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.
Received for publication December 27, 2001.
Revision received October 24, 2002.
Accepted for publication January 10, 2003.
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Journal of Dental Research, Vol. 82, No. 4,
312-317 (2003)
DOI: 10.1177/154405910308200414
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ABSTRACT
INTRODUCTION