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RESEARCH REPORT |
1 Dept. of Anesthesiology, Box 356540 , and
2 Dept. of Endodontics, Univ. of Washington, Seattle, WA 98195-6540;
*corresponding author, Byersm{at}u.washington.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: odontoblasts • aging • innervation • injury • reparative dentin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). 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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). 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).
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, 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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, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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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FOOTNOTES |
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Received December 27, 2001; Last revision October 24, 2002; Accepted January 10, 2003
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REFERENCES |
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