- Email: [email protected]
Traumatic root resorption in dentine-immunized mice
Traumatic root resorption in dentine-immunized mice Timothy T. Wheeler, DMD, PhD, ~ and Suzanne E. Stroup, BS ~
Gainesville, Fla. Traumatic root resorption in a mouse model has been shown to coincide with a decline in naturally occurring serum antibody levels to dentin. It has been proposed that root resorption may be dentin antibody mediated. The purpose Of this study was to examine the traumatic root resorption response in mice after hyperimmunization with a crude tooth extract (dentin). The hypothesis of this study was that elevated dentin antibody titers would Positively correlate with root resorption. Mice were immunized with mouse dentin and controls were sham immunized. All mice were boosted 4 weeks later with or without mouse dentin as appropriate. All mice were then boosted two more times at Weekly intervals with mouse dentin and then twice at weekly intervals with rat dentin. The change to rat dentin was made to increase mouse serum antibody titers to dentin. Serum samples were obtained before the initial immunization and weekly after each boost and were examined for antibody-to-derltin antigen by the enzyme-linked immune sorbent assay (ELISA). One week after the second boost with rat dentin, all animals were exposed to the cryoprobe procedure. Mice were killed 10 days later and serum tested for antibody to dentin antigen. The incisors were examined by scanning electron microscopy, and root resorPtion quantified. Root resorption was observed on the incisors in the Sham-immunized mice but not in the dentin-immunized mice. A trend toward increased serum antibody titers to dentin in immunized mice was observed over time. However, only the serum antibody titers to 9 from preimmune mice and bleed 5 were statistically Significant. Sham-immunized animals showed no significant changes in dentin antibody titers throughout the experiment. The data indicate that antibody does not mediate the traumatic root resorption process as originally hypothesized and suggest that hyperimmunization with dentin may protect against traumatic root resorption. (AMJ ORTHoDDENTOFACORTHoP 1993;103;352-7.)
T h e mechanism of traumatic tooth root resorption is poorly understood. Resorption of the tooth root has been shown to occur in deciduous teeth, traumatized teeth, and orthodontically treated teeth. Similarities or dissimilarities of these resorption processes, however, are unknown. Furthermore, despite reports of high prevalence of root resorption in reimplanted t and orthodontically treated teeth, 24 effective means of prevention or treatment is still lacking perhapsbecause of a lack of understanding of mechanisms regulating this process. King and Courts 5 suggested that, in the dog, the humoral immune system and root resorption may be correlated. They Showed that there was a decline in
From the Department of Orthodontics, University of Horida College of Dentistry. This work was funded by a research development grant from the Division of Sponsored Research at the University of Florida and by the Southern Association of Orthodontists. 'Assistant Professor. bSenior Biol~ical Scientist. Copyright 9 7993 by the American Association of Orthodontists. 0889-5406/93/$1.00 + 0.10 811134464
352
serum antibody titers to dentin antigen during active root resorption suggesting either that the resorbing root surface may act as an dntigen sink and absorb antibody from the serum or that a suppressor T-cell population may have been activated to down regulate the production of antibody. In addition, Ng and King 6 have recently shown that after inducing incisor root resorption in a mouse model, there is a coincident decrease in dentin serum antibody titers along with root resorption. This also suggests either an antigen sink type of mechanism or a suppression of antibody production with the possibility of the immune system playing a role in root resorption. No other studies have been done to examine further the relationship of the immune system with traumatic root resorption. The studies by King and Courts 5 and Ng and King 6 implicated that dentin antibody may play some role in root resorption. Therefore it would be of great interest to determine whether elevated antibody levels obtained by hyperimmunization with dentin would affect tooth root resorption. If resorbing root surface acted as an antigen sink, antibody levels should decrease proportionally with the amount of root resorption. If a Sup-
American Journal of Orthodontics and Dentofacial Orthopedics Volume 103, NO. 4
Wheeler and Stroltp
I Boost 1 II Boost 2 I B~ 3 ]l Mous~ Dentinell Mouse Dentine Mouse Dentine ll Mouse Pre-bleed &::-!i!!!::!!!:.!!I
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353
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Fig. 1. Experimental design: Six NIH Swiss female mice were immunized intraperitoneally with mouse dentine and six controls were sham-immunized without mouse dentine (time point 0). All mice were boosted intraperitoneally 4 weeks later with or without mouse dentine as appropriate (boost 1). All mice were then boosted intraperiotoneally two more times at weekly intervals with mouse dentine (boosts 2 and 3) and then twice at weekly intervals with rat dentine (boosts 4 and 5). Serum samples were obtained before the initial immunization and weekly after each boost (immune bleeds 1 to 5). One week after the second boost with rat dentine (week 9), all animals were exposed to the cryoprobe root resorption procedure. Mice were killed after 10 days, serum collected, and incisors removed.
presser T-cdll down regulated dentin antibody production, a general decrease in antibody levels would be expected that would not necessarily correlate with the amount of root resorption. Elevating dentin antibody levels above normal would allow the determination, if antibody mediated traumatic tooth root resorption. The hypothesis Of this study was that elevated dentin antibody titers Would correlate with increased traumatic root resorption. The purpose of this study was to examine a strain of mice that initially had very low levels of dentin antibody and examine the effect that hyperimmunization with dentin had on antibody titers and trauma-induced root resorption. This would provide further information for possible involvement of antibody in the traumatic root resorption process.
MATERIALS AND METHODS Experimental design As shown in Fig. I, six NIH Swiss female mice were immunized intrapefitoneallywith 100 I.tg of NIH Swiss mouse dentin protein in 200 I.tl of Complete Freund's Adjuvant (Sigma, St. Louis, Me.), and six controls were sham immunized with Complete Freund's Adjuvant without mouse dentin (time point 0). All mice wereboosted intraperitoneally 4 weeks later with Incomplete Freund's Adjuvant (Sigma) either with or without mouse dentin as appropriate (boost 1). All mice were then boosted intrapefitoneally two more times at weekly intervals with mouse dentin (boosts 2 and 3) and then twice at weekly intervals with Sprague-Dawley strain rat dentin (boosts 4 and 5). Serum samples were obtained by retroorbital bleeding before the initial immunization and weekly after each boost (immune bleeds 1 to 5) and were examined for antibody-todentin antigen by the enzyme-linked immune sorbent assay (ELISA) as described later. One week after the second boost with rat dentin (week 9), the incisors of all animals were exposed to the cryoprobe
procedure. Mice were killed 10 days after exposure to the cryoprobe by exsanguination after anesthesia, and serum collected and stored at - 7 0 ~ C until tested by ELISA for antib0dy-t0-dentin antigen. The incisors were removed from the mandible, prepared and examined by scanning electron microscopy (SEM). These procedures are described later in the article.
Cryoprobe procedure The cryoprobe procedure used has bccn described by Ng and King.6 Mice were anesthetized with Ketamine Hydrochloride (Ketaset, Bristol Laboratories, Syracuse, N.Y.) (87 mg/kg body weight) and Xylazine (Rompun, BAYVET Division Miles Laboratories, Inc., Shawnee, Kas.) (13 mg/kg body weight). A midline incision was made below the symphysis of the mandible, and the over!ying skin and fascia were reflected on each side to expose the lateral surface of the mandible. A 1.5 mm diameter cryoprobe (Cylinder TopUnit Cryosurgical system, Keeler Instruments, Inc., Broomall, Pa.) was applied to the exposed bone surface of the left incisor for 5 min at - 80~ C. After freezing, the probe and the tissue were allowed to defrost while in contact, so that frozen tissue was not fractured off on probe removal. The incision then was closed with 6-0 silk sutures. Sutures were removed after 1 week. The fight incisor was used as an untreated control.
Scanning electron microscopy Mice mandibles were dissected and then fixed in 10% buffered formalin for 24 hours. Mandibles were then digested in 5.25% sodium hypochlorite until the incisors could be easily removed from the jaw. The intact incisors were then dehydrated in a graded series of acetone solutions (75% to 100%) and stored in an oven at 27 ~ C. Each incisor was mounted on a stub the same way, so that the lateral surface of the tooth could be viewed. Incisors were coated with gold palladium in a high vacuum coating unit (Hummer I, Anatech, Ltd. Alexandria, Va.) for 2.5 minutes and viewed with the SEM (Hitachi S-450, Rockville, Md.). Micrographs were
354 Wheeler (tnd Slrottp
taken of the lateral surface of the tooth root. Areas of root surface containing resorption were quantified manually with the principle of point-counting volumetry.' In this technique, micrographs were placed under a regular point lattice, and points coincident with resorption lacunae were scored. The total points scored, corrected to a standard magnification, gave a measure of the relative extent of the resorption areas and are referred to as resorption indices.
Antigen preparation A very crude antigen was prepared from extracted N1H Swiss mouse incisors and from Sprague-Dawley strain rats. All incisors were cleaned of bone and all visible soft tissue by scraping with periodontal scalers under a dissecting microscope. The small size and fragility of the mouse incisors did not allow any further cleaning. The teeth were extracted with 5M guanidine hydrochloride containing 10% EDTA for 48 hrs at 4" C. Extracts were centrifuged (1100 • g) and renatured by exhaustive dialysis against distilled and deioni~ed water for 1 week at 4 ~ C. Extracts were then lyophilized and resuspended in PBS. Protein concentrations were determined by the bicinchoninic acid protein assay procedure (Sigma, St. Louis, Mo.). This extract is considered to be a very crude antigen preparation containing pulp, enamel, cementum, and dentin proteins. It has been prepared identically to that used in other studies examining root resorption. ~6 It is recognized that this preparation is not highly refined and contains many contaminating proteins. However, since the majority of the material is dentin, and to keep consistent with published literature, it will be considered a crude dentin extract.
ELISA Antibody activity against dentin antigen was detected using the ELISA. 8 Dentin, extracted by guanidine-EDTA from mouse incisors, was used as the source of antigen. Mouse dentin at I I.tg/ml protein was diluted in carbonate-bicarbonate buffer pH 9.6. Rat dentin at the same concentrations and treated identically was used for detection of mouse antirat dentin antibody. To each well of a polystyrene microtiter plate (Linbro E.I.A. Microtitration plate, Flow Laboratories, Inc. McLean, Va.), 100 mm 3 of antigen was added and allowed to incubate for 16 hours at 4" C. Unbound antigen was removed by washing the wells with 0.15 M PBS, pH 7.2, containing 0.05% Tween 20, using an automated rinser (Multiwash, Bio-Rad Laboratories, Richmond, Calif.). Two hundred microliters of I% skim milk (SM) in 0.15 M PBS, pH 7.2, was added to each well and incubated for two hours at room temperature. After removal of the 1% SM solution and washing of the plate as previously described, 100 mm 3 of mouse serum, diluted 1:5 in 1% SM-PBS, was added to the appropriate wells. Antibody was allowed to bind to the antigen for I hour at room temperature. Unbound antibody was removed by rinsing, and 0.1 ml of a predetermined dilution (usually 1 : 1000) of affinity-purified, peroxidase conjugated goat antimouse lg (lgA, lgM, lgG, H- and L-chain stral~" specific, Organon Teknika Laboratories, Durham, N.C.) was added. The peroxidase-conjugated antibody was incubated on
American Journal of Orthodontics and Dentofacial Orthopedics April 1993
the plates for 1 hour at room temperature. Unbound second antibody was removed by rinsing as previously described. Two hundred microliters of peroxidase enzyme substrate were added to each well. The substrate solution consisted of 10 " mg O-phenylenediamine, and 12 mm 3 H_,O_,(30%) in 20 ml of 0.1 M citrate buffer, pH 4.5, made fresh immediately before use. Optical density (OD) of the enzyme substrate solution was monitored after 1 hour of incubation at room temperature at 492 nm with a Multiskan (Flow Laboratories, Inc. McLean, Va.). Negative control sera were obtained from nonimmunized mice. Bacteroides ghzgivalis was used to coat wells to check for antibody specificity. Additional controls were wells with only enzyme substrate, and wells with antigen and peroxidase-conjugated antibody only.
Statistical methods The means and standard errors of the serum dentin antibody ELISA ODs were calculated for each serum sampling point. The means and standard errors of both the root resorption areas, as well as the ELISA OD at the final bleeding, were also calculated. Root resorption between immunized and sham-immunized groups was compared with a one-tailed paired t test. An analysis of variance (ANOVA) was performed to examine differences between immunized and sham-immunized groups ,at each time point and across times within each treatment group. Pairwise (Scheffr) comparisons were performed between the individual treatment groups when the ANOVA indicated that significant differences existed (p < 0.05).
RESULTS Root resorption was observed on the lateral surface of the left incisors in the sham-immunized mice (Figs. 2, A, and 3) but not in the dentin-immunized mice ( p = 0.01). No root resorption was observed on the contralateral control incisors o f either group (Fig. 2, B). A trend toward increased serum antibody titers to dentin in immunized mice was observed over time (Fig. 4). However, only the serum antibody titers to dentin from preimmune mice and bleed 5 were statistically significant ( p < 0.05). A slight drop in dentin antibody titers in immunized mice was observed from bleed 5 and postcryotrauma, however, this was not significant. Sham-immunized animals showed no significant changes in dentin antibody titers throughout the experiment.
DISCUSSION As previously reported, 6"9 trauma by application o f cold to the lateral side o f the mandible stimulates an observable tooth root resorption lesion by day 10 in normal mice. It may be argued that root resorption in the i m m u n i z e d ' a n i m a l s may begin later than day 10. However, Ng and King 6 have shown that cryoprobe
Wheeler attd Stroup
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355
% Root R e s o r p t i o n (_+SEM) 7-
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Fig. 3. Root resorption of mouse incisor after exposure to cryoprobe at - 8 0 ~ C for 5 minutes. Each bar represents the mean of six observati0ns, and the vertical bar represents the SEM (p ~- 0.01).
Serum Ab to Dentine (Mean _+SEM) 0.04
0.03
Fig. 2. Scanning electron micrographs of (A) representative root resorption lesion of cryoprobe-treated left incisor of sham immunized mouse and (B) contralateral incisor of sham-immunized animals showing no root resorption. Incisors from animals immunized with dentin were identical to those pictured in (B). Bar = 50 FM.
c) o <1: CO ,.J t/J
0.02
0.01
induced root resorption was greatest on day 10, and that by day 21 lesions may erupt into the mouth. Therefore day 10 was chosen in this experiment to examine the tooth for root resorption. To increase dentin antibody titers, the homologous mouse dentin preparation was replaced with a heterologous rat dentin preparation. It is ,,,,,ell documented that to obtain elevated antibody titers when immunizing with "self" or homologous antigens is very difficult. To increase titers, heterologous antigen preparations of similar tissues can be used to increase antibody levels. 1o Therefore the change to rat dentin was made to increase mouse serum antibody titers to dentin. ,~ Quantitative SEM has been used to document ortho- 9 dontic root resorption in rodents 6"1~ and is considered to be reliable and valid. Resorption lacunae spread lat-
0.00
Pie-immune
1
2
3 Immune BIeed
4
5
I Post-Cryo
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Fig. 4. Mouse antibody titers (ELISA OD at 494 nm) before immunization (preimmune), after boosts with mouse dentine (immune bleed 1, 2, and 3) and with rat dentine (immune bleed 4 and 5), and at time of death (postcryo) of mice immunized with dentine and sham-immunized animals. Each bar represents the mean of six observations and the vertical bar represents the SEMs. *Indicates significance p < 0.05.
erally and remain very shallow in comparison with the extefft of their lateral spread.. Ideally, absolute measures of lesion volumes may be more specific, however, a two-dimensional depiction of a shallow object is not
356
Wheeler and Stroup
expected to introduce systematic errors during quantification with relative measures. A question may be raised as to the similarity of root resorption as the result of orthodontic tooth movement compared with cryoprobe stimulated injury. The resorption lacunae observed in this experiment are structurally identical to those observed during orthodontic tooth movement. 12.,3Histomorphometric studies of root resorption initiated by these different procedures will produce a more definitive answer to this question. The cryoprobe alone should not cause any injury since the tooth is never exposed. After freezing, the probe and tissue were allowed to defrost while in contact, so that frozen tissue was not fractured off on probe removal. The lateral border of the mandible only is exposed by the surgery and then touched by the cryoprobe. ~A slight decrease in antibody titer to dentin was observed in the sham-immunized animals, however, it was not statistically significant. This is in contrast to the findings of King et al. 5'6 and the observation t h a t aut0antibody titers to dentin significantly decreased i n animals during the process of root resorption. The stud: ies by King et a12 "6 examined naturally occurring autoantibody titers to the dentin antigen, whereas this study examined the effect on increased antibody levels caused by hyperimmunization. It is very possible that the immunization procedure altered the cellular immune response, as well as the humoral response, of the animal to dentin thereby protecting the root from resorption on injury. The role of the various components of the immune system in the protection to cryo-induced root resorption is now being investigated. The decrease in the antibody titers observed by King s'6 could be explained by trauma-induced immunosuppression. Trauma has been shown to alter the immune system of both humans and animals. '4'8 The T-cell dependent antibody production in humans to tetanus toxoid has been shown to decrease after thermal injury? 8 The cell-mediated immune system has been shown to be depressed in patients suffering from chronic posttraumatic osteomyelitis, whereas the humoral immune response appeared to be less affected.~5 If a generalized antibody production decrease occurs in traumatized animals, this could also explain the decrease observed in dentin antibody by King et a l ) "6 Therefore the antibody suppression observed by King et al. does not necessarily indicate that the immune system contributes to or mediates the resorption process. It has been suggested that osteoclast attachment-is_ in part mediated by arginine-glycine-aspartic acid (RGD) peptide sequences in bone matrix proteins.19:-~
American Journal of Orthodontics and Dentofacial Orthopedics April 1993
Proteins that contain these tripeptides include fibronectin, vitronectin, osteopontin, collagens, thrombospondin, fibrinogen, and von Willebrand factor. 21 Recently, it has been shown that in vitro osteoclast-mediated dentin resorption can be inhibited by antivitronectin receptor antibody. 2-" In addition, synthetic RGD peptides, have been shown to inhibit dentin resorption in vitro, 2-" as well as in vitro bone resorption? 9,z~ If the crude dentin preparation used in the present study contained an RGD peptide, it is possible that immunization with this material could induce an antibody response that could inhibit root resorption in vivo. The dentin preparation is now being purified, and active components identified. It was originally hypothesized that antibody played some role in the root resorption process. Therefore it was expected that the immunized mice, with increased dentin antibody titers, would result in greater root resorption than sham-immunized mice. However, the ob9servation that incisor roots from dentin immunized mice did not resorb was very surprising. In addition, the sham-immunized mice did present with root resorption. These findings.do, not suggest that antibody plays a role in the process of root resorption as hypothesized by King et a l ) "6 In contrast, the data presented here suggest that the immunization procedure prevented root resorption and that increased dentin antibody titers may protect mice from traumatic root resorption. The role that the dentin immunization procedure had on the cellular immune system is not known. Since trauma is known to suppress the immune system, and if this suppression induces .root resorption, it is possible that the immunization procedure prevented the suppression and the resultant root resorption. It would be of interest to determine whether the dentin immunization procedure induced any type of cellular immune response that may be protective against trauma induced root resorption. This also is now being investigated. We thank Dr. Greg Erdos and the Electron Microscope Core Facility for their assistance with the SEM examinations. We also thank Dr. Greg King for his helpful advice. REFERENCES
1. AndreasenJO. External root resorption: its implicationin dental traumatol~y, paedodontics, periodontics,orthodontics and endodontics. Int Endod J 1985;18(2):109-18. 2. HarrisEF, BakerWC. Lossof root lengthand crestalboneheight before and during treatment in adolescent and adult orthodontic patients9AM J ORTIIOD DENTOFACORTHOP 1990;98(5):463-9. 3. DermautLR, De MA. Apical root resorptionof upper incisors caused by intrusive tooth movement: a radi~raphic study. A.~1 J ORTtIOD DENTOFACORTIIOP 1986;90(4):321-6. 4. Goldie RS, King GJ. Root resorption and tooth movement in orthodontically treated, calcium-deficient, and lactating rats. AM J OR'ntOD 1984;85(5):424-309
American Journal of Orthodontics and Dentofacial Orthopedics Volume 103, No. 4
5. King GJ, Courts F. The biol~y of tooth movement. Boca Raton, Florida: CRC Press, 1989:275-285. 6. Ng KT, King GJ, Courts FJ. Humoral immune response to active root resorption with a murine model. AM J ORT|IOD DEN'rOFAC OR'ntor 1990;98(5):456-62. 7. Weibel ER, Kestler GS, Scherle WF. Practical sterological methods for morphometric cytology. J Cell Biol 1966;30:23-38. 8. Voller A, Bidwell D, Bartlett A. Enzyme-linked immunosorbent assay. In: Rose NR, Friedman H, ed. Manual of Clinical Immunology. (2nd ed.) Washington, D.C.: American Society for Microbiol~y, 1980:359-371. 9. Wesselink PR. Resorption of the mouse incisor after the application of cold to the periodontal attachment apparatus. Calcif Tissue Int 1986;39:11-21. 10. Charreire J. Immune mechanisms in autoimmune thyroiditis. Adv Immunol 1989;46:263-334. 11. King GJ, Fishlschweiger W. The effect of force magnitude on extractable bone resorptive activity and cemental cratering in orthodontic tooth movement. J Dent Res 1982;61:775-9. 12. Kvam E. Scanning electron microscopy of tissue changes on the pressure .Surface of human premolars following tooth movement. Scand J Dent Res 1972;80:357-68. 13. Reitan K. Initial tissue behavior during apical root resorption. Angle Orthod 1974;44(1):68-82. 14. Pretus HA, Browder IW, Lucore P, McNamee RB, Jones.EL, Williams DL. Maerophage activation decreases macrophage prostaglandin E2 release in experimental trauma. Trauma 1989;29:1152-7. 15. Schliiter B, Bergmannn U, Josten C, Walz M, K6nig W. Impairment of specific host defense mechanisms in patients with chronic post-traumatic osteomyelitis. Trauma 1991;3 !:68-73.
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16. O'Mahoney JB, Palder SB, Wood JJ, et al. Depression of cellular immunity after multiple trauma in the absence of sepsis. Trauma 1984;24:869-75. 17. Faist E, Mewes A, Baker CC, et al. Prostaglandin E2 (PGE2) -dependent suppression of interleukin alpha (IL-2) production in patients with major trauma. Trauma 1987;27:837-47. 18. x,Vood JJ, O'Mahony JB, Rodrick ML, Eaton R, Demling RH, Mannick JA. Abnormalities of antibody production after thermal injury. Arch Surg 1986;121:108-15. 19. van der Pluijm G, Mouthaan H, Papapoulos S, Lfwik C. Inhibition of osteoclast precursor attachment and subsequent bone resorption by synthetic RGD-peptides. J Bone Miner Res 1991;6(Suppl. 1):S 146. 20. Bertolini DR, Stadel J, Samanen J, et al. Inhibition of bone resorption by synthetic RGD containing peptides. J Bone Miner Res 1991;6(Suppl. I):S146. 21. Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987;238:491-7. 22. Horton MA, Taylor ML, Arnett TR, Helfrich MH. Arg-Gly-Asp (RGD) peptides and the anti-vitronectin receptor antibody 23C6 inhibit dentine resorption and cell spreading by osetoclasts. Exp Cell Res 1991;195(2):368-75. Reprint requests to: Dr. Timothy T. Wheeler Box J-444, JHMHC Department of Orthodontics University of Florida College Of Dentistry Gainesville, FL 32610
Gainesville, Fla. Traumatic root resorption in a mouse model has been shown to coincide with a decline in naturally occurring serum antibody levels to dentin. It has been proposed that root resorption may be dentin antibody mediated. The purpose Of this study was to examine the traumatic root resorption response in mice after hyperimmunization with a crude tooth extract (dentin). The hypothesis of this study was that elevated dentin antibody titers would Positively correlate with root resorption. Mice were immunized with mouse dentin and controls were sham immunized. All mice were boosted 4 weeks later with or without mouse dentin as appropriate. All mice were then boosted two more times at Weekly intervals with mouse dentin and then twice at weekly intervals with rat dentin. The change to rat dentin was made to increase mouse serum antibody titers to dentin. Serum samples were obtained before the initial immunization and weekly after each boost and were examined for antibody-to-derltin antigen by the enzyme-linked immune sorbent assay (ELISA). One week after the second boost with rat dentin, all animals were exposed to the cryoprobe procedure. Mice were killed 10 days later and serum tested for antibody to dentin antigen. The incisors were examined by scanning electron microscopy, and root resorPtion quantified. Root resorption was observed on the incisors in the Sham-immunized mice but not in the dentin-immunized mice. A trend toward increased serum antibody titers to dentin in immunized mice was observed over time. However, only the serum antibody titers to 9 from preimmune mice and bleed 5 were statistically Significant. Sham-immunized animals showed no significant changes in dentin antibody titers throughout the experiment. The data indicate that antibody does not mediate the traumatic root resorption process as originally hypothesized and suggest that hyperimmunization with dentin may protect against traumatic root resorption. (AMJ ORTHoDDENTOFACORTHoP 1993;103;352-7.)
T h e mechanism of traumatic tooth root resorption is poorly understood. Resorption of the tooth root has been shown to occur in deciduous teeth, traumatized teeth, and orthodontically treated teeth. Similarities or dissimilarities of these resorption processes, however, are unknown. Furthermore, despite reports of high prevalence of root resorption in reimplanted t and orthodontically treated teeth, 24 effective means of prevention or treatment is still lacking perhapsbecause of a lack of understanding of mechanisms regulating this process. King and Courts 5 suggested that, in the dog, the humoral immune system and root resorption may be correlated. They Showed that there was a decline in
From the Department of Orthodontics, University of Horida College of Dentistry. This work was funded by a research development grant from the Division of Sponsored Research at the University of Florida and by the Southern Association of Orthodontists. 'Assistant Professor. bSenior Biol~ical Scientist. Copyright 9 7993 by the American Association of Orthodontists. 0889-5406/93/$1.00 + 0.10 811134464
352
serum antibody titers to dentin antigen during active root resorption suggesting either that the resorbing root surface may act as an dntigen sink and absorb antibody from the serum or that a suppressor T-cell population may have been activated to down regulate the production of antibody. In addition, Ng and King 6 have recently shown that after inducing incisor root resorption in a mouse model, there is a coincident decrease in dentin serum antibody titers along with root resorption. This also suggests either an antigen sink type of mechanism or a suppression of antibody production with the possibility of the immune system playing a role in root resorption. No other studies have been done to examine further the relationship of the immune system with traumatic root resorption. The studies by King and Courts 5 and Ng and King 6 implicated that dentin antibody may play some role in root resorption. Therefore it would be of great interest to determine whether elevated antibody levels obtained by hyperimmunization with dentin would affect tooth root resorption. If resorbing root surface acted as an antigen sink, antibody levels should decrease proportionally with the amount of root resorption. If a Sup-
American Journal of Orthodontics and Dentofacial Orthopedics Volume 103, NO. 4
Wheeler and Stroltp
I Boost 1 II Boost 2 I B~ 3 ]l Mous~ Dentinell Mouse Dentine Mouse Dentine ll Mouse Pre-bleed &::-!i!!!::!!!:.!!I
[
4
5
6
Boost4 II Rat Dentine
353
Boost5 Rat Dentine
I R~:~'I::I!I 7
8
9 .~10
i
Days
I
Weeks
Fig. 1. Experimental design: Six NIH Swiss female mice were immunized intraperitoneally with mouse dentine and six controls were sham-immunized without mouse dentine (time point 0). All mice were boosted intraperitoneally 4 weeks later with or without mouse dentine as appropriate (boost 1). All mice were then boosted intraperiotoneally two more times at weekly intervals with mouse dentine (boosts 2 and 3) and then twice at weekly intervals with rat dentine (boosts 4 and 5). Serum samples were obtained before the initial immunization and weekly after each boost (immune bleeds 1 to 5). One week after the second boost with rat dentine (week 9), all animals were exposed to the cryoprobe root resorption procedure. Mice were killed after 10 days, serum collected, and incisors removed.
presser T-cdll down regulated dentin antibody production, a general decrease in antibody levels would be expected that would not necessarily correlate with the amount of root resorption. Elevating dentin antibody levels above normal would allow the determination, if antibody mediated traumatic tooth root resorption. The hypothesis Of this study was that elevated dentin antibody titers Would correlate with increased traumatic root resorption. The purpose of this study was to examine a strain of mice that initially had very low levels of dentin antibody and examine the effect that hyperimmunization with dentin had on antibody titers and trauma-induced root resorption. This would provide further information for possible involvement of antibody in the traumatic root resorption process.
MATERIALS AND METHODS Experimental design As shown in Fig. I, six NIH Swiss female mice were immunized intrapefitoneallywith 100 I.tg of NIH Swiss mouse dentin protein in 200 I.tl of Complete Freund's Adjuvant (Sigma, St. Louis, Me.), and six controls were sham immunized with Complete Freund's Adjuvant without mouse dentin (time point 0). All mice wereboosted intraperitoneally 4 weeks later with Incomplete Freund's Adjuvant (Sigma) either with or without mouse dentin as appropriate (boost 1). All mice were then boosted intrapefitoneally two more times at weekly intervals with mouse dentin (boosts 2 and 3) and then twice at weekly intervals with Sprague-Dawley strain rat dentin (boosts 4 and 5). Serum samples were obtained by retroorbital bleeding before the initial immunization and weekly after each boost (immune bleeds 1 to 5) and were examined for antibody-todentin antigen by the enzyme-linked immune sorbent assay (ELISA) as described later. One week after the second boost with rat dentin (week 9), the incisors of all animals were exposed to the cryoprobe
procedure. Mice were killed 10 days after exposure to the cryoprobe by exsanguination after anesthesia, and serum collected and stored at - 7 0 ~ C until tested by ELISA for antib0dy-t0-dentin antigen. The incisors were removed from the mandible, prepared and examined by scanning electron microscopy (SEM). These procedures are described later in the article.
Cryoprobe procedure The cryoprobe procedure used has bccn described by Ng and King.6 Mice were anesthetized with Ketamine Hydrochloride (Ketaset, Bristol Laboratories, Syracuse, N.Y.) (87 mg/kg body weight) and Xylazine (Rompun, BAYVET Division Miles Laboratories, Inc., Shawnee, Kas.) (13 mg/kg body weight). A midline incision was made below the symphysis of the mandible, and the over!ying skin and fascia were reflected on each side to expose the lateral surface of the mandible. A 1.5 mm diameter cryoprobe (Cylinder TopUnit Cryosurgical system, Keeler Instruments, Inc., Broomall, Pa.) was applied to the exposed bone surface of the left incisor for 5 min at - 80~ C. After freezing, the probe and the tissue were allowed to defrost while in contact, so that frozen tissue was not fractured off on probe removal. The incision then was closed with 6-0 silk sutures. Sutures were removed after 1 week. The fight incisor was used as an untreated control.
Scanning electron microscopy Mice mandibles were dissected and then fixed in 10% buffered formalin for 24 hours. Mandibles were then digested in 5.25% sodium hypochlorite until the incisors could be easily removed from the jaw. The intact incisors were then dehydrated in a graded series of acetone solutions (75% to 100%) and stored in an oven at 27 ~ C. Each incisor was mounted on a stub the same way, so that the lateral surface of the tooth could be viewed. Incisors were coated with gold palladium in a high vacuum coating unit (Hummer I, Anatech, Ltd. Alexandria, Va.) for 2.5 minutes and viewed with the SEM (Hitachi S-450, Rockville, Md.). Micrographs were
354 Wheeler (tnd Slrottp
taken of the lateral surface of the tooth root. Areas of root surface containing resorption were quantified manually with the principle of point-counting volumetry.' In this technique, micrographs were placed under a regular point lattice, and points coincident with resorption lacunae were scored. The total points scored, corrected to a standard magnification, gave a measure of the relative extent of the resorption areas and are referred to as resorption indices.
Antigen preparation A very crude antigen was prepared from extracted N1H Swiss mouse incisors and from Sprague-Dawley strain rats. All incisors were cleaned of bone and all visible soft tissue by scraping with periodontal scalers under a dissecting microscope. The small size and fragility of the mouse incisors did not allow any further cleaning. The teeth were extracted with 5M guanidine hydrochloride containing 10% EDTA for 48 hrs at 4" C. Extracts were centrifuged (1100 • g) and renatured by exhaustive dialysis against distilled and deioni~ed water for 1 week at 4 ~ C. Extracts were then lyophilized and resuspended in PBS. Protein concentrations were determined by the bicinchoninic acid protein assay procedure (Sigma, St. Louis, Mo.). This extract is considered to be a very crude antigen preparation containing pulp, enamel, cementum, and dentin proteins. It has been prepared identically to that used in other studies examining root resorption. ~6 It is recognized that this preparation is not highly refined and contains many contaminating proteins. However, since the majority of the material is dentin, and to keep consistent with published literature, it will be considered a crude dentin extract.
ELISA Antibody activity against dentin antigen was detected using the ELISA. 8 Dentin, extracted by guanidine-EDTA from mouse incisors, was used as the source of antigen. Mouse dentin at I I.tg/ml protein was diluted in carbonate-bicarbonate buffer pH 9.6. Rat dentin at the same concentrations and treated identically was used for detection of mouse antirat dentin antibody. To each well of a polystyrene microtiter plate (Linbro E.I.A. Microtitration plate, Flow Laboratories, Inc. McLean, Va.), 100 mm 3 of antigen was added and allowed to incubate for 16 hours at 4" C. Unbound antigen was removed by washing the wells with 0.15 M PBS, pH 7.2, containing 0.05% Tween 20, using an automated rinser (Multiwash, Bio-Rad Laboratories, Richmond, Calif.). Two hundred microliters of I% skim milk (SM) in 0.15 M PBS, pH 7.2, was added to each well and incubated for two hours at room temperature. After removal of the 1% SM solution and washing of the plate as previously described, 100 mm 3 of mouse serum, diluted 1:5 in 1% SM-PBS, was added to the appropriate wells. Antibody was allowed to bind to the antigen for I hour at room temperature. Unbound antibody was removed by rinsing, and 0.1 ml of a predetermined dilution (usually 1 : 1000) of affinity-purified, peroxidase conjugated goat antimouse lg (lgA, lgM, lgG, H- and L-chain stral~" specific, Organon Teknika Laboratories, Durham, N.C.) was added. The peroxidase-conjugated antibody was incubated on
American Journal of Orthodontics and Dentofacial Orthopedics April 1993
the plates for 1 hour at room temperature. Unbound second antibody was removed by rinsing as previously described. Two hundred microliters of peroxidase enzyme substrate were added to each well. The substrate solution consisted of 10 " mg O-phenylenediamine, and 12 mm 3 H_,O_,(30%) in 20 ml of 0.1 M citrate buffer, pH 4.5, made fresh immediately before use. Optical density (OD) of the enzyme substrate solution was monitored after 1 hour of incubation at room temperature at 492 nm with a Multiskan (Flow Laboratories, Inc. McLean, Va.). Negative control sera were obtained from nonimmunized mice. Bacteroides ghzgivalis was used to coat wells to check for antibody specificity. Additional controls were wells with only enzyme substrate, and wells with antigen and peroxidase-conjugated antibody only.
Statistical methods The means and standard errors of the serum dentin antibody ELISA ODs were calculated for each serum sampling point. The means and standard errors of both the root resorption areas, as well as the ELISA OD at the final bleeding, were also calculated. Root resorption between immunized and sham-immunized groups was compared with a one-tailed paired t test. An analysis of variance (ANOVA) was performed to examine differences between immunized and sham-immunized groups ,at each time point and across times within each treatment group. Pairwise (Scheffr) comparisons were performed between the individual treatment groups when the ANOVA indicated that significant differences existed (p < 0.05).
RESULTS Root resorption was observed on the lateral surface of the left incisors in the sham-immunized mice (Figs. 2, A, and 3) but not in the dentin-immunized mice ( p = 0.01). No root resorption was observed on the contralateral control incisors o f either group (Fig. 2, B). A trend toward increased serum antibody titers to dentin in immunized mice was observed over time (Fig. 4). However, only the serum antibody titers to dentin from preimmune mice and bleed 5 were statistically significant ( p < 0.05). A slight drop in dentin antibody titers in immunized mice was observed from bleed 5 and postcryotrauma, however, this was not significant. Sham-immunized animals showed no significant changes in dentin antibody titers throughout the experiment.
DISCUSSION As previously reported, 6"9 trauma by application o f cold to the lateral side o f the mandible stimulates an observable tooth root resorption lesion by day 10 in normal mice. It may be argued that root resorption in the i m m u n i z e d ' a n i m a l s may begin later than day 10. However, Ng and King 6 have shown that cryoprobe
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% Root R e s o r p t i o n (_+SEM) 7-
..'U T . . . . ~.
__
1
9 .~
I X
6:
jl
5
etO
4
~
a
0 n," 2
A
Sham
Immunized
Fig. 3. Root resorption of mouse incisor after exposure to cryoprobe at - 8 0 ~ C for 5 minutes. Each bar represents the mean of six observati0ns, and the vertical bar represents the SEM (p ~- 0.01).
Serum Ab to Dentine (Mean _+SEM) 0.04
0.03
Fig. 2. Scanning electron micrographs of (A) representative root resorption lesion of cryoprobe-treated left incisor of sham immunized mouse and (B) contralateral incisor of sham-immunized animals showing no root resorption. Incisors from animals immunized with dentin were identical to those pictured in (B). Bar = 50 FM.
c) o <1: CO ,.J t/J
0.02
0.01
induced root resorption was greatest on day 10, and that by day 21 lesions may erupt into the mouth. Therefore day 10 was chosen in this experiment to examine the tooth for root resorption. To increase dentin antibody titers, the homologous mouse dentin preparation was replaced with a heterologous rat dentin preparation. It is ,,,,,ell documented that to obtain elevated antibody titers when immunizing with "self" or homologous antigens is very difficult. To increase titers, heterologous antigen preparations of similar tissues can be used to increase antibody levels. 1o Therefore the change to rat dentin was made to increase mouse serum antibody titers to dentin. ,~ Quantitative SEM has been used to document ortho- 9 dontic root resorption in rodents 6"1~ and is considered to be reliable and valid. Resorption lacunae spread lat-
0.00
Pie-immune
1
2
3 Immune BIeed
4
5
I Post-Cryo
I
Fig. 4. Mouse antibody titers (ELISA OD at 494 nm) before immunization (preimmune), after boosts with mouse dentine (immune bleed 1, 2, and 3) and with rat dentine (immune bleed 4 and 5), and at time of death (postcryo) of mice immunized with dentine and sham-immunized animals. Each bar represents the mean of six observations and the vertical bar represents the SEMs. *Indicates significance p < 0.05.
erally and remain very shallow in comparison with the extefft of their lateral spread.. Ideally, absolute measures of lesion volumes may be more specific, however, a two-dimensional depiction of a shallow object is not
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expected to introduce systematic errors during quantification with relative measures. A question may be raised as to the similarity of root resorption as the result of orthodontic tooth movement compared with cryoprobe stimulated injury. The resorption lacunae observed in this experiment are structurally identical to those observed during orthodontic tooth movement. 12.,3Histomorphometric studies of root resorption initiated by these different procedures will produce a more definitive answer to this question. The cryoprobe alone should not cause any injury since the tooth is never exposed. After freezing, the probe and tissue were allowed to defrost while in contact, so that frozen tissue was not fractured off on probe removal. The lateral border of the mandible only is exposed by the surgery and then touched by the cryoprobe. ~A slight decrease in antibody titer to dentin was observed in the sham-immunized animals, however, it was not statistically significant. This is in contrast to the findings of King et al. 5'6 and the observation t h a t aut0antibody titers to dentin significantly decreased i n animals during the process of root resorption. The stud: ies by King et a12 "6 examined naturally occurring autoantibody titers to the dentin antigen, whereas this study examined the effect on increased antibody levels caused by hyperimmunization. It is very possible that the immunization procedure altered the cellular immune response, as well as the humoral response, of the animal to dentin thereby protecting the root from resorption on injury. The role of the various components of the immune system in the protection to cryo-induced root resorption is now being investigated. The decrease in the antibody titers observed by King s'6 could be explained by trauma-induced immunosuppression. Trauma has been shown to alter the immune system of both humans and animals. '4'8 The T-cell dependent antibody production in humans to tetanus toxoid has been shown to decrease after thermal injury? 8 The cell-mediated immune system has been shown to be depressed in patients suffering from chronic posttraumatic osteomyelitis, whereas the humoral immune response appeared to be less affected.~5 If a generalized antibody production decrease occurs in traumatized animals, this could also explain the decrease observed in dentin antibody by King et a l ) "6 Therefore the antibody suppression observed by King et al. does not necessarily indicate that the immune system contributes to or mediates the resorption process. It has been suggested that osteoclast attachment-is_ in part mediated by arginine-glycine-aspartic acid (RGD) peptide sequences in bone matrix proteins.19:-~
American Journal of Orthodontics and Dentofacial Orthopedics April 1993
Proteins that contain these tripeptides include fibronectin, vitronectin, osteopontin, collagens, thrombospondin, fibrinogen, and von Willebrand factor. 21 Recently, it has been shown that in vitro osteoclast-mediated dentin resorption can be inhibited by antivitronectin receptor antibody. 2-" In addition, synthetic RGD peptides, have been shown to inhibit dentin resorption in vitro, 2-" as well as in vitro bone resorption? 9,z~ If the crude dentin preparation used in the present study contained an RGD peptide, it is possible that immunization with this material could induce an antibody response that could inhibit root resorption in vivo. The dentin preparation is now being purified, and active components identified. It was originally hypothesized that antibody played some role in the root resorption process. Therefore it was expected that the immunized mice, with increased dentin antibody titers, would result in greater root resorption than sham-immunized mice. However, the ob9servation that incisor roots from dentin immunized mice did not resorb was very surprising. In addition, the sham-immunized mice did present with root resorption. These findings.do, not suggest that antibody plays a role in the process of root resorption as hypothesized by King et a l ) "6 In contrast, the data presented here suggest that the immunization procedure prevented root resorption and that increased dentin antibody titers may protect mice from traumatic root resorption. The role that the dentin immunization procedure had on the cellular immune system is not known. Since trauma is known to suppress the immune system, and if this suppression induces .root resorption, it is possible that the immunization procedure prevented the suppression and the resultant root resorption. It would be of interest to determine whether the dentin immunization procedure induced any type of cellular immune response that may be protective against trauma induced root resorption. This also is now being investigated. We thank Dr. Greg Erdos and the Electron Microscope Core Facility for their assistance with the SEM examinations. We also thank Dr. Greg King for his helpful advice. REFERENCES
1. AndreasenJO. External root resorption: its implicationin dental traumatol~y, paedodontics, periodontics,orthodontics and endodontics. Int Endod J 1985;18(2):109-18. 2. HarrisEF, BakerWC. Lossof root lengthand crestalboneheight before and during treatment in adolescent and adult orthodontic patients9AM J ORTIIOD DENTOFACORTHOP 1990;98(5):463-9. 3. DermautLR, De MA. Apical root resorptionof upper incisors caused by intrusive tooth movement: a radi~raphic study. A.~1 J ORTtIOD DENTOFACORTIIOP 1986;90(4):321-6. 4. Goldie RS, King GJ. Root resorption and tooth movement in orthodontically treated, calcium-deficient, and lactating rats. AM J OR'ntOD 1984;85(5):424-309
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5. King GJ, Courts F. The biol~y of tooth movement. Boca Raton, Florida: CRC Press, 1989:275-285. 6. Ng KT, King GJ, Courts FJ. Humoral immune response to active root resorption with a murine model. AM J ORT|IOD DEN'rOFAC OR'ntor 1990;98(5):456-62. 7. Weibel ER, Kestler GS, Scherle WF. Practical sterological methods for morphometric cytology. J Cell Biol 1966;30:23-38. 8. Voller A, Bidwell D, Bartlett A. Enzyme-linked immunosorbent assay. In: Rose NR, Friedman H, ed. Manual of Clinical Immunology. (2nd ed.) Washington, D.C.: American Society for Microbiol~y, 1980:359-371. 9. Wesselink PR. Resorption of the mouse incisor after the application of cold to the periodontal attachment apparatus. Calcif Tissue Int 1986;39:11-21. 10. Charreire J. Immune mechanisms in autoimmune thyroiditis. Adv Immunol 1989;46:263-334. 11. King GJ, Fishlschweiger W. The effect of force magnitude on extractable bone resorptive activity and cemental cratering in orthodontic tooth movement. J Dent Res 1982;61:775-9. 12. Kvam E. Scanning electron microscopy of tissue changes on the pressure .Surface of human premolars following tooth movement. Scand J Dent Res 1972;80:357-68. 13. Reitan K. Initial tissue behavior during apical root resorption. Angle Orthod 1974;44(1):68-82. 14. Pretus HA, Browder IW, Lucore P, McNamee RB, Jones.EL, Williams DL. Maerophage activation decreases macrophage prostaglandin E2 release in experimental trauma. Trauma 1989;29:1152-7. 15. Schliiter B, Bergmannn U, Josten C, Walz M, K6nig W. Impairment of specific host defense mechanisms in patients with chronic post-traumatic osteomyelitis. Trauma 1991;3 !:68-73.
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16. O'Mahoney JB, Palder SB, Wood JJ, et al. Depression of cellular immunity after multiple trauma in the absence of sepsis. Trauma 1984;24:869-75. 17. Faist E, Mewes A, Baker CC, et al. Prostaglandin E2 (PGE2) -dependent suppression of interleukin alpha (IL-2) production in patients with major trauma. Trauma 1987;27:837-47. 18. x,Vood JJ, O'Mahony JB, Rodrick ML, Eaton R, Demling RH, Mannick JA. Abnormalities of antibody production after thermal injury. Arch Surg 1986;121:108-15. 19. van der Pluijm G, Mouthaan H, Papapoulos S, Lfwik C. Inhibition of osteoclast precursor attachment and subsequent bone resorption by synthetic RGD-peptides. J Bone Miner Res 1991;6(Suppl. 1):S 146. 20. Bertolini DR, Stadel J, Samanen J, et al. Inhibition of bone resorption by synthetic RGD containing peptides. J Bone Miner Res 1991;6(Suppl. I):S146. 21. Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987;238:491-7. 22. Horton MA, Taylor ML, Arnett TR, Helfrich MH. Arg-Gly-Asp (RGD) peptides and the anti-vitronectin receptor antibody 23C6 inhibit dentine resorption and cell spreading by osetoclasts. Exp Cell Res 1991;195(2):368-75. Reprint requests to: Dr. Timothy T. Wheeler Box J-444, JHMHC Department of Orthodontics University of Florida College Of Dentistry Gainesville, FL 32610