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The effect of partial damage to the enamel-related periodontium combined with root resection on eruption of the rat incisor eruption
Archives of Oral Biology (2004) 49, 209—216
The effect of partial damage to the enamel-related periodontium combined with root resection on eruption of the rat incisor eruption ´ Merzel*, Silvana F. Nunes, Pedro D. Novaes Jose Department of Morphology, Faculty of Odontology of Piracicaba, State University of Campinas (Unicamp), P.O. Box 52, 13414-903 Piracicaba, SP, Brazil Accepted 24 September 2003
KEYWORDS Rat incisor; Enamel-related periodontium; Dental follicle; Tooth eruption
Summary Previous work has indicated that the enamel-related periodontium (ERP) has a role in the eruptive process of the rat lower incisor. By combining partial damage of this tissue with resection of the odontogenic organ, we examined the effect of the damage on subsequent incisor eruption. The connective tissue of the enamel-related periodontium was regenerated in less than 2 weeks, showing morphology close to normal. The injured part of the enamel organ was neither regenerated nor repaired, and a cement-like tissue, continuous with the true acellular cement, was formed on the denuded enamel. Before tooth exfoliation, the operated teeth erupted at a slower rate compared with rootresected and sham-operated incisors, probably because of the absence of a substantial part of the enamel organ due to surgical damage. As with the coronal dental follicle and the enamel organ in rat molars, the enamel-related periodontium and the enamel organ of rat incisors may have some control on their eruptive process. ß 2003 Elsevier Ltd. All rights reserved.
Introduction Tooth eruption is a multifactorial process that depends on the generation of a force to move the tooth, on the translation of that force into movement through the surrounding tissues, and on the resistance and remodelling of such tissues. The mechanism of eruption, particularly the nature and origin of the eruptive force or forces, is still a matter of controversy, as is the question of which tissues (periodontal tissues or their precursor, the dental follicle) control the process.29 The role of the coronal part of the dental follicle together with the enamel organ in the intra-osseous eruptive phase of teeth of limited growth and eruption, such as dog premolars and rat molars, has been convincingly demonstrated.13,23 *
Corresponding author. Fax: þ55-19-430-5218. E-mail address: [email protected] (J. Merzel).
The enamel-related periodontium (ERP), located on the labial and part of the distal aspects of the rat incisor socket, is formed by a loose, highly-vascularised connective tissue that is continuous with the dental follicle at the tooth odontogenic base. Partial damage to the ERP and to the related enamel organ causes a variable period of retardation, and sometimes even a temporary cessation, of the eruptive movement. Similar damage to the periodontal ligament has no effect on eruption.27 The ERP, which corresponds embryologically with tissues related to the crowns of rat molars in the intraosseous stage of development, has not yet been implicated in the eruptive process.30 After surgical removal of the odontogenic basal extremity (root resection), or when the root is transected, the rat incisor continues no erupt until the tooth loses the ERP.26 The aim of the present study was to examine the effect of damage to the ERP combined with root
0003–9969/$ — see front matter ß 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2003.09.009
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resection on eruption of the rat incisor. By removing the odontogenic organ, the main source of all dental and periodontal cells, we intended to prevent the regeneration of the damaged tissues, thereby mimicking the removal of the dental follicle (together with the enamel organ) of teeth showing limited growth and eruption.20 If there were any regeneration, it would be restricted to the ERP connective tissue, due to the proliferative capacity of this tissue. The enamel organ alone cannot support the eruption of teeth of limited growth, but there is no evidence regarding whether the dental follicle would be able to do so without the associated enamel organ.18
Material and methods
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through a skin incision following a plane between the angle of the mouth and the external auditory meatus, and by a second incision of the anterior half of the masseter muscle above the facial nerve.6 The bone was perforated with a no. 8859 diamond bur (Sorensen, KG, Brazil), and the soft tissues of the incisor odontogenic base were removed with a curete. Sham operation The aforementioned bone surfaces of the left hemimandible were exposed, but the bone, dental and paradental tissues were not damaged. After these procedures, the skin was sutured and the rats were given a single antibiotic cover of penicillin (60 000 units) and streptomycin (33.3 mg) (Pentabiotico Vet, Wyeth Ltd., Brazil). For the first week after surgery, solid food was offered as ground pellets. Thereafter, regular pellets Nuvital, PR, Brazil were offered.
Animals Histology Adult male Wistar rats (300—400 g body weight) were acquired from CEMIB-UNICAMP and kept on a 12 h light/dark cycle at 25—30 8C, with rodent chow and tap water ad libitum. The experimental protocol was approved by the university’s Committee for Ethics in Animal Research (CEEA-UNICAMP).
Surgical procedures The rats were anesthetized with an intramuscular injection of ketamine (80 mg/kg) and xylasine (8 mg/kg) (Francotar and Virbaxyl, respectively, Virbac, Brazil). Damage to the enamel-related periodontium The lower border of the left hemimandible was exposed through a skin incision in a plane parallel to the border followed by reflection of the masseter and digastric muscles. Using a small osteotome, the bone of the lower border was removed, beginning behind the insertion of the anterior belly of the digastric muscle up to the level of the second or third lower molars. The exposed labial periodontal tissues of the incisor were removed by scrubbing the enamel with a piece of gauze and by scraping part of the distal face of the tooth with a dental excavator. The damage was completed introducing a no. 15 endodontic file into the incisal part of the labial periodontal space until its tip appeared through the gingival margin.27 Root resection The bluish protuberance on the lateral surface of the left mandibular ramus, which is related to the odontogenic base of the incisor, was exposed
The ERP of the left lower incisors of 12 rats was injured and the odontogenic region was resected. After surgery, notches were made with a no. 8859 diamond bur on the labial face of both lower incisors, close to the gingival margin. The behaviour of the eruption was followed twice a week through these notches by comparing the operated tooth with the intact contralateral tooth. The marks were repeated, under light ether anaesthesia, as the previous mark came closer to the incisal border. The rats were killed in pairs 1, 2, 4, 6, 8 and 10 weeks after surgery. The left hemimandible was removed, fixed in 10% buffered formalin (0.1 M phosphate buffer, pH 7.2—7.4) for 48 h at 4 8C, and radiographed while in the fixative. The jaws were demineralized with 5% nitric acid in 10% formalin and divided into six transversal segments from the alveolar crest to the basal end of the incisor. The pieces were embedded in paraplast, and semiserial cross-sections, 5 mm thick, were stained with haematoxylin and eosin or periodic acid-Schiff (PAS) and haematoxylin.
Eruption rate Four groups of eight rats were used. One rat in each group was allotted to one of the following procedures: (i) damage to the ERP plus resection of the basal end of the incisor, as in the previous experiment (PR group); (ii) damage to the ERP and sham operation at the basal end of the incisor (PS group); (iii) root resection and sham operation at the mandibular lower border (SR group) of the incisor and (iv) sham operation at both sites (SS group). In
Eruptive process of lower incisor
previous experiments, we have shown that surgical damage to the ERP is prone to error, resulting in permanent cessation of the eruptive movement.27 Thus, to have enough animals for statistical analysis, three additional rats were operated in the PR group and three more in the PS group. A trained examiner, unaware of the group to which a rat belonged, made the measurements once a week, at the same time of day, for 16 weeks after surgery. Using a calibrated grid in a microscope eyepiece at 8 magnification, the distance from the gingival margin to a mark at labial face of the left lower incisor (notches made as described above) was recorded and the eruption rate was expressed as mm per day. The marks on the contralateral incisors were also used as a visual control of the eruption rate. For these procedures, the animals were lightly anaesthetised with ether. The rats in the PR and PS groups in which eruption of the operated incisor ceased and did not recover by the end of the experiment were radiographed; this was also done for the lower left incisor of rats in the PR and SR groups which had not exfoliated by the end of the experiment. When the radiograph indicated a possible surgical error, the tooth measurements were excluded from the statistical analysis.
Statistical analysis The data obtained for each week were analysed by ANOVA, and pairwise comparisons between groups were made using Tukey’s (HSD) test and a significance level of 0.05.
Results Histology The left lower incisors, which were root-resected and had their ERP partially removed, erupted more slowly than the contralateral controls. The resected base was at the level of the third lower molar 1 week after surgery, and at the level of the labial alveolar crest 8—10 weeks after surgery. One week after surgery, the damaged tissues of the mandibular lower border showed some signs of repair, i.e. new bone and new blood vessels being formed. One week later, the tooth base was at the level between the second and third lower molars, and the repair was almost complete. Newly formed bone replaced most of the removed bone. The labial periodontal space, in an extension from the first lower molar up to the level of the mesial root of the second lower molar, was filled with connective
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tissue in direct contact with the enamel without the presence of epithelial tissues of the enamel organ (Fig. 1A and B). This connective tissue, although denser than normal, showed the distribution and size of the blood vessels similar to those of normal ERP. A considerable amount of young enamel was seen in the region where the enamel organ was absent (Fig. 1B) and a cement-like tissue, continuous with the true acellular cement, lined the inner face of the labial periodontal connective tissue facing the denuded enamel. Mature enamel, represented by an empty space, was present in the regions in front of and behind the damaged zone. Incisally to the damaged zone, the enamel organ was reduced to a discontinuous, mostly single, layer of cuboidal or squamous epithelial cells. Behind the damaged zone, the enamel organ was at the maturation stage and some young enamel was present. The pulp of the resected teeth showed infiltration of mononuclear cells, large areas of necrosis (Fig. 1A) and, at later time intervals, calcified areas resembling osteodentin. In the fourth week after surgery, the tooth-resected base was at the level of the mandibular diastema, no enamel organ was seen and the cement-like tissue was more conspicuous (Fig. 1C). The healing of the tooth-free socket followed a sequence that has been described previously.4 As the resected tooth erupted, the alveolar bone remained invested by connective tissue, and the tooth space was filled by an amorphous, cell-free material seldom preserved after processing for histology. In the tooth-free socket, intense osteoblastic activity was present on the surfaces of the alveolar bone related to the periodontal ligament, with trabecular bone growing around the blood vessels, thereby reducing the empty space. On the labial face, the osteogenic activity was less intense, the smoothness of the alveolar bone was preserved, and the appearance of the highly vascularised connective tissue was similar to that of normal ERP. Two of the 12 rats used in this experiment showed surgical failures. In one of the rats killed in the second week, the hard dental tissues were transfixed and the pulp was connected with the labial periodontal tissue. This event would probably cause cessation of the eruptive movement at later time intervals. In another rat killed 10 weeks after surgery, the root resection was only partial, giving rise to a dentin tooth as described by Berkovitz.3
Eruption rate Pairwise comparisons of the eruption rates between the groups are shown in Fig. 2. The data of five rats
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(three of the PS group and two of the PR group) in which eruption of the left incisor ceased and did not recover by the end of the experiment have been excluded. The clinical crown of these incisors showed a visible decrease in height. One rat of the SR group died during anaesthesia in Week 1, and this also occurred with two rats of the PS group in 14 and 15. The left incisors of rats from the SR and PR groups started to exfoliate from Week 7 onwards. The control group (SS) had a mean eruption rate of about 0.45 mm per day, considered normal for impeded lower incisors of rats, and did not differ visually from the eruption of the contralateral incisors. After a slight non-significant slowdown in the week 1, the incisors of the SR group showed normal rates for the next 3 weeks, but thereafter the eruption rate started to decrease, becoming close to zero in Week 8. The resected teeth of the SR group started to exfoliate at Week 8 and only one rat retained its left incisor after the Week 13. The eruption rate of the PS group was slower than that of the control group (SS) from Weeks 1 to 5 (significantly less on Weeks 1, 4 and 5). At later time intervals, the rate was variable but not significantly different from normal, tending to stabilise after Week 12. The left incisor of four PS rats showed a transitory cessation of eruption: two rats for 1 week (one rat at Week 4 and another at the Week 6), one rat for 2 weeks (during Weeks 2 and 3) and one rat for 7 weeks (from the Weeks 3 to 9). The eruption in the PR group, with the exception of the second week, never reached the normal rate and started to decrease in Week 6, becoming close to zero after Week 8. The rate of exfoliation of the left incisors in the PR group was slower than that of the SR group, with four rats retaining their teeth until the end of the experiment. In the first 5 weeks of the experiment, there were no significant differences in the mean eruption rates between PR and PS incisors. However, the former did not show a temporary cessation of eruption throughout the experiment.
Figure 1 Sections of rat incisors submitted to damage of the enamel-related periodontium tissues plus root resection. (A) Two weeks after surgery, at the level of
the second molar, showing the recovery of the alveolar bone (AB) and the dental follicle (DF) while the enamel organ is absent. (B) Higher magnification of the region marked () in (A). (C) At 4 weeks after surgery, in a region close to the alveolar crest, showing the cementlike structure formed between the dental follicle and the enamel surface (arrow). P, pulp; D, dentin; E, young enamel; ES, enamel space; PL, periodontal ligament. (A and B) HE, (C) PAS—haematoxylin. Scale bars ¼ 200 mm in (A) and 50 mm in (B and C).
Eruptive process of lower incisor
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SS X PR
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Weeks Figure 2 Pairwise comparisons of the eruption rates of the lower left incisor between experimental groups: PR (-!!-) damage to the ERP plus root resection; PS (-D-D-) damage to the ERP plus sham operation at the odontogenic base; SR (-*-*-) sham operation at the mandible lower border and root resection; and SS (-*-*-) sham operation at both sites. The number of animals used in the first week and the number of animals remaining at later time intervals (see text for details) are indicated in brackets. Vertical bars represent the SEM; indicates significant differences (P < 0:05) at each time interval.
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Discussion Most root-resection experiments have been performed in unimpeded rat incisors. The eruption rate of these teeth shows three successive phases: an initial slowdown followed by a period of normal eruption rate and a final slowdown before the tooth is exfoliated.6,8 Root-resected impeded (normal) incisors show a similar pattern of eruption7 and this was confirmed by our results with SR incisors. The incisors of the PR group did not have a period where the tooth erupted at normal rate. The initial slowdown phase lasted for about 5 weeks, followed by a sharp decline Week 6. Most of the PR incisors lasted longer than SR incisors before the teeth were exfoliated. The incisors of the PR group did not show temporary cessation of eruption throughout the experiment, in contrast to some teeth in the PS group, according to our present and previous results.27 The main difference between the two groups was the absence of the odontogenic region and an initial part of the region of enamel secretion. This absence may indicate that these structures have a modulatory effect on eruption. Granule proteins, isolated from stratum intermedium and dental follicle of rat molars, delay the eruption of incisors and molars, as well as, being responsible for eyelid opening.19 The level of these proteins would be lower in root-resected teeth, and this could explain why the PR incisors did not show any cessation of eruption. The connective tissue of the enamel-related periodontium is equivalent to the coronal part of the dental follicle of teeth with limited growth and eruption. The histology of this tissue in the rat incisor5,25 is comparable to the coronal dental follicle described in dog premolars.24 Both tissues have an inner layer related to the enamel organ, which contains fibroblasts, densely packed collagen and a capillary network, considered to be the true dental follicle.24 The outer layer, related to the alveolar bone, is formed by loose connective tissue, is rich in ground substance and is well vascularised. In incisors, this vascularisation involves large blood vessels. Where denuded enamel is in contact with the dental follicle, it induces the formation of a cement-like tissue as shown by our results and also in rat molars.32 Removal of the dental follicle (with the enamel organ) prevents the eruption of dog premolars.20 Larson et al.18 were unable to confirm whether the dental follicle without the associated enamel organ could support the eruption of dog teeth. When partial damage to the enamel-related periodontal tissues was associated with resection of the incisor odontogenic region (PR group), the dental follicle
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was completely regenerated in less than 2 weeks whereas the enamel organ was not. The morphology of the ERP seems to recover in less than 2 weeks; however, the eruption rate takes at least 3 more weeks to reach normal values in the PS group. Thus, it can be hypothesised that the functional recovery of ERP, as the production of the putative molecules involved in the eruptive process, takes more time. In rat molars, the source of most of these molecules is the coronal dental follicle and some are produced in the stellate reticulum of the enamel organ.36 Some of these molecules peak at the time when the tooth crown is being completed, earlier than the start of the eruptive process.34,36 The lack of a substantial part of the enamel organ at a corresponding stage of development of the rat incisor (at the level of the third and second lower molars) could explain the decrease in the eruption rate of the PS and PR groups compared with SS and SR groups during the first 5 weeks of experiment. It also took some weeks for the ERP to acquire cementogenic ability. The odontogenic tissues at earlier stages of tooth development have never been implicated in the eruptive process. This may be the reason why root-resected incisors (SR group) showed normal eruption rates in the first 4 weeks after surgery. The coronal aspect of the dental follicle in molars and the ERP connective tissue in rat incisors are both related to bone resorption. Preceding the eruptive movement in dog premolars or rat molars, there is an influx of mononuclear cells into the dental follicle followed by an increase in osteoclasts22,35 that promote the resorption of bone close to the dental crown, thereby creating the eruption pathway.20 The inner surface of the labial wall of the incisor socket is carpeted with osteoclasts17 and resorption activity starts somewhere in the region of the lower molars and increases towards the alveolar crest.12 Bone resorption controlled by the coronal part of the dental follicle is a key factor in the intra-osseous phase of the eruptive process of teeth with limited growth.34,36 Bone resorption of the labial aspect of the socket may have an equivalent role in rodent incisors, which are considered to be in continuous supra-osseous eruption. Drugs that inhibit bone resorption by osteoclasts delay eruption, such as bafilomycin A in dog premolars33 and bisphosphonate in rat incisors and molars.15 Osteopetrotic rats, which show a total absence of erupted teeth, treated with CSF-1 from birth, have the eruption of the incisors and molars restored.16 The bone resorption and probably the bone remodeling promoted by the dental follicle are apparently necessary for tooth growth and eruption. When bone resorption is impaired, eruption is retarded
Eruptive process of lower incisor
or interrupted but growth may change direction or become anomalous as in impacted teeth, in molar teeth after removal of the coronal part of the dental follicle,21 or in the proximal segment of root-transected rodent incisors.26 In teeth of limited growth and eruption, bone resorption to form the eruptive pathway is a result of the interaction between cells of the enamel organ and the dental follicle. A cascade of growth factors (EGF, TGF-b) and cytokines (IL-1a) enhances the production of CSF-1 by dental follicle cells. CSF1 is responsible for the influx of mononuclear blood cells (monocytes) into the dental follicle, which in turn are the probable precursors of osteoclasts seen at the bone surface of the crypt.36 Monocyte chemotatic protein-1 (MCP-1)) which is expressed and secreted by dental follicle cells, has the same effects as CSF-1.31 In PR rats, the remaining part of the incisors, enamel organ may have been enough to provide the necessary signals, but in an insufficient amount to maintain a normal eruption rate. There are indications that these signals in rats may be different for molars and incisors. Neonatal injections of EGF accelerate the eruption of incisors and have no significant effect on the eruption of the first molar, while CSF-1 accelerates molar eruption more than incisor eruption.11 The effects of dexamethasone, which stimulates bone resorption, parallel those of EGF.37 Nevertheless, there are some reported similarities on the eruptive behaviour of both types of teeth, as already mentioned in relation to the effects of granule proteins,19 of drugs that inhibit bone resorption15 and of CSF-1 in osteopetrotic rats.16 The controversies on the mechanism of tooth eruption are still no settled.23,29,10 Most authors consider the periodontal ligament, which is derived from the dental follicle, as the tissue responsible for the generation of the eruptive force(s) in continuously erupting teeth.1,2,28,29 The available evidence does not favour the hypothesis that the forces are tractional, either through collagen contraction or periodontal fibroblast traction.29 There is stronger evidence that one of the factors responsible for eruption is related to the vascular/periodontal tissue, hydrostatic pressure.28,29 In this regard, surgical disruption of the vascular entwork of the enamel-related periodontal tissues could be responsible for the decreased eruption rate of PS and PR incisors. However, in root-resected and root-transected incisors, the eruption ceases for several weeks before exfoliation when the base of the erupting tooth surpasses the labial level of the alveolar crest. In this situation, all the ERP is lost and some functional periodontal ligament is still present.26 Rootless teeth of limited growth,
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and therefore without a periodontal ligament, do erupt.9,14 Thus, whatever the mechanism, some evidence indicates that the dental follicle together with the enamel organ are probably the tissues in control of the eruptive process.
Acknowledgements The authors thank Maria A. Varela, Eliene A.N. Romani and Waldek R. Moreira for technical assistance, and Dr. Glaucia M.B. Ambrosano for statistical advice. This work was partially supported by FAPESP (Proc. 96/07166-4).
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16. Iizuka T, Cielinski M, Aukerman SL, Marks Jr SC. The effects of colony-stimulating factor-1 on tooth eruption in the toothless (osteopetrotic) rat in relation to the critical periods for bone resorption during tooth eruption. Arch Oral Biol 1992;37:629—36. 17. Irie K, Ozawa H. Relationships between tooth eruption, occlusion and alveolar bone resorption: cytological and cytochemical studies of bone resorption on rat incisor alveolar facing the enamel. Arch Histol Cytol 1990;53: 497—509. 18. Larson EK, Cahill DR, Gorski JP, Marks Jr SC. The effect of removing the true dental follicle on premolar eruption in the dog. Arch Oral Biol 1994;39:271—5. 19. Lin F, Fan W, Wise GE. Granule proteins of the dental follicle and stellate reticulum inhibit tooth eruption and eyelid opening in postnatal rats. Arch Oral Biol 1992;37: 841—7. 20. Marks Jr SC, Cahill DR. Experimental study in the dog of the non-active role of the tooth in the eruptive process. Arch Oral Biol 1984;29:311—22. 21. Marks Jr SC, Cahill DR. Regional control by the dental follicle of alterations in the alveolar bone metabolism during tooth eruption. J Oral Pathol 1987;16:164—9. 22. Marks Jr SC, Grolman M-L. Tartrate-resistant acid phosphatase in mononuclear and multinuclear cells during the bone resorption of tooth eruption. J Histochem Cytochem 1987; 35:1227—30. 23. Marks Jr SC, Schroeder HE. Tooth eruption: theories and facts. Anat Rec 1996;245:374—93. 24. Marks Jr SC, Cahill DR, Wise GE. The cytology of the dental follicle and adjacent alveolar bone during tooth eruption in the dog. Am J Anat 1983;168:277—89. 25. Matena V. The periodontium of the enamel aspect of the rat incisor. J Periodont 1972;43:311—5. 26. Merzel J, Novaes PD, Furlan S. A histological study of rootresected and root-transected rat incisors when eruption ceases, shortly before they are exfoliated from the socket. Arch Oral Biol 2000;45:315—22.
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27. Merzel J, Novaes PD, Furlan S. The effects of local trauma to the enamel-related periodontal tissues in the eruption of the rat incisor. Arch Oral Biol 2000;45:323—33. 28. Moxham BJ. What the structure and the biochemistry of the periodontal ligament tell us about the mechanism of tooth eruption. In: Davidovitch Z, editor. The biological mechanisms of tooth eruption, root-resorption and replacement of implants. Boston, MA: Harvard Society for the Advancement of Orthodontics; 1994. p. 437—50. 29. Moxham BJ, Berkovitz BKB. The periodontal ligament and physiological tooth movements. In: Berkovitz BKB, Moxham BJ, Newman HN, editors. The periodontal ligament in health and disease. London: Mosby-Wolfe; 1995. p. 183—214. 30. Moxham BJ, Shore RC, Berkovitz BKB. A quantitative study of the ultrastructure of fibroblasts within the enamelrelated connective tissue of the rat incisor. J Biol Buccale 1991;19:135—40. 31. Que BG, Wise GE. Colony-stimulating factor-1 and monocyte chemotactic protein-1 chemotaxis for monocytes in the rat dental follicle. Arch Oral Biol 1997;42:860—5. 32. Spahr A, Hammarstrom L. Response of dental follicular cells to the exposure of denuded enamel matrix in rat molars. Eur J Oral Sci 1999;107:360—7. 33. Sundquist KT, Marks Jr SC. Bafilomycin A1 inhibits bone resorption and tooth eruption in vivo. J Bone Min Res 1994;9:1575—82. 34. Wise GE. The biology of tooth eruption. J Dent Res 1998; 77:1576—9. 35. Wise GE, Fan W. Changes in the tartrate-resistant acid phosphatase cell population in dental follicles and bony crypts of rat molars during tooth eruption. J Dent Res 1989;68:150—6. 36. Wise GE, Frazier-Bowers S, D’Souza RN. Cellular, molecular, and genetic determinants of tooth eruption. Critic Ver Oral Biol Med 2002;13:323—34. 37. Wise GE, Grier IV RL, Lumpkin SJ, Zhang O. Effects of dexamethasone on tooth eruption in rats: differences in incisor and molar eruption. Clin Anat 2001;14:204—9.
The effect of partial damage to the enamel-related periodontium combined with root resection on eruption of the rat incisor eruption ´ Merzel*, Silvana F. Nunes, Pedro D. Novaes Jose Department of Morphology, Faculty of Odontology of Piracicaba, State University of Campinas (Unicamp), P.O. Box 52, 13414-903 Piracicaba, SP, Brazil Accepted 24 September 2003
KEYWORDS Rat incisor; Enamel-related periodontium; Dental follicle; Tooth eruption
Summary Previous work has indicated that the enamel-related periodontium (ERP) has a role in the eruptive process of the rat lower incisor. By combining partial damage of this tissue with resection of the odontogenic organ, we examined the effect of the damage on subsequent incisor eruption. The connective tissue of the enamel-related periodontium was regenerated in less than 2 weeks, showing morphology close to normal. The injured part of the enamel organ was neither regenerated nor repaired, and a cement-like tissue, continuous with the true acellular cement, was formed on the denuded enamel. Before tooth exfoliation, the operated teeth erupted at a slower rate compared with rootresected and sham-operated incisors, probably because of the absence of a substantial part of the enamel organ due to surgical damage. As with the coronal dental follicle and the enamel organ in rat molars, the enamel-related periodontium and the enamel organ of rat incisors may have some control on their eruptive process. ß 2003 Elsevier Ltd. All rights reserved.
Introduction Tooth eruption is a multifactorial process that depends on the generation of a force to move the tooth, on the translation of that force into movement through the surrounding tissues, and on the resistance and remodelling of such tissues. The mechanism of eruption, particularly the nature and origin of the eruptive force or forces, is still a matter of controversy, as is the question of which tissues (periodontal tissues or their precursor, the dental follicle) control the process.29 The role of the coronal part of the dental follicle together with the enamel organ in the intra-osseous eruptive phase of teeth of limited growth and eruption, such as dog premolars and rat molars, has been convincingly demonstrated.13,23 *
Corresponding author. Fax: þ55-19-430-5218. E-mail address: [email protected] (J. Merzel).
The enamel-related periodontium (ERP), located on the labial and part of the distal aspects of the rat incisor socket, is formed by a loose, highly-vascularised connective tissue that is continuous with the dental follicle at the tooth odontogenic base. Partial damage to the ERP and to the related enamel organ causes a variable period of retardation, and sometimes even a temporary cessation, of the eruptive movement. Similar damage to the periodontal ligament has no effect on eruption.27 The ERP, which corresponds embryologically with tissues related to the crowns of rat molars in the intraosseous stage of development, has not yet been implicated in the eruptive process.30 After surgical removal of the odontogenic basal extremity (root resection), or when the root is transected, the rat incisor continues no erupt until the tooth loses the ERP.26 The aim of the present study was to examine the effect of damage to the ERP combined with root
0003–9969/$ — see front matter ß 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2003.09.009
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resection on eruption of the rat incisor. By removing the odontogenic organ, the main source of all dental and periodontal cells, we intended to prevent the regeneration of the damaged tissues, thereby mimicking the removal of the dental follicle (together with the enamel organ) of teeth showing limited growth and eruption.20 If there were any regeneration, it would be restricted to the ERP connective tissue, due to the proliferative capacity of this tissue. The enamel organ alone cannot support the eruption of teeth of limited growth, but there is no evidence regarding whether the dental follicle would be able to do so without the associated enamel organ.18
Material and methods
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through a skin incision following a plane between the angle of the mouth and the external auditory meatus, and by a second incision of the anterior half of the masseter muscle above the facial nerve.6 The bone was perforated with a no. 8859 diamond bur (Sorensen, KG, Brazil), and the soft tissues of the incisor odontogenic base were removed with a curete. Sham operation The aforementioned bone surfaces of the left hemimandible were exposed, but the bone, dental and paradental tissues were not damaged. After these procedures, the skin was sutured and the rats were given a single antibiotic cover of penicillin (60 000 units) and streptomycin (33.3 mg) (Pentabiotico Vet, Wyeth Ltd., Brazil). For the first week after surgery, solid food was offered as ground pellets. Thereafter, regular pellets Nuvital, PR, Brazil were offered.
Animals Histology Adult male Wistar rats (300—400 g body weight) were acquired from CEMIB-UNICAMP and kept on a 12 h light/dark cycle at 25—30 8C, with rodent chow and tap water ad libitum. The experimental protocol was approved by the university’s Committee for Ethics in Animal Research (CEEA-UNICAMP).
Surgical procedures The rats were anesthetized with an intramuscular injection of ketamine (80 mg/kg) and xylasine (8 mg/kg) (Francotar and Virbaxyl, respectively, Virbac, Brazil). Damage to the enamel-related periodontium The lower border of the left hemimandible was exposed through a skin incision in a plane parallel to the border followed by reflection of the masseter and digastric muscles. Using a small osteotome, the bone of the lower border was removed, beginning behind the insertion of the anterior belly of the digastric muscle up to the level of the second or third lower molars. The exposed labial periodontal tissues of the incisor were removed by scrubbing the enamel with a piece of gauze and by scraping part of the distal face of the tooth with a dental excavator. The damage was completed introducing a no. 15 endodontic file into the incisal part of the labial periodontal space until its tip appeared through the gingival margin.27 Root resection The bluish protuberance on the lateral surface of the left mandibular ramus, which is related to the odontogenic base of the incisor, was exposed
The ERP of the left lower incisors of 12 rats was injured and the odontogenic region was resected. After surgery, notches were made with a no. 8859 diamond bur on the labial face of both lower incisors, close to the gingival margin. The behaviour of the eruption was followed twice a week through these notches by comparing the operated tooth with the intact contralateral tooth. The marks were repeated, under light ether anaesthesia, as the previous mark came closer to the incisal border. The rats were killed in pairs 1, 2, 4, 6, 8 and 10 weeks after surgery. The left hemimandible was removed, fixed in 10% buffered formalin (0.1 M phosphate buffer, pH 7.2—7.4) for 48 h at 4 8C, and radiographed while in the fixative. The jaws were demineralized with 5% nitric acid in 10% formalin and divided into six transversal segments from the alveolar crest to the basal end of the incisor. The pieces were embedded in paraplast, and semiserial cross-sections, 5 mm thick, were stained with haematoxylin and eosin or periodic acid-Schiff (PAS) and haematoxylin.
Eruption rate Four groups of eight rats were used. One rat in each group was allotted to one of the following procedures: (i) damage to the ERP plus resection of the basal end of the incisor, as in the previous experiment (PR group); (ii) damage to the ERP and sham operation at the basal end of the incisor (PS group); (iii) root resection and sham operation at the mandibular lower border (SR group) of the incisor and (iv) sham operation at both sites (SS group). In
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previous experiments, we have shown that surgical damage to the ERP is prone to error, resulting in permanent cessation of the eruptive movement.27 Thus, to have enough animals for statistical analysis, three additional rats were operated in the PR group and three more in the PS group. A trained examiner, unaware of the group to which a rat belonged, made the measurements once a week, at the same time of day, for 16 weeks after surgery. Using a calibrated grid in a microscope eyepiece at 8 magnification, the distance from the gingival margin to a mark at labial face of the left lower incisor (notches made as described above) was recorded and the eruption rate was expressed as mm per day. The marks on the contralateral incisors were also used as a visual control of the eruption rate. For these procedures, the animals were lightly anaesthetised with ether. The rats in the PR and PS groups in which eruption of the operated incisor ceased and did not recover by the end of the experiment were radiographed; this was also done for the lower left incisor of rats in the PR and SR groups which had not exfoliated by the end of the experiment. When the radiograph indicated a possible surgical error, the tooth measurements were excluded from the statistical analysis.
Statistical analysis The data obtained for each week were analysed by ANOVA, and pairwise comparisons between groups were made using Tukey’s (HSD) test and a significance level of 0.05.
Results Histology The left lower incisors, which were root-resected and had their ERP partially removed, erupted more slowly than the contralateral controls. The resected base was at the level of the third lower molar 1 week after surgery, and at the level of the labial alveolar crest 8—10 weeks after surgery. One week after surgery, the damaged tissues of the mandibular lower border showed some signs of repair, i.e. new bone and new blood vessels being formed. One week later, the tooth base was at the level between the second and third lower molars, and the repair was almost complete. Newly formed bone replaced most of the removed bone. The labial periodontal space, in an extension from the first lower molar up to the level of the mesial root of the second lower molar, was filled with connective
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tissue in direct contact with the enamel without the presence of epithelial tissues of the enamel organ (Fig. 1A and B). This connective tissue, although denser than normal, showed the distribution and size of the blood vessels similar to those of normal ERP. A considerable amount of young enamel was seen in the region where the enamel organ was absent (Fig. 1B) and a cement-like tissue, continuous with the true acellular cement, lined the inner face of the labial periodontal connective tissue facing the denuded enamel. Mature enamel, represented by an empty space, was present in the regions in front of and behind the damaged zone. Incisally to the damaged zone, the enamel organ was reduced to a discontinuous, mostly single, layer of cuboidal or squamous epithelial cells. Behind the damaged zone, the enamel organ was at the maturation stage and some young enamel was present. The pulp of the resected teeth showed infiltration of mononuclear cells, large areas of necrosis (Fig. 1A) and, at later time intervals, calcified areas resembling osteodentin. In the fourth week after surgery, the tooth-resected base was at the level of the mandibular diastema, no enamel organ was seen and the cement-like tissue was more conspicuous (Fig. 1C). The healing of the tooth-free socket followed a sequence that has been described previously.4 As the resected tooth erupted, the alveolar bone remained invested by connective tissue, and the tooth space was filled by an amorphous, cell-free material seldom preserved after processing for histology. In the tooth-free socket, intense osteoblastic activity was present on the surfaces of the alveolar bone related to the periodontal ligament, with trabecular bone growing around the blood vessels, thereby reducing the empty space. On the labial face, the osteogenic activity was less intense, the smoothness of the alveolar bone was preserved, and the appearance of the highly vascularised connective tissue was similar to that of normal ERP. Two of the 12 rats used in this experiment showed surgical failures. In one of the rats killed in the second week, the hard dental tissues were transfixed and the pulp was connected with the labial periodontal tissue. This event would probably cause cessation of the eruptive movement at later time intervals. In another rat killed 10 weeks after surgery, the root resection was only partial, giving rise to a dentin tooth as described by Berkovitz.3
Eruption rate Pairwise comparisons of the eruption rates between the groups are shown in Fig. 2. The data of five rats
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(three of the PS group and two of the PR group) in which eruption of the left incisor ceased and did not recover by the end of the experiment have been excluded. The clinical crown of these incisors showed a visible decrease in height. One rat of the SR group died during anaesthesia in Week 1, and this also occurred with two rats of the PS group in 14 and 15. The left incisors of rats from the SR and PR groups started to exfoliate from Week 7 onwards. The control group (SS) had a mean eruption rate of about 0.45 mm per day, considered normal for impeded lower incisors of rats, and did not differ visually from the eruption of the contralateral incisors. After a slight non-significant slowdown in the week 1, the incisors of the SR group showed normal rates for the next 3 weeks, but thereafter the eruption rate started to decrease, becoming close to zero in Week 8. The resected teeth of the SR group started to exfoliate at Week 8 and only one rat retained its left incisor after the Week 13. The eruption rate of the PS group was slower than that of the control group (SS) from Weeks 1 to 5 (significantly less on Weeks 1, 4 and 5). At later time intervals, the rate was variable but not significantly different from normal, tending to stabilise after Week 12. The left incisor of four PS rats showed a transitory cessation of eruption: two rats for 1 week (one rat at Week 4 and another at the Week 6), one rat for 2 weeks (during Weeks 2 and 3) and one rat for 7 weeks (from the Weeks 3 to 9). The eruption in the PR group, with the exception of the second week, never reached the normal rate and started to decrease in Week 6, becoming close to zero after Week 8. The rate of exfoliation of the left incisors in the PR group was slower than that of the SR group, with four rats retaining their teeth until the end of the experiment. In the first 5 weeks of the experiment, there were no significant differences in the mean eruption rates between PR and PS incisors. However, the former did not show a temporary cessation of eruption throughout the experiment.
Figure 1 Sections of rat incisors submitted to damage of the enamel-related periodontium tissues plus root resection. (A) Two weeks after surgery, at the level of
the second molar, showing the recovery of the alveolar bone (AB) and the dental follicle (DF) while the enamel organ is absent. (B) Higher magnification of the region marked () in (A). (C) At 4 weeks after surgery, in a region close to the alveolar crest, showing the cementlike structure formed between the dental follicle and the enamel surface (arrow). P, pulp; D, dentin; E, young enamel; ES, enamel space; PL, periodontal ligament. (A and B) HE, (C) PAS—haematoxylin. Scale bars ¼ 200 mm in (A) and 50 mm in (B and C).
Eruptive process of lower incisor
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SS X PR
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Weeks Figure 2 Pairwise comparisons of the eruption rates of the lower left incisor between experimental groups: PR (-!!-) damage to the ERP plus root resection; PS (-D-D-) damage to the ERP plus sham operation at the odontogenic base; SR (-*-*-) sham operation at the mandible lower border and root resection; and SS (-*-*-) sham operation at both sites. The number of animals used in the first week and the number of animals remaining at later time intervals (see text for details) are indicated in brackets. Vertical bars represent the SEM; indicates significant differences (P < 0:05) at each time interval.
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Discussion Most root-resection experiments have been performed in unimpeded rat incisors. The eruption rate of these teeth shows three successive phases: an initial slowdown followed by a period of normal eruption rate and a final slowdown before the tooth is exfoliated.6,8 Root-resected impeded (normal) incisors show a similar pattern of eruption7 and this was confirmed by our results with SR incisors. The incisors of the PR group did not have a period where the tooth erupted at normal rate. The initial slowdown phase lasted for about 5 weeks, followed by a sharp decline Week 6. Most of the PR incisors lasted longer than SR incisors before the teeth were exfoliated. The incisors of the PR group did not show temporary cessation of eruption throughout the experiment, in contrast to some teeth in the PS group, according to our present and previous results.27 The main difference between the two groups was the absence of the odontogenic region and an initial part of the region of enamel secretion. This absence may indicate that these structures have a modulatory effect on eruption. Granule proteins, isolated from stratum intermedium and dental follicle of rat molars, delay the eruption of incisors and molars, as well as, being responsible for eyelid opening.19 The level of these proteins would be lower in root-resected teeth, and this could explain why the PR incisors did not show any cessation of eruption. The connective tissue of the enamel-related periodontium is equivalent to the coronal part of the dental follicle of teeth with limited growth and eruption. The histology of this tissue in the rat incisor5,25 is comparable to the coronal dental follicle described in dog premolars.24 Both tissues have an inner layer related to the enamel organ, which contains fibroblasts, densely packed collagen and a capillary network, considered to be the true dental follicle.24 The outer layer, related to the alveolar bone, is formed by loose connective tissue, is rich in ground substance and is well vascularised. In incisors, this vascularisation involves large blood vessels. Where denuded enamel is in contact with the dental follicle, it induces the formation of a cement-like tissue as shown by our results and also in rat molars.32 Removal of the dental follicle (with the enamel organ) prevents the eruption of dog premolars.20 Larson et al.18 were unable to confirm whether the dental follicle without the associated enamel organ could support the eruption of dog teeth. When partial damage to the enamel-related periodontal tissues was associated with resection of the incisor odontogenic region (PR group), the dental follicle
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was completely regenerated in less than 2 weeks whereas the enamel organ was not. The morphology of the ERP seems to recover in less than 2 weeks; however, the eruption rate takes at least 3 more weeks to reach normal values in the PS group. Thus, it can be hypothesised that the functional recovery of ERP, as the production of the putative molecules involved in the eruptive process, takes more time. In rat molars, the source of most of these molecules is the coronal dental follicle and some are produced in the stellate reticulum of the enamel organ.36 Some of these molecules peak at the time when the tooth crown is being completed, earlier than the start of the eruptive process.34,36 The lack of a substantial part of the enamel organ at a corresponding stage of development of the rat incisor (at the level of the third and second lower molars) could explain the decrease in the eruption rate of the PS and PR groups compared with SS and SR groups during the first 5 weeks of experiment. It also took some weeks for the ERP to acquire cementogenic ability. The odontogenic tissues at earlier stages of tooth development have never been implicated in the eruptive process. This may be the reason why root-resected incisors (SR group) showed normal eruption rates in the first 4 weeks after surgery. The coronal aspect of the dental follicle in molars and the ERP connective tissue in rat incisors are both related to bone resorption. Preceding the eruptive movement in dog premolars or rat molars, there is an influx of mononuclear cells into the dental follicle followed by an increase in osteoclasts22,35 that promote the resorption of bone close to the dental crown, thereby creating the eruption pathway.20 The inner surface of the labial wall of the incisor socket is carpeted with osteoclasts17 and resorption activity starts somewhere in the region of the lower molars and increases towards the alveolar crest.12 Bone resorption controlled by the coronal part of the dental follicle is a key factor in the intra-osseous phase of the eruptive process of teeth with limited growth.34,36 Bone resorption of the labial aspect of the socket may have an equivalent role in rodent incisors, which are considered to be in continuous supra-osseous eruption. Drugs that inhibit bone resorption by osteoclasts delay eruption, such as bafilomycin A in dog premolars33 and bisphosphonate in rat incisors and molars.15 Osteopetrotic rats, which show a total absence of erupted teeth, treated with CSF-1 from birth, have the eruption of the incisors and molars restored.16 The bone resorption and probably the bone remodeling promoted by the dental follicle are apparently necessary for tooth growth and eruption. When bone resorption is impaired, eruption is retarded
Eruptive process of lower incisor
or interrupted but growth may change direction or become anomalous as in impacted teeth, in molar teeth after removal of the coronal part of the dental follicle,21 or in the proximal segment of root-transected rodent incisors.26 In teeth of limited growth and eruption, bone resorption to form the eruptive pathway is a result of the interaction between cells of the enamel organ and the dental follicle. A cascade of growth factors (EGF, TGF-b) and cytokines (IL-1a) enhances the production of CSF-1 by dental follicle cells. CSF1 is responsible for the influx of mononuclear blood cells (monocytes) into the dental follicle, which in turn are the probable precursors of osteoclasts seen at the bone surface of the crypt.36 Monocyte chemotatic protein-1 (MCP-1)) which is expressed and secreted by dental follicle cells, has the same effects as CSF-1.31 In PR rats, the remaining part of the incisors, enamel organ may have been enough to provide the necessary signals, but in an insufficient amount to maintain a normal eruption rate. There are indications that these signals in rats may be different for molars and incisors. Neonatal injections of EGF accelerate the eruption of incisors and have no significant effect on the eruption of the first molar, while CSF-1 accelerates molar eruption more than incisor eruption.11 The effects of dexamethasone, which stimulates bone resorption, parallel those of EGF.37 Nevertheless, there are some reported similarities on the eruptive behaviour of both types of teeth, as already mentioned in relation to the effects of granule proteins,19 of drugs that inhibit bone resorption15 and of CSF-1 in osteopetrotic rats.16 The controversies on the mechanism of tooth eruption are still no settled.23,29,10 Most authors consider the periodontal ligament, which is derived from the dental follicle, as the tissue responsible for the generation of the eruptive force(s) in continuously erupting teeth.1,2,28,29 The available evidence does not favour the hypothesis that the forces are tractional, either through collagen contraction or periodontal fibroblast traction.29 There is stronger evidence that one of the factors responsible for eruption is related to the vascular/periodontal tissue, hydrostatic pressure.28,29 In this regard, surgical disruption of the vascular entwork of the enamel-related periodontal tissues could be responsible for the decreased eruption rate of PS and PR incisors. However, in root-resected and root-transected incisors, the eruption ceases for several weeks before exfoliation when the base of the erupting tooth surpasses the labial level of the alveolar crest. In this situation, all the ERP is lost and some functional periodontal ligament is still present.26 Rootless teeth of limited growth,
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and therefore without a periodontal ligament, do erupt.9,14 Thus, whatever the mechanism, some evidence indicates that the dental follicle together with the enamel organ are probably the tissues in control of the eruptive process.
Acknowledgements The authors thank Maria A. Varela, Eliene A.N. Romani and Waldek R. Moreira for technical assistance, and Dr. Glaucia M.B. Ambrosano for statistical advice. This work was partially supported by FAPESP (Proc. 96/07166-4).
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16. Iizuka T, Cielinski M, Aukerman SL, Marks Jr SC. The effects of colony-stimulating factor-1 on tooth eruption in the toothless (osteopetrotic) rat in relation to the critical periods for bone resorption during tooth eruption. Arch Oral Biol 1992;37:629—36. 17. Irie K, Ozawa H. Relationships between tooth eruption, occlusion and alveolar bone resorption: cytological and cytochemical studies of bone resorption on rat incisor alveolar facing the enamel. Arch Histol Cytol 1990;53: 497—509. 18. Larson EK, Cahill DR, Gorski JP, Marks Jr SC. The effect of removing the true dental follicle on premolar eruption in the dog. Arch Oral Biol 1994;39:271—5. 19. Lin F, Fan W, Wise GE. Granule proteins of the dental follicle and stellate reticulum inhibit tooth eruption and eyelid opening in postnatal rats. Arch Oral Biol 1992;37: 841—7. 20. Marks Jr SC, Cahill DR. Experimental study in the dog of the non-active role of the tooth in the eruptive process. Arch Oral Biol 1984;29:311—22. 21. Marks Jr SC, Cahill DR. Regional control by the dental follicle of alterations in the alveolar bone metabolism during tooth eruption. J Oral Pathol 1987;16:164—9. 22. Marks Jr SC, Grolman M-L. Tartrate-resistant acid phosphatase in mononuclear and multinuclear cells during the bone resorption of tooth eruption. J Histochem Cytochem 1987; 35:1227—30. 23. Marks Jr SC, Schroeder HE. Tooth eruption: theories and facts. Anat Rec 1996;245:374—93. 24. Marks Jr SC, Cahill DR, Wise GE. The cytology of the dental follicle and adjacent alveolar bone during tooth eruption in the dog. Am J Anat 1983;168:277—89. 25. Matena V. The periodontium of the enamel aspect of the rat incisor. J Periodont 1972;43:311—5. 26. Merzel J, Novaes PD, Furlan S. A histological study of rootresected and root-transected rat incisors when eruption ceases, shortly before they are exfoliated from the socket. Arch Oral Biol 2000;45:315—22.
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27. Merzel J, Novaes PD, Furlan S. The effects of local trauma to the enamel-related periodontal tissues in the eruption of the rat incisor. Arch Oral Biol 2000;45:323—33. 28. Moxham BJ. What the structure and the biochemistry of the periodontal ligament tell us about the mechanism of tooth eruption. In: Davidovitch Z, editor. The biological mechanisms of tooth eruption, root-resorption and replacement of implants. Boston, MA: Harvard Society for the Advancement of Orthodontics; 1994. p. 437—50. 29. Moxham BJ, Berkovitz BKB. The periodontal ligament and physiological tooth movements. In: Berkovitz BKB, Moxham BJ, Newman HN, editors. The periodontal ligament in health and disease. London: Mosby-Wolfe; 1995. p. 183—214. 30. Moxham BJ, Shore RC, Berkovitz BKB. A quantitative study of the ultrastructure of fibroblasts within the enamelrelated connective tissue of the rat incisor. J Biol Buccale 1991;19:135—40. 31. Que BG, Wise GE. Colony-stimulating factor-1 and monocyte chemotactic protein-1 chemotaxis for monocytes in the rat dental follicle. Arch Oral Biol 1997;42:860—5. 32. Spahr A, Hammarstrom L. Response of dental follicular cells to the exposure of denuded enamel matrix in rat molars. Eur J Oral Sci 1999;107:360—7. 33. Sundquist KT, Marks Jr SC. Bafilomycin A1 inhibits bone resorption and tooth eruption in vivo. J Bone Min Res 1994;9:1575—82. 34. Wise GE. The biology of tooth eruption. J Dent Res 1998; 77:1576—9. 35. Wise GE, Fan W. Changes in the tartrate-resistant acid phosphatase cell population in dental follicles and bony crypts of rat molars during tooth eruption. J Dent Res 1989;68:150—6. 36. Wise GE, Frazier-Bowers S, D’Souza RN. Cellular, molecular, and genetic determinants of tooth eruption. Critic Ver Oral Biol Med 2002;13:323—34. 37. Wise GE, Grier IV RL, Lumpkin SJ, Zhang O. Effects of dexamethasone on tooth eruption in rats: differences in incisor and molar eruption. Clin Anat 2001;14:204—9.