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Focal hyalinization during experimental tooth movement in beagle dogs
ORIGINAL ARTICLE
Focal hyalinization during experimental tooth movement in beagle dogs Martina von Bo¨hl, DDS,a Jaap C. Maltha, PhD,b Johannes W. Von Den Hoff, PhD,c and Anne Marie KuijpersJagtman, DDS, PhD, FDSRCSEngd Nijmegen, The Netherlands The aim was to study morphological differences between the periodontal structures of beagle dogs showing different rates of tooth movement under identical experimental conditions. An orthodontic appliance was placed on the mandibular second premolar and the first molar to exert a continuous and constant reciprocal force of 25 cN. Tooth movement was recorded weekly. The dogs were killed after 1, 4, 20, 40, and 80 days for histological evaluation. Haematoxylin and eosin staining was used for tissue survey, alkaline phosphatase staining was used as a marker for active osteoblasts, and tartrate resistant acid phosphatase staining was used for osteoclasts. After 24 hours, osteoclastic and osteoblastic activity had already increased at the pressure and tension sides, respectively, and, in some samples, hyalinization was found. In case of fast-moving teeth, areas of direct bone resorption at the pressure side and deposition of trabecular bone at the tension side were found throughout the experimental period. In the periodontal ligaments of teeth showing little movement, small patches of hyalinization were found at the pressure side, mostly located buccally or lingually of the mesiodistal plane. These phenomena were found in both molars and premolars and at all time points. It is concluded that small focal hyalinizations might be a factor that could explain individual differences in the rate of tooth movement. (Am J Orthod Dentofacial Orthop 2004;125:615-23)
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rthodontic tooth movement has been defined as the result of a biological response to an interference in the physiological equilibrium in the dentofacial complex by an externally applied force.1 Previous studies on experimental tooth movement have shown that orthodontic tooth displacement follows a specific pattern in time. In beagle dogs, it was demonstrated that the time-displacement curves for mandibular second premolars could be divided into 4 phases.2,3 The first phase takes 24 hours to 2 days and represents the initial movement of the tooth in the bony socket; it is followed by the second phase of arrest in tooth movement lasting 20 to 30 days. This standstill period is generally attributed to hyalinization. The third and fourth phases comprise the real tooth movement. After the removal of the necrotic tissue, tooth movement accelerates in the third phase and continues in the fourth. This tooth movement pattern in dogs generally From the Department of Orthodontics and Oral Biology, College of Dental Science, University Medical Centre, Nijmegen, The Netherlands. a Researcher. b Associate professor of Oral Biology. c Assistant professor of Oral Biology. d Head and professor of Orthodontics. Reprint requests to: Prof A. M. Kuijpers-Jagtman, Department of Orthodontics and Oral Biology, University Medical Centre Nijmegen, PO Box 9101, NL 6500 HB Nijmegen, The Netherlands; e-mail, [email protected]. Submitted, November 2002; revised and accepted, August 2003. 0889-5406/$30.00 Copyright © 2004 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2003.08.023
agrees with that described by Proffit as an initial phase, a lag, and a post-lag phase in human clinical tooth movement.1 As for most biological reactions to mechanical stimuli, one would expect some dose-response relationship in this system. Quinn and Yoshikawa4 supported this idea when they hypothesized that a dose-response relationship would exist in the lower force range. At increasing forces, a constant rate of movement would be reached for a broad range of forces, suggesting that, in that range, force magnitude plays only a subordinate role. This idea is supported by data from animal and clinical studies.2,3,5-7 Pilon et al2 showed in their experimental work with dogs that 2 different forces (eg, 50 and 100 cN) applied to the second premolar at the left and right sides of the mandible in the same dog very often led to the same rate of tooth movement. These findings agree with the results of clinical studies8 that showed that, in humans, the mean rate of tooth movement during buccal tipping of premolars was the same with forces of 50 and 200 cN. The data found by both research groups fit into the model of Quinn and Yoshikawa,4 which is currently used as a theoretical framework,1,9 assuming that the forces are beyond the range in which a dose-response relationship is to be expected. According to this model, this means that the optimal force must be 615
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lower than 50 cN. This is true for bodily movement of dog premolars and for tipping movement of human premolars. On the other hand, many data show large interindividual differences in the rate of tooth movement under identical conditions in both animals and humans.2,3,8,10 These interindividual differences led Pilon et al2 to state that some animals could be considered as typical slow movers and others as typical fast movers. The underlying biological differences between these 2 types are possibly related to individual variation in bone density or the metabolism of bone and the periodontal ligament (PDL). Another important factor might be that the levels of cytokines and growth factors such as PGE2, IL-1 and TGF-1, which are involved in the remodelling process during tooth movement, show significant differences between individuals, both in controls and during orthodontic tooth movement.11-15 Also, morphological and biomechanical differences in teeth or the PDL, which result in differential distribution of stress and strain in the PDL, could play a role. The purpose of this study was to relate local morphological changes in the periodontal structures to individual differences in rate of tooth movement induced by standardized orthodontic appliances. MATERIAL AND METHODS
A group of 15 young adult beagles with complete permanent dentition was used. The experiment was approved by the Board for Animal Experiments of the University Medical Centre Nijmegen, The Netherlands. Three months before the start of the experiment, the third and fourth premolars at the left and right sides of each mandible were extracted after hemisection of their 2 roots. The maxillary third and fourth premolars were also extracted to avoid functional interferences with the orthodontic appliance. Before extraction, the dogs were premedicated with 1.5 ml Thalamonal (Fentanyl 0.05 mg/ml and Droperidol 2.5 mg/ml; Janssen Pharmaceutica, Beerse, Belgium) and anaesthetized with 15 mg/kg Nesdonal (Thiopental sodium 50 mg/ml; RhonePoulenc Pharma, Amstelveen, The Netherlands). Radiographic evaluation 3 months after extraction showed complete healing of the wounds and the alveolar bone. Then burr holes were prepared in the mandibular extraction diastemata, and custom-made titanium implants (height 10 mm, outer diameter 3.1 mm, sandblasted), with a locking screw on top, were placed press fit into these holes. Soft tissues were closed with sutures (Vicryl absorbable 3-0; Ethicon, Brussels, Belgium) over the implants. Vinylpolysi-
American Journal of Orthodontics and Dentofacial Orthopedics May 2004
Fig 1. Experimental appliance inducing bodily displacement of premolar and molar in direction of arrows by closed coil spring producing reciprocal force of 25 cN. C, canine; P2, second premolar; M1, first molar.
loxane impressions (Express STD; 3M, St Paul, Minn) were made for the construction of orthodontic appliances. The dogs were fed their usual diet, but the food particles were smaller to avoid damage to the appliances. Three months after implant placement, the implants were uncovered, the locking screws were removed, and a supra-structure was placed on the implants. A holder for a stainless steel sliding-bar (Ø 2.0 mm H6 type 316; Rijnvis, Rotterdam, The Netherlands) was attached with glass ionomer-cement (Ketac-Cem, ESPE, Seefeld, Germany) to this supra-structure. The rigid sliding bar was fixed into its holder by a small locking screw. Custom-made cobalt-chromium alloy crowns (Heraeus Kulzer, Hanau, Germany) were cemented on the mandibular second premolars and first molars with PanaviaEx Dental adhesive (Kuraray, Osaka, Japan). Polyacetal homopolymer tubes Ø 2.0 mm H7 (Vink Kunststof, Didam, The Netherlands), used as bearings, were glued into metal cylinders with bonding (Vitermer; 3M Dental Products). These cylinders in turn were soldered onto the crowns. The sliding bar, which was fixed to the implant supra-structure, ran freely through the low-friction polyacetal homopolymer tubes on the premolar and molar.3 To produce bodily displacement of the second premolar and the first molar, a Sentalloy Closed Coil Spring (GAC International, New York, NY) producing a force of 25 cN was attached to buccal hooks on the crowns on the second premolar and the first molar (Fig 1). These springs exert a constant, continuous, reciprocal force on both teeth over a wide range of activation3; this means that reactivation is not needed. For each session, the dogs were sedated with 3 ml of a generic preparation containing 10 mg/ml oxycodon HCL, 1 mg/ml acepromazine (Vetimex Animal Health, Bladel, The Netherlands) and 0.5 mg/ml
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Fig 2. Schematic drawing of general time-displacement curve. Arrows indicate time points of killing for histological evaluation.
atropine sulphate (Centrafarm, Etten-Leur, The Netherlands). Once a week, the positions of the experimental teeth were measured with a digital calliper as the distance between a reference point on the tooth in question and a reference point on the implant construction. This technique has been shown to be accurate. In previous studies,2 the intraobserver differences appeared to be about 0.01 mm, and the standard deviation of the mean differences between 2 observers was 0.02 mm. At each session, the coil spring and the sliding bar were removed to facilitate cleaning of the oral mucosa and the dentition with a toothbrush and 0.4 mg/ml chlorhexidine digluconate in water (Astra Chemicals, Rijswijk, The Netherlands). Then the sliding bars were cleaned and polished (Abraso-Star K50, Bredent, Senden, Germany) outside the oral cavity, and petroleum jelly was put into the polyacetal homopolymer tubes and on the sliding bar to keep friction to a minimum. Then the sliding bar and the coil springs were replaced and checked for friction and force delivery. Time-displacement curves of each tooth were constructed, based on the weekly intraoral measurements. For histological analysis, groups of 3 dogs were killed after 1, 4, 20, 40, and 80 days. Figure 2 shows an example of a general time displacement curve with the times of killing indicated by arrows. The means and standard deviations of the rate of movement of the experimental teeth over the preceding period (4-7 days) were calculated for all animals when they were killed for histological evaluation. Furthermore, individual data are given for all experimental teeth in dogs that were killed at a certain time (Fig 3).
Fig 3. Graph showing individual rates of displacement of experimental teeth (small symbols) at killing, and means and SDs of these rates for all animals. Fig 7, 8, 9, 10 in graph refer to histological photomicrographs of Figs 7, 8, 9, and 10.
Groups of 3 dogs were killed after general anaesthesia by a lethal dose of Narcovet (sodium pentobarbital 60 mg/ml, Apharmo, Arnhem, The Netherlands) after 1, 4, 20, 40, and 80 days. The mandibles were dissected, and each tooth and its surrounding bone were split into a mesial and a distal part, each containing 1 root for different ways of processing. All mesial roots were stained for paraffin sections, while the distal roots were used for cryosections. Paraffin sections were prepared parallel to the long axis for normal histological evaluation, and cryosections were prepared for enzyme histochemistry. The tissues for paraffin sections were fixed in a 4% buffered formaldehyde solution in 0.1 mol/L PBS for 2 weeks and then decalcified in 20% formic acid and 5% sodium citrate for approximately 4 weeks, dehydrated and embedded in Paraplast (Monoject Scientific, Athy, Ireland). Serial mesiodistal sections of 7 m from the buccal to the lingual side were prepared and stained with haematoxylin and eosin. The material for cryosections and subsequent enzyme histochemical evaluations were rinsed in cold
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Tris-HCl-PVP buffer, containing 0.1 mol/L TRIS and 6% polyvinylpyrolidine at pH 7.4. Then the material was decalcified in cold 10% EDTA in Tris-HCl-PVP at pH 7.4. Decalcification was performed at 4°C, and the endpoint was determined by radiography (Philips Oralix, Eindhoven, The Netherlands). After 10 to 14 weeks of decalcification, the tissue was embedded in Tissue-Tek (Sakura, Zoeterwoude, The Netherlands) and kept at ⫺80°C until sectioning. Serial mesiodistal sections of 7 m were cut at ⫺20°C on a cryostat microtome HM 500 7 (Adamas Instrumenten, Leersum, The Netherlands) parallel to the long axis of the root. Selected sections were stained for alkaline phosphatase as a marker for active osteoblasts or tartrate resistant acid phosphatase (TRAP) for differentiated osteoclasts and osteoclast precursors. For both enzymes, the modified staining techniques according to Van de Wijngaert and Burger16 were used. RESULTS
All appliances were checked weekly, and, when the animals were killed, all appliances were still in place and in good order. Based on the weekly measurements, the time-displacement curves were constructed. The curves for most teeth could be divided into 4 phases of movement as described previously.2,3 In some curves, the transition from phase 1 to phase 2 was difficult to distinguish because of lack of data for the first phases. The rate of tooth movement in the different phases showed big differences as shown by the large standard deviations in Figure 3. Interindividual differences of tooth movement were much bigger than the intraindividual differences between a premolar and a molar in 1 dog. In all animals killed after 24 hours of force application, in the phase of initial tooth movement, cellular and tissue reactions had already started. At the tension side, the fibers were stretched (Fig 4), and, at the pressure side, the fibers of the PDL were compressed (Fig 5). Osteoclastic and osteoblastic activity was already increased at the pressure and tension sides (not shown). After 4 and 20 days of force application, most animals were in the second phase of tooth movement. At the pressure side, areas of hyalinization were found; in some cases, pyknotic nuclei were present (Fig 6). In these regions, distortion of the normal periodontal fiber arrangement was encountered. Deviating periodontal fiber arrangement was also seen in areas without apparent hyalinization. Some sections showed a normal periodontal structure, but, in other sections, the periodontal fibers were oriented parallel to the root or even completely disorganized. Adjacent to hyalinized areas,
Fig 4. Photomicrograph showing stretched fibers of PDL of second premolar at tension side (center region of root) after force of 25 cN was applied for 24 hours; haematoxylin and eosin staining. B, bone; PDL, periodontal ligament; T, tooth.
tartrate-resistant-acid-phosphatase–positive cells were often found, in either the periodontal space or the bone marrow cavities, related to direct or undermining resorption, respectively (Fig 7). At the tension sides, in most cases, only a few osteoblastic cells were present (not shown). Generally after 20, 40, and 80 days of orthodontic force application, when tooth movement had reached its linear phase, many pressure sides showed irregular bone surfaces due to direct bone resorption (not shown). At other pressure sides, however, hyalinized areas were present. In those areas, the structure of the PDL was completely lost, and no cells were present. The dimensions of these hyalinized areas differed considerably. Large areas covering 1-2 mm of the length of the root surface (Fig 8), as well as focal patches measuring less that 50 m (Figs 9 and 10) were found. These areas were generally not located at the
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Fig 5. Photomicrograph showing compressed PDL at pressure side of second premolar (center region of root) after force of 25 cN was applied for 24 hours; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone.
Fig 6. Photomicrograph showing hyalinization with pyknotic nuclei of PDL at pressure side of second premolar after force of 25 cN was applied for 4 days; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone.
pressure side, but more to the buccal or to the lingual (Fig 11). Figures 8-10 and 12, for example, depict, respectively, sections taken 100 m lingually, 150 m lingually, 400 m buccally, and 450 m buccally from the central section. The focal patches of hyalinization were in most cases related to a remaining bone spicula (Figs 9 and 10). In general, at the pressure side, an accumulation of osteoclasts was present near the hyalinization areas, which led in some samples to direct bone resorption. But more often these cells appeared to be involved in root resorption (Fig 10). At the tension sides, bone deposition had taken place as more or less compact or cancellous bone. The bone surface was mainly covered with alkaline-phosphatase–positive osteoblastic cells (Fig 13). With respect to the rate of tooth movement, direct osteoclastic bone resorption was mainly found at the pressure sides of rapidly moving teeth. The tension
sides of these teeth showed usually active bone deposition by osteoblastic cells. Focal sites of hyalinized tissue in the PDL were mostly found at slow moving teeth. For example, Figure 8 shows a section of a premolar’s PDL after 40 days. This dog was an extremely slow mover; its movement rate over the last period was ⫺17.5 m/d, whereas the mean for the group was 17.6 ⫾ 17.4 m/d (Fig 3). Figure 9 shows a premolar’s PDL after 20 days. Its movement rate over the last period before killing was ⫺13.0 m/d, whereas the mean for the group was 8.8 ⫾ 21.6 m/d (Fig 3). In some cases, the PDL of a root of a slow-moving tooth did not show any hyalinization. The PDL at the pressure side in those cases contained almost no osteoclasts, and only a few osteoblastic cells were found at the tension side. The other root of such a tooth, however, which was evaluated separately, always showed focal hyalinization areas.
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Fig 7. Photomicrograph showing osteoclasts (OC) in vicinity of hyalinized area of PDL at pressure side of second premolar to which force of 25 cN was applied for 40 days; TRAP staining. T, tooth; PDL, periodontal ligament; B, bone.
American Journal of Orthodontics and Dentofacial Orthopedics May 2004
Fig 8. Photomicrograph showing large area of hyalinization in PDL at pressure side of second premolar to which force of 25 cN was applied for 40 days. Section was taken 100 m away from central section lingually of mesiodistal plane; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone; H, hyalinization.
DISCUSSION
Several authors1,3,17-19 have argued that the efficiency of tooth movement might be improved by preventing hyalinization, thus eliminating the lag phase, and they advocated using light forces for this purpose. In our study, we used a continuous force of 25 cN, not only on the dogs’ second premolars but also on the first molars. The surface area of a first molar’s root is about 10 times larger than that of a premolar. Therefore, the biological effect of a force of 25 cN on a molar would be equivalent to the biological effect of a force of 2.5 cN on a premolar. Even with this very low force on a molar, a phase of arrest after the initial phase was found in most cases. Histological evaluation of the samples taken after 4 days of force application confirmed the presence of hyalinized areas in the PDL of both premolars and
molars. This indicates that these light forces do not prevent hyalinization of the PDL. In the samples where hyalinization was found at the pressure side, little osteoblastic activity was seen at the tension side. Removal of the hyalinized tissues appeared to be essential for the start of the real tooth movement, but, even after this real tooth movement had started, a large variation in its rate was found. In the literature, these differences are often related to individual variations in bone metabolism, bone density, and the amount of osteogenic or osteoclastic cells.6,20 The results of our previous21 and present studies indicate, however, that the formation of patches of hyalinized tissue during later phases of tooth movement might also play a role. Possibly, during orthodontic tooth movement, high stresses or
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Fig 9. Photomicrograph showing focal hyalinization of PDL at pressure side of second premolar to which force of 25 cN was applied for 20 days. Section was taken 150 m away from central section lingually of mesiodistal plane; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone; H, hyalinization; OC, osteoclast.
strains are generated locally due to irregularities in the outline of the alveolar bone; this causes local hyalinization.10,22 The hyalinized patches described in this study, however, were not present in front of the moving teeth, but more lingually or buccally where compressive stress will be less important than shear stress. Hyalinized patches at these localizations have not been described in the literature before. They probably have been overlooked because of their unexpected localization. An explanation for this contrasting outcome might be the way the sections were cut. In nearly all previous studies, the teeth that were moved were sectioned vertically through the central plane, or only a few sections from the entire tooth were evaluated.2,23,24 However, in the present study, the teeth were serially sectioned in 7-m
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Fig 10. Photomicrograph showing osteoclasts (OC) near small patch of hyalinization in PDL at pressure side of first molar to which force of 25 cN was applied for 80 days. Section was taken 400 m away from central section buccally of mesiodistal plane; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone; H, hyalinization.
Fig 11. Schematic drawing of horizontal section of 2 roots of premolar. Horizontal arrow marks direction of tooth movement; 4 small arrows indicate localization of hyalinization areas of both roots, which were mainly found buccally or lingually of root.
slices from the mesial to the distal side. By using this protocol, a systematic overview of the changes of the PDL during tooth movement was possible.
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Fig 12. Photomicrograph showing hyalinization next to area of intact PDL at pressure side of first molar to which force of 25 cN was applied for 40 days. Section was taken 450 m away from central section buccally of mesiodistal plane; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone; H, hyalinization.
CONCLUSIONS
The consequence of these findings is that the present models that describe the relationships between force magnitude and rate of tooth movement probably are not valid. Hyalinization is most likely one of the many known and yet to be elucidated factors that can affect tooth movement. Focal hyalinizations, which counteract tooth movement, probably do not directly depend on the applied force but on local stresses or strains that might be quite individually and locally determined due to irregularities in periodontal and bone morphology. Such stress concentrations and especially shear stress concentrations at the buccal and lingual aspects of moving teeth complicate the system further. This means that an appropriate model cannot be de-
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Fig 13. Photomicrograph showing osteoblasts (OB) covering bone surface at tension side of second premolar to which force of 25 cN was applied for 80 days; alkaline phosphatase staining. T, tooth; PDL, periodontal ligament; B, bone.
signed with present knowledge. Far more biomechanical information on local effects of force application and insight into the individual variation in bone density and the metabolism of bone and the PDL and even the level of cytokines and growth factors is needed to construct such a model. REFERENCES 1. Proffit WR. Biomechanics and mechanics. In: Proffit WR, editor. Contemporary orthodontics. St Louis: Mosby; 2000. 296-361. 2. Pilon JJGM, Kuijpers-Jagtman AM, Maltha JC. Magnitude of orthodontic forces and rate of bodily tooth movement: an experimental study in beagle dogs. Am J Orthod Dentofacial Orthop 1996;110:16-23. 3. Van Leeuwen EJ, Maltha JC, Kuijpers-Jagtman AM. Tooth movement with light continuous and discontinuous forces in beagle dogs. Eur J Oral Sci 1999;107:468-74. 4. Quinn RS, Yoshikawa DK. A reassessment of force magnitude in orthodontics. Am J Orthod 1985;88:252-60.
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5. Sandstedt C. Einige Beitra¨ ge zur Theorie der Zahnregulierung. Nord Tandlæk Tidsskr 1904;5:236-56. 6. Reitan K. The initial tissue reaction incident to orthodontic tooth movement as related to the influence of function: a histological study on animal and human material. Acta Odontol Scand 1951;9(Suppl 6):9-240. 7. Owman-Moll P, Kurol J, Lundgren D. Effects of a doubled orthodontic force magnitude on tooth movement and root resorptions: an inter-individual study in adolescents. Eur J Orthod 1996;18:141-50. 8. Owman-Moll P, Kurol J, Lundgren D. The effects of a four-fold increased orthodontic force magnitude on tooth movement and root resorptions: an inter-individual study in adolescents. Eur J Orthod 1996;18:287-94. 9. Lindauer SJ, Britto AD. Biological response to biomechanical signals: orthodontic mechanics to control tooth movement. Semin Orthod 2000;6:145-54. 10. Melsen B. Biological reaction of alveolar bone to orthodontic tooth movement. Angle Orthod 1999;69:151-8. 11. Davidovitch Z, Nicolay OF, Ngan P, Shanfeld J. Neurotransmitters, cytokines and the control of alveolar bone remodeling in orthodontics. Dent Clin North Am 1988;32:411-35. 12. Shanfeld J, Jones JL, Laster L, Davidovitch Z. Biochemical aspects of orthodontic tooth movement; cyclic-nucleotide and prostaglandin concentrations in tissues surrounding orthodontically treated teeth. Am J Orthod Dentofacial Orthop 1986;90: 139-41. 13. Bartold PM, Walsh LJ, Narayanan AS. Molecular and cell biology of the gingiva. Perio 2000 2000;24:28-55. 14. Wang LL, Zhu H, Liang T. Changes of transforming growth factor beta 1 in rat periodontal tissue during orthodontic tooth movement. Chin J Dent Res 2000;3:19-22. 15. Ren Y, Maltha JC, Von den Hoff JW, Kuijpers-Jagtman AM,
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Ding Z. Cytokine levels in crevicular fluid are less responsive to orthodontic forces in adults than in juveniles. J Clin Periodontol 2002;29:400-5. Van de Wijngaert FP, Burger EH. Demonstration of tartrateresistant acid phosphatase in undecalcified, glycolmethacrylateembedded mouse bone: a possible marker for (pre)osteoclast identification. J Histochem Cytochem 1986;34:1317-23. Van Driel WD, Van Leeuwen EJ, Blankevoort L, Von den Hoff JW, Maltha JC. Time dependent displacement of the tooth in response to orthodontic forces. J Biomech 1998;31(suppl 1):35. Van Driel WD, Van Leeuwen EJ, Von den Hoff JW, Maltha JC, Kuijpers-Jagtman AM. Time-dependent mechanical behaviour of the periodontal ligament. Proc Inst Mech Eng 2000;241:497-504. Jarabak JR, Fizzell JA. Technique and treatment with light-wire appliances, light differential forces in clinical orthodontics. St Louis: Mosby; 1963. Sodek J. A new approach to assessing collagen turnover by using a micro-assay: a highly efficient and rapid turnover of collagen in rat periodontal tissues. J Biomech 1976;160:243-6. Von Bo¨ hl M, Kuijpers-Jagtman AM, Maltha JC, Von den Hoff JW. Changes in the periodontal ligament after experimental tooth movement using high and low continuous forces in beagle dogs. Angle Orthod 2004; in press. Katona TR, Paydar NH, Akay HU, Roberts WE. Stress analysis of bone modeling response to rat molar orthodontics. J Biomech 1995;28:27-38. Rygh P. Ultrastructural changes in pressure zones of human periodontium to orthodontic tooth movement. Acta Odontol Scand 1973;31:109-22. Owman-Moll P. Orthodontic tooth movement and root resorption with special reference to force magnitude and duration. Swed Dent J 1995; Suppl 105.
Focal hyalinization during experimental tooth movement in beagle dogs Martina von Bo¨hl, DDS,a Jaap C. Maltha, PhD,b Johannes W. Von Den Hoff, PhD,c and Anne Marie KuijpersJagtman, DDS, PhD, FDSRCSEngd Nijmegen, The Netherlands The aim was to study morphological differences between the periodontal structures of beagle dogs showing different rates of tooth movement under identical experimental conditions. An orthodontic appliance was placed on the mandibular second premolar and the first molar to exert a continuous and constant reciprocal force of 25 cN. Tooth movement was recorded weekly. The dogs were killed after 1, 4, 20, 40, and 80 days for histological evaluation. Haematoxylin and eosin staining was used for tissue survey, alkaline phosphatase staining was used as a marker for active osteoblasts, and tartrate resistant acid phosphatase staining was used for osteoclasts. After 24 hours, osteoclastic and osteoblastic activity had already increased at the pressure and tension sides, respectively, and, in some samples, hyalinization was found. In case of fast-moving teeth, areas of direct bone resorption at the pressure side and deposition of trabecular bone at the tension side were found throughout the experimental period. In the periodontal ligaments of teeth showing little movement, small patches of hyalinization were found at the pressure side, mostly located buccally or lingually of the mesiodistal plane. These phenomena were found in both molars and premolars and at all time points. It is concluded that small focal hyalinizations might be a factor that could explain individual differences in the rate of tooth movement. (Am J Orthod Dentofacial Orthop 2004;125:615-23)
O
rthodontic tooth movement has been defined as the result of a biological response to an interference in the physiological equilibrium in the dentofacial complex by an externally applied force.1 Previous studies on experimental tooth movement have shown that orthodontic tooth displacement follows a specific pattern in time. In beagle dogs, it was demonstrated that the time-displacement curves for mandibular second premolars could be divided into 4 phases.2,3 The first phase takes 24 hours to 2 days and represents the initial movement of the tooth in the bony socket; it is followed by the second phase of arrest in tooth movement lasting 20 to 30 days. This standstill period is generally attributed to hyalinization. The third and fourth phases comprise the real tooth movement. After the removal of the necrotic tissue, tooth movement accelerates in the third phase and continues in the fourth. This tooth movement pattern in dogs generally From the Department of Orthodontics and Oral Biology, College of Dental Science, University Medical Centre, Nijmegen, The Netherlands. a Researcher. b Associate professor of Oral Biology. c Assistant professor of Oral Biology. d Head and professor of Orthodontics. Reprint requests to: Prof A. M. Kuijpers-Jagtman, Department of Orthodontics and Oral Biology, University Medical Centre Nijmegen, PO Box 9101, NL 6500 HB Nijmegen, The Netherlands; e-mail, [email protected]. Submitted, November 2002; revised and accepted, August 2003. 0889-5406/$30.00 Copyright © 2004 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2003.08.023
agrees with that described by Proffit as an initial phase, a lag, and a post-lag phase in human clinical tooth movement.1 As for most biological reactions to mechanical stimuli, one would expect some dose-response relationship in this system. Quinn and Yoshikawa4 supported this idea when they hypothesized that a dose-response relationship would exist in the lower force range. At increasing forces, a constant rate of movement would be reached for a broad range of forces, suggesting that, in that range, force magnitude plays only a subordinate role. This idea is supported by data from animal and clinical studies.2,3,5-7 Pilon et al2 showed in their experimental work with dogs that 2 different forces (eg, 50 and 100 cN) applied to the second premolar at the left and right sides of the mandible in the same dog very often led to the same rate of tooth movement. These findings agree with the results of clinical studies8 that showed that, in humans, the mean rate of tooth movement during buccal tipping of premolars was the same with forces of 50 and 200 cN. The data found by both research groups fit into the model of Quinn and Yoshikawa,4 which is currently used as a theoretical framework,1,9 assuming that the forces are beyond the range in which a dose-response relationship is to be expected. According to this model, this means that the optimal force must be 615
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lower than 50 cN. This is true for bodily movement of dog premolars and for tipping movement of human premolars. On the other hand, many data show large interindividual differences in the rate of tooth movement under identical conditions in both animals and humans.2,3,8,10 These interindividual differences led Pilon et al2 to state that some animals could be considered as typical slow movers and others as typical fast movers. The underlying biological differences between these 2 types are possibly related to individual variation in bone density or the metabolism of bone and the periodontal ligament (PDL). Another important factor might be that the levels of cytokines and growth factors such as PGE2, IL-1 and TGF-1, which are involved in the remodelling process during tooth movement, show significant differences between individuals, both in controls and during orthodontic tooth movement.11-15 Also, morphological and biomechanical differences in teeth or the PDL, which result in differential distribution of stress and strain in the PDL, could play a role. The purpose of this study was to relate local morphological changes in the periodontal structures to individual differences in rate of tooth movement induced by standardized orthodontic appliances. MATERIAL AND METHODS
A group of 15 young adult beagles with complete permanent dentition was used. The experiment was approved by the Board for Animal Experiments of the University Medical Centre Nijmegen, The Netherlands. Three months before the start of the experiment, the third and fourth premolars at the left and right sides of each mandible were extracted after hemisection of their 2 roots. The maxillary third and fourth premolars were also extracted to avoid functional interferences with the orthodontic appliance. Before extraction, the dogs were premedicated with 1.5 ml Thalamonal (Fentanyl 0.05 mg/ml and Droperidol 2.5 mg/ml; Janssen Pharmaceutica, Beerse, Belgium) and anaesthetized with 15 mg/kg Nesdonal (Thiopental sodium 50 mg/ml; RhonePoulenc Pharma, Amstelveen, The Netherlands). Radiographic evaluation 3 months after extraction showed complete healing of the wounds and the alveolar bone. Then burr holes were prepared in the mandibular extraction diastemata, and custom-made titanium implants (height 10 mm, outer diameter 3.1 mm, sandblasted), with a locking screw on top, were placed press fit into these holes. Soft tissues were closed with sutures (Vicryl absorbable 3-0; Ethicon, Brussels, Belgium) over the implants. Vinylpolysi-
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Fig 1. Experimental appliance inducing bodily displacement of premolar and molar in direction of arrows by closed coil spring producing reciprocal force of 25 cN. C, canine; P2, second premolar; M1, first molar.
loxane impressions (Express STD; 3M, St Paul, Minn) were made for the construction of orthodontic appliances. The dogs were fed their usual diet, but the food particles were smaller to avoid damage to the appliances. Three months after implant placement, the implants were uncovered, the locking screws were removed, and a supra-structure was placed on the implants. A holder for a stainless steel sliding-bar (Ø 2.0 mm H6 type 316; Rijnvis, Rotterdam, The Netherlands) was attached with glass ionomer-cement (Ketac-Cem, ESPE, Seefeld, Germany) to this supra-structure. The rigid sliding bar was fixed into its holder by a small locking screw. Custom-made cobalt-chromium alloy crowns (Heraeus Kulzer, Hanau, Germany) were cemented on the mandibular second premolars and first molars with PanaviaEx Dental adhesive (Kuraray, Osaka, Japan). Polyacetal homopolymer tubes Ø 2.0 mm H7 (Vink Kunststof, Didam, The Netherlands), used as bearings, were glued into metal cylinders with bonding (Vitermer; 3M Dental Products). These cylinders in turn were soldered onto the crowns. The sliding bar, which was fixed to the implant supra-structure, ran freely through the low-friction polyacetal homopolymer tubes on the premolar and molar.3 To produce bodily displacement of the second premolar and the first molar, a Sentalloy Closed Coil Spring (GAC International, New York, NY) producing a force of 25 cN was attached to buccal hooks on the crowns on the second premolar and the first molar (Fig 1). These springs exert a constant, continuous, reciprocal force on both teeth over a wide range of activation3; this means that reactivation is not needed. For each session, the dogs were sedated with 3 ml of a generic preparation containing 10 mg/ml oxycodon HCL, 1 mg/ml acepromazine (Vetimex Animal Health, Bladel, The Netherlands) and 0.5 mg/ml
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Fig 2. Schematic drawing of general time-displacement curve. Arrows indicate time points of killing for histological evaluation.
atropine sulphate (Centrafarm, Etten-Leur, The Netherlands). Once a week, the positions of the experimental teeth were measured with a digital calliper as the distance between a reference point on the tooth in question and a reference point on the implant construction. This technique has been shown to be accurate. In previous studies,2 the intraobserver differences appeared to be about 0.01 mm, and the standard deviation of the mean differences between 2 observers was 0.02 mm. At each session, the coil spring and the sliding bar were removed to facilitate cleaning of the oral mucosa and the dentition with a toothbrush and 0.4 mg/ml chlorhexidine digluconate in water (Astra Chemicals, Rijswijk, The Netherlands). Then the sliding bars were cleaned and polished (Abraso-Star K50, Bredent, Senden, Germany) outside the oral cavity, and petroleum jelly was put into the polyacetal homopolymer tubes and on the sliding bar to keep friction to a minimum. Then the sliding bar and the coil springs were replaced and checked for friction and force delivery. Time-displacement curves of each tooth were constructed, based on the weekly intraoral measurements. For histological analysis, groups of 3 dogs were killed after 1, 4, 20, 40, and 80 days. Figure 2 shows an example of a general time displacement curve with the times of killing indicated by arrows. The means and standard deviations of the rate of movement of the experimental teeth over the preceding period (4-7 days) were calculated for all animals when they were killed for histological evaluation. Furthermore, individual data are given for all experimental teeth in dogs that were killed at a certain time (Fig 3).
Fig 3. Graph showing individual rates of displacement of experimental teeth (small symbols) at killing, and means and SDs of these rates for all animals. Fig 7, 8, 9, 10 in graph refer to histological photomicrographs of Figs 7, 8, 9, and 10.
Groups of 3 dogs were killed after general anaesthesia by a lethal dose of Narcovet (sodium pentobarbital 60 mg/ml, Apharmo, Arnhem, The Netherlands) after 1, 4, 20, 40, and 80 days. The mandibles were dissected, and each tooth and its surrounding bone were split into a mesial and a distal part, each containing 1 root for different ways of processing. All mesial roots were stained for paraffin sections, while the distal roots were used for cryosections. Paraffin sections were prepared parallel to the long axis for normal histological evaluation, and cryosections were prepared for enzyme histochemistry. The tissues for paraffin sections were fixed in a 4% buffered formaldehyde solution in 0.1 mol/L PBS for 2 weeks and then decalcified in 20% formic acid and 5% sodium citrate for approximately 4 weeks, dehydrated and embedded in Paraplast (Monoject Scientific, Athy, Ireland). Serial mesiodistal sections of 7 m from the buccal to the lingual side were prepared and stained with haematoxylin and eosin. The material for cryosections and subsequent enzyme histochemical evaluations were rinsed in cold
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Tris-HCl-PVP buffer, containing 0.1 mol/L TRIS and 6% polyvinylpyrolidine at pH 7.4. Then the material was decalcified in cold 10% EDTA in Tris-HCl-PVP at pH 7.4. Decalcification was performed at 4°C, and the endpoint was determined by radiography (Philips Oralix, Eindhoven, The Netherlands). After 10 to 14 weeks of decalcification, the tissue was embedded in Tissue-Tek (Sakura, Zoeterwoude, The Netherlands) and kept at ⫺80°C until sectioning. Serial mesiodistal sections of 7 m were cut at ⫺20°C on a cryostat microtome HM 500 7 (Adamas Instrumenten, Leersum, The Netherlands) parallel to the long axis of the root. Selected sections were stained for alkaline phosphatase as a marker for active osteoblasts or tartrate resistant acid phosphatase (TRAP) for differentiated osteoclasts and osteoclast precursors. For both enzymes, the modified staining techniques according to Van de Wijngaert and Burger16 were used. RESULTS
All appliances were checked weekly, and, when the animals were killed, all appliances were still in place and in good order. Based on the weekly measurements, the time-displacement curves were constructed. The curves for most teeth could be divided into 4 phases of movement as described previously.2,3 In some curves, the transition from phase 1 to phase 2 was difficult to distinguish because of lack of data for the first phases. The rate of tooth movement in the different phases showed big differences as shown by the large standard deviations in Figure 3. Interindividual differences of tooth movement were much bigger than the intraindividual differences between a premolar and a molar in 1 dog. In all animals killed after 24 hours of force application, in the phase of initial tooth movement, cellular and tissue reactions had already started. At the tension side, the fibers were stretched (Fig 4), and, at the pressure side, the fibers of the PDL were compressed (Fig 5). Osteoclastic and osteoblastic activity was already increased at the pressure and tension sides (not shown). After 4 and 20 days of force application, most animals were in the second phase of tooth movement. At the pressure side, areas of hyalinization were found; in some cases, pyknotic nuclei were present (Fig 6). In these regions, distortion of the normal periodontal fiber arrangement was encountered. Deviating periodontal fiber arrangement was also seen in areas without apparent hyalinization. Some sections showed a normal periodontal structure, but, in other sections, the periodontal fibers were oriented parallel to the root or even completely disorganized. Adjacent to hyalinized areas,
Fig 4. Photomicrograph showing stretched fibers of PDL of second premolar at tension side (center region of root) after force of 25 cN was applied for 24 hours; haematoxylin and eosin staining. B, bone; PDL, periodontal ligament; T, tooth.
tartrate-resistant-acid-phosphatase–positive cells were often found, in either the periodontal space or the bone marrow cavities, related to direct or undermining resorption, respectively (Fig 7). At the tension sides, in most cases, only a few osteoblastic cells were present (not shown). Generally after 20, 40, and 80 days of orthodontic force application, when tooth movement had reached its linear phase, many pressure sides showed irregular bone surfaces due to direct bone resorption (not shown). At other pressure sides, however, hyalinized areas were present. In those areas, the structure of the PDL was completely lost, and no cells were present. The dimensions of these hyalinized areas differed considerably. Large areas covering 1-2 mm of the length of the root surface (Fig 8), as well as focal patches measuring less that 50 m (Figs 9 and 10) were found. These areas were generally not located at the
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Fig 5. Photomicrograph showing compressed PDL at pressure side of second premolar (center region of root) after force of 25 cN was applied for 24 hours; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone.
Fig 6. Photomicrograph showing hyalinization with pyknotic nuclei of PDL at pressure side of second premolar after force of 25 cN was applied for 4 days; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone.
pressure side, but more to the buccal or to the lingual (Fig 11). Figures 8-10 and 12, for example, depict, respectively, sections taken 100 m lingually, 150 m lingually, 400 m buccally, and 450 m buccally from the central section. The focal patches of hyalinization were in most cases related to a remaining bone spicula (Figs 9 and 10). In general, at the pressure side, an accumulation of osteoclasts was present near the hyalinization areas, which led in some samples to direct bone resorption. But more often these cells appeared to be involved in root resorption (Fig 10). At the tension sides, bone deposition had taken place as more or less compact or cancellous bone. The bone surface was mainly covered with alkaline-phosphatase–positive osteoblastic cells (Fig 13). With respect to the rate of tooth movement, direct osteoclastic bone resorption was mainly found at the pressure sides of rapidly moving teeth. The tension
sides of these teeth showed usually active bone deposition by osteoblastic cells. Focal sites of hyalinized tissue in the PDL were mostly found at slow moving teeth. For example, Figure 8 shows a section of a premolar’s PDL after 40 days. This dog was an extremely slow mover; its movement rate over the last period was ⫺17.5 m/d, whereas the mean for the group was 17.6 ⫾ 17.4 m/d (Fig 3). Figure 9 shows a premolar’s PDL after 20 days. Its movement rate over the last period before killing was ⫺13.0 m/d, whereas the mean for the group was 8.8 ⫾ 21.6 m/d (Fig 3). In some cases, the PDL of a root of a slow-moving tooth did not show any hyalinization. The PDL at the pressure side in those cases contained almost no osteoclasts, and only a few osteoblastic cells were found at the tension side. The other root of such a tooth, however, which was evaluated separately, always showed focal hyalinization areas.
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Fig 7. Photomicrograph showing osteoclasts (OC) in vicinity of hyalinized area of PDL at pressure side of second premolar to which force of 25 cN was applied for 40 days; TRAP staining. T, tooth; PDL, periodontal ligament; B, bone.
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Fig 8. Photomicrograph showing large area of hyalinization in PDL at pressure side of second premolar to which force of 25 cN was applied for 40 days. Section was taken 100 m away from central section lingually of mesiodistal plane; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone; H, hyalinization.
DISCUSSION
Several authors1,3,17-19 have argued that the efficiency of tooth movement might be improved by preventing hyalinization, thus eliminating the lag phase, and they advocated using light forces for this purpose. In our study, we used a continuous force of 25 cN, not only on the dogs’ second premolars but also on the first molars. The surface area of a first molar’s root is about 10 times larger than that of a premolar. Therefore, the biological effect of a force of 25 cN on a molar would be equivalent to the biological effect of a force of 2.5 cN on a premolar. Even with this very low force on a molar, a phase of arrest after the initial phase was found in most cases. Histological evaluation of the samples taken after 4 days of force application confirmed the presence of hyalinized areas in the PDL of both premolars and
molars. This indicates that these light forces do not prevent hyalinization of the PDL. In the samples where hyalinization was found at the pressure side, little osteoblastic activity was seen at the tension side. Removal of the hyalinized tissues appeared to be essential for the start of the real tooth movement, but, even after this real tooth movement had started, a large variation in its rate was found. In the literature, these differences are often related to individual variations in bone metabolism, bone density, and the amount of osteogenic or osteoclastic cells.6,20 The results of our previous21 and present studies indicate, however, that the formation of patches of hyalinized tissue during later phases of tooth movement might also play a role. Possibly, during orthodontic tooth movement, high stresses or
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Fig 9. Photomicrograph showing focal hyalinization of PDL at pressure side of second premolar to which force of 25 cN was applied for 20 days. Section was taken 150 m away from central section lingually of mesiodistal plane; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone; H, hyalinization; OC, osteoclast.
strains are generated locally due to irregularities in the outline of the alveolar bone; this causes local hyalinization.10,22 The hyalinized patches described in this study, however, were not present in front of the moving teeth, but more lingually or buccally where compressive stress will be less important than shear stress. Hyalinized patches at these localizations have not been described in the literature before. They probably have been overlooked because of their unexpected localization. An explanation for this contrasting outcome might be the way the sections were cut. In nearly all previous studies, the teeth that were moved were sectioned vertically through the central plane, or only a few sections from the entire tooth were evaluated.2,23,24 However, in the present study, the teeth were serially sectioned in 7-m
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Fig 10. Photomicrograph showing osteoclasts (OC) near small patch of hyalinization in PDL at pressure side of first molar to which force of 25 cN was applied for 80 days. Section was taken 400 m away from central section buccally of mesiodistal plane; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone; H, hyalinization.
Fig 11. Schematic drawing of horizontal section of 2 roots of premolar. Horizontal arrow marks direction of tooth movement; 4 small arrows indicate localization of hyalinization areas of both roots, which were mainly found buccally or lingually of root.
slices from the mesial to the distal side. By using this protocol, a systematic overview of the changes of the PDL during tooth movement was possible.
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Fig 12. Photomicrograph showing hyalinization next to area of intact PDL at pressure side of first molar to which force of 25 cN was applied for 40 days. Section was taken 450 m away from central section buccally of mesiodistal plane; haematoxylin and eosin staining. T, tooth; PDL, periodontal ligament; B, bone; H, hyalinization.
CONCLUSIONS
The consequence of these findings is that the present models that describe the relationships between force magnitude and rate of tooth movement probably are not valid. Hyalinization is most likely one of the many known and yet to be elucidated factors that can affect tooth movement. Focal hyalinizations, which counteract tooth movement, probably do not directly depend on the applied force but on local stresses or strains that might be quite individually and locally determined due to irregularities in periodontal and bone morphology. Such stress concentrations and especially shear stress concentrations at the buccal and lingual aspects of moving teeth complicate the system further. This means that an appropriate model cannot be de-
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Fig 13. Photomicrograph showing osteoblasts (OB) covering bone surface at tension side of second premolar to which force of 25 cN was applied for 80 days; alkaline phosphatase staining. T, tooth; PDL, periodontal ligament; B, bone.
signed with present knowledge. Far more biomechanical information on local effects of force application and insight into the individual variation in bone density and the metabolism of bone and the PDL and even the level of cytokines and growth factors is needed to construct such a model. REFERENCES 1. Proffit WR. Biomechanics and mechanics. In: Proffit WR, editor. Contemporary orthodontics. St Louis: Mosby; 2000. 296-361. 2. Pilon JJGM, Kuijpers-Jagtman AM, Maltha JC. Magnitude of orthodontic forces and rate of bodily tooth movement: an experimental study in beagle dogs. Am J Orthod Dentofacial Orthop 1996;110:16-23. 3. Van Leeuwen EJ, Maltha JC, Kuijpers-Jagtman AM. Tooth movement with light continuous and discontinuous forces in beagle dogs. Eur J Oral Sci 1999;107:468-74. 4. Quinn RS, Yoshikawa DK. A reassessment of force magnitude in orthodontics. Am J Orthod 1985;88:252-60.
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5. Sandstedt C. Einige Beitra¨ ge zur Theorie der Zahnregulierung. Nord Tandlæk Tidsskr 1904;5:236-56. 6. Reitan K. The initial tissue reaction incident to orthodontic tooth movement as related to the influence of function: a histological study on animal and human material. Acta Odontol Scand 1951;9(Suppl 6):9-240. 7. Owman-Moll P, Kurol J, Lundgren D. Effects of a doubled orthodontic force magnitude on tooth movement and root resorptions: an inter-individual study in adolescents. Eur J Orthod 1996;18:141-50. 8. Owman-Moll P, Kurol J, Lundgren D. The effects of a four-fold increased orthodontic force magnitude on tooth movement and root resorptions: an inter-individual study in adolescents. Eur J Orthod 1996;18:287-94. 9. Lindauer SJ, Britto AD. Biological response to biomechanical signals: orthodontic mechanics to control tooth movement. Semin Orthod 2000;6:145-54. 10. Melsen B. Biological reaction of alveolar bone to orthodontic tooth movement. Angle Orthod 1999;69:151-8. 11. Davidovitch Z, Nicolay OF, Ngan P, Shanfeld J. Neurotransmitters, cytokines and the control of alveolar bone remodeling in orthodontics. Dent Clin North Am 1988;32:411-35. 12. Shanfeld J, Jones JL, Laster L, Davidovitch Z. Biochemical aspects of orthodontic tooth movement; cyclic-nucleotide and prostaglandin concentrations in tissues surrounding orthodontically treated teeth. Am J Orthod Dentofacial Orthop 1986;90: 139-41. 13. Bartold PM, Walsh LJ, Narayanan AS. Molecular and cell biology of the gingiva. Perio 2000 2000;24:28-55. 14. Wang LL, Zhu H, Liang T. Changes of transforming growth factor beta 1 in rat periodontal tissue during orthodontic tooth movement. Chin J Dent Res 2000;3:19-22. 15. Ren Y, Maltha JC, Von den Hoff JW, Kuijpers-Jagtman AM,
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Ding Z. Cytokine levels in crevicular fluid are less responsive to orthodontic forces in adults than in juveniles. J Clin Periodontol 2002;29:400-5. Van de Wijngaert FP, Burger EH. Demonstration of tartrateresistant acid phosphatase in undecalcified, glycolmethacrylateembedded mouse bone: a possible marker for (pre)osteoclast identification. J Histochem Cytochem 1986;34:1317-23. Van Driel WD, Van Leeuwen EJ, Blankevoort L, Von den Hoff JW, Maltha JC. Time dependent displacement of the tooth in response to orthodontic forces. J Biomech 1998;31(suppl 1):35. Van Driel WD, Van Leeuwen EJ, Von den Hoff JW, Maltha JC, Kuijpers-Jagtman AM. Time-dependent mechanical behaviour of the periodontal ligament. Proc Inst Mech Eng 2000;241:497-504. Jarabak JR, Fizzell JA. Technique and treatment with light-wire appliances, light differential forces in clinical orthodontics. St Louis: Mosby; 1963. Sodek J. A new approach to assessing collagen turnover by using a micro-assay: a highly efficient and rapid turnover of collagen in rat periodontal tissues. J Biomech 1976;160:243-6. Von Bo¨ hl M, Kuijpers-Jagtman AM, Maltha JC, Von den Hoff JW. Changes in the periodontal ligament after experimental tooth movement using high and low continuous forces in beagle dogs. Angle Orthod 2004; in press. Katona TR, Paydar NH, Akay HU, Roberts WE. Stress analysis of bone modeling response to rat molar orthodontics. J Biomech 1995;28:27-38. Rygh P. Ultrastructural changes in pressure zones of human periodontium to orthodontic tooth movement. Acta Odontol Scand 1973;31:109-22. Owman-Moll P. Orthodontic tooth movement and root resorption with special reference to force magnitude and duration. Swed Dent J 1995; Suppl 105.