ORIGINAL ARTICLE

Morphologic and hemodynamic analysis of dental pulp in dogs after molar intrusion with the skeletal anchorage system
Yuichi Konno,a Takayoshi Daimaruya,b Masahiro Iikubo,c Reiko Kanzaki,b Ichiro Takahashi,b Junji Sugawara,d and Takashi Sasanoe Sendai, Japan Introduction: We have successfully treated skeletal open bite by intruding posterior teeth with the skeletal anchorage system. Our aim in this study was to morphologically and hemodynamically evaluate the changes in pulp tissues when molars are radically intruded. Methods: The mandibular fourth premolars of 9 adult beagle dogs were divided into 3 groups: a sham operated group (n 6, 3 dogs), 4-month intrusion group (n 6, 3 dogs), and a further 4-month retention group (n 6, 3 dogs). We evaluated the morphological changes of the pulp and dentinthe amount of vacuolar degeneration in the odontoblast layer, the predentin width and nervous continuity in the pulp tissue, and the pulpal blood-ow response evoked by electrical stimulation in the dental pulp. Results: Extreme molar intrusion with the skeletal anchorage system caused slight degenerative changes in the pulp tissue, followed by recovery after the orthodontic force was released. Circulatory system and nervous functions were basically maintained during the intrusion, although a certain level of downregulation was observed. These morphologic and functional regressive changes in the pulp tissue after molar intrusion improved during the retention period. Conclusions: Histologic changes and changes in pulpal blood ow and function are reversible, even during radical intrusion of molars. (Am J Orthod Dentofacial Orthop 2007;132:199-207)

ntrusive force application is thought to cause several pulpal tissue changes due in part to the compression force on periapical blood vessels created by the apical displacement of the tooth. Histologic studies showed that the application of continuous intrusive force causes pulp tissue changes such as disruption of the odontoblastic layer,1 vacuolization, and circulatory disturbances.2 Previous information is available on the effect of brief intrusive forces on pulpal blood ow. Orthodontic intrusion of teeth evoked a temporary reduction in pulpal blood ow
From the Graduate School of Dentistry, Tohoku University, Sendai, Japan. a Research fellow, Division of Orthodontics and Dentofacial Orthopedics, Department of Oral Health and Development Sciences. b Research associate, Division of Orthodontics and Dentofacial Orthopedics, Department of Oral Health and Development Sciences. c Research associate, Division of Oral Diagnosis, Department of Oral Medicine and Surgery. d Associate professor, Division of Orthodontics and Dentofacial Orthopedics, Department of Oral Health and Development Sciences. e Professor and chairman, Division of Oral Diagnosis, Department of Oral Medicine and Surgery. Reprint requests to: Yuichi Konno, Division of Orthodontics and Dentofacial Orthopedics, Department of Oral Health and Development Sciences, Tohoku University, Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; e-mail, yuuichi@mail.tains.tohoku.ac.jp. Submitted, April 2005; revised and accepted, July 2005. 0889-5406/$32.00 Copyright 2007 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2005.07.029

during the rst minute after force application. Then pulpal blood ow gradually increased toward preloading ow values for the next 4 minutes and returned to the prestimulus level 3 minutes after unloading.3,4 These previous studies simply evaluated the short-term changes in the blood ow of an incisor when intrusive force was applied and did not conrm histologic changes in tooth movement accompanied by bone remodeling. It was difcult to provide absolute anchorage for molar intrusion by using traditional orthodontic mechanotherapy. We developed a skeletal anchorage system (SAS) to provide intraoral absolute anchorage for orthodontic tooth movement.5,6 We demonstrated molar intrusion to achieve counterclockwise rotation of the mandibular and occlusal planes to improve open bites without orthognathic surgery.7,8 Clinically, we observed no signs of iatrogenic problems in the dental pulp of molars intruded using the SAS. In a previous study, we demonstrated remarkable mandibular fourth premolar intrusion using the SAS in beagle dogs and reported that neither nerves nor blood vessels of the inferior alveolar neurovascular bundles were damaged.9 Few studies have investigated pulp responses for noxious stimulation to evaluate the vitality and reactivity of the dental pulp during and after molar intrusion.
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The antidromic activation of sensory nerves was evoked by stimulation in the peripheral direction, and it followed that blood ow increased in the tissues they innervate.10,11 Moreover, vasodilatation in the pulp of cats can also be elicited by electrical stimulation of the mental nerve or the pulp of an adjacent tooth.12 These results suggested that axon reex vasodilatation in the dental pulp might be mediated by stimulation. In this study, the pulpal nerves of the experimental teeth were electrically stimulated at the threshold level, and blood ow was assessed with a laser Doppler ow meter. We carried out experiments to determine whether detectable changes in the pulpal blood ow could be evoked by electrical stimulation as a painproducing stimulus of the pulpal nerve before and after molar intrusion, and after mechanical retention. Additionally, we evaluated the pulpal nerve continuity after extreme molar intrusion using wheat-germ agglutinin conjugated with horseradish peroxidase (WGA-HRP, type IV, Sigma, St Louis, Mo) as an anterograde and retrograde axonal tracer. If the pulpal nerve is not damaged and normally continuous, it will be transported to the pulpal nerve through the mental nerve. Our aim in this study was to evaluate the morphologic and functional changes in pulp tissuee when molars are intruded and after they have been mechanically retained, using morphologic and hemodynamic analyses.
MATERIAL AND METHODS

Nine adult female beagles (age, 12 months) were used in this investigation. The bilateral mandibular fourth premolars were divided into 3 groups: a sham operated group (C group, n 6, 3 dogs), a 4-month intrusion group (I group, n 6, 3 dogs), and a 4-month retention group after 4 months of intrusion (R group, n 6, 3 dogs). In the dogs in the C group, the anchor plate was implanted, and no orthodontic force was applied; they served as controls. In the I group and the R group, the premolars were intruded by using the anchor plate for 4 months, and an additional 4 months of retention was carried out for the intruded molars in the R group. The anchor plates (ORTHOANCHOR plate, T-S-L and T-S-R, 18.85 mm long; pure titanium) and bone screws (ORTHOANCHOR screw, 0.2 mm 5 mm, pure titanium) (Dentsply Sankin, Tokyo, Japan) used for anchorage of tooth movement were specically designed for orthodontic treatment, as shown in Figure 1, a. The surgical procedures for the implantation of anchor plates were described previously.9 Briey, the animals were anesthetized with sodium pentobarbital (Wako Chemicals, Tokyo, Japan) at a dose of 25 mg

per kilogram of body weight. Local anesthesia (2% lidocanine, Fujisawa Yakuhin, Tokyo, Japan) was applied at the implantation sites of the anchor plates. A 2-cm mucosal incision was made in the buccal mandible, and the mucoperiosteal ap was reected inferiorly to the lower border of the mandible. The anchor plates were xed on the buccal side of the mandible with 3 titanium bone screws, and the hook was placed in the middle area of the mandibular third and fourth premolars (Fig 1, c). Another bone screw was xed on the lingual alveolar bone between the fourth premolar and the rst molar (Fig 1, d). The mucoperiosteal ap was repositioned, and the surgical wound was sutured with the long arm of the anchor plate intraorally exposed. Additionally, bone markers were inserted into the buccal cortical bone around the anchor plate as reference points to measure tooth movement by superimposing the tracings of radiographs. After a 3-week healing period, an intrusive force (100-150 g) was applied to each mandibular premolar for 4 months. During the experimental period, the tooth and the transmucosal portions of the anchor plates were cleaned by brushing and irrigation with 0.2% aqueous chlorhexidine. To evaluate the amount of molar intrusion, standardized dental radiographs were taken by using a lm holder attached to the titanium miniplates and the third premolar. Standardized radiographs, with 200-mm focus-to-object distance and 2 mm object-to-lm distance, were taken every 4 weeks. The radiographs were magnied 5 times with a mounted slide projector. The titanium miniplate and screws, teeth, lower border of the mandible, inferior alveolar neurovascular bundle, and bone markers were traced. The amount of tooth movement of the 18 fourth premolars, dened as the distance between the 2 lines connecting the mesial and distal root apices, was measured by superimposing those tracings on the titanium bone markers and the anchor plate. Actual tooth movement was calculated by dividing the measured values by 5. As described in our previous study,9 the intraobserver difference was about 0.015 mm, and the standard deviation of the mean differences between 2 observers was 0.03 mm. To prevent damage on the nerve by WGA-HRP injection, pulpal blood ow was measured 2 days before the experimental period of each group. The animals were anesthetized with sodium pentobarbital at an initial dose of 30 mg per kilogram intravenously, supplemented, when necessary, with additional doses of 20 to 30 mg per kilogram. Because our previous study indicated that pulpal blood ow strongly depended on systemic blood pressure,13 we continuously

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Fig 1. SAS components, surgical procedures, and monitoring system for pulpal blood ow and systemic blood pressure. a, Orthodontic anchor plates and monocortical bone screws and b, placement of anchor plate on cortical bone under mucoperiosteal ap. c and d, Orthodontic mechanics using orthodontic anchor plate and lingual bone screw. e and f, Schematic representation of and monitoring system for pulpal blood ow and systemic blood pressure.

monitored blood pressure in the femoral artery with a pressure transducer and recorded it on a polygraph (Polygraph 360 system, NEC, Tokyo, Japan). Thus, we veried that there was no inuence from the center. The animals were intubated and paralyzed by intravenous injection of pancuronium bromide (Mioblock, Sankyo, Tokyo, Japan; 0.4 mg per kilogram initially, then constant infusion of approximately 0.2 mg per kilogram per hour), and articial ventilation was maintained with a respirator (model SN-480-3, Shimano, Tokyo, Japan) with a mixture of 25% pure oxygen and

75% room air at a frequency of 20 to 24 times per minute. Each animal lay on a warmer table, with the temperature thermostatically maintained at 42C. The head was xed to a steel table with a magnetic clamp (MB-B, Kanetue, Tokyo, Japan) with a bar attached to the frontal sinus. The jaws were held open with silicone putty. The pulpal blood ow of the fourth mandibular premolar was recorded noninvasively with a modied conventional laser Doppler ow meter (ALF 21D; Advance, Tokyo, Japan), which emits a 5 mW intensity

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beam of monochromatic light from a laser diode. The ow meters probe (outer diameter: 1.5 mm) is composed of 2 glass graded-index optical bers, 1 transmitting and the other receiving, each with a core diameter of 100 m. With transmitted laser light, we used 2 probes, 1 ber of which served as the transmitter on the labial side of the tooth, and the other (having been placed to the lingual side) served as the receiver. The method used to monitor pulpal blood ow was similar to that reported previously.14 This equipment gives no absolute values but permits relative changes in blood ow to be assessed. To enable a reliable interpretation of the measurements, the 2 probes of the ow meter were stabilized by inserting them into a stainless-steel tube (inner diameter; 1.5 mm) attached to the buccolingual sides of the acrylic stent, with its center positioned at the central long axis and 4 mm below the premolar cusp (Fig 1, f). To verify the reliability of the transmitted laser light at a laser power of 5 mW, a pilot study was conducted in the bilateral fourth premolars in 4 dogs. Before treatment of the pulp to induce necrosis, pulpal blood ow and systemic blood pressure were measured in 4 subjects. After monitoring in each experimental subject, the pulp of the left premolar was exposed with a high-speed water-cooled diamond bar, and Periodon was applied (Neo Dental Chemical Products, Tokyo, Japan) to cause necrotic changes of the pulp tissue. A week later, the pulpal blood ow of the bilateral fourth premolar was monitored again. The output signals measured with transmitted laser light registered 0 aubitory units (a.u.) in all nonvital teeth. On the other hand, vital teeth before Periodon treatment showed 3.21 a.u. on average. The transmitted laser with the laser power increased to 5 mW monitored pulpal blood ow and assessed tooth pulp vitality. Blood ow signals from outside the pulp were not detectable. We carried out experiments to determine whether detectable changes in pulpal blood ow could be evoked by electrical stimulation of a pulpal nerve. The electrical stimulation was applied to the pulp in the experimental teeth in each of the 3 groups by using a noninvasive tooth pulp stimulation test; in a stepwise manner, we increased the electrical stimuli delivered with an electric pulp tester (Analytic Technologies, Redmond, Wash). Before stimulation, each tooth was carefully cleaned with alcohol, dried with a stream of air, and isolated with cotton wool. The cathode of the stimulator was attached so that it made rm contact with the buccal surface of the tooth at least 3.5 mm from the gingival margin. Pulpal blood ow and sys-

temic blood pressure were recorded before, during, and after the stimulation.
Data analysis

Pulpal blood-ow signals were analyzed by using MacLab (ADInstruments, Castle Hill, NSW, Australia). The peak values of the pulpal blood-ow changes were expressed as percentages of the baseline blood ow (baseline, mean of the prestimulation ow for 3 minutes; peak, mean of the maximum ow for 30 seconds during or after stimulation in the same animal) and given as mean standard deviaiton.15 The calculated peak values after electrical stimulation in the I group were compared with those in the C and the R groups. Statistical analysis was undertaken with SPSS for Windows (SPSS, Chicago, Ill). Differences were evaluated with the Mann-Whitney U test, and P values less than .05 were considered signicant. To evaluate nerve continuity, WGA-HRP in physiologic saline solution was injected into the mental nerve as an antidromic and anterograde neural tracer. After recording the pulpal blood ow and axon reex vasodilatation, topical injection of WGA-HRP (Toyobo, Osaka, Japan) into the mental nerve was carried out as described by Marfurt and Turner.16 The bilateral mental foramen and nerve were exposed by removing the surrounding tissues, and 1 L of 5% WGA-HRP in saline solution was injected through a glass micropipette (diameter, 50 to 70 m) connected to micro syringe. Total injection (1 L 5 times) required at least 10 minutes, and the micropipette was left in place for an additional 5 minutes before being withdrawn. This injection was carefully performed to prevent any diffusion of the tracer. All animals were killed 48 hours after the end of the experiment. Each animal was anesthetized with sodium pentobarbital and perfused with paraformaldehyde through both common carotid arteries. The middle part of the mandibular bodies including the fourth premolars was dissected. The specimens were decalcied in ethylene diamine tetra-acetic acid for 16 weeks. Then they were divided into the mesial area (including mesial root) for histologic evaluation of the pulp with surrounding tissues and the distal area (including distal root) for evaluation of the pulpal nervous tissue. The mesial area of each specimen was embedded in parafn after decalcication. Five-micrometer thick serial sections through the pulp in an axio-buccolingual direction were prepared. The sections were stained with hematoxylin and eosin for histologic observation of the pulp tissue. Additionally, the distal root of the experimental premolar was dissected out and stored overnight in 20% sucrose solution. The distal root was cryoembedded in

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Fig 2. Radiographic images of fourth premolars: a, before intrusion; b, after intrusion; and c, after retention. d, Amount and time course of tooth movement. Broken line, I group; solid line, R group. Mean and SD bars are indicated at each time point (n 9).

OCT compound (Sakura seiki Products, Osaka, Japan) and then sectioned at 20 m with a cryostat. The sections were reacted according to a tungstate modication of the tetramethylbenzidine technique for the visualization of horseradish peroxidase.17 After completing the reaction, all sections were air-dried and counterstained with 0.1% neutral red. The sections were then observed with a phase contrast microscope.
RESULTS

The results of the radiographic analysis are summarized in Figure 2. The amount of tooth movement during the experimental period was 2.9 0.5 mm on average in the I group and 3.0 0.5 mm in the R group. There was no statistically signicant difference between the 2 groups. The fourth premolars were intruded in 4 months without lingobuccal and mesiodistal tipping, and the root apices had penetrated into

the alveolar bone wall (Fig 2, b). The intruded fourth premolars of the R group were stable through the 4-month retention period as shown in Figure 2, c. Typical recordings of the 3 experimental groups are shown in Figure 3, a-c. No changes in systemic arterial blood pressure were induced by the electrical stimulation throughout the experiment (upper traces, Fig 3, a-c). The amounts of increase in pulpal blood ow evoked by the electrical stimulation, expressed as a percentage of resting, were 347.8% 59.8% (n 6) in the C group, 157.8% 23.3% (n 6) in the I group, and 330.3% 54.6% (n 6) in the R group (MannWhitney U test, Fig 3, d). Because this response was not associated with increased systemic arterial blood pressure, it was assumed to be due to pulpal vasodilation. The pulpal blood-ow responses were signicantly lower in the I and C groups (P .05), and the signal reaction, reduced during intrusion, was restored

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the periodontal ligament below the apical foramen and in the size and number of odontoblast and broblasts compared with the C group were found immediately after intrusion (Fig 4, b and h). At the end of the retention period, the amount of vacuolization in the odontoblast layer was reduced in the R group compared with that in the I group. Moreover, the predentin width was slightly larger in the R group than in the I group (Fig 4, c). There was a noticeable increase in the concentration of capillary blood vessels in the periodontal ligament below the apical foramen region in the R group compared with the I group (Fig 4, i). The intensity of WGA-HRP labeling varied among dogs, but no great difference in distribution was observed. The WGA-HRP reaction products indicated rich innervations in dental pulp. The labeled nerve bundle entered the root pulp through the apical dental foramen with the blood vessel parallel to the long axis of the tooth. The nervous continuity was maintained in the 3 groups (Fig 4, d-f).
DISCUSSION

Fig 3. Systemic blood pressure and responses in pulpal blood ow in electrically stimulated control and experimental fourth premolars: a, C group; b, I group; c, R group. d, Pulpal blood-ow response in each group was quantitated. Pulpal blood response in I group was signicantly lower than in other groups (P .05). BP, blood pressure; PBF, pulpal blood ow. Arrows indicate electrical stimulation in time course.

to near the control value by the end of the retention period. There was a signicantly greater increase in the R group compared with the I group (P .05). The pulp tissues in the C group exhibited normal histologic appearance (Fig 4, a). Odontoblasts had the characteristically columnar shape in the coronal portion. Most teeth in the I group showed marked changes in the pulp and dentin (Fig 4, b). Higher vacuolization of the odontoblast layer than that of controls and reduction in the width of predentin layer were seen in the I group. Inammatory responses were not generally seen, and a slight cellular inltration was discerned. Reductions in the number of capillary blood vessels in

The laser Doppler ow meter, developed in the 1970s as a noninvasive electro-optical technique to measure the velocity of red cells in skin capillaries, has been used for the diagnosis of pulp vitality in human teeth.18 It was originally developed to measure blood ow in the skin, in which capillaries are much closer to the surface than in the dental pulp. Hence, the widely used laser power (2 mW) is insufcient for detecting pulpal blood ow with transmitted laser light (at least with the current type of apparatus). We developed a transmitted laser light ow meter apparatus and demonstrated that high-powered (5 mW) transmitted laser light could be a better tool for both monitoring the pulpal blood ow of the teeth and assessing tooth-pulp vitality than the conventional back-scattered light ow meter apparatus.19,20 Therefore, the transmitted laser light was found to be useful for the assessment of tooth pulp vitality both because blood-ow signals did not include ow of nonpulpal origin, and also because the output signals and responses to blood-ow changes were clear and could easily be monitored. In previous studies, researchers reported that degenerative changes, vacuolization of the odontoblast layer, circulatory disturbances,2 and transient reduction of the amount of pulpal blood ow21 were induced in pulp tissues after short-term tooth intrusion. Based on our histologic ndings, the pressure generated by the intrusive force reduced the number of capillary blood vessels below the apical foramen. In addition, we demonstrated that it was possible to record the increase and decrease in pulpal blood ow caused by electrical

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Fig 4. Photmicrographs of sections from pulp tissue from fourth premolars: a and d, C group; b and e, I group; c and f, R group. Vacuolization in odontoblast layer in I group was outstanding in hematoxylin and eosin staining (a-c). Nervous continuity in all experimental groups indicated by WGA-HRP labeling (white arrows). Photomicrographs from apical tissue: g, C group; h, I group; i, R group. Arrowheads indicate capillary vessels. Original magnication 400 (a-c), 400 (d-f), and 200 (g-i). Black arrows, Vacuolization in odontoblast layer; D, Dentin; P, pulp tissue; ob, odontoblasts.

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stimulation of the pulpal nerve. Because the innervation was maintained in the 3 groups and there was no associated change in femoral blood pressure, it can be assumed that these effects were due to pulpal vasodilatation and vasoconstriction. Previous research suggested that a parasympathetic vasodilator mechanism is not present in feline dental pulp.20 Therefore, it can be considered that the sensory and sympathetic nerves in the pulp were not damaged, and the peripheral circulatory mechanism was maintained, whereas the apparent reduction in blood ow in response to electrical stimulation suggested actual suppression of the blood supply to the pulp tissue. Taking these into consideration, pulpal tissues might have been exposed to a reduction in nutrients or oxygen when the intrusive force was applied to the teeth. The greatly reduced width of the predentin layer observed in the pulp of the teeth in the I group, in which the vacuolization was the most marked of the 3 groups, suggested that the matrix formation was retarded or inhibited by the low blood supply. Because more capillaries in the apical area and reduction of the vacuolization in the pulp were found in the R group, repair of the odontoblast layer might have occurred because of the improved circulatory pattern that resulted from angiogenesis in the apical area after the mechanical retention. Additionally, no signicant differences in the extent of vasodilation evoked by electrical stimulation were found between the C and R groups. It is suggested that alteration in the circulatory pattern and reparative changes occurred in the pulp as a result of the retention. Therefore, it can be considered that the encapsulated pulp tissue was severely disrupted during the application of intrusive force, although it did not undergo necrotic changes, and that recovery from the degenerative changes can occur after the release from compression. The activation of sensory nerves in the pulp by direct electrical stimulation induces a long-lasting blood-ow increase in the pulp in normal conditions as shown in this and previous studies.22,23 Upon activation of these nerves, vasoactive neurokinins, such as substance P (SP) and calcitonin gene-related peptide (CGRP), might be released from sensory nerve terminals. The initial component of axon reex-mediated vasodilation is mediated by SP, whereas the continuing long-lasting rise in blood ow depends on CGRP.24,25 SP might lead to endogenous activation of inammatory mediators released in the pulp. This aspect became obvious when it was found that pulpal vasodilation in response to the local application of bradykinin occurred, to a great extent, via the activation of sensory nerves.26 On the other hand, sympathetic vasoconstric-

tion is counteracted by the local activation of sensory nerves and the release of vasodilator neuropeptides. Reex activation of sympathetic nerves and local release of noradrenaline attenuates the release of CGRP and SP. Overall, the process is regarded as an appropriate defense response that enhances the transportation of nutrients and metabolites to the pulp tissue. Taken together, the blood-ow response to the electrical stimulation to the pulp could be regulated by SP or CGRP. In this study, we demonstrated the effects of longterm, radical molar intrusion using SAS on morphologic and hemodynamic changes in pulp tissues. Although radical molar intrusion caused slight regressive changes in the pulp tissue in the short term, blood ow and innervation were maintained in the pulp chamber, and the axon reex vasodilatation could be detected. Therefore, we concluded that the histologic changes and the changes in pulpal blood ow and function are reversible even during radical intrusion of molars. However, in humans, it is still unclear whether the application of intrusive forces for longer than 4 months affects pulp viability. Further experiments are needed to clarify the biologic effects of intrusion of molars with SAS.
CONCLUSIONS

Although remarkable molar intrusion with SAS caused slight regressive changes in pulp tissues, the blood ow and the nervous system were kept in the dental pulp, and the axon reex vasodilatation was detected, indicating that functions of the blood vessels and the nerves were maintained. Furthermore, these morphologic and functional regressive changes in pulp tissue after molar intrusion improved during the retention period. These results indicated that changes in dental pulp after radical intrusion with SAS are less and reversible.
REFERENCES 1. Anstendig HS, Kronman JH. A histologic study of pulpal reaction to orthodontic tooth movement in dogs. Angle Orthod 1972;42:50-5. 2. Stenvik A, Mjor IA. Pulp and dentine reactions to experimental tooth intrusion. A histologic study of the initial changes. Am J Orthod 1970;57:370-85. 3. Brodin P, Linge L, Aars H. Instant assessment of pulpal blood ow after orthodontic force application. J Orofac Orthop 1996; 57:306-9. 4. Sano Y, Ikawa M, Sugawara J, Horiuchi H, Mitani H. The effect of continuous intrusive force on human pulpal blood ow. Eur J Orthod 2002;24:159-66. 5. Sugawara J. Dr. Junji Sugawara on the skeletal anchorage system. Interview by Dr. Larry W. White. J Clin Orthod 1999;33:689-96.

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6. Sugawara J, Baik UB, Umemori M, Takahashi I, Nagasaka H, Kawamura H, et al. Treatment and posttreatment dentoalveolar changes following intrusion of mandibular molars with application of a skeletal anchorage system (SAS) for open bite correction. Int J Adult Orthod Orthognath Surg 2002;17:243-53. 7. Umemori M, Sugawara J, Mitani H, Nagasaka H, Kawamura H. Skeletal anchorage system for open-bite correction. Am J Orthod Dentofacial Orthop 1999;115:166-74. 8. Sugawara J, Daimaruya T, Umemori M, Nagasaka H, Takahashi I, Kawamura H, et al. Distal movement of mandibular molars in adult patients with the skeletal anchorage system. Am J Orthod Dentofacial Orthop 2004;125:130-8. 9. Daimaruya T, Nagasaka H, Umemori M, Sugawara J, Mitani H. The inuences of molar intrusion on the inferior alveolar neurovascular bundle and root using the skeletal anchorage system in dogs. Angle Orthod 2001;71:60-70. 10. Lembeck F, Holzer P. Substance P as neurogenic mediator of antidromic vasodilation and neurogenic plasma extravasation. Naunyn Schmiedebergs Arch Pharmacol 1979;310:175-83. 11. Couture R, Cuello AC. Studies on the trigeminal antidromic vasodilatation and plasma extravasation in the rat. J Physiol 1984;346:273-85. 12. Vongsavan N, Matthews B. Antidromic vasodilatation in the dental pulp and lip of cats (abstract). J Dent Res 1992;71(Spec iss):534. 13. Sasano T, Kuriwada S, Sanjo D. Arterial blood pressure regulation of pulpal blood ow as determined by laser Doppler. J Dent Res 1989;68:791-5. 14. Sasano T, Onodera D, Hashimoto K, Iikubo M, Satoh-Kuriwada S, Shoji N, et al. Possible application of transmitted laser light for the assessment of human pulpal vitality. Part 2. Increased laser power for enhanced detection of pulpal blood ow. Dent Traumatol 2005;21:37-41. 15. Hikiji A, Yamamoto H, Sunakawa M, Suda H. Increased blood ow and nerve ring in the cat canine tooth in response to stimulation of the second premolar pulp. Arch Oral Biol 2000; 45:53-61.

16. Marfurt CF, Turner DF. Sensory nerve endings in the rat oro-facial region labeled by the anterograde and transganglionic transport of horseradish peroxidase: a new method for tracing peripheral nerve bers. Brain Res 1983;261:1-12. 17. Weinberg RJ, Van Eyck SL. A tetramethylbenzidine/tungstate reaction for horseradish peroxidase histochemistry. J Histochem Cytochem 1991;39:1143-8. 18. Gazelius B, Olgart L, Edwall B, Edwall L. Non-invasive recording of blood ow in human dental pulp. Endod Dent Traumatol 1986;2:219-21. 19. Sasano T, Nakajima I, Shoji N, Kuriwada S, Sanjo D, Ogino H, et al. Possible application of transmitted laser light for the assessment of human pulpal vitality. Endod Dent Traumatol 1997;13:88-91. 20. Sasano T, Shoji N, Kuriwada S, Sanjo D, Izumi H, Karita K. Absence of parasympathetic vasodilatation in cat dental pulp. J Dent Res 1995;74:1665-70. 21. Ikawa M, Fujiwara M, Horiuchi H, Shimauchi H. The effect of short-term tooth intrusion on human pulpal blood ow measured by laser Doppler owmetry. Arch Oral Biol 2001;46:781-7. 22. Sasano T, Kuriwada S, Shoji N, Sanjo D, Izumi H, Karita K. Axon reex vasodilatation in cat dental pulp elicited by noxious stimulation of the gingiva. J Dent Res 1994;73:1797-802. 23. Andrew D, Matthews B. Properties of single nerve bres that evoke blood ow changes in cat dental pulp. J Physiol 2002; 542(Pt 3):921-8. 24. Kerezoudis NP, Olgart L, Edwall L. CGRP (8-37) reduces the duration but not the maximal increase of antidromic vasodilation in dental pulp and lip of the rat. Acta Physiol Scand 1994;151: 73-81. 25. Kerezoudis NP, Olgart L, Edwall L. Involvement of substance P but not nitric oxide or calcitonin gene-related peptide in neurogenic plasma extravasation in rat incisor pulp and lip. Arch Oral Biol 1994;39:769-74. 26. Olgart L, Edwall L, Gazelius B. Involvement of afferent nerves in pulpal blood-ow reactions in response to clinical and experimental procedures in the cat. Arch Oral Biol 1991;36:575-81.