European Journal of Orthodontics 13 (1991) 255-263
> 1991 European Orthodontic Society
Root resorption after local injection of prostaglandin E2 during experimental tooth movement Pongsri Brudvik and Per Rygh Department of Orthodontics and Facial Orthopedics, Faculty of Dentistry, University of Bergen, Norway
The purpose of this study was to investigate the occurrence of orthodontic root resorption in connection with local injection of prostaglandin E2 (PGE2). The material consisted of 25 male Wistar rats. The control group comprised six animals where no force was applied. In five animals 0.1 ml of 0.1 /jg//*l PGE2 was injected in the gingival area of the upper right first molar. In one animal no PGE2 was injected. The animals were killed after 3 days. The experimental tooth movement groups consisted of 19 animals. Duration of experiments was 3 days, 7 days, and 10 days. The maxillary first molars on both sides were each moved mesially by means of a coil spring. On the right side 0.1 ml of PGE2 0.1 /xg///l was injected in the gingiva on the buccal side of the upper first molar on days 0, 3, 5, and 7. On the left side no injection of PGE2 was performed. In three animals in the 7-day group the vehicle (Waymouth medium) was injected. There was no significant difference in root resorption between the experimentally moved teeth with and without local injection of PGE2, but a trend towards more root resorption was registered on the teeth where such injections had been performed. SUMMARY
In tooth movement, the primary stimulus to evoke bone resorption and apposition is physical, but the mechanism for transcription of the forces has not been established. It seems that the responses to mechanical stress are a result of a number of factors acting in concert: neural, vascular, and bioelectric (Davidovitch et al., 1989). The involvement of prostaglandins and the cyclic AMP pathway is well established (Harell et al., 1977; Somjen et al., 1980; Yeh and Rodan 1984; Ngan et al., 1988). It has been suggested that mechanical distortion of the cell initiates PGE2 synthesis from membrane phospholipids; subsequent binding of extracellular PGE2 to cell surface receptors activates adenylate cyclase and the cAMP pathway. This mechanism enables PGE2 to reactivate the cell of origin as well as adjacent cells, thereby amplifying the initial signal. Evidence has been presented indicating that prostaglandin plays an important role in orthodontic tooth movement. Based on measurements on treated humans and monkeys, Yamasaki et al. (1982, 1984) reported that local
injection of prostaglandin (PGEi or PGE2), accelerates the rate of tooth movement. It was assumed that bone resorption was facilitated. This was based on the observation that more osteoclasts were seen in the inter-radicular area after local injections of PGEj or PGE2 near the first rat molars that had not been moved experimentally (Yamasaki et al., 1980). Sandy and Harris (1984) found significant decrease in the number of osteoclasts and a trend towards decreased tooth movement in rabbits where prostaglandin synthesis was inhibited by flurbiprofen. Blocking prostaglandin synthesis by indomethacin resulted in significantly slower tooth movement in cats (Chumbley and Tuncay, 1986). Besides the remodelling of supporting structures (Reitan 1951; Macapanpan et al., 1954; Roberts et al., 1981) dental tissues may also be involved during orthodontic tooth movement. A serious sequel which occurs in tooth movement is resorption of cementum and dentine (Oppenheim, 1936; Kvam, 1969, 1972; Rygh, 1974, 1977; Reitan, 1974; King and Fischlschweiger, 1982; Williams, 1984). While the influence of local injection of pros-
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Introduction
256
P. BRUDVIK AND PER RYGH
taglandins on bone resorption and the rate of orthodontic tooth movement has been quite extensively studied, the possible influence on root resorption has not been clearly documented. The purpose of this study was to investigate the occurrence of root resorption after local injection of PGE 2 during experimental tooth movement.
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was no further activation of force during the experimental period. Immediately after the force had been applied, PGE 2 , the same dose as used for the control group, was injected into the buccal gingival area of upper right first molar. In the 3-day group PGE 2 was injected only on the operation day (day zero). In the 7-day group PGE 2 was injected on day 0, 3, and 5. In the 10-day group PGE 2 was injected on days 0, 3, 5, and 7. Material and methods No injection was performed on the left side of animals in the 3- and 10-day groups. The material consisted of 25 male Wistar rats, weighing 165 ± 20 g. In order to study the effect of the injection per se Waymouth medium (used as vehicle in The control group without force or tooth the PGE 2 solutions) was injected into the left movement comprised six animals. In one animal side of three out of seven animals from the where no PGE 2 was injected, both left and right 7-day group. The injections of vehicle were done molars were used as pure control specimens. In at the same time as the PGE 2 injection on the five animals 0.1 ml of 0.1 //g//zl PGE 2 solution right side (day 0, 3, 5). In the remaining four was injected into the buccal gingival area of maxillary right first molar. The PGE 2 was dis- < animals, no injection was performed on the left side. solved in ethanol initially and then diluted in Waymouth's medium at concentration 0.1 ng/ All operations were performed under general /A (Yen et al., 1987). The left sides were used anaesthesia [Dormicum (F. Hoffmann-La as control. Roche & Co. AG, Basel, Switzerland)/Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) dosPGE 2 injection was performed on day zero age 0.15-0.2 ml/100 g body weight]. The and the animals were killed after 3 days. animals were killed by perfusion under an overThe material with experimental tooth movedose of Dormicum/Hypnorm (Grevstad, 1987). ment consisted of 19 animals. Experimental After perfusion, the right and left maxillary periods were 3, 7, and 10 days. These groups molar segments were dissected out and fixed. contained six, seven, and six animals, After decalcification in 0.25 M EDTA, pH 7.2, respectively. each specimen was split mesio-distally by a Both maxillary first molars were moved mesirazor blade. The buccal part was prepared for ally by means of coil spring ligated to each light microscopy and the palatal part was premolar and through eyelets soldered to an incisor pared for electron microscopy. In this paper, band (Fig. 1). The applied force was 50 g. There only the results from light microscopy will be presented. Parasagittal sections of the mesiodistal aspect of the teeth were cut at 6 fim, and stained with haematoxylin and eosin (Fig. 2a). The middle section containing an interradicular area of 855 x 380 fim2 on the mesial surface of the middle or disto-buccal root of the first molar was chosen as a basis for the histological investigation (Fig. 2a). The specimens where root length was less than 855 fim were omitted. Out of 25 animals (50 specimens), 41 specimens complied with our criteria (Table 1). For each specimen, every second section of Figure 1 The appliance consisting of a closed coil spring an overall thickness of 420 fim, 210 ^m apart ligated between upper first molar and an eyelet of an incisor band. from the base section on each side, was exam-
257
ROOT RESORPTION AFTER INJECTION OF PGE 2
Table 1 Mean root absorbed areas (Res. area, Aim2) and the depth of the resorbed cavities (/jm) in the groups without PGE 2 injection (non-PGE 2 ) and with PGE 2 injection (PGE 2 ). N= total number of specimens. Res. spec. = number of specimens with resorption. SD = standard deviation. Contr. = control. Exp. = Experimental tooth movement. Non-PGE 2 . N
Res. spec.
Contr. 3d
6
0
Exp. 3d 7d lOd
5 5 4
0 4 1
Res. area JP(SD)
13 712 (20642) 31637(63275)
PGE 2 Depth AXSD)
30.9 (30.9) 8.0 (17.0)
/'-value
N
Res. spec.
5
0
5 6 5
2 6 4
Res. area JP(SD)
Depth JP(SD)
Area
Depth
138 (206) 37 452(34339) 74000(85249)
6.4 (9.2) 29.9 (10.2) 17.8 (11.7)
ns1 ns3
ns 2 ns*
ns = Not significant (/" = 0.1709; P 2 = 0.7150; P3 = 0.2207; P* = 0.4724).
M b. Figure 2 Areas examined histologically. D: distal; M: mesial; B: buccal; L: lingual; R: resorption. (a) The designed length (855 fim), width (380 pun) at the inter-radicular area. The total length (L, + L 2 ) and depth (d.) of resorption lacunae. Direction of tooth movement (big arrow), (b) Overall width of area being examined (420 fim). Total resorbed area (R) = YJ=* LaC W,(fim2), where Z.ol = total resorbed length from each investigated section; wt = thickness between each investigated section (12 fim).
ined (Fig. 2b). This meant that the overall thickness included 70 sections out of which 35 were examined. In specimens where resorption occurred, the total length of resorbed surfaces from every second section (at 12//m interval) in the given area were measured directly by means of a micrometer. The length was measured parallel to the root surface. The depth of each resorption lacuna was measured at the deepest point by using the distance from the bottom of the cavity tangent to the line passing the intact root surface from both sides of the
[zd1
tions. The statistics T= /—— and the paired
V In
Mest were used. Results The value of 7(7=0.798) indicated an acceptable method error. Similarly, the small /-value (J=0.02, SD= 1.13, * = 0.17, P=0.86) indicated only insignificant systematic errors. Control group (Fig. 3a, b) In the control group where no force was applied, neither active resorption nor hyalinized tissue was registered. There was no apparent histological difference between the non-injected control group and the group with PGE2 injection (Fig. 3a, b). 3-day group (Fig. 3c, d) In all specimens where orthodontic force had been applied, hyalinized tissue was observed.
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B
cavity (Fig. 2a). For each specimen, only the deepest lacuna was recorded. A total of 1435 sections were examined. Resorbed cavities were recorded in 251 sections in 17 specimens (Table 1). The total resorbed area for each specimen was computerized by adding all resorbed surface areas from each registration. Since the distance between each registration was small (12 /mi), only rectangular areas were calculated. A hyalinized zone was recorded as being present or absent. Method error and the systematic error were evaluated by double measurements in 102 sec-
258
P. BRUDVIK AND PER RYGH
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Figure 3 Inter-radicular area of upper first molars in the 3-day specimens. B, bone; PL, periodontal ligament; H, hyalinized tissue, C, cementum, D, dentin; R, resorption. Big arrows indicate the direction of tooth movement, (a) control group without injection, (b) control group with PGE2 injection, (c) 3-day group with only orthodontic experimental force, (d) 3-day group with force + PGE2 injection.
No root resorption was observed in specimens where only force had been applied (Fig. 3c). In two of the specimens with both force and PGE2, a small resorbed cavity had developed at the periphery of the hyalinized tissue (Fig. 3d). Cavity depth was small (Fig. 7). 7-day group (Fig. 4a-d) Most specimens demonstrated pronounced resorptive activity on bone as well as on root surfaces. Root resorption was found in all specimens, except one, in varying degrees (Table 1). The total areas of root resorption of the specimens with and without vehicle injection (non-PGE2 injected specimens) in the 7-day group were compared. The amount of resorp-
tion found in specimens where vehicle had been injected were not different from specimens without such injection. The results indicated that trauma from injection did not influence the resorptive processes. Based upon this finding, the specimens with only vehicle injection were included in the group without PGE2 injection. The mean area of root resorption was 13 172 /an2 for non-PGE2 injected specimens. The corresponding area of PGE2 injected specimens was 37 542 /on2. Contra-lateral specimens could be compared in four animals. Among these, three animals demonstrated more extensive resorption on the PGE2 injected sides. Furthermore, two of the non-PGE2 injected specimens showed very small
ROOT RESORPTION AFTER INJECTION OF PGE 2
259
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Figure 4 Inter-radicular area of first molars in the 7-day group. B, bone; PL, periodontal ligament; H, hyalinized tissue; C, cementum; D, dentin. Big arrows indicate direction of tooth movement. Odontoclast-like cells (small arrows) in the resorption lacunae, (a) only force, (b) force+ PGE 2 injection, (c) force combined with vehicle injection, (d) force+ PGE 2 injection. N.B. (a) and (b) are contralateral specimen of the same animal.
resorbed areas (350 and 1160/nn2). However, one animal demonstrated more resorption on the non-PGE2- than on the PGE2-injected side. Hyalinized/semihyalinized tissue was observed in 40 and 28 per cent of the cases in non-PGE2- and PGE2-injected groups, respectively. The mean depth of cavities was greatest in the 7-day group (Fig. 7). There was no difference in cavity depth between specimens with and without PGE2 injection (Table 1). 10-day group (Fig. 5a-c) The mean area of root resorption was higher than in the 7-day group (Table 1). In specimens
where only force was applied, extensive resorption was observed in only one specimen. No root resorption was observed on the rest of the specimens without PGE2 injection. In specimens where both force and PGE2 had been applied, root resorption was observed in all specimens except one. The mean area of root resorption was 31 637 and 74000/im2 in the non-PGE2- and PGE2injected groups, respectively (Table 1). Hyalinized tissue was still observed in some specimens in both non-PGE2 and PGE2 injected groups. In the 10-day group, contralateral specimens were compared in three animals. No root resorption occurred on the non-PGE2-injected
260
P. BRUDVIK AND PER RYGH
sides. Unfortunately, in the only animal with root resorption on the non-PGE2-injected side, no contralateral specimen was available. The results indicated that the resorbed areas had increased with longer experimental periods (Fig. 6). The mean value of the resorbed areas was higher in groups with local application of PGE2 combined with orthodontic tooth movement than in groups with only orthodontic mechanical force. However, the differences were not statistically significant (Mann-Whitney P = 0.\7 and P=0.22 for the 7- and 10-day group, respectively; Table 1). In the 10-day group, the cavities were about two times deeper in PGE2-injected specimens than in non-PGE2 injected specimens. However, the differences were not significant (Table 1).
'1M000
g^ |
Fore. |
Only
• force
10000 ?
JI
~i r
a
0
I 0
10000
Q
Discussion
The present investigation demonstrated that with the experimental model used, root resorption first occurred on the mesial aspect of the distal root both with and without local application of PGE2. The rate of removal of necrotic
03 0 DURATION
0 70 OF
01O0
FORCE
Idsyil
Figure 6 Histograms showing the mean resorbed area (A") in fim2 from the different experimental groups. Bar lines represent standard deviation (SD).
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Figure 5 Inter-radicular area of first molars in the 10-day group. B, bone; PL, periodontal ligament; H, hyalinized tissue; C, cementum; D, dentin. Big arrows indicate direction of tooth movement. Odontoclast-like cells (small arrows) in the resorptive lacunae, (a) Only force: no root resorption; (b) contra-lateral specimen of animal in (a) where both force and PGE 2 being applied. This specimen represents the most advanced resorption. (c) Only force with extensive root resorption.
ROOT RESORPTION AFTER INJECTION OF PGE2
IMort U.,n
PCE2 vlu.
fO ) I*)
a o
Figure 7 Curve showing the mean depth of the resorbed cavities in the different experimental groups. Each sign represents the depth value for each individual specimen.
Root resorption did not occur in the present control material, but cannot be excluded in rats under physiologic conditions (Grevstad, 1987). Trauma from orthodontic force on pre-existing resorbing lesions on the root surface might enhance the activity resulting in more extensive root resorption. This may explain the extensive resorption which occurred in one of the nonPGE2-injected specimens in the 10-day group. The involvement of prostaglandins in orthodontic tooth movement which has been reported earlier by Yamasaki et al. (1982, 1984), Sandy and Harris (1984), and Chumbley and Tuncay (1986) may be due to a stimulation of precursor cells to differentiate into resorptive cells (Klein and Raisz, 1970; Harris, 1973; Goodson et al., 1974; Yu et al., 1976; Yamasaki et al., 1980; Yen et al., 1987). Since the presence of hyalinized tissue stops orthodontic tooth movement (Reitan, 1952; Kvam, 1972; Rygh, 1974), any increase of the rate of dissolution of hyalinized tissue by degeneration or by phagocytic cell activity through stimulation by an agent such as PGE2, would facilitate tooth movement. When Yamasaki et al. (1984) investigated possible accelerated tooth movement by local injection of PGE2 in orthodontic patients, a wide range of individual response was registered. In general, the individual biological response may vary (Reitan, 1951). Individual tissue response to periodontal trauma incident to the experimental force may explain the wide range of resorption demonstrated in this study. In the present experiments, local injection of PGE2 was found to be technically difficult. Some leakage occurred and the full effect may not have been achieved in all cases. Both resorptive as well as reparative phases were observed. In the lacunae in 10-day group specimens, cementum deposited in the resorbed lacunae during the reparative processes (King and Fischlschweiger, 1982; Grevstad, 1987), may be the reason for the smaller cavity depth in the 10-day experimental period than in the 7-day group. It is possible that in the non-PGE2-injected groups, where there was no chemical stimulation of the resorbed cells, some small resorbed lesions which occurred in the earlier period may have been completely repaired without showing any lesion in the 10-day group. This may be the explanation for the smaller number of resorbed specimens in the 10-day than in the 7-day nonPGE2 group.
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tissue was higher on the side where PGE2 was injected. The resorbed cavities on the root surfaces found in this study occurred at the same time as the removal of hyalinized tissue by osteoclastic activity as described by Kvam (1972), Reitan (1974), Rygh (1974), and King and Fischlschweiger (1982). Furthermore, the resorbed areas increased with longer experimental periods. The largest resorbed area occurred in the 10-day group in both PGE2and non-PGE2-injected groups. The observation that the mean resorbed area seen in specimens with local application of PGE2 combined with orthodontic tooth movement was not significantly different from the group where only force was applied, supports the work by Kalange et al. (1988). However, the present investigation indicated a trend towards more root resorption in animals with local application of PGE2. In the present investigation, the number of animals in each group was relatively small and the results from the statistical analysis should be evaluated with care. Trauma to periodontal tissue during orthodontic tooth movement results in vascular changes and damage to blood vessels (Rygh, 1973). It has been assumed that the inflammatory reactions cause release of prostaglandins and other factors (Harris, 1973; Davidovitch and Shanfield, 1975, 1980; King and Thiems, 1979; Yamasaki, 1983; Yamasaki et al., 1980).
261
262 In conclusion, local administration of PGE2 in connection with orthodontic tooth movement does not give less tissue damage in the form of root resorption. On the contrary, local application of PGE2 combined with orthodontic tooth movement, although not significantly increasing root resorption, seems to involve a higher risk. Address for correspondence
Dr P. Brudvik Department of Orthodontics and Facial Orthopedics School of Dentistry University of Bergen Arstadveien 17 N-5009 Bergen Norway Acknowledgements
References Chumblay A B, Tuncay O C 1986 The effect of indomethacin (an aspirin-like drug) on the rate of orthodontic tooth movement. American Journal of Orthodontics 89: 312-314 Davidovitch Z, Shanfeld J 1975 Cyclic AMP levels in alveolar bone of orthodontically treated cats. Archives of Oral Biology 20: 567-574 Davidovitch Z, Shanfeld J L 1980 Prostaglandin E2 (PGE2) in alveolar bone of orthodontically treated cats. Journal of Dental Research 59: 977 Davidovitch Z, Nicolay O, Alley K, Zwilling B, Lanese B, Shanfeld J L 1989 First and second messenger interactions in stressed connective tissue in vivo. In: Norton L A, Burstone C F (eds) The biology of tooth movement. CRC Press, Boca Raton, Florida, p. 97 Goodson J M, McClatchy K, Revell C 1974 Prostaglandin induced resorption of the adult rat calvarium. Journal of Dental Research 53: 670-677 Grevstad J H 1987 Experimentally induced resorption cavities in rat molars. Scandinavian Journal of Dental Research 95: 428-440 Harell A, Dekel S, Bindermann I 1977 Biochemical effect of mechanical stress on cultured bone cells. Calcified Tissue Research 22: 5202-5209
Harris M 1973 Prostaglandin production and bone resorption by dental cysts. Nature 245: 213-215 Kalange J T, Ferguson D J, Meyer R A 1988 The effect of prostaglandin E2 and L-thyroxine on experimental orthodontic tooth movement in Cavia procellus. Thesis abstract, Marquette University, Milwaukee King GJ, Thiems S 1979 Chemical mediation of bone resorption induced by tooth movement in rat. Archives of Oral Biology 24: 811-815 King GJ, Fischlschweiger W 1982 The effect of force magnitude on extractable bone resorptive activity and cemental cratering in orthodontic tooth movement. Journal of Dental Research 61: 775-779 Klein D C, Raisz L G 1970 Prostaglandins: stimulation of bone resorption in tissue culture. Endocrinology 86: 1436-1440 Kyam E 1969 Study of the cell-free zone following experimental tooth movement in the rat. Transactions of the European Orthodontic Society 419-434 Kvam E 1972 Cellular dynamics on the pressure side of the rat periodontium following experimental tooth movement. Scandinavian Journal of Dental Research 80: 369-383 Macapanpan L C, Weinmann J P, Brodie A G 1954 Early tissue changes following tooth movement in rats. The Angle Orthodontist 24: 79-95 Ngan P W, Crock B, Varghese J, Lanese R, Shanfield J 1, Davidovitch Z 1988 Immunohistochemical assessment of the effect of chemical and mechanical stimuli on cAMP and prostaglandin E levels in human gingival fibroblasts in vitro. Archives of Oral Biology 33: 163-174 Oppenheim A 1936 Biological orthodontic therapy and reality. The Angle Orthodontist 6: 153-183 Reitan K 1951 The initial tissue reaction incident to orthodontic tooth movement as related to the influence of function. Thesis, University of Oslo Reitan K 1952 Tissue changes following experimental tooth movement as related to the time factor. Transactions of the European Orthodontic Society 176-189 Reitan K 1974 Initial tissue behavior during apical root resorption. The Angle Orthodontist 44: 68-82 Roberts WE, Goodwin W C, Heiner SR 1981 Cellular response to orthodontic forces. Dental Clinic of North America 25: 3-17 Rygh P 1973 Ultrastructural changes in pressure zones of human periodontium incident to orthodontic tooth movement. Acta Odontologica Scandinavica 31: 109-122 Rygh P 1974 Hyalinization of the periodontal ligament incident to orthodontic tooth movement. Thesis, University of Bergen Rygh P 1977 Orthodontic root resorption studied by electron microscopy. The Angle Orthodontist 47: 1-16 Sandy J R, Harris M 1984 Prostaglandins and tooth movement. European Journal of Orthodontics 6: 175-182 Somjen D, Binderman I, Berger E, Harell A 1980 Bone remodelling induced by physical stress is prostaglandin mediated. Biochimica Biophysica Acta 627: 91-100 Williams S 1984 A histomorphometric study of orthodontically induced root resorption. European Journal of Orthodontics 6: 35-47
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This study was supported by a grant from The Norwegian Council for Science and The Humanities (NAVF). The authors wish to express their gratitude to engineer Rita Eriksen for histological tissue processing, to Dr odont. Nils Roar Gjerdet for his aid in developing a computer program for area calculation and to Dr Olav Bee for advice on the statistical analysis.
P. BRUDVIK AND PER RYGH
ROOT RESORPTION AFTER INJECTION OF PGE2 Yamasaki K. 1983 The role of cyclic AMP, calcium and prostaglandins in the induction of osteoclastic bone resorption associated with experimental tooth movement. Journal of Dental Research 62: 877-881 Yamasaki K, Miura F, Suda T 1980 Prostaglandin as a mediator of bone resorption induced by experimental tooth movement in rats. Journal of Dental Research 59: 1635-1642 Yamasaki K, Shibata Y, Fukuhara T 1982 The effect of prostaglandins on experimental tooth movement in monkeys (Macaca fuscata). Journal of Dental Research 61: 1444-1446 Yamasaki K, Shibata Y, Imai S, Tani Y, Shibasaki Y, Fukhara T 1984 Clinical application of prostaglandin E,
263 (PGE,) upon orthodontic tooth movement. American Journal of Orthodontics 85: 508-518 Yeh C K, Rodan G A 1984 Tensile forces enhance prostaglandin E synthesis in osteoblastic cell grown on collagen ribbons. Calcifed Tissue International 36: 567-571 Yen EHK, Rygh P, Suga DM 1987 Increase of osteoclast number by prostaglandin E2 in orthodontically stress periodontium in vitro. Journal of Dental Research 66: Abstract 321 Yu J H, Wells H, Ryan W J, Lloyd WS Jr 1976 Effect of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells. Prostaglandins 12: 501-513
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> 1991 European Orthodontic Society
Root resorption after local injection of prostaglandin E2 during experimental tooth movement Pongsri Brudvik and Per Rygh Department of Orthodontics and Facial Orthopedics, Faculty of Dentistry, University of Bergen, Norway
The purpose of this study was to investigate the occurrence of orthodontic root resorption in connection with local injection of prostaglandin E2 (PGE2). The material consisted of 25 male Wistar rats. The control group comprised six animals where no force was applied. In five animals 0.1 ml of 0.1 /jg//*l PGE2 was injected in the gingival area of the upper right first molar. In one animal no PGE2 was injected. The animals were killed after 3 days. The experimental tooth movement groups consisted of 19 animals. Duration of experiments was 3 days, 7 days, and 10 days. The maxillary first molars on both sides were each moved mesially by means of a coil spring. On the right side 0.1 ml of PGE2 0.1 /xg///l was injected in the gingiva on the buccal side of the upper first molar on days 0, 3, 5, and 7. On the left side no injection of PGE2 was performed. In three animals in the 7-day group the vehicle (Waymouth medium) was injected. There was no significant difference in root resorption between the experimentally moved teeth with and without local injection of PGE2, but a trend towards more root resorption was registered on the teeth where such injections had been performed. SUMMARY
In tooth movement, the primary stimulus to evoke bone resorption and apposition is physical, but the mechanism for transcription of the forces has not been established. It seems that the responses to mechanical stress are a result of a number of factors acting in concert: neural, vascular, and bioelectric (Davidovitch et al., 1989). The involvement of prostaglandins and the cyclic AMP pathway is well established (Harell et al., 1977; Somjen et al., 1980; Yeh and Rodan 1984; Ngan et al., 1988). It has been suggested that mechanical distortion of the cell initiates PGE2 synthesis from membrane phospholipids; subsequent binding of extracellular PGE2 to cell surface receptors activates adenylate cyclase and the cAMP pathway. This mechanism enables PGE2 to reactivate the cell of origin as well as adjacent cells, thereby amplifying the initial signal. Evidence has been presented indicating that prostaglandin plays an important role in orthodontic tooth movement. Based on measurements on treated humans and monkeys, Yamasaki et al. (1982, 1984) reported that local
injection of prostaglandin (PGEi or PGE2), accelerates the rate of tooth movement. It was assumed that bone resorption was facilitated. This was based on the observation that more osteoclasts were seen in the inter-radicular area after local injections of PGEj or PGE2 near the first rat molars that had not been moved experimentally (Yamasaki et al., 1980). Sandy and Harris (1984) found significant decrease in the number of osteoclasts and a trend towards decreased tooth movement in rabbits where prostaglandin synthesis was inhibited by flurbiprofen. Blocking prostaglandin synthesis by indomethacin resulted in significantly slower tooth movement in cats (Chumbley and Tuncay, 1986). Besides the remodelling of supporting structures (Reitan 1951; Macapanpan et al., 1954; Roberts et al., 1981) dental tissues may also be involved during orthodontic tooth movement. A serious sequel which occurs in tooth movement is resorption of cementum and dentine (Oppenheim, 1936; Kvam, 1969, 1972; Rygh, 1974, 1977; Reitan, 1974; King and Fischlschweiger, 1982; Williams, 1984). While the influence of local injection of pros-
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Introduction
256
P. BRUDVIK AND PER RYGH
taglandins on bone resorption and the rate of orthodontic tooth movement has been quite extensively studied, the possible influence on root resorption has not been clearly documented. The purpose of this study was to investigate the occurrence of root resorption after local injection of PGE 2 during experimental tooth movement.
Downloaded from by guest on January 3, 2015
was no further activation of force during the experimental period. Immediately after the force had been applied, PGE 2 , the same dose as used for the control group, was injected into the buccal gingival area of upper right first molar. In the 3-day group PGE 2 was injected only on the operation day (day zero). In the 7-day group PGE 2 was injected on day 0, 3, and 5. In the 10-day group PGE 2 was injected on days 0, 3, 5, and 7. Material and methods No injection was performed on the left side of animals in the 3- and 10-day groups. The material consisted of 25 male Wistar rats, weighing 165 ± 20 g. In order to study the effect of the injection per se Waymouth medium (used as vehicle in The control group without force or tooth the PGE 2 solutions) was injected into the left movement comprised six animals. In one animal side of three out of seven animals from the where no PGE 2 was injected, both left and right 7-day group. The injections of vehicle were done molars were used as pure control specimens. In at the same time as the PGE 2 injection on the five animals 0.1 ml of 0.1 //g//zl PGE 2 solution right side (day 0, 3, 5). In the remaining four was injected into the buccal gingival area of maxillary right first molar. The PGE 2 was dis- < animals, no injection was performed on the left side. solved in ethanol initially and then diluted in Waymouth's medium at concentration 0.1 ng/ All operations were performed under general /A (Yen et al., 1987). The left sides were used anaesthesia [Dormicum (F. Hoffmann-La as control. Roche & Co. AG, Basel, Switzerland)/Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) dosPGE 2 injection was performed on day zero age 0.15-0.2 ml/100 g body weight]. The and the animals were killed after 3 days. animals were killed by perfusion under an overThe material with experimental tooth movedose of Dormicum/Hypnorm (Grevstad, 1987). ment consisted of 19 animals. Experimental After perfusion, the right and left maxillary periods were 3, 7, and 10 days. These groups molar segments were dissected out and fixed. contained six, seven, and six animals, After decalcification in 0.25 M EDTA, pH 7.2, respectively. each specimen was split mesio-distally by a Both maxillary first molars were moved mesirazor blade. The buccal part was prepared for ally by means of coil spring ligated to each light microscopy and the palatal part was premolar and through eyelets soldered to an incisor pared for electron microscopy. In this paper, band (Fig. 1). The applied force was 50 g. There only the results from light microscopy will be presented. Parasagittal sections of the mesiodistal aspect of the teeth were cut at 6 fim, and stained with haematoxylin and eosin (Fig. 2a). The middle section containing an interradicular area of 855 x 380 fim2 on the mesial surface of the middle or disto-buccal root of the first molar was chosen as a basis for the histological investigation (Fig. 2a). The specimens where root length was less than 855 fim were omitted. Out of 25 animals (50 specimens), 41 specimens complied with our criteria (Table 1). For each specimen, every second section of Figure 1 The appliance consisting of a closed coil spring an overall thickness of 420 fim, 210 ^m apart ligated between upper first molar and an eyelet of an incisor band. from the base section on each side, was exam-
257
ROOT RESORPTION AFTER INJECTION OF PGE 2
Table 1 Mean root absorbed areas (Res. area, Aim2) and the depth of the resorbed cavities (/jm) in the groups without PGE 2 injection (non-PGE 2 ) and with PGE 2 injection (PGE 2 ). N= total number of specimens. Res. spec. = number of specimens with resorption. SD = standard deviation. Contr. = control. Exp. = Experimental tooth movement. Non-PGE 2 . N
Res. spec.
Contr. 3d
6
0
Exp. 3d 7d lOd
5 5 4
0 4 1
Res. area JP(SD)
13 712 (20642) 31637(63275)
PGE 2 Depth AXSD)
30.9 (30.9) 8.0 (17.0)
/'-value
N
Res. spec.
5
0
5 6 5
2 6 4
Res. area JP(SD)
Depth JP(SD)
Area
Depth
138 (206) 37 452(34339) 74000(85249)
6.4 (9.2) 29.9 (10.2) 17.8 (11.7)
ns1 ns3
ns 2 ns*
ns = Not significant (/" = 0.1709; P 2 = 0.7150; P3 = 0.2207; P* = 0.4724).
M b. Figure 2 Areas examined histologically. D: distal; M: mesial; B: buccal; L: lingual; R: resorption. (a) The designed length (855 fim), width (380 pun) at the inter-radicular area. The total length (L, + L 2 ) and depth (d.) of resorption lacunae. Direction of tooth movement (big arrow), (b) Overall width of area being examined (420 fim). Total resorbed area (R) = YJ=* LaC W,(fim2), where Z.ol = total resorbed length from each investigated section; wt = thickness between each investigated section (12 fim).
ined (Fig. 2b). This meant that the overall thickness included 70 sections out of which 35 were examined. In specimens where resorption occurred, the total length of resorbed surfaces from every second section (at 12//m interval) in the given area were measured directly by means of a micrometer. The length was measured parallel to the root surface. The depth of each resorption lacuna was measured at the deepest point by using the distance from the bottom of the cavity tangent to the line passing the intact root surface from both sides of the
[zd1
tions. The statistics T= /—— and the paired
V In
Mest were used. Results The value of 7(7=0.798) indicated an acceptable method error. Similarly, the small /-value (J=0.02, SD= 1.13, * = 0.17, P=0.86) indicated only insignificant systematic errors. Control group (Fig. 3a, b) In the control group where no force was applied, neither active resorption nor hyalinized tissue was registered. There was no apparent histological difference between the non-injected control group and the group with PGE2 injection (Fig. 3a, b). 3-day group (Fig. 3c, d) In all specimens where orthodontic force had been applied, hyalinized tissue was observed.
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B
cavity (Fig. 2a). For each specimen, only the deepest lacuna was recorded. A total of 1435 sections were examined. Resorbed cavities were recorded in 251 sections in 17 specimens (Table 1). The total resorbed area for each specimen was computerized by adding all resorbed surface areas from each registration. Since the distance between each registration was small (12 /mi), only rectangular areas were calculated. A hyalinized zone was recorded as being present or absent. Method error and the systematic error were evaluated by double measurements in 102 sec-
258
P. BRUDVIK AND PER RYGH
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Figure 3 Inter-radicular area of upper first molars in the 3-day specimens. B, bone; PL, periodontal ligament; H, hyalinized tissue, C, cementum, D, dentin; R, resorption. Big arrows indicate the direction of tooth movement, (a) control group without injection, (b) control group with PGE2 injection, (c) 3-day group with only orthodontic experimental force, (d) 3-day group with force + PGE2 injection.
No root resorption was observed in specimens where only force had been applied (Fig. 3c). In two of the specimens with both force and PGE2, a small resorbed cavity had developed at the periphery of the hyalinized tissue (Fig. 3d). Cavity depth was small (Fig. 7). 7-day group (Fig. 4a-d) Most specimens demonstrated pronounced resorptive activity on bone as well as on root surfaces. Root resorption was found in all specimens, except one, in varying degrees (Table 1). The total areas of root resorption of the specimens with and without vehicle injection (non-PGE2 injected specimens) in the 7-day group were compared. The amount of resorp-
tion found in specimens where vehicle had been injected were not different from specimens without such injection. The results indicated that trauma from injection did not influence the resorptive processes. Based upon this finding, the specimens with only vehicle injection were included in the group without PGE2 injection. The mean area of root resorption was 13 172 /an2 for non-PGE2 injected specimens. The corresponding area of PGE2 injected specimens was 37 542 /on2. Contra-lateral specimens could be compared in four animals. Among these, three animals demonstrated more extensive resorption on the PGE2 injected sides. Furthermore, two of the non-PGE2 injected specimens showed very small
ROOT RESORPTION AFTER INJECTION OF PGE 2
259
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Figure 4 Inter-radicular area of first molars in the 7-day group. B, bone; PL, periodontal ligament; H, hyalinized tissue; C, cementum; D, dentin. Big arrows indicate direction of tooth movement. Odontoclast-like cells (small arrows) in the resorption lacunae, (a) only force, (b) force+ PGE 2 injection, (c) force combined with vehicle injection, (d) force+ PGE 2 injection. N.B. (a) and (b) are contralateral specimen of the same animal.
resorbed areas (350 and 1160/nn2). However, one animal demonstrated more resorption on the non-PGE2- than on the PGE2-injected side. Hyalinized/semihyalinized tissue was observed in 40 and 28 per cent of the cases in non-PGE2- and PGE2-injected groups, respectively. The mean depth of cavities was greatest in the 7-day group (Fig. 7). There was no difference in cavity depth between specimens with and without PGE2 injection (Table 1). 10-day group (Fig. 5a-c) The mean area of root resorption was higher than in the 7-day group (Table 1). In specimens
where only force was applied, extensive resorption was observed in only one specimen. No root resorption was observed on the rest of the specimens without PGE2 injection. In specimens where both force and PGE2 had been applied, root resorption was observed in all specimens except one. The mean area of root resorption was 31 637 and 74000/im2 in the non-PGE2- and PGE2injected groups, respectively (Table 1). Hyalinized tissue was still observed in some specimens in both non-PGE2 and PGE2 injected groups. In the 10-day group, contralateral specimens were compared in three animals. No root resorption occurred on the non-PGE2-injected
260
P. BRUDVIK AND PER RYGH
sides. Unfortunately, in the only animal with root resorption on the non-PGE2-injected side, no contralateral specimen was available. The results indicated that the resorbed areas had increased with longer experimental periods (Fig. 6). The mean value of the resorbed areas was higher in groups with local application of PGE2 combined with orthodontic tooth movement than in groups with only orthodontic mechanical force. However, the differences were not statistically significant (Mann-Whitney P = 0.\7 and P=0.22 for the 7- and 10-day group, respectively; Table 1). In the 10-day group, the cavities were about two times deeper in PGE2-injected specimens than in non-PGE2 injected specimens. However, the differences were not significant (Table 1).
'1M000
g^ |
Fore. |
Only
• force
10000 ?
JI
~i r
a
0
I 0
10000
Q
Discussion
The present investigation demonstrated that with the experimental model used, root resorption first occurred on the mesial aspect of the distal root both with and without local application of PGE2. The rate of removal of necrotic
03 0 DURATION
0 70 OF
01O0
FORCE
Idsyil
Figure 6 Histograms showing the mean resorbed area (A") in fim2 from the different experimental groups. Bar lines represent standard deviation (SD).
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Figure 5 Inter-radicular area of first molars in the 10-day group. B, bone; PL, periodontal ligament; H, hyalinized tissue; C, cementum; D, dentin. Big arrows indicate direction of tooth movement. Odontoclast-like cells (small arrows) in the resorptive lacunae, (a) Only force: no root resorption; (b) contra-lateral specimen of animal in (a) where both force and PGE 2 being applied. This specimen represents the most advanced resorption. (c) Only force with extensive root resorption.
ROOT RESORPTION AFTER INJECTION OF PGE2
IMort U.,n
PCE2 vlu.
fO ) I*)
a o
Figure 7 Curve showing the mean depth of the resorbed cavities in the different experimental groups. Each sign represents the depth value for each individual specimen.
Root resorption did not occur in the present control material, but cannot be excluded in rats under physiologic conditions (Grevstad, 1987). Trauma from orthodontic force on pre-existing resorbing lesions on the root surface might enhance the activity resulting in more extensive root resorption. This may explain the extensive resorption which occurred in one of the nonPGE2-injected specimens in the 10-day group. The involvement of prostaglandins in orthodontic tooth movement which has been reported earlier by Yamasaki et al. (1982, 1984), Sandy and Harris (1984), and Chumbley and Tuncay (1986) may be due to a stimulation of precursor cells to differentiate into resorptive cells (Klein and Raisz, 1970; Harris, 1973; Goodson et al., 1974; Yu et al., 1976; Yamasaki et al., 1980; Yen et al., 1987). Since the presence of hyalinized tissue stops orthodontic tooth movement (Reitan, 1952; Kvam, 1972; Rygh, 1974), any increase of the rate of dissolution of hyalinized tissue by degeneration or by phagocytic cell activity through stimulation by an agent such as PGE2, would facilitate tooth movement. When Yamasaki et al. (1984) investigated possible accelerated tooth movement by local injection of PGE2 in orthodontic patients, a wide range of individual response was registered. In general, the individual biological response may vary (Reitan, 1951). Individual tissue response to periodontal trauma incident to the experimental force may explain the wide range of resorption demonstrated in this study. In the present experiments, local injection of PGE2 was found to be technically difficult. Some leakage occurred and the full effect may not have been achieved in all cases. Both resorptive as well as reparative phases were observed. In the lacunae in 10-day group specimens, cementum deposited in the resorbed lacunae during the reparative processes (King and Fischlschweiger, 1982; Grevstad, 1987), may be the reason for the smaller cavity depth in the 10-day experimental period than in the 7-day group. It is possible that in the non-PGE2-injected groups, where there was no chemical stimulation of the resorbed cells, some small resorbed lesions which occurred in the earlier period may have been completely repaired without showing any lesion in the 10-day group. This may be the explanation for the smaller number of resorbed specimens in the 10-day than in the 7-day nonPGE2 group.
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tissue was higher on the side where PGE2 was injected. The resorbed cavities on the root surfaces found in this study occurred at the same time as the removal of hyalinized tissue by osteoclastic activity as described by Kvam (1972), Reitan (1974), Rygh (1974), and King and Fischlschweiger (1982). Furthermore, the resorbed areas increased with longer experimental periods. The largest resorbed area occurred in the 10-day group in both PGE2and non-PGE2-injected groups. The observation that the mean resorbed area seen in specimens with local application of PGE2 combined with orthodontic tooth movement was not significantly different from the group where only force was applied, supports the work by Kalange et al. (1988). However, the present investigation indicated a trend towards more root resorption in animals with local application of PGE2. In the present investigation, the number of animals in each group was relatively small and the results from the statistical analysis should be evaluated with care. Trauma to periodontal tissue during orthodontic tooth movement results in vascular changes and damage to blood vessels (Rygh, 1973). It has been assumed that the inflammatory reactions cause release of prostaglandins and other factors (Harris, 1973; Davidovitch and Shanfield, 1975, 1980; King and Thiems, 1979; Yamasaki, 1983; Yamasaki et al., 1980).
261
262 In conclusion, local administration of PGE2 in connection with orthodontic tooth movement does not give less tissue damage in the form of root resorption. On the contrary, local application of PGE2 combined with orthodontic tooth movement, although not significantly increasing root resorption, seems to involve a higher risk. Address for correspondence
Dr P. Brudvik Department of Orthodontics and Facial Orthopedics School of Dentistry University of Bergen Arstadveien 17 N-5009 Bergen Norway Acknowledgements
References Chumblay A B, Tuncay O C 1986 The effect of indomethacin (an aspirin-like drug) on the rate of orthodontic tooth movement. American Journal of Orthodontics 89: 312-314 Davidovitch Z, Shanfeld J 1975 Cyclic AMP levels in alveolar bone of orthodontically treated cats. Archives of Oral Biology 20: 567-574 Davidovitch Z, Shanfeld J L 1980 Prostaglandin E2 (PGE2) in alveolar bone of orthodontically treated cats. Journal of Dental Research 59: 977 Davidovitch Z, Nicolay O, Alley K, Zwilling B, Lanese B, Shanfeld J L 1989 First and second messenger interactions in stressed connective tissue in vivo. In: Norton L A, Burstone C F (eds) The biology of tooth movement. CRC Press, Boca Raton, Florida, p. 97 Goodson J M, McClatchy K, Revell C 1974 Prostaglandin induced resorption of the adult rat calvarium. Journal of Dental Research 53: 670-677 Grevstad J H 1987 Experimentally induced resorption cavities in rat molars. Scandinavian Journal of Dental Research 95: 428-440 Harell A, Dekel S, Bindermann I 1977 Biochemical effect of mechanical stress on cultured bone cells. Calcified Tissue Research 22: 5202-5209
Harris M 1973 Prostaglandin production and bone resorption by dental cysts. Nature 245: 213-215 Kalange J T, Ferguson D J, Meyer R A 1988 The effect of prostaglandin E2 and L-thyroxine on experimental orthodontic tooth movement in Cavia procellus. Thesis abstract, Marquette University, Milwaukee King GJ, Thiems S 1979 Chemical mediation of bone resorption induced by tooth movement in rat. Archives of Oral Biology 24: 811-815 King GJ, Fischlschweiger W 1982 The effect of force magnitude on extractable bone resorptive activity and cemental cratering in orthodontic tooth movement. Journal of Dental Research 61: 775-779 Klein D C, Raisz L G 1970 Prostaglandins: stimulation of bone resorption in tissue culture. Endocrinology 86: 1436-1440 Kyam E 1969 Study of the cell-free zone following experimental tooth movement in the rat. Transactions of the European Orthodontic Society 419-434 Kvam E 1972 Cellular dynamics on the pressure side of the rat periodontium following experimental tooth movement. Scandinavian Journal of Dental Research 80: 369-383 Macapanpan L C, Weinmann J P, Brodie A G 1954 Early tissue changes following tooth movement in rats. The Angle Orthodontist 24: 79-95 Ngan P W, Crock B, Varghese J, Lanese R, Shanfield J 1, Davidovitch Z 1988 Immunohistochemical assessment of the effect of chemical and mechanical stimuli on cAMP and prostaglandin E levels in human gingival fibroblasts in vitro. Archives of Oral Biology 33: 163-174 Oppenheim A 1936 Biological orthodontic therapy and reality. The Angle Orthodontist 6: 153-183 Reitan K 1951 The initial tissue reaction incident to orthodontic tooth movement as related to the influence of function. Thesis, University of Oslo Reitan K 1952 Tissue changes following experimental tooth movement as related to the time factor. Transactions of the European Orthodontic Society 176-189 Reitan K 1974 Initial tissue behavior during apical root resorption. The Angle Orthodontist 44: 68-82 Roberts WE, Goodwin W C, Heiner SR 1981 Cellular response to orthodontic forces. Dental Clinic of North America 25: 3-17 Rygh P 1973 Ultrastructural changes in pressure zones of human periodontium incident to orthodontic tooth movement. Acta Odontologica Scandinavica 31: 109-122 Rygh P 1974 Hyalinization of the periodontal ligament incident to orthodontic tooth movement. Thesis, University of Bergen Rygh P 1977 Orthodontic root resorption studied by electron microscopy. The Angle Orthodontist 47: 1-16 Sandy J R, Harris M 1984 Prostaglandins and tooth movement. European Journal of Orthodontics 6: 175-182 Somjen D, Binderman I, Berger E, Harell A 1980 Bone remodelling induced by physical stress is prostaglandin mediated. Biochimica Biophysica Acta 627: 91-100 Williams S 1984 A histomorphometric study of orthodontically induced root resorption. European Journal of Orthodontics 6: 35-47
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This study was supported by a grant from The Norwegian Council for Science and The Humanities (NAVF). The authors wish to express their gratitude to engineer Rita Eriksen for histological tissue processing, to Dr odont. Nils Roar Gjerdet for his aid in developing a computer program for area calculation and to Dr Olav Bee for advice on the statistical analysis.
P. BRUDVIK AND PER RYGH
ROOT RESORPTION AFTER INJECTION OF PGE2 Yamasaki K. 1983 The role of cyclic AMP, calcium and prostaglandins in the induction of osteoclastic bone resorption associated with experimental tooth movement. Journal of Dental Research 62: 877-881 Yamasaki K, Miura F, Suda T 1980 Prostaglandin as a mediator of bone resorption induced by experimental tooth movement in rats. Journal of Dental Research 59: 1635-1642 Yamasaki K, Shibata Y, Fukuhara T 1982 The effect of prostaglandins on experimental tooth movement in monkeys (Macaca fuscata). Journal of Dental Research 61: 1444-1446 Yamasaki K, Shibata Y, Imai S, Tani Y, Shibasaki Y, Fukhara T 1984 Clinical application of prostaglandin E,
263 (PGE,) upon orthodontic tooth movement. American Journal of Orthodontics 85: 508-518 Yeh C K, Rodan G A 1984 Tensile forces enhance prostaglandin E synthesis in osteoblastic cell grown on collagen ribbons. Calcifed Tissue International 36: 567-571 Yen EHK, Rygh P, Suga DM 1987 Increase of osteoclast number by prostaglandin E2 in orthodontically stress periodontium in vitro. Journal of Dental Research 66: Abstract 321 Yu J H, Wells H, Ryan W J, Lloyd WS Jr 1976 Effect of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells. Prostaglandins 12: 501-513
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