0003-9969190 $3.00f0.00
Archs oral Bid. Vol. 35,No. IO,pp. 801406, 1990 Printed in Great Britain. A.11 rights reserved
Copyright 0 1990Pergamon Press plc
THE EFFECT OF CYCLOPHOSPHAMIDE ERUPTION OF IMPEDED AND RESECTED IN RATS
ON THE INCISORS
R. A. BURN-MURDOCH Physiology Division, United Medical and Dental Schools of Guy’s and St Thomas’ Hospitals, St Thomas’ Campus, Lambeth Palace Road, London SE1 7EH, England (Received 18 January 1990; accepted 23 May 1990)
Summary-It
is known that a single dose of cyclophosphamide (40mg’kg-’ intraperitoneal) slows the eruption of unimpeded incisors but has no effect on impeded incisors. In this study incisors that were impeded when cyclophosphamide was given were subsequently made unimpeded. When they were unimpeded they erupted more slowly than the controls. Therefore the changes cyclophosphamide produces which slow eruption are formed in impeded incisors but have an effect only in unimpeded incisors. It has been reported that cyclophosphamide slows the eruption of resected incisors when it is given before the resection. In a second experiment cyclophosphamide was given after the resection and had no effect on eruption rates. It is suggested from these and earlier findings that cyclophosphamide slows eruption by imposing a maximum on the rate at which it can occur and that it does so through the changes it produces in the periodontal ligament. The periodontal changes make it harder for the tooth to move rather than altering the forces moving the tooth. This explanation of how cyclophosphamide slows eruption produces an alternative interpretation of some, but not all, of the findings that have been cited as evidence that there are two mechanisms of tooth eruption. Key words: cyclophosphamide,
tooth eruption.
INTRODUCTION
that are repeatedly shortened to stop them being bitten upon (unimpeded) erupt at 2-3 times the rate of unshortened (impeded) incisors. A single injection of cyclophosphamide at a dose of 40 rng. kg-’ (intraperitoneal) produces a prolonged slowing of the unimpeded eruption rate of rodent incisors without affecting impeded rates (Adatia and Berkovitz, 1981; Burn-Murdoch, 1988). The absence of an effect on impeded rates could be either because cyclophosphamide-induced changes which slow eruption do not occur in impeded incisors or because the changes are present in impeded incisors but effective only in unimpeded incisors. To distinguish between these possibilities, I have now examined incisors that were impeded when cyclophosphamide was given and were subsequently Irendered unimpeded. Cyclophosphamide also slows the eruption of resected incisors (incisors whose growing ends have been removed) when it is given before the resection (Burn-Murdoch, 1988). I have also now examined the effect of giving cyclophosphamide after the resection. Rat incisors
METHODS Unimpeded and impeded eruption rates of the lower incisors of rats were measured by recording the distance of marks on the lower incisor teeth of rats from various anatomical landmarks (reference points) at 2-day intervals, using a calibrated graticule
in a microscope eyepiece under 10 x magnification. The reference points used were the gingival margins of the two incisors and the incisal edge of the impeded
incisors. For all prodedures the rats were anaesthetized by ether. The technique has been described in more detail by Aladdin and Burn-Murdoch (1985b). The weight of the rats was recorded every 2 days. The rats were housed in pairs and the food eaten (taken as the weight of food lost from the feeding bowls and not readily recoverable from the floor of the cages) over each 2-day period was measured. Impeded incisor experiment
The right incisor was made unimpeded by repeatedly shortening it from day 0, the last shortening being made on day 24. The left incisor was unimpeded from day 30 to the end of the experiment (day 40). Cyclophosphamide (40 mg. kg-’ intraperitoneal in 2 ml isotonic saline; Sigma, Poole, England) was given to the 8 experimental rats (initial weight 284 $8(SD) g) on day 8, the 8 controls (285 f 11 g) receiving 2 ml isotonic saline. Resected incisor experiment
The right incisors of all rats were resected by the technique described earlier (Aladdin and BurnMurdoch, 1985a) on day 0 under ketamine anaesthesia (175 mg. kg-’ intraperitoneal; Parke-Davis, Pontypool, England) and erythromycin antibiotic cover (10 mg erythromycin activity as the lactobionate, intraperitoneal; Abbott, Queensborough, England). The right incisor was unimpeded and the left impeded throughout the experiment, which ended on day 20. Cyclophosphamide (40 mg. kg-’ intraperitoneal in 2 ml isotonic saline) was given to the 8 801
R. A. BURN-MUIUKJCH
802
experimental rats (initial weight 283 f 16 g) on day 8, the 8 control rats (weight 286 f 18 g) receiving 2 ml isotonic saline. RESULTS Impeded incisor experiment
According to the criteria of Aladdin and BurnMurdoch (1985b) and their later expansion (BurnMurdoch, 1988), the gingival margin of the left (impeded incisor) gave better estimates of the eruption rates than the other reference points before day 30. After day 30 the gingival margin of the right incisor became the best reference point for the right
Eruption rate of the right incisor (mm day-11
incisor, the margin of the left incisor remaining the best reference point for the left incisor. These are the reference points that are used for the results in Fig. 1, but generally the conclusion of the experiments was not affected by which reference point was used. The results for each 48-h interval were compared by t-tests and, where t-tests were precluded by significant differences in variances, by rank sum tests. Significant differences are indicated by an asterisk in Fig. 1. Comparison of averages taken over several intervals and analysis of variance of all the data allowing for repeated observations in individual rats (Winer, 1971) did not reveal any other significant differences.
0.5
**xx*%
1.5
I
Eruption rate of the left incisor (mm day-II
,,. _
05
-
OO-
Length of the impeded
10
Oistdnce 5.0from the end of the unimpeded 2.5 _ Incisor to the incisal edge 0.0; (mm1
I
xx
LLIT-T
10 9
DAYS RIGHT
I7 1
00
30
20
1
I
LEFT
I
Fig. 1. The results of the impeded incisor experiment. They are the mean f 1 SD, with x representing the 8 experimental rats and 0 the 8 controls. An asterisk indicates there was a significant difference between the two groups. The arrow beneath the time axis indicates when cyclophosphamide, or saline for the controls, was given; the bars indicate which incisor was unimpeded. For the bottom two graphs the right incisor was taken as being unimpeded until day 30. On day 30, two pairs of values for the length of the impeded incisor are given, the first for the left incisor before they were shortened and the second, which shows a significant difference, for the right incisor.
How cyclophosphamide slows eruption
The eruption rate of the left incisor, which was impeded when the cyclophosphamide was given, was slower in the experimental group than in the controls after the incisor was made unimpeded (Fig. 1). The distance between the cut surface of the unimpeded incisor and the incisal edge of the impeded incisor immediately after shortening the incisors on day 30 was significamly less in the experimental than in the control rats (3.6 + 0.5,4.6 f 0.2 mm,p < O.Ol), but there were no significant differences in this measurement after shortening the incisor on any occasion from day 32 to the end of the experiment. The distance between the cut surface of the unimpeded incisor and the incisal edge at the end of each 48-h period was significantly greater in the experimental than in the control rats at each measurement from day 32 to the end of the experiment. The mean distance for each 48-h interval was taken as the average of the distance immediately after shortening the incisor at one measurement and the distance before shortening the incisor at the next measurement. The mean distance showed no significant differences between the groups for any interval after day 30 (Fig. 1). There were no significant differences between the body weights of the two groups, but the gain in weight between days 8 and 10 was significantly less in the experimentals than the controls (5.6 + 3.0, 10.0 &-3.5 g, p < 0.09). The experimental rats ate less food than the controls between days 8 and 10 (19.7 f 1.0, 26.6 &-3.4g.rat-‘.day-‘), but this was not statistically significant. Analysis of variance of the early part of the (experiment revealed a significant (p < 0.05) interaction between days and groups for the amount of food eaten. Apart from this there were no significant differences in the amount of food eaten.
803
TT
Resected incisor experiment
The gingival margins were better reference points than the incisal edge of the impeded incisor. For the left incisor the gingival margin of the left incisor was the best reference point, but the criteria of Aladdin and Burn-Murdoch (1985b) did not significantly distinguish between the two gingival margins as a reference point for the right incisor. The average eruption rate of the resected incisors from days 0 to 20, which includes the slow phases, in 15 of the 16 rats lay between 0.67 and 0.85mm.dayy’. One rat in the experimental group had an average rate of 0.14 mm.day-’ and has been eliminated from the results, though the conclusions are not affected if the findings from this rat are used. The eruption rates are given in Fig. 2 where the margin of the left incisor was taken as the reference point for both incisors. Statistical analysis was as in the first experiment and there were no significant differences between the groups even when averages over several days or analysis of variance were used. There were no signilicant differences when using the measurements from the gingival margin of the right incisor or incisal edge. There were no significant differences between the groups in the amount of food eaten. The rats gained weight throughout the experiment and from days 0 to 8 the weight gains did not differ significantly between the groups (38 + 8, 34 k 5 g) though the experimental
=: 0.0 L f
0
10
20
DAYS
Fig. 2. The results of the resected incisor experiment. The symbols are as in Fig. 1 but there were only 7 experimental rats and the right incisor was the unimpeded one throughout the experiment.
rats gained significantly more weight than the controls between days 6 and 8 (13 + 2,9 + 3 g, p < 0.05). After the cyclophosphamide was given on day 8, the experimental rats gained weight more slowly than the controls, but the change in weight since day 8 showed significant differences between the groups only on days 16 (19 f 7, 33 f 5 g, p < 0.01) and 18 (23 f 8, 36 f 8 g, p < 0.05) and not on days 12, 14 and 20. The difference was not significant on day 10, but one of the control rats was significantly (p < 0.05) outside the normal distribution given by data from the other rats in the group (Lentner, 1982) and if this outlier was excluded the difference became significant (2 + 5, 10 * 5 g, p < 0.05). DISCUSSION
The incisors that were impeded when cyclophosphamide was given erupted more slowly than the controls when they were made unimpeded. This is
804
R. A. BURN-MURDOCH
consistent with the changes produced by cyclophosphamide being present in impeded incisors, but effective only in unimpeded incisors. However, another explanation of the decreased unimpeded rate from days 30 to 40 needs to be considered. When the shortening of the right incisors was stopped they came into occlusion more rapidly in the control rats than in the experimental, whose eruption rate was lower. Therefore the right incisor was shorter in the experimental group than in the controls on day 30, so when the left incisors were shortened the cut surface was nearer to the incisal edge of the right incisor in the experimental than in the control rats. Consequently more force may have been exerted on the unimpeded incisor during oral functions in the experimental group than in the controls. This is not the reason for the reduced unimpeded rate from days 30 to 40 because there was no difference in the length of the impeded (right) incisors from day 32, nor in the distance between the cut surface of the unimpeded (left) incisor and the incisal edge of the impeded incisor (Fig. 1). Also, at the ends of the 48-h intervals the distance between the cut surface and the incisal edge was less in the controls than in the experimental rats because of the difference in eruption rates. The unimpeded rate in the experimental rats after day 30 was less than the rate of the right incisors in the controls from days 28 to 30 (Fig. 1) when the latter were almost fully back into occlusion with extensive wear facets present on all the incisors. Wear facets were not seen on the unimpeded incisors in the experimental group after day 32. Two aspects of the impeded eruption rates require discussion. Firstly, after day 30 there was a significant difference in impeded eruption rates between the groups (Fig. 1) but cyclophosphamide generally does not slow impeded rates (Adatia and Berkovitz, 198 1; Burn-Murdoch, 1988). Secondly, the eruption rate of the left incisor was significantly less in the experimental than the control rats from days 28 to 30, when the left incisor was still impeded. Both the impeded and the unimpeded rates were lower after day 30 than they were between days 20 and 26 and the difference for the left incisor began on day 28 before it was shortened; the low rates may thus be an effect of time, but other explanations are possible. At the end of periods of unimpeded eruption in rabbits and mice there are changes in the eruption rates and lengths of the adjacent impeded incisor (Ness, 1956, 1965). As the unimpeded incisors in my experiments returned to occlusion at different rates, the impeded rate may also have changed at different rates, so explaining the difference in eruption rates for days 28 to 30. During these 2 days the left incisors lengthened more in the control rats than in the experimental (Fig. 1) though this was not statistically significant. The difference in eruption rates was significant only in the measurements taken from the gingival margin of the left incisor, and not in the measurements from the other reference points. A lengthening of the tooth associated with an apparent increase in eruption rates could have been an artefact produced by movement of the gingival margin but the criteria of Aladdin and Burn-Murdoch (1985b) suggested that the margin of the left incisor was generally the most stable reference point. The impeded incisors were shorter after day 30
than the impeded incisors were from days 12 to 26 (Fig. l), which might alter the magnitude of the forces exerted on the teeth or their leverage, but the difference in lengths was the same in the two groups so this is unlikely to be the cause of the difference in impeded eruption rates. The conclusion from the first experiment is that the changes by which cyclophosphamide slows eruption are present in impeded incisors but are effective only in the more rapidly erupting unimpeded incisors. The unimpeded rate is reduced by more than 50% and a reduction of the impeded rate of this size would have produced a significant difference. As repeated injections of cyclophosphamide slow the unimpeded rate more than a single injection and also slow the impeded rate (Adatia and Berkovitz, 1981), I suggest that cyclophosphamide slows eruption by placing a maximum on the rate at which it can occur. The higher is the cumulative dose of cyclophosphamide, the lower is the maximum rate. Incisors that were erupting faster than the maximum will be slowed; those that were erupting more slowly will be unaffected. An alternative hypothesis is that two mechanisms produce eruption, only one of which is sensitive to cyclophosphamide (Moxham and Berkovitz, 1983). This hypothesis explains fewer observations than the maximum speed theory. The two-mechanisms hypothesis was produced to explain the observation that steroids accelerate unimpeded eruption yet after cyclophosphamide is given the slower eruption that is produced is not accelerated by steroids (Moxham and Berkovitz, 1983): one mechanism, it was suggested, is sensitive to cyclophosphamide and steroids and the other is insensitive to these agents. The interaction between cyclophosphamide and steroids is also consistent with the maximum speed hypothesis. After the administration of cyclophosphamide, the incisors would have been erupting at the maximum rate permitted by cyclophosphamide, so no acceleration in response to steroids would have been possible. The absence of an effect of cyclophosphamide on impeded incisors is consistent with the maximum speed hypothesis but could be explained by the two-mechanism hypothesis only if impeded eruption were produced solely by the insensitive mechanism; but steroids would have no effect on impeded eruption which, in fact, they accelerate (Domm and Wellband, 1960; Garren and Greep, 1960; Chiba, Kuroda and Ohshima, 1981; Burn-Murdoch, 1988). After the removal of their growing ends (resection), rat incisors continue to erupt, slowly for a few days, then returning to normal rates (Berkovitz and Thomas, 1969). During the phase of normal rates, resected incisors respond to some drugs in the same way as normal incisors (Berkovitz, 1972; Berkovitz, Migdalski and Solomon, 1972; Chiba and Ohshima, 1985; Burn-Murdoch, 1988), suggesting that their eruption is normal. Cyclophosphamide, given before the resection, has an effect during the phase of normal rates, but not during the initial slow phase (BurnMurdoch, 1988). This is consistent with the maximum speed hypothesis: in the slow phase the eruption rate is less than the maximum so would be unaffected. In the normal phase the eruption would have exceeded the speed limit, so is slowed by cyclophos-
How cyclophosphamide
phamide. According to the two-mechanism hypothesis, the cyclophosphamideand steroid-sensitive mechanism would work only during the normal phase, with the insensitive mechanism producing the slow phase. Procedures intended to alter interstitial pressures in the socket of resected incisors also had an effect only during the normal phase (Aladdin and Burn-Murdoch, 198!ia), leading to the suggestion that interstitial pressures contributed to eruption only in the normal phase and that the periodontal ligament produced the slow phases. The cyclophosphamide-sensitive mechanism then corresponds to interstitial pressure and the insensitive mechanism is in the ligament. Histological studies have shown that cyclophosphamide produces changes in the dentine, pulp and periodontal. ligament (see Koppang, 1973; Adatia and Berkovitz, 198 I). Cyclophosphamide slows eruption in resected incisors (Burn-Murdoch, 1988) in which the pulp is an empty cavity (Berkovitz and Thomas, 1969), which suggests that the cyclophosphamide-sensitive mechanism is in the ligament. This is contrary to what has just been deduced, but this discrepancy assumes that the two mechanisms in the resected incisors have been correctly identified. The observations that procedures intended to alter interstitial pressures and steroids have an effect during the normal hut not the slow phases are still consistent with there being two mechanisms (Aladdin and Burn-Murdoch, 1985a; Burn-Murdoch, 1988). It is possible that both the maximum speed and twomechanisms hypotheses might be correct, but the two would not be cyclophosphamide-sensitive and -insensitive, respectively. The periodontal lesion produced by cyclophosphamide consists of a small region of the ligament where the arrangement of cells and fibres is abnormal (Koppang, 1973; Adatia and Berkovitz, 1981). The lesion is initially near the growing end of the incisor and moves along the ligament as the tooth erupts. There are two ways in which the lesion may be produced and move along the ligament. It has been proposed that the half of the ligament against the tooth moves with the tooth as it erupts and new ligament is constantly formed at the growing end of the incisor (see Beertsen and Hoeben, 1987). The lesion is an abnormal area of ligament formed under the influence of cyclophosphamide which moves because the ligament moves. After resection, cyclophosphamide would not produce any periodontal lesions, but lesions formed before the resection would persist. Berkovitz, Shore and Sloan (1980) suggested that the periodontal ligament did not move. The change produced by cyclophosphamide is a response of the ligament to the abnormal dentine formed under the influence of cyclophosphamide. As the abnormal dentine moves it induces the adjacent ligament to become disorganized while the ligament it has passed returns to normal (Adatia and Berkovitz, 1981). After resection, cyclophosphamide would not produce any changes but abnormal dentine and periodontal changes formed before the resection would persist. This is the same prediction as that of Beertsen and Hoeben’s hypothesis (1987). When cyclophosphamide is given before the resection, the eruption ral:e after the resection is reduced
slows eruption
805
(Burn-Murdoch, 1988). In my second experiment, cyclophosphamide was given after the resection and had no effect on unimpeded eruption rates (Fig. 2). The weight changes show that the cyclophosphamide had an effect on the rats. The dose of cyclophosphamide used would normally have slowed eruption by more than 50% (Adatia and Berkovitz, 1981; Moxham and Berkovitz, 1983; Burn-Murdoch, 1988), which would have produced a significant difference. The latency of cyclophosphamide’s effects on eruption is 2-4 days, so any effects would have occurred while the tooth was still in its normal phase. This is further evidence that cyclophosphamide slows eruption through the periodontal changes. The lesion occupies only a small proportion of the ligament at any time (Adatia and Berkovitz, 1981). When it is formed it is at the growing end and reduces the eruption rate by more than 50%. If the lesion worked by reducing the forces moving the tooth, and assuming that the lesion is no larger than its microscopical appearances indicate, most of the forces moving the tooth would be produced near the growing end of the tooth. Therefore the effect of cyclophosphamide would be lost as the lesion moved along the ligament. but, in fact, its effect persists. Therefore the lesion does not work by reducing the forces moving the tooth but works by making it more difficult for the tooth to move through the tissues, i.e. it increases the resistance to eruption, possibly by interfering with the remodelling of the ligament. If this hypothesis is confirmed it may have wider implications. Many agents are known that alter the rate of eruption, but it is not generally known whether they do so by changing the forces making the tooth erupt or by varying the resistance exerted by the surrounding tissues. It is possible that this distinction may be made by studying interactions between cyclophosphamide and other agents that slow eruption rates. Acknowledgemenfs--I
am grateful to the H. and E. Stroyan Fund for financial support, Ken Applebee and his staff for the care of the rats, Lynn Forrester for typing the manuscript and Bob Burton for the illustrations. REFERENCES
Adatia A. K. and Berkovitz B. K. B. (1981) The effects of cyclophosphamide on eruption of the continuously growing mandibular incisor of the rat. Archs oral Biol. 26, 607413.
Aladdin Q. I. and Burn-Murdoch R. A. (1985a) The effect of procedures intended to alter the interstitial fluid pressure in the sockets of resected rat incisors on their eruption rate. Archs oral Biol. 30, 525-530. Aladdin Q. I. and Burn-Murdoch R. A. (1985b) Techniques for choosing reference points from which to measure eruption rates of rat incisors. Archs oral Biol. 30, 531-537.
Beertsen W. and Hoeben K. A. (1987) Movement of fibroblasts in the periodontal ligament of the mouse incisor is related to eruption. J. dent. Res. 66, 100~1010. Berkovitz B. K. B. (1972) The effect of demecolcine and of triethanomelamine on the unimpeded eruption rate of normal and root-resected incisor teeth in rats. Archs oral Biol. 17, 937-947.
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Berkovitz B. K. B. and Thomas N. R. (1969) Unimpeded eruption in the root-resected lower incisor of the rat with a preliminary note on root transection. Archs oral Biol. 14, 771-780.
Berkovitz B. K. B., Migdalski A. and Solomon M. (1972) The effect of the lathyritic agent aminoacetonitrile on the unimpeded eruption rate in normal and root-resected rat lower incisors. Archs oral Biol. 17, 175551763. Berkovitz B. K. B., Shore R. C. and Sloan P. (1980) Histology of the periodontal ligament of the rat mandibular incisor following root resection, with special reference to the zone of shear. Archs oral Biol. 25, 235-244. Burn-Murdoch R. A. (1988) The effect of corticosteroids and cyclophosphamide on the eruption of resected incisor teeth in the rat. Archs oral Biol. 33, 661-667. Chiba M. and Ohshima S. (1985) Effects of colchicine and hydrocortisone on unimpeded eruption rates of rootresected mandibular incisors of rats. Archs oral Biol. 30, 147-153.
Chiba M., Kuroda T. and Ohshima S. (1981) Effects of adrenocorticoids on impeded and unimpeded eruption rates and on the mechanical properties of the periodontium in the rat mandibular incisor. Archs oral Biol. 26, 577-583.
Domm L. V. and Wellband W. A. (1960) Effect of adrenalectomy and cortisone on eruption rate of incisors in young female albino rats. Proc. Sot. exp. Biol. Med. 104, 582-584. Garren L. and Greep R. 0. (1960) Effect of adrenal cortical hormones on eruption rate of incisor teeth in the rat. Endocrinology 66, 625428.
Koppang H. S. (1973) Histomorphologic investigations on the effect of cyclophosphamide on dentinogenesis of the rat incisor. Stand. J. dent. Res. 81, 383-396. Lentner C. (1982) Geigy ScientiJc Tables, Vol. 2. Ciba Geigy, Basle. Moxham B. J. and Berkovitz B. K. B. (1983) Interactions between thyroxine, hydrocortisone and cyclophosphamide in their effects on the eruption of the rat mandibular incisor. Archs oral Biol. 28, 1083-1087. Ness A. R. (1956) The response of the rabbit mandibular incisor to experimental shortening and to the prevention of its eruption. Proc. R. Sot. B146, 129-154. Ness A. R. (1965) Eruption rates of impeded and unimpeded mandibular incisors of the adult laboratory mouse. Archs oral Biol. 10, 43945 1. Winer B. J. (1971) Statistical Principles in Experimental Design, 2nd edn. McGraw-Hill, New York.
Archs oral Bid. Vol. 35,No. IO,pp. 801406, 1990 Printed in Great Britain. A.11 rights reserved
Copyright 0 1990Pergamon Press plc
THE EFFECT OF CYCLOPHOSPHAMIDE ERUPTION OF IMPEDED AND RESECTED IN RATS
ON THE INCISORS
R. A. BURN-MURDOCH Physiology Division, United Medical and Dental Schools of Guy’s and St Thomas’ Hospitals, St Thomas’ Campus, Lambeth Palace Road, London SE1 7EH, England (Received 18 January 1990; accepted 23 May 1990)
Summary-It
is known that a single dose of cyclophosphamide (40mg’kg-’ intraperitoneal) slows the eruption of unimpeded incisors but has no effect on impeded incisors. In this study incisors that were impeded when cyclophosphamide was given were subsequently made unimpeded. When they were unimpeded they erupted more slowly than the controls. Therefore the changes cyclophosphamide produces which slow eruption are formed in impeded incisors but have an effect only in unimpeded incisors. It has been reported that cyclophosphamide slows the eruption of resected incisors when it is given before the resection. In a second experiment cyclophosphamide was given after the resection and had no effect on eruption rates. It is suggested from these and earlier findings that cyclophosphamide slows eruption by imposing a maximum on the rate at which it can occur and that it does so through the changes it produces in the periodontal ligament. The periodontal changes make it harder for the tooth to move rather than altering the forces moving the tooth. This explanation of how cyclophosphamide slows eruption produces an alternative interpretation of some, but not all, of the findings that have been cited as evidence that there are two mechanisms of tooth eruption. Key words: cyclophosphamide,
tooth eruption.
INTRODUCTION
that are repeatedly shortened to stop them being bitten upon (unimpeded) erupt at 2-3 times the rate of unshortened (impeded) incisors. A single injection of cyclophosphamide at a dose of 40 rng. kg-’ (intraperitoneal) produces a prolonged slowing of the unimpeded eruption rate of rodent incisors without affecting impeded rates (Adatia and Berkovitz, 1981; Burn-Murdoch, 1988). The absence of an effect on impeded rates could be either because cyclophosphamide-induced changes which slow eruption do not occur in impeded incisors or because the changes are present in impeded incisors but effective only in unimpeded incisors. To distinguish between these possibilities, I have now examined incisors that were impeded when cyclophosphamide was given and were subsequently Irendered unimpeded. Cyclophosphamide also slows the eruption of resected incisors (incisors whose growing ends have been removed) when it is given before the resection (Burn-Murdoch, 1988). I have also now examined the effect of giving cyclophosphamide after the resection. Rat incisors
METHODS Unimpeded and impeded eruption rates of the lower incisors of rats were measured by recording the distance of marks on the lower incisor teeth of rats from various anatomical landmarks (reference points) at 2-day intervals, using a calibrated graticule
in a microscope eyepiece under 10 x magnification. The reference points used were the gingival margins of the two incisors and the incisal edge of the impeded
incisors. For all prodedures the rats were anaesthetized by ether. The technique has been described in more detail by Aladdin and Burn-Murdoch (1985b). The weight of the rats was recorded every 2 days. The rats were housed in pairs and the food eaten (taken as the weight of food lost from the feeding bowls and not readily recoverable from the floor of the cages) over each 2-day period was measured. Impeded incisor experiment
The right incisor was made unimpeded by repeatedly shortening it from day 0, the last shortening being made on day 24. The left incisor was unimpeded from day 30 to the end of the experiment (day 40). Cyclophosphamide (40 mg. kg-’ intraperitoneal in 2 ml isotonic saline; Sigma, Poole, England) was given to the 8 experimental rats (initial weight 284 $8(SD) g) on day 8, the 8 controls (285 f 11 g) receiving 2 ml isotonic saline. Resected incisor experiment
The right incisors of all rats were resected by the technique described earlier (Aladdin and BurnMurdoch, 1985a) on day 0 under ketamine anaesthesia (175 mg. kg-’ intraperitoneal; Parke-Davis, Pontypool, England) and erythromycin antibiotic cover (10 mg erythromycin activity as the lactobionate, intraperitoneal; Abbott, Queensborough, England). The right incisor was unimpeded and the left impeded throughout the experiment, which ended on day 20. Cyclophosphamide (40 mg. kg-’ intraperitoneal in 2 ml isotonic saline) was given to the 8 801
R. A. BURN-MUIUKJCH
802
experimental rats (initial weight 283 f 16 g) on day 8, the 8 control rats (weight 286 f 18 g) receiving 2 ml isotonic saline. RESULTS Impeded incisor experiment
According to the criteria of Aladdin and BurnMurdoch (1985b) and their later expansion (BurnMurdoch, 1988), the gingival margin of the left (impeded incisor) gave better estimates of the eruption rates than the other reference points before day 30. After day 30 the gingival margin of the right incisor became the best reference point for the right
Eruption rate of the right incisor (mm day-11
incisor, the margin of the left incisor remaining the best reference point for the left incisor. These are the reference points that are used for the results in Fig. 1, but generally the conclusion of the experiments was not affected by which reference point was used. The results for each 48-h interval were compared by t-tests and, where t-tests were precluded by significant differences in variances, by rank sum tests. Significant differences are indicated by an asterisk in Fig. 1. Comparison of averages taken over several intervals and analysis of variance of all the data allowing for repeated observations in individual rats (Winer, 1971) did not reveal any other significant differences.
0.5
**xx*%
1.5
I
Eruption rate of the left incisor (mm day-II
,,. _
05
-
OO-
Length of the impeded
10
Oistdnce 5.0from the end of the unimpeded 2.5 _ Incisor to the incisal edge 0.0; (mm1
I
xx
LLIT-T
10 9
DAYS RIGHT
I7 1
00
30
20
1
I
LEFT
I
Fig. 1. The results of the impeded incisor experiment. They are the mean f 1 SD, with x representing the 8 experimental rats and 0 the 8 controls. An asterisk indicates there was a significant difference between the two groups. The arrow beneath the time axis indicates when cyclophosphamide, or saline for the controls, was given; the bars indicate which incisor was unimpeded. For the bottom two graphs the right incisor was taken as being unimpeded until day 30. On day 30, two pairs of values for the length of the impeded incisor are given, the first for the left incisor before they were shortened and the second, which shows a significant difference, for the right incisor.
How cyclophosphamide slows eruption
The eruption rate of the left incisor, which was impeded when the cyclophosphamide was given, was slower in the experimental group than in the controls after the incisor was made unimpeded (Fig. 1). The distance between the cut surface of the unimpeded incisor and the incisal edge of the impeded incisor immediately after shortening the incisors on day 30 was significamly less in the experimental than in the control rats (3.6 + 0.5,4.6 f 0.2 mm,p < O.Ol), but there were no significant differences in this measurement after shortening the incisor on any occasion from day 32 to the end of the experiment. The distance between the cut surface of the unimpeded incisor and the incisal edge at the end of each 48-h period was significantly greater in the experimental than in the control rats at each measurement from day 32 to the end of the experiment. The mean distance for each 48-h interval was taken as the average of the distance immediately after shortening the incisor at one measurement and the distance before shortening the incisor at the next measurement. The mean distance showed no significant differences between the groups for any interval after day 30 (Fig. 1). There were no significant differences between the body weights of the two groups, but the gain in weight between days 8 and 10 was significantly less in the experimentals than the controls (5.6 + 3.0, 10.0 &-3.5 g, p < 0.09). The experimental rats ate less food than the controls between days 8 and 10 (19.7 f 1.0, 26.6 &-3.4g.rat-‘.day-‘), but this was not statistically significant. Analysis of variance of the early part of the (experiment revealed a significant (p < 0.05) interaction between days and groups for the amount of food eaten. Apart from this there were no significant differences in the amount of food eaten.
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Resected incisor experiment
The gingival margins were better reference points than the incisal edge of the impeded incisor. For the left incisor the gingival margin of the left incisor was the best reference point, but the criteria of Aladdin and Burn-Murdoch (1985b) did not significantly distinguish between the two gingival margins as a reference point for the right incisor. The average eruption rate of the resected incisors from days 0 to 20, which includes the slow phases, in 15 of the 16 rats lay between 0.67 and 0.85mm.dayy’. One rat in the experimental group had an average rate of 0.14 mm.day-’ and has been eliminated from the results, though the conclusions are not affected if the findings from this rat are used. The eruption rates are given in Fig. 2 where the margin of the left incisor was taken as the reference point for both incisors. Statistical analysis was as in the first experiment and there were no significant differences between the groups even when averages over several days or analysis of variance were used. There were no signilicant differences when using the measurements from the gingival margin of the right incisor or incisal edge. There were no significant differences between the groups in the amount of food eaten. The rats gained weight throughout the experiment and from days 0 to 8 the weight gains did not differ significantly between the groups (38 + 8, 34 k 5 g) though the experimental
=: 0.0 L f
0
10
20
DAYS
Fig. 2. The results of the resected incisor experiment. The symbols are as in Fig. 1 but there were only 7 experimental rats and the right incisor was the unimpeded one throughout the experiment.
rats gained significantly more weight than the controls between days 6 and 8 (13 + 2,9 + 3 g, p < 0.05). After the cyclophosphamide was given on day 8, the experimental rats gained weight more slowly than the controls, but the change in weight since day 8 showed significant differences between the groups only on days 16 (19 f 7, 33 f 5 g, p < 0.01) and 18 (23 f 8, 36 f 8 g, p < 0.05) and not on days 12, 14 and 20. The difference was not significant on day 10, but one of the control rats was significantly (p < 0.05) outside the normal distribution given by data from the other rats in the group (Lentner, 1982) and if this outlier was excluded the difference became significant (2 + 5, 10 * 5 g, p < 0.05). DISCUSSION
The incisors that were impeded when cyclophosphamide was given erupted more slowly than the controls when they were made unimpeded. This is
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consistent with the changes produced by cyclophosphamide being present in impeded incisors, but effective only in unimpeded incisors. However, another explanation of the decreased unimpeded rate from days 30 to 40 needs to be considered. When the shortening of the right incisors was stopped they came into occlusion more rapidly in the control rats than in the experimental, whose eruption rate was lower. Therefore the right incisor was shorter in the experimental group than in the controls on day 30, so when the left incisors were shortened the cut surface was nearer to the incisal edge of the right incisor in the experimental than in the control rats. Consequently more force may have been exerted on the unimpeded incisor during oral functions in the experimental group than in the controls. This is not the reason for the reduced unimpeded rate from days 30 to 40 because there was no difference in the length of the impeded (right) incisors from day 32, nor in the distance between the cut surface of the unimpeded (left) incisor and the incisal edge of the impeded incisor (Fig. 1). Also, at the ends of the 48-h intervals the distance between the cut surface and the incisal edge was less in the controls than in the experimental rats because of the difference in eruption rates. The unimpeded rate in the experimental rats after day 30 was less than the rate of the right incisors in the controls from days 28 to 30 (Fig. 1) when the latter were almost fully back into occlusion with extensive wear facets present on all the incisors. Wear facets were not seen on the unimpeded incisors in the experimental group after day 32. Two aspects of the impeded eruption rates require discussion. Firstly, after day 30 there was a significant difference in impeded eruption rates between the groups (Fig. 1) but cyclophosphamide generally does not slow impeded rates (Adatia and Berkovitz, 198 1; Burn-Murdoch, 1988). Secondly, the eruption rate of the left incisor was significantly less in the experimental than the control rats from days 28 to 30, when the left incisor was still impeded. Both the impeded and the unimpeded rates were lower after day 30 than they were between days 20 and 26 and the difference for the left incisor began on day 28 before it was shortened; the low rates may thus be an effect of time, but other explanations are possible. At the end of periods of unimpeded eruption in rabbits and mice there are changes in the eruption rates and lengths of the adjacent impeded incisor (Ness, 1956, 1965). As the unimpeded incisors in my experiments returned to occlusion at different rates, the impeded rate may also have changed at different rates, so explaining the difference in eruption rates for days 28 to 30. During these 2 days the left incisors lengthened more in the control rats than in the experimental (Fig. 1) though this was not statistically significant. The difference in eruption rates was significant only in the measurements taken from the gingival margin of the left incisor, and not in the measurements from the other reference points. A lengthening of the tooth associated with an apparent increase in eruption rates could have been an artefact produced by movement of the gingival margin but the criteria of Aladdin and Burn-Murdoch (1985b) suggested that the margin of the left incisor was generally the most stable reference point. The impeded incisors were shorter after day 30
than the impeded incisors were from days 12 to 26 (Fig. l), which might alter the magnitude of the forces exerted on the teeth or their leverage, but the difference in lengths was the same in the two groups so this is unlikely to be the cause of the difference in impeded eruption rates. The conclusion from the first experiment is that the changes by which cyclophosphamide slows eruption are present in impeded incisors but are effective only in the more rapidly erupting unimpeded incisors. The unimpeded rate is reduced by more than 50% and a reduction of the impeded rate of this size would have produced a significant difference. As repeated injections of cyclophosphamide slow the unimpeded rate more than a single injection and also slow the impeded rate (Adatia and Berkovitz, 1981), I suggest that cyclophosphamide slows eruption by placing a maximum on the rate at which it can occur. The higher is the cumulative dose of cyclophosphamide, the lower is the maximum rate. Incisors that were erupting faster than the maximum will be slowed; those that were erupting more slowly will be unaffected. An alternative hypothesis is that two mechanisms produce eruption, only one of which is sensitive to cyclophosphamide (Moxham and Berkovitz, 1983). This hypothesis explains fewer observations than the maximum speed theory. The two-mechanisms hypothesis was produced to explain the observation that steroids accelerate unimpeded eruption yet after cyclophosphamide is given the slower eruption that is produced is not accelerated by steroids (Moxham and Berkovitz, 1983): one mechanism, it was suggested, is sensitive to cyclophosphamide and steroids and the other is insensitive to these agents. The interaction between cyclophosphamide and steroids is also consistent with the maximum speed hypothesis. After the administration of cyclophosphamide, the incisors would have been erupting at the maximum rate permitted by cyclophosphamide, so no acceleration in response to steroids would have been possible. The absence of an effect of cyclophosphamide on impeded incisors is consistent with the maximum speed hypothesis but could be explained by the two-mechanism hypothesis only if impeded eruption were produced solely by the insensitive mechanism; but steroids would have no effect on impeded eruption which, in fact, they accelerate (Domm and Wellband, 1960; Garren and Greep, 1960; Chiba, Kuroda and Ohshima, 1981; Burn-Murdoch, 1988). After the removal of their growing ends (resection), rat incisors continue to erupt, slowly for a few days, then returning to normal rates (Berkovitz and Thomas, 1969). During the phase of normal rates, resected incisors respond to some drugs in the same way as normal incisors (Berkovitz, 1972; Berkovitz, Migdalski and Solomon, 1972; Chiba and Ohshima, 1985; Burn-Murdoch, 1988), suggesting that their eruption is normal. Cyclophosphamide, given before the resection, has an effect during the phase of normal rates, but not during the initial slow phase (BurnMurdoch, 1988). This is consistent with the maximum speed hypothesis: in the slow phase the eruption rate is less than the maximum so would be unaffected. In the normal phase the eruption would have exceeded the speed limit, so is slowed by cyclophos-
How cyclophosphamide
phamide. According to the two-mechanism hypothesis, the cyclophosphamideand steroid-sensitive mechanism would work only during the normal phase, with the insensitive mechanism producing the slow phase. Procedures intended to alter interstitial pressures in the socket of resected incisors also had an effect only during the normal phase (Aladdin and Burn-Murdoch, 198!ia), leading to the suggestion that interstitial pressures contributed to eruption only in the normal phase and that the periodontal ligament produced the slow phases. The cyclophosphamide-sensitive mechanism then corresponds to interstitial pressure and the insensitive mechanism is in the ligament. Histological studies have shown that cyclophosphamide produces changes in the dentine, pulp and periodontal. ligament (see Koppang, 1973; Adatia and Berkovitz, 198 I). Cyclophosphamide slows eruption in resected incisors (Burn-Murdoch, 1988) in which the pulp is an empty cavity (Berkovitz and Thomas, 1969), which suggests that the cyclophosphamide-sensitive mechanism is in the ligament. This is contrary to what has just been deduced, but this discrepancy assumes that the two mechanisms in the resected incisors have been correctly identified. The observations that procedures intended to alter interstitial pressures and steroids have an effect during the normal hut not the slow phases are still consistent with there being two mechanisms (Aladdin and Burn-Murdoch, 1985a; Burn-Murdoch, 1988). It is possible that both the maximum speed and twomechanisms hypotheses might be correct, but the two would not be cyclophosphamide-sensitive and -insensitive, respectively. The periodontal lesion produced by cyclophosphamide consists of a small region of the ligament where the arrangement of cells and fibres is abnormal (Koppang, 1973; Adatia and Berkovitz, 1981). The lesion is initially near the growing end of the incisor and moves along the ligament as the tooth erupts. There are two ways in which the lesion may be produced and move along the ligament. It has been proposed that the half of the ligament against the tooth moves with the tooth as it erupts and new ligament is constantly formed at the growing end of the incisor (see Beertsen and Hoeben, 1987). The lesion is an abnormal area of ligament formed under the influence of cyclophosphamide which moves because the ligament moves. After resection, cyclophosphamide would not produce any periodontal lesions, but lesions formed before the resection would persist. Berkovitz, Shore and Sloan (1980) suggested that the periodontal ligament did not move. The change produced by cyclophosphamide is a response of the ligament to the abnormal dentine formed under the influence of cyclophosphamide. As the abnormal dentine moves it induces the adjacent ligament to become disorganized while the ligament it has passed returns to normal (Adatia and Berkovitz, 1981). After resection, cyclophosphamide would not produce any changes but abnormal dentine and periodontal changes formed before the resection would persist. This is the same prediction as that of Beertsen and Hoeben’s hypothesis (1987). When cyclophosphamide is given before the resection, the eruption ral:e after the resection is reduced
slows eruption
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(Burn-Murdoch, 1988). In my second experiment, cyclophosphamide was given after the resection and had no effect on unimpeded eruption rates (Fig. 2). The weight changes show that the cyclophosphamide had an effect on the rats. The dose of cyclophosphamide used would normally have slowed eruption by more than 50% (Adatia and Berkovitz, 1981; Moxham and Berkovitz, 1983; Burn-Murdoch, 1988), which would have produced a significant difference. The latency of cyclophosphamide’s effects on eruption is 2-4 days, so any effects would have occurred while the tooth was still in its normal phase. This is further evidence that cyclophosphamide slows eruption through the periodontal changes. The lesion occupies only a small proportion of the ligament at any time (Adatia and Berkovitz, 1981). When it is formed it is at the growing end and reduces the eruption rate by more than 50%. If the lesion worked by reducing the forces moving the tooth, and assuming that the lesion is no larger than its microscopical appearances indicate, most of the forces moving the tooth would be produced near the growing end of the tooth. Therefore the effect of cyclophosphamide would be lost as the lesion moved along the ligament. but, in fact, its effect persists. Therefore the lesion does not work by reducing the forces moving the tooth but works by making it more difficult for the tooth to move through the tissues, i.e. it increases the resistance to eruption, possibly by interfering with the remodelling of the ligament. If this hypothesis is confirmed it may have wider implications. Many agents are known that alter the rate of eruption, but it is not generally known whether they do so by changing the forces making the tooth erupt or by varying the resistance exerted by the surrounding tissues. It is possible that this distinction may be made by studying interactions between cyclophosphamide and other agents that slow eruption rates. Acknowledgemenfs--I
am grateful to the H. and E. Stroyan Fund for financial support, Ken Applebee and his staff for the care of the rats, Lynn Forrester for typing the manuscript and Bob Burton for the illustrations. REFERENCES
Adatia A. K. and Berkovitz B. K. B. (1981) The effects of cyclophosphamide on eruption of the continuously growing mandibular incisor of the rat. Archs oral Biol. 26, 607413.
Aladdin Q. I. and Burn-Murdoch R. A. (1985a) The effect of procedures intended to alter the interstitial fluid pressure in the sockets of resected rat incisors on their eruption rate. Archs oral Biol. 30, 525-530. Aladdin Q. I. and Burn-Murdoch R. A. (1985b) Techniques for choosing reference points from which to measure eruption rates of rat incisors. Archs oral Biol. 30, 531-537.
Beertsen W. and Hoeben K. A. (1987) Movement of fibroblasts in the periodontal ligament of the mouse incisor is related to eruption. J. dent. Res. 66, 100~1010. Berkovitz B. K. B. (1972) The effect of demecolcine and of triethanomelamine on the unimpeded eruption rate of normal and root-resected incisor teeth in rats. Archs oral Biol. 17, 937-947.
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Berkovitz B. K. B. and Thomas N. R. (1969) Unimpeded eruption in the root-resected lower incisor of the rat with a preliminary note on root transection. Archs oral Biol. 14, 771-780.
Berkovitz B. K. B., Migdalski A. and Solomon M. (1972) The effect of the lathyritic agent aminoacetonitrile on the unimpeded eruption rate in normal and root-resected rat lower incisors. Archs oral Biol. 17, 175551763. Berkovitz B. K. B., Shore R. C. and Sloan P. (1980) Histology of the periodontal ligament of the rat mandibular incisor following root resection, with special reference to the zone of shear. Archs oral Biol. 25, 235-244. Burn-Murdoch R. A. (1988) The effect of corticosteroids and cyclophosphamide on the eruption of resected incisor teeth in the rat. Archs oral Biol. 33, 661-667. Chiba M. and Ohshima S. (1985) Effects of colchicine and hydrocortisone on unimpeded eruption rates of rootresected mandibular incisors of rats. Archs oral Biol. 30, 147-153.
Chiba M., Kuroda T. and Ohshima S. (1981) Effects of adrenocorticoids on impeded and unimpeded eruption rates and on the mechanical properties of the periodontium in the rat mandibular incisor. Archs oral Biol. 26, 577-583.
Domm L. V. and Wellband W. A. (1960) Effect of adrenalectomy and cortisone on eruption rate of incisors in young female albino rats. Proc. Sot. exp. Biol. Med. 104, 582-584. Garren L. and Greep R. 0. (1960) Effect of adrenal cortical hormones on eruption rate of incisor teeth in the rat. Endocrinology 66, 625428.
Koppang H. S. (1973) Histomorphologic investigations on the effect of cyclophosphamide on dentinogenesis of the rat incisor. Stand. J. dent. Res. 81, 383-396. Lentner C. (1982) Geigy ScientiJc Tables, Vol. 2. Ciba Geigy, Basle. Moxham B. J. and Berkovitz B. K. B. (1983) Interactions between thyroxine, hydrocortisone and cyclophosphamide in their effects on the eruption of the rat mandibular incisor. Archs oral Biol. 28, 1083-1087. Ness A. R. (1956) The response of the rabbit mandibular incisor to experimental shortening and to the prevention of its eruption. Proc. R. Sot. B146, 129-154. Ness A. R. (1965) Eruption rates of impeded and unimpeded mandibular incisors of the adult laboratory mouse. Archs oral Biol. 10, 43945 1. Winer B. J. (1971) Statistical Principles in Experimental Design, 2nd edn. McGraw-Hill, New York.
