SCIENTIFIC ARTICLES
Changes in Tooth Mobility after Experimental Replantation
F. H. Barbakow, BDS, HDD; P. E. Cleaton-Jones, BDS, MB, BCh, PhD, DA; J. C. Austin, BVSc; J. O. Andreasen, DDS; and E. Vieira, Dip Micro, Johannesburg, South Africa
Maxillary central incisors were r e p l a n t e d in ten v e r v e t m o n k e y s after an extra-alveolar time (EAT) of two h o u r s d u r i n g w h i c h the teeth were kept moist with a saline solution. I n t r a m u s c u l a r t h y r o c a l c i t o n i n was a d m i n i s t e r e d to the e x p e r i m e n t a l g r o u p , b u t not to the control g r o u p to assess the effect of t h y r o c a l c i t o n i n o n the p o s t o p e r a t i v e ankylosis, which was e x p e c t e d to o c c u r after an E A T of two hours. T h e p r e o p e r a t i v e m o b i l i t y was n o t e d in all of the t e e t h that w e r e p l a n n e d for r e p l a n t a t i o n b y a t e c h n i q u e k n o w n as p e r i o d o n t o m e t r y . T h e p o s t o p e r a t i v e changes in m o b i l i t y w e r e similarly r e c o r d e d at w e e k l y intervals for eight weeks. T h y r o c a l c i t o n i n did not alter the mobility of the teeth after r e p l a n t a t i o n w h e n c o m p a r e d to the teeth r e p l a n t e d in the control group.
Replantation is the act of replacing
one or more teeth into their original rockets after accidental avulsion or extraction of the teeth. The procedure has probably been practiced in one form or another for as long as man has suffered the accidental loss of teeth; it is also possible that teeth were the first organs to have been transplanted in humans. The prognosis after replantation of teeth is greatly influenced by two factors, namely, the onset and progression of resorption and ankylosis. Andreasen and Hj~rting-Hansen,1~ and other investigators :~-7have described various forms of root resorption that may occur after replantation of teeth, one of which is replacement resorption (ankylosis). Replacement resorption, which may he seen radiographically, results in the limitation of normal mobility of
the tooth. Its presence has been confirmed in histological investigations.-'.~-,: In addition to histological examination with use of the light microscope, periodontometry may be used to demonstrate replacement resorption. Periodontometry was developed by Mfihlemann,':' who studied the periodontal movement of monkeys' teeth. Subsequently, Miihlem a n n " described the clinical application of periodontometry in man as an aid in the diagnosis of periodontal disease. More recently, Andreasen" has documented how periodontometry may be used to determine the condition of periodontal healing in cases of replantation of human teeth after accidental avulsion. However, the analysis of periodontal healing by periodontometry in experimental animals who are undergoing replan-
tation of teeth has not previously been documented, although preliminary findings of changes in mobility of teeth were reported in vervet monkeys that had been given thyrocalcitonin after replantation of teeth?:' In the study, teeth were replanted after an extra-alveolar time (EAT) of 30 and 60 minutes. The mobility of the replanted teeth was greatly reduced but there was no significant difference between the two time groups. Thyrocalcitonin was administered because Aliapoulius and others TM reported that the addition of the hormone reduced bone resorption, in tissue culture, that had been induced by the parathyroid hormone. In addition, Foster ~7 documented that thyrocalcitonin reduced osteoclastic activity and thus reduced bone resorption.
265
Fig 3-CarboxyIate cement within aqylic tray which has been removedfrom mouth of monkey after mobility changes were recorded.
Fig 1--Miihlemann periodontometer modified with aqylic tray molded to fit hard palate and posterior maxillary teeth.
Fig 2 Modified periodontometer cemented onto hard palate and posterior maxillary teeth. Pointer positioned at midpoint of buccal surface.
would more likely develop when the EAT was extended beyond 60 minlltes. ~
MATERIALS AND METHODS
Fig 4-Periodontometer cemented into place with pointer of dial indicator on buccal with force meter applying force from palatal surface
(arrow). The current investigation, an extension of previous work, was undertaken to examine the changes in mobility that would occur after experimental replantation, with an extra-alveolar time of two hours, in vervet monkeys given thyrocalcitonin. An EAT of two hours was decided on because it has been shown that replacement resorption or ankylosis 266
Ten vervet monkeys (Cercopithecus aethiops), six females and four males, in which second permanent molars had erupted, were immobilized with phencyelidine hydrochloride (sernylan, 0.2 m g / k g body weight) and anesthetized with intravenous sodium pentobarbital. The preoperative mobility of the central maxillary incisors was determined with use of a Miihlemann periodontometer ~ modified to fit the mouth of the vervet monkey. A premolded tray that had been made with a rapid-curing acrylic was attached to the periodontometer (Fig 1). The acrylic tray was shaped to conform to the hard palate and to the maxillary molars and premolars on a dried monkey's skull. During the periodontometry, the acrylic tray was cemented onto the posterior maxillary, teeth with a carboxylate
cement (Durelon*) (Fig 2). This produced a stable form of retention of the periodontometer to minimize movement of the instrument itself while the mobility of the experimental teeth was determined (Fig 3). After the carboxylate cement had set, the pointer of the dial indicator was positioned at a right angle to the midpoint of the buccal surface of the maxillary central incisors. A force of 100 gm was applied during two tO three seconds by means of a forcemeter (Corex gauge, Haag-Streit) placed approximately 1 m m above the incisal edge in the midline of the buccal and palatal surfaces of the tooth (Fig 4). The deflection of the tooth was noted and three successive readings were made with the force applied from the buccal surface and then from the palatal surfacel Between each reading, the force gauge was removed from the surface of the tooth and the tooth was allowed to return to the rest position. It was noted, however, in the vast majority of readings that the dial gauge did not return to the initial starting positions. It was thus decided to read all subsequent changes in mobility from the position that the tooth assumed after the force had been removed. Stability usually occurred within five seconds after the force was removed from the tooth. Before extraction of the teeth,
JOURNAL OF ENDODONTICS I VOL 4, NO 9, SEPTEMBER 1978
jhallow grooves were cut into the inesial and distal interstitial surfaces ~0fall the maxillary incisors to facilii~te splinting after replantation. The ulp chambers of the two central incisors were opened from the palatal ~spect, after which these two teeth were extracted from each of the experimental animals with use of conventional extraction forceps. The lime taken for each extraction was irecorded. The extracted teeth were ~ p t moist in physiologic saline solulion for two hours and then ~eplanted. During the two-hour rAT, the endodontic treatment was ~mpleted on the extracted teeth and .tgeat care was taken to avoid chemiiml or mechanical damage to the /i~eriodontal remnants on the root I~rfaces. The root canals were filled with gutta-percha cones and a zincoxide and eugenol mixture (Gross~ n ' s paste) using the lateral condensation technique. The excess of ~ones and paste that protruded through the apices were carefully trimmed tlush with the apices with a warmed scalpel blade. The access cavities were closed with zincoxyphosphate cement and the teeth were replanted into their original sockets without washing out any of the blood clots within the sockets. All the replanted teeth were splinted with figure-eight interdental wiring with use of no. 4-0 braided multi filament stainless steel sutures placed into the previously cut interstitial grooves (Fig 5). Each monkey was given an intramuscular injection of long-acting penicillin (Compropen V'[') and, finally, the weight of each monkey was noted. The monkeys were fed a soft diet of cut fruits and bread with various spreads to minimize the masticatory trauma to the replanted teeth. Splinting was maintained for two weeks.
~
At this stage, the monkeys were classified in two groups of five monkeys each. In the control group, no further medication was given. The five monkeys of the experimental group, however, were given daily intramuscular injections of 20 Medical Research Council (MRC) units of thyrocalcitonin{ between 6 and 8 PM. Each of the ten monkeys was immobilized at weekly intervals with phencyclidine hydrochloride and the mobility of the replanted teeth was recorded as previously described. Samples of venous blood were taken from the femoral veins of the experimental monkeys and from a control monkey at the same time. The serum calcium, serum phosphate, and serum alkaline phosphatase were determined with use of a photometric technique (Boehringer Mannheim). Where possible, the samples of blood were taken 12 hours after the thyrocalcitonin was given. When the measurements of mobility and venipuncture were completed at the eighth postoperative week, the monkeys were killed and their heads were fixed with use of the perfusion technique of Retief and Austin 1" for histological examination. Finally, postmortem radiographs of the replanted teeth were taken after the fixed premaxillary blocks had been removed from the heads of the monkeys. All the recorded data was placed onto computer punch cards and analyzed by an IBM 370/158 computer with use of the Statistical Package for the Social Sciences)'
experimental group. Three teeth in the experimental group could not be evaluated for the entire eight-week period. One of these three teeth was fractured during the extraction and another monkey died after the fifth postoperative week. A postmortem examination on this monkey disclosed no obvious organic disease but the tongue was badly macerated, possibly indicating some convulsive condition, such as tetany, before death. The ten teeth in the control group and the seven teeth in the experimental group were all clinically firm, and the surrounding gingivae were healthy with no obvious sign of clinical abnormality. During the experiment, all the monkeys showed loss of weight which was not statistically significant. The Table lists the mean extraction times for the teeth that were investigated. No statistically significant differences in extraction times were found between right and left teeth within each group and between the experimental and control groups. The values for serum calcium, serum phosphate, and serum alkaline phosphatase are graphically shown in Figure 6. The serum calcium levels of the five experimental monkeys ranged from 8 to 10 mg/100 ml in
RESULTS
There were 20 teeth planned for replantation in this investigation, ten in the control group and ten in the
Fig 5--Figure-eight interdental splint in situ. 267
T a b l e 9 Extraction T i m e in S e c o n d s .
Experiment Saline Thyrocaleitonin Saline Thyrocalcitonin n = no. of teeth; R = mean;
Tooth
n
Left Right Left Right Left, right Left, right
5 5 5 4 10 9
t = Student's
.~ +_ SD 17.8 20.8 20.2 17.3 19.3 18.9
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Fig 6-Results of serum investigations. Dotted lines indicate that twoweek specimen was not analyzed. Interrupted line betweenfour and five weeks in alkaline phosphatase results indicates that maximum value of system used of 500 IU had been reached. Possibly results from five weeks onward could have been greater than 500 IU.
/
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WEEKS
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Fig 7 Mean mobility changes when load of 100 gm was applied to buccal surfaces of replanted teeth in control (D) and experimental (0) groups. g
I
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TIME IN WEEKS
268
6.00
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comparison with a range of 10 to 11 mg/100 ml in the control monkey. An analysis of variance showed the differences to be statistically signifi. cant at all periods except week five ( P < .001). The serum phosphate levels were slightly higher in the experimental monkeys in comparison with the control monkey. The serum alkaline phosphatase levels after the third postoperative week were greatly raised in the five experimental monkeys. This was in contrast to the serum alkaline phosphatase of the control monkey, which remained constant throughout the experiment (P < .001). The mean changes in mobility after the buccal and the palatal loading, and the combined values of these are graphically shown in Figures 7 to 9, respectively. The general trend from the aforementioned three graphs shows that, after an increased mobility at one week, there was a substantial decrease in mobility. Thus, by the third or fourth week, the basal mobility value was reached but the decrease in amount of mobility continued until the termination of the experiment. When the force or load was applied to the buccal surfaces of the control and experimental teeth (Fig 7), the initial mean mobility of all the teeth in each group was 0.012 mm and 0.013 mm, respectively. The greatest mobility reached was 0.027 mm and 0.028 mm at the first and second week, respectively, for the control and experimental groups9 The mobility decreased to equal the basal values between the third and fourth week but, by the eighth week, had decreased to 0.006 mm and 0.002 mm for the control and experimental groups. When the force waS applied to the palatal surfaces (Fig
cc
8), the initial mean mobility was 0.079 m m and 0.078 m m for the control and experimental groups. This increased to 0.102 m m and 0.153 m m for the two groups which, after eight weeks, decreased to 0.007 mm and 0.005 ram, respectively, for the control and experimental groups. The total mobility of each tooth was derived by combining the displacement produced when the force was applied from both the buccal and palatal surfaces. The mean initial total mobility (Fig 9) for the control and experimental groups was 0.071 m m and 0.087 ram, respec~vely, which reached peak levels of ~.137 m m and 0.173 m m at the first ~eek. Between the second and third peek the initial basal levels were ireached, but the mobility decreased that, by the eighth week, the ivalues reached were 0.013 m m and 9.007 m m for the control and experhnental groups, respectively. Figures 10 to I2 graphically ~represent the percentage change in !mobility at the weekly periods of :-:0bservation for the buccal and palatal loading, and for the combined total of the buccal plus the palatal loading, respectively. The mean per~ n t a g e changes relative to the mean ~reoperative levels were chosen in~ead of the real values because individual variation accounted for a wide range of preoperative mobility levels. Furthermore, it was not possible to control either the force or the trauma inflicted on the root and alveolar socket during the extraction procedure before replantation. The Student's t test was used to deterraine any statistical significance Within the results. In the buccal loading (Fig 10) the taobility increased by approximately
~~
Fig 8 Mean mobility changes when the load of 100 gm was applied from palatal surfaces of replanted teeth in control ([3) and experimental (0) groups.
2~
m I
0.00
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Fig 9 Mean changes of total mobility (buccal mobility + palatal mobility) of each replanted tooth in the control (El) and experimental (0) groups.
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140.0% in both groups but decreased by 46.4% in the control group and 80.4% in the experimental group after eight weeks. This difference, as well as the difference in the two preceding weeks, was statistically significant at the 5% level. This indicates that the thyrocalcitonin may
have allowed a greater decrease in mobility change. When the load was applied from the palatal surface (Fig 11), the mobility at one week showed a 97.7% and 481.6% increase in the control and experimental groups, respectively. This decreased to 88.3% and 71.3% below the baseline for the 269
i VOL 4, N O 9, SEPTEMBER 1978
JOURNAL OF E N D O D O N T I C S
+300
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F~g lO--Mean percentage changes with load on buccal surfaces of control and experimental groups.
Fig l 1-Mean percentage changes with load on palatal surfaces of control and experimental groups.
two groups respectively, which was not statistically significant. Figure 12 graphically indicates the mean percentage change for the total of the buccal and the palatal loading, and shows an increased mobility of 103% and 233% for the control and experimental groups, respectively. This decreased by 81.0% and 80.4%, respectively, with no statistical significance after eight weeks.
probably related to healing of the socket after extraction of the teeth, and to tooth resorption. The unusual serum calcium and serum phosphate levels noted at five weeks appears inexplicable. The M/ihlemann technique la~~ of quantitating the mobility of teeth appears to be a reliable method of assessing the state of periodontal repair after the replantation of teeth in the vervet monkey. The retention of the periodontometer to the hard palate and posterior teeth afforded a particularly stable situation for the accurate determination of the displacement of the teeth when the load of i00 gm was applied to the teeth under observation.
DISCUSSION In this study, a significant decrease in the serum calcium levels of the experimental animals was observed, which we consider to be indicative of thyrocalcitonin activity. The distinct changes in alkaline phosphatase are 270
Fig 12--Mean percentage changes of total mobility (buccal mobility + palatal mobility) of control and experimental groups.
It was observed that when the load was removed from the surface of the tooth, the pointer of the dial gauge often would not return to the initial resting levels. This could be attributed to either the inability of the compressed and stretched periodontal ligament to return to the original rest position immediately, or to some inherent friction of the measuring device, or both. Furthermore, the three mobility recordings were first noted when the load was applied from the one surface and then noted again when the load was applied three times from the opposite surface. This technique was not the same as that advocated by Mfihlem a n n 13 who alternated his record"
JOURNAL OF ENDODONTICS [ VOL 4, NO 9, SEPTEMBER 1978
hags between the o p p o s i t e surfaces of the teeth. By a p p l y i n g a load of 100 g m to these teeth, the values o b t a i n e d were within the limits of initial m o b i l i t y as described by M f i h l e m a n n ? ~ This initial mobility is said to be t h a t movement allowed to occur by virtue of the elasticity or c o m p r e s s i b i l i t y of the p e r i o d o n t a l l i g a m e n t only. L o a d s greater than 100 g m cause some distortion of the a l v e o l a r walls; this mobility has been t e r m e d s e c o n d a r y mobility. T h e findings of this investigation show that the d i s p l a c e m e n t is far greater when the load was applied from the p a l a t a l surface t h a n when the load was a p p l i e d from the buccal surface. It is not clear w h y this difference of degree of m o b i l i t y exists. Possible reasons might be t h a t there are some microanatomical differences between the buccal a n d p a l a t a l periodontal m e m b r a n e constituents. F u r thermore, the denser p a l a t a l shelf of bone m a y offer g r e a t e r resistance to displacement when c o m p a r e d with the t h i n n e r buecal p l a t e of bone. O n the other h a n d , after the a p p l i c a t i o n of the p e r i o d o n t o m e t e r to the buccal surface of a tooth, the pressure of the instrument on the crown m a y displace the tooth to the p a l a t a l side of the rest position. By decreasing the serum c a l c i u m levels with thyrocalcitonin, it was hoped to interfere with the ankylosis, which was expected to develop in replantations p e r f o r m e d with an EAT of two hours. Also, osteoclastic activity might have been inhibited, thus decreasing the degree of root resorption. The a m o u n t of m o b i l i t y of the replanted teeth in b o t h groups a p p r o x i m a t e d a n d then d r o p p e d below the basal or p r e o p e r a t i v e
m o b i l i t y levels between the t h i r d a n d fourth postoperative weeks. T h e decreased m o b i l i t y was progressive until the t e r m i n a t i o n of the experiment. This l i m i t a t i o n of m o b i l i t y is indicative of ankylosis b u t histological evidence is required for c o n f i r m a tion. Andreasen, TM by use of the same m e t h o d of p e r i o d o n t o m e t r y in cases of r e p l a n t a t i o n of teeth in m a n , was also able to detect a r a p i d h e a l i n g of the p e r i o d o n t a l m e m b r a n e and found t h a t n o r m a l values of m o b i l i t y were o b t a i n e d three weeks after replantation. T h e results of the c u r r e n t s t u d y tend to indicate t h a t 20 M R C units of t h y r o c a l c i t o n i n given once d a i l y to the vervet monkeys d i d not influence the onset of ankylosis or m o d i f y the progression of ankylosis, b u t this must be confirmed histologically. Certainly, it a p p e a r s as t h o u g h t h y r o c a l c i t o n i n tended to decrease further the m o b i l i t y of the r e p l a n t e d teeth after the sixth week, c o m p a r e d with the r e p l a n t e d teeth in the monkeys not given thyrocalcitonin. T h e fact t h a t one of the five m o n k e y s given t h y r o c a l c i t o n i n died indicates the c a u t i o n t h a t must be exercised before the a d m i n i s t r a t i o n of such a drug. F u r t h e r m o r e , the vervet m o n k e y can be used as an excellent m o d e l system for studies of r e p l a n t a t i o n . This is because the t i m i n g of the healing of the p e r i o d o n t a l m e m b r a n e before the onset of ankylosis in the vervet m o n k e y is similar to t h a t described by A n d r e a s e n 1~ in clinical studies of humans. In the future, it is p l a n n e d to histologically q u a n t i t a t e the postrep l a n t a t i o n ankylosis a n d to relate the a m o u n t of ankylosis to the c u r r e n t findings of decreased mobility.
CONCLUSIONS T h e a d m i n i s t r a t i o n of i n t r a m u s c u lar injections of t h y r o c a l c i t o n i n to the vervet m o n k e y after r e p l a n t a t i o n did not alter the m o b i l i t y c o m p a r e d with teeth in the control group. Changes in m o b i l i t y indicative of ankylosis were evident three to four weeks after r e p l a n t a t i o n in b o t h the e x p e r i m e n t a l a n d control groups as measured by p e r i o d o n t o m e t e r y . *ESPE, Seefeld/Oberbay, West Germany tGlaxo, Wadeville, South Africa ~Sandoz Co., Rasdburg, South Africa The authors thank the Sandoz Company and Dr. John Glynn of Johannesburg for the generous supply of thyrocalcitonin, and the staff of the Dental Research Unit of the South African Medical Research Council and the University of the Witwatersrand for the photographic preparation. This project was supported financially by Elida-Gibbs and by the Research, Education and Development Fund of the Dental Association of South Africa. Dr. Barbakow is in private practice and is an honorary research fellow, Prof. CleatonJones is the director, Dr. Austin is a senior research officer, and Miss Vieira is a technician, Dental Research Unit of the South African Medical Research Council and the University of the Witwatersrand, Johannesburg, South Africa. Dr. Andreasen is the associate director of the Afdelingen for Tand-, Mund-, Og Kaebesygdomme Z of the Rigshospitalet, Copenhagen, Denmark. Requests for reprints should be directed to Dr. F. H. Barbakow, Dental Research Unit, School of Dentistry, University of the Witwatersrand, 1 Jan Smuts Ave, Johannesburg, 2001, South Africa.
References
1. Andreasen, J.O., and Hj~rting-Hansen, E. Replantation of teeth. Radiographic and clinical study of 110 human teeth replanted after accidental loss. Acta Odont Scand 24: 263 Nov 1966. 2. Andreasen, J.O., and Hjqlrting-Hansen, E. Replantation of teeth. Histological study of 22 replanted anterior teeth in humans. Acta Odont Scand 24:287 Nov 1966. 271
JOURNAL OF E N D O D O N T I C S ] VOL 4, N O 9, SEPTEMBER 1978
3. Sherman, P. Intentional replantation of teeth in dogs and monkeys. J Dent Res 47:1066 Nov-Dec 1968. 4. Grossman, LT, and Ship, I.I. Survival rate of replanted teeth. Oral Surg 29:899 June 1970. 5. Guralnick, W.C. Autogenous and allogeneie transplantation of teeth. J Oral Surg 28:575 Aug 1970. 6. Shulman, L.B. The current status of allogenic tooth transplantation: attachment, repair, regeneration and development in dental replants, transplants and implants. Ciba Foundation Symposium 11. Hard tissue growth, repair and remineralisation, p 91, 1973. 7. Heithersay, G.S. Replantation of avulsed teeth. A review. Aust Dent J 20:63 April 1975. 8. Anderson, A.W.; Sharav, Y.; and Massler, M. Periodontal reattachment after tooth replantation. Periodontics 6:161 Aug 1968. 9. Andreasen, J.O. Traumatic injuries of
272
the teeth. Copenhagen, Munksgaard, 1972, p 193. 10. Andreasen, J.O. Periodontal healing after replantation of traumatically avu[sed human teeth. Assessment by mobility testing and radiography. Acta Odont Scand 33:325 Dec 1975. 11. Hurst, R.V. Regeneration of periodontal and transseptal fibers after autografts in Rhesus monkeys: a qualitative approach. J Dent Res 51:1183 Sept-Oct 1972. 12. Barbakow, F.H.; Austin, J.C.; and Cleaton-Jones, P.E. Experimental replantation of root-canal-filled and untreated teeth in the vervet monkey. J Endod 3:89 March 1977. 13. Miihlemann, H.R. Tooth mobility: The measurements method, initial and secondary tooth mobility. J Periodont 25:22 Jan 1954. 14. Mfihlemann, H.R. 10 years of toothmobility measurements. J Periodont 31:110 April 1960.
15. Barbakow, F.H.; Austin, J.C.; Cleaton. Jones, P.E.; and Vieira, E. The effect of calcitonin on replanted teeth. Proe South African Div of IADR, Durban, 1976. 16. Aliapoulius, M.A.; Goldhaber, P.; and Munson, P.L. Thyrocalcitonin inhibition of bone resorption induced by parathyroid hormone in tissue culture. Science 151:330 Jan 21, 1966. 17. Foster, G.V. Calcitonin. A review of experimental and clinical investigations. Postgrad M e d J 44:411 May 1968. 18. Retief, D.H., and Austin, J.C. The vervet monkey (Cercopithecus aethiops) as an experimental model for pulpal studies. J Dent Assoc S Africa 28:98 Feb 1973. 19. Nie, N.H.; Hull, C.H.; Jenkins, J.G.; Steinbrenner, R.; and Bent, D.A. SSPS: statistical package for the social sciences, ed 2. New York, McGraw-Hill, 1975. 20. Mfihlemann, H.R. Tooth mobility: a review of clinical aspects and research findings. J Periodont 38:(suppl)686 Nov-Dee 1967.
Changes in Tooth Mobility after Experimental Replantation
F. H. Barbakow, BDS, HDD; P. E. Cleaton-Jones, BDS, MB, BCh, PhD, DA; J. C. Austin, BVSc; J. O. Andreasen, DDS; and E. Vieira, Dip Micro, Johannesburg, South Africa
Maxillary central incisors were r e p l a n t e d in ten v e r v e t m o n k e y s after an extra-alveolar time (EAT) of two h o u r s d u r i n g w h i c h the teeth were kept moist with a saline solution. I n t r a m u s c u l a r t h y r o c a l c i t o n i n was a d m i n i s t e r e d to the e x p e r i m e n t a l g r o u p , b u t not to the control g r o u p to assess the effect of t h y r o c a l c i t o n i n o n the p o s t o p e r a t i v e ankylosis, which was e x p e c t e d to o c c u r after an E A T of two hours. T h e p r e o p e r a t i v e m o b i l i t y was n o t e d in all of the t e e t h that w e r e p l a n n e d for r e p l a n t a t i o n b y a t e c h n i q u e k n o w n as p e r i o d o n t o m e t r y . T h e p o s t o p e r a t i v e changes in m o b i l i t y w e r e similarly r e c o r d e d at w e e k l y intervals for eight weeks. T h y r o c a l c i t o n i n did not alter the mobility of the teeth after r e p l a n t a t i o n w h e n c o m p a r e d to the teeth r e p l a n t e d in the control group.
Replantation is the act of replacing
one or more teeth into their original rockets after accidental avulsion or extraction of the teeth. The procedure has probably been practiced in one form or another for as long as man has suffered the accidental loss of teeth; it is also possible that teeth were the first organs to have been transplanted in humans. The prognosis after replantation of teeth is greatly influenced by two factors, namely, the onset and progression of resorption and ankylosis. Andreasen and Hj~rting-Hansen,1~ and other investigators :~-7have described various forms of root resorption that may occur after replantation of teeth, one of which is replacement resorption (ankylosis). Replacement resorption, which may he seen radiographically, results in the limitation of normal mobility of
the tooth. Its presence has been confirmed in histological investigations.-'.~-,: In addition to histological examination with use of the light microscope, periodontometry may be used to demonstrate replacement resorption. Periodontometry was developed by Mfihlemann,':' who studied the periodontal movement of monkeys' teeth. Subsequently, Miihlem a n n " described the clinical application of periodontometry in man as an aid in the diagnosis of periodontal disease. More recently, Andreasen" has documented how periodontometry may be used to determine the condition of periodontal healing in cases of replantation of human teeth after accidental avulsion. However, the analysis of periodontal healing by periodontometry in experimental animals who are undergoing replan-
tation of teeth has not previously been documented, although preliminary findings of changes in mobility of teeth were reported in vervet monkeys that had been given thyrocalcitonin after replantation of teeth?:' In the study, teeth were replanted after an extra-alveolar time (EAT) of 30 and 60 minutes. The mobility of the replanted teeth was greatly reduced but there was no significant difference between the two time groups. Thyrocalcitonin was administered because Aliapoulius and others TM reported that the addition of the hormone reduced bone resorption, in tissue culture, that had been induced by the parathyroid hormone. In addition, Foster ~7 documented that thyrocalcitonin reduced osteoclastic activity and thus reduced bone resorption.
265
Fig 3-CarboxyIate cement within aqylic tray which has been removedfrom mouth of monkey after mobility changes were recorded.
Fig 1--Miihlemann periodontometer modified with aqylic tray molded to fit hard palate and posterior maxillary teeth.
Fig 2 Modified periodontometer cemented onto hard palate and posterior maxillary teeth. Pointer positioned at midpoint of buccal surface.
would more likely develop when the EAT was extended beyond 60 minlltes. ~
MATERIALS AND METHODS
Fig 4-Periodontometer cemented into place with pointer of dial indicator on buccal with force meter applying force from palatal surface
(arrow). The current investigation, an extension of previous work, was undertaken to examine the changes in mobility that would occur after experimental replantation, with an extra-alveolar time of two hours, in vervet monkeys given thyrocalcitonin. An EAT of two hours was decided on because it has been shown that replacement resorption or ankylosis 266
Ten vervet monkeys (Cercopithecus aethiops), six females and four males, in which second permanent molars had erupted, were immobilized with phencyelidine hydrochloride (sernylan, 0.2 m g / k g body weight) and anesthetized with intravenous sodium pentobarbital. The preoperative mobility of the central maxillary incisors was determined with use of a Miihlemann periodontometer ~ modified to fit the mouth of the vervet monkey. A premolded tray that had been made with a rapid-curing acrylic was attached to the periodontometer (Fig 1). The acrylic tray was shaped to conform to the hard palate and to the maxillary molars and premolars on a dried monkey's skull. During the periodontometry, the acrylic tray was cemented onto the posterior maxillary, teeth with a carboxylate
cement (Durelon*) (Fig 2). This produced a stable form of retention of the periodontometer to minimize movement of the instrument itself while the mobility of the experimental teeth was determined (Fig 3). After the carboxylate cement had set, the pointer of the dial indicator was positioned at a right angle to the midpoint of the buccal surface of the maxillary central incisors. A force of 100 gm was applied during two tO three seconds by means of a forcemeter (Corex gauge, Haag-Streit) placed approximately 1 m m above the incisal edge in the midline of the buccal and palatal surfaces of the tooth (Fig 4). The deflection of the tooth was noted and three successive readings were made with the force applied from the buccal surface and then from the palatal surfacel Between each reading, the force gauge was removed from the surface of the tooth and the tooth was allowed to return to the rest position. It was noted, however, in the vast majority of readings that the dial gauge did not return to the initial starting positions. It was thus decided to read all subsequent changes in mobility from the position that the tooth assumed after the force had been removed. Stability usually occurred within five seconds after the force was removed from the tooth. Before extraction of the teeth,
JOURNAL OF ENDODONTICS I VOL 4, NO 9, SEPTEMBER 1978
jhallow grooves were cut into the inesial and distal interstitial surfaces ~0fall the maxillary incisors to facilii~te splinting after replantation. The ulp chambers of the two central incisors were opened from the palatal ~spect, after which these two teeth were extracted from each of the experimental animals with use of conventional extraction forceps. The lime taken for each extraction was irecorded. The extracted teeth were ~ p t moist in physiologic saline solulion for two hours and then ~eplanted. During the two-hour rAT, the endodontic treatment was ~mpleted on the extracted teeth and .tgeat care was taken to avoid chemiiml or mechanical damage to the /i~eriodontal remnants on the root I~rfaces. The root canals were filled with gutta-percha cones and a zincoxide and eugenol mixture (Gross~ n ' s paste) using the lateral condensation technique. The excess of ~ones and paste that protruded through the apices were carefully trimmed tlush with the apices with a warmed scalpel blade. The access cavities were closed with zincoxyphosphate cement and the teeth were replanted into their original sockets without washing out any of the blood clots within the sockets. All the replanted teeth were splinted with figure-eight interdental wiring with use of no. 4-0 braided multi filament stainless steel sutures placed into the previously cut interstitial grooves (Fig 5). Each monkey was given an intramuscular injection of long-acting penicillin (Compropen V'[') and, finally, the weight of each monkey was noted. The monkeys were fed a soft diet of cut fruits and bread with various spreads to minimize the masticatory trauma to the replanted teeth. Splinting was maintained for two weeks.
~
At this stage, the monkeys were classified in two groups of five monkeys each. In the control group, no further medication was given. The five monkeys of the experimental group, however, were given daily intramuscular injections of 20 Medical Research Council (MRC) units of thyrocalcitonin{ between 6 and 8 PM. Each of the ten monkeys was immobilized at weekly intervals with phencyclidine hydrochloride and the mobility of the replanted teeth was recorded as previously described. Samples of venous blood were taken from the femoral veins of the experimental monkeys and from a control monkey at the same time. The serum calcium, serum phosphate, and serum alkaline phosphatase were determined with use of a photometric technique (Boehringer Mannheim). Where possible, the samples of blood were taken 12 hours after the thyrocalcitonin was given. When the measurements of mobility and venipuncture were completed at the eighth postoperative week, the monkeys were killed and their heads were fixed with use of the perfusion technique of Retief and Austin 1" for histological examination. Finally, postmortem radiographs of the replanted teeth were taken after the fixed premaxillary blocks had been removed from the heads of the monkeys. All the recorded data was placed onto computer punch cards and analyzed by an IBM 370/158 computer with use of the Statistical Package for the Social Sciences)'
experimental group. Three teeth in the experimental group could not be evaluated for the entire eight-week period. One of these three teeth was fractured during the extraction and another monkey died after the fifth postoperative week. A postmortem examination on this monkey disclosed no obvious organic disease but the tongue was badly macerated, possibly indicating some convulsive condition, such as tetany, before death. The ten teeth in the control group and the seven teeth in the experimental group were all clinically firm, and the surrounding gingivae were healthy with no obvious sign of clinical abnormality. During the experiment, all the monkeys showed loss of weight which was not statistically significant. The Table lists the mean extraction times for the teeth that were investigated. No statistically significant differences in extraction times were found between right and left teeth within each group and between the experimental and control groups. The values for serum calcium, serum phosphate, and serum alkaline phosphatase are graphically shown in Figure 6. The serum calcium levels of the five experimental monkeys ranged from 8 to 10 mg/100 ml in
RESULTS
There were 20 teeth planned for replantation in this investigation, ten in the control group and ten in the
Fig 5--Figure-eight interdental splint in situ. 267
T a b l e 9 Extraction T i m e in S e c o n d s .
Experiment Saline Thyrocaleitonin Saline Thyrocalcitonin n = no. of teeth; R = mean;
Tooth
n
Left Right Left Right Left, right Left, right
5 5 5 4 10 9
t = Student's
.~ +_ SD 17.8 20.8 20.2 17.3 19.3 18.9
+ 9.5 _+ 16.5 -- 15.2 + 13.4 +_ 12.8 -+ 13.6
t
P
0.32
NS
0.28
NS
0.06
NS
test; P = probability.
12. lO~
8.
g
6-
==
Control 4.
0 Calcit~nin
2.
3
E o o
2
500
~
0
0
400 ./ 300
E
/ /
200
/ /
I00
Fig 6-Results of serum investigations. Dotted lines indicate that twoweek specimen was not analyzed. Interrupted line betweenfour and five weeks in alkaline phosphatase results indicates that maximum value of system used of 500 IU had been reached. Possibly results from five weeks onward could have been greater than 500 IU.
/
1
2
4
3
5
6
7
8
WEEKS
g
no
z~
c3
Fig 7 Mean mobility changes when load of 100 gm was applied to buccal surfaces of replanted teeth in control (D) and experimental (0) groups. g
I
'0~00
29
4.00
TIME IN WEEKS
268
6.00
89
I
10.00
comparison with a range of 10 to 11 mg/100 ml in the control monkey. An analysis of variance showed the differences to be statistically signifi. cant at all periods except week five ( P < .001). The serum phosphate levels were slightly higher in the experimental monkeys in comparison with the control monkey. The serum alkaline phosphatase levels after the third postoperative week were greatly raised in the five experimental monkeys. This was in contrast to the serum alkaline phosphatase of the control monkey, which remained constant throughout the experiment (P < .001). The mean changes in mobility after the buccal and the palatal loading, and the combined values of these are graphically shown in Figures 7 to 9, respectively. The general trend from the aforementioned three graphs shows that, after an increased mobility at one week, there was a substantial decrease in mobility. Thus, by the third or fourth week, the basal mobility value was reached but the decrease in amount of mobility continued until the termination of the experiment. When the force or load was applied to the buccal surfaces of the control and experimental teeth (Fig 7), the initial mean mobility of all the teeth in each group was 0.012 mm and 0.013 mm, respectively. The greatest mobility reached was 0.027 mm and 0.028 mm at the first and second week, respectively, for the control and experimental groups9 The mobility decreased to equal the basal values between the third and fourth week but, by the eighth week, had decreased to 0.006 mm and 0.002 mm for the control and experimental groups. When the force waS applied to the palatal surfaces (Fig
cc
8), the initial mean mobility was 0.079 m m and 0.078 m m for the control and experimental groups. This increased to 0.102 m m and 0.153 m m for the two groups which, after eight weeks, decreased to 0.007 mm and 0.005 ram, respectively, for the control and experimental groups. The total mobility of each tooth was derived by combining the displacement produced when the force was applied from both the buccal and palatal surfaces. The mean initial total mobility (Fig 9) for the control and experimental groups was 0.071 m m and 0.087 ram, respec~vely, which reached peak levels of ~.137 m m and 0.173 m m at the first ~eek. Between the second and third peek the initial basal levels were ireached, but the mobility decreased that, by the eighth week, the ivalues reached were 0.013 m m and 9.007 m m for the control and experhnental groups, respectively. Figures 10 to I2 graphically ~represent the percentage change in !mobility at the weekly periods of :-:0bservation for the buccal and palatal loading, and for the combined total of the buccal plus the palatal loading, respectively. The mean per~ n t a g e changes relative to the mean ~reoperative levels were chosen in~ead of the real values because individual variation accounted for a wide range of preoperative mobility levels. Furthermore, it was not possible to control either the force or the trauma inflicted on the root and alveolar socket during the extraction procedure before replantation. The Student's t test was used to deterraine any statistical significance Within the results. In the buccal loading (Fig 10) the taobility increased by approximately
~~
Fig 8 Mean mobility changes when the load of 100 gm was applied from palatal surfaces of replanted teeth in control ([3) and experimental (0) groups.
2~
m I
0.00
2,00
4.00
8.00
I
~0.00
8.00
JIME IN WEEKS
Fig 9 Mean changes of total mobility (buccal mobility + palatal mobility) of each replanted tooth in the control (El) and experimental (0) groups.
~L ~.00
I
t 2.00
f
I ~.00
~
I 8.00
J
I 8.00
;
I
10~
TIME IN WEEKS
140.0% in both groups but decreased by 46.4% in the control group and 80.4% in the experimental group after eight weeks. This difference, as well as the difference in the two preceding weeks, was statistically significant at the 5% level. This indicates that the thyrocalcitonin may
have allowed a greater decrease in mobility change. When the load was applied from the palatal surface (Fig 11), the mobility at one week showed a 97.7% and 481.6% increase in the control and experimental groups, respectively. This decreased to 88.3% and 71.3% below the baseline for the 269
i VOL 4, N O 9, SEPTEMBER 1978
JOURNAL OF E N D O D O N T I C S
+300
+3o0.
BUCCAL LOADING
+200
PALATAL LOADING
?../~?
BUCCAL + PALATALLOADING
+300_
I
'
I
I I L
9
Saline
0
Calcitonin
+200.
I
0
9 Saline 0 Calcitonin
+200
9 Saline
I
Calcitonin
\
J
z
z
N
N +lOO_
+IOO.
~ §
I
f,
N
0
,
.
, ~\
1
2
3
~ /
6
7
8
o,
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"
"
5
6
~ a WEEKS
I
2
3** 4
5
6
7
8
WEEKS
A \\
0~.
"x~ "o
-I00
-lOI
F~g lO--Mean percentage changes with load on buccal surfaces of control and experimental groups.
Fig l 1-Mean percentage changes with load on palatal surfaces of control and experimental groups.
two groups respectively, which was not statistically significant. Figure 12 graphically indicates the mean percentage change for the total of the buccal and the palatal loading, and shows an increased mobility of 103% and 233% for the control and experimental groups, respectively. This decreased by 81.0% and 80.4%, respectively, with no statistical significance after eight weeks.
probably related to healing of the socket after extraction of the teeth, and to tooth resorption. The unusual serum calcium and serum phosphate levels noted at five weeks appears inexplicable. The M/ihlemann technique la~~ of quantitating the mobility of teeth appears to be a reliable method of assessing the state of periodontal repair after the replantation of teeth in the vervet monkey. The retention of the periodontometer to the hard palate and posterior teeth afforded a particularly stable situation for the accurate determination of the displacement of the teeth when the load of i00 gm was applied to the teeth under observation.
DISCUSSION In this study, a significant decrease in the serum calcium levels of the experimental animals was observed, which we consider to be indicative of thyrocalcitonin activity. The distinct changes in alkaline phosphatase are 270
Fig 12--Mean percentage changes of total mobility (buccal mobility + palatal mobility) of control and experimental groups.
It was observed that when the load was removed from the surface of the tooth, the pointer of the dial gauge often would not return to the initial resting levels. This could be attributed to either the inability of the compressed and stretched periodontal ligament to return to the original rest position immediately, or to some inherent friction of the measuring device, or both. Furthermore, the three mobility recordings were first noted when the load was applied from the one surface and then noted again when the load was applied three times from the opposite surface. This technique was not the same as that advocated by Mfihlem a n n 13 who alternated his record"
JOURNAL OF ENDODONTICS [ VOL 4, NO 9, SEPTEMBER 1978
hags between the o p p o s i t e surfaces of the teeth. By a p p l y i n g a load of 100 g m to these teeth, the values o b t a i n e d were within the limits of initial m o b i l i t y as described by M f i h l e m a n n ? ~ This initial mobility is said to be t h a t movement allowed to occur by virtue of the elasticity or c o m p r e s s i b i l i t y of the p e r i o d o n t a l l i g a m e n t only. L o a d s greater than 100 g m cause some distortion of the a l v e o l a r walls; this mobility has been t e r m e d s e c o n d a r y mobility. T h e findings of this investigation show that the d i s p l a c e m e n t is far greater when the load was applied from the p a l a t a l surface t h a n when the load was a p p l i e d from the buccal surface. It is not clear w h y this difference of degree of m o b i l i t y exists. Possible reasons might be t h a t there are some microanatomical differences between the buccal a n d p a l a t a l periodontal m e m b r a n e constituents. F u r thermore, the denser p a l a t a l shelf of bone m a y offer g r e a t e r resistance to displacement when c o m p a r e d with the t h i n n e r buecal p l a t e of bone. O n the other h a n d , after the a p p l i c a t i o n of the p e r i o d o n t o m e t e r to the buccal surface of a tooth, the pressure of the instrument on the crown m a y displace the tooth to the p a l a t a l side of the rest position. By decreasing the serum c a l c i u m levels with thyrocalcitonin, it was hoped to interfere with the ankylosis, which was expected to develop in replantations p e r f o r m e d with an EAT of two hours. Also, osteoclastic activity might have been inhibited, thus decreasing the degree of root resorption. The a m o u n t of m o b i l i t y of the replanted teeth in b o t h groups a p p r o x i m a t e d a n d then d r o p p e d below the basal or p r e o p e r a t i v e
m o b i l i t y levels between the t h i r d a n d fourth postoperative weeks. T h e decreased m o b i l i t y was progressive until the t e r m i n a t i o n of the experiment. This l i m i t a t i o n of m o b i l i t y is indicative of ankylosis b u t histological evidence is required for c o n f i r m a tion. Andreasen, TM by use of the same m e t h o d of p e r i o d o n t o m e t r y in cases of r e p l a n t a t i o n of teeth in m a n , was also able to detect a r a p i d h e a l i n g of the p e r i o d o n t a l m e m b r a n e and found t h a t n o r m a l values of m o b i l i t y were o b t a i n e d three weeks after replantation. T h e results of the c u r r e n t s t u d y tend to indicate t h a t 20 M R C units of t h y r o c a l c i t o n i n given once d a i l y to the vervet monkeys d i d not influence the onset of ankylosis or m o d i f y the progression of ankylosis, b u t this must be confirmed histologically. Certainly, it a p p e a r s as t h o u g h t h y r o c a l c i t o n i n tended to decrease further the m o b i l i t y of the r e p l a n t e d teeth after the sixth week, c o m p a r e d with the r e p l a n t e d teeth in the monkeys not given thyrocalcitonin. T h e fact t h a t one of the five m o n k e y s given t h y r o c a l c i t o n i n died indicates the c a u t i o n t h a t must be exercised before the a d m i n i s t r a t i o n of such a drug. F u r t h e r m o r e , the vervet m o n k e y can be used as an excellent m o d e l system for studies of r e p l a n t a t i o n . This is because the t i m i n g of the healing of the p e r i o d o n t a l m e m b r a n e before the onset of ankylosis in the vervet m o n k e y is similar to t h a t described by A n d r e a s e n 1~ in clinical studies of humans. In the future, it is p l a n n e d to histologically q u a n t i t a t e the postrep l a n t a t i o n ankylosis a n d to relate the a m o u n t of ankylosis to the c u r r e n t findings of decreased mobility.
CONCLUSIONS T h e a d m i n i s t r a t i o n of i n t r a m u s c u lar injections of t h y r o c a l c i t o n i n to the vervet m o n k e y after r e p l a n t a t i o n did not alter the m o b i l i t y c o m p a r e d with teeth in the control group. Changes in m o b i l i t y indicative of ankylosis were evident three to four weeks after r e p l a n t a t i o n in b o t h the e x p e r i m e n t a l a n d control groups as measured by p e r i o d o n t o m e t e r y . *ESPE, Seefeld/Oberbay, West Germany tGlaxo, Wadeville, South Africa ~Sandoz Co., Rasdburg, South Africa The authors thank the Sandoz Company and Dr. John Glynn of Johannesburg for the generous supply of thyrocalcitonin, and the staff of the Dental Research Unit of the South African Medical Research Council and the University of the Witwatersrand for the photographic preparation. This project was supported financially by Elida-Gibbs and by the Research, Education and Development Fund of the Dental Association of South Africa. Dr. Barbakow is in private practice and is an honorary research fellow, Prof. CleatonJones is the director, Dr. Austin is a senior research officer, and Miss Vieira is a technician, Dental Research Unit of the South African Medical Research Council and the University of the Witwatersrand, Johannesburg, South Africa. Dr. Andreasen is the associate director of the Afdelingen for Tand-, Mund-, Og Kaebesygdomme Z of the Rigshospitalet, Copenhagen, Denmark. Requests for reprints should be directed to Dr. F. H. Barbakow, Dental Research Unit, School of Dentistry, University of the Witwatersrand, 1 Jan Smuts Ave, Johannesburg, 2001, South Africa.
References
1. Andreasen, J.O., and Hj~rting-Hansen, E. Replantation of teeth. Radiographic and clinical study of 110 human teeth replanted after accidental loss. Acta Odont Scand 24: 263 Nov 1966. 2. Andreasen, J.O., and Hjqlrting-Hansen, E. Replantation of teeth. Histological study of 22 replanted anterior teeth in humans. Acta Odont Scand 24:287 Nov 1966. 271
JOURNAL OF E N D O D O N T I C S ] VOL 4, N O 9, SEPTEMBER 1978
3. Sherman, P. Intentional replantation of teeth in dogs and monkeys. J Dent Res 47:1066 Nov-Dec 1968. 4. Grossman, LT, and Ship, I.I. Survival rate of replanted teeth. Oral Surg 29:899 June 1970. 5. Guralnick, W.C. Autogenous and allogeneie transplantation of teeth. J Oral Surg 28:575 Aug 1970. 6. Shulman, L.B. The current status of allogenic tooth transplantation: attachment, repair, regeneration and development in dental replants, transplants and implants. Ciba Foundation Symposium 11. Hard tissue growth, repair and remineralisation, p 91, 1973. 7. Heithersay, G.S. Replantation of avulsed teeth. A review. Aust Dent J 20:63 April 1975. 8. Anderson, A.W.; Sharav, Y.; and Massler, M. Periodontal reattachment after tooth replantation. Periodontics 6:161 Aug 1968. 9. Andreasen, J.O. Traumatic injuries of
272
the teeth. Copenhagen, Munksgaard, 1972, p 193. 10. Andreasen, J.O. Periodontal healing after replantation of traumatically avu[sed human teeth. Assessment by mobility testing and radiography. Acta Odont Scand 33:325 Dec 1975. 11. Hurst, R.V. Regeneration of periodontal and transseptal fibers after autografts in Rhesus monkeys: a qualitative approach. J Dent Res 51:1183 Sept-Oct 1972. 12. Barbakow, F.H.; Austin, J.C.; and Cleaton-Jones, P.E. Experimental replantation of root-canal-filled and untreated teeth in the vervet monkey. J Endod 3:89 March 1977. 13. Miihlemann, H.R. Tooth mobility: The measurements method, initial and secondary tooth mobility. J Periodont 25:22 Jan 1954. 14. Mfihlemann, H.R. 10 years of toothmobility measurements. J Periodont 31:110 April 1960.
15. Barbakow, F.H.; Austin, J.C.; Cleaton. Jones, P.E.; and Vieira, E. The effect of calcitonin on replanted teeth. Proe South African Div of IADR, Durban, 1976. 16. Aliapoulius, M.A.; Goldhaber, P.; and Munson, P.L. Thyrocalcitonin inhibition of bone resorption induced by parathyroid hormone in tissue culture. Science 151:330 Jan 21, 1966. 17. Foster, G.V. Calcitonin. A review of experimental and clinical investigations. Postgrad M e d J 44:411 May 1968. 18. Retief, D.H., and Austin, J.C. The vervet monkey (Cercopithecus aethiops) as an experimental model for pulpal studies. J Dent Assoc S Africa 28:98 Feb 1973. 19. Nie, N.H.; Hull, C.H.; Jenkins, J.G.; Steinbrenner, R.; and Bent, D.A. SSPS: statistical package for the social sciences, ed 2. New York, McGraw-Hill, 1975. 20. Mfihlemann, H.R. Tooth mobility: a review of clinical aspects and research findings. J Periodont 38:(suppl)686 Nov-Dee 1967.
