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Effect of daily or 4-hourly administrations of lathyrogens on the eruption rates of impeded and unimpeded mandibular incisors of rats
EFFECT OF DAILY OR 4-HOURLY ADMINISTRATIONS OF LATHYROGENS ON THE ERUPTION RATES OF IMPEDED AND UNIMPEDED MANDIBULAR INCISORS OF RATS M. Ts( KI’I.\.* K. Erot
and M.
CHIBA:
*Departments of Orthodontics and tPedodontics. Tokyo Medical and Dental Universit). Bunkqo-ku. and ZDepartment of Pharmacology. Tsuruml I,‘nivcrsity. Tsuruml-ku. Yokohama. Japan
Tokyo.
Summary-Following daily administration of aminoacctonitrile for 10 days. eruption rates measured over 2 days in unimpeded right mandibular incisors did not show retardation related to the lathyritic changes but those in impcdcd left mandibular incisors showed gradual decreases in proportion to the time and dose. Acute effects of repeated injection of aminoacetonitrile. and of single injection of a large dose of aminoacetonitrile. p-aminopropionitrile or cysteamine on the eruption rates/4 hr had no marked effect on either ummpeded or impeded mandibular incisors within 24 hr of drug administration. It seems that the effect of the lathyrogens on the eruptlon rates of rat mandibular incisors is mainly indirect and that the retardation of the eruption rates is mediated by the degradation of the periodontal collagen fibres or by the retardation of the resisting force in the periodontal ligament. INTRODUCTION It has been suggested that the collagen fibres of the periodontium play an important role in the process of the eruption of continuously growing incisors of rats (Thomas. 1964, 1967a.b). To support this hypothesis. Thomas carried out an experiment in which both impeded and unimpeded eruption rates of weanling rat incisors decreased progressively when collagen “maturation” was interfered with following the administration of a lathyrogen (Thomas. 1965, 1967~). Sarnat and Sciaky (1965) have also reported a retardation in impeded eruption rates following the administration of aminoacctonitrile. Berkovitz. Migdalski and Solomon (1972) have examined the effect of the lathyritic agent, aminoacetonitrile. on the unimpeded eruption rate in normal and root-resected rat lower incisors. They found that unimpeded eruption rates in the lathyrogen-treated animals were not significantly lower than the respective control values. They concluded lhat their findings do not support the view that traction within “maturing” collagen fibres of the periodontium is the prime mover in tooth eruption. To test their conclusion. it was thought to be important to discover whether impeded eruption rates are affected differently from unimpeded ones under comparable experimental conditions. It was also considered worthwhile to seek an acute effect of lathyrogens on eruption rates as it is known that the rates of elimination of labclled aminoacetonitrile is fairly rapid
* Present Address: Department of Orthodontics, University, Tsurumi-ku. Yokohama. Japan.
Tsurumi
(Ponseti (‘t trl.. 1956b). Also the effects of the drugs on connective tissues appear fairly rapidly after drug administration (Suzuki. 1970; Suzuki. Matsumoto and rtoga. 1971). To this end. the relationship between the administration of lathyrogens and the response in eruption measured at 4 hr intervals for 24 hr or at daily intervals for IO days in both impeded and unimpeded mandibular incisors of adult male rats wcrc examined in the present stud!.
MATER19LS
4ND METHODS
Seventy-nine malt adult rats of the Wistar strain aged from 8 to 12 weeks were used. They were kept in metal cages. were fed a pellet diet of CE-2 (Nippon Clea Co. Ltd.. Tokyo) and took water UI lihirm from bottles with metal delivery tubes. The eruption rates of the mandibular incisors were measured by a photographic method (Chiba. Tsuruta and Eto. 1973). Three experiments were carried out as follows.
Thirty-seven rats. 8 weeks of age and weighing 774 g on the average were divided into four sub-groups of 9--lO animals. The initial number of anitnals was 38 but one animal given daily injections of AAN (30 mg.‘lOO g, day) died. apparently of an overdose of ether on the 3rd day of the experiment. The animals of three experimental groups received dail) subcutaneous injections of IO. 20 or 30 mg of AAN 100 g of body weight, respectively during the 10
da!5 ofthc eq7crimcntal period. Aminoac~tonitril~ hydrogcn sulpha~c (Tokyo Kascikogyo (‘0. Ltd., Tokyo) V,;IS dis\ol\cd in distilled water and the pH W;IX aci_iustcd to ncutralit~, The volume of injection solutions \\;Is 0.3 ml 100 g of body cjcight in all cxpcrimcnts. One group of animals was used as the control and rccci\cd suhcutancous injections of the same \olunic of saline diirin! the cxpcrimcntal period. .ILIS~ bcforc the lirst Il+ction. the right mandibular inclsol- of each rat uxs shortcncd at the Ic\cl of the in(cl--in&al crest using ;I dcntnl diamond disc. undcl cthcr anacsthesia. The same procedures Mcrc repeated cccrq’ two da~j during the cxpcrimcntal period. when the eruption of both unimpeded right and impeded left mandibular incisors Lverc recorded.
Twet\c rats. X weeks of age and weighing 227 g the a\crape wcrc divided into t\vo cqual sub-groups. One
PI-wp
30 mg
of rats
of AAN
subcutaneous
injections
on
of
100 g of hod!
inlcr\,als. :Inothcr trol and rcceivcd cxlxrimcntal
received
bvcight siu times at 4 hl group of rats mas used as die con-
repcatcd
injections
of saline
as in the
group.
The right mandilxhr incisors of all ratj wcrc shortencti \z ith ;I dental diamond disc tw’icc at 3 days and I da! bclorc the ilijcctions. The eruption rates of both unimpcdcd right and impcdcd Icft mandibular incisors \+crc mcasurcd at 4 hr intervals for 24 hr following the inicction\.
‘Thirt) rat\. I2 \+\ccks of age and L\cighing 3.39 p on the a\c~~gc Marc divided into four subgroups of 7 8 animals. The animals of three csperimcntal groups rccCi\ai ;I 5inglc subcutaneous ilijcction of X0 mg of
0.600
I
I I
2
3
AAN. 100 mg of /I-aminopropionitrile (BAPN. Tokyo Kaseikogyo <‘(I. Ltd.. Tokyo) or 30 mg of cystcamine (/~-mcrcaptoeth! laminc hydrochloride. Tokyo Kaseikogqo (‘0. Ltd.. Tokyo) per I00 g of body weight respectively. The drugs Lvcrc dissolved in distilled water and then the pH M;IS ad,justcd to neutrality. One rat died in between X I2 hr after an injection of cysteamine but other animals survived during the euperimental period. One group of animals was used as the control and rccci\cd a Gngle subcutaneous injection ol saline. The right mandibular incisors of all rats were shortcncd as in the Expcrlmcnt 2. The eruption rates of both unimpedai Iright and impeded left mandibular incisors were also measured at 3 hr intervals for 24 hr after the drug adlninislration.
I 4
~h~cwr/ oh.sc’/~r~trtio~~s. As shown in Fig. I. the body \+eight of the rats in the experimental groups showed ;I gradual decrcasc during the experimental period. Apparently the larger the dose of AAN the more severe the Gght loss. On the’ other hand. the control animals showed ;I gradual incrcasc in body weight. The animals given 20 or 30 mg of AAN: I00 g/‘daq for IO days bccamc \crc ill. particularly later in the cxpcrimentnl period. Nasal bleeding. inflammatory or oedomatous changes in the inter-incisal papilla and in the gingival tissues. and swellings at the mandibular ,joints due to cxostosis. which was confirmed at autopsy. Nerc occasionally observed. In all experimental animals. it WIS noticed that their connective tissues such as skin. tendon and blood vcsscls were fragile when thq wcrc killed. Such changes were most promincnt in the 30 mg-group.
I 5
I
6
I
7
I
6
I
9
I
10
Days
Fig. I. Rclatlve changes in body weight following administration of various doses of AAN. Average values m each gl-oup arc shown. Smce the average body acights in the group of animals at the beginning 01 111~‘ cxpcriment were not very uniform and it was difficult to xc the changes m the body weights in proportion to the dosage, their values relative to the initial body weights are shonn. Solid circles. crosses. open circles nnd triangles represent: control. IO mg-. 20 mg- and .X1mg-group respectively.
Effect
of lathyogens
I x.1
on crt~pt~on rate\
Impeded eruption rates measured at 2 day inter\als (Fig. 2) in the experimental groups were also compared with the respective control values. Significant decreases in the eruption rates were found from the 4th to 6th and 6th to 8th day (p < 0.05 002) cvcn III the IO mg-group following the drug administration. Except for the values in the initial two days. the inpedcd eruption rate\ in 70 mg- and 30 mg-groups \\ere all sipnificantl~ smaller than those in the control group (p < 0.0 1400 I ).
L------
2
l 6
4
I
I 6
IO
Days Fig. 2. I n~~npcdcd (solid lines) and impeded (dotted lines) eruption ra1es.2 days following administration of various doses of AAN. .AwI-age values in each group are shown. Solid circlej. crosses. open circles and triangles represent: control. IO me-. 20 mg- and 30 mg-group respectively.
CW/U~OI,rrrtcs. Unimpeded eruption rates measured at Z-da! intervals (Fig. 2) in the experimental groups LICI’L‘ compared with the respective control values. No significant ditference was found between the control and IO mg-group at any measured periods. Significant decreases m the eruption rates were found from the 7nd to 4th. 4th to 6th and 8th to 10th day (p < 0.05% 002) in 20 mg-group and from the 8th to 10th day (11< OWI ) in 30 mg-group following the drug administration.
Table I. Eruption
Impcdcd <‘ontrol
group AAN group
incisors Mean SD. Mean S.D.
The general appearance and the nature of conncctivc tissues of the rats given AAN or BAPN i@ction were also not appreciably diRerent from those of the
rates (pm):‘4 hr following repeated
-4~ 0 I’ninipcdcd (‘ontrol group AAN group
The animals given &hourly injections of AAN did not stem to be \crc ill during the eapcrimcntal period. The skin. tendons and blood vessels wc’rc not fragile when thq \\err: killed. Both unimpeded and impeded eruption rates measured at 1 hr intervals for 24 hr followmg the rcpcatcd in.jections of AAiX (Table I) wcrc compared \\\ith the respective control values. Significant dccreasc of Lhc unimpeded eruption rate was found from 20 to 7-l 111 follo\4 ing the drug administration. Differcnccs \+crt‘ not significant at an) other measured period in clther unimpeded or impeded eruption rates. Means + S.D. of the total unimpeded eruption rates 24 hr aerc 1087 k 68 ,~rn in the experimental group and IO46 I 17X pm in the control group rcspectively. Those of the total impeded eruption rates’ 34 hr were 795 + 90 /nn in the exprrimcntal group and 707 + 217 Itm % the control group rcspectivcl!. No significant differsncc of the eruption rates 24 hr ~\as found between the control and experimental groups in both unimpeded and impeded incisors.
injections of 70 mg of AAN 100 g 4 ht
Periods (hr) following the injections I2 I6 4x x I2 @4
20 2-z
I6 10 _
154 I ox I51 64
IO5 XI 176 71
I% 9s IX? .cl
I67 x3 IXX 20
21X 90 233 45
I71 114 19s
IX7 69
10
45
IX 57 II7 85
101 52 I36 78
I’8 59 I55 59
I04 37 I54 50
I IO 31 132 31
I27 70 I IO 5’
IJ2 75
111%
incisors
Mean S.D. Mean SD.
I ox 3-l
Means and standard deviations (S.D.) of six rats in both control and AAN groups arc shown, Initial injection was made at time 0. ” Significant dilrerence from the control value (p < 0.05).
1224
M. Tsuruta, K. Eto and M. Chiba
Table 2. Eruption
rates (urn),;4 hr after single injection of 80 mg of AAN. 100 mg of BAPN or 30 mg oicysteamine? IO0 g of body weight
-4 Unimpeded Control group AAN group BAPN group Cysteamine group
incisors Mean SD. Mean S.D. Mean S.D. Mean S.D.
Impeded incisors Control group AAN group BAPN group Cystcamine group
Mean S.D. Mean S.D. Mean S.D. Mean SD.
0
Periods (hr) after the injection t&4 4X X -i2
12 I6
16 20
20 23
193 X9 174 X7 189 X3 181 X3
266 93 201 X7 I78 125 201 61
214 x5 153 45 152 73 16X 49
I96 5X 241 I06 ‘47 -6; 215 9X
I64 102 205 X6 I74 XX 237
‘71 X3 18X 09 267 45 253
I I’)
Xl
I 64 64 219 69 I95 56 315 -__ 51
142 53 13X 59 129 64 I 06 66
201 93 14Y x4 130 94 108’ 28
118 91 I31 5X I26 41 159 49
Id2 43 I63 63 15X 44 I71 X4
IO3 45 I26 37 I49 61 174* 60
155 35 161 63 I60 63 142 34
IO1 55 I39 .32 I 36 11 124 37
Means and standard deviations (SD.) in each group (basically 778 rats: the values from a rat of the cystzamine group which died in the middle of the experimental period were included) are shown. Injection of the drugs was made at time 0. * Significant difference from the respective control value (/I < 005).
control animals as in Exp. 2. But the rats given cystcamine injection seemed to be very ill, particularly from about 6 to 12 hr after the drug administration. Their connective tissue elements were not fragile when they were killed. Unimpeded eruption rates measured at 4 hr intervals for 24 hr after a large dose of AAN. BAPN or cysteamine (Table 2) were compared with the respective control values. No significant difference was found at any measured periods in any groups. Impeded eruption rates (Table 2) were also compared between the control and each of the experimental groups. No significant changes in the eruption rates were found after an injection of a large dose of AAN or BAPN. However. impeded eruption rates in the group of rats given an injection of cysteamine were significantly smaller (p < 0.05) from 0 to 4 hr and larger (/I < 0.05) from I2 to I6 hr after the injection than those in the control group. Means k S.D. of the total unimpeded eruption rates;24 hr were 1206 f 233 ltrn in AAN-group. 1207 + 1301 k 164 pm in _ I52 Ann in BAPN-group. cysteamine-group and 1277 + 172 L(rn in the control group respectively. Those of the total impeded eruption rates;24 hr were X69 & 152 Lnn in AAN-group, X59 $ I84 pm in BAPN-group. X97 + 142 pm in cystcamme-group and 726 f 379 /lrn in the control group respectively. No significant difference of the eruption rates.‘24 hr was found between the control and experi-
mental groups sors
in both unimpeded
and impeded
inci-
DISCCSSIOI*; When the animals were given daily injections of various doses of AAN for IO days, typical lathyritic reactions. proportional to the dosage. w’crc obscrvcd like those described by other investigators (Daslcr. 1954; Selye, 1957; Gardner, Dasler and Wcinmann, 1958; Suzuki, 1959: Sciaky and Unger. I96la and b: Salnat and Sciaky. 1965). Differences of route of the drug administration did not seem to affect the lathyritic reaction when the dose and period of drug administration were appropriate. If periodontal collagen fibres produce the eruptive force of the rat incisor, it would be expected that the unimpeded eruption rates would decrease proportionally to the lathyritic reaction. But such retardation of eruption rate was not found either in the present or in previous (Berkovitz er LIP.,1972) experiments. Unimpeded eruption rates per 2 days. however. showed significant decreases at some measured periods (Fig. 2). This effect is probably due to the incomplete elimination of occlusal contact or to the fact that the drug might have exerted its effect directly on the source 01 the eruptive force or both. Decreases of impeded eruption rate following administration of the drug. which have also been seen
E&t
of lathyrogens
and Sciaky, 1965; Berkovitz et al., 1972). have been considered to be caused by the gradual degradation of the periodontal ligament as a tooth supportive organ (Krikos et al.. 1958; Krikos, 1959, 1964: Krikos. Bertran and Cohen. 1965). It is likely that the impeded incisors kept occlusal contact with their opponent teeth during the experimental period and, with the gradual degradation of the periodontal collapcn fibres. biting exerted its force more markedly on those basal soft tissues of the incisor which might play an important role in the process of the tooth eruption. The impcdcd eruption rates. particularly those in Fxperimcnts 2 and 3. arc relatively high. The explanation is probably that. when one incisor is shortened. the other becomes shorter by as much as I mm and its eruption rate increases greatly for a while (M. Chiba. .I. M. Narraway and A. R. Ness. unpublished). BAPN and of qsteaminc have been shown to have lath!rogcnic potency (Wawzonek ct rd.. 1955: Ponscti c’t r/l.. 1956a; Ramamurti and Taylor. 1958; Dasler and Milliser. 1958: Levene, 1961). Following administration of these substances, the urinary excretion and serum content of hydroxyproline Increase (.lasin and Ziff, 1962: Orbison or al., 1965). suggesting that there is an increase in the size of the metabolicallyacti>c soluble collagen pool. Levene and Gross (1959) and Gross. Levenc and Orloff (1960) have shown that ;I lathyrogcn induces a loosening of the intermolecular structure \z ithin collagen librils allowing them to dissol\c in cold neutral saline. More recently, Siegel and Martin (1970) have shown that /Gaminopropionitrile inhibits l\bvI oxidase which is necessary for the first step in the cross-linking of collagen. Suzuki et (I/. (I 97 I ) and Suzuki (1972) found that in most cases AAN. BAPN or qstcamine increased the urinary and serum contents of hydroxyproline in rats within 24 hr following single iqjection of the drugs. Ifci-o\s-linking of the newly synthesized periodontal collagen lihrcs produces the eruptive force in the rat incisor. ah suggested by Thomas (I 967b). and if the crosslinking W;I~ cfl’cctivel! inhibited by the drugs fairly rapidly after the drug administration. say within 24 hr. almost immediate retardation of incisor eruption should ha\c occurred in the present short-term experimcnts. Although. in some measured periods, the eruption rates per 1 hr showed significant changes from the rcspcctibc control values. they did not seem to be related \+ ith the time sequence of the changes in the urinar> and serum lqtlrouyproline (Suzuki et al.. 1971 ; Sucukl, 1972)c)r an inhibition of collagen cross-linking. The drugs might have had a different pharmacological action. If the Inhibition of collagen cross-linking occurs mainI! in the newly synthesized fibrils and the turno\‘er rate of collagen is fairly slow. the degradation of whole collagen fibres in the connective tissue would not occur within a day or so after the drug administration. This would probably explain why an apprcciablc degradation of connective tissues was not found in the Experiments 2 and 3. (Salnat
on eruption
rates
1735
From the present experimental results. it seems that the effect of lathyrogens on tooth eruption is indirect. The gradual degradation of the periodontal collagen fibres causes a retardation of the resisting force in the tooth when the drug is given for rather longer period. In a tooth with occlusal contact, the force might affect the tooth base more severely, thus resulting in the decrcasc of growth rate. and this might affect the cruption rate. .4CXWlCllYkJlwlm We wish to thank Prof. A. Suruki. Niigata Llm\ersity. for his helpful advice as to the determination of the dosage of lathyrogens and Mr. Alan R. Ness. Universit! College London. for his constructive criticism 01 this Mark.
REFERENCES
Berkovitz B. K. B.. Migdalski A. and Solomon M. 1972. The effect of the lathyrltic agent aminoacetonitrile on the unimpeded eruption rate in normal and root-resected rat lower Incisors. drc~/is orcd Bid. 17, 1755- 176i. Chiba M.. Tsuruta M. and Eto K. 1973. .A photographic method of measuring eruption rates of rat mandibular incisors. ,4r,ch* owl Biol. 18, 1003 1010. Dasher W. 1954. Incisor ash versus femur ash in S\VC;IIpea lathyrism (odoratism). .I. C\;w. 54, 397 302. Da&r W. and Mill&r R. V. 195X. Ostcolathyrogenic action of mercaptoethylamine and of cystaminc. Proc~. Sot C’Y~ Bid. 5frd. 98, 759-762. Gardner A. F.. Daslcr W. and Weinmann .I. P. 195% Masticatory apparatus ofalbino rats in esperlmcntal lath!rism. .1. de/~/. Rcs. 37, 492-~5 15. Gross .I.. Levcne C. I. and Orloff S. 1960. Fraglllt! and cxtractable collagen in the lathyritic chick emhrho. An arsa> for lathyrogenic :lgcnts. Proc. Sot. VY,I.Hid. !\Ict/. 105, Ia 151. Jasin H. E. and ZitT M. 1962. Relationship hetl\cen soluhlc collagen and urinary hydroxyproline in the lathlriric rat. Proc. sot. I>Y,‘.Bioi. .ticrl. 110, 837 ~841. Krikos G. A., Morris A. L.. Hammond W. S. and McClure H. H. 1958. Oral changes in experimental Iath\rism (odoratism), Orul Swq. 1 I, 309-32 I. Krikos G. A. 1959. The effects of Beta-aminopropionltrilc upon the molar teeth of the rat. J. c/cur. RL,,Y 38, 27 35. Krikos C. A. 1964. Histochemical studies on the periodontal ligament in lathyritic rats. .-lwhv orul Bid. 9, Ii 5 120. Krikos G. A.. Beltran R. and Cohen A. 196.5. Signlticance of mechanical stress on the development of periodontal Icsions in lathyritic rats. J. tlent. Rex 44, 600 607. Lcvene C. 1. and Gross J. 1959. Alterations in state of molccu1ar aggregation of collagen induced in chick cmhryos bq /I-aminopropiomtrile (Lathyrus factor). J. (‘_Y,‘.,Zlcil. 110.771 790. Lcvene C. I. 1961. Structural requwmcnts for lathwoncnic _ _ agents. J. r’~,‘. MC& 114, 295--? IO. Orbison J. L.. McCrarv C. and Callahan M. 1965. L‘rinar\ excretion and serum composition of lathyrltic rats. .lrcht Put/i. 79, 292 298. Ponseti 1. V.. Wawsonek S.. Shepard R. S.. Evans T. (‘. and Stearns G. 1956a. Further studies on lathyrlsm in the rat. P,oc~. So<. e Y/J. Bioi. Mrd. 92, 366 369. Ponsctl I. V.. Wawronek S., Franklin W. E.. Winnick R. E. and hmnick T 19ihb. Distribution of C”-labeled
I276
M. Tsuruta,
K. Eto and M. Chiba
aminoacetonltrile in tissues of rat. Metabolism and mode of elimination. Pwc. Sot. cxp. Rio/. Mctl. 93, 5 15 5 19. Ramamurtl P. and Tavlor H. E. 195X. Skeletal lesions nro duccd in rats by feeding beta-mcrcaptocthylan;inc. A.M.A. lwh\ Ptrrll. 66, 270 ‘77 Sarnat H. and Sciak! I. 1965. Experimental lathyrism in rats: EtYccts of removing incrsal stress. Pwior/o/7tic~s 3, 12x 13-1. Sciak> I. and I;nger H. 1961x. Osteolathyrlsm in the incihors of rats. liiri. Dc~fl. 20, 33 50. S&k! 1. and Ilngcr H. 19611~. EITccts of cxperimcntal lathprism on the suspensory apparatus of incisors and molars in rats. IIIII. DCII~. 20, 90 99. SelJc H. 1957. Lathyism. Rcr. (‘OJI. Bid. 16, I X2. Sicgcl R. c‘. and Martin. G. R. 1970. Collagen cross-linking. I‘n/!matic bynthcais of Iksinc-dorived aldehqdes and the productmn of cross-linked componentc. J. hiol. C/~/II. 245, I653 IhiS. Su/uhl A. 1959. Pathological changes of hard tissues folloalng Illjcctlon of alllinoacetonltrile in rats. Oc/itr~w~~iz~i lrll~rl. .I. I. 2-w 14x3. Swuki A 1070. Phal-macology ofconnscti\c tissues: On the ~ncrcascd cxcrction of urinary hydrozqpl-oline and glycosuria produced I~! lath~rogen. Co//trcqc,~~S!mpo,\ium 8.
I .II I19
Suzuki A., Matsumoto A. and Itoga M. 1971. Studicb on the mechanisms of action of lathqrogens: relationship between urinary and sxum hqdroxyprolinc and n\tcolathvritic changes. Bow .!4~tcho/. 4, 33 13. Suzuki A. 1977. Further studies on the muchanlsms 01 action oflathyrogcns: Changes of plasma h~dt-os)I”‘olin~ in ncphrectomized rats. Bow ,‘Mcrrr/~ol. 5. 32.3 2X I. Thomas N. R. 1964. The role of collagen maturation 111 alveolar hone growth and tooth eruption .I. r/c,r~~ /ic,,\. 43. 947. Thomas N. R. 1965. The etTect of inhibition 01 collagen maturation on eruption in rats. J. dc~r/. Kc\. 44, I 159. Thomas N. R. 1967a. The turnoccr of collagen and no11-colagenous protcm and tibroblast migration in the pcriodontia of rats. .I. r/c~~r. RCA. 46, I707. Thomas N. R. l967b. The properties of collagen 111the pcllodontium of an erupting tooth. In: 7‘/1(, !\l~~/w~,wr\ (I/ 7‘oorh .‘Gtp/wr~ A Symposium (Edited b! Andcrwll II. J.. Eastoe J. E. and Picton D.). pp. 102 106. Wright Kc Son\ Ltd.. Bristol. Thomas N. R. 1967~. The tension thcoq of eruption. ./. rlwr. 102, 14.3. Wawzonek S.. Ponseti I. V.. Shepard R. S. and Wlcdcnmann L. G. 1955. FpiphJseal plate Icsions. degcncl-ati\c arthritis. and dissecting aneurysm of the aorta pt-oducctl I~! aminonitrilcs. Sc~icwc, 121, 6.7 65.
and M.
CHIBA:
*Departments of Orthodontics and tPedodontics. Tokyo Medical and Dental Universit). Bunkqo-ku. and ZDepartment of Pharmacology. Tsuruml I,‘nivcrsity. Tsuruml-ku. Yokohama. Japan
Tokyo.
Summary-Following daily administration of aminoacctonitrile for 10 days. eruption rates measured over 2 days in unimpeded right mandibular incisors did not show retardation related to the lathyritic changes but those in impcdcd left mandibular incisors showed gradual decreases in proportion to the time and dose. Acute effects of repeated injection of aminoacetonitrile. and of single injection of a large dose of aminoacetonitrile. p-aminopropionitrile or cysteamine on the eruption rates/4 hr had no marked effect on either ummpeded or impeded mandibular incisors within 24 hr of drug administration. It seems that the effect of the lathyrogens on the eruptlon rates of rat mandibular incisors is mainly indirect and that the retardation of the eruption rates is mediated by the degradation of the periodontal collagen fibres or by the retardation of the resisting force in the periodontal ligament. INTRODUCTION It has been suggested that the collagen fibres of the periodontium play an important role in the process of the eruption of continuously growing incisors of rats (Thomas. 1964, 1967a.b). To support this hypothesis. Thomas carried out an experiment in which both impeded and unimpeded eruption rates of weanling rat incisors decreased progressively when collagen “maturation” was interfered with following the administration of a lathyrogen (Thomas. 1965, 1967~). Sarnat and Sciaky (1965) have also reported a retardation in impeded eruption rates following the administration of aminoacctonitrile. Berkovitz. Migdalski and Solomon (1972) have examined the effect of the lathyritic agent, aminoacetonitrile. on the unimpeded eruption rate in normal and root-resected rat lower incisors. They found that unimpeded eruption rates in the lathyrogen-treated animals were not significantly lower than the respective control values. They concluded lhat their findings do not support the view that traction within “maturing” collagen fibres of the periodontium is the prime mover in tooth eruption. To test their conclusion. it was thought to be important to discover whether impeded eruption rates are affected differently from unimpeded ones under comparable experimental conditions. It was also considered worthwhile to seek an acute effect of lathyrogens on eruption rates as it is known that the rates of elimination of labclled aminoacetonitrile is fairly rapid
* Present Address: Department of Orthodontics, University, Tsurumi-ku. Yokohama. Japan.
Tsurumi
(Ponseti (‘t trl.. 1956b). Also the effects of the drugs on connective tissues appear fairly rapidly after drug administration (Suzuki. 1970; Suzuki. Matsumoto and rtoga. 1971). To this end. the relationship between the administration of lathyrogens and the response in eruption measured at 4 hr intervals for 24 hr or at daily intervals for IO days in both impeded and unimpeded mandibular incisors of adult male rats wcrc examined in the present stud!.
MATER19LS
4ND METHODS
Seventy-nine malt adult rats of the Wistar strain aged from 8 to 12 weeks were used. They were kept in metal cages. were fed a pellet diet of CE-2 (Nippon Clea Co. Ltd.. Tokyo) and took water UI lihirm from bottles with metal delivery tubes. The eruption rates of the mandibular incisors were measured by a photographic method (Chiba. Tsuruta and Eto. 1973). Three experiments were carried out as follows.
Thirty-seven rats. 8 weeks of age and weighing 774 g on the average were divided into four sub-groups of 9--lO animals. The initial number of anitnals was 38 but one animal given daily injections of AAN (30 mg.‘lOO g, day) died. apparently of an overdose of ether on the 3rd day of the experiment. The animals of three experimental groups received dail) subcutaneous injections of IO. 20 or 30 mg of AAN 100 g of body weight, respectively during the 10
da!5 ofthc eq7crimcntal period. Aminoac~tonitril~ hydrogcn sulpha~c (Tokyo Kascikogyo (‘0. Ltd., Tokyo) V,;IS dis\ol\cd in distilled water and the pH W;IX aci_iustcd to ncutralit~, The volume of injection solutions \\;Is 0.3 ml 100 g of body cjcight in all cxpcrimcnts. One group of animals was used as the control and rccci\cd suhcutancous injections of the same \olunic of saline diirin! the cxpcrimcntal period. .ILIS~ bcforc the lirst Il+ction. the right mandibular inclsol- of each rat uxs shortcncd at the Ic\cl of the in(cl--in&al crest using ;I dcntnl diamond disc. undcl cthcr anacsthesia. The same procedures Mcrc repeated cccrq’ two da~j during the cxpcrimcntal period. when the eruption of both unimpeded right and impeded left mandibular incisors Lverc recorded.
Twet\c rats. X weeks of age and weighing 227 g the a\crape wcrc divided into t\vo cqual sub-groups. One
PI-wp
30 mg
of rats
of AAN
subcutaneous
injections
on
of
100 g of hod!
inlcr\,als. :Inothcr trol and rcceivcd cxlxrimcntal
received
bvcight siu times at 4 hl group of rats mas used as die con-
repcatcd
injections
of saline
as in the
group.
The right mandilxhr incisors of all ratj wcrc shortencti \z ith ;I dental diamond disc tw’icc at 3 days and I da! bclorc the ilijcctions. The eruption rates of both unimpcdcd right and impcdcd Icft mandibular incisors \+crc mcasurcd at 4 hr intervals for 24 hr following the inicction\.
‘Thirt) rat\. I2 \+\ccks of age and L\cighing 3.39 p on the a\c~~gc Marc divided into four subgroups of 7 8 animals. The animals of three csperimcntal groups rccCi\ai ;I 5inglc subcutaneous ilijcction of X0 mg of
0.600
I
I I
2
3
AAN. 100 mg of /I-aminopropionitrile (BAPN. Tokyo Kaseikogyo <‘(I. Ltd.. Tokyo) or 30 mg of cystcamine (/~-mcrcaptoeth! laminc hydrochloride. Tokyo Kaseikogqo (‘0. Ltd.. Tokyo) per I00 g of body weight respectively. The drugs Lvcrc dissolved in distilled water and then the pH M;IS ad,justcd to neutrality. One rat died in between X I2 hr after an injection of cysteamine but other animals survived during the euperimental period. One group of animals was used as the control and rccci\cd a Gngle subcutaneous injection ol saline. The right mandibular incisors of all rats were shortcncd as in the Expcrlmcnt 2. The eruption rates of both unimpedai Iright and impeded left mandibular incisors were also measured at 3 hr intervals for 24 hr after the drug adlninislration.
I 4
~h~cwr/ oh.sc’/~r~trtio~~s. As shown in Fig. I. the body \+eight of the rats in the experimental groups showed ;I gradual decrcasc during the experimental period. Apparently the larger the dose of AAN the more severe the Gght loss. On the’ other hand. the control animals showed ;I gradual incrcasc in body weight. The animals given 20 or 30 mg of AAN: I00 g/‘daq for IO days bccamc \crc ill. particularly later in the cxpcrimentnl period. Nasal bleeding. inflammatory or oedomatous changes in the inter-incisal papilla and in the gingival tissues. and swellings at the mandibular ,joints due to cxostosis. which was confirmed at autopsy. Nerc occasionally observed. In all experimental animals. it WIS noticed that their connective tissues such as skin. tendon and blood vcsscls were fragile when thq wcrc killed. Such changes were most promincnt in the 30 mg-group.
I 5
I
6
I
7
I
6
I
9
I
10
Days
Fig. I. Rclatlve changes in body weight following administration of various doses of AAN. Average values m each gl-oup arc shown. Smce the average body acights in the group of animals at the beginning 01 111~‘ cxpcriment were not very uniform and it was difficult to xc the changes m the body weights in proportion to the dosage, their values relative to the initial body weights are shonn. Solid circles. crosses. open circles nnd triangles represent: control. IO mg-. 20 mg- and .X1mg-group respectively.
Effect
of lathyogens
I x.1
on crt~pt~on rate\
Impeded eruption rates measured at 2 day inter\als (Fig. 2) in the experimental groups were also compared with the respective control values. Significant decreases in the eruption rates were found from the 4th to 6th and 6th to 8th day (p < 0.05 002) cvcn III the IO mg-group following the drug administration. Except for the values in the initial two days. the inpedcd eruption rate\ in 70 mg- and 30 mg-groups \\ere all sipnificantl~ smaller than those in the control group (p < 0.0 1400 I ).
L------
2
l 6
4
I
I 6
IO
Days Fig. 2. I n~~npcdcd (solid lines) and impeded (dotted lines) eruption ra1es.2 days following administration of various doses of AAN. .AwI-age values in each group are shown. Solid circlej. crosses. open circles and triangles represent: control. IO me-. 20 mg- and 30 mg-group respectively.
CW/U~OI,rrrtcs. Unimpeded eruption rates measured at Z-da! intervals (Fig. 2) in the experimental groups LICI’L‘ compared with the respective control values. No significant ditference was found between the control and IO mg-group at any measured periods. Significant decreases m the eruption rates were found from the 7nd to 4th. 4th to 6th and 8th to 10th day (p < 0.05% 002) in 20 mg-group and from the 8th to 10th day (11< OWI ) in 30 mg-group following the drug administration.
Table I. Eruption
Impcdcd <‘ontrol
group AAN group
incisors Mean SD. Mean S.D.
The general appearance and the nature of conncctivc tissues of the rats given AAN or BAPN i@ction were also not appreciably diRerent from those of the
rates (pm):‘4 hr following repeated
-4~ 0 I’ninipcdcd (‘ontrol group AAN group
The animals given &hourly injections of AAN did not stem to be \crc ill during the eapcrimcntal period. The skin. tendons and blood vessels wc’rc not fragile when thq \\err: killed. Both unimpeded and impeded eruption rates measured at 1 hr intervals for 24 hr followmg the rcpcatcd in.jections of AAiX (Table I) wcrc compared \\\ith the respective control values. Significant dccreasc of Lhc unimpeded eruption rate was found from 20 to 7-l 111 follo\4 ing the drug administration. Differcnccs \+crt‘ not significant at an) other measured period in clther unimpeded or impeded eruption rates. Means + S.D. of the total unimpeded eruption rates 24 hr aerc 1087 k 68 ,~rn in the experimental group and IO46 I 17X pm in the control group rcspectively. Those of the total impeded eruption rates’ 34 hr were 795 + 90 /nn in the exprrimcntal group and 707 + 217 Itm % the control group rcspectivcl!. No significant differsncc of the eruption rates 24 hr ~\as found between the control and experimental groups in both unimpeded and impeded incisors.
injections of 70 mg of AAN 100 g 4 ht
Periods (hr) following the injections I2 I6 4x x I2 @4
20 2-z
I6 10 _
154 I ox I51 64
IO5 XI 176 71
I% 9s IX? .cl
I67 x3 IXX 20
21X 90 233 45
I71 114 19s
IX7 69
10
45
IX 57 II7 85
101 52 I36 78
I’8 59 I55 59
I04 37 I54 50
I IO 31 132 31
I27 70 I IO 5’
IJ2 75
111%
incisors
Mean S.D. Mean SD.
I ox 3-l
Means and standard deviations (S.D.) of six rats in both control and AAN groups arc shown, Initial injection was made at time 0. ” Significant dilrerence from the control value (p < 0.05).
1224
M. Tsuruta, K. Eto and M. Chiba
Table 2. Eruption
rates (urn),;4 hr after single injection of 80 mg of AAN. 100 mg of BAPN or 30 mg oicysteamine? IO0 g of body weight
-4 Unimpeded Control group AAN group BAPN group Cysteamine group
incisors Mean SD. Mean S.D. Mean S.D. Mean S.D.
Impeded incisors Control group AAN group BAPN group Cystcamine group
Mean S.D. Mean S.D. Mean S.D. Mean SD.
0
Periods (hr) after the injection t&4 4X X -i2
12 I6
16 20
20 23
193 X9 174 X7 189 X3 181 X3
266 93 201 X7 I78 125 201 61
214 x5 153 45 152 73 16X 49
I96 5X 241 I06 ‘47 -6; 215 9X
I64 102 205 X6 I74 XX 237
‘71 X3 18X 09 267 45 253
I I’)
Xl
I 64 64 219 69 I95 56 315 -__ 51
142 53 13X 59 129 64 I 06 66
201 93 14Y x4 130 94 108’ 28
118 91 I31 5X I26 41 159 49
Id2 43 I63 63 15X 44 I71 X4
IO3 45 I26 37 I49 61 174* 60
155 35 161 63 I60 63 142 34
IO1 55 I39 .32 I 36 11 124 37
Means and standard deviations (SD.) in each group (basically 778 rats: the values from a rat of the cystzamine group which died in the middle of the experimental period were included) are shown. Injection of the drugs was made at time 0. * Significant difference from the respective control value (/I < 005).
control animals as in Exp. 2. But the rats given cystcamine injection seemed to be very ill, particularly from about 6 to 12 hr after the drug administration. Their connective tissue elements were not fragile when they were killed. Unimpeded eruption rates measured at 4 hr intervals for 24 hr after a large dose of AAN. BAPN or cysteamine (Table 2) were compared with the respective control values. No significant difference was found at any measured periods in any groups. Impeded eruption rates (Table 2) were also compared between the control and each of the experimental groups. No significant changes in the eruption rates were found after an injection of a large dose of AAN or BAPN. However. impeded eruption rates in the group of rats given an injection of cysteamine were significantly smaller (p < 0.05) from 0 to 4 hr and larger (/I < 0.05) from I2 to I6 hr after the injection than those in the control group. Means k S.D. of the total unimpeded eruption rates;24 hr were 1206 f 233 ltrn in AAN-group. 1207 + 1301 k 164 pm in _ I52 Ann in BAPN-group. cysteamine-group and 1277 + 172 L(rn in the control group respectively. Those of the total impeded eruption rates;24 hr were X69 & 152 Lnn in AAN-group, X59 $ I84 pm in BAPN-group. X97 + 142 pm in cystcamme-group and 726 f 379 /lrn in the control group respectively. No significant difference of the eruption rates.‘24 hr was found between the control and experi-
mental groups sors
in both unimpeded
and impeded
inci-
DISCCSSIOI*; When the animals were given daily injections of various doses of AAN for IO days, typical lathyritic reactions. proportional to the dosage. w’crc obscrvcd like those described by other investigators (Daslcr. 1954; Selye, 1957; Gardner, Dasler and Wcinmann, 1958; Suzuki, 1959: Sciaky and Unger. I96la and b: Salnat and Sciaky. 1965). Differences of route of the drug administration did not seem to affect the lathyritic reaction when the dose and period of drug administration were appropriate. If periodontal collagen fibres produce the eruptive force of the rat incisor, it would be expected that the unimpeded eruption rates would decrease proportionally to the lathyritic reaction. But such retardation of eruption rate was not found either in the present or in previous (Berkovitz er LIP.,1972) experiments. Unimpeded eruption rates per 2 days. however. showed significant decreases at some measured periods (Fig. 2). This effect is probably due to the incomplete elimination of occlusal contact or to the fact that the drug might have exerted its effect directly on the source 01 the eruptive force or both. Decreases of impeded eruption rate following administration of the drug. which have also been seen
E&t
of lathyrogens
and Sciaky, 1965; Berkovitz et al., 1972). have been considered to be caused by the gradual degradation of the periodontal ligament as a tooth supportive organ (Krikos et al.. 1958; Krikos, 1959, 1964: Krikos. Bertran and Cohen. 1965). It is likely that the impeded incisors kept occlusal contact with their opponent teeth during the experimental period and, with the gradual degradation of the periodontal collapcn fibres. biting exerted its force more markedly on those basal soft tissues of the incisor which might play an important role in the process of the tooth eruption. The impcdcd eruption rates. particularly those in Fxperimcnts 2 and 3. arc relatively high. The explanation is probably that. when one incisor is shortened. the other becomes shorter by as much as I mm and its eruption rate increases greatly for a while (M. Chiba. .I. M. Narraway and A. R. Ness. unpublished). BAPN and of qsteaminc have been shown to have lath!rogcnic potency (Wawzonek ct rd.. 1955: Ponscti c’t r/l.. 1956a; Ramamurti and Taylor. 1958; Dasler and Milliser. 1958: Levene, 1961). Following administration of these substances, the urinary excretion and serum content of hydroxyproline Increase (.lasin and Ziff, 1962: Orbison or al., 1965). suggesting that there is an increase in the size of the metabolicallyacti>c soluble collagen pool. Levene and Gross (1959) and Gross. Levenc and Orloff (1960) have shown that ;I lathyrogcn induces a loosening of the intermolecular structure \z ithin collagen librils allowing them to dissol\c in cold neutral saline. More recently, Siegel and Martin (1970) have shown that /Gaminopropionitrile inhibits l\bvI oxidase which is necessary for the first step in the cross-linking of collagen. Suzuki et (I/. (I 97 I ) and Suzuki (1972) found that in most cases AAN. BAPN or qstcamine increased the urinary and serum contents of hydroxyproline in rats within 24 hr following single iqjection of the drugs. Ifci-o\s-linking of the newly synthesized periodontal collagen lihrcs produces the eruptive force in the rat incisor. ah suggested by Thomas (I 967b). and if the crosslinking W;I~ cfl’cctivel! inhibited by the drugs fairly rapidly after the drug administration. say within 24 hr. almost immediate retardation of incisor eruption should ha\c occurred in the present short-term experimcnts. Although. in some measured periods, the eruption rates per 1 hr showed significant changes from the rcspcctibc control values. they did not seem to be related \+ ith the time sequence of the changes in the urinar> and serum lqtlrouyproline (Suzuki et al.. 1971 ; Sucukl, 1972)c)r an inhibition of collagen cross-linking. The drugs might have had a different pharmacological action. If the Inhibition of collagen cross-linking occurs mainI! in the newly synthesized fibrils and the turno\‘er rate of collagen is fairly slow. the degradation of whole collagen fibres in the connective tissue would not occur within a day or so after the drug administration. This would probably explain why an apprcciablc degradation of connective tissues was not found in the Experiments 2 and 3. (Salnat
on eruption
rates
1735
From the present experimental results. it seems that the effect of lathyrogens on tooth eruption is indirect. The gradual degradation of the periodontal collagen fibres causes a retardation of the resisting force in the tooth when the drug is given for rather longer period. In a tooth with occlusal contact, the force might affect the tooth base more severely, thus resulting in the decrcasc of growth rate. and this might affect the cruption rate. .4CXWlCllYkJlwlm We wish to thank Prof. A. Suruki. Niigata Llm\ersity. for his helpful advice as to the determination of the dosage of lathyrogens and Mr. Alan R. Ness. Universit! College London. for his constructive criticism 01 this Mark.
REFERENCES
Berkovitz B. K. B.. Migdalski A. and Solomon M. 1972. The effect of the lathyrltic agent aminoacetonitrile on the unimpeded eruption rate in normal and root-resected rat lower Incisors. drc~/is orcd Bid. 17, 1755- 176i. Chiba M.. Tsuruta M. and Eto K. 1973. .A photographic method of measuring eruption rates of rat mandibular incisors. ,4r,ch* owl Biol. 18, 1003 1010. Dasher W. 1954. Incisor ash versus femur ash in S\VC;IIpea lathyrism (odoratism). .I. C\;w. 54, 397 302. Da&r W. and Mill&r R. V. 195X. Ostcolathyrogenic action of mercaptoethylamine and of cystaminc. Proc~. Sot C’Y~ Bid. 5frd. 98, 759-762. Gardner A. F.. Daslcr W. and Weinmann .I. P. 195% Masticatory apparatus ofalbino rats in esperlmcntal lath!rism. .1. de/~/. Rcs. 37, 492-~5 15. Gross .I.. Levcne C. I. and Orloff S. 1960. Fraglllt! and cxtractable collagen in the lathyritic chick emhrho. An arsa> for lathyrogenic :lgcnts. Proc. Sot. VY,I.Hid. !\Ict/. 105, Ia 151. Jasin H. E. and ZitT M. 1962. Relationship hetl\cen soluhlc collagen and urinary hydroxyproline in the lathlriric rat. Proc. sot. I>Y,‘.Bioi. .ticrl. 110, 837 ~841. Krikos G. A., Morris A. L.. Hammond W. S. and McClure H. H. 1958. Oral changes in experimental Iath\rism (odoratism), Orul Swq. 1 I, 309-32 I. Krikos G. A. 1959. The effects of Beta-aminopropionltrilc upon the molar teeth of the rat. J. c/cur. RL,,Y 38, 27 35. Krikos C. A. 1964. Histochemical studies on the periodontal ligament in lathyritic rats. .-lwhv orul Bid. 9, Ii 5 120. Krikos G. A.. Beltran R. and Cohen A. 196.5. Signlticance of mechanical stress on the development of periodontal Icsions in lathyritic rats. J. tlent. Rex 44, 600 607. Lcvene C. 1. and Gross J. 1959. Alterations in state of molccu1ar aggregation of collagen induced in chick cmhryos bq /I-aminopropiomtrile (Lathyrus factor). J. (‘_Y,‘.,Zlcil. 110.771 790. Lcvene C. I. 1961. Structural requwmcnts for lathwoncnic _ _ agents. J. r’~,‘. MC& 114, 295--? IO. Orbison J. L.. McCrarv C. and Callahan M. 1965. L‘rinar\ excretion and serum composition of lathyrltic rats. .lrcht Put/i. 79, 292 298. Ponseti 1. V.. Wawsonek S.. Shepard R. S.. Evans T. (‘. and Stearns G. 1956a. Further studies on lathyrlsm in the rat. P,oc~. So<. e Y/J. Bioi. Mrd. 92, 366 369. Ponsctl I. V.. Wawronek S., Franklin W. E.. Winnick R. E. and hmnick T 19ihb. Distribution of C”-labeled
I276
M. Tsuruta,
K. Eto and M. Chiba
aminoacetonltrile in tissues of rat. Metabolism and mode of elimination. Pwc. Sot. cxp. Rio/. Mctl. 93, 5 15 5 19. Ramamurtl P. and Tavlor H. E. 195X. Skeletal lesions nro duccd in rats by feeding beta-mcrcaptocthylan;inc. A.M.A. lwh\ Ptrrll. 66, 270 ‘77 Sarnat H. and Sciak! I. 1965. Experimental lathyrism in rats: EtYccts of removing incrsal stress. Pwior/o/7tic~s 3, 12x 13-1. Sciak> I. and I;nger H. 1961x. Osteolathyrlsm in the incihors of rats. liiri. Dc~fl. 20, 33 50. S&k! 1. and Ilngcr H. 19611~. EITccts of cxperimcntal lathprism on the suspensory apparatus of incisors and molars in rats. IIIII. DCII~. 20, 90 99. SelJc H. 1957. Lathyism. Rcr. (‘OJI. Bid. 16, I X2. Sicgcl R. c‘. and Martin. G. R. 1970. Collagen cross-linking. I‘n/!matic bynthcais of Iksinc-dorived aldehqdes and the productmn of cross-linked componentc. J. hiol. C/~/II. 245, I653 IhiS. Su/uhl A. 1959. Pathological changes of hard tissues folloalng Illjcctlon of alllinoacetonltrile in rats. Oc/itr~w~~iz~i lrll~rl. .I. I. 2-w 14x3. Swuki A 1070. Phal-macology ofconnscti\c tissues: On the ~ncrcascd cxcrction of urinary hydrozqpl-oline and glycosuria produced I~! lath~rogen. Co//trcqc,~~S!mpo,\ium 8.
I .II I19
Suzuki A., Matsumoto A. and Itoga M. 1971. Studicb on the mechanisms of action of lathqrogens: relationship between urinary and sxum hqdroxyprolinc and n\tcolathvritic changes. Bow .!4~tcho/. 4, 33 13. Suzuki A. 1977. Further studies on the muchanlsms 01 action oflathyrogcns: Changes of plasma h~dt-os)I”‘olin~ in ncphrectomized rats. Bow ,‘Mcrrr/~ol. 5. 32.3 2X I. Thomas N. R. 1964. The role of collagen maturation 111 alveolar hone growth and tooth eruption .I. r/c,r~~ /ic,,\. 43. 947. Thomas N. R. 1965. The etTect of inhibition 01 collagen maturation on eruption in rats. J. dc~r/. Kc\. 44, I 159. Thomas N. R. 1967a. The turnoccr of collagen and no11-colagenous protcm and tibroblast migration in the pcriodontia of rats. .I. r/c~~r. RCA. 46, I707. Thomas N. R. l967b. The properties of collagen 111the pcllodontium of an erupting tooth. In: 7‘/1(, !\l~~/w~,wr\ (I/ 7‘oorh .‘Gtp/wr~ A Symposium (Edited b! Andcrwll II. J.. Eastoe J. E. and Picton D.). pp. 102 106. Wright Kc Son\ Ltd.. Bristol. Thomas N. R. 1967~. The tension thcoq of eruption. ./. rlwr. 102, 14.3. Wawzonek S.. Ponseti I. V.. Shepard R. S. and Wlcdcnmann L. G. 1955. FpiphJseal plate Icsions. degcncl-ati\c arthritis. and dissecting aneurysm of the aorta pt-oducctl I~! aminonitrilcs. Sc~icwc, 121, 6.7 65.