In situ hybridization $BK!$rMQ$$$?%i%C%H@Z;u%(%J%a%k
-Bisphosphonate$B$N:nMQ$K$D$$$F(J-
$B;3(J $BED(J $B2E=E!!(J $B9b(J $BLZ(J $BM5(J $B;0(J $B!!2<(J $B@n(J $B?N(J $BLo(J $BB@(J* $B>.(J $BLn(J $BGn(J $B;V(J $B!!Bg(J $BC+(J
$B7<(J $B0l(J **
$BEl5~0e2J;u2JBg3X;u3XIt>.;y;u2J3X9V:B(J
*$BEl5~0e2J;u2JBg3X;u3XIt@82=3X9V:B(J
**$BEl5~0e2J;u2JBg3X;u3XIt;u2JLtM}3X9V:B(J
The study of enamel formation of rats incisors by in situ hybridization
$B!!!!!!(J -The effect of bisphosphonate -
Yoshishige Yamada, Yuzou Takagi, Hitoyata Shimokawa*,
Hiroshi Ono and Keiichi Ohya**
Department of Pedodontics, *Biochemistry and **Dental Pharmacology,
Faculty of Dentistry, Tokyo Medical and Dental University,
1-5-45 Yushima Bunkyo-ku,
Tokyo 113, Japan
Key word: In situ hybridization /enamel hypoplasia /bisphosphonate /amelogenin
Abstract:
The successive injection of 1-hydroxyethylidene-1, 1-bisphosphonate
(HEBP) induced enamel hypoplasia in rat incisor (Jpn J Oral Biol, 33, 82-96,
1991). The present study was undertaken to investigate the amelogenin gene
expression in ameloblast of enamel hypoplasia by in situ hybridization.
Male Wistar rats, weighing 120-130g, were injected subcutaneously with HEBP
at a dose of 8 mgP/kg/day. The control rats were injected with a physiological
saline. They were sacrificed under anesthesia 24 hours after the seventh
injection, and their maxillary incisors were used for in situ hybridization.
In the light microscopic observation, the islets of enamel appeared along
the incisor surface and enamel-free zone were observed between these islets.
Contact microradiographs indicated that mineralization of the enamel had
gradually developed in these islets of enamel, but several hypoplastic bands
were observed in the enamel produced during the experimental period.
In situ hybridization using amelogenin RNA prove showed that amelogenin
gene expression was uniformly observed in ameloblasts of the secretory stage
in the control rat. In the HEBP injected rats, amelogenin gene expression
was observed almost same level of the control rats and their expression
in ameloblasts were not different between The region of enamel hypoplasia
and islet enamel. These results indicated that the successive injection
of HEBP did not alter the amelogenin gene expression in ameloblasts, but
inhibited the process after gene transcription, resulting the enamel hypoplasia.
$B!Z=o8@![(J
$B!!(JBisphosphonate$B$OL55!%T%m%j%s;@$N9=B$N`;wBN$G$"$j!"$=$NCf?4:?$,%T%m%j%s;@(JP-O-P
bond$B$NBe$j$K(JP-C-P bond$B$+$i$J$k2=9gJ*$G$"$k(J4, 5$B!K!#(JBisphosphonate$B$O%T%m%j%s;@$H0c$$%U%)%9%U%!%?!<%<$J$I$K$h$k9ZAGE*$J2C?eJ,2r$r=$KBP$7$F6/$$?FOB@-$rM-$7$F$$$k(J11)$B!#(JIn
vitro $B$K$F(Jbisphosphonate$B$ONU;@%+%k%7%&%`7k>=$N3K7A@.!"6E=8!"@.D9$KBP$7$F>c32:nMQ$r<($7(J8,
9, 11, 15)$B!"$^$?%O%$%I%m%-%7%"%Q%?%$%H$NMO2rM^@):nMQ(J2, 3, 23)$B$r<($9!#$5$i$K(Jin
vivo$B$G(Jbisphosphonates$B$O9|(J13, 16, 21, 22, 24, 25)$B!">]2gc32:nMQ$J$i$S$K9|5[<}M^@):nMQ$r<($9$3$H$,L@$i$+$K$5$l!"FC$K9|5[<}M^@)8z2L$rMQ$$$?Be
$B!!2f!9$O(JHEBP$B$r%i%C%H$K#7F|4VO"B3EjM?$7!":G=*EjM?(J24$B;~4V8e$K>e3\@Z;u$N%(%J%a%kuBV$r4Q;!$7$?$H$3$m!"%(%J%a%k]$rJs9p$7$?(J18,
30)$B!#$5$i$K$3$N%(%J%a%k]2geHi!<4VMU7O$NAj8_:nMQ(J(epithelio
-mesenchymal interaction$B!K$,(JHEBP$B$K$h$jAK32$5$l$k$3$H$K$"$k$N$G$O$J$$$+$H;XE&$7$F$-$?!#(J
$B!!:#2s(Jbisphosphonate$BEjM?$K$h$j=P8=$9$k%(%J%a%keHi!<4VMU7O$NAj8_:nMQ$K5Z$\$9(JHEBP$B$N1F6A$r8!:w$7$?!#(J
$B!Z:`NA5Z$SJ}K!![(J
1. $B
$BH72%i%C%H$K$OF1MFNL$N@8M}?)1v1U$rEjM?$7$?!#LtJ*:G=*EjM?(J24$B;~4V8e$K%M%s%V%?!<%kKc?l2<$K$F!"(J4%$B%Q%i%[%k%`%"%k%G%R%IMO1U$rMQ$$?4B!$h$j(J15$BJ,4V4CN.8GDj$r9T$C$?!#$5$i$K>e3\3\9|$r
2. Contact microradiograph(CMR)$B$N;#1F(J
$B!!8GDj=*N;$N8e!":8B&>e3\@Z;u$rCm0U?<$/3\9|$h$joK!$K=>$$%]%j%(%9%F%k&;v!K$KJqKd$7$?!#JqKd$7$?>e3\@Z;u$O=DCGJ}8~$K%@%$%d%b%s%I%+%C%?!r7o2<$G;#1F$r9T$C$?!#%U%$%k%`$O(JKodak
D-19$B8=A|1U$G(J20$B!n$G(J4$BJ,4V8=A|$7!"(JFujifix$B$G(J20$B!n!"(J20$BJ,4VDjCe$r9T$C$?!#(J
3. In situ hybridization$BK!(J
1$B!K;nNA:n@=!!(J
$B!!8GDj=*N;$N8e!">e3\1&B&@Z;u$r3\9|$h$j:Ne$KE=$jIU$1$?!#(J
$B!!(J2$B!K%W%m!<%V:n@=(J
$B!!(JIn situ hybridization$B$K;HMQ$7$?%W%m!<%V$O!"(Jpspt18 transcription vector
plasmid $B$KAH$_9~$^$l$?%&%7(J amelogenin cDNA$B$N(JEcoR1 fragment$B$h$jD4@=$7$?!#(JPlasmid
$B$r@)8B9ZAG(J BamH1$B$G>C2=$7!"%F%s%W%l!<%H(JDNA$B$r:n@=$7!"(JDIG RNA Labeling Kit
(Boehringer$B!"FH(J)$B$rMQ$$$F(Jamelogenin RNA$BE>
3$B!K%O%$%V%j%@%$%
$B!!(JIn situ hybridization$B$OLnB<$NJ}K!$K=`$8$F9T$C$?(J31, 32)$B!#$9$J$o$A!"%W%m!<%V$r%O%$%V%j%@%$%e$N3F@ZJR$K$*$h$=(J80$B&L(Jl$B$:$DE)2<$7!"%Q%i%U%#%k%`$GJ$$$!"(J50$B!s%[%k%`%"%_%I$G<>=a$5$;$?L)JDH"Fb$K$F(J16$B;~4V!"(J50$B!n$GH?1~$5$;$?!#(J
4$B!K@v>t5Z$S8!=P(J
$B!!%O%$%V%j%@%$%
$B!Z7k2L![(J
1$B!K(JCMR$B$K$h$k4Q;!(J
$B!!BP>H72$N4p]2g]2g
$B!!(JHEBP$B$r(J8 mgP/kg/day 7$BF|4VEjM?$7$?u%(%J%a%ke$K=P8=$7$?!#$^$?%(%J%a%k]2g]2gu$K7A@.$5$l$?%(%J%a%k
2$B!K7ABV3XE*4Q;!$J$i$S$K(Jin situ hybridization$B$K$h$k4Q;!(J
$B!!(J $BBP>H72%i%C%H$N@Z;u;ufuIt$G$O!"4p$$%(%J%a%k]2g]2g$$=y!9$KHn8|$7$F$$$kA|$,4Q;!$5$l$?!#(JIn
situ hybridization$B$K$h$j%"%a%m%8%'%K%sCAGr0dEA;R6I:_$r8!F$$7$?$H$3$m!"Cc?'$K@w?'$5$l$k0dEA;R$NH/8=It0L$,4p
$B!!(J HEBP$BEjM?72%i%C%H$G$O%(%J%a%ku$K?t%u=jB8:_$7!"$=$N4V$K%(%J%a%ku$K=P8=$7$?%(%J%a%k]2g6-$HJ?9T$K?tK\$N7A@.ITA4@~$,B8:_$7$?!#%(%J%a%ko$,8+$i$l$k>l9g$,B?$/!"GX>f$,Dc$/$J$C$?$j!"7ABV$K0[>o$r<($7$F$$$k:YK&$,4Q;!$5$l$?!#0lJ}!"%(%J%a%k
$BK&$OHf3SE*@5>o$K6a$$7ABV$rJ]$C$F$$$?!#$5$i$K>]2g]2gA0u$r<($7$F$$$?!#$7$+$7!"%(%J%a%k]2gH72$K8+$i$l$k@P3%2=>]2g
$B!!(JIn situ hybridization$B$N4Q;!$G$O!"Cc?'$K@w?'$5$l$k%"%a%m%8%'%K%sCAGr0dEA;R$NH/8=It0L$O%(%J%a%k2j:YK&A4$F$K$[$\F1DxEY$K4Q;!$5$l$?!#$=$NH/8=$OEg>u$K=P8=$9$k%(%J%a%k
$B!Z9M;!![(J
$B!!0lHL$K%i%C%H@Z;u;ufu$G$O30Ee>]2geHi!<4VMU7OAj8_:nMQ(J(epithelio-mesenchymal
interaction$B!K$K$h$j;YG[$5$l$F$*$j!"J#;($J;u$N7A@.%a%+%K%:%`$K$*$1$k:G=i$N%9%F%C%W$H$7$F=EMW$G$"$k(J27,
29)$B!#(JHEBP$B$O>]2g=$KBP$7D>@\:nMQ$9$k$3$H$K$h$j30Ee>]2g]2g]$KL)@\$K4XO"$7$F$$$k$H;W$o$l$k!#(J
$B!!(JHEBP$B$OEjM?D>8e$N7lCf$KHf3SE*9bG;EY$G0];}$5$l$F$$$k4V$K!"30Ee>]2g]2g]2geHi!<4VMU7OAj8_:nMQ$N:nMQ$,AK32$5$l$k$H9M$($i$l$k!#$=$N7k2L!"%(%J%a%k2j:YK&$NJ,2=$rM6F3$9$k$3$H$,$G$-$:!"%(%J%a%k2j:YK&$N2L$?$9%(%J%a%kCAGr$N9g@.!"J,Hg$H$=$l$KB3$/%(%J%a%kc32$5$l$k$b$N$H?d;!$G$-$k!#(JHEBP$BEjM?8e!";~4V$,7P2a$9$k$K$7$?$,$$!"LtJ*$N7lCfG;EY$,Dc2<$9$k$H!"30Ee>]2geHi!<4VMU7OAj8_:nMQ$,2zI|$7!"%(%J%a%k2j:YK&$NJ,2=$,?J9T$7$F:FEY%(%J%a%k
$B!!$3$l$^$G8!:w$K$h$j!"(JHEBP$BEjM?$K$h$k%(%J%a%ke5-$N$h$&$K>eHi!<4VMU7OAj8_:nMQ$N>c32$HL)@\$K4X$o$C$F$$$k$3$H$,?d;!$G$-$?$,(J18,
30)$B!"%(%J%a%k2j:YK&$N5!G=H/8=$X$N1F6A$Nc32$K4p$:$/$b$N$G$O$J$/!"0dEA;RE>c32$r\:Y$O:#8e$N8&5f$K$h$jL@$i$+$K$9$kI,MW$,$"$k$,!"4v$D$+$N2DG=@-$r<($9$H!"(J1$B!K0dEA;R$NE>c32!"(J2$B!K(JHEBP$BEjM?$K$h$C$F$b%"%a%m%8%'%K%sCAGr9g@.G=$O>c32$5$l$J$$$,!"$=$N8e$NCAGrJ,HgG=$N>c32!"(J3$B!K%"%a%m%8%'%K%sCAGr$N9g@.J,Hg$O>c32$5$l$J$$$,!"CAGr9g@.0J9_$N(Jmodification$B$,IT40A4$G4p
$B!!$^$?c32A|$,4Q;!$G$-$k$3$H$+$i!"$$$C$?$s4pc32$r5/$3$9$b$N$H9M$($i$l$k!#$3$N:nMQ$O!"%(%J%a%k
$B!!K\8&5f$GL@$i$+$K$J$C$?(JHEBP$BEjM?$K$h$k%i%C%H@Z;uI=AX$K=P8=$9$k%(%J%a%k]$O!"@Z;u$N7A@.2aDx$r9M$($k>e$G6=L#?<$$8=>]$H;W$o$l$k!#FC$K>eHi!<4VMU7OAj8_:nMQ$N;u$N7A@.2aDx$K$*$1$kK\BV$r5fL@$9$k>e$GM-MQ$J\:Y$J8!:w$r9T$$!"(JHEBP$BEjM?$K$h$k%(%J%a%k
$B!ZJ88%![(J
1) Beertsen, W., Niehof, A. and Everts, V. : Effect of 1-hydroxyethylidene-1,1-bisphosphonate
(HEBP) on the formation of dentin and the periodontal attachment apparatus
in the mouse. Am. J.
Anat. 174 : 83-103, 1985.
2) Fleisch, H., Russell, R. G. G., Bisaz, S., Casey, P. A. and Mulbauer,
R. C. : The influence of pyrophosphate analogues (diphosphonates ) on the
precipitation and dissolution of calcium phosphate in vitro and in vivo.
Calcif. Tissue Int. 2 (suppl.) : 10-10 A, 1968.
3) Fleisch, H., Russell, R. G. G. and Francis, M. D. : Diphosphonates inhibit
hydroxyapatite
dissolution in vitro and bone resorption in tissue culture and in vivo.
Science 65 : 1262-1264, 1968.
4) Fleisch,H. and Russell, R. G. G. : A review of the physiological and
pharmacological effects of pyrophosphate and diphosphonates on bones and
teeth. J. Dent. Res. 51 : 324-332, 1974.
5) Fleisch, H.: Bisphosphonates : Mechanism of action and clinical applications.
In Bone and Mineral Research Annual I. (ed. by Peck, W. A.) pp. 319-357,
Excerpta Medica, Amsterdam, 1983.
6) Fleisch, H.: New bisphosphonates in osteoporosis. Osteoporosis Int. 2
(Suppl.): S15-22, 1993.
7) Fouda, N., Caracatsanis, M. and Hammarstrom, L. : Developmental disturbances
of the rat molar induced by two diphosphonates. Adv. Dent. Res. 3 : 234-240,
1989.
8) Francis, M. D., Russell, R. G. G. and Fleisch, H. : Diphosphonates inhibit
formation of calcium phosphate crystals in vitro and pathological calcification
in vivo. Science 65 : 1264-1266, 1969.
9) Francis, M. D. : The inhibition of calcium hydroxyapatite crystal growth
by polyphosphonates and polyphosphates. Calcif. Tissue Int. 3 : 151-162,
1969.
10) Gasser, A. B., Morgan, D. B., Fleisch, H. and Richelle, L. J. : The
influence of two diphosphonates on calcium metabolism in the rat. Clin.
Sci. 43 : 31-45, 1972.
11) Hansen, N. M., Felix, R.,Bisaz, S. and Fleisch, H. : Aggregation of
hydroxyapatite crystals. Biochem. Biophys. Acta 451 : 549-559, 1976.
12) Jung, A., Bisaz, S. and Fleisch, H. : The binding of pyrophosphate
and two diphosphonates by hydroxyapatite crystals. Calcif. Tissue Int.
11 : 269-280, 1973.
13) King, W.R., Francis, M. D. and Michaael, W. R. : Effect of disodium
ethane-1-hydroxy-1, 1-diphosphonate on bone formation. Clin. Orthop. 78
: 251-270, 1971.
14) Larsson, A : The short-term effects of high dose of ethane-1-hydroxy-1,1-diphosphonate
upon early dentin formation. Calcif. Tissue Int. 16 : 109-127, 1974.
15) Meyer, J. L. and Nancollas, G. H. : The influence of multidentate organic
phosphonates on the crystal growth of hydroxyapatite. Calcif. Tissue Int.
13 : 295-303, 1973.
16) Michael, W. R., King, W. R. and Francis, M. D. : Effectiveness of diphosphonates
in preventing "osteoporosis" of disuse in the rat. Clin. Orthop.
78 : 271-276, 1971.
17) Ogawa, Y., Adachi, Y., Hong, S. and Yagi T.:1-hydroxyethylidene-1, 1-bisphosphonate
(HEBP) simultaneously induces two distinct types of hypomineralization in
the rat incisor dentin. Calcif. Tissue Int. 44 : 46-60, 1989.
18) Ohya, K., Mataki, S., Wakamatsu H. and Ogura H.: Effects of successive
injection of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on the rat incisor
enamel. In Tooth Enamel V( edit. R. W. Fearnhead) pp 227-234, Florence
Publishers, 1989.
19) Reith, E. J. : The early stage of amelogenesis as observed in molar
teeth of young rats. J. Ultrastruct. Res. 17 : 503-526, 1967.
20) Resenblum, I. Y. : The effect of disodium ethane-1-hydroxy-1, 1-diphosphonate
(EHDP) on bone and serum chemistry in rabbits. Calcif. Tissue Int. 16 :
145-152, 1974.
21) Reynolds, J. J., Minkin, C., Morgan, D. B., Spycher, D. and Fleisch,
H. : The effect of two diphosphonates on the resorption of mouse calvaria
in vitro. Calcif. Tissue Int. 10 : 302-313, 1972.
22) Reynolds, J. J., Murphy, H., Mulbauer, R. C., Morgan, D. B. and Fleisch,
H. : Inhibition by
diphosphonates of bone resorption in mice and comparison with gray-lethalosteoporosis.
Calcif. Tissue Int. 12 : 59-71, 1973.
23) Russell, R. G. G., Mulbauer, R. C., Bisaz, S., Williams, D. A. and
Fleisch, H. : The influence of pyrophosphate, condensed phosphates, phosphonates
and other phosphate compounds on the dissolution of hydroxyapatite in vitro
and on bone resorption induced by parathyroid hormone in tissue culture
and in thyroparathyroidectomised rats. Calcif. Tissue Int. 6 : 183-196,
1970.
24) Schenk, R., Merz, W. A., Mulbauer, R., Russell, R. G. G. and Fleisch,
H. : Effect of ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and dichloromethylene
diphosphonate (Cl2MDP) on the calcification and resorption of cartilage
and bone in the tibial epiphysis and metaphysis of rats. Calcif. Tissue
Int. 11 : 196-214, 1973.
25) Shinoda, H., Adamek, G., Felix, R.: Fleisch, H., Schenk, R. and Hagan,
P. : Structure- activity relationships of various bisphosphonates. calcif.
Tissue Int. 35 : 87-99, 1983.
26) Simmelink, J. W. : Ultrastructural effects diphosphonates on dental
enamel. Adv. Dent. Res. 1 : 356-365, 1987.
27) Slavkin, H. C. and Bringas, Jr. P.: Epithelial-mesenchyme interactions
odontogenesis 4. Morphological evidence for direct heterotypic cell-cell
contacts. Dev. Biol. 50 :428-442, 1976.
28) Taya, Y. and Suga, S. : Disturbed mineralization of developing enamel
by the single injection of HEBP. In tooth Enamel V. (ed. by Fearnhead R.
W.) pp 219-223, Florence Publishers, Yokohama, 1989.
29) Thesleff, I. and Hurmerinta, K. : Tissue interactions in tooth development.
Differentiation 18 : 75-88, 1981.
30) Wakamatsu, H. : A study of the effects of successive injections of 1-hydroxyethylidene-1,
1-bisphosphonate(HEBP) on the enamel formation in the rat incisor. Jpn.
J. Oral Biol. 33: 82-96, 1991.
31) $BLnB5B@O:(J : in situ $B%O%$%V%j%@%$%
32) $BLnB5B@O:(J : $B9EAH?%$rMQ$$$?(Jin situ $B%O%$%V%j%@%$%
{$B!!(JSorry, Figures will be installed soon.$B!!(J}
Fig. 1
Contact microradiographies of the longitudinal ground surface of enamel
in the ratmaxillary . (a) The HEBP 8 mgP/kg/day-injected group. Several
islets of mineralized enamel are observed in the secretory stage. ( b) High
power magnification of (a). The mineralization of dentin has occurred only
under the islets of enamel. E : enamel, D : dentin.
Fig. 2
Light microscopic photographs of the longitudinal section of the rat maxillaryincisor.
(a) The control group. In situ hybridization showed the expression of amelogenin
gene at the
secretory stage of ameloblasts. (b) -(d) The HEBP 8 mgP/kg/day-injected
group. (b)Normal light
microscopic section. Several islets of enamel (*) appear along the dentin
surfaceand enamel-free zones are observed between these islets. The dentin
under the islets of enamel is more strongly stained with hematoxylin-eosin
than the dentin under the enamel-free zone. The ameloblasts facing the enamel-free
zone seem to be normal in form. The odontoblasts shows an unusual cell-arrangement.
(c) Expression of amelogenin gene by in situ hybridization using anti-sense
prove. Amelogenin gene expression are observed in the ameloblast between
enamel-free area and islets of enamel. The level of expression is similar
between enamel-free area and islets of enamel. (d) Expression of amelogenin
gene using sense prove. The expression of amelogenin could not be observed
in the ameloblast. E : enamel, D : dentin.