The Effects of Hypocalcemia/Hypophosphatemia on Porcine Bone and Dental Hard Tissues in an Inherited Form of Type 1 Pseudo-Vitamin D Deficiency Rickets H. LIMEBACK, C. SCHLUMBOHM1 A. SEN, and G. NIKIFORUK Faculty ofDentistry, University of Toronto, 124 Edward St., Room 455, Toronto, ON, Canada M5G 1G6; and 'Department of Physiology, School of Veterinary Medicine, Hannover, Germany The effects of Vitamin D deficiency rickets on the formation of mineralized dental tissues were studied in a breed of pigs which had moderate and marked hypocalcemia because of an inherited defect in the renal production of the biologically-active Vitamin D metabolites. Affected piglets developed classical symptoms of rickets which were fatal unless Vitamin D supplements were given. The dissected mandibles of homozygous (rickets) and heterozygous (normal) pigs were photographed and radiographed. Compared with- those of normal pigs, the mandibles of homozygous pigs demonstrated slowed development/eruption of permanent teeth, under-mineralized bone, underdeveloped dentin (enlarged pulp chambers), interglobular dentin, and enamel hypoplasia. Enamel defects in rachitic pig teeth were difficult to observe radiographically, but could be detected visually and by SDS-PAGE analysis ofthe enamel protein components of developing and maturing enamel. There was significant retention of amelogenins in the enamel maturation zones of developing molars taken from rachitic pigs, but there was no obvious difference in the patterns of amelogenin processing. J Dent Res 71(2):346-352, February, 1992
Introduction. Abnormalities in the mineralization of bone in human subjects with rickets have been well-documented, but the effects of rickets on dental tissues are less well-known. Dentin seems to be affected more frequently than enamel: The usual dental findings in rickets are large pulp chambers and pulp horns, loss of lamina dura, interglobular dentin, and premature loss ofprimary teeth (Nikiforuk and Fraser, 1979). Enamel lesions in humans can, however, result from disorders affecting calcium metabolism (Nikiforuk and Fraser, 1981). In general, hypocalcemia in the presence of normo- or hyperphosphatemia does not produce characteristic rachitic lesions in bone or dentin. Furthermore, hypophosphatemia alone does not produce enamel lesions. These observations led Nikiforuk and Fraser (1979, 1981) to propose a unifying concept on the etiology of dental abnormalities in rickets. It was suggested by these authors that enamel hypoplasia results from any disease or condition that produces periods of prolonged hypocalcemia during the development of teeth. Because of the difficulties inherent in the use of human experimental subjects, animal model systems have been sought for studies of the effects of serum calcium and phosphate changes on hardtissue formation. A breed of pigs with a genetic defect of renal calcitriol formation, resulting in pseudo-Vitamin D deficiency type 1 rickets (Harmeyer et al., 1982; Kaune and Harmeyer, 1985, 1987; Winkler et al., 1986), was used in this study to examine the effects of rickets on formation of the hard tissues in the mandible and to examine the effects of hypocalcemia on enamel formation. The features and clinical symptoms observed in the homozygous offReceived for publication May 17, 1991 Accepted for publication September 12, 1991 This collaborative study was supported by the Medical Research Council of Canada.
346
spring of this breed of pigs include: (a) autosomal recessive inheritance pattern; (b) onset of symptoms of rickets 4-7 weeks post-natal;
(c) low plasma calcium and phosphate levels; (d) excessive urinary excretion of phosphates, amino acids, and cAMP; (e) high blood levels ofalkaline phosphatase and PTH; (f) reduced rates of calcium and phosphate transport in the gut; (g) reduced activities of renal 25-hydroxycholecalciferol-1 and 24-hydroxylases; (h) classic skeletal changes (increased rates of bone mineral mobilization and decreased rates of bone mineral accretion); and (i) reversal of symptoms with injected Vitamin D. While the effects of rickets on bone have been studied in various animal model systems, they have not been used extensively to study the effects on dental tissues. We have made use of the Hannover Pig Model to examine the effects of rickets on dental tissues including enamel. Nucleation ofthe initial mineral deposits in hard tissues, including enamel, occurs in collagen-based matrix. Enamel is different from the other hard tissues in that the overall mineralization process is governed by a non-collagenous matrix which is almost completely replaced by mineral (see Fearnhead and Suga, 1990, for a recent review). The factors that control hard-tissue mineralization are still unknown. An animal rickets model system should prove useful for studying how reduced blood levels of calcium, phosphate, and Vitamin D metabolites affect formation and mineralization of dental tissues.
Materials and methods. Preparation ofpig litters for the study.-The model system used in this study was the Hannover Pig Strain (Harmeyer and Kaune, 1990; Harmeyer, 1991). The breed selected was a cross between homozygous Landrace pigs and heterozygous Minipigs. Three litters of 3-6 piglets each were produced and maintained on a commercial pig starter diet with 0.8% calcium and 0.6% phosphorus. The untreated homozygous offspring developed symptoms of rickets by the 4th to 7th weeks and were given from 0.5 to 3.0 mg Vitamin D3 i.m. injections to keep them alive. Heterozygous normal pigs and homozygous rachitic pigs were killed 4-6 months after birth and processed as outlined below. Plasma calcium, phosphate, and alkaline phosphatase determinations.-Plasma calcium, phosphate, and alkaline phosphatase levels were determined during the supplementation periods and just prior to death, according to previously described methods (Winkler et al., 1986). Tissue preparation.-The mandibles of each group of offspring were dissected, shock-frozen in liquid nitrogen, stored at -70'C, and transported on solid CO2 by air freight from Hannover to Toronto. After being thawed, the jaws were photographed and radiographed. In one group of pigs, both right and left unerupted permanent second mandibular molars (M2) and corresponding canines were carefully dissected and photographed. The transitional enamel on the mesolingual surface ofeach M2 was visualized under U.V. light and marked with a scalpel blade. A strip of developing enamel (D), approximately 2 mm x 4 mm, was scraped off immediately cervical to the transitional enamel, avoiding the underlying dentin, and a corresponding strip of mature enamel (M) was scraped off immedi-
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Vol. 71 No. 2
THE EFFECTS OF RICKETS ON BONE AND TEETH
347
TABLE TIME OF VITAMIND THERAPYAND PLASMA CALCIUMAND PHOSPHATE DETERMINATIONS INTHE PIGS USED IN THIS STUDY
Group Specimen Calcium Week
I N N
I R S
I R S
II N N
II N N
II R M
II R S
II R S
5
0.50 D3
6
1.25 D3 1.25 D3 1.25 D3 5.0 D3 5.0 D3
7
III N N
III R S
III R S
Development of the 2nd mandibular molar cusp tips starting to mature
1.25 D3 1.25 D3 1.25 D3
8
1.25 D3
9
1.25 D3 1.25 D3 2.5 D3
10
2.5 D3 2.5 D3 crown form completed 3.0 Ca 1.7 Ca 2.0 Ca
16
3.6 Ca
2.5D3
3.2P
2.7 Ca
2.6P 17
3.2 Ca
3.75D3 2.5D3
3.0 P
2.2 Ca 2.4 Ca
2.3P 2.5P 23
3.1 Ca
maturation of crown at cervical margin
3.3P 25 26
1.7 Ca 3.1 Ca
2.5 Ca
1.2P 2.9P
2.2P
2.1Ca 2.0Ca 2.0P 1.7P 1.6 Ca
30
1.2P 34
2.3 Ca
2.3P N - Normal, R - rachitic, S - severe hypocalcemia, M - moderate hypocalcemia, D3 - Vitamin D3 supplement (mg), Ca - plasma calcium (mmol/L), and P - plasma phosphorus (mmol/L). ately apical to the transitional enamel. The enamel scrapings were allowed to dry at 25°C in air for four h. For examination of rachitic dentin, selected mandibular first molars that were removed from a homozygous pig jaw were sectioned with a rotary saw. The sections were ground with fine sandpaper and photographed under a microscope. Enamel protein analysis. -Samples of enamel scrapings, taken from both the right and left molars, were weighed and extracted directly in SDS-PAGE sample buffer by being heated at 900C for 20 min. The ratio of enamel biopsy to extraction buffer (5.0 mg enamel! mL buffer) was the same for all samples analyzed. Following removal of insoluble material by centrifugation, the extracted enamel proteins were analyzed on 12.5% polyacrylamide slab gels and then stained with silver as described elsewhere (Limeback and Simic, 1990).
For quantification of proteins in the extracts, the 20-kDa amelogenin bands in each enamel extract were scanned and the resulting peaks integrated. Limiting the densitometry to this protein in samples separated in the same SDS-PAGE gel and stained by the same solutions avoided problems with variation in silver-staining intensities. The total amount of 20-kDa protein was estimated by combining the data from the bands for developing and mature enamel for each tooth. The mean percentages of 20-kDa amelogenin in both the developing and mature enamel compartments were calculated and compared by Student's t test.
Results. Blood chemistry.-The results of the plasma calcium, phosphate, and alkaline phosphatase levels of the three groups of pigs studied
Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on March 23, 2015 For personal use only. No other uses without permission.
JDent Res Fbruy 199
A
Nrmnal,
(heterozygous B
(homozy0
ou
C
rID
ri k
lature ofthe
H
sytmi nerte* tos
:dactdob perante
tmanph the p used iths stu shwe th those of oer dgated pig stair 0
VL
GeneaI
1
tMzyo
reda
ladogr £e epanry depnd
10 209 smaller.
en
1Ihe effects orfckets on toh devlopmet
thlat we
cleh wras eerozygous pigs. This The pig wi rckets w small( v~elop more slowly than heru1n
jaws Thi
rdnhibles
h re
under identical conitons, te rachitic jaws showed radiographed a p bounced rdction in bone Ts was also observed densitr defcien and dow dringissecton of unrpted teet in the racihtichone was dissected wth less it. Radographs oGo I pg jaws are
erdeveloped skeletal systems a
,r
Padio
[t
g crown foration ese teeth are most
rst six mor
r
iand rahite pig mandie h1c compw uou rts) and heterogous (noal) aws om GrupI ensng * at idential codtions ofX-a~diogaphed togther, fim proessn, and p vtogaph sy wr aistrl spcmens. 30 ws; and (C) jaw with 33 eeks (B) jaw ii sw
Fig
fteCl
syte
he
Aas
TI
lev
the
th
Not
ke smaller pges w rts n th]
o
the pi
shwn in Fig. 2. here wev no alteratons tenumbers lhat developed a)r their positi in the ante However, ;itydomiral iin allIof t pervan teth was clearly om pi wih ikets Frerore he i nthe pe et teth as restricted in e o0r t ris was e yint b e great elaed pip en e seco nd adthird reseng o ne showed eiad pulp chamber n e affected
Toot re om
eruption tme was afetd in ritkets WVhen ittermates ioo he first pemanen mola was taped t
ot only was ess deir forced rickets but te groud sertn in Ft. 3 the dentin was Tahzed. Irte globular derntn was sered nthe tet
esronsr
aso, as den poo0y mnli
onsated
cited w Gh rickets.
dIesI Defcsan the erareal primary teeth :rved in] rachitc mBandilbles, bu1t enamel dlefecs, caraclties in e enaml s ac, were teize~d by nittin or evi ent on te eraptddirst perant molars (Fig 4). The xct of CIIe lesion depended on the stage development of the tooth du:n'n ,grids f 90poa1ema/bypophosp1hartema Further* o
were
ename
mrore vitamAn D supple ents appeared to restore normal eramel kt theextent fte ena llieson (ee sample B ma1urati1 andt
The ana dcts t Ieh In the teeth, t However
wile
r esas pronounced tn nerupted rachttic enre' ca fre was generally still intact
normaia
epted
teeth
had
enamel thatwas
smooth and shiny, the enamel of rachitc teeth wasrrul ad dull we earned at deti stages of develop(tg 5) meIt id not wever, show morph11otical differenrs Neverthe a
moar. A rachVit d seion of a Fig. tg second ofIa f ast permanent manibuar modarlissect daw anF 2 was pd ad photogaed (X lf
dentin is makd, wit
an arw.
thik v
romthrathit Pig The inerltol Oar
less, the lower mineralconte1 t
darer appearnc.m
Lrcmlpro ep crptr takerhro
ave te ctteetha overall
rv By compa nxg eqal portions ofename xac the same stag f enamel develop
Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on March 23, 2015 For personal use only. No other uses without permission.
l~~~o VolVol 71 No THRE E LCT OF RIMV m
341,11
Fig Enam toplasai -achitipis, ese are photo hs wheas theist martndibla mor s had deistint rons of enamel hypoph ua be noted on the molar in B by thE appearance of rl enamel tissue cr ment it was pos1ble to make stations oner the d of rockets on the tnamer Iprotei patterns of dissectd molars(sg 6A). In this srain ofpig te name protein patem n tie dvelopig enmel of both normal and rachtic animals was similar that described
doe pg (Overall and LimebackE 1988) ndi enamel proeins prepessedby te name Vrelenirs wer lael proteinases m a smiar manre movAed in the mure enamel, whereas serum album appeared to
preously catig be
n te
enamel
o
te tee
severehypocacenia/iyophospnte1ia had
elni
orea
densitometry (Fig.
B).
prporton of tie
amelogenin that was recorded in significantly great in e rachitc te normal teeth
total
amou
mature enamel
t
(p
Introduction. Abnormalities in the mineralization of bone in human subjects with rickets have been well-documented, but the effects of rickets on dental tissues are less well-known. Dentin seems to be affected more frequently than enamel: The usual dental findings in rickets are large pulp chambers and pulp horns, loss of lamina dura, interglobular dentin, and premature loss ofprimary teeth (Nikiforuk and Fraser, 1979). Enamel lesions in humans can, however, result from disorders affecting calcium metabolism (Nikiforuk and Fraser, 1981). In general, hypocalcemia in the presence of normo- or hyperphosphatemia does not produce characteristic rachitic lesions in bone or dentin. Furthermore, hypophosphatemia alone does not produce enamel lesions. These observations led Nikiforuk and Fraser (1979, 1981) to propose a unifying concept on the etiology of dental abnormalities in rickets. It was suggested by these authors that enamel hypoplasia results from any disease or condition that produces periods of prolonged hypocalcemia during the development of teeth. Because of the difficulties inherent in the use of human experimental subjects, animal model systems have been sought for studies of the effects of serum calcium and phosphate changes on hardtissue formation. A breed of pigs with a genetic defect of renal calcitriol formation, resulting in pseudo-Vitamin D deficiency type 1 rickets (Harmeyer et al., 1982; Kaune and Harmeyer, 1985, 1987; Winkler et al., 1986), was used in this study to examine the effects of rickets on formation of the hard tissues in the mandible and to examine the effects of hypocalcemia on enamel formation. The features and clinical symptoms observed in the homozygous offReceived for publication May 17, 1991 Accepted for publication September 12, 1991 This collaborative study was supported by the Medical Research Council of Canada.
346
spring of this breed of pigs include: (a) autosomal recessive inheritance pattern; (b) onset of symptoms of rickets 4-7 weeks post-natal;
(c) low plasma calcium and phosphate levels; (d) excessive urinary excretion of phosphates, amino acids, and cAMP; (e) high blood levels ofalkaline phosphatase and PTH; (f) reduced rates of calcium and phosphate transport in the gut; (g) reduced activities of renal 25-hydroxycholecalciferol-1 and 24-hydroxylases; (h) classic skeletal changes (increased rates of bone mineral mobilization and decreased rates of bone mineral accretion); and (i) reversal of symptoms with injected Vitamin D. While the effects of rickets on bone have been studied in various animal model systems, they have not been used extensively to study the effects on dental tissues. We have made use of the Hannover Pig Model to examine the effects of rickets on dental tissues including enamel. Nucleation ofthe initial mineral deposits in hard tissues, including enamel, occurs in collagen-based matrix. Enamel is different from the other hard tissues in that the overall mineralization process is governed by a non-collagenous matrix which is almost completely replaced by mineral (see Fearnhead and Suga, 1990, for a recent review). The factors that control hard-tissue mineralization are still unknown. An animal rickets model system should prove useful for studying how reduced blood levels of calcium, phosphate, and Vitamin D metabolites affect formation and mineralization of dental tissues.
Materials and methods. Preparation ofpig litters for the study.-The model system used in this study was the Hannover Pig Strain (Harmeyer and Kaune, 1990; Harmeyer, 1991). The breed selected was a cross between homozygous Landrace pigs and heterozygous Minipigs. Three litters of 3-6 piglets each were produced and maintained on a commercial pig starter diet with 0.8% calcium and 0.6% phosphorus. The untreated homozygous offspring developed symptoms of rickets by the 4th to 7th weeks and were given from 0.5 to 3.0 mg Vitamin D3 i.m. injections to keep them alive. Heterozygous normal pigs and homozygous rachitic pigs were killed 4-6 months after birth and processed as outlined below. Plasma calcium, phosphate, and alkaline phosphatase determinations.-Plasma calcium, phosphate, and alkaline phosphatase levels were determined during the supplementation periods and just prior to death, according to previously described methods (Winkler et al., 1986). Tissue preparation.-The mandibles of each group of offspring were dissected, shock-frozen in liquid nitrogen, stored at -70'C, and transported on solid CO2 by air freight from Hannover to Toronto. After being thawed, the jaws were photographed and radiographed. In one group of pigs, both right and left unerupted permanent second mandibular molars (M2) and corresponding canines were carefully dissected and photographed. The transitional enamel on the mesolingual surface ofeach M2 was visualized under U.V. light and marked with a scalpel blade. A strip of developing enamel (D), approximately 2 mm x 4 mm, was scraped off immediately cervical to the transitional enamel, avoiding the underlying dentin, and a corresponding strip of mature enamel (M) was scraped off immedi-
Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on March 23, 2015 For personal use only. No other uses without permission.
Vol. 71 No. 2
THE EFFECTS OF RICKETS ON BONE AND TEETH
347
TABLE TIME OF VITAMIND THERAPYAND PLASMA CALCIUMAND PHOSPHATE DETERMINATIONS INTHE PIGS USED IN THIS STUDY
Group Specimen Calcium Week
I N N
I R S
I R S
II N N
II N N
II R M
II R S
II R S
5
0.50 D3
6
1.25 D3 1.25 D3 1.25 D3 5.0 D3 5.0 D3
7
III N N
III R S
III R S
Development of the 2nd mandibular molar cusp tips starting to mature
1.25 D3 1.25 D3 1.25 D3
8
1.25 D3
9
1.25 D3 1.25 D3 2.5 D3
10
2.5 D3 2.5 D3 crown form completed 3.0 Ca 1.7 Ca 2.0 Ca
16
3.6 Ca
2.5D3
3.2P
2.7 Ca
2.6P 17
3.2 Ca
3.75D3 2.5D3
3.0 P
2.2 Ca 2.4 Ca
2.3P 2.5P 23
3.1 Ca
maturation of crown at cervical margin
3.3P 25 26
1.7 Ca 3.1 Ca
2.5 Ca
1.2P 2.9P
2.2P
2.1Ca 2.0Ca 2.0P 1.7P 1.6 Ca
30
1.2P 34
2.3 Ca
2.3P N - Normal, R - rachitic, S - severe hypocalcemia, M - moderate hypocalcemia, D3 - Vitamin D3 supplement (mg), Ca - plasma calcium (mmol/L), and P - plasma phosphorus (mmol/L). ately apical to the transitional enamel. The enamel scrapings were allowed to dry at 25°C in air for four h. For examination of rachitic dentin, selected mandibular first molars that were removed from a homozygous pig jaw were sectioned with a rotary saw. The sections were ground with fine sandpaper and photographed under a microscope. Enamel protein analysis. -Samples of enamel scrapings, taken from both the right and left molars, were weighed and extracted directly in SDS-PAGE sample buffer by being heated at 900C for 20 min. The ratio of enamel biopsy to extraction buffer (5.0 mg enamel! mL buffer) was the same for all samples analyzed. Following removal of insoluble material by centrifugation, the extracted enamel proteins were analyzed on 12.5% polyacrylamide slab gels and then stained with silver as described elsewhere (Limeback and Simic, 1990).
For quantification of proteins in the extracts, the 20-kDa amelogenin bands in each enamel extract were scanned and the resulting peaks integrated. Limiting the densitometry to this protein in samples separated in the same SDS-PAGE gel and stained by the same solutions avoided problems with variation in silver-staining intensities. The total amount of 20-kDa protein was estimated by combining the data from the bands for developing and mature enamel for each tooth. The mean percentages of 20-kDa amelogenin in both the developing and mature enamel compartments were calculated and compared by Student's t test.
Results. Blood chemistry.-The results of the plasma calcium, phosphate, and alkaline phosphatase levels of the three groups of pigs studied
Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on March 23, 2015 For personal use only. No other uses without permission.
JDent Res Fbruy 199
A
Nrmnal,
(heterozygous B
(homozy0
ou
C
rID
ri k
lature ofthe
H
sytmi nerte* tos
:dactdob perante
tmanph the p used iths stu shwe th those of oer dgated pig stair 0
VL
GeneaI
1
tMzyo
reda
ladogr £e epanry depnd
10 209 smaller.
en
1Ihe effects orfckets on toh devlopmet
thlat we
cleh wras eerozygous pigs. This The pig wi rckets w small( v~elop more slowly than heru1n
jaws Thi
rdnhibles
h re
under identical conitons, te rachitic jaws showed radiographed a p bounced rdction in bone Ts was also observed densitr defcien and dow dringissecton of unrpted teet in the racihtichone was dissected wth less it. Radographs oGo I pg jaws are
erdeveloped skeletal systems a
,r
Padio
[t
g crown foration ese teeth are most
rst six mor
r
iand rahite pig mandie h1c compw uou rts) and heterogous (noal) aws om GrupI ensng * at idential codtions ofX-a~diogaphed togther, fim proessn, and p vtogaph sy wr aistrl spcmens. 30 ws; and (C) jaw with 33 eeks (B) jaw ii sw
Fig
fteCl
syte
he
Aas
TI
lev
the
th
Not
ke smaller pges w rts n th]
o
the pi
shwn in Fig. 2. here wev no alteratons tenumbers lhat developed a)r their positi in the ante However, ;itydomiral iin allIof t pervan teth was clearly om pi wih ikets Frerore he i nthe pe et teth as restricted in e o0r t ris was e yint b e great elaed pip en e seco nd adthird reseng o ne showed eiad pulp chamber n e affected
Toot re om
eruption tme was afetd in ritkets WVhen ittermates ioo he first pemanen mola was taped t
ot only was ess deir forced rickets but te groud sertn in Ft. 3 the dentin was Tahzed. Irte globular derntn was sered nthe tet
esronsr
aso, as den poo0y mnli
onsated
cited w Gh rickets.
dIesI Defcsan the erareal primary teeth :rved in] rachitc mBandilbles, bu1t enamel dlefecs, caraclties in e enaml s ac, were teize~d by nittin or evi ent on te eraptddirst perant molars (Fig 4). The xct of CIIe lesion depended on the stage development of the tooth du:n'n ,grids f 90poa1ema/bypophosp1hartema Further* o
were
ename
mrore vitamAn D supple ents appeared to restore normal eramel kt theextent fte ena llieson (ee sample B ma1urati1 andt
The ana dcts t Ieh In the teeth, t However
wile
r esas pronounced tn nerupted rachttic enre' ca fre was generally still intact
normaia
epted
teeth
had
enamel thatwas
smooth and shiny, the enamel of rachitc teeth wasrrul ad dull we earned at deti stages of develop(tg 5) meIt id not wever, show morph11otical differenrs Neverthe a
moar. A rachVit d seion of a Fig. tg second ofIa f ast permanent manibuar modarlissect daw anF 2 was pd ad photogaed (X lf
dentin is makd, wit
an arw.
thik v
romthrathit Pig The inerltol Oar
less, the lower mineralconte1 t
darer appearnc.m
Lrcmlpro ep crptr takerhro
ave te ctteetha overall
rv By compa nxg eqal portions ofename xac the same stag f enamel develop
Downloaded from jdr.sagepub.com at UCSF LIBRARY & CKM on March 23, 2015 For personal use only. No other uses without permission.
l~~~o VolVol 71 No THRE E LCT OF RIMV m
341,11
Fig Enam toplasai -achitipis, ese are photo hs wheas theist martndibla mor s had deistint rons of enamel hypoph ua be noted on the molar in B by thE appearance of rl enamel tissue cr ment it was pos1ble to make stations oner the d of rockets on the tnamer Iprotei patterns of dissectd molars(sg 6A). In this srain ofpig te name protein patem n tie dvelopig enmel of both normal and rachtic animals was similar that described
doe pg (Overall and LimebackE 1988) ndi enamel proeins prepessedby te name Vrelenirs wer lael proteinases m a smiar manre movAed in the mure enamel, whereas serum album appeared to
preously catig be
n te
enamel
o
te tee
severehypocacenia/iyophospnte1ia had
elni
orea
densitometry (Fig.
B).
prporton of tie
amelogenin that was recorded in significantly great in e rachitc te normal teeth
total
amou
mature enamel
t
(p
