See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/9067618

The Influence of Fluoride Exposure on Dentin Mineralization

Using an in Vitro Organ Culture Model

Article  in  Calcified Tissue International · December 2003

DOI: 10.1007/s00223-003-0022-8 · Source: PubMed

CITATIONS READS
18 61

6 authors, including:

Ryan Moseley Rachel Waddington

Cardiff University Cardiff University
57 PUBLICATIONS   1,524 CITATIONS    130 PUBLICATIONS   2,773 CITATIONS   

SEE PROFILE SEE PROFILE

Alastair J Sloan
Cardiff University
138 PUBLICATIONS   2,749 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Novel antimicrobial coating for dental abutments to prevent peri-implantitis View project

All content following this page was uploaded by Ryan Moseley on 25 March 2016.

The user has requested enhancement of the downloaded file.

Calcif Tissue Int (2003) 73:470–475
DOI: 10.1007/s00223-003-0022-8 Calcified
Tissue
International

2003 Springer-Verlag New York Inc.

Laboratory Investigations
The Influence of Fluoride Exposure on Dentin Mineralization Using
an in Vitro Organ Culture Model
R. Moseley,1,4 R. J. Waddington,2 A. J. Sloan,2 A. J. Smith,2 R. C. Hall,3 G. Embery3
1
Matrix Biology & Tissue Repair Research Unit, Department of Dental Health & Biological Sciences, Dental School,
University of Wales College of Medicine, Heath Park, Cardiff, UK
2
Oral Biology, School of Dentistry, St. Chads Queensway, Birmingham, UK
3
Department of Clinical Dental Sciences, Edwards Building, School of Dentistry, University of Liverpool, Daulby Street, Liverpool, UK
4
Department of Oral Surgery, Medicine & Pathology, Dental School, University of Wales College of Medicine, Cardiff, UK

Received: 22 January 2002 / Accepted: 2 March 2003 / Online publication: 6 October 2003

Abstract. This study aimed to characterize fluoride-in-
duced alterations in dentin mineralization within a dentin-
pulp organ culture system. Tooth sections derived from
male Wistar rat incisors were cultured in Trowel-type
culture for 14 days, in the presence of 0 mM, 1 mM, 3 mM
and 6 mM sodium fluoride. Tooth sections were processed
and analyzed for uptake of fluoride, its subsequent effect
on dentin mineralization by tetracycline hydrochloride
incorporation and mineral composition, expressed as
calcium/phosphorous (Ca/P) ratios. Tetracycline hydro-
chloride incorporation was demonstrated to decrease with
increased fluoride exposure, accompanied by significant
increases in both Ca/P ratios and fluoride incorporation.
These findings provide further evidence that the estab-
lished alterations in dentin formation during fluorosis are
a consequence of disruption to the mineralization process,
and provide a model system with which to investigate
further the potential role the extracellular matrix plays in
inducing the apparent changes in mineral composition.
Key words: Dentin — Mineralization — Fluoride —
Odontoblasts
Dentinogenesis involves the secretion of an extracellular
matrix, which is subsequently mineralized. The forma-
tive cells, the odontoblasts, are derived from the ecto-
mesenchymal cells of the dental papilla and lie on the
pulpal interface of dentin, maintaining communication
with the matrix through the odontoblast process. The
extracellular matrix of dentin is rich in collagenous and
noncollagenous components. Initial secretion of the un-
mineralized dentin matrix is followed by proteolytic
matrix remodeling, together with secretion of further
matrix components, which permit and regulate the
mineralization process in the formation of dentin [1]. As
Correspondence to: R. J. Waddington; E-mail: wadding-
tonrj@cardiff.ac.uk
well as a fundamental role in the regulation of matrix
synthesis and secretion, the odontoblast is also respon-
sible for the channeling of calcium and phosphate to the
mineralization front at the predentin/dentin interface [2].
It is now clear that fluoride is capable of influencing
the mineralization process of calcified tissues [3]. Lower
concentrations of fluoride can reduce acid dissolution of
mineral during dental caries by stabilization of the hy-
droxyapatite crystal lattice through fluoride substitution
[4, 5]. However, higher concentrations can give rise to
fluorosis characterized by abnormalities in mineraliza-
tion [6, 7]. Though ionic substitution of the crystal
lattice represents a predominantly physicochemical
process, the tissue changes observed during fluorosis
probably have a greater cellular basis. The characteristic
mottled appearance of enamel during fluorosis does not
appear to be paralleled by such obvious tissue changes
in dentin and bone, although small hypomineralized
interglobular spaces have been reported in the dentin
matrix of fluorotic teeth [8].
At the molecular level, several studies have suggested
that fluoride leads to extracellular matrix alterations in
dentin [9–15]. Both collagenous and noncollagenous
components appear to undergo structural alterations
during fluorosis, although the precise mechanisms are
unclear. Focus has been directed to the noncollagenous
components, particularly the small leucine-rich prote-
oglycans, decorin and biglycan [16] and dentin phos-
phoprotein [17], in view of the putative roles of these
molecules in mineralization. In vivo studies have indi-
cated that fluoride exerts an increase in the levels of
dermatan sulfate-substituted forms of decorin and big-
lycan with an overall increase in the charge density of
the proteoglycans [13]. This later study confirmed pre-
vious in vitro studies investigating the influence of fluo-
ride on proteoglycan synthesis within a rat incisor organ
R. Moseley et al.: Effects of Fluoride on Dentin Mineralization In Vitro 471

Table 1. Incorporation of fluoride into tooth sections following incubation within fluoride supplemented media

Uncorrected P value Bonferroni P value

Mean (ppm) SD vs 1 mM F) vs 3 mM F) vs 6 mM F) vs 1 mM F) vs 3 mM F) vs 6 mM F)

No F) 2.775 0.79 <0.0001 <0.0001 <0.0001 <0.001 <0.001 <0.001

1 mM F) 360.05 73.70 >0.8 0.0698 >0.05 >0.05
3 mM F) 359.07 77.53 0.0664 >0.05
6 mM F) 425.88 34.30
Results are mean values of fluoride present in ashed tooth sections (n = 6) together with statistical determination for significant
differences between group means. Statistical differences between groups were determined using ANOVA and P values corrected
using the Bonferroni method

Table 2. Calcium phosphate ratio within newly formed dentin following incubation in fluoride supplemented media

Uncorrected P value Bonferroni P value

Ca/P ratio SD vs 1 mM F) vs 3 mM F) vs 6 mM F) vs 1 mM F) vs 3 mM F) vs 6 mM F)

No F) 0.909 0.06 <0.0001 <0.0001 <0.0001 <0.001 <0.001 <0.001

1 mM F) 1.002 0.07 >0.8 <0.0001 >0.05 <0.001
3 mM F) 1.007 0.07 <0.0001 <0.001
6 mM F) 1.099 0.15
Results are presented as mean values from 30 fields of view at a magnification of ·l6,000. Statistical differences between groups
were determined using ANOVA and P values corrected using the Bonferroni method

culture system, suggesting a reduction in sulfation [9]
and a reduction in the length of the glycosaminoglycan
chains as a result of posttranslational influences, hence
leading to an overall reduction in the molecular size of
the proteoglycans synthesized [12]. In vivo studies in rats
have also shown that prolonged ingestion of high levels
of fluoride (>20 ppm) results in a reduction in the
phosphate content of the dentin phosphoproteins [14].
In view of the putative role of these noncollagenous
components of dentin in mineralization, the aim of the
present study was to investigate the influence of excess
fluoride upon the mineralization process in dentin. An in
vitro organ culture model [18] was adopted for the study
enabling the normal tissue architecture of the dentin-
pulp complex to be maintained together with direct
application of known concentrations of fluoride to the
cells. This model also allows us to study differences in
mineralization over periods of 14 days, which were not
possible in previous in vitro studies [12]. Both analytical
and morphological approaches were undertaken with a
particular focus on the transition of predentin to dentin
where noncollagenous matrix modifications are impor-
tant in the mineralization of dentin [1].
Materials and Methods
Rat Incisor Organ Culture
The organ culture of rat incisor tooth sections was performed
according to the method of Sloan et al. [18]. This established
culture model preserves the normal tissue architecture of the
dentin- pulp complex with the odontoblasts lining the formative
surface of the dentin matrix and maintenance of the underlying
fibroblasts and other pulpal cells. Briefly, upper and lower incisor
teeth were dissected from 28-day-old male Wistar rats sacrificed
by cervical dislocation. Animals were maintained and humanely
treated within the University’s animal facility in accordance with
good practice and Government regulatory procedures. The in-
cisors were placed in sterile washing medium, consisting of
DMEM (Sigma, Dorset, UK), supplemented with 4 mM L-
glutamine and penicillin/streptomycin/amphotericin (100 U/ml,
100 lg/ml and 25 lg/ml, respectively). Then, 2 mm of the growing
root were removed and discarded and 2–3 transverse sections of
rat incisors, approximately l–2 mm in thickness were cut with a
segmented, diamond-edged rotary saw (TAAB Laboratories
Equipment Ltd., Berkshire, U.K.), cooled with washing medi-
um. The tooth sections were immediately placed in several
changes of washing medium at 37C prior to culture.
The tooth sections were transferred to individual wells of a
plastic, 96-well plate containing 100 ll of embedding medium,
consisting of DMEM, 4 mM L-glutamine, penicillin/strepto-
mycin/amphotericin (100 U/ml, 100 lg/ml and 25 lg/ml, re-
spectively), 0.15 mg/ml L-ascorbic acid, 10% heat inactivated
fetal calf serum and 1% low melting point agarose. Culture
medium (same composition as embedding medium with the
omission of the agarose) was also supplemented with either 0
mM, 1 mM, 3 mM or 6 mM sodium fluoride. Such concen-
trations were used as these have previously been demonstrated
not to be lethal to odontoblasts and to allow ECM synthesis to
continue [12]. The embedding medium was allowed to cool
until semisolid and each embedded tooth section was trans-
ferred to a sterile Millipore filter on a sterile plastic support, in
a 35 mm Petri dish. The filters were suspended on the surface
of 4 ml of fluoride-free or fluoride-supplemented (1 mM, 3 mM
or 6 mM) culture media in Trowel-type cultures. Tooth sec-
tions were cultured at 37C in 5% CO2 in air for 14 days, with
the culture medium being changed every 2 days.
Quantification of Fluoride Incorporation into Dentin
The levels of fluoride incorporation into the dentin of tooth
sections following culture in the absence and presence of 1
472
Fig. 1. Percentage increase in calcium/phosphorus (Ca/P) ra-
tios of dentin of tooth sections following culture in the pres-
ence of 1, 3 and 6 mM sodium fluoride for 14 days compared
with no fluoridecontrols (determined at 30 random points).
mM, 3 mM and 6 mM sodium fluoride were determined by
established techniques for the quantification of fluoride in
mineralized tissues [19]. Tooth sections (6 sections per fluoride
concentration) were cultured at 37C in 5% CO2 in air for 14
days, as described above. Following removal from culture,
washing and blotting on a tissue, whole tooth sections were
ashed at 600C for 18 h and weighed. The ashed tooth sections
were dissolved in 400 ll 1 M perchloric acid prior to the ad-
dition of 1200 ll 1 M sodium acetate, to give a final pH of 5.2.
An equal volume of Total Ionic Strength Adjustment Buffer II
(TISAB II) (Orion Research Incorporated, Massachusetts,
USA) was added to each solubilized tooth section and the
fluoride concentration was determined by a combined fluoride/
reference electrode (Orion Research Incorporated), against
known fluoride standards (0.1–200 ppm). Tooth sections
(n = 6) cultured at 37C in 5% CO2 in air for 14 days in the
absence of sodium fluoride served as controls. The standard
electrode potentials (mV) obtained were plotted against log10
of the fluoride concentrations of the standard solutions and the
concentration of fluoride within the ashed tooth samples were
expressed as parts per million (ppm). Levels of fluoride within
the tooth sections following incubation in the different fluoride
concentrations were compared and statistically analyzed by
one-way ANOVA.
Tetracycline Hydrochloride Incorporation as an Indication of
Dentin Mineralization
Analysis of dentin mineralization during culture was per-
formed examining tetracycline hydrochloride incorporation
into dentin in the absence and presence of sodium fluoride,
through pulse labeling of the marker and microscopic visual-
ization. The tooth sections were cultured in semisolid culture
media and liquid culture media for 24 h, supplemented with 0
mM, 1 mM, 3 mM and 6 mM sodium fluoride, as described
above. The tooth sections were removed from the semisolid
embedding medium after 24 h and re-embedded in fluoride-
free or fluoride-supplemented semisolid medium containing l
lg/ml tetracycline hydrochloride (Sigma) (6 tooth slices per
fluoride concentration). The re-embedded tooth sections were
cultured for a further 24 h at 37C in 5% CO2 in air with 4 ml
fluoride-free or fluoride-supplemented liquid culture media
also containing 1 lg/ml tetracycline hydrochloride. Tooth
sections were then removed from the tetracycline hydrochlo-
ride-supplemented embedding and culture media after 24 h
and re-embedded in fresh, tetracycline-free embedding and
culture (fluoride-free or supplemented) medium for a further
12 days at 37C in 5% CO2 in air.
Following 14 days in culture, tooth sections were removed,
washed in Sorensens phosphate buffer (4 · 30 min), fixed and
R. Moseley et al.: Effects of Fluoride on Dentin Mineralization In Vitro
dehydrated through graded ethanols (3 · 15 min). The tooth
sections were washed in LR White resin (4 · 30 min) and
subsequently embedded in LR White resin for 24 h. Thin
(approx 500 lm) slices of the embedded tooth sections were
cut with a Microslice (Cambridge Scientific Instruments,
Cambridgeshire, UK). Sections were then successively polished
with graded aluminium oxide abrasive strips (Agar Scientific,
Essex, UK) in distilled water to a thickness where predentin,
dentin and enamel were identifiable when the ground section
was examined under a light microscope. Sections were then
examined with an Olympus Provis AX70A light/fluorescent
microscope (Olympus Microscopes, Middlesex, UK) at ·400
magnification.
EDAX Analysis of Calcium and Phosphorus Ratios within
Fluoride-Exposed Dentin
Following removal from organ culture, the tooth sections ex-
posed to 0 , 1 , 3 and 6 mM sodium fluoride (n = 6/fluoride
concentration) were fixed in 2.5% gluteraldehyde for 24 h and
dehydrated through graded ethanol washes (4 · 15 min).
Tooth sections were critical-point dried on a Polaron E3000
Critical Point Drying Apparatus (Thermo Electron Company,
West Sussex, UK) and ion etched for 2 h using a B304/404
Microlap ion beam in an argon atmosphere (Ion Tech. Ltd.,
Middlesex, UK). The tooth sections were then carbon-coated
on an EMScope SC 5000 Sputter Coater (Thermo Electron
Company).
Calcium and phosphorus (Ca/P) ratios were determined at
30 random points in each section within the dentin along a
region representing newly deposited dentin matrix, immedi-
ately above the identifiable predentin-dentin transition area,
located approximately 10–20 lm from the pulpal border.
Measurements were made with a Jeol Scanning Electron Mi-
croscope (SEM) with X-ray Microanalyser (JEOL Ltd.,
Hertfordshire, U.K.), at 15 KeV for 100 sec (2000 counts per
second), counting the whole frame at a magnification of
·16,000 and quantified by comparison with an apatite stand-
ard (Agar Scientific). The changes in the Ca/P ratios obtained
for each fluoride concentration were statistically analyzed by
one-way ANOVA.
Results
Quantification of Fluoride Incorporation into Dentin
The levels of fluoride incorporation into the dentin of
tooth sections following culture for 14 days in the ab-
sence and presence of 1, 3 and 6 mM sodium fluoride are
shown in Table 1. The mean levels of fluoride within
tooth sections culture at differing fluoride concentra-
tions are given. Statistical analysis of the data using one-
way ANOVA yielded a P value of <0.001, indicating
that the difference among group means is extremely
significant P values were then calculated for statistical
differences between the fluoride conditions. However,
this test does not take into account that same sets of
data are used in multiple comparisons where there is a
5% probability that random chance would cause each P
value derived to be less than 0.05. Therefore, P values
were corrected using the Bonferroni method. Significant
differences in the level of fluoride incorporation were
evident on comparing those tooth sections cultured in
fluoride (1, 3 and 6 mM) with those that received no
fluoride supplementation (P < 0.001). However, no
R. Moseley et al.: Effects of Fluoride on Dentin Mineralization In Vitro 473

Fig. 2. Tetracycline hydrochloride incorporation into rat incisor tooth sections following culture in the absence or presence of 1, 3
or 6 mM sodium fluoride for 14 days (·400). The fluorescent zone of incorporation at the predentin/dentin interface is indicated by
arrows. Areas marked indicate the predentin matrix (PD), dentin matrix (D) and autofluorescence within the enamel layer (E).

significant differences in levels of fluoride incorporation
were observed when comparing tooth sections cultured
at the various fluoride concentrations of 1 mM, 3 mM
and 6 mM.
Calcium/Phosphorous (Ca/P) Ratios within Fluoride-Exposed
Dentin Mineral
The Ca/P ratios of the dentin mineral in tooth sections
following culture for 14 days in the absence and pres-
ence of 1, 3 and 6 mM sodium fluoride are shown in
Table 2. Percentage increases in the Ca/P ratios com-
pared with the no fluoride control are shown in Figure 1.
Statistical analysis using one way ANOVA indicated an
extremely significant difference between groups (P <
0.0001). Table 2 shows P values calculated when com-
paring individual fluoride conditions, which were cor-
rected to allow for multiple analyses using the
Bonferroni method. All Ca/P ratios were demonstrated
to significantly increase as a function of fluoride con-
centration, compared to tooth sections unexposed to
sodium fluoride (P < 0.0001). No significant increase in
Ca/P ratio was evident on comparing tooth sections
exposed to 1 and 3 mM sodium fluoride (P > 0.8), 1 and
6 mM sodium fluoride (P > 0.05) or 3 and 6 mM flu-
oride (P > 0.05).
Tetracycline Hydrochloride Incorporation During Dentin
Mineralization
Tetracycline hydrochloride incorporation into the den-
tin of tooth sections was examined following culture in
the absence and presence of sodium fluoride for 14 days,
and typical images obtained under the fluorescent mi-
croscope are shown in Figure 2. For tooth sections
474
cultured in the absence of fluoride, a narrow and dis-
crete fluorescent band was evident, as indicated by the
arrows, and corresponds to the incorporation of tetra-
cycline hydrochloride into newly mineralized dentin
formed over the 14-day culture period. For all tooth
sections incubated in the presence of 1, 3 or 6 mM flu-
oride, the fluorescence from the tetracycline incorpora-
tion in the predentin-dentin transition area appeared
much more diffuse in nature (indicated by an arrow) and
a narrow, discrete fluorescent band was not identifiable.
Discussion
The present study has demonstrated altered patterns of
mineralization in the transition of predentin to dentin
during the in vitro culture of tooth slices in the presence
of fluoride, which shows correlations with the extracel-
lular matrix changes previously reported in this region
of the tissue [12–14]. Specifically, it has been possible to
demonstrate incorporation of fluoride into the dentin
matrix during culture, changes in the Ca/P ratios of the
mineral and a more diffuse pattern of tetracycline in-
corporation, implying alterations in the regulation of the
mineralization process.
The concentrations of fluoride adopted in the study
were in the millimolar range to allow direct comparison
with previous studies investigating extracellular matrix
changes [12]. At these concentrations of fluoride, the
morphological appearance of mature odontoblasts in
culture is unaffected, but total collagen synthesis is re-
duced [15], confirming the previously reported effects of
fluoride on the extracellular matrix [12–14]. These ob-
servations are at variance with data from exposure of
cultured tooth germs to fluoride where de novo dentin
formation and mineralization was reported to be unaf-
fected [20, 21]. Such differences may reflect the stages of
development of these tissues since matrix-vesicle-medi-
ated mineralization [22] would be predominant in the
tooth germ cultures; in contrast, dentinogenesis would
be proceeding through matrix-mediated mineralization
[23] in the cultured mature tooth slices.
Though there appeared to be a dose dependency in
Ca/P ratios at the highest fluoride concentration studied
(6 mM), this was not reflected in the fluoride uptake into
the tissue or the altered patterns of tetracycline incor-
poration. In vivo, fluoride concentrations have been re-
ported to be highest at or near to the pulpal surface [24],
probably reflecting the continued apposition of matrix
at this site. Previous studies have identified that fluoride
exposure leads to reductions in both Ca2+ and PO43)
(including incorporation into phosphoproteins) uptake
and transport in dentin and enamel tissues, following
chronic fluoride exposure [20, 25]. The increase in the
Ca/P ratio in the newly formed dentin, reported herein,
is in agreement with previous studies which have iden-
R. Moseley et al.: Effects of Fluoride on Dentin Mineralization In Vitro
tified similar trends within enamel and whole tooth germ
explants [25–27]. These studies have suggested that flu-
oride influences the cellular activities of both odonto-
blasts and ameloblasts, and the alterations in Ca/P
ratios with fluoride exposure may reflect alterations in
the substitution of hydroxyl ions in the hydroxyapatite
crystal lattice by fluoride ions [24]. Phosphate ions may
also be substituted by fluoride ions, in conjunction with
carbonate ions [24]. Such substitutions would provide a
sufficient localized fluoride concentration to disrupt
normal apatite nucleation and crystal growth, forming
hypomineralized regions and also increasing Ca/P ratios
[27]. Loss and substitution of hydroxyl and phosphate
ions at fluoride concentrations above 100 ppm can lead
to nonspecific deposition of calcium fluoride, giving rise
to increased Ca/P ratios [24, 25]. This may partly explain
the dose dependency of the Ca/P ratios in the cultured
tooth slices at the highest fluoride concentration studied.
The rather diffuse incorporation of tetracycline
within the dentin matrix of tooth sections exposed to
fluoride implies alterations in the regulation of the
mineralization process and would be in accord with the
observations of small hypomineralized interglobular
spaces in fluorotic dentin [28]. Changes in the pattern of
dentin mineralization after a single acute dose of fluo-
ride have also been reported [27] and decreases in tet-
racycline incorporation and dentin apposition have been
observed during exposure to high fluoride concentra-
tions [29–31]. The more diffuse appearance of tetracy-
cline incorporation into the dentin matrix of tooth
sections cultured in the presence of fluoride in the pre-
sent study suggest that epitactic nucleation sites involved
in matrix-mediated mineralization may have become
disrupted. This would be in accord with the extracellular
matrix changes previously reported in this region of the
tissue [12–15] and reinforces the importance of these
extracellular matrix changes in the regulation of min-
eralization [1].
Thus, the present study has demonstrated that ex-
posure of the dentin-pulp complex to higher concen-
trations of fluoride influences the mineralization process
in the transition from predentin to dentin, which may
well have a mechanistic basis in the fluoride-induced
extracellular matrix changes arising in this region of the
tissue. In view of the continued apposition of dentin
throughout life, these observations indicate that expo-
sure to high levels of fluoride may exert effects at the
cellular level well beyond tooth development during
primary, physiological secondary and tertiary dentino-
genesis. There are also implications for the regeneration
and repair of dentin after injury. The critical importance
of growth factors sequestered within the dentin matrix
[32] in dentin repair and their association with extra-
cellular matrix components [33] imply that biological
repair processes may also be susceptible to the effects of
excessive fluoride exposure.
R. Moseley et al.: Effects of Fluoride on Dentin Mineralization In Vitro
Acknowledgements. We gratefully acknowledge the funding of
this research by The Wellcome Trust (Grant N 054420) and
for a Research Fellowship to support Dr. R. Moseley. We also
wish to express our gratitude to Mrs. G. Smith and Mrs. W. G.
Rowe for their assistance during the course of this study.
References
1. Embery G, Hall RC, Waddington RJ, Septier D, Gold-
berg M (2001) Proteoglycans in dentinogenesis. Crit Rev
Oral Biol Med 12:331–349
2. Lundgren T, Engstrom EU, Levi-Setti R, Linde A, Noren
JG (1994) The use of stable isotope 44Ca in studies of
calcium incorporation into dentine. J Microscropy
173:149–154
3. Robinson C, Kirkham J (1990) The effect of fluoride on
the developing mineralized tissues. J Dent Res 69:685–691
4. Murray JJ, Rugg-Gunn AJ, Jenkins GN (1991) Fluorides
in caries prevention (3rd ed). Wright, Oxford
5. ten Gate JM (1999) Current concepts on the theories of the
mechanism of action of fluoride. Acta Odont Scand
57:325–329
6. Fejerskov O, Kragstrup J, Richards A (1988) Fluorosis of
Teeth and Bone In: Ekstrand J, Fejerskov O, Silverstone LM
(Eds.) Fluoride in dentistry. Munksgaard, pp 190–228
7. Whitford GM (1997) Determinants and mechanisms of
enamel fluorosis. Ciba Foundation Symposium 205:226–
241
8. Appleton J (1994) Formation and structure of dentine in
the rat incisor after chronic fluoride exposure to sodium
fluoride. Scan Microsc 8:711–719
9. Smalley JW, Embery G (1980) The influence of fluoride
administration on the structure of proteoglycans in the
developing rat incisor. Biochem J 190:263–272
10. Susheela AK, Sharma K (1988) Fluoride-induced changes
in the tooth glycosaminoglycans: an in vivo study in the
rabbit. Arch Toxicol 62:328–330
11. Susheela AK, Sharma K, Rajan BP, Gnarasundaram N
(1988) The status of sulfated isomers of glycosaminogly-
cans in fluorosed human teeth. Archs Oral Biol 33:765–767
12. Waddington RJ, Embery G, Hall RC (1993) The influence
of fluoride on proteoglycan structure using a rat odonto-
blast in vitro system. Calcif Tissue Int 52:392–398
13. Hall RC, Embery G, Waddington RJ (1996) Modification
of the proteoglycans of rat incisor dentine-predentine
during in vivo fluorosis. Eur J Oral Sci 104:285–291
14. Milan AM, Waddington RJ, Embery G (1998) Altered
phosphorylation of rat dentine phosphoproteins by fluo-
ride in vivo. Calcif Tissue Int 64:234–238
15. Moseley R, Sloan AJ, Waddington RJ, Smith AJ, Hall
RC, Embery G (2003) The influence of fluoride on the
cellular morphology and synthetic activity of the rat den-
tine-pulp complex in vitro. Archs Oral Biol (in press)
16. Iozzo RV (1999) The biology of the small leucine-rich
proteoglycans. Functional network of interactive proteins.
J Biol Chem 274:18843–18846
View publication stats
475
17. Butler WT, Ritchie H (1995) The nature and functional
significance of dentin extracellular matrix proteins. Int J
Dev Biol 39:169–179
18. Sloan AJ, Shelton RM, Hann AC, Moxham BJ, Smith AJ
(1998) An in vitro approach for the study of dentinogenesis
by organ culture of the dentine-pulp complex from rat
incisor teeth. Archs Oral Biol 43:421–430
19. Venkateswarlu P (1990) Evaluation of analytical methods
for fluorine in biological and related materials. J Dent Res
69:514–521
20. Bronckers ALJJ, Jansen LL, Wöltgens JHM (1984) Long-
term (8 days) effects of exposure to low concentrations of
fluoride on enamel formation in hamster tooth-germs in
organ culture in vitro. Archs Oral Biol 29:811–819
21. Bronckers ALJJ, Jansen LL, Wöltgens JHM (1984) A
histological study of the short-term effects of fluoride on
enamel and dentine formation in hamster tooth-germs in
organ culture in vitro. Archs Oral Biol 29:803–810
22. Katchburian E (1973) Membrane-bound bodies as initia-
tors of mineralization of dentine. J Anat 116:285–302
23. Linde A, Goldberg M (1993) Dentinogenesis. Crit Rev
Oral Biol Med 4:679–728
24. Robinson C, Kirkham J, Weatherall JA (1996) Fluoride in
teeth and bones. In: Ferjerskov O, Ekstrand J, Burt BA
(Eds.) Fluoride in dentistry, 2nd ed, Munksgaard, pp 69–
87
25. Susheela AK, Bhatnagar M (1993) Fluoride toxicity: a
biochemical and scanning electron microscopic study of
enamel surface of rabbit teeth. Arch Toxicol 67:573–579
26. Bronkers ALJJ, Wöltgens JHM (1985) Short-term effects
of fluoride on biosynthesis of enamel-matrix proteins and
dentine collagens and on mineralisation during hamster
tooth-germ development in organ culture. Archs Oral Biol
30:181–191
27. Fejerskov O, Yaeger JA, Thylstrup A (1979) Microradi-
ography of the effect of acute and chronic administration
of fluoride on human and rat dentine and enamel. Archs
Oral Biol 24:123–130
28. Appleton J (1994) Formation and structure of dentine in
the rat incisor after chronic exposure to sodium fluoride.
Scanning Microsc 8:711–719
29. Yaeger JA, Hinrichsen CFL, Cohen MJ (1964) Develop-
ment of the response in rat incisor dentine to injected
strontium and fluoride. Am J Anat 114:255–272
30. Meffert O, Kämmerer H (1973) The influence of fluoride
on the mineralisation of rat teeth. Calcif Tissue Int 11:176–
178
31. Kortelainen S, Larmas M (1990) Effects of low and high
fluoride levels on rat dental caries and simultaneous den-
tine apposition. Archs Oral Biol 35:229–234
32. Smith AJ, Lesot H (2001) Induction and regulation of
crown dentinogenesis—embryonic events as a template for
dental tissue repair. Crit Rev Oral Biol Med 12:425–437
33. Smith AJ, Matthews JB, Hall RC (1998) Transforming
growth factor-b1 (TGF-b1) in dentine matrix: ligand ac-
tivation and receptor expression. Eur J Oral Sci 106:179–
184