Normalisation of calcium status reverses the phenotype indentin, but not in enamel of VDR-deficient mice

Xueming Zhang a,*, Firoz Rahemtulla a, Ping Zhang b, Xingsheng Li c, Preston Beck a,Huw F. Thomas b

aDepartment of Prosthodontics, School of Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294-2170, USAbDepartment of Pediatric Dentistry, School of Dentistry, University of Alabama at Birmingham, Birmingham, AL, USAcDepartment of Nutrition Science, School of Health Related Professional, University of Alabama at Birmingham, Birmingham, AL, USA

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 1 1 0 5 – 1 1 1 0

a r t i c l e i n f o

Article history:

Accepted 17 September 2009

Keywords:

Vitamin D receptor

Enamel

Dentin

Mineralisation

a b s t r a c t

Objective: To determine the effects of vitamin D receptor (VDR) deficiency on mouse dentin

and enamel mineralisation, and how normalisation of serum calcium level affects dentin

and enamel phenotypes in VDR knockout mice.

Materials and methods: Groups of VDR wild-type (VDR+/+), VDR deficient (VDR�/�) and

VDR�/� rescued mice were sacrificed at 70.5 days of life. The rescued group was established

by a high-calcium diet feeding the VDR�/�mice from postnatal 19 days. Micro-CT was used

to compare enamel and dentin mineralisation density (MD) at different levels of mandibular

incisors among the groups. The scanning electron microscope (SEM) was used to examine

the ultrastructure of the enamel and dentin in the corresponding levels and of surface

enamel after acidic treatment.

Results: Micro-CT showed that in VDR�/� rescued group, dentin phenotype was reversed

and dentin MD was reversed to normal; however, enamel mineralisation was not reversible,

and remained as hypermineralisation in molar region and apical region of the incisors. SEM

also revealed enamel hypermineralisation in the VDR�/� rescued group. This early enamel

hypermineralisation was more susceptible to acidic erosion.

Conclusion: Vitamin D affects dentin mineralisation systemically, and it regulates enamel

mineralisation locally.

Published by Elsevier Ltd.

avai lab le at www.sc iencedi rect .com

journal homepage: www.intl.elsevierhealth.com/journals/arob

The vitamin D endocrine system is essential for calcium

homeostasis in mammals. Mineral homeostasis is system-

atically regulated through intestinal calcium absorption and

renal re-absorption into the circulatory system. The classic

pathway of vitamin D function is through binding with the

vitamin D receptor (VDR) in the intestine and the kidney that

govern calcium transport in these organs. The fluctuation of

serum calcium level affects bone mineralisation density (BMD)

through trapping and mobilisation of calcium and phosphorus

in bone.1

Mineralised tissue results from a complex series of well-

coordinated processes that requires optimal function of

* Corresponding author. Tel.: +1 205 934 7823; fax: +1 205 934 6108.E-mail address: zhangxm@uab.edu (X. Zhang).

0003–9969/$ – see front matter . Published by Elsevier Ltd.doi:10.1016/j.archoralbio.2009.09.004

numerous genes and proteins. Enamel, dentin and bone are

major tissues in mammals that develop through matrix-

mediated mineralisation processes. However, the origin and

composition, as well as the remodelling potential of these

tissues are different. For example, the mineralisation density

(MD) of the bone relies on a delicate balance between

osteoblast and osteoclast activity, whereas resorption of

enamel and dentin are processes unrelated to their forma-

tion. Furthermore, BMD varies with changes in serum

calcium levels, while the MD in unerupted enamel and

dentin remains at the level attained during their develop-

ment.2,3

Fig. 2 – Magnification of micro-CT examination on enamel

and dentin mineralisation at erupted level and third molar

level of VDR+/+, VDRS/S and VDRS/S rescued mouse

incisors (En: enamel; Dt: dentin).

Fig. 1 – Micro-CT examination on enamel and dentin

mineralisation at different levels in VDR+/+, VDRS/S and

VDRS/S rescued mouse incisors (En: enamel; Dt: dentin).

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 1 1 0 5 – 1 1 1 01106

VDR expression has been identified in virtually every

mammalian tissue.4,5 The universal presence of VDR indicates

a wide spectrum of activities for the vitamin D–VDR endocrine

system. The early evolutionary origin of VDR in invertebrates

has indicated that the vitamin D system is not simply a major

player in calcium homeostasis.6 In mineralised tissues, VDR has

been reported in osteoblasts, osteoclasts, ameloblasts and

odontoblasts,4,5 and it is believed to play different roles in these

cells.6

The precise mode and the full spectrum of activities of the

vitamin D hormone can now be better evaluated by the critical

analysis of mice with engineered deletion of VDR.1 VDR

deficiency was reported to result in hypocalcaemia, hypopho-

sphataemia and secondary hyperparathyroidism.7,8 In our

previous studies, we reported that dentin and enamel present

different mineralisation patterns in VDR�/�mice,2,9 suggest-

ing a differential regulation of vitamin D pathway in enamel

and dentin mineralisation. We speculated that vitamin D

regulates dentin mineralisation mainly through its regulation

of serum calcium level, just as it occurs in bone.7,8 However,

enamel mineralisation follows a different pattern; and we

hypothesised that the removal of the local inhibitory function

of VDR in ameloblasts causes the hypermineralisation of

enamel seen in VDR-deficient animals.2,3

To gain insights into the underlying mechanisms regulat-

ing enamel and dentin mineralisation and to evaluate how

serum calcium level affects enamel and dentin mineralisation

in VDR-deficient mice, we normalised serum calcium level by

providing a high-calcium diet.

1. Materials and methods

1.1. Animal maintenance

All experimental procedures involving the use of animals were

reviewed andapproved bytheInstitutional Animal CareandUse

Committee. A pair of heterozygous mice (vdr+/�) was kindly

provided by Dr. Marie Demay (Massachusetts General Hospital,

Boston, MA, USA). VDR+/+ and VDR�/�mice were grouped by

genotyping as previously reported.2 The VDR�/� rescued mice

were fed with autoclaved TD96348 diet (Harlan Teklad, USA)

containing 20% lactose, 2.0% calcium and 1.25% phosphorus

supplemented with 2.2 IU vitamin D g�1 from 19 days of age.10,11

These mice are referred to as rescued mice in the following text.

1.2. Micro-computed tomography (micro-CT)

Groups of postnatal 70.5-day-old VDR+/+, VDR�/� and the

VDR�/� rescued mice were sacrificed by cervical dislocation

under CO2 anaesthesia, and the lower jaws from each

individual mouse were dissected free of soft tissues and fixed

in 100% alcohol. After desiccation, the mandibles were

mounted on a square stage at matched positions for micro-

CT examination as reported previously.2,3 The cross-sectional

images were collected starting from the incisal edge of the

mandible to the distal surface of the mandibular condyle. A

plot profile describing the grey scale–distance relationship in

micro-CT images was achieved by NIH Image 1.63 software

through a line bisecting the incisor. Furthermore, the enamel

and dentin MD was measured and compared. A Student’s t-

test was performed using GraphPad Instat 3.0 software

(GraphPad Software, San Diego, CA, USA) and the significant

level was set as p < 0.05.

1.3. Scanning electronic microscope (SEM)

Groups of 70.5-day-old VDR+/+, VDR�/� and VDR�/� rescued

mice were sacrificed; the lower jaws from each individual

mouse were dissected free of the soft tissues and fixed in 4%

paraformaldehyde for 24 h. The samples were then processed

through distilled water, dehydrated in a series of 70%, 90% and

100% alcohol and xyline and embedded inmethyl methacrylate.

The blocks were cut transversely at eruption level and second

molar level of the incisor. Each section was polished and then

ultrasonically cleaned with 5% sodium hypochlorite solution

for 10 min. The cleaned sections were repeatedly rinsed with

distilled water and then etched with 75% H3PO4 for 15 s. The

sections were cleaned with hypochlorite solution for another

10 min and rinsed in distilled water. The treated specimens

were air-dried at room temperature and mounted on SEM (SX-

30, ISI Ltd., UK) stubs. The section was sputter-coated (Hummer

Sputter System, Anatech, Hayward, CA, USA) with gold–

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 1 1 0 5 – 1 1 1 0 1107

palladium, and viewed under SEM with an accelerating 15 kV.

The images were taken at 4000� magnification, and then

compared at the matched locations among the groups.

1.4. Surface erosion

Groups of 70.5-day-old VDR+/+, VDR�/� and VDR�/� rescued

mice were sacrificed, the lower jaws from each individual

mouse was dissected free of the soft tissues and fixed in 4%

paraformaldehyde for 24 h. The samples were then ultra-

sonically cleaned in 5% sodium hypochlorite solution. The

cleaned incisors were repeatedly rinsed in distilled water and

then etched with 75% H3PO4 for 15 s. They were cleaned with

hypochlorite solution again and rinsed in distilled water. The

treated specimens were air-dried at room temperature and

mounted for SEM examination as mentioned above.

Fig. 3 – Grey scale–distance lot profiles along the line bisecting th

rescued mice (En: enamel; Dt: dentin).

2. Results

Our initial study compared the enamel and dentin miner-

alisation pattern in the VDR+/+, VDR�/� and VDR�/� rescued

mice by micro-CT. The VDR�/� and VDR+/+ mice showed

distinctive mineralisation patterns at different levels of the

incisors (Figs. 1 and 2). All three groups show progressive

dentin mineralisation with tooth development. However,

dentin mineralisation was noticeably lower at each level in

the VDR�/� group compared with the VDR+/+ mice. The

rescued �/�mice attained the same degree of mineralisation

as the VDR+/+ mice. We observed a gradual mineralisation of

enamel with tooth development in the VDR+/+ mice. Both the

VDR�/� and rescued animals attained high mineralisation

levels at an early stage with only gradual increase during

development. Enamel hypermineralisation was observed in

e incisor at different levels in VDR+/+, VDRS/S and VDRS/S

Fig. 4 – Statistical comparison of enamel and dentin

mineralisation density at different levels in VDR+/+,

VDRS/S and VDRS/S rescued mice (n = 6).

Fig. 6 – SEM observation on the surface enamel of VDR+/+,

VDRS/S and VDRS/S rescued mouse incisors (En: enamel).

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 1 1 0 5 – 1 1 1 01108

the molar region and apical region of the incisors. Greater

porosity was observed in the VDR�/� dentin, and the pulp

space appeared larger with a thinner dentin wall. The VDR�/�phenotype of both alveolar bone and dentin was reversible in

the VDR�/� rescued mice and showed increased bone and

dentin MD, this being comparable with the VDR+/+ group. The

Fig. 5 – SEM observation on the enamel and dentin at

different level in VDR+/+, VDRS/S and VDRS/S rescued

mouse incisors (En: enamel; Dt: dentin).

dentin wall and pulp space were also normal in the rescued

group (Fig. 1). However, the enamel hypermineralisation seen

in the molar region and apical region of the incisors was

unchanged in the VDR�/� rescued mice.

Statistical comparison of the enamel and dentin MD at

different levels of incisor in three groups indicated that there

was no significant difference in dentin MD between the VDR�/

� rescued mice and the VDR+/+ mice ( p > 0.05), but the value

is significantly higher in the rescued animals than that in the

VDR�/� group (Fig. 3). By contrast, the enamel mineralisation

showed significant difference among the three groups

( p < 0.05), and the VDR�/� rescued animals had high MD at

the second molar and the third molar levels. There was no

significant difference of enamel MD between the VDR�/� and

the VDR�/� rescued groups (p > 0.05).

We also examined by SEM the ultrastructure of the enamel

and dentin at the eruption level and at the second molar level

along the incisors (Fig. 4). Different prism patterns were

observed among the three groups. The VDR�/� group and

VDR�/� rescued group had smaller prisms, and the inter-

prismal space was observed to be a tightly packed pattern of

deposition. Dentin showed an irregular hypomaturation at

eruption level. At the second molar level, early enamel

maturation was seen in the VDR�/� and VDR�/� rescued

groups.

Finally, we examined the surface enamel of the three

groups after acid treatment to determine susceptibility of this

early enamel hypermineralisation to acidic erosion (Fig. 5).

Structureless outer enamel layer was much thinner in the

VDR�/� and VDR�/� rescued mice. The surface enamel in the

VDR�/� and VDR�/� rescued mice showed increased eroded

area and more open space exposed at the incisal and cervical

regions. Larger-size crystal structure was revealed in the outer

layer of the VDR�/� and VDR�/� rescued mice (Fig. 6).

3. Discussion

It is now known that 1,25(OH)2D3 functions through its widely

distributed VDR in regulation of gene transcription or through

a putative membrane receptor that induces a variety of rapid

responses.12,13 Although VDR exists in osteoblasts and

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osteoclasts, bone mineralisation and bone remodelling are

believed to be independent of VDR local expression; rather, it is

regulated by the serum calcium level.14 Several studies have

shown that serum calcium level in the VDR�/� mice can be

reversed by feeding with a high-calcium diet.10,11 It has been

reported that 1,25(OH)2D3 down-regulates cadherin-17 and

aquaporin-8. Thus, the accumulation of 1,25(OH)2D3 can route

calcium absorption through a paracellular path in the intestine,

which contributes to normalisation of serum calcium level.15,16

Our previous studies found that both bone and dentin

exhibit a hypomineralisation phenotype in VDR�/� mice,

suggesting that dentin shares a similar mechanism to bone in

the mineralisation process.2,9 The present study has demon-

strated that dentin hypomineralisation phenotype in VDR�/�mice can be reversed by feeding with a high-calcium diet early

in life. This indicates that dentin mineralisation is indeed

regulated by serum calcium level and that VDR is not required

for normal dentin mineralisation.

In contrast to dentin, our study found hypermineralisation

patterns in VDR�/� enamel. More importantly, we found that

the enamel hypermineralisation phenotype could not be

reversed by a high-calcium diet. Enamel differs from bone

and dentin in its unique epithelial origin.17 In epithelium,

calcium ions are carried through the capillaries into cells. Such

is the case with enamel. During enamel mineralisation,

enamel organ is required to supply calcium in bulk and to

quickly move the calcium and phosphate ions from blood

stream into the enamel layer. This process takes place despite

the presence of many ionic barriers possibly created by

intervening capillary endothelial cells, papillary layer cells and

ameloblasts. Furthermore, ameloblasts maintain low free

cytosolic calcium concentrations to avoid toxicity and to

retain cell responsiveness to intracellular calcium signalling

cascades that use calcium transiently.17–19 Transcellular cal-

cium transfer across the ameloblast barrier consists of a passive

concentration gradient-dependent transportationandanactive

ATP-dependent transportation.20 Transcellular calcium is

responsible for transferring calcium ions through the tightly

sealed ruffle-ended ameloblasts whereas paracellular calcium

transportation is believed to occur between leaky smooth-

ended ameloblasts.19 Disruption of calcium transportation

could contribute to a variety of developmental defects in the

enamel. Removal of the VDR may open the tight junction in the

ruffle-ended ameloblasts and enhance paracellular calcium

transport as observed in the intestines.15,16 Further studies need

to be pursued in future to determine the relative change

between the transcellular and the paracellular calcium trans-

portation in VDR-deficient mouse ameloblasts. Understanding

underlying cellular mechanisms in VDR-mediated disruption of

calcium transportation will provide potential targets for drug

development to improve enamel quality.

The surface enamel serves as a barrier to block bacterial

penetration and resist abrasion and attrition. In our study, we

found that the surface enamel layer is much thinner in VDR�/

� mice and is more susceptible to acid erosion. This reduced

surface enamel layer is not reversible by a high-calcium diet.

After enamel maturation, the thickness of surface enamel is

maintained by the sum of two distinct and opposing

processes, mineralisation and demineralisation.21 The pH,

titratable acidity, phosphate and calcium concentrations as

well as fluoride content of enamel determine the degree of

tooth mineral saturation, and this degree of saturation is the

driving force of its demineralisation and remineralisation.22

Continuously erupting mouse incisors share some char-

acteristic features with human primary teeth, and acidic

erosion on the mouse incisors could be viewed as a good

simulator of the development of early childhood caries (ECC)

in American children. Despite efforts to intervene in the

transmission of the microbes and practices to correct

unhealthy eating habits, the average number of decayed

and filled teeth among 2- to 4-year-olds has remained

unchanged over the past 25 years.23 Some individuals appear

to be more susceptible to caries, regardless of environmental

risk factors to which they are exposed, indicating that genetic

factor may play an essential role in the development of ECC.24

The VDR gene polymorphisms were first reported to be

associated with variation in BMD in 1994.25 Since then, much

attention has been focussed on this relationship to resolve

controversies regarding the association of such polymorph-

isms and BMD.26–28 The conflicting results may result from

some compensation in calcium homeostasis since the bone

mineralisation is mainly regulated by systemic serum calcium

level. However, enamel mineralisation is associated with VDR

local expression and function. VDR could be a good candidate

gene to be investigated in the relationship between genetic

factors and caries susceptibility.

4. Conclusion

In this study, we used a high-calcium diet administration to

normalise serum calcium level and found that high calcium

supplementation can reverse dentin hypomineralisation, but

not enamel hypermineralisation. The result demonstrated

that dentin mineralisation is regulated systemically by serum

calcium level; however, VDR affects enamel mineralisation

locally.

Acknowledgements

We thank Dr. Marie Demay of Massachusetts General Hospital,

Harvard Medical School, for providing us with breeding pairs

of VDR heterozygous mice.

Funding: UABSOD Dean’s Research Fund.

Competing interests: None declared.

Ethical approval: Not required.

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