archives of oral biology 58 (2013) 975980

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Regeneration of biomimetic hydroxyapatite on etched

human enamel by anionic PAMAM template in vitro
Liang Chen a,b, Kunneng Liang a,b, Jianshu Li c, Duo Wu c, Xuedong Zhou a,b, Jiyao Li a,b,*
a

State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China
Department of Endodontics and Operative Dentistry, West China School of Stomatology, Sichuan University, Chengdu, China
c
Department of Biomedical Polymers and Artificial Organs, College of Polymer Science and Engineering, Sichuan University, Chengdu, China
b

article info

abstract

Article history:

Objective: To repair the demineralized enamel by biomimetic method, and the effect of Poly

Accepted 17 March 2013

(amido amine) (PAMAM) dendrimers on the crystallization of hydroxyapatite on etched

Keywords:

Design: PAMAM dendrimers were synthesized step by step following the classical method

Dental caries

and modified with the carboxylic acid groups (COOH) on the surface. Demineralized human

Biomimetic mineralization

enamel samples were immersed in 10,000 ppm PAMAM-COOH solution for 30 min and then

enamel surface is investigated.

Human enamel

in calcium phosphorous solution with or without fluoride under near-clinical conditions for

PAMAM

20 h. Other samples without PAMAM-COOH were immersed in calcium phosphorous solu-

Remineralization

tion as the control group. After the immersion, the micro structure, morphology and
composition of the regrown crystals on the longitudinal and transversal enamel surfaces
were investigated by SEM, XRD and FTIR, and the results were compared with etched enamel
and intact enamel.
Results: With the PAMAM-COOH templates, well-arranged rod-like crystals were formed
and they were parallel to the long axis of enamel crystals, which was more obvious on the
longitudinal enamel surface. Otherwise, irregular flake-like crystals were obtained without
PAMAM-COOH. Fluorapatite was not influenced by the PAMAM-COOH but its specific
distribution also shown the patterns of the PAMAM-COOH temples XRD spectra showed
that the main phase of the obtained crystals with PAMAM-COOH was hydroxyapatite and
their morphology and structure were close to the intact enamel. Amide I band and two
bands of methylene groups of PAMAM-COOH detected by FTIR demonstrated the presence
of PAMAM-COOH within the biomimetic coating.
Conclusions: It was concluded that PAMAM-COOH can play as the organic template on the
demineralized enamel surface to induce the formation of HAP crystals with the same
structure, orientation and mineral phase of the intact enamel in relatively short time.
# 2013 Elsevier Ltd. All rights reserved.

1.

Introduction

Dental enamel is the hardest mineralized tissue in the human

body.1,2 This hard tissue comprises 96% inorganic materials

and 4% organic materials and water by weight. The inorganic

content is nanorod-like hydroxyapatite crystals arranged into
highly organized hierarchical microstructures. These special
structures play an important role in determining the unique
physicochemical properties of dental enamel. Recent studies

* Corresponding author at: Department of Operative Dentistry and Endodontics, West China Hospital of Stomatology, No. 14, Unit 3,
Renmin Nan Road, Chengdu City 610041, Sichuan Province, China. Tel.: +86 28 85501439; fax: +86 28 85582167.
E-mail addresses: jiyao_li@yahoo.com, chenliang0125@gmail.com (J. Li).
00039969/$ see front matter # 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.archoralbio.2013.03.008

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archives of oral biology 58 (2013) 975980

have reported that extracellular matrix proteins, such as

amelogenin, are essential for the control and modulation of
these special structures during the biomineralization of
enamel.310 However, these proteins that induce and control
the crystallization of apatite are almost completely degraded
or removed during enamel maturation.4,6 Therefore, unlike
dentine and bone, mature enamel has no ability to reform
highly organized crystals. Conventional treatments replace
the defected enamel with substitute materials, such as resin
and amalgam. These substitute materials are quite different
from the normal enamel in chemical composition and crystal
structure. Moreover, these materials require the sacrifice of
healthy tooth tissue. Thus, the materials are not ideal for
repairing defective enamel.
Presently, the biomimetic synthesis of enamel-like hydroxyapatite (HAP) has attracted much interest from research
groups. Many methods have been used in vitro to form
enamel-like crystals, including amelogenin, calcium phosphate solution and nano-hydroxyapatite. Some have used
amelogenin to mimic the biomineralization process of
enamel.1118 However, the structure of amelogenin and
mechanisms of amelogenin-mediated mineralization have
not yet been fully elucidated. Additionally, amelogenin is
difficult to extract and store, features that make it relatively
unsuitable for further clinical use.
Poly(amido amine) (PAMAM) dendrimers are highly
branched polymers. Unlike conventional linear polymers, they
are characterized by the presence of internal cavities, a large
number of reactive end groups and well-defined size and shape.
These structures allow it to be used as biomimetic macromolecules for proteins. Zhou et al.19 and Yan et al.20 demonstrated that PAMAM dendrimers with carboxylic groups have an
effective influence on the size and shape of hydroxyapatite
nanostructures. Zhang et al.21 reported that PAMAM dendrimers appeared to be an inhibitor for crystal formation and
affected crystal morphology and particle size during mineralization in vitro. Chen et al.22,23 demonstrated that PAMAM
dendrimers capped with carboxylic acid can be absorbed to
enamel crystals. In addition, Sheng Yang et al.24 reported an
amphiphilic PAMAM dendrimer that mimics the self-assembly
behaviour of amelogenins in vitro. They observed that SaPAMAM-Asp self-assembled into nanospheres initially and
further translated into linear chains either by increasing the
concentration or by adding calcium ion at pH 7.4 and 37 8C in
aqueous solution. The critical aggregation concentration for
this translation was 5.5  106 M, and linear chains of 100 nm to
1.5 mm in length were finally observed. Therefore, the effect of
PAMAM on HAP synthesis has casted some light on the
biomimetic mineralization of enamel crystals.
Based on previous studies, the aim of the present study was
to investigate the effect of G3.0 PAMAM dendrimers with
carboxylic acid (PAMAM-COOH) on crystal growth on etched
enamel at 37 8C and pH 7.0. The morphology and mineral
phase of the regrown crystal are analyzed by scanning
electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), and a possible
mechanism is explored explaining how the enamel-like
structures are formed. The hypothesis is that the PAMAM
dendrimers could act as an organic template to control the
growth of crystals on the enamel surface.

2.

Materials and methods

2.1.

PAMAM synthesis

The divergent synthesis of PAMAM dendrimers included a

two-step interactive sequence to produce amine-terminated
structures. Iterative sequencing involved alkylation with
methyl acrylate (MA) followed by amidation with excess 1,2ethylenediamine (EDA). The alkylation step produced esterterminated intermediates that were referred to as halfgenerations. The second step involved amidation of the
ester-terminated intermediates with a large excess of EDA to
produce amine terminated intermediates, which are referred
to as full-generations. G3.0 PAMAM dendrimers were
synthesized for further carboxyl modification. 2 g of dry
G3.0 PAMAM was dissolved in 25 mL dimethyl sulfoxide
(DMSO) in a round-bottom flask. To this dendrimer solution,
15 mL of the DMSO solution containing 5.15 g succinic
anhydride (molar ratio of SAH/NH2 = 5:1) was added under
vigorous stirring and was reacted for 24 h without oxygen. The
DMSO solution was dialyzed against water to remove the
excess succinic anhydride as well as the organic solvent. The
aqueous retentate was filtered using a 0.45 mm diameter
membrane, and then was lyophilized.

2.2.

Tooth sample preparation

Human third molars were obtained from subjects aged

between 18 and 30 years at the West China Hospital of
Stomatology, Sichuan University, on the criterion that the
enamel of the teeth had matured without caries, cracks or
other defects. The study was approved by the hospitals
Institutional Review Board. The roots were removed, and the
enamels were cut longitudinally or transversely using a lowspeed diamond saw (Minitom, Struers, Denmark) with water
to obtain enamel samples that are approximately
3  3  1 mm, and then the test surfaces were polished using
600-, 1000-, 2000-, 4000-grit SiC wet paper in sequence with the
machine wheel set to 250 rpm. All the enamel samples were
stored in 0.1% thymol solution at 4 8C before use.

2.3.

PAMAM coating preparation

The enamel samples were etched by 3% HNO3 solution for 50 s,

ultrasonically cleaned for 10 min, and then rinsed with
sufficient de-ionized water. A 10,000-ppm PAMAM solution
was prepared and statically placed for 18 h to ensure the selfassembly process was completed. The PAMAM coating was
obtained by immersing the etched enamel samples in the
PAMAM solution. After 30 min, the enamel samples were
removed from the solution, rinsed with running de-ionized
water for 50 s and air-dried.

2.4.

Mineralization solution

The samples were immersed in 3 mL of calcification solution

containing 2.58 mM CaCl2 and 1.55 mM KH2PO4 at 37 8C,
buffered by 20 mM HEPES and 180 mM NaCl. The concentration of the mineralization solution was referred from previous

archives of oral biology 58 (2013) 975980

studies, in which amelogenin was used as the template to

form the biomimetic HAP on the etched enamel.15,18 The pH of
the mineralization solution was adjusted to 7.00 using 1 M
NaOH. NaF was added to obtain 1 mg/L fluoride, immediately
before immersing tooth samples. The mineralization solution
was sealed air-tight in a 5-mL microcentrifuge tube and
incubated at 37 8C statically for 20 h. After the desired time, the
tooth samples were removed from the solution, rinsed with
running de-ionized water for 50 s and air-dried.

2.5.

Characterization

The enamel surfaces were analyzed by SEM (Inspect F50; FEI,

USA). The surfaces were sputter coated with Au before
observation. The crystal sizes in the digital SEM images were
measured using Photoshop software (n > 15). ATR-IR (NICOLET iS10; Thermo Scientific, USA) spectra were used for
compositional analysis of the regrown crystals. X-ray diffraction analysis (XPert PRO MPD; PANalytical, Netherlands) was
performed to detect the crystal orientation and mineral phase
of the new crystals. The samples for XRD analysis were freshly
prepared to prevent hydrolysis of octacalcium phosphate. The
average crystallite sizes along the c and a axes were calculated

977

from the (0 0 2) and (2 1 1) indexed XRD peaks, respectively,

using the Scherrer equation as follows:
Kl
D nm
n cos u
where D is the crystallite size in nm, K is the Scherrer constant
(here, K = 0.9), l is the X-ray wavelength in nm (here, l = 0.154),
b is the half-maximum intensity of the diffraction peaks, and u
is the diffraction angle for the diffraction peaks.

3.

Results

The SEM results of the longitudinal and transversal enamel

surfaces showed that, after the etching, peripheral regions of
enamel prisms were more demineralized than central regions,
so the outline of the prisms became more distinct. After
soaking in the calcium phosphate solution, irregularly
arranged flake-like crystals with a thickness of 2 mm formed
on the enamel surface. There were large gaps between the
flakes. With 1 ppm NaF in the remineralization solutions, the
morphology of the crystals changed to a needle-like morphology with lengths of 12 mm. In the presence of PAMAM-COOH,

Fig. 1 SEM images of the enamel surface after acidic etching (A). SEM images of crystals grown on acid-etched enamel for
20 h at 37 8C in HEPES-buffered calcium phosphate solution without the PAMAM-COOH template and fluoride (B), with
1 ppm sodium fluoride (C), with the PAMAM-COOH template (D), and with both 1 ppm sodium fluoride and the PAMAMCOOH template (E) (Group 1 for transversal surface and Group 2 for longitudinal surface).

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archives of oral biology 58 (2013) 975980

Fig. 2 SEM images of crystals grown on longitudinal

enamel with 1 ppm sodium fluoride and the PAMAMCOOH template. The distribution of F-HAP was
significantly influenced by the PAMAM-COOH template.

the gaps between enamel prisms were restored by rod-like

crystals on the transversal section, and the new crystals
preferred to grow in the peripheral regions than in the central
regions of enamel prisms. On the longitudinal section, short
rod-like crystals were formed on the enamel surface, interprismatic gaps disappeared, and the direction of the new
crystals was parallel to the long axis of the enamel crystals
Figs. 14.
The results of ATR FT-IR indicated that the amide I band at
1650 cm1 appeared on the biomimetic coating. On the etched

Fig. 4 The ATR FT-IR spectrum of the biomimetic coating

on enamel prepared after 20 h compared with intact
enamel and etched enamel. The presence of an amide I
band at 1650 cmS1, and two bands at 2926 cmS1 and
2854 cmS1 resulting from the methylene groups in
PAMAM-COOH were significant.

enamel surface, there were no characteristic peaks detected

for organics.
The XRD results showed that the length of the particles was
37.05 nm for intact enamel, 36.40 nm for etched enamel and
38.27 nm for the biomimetic coating; the width was 24.01 nm
for intact enamel, 26.65 nm for etched enamel and 21.67 nm
for the biomimetic coating. The aspect ratio was 1.54, 1.36 and
1.76, respectively. The results revealed that the average crystal
sizes and preferential orientation of the biomimetic coating
were almost the same with the intact enamel.25

4.

Fig. 3 The XRD spectrum of the biomimetic coating on

enamel with the PAMAM-COOH template. The presence of
a hydroxyapatite diffraction band (0 0 2) at 25.9, (2 1 1) at
31.8, (1 1 2) at 32.2, and (3 0 0) at 32.9 was clearly detected.
The PAMAM-COOH template did not alter the mineral
phase of the calcium phosphate coating significantly,
compared with the intact enamel.

Discussion

Chen et al. have demonstrated that PAMAM-COOH can be

bound to single crystals from enamel through the interaction
between anionic branches of the dendrimers and cationic
calcium on the crystal surface.22,23 In the present study, FTIR
showed an amide I band at 1650 cm1 at the surface of intact
enamel, which may be induced by residual proteins15; after the
etching and rinsing, all the proteins were supposed to be
removed. However, the amide I band at 1650 cm1 reappeared
at the remineralized surface due to the PAMAM template.
Additionally, two characteristic bands of methylene groups in
PAMAM-COOH at 2926 and 2754 cm1 were also detected. Both
results demonstrated the presence of PAMAM-COOH within
the biomimetic coating.26
The interaction between proteins and hydroxyapatite
indicate that acidic proteins are preferentially adsorbed on
the crystal face along the c axis because the crystal face is more
positively charged than other faces.2729 PAMAM-COOH
demonstrated the same characteristic through AFM observation.22 In the present study, due to demineralization, peripheral regions of enamel prisms were suggested to be more
exposed areas along with the c axis than at central regions,

archives of oral biology 58 (2013) 975980

which can absorb more PAMAM self-assemblies to increase

nucleation and growth of new crystals around enamel prisms.
Therefore, Fig. 1 shows processes of crystals surrounding the
prisms on the transversal surface. This finding also explains
why the phenomenon was more obvious in SEM of the
longitudinal section.
In recent studies, many methods in vitro have been used to
mimic the biomineralization of human enamel, including
amelogenin, calcium phosphate solution and nano-hydroxyapatite. These results show the successful formation of
enamel-like crystals. However, the experimental conditions
are too harsh for clinics, and the retained crystals are
perpendicular to the enamel surface without consideration
of the direction of the original enamel prisms. The original
crystals are very important because we believe that the
principle of caries treatment, which require complete removal
of infected enamel, must also be suitable for the future
application of enamel biomineralization in clinics. Cavities
surrounded by healthy enamel with prisms of different
directions must be prepared, and if the regrown enamel
layers are perpendicular to the cavity walls particularly the
axial walls the micro structure of the final repaired enamel
will be totally different from the original enamel. In the
present study, we demonstrated that with the effect of the
PAMAM-COOH template, the biomimetic crystals can be
formed in a relatively short time and under near-clinical
conditions, and the direction of the regrown crystals can be
consistent with the enamel prisms on both longitudinal and
transversal surfaces. This result has not been reported in any
previous study.
Conclusively, we proposed that PAMAM self-assemblies
play as an organic template between the regrown crystals and
etched enamel regulating both mineral nucleation and crystal
growth. First, the chain-like PAMAM self-assemblies were
adsorbed on the surface of the enamel crystals by electrostatic
interaction between carboxylic terminates and enamel crystals. When in the calcium phosphate solution, the template
served as a nucleation site due to calcium binding on the
carboxylic acid terminals. The interaction led to a high local
concentration of calcium ions, and the deposition of HAP
occurs when the phosphate ions arrived. As the deposition
continued, HAP crystals started growing at specific sites along
the long axis of enamel crystals and finally formed a rod-like
morphology, parallel to the enamel prisms. Although the FHAP
crystals were not parallel with the enamel prism, and the
underlying cause warrants further study, Fig. 2 shows that the
distribution of F-HAP was significantly influenced by the
PAMAM-COOH template. We can obviously see the patterns of
enamel prisms through the F-Hap layer. It is assumed that the
nucleation of F-HAP was controlled by the PAMAM-COOH
template. However, subsequently, F-HAP crystals grew too fast
in length to be controlled by the PAMAM-COOH template.
Therefore, needle-like crystals are still observed even with the
template.

5.

Conclusions

Previous studies of biomimetic remineralization of enamel

only focused on the morphology and structure of the

979

newborn crystals, but few studies were concerned with

their relationship with the original enamel crystals. This
biomimetic structure is very important for the regeneration
of teeth in reparative and restorative dentistry. Initially, in
the present study, we successfully formed new rod-like
crystals with the same structure, orientation and mineral
phase of intact enamel, and the hydroxyapatite nanorods
were closely paralleled to the original prisms. Therefore, we
conclude that G3-PAMAM-COOH can be absorbed on the
exposed enamel prisms and can function as the organic
template for biomimetic remineralization on etched
enamel.

Funding
National Natural Science Foundation of China (Grant No.
81170958 and 51073102).
Specialized Research Fund for the Doctoral Program of
Higher Education (20100181110056).

Competing interest
The authors do not have any possible conflicts of interest.

Ethical approval statement

None.

Acknowledgments
This research was supported by the National Natural Science
Foundation of China (Grant Nos. 81170958 and 51073102) and
the Specialized Research Fund for the Doctoral Program of
Higher Education (Grant No. 20100181110056).

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