Oriented crystallization of hydroxyapatite by the biomimetic amelogenin nanospheres from self-assemblies of amphiphilic dendrons

Article abstract of DOI:10.1039/c1cc13661e

An amphiphilic PAMAM dendron with aspartic acids on the periphery and an aliphatic chain at the focal point was synthesized. The dendrons initially self-assembled to nanospheres in aqueous solution and further translated to linear chains that showed a function similar to amelogenin in the oriented growth of HAP in vitro. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1cc13661e

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COMMUNICATION
Oriented crystallization of hydroxyapatite by the biomimetic amelogenin
nanospheres from self-assemblies of amphiphilic dendronsw
Sheng Yang,a Hai He,b Lei Wang,*a Xinru Jia*b and Hailan Feng*a
Received 20th June 2011, Accepted 22nd July 2011
DOI: 10.1039/c1cc13661e
An amphiphilic PAMAM dendron with aspartic acids on the
periphery and an aliphatic chain at the focal point was synthe-
sized. The dendrons initially self-assembled to nanospheres in
aqueous solution and further translated to linear chains that
showed a function similar to amelogenin in the oriented growth
of HAP in vitro.
self-assembly of peptide amphiphiles into cylindrical micelles
that functioned as a template to direct the formation of thin
HAP crystals. J. Moradian-Oldak et al.4 reported the dependence
of c-axial orientation of hydroxyapatite on the hierarchical
self-assembly of birefringent ‘‘microribbons’’ from porcine
amelogenins. They suggested that the transition from nano-
spheres to long chains might be involved in the mechanism of
amelogenin as a scaffold in the oriented and elongated growth
of apatite crystals.
As is well known, the mineralized tissues, including cartilage,
bone and tooth with special morphologies and excellent
biomechanical properties, are developed by the deposition of
calcium phosphates on ordered self-assembled proteins.1,2 For
example, in the oriented growth of enamel crystals, amelogenin, a
hydrophobic protein, plays a key role as a highly orchestrated
extracellular matrix in the initial step of dental enamel formation
(amelogenesis).3 The amelogenin is secreted by ameloblasts
and self-organizes into nanospheres that regulate the parallel
arrangement of rod-like nano-hydroxyapatite (HAP) crystals.
Enamel is the hardest mineralized tissue in vertebrates and
cannot regenerate once it is damaged after the elimination of
ameloblasts at enamel maturation. Currently, defects in
enamel are usually repaired by utilizing many unstructured
substitutes like synthetic resins, ceramics and metals which not
only do not function as well as the natural one but also have
problems integrating with the tissues of the tooth, with ageing,
adhesion failure and the materials fracturing. Therefore,
the ability to create artificially synthesized materials with
enamel-like microstructures for dental therapy is urgently
needed.4,5
Inspired by the pioneers’ findings, we have been trying to
design a PAMAM-based dendritic molecule, with a simple
structure and simple synthetic procedures but a facile assembly
property, to show novel functions similar to the amelogenins
for the oriented and elongated growth of apatite crystals. The
obvious advantages are that the dendritic molecules, such as
PAMAM dendrimers, closely resemble the topologies and
dimensions in the proteins, and the layered structures make
them easily modifiable in order to tailor the properties to
match various requirements.13,14 In addition, our previous
studies on the PAMAM dendrons demonstrate their versatile
self-assembly behavior with different packing modes caused by
changing the focal or periphery groups.15,16 Crystallization of
HAP in the presence of PAMAM dendrimers has been
reported in recent years. For instance, Yang and coworkers17,18
reported that the size and shape of HAP nanostructures were
effectively influenced by PAMAM with different surface
groups, generations and concentrations. Xie et al.19 reported
that PAMAM with L-glutamic acid groups on the periphery
could affect the crystallization of HAP. However, self-
assembly structures from dendritic molecules functioning as
amelogenins to direct HAP crystallization are rarely reported.
Herein, we report a novel amphiphilic PAMAM dendritic
molecule modified with L-aspartic acids on the periphery and
an aliphatic chain at the focal point (Sa-PAMAM-Asp)
(Fig. 1). In such a way, we aim to mimic the self-assembly
behavior, the function of amelogenins, and the interaction of
In order to mimic the biomineralization, many helpful ways,
such as peptides,6 proteins,7 polymer microgels8,9 and self-
assembling amphiphiles10,11 have been developed to ascertain
the oriented arrangement of hydroxyapatite crystals in the
hard tissues. For example, Stupp and coworkers12 reported the
a Department of Prosthodontics, Peking University School and
Hospital of Stomatology, 22 Zhongguancun Nandajie,
Haidian District, Beijing 100081, P. R. China.
E-mail: kqfenghl@bjmu.edu.cn; Fax: +86 10 62173402;
Tel: +86 10 82195232
b Beijing National Laboratory for Molecular Sciences, and Key
Laboratory of Polymer Chemistry and Physics of the Ministry of
Education, College of Chemistry and Molecular Engineering, Peking
University, Beijing 100871, P. R. China. E-mail: xrjia@pku.edu.cn;
Fax: +86 10 62751708; Tel: +86 10 62752102
Fig. 1 The modification of PAMAM dendron with aspartic acid and
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13661e
stearic acid.
c
10100 Chem. Commun., 2011, 47, 10100–10102
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aspartic acid residues on the C terminus of amelogenin with
apatite in vitro.
resulted from the fusion of nanospheres (40 nm in diameter,
inset of Fig. 2c) could also be obtained in the presence of
0.1 M Ca(NO3)2 and 1.6 Â 10À4 M Sa-PAMAM-Asp (Fig. 2c).
Such self-assembly behavior is similar to our previous study on
the PAMAM dendrons focally modified with aromatic
chromophores.16 The driving forces in the assemblies include
H-bonding of amides in the branches, carboxyl groups on the
periphery, and hydrophobic interactions of the aliphatic chain
at the focal point. Structurally, flexible hydrophilic dendritic
branches and the carboxyl groups are located in the outer
layer of the aggregates, leading to nanospheres that are very
adhesive. They are readily attached by H-bonding interaction
and the entanglement of the branches, which leads to the
formation of linear chains by the fusion of several individuals.
The entanglement of branches, the H-bonding of carboxyl
groups and amide groups in the branches would strengthen the
contact of the assemblies, leading to fusion of nanospheres to
form linear chains (Fig. S3w).
The resulting assemblies resemble amelogenin in morphology
and structural residues of aspartic acid, which may create an
ideal environment for the oriented growth of apatite crystals
in vitro. Encouraged by this, we then used the assemblies of
Sa-PAMAM-Asp in aqueous solution for the mineralization
of HAP (for detailed procedures see ESIw) at pH = 7.4 and
37 1C. It was found that the HAP crystals displayed quite a
different morphology as compared to the plate-like crystals
(100–600 nm in length and 50–100 nm in wide) developed in
the absence of the aggregates (Fig. S4 in the ESIw). As showed
in Fig. 3a, bundles of oriented crystals of 10–50 nm in width
and 110–700 nm in length were observed in the TEM images.
Remarkably, the crystals arrayed approximately along the
linear chains and each bundle contained several parallel filaments
of 3–4 nm in width and 100–500 nm in length (Fig. 3b and Fig. S7
in the ESIw). Such structures are consistent with reported
parallel filaments obtained by adding the recombination
amelogenin into calcifying solution.3,22,23
The PAMAM dendrons (G = 1) were synthesized by
divergent method and further modified with aspartic acids
on the periphery and an aliphatic chain at the focal point
(Fig. 1, Sa-PAMAM-Asp). Nuclear magnetic resonance
(NMR) technique, electrospray ionization mass spectrometry
(ESI-MS) and Fourier transform infrared spectroscopy
(FTIR) were used to verify the structure and purity of the
obtained compound. The results were in good agreement with
the proposed structures and the detailed materials including
synthetic procedures and characterization of the amphiphilic
dendrons are described in the ESI.w
The aggregates of Sa-PAMAM-Asp were prepared by
dissolving the sample in dimethyl sulfoxide (DMSO) at room
temperature, and then dropping it into aqueous solution at
pH 7.4 and 37 1C. The critical aggregation concentration (CAC)
of Sa-PAMAM-Asp in aqueous solution was determined by
monitoring the change of fluorescence intensity with an
increase of the concentration of modified dendrons (Fig. S1 in
the ESIw). The CAC value was very low (5.5 Â 10À6 M), which
coincided with our previous observation for PAMAM
dendrimers containing aromatic chromophores on the
periphery.15
The self-assembly of the resulting amphiphilic dendrons in
aqueous solution was studied by means of transmission elec-
tron microscopy. Fig. 2 shows typical TEM images of the
aggregates from Sa-PAMAM-Asp at different concentrations
in aqueous solution. Nanospheres of ca. 20–50 nm in diameter
were initially observed from the samples with concentrations
around the CAC, such as 6.0 Â 10À6 M (Fig. 2a). However, the
nanospheres were not thermodynamically stable. After the
sample was kept in aqueous solution at 37 1C for 2 days,
short linear chains of ca. 20–30 nm in width and 100–200 nm
in length were clearly observed, indicating a process of fusion
of the nanospheres, resulting in a stable state (Fig. 2b).
In addition, linear chains of 30–50 nm in diameter and
20–500 nm in length could also be generated with the increase
of concentration (e.g. up to 1.6 Â 10À4 M). In this case,
nanospheres could not be observed from the freshly prepared
samples (Fig. S2 in the ESIw). In order to facilitate the linear
fabrication of the dendrons, 0.1 M Ca(NO3)2 was added as an
additive into the system for the reason that it was an effective
way to alter the morphology of supramolecular assemblies by
changing the microenvironment as reported in the
literature.20,21 The longer linear chains (300 nm–1.5 mm in length)
The calcium-phosphorus molar ratio (Ca/P) of the resulting
crystals was analyzed by energy dispersion from an X-ray
detector (EDX). By careful observation, we found that a
metastable amorphous calcium phosphate (ACP) with a Ca/P
molar ratio of ca. 1.5 took shape rapidly in the initial stage
(in 7 days, inset of Fig. 3a), and it transformed to the stable
apatite crystals in 14 days with a Ca/P molar ratio of ca. 1.67
(inset of Fig. 3d). Fig. 3c and f show the electron diffraction
patterns of regions in images (a) and (b) respectively, and the
typical reflections of HAP are marked with (002), (211) and
(210). The electron diffraction patterns indicated the parallel
alignment of oriented and elongated apatite crystals, similar to
the enamel rods.24 As shown in Fig. 3e, the spacing of crystal
lattice along the long axis is 0.346 nm (selected region from
Fig. 3d, marked with red frame) which corresponded to the
(002) face of HAP crystals as reported in the literature.23 To
develop a better understanding of the crystals, XRD measurement
was used to further verify its structure. A peak appeared at 2y =
25.91 which was the characteristic (002) diffraction of HAP
(Fig. S9a in the ESIw). This peak was enhanced as compared
with that of the crystals formed without assemblies (Fig. S9b in
the ESIw), suggesting that the apatite developed preferentially
along the c-axis to some extent.
Fig. 2 TEM images of the aggregates of Sa-PAMAM-Asp: (a) the
nanospheres at a concentration of 6.0 Â 10À6 M; (b) the short chains
of fusing nanospheres as time goes on (2 days); (c) the linear chains of
the amphiphilic dendrons at a concentration of 1.6 Â 10À4 M in the
presence of 0.1 M Ca(NO3)2.
c
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fabricating the oriented HAP with synthetic dendritic
molecules. The present work provides direct experimental
evidence for the protein-mediated HAP nucleation and growth
and it may also reveal the mechanism involved in enamel
biomineralization.
The authors thank Jing Chen, Mingjun Teng for TEM and
EDS characterization, Fuhui Liao for XRD analysis, and
Ming Gao for CAC tests. The authors are most grateful to
all reviewers of this article for their comments and suggestions.
The work is supported by the National Natural Science
Foundation of China (NSFC Grant 30572063 and 30600717)
and the Doctoral Fund of Ministry of Education of China
(20070001726).
Fig. 3 The high-resolution TEM images of (a) the oriented growth of
HAP crystals in the present of linear chains from Sa-PAMAM-Asp
(7 days). (b) TEM image of a mineralized bundle of filaments assembled
parallel to their c-axis (selected from image (a), marked with red
frame); (c) electron diffraction pattern of a region taken from image
(a), typical reflections of HAP are marked; (d) HAP crystals in the
presence of linear chains from Sa-PAMAM-Asp (14 days). (e) The
spacing of the crystal lattice along the long axis (selected from image
(d), marked with red frame). Long white arrow indicates the long axis
direction of the microstructures. (f) Electron diffraction pattern of a
region taken from image (d), typical reflections of HAP are marked.
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In conclusion, the self-assembly behavior of a novel amphi-
philic dendron Sa-PAMAM-Asp in aqueous solution was
investigated. Sa-PAMAM-Asp self-assembled into nano-
spheres initially and further translated into linear chains either
by increasing the concentration or by adding an additive. The
linear assemblies showed a function similar to amelogenin in
the oriented growth of HAP. It was found that the apatite
developed in the presence of the linear assemblies resembled
some of the features of the lowest level of the hierarchical
structure of enamel, such as the preferential orientation of the
c-axis of the HAP crystals along the amelogenin aggregates.
To the best of our knowledge, this is a rare example of
26 H. C. Margolis, E. Beniash and C. E. Fowler, J. Dent. Res., 2006,
85, 775–793.
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10102 Chem. Commun., 2011, 47, 10100–10102
This journal is The Royal Society of Chemistry 2011