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

Biomimetic transformation of polyphosphate

microparticles during restoration of damaged teeth

Maximilian Ackermann a , Emad Tolba b,c , Meik Neufurth b ,

Shunfeng Wang b , Heinz C. Schröder b , Xiaohong Wang b ,
Werner E.G. Müller b,∗
a Institute of Functional and Clinical Anatomy, University Medical Center of the Johannes Gutenberg University,
Johann Joachim Becher Weg 13, D-55099 Mainz, Germany
b ERC Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry, University Medical

Center of the Johannes Gutenberg University, Duesbergweg 6, D-55128 Mainz, Germany

c Polymers and Pigments Department, National Research Center, 33 El Buhouth St, Dokki, 12311 Cairo, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Objective. In the present study, we investigated the fusion process between amorphous
Received 29 July 2018 microparticles of the calcium salt of the physiological polymer comprising orthophosphate
Received in revised form units, of inorganic polyphosphate (polyP), and enamel.
1 November 2018 Methods. This polymer was incorporated as an ingredient into toothpaste and the fusion
Accepted 14 November 2018 process was studied by electron microscopy and by synchrotron-based X-ray tomography
Available online xxx microscopy (SRXTM) techniques.
Results. The data showed that toothpaste, supplemented with the amorphous Ca-polyP
Keywords: microparticles (aCa-polyP-MP), not only reseals tooth defects on enamel, like carious lesions,
Inorganic polyphosphate and dentin, including exposed dentinal tubules, but also has the potential to induce re-
Microparticles mineralization in the enamel and dentin regions. The formation of a regeneration mineralic
Dentifrice zone on the tooth surface induced by aCa-polyP-MP was enhanced upon exposure to artifi-
Alkaline phosphatase cial saliva, as demonstrated by SRXTM. Energy dispersive X-ray analysis revealed an increase
Coavervate in the calcium/phosphorus atomic ratio of the enamel deposits to values characteristic for
Regeneration the particles during the treatment with polyP applied in the toothpaste, indicating a fusion
Enamel/dentin damage repair of the particles with the tooth mineral.
Caries Significance. Our results suggest that toothpaste enriched with aCa-polyP-MP is a promising
biomimetic material for accelerating enamel and dentin restoration.
© 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

ture occurring as a consequence of caries or of external

1. Introduction trauma. Such a closing of space, where bacteria could enter,
also helps to prevent further decay. At present the materials
Dental restoration/dental filling aims to treat and to restore
used for fillings include gold, porcelains, defined composite
the function, integrity, and morphology of missing tooth struc-
resins and amalgams. Those components are regeneratively
inert, since they are not included in a repair or a remodel-
ing process of the decayed tooth, or of the surrounding tissue
∗ (reviewed in Ref.: [1]). However, there are nanotechnology-
Corresponding author.
E-mail address: wmueller@uni-mainz.de (W.E.G. Müller).
https://doi.org/10.1016/j.dental.2018.11.014
0109-5641/© 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

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based restorative materials that are currently intensively from which phosphorylation to ATP is likely to occur via the
developed [2], as well as regeneratively active biomaterials [3] adenylate kinase [25,26]. The encapsulation of polyP as amor-
that are advanced rapidly in the field of repair of bone and phous particles [27], as in the acidocalcisome [28], opens a new
bone-derived rigid organs. These developments are driven by avenue for the application of polyP as a smart regeneratively
new spectacular discoveries in the field of biomaterial sci- active biomaterial for tissue engineering [29].
ences. ACP [Ca3 (HPO4 )3 (PO4 )3y ·zH2 O], the putative precursor
Minerals are ubiquitous components of living systems for bone HA readily occurs in aqueous solution by pre-
from bacteria to plants and the different animal phyla where cipitation through multiple intermediates, often with a
they accomplish an array of crucial structural and functional further ACP-stage and an octacalcium phosphate phase
roles. While minerals in the non-living world are usually [Ca8 H2 (PO4 )6 ·5H2 O] until the formation of the crystalline cal-
formed at extreme temperatures and pressures and over cium phosphate (reviewed in Ref.: [30]). In vitro, the solid
long geological timespans, biogenic minerals are synthe- phase ACP precipitates from a highly supersaturated phos-
sized at physiological, ambient temperatures and pressures, phate solution and is then readily converted to octacalcium
eventually leading to crystal formation. For these biological phosphate or apatite (Ca10-x (HPO4 )x (PO4 )6-y (OH)2-x ). Applying
processes, the term biomineralization has been coined [4,5]. In a variety of techniques, including cryo-TEM, in situ as well
1988 [6], it has been postulated that proteinaceous templates as ex situ procedures, ACP has been shown to crystallize
control crystal growth via their functional groups through forming nanometre-sized units of calcium triphosphate com-
precise stereochemical coordination of metal atoms. Other plexes. During this apatite formation process, the precursors
organic templates, like complex carbohydrates, have also been take up an additional Ca2+ ion and form fractal clusters of
identified [7]. Since (almost) any kind of process in living Ca2 (HPO4 )3 2− . From this stage, the crystal structure of octa-
organisms is enzymatically controlled, it was a matter of time calcium and apatite maturates [31]. Unlike the chemically
to disclose these biocatalysts as causative factors involved in synthesized apatite (geological apatite), bone HA is formed
biomineralization. around a collagen scaffold [32] and does not have a hexago-
The first enzyme found to be involved in biomineraliza- nal crystal morphology; it is described as a monoclinic apatite
tion was silicatein, a cathepsin-related enzyme that forms [33,34]. Moreover, the bone HA is only poorly crystalline due to
the basis for the synthesis of biosilica in sponges ([8,9]; the small size of the crystals, the residual stresses in the crys-
reviewed in Refs.: [10,11]). In addition, it has been demon- tal lattice, and ionic substitutions, such as of PO4 3− by HPO4 2− ,
strated that silicatein comprises not only an enzymatic or of Ca2+ by Na+ , Sr2+ , Mg2+ , or Zn2+ [35,36].
activity but also a structure-guiding function during the min- It has been proposed that inorganic pyrophosphate is a
eralization process [12,13]. More recently, the first enzymes regulator of bone HA synthesis both under pathological and
involved in the formation of bone and dentin mineral, car- physiological conditions [37] and presumably inhibits the ALP
bonated apatite/hydroxyapatite, have been identified. Since [38]; a large amount of data has been gathered indicating that
hydroxyapatite [HA] contains a considerable amount of car- the enzyme promotes calcification by lowering pyrophosphate
bonate besides of 50% octacalcium phosphate (47% carbonate levels ([39]; reviewed in Ref.: [40]). This view is supported by
apatite with about 2% calcium carbonate), especially during the demonstration that cells present in calcifying cartilage
embryogenesis [14], the enzyme carbonic anhydrase has been and resorbing bone are rich in ALP [41]. Using the biomimetic
proposed to be the initiating enzyme during bone formation approach but using polyP instead of pyrophosphate, we pre-
[15]. Considering the basic premise that crystalline biomate- pared stable ACP in the presence of <10 wt.% of the polymer
rials are formed in vivo from amorphous precursors [16], it is [42]. So far, no data are available on the possible presence of
proposed that the amorphous calcium carbonate [ACC] in the polyP in the HA biomaterial in vivo. However, the accumulation
bone mineral is a product of the carbonic anhydrase reaction of blood platelets at sites of bone damage or injury (reviewed in
[17], a view also shared by Weiner et al. [16]. ACC is converted Ref.: [43]) and the observation that platelet-rich plasma accel-
into the calcium phosphate mineral, again in the amorphous erates the healing of musculoskeletal injuries (reviewed in
phase [17], a process that is driven non-enzymatically and Ref.: [44]) support the proposition that polyP released from the
exclusively thermodynamically. The product formed is amor- blood platelets contributes to HA formation. In the extracel-
phous calcium phosphate [ACP], from which the crystalline lular space, polyP is hydrolyzed by ALP [23]. The existence of
HA maturates [18]. this enzyme in the saliva is well known [45].
ß-Glycerophosphate has been shown to be a potent phos- In previous studies, we reported that amorphous polyP,
phate donor in vitro [19]. However, in vivo this metabolite encapsulated into microparticles as an amorphous Ca2+ salt,
is unlikely to be an efficient source for phosphate as it is aCa-polyP-MP, efficiently seals open dentinal tubules exposed
rapidly hydrolyzed by extracellular alkaline phosphatase [ALP] at the tooth surface [46]. In subsequent analyses, we have
[20]. Another phosphate source for bone mineralization is formulated a toothpaste composition containing aCa-polyP-
polyphosphate [polyP] [21], an abundant inorganic polymer MP that efficiently reduced dental biofilm formation [47] and
found in human/animal body fluids as well as intracellularly, induced the remineralization process [48]. In the present
particularly in the megakaryocyte-derived cell fragments, the study, we apply the technique of synchrotron-based X-ray
blood platelets [22]. Physiologically, this polymer consists of tomographic microscopy (SRXTM) [49,50] to demonstrate the
about 40–100 phosphate units which are linked by high-energy fusion process between the microparticles and the tooth
phosphoanhydride bonds. The main polyP-degrading enzyme enamel. It is found that the enamel becomes covered by an
is the ALP [23,24]. Recent studies have shown that enzymatic induced mineralization material when the polyP-treated paste
cleavage of polyP by ALP transfers metabolic energy to ADP, is submersed in an artificial saliva fluid. This induced miner-

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alization process does not occur on non-treated enamel. The 2.6. Exposure to artificial saliva
amalgamation of aCa-polyP-MP with the tooth mineral is also
supported by energy dispersive X-ray analysis [EDX] which The microparticles, aCa-polyP-MP, were applied as a glycerol
revealed that with increasing duration of the polyP treatment, suspension. The microparticles were suspended at a concen-
applied in the dentifrice, the calcium/phosphorus atomic ratio tration of 3% [w/w] in 86% (v/v) aqueous glycerol, containing
increased from 0.72 ± 0.13 to 1.50 ± 0.23 (6 d treatment), a value 2.5% xanthan gum (Xanthomonas campestris; #G1253 Sigma).
close to that measured for human enamel (Ca/P atomic ratio, This suspension was homogenized in a mortar and pestle to
1.43 ± 0.14). obtain a uniform mixing. From this 0.2 g were applied per tooth
specimen for 2 d, and brushed twice daily. Then the specimens
were transferred into the artificial saliva. In the control series,
2. Materials and methods the specimens were treated with the plain glycerol suspension
only.
2.1. Materials The composition of the artificial saliva was chosen from
the literature [51,52]. It contained 1 mM CaCl2 , 0.2 mM MgCl2 ,
Sodium polyphosphate (Na-polyP) with an average chain 4.0 mM KH2 PO4 , 30.0 mM KCl, and 20.0 mM HEPES [(4-(2-
length of ≈40 phosphate units was obtained from Chemische hydroxyethyl)-1-piperazineethanesulfonic acid]). The pH was
Fabrik Budenheim (Budenheim, Germany). adjusted to 7.0 (KOH). The tooth samples were submersed in
an excess of solution.
2.2. Preparation of polyP microparticles
2.7. Microscopy
aCa-polyP-MP, Ca2+ -polyP microparticles, were prepared from
Na-polyP as described [27]. Na-polyP was dissolved in distilled 2.7.1. Digital light microscopy
water, then treated with a 2-fold weight ratio of CaCl2 ·2H2 O The analyses were performed with a VHX-600 Digital Micro-
(#223506; Sigma-Aldrich, Taufkirchen, Germany) at pH10 scope (Keyence, Neu-Isenburg; Germany), equipped with a
(room temperature) and stirred for 12 h. The microparticles VH-Z100 zoom lens.
formed were collected by filtration, washed with ethanol, and
dried at 50 ◦ C. 2.7.2. Electron microscopy
For high-resolution scanning electron microscopic (SEM) anal-
2.3. Composition of toothpaste ysis, a HITACHI SU 8000 (Hitachi High-Technologies Europe
GmbH, Krefeld, Germany) equipped with a low voltage (<1 kV)
The complete procedure used for the preparation of the tooth- near-surface organic surfaces detector was used. The samples
paste has been given recently [47]. Diatomaceous earth was were processed without further sputtering. Lower-resolution
added as an abrasive and xylitol, xanthan, ␬-carrageenan and inspections were made with an ESEMXL-30 machine (Philips,
glycerol were used as components of the dentifrice base. Eindhoven; The Netherlands). The teeth were dehydrated
Where indicted, 3% [w/w] of aCa-polyP-MP (final concentra- in ethanol, freeze-dried, mounted onto specimen holders
tion) was added. and finally sputtered with gold in an argon atmosphere as
described.
2.4. Tooth samples
2.8. Synchrotron-based X-ray tomographic microscopy
Human teeth (molar and premolar) were used as samples for
the treatment with the experimental dentifrice. They were The non-destructive high-resolution technique of
provided by the Institute of Functional and Clinical Anatomy, synchrotron-based X-ray tomographic microscopy (SRXTM)
University Medical Center of the Johannes Gutenberg Univer- was used as described before [49,50]. Imaging recording was
sity, Mainz, Germany, following the ethical guidelines of the performed at the TOMCAT-beamline of the Swiss Light Source
University Medical Center Mainz. The specimens were cleaned at the Paul Scherrer Institute (Villigen; Switzerland).
from organic material by incubation in 4% sodium hypochlo-
rite solution for 4 h. Then the samples were thoroughly rinsed 2.9. Energy dispersive X-ray spectroscopy
with distilled water and air dried.
For the experiments, an EDAX Genesis EDX System attached
2.5. Treatment of the teeth to a scanning electron microscope (Nova 600 Nanolab, FEI,
Eindhoven; The Netherlands) and operating at 10 kV with a
The teeth samples were brushed with an electric toothbrush collection time of 30–45 s was applied. The system was cali-
(Braun Oral-B PRO 6000; Procter & Gamble, Cincinnati; OH) at brated with standard samples, allowing measurements with
8000 rpm and 100 × g force for 3 min at room temperature [47]. an error of approximately 10% [53].
An amount of ≈0.2 g of dentifrice was applied on top of the
respective enamel and dentin surfaces. Routinely the speci- 2.10. Statistical analysis
mens were brushed twice a day for 5 min each. Subsequently,
the specimens were thoroughly rinsed. During the inter-brush The results were statistically evaluated using the paired Stu-
periods, the teeth remained in a humid chamber. dent’s t-test [54].

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Fig. 1 – Brushing of the enamel surface of human teeth with the toothpaste without polyP (A–C), or with the paste
supplemented with 3% [wt/wt] of aCa-polyP-MP (D–F). Onto the surface areas of teeth brushed with the
aCa-polyP-MP-containing toothpaste, extensive areas remained covered with dentifrice (df) onto the carious, decayed
regions (cl). The paste was applied for a period of 6 d.

are covered with a layer of amorphous material (Fig. 2D). This

3. Results material was identified by EDX as Ca-polyP (data not shown).

3.1. Sealing activity of the polyP-toothpaste: enamel

3.2. Increase of the Ca/P atomic ratio of the dentifrice
The fabricated toothpaste containing 3% [w/w] of aCa-polyP- deposits with the duration of the application
MP was applied to the enamel of the teeth and processed
as described under Section 2. In parallel, the dentifrice The Ca/P atomic ratio within the Ca-polyP microparticles has
without polyP was used as a control. After a 6 days’ appli- been previously determined to be 0.62 [26,27]. The deposits
cation/brushing (twice daily) on the tooth surfaces and after on the enamel of the aCa-polyP-MP-dentifrice after a one-day
thorough rinsing the specimens with PBS [phosphate buffered application measured a Ca/P atomic ratio of 0.72 ± 0.13 (num-
saline], no extensive plaques on the decayed carious teeth that ber of independent measurements: 6); a representative EDX
might have originated from the paste adhered to the enamel in spectrum is given in Fig. 3A. This ratio increased significantly
the series with dentifrice without polyP (Fig. 1A–C). In contrast, to 0.93 ± 0.18 (p < 0.05) after a 3 days’ treatment (Fig. 3B) and
large areas of dentifrice remained on comparable surfaces of even further to 1.50 ± 0.23 (p < 0.01) after a 6 days’ applica-
teeth treated with the aCa-polyP-MP-containing toothpaste tion (Fig. 3C). This value is consistent with those published
(Fig. 1D–F) when analyzed with light microscopy. for pig enamel (1.52; [56]) and human enamel 1.92 [57]. In
Closer inspection of the enamel surfaces by SEM revealed our approach, using the EDX (semi-)quantitative technique,
that in the tooth series treated with dentifrice without polyP we measured a value of 1.43 ± 0.14 for human enamel, a figure
the vast majority of the cracks remained unsealed and had which matches the published data obtained by using the same
the appearance of cracks not treated at all (Fig. 2A). In con- analysis technique [58]. In contrast, when the scarcely occur-
trast, most of the cracks within the enamel at the beginning ring deposits from the polyP-lacking dentifrice were measured
of the treatment were sealed with tightly fitting material after 6 d, a Ca/P atomic ratio of 0.78 ± 0.17 was recorded.
after the 6 days’ brushing period (Fig. 2B). At high-resolution
SEM inspection of the carious areas, plain highly-oriented HA 3.3. Sealing of the carious lesions/dentinal tubules
crystals that are not covered by additional material are vis- with Ca-polyP-containing dentifrice
ible in the control samples (Fig. 2C). Fully developed mature
dental enamel is seen, which comprises highly organized Carious lesions appear in the enamel and may gradually
structures of enamel prisms consisting of bundles of nanorod- spread to the dentin [59], exposing the HA crystals; Fig. 4A,B.
like HA crystals. The ribbon-like crystals measure 60–70 nm In the deeper regions, the dentinal tubules are seen that
in width and 20–30 nm in thickness [55]. In contrast, the HA extend from the dentino-enamel junction, or the dentino-
crystals in the carious lesions in the enamel of teeth sam- cemental junction in the root area, to the outer wall. These
ples brushed with the aCa-polyP-MP-containing toothpaste tubules follow a radial or S-shaped path (Fig. 4C). In a com-
parative study, these carious lesions were treated either with
the polyP-free control dentifrice (Fig. 4A,C,E) or the aCa-polyP-

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Fig. 2 – Sealing of carious areas on the enamel; high-resolution SEM. Tooth samples were treated with control dentifrice
lacking polyP (A and C), or with dentifrice supplemented with 3% aCa-polyP-MP (B and D). In the samples treated with
control toothpaste the cracks remained non-modified (> <) (A) and the mineral shows the characteristic, highly-oriented HA
crystals (C). In contrast, in the test series with the aCa-polyP-MP-containing dentifrice the cracks are sealed (> <) (B) and the
mineralic material is covered and does not expose the nanorod-like HA crystals (D).

Fig.3 – Increase of the Ca/P atomic ratio in the deposits, formed after treatment with aCa-polyP-MP-containing dentifrice on
enamel for 1 d (A), 3 d (B) and 6 d (C). The prominent peaks, including those for Ca and P are marked.

MP-supplemented dentifrice (Fig. 4B,D,F). Already after a 1 d The size of these tubules measures between 2 and 3 ␮m in
application with the polyP-containing dentifrice, a relatively diameter, depending on the demineralization stage [60]. If the
homogenous layer can be seen in the deeper regions of the tooth dentin regions are treated for 5 d with the control paste,
lesions (Fig. 4B), which increases in continuity after 3 d and the openings of the dentinal tubules are clearly visible (Fig. 5-
6 d (Fig. 4D and F). In comparison, residual dentifrice areas are IA,C,E) and their margins are sharp-edged (Fig. 5-IE). However,
only occasionally seen in the polyP-lacking paste (Fig. 4A,C, if the teeth are treated with polyP-enriched dentifrice for the
and E). same period of time, most of the tubules are either partially
The tooth dentin is traversed/radiated outwards with or completely sealed (Fig. 5-IB,D, and F). To demonstrate that
microscopic channels, the dentinal tubules that originate from the tubule is indeed sealed by polyP, an EDX scan was run
the pulp and reach the exterior enamel-cementum border.

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Fig. 4 – Protection of carious lesions on human teeth after treatment with polyP-lacking (A, C, E) and polyP-containing
dentifrice (B, D, F). The pastes were applied for 1 d, 3 d, or 6 d, as indicated. In the series with polyP-lacking dentifrice, the
morphology of the teeth could be delimited, while in the series with the aCa-polyP-MP-containing paste the developed
layer covered the lesions. In E, the openings of the dentinal tubules (dt) are visible.

over the occlusion (Fig. 5II). The scan shows distinct peaks non-pretreated control samples, the mineralic deposits are
corresponding to Ca and P. individually layered onto the top of the enamel (Fig. 6D),
continuous layers of deposits are formed on the enamel pre-
treated with polyP (Fig. 6E).
3.4. Mineralization induction effects of polyP onto
Synchrotron-based tomographic imaging was applied to
enamel surface
study the crystal organization on the border between the
enamel and the mineral deposits formed during the incu-
It is known that artificial saliva can induce mineralization on
bation with the artificial saliva (Fig. 7). In the controls not
the enamel surface [61]. For the study reported here, the arti-
treated with polyP, only the genuine enamel layer is visible
ficial saliva was supplemented with 1 mM CaCl2 and 4.0 mM
(Fig. 7A). However, onto the enamel samples brushed with aCa-
KH2 PO4 as described under Section 2.
polyP-MP glycerol, an additional layer composed of “induced
To determine if polyP could cause any effect on the arti-
material”, as termed here, appeared (Fig. 7B–D). Furthermore,
ficial mineralization process, the enamel surface remained
bulky deposits originating from the artificial saliva can be
either untreated (Fig. 6A and D) or was pretreated with polyP,
distinguished. Interestingly, this material is not seen in the
applied as aCa-polyP-MP (Fig. 6B,C,E, and F). The microparti-
controls, underscoring the mineralization inducing property
cles were suspended in glycerol and then applied for 2 d onto
of polyP.
the enamel. Already at this stage an almost homogeneous
layer can be discerned (Fig. 6B); at higher magnification, the
individual microparticles can be visualized (Fig. 6C and F). 4. Discussion
If the samples, non-treated or polyP-treated, are immersed
in artificial saliva and incubated there for 5 d, a striking dif- Dental enamel is the hardest tissue in mammals and consists
ference in the mineralization pattern is seen. While in the to ≈95% of carbonated hydroxyapatite. The inorganic deposits

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Fig.5 – Sealing of the openings of the dentinal tubules with polyP. (I) REM analysis. (A, C, E) The surfaces of the exposed
dentinal tubules (dt) traversing the dentin region to the enamel border are treated for 5 d with polyP-free dentifrice. The
dentin surface and the openings of the dentinal tubules are clearly visible. (B, D, F) Treatment of those surfaces for the same
period of time (5 d) with the aCa-polyP-MP-containing dentifrice results in a sealing of the openings. (II) EDX analysis. The
spectroscopic scan was performed over one sealed dentinal tubule (marked in F, ****).

are formed during the biomineralization process onto matrix on the surfaces of the HA crystals. This property allows the
proteins (amelogenin, ameloblastin and enamelin) which are incorporation of carbonate substitutions that result in the
secreted by ameloblasts and serve as guidance. This matrix transition of the poorly crystalline HA with high HPO4 2− con-
allows the formation of individual HA “rods” or “prisms” that tent to a biomineral of higher crystallinity, lower phosphate
grow appositionally away from the dentino-enamel junction content, and a more complex crystal organization that still
[62]. Finally, most of the organic matrix is metabolized by pro- contains carbonate substitutions [68,69]. Taken together, the
teases. The basic units of tooth enamel, the rods, measure biomineralization of bone tissue, in contrast to the geological
≈4 ␮m in width to ≈8 ␮m in height and consist of highly orga- HA mineralization, can undergo a remodeling which is com-
nized HA [63]. In the dentin, the crystals are about 50 nm in paratively high in bone and dentin and very low in enamel.
length, 20 nm in width and 2–5 nm in thickness [64]. As in bone, Especially in the latter material, HA is prone exclusively to
dentin collagen and its associated proteins play the dominant physical dissolution processes by bacterial and environmental
role in determining the mineralization process. The content acids [70].
of organic materials within the dentin is larger and amounts Critical for the durability of the mineralic matrix of teeth
to ≈33% (by weight), while the content of HA is about 45%. is the pH, which is at pH 5.5. Above this value, the mineral
Because of these factors, especially the low content of cells environment is supersaturated with the mineral, and the addi-
and organic matrices in the enamel, the enamel acid cannot tional mineral tends to precipitate [71]. Conversely, the milieu
re-calcify [65]. is unsaturated at lower pH values in the surroundings, result-
Until now, the controversy over the presence of an ACP has ing in dissolution processes. As outlined in the Section 1,
not been solved, since complex structural analyses have failed polyP is a promising inorganic and physiological polymer for
to detect the presence of ACP in young bone [66]. Nonethe- potential use in restoration of HA in teeth [26,46,47]. In particu-
less, the physiological HA differs from the geological form by lar, when formulated in microparticles such as aCa-polyP-MP,
the smaller crystal size, the high degree of carbonate substi- release of the polymer from the biomimetically fabricated par-
tution combined with a substantial OH deficiency, and the ticles and subsequent hydrolytic cleavage by the ALP is slower
presence of lattice vacancies that render the bone and teeth than with soluble Na-polyP. The following properties of polyP
HA more soluble (see Ref.: [67]). The smaller size of the physio- qualify this polymer. As outlined in the Section 1, polyP is a
logical HA makes it more soluble, as more atoms are exposed readily available component for HA formation after enzymatic

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Fig. 6 – Homogeneous layer-formation on enamel after a pretreatment with polyP. The polymer was added as aCa-polyP-MP.
The enamel remained either untreated (- polyP) (A and D) or was treated with polyP, as described under Section 2 (B, C, E
and F). After 2 d of application the particles already coalesce onto the surface of the enamel (B). If the samples were further
processed by submersing into artificial saliva, additional crystals formed onto non-pretreated enamel remain separate and
craggy (D), while the deposits onto the polyP-pretreated specimens form a continuous layer (E). The two images C and F
show the microparticles on the enamel after the 2 d application.

Fig. 7 – Induced mineralization on enamel specimens. The teeth samples were treated either with the plain glycerol
solution only (A), or with the aCa-polyP-MP-containing glycerol solution (B–D) for 2 d, and then transferred to artificial
saliva. After incubation for 5 d the specimens were inspected by synchrotron-based X-ray tomographic microscopy. In the
control sample (A) the enamel surface is distinctly to discern in addition to the brushing area (ba). No additional layer is
seen. In contrast, in the polyP-treated samples (B–D), the enamel zone is clearly and additionally over-layed by an induced
material (im) which is most likely attributable to polyP. The specimens are surrounded by deposits which are contributed by
the polyP-artificial saliva (polyP-as).

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Fig. 8 – Application of polyP microparticles as material applicable for enamel reconstitution and dentin or dentinal tubules
restoration (Scheme). The region(s) of caries decay and dentinal defects are overlayed by aCa-polyP-MP particles. Introduced
into the biological environment the microparticles undergo coacervate formation a process during which polyP gains its
beneficial ameliorating property.

cleavage to ortho-phosphate by the ALP. The polymer can also known to be synthesized and metabolized by both caries-
stabilize the precursor of bone-mineral ACC, which certainly active and caries-inactive Streptococci bacteria living in the
promotes the synthesis of the Ca-polyP mineral [17]. In addi- oral cavity [79–81]. In these studies, no direct causal relation-
tion, amorphous polyP releases metabolic energy during and ship between the biological polyP turnover and the dental
after hydrolytic cleavage by the ALP [26], an enzyme which caries process has been addressed. Recently, it has been pro-
is abundant in saliva [72]. The ALP is a processing enzyme posed that polyP-accumulating bacteria that are also present
[73] that, like in ATP, cleaves off one terminal hydrogen phos- in the oral biofilm contribute to undersaturated phosphate
phate and one additional proton per anhydride linkage from conditions that can lead to mineral dissolution and caries pro-
polyP [74–76]. This finding implies that during polyP degra- gression [82]. While the correlations between polyP-producing
dation a local pH shift occurs, which can lead to a localized bacteria and acid production and a relative phosphate depri-
demineralization of the HA and thus to an increase micror- vation could indeed occur locally, the beneficial/ameliorating
oughness of the respective surface [77]. The concentration of effect of polyP on HA integrity was not the task of these
Ca2+ in human saliva is ≈1 mM and the phosphate concen- papers. There is experimental evidence demonstrating that
tration is ≈ 3.5 mM [51]. However, the extent of mineralization an increase in bacterial polyP metabolism is also associated
in the saliva is also dependent on the protein content. Here with an increased sequestering of the polymer [83], which has
polyP provides a distinguished feature. In an aqueous sys- a beneficial effect on the HA dissolution rate [84]. It has been
tem without protein, the aCa-polyP-MP are not undergoing found that the polymer reduces the baseline dissolution rate
dissolution [78]. However, after exposure to protein, polyP of HA, an effect that is even enhanced by fluoride.
undergoes rapid coacervation under concomitant release of It has been suggested that polyP is released by bacte-
the biological potency. In turn, polyP is activated in parallel ria in the form of particles [85]. In the mammalian system,
with an increase in protein levels. polyP is stored and released in the form of nanoparti-
polyP as an “ancient” molecule found in all living organ- cles/microparticles (reviewed in Ref.: [22]) in every type of
isms from bacteria to mammals (reviewed in Ref.: [22]) is cells, and particularly in blood platelets (see Refs.: [25,86]).

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It has recently been shown that these particles released on in this study, accumulates that polyP, particularly when pro-
the surface of platelets are the causative components of the duced in the form of microparticles, aCa-polyP-MP, induces
procoagulant activity of these cells [87]. the formation of a resealing layer on teeth decay regions. Since
It has been possible to fabricate, in a biomimetic way, the the material accelerates the restoration process not only on
physiologically occurring polyP with a chain length of ≈40 the enamel but also on the dentin, the polyP appears to be
phosphate units as amorphous aCa-polyP-MP [27]. These par- a promising dental filling material which elicits regenerative
ticles, when packed into a dentifrice [26,46,47], contribute activity (Fig. 8).
to the resealing of teeth defects and are even toxic for the As outlined in the present study, the amorphous particles,
cariogenic bacterium Streptococcus mutans [47]. As shown in when packed in a dentifrice, form a mineralic layer on both
the present contribution the HA-promoting activity of polyP mineral zones of the teeth. This process is further enhanced by
is seen in both the enamel and dentin regions of human the ions present in saliva, as shown here. In addition, the saliva
teeth. Data are summarized demonstrating that toothpaste contains proteins/mucoproteins in the range of 1 mg/ml [93].
supplemented with 3% [w/w] of aCa-polyP-MP shows a pro- In light of our recent study [78], these proteinaceous compo-
longed retention time compared to the paste without polyP nents of the saliva will surely reduce the zeta potential of the
on the surface of the enamel. While the paste without polyP particles and promote the conversion of aCa-polyP-MP into the
is almost completely removed after the 6 d treatment period, coacervate form, which is the biocompatible and ultimately
a substantial amount of deposits on the enamel brushed with bioactive form of the microparticles. During this process, polyP
polyP-containing dentifrice has been identified. By analysis is enzymatically cleaved by the ALP, resulting in the release of
of the remaining deposits with EDX, this material could be the ortho-phosphate building unit for HA and of metabolic
identified as Ca-polyP. Moreover, by applying the quantita- energy required for the energy-consuming anabolic metabolic
tive approach, it could be shown that the Ca/P atomic ratio processes that likely involve a second enzyme, the carbonic
increased from 0.6, found within the starting polyP-containing anhydrase IX, to synthesize the amorphous calcium carbon-
paste to 0.93 ± 0.18 after a treatment of the enamel for 3 d and ate bio-seeds which trigger calcium phosphate bone mineral
finally to 1.50 ± 0.23, after the 6 d incubation period. The lat- deposition ([94]; reviewed in Ref.: [29]). Based on the avail-
ter figures support the view that polyP present on the enamel able data, an inlay or onlay material containing aCa-polyP-MP,
accumulates Ca2+ with increasing duration of treatment. This as outlined in Fig. 8, will shift to the bioactive form of polyP
supports the view that the microparticles tend to accumulate through the metabolic exchange processes mediated by the
Ca2+ over phosphate to over-stoichiometric ratio values of >2 dentin tubules after filling into the decay region, and by inte-
[29]. The Ca/P atomic ratio of 1.50 is close to that characteris- gration processes of the polyP into the HA material.
tic for geological HA, which is around 1.67 (reviewed in Ref.: In addition, if this approach proves successful, the use of
[88,89]. The polyP-mediated sealing effect on tooth damages is amorphous polyP particles as fillers for caries lesions can be
observed both on the enamel (covering carious lesions) and on anticipated
the dentin (closing of the dentinal tubules). As shown before,
these polyP-mediated deposits are sustainable and resistant
to ultrasonication [26]. Acknowledgements
In the focus of the present study is the observation that
polyP, applied as aCa-polyP-MP, induces a regeneration min- W.E.G.M. is the holder of an ERC Advanced Investigator Grant
eralic zone on the surface of the enamel, as visualized by (Grant No. 268476) and has received three ERC-PoC grants (Si-
SRXTM. The polyP particles embedded in a glycerol solvent Bone, Grant No. 324564; MorphoVES-PoC, Grant No. 662486;
initiate this induced mineralization process. While the polyP- and ArthroDUR Grant No. 767234). In addition, this work was
lacking glycerol samples do not have a promoting effect on supported by a grant from the Federal Ministry for Educa-
mineralization process during incubation with artificial saliva, tion and Research (NanoOsMed; Grant No. 01DH17034A), the
the polyP-glycerol-treated enamel samples are covered with a BiomaTiCS research initiative of the University Medical Center
≈20 ␮m thick “induced mineral deposit”. This result supports Mainz and the International Human Frontier Science Program.
the view that polyP accelerates the natural maturation phases
of HA derived from ACP [90].
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Please cite this article in press as: Ackermann M, et al. Biomimetic transformation of polyphosphate microparticles during restoration of
damaged teeth. Dent Mater (2018), https://doi.org/10.1016/j.dental.2018.11.014