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Journal of the Formosan Medical Association xxx (xxxx) xxx

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Original Article

Crystal growth in dentinal tubules with bio-

calcium carbonate-silica sourced from
equisetum grass
Hao-Hueng Chang a, Chun-Liang Yeh a, Yin-Lin Wang a,
Guan-Wen Liu b, Hong-Ping Lin b,**, Chun-Pin Lin a,*

a
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, No. 1,
Changde Street, Taipei, 10048, Taiwan
b
Department of Chemistry, National Cheng Kung University, No.1, Daxue Rd., Tainan City, 701, Taiwan

Received 15 January 2020; received in revised form 19 February 2020; accepted 23 February 2020

KEYWORDS Background/Purpose: One effective way to deal with dentin hypersensitivity is to develop ma-
Biocompatible terials to seal the tubules. The porous bio-calcium carbonate-silica (BCCS) contained well-
materials; dispersed CaCO3 would form calcium phosphates to seal the dentinal tubules when mixed with
Dental seal; an acidic solution. The acidic hydrothermal treatment and calcination to isolate the BCCS from
Dentin sensitivity; the agricultural waste like equisetum grass was used, which would be more environmentally
Dentin permeability; friendly than chemically synthesized mesoporous biomaterials. The aim of this study was to
Green chemistry develop mesoporous materials from natural resources to occlude the dentinal tubules which
technology could be more environmentally-friendly.
Methods: Dentin disc samples were prepared and treated with different methods as follows:
(1) BCCS mixed with H3PO4; (2) BCCS mixed with KH2PO4; (3) Seal & Protect
was used as a
comparison group. Sealing efficacy was evaluated by measuring the depths and percentages
of precipitate occlusion in dentinal tubules with SEM.
Results: The N2 adsorptionedesorption isotherm of the BCCS demonstrated a pore size of
around 15.0 nm and a surface area of 61 m2g-1. From the results of occlusion percentage
and depth, the BCCS treated with H3PO4 or KH2PO4 demonstrated promising sealing efficacy
than the commercial product.
Conclusion: This synthetic process used the agricultural waste equisetum grass to produce bio-
calcium carbonate-silica would be environmentally friendly, which has great potential in treat-
ing exposed dentin related diseases.

* Corresponding author. Department of Dentistry, National Taiwan University Hospital, No.1, Changde Street, Taipei, 10048, Taiwan.
Fax: þ886 2 23831346.
** Corresponding author.
E-mail addresses: hplin@mail.ncku.edu.tw (H.-P. Lin), chunpinlin@gmail.com (C.-P. Lin).

https://doi.org/10.1016/j.jfma.2020.02.010
0929-6646/Copyright ª 2020, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Chang H-H et al., Crystal growth in dentinal tubules with bio-calcium carbonate-silica sourced from equisetum
grass, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.02.010
 + MODEL
2 H.-H. Chang et al.
Copyright ª 2020, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Introduction
Dentin hypersensitivity is a common clinical condition that
can be characterized by a short, sharp pain caused by
exposed dentinal surface in response to stimuli, typically
thermal, evaporative, tactile, osmotic, or chemical. Based
on Brannstrom’s hydrodynamic theory, the occurrence of
pain could be explained by the fluid movement within the
dentinal tubules.1,2 The reduction of dentin permeability
could be achieved by the occlusion of tubules, which is quite
effective for treating dentin hypersensitivity.3e5 The current
treatments for dentinal tubule occlusion include the use of
strontium chloride,6 potassium oxalate,7 iontophoresis/so-
dium fluoride, dentin bonding agents,8,9 glass-ionomer
cement, and laser treatment.10 The dentinal permeation
and demineralization can be reduced by sealing or occluding
the exposed dentinal tubules, but the effects are not long-
standing.11 Recently, a number of home-use dental care
commercial products containing arginine and calcium car-
bonate, have claimed to occlude exposed dentinal
tubules.12e14 Unfortunately, the penetration depth of the
precipitation into the dentinal tubules is less than 10 mm,
which may not combat the erosion and tooth wear resulting
from chewing, brushing, and mechanical force.
The currently in-office or home care products could not
withstand the stresses of the oral environment and then
degrade over time.15,16 It is desirable to develop materials
that are efficient and enduring for dentinal tubule occlu-
sion.17 Several commercial products have been proven to
be effective in clinical performance for tooth desensitiza-
tion, but with limited durations.18 Our previous studies
have shown that a calcium carbonate@mesoporous silica
biomaterial (CCMS) mixed with 30% phosphoric acid can
produce dicalcium phosphate dihydrate (DCPD), tricalcium
phosphate (TCP) or hydroxyapatite (HAp) to occlude
dentinal tubules to a depth of around 40 mm.19 Although the
biocompatibility of CCMS paste was highly advantageous,
the complexity of the synthesis process could limit the
practicality of CCMS.
The porous material can be isolated from the plant as a
raw material to obtain a micron or nanoscale material. Eq-
uisetum is an ancient hollow plant characterized by a rela-
tively high amount of silica, up to 25% by dry weight.20 The
components of silica are mainly distributed in the epidermis
layer and exist in an amorphous form. It creates a so-called
silica/biopolymer nanocomposite with organic components.
Biogenic silica can be obtained by some means to remove
organic substances. Calcination can be used to remove
organic substances, but when the temperature exceeds

750 C, silica may be converted into a crystalline form that
lacks a porous structure. Sapei et al. found that the struc-
ture of porous silica could be obtained by treating firstly with
hydrochloric acid, then performing calcination.21 However,
Please cite this article as: Chang H-H et al., Crystal growth in dentinal tubules with bio-calcium carbonate-silica sourced from equisetum
grass, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.02.010
the calcination temperature must be carefully controlled to
avoid a severe shrinkage of the pore structure and phase
transformation in the silica. In addition to silica, equisetum
is also high in calcium content, which can reach up to a
quarter of the silica content. By using a hydrothermal
treatment to retain the calcium component, the porous sil-
ica structure could be obtained from equisetum. This ma-
terial with a similar porous matrix for well-dispersion of the
calcium component therefore has great potential to be used
for the sealing of dentinal tubules.
The aim of the study was to report a simple and green
method via an oxalic acid-hydrothermal treatment and
calcination to isolate bio-calcium carbonate-silica (BCCS)
with high calcium content from the equisetum grass. In the
BCCS, the CaCO3 composition dispersed well within the
porous silica matrix. When mixed with KH2PO4 solution, the
BCCS-phosphate paste (denoted as BCCS-P) was formed to
create a supersaturated environment of calcium and
phosphate ions on the exposed dentin surface as effective
as that of CCMS and phosphoric acid.22 Consequently, the
calcium phosphate crystals grew heterogeneously in an
efficient manner within open dentinal tubules to form an
integrated protective barrier.
Materials and methods
Preparation of BCCS
Equisetum grass harvested in Taiwan was employed as a raw
material. 7.0 g of equisetum grass was hydrothermally
treated in 300 mL of oxalic acid solution at pH of 2.5 and
100  C for 24 h to form CaC2O4 precipitates wherein un-
desired metal ions needed to be removed. After acidic
hydrothermal treatment, the equisetum grass was filtrated
and dried. After calcination at 600  C for 3 h in the air to
remove the organic components and thermally decomposed
CaC2O4 to CaCO3, the porous bio-calcium carbonate-silica
was generated.
Characterization
Scanning electron microscopy (SEM)/energy dispersive X-
ray (EDX) elemental mapping and quantitative analysis of
elements in the BCCS were taken on TM3000 (Hitachi,
Tokyo, Japan) with Bruker XFlash 5060FQ detector oper-
ated at 10 kV. The N2 adsorptionedesorption isotherm was
obtained at 77 K on a Micromeritics ASAP 2020 apparatus
(Micromeritics Instrument Corporation, GA, USA) to esti-
mate the pore size and surface area. The phase of crystal
formation of BCCS and BCCS-P were determined by a
powder X-ray diffraction (XRD) using Cu Ka radiation at
2q Z 10 to 70 .
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Crystal growth in dentinal tubules from equisetum grass
Sealing efficacy in dentinal tubules
With the NTUH IRB (National Taiwan University Hospital
Institutional Review Board) approval and informed patient
consent, a total of 12 intact human molars were collected
and stored at 4  C of 0.4% thymol solution (w/v). All human
teeth used in this study were obtained from 20 to 25 years
old patient for wisdom tooth extraction after their
approval, and informed consent was signed. A precision
diamond saw (ISOMET
2000, BUEHLER
LTD., Lake Bluff,
IL, USA) was used to expose the flat dentin surface via
horizontal cuts perpendicular to the long axis of the tooth
following embedded in resin, therefore 1-mm-thick dentin
discs were obtained (Fig. 1). All discs were pretreated with
37 wt.% phosphoric acid for 20 s to expose the opening of
the dentinal tubules before surface treatment. The dentin
discs were divided into four groups. Treatments of the
dentin discs with different methods were as follows: (1)
0.1 g of BCCS was mixed with 0.68 mL of 37 wt.% H3PO4
solution to form a paste with a pH value of about 2.0
(denoted as BCCS-P2.0). This paste was immediately
applied to 1 mm-thick dentin discs using a micro-brush,
then rinsed the dentin surfaces with distilled water after
10 min; (2) 0.1 g of BCCS was mixed with the KH2PO4 so-
lution (0.42 g KH2PO4 and 0.83 g of water) to form a paste
with a pH value of about 4.0 (denoted as BCCS-P4.0). This
paste was immediately applied to dentin discs using a
micro-brush, then rinsed the dentin surfaces with distilled
water after 10 min; (3) Seal & Protect
(Dentsply DeTrey,
Konstanz, Germany) was applied to dentin discs according
to the manual instructions. Treated dentin discs were
stored for 24 h at 37  C with 100% relative humidity. Dentin
discs were split following different treatments. Split dentin
discs were used for studying the depth of crystallization in
the dentinal tubules (Fig. 1). The depths and percentages
of precipitate occlusion in dentinal tubules were examined
by SEM. It was analyzed with Image J software (NIH, USA).
We examined three areas randomly in each sample and
averaged the obtained occlusion depths and percentages
(n Z 3). Statistical comparisons of the obtained occlusion
depths and percentages were performed using one-way
Fig. 1 Preparation dentin disc for studying the depth of
crystallization and sealing efficacy.
Please cite this article as: Chang H-H et al., Crystal growth in dentinal tubules with bio-calcium carbonate-silica sourced from equisetum
grass, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.02.010
3
ANOVA and post hoc Tukey’s test, with the statistical sig-
nificance set as a Z 0.05.
Results
SEM and EDX examination
From the EDX analysis (Fig. 2a), the major inorganic ele-
ments in BCCS were silicon and calcium (the element per-
centage of Ca and Si is about 97%), which demonstrated the
effectiveness in removing other metal ions from equisetum
grass using oxalic acid. The Ca/Si ratio in the resulted BCCS
was about 0.25. The EDX element mapping in the BCCS
revealed that Ca elements were well-dispersed in the silica
matrix (Fig. 2bed). Fig. 3 shows the XRD patterns of the
BCCS and BCCS-P paste. The BCCS (Fig. 3a) was composed
of mainly amorphous silica and calcium carbonate, which
presented significant characteristic peak at 2q Z 22.7
accompanying with other peaks at 2q Z 29.4 and 39.5 ,
and 48.5 .
N2 adsorptionedesorption isotherm
To measure the porosity and surface area of a material, the
N2 adsorptionedesorption isotherm was typically used. The
BBCS showed a hysteresis loop at a relative pressure (p/p0)
around 0.7 to 0.9 (Fig. 4a), which indicated the presence of
mesoporosity in the BCCS. The pore size calculated using
the BJH (Barrett, Joyner, and Halenda) method was around
15 nm, and the high specific surface area was 61 m2g-1. The
pore size distribution obtained from the BJH analysis of the
porous bio-calcium carbonate-silica centered at around
10.0 nm. This result indicates that the porous bio-calcium
carbonate-silica is ascribed to the mesoporous materials
(Fig. 4b).
Sealing efficacy in dentinal tubules
The dentin surface treated with the BCCS-P2.0 showed
prominent crystal growth in the dentinal tubules (Fig. 5).
Quantitative measurement of the sealing efficacy indicated
a significantly higher efficiency of the BCCS-P2.0 paste in
occluding the dentinal tubules. The occlusion percentage
was up to 92.1  3.8%. The depth of occlusion was
125  31 mm. When the acidity of the BCCS-P4.0, the oc-
clusion percentage dropped to 86  4.2% (p > 0.05), and
the depth was about 105  40 mm. Seal & Protect
pro-
duced a coated layer of about 10 mm in thickness only on
the dentin surface, with a sealing rate of 100%, but without
occlusion depth.
Discussion
Equisetum grass is a kind of wild plant that the major
inorganic components are silica, calcium, and other metal
ions (e.g., sodium, potassium, magnesium, and transition
metals). Therefore, a hydrothermal treatment in oxalic
acid solution was needed to remove other alkaline and
transition metal ions except for the calcium ion. In
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4 H.-H. Chang et al.

Fig. 2 Elemental composition analysis of the bio-calcium carbonate-silica (BCCS) isolated from equisetum, (a) EDX spectrum, (b)
SEM/EDX elemental mapping image of Ca & Si, (c) SEM/EDX elemental mapping image of Ca, (d) SEM/EDX elemental mapping image
of Si. Red Z [Ca], green Z [Si].

(eq. (1)) which maintained Ca content in the silica struc-

ture. After the calcination process at 600  C, the CaC2O4
released CO to form CaCO3 (eq. (2)). The equations are
shown as follows:

Ca2þ þ C2 O2
4 /CaC2 O4 ð1Þ

D
CaC2 O4 /CaCO3 þ CO ð2Þ

CaCO3ðsÞ þ 2H3 PO4ðaqÞ /Caþ 

2ðaqÞ þ 2H2 PO4ðaqÞ þ CO2ðgÞ þ H2 OðlÞ

ð3Þ

After mixing with the acidic KH2PO4 solution, CO2 was

Fig. 3 The XRD patterns of the materials, (a) the BCCS
powder indicating the amorphous structure and calcium car-
bonate; (b) the BCCS-P paste indicating the formation of
dicalcium phosphate dihydrate (DCPD) and KH2PO4. (C) DCPD,
CaHPO4$2H2O; (:) KH2PO4; (B) CaCO3.
addition, the Ca elements tended to distribute in the cen-
tral parts of the calcined equisetum grass.
Due to the high porosity, large surface area, and well
dispersion of CaCO3, the BCCS reacted with the H3PO4 or
KH2PO4 solution to form a paste containing plenty of Ca2þ
and HPO24 ions. Additionally, the high surface area of the
silica matrix could prevent the rapid precipitation of cal-
cium phosphate on the dentin surface to inhibit the pene-
tration of the solution into the open dentin tubules.
During the hydrothermal treatment in oxalic acid solu-
tion (H2C2O4), the C2O2 4 only precipitated with the
released Ca2þ ions from equisetum grass to form CaC2O4
released, and the BCCS-P paste was formed (eq. (3)). The
major crystal compound in the BCCS-P paste was DCPD
(CaHPO4$2H2O) with characteristic peaks at 2q Z 11.6 ,
21.0 , and 29.3 (Fig. 3).
When dentinal tubules are infected by bacteria, it could
cause dental caries, pulp diseases, and result in the
development of dentin hypersensitivity.23 Dentinal hyper-
sensitivity could be reduced by occlusion of dentinal tu-
bules or decreasing the permeability of dentinal tubules to
prevent the bacteria invasion. Bacterial invasion of the
dentinal tubules may induce the recurrence of periradicular
lesions and late failure of endodontic treatment.24,25
Several studies have used the calcium phosphates to block
dentinal tubules due to their similarities to the major
component of dentin.26e30 Disodium phosphate with calcium
chloride solution was applied on the surface of dentin to
obtain the accelerated precipitation of calcium phosphate
resulted in prompt relief from dental hypersensitivity.26
However, the crystals only formed on the surface but did not

Please cite this article as: Chang H-H et al., Crystal growth in dentinal tubules with bio-calcium carbonate-silica sourced from equisetum
grass, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.02.010
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Crystal growth in dentinal tubules from equisetum grass 5

Fig. 4 (a) Nitrogen adsorptionedesorption of the BCCS. Analyzed with the Barret-Joyner-Halenda method, the pore size is around
15 nm, and the area is 61 m2/g. (b) The pore size distribution obtained from the BJH analysis of the porous bio-calcium carbonate-
silica centered at around 10.0 nm. This result indicates that the porous bio-calcium carbonate-silica is ascribe to the mesoporous
materials.

Fig. 5 SEM micrographs of dentinal tubules treated with (a, b) the BCCS-P2.0; (c, d) the BCCS-P4.0; (e, f) Seal & Protect
.

Please cite this article as: Chang H-H et al., Crystal growth in dentinal tubules with bio-calcium carbonate-silica sourced from equisetum
grass, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.02.010
 + MODEL
6 H.-H. Chang et al.

Fig. 6 The crystallization mechanism of the BCCS-P paste in the dentinal tubules. The cube is represented as BCCS. When BCCS is
mixed with potassium dihydrogen phosphate, calcium ions are released from the BCCS, and phosphate ions (including HPO2- -
4 , H2PO4)
act to form a precipitate of calcium phosphate to seal the dentinal tubules. (green spots: calcium ions; red spots: phosphate ions).

grow in the dentinal tubules to become a sustained occlusion.
In order to prevent the fast precipitation on the dentin surface
from occluding the dentinal tubules, Ishikawa et al. proposed
to use a supersaturated acidic solution containing Ca2þ and
PO34 followed by neutralization to occlude the dentinal tu-
bules.27 The formation of apatite crystal only occluded the
exposed dentinal tubules to a depth in a range of 4e10 mm
which did not meet the requirement for clinical application. A
collagen/calcium dual-affinitive peptide and nano-
hydroxyapatite (nano-HAp) flocculi have been developed for
dentinal tubule occlusion.30 However, the penetration of the
collagen/calcium dual-affinitive peptide into the dentin
collagen matrix did not reach the promising depth, and it did
not form a deep occlusion of HAp precipitation in the tubules
either. The shallow depositions on the dentin surface would
not be able to provide a promising and efficient occlusion for a
positive clinical outcome.19
In the present study, the porous bio-calcium carbonate-
silica (BCCS) isolated from equisetum grass contained a
well-dispersed CaCO3 component. When mixed with the
H3PO4 solution, the porous BCCS with a high surface-to-
volume ratio could be quickly wetted and released plenty
of calcium and dihydrogen phosphate (H2POe 4 ) ions in the
paste at pH 2.0. After applying the paste on the dentin
tubules, the calcium and dihydrogen phosphate (H2POe 4)
ions could penetrate into the open dentin tubules. With the
pH value of the dentin disk being close to neutral, the
acidity of the penetrating solution of calcium and dihy-
drogen phosphate (H2POe 4 ) ions should also increase. It is an
established fact that Ksp values of calcium phosphates
decrease with the increase of the pH value. As the pH value
increases, the original solution of the calcium and dihy-
drogen phosphate (H2POe 4 ) ions becomes supersaturated.
The resulting calcium phosphates tend to heterogeneously
crystalize on the dentin tubules.19
By increasing the reaction time to 24 h, the calcium
phosphate crystals could seal almost all open dentin tubules.
However, the high acidity of the BCCS-P2.0 tended to cause
a detrimental erosive effect. In order to reduce the extent
of erosion, the BCCS-P4.0 was used. The occlusion depth and
percentage results showed that the BCCS-P4.0 still had a
high performance for the precipitation in the dentin tubules.
Unlike the BCCS-P2.0, there was still a tiny gap between the
precipitate and the wall of the dentinal tubules. The results
showed that in an acidic environment, BCCS-P had better
sealing properties for dentinal tubules but a higher accom-
panying erosive effect. When mixed with the KH2PO4 solu-
tion, the BCCS-P was formed to create a supersaturated
environment of calcium and phosphate ions on the exposed
dentin surface as effective as that of CCMS and phosphoric
acid.22 The high surface area of BCCS could also prevent fast
precipitation of DCPD on the dentin surface, which allowed
deeper penetration of the Ca2þ and HPO2 4 ions into the
dentinal tubules. With deeper penetration of the solution,
the length of the DCPD precipitate became longer. The
crystallization mechanism of the BCCD-P paste in the
dentinal tubules is illustrated in Fig. 6. We have investigated
and proposed the heterogenous-nucleation mechanism of
sealing dental tubule based on findings in crystallog-
raphy.19,22 Previous studies on crystallography proved the
supersaturation of solutions is crucial to overcome the
challenges associated with the permeation of ions into the
tiny dental tubules by precipitation of crystallites. Meso-
porous silicas own unique characteristics such as well-
defined structures, high surface areas, tunable pore sizes,
hydrocarbon sorption efficacy, and high thermal/hydrother-
mal stability; these materials are well suited for use as a
catalyst or support in electronic and optical materials, sep-
aration and sorption processes, and catalytic reactions. Our
previous findings have demonstrated the formation of DCPD
crystals can tightly occlude the dentine tubules and drove
depth of approximately 100 mm. Our previous investigation,
therefore, has shown that lattice matching of the DCPD- or
HA-like precipitates, rapid occlusion of the dentine tubules
proceeds via a heterogenous-nucleation mechanism rather
than via a homogenous-nucleation mechanism with higher
activation energy.19
In this study, a simple and green process to isolate
porous BCCS from equisetum grass had been provided.
Mixing the porous BCCS with H3PO4 or KH2PO4 solution
allowed the fast dissolution of the CaCO3 and produced a
paste consisting of a large quantity of calcium and

Please cite this article as: Chang H-H et al., Crystal growth in dentinal tubules with bio-calcium carbonate-silica sourced from equisetum
grass, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.02.010
 + MODEL
Crystal growth in dentinal tubules from equisetum grass
phosphoric ions. In the presence of a high surface area
silica matrix, the calcium and phosphoric ions in the paste
tended to promote deeper precipitation of crystals in the
dentinal tubules rather than precipitation on the dentin
surface. The resulting calcium-phosphate precipitation
with a length of one hundred micrometers in the dentinal
tubules could protect the exposed dentin and provide
promising occlusion percentage for dentinal tubules.
The porous materials could be obtained from natural
resources, and the synthetic process, which includes the
use of agricultural waste equisetum grass and non-toxic
oxalic acid for acidic hydrothermal treatment, could be a
more cost-effective and environmentally-friendly way to
produce bio-calcium carbonate-silica with great potential
in treating exposed dentin related diseases.
Declaration of Competing Interest
The authors have no conflicts of interest relevant to this
article.
Acknowledgements
This work was financially supported by the “Advanced
Research Center for Green Materials Science and Technol-
ogy” from The Featured Area Research Center Program
within the framework of the Higher Education Sprout
Project by the Ministry of Education (107L9006) and the
Ministry of Science and Technology in Taiwan (MOST107-
3017-F-002-001 and MOST 108-2622-B-002-002 -CC1) and
from National Taiwan University Hospital (NTUH 108-
004438).
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