Regenerative Endodontics

Enhanced Capability of Bone Morphogenetic

Protein 2–loaded Mesoporous Calcium Silicate
Scaffolds to Induce Odontogenic Differentiation
of Human Dental Pulp Cells
Kuo-Hao Huang, DDS, PhD,*† Yi-Wen Chen, PhD,‡§ Chen-Ying Wang, DDS, PhD,†
Yen-Hong Lin, MS,k¶ Yuan-Haw Andrew Wu, MD,k Ming-You Shie, PhD,k#** and
Chun-Pin Lin, DDS, PhD*†††

Abstract
Introduction: Calcium silicate bioceramics have been
broadly used as reparative or grafting materials with
good bioactivity and biocompatibility in dental applica-
tion. It has been shown that applying a mesoporous pro-
cess to calcium silicate gives it great potential as a
controlled drug delivery system. Methods: The aim of
this study was to investigate a novel osteoinductive
scaffold by loading bone morphogenetic protein 2
(BMP-2) to mesoporous calcium silicate (MesoCS) and
fabricating it as 3-dimensional scaffolds using fused
deposition modeling combined with polycaprolactone.
Results: The MesoCS/BMP-2 scaffold showed similar
patterns to that of a calcium silicate scaffold in releasing
calcium and silicon ions in a simulated body fluid (SBF)
immersion test for 7 days, but BMP-2 continued
releasing from the MesoCS/BMP-2 scaffold significantly
more than the CS scaffold from 48 hours to 7 days.
Adhesion and proliferation of human dental pulp cells
cultured on a MesoCS/BMP-2 scaffold were also more
significant than scaffolds without BMP-2 or mesoporous
as well as the results of the test on alkaline phosphatase
activity. Conclusions: The results support that the novel
3-dimensional–printed MesoCS scaffold performed well
as BMP-2 delivery system and would be an ideal odon-
toinductive biomaterial in regenerative endodontics. (J
Endod 2018;44:1677–1685)
Key Words
3D-printed scaffold, bone morphogenetic protein 2,
dental pulp cell, mesoporous calcium silicate, odonto-
genesis
F or therapy of an imma-
ture permanent tooth
with necrotic pulp, regen-
Significance
BMP-loaded mesoporous calcium silicate scaf-
folds were prepared by 3-dimensional printing
erative endodontics has
and were used as a drug carrier to release BMP-2.
become well developed in
The BMP-2–loaded MesoCS scaffold showed
recent years. This tech-
up-regulation of the odontogenic-related protein
nique includes removing
of hDPCs.
necrotic tissues, inducing
stem cells from apical
papilla by a revascularization procedure, and applying bioceramic repairing materials
(1). Calcium silicate (CS)-based bioceramics such as mineral trioxide aggregate have
been used in endodontic therapy for 20 years with trusted results (2–5). These
materials performed well in space maintenance with biocompatibility as a restorative
or grafting material. Numerous studies have reported successful odontoinductive and
osteoinductive processes during pulp and bone healing stimulated by CS-based mate-
rials (6, 7). The Si ions released from materials could promote odontogenesis and
angiogenesis of human dental pulp cells (hDPCs), cementogenesis of human
periodontal ligament cells, and proangiogenesis of human umbilical vein endothelial
cells, which are beneficial to hard tissue regeneration (8–10). However, dentin
regeneration is related to stem cells, morphogens, and scaffolds. The main
disadvantages of the current technique are the lack of designed scaffolds for hosting
stem cells in blood clots or releasing of growth factors and that bioceramics only
served as biocompatible restorative materials. In addition, the particle size of
traditional CS powder is usually at the micrometer level, making it difficult to inject,
and the solid powder is not favorable for drug delivery (11). Therefore, we prepared
CS with a mesoporous structure with pores in diameter of approximately 10 nm (9).
Bone morphogenetic proteins (BMPs) are a group of growth factors found during
the development of bone and cartilage (12). It has been shown that the progenitor cells
in dental pulp could differentiate into odontoblasts in response to BMPs. Recombinant
human bone morphogenetic protein 2 (BMP-2) is 1 of the growth factors approved by
the Food and Drug Administration in orthopedic and dental surgery for more than a
decade (13). The great capacity of BMP-2 combined with carriers or scaffolds to induce

From the *Graduate Institute of Clinical Dentistry, School of Dentistry, †Department of Dentistry, and ††Advanced Research Center for Green Materials Science and
Technology, National Taiwan University Hospital, Taipei, Taiwan; ‡Graduate Institute of Biomedical Sciences, ¶PhD Program for Medical Engineering and Rehabilitation
Science, #School of Dentistry, China Medical University, k3D Printing Medical Research Center, China Medical University Hospital, Taichung, Taiwan; §3D Printing Medical
Research Institute, Asia University, and **Department of Bioinformatics and Medical Engineering, Asia University, Taichung, Taiwan.
Ming-You Shie and Chun-Pin Lin contributed equally to this study.
Address requests for reprints to Dr Chun-Pin Lin, School of Dentistry, National Taiwan University, Taipei, Taiwan. E-mail address: chunpinlin@gmail.com
0099-2399/$ - see front matter
Copyright ª 2018 American Association of Endodontists.
https://doi.org/10.1016/j.joen.2018.08.008

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Regenerative Endodontics
odontoblast or osteoblast development has been broadly investigated in
various animal study and clinical applications (14, 15). However, the
traditional method of depositing BMP-2 on material surface showed
an early burst release after implantation and attenuation in a short
time. Hence, the development on carriers or scaffolds became a major
issue for controlling the release of growth factors (16).
Three-dimensional (3D) printing techniques supply new strate-
gies for the rapid fabrication of the porous scaffolds that had been
widely used for bone tissue engineering in recent years (17–19).
Therefore, 3D printing now can be easily fabricated into various
versatile, solid, free-form structures, providing unprecedented flexi-
bility in both material and geometry, thus creating a potential way to
manufacture customized 3D scaffolds for tissues regeneration (20–
22). Moreover, some clinical cases reported the successful use of a
3D-printed scaffold for treatment (17, 23). Polycaprolactone (PCL)
has already been approved for several medical and drug delivery
devices; it generally takes 6 months to 2 years to degrade in vivo
depending on its molecular weight. PCL-contained composites can be
melted and extruded in a computer-controlled pattern to construct scaf-
folds layer by layer by extrusion-based 3D printing (24–26). The
cytocompatibility of PCL scaffolds for dental pulp stem cells was also
tested in a previous study (26). Ceramic/PCL composites can also be
produced via 3D printing by dispersing ingredients as the second phase
in a polymer matrix via high heat or solvents (27). Several studies indi-
cated the denaturation of the drug or growth factor as well as aggrega-
tion and phase separation of additives during blending, which
compromise both the efficacy and consistency of functions. Therefore,
the use of low-temperature mixing is preferred but typically involves a
compromise between the maximum loading ability and homogeneity of
dispersion (28).
Our previous study has shown that mesoporous CS nanoparticles
possess excellent ability in releasing drugs and growth factors (9).
Therefore, we assume that mesoporous CS nanoparticles may also be
used to fabricate 3D scaffolds loaded with BMP-2, which supplies a suit-
able microenvironment for the odontogenic differentiation of hDPCs
and has drug-releasing properties. In this study, the mesoporous CS
nanoparticle–based 3D scaffolds were manufactured, and the cell
behavior and drug-releasing profile were observed.
Materials and Methods
Synthesis and Characterization of Mesoporous CS
Nanoparticles
Mesoporous CS (MesoCS) nanoparticles were prepared using a
template method that has been described elsewhere (9). Briefly,
3.3 g cetyltrimethylammonium bromide (CTAB; Sigma-Aldrich, St Louis,
MO) and 6 mL NH3  H2O were mixed in double-distilled water (ddH2O,
300 mL) and then stirred for 15 minutes at 60 C. Next, 15 mL tetraethyl
orthosilicate (Sigma-Aldrich) and 15.6 g calcium nitrate were added
with vigorous stirring for 3 hours. The precipitate products were
then collected by filtration and washed 3 times, each with 1 N hydro-
chloric acid and ethanol. After this, the collected powders were dried
at 60 C overnight and sintered at 800 C for 2 hours to remove the re-
maining traces of CTAB. As for the preparation of the solid CS powder,
the same process was performed without the addition of CTAB. The
mesoporous structure of particles was characterized using transmission
electron microscopy.
BMP-2 Loading
The BMP-2–loaded materials were prepared by immersing Mes-
oCS or CS powder in BMP-2 (MP Biomedicals, Solon, OH) solution
(0.5 mg/mL). This compound was then shaken for 24 hours at room
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temperature. The BMP-2–loaded materials were then separated using
centrifugation at 13,000 rpm for 15 minutes to remove unloaded
BMP-2. The BMP-2–loaded materials were then washed with ddH2O
several times and freeze-dried for 24 hours. The powder was placed
in the vacuum at 4 C.
Preparation of Scaffolds
A composite matrix was produced using the thermal pressing
method (29). First, reagent-grade PCL (molecular weight = 43,000–
50,000; Polysciences, Warrington, PA) was placed in a 100 C oven
for 2 hours. Then, the ceramic powder with or without BMP-2 loading
was suspended in absolute alcohol and dripped into PCL. The compos-
ite was then placed in an 80 C oven for 2 hours. The 3D printing of this
device was achieved through a precision 3-axis positioning system (Bio-
Scaffolder 3.1; GeSiM, Grosserkmannsdorf, Germany). In brief, the
printable composite pastes were loaded into a syringe and dispensed
through a steel nozzle at 90 C by applying a pressure of 400 kPa; seven
400-mm lines with a line height of 300 mm were printed parallel to each
other with a 600-mm gap in between every composite line. The 3D scaf-
fold was plotted layer by layer up to 16 layers through the extrusion of
the paste as a fiber.
Mechanical Properties of Scaffolds
The compressive strength of scaffolds was measured on an EZ Test
machine (Shimadzu, Kyoto, Japan) at a loading rate of 1 mm/min. The
maximal compression load producing breakage was obtained from the
recorded stress-strain curves. Six independent measurements were per-
formed, and the data were expressed as the mean  standard deviation.
In Vitro Soaking
The scaffolds were immersed in a simulated body fluid (SBF) so-
lution at 37 C. The SBF solution was similar to human blood plasma and
consisted of 7.9949 g NaCl, 0.2235 g KCl, 0.147 g K2HPO4, 0.3528 g
NaHCO3, 0.071 g Na2SO4, 0.2775 g CaCl2, and 0.305 g MgCl2  6H2O
in 1000 mL distilled H2O. The pH was adjusted to 7.4 with hydrochloric
acid and tris(hydroxymethyl)aminomethane. After immersion for
various time periods, the scaffolds were removed from SBF, and their
microstructure and strength were investigated. After immersion for
different time points, the Ca and Si ion concentrations released from
the specimens were analyzed using an inductively coupled plasma
atomic emission spectrometer (OPT 1 MA 3000DV; Perkin-Elmer, Shel-
ton, CT). Three samples were measured for each data point. The results
were obtained in triplicate from 3 separate samples for each test. The
specimens were coated with gold, and their morphologies were inves-
tigated under a scanning electron microscope (JSM-6700F; JEOL, To-
kyo, Japan) operated in the lower secondary electron image mode at
a 3-kV accelerating voltage.
BMP-2 Releases Kinetics
The drug-releasing kinetics of BMP-2–loaded scaffolds were
considered by soaking 150 mg of sample in 5 mL phosphate-buffered
saline (PBS) (pH = 7.4) at 37 C; fresh replacement PBS was substituted
for the old PBS at various time points. The release of BMP-2 was deter-
mined using an enzyme-linked immunosorbent assay kit (Invitrogen,
Carlsbad, CA) by following the manufacturer’s instruction kit. The con-
centration of BMP-2 was measured by comparing it with a standard
curve. Analysis of the blank scaffold was treated as a control. The prod-
ucts were determined using a multiwell spectrophotometer (Infinite Pro
M200; Tecan M€annedorf, Switzerland). All experiments were per-
formed in triplicate.
JOE — Volume 44, Number 11, November 2018

hDPC Isolation and Culture
hDPCs were freshly derived from caries-free, intact premolars that
had been extracted for orthodontic reasons as described previously
(30). Patients provided informed consent, and approval from the Ethics
Committee of the National Taiwan University Hospital, Taipei, Taiwan,
was obtained (no. 201612244RINC). After extraction, the tooth was
split sagittally with a chisel, and the pulp tissue was digested in PBS
(Caisson, North Logan, UT) solution containing 0.1% type I collagenase
(Sigma-Aldrich) for 30 minutes in the incubator. After being transferred
to a new plate, the cell suspension was cultured in Dulbecco modified
Eagle medium (DMEM, Caisson) supplemented with 20% fetal bovine
serum (GeneDireX, Taipei, Taiwan) and 1% penicillin (10,000 U/
mL)/streptomycin (10,000 mg/mL) (Caisson) in a humidified atmo-
sphere with 5% CO2 at 37 C; the medium was renewed every 2 days.
In the odontogenic differentiation assay, the medium was DMEM
supplemented with 108 mol/L dexamethasone (Sigma-Aldrich),
0.05 g/L L-Ascorbic acid (Sigma-Aldrich), and 2.16 g/L glycerol
2-phosphate disodium salt hydrate (Sigma-Aldrich).
Cell Adhesion and Proliferation
The 3D scaffolds were sterilized by soaking them in 75% ethanol
followed by directly exposing them to ultraviolet light for 20 minutes
before being used for cell cultures. After being directly cultured for
various time periods, cell viability was evaluated using the PrestoBlue
assay (Invitrogen), which detects mitochondrial activity. Thirty micro-
liters of PrestoBlue solution and 300 mL DMEM were added to each well
followed by 30 minutes of incubation. After incubation, 100 mL of the
solution in each well was transferred to a 96-well enzyme-linked immu-
nosorbent assay plate. The plates were analyzed in a multiwell spectro-
photometer at 570 nm with a reference wavelength of 600 nm. Cells
cultured on the tissue culture plate were used as a control.
Fluorescent Staining
After being cultured for 7 days, the specimens were washed with
cold PBS, fixed in 4% paraformaldehyde (Sigma-Aldrich) for 15 mi-
nutes, and then permeabilized with PBS containing 0.1% Triton X-
100 (Sigma-Aldrich) at room temperature. The F-actin filaments
were stained with phalloidin conjugated to Alexa Fluor 488 (Invitrogen)
for 1 hour. After washing, the morphology was obtained using a white
light laser confocal microscope (TCS SP8; Leica, Wetzlar, Germany).
Western Blot
After 1 day of culture, the cells on scaffolds were lysed with NP40
buffer (Thermo Fisher Scientific, Waltham, MA). The total protein
concentrations were determined using the BCA protein assay kit
(Bio-Rad Laboratories, CA). The cell lysates (40 mg protein) were sepa-
rated using sodium dodecyl sulfate polyacrylamide gel electrophoresis
and then transferred to polyvinylidene difluoride membranes (Millipore,
Burlington, MA). After blocking with 2% bovine serum albumin in tris-
buffered saline contained 0.1% Tween 20 for 1 hour, the membranes
were immunoblotted with primary anti-BMP2R, anti-pERK1/2, anti-
ERK1/2, anti-pSmad1/5, anti-Smad1/5, and beta-actin (GeneTex, San An-
tonio, TX) for 2 hours. The bands were then visualized after incubation
for 1 hour with horseradish peroxidase–conjugated secondary antibodies
by chemiluminescence using an ECL Detection Kit (Invitrogen). The
protein expression levels were normalized to beta-actin for each group.
Odontogenic Differentiation
The secretion of alkaline phosphatase (ALP) was determined on
days 3 and 7 after cell seeding on scaffolds. Briefly, the cells were lysed
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Regenerative Endodontics
using 0.2% NP40 and centrifuged at 6000 rpm for 15 minutes. ALP ac-
tivity was determined using p-nitrophenyl phosphate (Sigma-Aldrich)
as the substrate. Each sample was mixed with p-nitrophenyl phosphate
in 1 mol/L diethanolamine buffer. The reaction was stopped by the addi-
tion of 5 N NaOH and was quantified by a multiwell spectrophotometer
with absorbance at 405 nm. Osteocalcin (OC), dentin matrix protein 1,
and dentin sialophosphoprotein released from the hDPCs were cultured
on 3D scaffolds for 7 and 14 days. An enzyme-linked immunosorbent
assay kit (Invitrogen) was used to determine the protein content
following the manufacturer’s instructions. The concentration of OC pro-
teins was measured by comparing them with a standard curve. Analysis
of blank disks was treated as a control.
Calcium Deposition
The cells cultured on the scaffold surfaces for 7 and 14 days were
stained with alizarin red S to examine mineralized nodule formation and
calcium deposition (31). Briefly, the cells were fixed with 4% parafor-
maldehyde (Sigma-Aldrich). After 15 minutes, the cells were incubated
in 0.5% alizarin red S (Sigma-Aldrich) at a pH of 4.0 for 15 minutes at
room temperature. Then, the photographs of specimens were taken
with an optical microscope (BH2-UMA; Olympus, Tokyo, Japan) equip-
ped with a digital camera (Nikon, Tokyo, Japan) at a magnification of
200.
Statistical Analysis
One-way variance statistical analysis was used to evaluate the sig-
nificance of the differences between the means in the measured data.
The Scheffe multiple comparison test was used to determine the signif-
icance of the deviations in the data for each specimen. In all cases, the
results were considered statistically significant if the P value was <.05.
Results
Physicochemical Properties
Figure 1A shows the transmission electron microscopic micro-
graphs of MesoCS nanoparticles having an oval-like shape with a diam-
eter ranging from 70–100 nm, which is less than that of CS powder by
about 4 to 5 times. Mesoporous CS with a mesoscopic structure could
be observed and may bring a suitable environment for drug delivery
application. Homogeneous pore morphology and the porous structure
of CS and MesoCS scaffolds were well printed, with a 600-mm distance
between struts and a 400-mm strut width. The scaffold geometry was
designed as a shifted double-layer structure as can be seen from the op-
tical micrograph top view. Scanning electron microscopic images
showed a dense and uniform microstructure of the surface on both
the CS and MesoCS scaffolds. It is evident that some of the exposed par-
ticles on the surface were CS/mesoporous CS powders introduced into
PCL. After soaking in SBF for 1 day, both scaffolds presented apatite par-
ticles with an average particle size of about 250 nm uniformly deposited
on the surface. This result indicates that CS-based scaffolds have good
apatite formability, which can be expected in the bone biological activity
in vivo. Typical stress-strain profiles are also shown in Figure 1B.
Compression tests were performed on these composite scaffolds until
the specimen is compressed to about 20% of its deformation. The
compressive strength of CS and the MesoCS scaffold were 4.11 and
3.89 MPa, respectively. The compressive Young modulus of mesopo-
rous CS scaffolds was slightly increased when compared with that of
CS. However, there were no significant differences between the scaf-
folds.
BMP-2–loaded Mesoporous Calcium Silicate Scaffolds 1679
Regenerative Endodontics

Figure 1. (A) Transmission electron microscopic micrographs, overview graphs, and scanning electron microscopic micrographs of CS and MesoCS scaffold
before and after immersion in SBF. (B) The compressive strength of the composite scaffold. (C) Ca and Si ions and (D) BMP-2 release from an MesoCS/PCL
and CS/PCL after immersion in SBF at 37 C for different time points.

Ion and Drug Release
The change of Ca and Si ions in SBF with different immersion times
up to 7 days are shown in Figure 1C. The Si ions released from CS and
mesoporous CS rapidly increased within 12 hours of soaking in SBF.
After that, the release of Si ions slowed down. Still, the release of Si–
MesoCS blends reached 2.37 mmol/L on day 7, slightly higher than
the that observed in Si-CS blends (2.11 mmol/L). In contrast to Si,
the Ca ion concentration in SBF continuously decreased over 7 days
because of the formation of hydroxyapatite. The Si ion release from
the mesoporous CS scaffold showed faster release kinetics in compar-
ison with the CS scaffold after 72 hours and had a significant difference
(P < .05).
BMP-2–loaded CS and mesoporous CS were prepared by
immersing CS and mesoporous CS in BMP-2 solution to allow the
drug molecules to diffuse and adsorb onto the surface of the inner pores
of the inorganic particles. After that, the 3D scaffolds were printed using
the method described previously. The release profiles of the scaffolds
were conducted by measuring the amount of BMP-2 released in 37 C
PBS as a function of time (Fig. 1D). The results showed that the burst
release of BMP-2 was visible on the first 24 hours of the immersion
period for the CS scaffold and the MesoCS scaffold. There was 1.07
and 1.05 mg BMP-2 released from the CS scaffold and the mesoporous
CS scaffold after 24 hours, respectively. In addition, the release rate of
BMP-2 was decreased markedly in the CS scaffold group; the total
release amount of BMP-2 after 7 days was 1.28 mg. In contrast, the
amounts of BMP-2 released from the mesoporous CS scaffold on day
7 were approximately 2 times of that on day 1, implying that the sus-
tained release profile could be obtained in the mesoporous CS scaffold
acquired in the presence of mesopores, which appear to provide a
higher surface area for BMP-2 molecule adsorption.
Cell Adhesion and Proliferation
The adhesion, viability, and proliferation of hDPCs were consid-
ered after seeding on the surfaces of 4 different 3D scaffolds
(Fig. 2A). After culture for 4 hours, the results of the PrestoBlue assay
revealed that most cells on 4 scaffolds were viable, and the absorbance
was significantly higher (P < .05) compared with that of the normal tis-
sue culture plate (control). However, there was no significant difference
(P > .05) for hDPC adhesion on scaffolds with our without BMP-2. On
day 1s and 7, there were no differences in hDPC viability observed on the
mesoporous CS and CS scaffolds. As a result, mesoporous BMP-2–
loaded CS (CSB) showed the highest increase in cell proliferation after
1 and 7 days of culture. These results indicated that the addition of BMP-
2 had no positive effect on cell proliferation. At day 7, the cell prolifer-
ation of mesoporous CSB was significantly enhanced by 37%, 23%, and
30% compared with CS, CSB, and mesoporous CS, respectively. We also
used confocal microscopy to consider F-actin expression and to
observe cell proliferation after culturing for 7 days (Fig. 2B). This shows
that all scaffold groups can support initial cell adhesion and prolifera-
tion. In addition, F-actin expression was higher on CSB and mesoporous
CSB than the corresponding bare 3D ceramic scaffolds. Therefore,

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 Regenerative Endodontics

Figure 2. (A) Adhesion and proliferation of hDPCs cultured on BMP-2–loaded CS/PCL and MesoCS/PCL. *A significant difference (P < .05) from the other exam-
ined scaffolds. (B) Fluorescent images of cytoskeleton (green) staining in hDPCs cultured on various scaffolds for 7 days. Scale bar = 100 mm. Ctl, tissue cultured
plate; CS, calcium silicate/PCL scaffold; CSB, BMP-2–loaded calcium silicate/PCL scaffold; MesoCS, mesoporous calcium silicate/PCL scaffold; MesoCSB, BMP-2–
loaded mesoporous calcium silicate/PCL scaffold.
3D-printed CS scaffolds with BMP-2 loading enhanced cell behavior bet- shown that signaling transduction by BMP is through both
ter than bare 3D-printed CS scaffolds. Smad-dependent pathways and mitogen-activated protein kinase
(MAPK) signaling pathways. To investigate the role of BMP-2 in
affecting signaling in hDPCs, cells were cultured on CS and mes-
Signaling Pathway Analysis oporous CS scaffolds in the absence or presence of BMP-2. We
We knew that BMP signaling was involved in cell prolifera- performed immunodetection to examine the pathway signaling
tion, osteogenesis, and cell differentiation. Various studies have activation status of BMP2R, ERK, and SMAD1/5. We observed

Figure 3. (A) Immunodetection of BMP2R, ERK 1/2, p-ERK 1/2, SMAD 1/5, p-Smad 1/5, and beta-actin protein expression in hDPCs cultured with a BMP-2–
loaded scaffold for 1 day. Quantification of (B) BMP2R, (C) p-ERK 1/2, and (D) p-Smad1/5. *A a significant difference (P < .05) from the other examined scaffolds.
Ctl, tissue cultured plate; CS, calcium silicate/PCL scaffold; CSB, BMP-2–loaded calcium silicate/PCL scaffold; MesoCS, mesoporous calcium silicate/PCL scaffold;
MesoCSB, BMP-2–loaded mesoporous calcium silicate/PCL scaffold.

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Regenerative Endodontics
that BMP2R would be activated in a BMP-2–loaded scaffold
(Fig. 3A). In addition, strong activation of extracellular-signal-
regulated kinase (ERK)1/2 and drosophila mothers against de-
capentaplegic (SMAD)1/5 was observed. The relative protein
quantification levels are shown in Figure 3B–D. BMP-2 binding
to the BMP receptor would form heteromeric complexes and acti-
vate SMADs and the MAPK/ERK pathway by phosphorylation.
Therefore, the promotion effect of BMP-2–induced osteogenic ca-
pacity was via the SMAD pathway, which is the BMP canonical
signaling pathway.
Odontogenic Differentiation
The in vitro effect of the BMP-2–loaded ceramic scaffolds on os-
teogenesis differentiation was examined. ALP activity is known to be an
early marker for functionality during osteogenesis differentiation.

Figure 4. (A) ALP activity of hDPCs cultured on specimens for 3 and 7 days. (B) OC, (C) dentin matrix protein 1 (DMP-1), and (D) dentin sialophosphoprotein
concentration of hDPCs cultured on various scaffolds for 7 and 14 days. *A significant difference (P < .05) from other examined scaffolds. (E) Alizarin red S
staining of calcium mineral deposits of hDPCs cultured on different scaffolds for 7 and 14 days. The color of the alizarin red S staining ranged from light pink
to deep red, indicating the amounts of calcium mineral deposits of hDPCs was increased. Ctl, tissue cultured plate; CS, calcium silicate/PCL scaffold; CSB,
BMP-2–loaded calcium silicate/PCL scaffold; MesoCS, mesoporous calcium silicate/PCL scaffold; MesoCSB, BMP-2–loaded mesoporous calcium silicate/PCL scaf-
fold.

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Figure 4A shows the ALP activity of hDPCs after cultivating on a scaffold
without and with BMP-2 for 3 and 7 days. At day 3, the ALP activity of
BMP-2 loading was significantly higher than bare scaffolds (P < .05).
After 7 days, mesoporous CSB indicated the ALP activity was significantly
increased compared with other scaffolds. However, there was almost no
significant difference in ALP activities of hDPCs between BMP–loaded CS
and CS scaffold, indicating that BMP loading did not further promote
osteogenesis of the CS scaffold for long-term culture. The protein
expression levels of OC (Fig. 4B), dentin matrix protein 1 (Fig. 4C),
and dentin sialophosphoprotein (Fig. 4D), the biomarkers expressed
at the later stage of odontogenesis, at 7 and 14 days were analyzed using
the enzyme-linked immunosorbent assay. After culture for 7 and
14 days, the hDPCs cultured on mesoporous CSB showed significantly
higher (P < .05) odontogenic-related protein production than those of
CS, CSB, and mesoporous CS. This indicated that mesoporous CSB was
more beneficial than naked mesoporous CS scaffolds in inducing the
differentiation of hDPCs into osteoblasts and the promotion of cell
mineralization. Then, we considered the ability of hDPCs to deposit cal-
cium mineral nodules in vitro by alizarin red S staining in the presence
and absence of BMP-2 loading on CS and a mesoporous CS scaffold
(Fig. 4E). The color of the alizarin red S staining ranged from light to
deep pink, and the amounts of calcium mineral deposits increased
over time. At day 7, the hDPCs started to deposit small amounts of cal-
cium mineral nodules on CS and CSB, whereas more matrix deposition
and calcium mineral nodules were observed on mesoporous CS and
mesoporous CSB. At day 14, larger calcium mineral nodules were pre-
sent in BMP-2–loaded mesoporous CS compared with other scaffolds.
These data indicated that BMP-2–loaded mesoporous CS scaffolds
might stimulate mineralized nodule formation and calcium deposition.
Discussion
For the past few years, CS materials have attracted various attention
for their application in dental and bone regeneration topics because of
their excellent bioactivity and biocompatibility (29, 32). However,
conventional CS products are solid in physical property, making
them unsuitable for being used as drug delivery carriers (33). In the
previous study, we reported the successful synthesis of a MesoCS nano-
particle with superior textural properties and biocompatibility. More
specifically, the textural characteristics such as large surface area, uni-
form pore structure, and higher pore volume create a suitable platform
for excellent drug delivery and enhance the biomineralization process
(33). In addition, PCL was chosen as the binder for the fabrication of
ceramic paste, making the printable materials mechanically strong
and biodegradable.
The traditional mesoporous silica particles were synthesized
extensively for several biomedical applications because of their large
pore volume and tunable mesopore size (34). In our laboratory, we
synthesized MesoCS nanoparticles using the chemical precipitation
method that exhibited the ability to induce the formation of apatite
spherules (9). In the present study, we fabricated mesoporous CS scaf-
folds by 3D printing while avoiding the use of organic solvents. They
were covered with spherical aggregated minerals on CS and the meso-
porous CS scaffold after being immersed in SBF for 1 day. A previous
study showed that Ca ions released from CS-contained materials
possibly originated from less ordered hydration products that signifi-
cantly enhanced apatite formation by increasing local Ca concentration,
thereby raising the ionic product of the apatite in the surrounding envi-
ronment and enhancing the nucleation behavior of the apatite (31). The
main phenomenon could be attributed to the accessibility of nucleation
sites for the silicates (35). The loading efficiency of growth factors in
mesoporous nanoparticles is significantly better than that of conven-
JOE — Volume 44, Number 11, November 2018
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Regenerative Endodontics
tional nanoparticles because most proteins can be easily loaded into
the mesopores of the inner structure (36). The release profile clearly
indicates that the released BMP-2 from mesoporous CS scaffolds was
distinct compared with that of CS scaffolds. The BMP-2–loaded meso-
porous CS scaffold showed better cellular behavior than the CS scaffold.
Recently, the growth factor was incorporated into bioglass microparti-
cles and exhibited a slow release pattern from microparticles (37). The
observation of the proliferative potential of hDPCs reflects the biological
efficiency of BMP-2 release.
The BMP family is an important player in affecting odontoblast dif-
ferentiation in vitro and hard tissue regeneration in vivo (38, 39).
Most importantly, BMP-2 belongs to the transforming growth factor
family that has gained Food and Drug Administration approval for clin-
ical use, such as spinal fusion procedures, tibial shaft fracture treat-
ment, and oral and maxillofacial reconstruction (40). BMP-2
enhanced human dental pulp stem cell differentiation into odonto-
blast/osteoblast lineages and their formation of calcium deposits
(12). In addition, Nakashima (41) has shown that the progenitor cells
in dental pulp could differentiate into odontoblasts in response to BMPs
for hard tissue regeneration application. BMP-2 and its intracellular
transducers such as SMAD-related proteins serve as regulators of osteo-
genesis and odontogenesis (42). Recently, BMP-2 was incorporated
into bioglass microparticles and showed a slow release pattern of
growth factor from microparticles (37). The observation of the prolif-
erative potential of various cells was considered to reflect the biological
efficacy of the BMP-2 release. In addition, BMP-2 signals activated at the
cell membrane surface are transduced by the transcription factors of the
Smad family that directly regulate various gene expression and addi-
tional pathways such as MAPK and phosphoinositide 3-kinase (43).
On the other hand, a problem with this kind of administration is that
a large amount of BMP-2 protein was used because of its rapid
in vivo release (44). In a previous study, we not only showed that mes-
oporous CS nanoparticles promote hDPC behavior but also showed that
they prolong the growth factor releasing rate from mesoporous CS
nanoparticles compared with that of conventional CS cement (9). In
mesoporous CSB, greater amounts of p-ERK 1/2 and p-SMAD 1/5
were found in comparison with mesoporous CS after 1 day. To explain
the signaling molecules that may contribute to this effect, we proved that
the BMP-2 receptor and SMAD1/5 were constitutively phosphorylated in
mesoporous CS powder with or without BMP-2 loading, whereas the
activated BMP-2 receptor and phosphorylated SMAD1/5 were largely
dependent on BMP-2 released from ceramic scaffolds (45). In both
the CSB and mesoporous CSB groups, BMP-2 stimulation actually
increased the amount of p-ERK1/2 detected on Western blot analysis.
We have recently indicated that ERK1/2 down-regulation occurs in hu-
man mesenchymal stem cells undergoing odontogenic/osteogenic dif-
ferentiation (7). Our study has also previously reported that
fibroblast growth factor receptors/ERK activation promotes odonto-
genic/osteogenic differentiation of hDPCs in CS materials (46). In com-
bination, our current results and previous studies indicate that the
canonical BMPR signaling pathway is preferentially promoted during
odontogenic differentiation in 3D scaffold cultures across multiple pro-
genitor cell types (47, 48).
More recently, Tada et al (49) revealed increased messenger
RNA expression of BMP-2 via Ca channels and the ERK 1/2 pathway
in hDPCs, and this increase is modulated not only at a transcriptional
level but also at a posttranscriptional level. Our results confirmed that
BMP-2 release induces the protein expression of the BMP-2 receptor
in hDPCs and that SMAD1/5 is involved in inducing cell differentia-
tion. This phenomenon proved that the BMP-2 receptor/SMAD
pathway participates in the odontoblastic differentiation of hDPCs
but is not the only pathway involved in this process. The odontogenic
BMP-2–loaded Mesoporous Calcium Silicate Scaffolds 1683
Regenerative Endodontics
differentiation process is a multistep cascade of protein expression
initially supported by adhesion and proliferation (43). The early
important proliferative role of the ERK/MAPK and protein kinase B
pathway network could enhance the enlargement of the odontogenic
progenitor pool (42). In addition, the BMP-2–loaded mesoporous CS
scaffolds including mesoporous CS scaffolds promoted the activity of
hDPCs to a greater extent, and the results of ALP activity and alizarin
red S staining were similar to those found in BMP-2–loaded mesopo-
rous CS cement. Therefore, our results provide evidence proving that
BMP-2–loaded mesoporous CS scaffolds can increase ALP produc-
tion and mineralized nodule formation and may induce odontogenic
differentiation. Both BMP-2 and CS-based materials are widely
applied as regenerating biomaterials for tissue regeneration. We
believe that these growth factor–loaded ceramic 3D-printed scaffolds
could prove to be an innovative bioscaffold for the development of
hard tissue regeneration.
In summary, our data showed the combination of BMP-2 in mes-
oporous CS and loading them into 3D scaffolds can promote odonto-
genesis. The synergistic effect of these hard tissue growth factors in
combination with 3D-printed ceramic scaffolds has the potential to
be used for hard tissue formation. In future studies, we will consider
whether the sequence of BMP-2 delivery can be used to optimize the
level of new hard tissue regeneration. We believe that the BMP-2–loaded
mesoporous CS 3D scaffold could serve as an intracanal scaffold for
both the retaining blot clot and inducing odontogenesis in reparative
endodontic therapy.
Acknowledgments
Ming-You Shie and Chun-Pin Lin contributed equally to this
work.
Supported by the Advanced Research Center for Green Mate-
rials Science and Technology from the Featured Area Research Cen-
ter 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 (MOST 107-3017-F-002-001 and
107-2321-B-039-005).
The authors deny any conflicts of interest related to this study.
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