Keywords
osteoinduction - osteoconduction - regenerative endodontic
Introduction
Regenerative endodontic procedures are considered nowadays the ideal treatment for
necrotic immature permanent teeth. These procedures will allow for hard tissue formation
completing the root structure in length and thickness. Stem cells, growth factors,
and scaffolds are the three major components of regenerative endodontic procedures.[1]
Dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal
ligament stem cells, and stem cells of the apical papilla can now be isolated. Apical
papilla stem cells (SCAPs) are located at the apical part of immature teeth.[2] Osteogenic differentiation of SCAPs and the formation of osteoblast and osteoblast-like
cells have been demonstrated in addition to the formation of new hard tissue.[3]
Bioactive glass (BG) has been successfully used with implant placement and to treat
pathological periodontal bony defects owing to its superior biocompatibility, osteo-conductive
and -inductive properties. BG has the ability to modify osteoblastic gene expression
in a way that properly controls cell proliferation and differentiation.[4]
[5] Applying the BG in a nano-sized particles, 45S5 BG, has increased its osteoconduction,
osteoinduction properties and allowed for its use in bone tissue engineering.[6]
[7]
Hydroxyapatite shows excellent biocompatibility. Preparing it in the nanoscale yielded
superior biologic properties when used as a scaffold owing to particle size similar
to that of the natural hydroxyapatite. Nanohydroxyapatite has yielded promising results
in various regenerative procedures and led to the creation of various composite scaffolds.[8]
Chitosan, extracted from crustaceans, has been recently used and tested in various
endodontic applications owing to its excellent biologic behavior. It is a cationic
polymer that demonstrates good antimicrobial properties.[9]
[10]
[11] Composite scaffolds such as chitosan/hydroxyapatite have shown promising osteconductivity,
gaining the advantage of both materials used.[12]
The effect of hydroxyapatite coated by chitosan nanoparticles and BG nanoparticles
on the osteogenic differentiation and proliferation of stem cells of the apical papilla
has not yet been evaluated. Therefore, the aim of this study was to evaluate the effect
of hydroxyapatite coated by chitosan nanoparticles and BG nanoparticles on the osteogenic
differentiation and proliferation ability of stem cells of the apical papilla. The
null hypothesis tested is that there is no significant difference between the hydroxyapatite
coated by chitosan nanoparticles and BG nanoparticles on the osteogenic differentiation
and proliferation of stem cells of the apical papilla.
Materials and Methods
Preparation of Nanomaterials
Bioactive Glass 45S5 Nanoparticles
The sol-gel method was adopted to prepare BG 45S5 nanoparticles from a colloidal solution
of 45S5 composition (45 mol% SiO2, 24.5 mol% CaO, 24.5 mol% Na2O and 6 mol% P2O5).[13] Ceramic powder was produced from the gel after heating.
Hydroxyapatite Nanoparticles
Ammonium hydroxide and calcium nitrate were used to synthesize the hydroxyapatite
nanoparticles following the methodology previously described by Cengiz et al.[14]
Chitosan-Coated Nanohydroxyapatite
Preparation of the composite chitosan-coated nanohydroxyapatite was done following
Nikpour et al methodology.[15] Powder was obtained after freeze-drying of the mixture.
Characterization of all of the prepared nanoparticles was done using high-resolution
transmission electron microscopy (TEM) and X-ray Powder Diffraction (XRD) with 2 thetas
(10ᴼ-70ᴼ), with a scanning speed of 1°/min and minimum step size 2Theta: 0.001 at wavelength
(Kα) = 1.54614ᴼ[16] as shown in [Fig. 1].
Fig. 1 Transmission electron microscopic image of (A) bioactive glass 45S5 nanoparticles, (B) hydroxyapatite nanoparticles, and (C) chitosan-coated nanohydroxyapatite nanoparticles.
Stem Cells Harvesting and Culture
SCAPs were harvested and cultured from freshly extracted wisdom teeth of three patients
after obtaining an informed consent. Inverted phase contrast microscope was used to
check for growth and/or contamination.[17]
SCAP Characterization
Flow cytometric analysis was performed using the protocol published earlier on Navios
software.[17]
SCAP Culture
Cells cultured in complete culture media and harvested after the third passage. The
harvested SCAPs were cryopreserved at °80°C for further analysis.[17]
All tested nanomaterials were mixed according to manufacturer instructions. The samples
were classified to five equal groups:
-
Negative control group: SCAP with Dulbecco's Modified Eagle Medium (DMEM).
-
Positive control group: SCAP with inductive media (OM).
-
Group I: Nanohydroxyapatite (NHAP 10 µg/mL) with SCAP.
-
Group II: Chitosan-coated nanohydroxyapatite (NHAP/chitosan 10 µg/mL) with SCAP.
-
Group III: BG nanoparticles (NBG 500 µg/mL) with SCAP.
For osteoblastic differentiation, six-well plates were used to culture stem cells
of the apical papilla OM seeded at 4.5 × 105 cells/well. Plates were incubated for a period of 72 hours at 37°C and 5% CO2. The activity of alkaline phosphatase (ALP) was measured using enzymatic dephosphorylation
by ALP assay kit. For testing the expression of RANKL for SCAP, the cells were examined
using specific polyclonal antibody by fluorescence microscope.
Regarding evaluation of the proliferation, the SCAPs were stained by trypan blue and
counted by hemocytometer to estimate the number of dead cells. The MTT assay was performed
using the Vybrant MTT Cell Proliferation Assay Kit. Cell viability was determined
by measuring the optical density at 570 nm on a spectrophotometer.
Statistical Analysis
Mean and standard deviation values of each group were calculated. Shapiro–Wilk test
and Levene's test were used to test for normality of the data. One-way analysis of
variance test was run followed by Tukey's post hoc test as the data was normally distributed.
The significance level was set at p-value less than0.05. Statistical analysis was performed with Statistical package
for Social Science software.
Results
The observed results of the characterized SCAP revealed that the cells showed double
bright surface expression of CD44/CD73 and failed to express CD45, indicating a nonhematopoietic
origin as shown in [Fig. 2].
Fig. 2 Flow cytometry (FCM) dot plots showing the gate protocol for apical papilla stem
cells (SCAPs). The SCAPs were stained with stem cell markers (CD73, CD44, and CD45).
The CD73 and CD44 positive cells were gated in corresponding to CD45.
NHAP/chitosan showed the highest ALP concentration followed by NBG, NHAP, and DMEM-NC
as shown in [Table 1]. RANKL expression results are shown in [Table 2] and [Fig. 3] where NHAP/chitosan showed the highest H score followed by NBG, NHAP, and DMEM-NC.
NHAP/chitosan showed the highest viable cell count as shown in [Table 3]. NHAP/chitosan showed the highest viable count also using the MTT assay, although
the difference was not statistically significant as shown in [Table 4].
Table 1
Mean ± SD and p-values of ALP concentration of all tested groups
|
(OM-PC)
|
(NHAP 10 µg/mL)
|
NHAP/chitosan (10 µg/mL]
|
NBG
(500 µg/mL)
|
(DMEM-NC)
|
|
Mean ± SD
|
77.86 ± 0.15b
|
68.72 ± 0.13c
|
82.90
± 0.10a
|
70.36 ± 0.10d
|
55.18 ± 0.8e
|
|
p-Value
|
<0.001
|
Abbreviations: ALP, alkaline phosphatase; NBG, bioactive glass nanoparticles; NHAP,
nanohydroxyapatite; SD, standard deviation.
Means with different letters were statically significant.
Table 2
Mean ± SD and p-values of IF assay of all tested groups
|
(OM-PC)
|
(NHAP 10 µg/mL)
|
NHAP/chitosan (10 µg/mL)
|
NBG
(500 µg/mL)
|
(DMEM-NC)
|
|
Mean ± SD
|
82.67 ± 1.53c
|
67.67 ± 2.52d
|
180.67 ± 4.04a
|
154.67 ± 4.16b
|
17.68 ± 1.52e
|
|
p-Value
|
<0.001
|
Abbreviations: IF, immunofluorescence; NBG, bioactive glass nanoparticles; NHAP, nanohydroxyapatite;
SD, standard deviation.
Means with different letters were statically significant.
Table 3
Total, dead, viable cell counts, and mean ± SD values for tested groups
|
Total cell count
|
Dead cell count
|
Viable cell count
|
% Viability
|
|
(OM-PC)
|
60.7 × 105 ± 34.2 × 105 b
|
7.34 × 103
± 1.89 × 103b
|
60.6 × 105 ± 34.2 × 105 b
|
99.879
|
|
NHAP (10 µg/mL)
|
157 × 105 ± 33.8 × 105 a
|
5.28 × 103
± 0.88 × 103 b
|
157 × 105
± 33.8 × 105 a
|
99.966
|
|
NHAP/Ch (10 µg/mL)
|
218 × 105 ± 7.21 × 105 b
|
1.64 × 103
± 0.12 × 103 b
|
218 × 105
± 7.21 × 105 a
|
99.996
|
|
NBG (500 µg/mL]
|
198.7 × 105 ± 69.8 × 105 a
|
18.9 × 103 ± 7.80 × 103 a
|
198.7 × 105 ± 69.8 × 105 a
|
99.905
|
|
(DMEM-NC)
|
4.647 × 105 ± 1.147 × 105 b
|
4.11 × 103 ± 4.41 × 103 a
|
4.6 × 105
± 1.1 × 105 b
|
99.116
|
|
p-Value
|
<0.001
|
0.004
|
<0.001
|
|
Abbreviations: Ch, chitosan; NBG, bioactive glass nanoparticles; NHAP, nanohydroxyapatite;
SD, standard deviation.
Means with different letters were statically significant.
Table 4
Mean ± SD and p-values of viability test (MTT assay)
|
(OM-PC)
|
(NHAP 10 µg/mL)
|
NHAP/chitosan (10 µg/mL)
|
NBG
(500 µg/mL)
|
(DMEM-NC)
|
|
Mean ± SD
|
0.930 ± 0.042b
|
1.502 ± 0.243a
|
1.645
± 0.23a
|
1.551 ± 0.292a
|
0.821 ± 0.019b
|
|
p-Value
|
<0.001
|
Abbreviations: NBG, bioactive glass nanoparticles; NHAP, nanohydroxyapatite; SD, standard
deviation.
Means with different letters were statically significant.
Fig. 3 Photomicrograph showing expression of RANKL protein in differentiated apical papilla
stem cells (SCAPs), the photos were captured by LABOMED Immunofluorescence microscopes.
(A) Negative control cells shows small colonies of cells that showed a homogenous faint
expression of RANKL, the expression was localized to the cell membrane. (B) Positive control cells with increased number of osteoblasts like colonies which
are presented with dense homogenous expression of RANKL. However, the SCAP cultured
with nanohydroxyapatite (NHAP) (C), NHAP/chitosan (D) and bioactive glass nanoparticles (E), showed a merged large colony of osteoblast like cells with dense homogenous membranous
and nuclear expression of RANKL. The magnification power is 10x.The white circles
highlight the osteoblast like colonies, white arrow: membranous expression of RANKL,
yellow arrow: dense nuclear expression of RANKL.
Discussion
Proper management and long-term success of nonvital immature permanent teeth continue
to be a challenge for clinicians. The ability to regenerate a pulp or pulp-like structure
that can lay down hard tissue structure increasing the root length and thickness will
increase the tooth's fracture resistance and maintain it in function for a longer
duration.[18]
During various regenerative endodontic procedures, mesenchymal stem cells have been
demonstrated related to immature teeth in addition to mature ones. The origin of such
mesenchymal stem cells is believed to be the apical papilla, bone, Periodontal ligament
(PDL), and/or granulomas.[19]
Apical papilla can be defined as the loosely attached soft tissue related to the root
end of immature teeth. This apical papilla is separated from the dental pulpal tissue
by a cell rich zone. The dental pulpal tissue shows more cellular and vascular elements
than does the apical papilla.[2]
SCAPs were first isolated by Sonoyama et al. SCAPs are derived from an embryonic neural
crest-like tissue, located at the root end of immature teeth. In contrast to other
isolated types of stem cells, SCAPs demonstrate impressive odontogenic differentiation
and proliferation in addition to massive dentinogensis.[2]
[20]
Under favorable conditions, mineral trioxide aggregates have the ability to stimulate
the proliferation and differentiation of SCAPs resulting in hard tissue formation.
However, the effect of other materials on SCAPs is not well studied.[21] BG, hydroxyapatite, and chitosan have shown promising results when tested for their
biologic effect on dental pulp stem cells and mesenchymal stem cells.[22-27] The aim of the current study was to investigate the effect of nanohydroxyapatite
coated by chitosan and nano-BG on osteogenic differentiation and proliferation of
stem cells of the apical papilla.
Trypan blue was used for counting the viable cells in the current study owing to its
characteristic ability to stain only the dead cells following penetration of its cell
membrane.[28]
[29] The MTT assay was used due to its capability to determine the mitochondrial activity.[30]
[31] ALP enzyme activity assays were used as a measure of SCAP differentiation into osteoblast-like
cell as it is considered as a characteristic marker for bone-forming cell differentiation.[32] Immunofluorescence assay is considered as one of the most reliable tests that helps
elaborate specific protein of interest through antigen-antibody reaction.[33] The RANK-L concentration is proportional to the number of osteogenic cells because
it is deemed mandatory for its differentiation.[34]
[35]
Our results of SCAP characterization come in full agreement with Kang et al who also
confirmed the nonhemopoietic origin of the stem cells by lack of CD45 expression.[36]
The superior results of NHAP/chitosan group regarding the ALP and RANKL come in full
agreement with Kong et al.[37] This superiority could be attributed to the composite nature of this group that
allowed for better mineralization and differentiation. This can be explained by the
increased levels of calcium phosphate and calcium carbonate.[38] This also comes in agreement with Ge et al[39] who tested this composite on periodontal ligament stem cells differentiation. They
attributed their results to increased concentration of the calcium and phosphate ions.
The NHAP/chitosan group also showed superior results in osteogenic differentiation
and proliferation potential of SCAP. This might be because of the chitosan coating
that directly stimulates progenitor cell differentiation at the mRNA level of ALP
enzyme.[40]
[41]
[42] This superiority of the composite group comes in full agreement with Kong et al[37] who attributed this to the topography and quantity. Also, this finding is similar
to that obtained by Ge et al[39] on periodontal ligament stem cells explained on basis of surface chemistry and geometry.
Similar results were also obtained by Tondnevis et al[43] on dental pulp stem cells.
BG showed significant effect on SCAP viability and osteogenic differentiation compared
to the negative control group. This is consistent with Wang et al[44] who tested BG on bone marrow stem cells. This could be simply explained by the increased
ion release, specifically calcium ions that attracts different cells.
The nanohydroxyapatite group also showed significant effect on SCAP viability and
osteogenic differentiation in compared to the negative control group. This finding
is in agreement with Yang et al[25] who tested it on mesenchymal stem cells. This could be explained on the basis of
the nanoparticle size that greatly affects its behavior in addition to the increased
calcium ions release that increases cell mineralization.
Nanohydroxyapatite coated by chitosan, nanobioactive glass and nanohydroxyapatite
as biomaterials, proved to enhance the osteogenic differentiation and proliferation
of SCAP. This could improve the regenerative procedure in endodontic as osteogenic
differentiation enhances lesion healing and laying down hard tissue structure that
might be dentin like.
Conclusion
Within the limitations of this in vitro study, it can be concluded that isolation of SCAP can be done from extracted fully
impacted immature third molars. All tested biomaterials have the ability to induce
osteogenic differentiation and proliferation of SCAP. Chitosan-coated nanohydroxyapatite
biomaterial has increased ability for differentiation of SCAP to osteoblasts. Chitosan-coated
nanohydroxyapatite biomaterial has increased ability for proliferation of SCAP proved
by upregulated cell viability.
