Life Sciences 258 (2020) 118143

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Kaempferol promotes proliferation and osteogenic differentiation of T

periodontal ligament stem cells via Wnt/β-catenin signaling pathway
⁎
Fujiao Niea, Wenjuan Zhangb, Qun Cuia, Yajing Fua, Hongkun Lia, Jun Zhanga,
a
Department of Orthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue
Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, Jinan, Shandong Province 250012, China
b
Department of Stomatology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, Shandong Province 250012, China

A R T I C LE I N FO A B S T R A C T

Keywords: Aims: Kaempferol, a type of flavonoid, is widely present in fruits, vegetables and medicinal herbs. This study was
Kaempferol designed to investigate the effects of kaempferol on proliferation and osteogenesis of periodontal ligament stem
Periodontal ligament stem cells (PDLSCs) cells (PDLSCs) and to identify the related pathway.
Osteogenic differentiation Materials and methods: PDLSCs were isolated from extracted premolars and cultured in vitro. Cell-counting kit-8
β-Catenin
(CCK-8) and colony formation assays were performed to determine the effect of kaempferol, at various con-
centrations, on the proliferation of PDLSCs. Alkaline phosphatase (ALP) activity was analyzed both quantita-
tively and qualitatively, and extracellular matrix mineralization was examined by alizarin red-S staining. In
addition, the mRNA and protein expression levels of ALP, RUNX Family Transcription Factor 2 (RUNX2), Sp7
Transcription Factor (SP7; Osterix), Bone Gamma-Carboxyglutamate Protein (BGLAP; osteocalcin) and catenin
beta 1 (CTNNB1; β-catenin) were monitored by qPCR and Western blot analysis. Additionally, the tankyrase
inhibitor, XAV939, was used to determine the role of the Wnt/β-catenin pathway.
Key findings: The results illustrated that 10−6 M kaempferol markedly promoted the proliferation, ALP activity
and mineral deposition of PDLSCs (P < 0.05). The expression levels of ALP, RUNX2, SP7, BGLAP and β-catenin
were all upregulated (P < 0.05). After blocking the Wnt/β-catenin pathway with XAV939, the effects of
kaempferol were apparently reversed.
Significance: kaempferol enhanced proliferation and osteogenesis of PDLSCs by activating the Wnt/β-catenin
signaling, which suggests the potential application of kaempferol for periodontal tissue regeneration.

1. Introduction pronunciation function and appearance. Currently, periodontitis has

Periodontitis, an inflammatory and destructive disease involving
periodontal support tissue, is associated not only to the health of per-
iodontal tissue, but also the occurrence, development and prognosis of
certain systemic diseases. One of the major pathological changes in
periodontitis is the resorption of alveolar bone, which is the most active
process in human bone remodeling. The development of the disease
may lead to spontaneous tooth loosening and tooth displacement. More
seriously, the teeth affected by periodontitis may fall out in severe
cases, which have negative effects on the patients' chewing,
become a global burden [1], and as a result periodontal tissue re-
generation has attracted extensive attention worldwide.
Although the current clinical periodontitis treatments, such as per-
iodontal scaling and root planing, can control the progression of peri-
odontal inflammation and prevent further loss of periodontal support
tissue, they cannot achieve the desired regeneration of the lost tissue.
Guided tissue regeneration and bone grafting bring new hopes for the
treatment of periodontitis. However, the narrow range of adaptation,
unpredictable efficacy, and low expectations of these methods do not
meet people's requirements [2,3]. With the rapid development of

Abbreviations: PDLSCs, periodontal ligament stem cells; CCK-8, cell counting kit-8; ALP, alkaline phosphatase; RUNX2, RUNX Family Transcription Factor 2; SP7,
Sp7 Transcription Factor; BGLAP, Bone Gamma-Carboxyglutamate Protein; CTNNB1, catenin beta 1; PBS, phosphate-buffered Saline; α-MEM, α-minimum essential
medium; FBS, fetal bovine serum; BCA, bicinchoninic acid assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CPC, cetylpyridinium chloride; SDS-PAGE,
sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidenefluoride; MSC, mesenchymal stromal cell
⁎
Corresponding author at: Department of Orthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key
Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, No. 44-1, Wenhua West Road, Jinan,
Shandong Province 250012, China.
E-mail address: zhangj@sdu.edu (J. Zhang).

https://doi.org/10.1016/j.lfs.2020.118143
Received 14 May 2020; Accepted 21 July 2020
Available online 25 July 2020
0024-3205/ © 2020 Elsevier Inc. All rights reserved.
F. Nie, et al.
molecular biology, tissue engineering and stem cell technology, peri-
odontal tissue regeneration has become a research hotspot in the
treatment of periodontitis. Periodontal ligament stem cells (PDLSCs)
were isolated from the periodontal ligament tissue and cultured for the
first time by Seo et al. [4] in 2004, providing a new type of seed cell for
periodontal tissue regeneration research. Subsequent studies verified
that PDLSCs had a high proliferative capacity and could differentiate
into different cell lineages under appropriate induction conditions both
in vitro and in vivo, such as osteoblast-like cells, cementoblast-like cells,
adipocytes and fibroblast-like cells [5,6]. Furthermore, PDLSCs are
extremely important for maintaining periodontal ligament stability,
repairing cementum tissue damage and achieving functional cell re-
newal. In summary, PDLSCs have a promising application prospect in
the field of periodontal tissue regeneration engineering.
Kaempferol, a type of flavonoid, is widely found in vegetables, fruits
and herbs, such as Kaempferia galanga L, Hawthorn and Polygonum
tinctorium Lour, etc. It has been reported that kaempferol has many
effects, such as anticancer [7,8], anti-inflammatory [9,10], anti-
bacterial [11,12], antiviral [13] and neuroprotective [14]. In addition,
it has been reported that kaempferol can effectively prevent female
menopausal osteoporosis [15,16]. Earlier studies have shown that
kaempferol has the ability to stimulate the differentiation of osteoblasts
[17–19] and repress the resorption of bone [20,21]. However, whether
kaempferol has osteogenic promoting effects on PDLSCs and the un-
derlying mechanisms of such effects are not fully understood.
Various biological processes involved in individual growth and de-
velopment are regulated by the Wnt signaling pathway [22]. The ca-
nonical pathway Wnt/β-catenin pathway is particularly essential for
skeletal system development compared with many other Wnt pathways
[23,24], and β-catenin plays a major role in the processes of cell pro-
liferation, directional differentiation and maturation. Previous research
has found that Wnt/β-catenin pathway could regulate bone formation
at various stages of skeletongenesis by enhancing osteoblast differ-
entiation and inhibiting osteoclast differentiation [25]. Also, Wnt/β-
catenin pathway regulates the conversion of progenitor cells to osteo-
blasts, chondrocytes and adipocytes in bone development [26,27].
Several studies have demonstrated that the activation of Wnt/β-catenin
pathway mediates the osteogenic differentiation of PDLSCs [28–30].
Accordingly, we assumed that activation of Wnt/β-catenin pathway
might contribute to osteogenesis of PDLSCs.
The objectives of this study were to investigate the effects of
kaempferol on osteogenesis of PDLSCs and determine the potential
mechanisms underlying such effects, which were hypothesized to be
mediated by activation of the Wnt/β-catenin pathway. We anticipate
that this research will generate data which can be used as valuable
reference in the clinical application of kaempferol and PDLSCs in per-
iodontal tissue regeneration engineering.
2. Materials and methods
2.1. Isolation, cultivation and characterization of PDLSCs
Primary PDLSCs were prepared from healthy and non-carious pre-
molars (n = 20) extracted for orthodontic purpose (patients' age:
16–25 years old). All subjects gave their informed consent for inclusion
before they participated in the study. The study was conducted in ac-
cordance with the Declaration of Helsinki, and the protocol (No.
20190208) was approved and supervised by the Ethics Committee of
the Hospital of Stomatology, Shandong University (Jinan, China). The
primary PDLSCs were cultured using the tissue block method at 37 °C in
a humidified atmosphere with 95% air and 5% CO2. First, the extracted
teeth were rinsed with phosphate-buffered saline (PBS) containing 5%
penicillin/streptomycin antibiotics solution (Thermo Fisher Scientific
Inc., Waltham, MA, USA). Then, the periodontal ligament from the
middle third of the tooth root was gently scraped and cut into small
pieces with a sterile blade. The tissue pieces were arrayed at the bottom
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Life Sciences 258 (2020) 118143
of the cell culture flask. PDLSCs were cultured in complete medium,
consisting of α-minimum essential medium (α-MEM; Hyclone, Logan,
UT, USA) supplemented with 10% fetal bovine serum (FBS; Thermo
Fisher Scientific Inc.) and 1% antibiotics, which was renewed every two
or three days. Once the cells reached 80% confluence, they were rinsed
with PBS, digested with a 0.25% trypsin-EDTA solution (Thermo Fisher
Scientific Inc.) and passed on. The obtained cells were isolated and
purified using the limited dilution cloning method. Single cell-derived
colonies between passages three to five were used in subsequent ex-
periments. Eventually, the isolated cells were characterized by de-
tecting surface molecular markers of PDLSCs, including CD34, CD44,
CD45 and CD90, by flow cytometry analysis using a flow cytometer
(Beckman Coulter, Brea, CA, USA) to detect cell fluorescence.
2.2. Osteogenic and adipogenic induction
To identify the multi-directional differentiation capacity of PDLSCs,
the cells (passage three) were seeded in 6-well plates at a density of
1.5 × 105 cells per well. After incubating in complete medium for 24 h,
the medium was changed to osteogenic inducing medium (α-MEM
containing 8% FBS, 50 μg·ml−1 ascorbic acid, 10 mM β-glyceropho-
sphate and 0.01 μM dexamethasone) or adipogenic inducing medium
(α-MEM containing 8% FBS, 500 μM 3-isobutyl-1-methylxanthine
(IBMX), 200 μM indomethacin, 1 μM dexamethasone and 10 μg·ml−1
insulin). After induction for three weeks, the cells were fixed with 4%
paraformaldehyde for 30 min, and stained with Alizarin Red-S or Oil
Red O (Solarbio, Beijing, china).
2.3. Cell proliferation assay and colony formation assay
The Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto,
Japan) was used to determine the effects of kaempferol on the pro-
liferation of PDLSCs. PDLSCs were cultured in 96-well plates (3 × 103
cells per well) with complete medium for 24 h. Then, the medium was
replaced by complete medium supplemented with kaempferol at var-
ious concentrations (0, 10−8, 10−7, 10−6, 10−5 and 10−4 M), using
five duplicate wells for each concentration group. After culturing for
one, three and five day(s), the original medium was aspirated and then
100 μl of CCK-8 solution (α-MEM and CCK-8 reagent mixing in a ratio
of nine to one) was added to every tested well. Ultimately, the absor-
bance was measured at 450 nm on a SPECTROstar Nano microplate
reader (BMG Labtech Inc., Ortenberg, Germany) after incubation for 2 h
at 37 °C in a dark room. Wells containing 100 μl CCK-8 solution without
seeding cells were used as blank control.
For colony formation assay, a total of 1 × 103 cells were plated into
10 cm diameter culture dishes in the absence or presence of kaempferol
(10−6 M). After culturing for 10 days, cells were stained by the crystal
violet (Solarbio) staining methods. Cell colonies (clusters with 50 or
more cells originated from the same cell) were counted to determine the
ability of PDLSCs to proliferate and form colonies.
2.4. Alkaline phosphatase (ALP) activity and staining assay
The ALP activity assay is widely used to assess the early osteogen-
esis ability of stem cells. PDLSCs were plated in 6-well plates and
treated with osteogenic inducing medium containing different con-
centrations of kaempferol (0, 10−7, 10−6 and 10−5 M) for 7 and
14 days. ALP activity was quantified using an alkaline phosphatase
assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Cells were scraped off and solubilized in 1% Triton X-100 on ice. After
ultrasonic cell disruption for several seconds, the lysates were cen-
trifuged at 12,000 ×g for 5 min at 4 °C. Then, an aliquot of the su-
pernatant (30 μl per well) and working assay solution were added to 96-
well plates, followed by incubation for 15 min at 37 °C. Separately,
double distilled water and standard phenol solution were added as
negative control (NC) and standard control. Eventually, colorimetric
F. Nie, et al.
reagent was transferred to all wells and the absorbance values were
measured at 520 nm using a microplate reader. The ALP activity was
normalized to the respective total protein concentration detected by the
bicinchoninic acid (BCA) assay (Solarbio). In addition, the BCIP/NBT
Alkaline Phosphatase Color Development Kit (Beyotime, Shanghai,
China) was used to visualize ALP staining, and the depth of dark blue
reflects the activity of ALP. Images were captured by a scanner.
2.5. Assessment of mineralized nodules formation via alizarin red-S staining
assay
Alizarin red-S staining was performed to assess the formation of
mineralized nodules. The PDLSCs were cultured as described for the
osteogenic induction assay. After 21 days of induction with complete
medium (blank control group), osteogenic inducing medium (control
group) or osteogenic inducing medium with 10−6 M kaempferol (ex-
perimental group), the cells were fixed with 4% paraformaldehyde and
stained with alizarin red-S. Then, the stained plates were observed
under a light microscope and photographed to evaluate the mineralized
nodules. Additionally, in order to quantify the content of mineralized
matrix deposition, 10% cetylpyridinium chloride (CPC; Solarbio) was
added to the stained plates to dissolve the mineral nodules into solu-
tion, and the absorbance of the solution was measured at 562 nm on a
microplate reader.
2.6. XAV939 treatments
XAV939, a small molecule inhibitor, selectively inhibits Wnt/β-ca-
tenin signaling by inhibiting tankyrase 1/2 [31]. The PDLSCs were
cultured in four different media: i) simple osteogenic inducing medium;
ii) osteogenic inducing medium with 2.5 × 10–6 M XAV939 (Med-
ChemExpress LLC, Monmouth Junction, NJ, USA); iii) osteogenic in-
ducing medium with 10−6 M kaempferol; iv) osteogenic inducing
medium with XAV939 and kaempferol. After seven days of induction,
PDLSCs were harvested and protein expression was analyzed by Wes-
tern blot analysis.
2.7. qPCR analysis
The mRNA expression levels of ALP, RUNX Family Transcription
Factor 2 (RUNX2), Sp7 Transcription Factor (SP7; Osterix), Bone
Gamma-Carboxyglutamate Protein (BGLAP; osteocalcin) and catenin
beta 1 (CTNNB1; β-catenin) were measured by qPCR analysis. After
culturing the PDLSCs in the absence or presence of kaempferol
(10−6 M) for seven days, the total RNA was extracted using Trizol
(TaKaRa, Shiga, Japan) and reverse transcribed into cDNA with a
Primer Script® RT Reagent Kit (TaKaRa). Then qPCR analysis was
conducted using SYBR® Premix Ex Taq™ (TaKaRa). A reaction system of
10 μl volume was adopted and every RNA sample was tested in tripli-
cate. The mRNA expression level was calculated by the 2-△△Ct method
[32] using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a
control. The primer sequences used in the present study were as fol-
lows: ALP: 5′-CCACGTCTTCACATTTGGTG-3′ and 5′-AGACTGCGCCTG
GTAGTTGT-3′; RUNX2: 5′-CGAATGGCAGCACGCTATTAA-3′ and
5′-GTCGCCAAACAGATTCATCCA-3′; SP7: 5′-GCTTGAGGAGGAAGTTC
ACTA-3′ and 5′-GAGTTGTTGAGTCCCGCAGA-3′; BGLAP: 5′-TAGTGAA
GAGACCCAGGCGCT-3′ and 5′-ATAGGCCTCCTGAAAGCCGA-3′;
CTNNB1: 5′-GGCTTGGAATGAGACTGCTG-3′ and 5′-GGTCCATACCCA
AGGCATCC-3′; GAPDH: 5′-GCACCGTCAAGGCTGAGAAC-3′ and
5′-TGGTGAAGACGCCAGTGGA-3′.
2.8. Western blot analysis
The protein expression levels of ALP, RUNX2, SP7, BGLAP and β-
catenin were measured by Western blot analysis. The PDLSCs were
treated as described for the qPCR analysis. Total proteins were
3
Life Sciences 258 (2020) 118143
extracted from the cells using the RIPA buffer (Beyotime) with 1%
phenylmethylsulfonyl fluoride (PMSF; Solarbio). Then the protein
concentration was determined by the BCA method. The obtained pro-
teins (20 μg) were denatured and separated by 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then
transferred onto 0.45-μm polyvinylidene difluoride membranes (PVDF;
Millipore, Billerica, MA, USA). Afterwards, the membranes were
blocked with 5% skimmed milk at room temperature for 1 h, and in-
cubated with the following primary antibodies at 4 °C overnight: ALP,
RUNX2, SP7 and BGLAP (Abcam, Cambridge, UK), and β-catenin,
GSK3β, p-GSK3β (Ser9), LEF and GAPDH (Cell Signaling Technology,
MA, Danvers, USA). After washing in Tris-buffered saline with 0.1%
Tween 20 (TBST), the membranes were incubated with a secondary
antibody (Absin, Shanghai, China) solution at 37 °C for 1 h. Eventually,
the protein bands were visualized with an enhanced chemilumines-
cence kit. GAPDH was used as internal reference.
2.9. Statistical analysis
All experiments were performed in triplicates. Statistical analysis
was conducted using the SPSS 17.0 software (SPSS, Inc., Armonk, NY,
USA) as well as GraphPad Prism 6 (GraphPad Software, Inc., La Jolla,
CA, USA). The data are presented as the mean ± standard deviation,
and the significance of the differences was analyzed using Unpaired
Student's t-test and one-way analysis of variance (ANOVA). It was
considered statistically significant when P-value < 0.05.
3. Results
3.1. Cultivation and characterization of PDLSCs
Generally, primary cells grew from the edge of the explants after
five to ten days and rapidly proliferated around the explants (Fig. 1A).
Alizarin red-S staining and oil red O staining revealed that the cells had
differentiated into osteoblasts and adipocytes under osteogenic and
adipogenic induction in vitro, forming mineralized nodules and oil red
stained lipid droplets (Fig. 1B and C). To identify the mesenchymal
stem cell (MSC) properties of these cells, flow cytometry analysis was
performed to determine the expression levels of MSC surface markers.
The results showed that PDLSCs had high expression (99.5%) of MSC-
specific markers CD44 and CD90, while the expression of pan-leukocyte
marker CD45 and hematopoietic stem cell (HSC) marker CD34 (Fig. 2)
was negative.
3.2. Cell proliferation assay and colony formation assay
The results of the cell proliferation analysis using the CCK-8 assay
are shown in Fig. 3A and B. There were no significant differences
among the groups on day one and three, except that the 10−4 M
kaempferol suppressed the proliferation ability of PDLSCs to some ex-
tent on day three. On day five, significant promotion of proliferation of
PDLSCs was observed with 10−6 M and 10−7 M kaempferol
(P < 0.0001 for both concentrations), while high concentration of
kaempferol (10−4 M) markedly inhibited the proliferation of PDLSCs.
In addition, the colony formation assay showed that after cultivation for
10 days the cell colonies of the kaempferol group (10−6 M) were clearly
more numerous and larger than those of the control group (Fig. 3C and
D).
3.3. ALP activity and mineralized nodule deposition of PDLSCs
Compared with the control group, the ALP activity was significantly
increased in the 10−6 M kaempferol group (P < 0.01) both on day 7
and day 14 (Fig. 4B). Also, a similar trend was observed in the results of
the ALP staining assay (Fig. 4A). Based on the results of the ALP activity
and CCK-8 assay, we concluded that 10−6 M was the optimal
F. Nie, et al.
Fig. 1. Cultivation and characterization of PDLSCs. (A) Primary cells exhibited spindle or long fusiform morphology. Scale bar: 200 μm. (B) Cells were stained with
alizarin red-S after osteogenic differentiation induction. Scale bars: 200 μm. (C) Cells were stained with oil red O after adipogenic induction. Scale bars: 200 μm.
concentration for the proliferation and osteogenesis of PDLSCs, so this
concentration was employed in subsequent experiments.
For the alizarin red-S staining assay (Fig. 4C and D), the kaempferol
group at the concentration of 10−6 M clearly accelerated the deposition
of mineralized nodules of PDLSCs (P < 0.001).
3.4. Osteogenesis-associated mRNA and protein expression
The mRNA expression levels of the osteogenesis-associated genes
ALP, RUNX2, SP7 and BGLAP were analyzed after osteogenic induction
for seven days (Fig. 5B). Compared with the control group, the
kaempferol-treated group showed upregulated expression of all of the
above-mentioned osteogenesis-associated genes. Additionally, the ex-
pression of CTNNB1 mRNA was also analyzed to determine the role of
Wnt/β-catenin signaling in this process. The kaempferol group ex-
hibited a remarkable increase in the expression level of CTNNB1 mRNA.
Furthermore, the protein expression levels of these osteogenesis-asso-
ciated genes were consistent with the results of the mRNA expression
analysis (Fig. 5A).
3.5. Blocking the Wnt/β-catenin pathway attenuated the Kaempferol-
induced promotion of osteogenesis of PDLSCs
To further elucidate the role of the Wnt/β-catenin pathway in
kaempferol-induced osteogenic promotion of PDLSCs, the specific tan-
kyrase inhibitor, XAV939, was used to block the Wnt/β-catenin
pathway, and the results are shown in Fig. 6. The protein expression
levels of the osteogenesis-associated genes RUNX2, SP7 and BGLAP
were decreased after treatment with XAV939, indicating that the in-
hibition of β-catenin could impair the kaempferol-induced osteogenic
promotion of PDLSCs. Moreover, the expression levels of β-catenin, p-
GSK3β and LEF, which are key effectors of Wnt/β-catenin signaling,
were also suppressed by XAV939. No significant differences were found
in the expression levels of GSK3β among the four groups in this study.
Additionally, the kaempferol-treated group showed the highest ex-
pression levels of osteogenesis-associated proteins and Wnt/β-catenin
pathway-related proteins among all groups. These results indicated that
kaempferol enhances the osteogenesis of PDLSCs by activating the Wnt/
β-catenin pathway, which was verified by the increase of protein ex-
pression levels of β-catenin, p-GSK3β and LEF. Also, this finding is
consistent with the observation that XAV939-induced inhibition of the
Wnt/β-catenin pathway led to the suppression of the osteogenic dif-
ferentiation of PDLSCs.
4. Discussion
The multi-directional differentiation potential and high periodontal
4
Life Sciences 258 (2020) 118143
correlation of PDLSCs make them the most dependable seed cells for the
regeneration of periodontal tissue. Indeed, PDLSCs are considered as
the cornerstone for research on oral tissue regeneration and re-
construction engineering [33,34]. Previous research has shown that
PDLSCs have the capabilities of osteogenic differentiation, expressing
osteogenesis-related proteins and forming calcium nodules [35,36].
Moreover, periodontal ligament-dentin complexes formed by PDLSCs
are similar in morphology and spatial arrangement to natural period-
ontal tissues [37], which also indicates that PDLSCs have unique ad-
vantages in periodontal tissue regeneration. Therefore, finding ways to
promote the proliferation and osteogenesis of PDLSCs could be of
considerable importance for periodontal tissue engineering. In our
study, the PDLSCs were isolated and cultured using a tissue block
method. We proved by flow cytometry analysis that the primary cells
were of mesenchymal origin, with no epithelial cell contamination. The
colony formation assay and the osteogenic and adipogenic induction
assays showed that the cultured PDLSCs showed a high degree of
clonality and multi-directional differentiation potential.
Estrogen plays a crucial role in maintaining the balance and
homeostasis of the human skeletal system. Lack of estrogen is the
leading cause of osteoporosis in menopausal women. Estrogen re-
placement therapy (ERT) is the traditional treatment for post-
menopausal osteoporosis [38,39]. Several studies have shown that es-
trogen can upregulate the expression of osteogenesis-related gene in
PDLSCs, and can promote the regeneration of cementum and the re-
construction of alveolar bone. However, ERT is no longer recommended
for long-term use due to its association with breast cancer development,
stroke and cardiovascular disease [40,41], etc. In contrast, due to the
absence of these unwanted side effects, flavonoids, a type of phytoes-
trogens, have been considered as potential drugs for the treatment of
pre-osteoporosis [42]. Kaempferol, as a type of flavonoids, has attracted
more and more attention for its role in promoting osteogenesis [17–21].
Therefore, it would be useful to determine whether kaempferol also
exerts its osteogenic-promoting effect on PDLSCs.
The enhancement of early ALP activity, deposition of mineralized
nodules as well as the upregulation of osteogenesis-related genes are
considered as indicators for identifying mineralization of cells. It is
generally accepted that ALP is a typical marker enzyme of osteoblasts,
and is closely related to bone matrix calcification. Alizarin red-S
staining can intuitively evaluate the mineralization capacity of cells by
forming mineralized nodules through the specific combination of ali-
zarin red-S and calcium ions [43]. In this research, we found that
10−6 M kaempferol can clearly promote the proliferation of PDLSCs. In
addition, since 10−6 M was also the optimal concentration of kaemp-
ferol to enhance ALP activity, this concentration was used in sub-
sequent experiments. The effects of kaempferol on the proliferation and
osteogenesis of PDLSCs were further confirmed by colony formation
 F. Nie, et al.

5
Fig. 2. Characterization of PDLSCs by cell surface markers. CD44 (A, B) and CD90 (C, D) were highly expressed, while the expression of CD34 (E, F) and CD45 (G, H) were negative.
Life Sciences 258 (2020) 118143
F. Nie, et al. Life Sciences 258 (2020) 118143

Fig. 3. Effects of kaempferol on the proliferation of PDLSCs. (A) The growth curves of the kaempferol-treated groups at different concentration and the control group
were drawn according to the results of CCK-8 assay. (B) The CCK-8 assay demonstrated the proliferation of PDLSCs was stimulated by 10−6 M and 10−7 M
kaempferol on day 5. Mean ± SD, ****P < 0.0001 (C) Colony formation assay was performed to test the colony forming capacity of PDLSCs. (D) In contrast to the
control group, the colony forming efficiency of kaempferol-treated group was significantly higher. ***P < 0.001.

assay and alizarin red-S staining. These results were consistent with the
findings reported by Wang Yang et al. showing that kaempferol can
enhance the proliferation, migration and osteogenesis of osteoblasts
[44].
In this study, four recognized osteogenesis-related genes (ALP,
RUNX2, SP7 and BGLAP) were selected for the evaluation of osteogenic
differentiation by qPCR and Western blot analysis, using GAPDH as a
standard reference. ALP is an early detection index of osteogenesis, and
its increased expression can trigger the mineralization of the matrix
[45]. RUNX2 is closely related to osteoblast differentiation as well as
chondrocyte maturation. RUNX2 also plays an essential role in pro-
moting the proliferation of osteoblast progenitor cells and regulating
the expression of major bone matrix proteins [46]. It has been reported
that the pathogenesis of cleidocranial dysplasia (CCD) is related to the
loss and pathogenic mutations of RUNX2 [47,48]. SP7 (or Osterix), a
protein closely involved in skeleton mineralization and tooth develop-
ment [49], can promote the mineralization ability of osteoblasts by
regulating the expression of a series of target genes. BGLAP, an osteo-
genesis-related protein, reflects the degree of osteogenic differentiation
and the potential for osteogenesis of stem cells [50]. In our experiment,
the mRNA and protein expression levels of osteogenesis-related genes,
such as ALP, RUNX2, SP7 and BGLAP in PDLSCs were all significantly
upregulated by kaempferol. Therefore, we concluded that kaempferol
can enhance the osteogenic potential of PDLSCs by regulating the ex-
pression of various osteogenesis-related genes. This finding could serve
as the experimental basis for the clinical use of kaempferol in period-
ontal tissue regeneration and bone remodeling.
The Wnt/β-catenin signaling pathway, which is evolutionarily
conserved, plays an important role in adjusting embryonic development
and adult tissue homeostasis [51]. Notably, this pathway is closely
connected with the maintenance of periodontal tissue homeostasis and
regulation of the development of periodontitis [52]. The key of this
pathway is the accumulation of β-catenin in the nucleus, which is
mainly affected by the phosphorylation status of GSK-3β. The specific
regulatory mechanism is described as follows [53–57] (Fig. 7). When
Wnt signaling is not expressed, β-catenin is phosphorylated by inter-
actions with a destruction complex made up of adenomatous polyposis
coli (APC), axin, and glycogen synthase kinase 3β (GSK-3β). Phos-
phorylated β-catenin is recognized by Beta-Transducin Repeat Con-
taining E3 Ubiquitin Protein Ligase (BTRC) to induce ubiquitination
and is eventually degraded. Conversely, when Wnt signaling ligands
bind to their coreceptors Frizzleds (Fz) and the lipoprotein receptor-
related protein (LRP) 5/6, Dishevelled (Dvl) protein will be activated,
which will impair the destruction complex and suppress the GSK-3β-
mediated phosphorylation of β-catenin. Afterwards, the β-catenin ac-
cumulated in the cytoplasm translocates to the nucleus, where it is
stabilized and interacts with T cell factor/lymphoid enhancer binding
factor (TCF/LEF), inducing the expression of downstream effector
genes. In previous studies, it was found that phosphorylation at Ser9
inhibits GSK3β activity, thus the activity of GSK3β is mainly evaluated
by the levels of phospho-Ser9 [58,59].
It has also been reported that some traditional Chinese medicine
formulations containing kaempferol can promote proliferation and
mineralization of osteoblast by stimulating the Wnt/β-catenin signaling
[60]. In this study, we found that kaempferol enhanced osteogenesis by
upregulating the expression of β-catenin and p-GSK3β (Ser9), as well as

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Fig. 4. Effects of kaempferol on ALP activity and mineralized nodule deposition of PDLSCs. a, b: complete medium; c, d: osteogenic inducing medium without
kaempferol; e, f: osteogenic inducing medium with 10−6 M kaempferol. (A) ALP staining was performed after inducing for seven days. Compared with the control
group, the color of ALP staining in kaempferol-treated group was significantly darker, and the blank control group was set to confirm the efficacy of the osteogenic
inducing medium. Scale bar: 100 μm. (B) ALP activity of PDLSCs was enhanced by 10−6 M kaempferol compared with the control group both on 7 and 14 days.
Mean ± SD, **P < 0.01. (C) For the alizarin red-S staining, larger and more mineralized nodules were observed in the kaempferol-treated group after inducing for
21 days. Scale bar: 100 μm. (D) The contents of extracellular matrix mineralization was relatively assessed by the absorbance at 562 nm. Mean ± SD,
***P < 0.001.

Fig. 5. Expression of osteogenesis-associated genes and proteins. ALP, RUNX2, SP7, BGLAP and CTNNB1 (β-catenin) were all enhanced by kaempferol in both mRNA
(B) and protein expression levels (A). **P < 0.01, ***P < 0.001, ****P < 0.0001.

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F. Nie, et al. Life Sciences 258 (2020) 118143

Fig. 6. Effects of XAV-939 on kaempferol-induced osteogenic promotion of PDLSCs. (A, B) The protein expression levels of the osteogenesis-associated genes SP7,
RUNX2 and BGLAP, and Wnt pathway-related proteins β-catenin, p-GSK3β and LEF were all down-regulated after treatment with XAV-939. The expression of GSK3β
showed no difference among four groups. Furthermore, the kaempferol-treated group showed the highest expression levels of osteogenesis-associated proteins and
Wnt/β-catenin pathway-related proteins.

Fig. 7. Wnt/β-catenin signaling transduction. When Wnt signaling is not activated, β-catenin is phosphorylated by a destruction complex made up of adenomatous
polyposis coli (APC), axin, and glycogen synthase kinase 3β (GSK-3β). Phosphorylated β-catenin is ubiquitinated by Beta-Transducin Repeat Containing E3 Ubiquitin
Protein Ligase (BTRC) and is eventually degraded. Conversely, when Wnt ligands bind to their coreceptors Frizzleds (Fz) and the lipoprotein receptor-related protein
(LRP) 5/6, Wnt signaling will be activated. Dishevelled (Dvl) protein will impair the destruction complex and suppress the GSK-3β-mediated phosphorylation of β-
catenin. Afterwards, the β-catenin accumulated in the cytoplasm translocates to the nucleus, where it is stabilized and interacts with T cell factor/lymphoid enhancer
binding factor (TCF/LEF), inducing the expression of downstream effector genes.

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the downstream signal factor LEF in PDLSCs. Wang Yang et al. also
demonstrated the importance of the Wnt/β-catenin pathway in the
promotion of osteoblast mineralization by kaempferol [43], which is in
agreement with our finding. Moreover, the promoting effect of
kaempferol on the osteogenesis of PDLSCs was clearly suppressed by
XAV939 by inhibiting the Wnt/β-catenin pathway. Based on the above
findings, we inferred that kaempferol stimulated Wnt signaling by ac-
tivating Ser9 phosphorylation and thereby damaging the destruction
complex, which in turn increased β-catenin translocation to the nucleus
and where it interacted with LEF, ultimately inducing the expression of
the osteogenesis-related genes. However, the precise mechanism of
action of kaempferol on PDLSCs is not fully understood, and the
pharmacological effects of kaempferol in vivo also remain unclear. More
research (such as experiments in vivo) is needed to elucidate the bio-
logical effects of kaempferol on PDLSCs.
5. Conclusion
In conclusion, our study demonstrated that kaempferol (10−6 M)
promotes the proliferation and osteogenesis of PDLSCs. The stimulation
of the Wnt/β-catenin pathway appears to play a significant role in
mediating these effects.
Declaration of competing interest
The authors declare that there are no conflicts of interest.
Acknowledgement
This work was supported by the Province Natural Science
Foundation of Shandong Province, China, grant numbers
ZR2019MH113.
Author contributions
Fujiao Nie: Conceptualization, Investigation, Formal analysis, Data
curation, Writing – original draft, Writing – review and editing;
Wenjuan Zhang: Methodology, Validation, Writing – review and
editing;
Qun Cui: Investigation, Writing – review and editing;
Yajing Fu: Investigation, Writing –review and editing;
Hongkun Li: Visualization, Writing – review and editing;
Jun Zhang: Conceptualization, Funding acquisition, Supervision,
Writing – review and editing.
References
[1] N.J. Kassebaum, E. Bernabe, M. Dahiya, et al., Global burden of severe periodontitis
in1990–2010: a systematic review and meta-regression, J. Dent. Res. 93 (11) (2014)
1045–1053.
[2] M. Shimono, T. Ishikawa, H. Ishikawa, et al., Regulatory mechanisms of periodontal
regeneration, Microsc. Res. Tech. 60 (5) (2003) 491–502.
[3] P.M. Bartold, C.A.G. Mcculloch, A.S. Narayanan, et al., Tissue engineering: a new
paradigm for periodontal regeneration based on molecular and cell biology,
Periodontology 24 (1) (2000) 253–269.
[4] Byoung-Moo Seo, Masako Miura, Stan Gronthos, et al., Investigation of multipotent
postnatal stem cells from human periodontal ligament, Lancet 364 (2004) 149–155.
[5] K. Kadar, M. Kiraly, B. Porcsalmy, et al., Differentiation potential of stem cells from
human dental origin - promise for tissue engineering, J. Physiol. Pharmacol. 60
Suppl 7 (4) (2009) 167–175.
[6] K. Mrozik, Gronthos, S. Shi, et al., A method to isolate, purify, and characterize
human periodontal ligament stem cells, Oral Biology, Humana Press, 2017, p. 1537.
[7] S.H. Kim, K.C. Choi, Anti-cancer effect and underlying mechanism(s) of kaempferol,
a phytoestrogen, on the regulation of apoptosis in diverse cancer cell models,
Toxicol. Res. 29 (4) (2013) 229.
[8] Imran Muhammad, Salehi Bahare, Sharifi-Rad Javad, et al., Kaempferol: a key
emphasis to its anticancer potential, Molecules (12) (2019 Jun 19) 24.
[9] K.P. Devi, D.S. Malar, S.F. Nabavi, et al., Kaempferol and inflammation: from
chemistry to medicine, Pharmacol. Res. 99 (2015) 1–10.
[10] J. Liberal, V. Francisco, G. Costa, A. Figueirinha, M.T. Amaral, C. Marques,
H. Girão, M.C. Lopes, M.T. Cruz, M.T. Batista, Bioactivity of Fragaria vesca leaves
9
Life Sciences 258 (2020) 118143
through inflammation, proteasome and autophagy modulation, J. Ethnopharmacol.
158 (2014) 113–122, https://doi.org/10.1016/j.jep.2014.09.043.
[11] Jayanta Kumar Patra, Eun Sil Kim, Kyounghee Oh, et al., Antibacterial effect of
crude extract and metabolites of Phytolacca americana on pathogens responsible for
periodontal inflammatory diseases and dental caries, BMC Complement. Altern.
Med. 14 (2014) 343.
[12] M. Kataoka, K. Hirata, T. Kunikata, et al., Antibacterial action of tryptanthrin and
kaempferol, isolated from the indigo plant (Polygonum tinctorium Lour.), against
Helicobacter pylori-infected Mongolian gerbils, J. Gastroenterol. 36 (1) (2001) 5–9.
[13] Wenjuan Dong, Xiuli Wei, Fayun Zhang, et al., A dual character of flavonoids in
influenza A virus replication and spread through modulating cell-autonomous im-
munity by MAPK signaling pathways, Sci. Rep. 4 (2014) 7237.
[14] G. Filomeni, I. Graziani, D. De Zio, L. Dini, D. Centonze, G. Rotilio, M.R. Ciriolo,
Neuroprotection of kaempferol by autophagy in models of rotenone-mediated acute
toxicity: possible implications for Parkinson’s disease, Neurobiol. Aging 33 (2012)
767–785, https://doi.org/10.1016/j.neurobiolaging.2010.05.021.
[15] M. Olszewska, Separation of quercetin, sexangularetin, kaempferol and iso-
rhamnetin for simultaneous HPLC determination of flavonoid agly-cones in in-
florescences, leaves and fruits of three Sorbus species, J. Pharm. Biomed. Anal. 48
(3) (2008) 629.
[16] L. Strauss, R. Santti, N. Saarinen, et al., Dietary phytoestrogens and their role in
hormonally dependent disease, Toxicol. Lett. s102–103 (102−103) (1998)
349–354.
[17] M. Miyake, N. Arai, S. Ushio, et al., Promoting effect of kaempferol on the differ-
entiation and mineralization of murine pre-osteoblastic cell line MC3T3-E1, Biosci.
Biotechnol. Biochem. 67 (6) (2003) 1199–1205.
[18] A.J. Guo, R.C. Choi, K.Y. Zheng, V.P. Chen, T.T. Dong, Z.-T. Wang, G. Vollmer,
D.T. Lau, K.W. Tsim, Kaempferol as a flavonoid induces osteoblastic differentiation
via estrogen receptor signaling, Chin. Med. 7 (2012) 10, https://doi.org/10.1186/
1749-8546-7-10.
[19] I.R. Kim, S.E. Kim, H.S. Baek, et al., The role of kaempferol-induced autophagy on
differentiation and mineralization of osteoblastic MC3T3-E1 cells, BMC
Complement. Altern. Med. 16 (1) (2016) 333.
[20] A. Wattel, S. Kamel, R. Mentaverri, F. Lorget, C. Prouillet, J.P. Petit, P. Fardelonne,
M. Brazier, Potent inhibitory effect of naturally occurring flavonoids quercetin and
kaempferol on in vitro osteoclastic bone resorption, Biochem. Pharmacol. 65 (2003)
35–42, https://doi.org/10.1016/S0006-2952(02)01445-4.
[21] Chang-Ju Kim, Sang-Hun Shin, Bok-Joo Kim, et al., The effects of kaempferol-in-
hibited autophagy on osteoclast formation, Int. J. Mol. Sci. 19 (1) (Jan 2, 2018),
https://doi.org/10.3390/ijms19010125 (pii: E125).
[22] K.M. Cadigan, R. Nusse, Wnt signaling: a common theme in animal development,
Genes Dev. 11 (1997) 3286–3305.
[23] H. Clevers, R. Nusse, Wnt/β-catenin signaling and disease, Cell 149 (6) (2012)
1192–1205.
[24] G. Rawadi, S. Roman-Roman, Wnt signalling pathway: a new target for the treat-
ment of osteoporosis, Expert Opin. Ther. Targets 9 (2005) 1063–1077.
[25] Donald A. Glass, Peter Bialek, Jong Deok Ahn, et al., Canonical Wnt signaling in
differentiated osteoblasts controls osteoclast differentiation, Dev. Cell 8 (2005)
751–764.
[26] L. Song, M. Liu, N. Ono, et al., Loss of wnt/β-catenin signaling causes cell fate shift
of preosteoblasts from osteoblasts to adipocytes, J. Bone Miner. Res. 27 (11) (2012)
2344–2358.
[27] T.F. Day, Y. Yang, Wnt and hedgehog signaling pathways in bone development, J.
Bone Joint Surg. Am. 90 (Suppl. 1) (2008) 19–24.
[28] F. Cao, J. Zhan, X. Chen, et al., miR-214 promotes periodontal ligament stem cell
osteoblastic differentiation by modulating Wnt/β-catenin signaling, Mol. Med. Rep.
16 (6) (2017, Dec) 9301–9308.
[29] J.S. Heo, S.Y. Lee, J.C. Lee, Wnt/b.catenin signaling enhances osteoblastogenic
differentiation from human periodontal ligament fibroblasts, Mol. Cell 30 (2010)
449–454.
[30] L.J. Chen, B.B. Hu, X.L. Shi, et al., Baicalein enhances the osteogenic differentiation
of human periodontal ligament cells by activating the Wnt/β-catenin signaling
pathway, Arch. Oral Biol. 78 (2017) 100–108, https://doi.org/10.1016/j.
archoralbio.2017.01.019.
[31] Shih-Min A. Huang, Yuji M. Mishina, Shanming Liu, et al., Tankyrase inhibition
stabilizes axin and antagonizes Wnt signalling, Nature 461 (7264) (2009) 614–620.
[32] K.J. Livak, D. Schmittgen Thomas, Analysis of relative gene expression data using
real- time quantitative PCR and the 2Ϫ⌬⌬CT method, Methods 25 (4) (2001)
402–408.
[33] S. Gronthos, K. Mrozik, S. Shi, et al., Ovine periodontal ligament stem cells: isola-
tion, characterization, and differentiation potential, Calcif. Tissue Int. 79 (5) (2006)
310–317.
[34] T. Akizuki, Application of periodontal ligament cell sheet for periodontal re-
generation: a pilot study in beagle dogs, J. Periodontal Res. 40 (2005).
[35] M. Mukai, Y. Yoshimine, A. Akamine, et al., Bone-like nodules formed in vitro by rat
periodontal ligament stem cells, Cell Tissue Res. 271 (3) (1993) 453–460.
[36] M.H. Choi, W.C. Noh, J.W. Park, et al., Gene expression pattern during osteogenic
differentiation of human periodontal ligament stem cells in vitro, J. Periodontal
Implant Sci. 41 (4) (2011) 167–175.
[37] S. Ivanovski, S. Gronthos, S. Shi, et al., Stem cells in the periodontal ligament, Oral
Dis. 12 (4) (2006) 358–363.
[38] X. Zheng, S.-K. Lee, O.K. Chun, Soy isoflavones and osteoporotic bone loss: a review
with an emphasis on modulation of bone remodeling, J. Med. Food 19 (1) (2016)
1–14.
[39] Rasime, Kalkan, Pinar, et al., The interactions between bone remodelling, estrogen
hormone and EPH family genes, Crit. Rev. Eukaryot. Gene Expr. 28 (2) (2018)
F. Nie, et al.
135–138.
[40] H.D. Nelson, L.L. Humphrey, P. Nygren, S.M. Teutsch, J.D. Allan, Postmenopausal
hormone replacement therapy: scientific review, JAMA 288 (7) (2002) 872–881.
[41] L. Schierbeck, Primary prevention of cardiovascular disease with hormone re-
placement therapy, Climacteric 18 (4) (2015) 492–497.
[42] M. Kruger, F. Wolber, Osteoporosis: modern paradigms for last century’s bones,
Nutrients 8 (6) (2016) 376.
[43] M. Yu, L. Wang, P. Ba, et al., Osteoblast progenitors enhance osteogenic differ-
entiation of periodontal ligament stem cells, J. Periodontol. 88 (10) (2017)
e159–e168.
[44] Yang Wang, Hongyu Chen, Hanyang Zhang, Kaempferol promotes proliferation,
migration and differentiation of MC3T3-E1 cells via up-regulation of microRNA-
101, Artif. Cells Nanomed. Biotechnol. 47 (2019) 1050–1056.
[45] X.T. Li, D. Zhang, M.K. Fu, Effects of continuous and intermittent forces on the
secretion of ALP by UMR106 osteoblast like cells in vitro, Chin. J. Orthod. 2 (2000)
55–57.
[46] Toshihisa Komori, Regulation of proliferation, differentiation and functions of os-
teoblasts by Runx2, Int. J. Mol. Sci. 20 (7) (2019) 1694.
[47] M. Bruderer, R.G. Richards, M. Alini, et al., Role and regulation of RUNX2 in os-
teogenesis, Eur. Cells Mater. 28 (28) (2014) 269–286.
[48] Ewa Hordyjewska-Kowalczyk, Anna Sowińska-Seidler, M. Olech Ewelina, et al.,
Functional analysis of novel RUNX2 mutations identified in patients with cleido-
cranial dysplasia, Clin. Genet. 96 (2019) 429–438.
[49] Zhongjian Chen, Zhiyun Song, Jinjing Yang, et al., Sp7/osterix positively regulates
and to affect tooth development and bone mineralization in zebrafish larvae, J.
Biosci. 44 (6) (Dec 2019).
[50] Jiayan Wu, Yunxiao Dou, Wangmi Liu, et al., Osteocalcin improves outcome after
10
Life Sciences 258 (2020) 118143
acute ischemic stroke, Aging (Albany NY) (Jan 5, 2020) 12.
[51] J. Gruber, Z. Yee, N.S. Tolwinski, Developmental drift and the role of Wnt signaling
in aging, Cancers 8 (2016) 73, https://doi.org/10.3390/cancers8080073.
[52] X. Yin, J. Li, B. Salmon, et al., Wnt signaling and its contribution to craniofacial
tissue homeostasis, J. Dent. Res. 94 (11) (2015) 1487–1493.
[53] C.Y. Logan, R. Nusse, The Wnt signaling pathway in development and disease,
Annu. Rev. Cell Dev. Biol. 20 (2004) 781–810.
[54] R.T. Moon, A.D. Kohn, G.V. De Ferrari, A. Kaykas, WNT and beta-catenin signalling:
diseases and therapies, Nat. Rev. Genet. 5 (2004) 691–701.
[55] Eric Hay, Chi Faucheu, Isabelle Suc-Royer, et al., Interaction between LRP5 and
Frat1 mediates the activation of the Wnt canonical pathway, J. Biol. Chem. 280
(2005) 13616–13623.
[56] Venkatesh Krishnan, Regulation of bone mass by Wnt signaling, J. Clin. Investig.
116 (5) (2006) 1202–1209.
[57] Bhukhai Kanit, Suksen Kanoknetr, Bhummaphan Narumol, et al., A phytoestrogen
diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3β pro-
tein-dependent activation of the Wnt/β-catenin signaling pathway, J. Biol. Chem.
287 (2012) 36168–36178.
[58] R.S. Jope, G.V. Johnson, The glamour and gloom of glycogen synthase kinase-3,
Trends Biochem. Sci. 29 (2004) 95–102, https://doi.org/10.1016/j. tibs. 2003. 12.
004.
[59] B.W. Doble, J.R. Woodgett, GSK-3: tricks of the trade for a multi-tasking kinase, J.
Cell Sci. 116 (2003) 1175–1186, https://doi.org/10.1242/jcs.00384.
[60] N.D. Zhang, T. Han, B.K. Huang, et al., Traditional Chinese medicine formulas for
the treatment of osteoporosis: implication for antiosteoporotic drug discovery, J.
Ethnopharmacol. 189 (2016) 61–80.