Keywords conditioned medium - mesenchymal stem cells - osteoblasts - osteoprotegerin - RANKL
- tumor necrosis factor-alpha
Introduction
Periodontitis is one of the chronic inflammatory diseases that affects quality of
life. Pathophysiology of this disease is caused by microbial challenge stimulating
the host immunoinflammatory response. Innate and adaptive immunity are stimulated
to release proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β
(IL-1β). These cytokines can upregulate other inflammatory mediators associated with
bone destruction including TNF-α, IL-1β, IL-6, and prostaglandin E2.[1 ]
TNF-α can stimulate osteoblasts to produce receptor activator of NF-κB ligand (RANKL).
When RANKL binds to its receptor, RANK, on osteoclast and preosteoclast cell surfaces,
it promotes osteoclast recruitment and stimulates osteoclast proliferation and differentiation.[2 ] This process is inhibited by osteoprotegerin (OPG) that acts as a decoy receptor
by binding to RANKL and blocking its interaction with RANK, and OPG is produced by
a variety of cell types including osteoblasts.[3 ] In addition, TNF-α can either activate or inhibit osteoblastic differentiation.
TNF-α upregulated ALP activity of osteoblasts in a dose-dependent manner. In contrast,
other studies showed that ALP activity was increased by low concentrations but decreased
at high concentrations of TNF-α.[2 ] Thus, osteoblast lineage cells may be an important therapeutic target in the prevention
of alveolar bone loss through the modulation of the RANKL/RANK/OPG axis.
Progenitor cells from bone and gingival connective tissue did not provide new connective
tissue attachment.[4 ]
[5 ] Instead, healing was characterized mainly by root resorption and ankylosis.[4 ] On the other hand, periodontal ligament cells can differentiate into cementum-forming
cells, bone-forming cells, or fibroblasts; therefore, they possess the ability to
reestablish connective tissue attachment with new cementum formation.[6 ]
Periodontal ligament stem cells (PDLSCs) are the mesenchymal stem cells (MSCs) derived
from periodontal ligament. They show the ability to regenerate periodontal tissue
through the formation of cementum/PDL-like structure and bone[7 ] and promote adhesion of collagen fibers with newly formed cementum-like structures,
mimicking physiological attachment of Sharpey's fibers in an animal study.[8 ] Transient paracrine actions from PDLSCs are strongly associated with tissue regeneration
and wound healing.[9 ] In addition, PDLSCs possess the ability to suppress immune reactions.[10 ] However, there are some limitations associated with the use of PDLSCs in tissue
regeneration including the risk of tumorigenesis, donor quality, and immune rejection.[11 ]
Research on the use of conditioned medium (CM) of MSCs is growing. The PDLSCs-derived
conditioned medium (PDLSCs-CM) contains various growth factors, proinflammatory and
anti-inflammatory cytokines, and tissue regenerative agents[11 ] secreted through either the autocrine or paracrine actions.[12 ] The advantages of CM are the ease of manufacturing and transportation and no need
of donor-recipient matching.[11 ] Recent studies found that PDLSCs-CM could reduce TNF-α and IL-1β gene expression
in lipopolysaccharide-challenged THP-1 cells (monocytoid human cell line) and MO3.13
(oligodendrocyte progenitor cells), as well as IL-1β-challenged chondrocytes, synoviocytes,
and meniscus.[13 ]
[14 ] Due to the role of TNF-α in alveolar bone resorption, we aimed to evaluate whether
PDLSCs-CM could alter the expression of genes related to bone homeostasis and differentiation
of TNF-α-challenged osteoblasts.
Materials and Methods
Cell Culture
The PDLSCs obtained from the previous study[15 ] were cultured in Dulbecco's modified Eagle's medium (DMEM: HyClone, Fisher Scientific,
Loughborough, UK) containing 10% fetal bovine serum (FBS: Biochrome, Berlin, DE) and
1% penicillin-streptomycin antimicrobial agent (Gibco, Thermo Fisher Scientific, Loughborough,
UK) at 37 °C and 5% CO2 . The culture medium was changed every other day. Cells were subcultured after 80
to 90% confluence using 0.25% trypsin/ethylenediaminetetraacetic acid (Gibco, Grand
Island, New York, US). The PDLSCs at passage 5-8 were used in this study.
Human osteoblastic cell line, human fetal osteoblastic (hFOB) 1.19, was purchased
from American Type Culture Collection (ATCC, Manassas, Virginia, US). According to
the manufacturer's instruction, the cells were cultured in a 1:1 mixture of Ham's
F12 Medium and Dulbecco's Modified Eagle's Medium without phenol red supplement with
2.5 mM L-glutamine (Gibco, Grand Island, New York, US), 10% FBS, and 0.3 mg/mL G418
(Gibco, Grand Island, New York, US). The hFOBs were seeded in 75 cm2 cell culture flasks (Thermo Fisher Scientific, Waltham, Massachusetts, US) under
the standard conditions of 34 °C and 5% CO2 . The culture medium was changed every 2 to 3 days.
Determination of Gene Expression in TNF-α-Challenged Osteoblasts
Osteoblasts (2 × 105 cells) were seeded in 6-well plates at least 24 hours to ensure proper attachment.
After that, cells were cultured with fresh DMEM mixed with 50 ng/mL TNF-α (R&D Systems,
Minneapolis, Minnesota, US) and incubated at 37°C and 5% CO2 for 24 and 48 hours. Cells cultured in fresh DMEM without TNF-α were served as control.
Expression of RANKL, OPG, and IL-1β was analyzed by quantitative reverse transcription
polymerase chain reaction (RT-qPCR). Briefly, total RNA was extracted using TRIzol
reagent (Invitrogen, Carlsbad, California, US) according to the manufacturer's instruction.
Purity and concentration of RNA were assessed using nanophotometer (Thermo Fisher
Scientific, Waltham, Massachusettes, US). To eliminate any contaminated DNA, DNase
I, RNase-free (Thermo Fisher Scientific, Waltham, Massachusettes, US) was used. The
purified RNA was reversed transcribed to cDNA using an iScript reverse transcription
supermix for RT-qPCR (Bio-Rad, Hercules, California, US) according to the manufacture's
instruction. Quantitative PCR was performed to compare the expression of the interested
genes using Luna Universal qPCR Master Mix (Luna, Ipswich, Massachusetts, US). Comparative
cycle threshold (CT ) was analyzed for relative gene expression with 2-ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control
gene. The primer sequences for RANKL, OPG, and IL-1β used in this study were shown
in [Table 1 ].
Table 1
Primer sequences for RT-qPCR
Genes
Sequences
Product length (bps)
Annealing temperature (°C)
Ref.
RANKL
F: 5′-TGATTCATGTAGGAGAATTAAA
CAGG-3′
R: 5′-GATGTGCTGTGATCCAACGA-3′
82
59
Zheng et al 2018[16 ]
OPG
F: 5′-TGAGGAGGCATTCTTCAGGT-3′,
R: 5′-CGCTGTTTTCACAGAGGTCA-3′
236
60
Yeom et al 2021[17 ]
IL-1β
F:5′-TGAGGATGACTTGTTCTTTGAAG-3′
R: 5′-GTGGTGGTCGGAGATTCG-3′
115
60
Ballerini et al 2017[14 ]
GAPDH
F: 5′-CTCATTTCCTGGTATGACACC-3′
R: 5′-CTTCCTCCTGTGCTCTTGCT-3′
122
60
Eslaminejad et al, 2010[18 ]
Abbreviations: bps, base pairs; IL-1β, interleukin-1 β; OPG, osteoprotegerin; RANKL,
receptor activator of NF-κB ligand; RT-qPCR, reverse transcription-quantitative polymerase
chain reaction.
Preparation of CM from Periodontal Ligament Stem Cells
The PDLSCs were cultured in 75 cm2 cell culture flasks to 80 to 90% confluence. They were washed twice with 10 mL of
phosphate buffer saline (PBS) and refreshed with 10 mL of serum-free DMEM. Culture
supernatant was collected after 48 hours of incubation and then centrifuged (1000 g,
5 min at 4°C) and filtered through a 0.2 μm syringe filter (Pall corporation, Port
Washington, New York, US) to remove cell debris. The CM was concentrated using ultrafiltration
with a cutoff of 10 kDa (Invitrogen, Carlsbad, California, US) at 5000 g for 40 minutes
and stored at −80°C until used.
Determination of Protein Concentration in the CM
Protein concentration in PDLSCs-CM was determined by Bradford assay. Briefly, protein
standards were prepared using bovine serum albumin (Merck, Darmstadt, DE). The protein
standards and unknown samples were added into each well and mixed with 200 μL of the
Bradford reagent (Bio-Rad, Hercules, California, US) and incubated at room temperature
for 5 to 10 minutes. The measurement of the absorbance was performed at a wavelength
of 595 nm. Protein concentration of the unknown samples was determined by calculating
the absorbance at 595 nm against the standard curve.
Determination of Gene Expression of TNF-α-Challenged Osteoblasts Cultured with PDLSCs-Derived
CM
The experiment was assigned into five groups as follows:
(1) Osteoblasts cultured in DMEM supplemented with 5% FBS (control group),
(2) Osteoblasts cultured in DMEM with 50 ng/mL TNF-α,
(3) Osteoblasts cultured in 50 ng/mL TNF-α and 1 μg/mL PDLSCs-CM,
(4) Osteoblasts cultured in 50 ng/mL TNF-α and 10 μg/mL PDLSCs-CM,
(5) Osteoblasts cultured in 50 ng/mL TNF-α and 100 μg/mL PDLSCs-CM.
Osteoblasts (2 × 105 cells) were seeded in 6-well plates for at least 24 hours. Then, fresh medium was
added as assigned and incubated at 37°C and 5% CO2 for 48 hours. Determination of gene expression in each group was performed as previously
described.
Determination of Osteoblastic Differentiation through Alkaline Phosphatase Activity
and Alizarin Red Staining
Osteoblasts (1 × 104 cells) seeded in 96-well plates were cultured in the assigned medium and incubated
at 37°C and 5% CO2 . Alkaline phosphatase (ALP) activity and alizarin red staining were evaluated at
1, 3, 6, 9, and 12 days.
For ALP activity, the medium was removed. The cells were washed three times with PBS.
Two hundred microliters of ALP assay buffer (Ab171729: Abcam, Cambridge, UK) were
added into the samples. In each group, 80 μL of the samples was added into 96-well
plate, followed by 50 μL of 5 mM p-nitrophenylphosphate (pNPP, Ab146203: Abcam, Cambridge,
UK). Standard of pNPP was prepared at the same time by diluting 5 mM of pNPP with
ALP buffer to obtain 1 mM of pNPP. Then, standard was placed into each well to produce
pNPP standards of 0, 4, 8, 12, 16, and 20 nmol/well. The final volume in each well
was adjusted to 120 μL by adding ALP assay buffer. Ten μL of ALP enzyme solution was
added and incubated for 1 hour at ambient temperature in dark condition. After incubation,
20 μL of stop solution was added. The absorbance was measured at 405 nm using microplate
reader. The optical density (OD) data of the samples were obtained by comparing with
standard curve following this formula:
B =Amount of p NP in sample that obtained from standard curve
ΔT = Reaction time (1 h)
V = Volume of original sample that added to the reaction (adjust to 80 μL)
D = Sample dilution factor
To obtain the relative ALP activity, the total ALP activity calculated from this formula
was normalized in a proportion of total protein calculated from Bradford assay.
The appearance of mineralization in osteoblasts was studied by alizarin red staining.
Briefly, 1% of alizarin red S solution (Sigma-Aldrich, St. Louis, Missouri, US) was
dissolved in distilled water and adjusted to the pH of 4.2, then filtered through
a 0.22 µm syringe filter (Pall corporation, Port Washington, New York, US). After
1, 3, 6, 9, and 12 days of incubation, old medium was removed. The cells were washed
three times with PBS, fixed with cold absolute methanol for 5 minutes, and then incubated
for 30 minutes at room temperature in the dark condition. After incubation, the excess
dye was carefully washed with distilled water. Finally, images of TNF-α-treated osteoblasts
cultured without or with PDLSCs-CM at different concentrations were captured under
an optical microscope.
Statistical Analysis
The distribution of all data was examined with Shapiro–Wilk test. Data were expressed
as median (P25, P75). The differences in gene expression of TNF-α-challenged osteoblasts
at 24 and 48 hours were analyzed with Mann–Whitney U test. The effect of PDLSCs-CM
on gene expression and ALP activity among all experimental groups were analyzed with
Kruskal–Wallis test. Then, Pairwise Comparison of Group was performed to compare the
difference between groups. The statistical analysis was performed using SPSS software
version 21.0 (IBM, Westchester County, New York, US). Statistical significance was
considered with p- value less than 0.05 in all analyzes.
Results
Gene Expression of TNF-α-Challenged Osteoblasts
There was an increase in gene expression in TNF-α-challenged osteoblasts as time passed.
Compared to the untreated control, TNF-α-challenged osteoblasts expressed significantly
higher expression of RANKL at 24 and 48 hours ([Fig. 1A ]), OPG at 48 hours ([Fig. 1B ]), and IL-1β at 24 and 48 hours ([Fig. 1C ]). When compared within group, OPG expression in TNF-α-challenged osteoblasts was
significantly increased from 24 to 48 hours (p < 0.05). ([Fig. 1B ]).
Fig. 1 Expression of mRNA level in tumor necrosis factor-alpha (TNF-α)-treated osteoblasts
and untreated control. (A ) Receptor activator of NF-κB ligand (RANKL), (B ) osteoprotegerin (OPG), and (C ) interleukin-1β (IL-1β) (n = 4, each). *Statistically significant differences between groups at 24 hours (p < 0.05). † Statistically significant differences between groups at 48 hours (p < 0.05). ‡ Statistically significant differences between 24 and 48 hours (p < 0.05).
Gene Expression of TNF-α -Challenged Osteoblasts Cultured without or with PDLSCs-Derived
CM
TNF-α increased the expression of RANKL in osteoblasts. The PDLSCs-CM at 1 μg/mL could
downregulate the expression of TNF-α activated RANKL [1.33 (0.97, 2.17) vs 2.2 (1.87,
3.75)], which was comparable to a level of the control group [1.00 (1.00, 1.00)] (p = 1.00). When PDLSCs-CM was increased to 10 and 100 μg/mL, the expression of RANKL
was increased. However, the difference did not reach statistical significance ([Fig. 2A ]).
Fig. 2 Expression of mRNA level in tumor necrosis factor alpha (TNF-α)-treated osteoblasts
with periodontal ligament stem cells-derived conditioned medium (PDLSCs-CM) at 0,
1, 10, and 100 µg/mL. (A ) receptor activator of NF-κB ligand (RANKL), (B ) osteoprotegerin (OPG), (C ) OPG/RANKL ratio, and (D ) interleukin-1β (IL-1β; n = 4, each). *p < 0.05.
Stimulation of OPG expression was seen in TNF-α-treated osteoblasts cultured without
or with PDLSCs-CM ([Fig. 2B ]). The PDLSCs-CM at 100 μg/mL significantly upregulated OPG expression compared to
the control group (p < 0.05) and closed to the osteoblasts treated with TNF-α. On the other hand, PDLSCs-CM
at 1 μg/mL was the CM-treated group that least stimulated OPG expression as compared
to the TNF-α group [10.58 (7.41, 22.11) vs 17.57 (9.66, 31.04)]. In addition, the
PDLSCs-CM at 1 μg/mL tended to increase OPG/RANKL ratio compared to the TNF-α-challenged
osteoblasts without PDLSCs-CM, but the difference did not reach statistical significance
([Fig. 2C ]).
There was an elevation of IL-1β gene expression in TNF-α-challenged osteoblasts cultured
without or with PDLSCs-CM. Between group comparison revealed a significant difference
between TNF-α-treated osteoblasts without PDLSCs-CM and the control group (p < 0.05). The group with 1 μg/mL PDLSCs-CM showed the most decreased expression of IL-1β
among PDLSCs-CM group when compared to the TNF-α group without PDLSCs-CM [75.22 (69.64,
102.84) vs. 108.41 (96.51, 140.23)]. However, there was no significant difference
between these two groups ([Fig. 2D ]).
Alkaline Phosphatase Activity of TNF-α-Challenged Osteoblasts
In the control group, ALP activity in osteoblasts significantly increased after 9
and 12 days of incubation compared to day 1 (p = 0.008 and p = 0.001, respectively). When compared to the control group, ALP activity in TNF-α-treated
osteoblasts decreased at day 3 until day 12, but significantly decreased at day 6
and 9 (p < 0.05).
As shown in [Table 2 ], when the effect of PDLSCs-CM on ALP activity of TNF-α-challenged osteoblasts was
evaluated, there was no significant difference between groups at day 1 and 3. A significant
difference in ALP activity was found between groups at day 6, 9, and 12 (p < 0.05). The PDLSCs-CM at 1 μg/mL showed a slightly elevated ALP activity [30.27
(14.72, 36.77)] compared to the TNF-α-challenged osteoblasts without PDLSCs-CM [18.16
(15.51, 40.88)] at day 12.
Table 2
Relative ALP activity in human osteoblasts of all experimental groups after 1, 3,
6, 9, and 12 days of incubation
Group
n
Relative ALP expression
Median (P25, P75)
Day 1
n
Day 3
n
Day 6
n
Day 9
n
Day 12
n
p- Value
Control
5
11.56
(9.00, 22.15)
5
71.09
(42.16, 111.61)
5
140.69
(111.43, 185.55)
5
187.87
(161.03, 328.15)[a ]
5
274.19
(224.20, 309.50)[a ]
5
<0.001†
TNF-α
5
14.34
(12.10, 26.29)
5
17.26
(14.76, 36.48)
5
11.63
(8.47, 17.51)[b ]
5
27.85
(13.98, 29.90)[b ]
5
18.16
(15.51, 40.88)
5
0.141
TNF-α + CM 1 μg/mL
5
15.11
(14.08, 25.58)
5
10.52
7.94, 41.02)
5
11.18
(9.93, 17.50)
5
26.27
(21.44, 29.80)
5
30.27
(14.72, 36.77)
5
0.138
TNF-α + CM 10 μg/mL
5
15.36
(11.88, 23.67)
5
15.55
(13.32, 42.67)
5
12.34
(9.66, 20.61)
5
30.56
(15.29, 35.32)
5
12.98
(8.69, 17.29)
5
0.139
TNF-α + CM 100 μg/mL
5
17.59
(14.05, 22.39)
5
18.09
(13.44, 36.44)
5
16.01
(11.20, 20.34)
5
27.46
(15.00, 37.96)
5
17.71
(15.13, 34.09)
5
0.613
p- Value
0.639
0.057
0.014*
0.016*
0.005*
Abbreviation: ALP, alkaline phosphatase; CM, conditioned medium; TNF-α, tumor necrosis
factor-alpha.
*, p < 0.05; † , p < 0.001 (n = 5, each).
a , Statistically significant difference when compared to the control group on day
1.
b , Statistically significant difference when compared to the control group in the
same day.
Alizarin Red Staining of TNF-α -Challenged Osteoblasts
At day 1, intracellular calcium formation observed as red deposits was not seen in
all groups. The calcium deposits could be found on day 3. The amount of alizarin red
S staining in the TNF-α-challenged groups cultured with PDLSCs-CM was comparable to
that without PDLSCs-CM on the same incubation day ([Fig. 3 ]).
Fig. 3 Late stage of osteoblast differentiation observed with alizarin red S staining at
day 1, 3, 6, 9, and 12. CM, conditioned medium; TNF-α, tumor necrosis factor alpha.
Discussion
In patients with periodontitis, TNF-α in gingival crevicular fluid was ranged from
0.10 to 700,000 pg/mL.[19 ] This cytokine has a paradoxical effect in inhibiting or activating osteoblastogenesis
depending on its concentration and exposure time as well as the differentiation stage
of the responding cells, that is, mediates early stage of osteogenic differentiation
and suppresses osteoblastogenesis when MSCs are ready for the differentiation process.[2 ] TNF-α also influences osteoclast precursor differentiation and bone resorption activity
through the induction of RANKL expression within osteogenic cells.[2 ] Recent studies found that 10 and 100 ng/mL of TNF-α could stimulate RANKL expression
of osteoblasts within 24 hours[16 ]
[20 ] and 3 days,[21 ] respectively. Therefore, we designed the model mimicking bone loss in periodontitis
by using TNF-α stimulated human osteoblasts and found that 50 ng/mL of TNF-α could
significantly upregulate RANKL expression at 24 and 48 hours.
Regarding OPG, our study showed that TNF-α significantly upregulated OPG mRNA level
after 48 hours of incubation. This might be due to the effect of TNF-α itself and
the permissive incubation temperature used in this study (34°C). It was shown that
the production of OPG by cultured osteoblasts increased with cell differentiation.[22 ] The hFOB 1.19 cells, which are human fetal osteoblastic cell line, are conditionally
coded with a temperature-sensitive mutant of the SV40 large T antigen (ts-SV40LTA ) gene. When the cells were cultured at permissive temperature (33.5°C), they proliferated
rapidly. On the other hand, they demonstrated less or no proliferation and instead
spontaneously differentiated into mature osteoblastic phenotype when cultured at restrictive
temperature (39.5°C).[23 ] Thus, our finding could be, in part, explained by changing of the incubation temperature
and thus, increasing osteoblast differentiation and expression of OPG.
To our knowledge, this study was the first to evaluate the effect of PDLSCs-CM on
gene expression of TNF-α-challenged osteoblasts. The concentrations of PDLSCs-CM were
selected based on the previous study in a mouse preosteoblasts model.[24 ] In that study, they investigated the protein concentration of RANKL and OPG in MC3T3-E1
osteoblasts treated with soybean extract.[24 ] They found that 1 and 100 μg/mL of soybean extract significantly increased the protein
level of OPG in a dose-dependent manner. On the other hand, RANKL was significantly
attenuated at 1 μg/mL, but slightly increased at 100 μg/mL of soybean extract.[24 ] Thus, the concentration of PDLSCs-CM at 1, 10, and 100 μg/mL was used in this experiment.
The results of this study indicated that PDLSCs-CM did not significantly alter the
expression of genes related to bone homeostasis in TNF- α-challenged osteoblasts.
The PDLSCs-CM at 1 and 10 μg/mL tended to downregulate OPG mRNA level of TNF-α-challenged
osteoblasts compared to the group without PDLSCs-CM, although the difference did not
reach statistical significance. As TNF-α itself significantly upregulated OPG mRNA
expression of osteoblasts, it seemed that PDLSCs-CM at low concentration could attenuate
the effect of TNF-α on the expression of OPG.
Besides the individual expression of OPG and RANKL, OPG/RANKL ratio is recommended
to use as a major determinant of bone homeostasis since the process is regulated by
RANK/RANKL/OPG system. In this study, the PDLSCs-CM at 1 μg/mL tended to increase
OPG/RANKL ratio compared to the TNF-α-challenged osteoblasts without PDLSCs-CM. It
was found that, in human periodontitis biopsies, RANKL mRNA expression levels were
increased, while OPG expression levels were decreased, thus reducing the OPG/RANKL
ratio.[25 ] Therefore, PDLSCs-CM at 1 μg/mL may demonstrate the benefits in reducing bone destruction
as indicated by an increased OPG/RANKL ratio.
IL-1β plays a role in bone resorption by inducing formation of new osteoclasts from
bone marrow precursors and activating osteoclasts to resorb bone through RANKL production
by osteoblasts.[26 ] When osteoblasts were stimulated under pathological condition, IL-1β were significantly
increased in 24 h.[27 ] Previous studies reported that CM from hPDLSCs decreased mRNA expression of IL-1β.[13 ]
[28 ] Similarly, the result from this study indicated that PDLSCs-CM at 1 μg/mL downregulated
mRNA expression of IL-1β.
In this study, the effect of PDLSCs-CM on gene expression of OPG was different from
that of RANKL and IL-1β. This could be explained by the different signaling pathways
since TNF-α was signaled via the p38 MAPK pathway to mediate RANKL and IL-1 gene expression
in murine marrow stromal cells and human mesenchymal stem cells (hMSCs),[29 ] or RANKL expression in osteocytes,[21 ] whereas Wnt pathway played a role in the mRNA expression of OPG in osteoblasts.[30 ]
The PDLSCs-CM contained various cytokines that could be grouped into growth factors,
proinflammatory and anti-inflammatory cytokines, and angiogenesis-related factors.[11 ]
[28 ] Several secretory proteins in PDLSCs-CM have been reported to exhibit immunomodulatory
actions.[31 ] They can reduce the expression of IL-1β and TNF-α.[32 ]
[33 ] Previous study found that the differences in culture medium and supplements, culture
duration and condition, as well as different passage and number of cells yielded the
different level of cytokine in CM.[11 ] Therefore, this might be the reason why the concentration of CM affected the level
of gene expression.
ALP is an enzyme involved in matrix maturation of early-stage bone formation.[34 ] In physiologic condition, ALP activity continued to increase in hFOB cells after
incubation at 37°C for 3 days and reached a peak at 6 days, then continued to decline
till day 12.[35 ] In contrast, our study observed an increased ALP activity in osteoblasts after 3
days of incubation and continued to increase for another 12 days. In terms of concentration,
TNF-α at less than 1 ng/mL promoted osteogenic differentiation by upregulating ALP
activity, while at higher concentrations of TNF-α (10 and 100 ng/mL), ALP activity
reduced to a level less than the control after 48 hours of incubation.[36 ]
[37 ] In addition, TNF-α could inhibit intracellular calcium formation[38 ] and induce apoptosis of osteocytes.[39 ] Consistent to our findings, it was shown that osteoblasts treated with 50 ng/mL
of TNF-α had lower ALP activity at day 3 and decreased in mineralization after 6 days
compared to the control.
The effect of PDLSCs-CM at 0, 1, 10, and 100 μg/mL on ALP activity of TNF-α-challenged
osteoblasts in our study showed no difference between groups. Only 1 μg/mL of PDLSCs-CM
slightly increased ALP activity at day 12 when compared with TNF-α-treated group,
but this different did not reach statistical significance. This might be due to the
effect of TNF-α that could induce apoptosis of the cells since the group with 1 μg/mL
PDLSCs-CM had more vital cells of hFOBs, while other groups showed an obvious decrease
in the cell number after day 6 (data not shown).
In this study, a small sample size was a limitation. The PDLSCs-CM at 1 μg/mL resulted
in an increased OPG/RANKL ratio of TNF-α-challenged osteoblasts, though not significant,
it may be a new approach for the treatment of alveolar bone resorption. To prove this,
larger sample size will be required in a further study. In addition, the components
in PDLSCs-CM and the pathways involved in the effect of PDLSCs-CM on gene expression
and ALP activity of osteoblasts should be explored.
Conclusion
TNF-α mediated gene expression related to bone homeostasis including RANKL, OPG, and
IL-1β, and diminished ALP activity in human osteoblasts. The PDLSCs-CM at 1 μg/mL
tended to downregulate RANKL, OPG, and IL-1β gene expression of TNF-α-challenged osteoblasts
compared to the TNF-α-challenged osteoblasts without PDLSCs-CM. Meanwhile, the PDLSCs-CM
did not improve ALP activity of TNF-α-treated osteoblasts.
