Bacterial species associated with persistent apical ... · both animal and clinical studies,...
Transcript of Bacterial species associated with persistent apical ... · both animal and clinical studies,...
Bacterial species associated with persistent apicalperiodontitis exert differential effects onosteogenic differentiation
A. T. Chow1, S. Y. Quah1, G. Bergenholtz2, K. C. Lim1, V. S. H. Yu1 & K. S. Tan1
1Faculty of Dentistry, National University of Singapore, Singapore, Singapore; and 2The Sahlgrenska Academy, University of
Gothenburg, G€oteborg, Sweden
Abstract
Chow AT, Quah SY, Bergenholtz G, Lim KC, Yu
VSH, Tan KS. Bacterial species associated with persistent
apical periodontitis exert differential effects on osteogenic
differentiation. International Endodontic Journal, 52, 201–210,
2019.
Aim To determine if bacteria associated with persis-
tent apical periodontitis induce species-specific pro-
inflammatory cytokine responses in macrophages,
and the effects of this species-specific microenviron-
ment on osteogenic differentiation.
Methodology Macrophages were exposed to Ente-
rococcus faecalis, Streptococcus oralis, Streptococcus mitis,
Fusobacterium nucleatum, Treponema denticola or Tan-
nerella forsythia, and levels of TNF-a and IL-1b elicited
were determined by immunoassay. Following treat-
ment of MG-63 pre-osteoblasts with conditioned
media from bacteria-exposed macrophages, osteogenic
differentiation and viability of osteoblasts were ana-
lyzed by Alizarin Red Staining and MTS assay, respec-
tively. Statistical analysis was carried out by one-way
ANOVA with the Tukey post-hoc test. Differences were
considered to be significant if P < 0.05.
Results Macrophages exposed to Gram-positive bac-
teria did not produce significant amounts of cytokines.
F. nucleatum-challenged macrophages produced up to
four-fold more TNF-a and IL-1b compared to T. denti-
cola or T. forsythia. Only conditioned media from
macrophages treated with Gram-negative bacteria
decreased mineralization and viability of osteoblasts.
Conclusions Gram-positive bacteria did not impact
osteogenic differentiation and appeared innocuous.
Gram-negative bacteria, in particular F. nucleatum eli-
cited an enhanced pro-inflammatory response in
macrophages, inhibited osteogenic differentiation and
reduced cell viability. The findings suggest that the
presence of this organism could potentially increase
the severity of persistent apical periodontitis.
Keywords: Fusobacterium nucleatum, inflammation,
macrophages, osteoblasts, persistent apical periodontitis.
Received 23 February 2018; accepted 6 August 2018
Introduction
Bacteria in the root canal system is the main cause
of persistent endodontic disease (Siqueira & Roc�as2008). In contrast to the more diverse microbiota of
primary apical periodontitis, the microflora in root-
filled teeth is less diverse and has been observed to
be dominated by only a few bacterial species
(Molander et al. 1998). Using bacterial culture,
Gram-positive facultative anaerobes such as Entero-
coccus faecalis and Streptococcus spp. have been
reported to be frequently present (Molander et al.
1998, Sundqvist et al. 1998, Cheung & Ho 2001,
Peciuliene et al. 2001). More recent observations
using molecular-based detection have reported that
fastidious Gram-negative anaerobes including
Fusobacterium nucleatum, Treponema denticola and Tan-
nerella forsythia also can be recovered (Blome et al.
2008, Roc�as & Siqueira 2012).
Whilst the goal of root canal treatment was to
eliminate microorganisms from the root canal system,
Correspondence: Kai Soo Tan, Faculty of Dentistry, National
University of Singapore, 11 Lower Kent Ridge Road, 119083
Singapore (Tel.: +65-6516-4635; fax: +65-6774-5701;
e-mail: [email protected]).
International Endodontic Journal, 52, 201–210, 2019© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
doi:10.1111/iej.12994
201
their complete removal is difficult even with contem-
porary debridement techniques (Nair et al. 2005,
Ricucci et al. 2009). Following a reduced microbial
burden, apical periodontitis may nevertheless heal in
certain cases (Siqueira & Roc�as 2008). Indeed, in
both animal and clinical studies, bacterial presence in
up to 45% of root filled teeth was without signs of
apical periodontitis (Molander et al. 1998, Fabricius
et al. 2006). However, when apical periodontitis is
persistent, the size of the lesion is not necessarily cor-
related with the total number of bacteria in the root
canals (Molander et al. 1998, Cheung & Ho 2001,
Martinho et al. 2010, Endo et al. 2012, Murad et al.
2014). Instead, high levels of Gram-negative species
and endotoxins have been observed in both untreated
and root filled teeth with larger radiolucent lesions
(Martinho et al. 2010, Endo et al. 2012, Murad et al.
2014). These findings suggest an important role of
Gram-negative bacteria in sustaining apical periodon-
titis. However, the impact of individual bacterial spe-
cies in the pathogenesis of persistent apical
periodontitis remains unclear.
Bacteria and their components elicit inflammation
in the periapical area leading to bone destruction.
Hence, apical periodontitis is attributed to bone
resorption that outpaces bone formation. Both pro-
cesses are under the regulation of osteoblasts. Osteo-
blasts produce matrix proteins and regulators that
are essential for matrix mineralization as well as
osteoclast differentiation (Yamashita et al. 2012, Rut-
kovskiy et al. 2016). Cellular behaviour of osteoblasts
is altered during inflammation via differential
responses to a number of locally acting cytokines
(Aubin 2001, Nanes 2003). TNF-a regulates various
genes that cause inhibition of osteoblast functions
and differentiation, whereas effects of IL-1b on osteo-
blasts may be both anabolic or catabolic depending
on their concentration (Tsuboi et al. 1999, Thamma-
sitboon et al. 2006, Huang et al. 2014). Certainly, a
host of other mediators also participate as well
(Aubin 2001). So the microenvironment in apical
periodontitis consists of a mixture of diverse cytokines
that may result in outcomes different from studies
investigating the effects of single cytokines on
osteoblast differentiation.
As the inflammatory microenvironment influences
osteogenic differentiation, it was hypothesized that
healing following root canal treatment may be
affected by the differential response of macrophages to
different bacterial species. In this study, the effects of
conditioned media produced by bacteria-treated
macrophages on osteoblasts were determined. The
conditioned media consists of different arrays of
cytokines that replicates the microenvironment dur-
ing bacterial infection. The production of TNF-a and
IL-1b in the microenvironment induced by macro-
phages in response to Gram-positive and Gram-nega-
tive bacteria were compared as well as the effects of
this microenvironment on osteogenic differentiation.
Materials and methods
Cell culture
The human monocytic cell THP-1 and osteoblast-like
cell MG-63 were obtained from the American Type
Culture Collection (ATCC) (Manassas, VA, USA).
THP-1 cells were cultured in Roswell Park Memorial
Institute (RPMI 1640) medium (Hyclone, Logan, UT,
USA) supplemented with 10% heat-inactivated Foetal
Bovine Serum (FBS) (Hyclone), 2 mmol L�1 L-gluta-
mine (Life Technologies, Carlsbad, CA, USA),
1 mmol L�1 sodium pyruvate and 100 mmol L�1
HEPES. MG-63 cells were maintained in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with
10% heat-inactivated FBS (Hyclone). Cells were incu-
bated in a humidified atmosphere with 5% CO2 at
37 °C (Thermo Fisher, Waltham, MA, USA).
Preparation of heat-killed bacteria
Reference strains of E. faecalis ATCC 29212, Streptococ-
cus mitis ATCC 49456, Streptococcus oralis ATCC
35037, F. nucleatum ATCC 25586, T. denticola ATCC
35405 and T. forsythia ATCC 43037 were purchased
from the ATCC. E. faecalis, S. mitis and S. oralis were
cultured in brain heart infusion (BHI) broth (Acume-
dia, Lansing, MI, USA). F. nucleatum was cultured in
BHI broth supplemented with 0.5% of yeast extract
(Acumedia), 5 lg mL�1 hemin and 1 lg mL�1 vita-
min K. T. denticola was cultured in modified new oral
spirochete (NOS) medium. T. forsythia was cultured in
BHI broth (Acumedia) supplemented with 10 lg mL�1
N-acetyl muramic acid (Sigma-Aldrich, St Louis, MO),
0.5% of yeast extract (Acumedia), 5 lg mL�1 hemin
(Sigma-Aldrich), 5% of FBS (Hyclone) and 1 lg mL�1
vitamin K (Sigma-Aldrich). Bacterial cultures were
incubated at 37 °C in a DG250 anaerobic workstation
(Don Whitley Scientific, Bingley, UK). Bacterial cultures
were pelleted by centrifugation at 8000 9 g for
10 min. Bacterial pellets were washed twice in sterile
PBS to remove residual culture media and resuspended
Inflammation and osteogenesis Chow et al.
© 2018 International Endodontic Journal. Published by John Wiley & Sons LtdInternational Endodontic Journal, 52, 201–210, 2019202
in PBS. Bacterial suspension was heated at 80 °C for
30 min. Complete killing of bacteria was determined by
inoculating an aliquot of the heat-treated bacteria in its
respective culture media and incubated anaerobically
at 37 °C for 7–10 days.
Preparation of conditioned media from infected
macrophages
THP-1 cells were seeded at a density of 1 9 106 cells
per well in a 12-well tissue culture plates (Nunc).
THP-1 cells were treated with 50 ng mL�1 phorbol
12-myristate 13-acetate (PMA) for 48 h to differenti-
ate the monocytic cells into macrophages. Differenti-
ated macrophages were characterized by adherence
to culture plates and expression of CD14, a macro-
phage-specific marker (Park et al. 2007). Following
media change, cells were treated with heat-killed bac-
teria at a multiplicity of infection (MOI) ratio of
10 : 1 (bacteria : cells) and incubated at 37 °C with
5% CO2 for 24 h. Conditioned media (culture super-
natant) was harvested and sterilized by passing
through a 0.2 lm syringe filter (Pall, Port Washing-
ton, NY, USA).
Enzyme-linked immunosorbent assay
The amount of TNF-a and IL-1b secreted by infected
macrophages in the culture supernatant was deter-
mined using enzyme-linked immunosorbent assay
(ELISA) kits (Biolegend, San Diego, CA, USA) and
used according to the manufacturer’s protocol.
Cell culture and treatment
MG-63 was seeded at a density of 2 9 103 cells/well in
a 96-well tissue culture plates (Nunc). Osteogenic
differentiation was induced by treating the cells with
osteogenic medium which consisted of DMEM
supplemented with 50 lg mL�1 ascorbic acid (Sigma-
Aldrich), 10 nmol L�1 dexamethasone (Sigma-Aldrich)
and 5 mmol L�1 b-glycerophosphate (Sigma-Aldrich).
Cells were treated with the respective conditioned
media at a ratio of 1:1. The positive control consisted of
osteogenic medium and conditioned media produced by
uninfected macrophages at a ratio of 1:1. The negative
control consisted of DMEM without osteogenic supple-
ment and conditioned media produced by uninfected
macrophages at the same ratio. The media were chan-
ged every 3 days.
Cell viability determination
The viability of THP-1 and MG-63 cells was determined
by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethox-
yphenyl)-2-(4-sulfophenyl)-2H-tatrazolium) (MTS) assay
using the CellTiter 96 Non-Radioactive Cell Proliferation
Assay kit (Promega, Madison, MI, USA). The assay was
carried out according to the manufacturer’s protocol.
Alkaline phosphatase assay
MG-63 cells were seeded as described above in 96-
well tissue culture plates. To assess the effects of
osteogenic differentiation on alkaline phosphatase
activity, cells were treated with DMEM medium with-
out osteogenic supplement (negative control) or with
osteogenic media and conditioned media from unin-
fected macrophages at a ratio of 1:1 for 1, 3, 5, 7
and 14 days; whilst the impact of conditioned media
from macrophages infected with bacteria on alkaline
phosphatase activity were determined on days 7 and
14 post-treatment. Cell lysates were harvested using
mammalian protein extraction reagent (M-PER,
Thermo Fisher). Cell debris was removed by centrifu-
gation at 10,000 9 g at 4 °C for 10 min. Total
amount of protein present in was assayed by Bradford
reagent (BioRad, Hercules, CA, USA). ALP activity
was determined using CSPD�substrate (Life Technolo-
gies, Carlsbad, CA, USA) according to the manufac-
turer’s instructions. Alkaline phosphatase activity was
expressed as fold change per lg of total protein.
Gene expression analysis
Osteogenic differentiation of MG-63 cells was carried
out as described above in the presence of the respec-
tive conditioned media from uninfected or infected
macrophages. Total RNA was harvested using RNeasy
Mini Kit (Qiagen) according to the manufacturer’s
protocol. RNA was treated with DNAse I (Promega)
to remove residual DNA in the sample prior to reverse
transcription. cDNA synthesis was carried out using
iScript Reverse Transcription SuperMix (BioRad). The
expression of collagen type I, osteonectin and osteo-
protegerin genes were determined by quantitative
PCR (qPCR). A reaction mixture consisted of 10 lLiTaq Universal SYBR Green Supermix (BioRad),
10 pmol each of the respective forward and reverse
primer in a final volume of 20 lL. qPCR was per-
formed in a CFX Connect real-time detection system
Chow et al. Inflammation and osteogenesis
International Endodontic Journal, 52, 201–210, 2019© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd 203
(BioRad). The expression of the respective target genes
was normalized to the relative abundance of glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) and
expressed as fold change using the DDCt method
(Livak & Schmittgen 2001). Sequences of primers
used were GAPDH (50-TGTTGCCATCAATGACCCCTTand 50-CTCCACGACGTACTCAGCG); collagen type I
(50-GAACGCGTGTCATCCCTTGT and 50-GAACGAGGTAGTCTTTCAGCAACA); osteonectin (50-AGCACCCCATTGACGGGTA and 50-GGTCACAGGTCTCGAAAAAGC);and osteoprotegerin: (50-TTCCGGAAACAGTGAATCAA and 50-CGCTGTTTTCACAGAGGTCA).
Assessment of osteogenic differentiation
Alizarin Red staining was carried out to determine
the degree of mineralization on day 14 following
osteogenic differentiation. Cells were fixed with 4%
formaldehyde (Sigma-Aldrich) and stained with
40 mmol L�1 Alizarin Red (Sigma-Aldrich) dissolved
in distilled water (pH 4.2). After washing, cells were
observed and photographed under an inverted-phase
contrast microscope (Olympus, Tokyo, Japan). To
quantify the amount of staining, the stain was dis-
solved in a solution of 10% acetic acid and measured
by spectrophotometric analysis at 405 nm (Biotek
Instruments, Winooski, VT, USA).
Statistical analysis
All experiments were carried out in triplicate and
repeated three times. Data were expressed as
mean � standard deviation. Statistical significance
was assessed by one-way analysis of variance with
the Tukey post-hoc test. Differences between groups
were considered significant if P < 0.05.
Results
Bacteria differ in ability to stimulate
pro-inflammatory response in macrophages
Macrophages exposed to Gram-negative bacteria that
is F. nucleatum, T. forsythia and T. denticola produced
significantly higher levels (P < 0.001) of TNF-a and
IL-1b (Fig. 1a–b), whereas Gram-positive bacteria had
no effect. Notably, macrophages exposed to F. nuclea-
tum produced up to four-fold more cytokines compared
to T. forsythia- or T. denticola-challenged macrophages.
The viability of macrophages was not affected by the
exposure to the bacteria tested (Fig. 1c).
Figure 1 Pro-inflammatory cytokine production in macro-
phages exposed to bacterial species associated with persistent
apical periodontitis. PMA-differentiated THP-1 cells were
exposed with the indicated heat-killed bacteria for 24 h.
Amounts of (a) TNF-a and (b) IL-1b proteins secreted in the
culture supernatant determined by ELISA. (c) Viability of
macrophages after treatment with the indicated heat-killed
bacteria for 24 h. **P < 0.01; ***P < 0.001.
Inflammation and osteogenesis Chow et al.
© 2018 International Endodontic Journal. Published by John Wiley & Sons LtdInternational Endodontic Journal, 52, 201–210, 2019204
Effects of conditioned media from bacteria-treated
macrophages on osteogenic differentiation
Osteogenic differentiation of MG-63 cells was evalu-
ated by measuring alkaline phosphatase activity.
Alkaline phosphatase is a well-recognized marker for
early osteoblasts (Zernik et al. 1990). As shown in
Fig. 2a, alkaline phosphatase activity increased signif-
icantly (P < 0.001) at day 1 following osteogenic dif-
ferentiation, peaking at day 7. Treatment of MG-63
cells with conditioned media produced by macro-
phages exposed to, E. faecalis, S. mitis and S. oralis did
not significantly affect alkaline phosphatase activity
and expression of key osteogenic genes, that is
collagen type I, osteonectin and osteoprotegerin
(Fig. 2b–e). By contrast, treatment of MG-63 cells with
conditioned media of macrophages challenged by
F. nucleatum, T. forsythia and T. denticola significantly
reduced (P < 0.001) alkaline phosphatase activity and
expression of collagen type I, osteonectin and osteopro-
tegerin (Fig. 2b–e). The degree of mineralization in
MG-63 cells treated with conditioned media from
macrophages infected with E. faecalis, S. mitis or
S. oralis did not differ significantly from the positive
control cells (Fig. 3a–e,i). However, treatment of MG-
63 cells with conditioned media of macrophages chal-
lenged by F. nucleatum, T. forsythia and T. denticola
inhibited osteogenic differentiation and mineralization
by up to 50% (Fig. 3f–h,i). In addition, viability of MG-
63 cells was reduced to 30% when treated with condi-
tioned media produced by macrophages exposed to the
three Gram-negative bacterial species (Fig. 4).
Discussion
The present study observed that Gram-positive bacte-
ria did not affect macrophage responses and had no
effect on osteogenic differentiation, whilst the Gram-
negative bacteria had an ability to stimulate both
these responses. F. nucleatum was an especially strong
instigator of these events. It is interesting to compare
the present findings with the observations conducted
in a study on monkeys by Fabricius et al. (2006). In
that study, a four or five strain combination of anaer-
obic and facultative bacterial strains were inoculated
in teeth to induce apical periodontitis. Subsequent to
root canal therapy, 15 of 17 root canals with healed
lesions 2–2.5 years later were found to contain strep-
tococci species and no anaerobes. In root canals
where anaerobes were isolated, 38 of 55 teeth did not
heal after root canal treatment. The present findings
also support the view expressed by Sp�angberg (2006)
that E. faecalis may just be an opportunistic bystander
and not a strong pathogen sustaining apical periodon-
titis. Even though E. faecalis has been frequently iso-
lated from root filled teeth, our findings confirm that
E. faecalis may not be a strong contributor to the
release of inflammatory mediators in apical periodon-
titis. In a prior study by Fabricius et al. (1982), this
view was supported by the observation that Strepto-
coccus milleri and E. faecalis caused only weak periapi-
cal reactions. In contrast, Gram-negative bacteria
dominated in the mixed infection of eight-strains and
resulted in more severe periapical tissue destruction.
Thus, the use of Gram-positive bacteria to evaluate
effectiveness of disinfection techniques or antimicro-
bial compounds should be reconsidered. Rather efforts
should be directed towards eradication of Gram-
negative bacteria in both single and mixed infections.
Macrophages are key inflammatory cells responsible
for the initiation and maintenance of inflammatory
responses during apical periodontitis (Stern et al.
1981). Following an encounter with bacteria or their
components, pro-inflammatory cytokines are produced.
In this study, levels of TNF-a and IL-1b that served as
proxies for the inflammatory response were deter-
mined. These mediators have been shown to have well-
established roles as inhibitors of bone formation and
have been frequently associated with large-sized radi-
olucent lesions (Stashenko et al. 1987, Atao�glu et al.
2002, Nanes 2003, Martinho et al. 2010). The levels
of TNF-a and IL-1b produced by macrophages differed
when exposed to the various bacterial species. Macro-
phages treated with Gram-negative bacteria namely
F. nucleatum, T. forsythia and T. denticola produced sig-
nificantly more pro-inflammatory cytokines compared
to macrophages treated with the Gram-positive bacte-
ria. The higher levels of pro-inflammatory cytokines eli-
cited by Gram-negative bacteria are likely to be
attributed to the presence of lipopolysaccharide (LPS),
which is the principal constituent of the outer surface
membrane of Gram-negative bacteria. In comparison
to lipoteichoic acid (LTA) that is the major immunos-
timulatory component of Gram-positive bacteria, LPS is
more potent and induces higher concentrations of
cytokines (Kimbrell et al. 2008). In T. denticola,
lipooligosaccharide (LOS) is analogous to LPS, and both
these membrane proteins have a lipid A moiety that is
responsible for their biological activity (Preston et al.
1996).
Chow et al. Inflammation and osteogenesis
International Endodontic Journal, 52, 201–210, 2019© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd 205
Figure 2 Effects of conditioned media from bacteria-infected macrophages on expression of osteogenic markers. (a) Analysis of
alkaline phosphatase (ALP) activity in MG-63 treated with osteogenic supplements over time. The effects of conditioned media
from bacterial-infected macrophages on (b) ALP activity, (c) collagen type I, (d) osteonectin and (e) osteoprotegerin expression.
*P < 0.05; ***P < 0.001 compared to conditioned media from uninfected macrophages (positive control).
Inflammation and osteogenesis Chow et al.
© 2018 International Endodontic Journal. Published by John Wiley & Sons LtdInternational Endodontic Journal, 52, 201–210, 2019206
The hyper-inflammatory response induced by F. nu-
cleatum could be a result of heterogeneity of the lipid A
structure in this organism and may help to explain the
association of this bacterium with clinical findings such
as history of pain, tenderness to percussion, wet canals
and purulent exudate (Gomes et al. 2004). Generally,
LPS derived from most Gram-negative anaerobes are
less immune-stimulatory compared to LPS of enter-
obacteria (Asai et al. 2007). However, Fusobacterium
spp. possesses a hexa-acylated lipid A with a chemical
structure similar to that of E. coli lipid A. Both lipid A
types possess two acyloxyacyl chains at the nonreduc-
ing terminal and two 3-hydroxyacyl chains at the
reducing terminal (Asai et al. 2007). Interestingly, the
number and length of side chain groups of lipid A
impact the strength of toll-like receptor (TLR) activa-
tion. Lipid A through its greater affinity for TLR-4
could modulate binding affinities for LPS-binding pro-
tein (LBP), transfer of LBP-LPS complex to CD14, and
influence the ability of lipid A to function as a TLR-4
agonist or antagonists (Cunningham et al. 1996, Asai
et al. 2007, Coats et al. 2011). The lowered biological
Figure 3 Effects of conditioned media from bacteria-challenged macrophages on osteogenic differentiation. Alizarin Red stain-
ing was carried out on day 14. (a) MG-63 cells were cultured in the basic culture medium without osteogenic supplements
(negative control). (b–h) MG-63 cells were cultured in the presence of osteogenic media with conditioned media from macro-
phages which were (b) uninfected (positive control), or infected with (c) E. faecalis, (d) S. mitis, (e) S. oralis, (f) F. nucleatum, (g)
T. forsythia and (h) T. denticola. The cells were visualized using a 209 objective. (i) The amount of Alizarin red stain was quan-
tified by dissolving the stain in acetic acid and measuring the amount of stain using a plate reader at 405 nm. **P < 0.01;
***P < 0.001.
Chow et al. Inflammation and osteogenesis
International Endodontic Journal, 52, 201–210, 2019© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd 207
response observed to T. forsythia and T. denticola could
be attributed to the number of phosphate groups or
altered lengths and positions of the fatty acids present
in lipid A (Dixon & Darveau 2005).
Differentiation and maturation of osteoblasts are
complex processes that involve proliferation, bone
matrix synthesis and mineralization (Aubin 2001).
The MG-63 cell line used in this study represents an
osteoblast of immature phenotype that can be induced
to differentiate to mature osteoblasts (Helfrich & Ral-
ston 2003). Treatment of MG-63 with conditioned
media from macrophages infected with Gram-negative
bacteria, in particular F. nucleatum led to significant
inhibition of osteogenic differentiation and reduction
of cell viability. TNF-a and IL-1b induce apoptosis of
both murine and human osteoblasts. It has been
reported that these cytokines induced up-regulation of
cell surface Fas expression leading to enhanced acti-
vation of caspases, culminating to programmed cell
death (Tsuboi et al. 1999, Thammasitboon et al.
2006). Indeed, TNF-a and IL-1b antagonists have
been demonstrated to increase bone regeneration by
inhibiting cell apoptosis and promoting bone forma-
tion activity (Thammasitboon et al. 2006, Nakachi
et al. 2012). Furthermore, higher levels of TNF-a are
associated with periapical lesions refractory to
endodontic treatment and larger periapical lesions
(Gazivoda et al. 2009, Henriques et al. 2011).
The present results further support the findings
from previous reports that TNF-a and IL-1b reduce
the production of type I collagen and alkaline phos-
phatase (Canalis 1986, Centrella et al. 1988). In fact,
regulatory regions within the promoters of COL1a-1
and COL1a-2 genes which confer TNF inhibitory
action have been identified in osteoblasts (Kouba et al.
1999). Type 1 collagen is the major protein compo-
nent of the bone extracellular matrix, which encom-
passes pro-collagen alpha-1 (COL1a-1) and alpha-2
(COLa-2) chains, whilst alkaline phosphatase elabo-
rates bone matrix that is mineralizable. TNF-a has
been reported to reduce ALP mRNA and protein
directly or in combination with various other growth
factors such as bone morphogenetic protein-2 (BMP-
2), bone morphogenetic protein-4 (BMP-4) and IFNc(Yoshihara et al. 1989, Kuroki et al. 1994, Nakase
et al. 1997). Osteonectin on the other hand is a phos-
phoprotein that is the most abundant noncollagenous
protein present in bone which allows organization of
the bone matrix as well as early stromal mineraliza-
tion. Both animal and patient studies have revealed
that osteonectin plays a critical role in maintaining
bone mass and bone quality (Delany et al. 2000,
2008). Osteoprotegerin, a soluble decoy receptor of
receptor activator of nuclear factor kappa-Β ligand
(RANKL) reduces bone resorption by inhibiting differ-
entiation of osteoclast via suppression of RANKL bind-
ing to its functional receptor RANK. Treatment of
bone marrow stromal cells with IL-1 and TNF-a stim-
ulated OPG secretion up to 500% compared to the
control (Br€andstr€om et al. 2001). Collectively, our
results suggest that Gram-negative bacteria attenuate
healing of periapical lesions via reduction of
maturation of osteoblasts and inhibition of osteoclast
differentiation.
Contrary to the present findings, Li et al. (2016)
reported increased osteogenic differentiation of MG-63
cells after treatment with conditioned media produced
by mesenchymal stem cells. In their study, MG-63
cells were only treated for 24 h, which could explain
the stimulatory effect of inflammatory cytokines on
bone formation when present transiently. However,
longer exposure of cytokines resulted in inhibition of
bone nodule formation (Ellies & Aubin 1990). In the
present experiment, MG-63 cells were exposed to the
cytokines for 14 days to mimic the continuous micro-
bial challenge during chronic inflammatory disease.
To summarize, conditioned media was prepared by
exposing macrophages to bacterial species recovered
from root canals of teeth with persistent apical peri-
odontitis, to allow study of the outcome of interac-
tions between intraradicular microbial factors and
periapical host defence, and subsequent impact of the
inflammatory microenvironment on differentiation of
osteoblasts.
Figure 4 Effects of conditioned media from bacteria-chal-
lenged macrophages on the viability of MG-63. ***P < 0.001.
Inflammation and osteogenesis Chow et al.
© 2018 International Endodontic Journal. Published by John Wiley & Sons LtdInternational Endodontic Journal, 52, 201–210, 2019208
Conclusions
Gram-positive bacteria did not impact osteogenic differ-
entiation and appeared innocuous. Hyper-inflammatory
responses triggered by certain microorganisms may
impact healing of periapical lesion through blocking the
maturation of osteoblasts. Thus, the presence of inflam-
mophilic bacteria can perpetuate tissue destruction.
Gram-negative bacteria, in particular F. nucleatum could
potentially contribute to the severity of persistent apical
periodontitis lesion to a more significant extent than
Gram-positive bacteria.
Acknowledgement
This study was funded by a postgraduate research
grant from the Faculty of Dentistry, National Univer-
sity of Singapore.
Conflict of interest
The authors have stated explicitly that there are no
conflict of interests in connection with this article.
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