The concentration of tumor necrosis factor alpha in periapical lesions

Abstract

Introduction. The balance between proinflammatory and anti-inflammatory cytokines plays an important role in the pathogenesis of chronic periapical lesions. The aim of this study was to determine the concentration of TNF-α in tissue homogenates of periapical lesions and analyze its levels in relation to the symptomatology and the size of lesions. Materials and Methods. 93 samples of chronic periapical lesions were obtained after extraction of teeth. Samples were classified according to the clinical presentation as symptomatic and asymptomatic, and according to the size as large and small. The concentration of TNF-α was analyzed using ELISA. Results. The results showed increased production of TNF-α in symptomatic lesions compared to asymptomatic. Higher concentration of TNF-α was demonstrated in large lesions compared to small. Large symptomatic lesions showed greater concentration of TNF-α compared to small symptomatic lesions, while bigger asymptomatic lesions demonstrated higher amount of the cytokines compared to small asymptomatic lesions. Conclusion. Higher concentration of TNF-α in large symptomatic lesions indicates that TNF-α is an important factor responsible for the progression of lesions.

Full text

Serbian Dental Journal, vol. 61, No 1, 2014
UDC: DOI:
ORIGINAL ARTICLE / ORIGINALNI RAD
7
UDC: 616.314.1-002:577.112 DOI: 10.2298/SGS1401007P
Address for correspondence: Jelena POPOVIĆ, Department for Restorative Dentistry and Endodontics, Dental Department,
Bulevar Dr Zorana Djindjića 52, 18000 Niš; jelenadp@gmail.com
7
The Concentration of Tumor Necrosis Factor Alpha in
Periapical Lesions
Jelena Popović1, Tatjana Cvetković2, Tanja Džopalić3, Aleksandar Mitić1, Marija Nikolić1,
Radomir Barac1, Slavoljub Živković4
1Department for Restorative Dentistry and Endodontics, Department of Dentistry, Faculty of Medicine,
University of Niš, Niš, Serbia;
2Institute of Biochemistry, Faculty of Medicine, University of Niš, Niš, Serbia;
3Institute of Immunology, Faculty of Medicine, University of Niš, Niš, Serbia;
4Department of Restorative Odontology and Endodontics, School of Dental Medicine, University of Belgrade,
Belgrade, Serbia
SUMMARY
Introduction The balance between proinflammatory and anti-inflammatory cytokines plays an important role in the
pathogenesis of chronic periapical lesions. The aim of this study was to determine the concentration of TNF-α in tis-
sue homogenates of periapical lesions and analyze its levels in relation to the symptomatology and the size of lesions.
Materials and Methods 93 samples of chronic periapical lesions were obtained after extraction of teeth. Samples were
classified according to the clinical presentation as symptomatic and asymptomatic, and according to the size as large
and small. The concentration of TNF-α was analyzed using ELISA.
Results The results showed increased production of TNF-α in symptomatic lesions compared to asymptomatic. Higher
concentration of TNF-α was demonstrated in large lesions compared to small. Large symptomatic lesions showed
greater concentration of TNF-α compared to small symptomatic lesions, while bigger asymptomatic lesions demon-
strated higher amount of the cytokines compared to small asymptomatic lesions.
Conclusion Higher concentration of TNF-α in large symptomatic lesions indicates that TNF-α is an important factor
responsible for the progression of lesions.
Keywords: periapical lesions; cytokines; TNF-α; symptomatic; asymptomatic
INTRODUCTION
Periapical lesions represent inflammatory and immune
diseases affecting periapical tissue and surrounding bone.
These periapical processes are primarily caused by bacterial
infection of the root canal. Their existence, progression to
chronic lesions and destruction of bony structures are the
result of host defense inability to eliminate infection [1, 2].
Cytokine network plays an important role in specific
and non-specific immune responses. A number of studies
have investigated cytokine production in periapical le-
sions at the level of gene expression, in tissue homogenate
or cell cultures and have found that in some cases the bal-
ance between pro-inflammatory and immunoregulatory
cytokines is disrupted [3, 4].
Tumor necrosis factor α (TNF-α) plays an important
role in the initiation and coordination of cellular events
in the response of immune system to infection. It is a
soluble mediator released by immune cells in the pro-
cess of inflammation. It is produced mainly by macro-
phages, as well as lymphoid cells, mastocysts, endothelial
cells, myocytes, adipocytes, fibroblasts and neural tissue.
Large amounts of TNF-α are released under the influ-
ence of lipopolysaccharide and other bacterial products
and interleukin-1 (IL-1) [5, 6]. TNF is the only molecule,
beside IL-1 that can activate osteoclasts. It induces cal-
cium release from bone in vitro and may play a role in
various stages of inflammatory diseases including bone
resorption. The presence of TNF-α has been shown in
human periapical lesions and exudates from root canals
with apical periodontitis [6, 7, 8].
The aim of this study was to determine the concen-
tration of TNF-α in the tissue homogenates of periapical
lesions and analyze its levels in relation to the symptom-
atology and the size of lesions.
MATERIAL AND METHODS
The study involved 93 patients from the Dental Clinic,
Faculty of Medicine, Nis, who had diagnosed chronic peri-
apical lesions using clinical and radiographic methods.
Periapical lesions were collected from teeth that were de-
termined as non treatable and extracted. Other inclusion
criteria were healthy patients not suffering from acute or
chronic diseases that could lead to immunodeficiency,

8Popović J. et al. The Concentration of Tumor Necrosis Factor Alpha in Periapical Lesions
who were not taking antibiotics and anti-inflammatory
medications previous two months. Only teeth with peri-
apical lesions which did not show moderate or severe
form of marginal periodontitis were included in the study.
According to subjective symptoms lesions were divided
into two groups- symptomatic and asymptomatic. Clinic-
ally symptomatic lesions were characterized by swelling,
pain, discomfort when chewing or sensitivity to percus-
sion and palpation whereas asymptomatic lesions showed
no symptoms. The size of periapical lesions was measured
in millimeters using a ruler and divided into two groups:
small (≤5 mm) and large (≥6 mm) (Table 1). Since peri-
apical lesions contain granulomatous inflammatory tissue
that replaces normal bone there was no equivalent tissue
that could be used as negative control.
Before administering local anesthetics, teeth, gingiva
and mucosa around the tooth were cleaned using 0.12%
chlorhexidine and a patient rinsed mouth with 0.12%
chlorhexidine for 30 seconds. Samples of periapical le-
sions removed from the root apex were collected im-
mediately after the extraction using sterile scalpel, then
washed in sterile saline, dried using sterile cotton, placed
in a sterile plastic Eppendorf tubes and frozen at -70°C.
Using teflon crusher in an iced phosphate buffer at pH
7.4 samples were homogenized with volume adapted to
weight of the tissue obtaining the final concentration of
10%. Larger debris was sedimented by centrifugation at
1400 rpm for 1 minute at -40°C. The supernatant was fro-
zen at -70°C until further analysis was performed.
The concentration of TNF-α was measured using
ELISA test (R&D Systems Inc. Minneapolis, USA) accord-
ing to the manufacturer’s instructions. The sensitivity of
ELISA test for TNF-α was from 0.5 to 5.5 pg/ml and the
concentration of cytokines was analyzed in relation to the
size and symptomatology of periapical lesions.
Statistical analysis was performed using the Mann-
Whitney Rank Sum test using software Sigmastat and Ori-
gin. The results were expressed as mean ± standard devia-
tion. P<0.05 was considered as statistically significant.
RESULTS
TNF-α was present in all analysed periapical lesions in
significant concentrations. Graph 1 shows the concentra-
tions of TNF-α in all samples, and the average values were
analyzed with respect to the size and symptomatology. In
the group of symptomatic lesions the average concentra-
tion was 15.08 pg/ml while in the group of asymptom-
atic lesions the average value was 8.24 pg/ml. There was a
statistically significant difference in the concentration of
TNF-α between symptomatic and asymptomatic lesions
(p<0.001). In the group of large lesions the average TNF-α
concentration was 13.24 pg/ml whereas in the group of
small lesions the average value was 9.61 pg/ml. There was
a statistically significant difference between TNF-α con-
centrations in large and small lesions (p<0.01).
Graph 2 shows the mean values of TNF-α within the
groups of symptomatic and asymptomatic lesions. In the
group of symptomatic lesions, the average concentration
of TNF-α was analyzed in large and small lesions. The
average concentration of TNF-α in symptomatic large
lesions was 17.67 pg/ml, while in small symptomatic le-
sions it was 12.07 pg/ml. The analysis of the average val-
ues showed significantly higher concentrations of TNF-α
in symptomatic large lesions (p<0.05). Also, in the group
of asymptomatic lesions there was statistically significant
Ta ble 1. Pe ri a pi cal le si ons ac cor ding to sympto ma to logy and si ze
Tabela 1. Podela ispitivanih lezija u grupe prema simptomatologiji
i veličini
Lesions
Lezije
Size
Veličina Total
Ukupno
Large
Velike
Small
Male
Symptomatology
Simptomatologija
Symptomatic
Simptomatske 23 23 46
Asymptomatic
Asimptomatske 23 24 47
Total
Ukupno 46 47 93
30
24
18
12
6
0
TNF – α (pg/ml)
Symptomatic lesions
Simptomatske lezije
Asymptomatic lesions
Asimptomatske lezije
Large lesions
Velike lezije
Small lesions
Male lezije
Graph 1. TNF-α concentration in tissue homogenates of periapical
lesions in relation to symptomatology and size
Grafikon 1. Koncentracija TNF-α u homogenatu tkiva periapeksnih
lezija u odnosu na njihovu simptomatologiju i veličinu
* p<0.01 vs. large lesions; ** p<0.001 vs. symptomatic lesions
* p<0,01 u odnosu na velike lezije; ** p<0,001 u odnosu na simptomatske lezije
30
24
18
12
6
0
TNF – α (pg/ml)
Large lesions
Velike lezije
Small lesions
Male lezije
Symptomatic lesions
Simptomatske lezije
Asymptomatic lesions
Asimptomatske lezije
Graph 2. TNF-α concentration of sympthomatic and asympthomatic peri-
apical lesions
Grafikon 2. Koncentracija TNF-α u okviru grupa simptomatskih i asimpto-
matskih lezija
* p<0.05 vs. large lesions; ** p<0.01 vs. large lesions
* p<0,05 u odnosu na velike lezije; ** p<0,01 u odnosu na velike lezije

9Stomatološki glasnik Srbije. 2014;61(1):7-13
difference in the concentrations of TNF-α in relation to
the size of lesions. The average concentration of TNF-α
in asymptomatic large lesions was 8.96 pg/ml, while it
was 7.58 pg/ml in asymptomatic small lesions (p<0.01).
Graph 3 shows the concentration of TNF-α within the
groups of large and small lesions where statistical signifi-
cance was analyzed in relation to the symptomatology.
The analysis showed statistically significant difference in
the concentration of TNF-α in large symptomatic lesions
(17.67 pg/ml) compared to large asymptomatic lesions
(8.96 pg/ml) (p<0.001). Statistically significant difference
was noticed in small lesions where the concentration of
TNF-α in small symptomatic lesions was 12.07 pg/ml, while
it was 7.58 pg/ml in small asymptomatic lesions (p<0.001).
DISCUSSION
Host response to antigen stimulation in chronic inflam-
matory processes is mainly controlled by the balance
between proinflammatory and anti-inflammatory cyto-
kines. While proinflammatory cytokines, such as IL-1, IL-
6, TNF-α, TNF-β, chemokines and Th1 cytokines promote
inflammation in the periapical tissue and activate osteo-
clastic bone resorption [1, 2], the role of anti-inflamma-
tory cytokines is important for the suppression of inflam-
matory processes and beginning of healing process [3,
9, 10]. This specific role of cytokines was studied in the
research of Gazivoda et al. [4] which showed that inflam-
matory cells from periapical lesions produced significant
levels of pro-inflammatory (IL-1β, IL-6, IL-8 and TNF-α)
and immunoregulatory (IL-10 and TGF-β) cytokines in
vitro. The authors investigated whether the production of
cytokines was associated with clinical symptoms and the
composition of infiltrating cells. In accordance with previ-
ous results [10,11] they found that symptomatic lesions
contained higher portion of neutrophils. The recruitment
of granulocytes in these lesions was probably caused by
re-infection of the root canal space and further reacti-
vation of chronic periapical process [1, 2]. Granulocytes
present together with activated and infiltrating macro-
phages produce a number of soluble mediators, including
proinflammatory cytokines [12].
IL-1, predominantly produced by mononuclear phago-
cytes, polymorphonuclear leukocytes and connective tis-
sue cells in periapical lesions, has been considered as pri-
mary stimulator of periapical bone destruction [9, 13, 14,
15]. Production of IL-1 in periapical lesions is regulated
by cytokines originating from Th1 cells, such as interferon
gamma (IFN-γ). IFN-γ as a potent activator of macro-
phages, stimulates the expression of IL-1 and TNF-α by
these cells. TNF-α induces the production of IL-1 whereas
IL-1 alone stimulates its own synthesis by positive feed-
back mechanism [2].
The current study analyzed the concentration of pro-
inflammatory cytokine TNF-α in the tissue homogenates of
chronic periapical lesions in relation to the symptoms and
the size of the lesions. Results demonstrated that the level
of TNF-α was significantly higher in symptomatic lesions
compared to asymptomatic and in large lesions compared
to small. Therefore, TNF-α can be considered responsible
for mediation of development and progression of periapical
lesions. The research of Gazivoda et al. [4] showed no sig-
nificant difference in the level of TNF-α between symp-
tomatic and asymptomatic lesions. According to their study,
increased secretion of TNF-α in large lesions may be associ-
ated with a different composition of infiltrating cells. Danin
et al. [3] suggested that activated macrophages may be the
main source of TNF-α. In contrast, Ma et al. [16] reported
decreased numbers of macrophages in large lesions. The
reason for this finding is not clear, however, the study of
Artese et al. [7] showed that although 41% of mononuclear
cells in periapical lesions are macrophages, only 2-3% of
them produced IL-1β and TNF-α. Danin et al. [3] found
significant levels of TNF-α in only two patients with peri-
apical lesions out of 25 while Safavi and Rossomando [6]
and Pezelj-Ribarić et al. [17] found TNF-α in all samples
of periapical exudates. Brekalo-Pršo et al. in their study [5]
demonstrated high concentrations of TNF-α in all symp-
tomatic and asymptomatic lesions. Slightly higher con-
centration of TNF-α was observed in symptomatic lesions
but the difference was not statistically significant. Such
differences may occur due to the different state of activat-
ing macrophages depending on the clinical case but also
as a result of different origin of produced cytokines due to
various experimental approaches (tissue extracts or culture
supernatants of inflammatory cells).
CONCLUSION
Symptomatic lesions showed increased production of
TNF-α compared to asymptomatic. Higher concentration
of TNF-α was demonstrated in large lesions compared
to small. Large symptomatic lesions showed greater con-
centration of TNF-α than small symptomatic lesions and
large asymptomatic lesions showed higher concentration
of cytokines compared to asymptomatic small lesions.
Based on these results it can be concluded that TNF-α
is an important factor responsible for the progression of
periapical lesions.
30
24
18
12
6
0
TNF – α (pg/ml)
Symptomatic lesions
Simptomatske lezije
Asymptomatic lesions
Asimptomatske lezije
Large lesions
Velike lezije
Small lesions
Male lezije
Graph 3. TNF-α concentration of large and small periapical lesions
Grafikon 3. Koncentracija TNF-α u okviru grupa velikih i malih lezija
* p<0.001 vs. symptomatic lesions
* p<0,001 u odnosu na simptomatske lezije

10
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Received: 01/11/2013 • Accepted: 10/01/2014
Popović J. et al. The Concentration of Tumor Necrosis Factor Alpha in Periapical Lesions

11
Ispitivanje koncentracije faktora nekroze tumora alfa u
tkivnim homogenatima periapeksnih lezija
Jelena Popović1, Tatjana Cvetković2, Tanja Džopalić3, Aleksandar Mitić1, Marija Nikolić1,
Radomir Barac1, Slavoljub Živković4
1Odeljenje za bolesti zuba i endodonciju, Klinika za stomatologiju, Medicinski fakultet, Univerzitet u Nišu, Niš, Srbija;
2Institut za biohemiju, Medicinski fakultet, Univerzitet u Nišu, Niš, Srbija;
3Institut za imunologiju, Medicinski fakultet, Univerzitet u Nišu, Niš, Srbija;
4Klinika za bolesti zuba, Stomatološki fakultet, Univerzitet u Beogradu, Beograd, Srbija
KRATAK SADRŽAJ
Uvod Ba lans iz me đu pro in fla ma tor nih i an ti in fla ma tor nih ci to ki na igra va žnu ulo gu u pa to ge ne zi hro nič nih pe ri a pek snih le zi ja.
Cilj ove stu di je je bio da se od re di kon cen tra ci ja fak to ra ne kro ze tu mo ra al fa (TNF-α) u ho mo ge na ti ma tki va pe ri a pek snih le zi ja i
re zul ta ti ana li zi ra ju u po gle du simp to ma to lo gi je tih le zi ja kod pa ci je na ta, od no sno ve li či ne le zi je.
Ma te ri jal i me to de ra da Is pi ta na su 93 uzor ka hro nič nih pe ri a pek snih le zi ja do bi je nih na kon eks trak ci je zu ba. Uzor ci le zi ja su,
pre ma simp to ma to lo gi ji pa ci je na ta, po de lje ni na simp to mat ske i asimp to mat ske, a pre ma ve li či ni na ve li ke i ma le. Kon cen tra ci ja
TNF-α u uzor ku is pi ti va na je po mo ću te sta ELI SA.
Re zul ta ti Kod kli nič ki simp to mat skih le zi ja uoče no je po ve ća no stva ra nje TNF-α u od no su na asimp to mat ske. Ve ća kon cen tra-
ci ja TNF-α je do ka za na i u ve li kim le zi ja ma u od no su na ma le. Ve li ke simp to mat ske le zi je su po ka za le ve ću kon cen tra ci ju TNF-α
u od no su na ma le simp to mat ske le zi je, dok su ve li ke asimp to mat ske le zi je ima le ve ću ko li či nu ovog ci to ki na u od no su na ma le
asimp to mat ske le zi je.
Za klju čak Ve ća kon cen tra ci ja TNF-α u ve li kim i u le zi ja ma s po ja ča nim kli nič kim simp to mi ma po ka zu je da je TNF-α va žan fak tor
od go vo ran za na pre do va nje le zi je.
Ključ ne re či: pe ri a pek sne le zi je; ci to ki ni; TNF-α; simp to mat ska; asimp to mat ska
UVOD
Pe ri a pek sne le zi je su in fla ma tor no-imun ska obo lje nja ko ja za-
hva ta ju pe ri a pek sna tki va i okol nu kost. Ova kvi pe ri ra dik sni
pro ce si su pri mar no iza zva ni bak te rij skom in fek ci jom iz ka na la
ko re na. Nji ho vo odr ža va nje, raz voj u hro nič ne le zi je i de struk ci-
ja ko šta nih struk tu ra po sle di ca su ne mo guć no sti me ha ni za ma
od bra ne do ma ći na da su zbi ju in fek ci ju [1, 2].
Mre ža ci to ki na igra zna čaj nu ulo gu u spe ci fič nim i ne spe-
ci fič nim imun skim od go vo ri ma. Mno ge stu di je su pro u ča va le
pro iz vod nju ci to ki na u pe ri a pek snim le zi ja ma na ni vou gen-
ske eks pre si je, tkiv nih ho mo ge na ta ili u će lij skim kul tu ra ma,
te usta no vi le da je u od re đe nim uslo vi ma ba lans iz me đu pro-
in fla ma tor nih i imu no re gu la tor nih ci to ki na po re me ćen [3, 4].
Fak tor ne kro ze tu mo ra al fa (TNF-α) igra va žnu ulo gu u ini-
ci ja ci ji i ko or di na ci ji će lij skih do ga đa ja i od go vo ru imu nskog
si ste ma na in fek ci ju. On je so lu bil ni me di ja tor ko jeg oslo ba đa-
ju imu no kom pe tent ne će li je u pro ce su za pa lje nja. Pro iz vo de
ga uglav nom ma kro fa gi, ali i lim fo id ne će li je, ma sto ci ti, en do-
tel ne će li je, mi o ci ti, adi po ci ti, fi bro bla sti i ner vno tki vo. Ve li ke
ko li či ne TNF-α se oslo ba đa ju pod uti ca jem li po po li sa ha ri da
i dru gih bak te rij skih pro izvoda i in ter le u ki na 1 (IL-1) [5, 6].
TNF-α je, po red IL-1, je di ni mo le kul ko ji ima funk ci ju oste o-
klast ne ak ti va ci je. On in du ku je oslo ba đa nje kal ci ju ma iz ko sti
in vi tro i mo že igra ti zna čaj nu ulo gu u raz li či tim sta di ju mi ma
in fla ma tor nih obo lje nja, uklju ču ju ći ko šta nu re sorp ci ju. Pri su-
stvo TNF-α je do ka za no u hu ma nim apek snim pa ro don tal nim
le zi ja ma i eks u da ti ma iz ka na la ko re na zu ba s apek snim pa ro-
don ti ti ma [6, 7, 8].
Cilj ove stu di je je bio da se od re di kon cen tra ci ja TNF-α u
ho mo ge na ti ma tki va pe ri a pek snih le zi ja i re zul ta ti ana li zi ra ju
u od no su na simp to ma to lo gi ju tih le zi ja kod pa ci je na ta, od no-
sno ve li či nu le zi je.
MATERIJAL I METODE RADA
U is tra ži va nje su uklju če na 93 pa ci jen ta Kli ni ke za sto ma to-
lo gi ju Me di cin skog fa kul te ta Uni ver zi te ta u Ni šu kod ko jih je
kli nič kim i ra di o graf skim me to da ma po sta vlje na di jag no za
hro nič ne pe ri a pek sne le zi je. Pe ri a pek sne le zi je su uzi ma ne sa
ko re no va zu ba ko ji su zbog ne mo guć no sti le če nja in di ko va ni
za eks trak ci ju. Po red pe ri a pek sne le zi je, uslov za uklju če nje
pa ci je na ta u stu di ju bio je da ne bo lu ju od akut nih ili hro nič nih
obo lje nja ko ja do vo de do sta nja imu no de fi ci jen ci je i da u pret-
hod na dva me se ca ni su uzi ma li an ti bi ot sku i an ti in fla ma tor nu
te ra pi ju. U is tra ži va nje su uklju či va ne pe ri a pek sne le zi je onih
zu ba ko ji ni su po ka zi va li ume ren ili te žak ob lik mar gi nal nog
pa ro don ti ti sa.
Is pi ti va ne le zi je su po de lje ne u dve gru pe pre ma su bjek tiv-
nim simp to mi ma pa ci je na ta: simp to mat ske i asimp to mat ske
le zi je. Kli nič ki su se simp to mat ske le zi je od li ko va le oto kom,
bo lom, ne la god no šću pri žva ka nju ili ose tlji vo šću na per ku si-
ju i pal pa ci ju, dok asimp to mat ske le zi je ni su po ka zi va le zna-
ke ili simp to me u vre me stu di je. Pe ri a pek sne le zi je su me re ne
mi li me tar skim le nji rom i u od no su na ve li či nu svr sta ne u dve
gru pe: ma le (≤ 5 mm) i ve li ke (≥6 mm) (Ta be la 1). Bu du ći
da pe ri a pek sne le zi je ob u hva ta ju re ak tiv no tki vo ko je se sa sto ji
uglav nom od gra nu lo ma to znog in fla ma tor nog tki va ko je za me-
nju je nor mal nu kost, ni je po sto jao pra vi tkiv ni ekvi va lent ko ji
bi slu žio kao ne ga tiv na kon tro la.
Pre pri me ne lo kal nog ane ste ti ka zu bi, gin gi va i slu zo ko ža
oko zu ba su oči šće ni hlor hek si di nom u kon cen tra ci ji od 0,12%,
a pa ci jent je is pi rao usta istim ras tvo rom 30 se kun di. Uzor ci pe-
ri a pek snih le zi ja su od mah po eks trak ci ji ste ril nim skal pe lom
od stra nje ni s vr ha ko re na zu ba, is pra ni u ste ril nom fi zi o lo škom
ras tvo ru, pro su še ni na ste ril noj va ti, sta vlje ni u ste ril nu pla stič-
nu epen dorf epru ve tu i za mr za va ni na -70°C. Ho mo ge ni za ci ja
Stomatološki glasnik Srbije. 2014;61(1):7-13

12
je vr še na te flon skim tuč kom u le de nom fos fat nom pu fe ru vred-
nosti pH od 7,4, či ja je za pre mi na pri la go đe na te ži ni tki va, ta ko
da fi nal na kon cen tra ci ja ho mo ge na ta iz no si 10%. Krup ni ji de-
tri tus je se di men ti ran cen tri fu gi ra njem na 1.400 obr ta ja to kom
jed nog mi nu ta na -4°C. Su per na tant je na kon to ga za mr znut na
-70°C do iz vo đe nja od go va ra ju ćih ana li za.
Kon cen tra ci ja TNF-α je od re đi va na ELI SA te stom (R&D
Systems Inc. Mi ne a po lis, SAD) pre ma uput stvu pro iz vo đa ča.
Sen zi tiv nost te sta ELI SA za TNF-α bi la je 0,5–5,5 pg/ml, a kon-
cen tra ci ja ci to ki na je ana li zi ra na u po gle du simp to ma to lo gi je i
ve li či ne pe ri a pek snih le zi ja.
Sta ti stič ka ana li za je ura đe na po mo ću Man–Vit ni je vog
(Mann–Whit ney) te sta su me ran go va ko ri šće njem pro gra ma
Sig ma stat i Ori gin. Re zul ta ti su iz ra ža va ni u vi du sred nje vred-
no sti sa stan dard nom de vi ja ci jom. Sta ti stič ki zna čaj nim raz li-
ka ma su sma tra ne one ko je su bi le pri p<0,05.
REZULTATI
Is pi ti va nje kon cen tra ci je TNF-α u tki vu pe ri a pek snih le zi ja
po ka za lo je sta ti stič ki zna čaj nu kon cen tra ci ju ci to ki na u svim
uzor ci ma. Na gra fi ko nu 1 pri ka za na je kon cen tra ci ja TNF-α
kod svih uzo ra ka, a pro seč ne vred no sti su ana li zi ra ne u od no-
su na simp to ma to lo gi ju i ve li či nu. U gru pi simp to mat skih le zi ja
pro seč na kon cen tra ci ja je bi la 15,08 pg/ml, a u gru pi asimp to-
mat skih le zi ja 8,24 pg/ml. Raz li ka je bi la sta ti stič ki zna čaj na
(p<0,001). U gru pi ve li kih le zi ja pro seč na kon cen tra ci ja TNF-α
je bi la 13,24 pg/ml, a u gru pi ma lih 9,61 pg/ml. Raz li ka je i iz-
me đu ove dve po sma tra ne gru pe uzo ra ka ta ko đe bi la sta ti stič ki
zna čaj na (p<0,01).
Na gra fi ko nu 2 pri ka za ne su pro seč ne vred no sti kon cen tra-
ci je TNF-α u okvi ru gru pa simp to mat skih i asimp to mat skih
le zi ja. U gru pi simp to mat skih le zi ja ana li zi ra na je pro seč na
kon cen tra ci ja TNF-α iz me đu ve li kih i ma lih le zi ja. Ona je kod
ve li kih le zi ja bi la 17,67 pg/ml, a kod ma lih 12,07 pg/ml. Raz li ka
je bi la sta ti stič ki zna čaj na (p<0,05). I u gru pi asimp to mat skih
le zi ja za pa že na je sta ti stič ki zna čaj na raz li ka u kon cen tra ci ja-
ma TNF-α u od no su na ve li či nu le zi ja. Pro seč na kon cen tra ci ja
TNF-α kod ve li kih le zi ja bi la je 8,96 pg/ml, a kod ma lih 7,58
pg/ml (p<0,01).
Na gra fi ko nu 3 pri ka za na je kon cen tra ci ja TNF-α u okvi ru
gru pa ve li kih i ma lih le zi ja, gde je sta ti stič ka zna čaj nost ana-
li zi ra na u od no su na simp to ma to lo gi ju. Ana li za je po ka za la
sta ti stič ki zna čaj no ve ću (p<0,001) kon cen tra ci ju TNF-α u
ve li kim simp to mat skim le zi ja ma (17,67 pg/ml) u od no su na
ve li ke asimp to mat ske lezije (8,96 pg/ml). Sta ti stič ki zna čaj na
raz li ka je uoče na i kod ma lih le zi ja, gde je kod simp to mat skih
kon cen tra ci ja TNF-α bi la 12,07 pg/ml, a kod asimp to mat skih
7,58 pg/ml (p<0,001).
DISKUSIJA
Ba lans iz me đu pro in fla ma tor nih i an ti in fla ma tor nih ci to ki na
u ve li koj me ri kon tro li še od go vo re do ma ći na na an ti ge nu sti-
mu la ci ju kod hro nič nih za pa ljenj skih pro ce sa. Dok pro in fla ma-
tor ni ci to ki ni, kao što su IL-1, IL-6, TNF-α, TNF-β, he mo ki ni i
Th1 ci to ki ni, pro pa gi ra ju in fla ma ci ju u pe ri a pek snim tki vi ma
i ak ti vi ra ju oste o klast nu re sorp ci ju ko sti [1, 2], ulo ga an ti in fla-
ma tor nih ci to ki na je va žna za su pre si ju in fla ma tor nih pro ce sa
i pro ce se za ra sta nja unu tar pe ri a pek snih le zi ja [3, 9, 10]. Ta kva
ulo ga ci to ki na je pro u ča va na u stu di ji Ga zi vo de i sa rad ni ka [4],
gde je do ka za no da in fla ma tor ne će li je iz pe ri a pek snih le zi ja
stva ra ju zna čaj ne ni voe pro in fla ma tor nih (IL-1β, IL-6, IL-8 i
TNF-α) i imu no re gu la tor nih (IL-10 i TGF-β) ci to ki na in vi tro.
Kao zna čaj ne in di vi du al ne raz li ke ko je su po sma tra ne, auto ri
su is pi ti va li da li je pro iz vod nja ci to ki na po ve za na s kli nič kim
od li ka ma le zi ja i sa sta vom in fil tri ra ju ćih će li ja. U skla du s pret-
hod nim re zul ta ti ma [10, 11] uoče no je da simp to mat ske le zi je
sa dr že ve ći pro ce nat ne u tro fil nih gra nu lo ci ta. Re gru to va nje gra-
nu lo ci ta u le zi ju je ve ro vat no iza zva no re in fek ci jom iz ka na la
ko re na i da ljom re ak ti va ci jom hro nič nog pe ri a pek snog pro ce sa
[1, 2]. Gra nu lo ci ti, za jed no s ak ti vi ra nim pri sut nim i in fil tri-
ra ju ćim ma kro fa gi ma, pro iz vo de broj ne so lu bil ne me di ja to re,
uklju ču ju ći pro in fla ma tor ne ci to ki ne [12].
Kao pri mar ni sti mu la tor pe ri a pek sne ko šta ne de struk ci je
na vo di se IL-1 [9, 13], ci to kin ko ji pre do mi nant no stva ra ju mo-
no nu kle ar ni fa go ci ti, po li mor fo nu kle ar ni le u ko ci ti i ve ziv not-
kiv ne će li je u pe ri a pek snim le zi ja ma [14, 15]. Pro iz vod nja IL-1
u pe ri a pek snim le zi ja ma je re gu li sa na ci to ki ni ma ko ji po ti ču
od Th1 će li ja, ka kav je in ter fe ron ga ma (IFN-γ). IFN-γ, kao
moć ni ak ti va tor ma kro fa ga, re gu li še eks pre si ju IL-1 i TNF-α
od stra ne ovih će li ja. TNF-α iza zi va stva ra nje IL-1, dok sam
IL-1 sti mu li še sop stve nu sin te zu me ha ni zmom po zi tiv ne po-
vrat ne spre ge [2].
U ovom is tra ži va nju ana li zi ra ne su kon cen tra ci je pro in fla-
ma tor nog ci to ki na TNF-α u tkiv nim ho mo ge na ti ma hro nič nih
pe ri a pek snih le zi ja u od no su na simp to ma to lo gi ju i ve li či nu
le zi ja. Re zul ta ti stu di je, ko ji po ka zu ju da su ni voi TNF-α bi li
sta ti stič ki zna čaj no vi ši u simp to mat skim le zi ja ma u od no su na
asimp to mat ske i u ve li kim le zi ja ma u od no su na ma le, go vo re
da je TNF-α me di ja tor od go vo ran za raz voj i na pre do va nje pe-
ri a pek snih le zi ja. U is tra ži va nju Ga zi vo de i sa rad ni ka [4] ni je
uoče na zna čaj na raz li ka u ni vou TNF-α iz me đu simp to mat skih
i asimp to mat skih le zi ja. Pre ma nji ho vom tu ma če nju, po ve ća no
lu če nje TNF-α u ve li kim le zi ja ma mo že bi ti u ve zi s raz li či tim
sa sta vom in fil tri ra ju ćih će li ja. Pre ma Da ni nu (Da nin) i sa rad-
ni ci ma [3], ak ti vi ra ni ma kro fa gi se sma tra ju glav nim iz vo rom
TNF-α. Su prot no to me, Ma (Ma) i sa rad ni ci [16] su ob ja vi li
da u ve li kim le zi ja ma ima ma nje ma kro fa ga. Raz log za ta kve
na la ze ni je ja san, ali stu di ja Ar te za (Ar te se) i sa rad ni ka [7] je
po ka za la da, iako 41% mo no nu kle ar nih in fla ma tor nih će li ja u
pe ri a pek snim le zi ja ma či ne ma kro fa gi, sa mo 2–3% njih pro-
iz vo di IL-1β i TNF-α. Da nin i sa rad ni ci [3] su pro na šli zna-
čaj ne ni voe TNF-α kod sa mo dva pa ci jen ta od 25 is pi ta ni ka
s pe ri a pek snim le zi ja ma, dok su Sa fa vi (Sa fa vi) i Ro so man do
(Ros so man do) [6] i Pe zelj-Ri ba ri će va i sa rad ni ci [17] ot kri li
TNF-α u svim uzor ci ma pe ri a pek snih eks u da ta. U is tra ži va nju
autor ke Bre ka lo-Pr šo i sa rad ni ka [5] utvr đe ne su vi so ke kon-
cen tra ci je TNF-α u svim simp to mat skim i asimp to mat skim
le zi ja ma. Ne što ve ća kon cen tra ci ja je uoče na u simp to mat skim
le zi ja ma, me đu tim, raz li ka ni je bi la sta ti stič ki zna čaj na. Ova-
kve raz li ke mo gu na sta ti usled raz li či tih ak ti vi ra ju ćih sta nja
ma kro fa ga u za vi sno sti od kli nič kih si tu a ci ja, ali i kao re zul tat
raz li či tog po re kla stvo re nog ci to ki na zbog raz li či tih eks pe ri-
men tal nih pri stu pa (tkiv ni eks trakt ili kul tur ni su per na tan ti
in fla ma tor nih će li ja).
Popović J. et al. The Concentration of Tumor Necrosis Factor Alpha in Periapical Lesions

13Stomatološki glasnik Srbije. 2014;61(1):7-13
ZAKLJUČAK
Kli nič ki simp to mat ske le zi je se ma ni fe stu ju po ve ća nom pro iz-
vod njom TNF-α u od no su na asimp to mat ske. Ve ća kon cen tra ci ja
TNF-α je do ka za na i u ve li kim le zi ja ma u od no su na ma le. Ve li ke
simp to mat ske le zi je su po ka za le ve ću kon cen tra ci ju TNF-α u od-
no su na ma le simp to mat ske le zi je, dok su ve li ke asimp to mat ske
le zi je ima le ve ću ko li či nu ovog ci to ki na u od no su na asimp to mat-
ske ma le le zi je. Na osno vu ova kvih re zul ta ta mo že se za klju či ti da
je TNF-α va žan fak tor od go vo ran za na pre do va nje le zi je.