in oral cavity , mechanical stress was generated in many situations , such as mastication , functional / parafunctional habits , orthodontic treatment , and occlusal trauma .
occlusal force plays a pivotal role in the regulation of periodontium homeostasis [ 13 ] .
the mechanical force in the range of physiological condition was involved in the maintaining of the periodontium system .
however , the force exceeding physiological limitation could lead to pathological change , such as periodontal ligament ( pdl ) space widening , periodontium destruction , and alveolar bone resorption [ 5 , 6 ] .
several lines of evidence demonstrated the effect of mechanical stress on cellular response , including periodontal ligament cells ( pdls ) [ 4 , 7 ] .
it has been illustrated both in vitro and in vivo that mechanical stress influenced pdl behavior .
several techniques were employed to investigate the effect of mechanical stress in vitro , for example , shear stress , cyclic tensile stress [ 9 , 10 ] , and static compressive stress .
the previous data showed that pdl responded to mechanical stress by releasing atp [ 12 , 13 ] , increasing intracellular calcium [ 11 , 14 ] , changing actin filament organization , and upregulating of several cytokines or growth factor , including insulin - like growth factor-1 ( igf-1 ) [ 1618 ] .
igf-1 plays a role in various cellular activities , including survival , proliferation , and differentiation [ 2026 ] .
igf-1 is involved in several kinds of cells and tissues   while igf-2 plays an important role mainly during prenatal development .
it has been illustrated that human pdl expressed the igf1 receptor , implying the ability to igf-1 stimulation .
previous report showed that igf-1 enhanced human periodontal ligament cells ( hpdls ) survival by inducing antiapoptotic molecules and downregulating proapoptotic molecules .
it was noted that the application of orthodontic force on rat teeth resulted in the upregulated igf-1 release in pdl in vivo [ 16 , 18 , 28 ] , though the molecular mechanism , by which mechanical stress stimulates igf-1 expression , is yet unclear .
therefore , the present study aimed to investigate molecular signaling mechanism of intermittent mechanical stress on the igf-1 expression in human pdls .
furthermore , the influence of hypoxia on the intermittent mechanical stress regulated igf-1 expression was examined .
cell culture medium was purchased from gibco brl ( brl , carlsbad , ca , usa ) .
culture dishes and plastic tubes were purchased from corning ( corning , ny , usa ) .
cyclohexylamine , genistein , monensin , tgf- receptor i inhibitor ( sb431542 ) , and recombinant human tgf-1 ( rhtgf-1 ) were purchased from sigma - aldrich ( st . louis , mo , usa ) .
p38 mapk inhibitor ( sb203580 ) was purchased from calbiochem ( merck chemicals , gibbstown , nj , usa ) . the tgf-1 antibody was purchased from r&d systems inc .
all protocol was approved by the ethics committee of the faculty of dentistry , chulalongkorn university .
third molars and premolars extracted for orthodontic reasons at the faculty of dentistry , chulalongkorn university , were collected for cell isolation .
the periodontal tissue was obtained from middle third of teeth 's root and the tissue was cultured in standard medium ( dulbecco 's modified eagle 's medium ( dmem ) containing 10% fetal bovine serum with 1% l - glutamine and 1%
ab / am ) and incubated at 37c in a humidified atmosphere of 5% co2 in air .
after the cells migrated from the tissue and became confluent , they were detached with 0.25% trypsin - edta and subcultured at a 1  :  3 ratio . in each experiment , cells from at least 3 donors were used .
cells were incubated with cocl2 at 150 or 300  m for 30  min prior to applying intermittent mechanical stress .
a cell compressive force loading apparatus ( thai patent i d : 1401006767 ) was designed and constructed to mechanically stimulate cells in a culture plate .
cells were seeded in 6-well culture plates at a density of 3  10 cells per well overnight .
the cells were starved with serum - free culture medium for 4  h before loading force .
compressive force generator v2.5 software was used to set times , loading type , and the amount of force . in brief , the loading cycle was set to press for 1  s and to unpress for 2  s to yield a loading cycle approximately 1/3 hertz and the force amount 1.5  g / cm . in some experiments , sb203580 ( 3.5  m ) , cocl2 ( 150  m ) , cyclohexylamine ( 10  m ) , genistein ( 92.5  m ) , monensin ( 100  m ) , sb431542 ( 10  m ) , rhtgf-1 (
2  ng / ml ) , or tgf-1 antibody ( 5  g / ml ) was added in the culture condition . hpdls were seeded in 6-well plates at a density of 3  10 cells per well for applying the force and 24-well plates at density of 5  10 cells per well for being treated with cocl2 .
subsequently cells were starved with serum - free media 4  h before treatment . at 24  h
, hpdls were incubated with 3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-diphenyltetrazolium bromide solution for 30  min .
formazan crystals were solubilized in dmso / glycine buffer solution ( 0.1  m  glycine/0.1  m sodium chloride ph10 ) .
the solution was further measured for an absorbance at 570  nm in a microplate reader ( elx800 , biotek , usa ) .
after 24  h , total cellular rna was extracted with trizol reagent ( molecular research center , cincinnati , ohio , usa ) according to the manufacturer 's instructions .
rna was quantified using a nanodrop 2000 spectrophotometer ( thermo scientific , wilmington , de , usa ) .
one microgram of rna sample was converted to cdna by improm - ii ( promega , madison , wi , usa ) .
subsequently , the real - time pcr reaction was using a lightcycler instrument ( roche diagnostics , usa ) with the lightcycler 480sybr green i master kit according to the manufacturer 's specifications .
the oligonucleotide sequences were as follows : igf-1 ( nm000618.3 ) , forward 5-catgcctgctcagaagggta-3 , reverse 5-gcctctgatccttgaggtga-3 ; 18s ( nr003286.2 ) , forward 5-ggcgtcccccaacttctta-3 , reverse 5-gggcatcacagacctgttatt-3. radioimmunoprecipitation assay ( ripa ) supplemented with protease inhibitors was used to extract cellular protein .
the amount of protein was measured by a bca protein assay kit ( pierce , rockford , il ) .
whole cell lysate and condition medium were collected at 80c for measuring the level of protein .
elisa was used for measuring the protein level according to the manuals of elisa kits ( quantikine immunoassay r&d systems ) .
the absorbance of elisa reaction product was measured at od 450  nm using microplate reader ( biotek , elx800 , usa ) .
statistical analyses were performed for two independent samples using the student t - test for two - group comparisons .
a one - way analysis of variance ( anova ) followed by turkey 's post hoc analysis ( spss , chicago , il , usa ) was employed for three or more group comparisons . the p value less than 0.05 was considered as statistically significant .
we began by investigating the effect of intermittent mechanical stress on hpdls viability and morphology using a microscope at 100x magnification .
hpdls morphology was similar in all groups ( see supplementary figure 1c in supplementary material available online at http://dx.doi.org/10.1155/2015/369874 ) and mechanical stress did not affect the hpdls viability ( supplementary figures 1a and 1b ) .
next , we investigated the effect of intermittent mechanical stress on igf-1 expression in hpdls at different time points ( figure 1 )
. there was no significant difference in igf-1 expression at 2  h , 4  h , or 8  h between the intermittent mechanical stress - treated group and the control group .
however , the igf-1 mrna levels were significantly increased at 24  h after exposing to mechanical stress .
thus , these results demonstrated intermittent mechanical stress - induced igf-1 expression in hpdls at 24  h. we started to pretreat hpdls with sb203580 which is p38 mapk inhibitor prior to applying the force .
our results demonstrated that p38 mapk inhibitor failed to block intermittent mechanical stress - induced igf-1 expression in hpdls ( supplementary figure 2 ) .
the results showed that cycloheximide pretreatment inhibited the intermittent compressive force - induced igf-1 mrna expression .
further , the mechanical force - induced igf-1 expression was also inhibited by the monensin , a protein transport inhibitor ( figure 2(b ) ) .
these results imply that the intermittent mechanical stress required the release of intermediate protein to induce igf-1 expression .
the intracellular mechanism was further identified using genistein , a tyrosine kinase inhibitor ( figure 2(c ) ) .
corresponding to the effect of cycloheximide and monensin , genistein abolished the intermittent mechanical stress - induced transcription of igf-1 . taken together , we concluded that intermittent mechanical stress required intermediate protein related to tyrosine kinase to induce igf-1 expression in hpdls .
as described above , the genistein inhibition blocked the intermittent mechanical stress - induced igf-1 expression .
thus , sb431542 ( tgf- receptor type i ( tri ) inhibitor ) was chosen to clarify mechanism ( figure 3(a ) ) .
the result demonstrated that sb431542 completely suppressed intermittent mechanical stress - induced igf-1 mrna expression . to confirm the tgf-1 role in this phenomenon , the neutralizing antibody against tgf-1
correspondingly with sb431542 treatment , the neutralizing antibody against tgf-1 reduced the igf-1 transcription under intermittent mechanical stress stimulation ( figure 3(b ) ) . finally , the addition of exogenous rhtgf-1 resulted in the upregulation of igf-1 mrna levels at 24  h ( figure 3(c ) )
however , to determine intermittent mechanical stress - induced igf-1 expression through tgf-1 protein secretion , we collected the cell culture medium from intermittent mechanical stress - treated group ( cms ) as well as the control group ( cmc ) and transferred it to another set of unstimulated hpdls for 24  h. surprisingly , igf-1 expression in those cells incubated with cms - treated group and cmc - treated group did not differ ( figure 3(d ) ) .
thus , we further measured the protein levels of tgf-1 in both condition mediums and found that tgf-1 protein levels in cms did not differ from cmc ( data not shown ) .
however , the whole cell lysate from intermittent mechanical stress - treated group expressed significantly higher tgf-1 protein levels than the control group ( figure 3(e ) ) .
such evidence may imply intermittent mechanical stress - induced tgf-1 protein to activate igf-1 expression in hpdls .
the results showed that cocl2 did not significantly affect igf-1 expression in normal culture ( figure 4(a ) ) .
however , cocl2 significantly inhibited igf-1 expression upon the intermittent stress treatment in a cocl2 dose - dependent manner ( figure 4(a ) ) .
therefore , we hypothesized that cocl2 affect the intracellular signaling of tgf-1 in order to induce igf-1 expression in hpdls .
however , in pathological condition ( i.e. , periodontal disease ) , the physiological force may lead to tissue destruction [ 30 , 31 ] .
igf-1 is an important growth factor regulating cell proliferation and differentiation in hpdls [ 23 , 24 ] .
further , the hypoxic mimic condition using cocl2 could attenuate the intermittent compressive stress - induced igf-1 expression , implying that occlusal force may not induce igf-1 expression in deep periodontal pocket , where it was considered as hypoxic microenvironment . the suggested model of intermittent mechanical stress - induced igf-1 expression in hpdls
igf-1 plays an important role in bone growth and development [ 32 , 33 ] and promotes cell proliferation and osteogenic differentiation in hpdls [ 20 , 24 ] .
in addition , the in vitro study demonstrated that igf-1 is a growth factor that responds early to mechanical stress . in the in vivo orthodontic tooth movement model , the orthodontic force or occlusal stimuli
thus , the present study was the first report which demonstrated that the intermittent mechanical stress promoted igf-1 expression by hpdls through tgf-1 pathway .
the present study showed that the intermittent compressive stress enhanced the increase of tgf-1 protein expression in cell lysate and the addition of rhtgf-1 resulted in the upregulation of igf-1 expression similar to those treated with the intermittent mechanical stress .
correspondingly , it was previously demonstrated that the tgf-1 treatment significantly increased igf-1 expression in dose- and time - dependent manner in human marrow stromal osteoblast precursor cells .
it was also shown that single - dose administration of tgf-1 promoted the osteogenic maker expression via the expression of igf-1 since the knockdown of insulin receptor substrate 1 could attenuate the tgf-1-induced osteogenic marker expression .
however , it should be noted that the repeat - dose of tgf-1 led to the inhibition of igf-1 expression and subsequently caused the suppression of osteogenic differentiation in hpdls , human mesenchymal stem cells , and murine preosteoblast ( mc3t3-e1 cells ) .
moreover , tgf- inhibited migration in c2cl2 skeletal muscle satellite cell and p19 embryonal carcinoma cell via decreasing igf-1 .
collectively , several lines of evidence indicated the close relationship between tgf-1 and igf-1 in a positive or negative regulator depending on cell types . the influence of hypoxia can be found in inflamed tissue including periodontitis .
the hypoxic condition is associated with imbalance between elevating the oxygen demand from inflammatory cells penetration and inadequate oxygen supply by poor perfusion [ 26 , 37 , 38 ] . in periodontitis ,
the hpdls respond to hypoxia by increasing the inflammatory mediator [ 39 , 40 ] and enhancement of alveolar bone loss [ 41 , 42 ] .
therefore , both intermittent mechanical stress and hypoxia are contributing factors to periodontal disease progression , leading us to investigate the effect of combining those two factors on hpdls . in this study , artificial hypoxic agent , cocl2 , abolished the intermittent mechanical stress - induced igf-1 expression in hpdls .
this condition represents the clinical situation , where the physiological force was loaded on periodontitis ' teeth .
therefore , this data assumed that hypoxia attenuated the intermittent mechanical stress - induced osteogenic differentiation through decrease in igf-1 expression in hpdls .
recently , it was demonstrated that the cyclic tensile stress under hypoxic condition regulated proliferation and osteogenic differentiation in hpdls via mapk pathway .
thus , this information could imply that the type , amount , and direction of force may play an important role in the hpdls ' response under hypoxic condition .
cocl2 , an inducer of hypoxia , is well known and commonly employed to establish the physical hypoxic - like condition in cell culture [ 4346 ] .
our previous study reported that cocl2 could stabilize hypoxia - inducible factor-1 alpha ( hif-1 ) , a key transcription factor for hypoxic condition in hpdls .
in contrast to some lines of evidence , there were some aspects of detailed different mechanisms between hypoxia and cocl2 [ 51 , 52 ] .
thus , the results from the present study may not be directly implied to those of physical hypoxia setting .
further experiment is required to fully investigate the role of hypoxia on the intermittent compressive stress - induced igf-1 expression in hpdls .
the present study showed that hypoxic mimic condition attenuated the intermittent mechanical stress - induced igf-1 expression in hpdls .
corresponding to previous studies , physical hypoxia and cocl2 attenuated igf-1 expression via the suppression of runx2 and the induction of c / ebp in rat osteoblasts .
runx2 could bind to the upstream element in igf-1 gene promoter and regulated igf-1 expression .
further , in systemic investigation , the serum igf levels were decreased in acute respiratory distress patients , which were a hypoxia state . on the contrary
further , in the present study , we demonstrated that cocl2 inhibited rhtgf-1-induced igf-1 expression in hpdls .
taken together , further study to evaluate the mechanism of hypoxic mimic condition on the inhibition of tgf-1-induced igf-1 expression in hpdls is necessitated . in conclusion , our results indicated the intermittent mechanical stress - induced igf-1 expression via tgf-1 signaling pathway in hpdls .
 mechanical force was shown to promote igf-1 expression in periodontal ligament both in vitro and in vivo . though the mechanism of this effect has not yet been proved , here we investigated the molecular mechanism of intermittent mechanical stress on igf-1 expression . 
 in addition , the role of hypoxia on the intermittent compressive stress on igf-1 expression was also examined . in this study , human periodontal ligament cells ( hpdls ) 
 were stimulated with intermittent mechanical stress for 24 hours . 
 igf-1 expression was examined by real - time polymerase chain reaction . 
 chemical inhibitors were used to determine molecular mechanisms of these effects . for hypoxic mimic condition , 
 the cocl2 supplementation was employed . 
 the results showed that intermittent mechanical stress dramatically increased igf-1 expression at 24  h. the pretreatment with tgf- receptor i or tgf-1 antibody could inhibit the intermittent mechanical stress - induced igf-1 expression . 
 moreover , the upregulation of tgf-1 proteins was detected in intermittent mechanical stress treated group . 
 correspondingly , the igf-1 expression was upregulated upon being treated with recombinant human tgf-1 . 
 further , the hypoxic mimic condition attenuated the intermittent mechanical stress and rhtgf-1-induced igf-1 expression . in summary , this study suggests intermittent mechanical stress - induced igf-1 expression in hpdls through tgf-1 and this phenomenon could be inhibited in hypoxic mimic condition . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion