periodontitis is defined as the inflammation of periodontal supporting tissue caused by specific pathogenic microorganisms , resulting in the advanced destruction of the periodontal ligament and the alveolar bone and subsequently leading to the formation of a gingival recession and a periodontal pocket . although the bacteria and plaque are the initiating factors of periodontitis , a simple bacterial infection is not sufficient to explain the range of severity of this condition in different individuals . in recent years , it has become accepted that susceptibility to periodontitis varies greatly between individuals who harbor the same pathogenic microflora . epidemiological studies have found a positive correlation between psychological stress and periodontal disease , suggesting that stress , depression , or anxiety , as systemic risk factors , may modify the host response and further contribute to the occurrence of periodontal disease [ 35 ] . periodontitis is a chronic inflammatory disease characterized by episodes of active destruction in which pronounced inflammatory infiltration , attachment loss , and alveolar bone loss occur as well as periods of quiescence in which periodontal regeneration takes place , including the restoration of the damaged periodontal ligament , the gingival tissue , the alveolar bone , and the cementum to their original levels [ 6 , 7 ] . inflammation of periodontal tissue is the main pathological feature in periodontitis and the tissue injury in periodontitis may be caused by the inability of the host to resolve the inflammation . studies also have shown that when periodontitis exists , some inflammatory cytokines , such as il-1 and tnf- , are significantly increased in periodontal tissue , gingival crevicular fluid , or peripheral blood [ 8 , 9 ] . the process of periodontal healing is regulated by multiple growth factors , among which bfgf is recognized as a key factor in periodontal ligament regeneration [ 1013 ] . as a typical member of the heparin - binding growth factor family that was first isolated from bovine pituitary extracts and shown to stimulate fibroblast proliferation , bfgf has been reported to exert a variety of effects on cell proliferation , differentiation , and angiogenesis . in animal studies , the topical application of bfgf can promote periodontal healing and regeneration without epithelial downgrowth , ankylosis , or root resorption in a surgically created furcation [ 16 , 17 ] . however , few studies have focused on the expression of endogenous bfgf in the periodontal ligament during the destruction and healing process of periodontitis . more recently , several animal studies have suggested the influence of psychological stress on the susceptibility to and progression of periodontal disease , indicating that the activation of the hypothalamic - pituitary - adrenal ( hpa ) axis seems to play an important role [ 1820 ] . additionally , it is known that psychological stress can delay wound healing by affecting the host immune response [ 21 , 22 ] . however , there is limited information about the effect of psychological stress on wound healing within the periodontal tissue . thus , we hypothesized that psychological stress could delay periodontal healing through the downregulation of bfgf expression . we tested this hypothesis by establishing an animal model of experimental periodontitis with psychological stress during periodontal healing . ninety - six adult male sprague - dawley rats ( provided by the laboratory animal center of the fourth military medical university , xi'an , china ) at 8 weeks of age and weighing 247.24  11.06  g were caged in a room with controlled temperature ( 22  1c ) , humidity ( 60  5% ) , and light - dark cycle ( light on 8:00 to 20:00  h ) . the rats were randomly distributed into three groups , with 32 rats in each group : control rats ; rats with periodontitis that underwent a natural healing process ( periodontitis group ) ; rats with periodontitis that were subjected to psychological stress during the healing process ( stress group ) . there were four subgroups in each group that were evaluated at different observation time points : baseline ( immediately after ligature removal ) , 1  w ( 1 week after ligature removal ) , 2  w ( 2 weeks after ligature removal ) , and 4  w ( 4 weeks after ligature removal ) . this study was performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health . the protocol was approved by the animal research ethics committee of the fourth military medical university ( xi'an , china ) and every effort was taken to avoid animal suffering at each stage of the experiment . the rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital ( 3.5  mg / kg ) prepared in sterile saline , and a 40 silk ligature was placed around the cervix of the right second maxillary molar to induce experimental periodontitis as previously described , except for the animals in the control group , which were submitted to the same procedure but without ligature placement . the ligatures were kept in place for four weeks and served as a retention device for oral microorganisms . then , the ligatures were removed for 1 , 2 , or 4 weeks of followup during the spontaneous natural healing process in the periodontitis group or healing under chronic unpredictable mild stress ( cums ) in the stress group . after ligature removal , the rats in the psychological stress group were subjected to cums which contains seven different mild psychological stressors including damp sawdust for 24 hours , food deprivation for 12 hours , water deprivation for 12 hours , inversion of the light - dark cycle , swimming in 4c cold water for 5  min , swimming in 45c hot water for 5  min , and 1  h of restraint stress ( the rats were kept in a restrainer made of an inflexible wire mesh with a sliding door to facilitate the restraint ; during the stress procedure , the rats were not allowed to move freely , but their bodies were not constricted ) [ 23 , 24 ] . over the course of each week of the cums procedure , one of the 7 stressors was applied each day on a random schedule . the same stressor appeared only once per week to avoid stress habituation in rats . at baseline and at weeks 1 , 2 , or 4 , the rats ( n = 8 rats each ) were weighed and prepared for the subsequent tests . the body weight gain was calculated according to the formula : [ ( body weight at time point t )  ( initial body weight)]/(initial body weight ) , where the time point is baseline or week 1 , 2 , or 4 . the open - field chamber ( rd 1412-of , shanghai mobile datum corporation , shanghai , china ) was placed inside a temperature - controlled chamber . this chamber consists of a 100  cm   100  cm   80  cm plexiglas box illuminated by one fluorescent light suspended over the chamber . the sides of the chamber were white , the bottom was brown , and the top was open to the air . the activities of each rat were automatically monitored using a digital video camera for 15  min in the open field . after each test , the maze was thoroughly cleaned with 20% alcohol to eliminate the odor and trace of the previously tested animal . the distance moved in center and the total distance moved the rats were anesthetized by intraperitoneal injections of 1% sodium pentobarbital ( 3.5  mg / kg ) prepared in sterile saline . next , blood samples were collected ( 1.52  ml from each animal ) by cardiac puncture . the blood was allowed to coagulate on ice for 1  h. thereafter , the serum was isolated following refrigerated centrifugation ( at 4c ) for 30  min at 3000  rpm . the right maxillae were immediately removed and fixed in 10% formalin for > 48 hours . subsequently , decalcification was carried out in 15% edta solution at room temperature for 6 weeks . serial paraffin sections that were 5  m thick were obtained in the mesial - distal direction and stained with hematoxylin and eosin ( h&e ) for histologic analysis . the serum aliquots were aspirated and stored in microcentrifuge tubes at 20c for measurement . the serum corticosterone ( cort ) and adrenocorticotropic hormone ( acth ) levels were determined with commercial enzyme - linked immunosorbent assay ( elisa ) kits ( shanghai xitang bioengineering institute , shanghai , china ) as previously described . the measurements included evaluation of the inflammatory infiltrate by measuring the surface of the connective tissue infiltrated with inflammatory cells ( si ) , evaluation of the alveolar bone loss ( abl ) by measuring the distance between the cement - enamel junction and the most coronal border of the alveolar bone , and evaluation of the attachment loss ( al ) by measuring the distance between the cement - enamel junction and the epithelial attachment   figure 1 . immunohistochemical staining was performed using a three - step avidin - biotin complex method as previously described [ 27 , 28 ] . the primary antibodies were rabbit polyclonal antibody to il-1 ( diluted at 1  :  200 , abcam biotechnology , cambridge , usa ) , tnf- ( diluted at 1  :  200 , abcam biotechnology , cambridge , usa ) , and bfgf ( diluted at 1  :  50 , abcam biotechnology , cambridge , usa ) . the observation area was photographed using a light microscope with a magnification of 200 and 400 . ( nikon microphoto - fxa , tokyo , japan ) . areas comprising 100  200  m sections of the periodontal ligament of the mesial root surface of the maxillary second molar , which were located just below the crest of the alveolar bone , were used for investigation . according to the histological observations and preliminary data , the number and the area of bfgf - immunoreactive cells per unit area were recorded   using image analysis software ( image - pro plus , media cybernetics , usa ) . the signal intensity was measured   using a high - resolution pathological image analysis system ( image - pro plus , media cybernetics , usa ) , and the optical density of each section was obtained . the experimental data were expressed as the means  standard error of means ( sem ) . differences among the groups were analyzed by one - way analysis of variance ( anova ) with the spss 13.0 software ( spss institute , chicago , il , usa ) followed by the snk - q test for multiple comparisons . the body weight gain of the rats in the stress group was significantly lower than that in the control group at weeks 1 , 2 , and 4 ( p < 0.05 , figure 2 ) , whereas no significant difference was observed between the periodontitis group and the control group at any time point ( p > 0.05 , figure 2 ) . in the open - field test , the distance moved in the center and total distance moved by the rats in the stress group were significantly less than the distances moved by the control group at weeks 2 and 4 , whereas no significant difference was observed between the stress group and the control group at week 1 ( p < 0.05 , figures 3(a ) and 3(b ) ) . there was also no significant difference between the periodontitis group and the control group at any point ( p > 0.05 , figures 3(a ) and 3(b ) ) . the serum corticosterone levels of the rats in the stress group were significantly higher in the control group at weeks 2 and 4 , but not at week 1 ( p < 0.05 , figure 4(a ) ) . such a difference was also observed in the serum acth levels ( p < 0.05 , figure 4(b ) ) . in contrast , no significant difference was observed between the periodontitis group and the control group at any time point ( p > 0.05 , figures 4(a ) and 4(b ) ) .   in the present study , the time point when the periodontitis group and periodontitis / stress group rats having experienced 4-week ligature placement was chosen as baseline to fully explore the effects of psychological stress on periodontitis healing . that is , at baseline , the periodontitis group contained the rats suffering periodontitis and , further to experience the natural healing process , the periodontitis / stress group contained the rats suffering periodontitis and further to experience the healing process under cums , and the blank control group contained the healthy rats without experimental periodontitis . the results showed that in the control group at each time point , the invasion of inflammatory cells was confined to the area beneath the epithelium . moreover , minimal alveolar bone loss and attachment loss were observed ( figures 5(a)5(d ) ) . in the periodontitis group and the stress group at the baseline observation point , there was a significant periodontal breakdown , including pronounced inflammatory infiltration , mostly composed of mononuclear cells with a few polymorphonuclear leukocytes , significant alveolar bone loss , and attachment loss ( figures 5(e ) and 5(i ) ) . the data analysis indicated that inflammatory infiltrate ( si ) , alveolar bone loss ( abl ) , and attachment loss ( al ) were significantly increased compared with the matched control group ( p < 0.05 , figures 5(m)5(o ) ) . after ligature removal , the rats in the periodontitis group showed spontaneous healing of the soft tissue and remodeling of the alveolar bone in the following four weeks , whereas psychological stress resulted in significantly delayed healing of the periodontal tissue ( figures 5(f)5(h ) and 5(j)5(l ) ) . histometric analysis showed that there was a significant reduction of si , abl , and al in the periodontitis group , although these values were still higher than those in the matched control group at all time points except for si at week 4 ( p < 0.05 , figures 5(m)5(o ) ) . moreover , si and abl were significantly higher in the stress group than in the periodontitis group at week 2 and week 4 ( p < 0.05 , figures 5(m ) and 5(n ) ) . additionally , a higher al was observed in the stress group than in the periodontitis group at week 4 ( p < 0.05 , figure 5(o ) ) . regarding il-1 and tnf- staining , little immunoreactive cells and low - level mean optical density were found in the control group at all time points ( figures 6 , 7(a)7(d ) , 7(m ) , and 7(n ) ) . at baseline , ligature placement resulted in a significant increase in the number of immunoreactive cells and the mean optical density in the periodontitis group and the stress group ( p < 0.05 , figures 6 , 7(e ) , 7(i ) , 7(m ) , and 7(n ) ) . after ligature removal , the expression of il-1 and tnf- significantly decreased in the periodontitis group at week 1 , 2 , and 4 compared with baseline level ( p < 0.05 , figures 6 , 7(m ) , and 7(n ) ) . for il-1 , the immunoreactive cell number and the mean optical density did not descend significantly in the stress group compared with baseline level ( p > 0.05 , figures 6(m ) and 6(n ) ) moreover , the cell number at week 1 , 2 , and 4 and the mean optical density at week 4 were higher in stress group than that in the periodontitis group ( p < 0.05 , figures 6(m ) and 6(n ) ) . for tnf- , although there was an expression downtrend , the immunoreactive cell number was still higher in the stress group than in the periodontitis group at week 2 and week 4 ( p < 0.05 , figure 7(m ) ) . as for bfgf staining , moderate immunoreactivity was found in the control group at all time points ( figures 8(a)8(d ) , 8(m ) , and 8(n ) ) . at baseline , ligature placement resulted in a significant reduction in the number of immunoreactive cells and the mean optical density in the periodontitis group and the stress group ( p < 0.05 , figures 8(e ) , 8(i ) , 8(m ) , and 8(n ) ) . in the following 4 weeks after ligature removal , bfgf expression in the periodontitis group prominently recovered when the cell number and mean od were significantly increased at weeks 1 , 2 , and 4 ( p < 0.05 , figures 8(f)8(h ) , 8(m ) and 8(n ) ) . furthermore , the mean od of the periodontitis group was significantly higher than that in the control group at week 2 ( p < 0.05 , figure 8(n ) ) . in contrast , psychological stress significantly delayed the recovery of bfgf expression , whereas the positive cell number and the mean od did not increase significantly ( p > 0.05 , figures 8(j)8(l ) , 8(m ) , and 8(n ) ) , with the exception of the cell number at week 4 ( p < 0.05 , figure 8(m ) ) . further , the ratios of immunoreactive cell numbers between inflammatory cytokines and bfgf , which indicated the homeostasis between inflammation and repair process , were calculated and statistically analyzed . it showed that both the il-1/bfgf ratio and tnf-/bfgf ratio were highest at baseline in periodontitis and periodontitis / stress group . as the healing time went on , the inflammatory cytokine / bfgf ratios decreased apparently ( p < 0.05 , figures 9(a ) and 9(b ) ) . compared with the ratios of periodontitis group , those of the periodontitis / stress group decreased significantly much slower ( p < 0.05 , figures 9(a ) and 9(b ) ) . in addition , the negative correlations between inflammatory cytokines and bfgf were observed ( r = 0.539,p < 0.0001 for il-1 and bfgf , and r = 0.542,p < 0.0001 for tnf- and bfgf ) . in contrast , the positive correlations between si and inflammatory cytokines were observed ( r = 0.494,p < 0.0001 for si and il-1 , and r = 0.519 , p < 0.0001 for si and tnf- ) . no significant correlation was found between si and bfgf ( r = 0.256 , p = 0.059 ) previous clinical studies have demonstrated a correlation between psychological stress and a more severe level of destruction during the progression of periodontitis . animal studies have also found a higher degree of attachment loss and alveolar bone resorption after exposing the animals to experimental periodontitis and stress at the same time [ 18 , 30 ] . however , from periodontitis to periodontal healing , the pathological condition of the periodontal tissue shifts from destruction to regeneration . whether psychological stress has an adverse impact on the healing process of periodontitis and whether bfgf plays a role in that process therefore , this study was designed to evaluate the effect of psychological stress on the healing process of periodontitis and the expression of bfgf in the periodontal ligament . the experimental model of periodontitis employed in the current study is induced by the local compression of a silk ligature that results in progressive accumulation of plaque and increased degradation of periodontal connective tissues and bone resorption [ 31 , 32 ] . as the previous studies have reported [ 19 , 30 , 33 ] , we also observed a significant increase in the surface of inflammatory infiltration , alveolar bone loss , and attachment loss in periodontitis group and periodontitis / stress group compared with those of control , which confirmed a successful establishment of periodontitis in rats . in addition , the results of the present work suggested an obvious downregulation of the expression of bfgf in the periodontal ligament associated with experimental periodontitis , which was demonstrated by the decreased immunoreactive cell number and mean optical density . these results may indicate that periodontal ligament repair is diminished in the destruction phase of periodontitis . our findings are similar to those of gao et al . , who identified , for the first time , the presence of bfgf in human periodontal ligament tissue and observed a significant decrease in the fibroblast number and staining intensity of bfgf in the periodontal ligament of patients with chronic periodontitis . moreover , the destruction of periodontal tissue may be attributed to the failure of recruitment and activation of macrophages , involving little bfgf expression . in contrast , as an important angiogenesis - associated factor , bfgf is upregulated in some other oral pathologies that involve the formation of a large number of new blood vessels , such as pyogenic granuloma . the stress model that we adopted in the current study is cums , which has been developed in recent years and is widely used to mimic chronic stress in daily life in studies [ 23 , 24 , 26 ] . the major advantage of cums is the avoidance of animal adaptation to invariant stressors by employing various physical and psychological stressors in a predetermined manner . we have found that the experimental animals showed a reduction in body weight gain and elevated plasma corticosterone and acth levels . moreover , the rats exposed to psychological stress showed stress - like behaviors based on the significant decrease observed in the distance moved in the center and total distance moved in the open - field test . these results are in accordance with previous studies [ 9 , 37 ] , indicating that the experimental animals were successfully stressed by the cums procedure . some clinical studies have demonstrated a link between psychological stress and periodontitis [ 35 ] , and several investigators have worked to determine the possible mechanisms behind it . reported the modulated expression of pro- and anti - inflammatory and proresorptive factors , suggesting that stress may alter the progression of periodontitis locally through its effect on the immune - inflammatory system . another study conducted with animal models   speculated on a correlation between periodontitis severity and psychological stress and periodontal tissue hypoxia by measuring the expression of hypoxia - induced factor-1. nevertheless , there is limited information regarding whether psychological stress has an adverse effect on the healing stage of periodontitis . thus , in our study , psychological stress was imposed on animals after ligature removal . on the one hand , in the periodontitis group , we observed spontaneous periodontal healing at 1 , 2 , or 4 weeks compared with baseline . this healing was characterized by a decreased surface of inflammatory infiltration , alveolar bone remodeling , and epithelial reattachment , confirming the results reported by coimbra et al . , who also observed significant repair of the soft tissue and regeneration of the alveolar bone in rats 15 days after ligature removal . on the other hand , we observed delayed periodontal healing in the stress group , and the periodontal breakdown was significantly more severe in the stress group than in the periodontitis group . periodontitis is a chronic infectious disease that the host inflammatory response to bacteria is the leading cause of tissue destruction . in our study , the expressions of two typical inflammatory cytokines , il-1 and tnf- , significantly increased in periodontal ligament after ligature placement , confirming the previous studies that in periodontitis the elevated levels of inflammatory cytokines can cause tissue injury and alveolar bone metabolism breakdown [ 29 , 39 ] . in fact , we did find the positive correlation between inflammatory cytokines expression and periodontal damage . in the natural healing process of periodontitis , in contrast , during periodontitis healing under conditions of psychological stress , the expression of inflammatory cytokines remained on a relatively higher level . the results were similar to our previous study , which demonstrated that psychological stress could induce upregulation of il-1 and tnf- and may further play a potential role in initiating the cartilage destruction . the present study suggested that psychological stress may also lead to disturbed inflammatory process , enhancing tissue damage in periodontal healing procedure . wound healing is a process consisting of overlapping stages , including clot formation , inflammation , proliferation , and remodeling [ 41 , 42 ] . bfgf has been proven that it can significantly promote the last three stages of wound healing process . normally , bfgf is produced primarily by periodontal ligament fibroblasts and endothelial cells in the periodontal tissue . in the healing stage of periodontitis , the basic cellular mechanisms of periodontal regeneration are similar to wound healing and involve proliferation and migration of fibroblasts in the periodontal ligament , the differentiation of osteoblasts and cementoblasts from undifferentiated precursor cells , and ultimately , the synthesis of extracellular matrix [ 43 , 44 ] . bfgf has been shown to be effective in promoting periodontal regeneration in a series of studies [ 10 , 11 , 16 , 17 ] , whereas the role bfgf plays in the development and recovery stage of periodontitis healing still keeps unknown . it was reported that bfgf levels may be decreased in tissue associated with chronic periodontal lesions , which could be related with reduced secretion of bfgf caused by periodontal ligament cells and endothelial cell damage during periodontitis . similarly , we also found the decreased bfgf secretion in periodontitis tissue , accompanied by an upside - down ratio between the inflammatory cytokines and bfgf secretion . we considered that it was associated not only with periodontal cell damage during periodontitis but also with the pathological feature of periodontitis , apparent failure of recruitment and activation of macrophage . during healing process , bfgf returned to control level , with which periodontal tissue repair and regeneration gradually took place and the inflammatory factors gradually fell back to slightly higher level compared to the control . negative correlation between bfgf and inflammatory factors during periodontitis confirmed by the present study further demonstrated the important roles that bfgf may play in the periodontal healing process . bosch et al .   confirmed a slower rate of mucosa healing of the oral hard palate in individuals with dysphoria compared with the median healing rate , suggesting delayed wound healing under stressed conditions . in addition , it was reported that delayed migration and differentiation of fibroblasts to smooth muscle myofibroblasts may contribute to the prolonged healing time of wounds on the backs of restraint - stressed mice . in the present study , the stressed rats showed significantly impaired periodontal healing and regeneration , along with a significant reduction in the number of bfgf - immunoreactive cells and bfgf staining intensity in the periodontal ligament compared with rats in the natural periodontitis healing group , indicating that psychological stress may delay periodontal healing through downregulation of bfgf expression , which is an essential factor in periodontal regeneration . psychological stress leads to a delay of periodontal healing in conditions of experimental periodontitis , which may be mediated in part by a downexpression of bfgf in periodontal ligament .
objective . to evaluate the effects of psychological stress on periodontitis healing in rats and the contribution of basic fibroblast growth factor ( bfgf ) expression to the healing process . methods . 
 ninety - six rats were randomly distributed into control group , periodontitis group , and periodontitis plus stress group . 
 then , the rats were sacrificed at baseline and week(s ) 1 , 2 , and 4 . 
 the periodontitis healing condition was assessed , and the expression of interleukin-1 ( il-1 ) , tumor necrosis factor- ( tnf- ) , and bfgf were tested by immunohistochemistry . results . 
 the stressed rats showed reduced body weight gain , behavioral changes , and increased serum corticosterone and acth levels ( p < 0.05 ) . the surface of inflammatory infiltrate , alveolar bone loss , attachment loss , and expression of il-1 and tnf- in the stress group were higher than those in the periodontitis group at weeks 2 and 4 ( p < 0.05 ) . 
 rats with experimental periodontitis showed decreased bfgf expression ( p < 0.05 ) , and the recovery of bfgf expression in the stress group was slower than that in the periodontitis group ( p < 0.05 ) . 
 negative correlations between inflammatory cytokines and bfgf were detected . 
 conclusion . psychological stress could delay periodontitis healing in rats , which may be partly mediated by downregulation of the expression of bfgf in the periodontal ligament .