various foods , particularly , vegetables , contain large amounts of sulfates that are converted into vscs after cooking or biological modification . trace amounts of h2s in vivo reportedly modify anti - inflammation , radical scavengers , signal transduction , proteins , ion channel regulation , and transition metal complexes , and are essential in maintaining homeostasis . approximately , 90% of vscs produced in the mouth consist of h2s and ch3sh and , in addition to halitosis , can cause discoloration of crown restorations , prostheses , and dentures . they can also increase the epithelial permeability and bacterial toxins in the soft gingival tissue , enhance the production of collagenase and prostaglandin , and induce apoptosis in gingival connective tissue , and are involved in the pathological modification and progression of periodontitis . hard tissue in the form of enamel functions inside the mouth in an exposed state , and thus is affected by direct exposure to vscs . this in turn triggers the deterioration and loss of dentin , which is believed to be involved in the onset and modification of tooth wear . however , the role of vscs in enamel changes and tooth wear remains unclear due to a lack of published literature . when tooth wear causes wedge - shaped defects and removal of crown restorations , the dentist may choose to perform a restorative procedure using adhesive composite resins . however , the enamel surface where the crown will be attached is exposed and therefore vulnerable to the effects of vscs , which may affect how well the composite resin bonds to the enamel . we investigated whether vscs in the oral cavity in the form of h2s are involved in tooth wear by observing its effect on enamel using an electron microscope ( transmission electron microscope [ tem ] and scanning electron microscopy [ sem ] ) . we also conducted a shear test to determine whether the effect of h2s on the enamel surface translated to an effect on the adhesive properties of composite resin restorations . extracted human teeth were sectioned at the cervical portion , and the crown portion was sealed with wax before coating half of these sections with nail polish . samples were then prepared from these sections using the uncoated portion as the experimental side and the coated portion as the control side . next , a saturated h2s solution was prepared by bubbling h2s in 50 ml physiological saline for 30 min . samples were immersed in saturated h2s solution in a sealed container , which was placed in an incubator for 14 days at 37c before observation under a tem . the h2s source was prepared by dissolving 50 g sodium hydrosulfide hydrate ( sigma - aldrich , st . louis , mo , usa ) in distilled pure water ( invitrogen corporation , carlsbad , ca , usa ) up to a volume of 100 ml . the h2s gas released from the aqueous solution of sodium hydrosulfide inside the sealed container was collected and dissolved in distilled water for the application to tooth samples . experimental side samples were placed in individual petri dishes with the aqueous solution of sodium hydrosulfide from inside the sealed container , and the control side samples were placed in individual petri dishes with sterilized distilled water . the aqueous solution of saturated hydrosulfide was replaced , and distilled water was added every 2 days , and samples were stored in a sealed container for 1 week . twenty bovine tooth root samples were sectioned , the pulp was removed , and the cervical portion was sealed with glass ionomer cement . teeth were then placed in a mold and embedded in self - curing resin before exposing the enamel using # 100 waterproof sandpaper and polishing from the cervical side to the crown side using # 600 waterproof sandpaper . teeth were then divided evenly into 10 h2s experimental group samples and 10 control samples . experimental group samples were immersed in distilled water and placed in individual petri dishes with an aqueous solution of sodium hydrosulfide from inside the sealed container , and the control group samples were immersed in distilled water and placed in individual petri dishes with sterilized distilled water . the aqueous solution of saturated hydrosulfide was replaced , and distilled water was added every 2 days , and samples were stored for 1 week . after 1 week , the polished surface of the tooth samples was treated with self - etching bonding system ( clearfil se bond , kuraray noritake dental , tokyo , japan ) , packed with resin composite ( clearfil ap - x a3 , kuraray noritake dental ) , and immersed in water for 24 h before undergoing a shear test with a cross - head speed of 1.0 mm / min ( model 444 , instron , darmstadt , germany ) . extracted human teeth were sectioned at the cervical portion , and the crown portion was sealed with wax before coating half of these sections with nail polish . samples were then prepared from these sections using the uncoated portion as the experimental side and the coated portion as the control side . next , a saturated h2s solution was prepared by bubbling h2s in 50 ml physiological saline for 30 min . samples were immersed in saturated h2s solution in a sealed container , which was placed in an incubator for 14 days at 37c before observation under a tem . the h2s source was prepared by dissolving 50 g sodium hydrosulfide hydrate ( sigma - aldrich , st . louis , mo , usa ) in distilled pure water ( invitrogen corporation , carlsbad , ca , usa ) up to a volume of 100 ml . the h2s gas released from the aqueous solution of sodium hydrosulfide inside the sealed container experimental side samples were placed in individual petri dishes with the aqueous solution of sodium hydrosulfide from inside the sealed container , and the control side samples were placed in individual petri dishes with sterilized distilled water . the aqueous solution of saturated hydrosulfide was replaced , and distilled water was added every 2 days , and samples were stored in a sealed container for 1 week . twenty bovine tooth root samples were sectioned , the pulp was removed , and the cervical portion was sealed with glass ionomer cement . teeth were then placed in a mold and embedded in self - curing resin before exposing the enamel using # 100 waterproof sandpaper and polishing from the cervical side to the crown side using # 600 waterproof sandpaper . teeth were then divided evenly into 10 h2s experimental group samples and 10 control samples . experimental group samples were immersed in distilled water and placed in individual petri dishes with an aqueous solution of sodium hydrosulfide from inside the sealed container , and the control group samples were immersed in distilled water and placed in individual petri dishes with sterilized distilled water . the aqueous solution of saturated hydrosulfide was replaced , and distilled water was added every 2 days , and samples were stored for 1 week . after 1 week , the polished surface of the tooth samples was treated with self - etching bonding system ( clearfil se bond , kuraray noritake dental , tokyo , japan ) , packed with resin composite ( clearfil ap - x a3 , kuraray noritake dental ) , and immersed in water for 24 h before undergoing a shear test with a cross - head speed of 1.0 mm / min ( model 444 , instron , darmstadt , germany ) . tem observation revealed distinctly hexagonal pillar - shaped hydroxyapatite crystals on the control side , but the crystals on the experimental h2s side were reduced in size , and their structure was not be clearly defined [ figure 1 ] . sem observation at low magnification indicated the greater surface coarseness on the h2s side than on the control side . observation of the control side at high magnification revealed concavo - convex patterns believed to be the ends of small enamel pillars . on the h2s side , however , the surface structure had collapsed , and the reticular pattern had changed [ figure 2 ] . bond strength between enamel and composite resin bonding applied with a self - etching system was 14.4  5.3 mpa in the experimental group samples ( n = 10 ) and 13.1  5.2 mpa in the h2s side samples ( n = 10 ) , but t - test did not reveal any significant differences [ table 1 ] . ( a ) control side transmission electron microscope , ( b ) experimental side transmission electron microscope , distinct hexagonal crystal structure can be seen on the control side ( a ) , but crystals are diminished and crystal structure can not be clearly seen on the experimental side ( b ) ( a and b ) control side scanning electron microscopy ; and ( d ) experimental side scanning electron microscopy on control side , the surface is generally consistent ( a ) and recesses believed to be the ends of small enamel pillars ( b ) can be seen . on experimental side , the surface appears coarse ( c ) and exhibits reticular changes ( d ) mean values of bond strength of enamel to composite resin in h2s and control groups h2s occurs in trace amounts in living organisms and is believed to be involved in gene expression via intracellular signal transduction and enzyme activation to maintain homeostasis . however , h2s occurring inside the mouth can cause localized halitosis , periodontal tissue disorders , and discoloration and corrosion of metal restorations . in the present study , we placed tooth samples exposed to h2s in distilled water and observed its effect on enamel surfaces using tem and sem . the major component is hydroxyapatite crystals with a hexagonal pillar configuration having a width of 40 - 50 nm , thickness of 25 - 60 nm , and height of 120 - 160 nm . in our study , the tooth samples exposed to h2s had a smaller , obscured crystal configuration when compared with control samples . the findings also showed what appeared to be disintegration of the reticular structure on the surface of the h2s samples . in other words , the sustained action of h2s on the enamel surface had caused the hydroxyapatite crystals to decay , triggering a reduction and loss of crystal structure , which in turn led to the progressive breakdown of dentin on the crown surface , ultimately resulting in deterioration of the enamel 's physical properties and loss of dentin . tooth surface loss that can not be attributed to tooth decay is known as tooth wear , which is a multifactorial disease that includes attrition due to occlusal contact ; wear caused by nonocclusal mechanical contact , acid - induced tooth decay , and abfraction . our study findings of h2s - induced decay of enamel surfaces suggest that h2s occurring inside the mouth is a cause of dentin surface loss . in clinical dentistry , our findings revealed changes to the crystal morphology and surface structure of the enamel to which these restorations are applied . although we initially suspected that these changes would affect the adhesive restoration , the results did not reveal any differences between the h2s - exposed samples and control samples in terms of the bond strength between the enamel and composite resin applied using a self - etching system . we attributed this finding to the effect of etching using a self - etching primer , whereby enamel exposed to h2s dissolved to reveal new enamel . although our findings suggest that h2s can cause tooth wear by triggering changes to enamel 's surface structure and crystal morphology , dental bonding by decalcification using a self - etching system does not impact the bond strength of enamel and composite resin when applying an adhesive restoration to enamel that has been affected by h2s .
background : volatile sulfur compounds ( vscs ) produced inside the mouth are a well - known cause of halitosis . 
 recent studies have suggested that vscs modify the pathology of periodontitis by encouraging the migration of bacterial toxins associated with increased permeability of gingival epithelia , and enhancing the production of matrix metalloproteinases in gingival connective tissue . 
 nonetheless , the effects on the enamel of direct exposure to vscs within the oral cavity remain unclear.in the present study , we observed the effects of vscs in the form of hydrogen sulfide ( h2s ) on enamel surfaces and determined their effects on restorations.materials and methods : extracted human tooth and bovine tooth samples were divided into the h2s experimental side and the control side . 
 we observed the effects of h2s on enamel surfaces using electron microscopy and conducted a shear test.results:we found that exposure to h2s obscured the enamel surface 's crystal structure . 
 the surface also exhibited coarseness and reticular changes . 
 shear testing did not reveal any differences in bond strength.conclusions:our findings suggested that h2s occurring inside the mouth causes changes to the crystal structure of the enamel surface that can lead to tooth wear , but that it does not diminish the effects of dental bonding in adhesive restorations .