the widespread use of fluoride ( f ) has contributed to a reduction in the prevalence and 
 incidence of dental caries worldwide . 
 however , along with the decline of tooth decay , an increase in dental fluorosis has been 
 observed , a side - effect resulting from exposure to f above the recommended levels during 
 the period of tooth formation . this 
 increased prevalence of dental fluorosis has been observed throughout the world , both in 
 fluoridated and in non - fluoridated areas . it is known that f interacts with mineralized tissues , and in high concentrations can 
 harm the process of mineralization . however , the molecular mechanisms involved in the pathogenesis of fluorosis are not 
 completely understood . enamel 
 formation is a complex process involving cell proliferation and differentiation through 
 epithelial - mesenchymal sequential secretion of matrix proteins , tissue - specific 
 transport of ions , including calcium and fluoride , and precipitation and alignment of 
 the crystals of enamel through interactions between organic and inorganic 
 molecules . increases in the roughness of apatite crystals surfaces in developing fluorosed enamel 
 have been reported . it was 
 suggested that these roughened crystals could cause fluorosis because they would impair 
 crystal growth due to changes in crystal surface chemistry and/or incomplete removal of 
 matrix proteins . additionally , rougher crystals have been shown to bind increased 
 amounts of protein in other tissues . several reasons related to the process of enamel development have been suggested for the 
 increased roughness , such as deposition of less soluble fluoridated mineral species 
 reflecting higher supersaturation level in tissue fluid and retention of matrix proteins 
 due to their increased interaction with the altered crystal surface . however , it was shown that increased roughness of enamel crystals 
 could also be observed after in vitro treatments with f in solutions 
 undersaturated with respect to apatite indicating that surface roughness should not 
 originate directly from growth - related phenomena , but from restructuring of the crystal 
 surface . despite the cause - effect relationship between fluoride exposure and dental fluorosis is 
 well established , other factors seem to account for the severity of dental 
 fluorosis . the observation 
 that certain ethnic groups are more susceptible to this disease led some researchers to investigate the genetic susceptibility of 
 different mice strains to dental fluorosis . it was observed that a / j mice are highly 
 susceptible to dental fluorosis , while 129p3/j mice are little affected , despite retaining more f in the body , 
 which leads to higher femur and plasma f concentrations . a recent study compared the profile of protein 
 expression in kidney of a / j and 129p3/j mice . an interesting observation was that the susceptible mice ( a / j ) presents greater 
 retention of proteins in the maturing enamel , even in the case of no exposure to 
 fluoride . this led us to the 
 assumption that differences in the profile of the crystals surfaces , regardless exposure 
 to f , would exist between a / j and 129p3/j mice strains and this could have an 
 implication on the mechanisms of dental fluorosis development . it was observed that the 
 length and width of bone crystals of the " resistant " 129p3/j strain mice was smaller 
 than the same parameter for the a / j strain , suggesting that the crystal growth rate of the " resistant " strain 
 is slower than that of the " susceptible " strain . however , differences in crystal 
 dimensions between the strains were not evaluated yet for enamel . slower crystal growth 
 rate would allow for an appropriate buffering action of amelogenins that under the 
 adequate ph would remain attached to the crystal surface , guiding its growth . thus , the aim of the present study was 
 to assess the overall apatite crystal profile in the enamel mineral matrix of mice 
 susceptible ( a / j ) or resistant ( 129p3/j ) to dental fluorosis through analyses by atomic 
 force microscopy ( afm ) . it was primarily hypothesized that a / j mice would have rougher 
 and wider crystals when compared with 129p3/j mice , which would lead to higher retention 
 of matrix proteins and this could help to explain their increased susceptibility to 
 dental fluorosis . the protocol of this study was approved by the ethics committee for animal research 
 of bauru school of dentistry , university of so paulo ( proc . 129p3/j and a / j mice ( 12 from each strain ) were obtained 
 from the jackson laboratory ( bar harbor , me , usa ) . the reason for the selection of 
 these two strains is based on the fact that the strain a / j is highly susceptible to 
 dental fluorosis , with a rapid and severe development of the disease when the animal 
 is exposed to f , while the strain 129p3/j is less affected , with a low rate of dental 
 fluorosis . in addition , the 
 phenotypic and genetic differences among animals , and their general availability as 
 standard laboratory strains , have also contributed to their selection . animals of the same strain were housed in plastic cages ( n=6 per 
 cage ) in the vivarium of bauru school of dentistry . the temperature and humidity in 
 the climate - controlled room , which had a 12-hour light / dark cycle , were 231c and 
 40 - 80% , respectively . the animals had free access to deionized water and a 
 semi - purified diet with low f concentration ( around 1 ppm ) that was prepared in our 
 laboratory . at the end of the experimental period ( age 12 weeks ) , animals were anesthetized with 
 ketamine and xylazine . the jaws were removed , the incisors extracted , carefully 
 dissected , washed in cold 0.1 m phosphate buffer ( ph 7.4 ) and allowed to dry for 
 about 3 min at room temperature until an opaque area was visible . it was then 
 possible to distinguish between the secretion and maturation phases of enamel 
 formation . with a scalpel , 
 the enamel matrix in the early stages of secretion and maturation was scraped in 
 pre - weighed and identified tubes of 1.5 ml until the enamel became too hard to be 
 cut . all detectable traces of matrix protein were removed from the enamel samples by a 
 sequential extraction procedure previously described . briefly , the enamel particles were first extracted 
 with 0.1 m phosphate buffer , ph 7.4 , to desorb mineral bound and freely mobile 
 species . the pelleted 
 material was then further extracted in 50 mm tris containing 4 m urea at ph 7.0 to 
 dissolve aggregated protein . the insoluble residue was then reextracted for a further 6 times with 0.1 m 
 phosphate buffer , ph 7.4 , to ensure final desorption of any mineral - bound components . the final extraction used distilled water with the ph adjusted to 7.0 in order to 
 remove all traces of buffer and urea . protein removal from the crystals was monitored by polyacrylamide gel electrophoresis 
 of the extraction solutions and of crystals obtained in a pilot study . part of the 
 sample from each group was also gold - sputtered and analyzed by sem to confirm the 
 presence of enamel crystals . the enamel crystals were sonicated for 40 min in hplc - grade methanol to prevent 
 aggregation . approximately 10 l of this suspension were then placed onto freshly 
 cleaved mica ( mica discs pelco , 9 mm diameter , ted pella , inc , ca , usa ) . the methanol evaporated rapidly leaving a spread of dispersed apatite crystals on the 
 surface that would be subsequently examined by afm . purified crystals from the mice incisors were imaged on a nanoscope iiia multimode 
 afm and controller ( digital instruments , santa barbara , ca , usa ) , equipped with a 
 jv - type scanner ( ~120x120 m maximum scan range in x - y direction ) . tapping mode in 
 air images were obtained using standard nsc15/aibs silicon single - rectangular 
 cantilever ( mikromasch , tallinn , estonia ) of quoted spring constant 46 n / m , radii of 
 curvature less than 10 nm , thickness of 3.5 - 4.5 m , tip height of 20 - 25 m , 
 cantilever length of 125 m and resonant frequency of 265 - 400 khz . cantilevers were 
 resonated at approximately 100 - 500 khz with drive amplitude in the range 110 - 270 mv 
 and images obtained at tapping amplitude of approximately 80% of free amplitude . multiple images for each sample were obtained with scan sizes ranging from 100x100 nm 
 to 1x1 m . surface roughness measurement ( ra ) was obtained over a 
 length of 30 - 80 nm in an approximately central line parallel to the crystal c - axis . for each crystal , a minimum of 5 measurements were obtained in 
 order to achieve a final mean roughness value for that crystal . ra roughness was calculated using a nanoscope iii 
 5.12r3 software and was defined as :   where f(x ) is the roughness curve relative to the center plane , and 
 l is the length of the roughness curve . therefore , the calculated surface roughness 
 in this case is the mean value of the surface relative to the center plane , and takes 
 into account any changes in the slope of the crystal surface . the measurement of crystal width was done using a nanoscope iii 5.12r3 software . 
 after drawing a line across the crystal the range 
 of detection of the jv - type scanner was 120x120 m in x - y direction and the vertical 
 range was 5 m . the software graphpad instat 4 version 3.0 for windows ( graphpad software , san diego , 
 ca , usa ) was used . data were checked for normality and homogeneity using 
 kolmogorov - smirnov and bartlett tests , respectively . since these criteria were 
 satisfied , data were analyzed by unpaired t test ( p<0.05 ) . the protocol of this study was approved by the ethics committee for animal research 
 of bauru school of dentistry , university of so paulo ( proc . 129p3/j and a / j mice ( 12 from each strain ) were obtained 
 from the jackson laboratory ( bar harbor , me , usa ) . the reason for the selection of 
 these two strains is based on the fact that the strain a / j is highly susceptible to 
 dental fluorosis , with a rapid and severe development of the disease when the animal 
 is exposed to f , while the strain 129p3/j is less affected , with a low rate of dental 
 fluorosis . in addition , the 
 phenotypic and genetic differences among animals , and their general availability as 
 standard laboratory strains , have also contributed to their selection . animals of the same strain were housed in plastic cages ( n=6 per 
 cage ) in the vivarium of bauru school of dentistry . the temperature and humidity in 
 the climate - controlled room , which had a 12-hour light / dark cycle , were 231c and 
 40 - 80% , respectively . the animals had free access to deionized water and a 
 semi - purified diet with low f concentration ( around 1 ppm ) that was prepared in our 
 laboratory . at the end of the experimental period ( age 12 weeks ) , animals were anesthetized with 
 ketamine and xylazine . the jaws were removed , the incisors extracted , carefully 
 dissected , washed in cold 0.1 m phosphate buffer ( ph 7.4 ) and allowed to dry for 
 about 3 min at room temperature until an opaque area was visible . it was then 
 possible to distinguish between the secretion and maturation phases of enamel 
 formation . with a scalpel , the enamel matrix in the early stages of secretion and maturation was scraped in 
 pre - weighed and identified tubes of 1.5 ml until the enamel became too hard to be 
 cut . all detectable traces of matrix protein were removed from the enamel samples by a 
 sequential extraction procedure previously described . briefly , the enamel particles were first extracted 
 with 0.1 m phosphate buffer , ph 7.4 , to desorb mineral bound and freely mobile 
 species . the pelleted 
 material was then further extracted in 50 mm tris containing 4 m urea at ph 7.0 to 
 dissolve aggregated protein . the insoluble residue was then reextracted for a further 6 times with 0.1 m 
 phosphate buffer , ph 7.4 , to ensure final desorption of any mineral - bound components . the final extraction used distilled water with the ph adjusted to 7.0 in order to 
 remove all traces of buffer and urea . protein removal from the crystals was monitored by polyacrylamide gel electrophoresis 
 of the extraction solutions and of crystals obtained in a pilot study . part of the 
 sample from each group was also gold - sputtered and analyzed by sem to confirm the 
 presence of enamel crystals . the enamel crystals were sonicated for 40 min in hplc - grade methanol to prevent 
 aggregation . approximately 10 l of this suspension were then placed onto freshly 
 cleaved mica ( mica discs pelco , 9 mm diameter , ted pella , inc , ca , usa ) . the methanol evaporated rapidly leaving a spread of dispersed apatite crystals on the 
 surface that would be subsequently examined by afm . purified crystals from the mice incisors were imaged on a nanoscope iiia multimode 
 afm and controller ( digital instruments , santa barbara , ca , usa ) , equipped with a 
 jv - type scanner ( ~120x120 m maximum scan range in x - y direction ) . tapping mode in 
 air images were obtained using standard nsc15/aibs silicon single - rectangular 
 cantilever ( mikromasch , tallinn , estonia ) of quoted spring constant 46 n / m , radii of 
 curvature less than 10 nm , thickness of 3.5 - 4.5 m , tip height of 20 - 25 m , 
 cantilever length of 125 m and resonant frequency of 265 - 400 khz . cantilevers were 
 resonated at approximately 100 - 500 khz with drive amplitude in the range 110 - 270 mv 
 and images obtained at tapping amplitude of approximately 80% of free amplitude . multiple images for each sample were obtained with scan sizes ranging from 100x100 nm 
 to 1x1 m . surface roughness measurement ( ra ) was obtained over a 
 length of 30 - 80 nm in an approximately central line parallel to the crystal c - axis . for each crystal , a minimum of 5 measurements were obtained in 
 order to achieve a final mean roughness value for that crystal . ra roughness was calculated using a nanoscope iii 
 5.12r3 software and was defined as :   where f(x ) is the roughness curve relative to the center plane , and 
 l is the length of the roughness curve . therefore , the calculated surface roughness 
 in this case is the mean value of the surface relative to the center plane , and takes 
 into account any changes in the slope of the crystal surface . the measurement of crystal width was done using a nanoscope iii 5.12r3 software . 
 after drawing a line across the crystal , width was automatically displayed . the range 
 of detection of the jv - type scanner was 120x120 m in x - y direction and the vertical 
 range was 5 m . the software graphpad instat 4 version 3.0 for windows ( graphpad software , san diego , 
 ca , usa ) was used . data were checked for normality and homogeneity using 
 kolmogorov - smirnov and bartlett tests , respectively . since these criteria were 
 satisfied , data were analyzed by unpaired t test ( p<0.05 ) . the mean ( sd ) ra of the crystals of a / j strain ( 0.580.15 
 nm ) was lower than the one found for the 129p3/j strain ( 0.660.21 nm ) but the 
 difference did not reach statistical significance ( t=1.187 , p=0.247 ) . figure 1 shows typical afm images of enamel crystals 
 of a / j and 129p3/j mice . crystals of the 129p3/j strain ( 70.426.79 nm ) were found to be 
 significantly narrower ( t with welch correction=4.013 , p=0.0013 ) than the same parameter 
 measured for the a / j strain ( 90.4215.86 nm ) . tapping mode afm images of enamel crystals on a mica surface , imaged in air . weanling animals had the free access to deionized water and 
 low - f diet until age 12 weeks . ( a)=left , height 
 image , z range , 100 nm ; right , phase image , z range , 107.1. ( b)=left , height 
 image , z range , 120 nm ; right , phase image , z range , 16.94 studies have shown that the a / j and 129p3/j strains present differences in water 
 metabolism ( a / j mice drink higher amounts of water ) and fluoride metabolism ( a / j mice 
 retain less fluoride ) , regardless f intake and key proteins possibly involved with this were recently identified 
 in kidney . also a / j mice present 
 greater retention of proteins in the maturing enamel , even in the case of no exposure to 
 f9 , which leads to lower enamel hardness . thus , since the main aim of this study was to evaluate differences 
 in the profile of the crystal surfaces of a / j and 129p3/j mice , regardless exposure to 
 f , animals were not treated with this ion . in previous studies , f concentrations were 
 evaluated both in plasma and enamel of a / j and 129p3/j mice exposed to 50 ppm f in the 
 drinking water or not ( control ; same condition of the present study ) . it was observed 
 that , both for plasmaand enamel 
 ( unpublished observations ) , no significant differences were detected among a / j and 
 129p3/j mice when the animals were not exposed to fluoride . only when the animals were 
 exposed to 50 ppm fluoride in the drinking water 129p3/j mice presented significantly 
 higher plasmaand enamel fluoride 
 levels when compared with a / j animals . thus , changes in enamel crystals profile found in 
 the present study can not be attributed to alterations in enamel f concentrations . this is the first study that compared surface roughness of enamel crystals of mice with 
 different susceptibilities to dental fluorosis . our results revealed similar values of 
 surface roughness ( ra ) for the enamel crystals from both the susceptible ( a / j ) and the 
 resistant ( 129p3/j ) mice strains . thus , the hypothesis that differences in surface 
 crystal roughness could lead to higher retention of matrix proteins and help to explain 
 the different susceptibility of these mice to dental fluorosis could not be confirmed , 
 at least without exposure to f. it is possible that if animals are exposed to f , surface 
 roughness of the enamel crystals is altered , as has been shown to occur for 
 rats . it would be interesting 
 to see if there are alterations in the pattern of surface roughness of a / j and 129p3/j 
 mice upon treatment with f. however , an interesting finding was the difference between the strains in the width of 
 the crystals . the crystals width of the " resistant " 129p3/j strain mice were found to be 
 narrower than the same parameter for the " susceptible " a / j strain , confirming our 
 hypothesis . such differences had already been shown to occur in bone crystals of mice 
 from these strains . it was observed using powder x - ray diffraction that bone crystals of 
 a / j mice were significantly longer and wider than those of 129p3/j mice even without 
 exposure to f. treatment with f ( 100 
 ppm in the drinking water ) did not significantly affect the crystal length but increased 
 the width for both strains , despite the resistant 129p3/j strain still had narrower bone 
 crystals . the authors proposed 
 that the overall shorter crystal size of 129p3/j mice suggests that the crystal growth 
 rate of this resistant strain is slower than that of the susceptible strain . this 
 reduced crystal growth rate may be due to stronger mineral - organic interfacial bond that 
 reduces the flux of calcium and phosphate ions to the apatite crystal surface . stronger 
 mineral - organic interfacial bond has previously been shown to play an important role in 
 bone quality , which may explain the 
 absence of bone mechanical properties alteration in the 129p3/j strain even under high 
 exposure to f. in enamel , the mineral - organic interface is equally important to guide crystal growth . the role of amelogenins is very important on this regard since these proteins form 
 nanospheres that coat crystal surfaces selectively in the secretory phase of 
 amelogenesis and act as spacer molecules between crystals . it makes crystals grow in length ( c - axis ) preventing their 
 lateral fusion . the role of the 
 mineral organic - interface on the mechanism of dental fluorosis development has been 
 emphasized in a new model to explain the development of dental fluorosis . it was proposed that f accelerates 
 crystal growth and the resulting rapid mineral deposition strongly increases protons 
 production that can no longer be buffered by the available amelogenins . upon acidic ph , 
 amelogenin nanospheres disaggregate and additionally , 
 newly secreted amelogenins are not able to form nanospheres , remaining monomeric . this 
 makes control of preferential crystal growth to be lost , leading to the well - known 
 characteristics of fluorosis . based on the above - mentioned observations and on our results , a model to explain the 
 decreased susceptibility to dental fluorosis of the 129p3/j strain could be proposed . the narrower enamel crystals observed for this strain are indicative of slower crystal 
 growth . this would allow an appropriate buffering action of amelogenins that under the 
 adequate ph would remain attached to the crystal surface , guiding crystal growth . the 
 proposed model would benefit from validation by studies that quantitatively analyze the 
 length and width of the enamel crystals formed by 129p3/j and a / j mice under chronic 
 exposure to f. despite this , it provides new insights into the mechanisms underlying the 
 genetic susceptibility to dental fluorosis . the mechanisms responsible for the 
 differential width of enamel crystals produced by a / j and 129p3/j mice strains surely 
 deserve investigation . the enamel crystals of the 129p3/j strain are narrower than a / j mice , and this fact can 
 be an indicative of slower crystal growth , which could contribute to increased 
 susceptibility to dental fluorosis .
objectivethis study aimed to assess the overall apatite crystals profile in the enamel 
 matrix of mice susceptible ( a / j strain ) or resistant ( 129p3/j strain ) to dental 
 fluorosis through analyses by atomic force microscopy ( afm).material and methodssamples from the enamel matrix in the early stages of secretion and maturation 
 were obtained from the incisors of mice from both strains . 
 all detectable traces 
 of matrix protein were removed from the samples by a sequential extraction 
 procedure . 
 the purified crystals ( n=13 per strain ) were analyzed 
 qualitatively in the afm . 
 surface roughness profile ( ra ) was measured.resultsthe mean ( sd ) ra of the crystals of a / j strain ( 0.580.15 nm ) was lower than the 
 one found for the 129p3/j strain ( 0.660.21 nm ) but the difference did not reach 
 statistical significance ( t=1.187 , p=0.247 ) . 
 crystals of the 129p3/j strain 
 ( 70.426.79 nm ) were found to be significantly narrower ( t=4.013 , p=0.0013 ) than 
 the same parameter measured for the a / j strain ( 90.4215.86 nm).conclusionenamel crystals of the 129p3/j strain are narrower , which is indicative of slower 
 crystal growth and could interfere in the occurrence of dental fluorosis .