we recently demonstrated that diabetes directly induced dental caries in rats and mice , although a direct association between the 2 was less evident and conflicting in humans [ 14 ] .
we demonstrated that dental caries were produced using noncariogenic diets with a low concentration of sugar in diabetic rodent models [ 57 ] , although many studies usually add large amounts of sugar to the diet to induce dental caries [ 8 , 9 ] .
in addition , early studies on the production of experimental dental caries have reported the effects of physical properties of cariogenic diets with high concentrations of sugar on dental caries .
these studies mainly used finely ground cariogenic diet [ 1013 ] because it is more cariogenic compared to a coarsely ground diet in rodents .
moreover , hard and coarse foods have the ability to prevent caries [ 1417 ] .
thus , modifying the dietary formulation may possibly enhance or reduce caries development in diabetic animals .
however , no reports have investigated the effects of the physical properties of non - cariogenic standard diets on dental caries in diabetic animals .  
currently , 2 types of non - cariogenic standard diet formulations ( i.e. , powdered and pelletized diets ) are widely used in experimental rodents . in the present study ,
we compared the effects of these diets on the development of dental caries in a diabetic rodent model focusing on the difference in hardness between the powdered and pelletized diets .
six - week - old female f344 rats were supplied by japan slc , inc .
the animals were housed in stainless - steel cages at a temperature of 2026c and a relative humidity of 4070% under a 12/12  h light / dark cycle , that were ventilated with filtered fresh air . to prevent infection ,
the animals were allowed free access to tap water and fed with a widely used standard pelletized or powdered diet ( charles river formula 1 ( crf-1 ) ; oriental yeast co. , ltd . ,
the animals were handled according to the principles for all experimental procedures outlined in the guide for the care and use of laboratory animals prepared by our institution ( setsunan university ) and the japanese association for laboratory animal science .
the glucose levels in the fresh urine were measured semiquantitatively using urine test paper ( wako pure chemical industries , osaka , japan ) daily from day 1 to day 3 after dosing , once every week for 1 month after the first week , and once every month thereafter .
the blood glucose levels in the tail vein samples were also measured semi - quantitatively using the glucose oxidase method ( glutest e ; sanwakagaku , aichi , japan ) once every month from the fourth week after alloxan injection .
blood samples obtained from the tail vein and fresh urine were collected from 1:00 to 4:00  p.m. the severity of hyperglycemia was defined as follows : normal , < 200  mg / dl ; mild , > 200  mg / dl ; moderate , > 300  mg / dl ; or severe , > 400  mg / dl . the severity of glycosuria was defined as follows : normal , < 100  mg / dl ; mild , > 100  mg / dl ; moderate , > 250  mg / dl ; or severe , > 500  mg / dl . the experimental design is shown in figure 1 .
ten rats , aged 7 weeks , were given a single dose ( 35  mg / kg body weight ) of alloxan ( sigma - aldrich japan , tokyo , japan ) via tail vein injection .
the dose of alloxan was determined as the given dose at which a rat survives for a long period after the onset of diabetic symptoms . after the confirmation of hyperglycemia and glucosuria following the dosing of alloxan ,
five rats were given a pelletized crf-1 diet ( diabetes - pelletized diet group ) and the remaining 5 rats were given a powdered crf-1 diet ( diabetes - powdered diet group ) .
as a control , each of the 5 intact rats was fed a pelletized or powdered crf-1 diet ( control - pelletized diet group and control - powdered diet group ) .
the animals were euthanized by exsanguination under deep anesthesia at the end of the observation period .
subsequently , the mandible was removed and fixed in 10% neutral - buffered formalin ( ph 7.4 ) .
after a 24  h fixation , the occlusal , buccolingual , and proximal surfaces of all of the molar teeth were intensively observed under a binocular stereoscope .
soft x - ray images of the mesiodistal plane were taken under conditions of 35  kv and 2  ma for 4  min .
the molar teeth were classified into 5 groups according to caries characteristics by observing and analyzing the radiographs : no radiolucent change ( grade 0 ) , radiolucent area only on the occlusal surface of the crown ( grade 1 ) , radiolucent areas on occlusal surface and either of the mesiodistal surfaces of the crown ( grade 2 ) , radiolucent areas over the entire surface of the crown ( grade 3 ) , and radiolucent areas over most of the surface of the dental root ( grade 4 ) .
the mean score of the caries was used as indicator for comparing the severity of the carious lesions between the groups .
after soft x - ray examination , a histopathological examination was performed on the mandible in all of the rats .
after fixation with 10% neutral - buffered formalin , the sample was decalcified in a 5% solution of ethylenediaminetetraacetic acid 4 na ( edta 4 na ) for 2 weeks at 4c .
after decalcification , the specimens were trimmed , dehydrated in a sequential ethanol series using an automated processor , and embedded in paraffin wax .
serial 7  m thick sections on the mesiodistal plane were made through the centers of all of the molars and then stained with hematoxylin and eosin for examination using light microscopy .
the severity of the caries lesion was graded as follows : slight , dentin caries localized in occlusal surface of dentin ; mild , dentin caries extended into the dental pulp with pulpitis and/or pulp necrosis ; moderate , dental crowns were partially decayed ; and severe , dental crowns were completely decayed ( only the molar roots remained ) .
the wilcoxon rank - sum test was employed to compare the differences in the mean scores of the caries lesions using soft x - ray examination between the groups .
the chi - square test was used to determine the caries incidence using soft x - ray examination and the incidence of histopathological lesions in each group of rats .
severe hyperglycemia ( > 400  mg / dl ) and glucosuria ( > 500  mg / dl ) continued from the day after alloxan injection to the last monitoring day in all of the rats in the alloxan - treated groups .
in addition , the blood glucose levels ranged from 78 to 120  mg / dl ( normoglycemia ) , and the urine glucose levels were less than 100  mg / dl in the control groups .  
typical macroscopic appearances of carious molars in diabetic rats are shown in figures 3(a ) , 3(b ) , and 3(c ) .
macroscopically , the dental caries developed mainly in occlusal fissures and were identified as partial coronal defects of the molars in alloxan - treated diabetic rats ( figure 3(b ) ) .
the carious lesions expanded horizontally , until the crown of the carious molar was completely invisible ( figures 3(b ) and 3(c ) ) . in contrast , control nondiabetic rats showed no changes in any of the molars ( figure 3(a ) ) .  
soft x - ray images of the carious lesion in diabetic rats are shown in figures 3(d ) , 3(e ) , and 3(f ) . in all of the alloxan - treated diabetic rats ,
radiographically , the dental caries progressed both horizontally and vertically . in diabetic rats fed with a pelletized diet ,
the carious lesion was mainly characterized as grade 2 ( figure 3(e ) ) or 4 type ( table 1 ) .
in addition , the diabetic rats fed a powdered diet demonstrated carious lesions that were nearly of grade 4 type ( figure 3(f ) ) . in the control
non - diabetic rats , there was no change in the molar teeth ( table 1 , figure 3(d ) ) .  
the histopathological characteristics of the carious lesion in diabetic rats are shown in figures 3(g ) , 3(h ) , and 3(i ) .
histopathological carious lesions were detected in the crown as eroded dentin with bacterial colonization in alloxan - treated diabetic rats . in diabetic rats fed with the pelletized diet , many of the molars were moderately affected and the dentin caries spread over a wide area of the dental crown ( figure 3(h ) ) . in the diabetic rats fed with the powdered diet , the dental caries were markedly worsened and the crowns were nearly completely decayed ( figure 3(i ) ) .  
the incidence of caries teeth on the basis of each scoring in the soft x - ray examination is shown in table 1 .
the incidence of dental caries in the diabetic rats fed on both the pelletized and powdered diets was apparently higher ( p < 0.01 ) compared to the non - diabetic control rats ( table 1 ) . in the diabetic rats fed with the pelletized diet , 96.7% of their molar teeth were affected with dental caries , and the mean caries score was 2.7 .
in addition , in the diabetic rats fed with the powdered diet , all of their molars ( 100% ) were affected with caries , and the mean caries score was 3.7 , which was significantly higher ( p < 0.01 ) than the diabetic rats fed with the pelletized diet ( table 1 , figure 4 ) .
no radiolucent lesions were observed in any of the molars in control non - diabetic rats .
the incidence of histopathological carious lesions on the basis of each grade is summarized in table 2 .
histopathologically , the severity of the carious lesions in the diabetic rats fed with a powdered diet was significantly enhanced compared to the diabetic rats fed with the pelletized diet ( table 2 ) . in control non - diabetic rats , slight carious lesions , which were macroscopically and radiographically normal ,
were detected in a few animals ; however , there were no differences observed between the non - diabetic rats on the pelletized and powdered diets ( table 2 ) .
the physical properties of foods such as hardness , adhesiveness , and cohesiveness are closely related with the caries - producing potential [ 19 , 20 ] . moreover ,
the development of caries is known to be profoundly affected by food hardness , and hard and coarse foods can exert a detergent activity during mastication , which is effective in the prevention of caries [ 17 , 19 ] . furthermore , a lower degree of caries was reported in animals fed with the hardest food diet . in the present study , the severity of caries in diabetic rats was enhanced in the powdered diet group compared to the pelletized diet group .
it was clear that food hardness affected the development of dental caries in diabetic rats fed on a non - cariogenic diet .
hard foods are known to help flush away or neutralize undesirable material within the dental plaque and exert a cleaning effect on smooth surfaces by direct mechanical friction .
therefore , cariogenicity will be higher in diabetic rats fed with a powdered diet than in those fed with a pelletized diet . in this study , a high incidence and severity of dental caries was confirmed in the alloxan - treated diabetic rats fed with 2 types of standard diets with a low concentration of sugar .
furthermore , the mean caries score of the diabetic rats fed on the pelletized diet at 52 weeks after alloxan dosing in this study was almost 2-fold compared to animals at 26 weeks after alloxan dosing described in a previous report , strongly suggesting that the duration of hyperglycemia may affect caries development in diabetic animals . in this study ,
dental caries developed and extended to all of the molars in diabetic rats fed on both the pelletized and powdered diets .
thus , the effect of food hardness on the different diets in caries development in diabetic rats may become clearer when the dental caries mildly develop during the more early stages . in conclusion
, food hardness could have an effect on the development of dental caries in diabetic rats , and appropriate consideration of the food formulation should be made in experimental caries studies using diabetic rodent models .
 we have previously shown that dental caries may be produced in diabetic rodent models fed with noncariogenic standard diets ; however , many studies usually add large amounts of sugar to the diet to induce dental caries . 
 moreover , the physical properties of cariogenic diets have been reported as an important factor in the formation of caries . 
 the aim of this study was to clarify the effect of the hardness of non - cariogenic diets on the development of dental caries in diabetic rodents . 
 seven - week - old female f344 rats were divided into 4 groups : intact rats fed with a standard pelletized or powdered diet and alloxan - induced diabetic rats fed with a standard pelletized or powdered diet . 
 all of the rats were sacrificed at 52 weeks of age for morphological examinations on their dental tissue . 
 dental caries had developed and extended to all the molars in the diabetic rats that were fed with both the pelletized and powdered diets . 
 moreover , the lesion was significantly enhanced in the powdered diet group compared to that in the pelletized diet group . in conclusion , food hardness is an important factor influencing the development of dental caries in diabetic rats . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion