studies of dental caries over historical periods allow us to know and follow the evolution of the frequency of the disease and also its association with environmental resources ,   especially the relationship ,   now well established ,   between formation of caries , type of food consumed , and lifestyle [ 13 ] .
observations around the aetiology of carious lesions identify the role played by carbohydrates ,   with sugar   as the main factor involved in the increased prevalence of carious lesions , which arises with the cultivation of cereals and the possibility of cooking them , because cooking makes the carbohydrates soft , thus allowing them to stick to teeth , and , moreover , modifies the carbohydrate makeup by cutting the chains into shorter pieces [ 5 , 6 ] .
overall , these modifications make the carbohydrate more cariogenic . nevertheless , multiple factors appear to be involved in dental caries , including oral ecosystem compounds and salivary gland function .
the keyes ' diagram summarizes the main areas of interactions involved in the dental carious process , that is , hygiene practices , oral environment , quality of food consumed [ 7 , 8 ] , and , above all , the time factor : how many times a day food intake occurs . our text is part of the history of carious lesions and the relationship between frequency of caries and quality of food intake [ 6 , 9 ] , which broadly depends on the socio - economic context , including access to food resources and means of cooking . taking these caries parameters into account ,
we propose to compare the frequency of caries ( i ) between two samples of populations who lived during two different historical periods , that is , the chalcolithic and the middle ages , and ( ii ) among three samples of populations , of which two were rural and one was urban , who lived in different socio - economic and geographical contexts in south - west france in the middle ages .
for the three medieval samples , we compare frequency of carious lesions , firstly among the two rural population groups , one that lived on the southwest mediterranean coast and the other that inhabited an inland region , and secondly rural and urban population samples [ 3 , 11 ] . given the age of all the collections studied , it is necessary to present the state of preservation of the jaws used to determine the carious lesion frequency . with this in mind , we chose to verify both of the following hypotheses : firstly , for two different historical periods , a thousand years apart , dental carious lesions are more frequent in population samples living in the middle ages than in the chalcolithic and , secondly , medieval population groups living in different socio - economic and geographical contexts show differences in caries frequency .
the present research was carried out on the skeletal remains of adults selected among the individuals excavated from the following 4 sites in southwest france ( figure 1 ) .
the les treilles cave , a rural inland burial ossuary site dating from the chalcolithic age ( 2 6001 700 bc ) , situated in a mountainous region where the soil is not suitable for the cultivation of cereals . the marsan - lasserre medieval cemetery ( 10th12th c. ) , situated at a rural inland site conducive to cereal cultivation . at this period
, people used to eat cooked cereal daily , the starch of which formed one of their main food resources . the saint michel medieval urban cemetery ( 12th14th c. ) situated in the toulouse suburb of saint michel , which was inhabited by very poor people who had difficulties in obtaining food because of limited access to food resources . the medieval rural cemetery of vilarnau ( 12th14th c. ) situated near the mediterranean coast , a region which enjoys a milder climate than the other three sites studied ; this geographical context allowed vines , fruits , and vegetables to be cultivated and gave access to sea foods . the samples studied in each of these four archaeological sites included ( i ) 24 mandibles for the les treilles ossuary , ( ii ) 33 pairs of jaws belonging to 33 individuals in the marsan collection , ( iii ) 41 pairs of jaws belonging to 41 individuals at the saint - michel cemetery , and ( iv ) 58 pairs of jaws belonging to 58 individuals at the vilarnau cemetery .
this provided a total of 103 maxillae , 127 mandibles , and 3316 teeth ( table 1 ) .
each tooth was recorded with reference to the maxillary state , with presence or absence of teeth and carious lesions .
data were recorded by the same team using a previously published method . to evaluate the degree of preservation and level of maxillary pathology in the four collections studied , we recorded the missing teeth as being lost antemortem or postmortem , which provided information regarding the condition and percentage of teeth lost because of diseases . when a part of the alveolar bone and the corresponding tooth were absent , we recorded them as undetermined .
antemortem tooth loss ( amtl ) was recorded if there were signs of bone remodelling at the level of the alveolar socket , and postmortem tooth loss ( pmtl ) if there was clear evidence of an empty alveolar socket . in the four archaeological collections ,
all jaws were selected according to the level of damage as defined by , and only those belonging to levels 1 and 3 were studied .
level 1 : indicating preservation of both maxilla and mandible and preservation of more than 50% of alveolar bones .
level 2 : indicating preservation of both maxilla and mandible but with preservation of less than 50% of alveolar bones .
level 3 : indicating preservation of only the maxilla or the mandible and preservation of more than 50% of alveolar bones .
level 4 : indicating preservation of only the maxilla or the mandible and preservation of less than 50% of alveolar bones . following these levels
overall , 103 maxillae and 127 mandibles with at least six teeth on the dental arch were studied ; all retained roots were recorded as remaining teeth .
a total of 3535 teeth were studied ( table 1 ) . to diagnose carious lesions we used simple stages to avoid subjectivity in the scoring and eliminate false diagnosis .
thus , a carious lesion was defined as a clear cavitation in tooth tissue   detected macroscopically under the right lighting with the naked eye and using a dental probe in case of doubt regarding lesion development .
we differentiated between carious lesions and other tooth surface defects like pits and deep fissures ; also colour changes of the enamel were not considered as caries unless there was cavitation underneath .
all sticky fissures and early decalcification without loss of enamel were disregarded because they could have introduced an element of doubt .
the carious lesions were recorded considering their topographical location according to tooth part , the surfaces affected , and whether or not the pulp chamber was penetrated , as follows : coronary location on occlusal , mesial , distal , lingual , buccal surfaces , radicular location on mesial , distal , lingual , and buccal surfaces , cervical location on mesial , distal , lingual , and buccal surfaces , all teeth were examined twice by direct inspection at an interval of two weeks with the same team and method . only surfaces that were considered to be carious at both examinations were taken into account .
we analysed results using discriminant analysis , a technique for classifying a set of observations in predefined classes .
the purpose is to determine the class of an observation based on a set of variables known as predictors or input variables .
the rows of the data matrix to be examined constitute points in a multidimensional space , as do the group mean vectors .
discriminating axes are determined in this space , in such a way that optimal separation of the predefined groups is attained .
the data recorded in table 1 concern the state of the dental arches for the four population samples from the four archaeological sites of southwest france . 
 table 1 shows the number of teeth expected , the numbers and percentages of teeth recorded present and absent , and the number of jaw samples from the four archaeological sites studied . for the chalcolithic collection , only 24 mandibles were present ; the remaining jawbones could not be examined because of their poor state of preservation .
the percentage of tooth presence was 40.8% , which was smaller than for marsan 74.9% , saint - michel 75.6% , and vilarnau 74.1% .
the percentage of teeth lost postmortem in les treilles was 47.3% , higher than that in marsan 13.2% , saint - michel 8.6% , and vilarnau 15.8% , and the there were not pulpar caries .
this poor state of preservation can be explained because the chalcolithic collection was discovered long ago , in 1939 in an ossuary , and the bones were handled many times at a period when anthropological considerations were different .
this observation led us to consider the limitations of the archaeological samples studied as follows .
( i ) in general , over time , bones and teeth undergo damage or taphonomic effects related to bad conditions of preservation , occurring , for instance , during long contact with the soil in the osteological series where graves were not individual or had deteriorated over time because of the basic coffin structure .
nevertheless we stress that teeth resist better than bone in extreme conditions of preservation because of their high degree of mineralization .
( ii ) in our study the limitation concerned ( 1 ) the pmtl ,   which most often affected incisors and premolars , due to the nonretentive shape of their roots as often found , and ( 2 ) the better state of preservation of mandibles than maxillae because the maxillary bone structure is more fragile than that of the mandibles .
that explains why maxillae were missing in the oldest collection whereas , in the medieval osteological collection , we were able to select paired maxillae and mandibles although the maxilla was generally in a poorer state than the mandible .
moreover it is difficult to have access to information about food intake , dental hygiene , and previous state of health . in table 2 , we give the frequency of presence or absence and the distribution of carious lesions according to tooth type : incisor , canine , premolar , and molar in the four population samples studied in les treilles , marsan , saint - michel , and vilarnau .
overall comparison between sites showed the teeth most affected by ( i ) antemortem tooth loss ( amtl ) were the molars in the four population samples and by ( ii ) postmortem tooth loss ( pmtl ) were the anterior teeth because of the nonretentive form of their roots . in both cases ,
as for the impact of amtl frequency on caries prevalence , we note that ( i ) the amtl frequency is low and so its influence is negligible and ( ii ) tooth loss can be due to wear level . indeed , in a study by lucas et al . , tooth wear , the main dental feature in historic populations ,
is reported to be associated , in case of heavy tooth wear , with periapical lesion development via pulpar necrosis .
thus , the first molars , the most worn teeth , are also the most often absent antemortem . considering the distribution of carious lesions
, we can see that , overall , the les treilles archaeological site was the least and marsan the most affected for all tooth types . also , for the four population samples , molars were the most affected tooth type and canines and incisors the least .
regarding the higher degree of severity of caries when root or pulp were affected , the four population samples showed that molars were the most often affected , followed by premolars .
canines and incisors were affected in the three medieval sites : marsan , vilarnau , and saint - michel .
regarding the types of carious lesions , marsan provided the population sample most affected by all types of caries and , moreover , showed the highest levels of severity , that is , pulpar and radicular cavities . bearing this in mind , we considered ( i ) the two historical periods studied , separated by more than 1000 years , and found that the les treilles population had the lowest percentage of teeth affected by caries compared to the three medieval populations , a finding consistent with other studies , and ( ii ) the geographical and socio - economic context , including food resources .
we explained the difference of caries incidence between the chalcolithic and middle ages by a diet with a lower consumption of carbohydrates and cooked food in the chalcolitic age , when cereal cultivation was less common than in the middle ages in general and especially for the population of les treilles , who lived in a mountainous area not conducive to cereal crop cultivation , which is still true today , cattle farming being the main resource in this inland region . on the other hand , data recorded for the three medieval population samples underlined the higher incidence of caries in the marsan samples than that in the saint - michel and vilarnau samples . in the light of the differences of access to dietary resources among these three population samples
, we can explain the percentage variation of caries : the marsan population lived in an inland region in which many silos for cereal crops have been discovered at archaeological sites , so the people 's daily food was based on cooked cereal , and cervical carious lesions gave evidence of cariogenic food intake .
in vilarnau , a coastal area , people had mediterranean food resources that included more fruits , green vegetables , and fish and less carbohydrate than in marsan . at the saint - michel archaeological site ,
a medieval suburb of toulouse in which poor people lived , who had difficulty in obtaining food , the food restriction that affected the population sample can be associated with the lower incidence of caries than that in marsan .
a survey conducted during the second world war , in a population subjected to dietary restrictions , also shows a decrease in caries frequency .
moreover , garcin 's comparative study , concerning medieval juveniles in four european countries , corresponds to our results regarding the geographical site and dietary resources : both coastal and , to a lesser extent , urban sample populations showed a lower dental caries frequency than the inland population , a result suggesting that the coastal population , who ate fish and few carbohydrates , had a less cariogenic diet . in figure 2 , the principal component analysis ( pca ) diagram displays the carious lesion distribution of the four population samples studied in relation to the tooth morphotype and type of carious lesion .
dimensions 1 and 2 include , respectively , 59.23% and 31.84% of information taking account of the five types of caries .
we can see that the chalcolithic sample from les treilles is characterized by cervical caries on molars and differs from the three medieval population samples ' distribution .
the marsan sample is related to pulpar and radicular caries on molars , premolars , canines , and incisors , thatis affected by the most severe type of caries .
the saint - michel and vilarnau samples show a low incidence of proximal and pulpar caries and occlusal caries on molars .
these french data regarding the comparison and evolution of the carious lesion processes in population samples belonging to two historical periods make an additional contribution and are a part of the history of dental caries .
our results also verify the hypothesis made for the effect of environment and lifestyle , including food preparation , on dental heath .
our findings are supported by works considering the area of dental caries history from the chalcolithic to the present time , which most often show an evident relationship between access to food resources , social level , and caries incidence .
studies considering archaeological human remains usually distinguish two main populations over time : the hunter - gatherers , with a very low percentage of dental caries , and the farmers , whose carious lesion incidence is increased relative to the first group .
so , the chalcolithic period , when diet was above all rich in hard fibrous vegetables and low in starch and sugars , was characterized by a low caries incidence .
for instance , caries frequency varied widely from none to 25% of the teeth for populations of the metal age and the islamic period in the arabian gulf .
this author points out differences between coastal and inland dwellers on the one hand and , on the other hand , a relationship between little evidence of calculus and low caries frequency and heavy calculus accumulation with high caries frequency because a fibrous diet , poor in carbohydrates , removes calculus deposits , unlike the soft , cooked carbohydrates which can not have this effect . in all cases , premolars and molars
were the teeth most often affected by the carious process . for eshed et al .
, changes in food - preparation techniques and nondietary use of teeth can explain the dental health differences before and after the agricultural revolution . in south america ,
. 's study   analysed patterns of carious lesions occurring in the coastal maya population of xcamb in northern yucatan in the classical period . to do this ,
the study investigated caries in the permanent dentition of adults from the early ( 250550 ad ) and late ( 550750 ad ) classical periods .
the results indicate an increase in caries from between 7.4% and 21.2% in the early classical to a mean of around 20% in the late classical period , but the author stresses on the limitations imposed by interpreting carious lesions solely in terms of single dietary components , such as maize consumption , without taking broader aspects of cultural and socioeconomic relevance into account .
following the period chronology , works studying population samples who lived over the early centuries ad noted variations in caries incidence related to access to dietary resources and socioeconomic level , as around the roman historical period . following the historic chronology , in the middle ages , caries incidence increased and affected around 20% of teeth in populations whose food was cooked and included carbohydrates [ 20 , 21 ] .
later , in the 18th c. , whittaker and molleson in england   drew a parallel between the increase of caries incidence and the increase in the importation of sugar . today , works on caries incidence report the impact of food resources on the risk of caries development [ 2328 ] .
for instance , during the first and second wars , the context of deprivation was related to a steady decline of caries incidence [ 1 , 29 ] .
moreover , recent studies in the different continents establish relationships between dental health and financial insecurity with low family income and poorly educated parents [ 24 , 2628 ] . in this context ,
very young children , less than one year old , are more often affected by caries because of frequent intake of sweet food and drink [ 23 , 2527 ] whereas , in caglar 's byzantine population , the caries prevalence in primary dentition was 0% .
these changes in daily eating and dietary models also have consequences on health and quality of life . to conclude , the population samples studied , over the historical period considered , displayed caries frequency differences related to the geographical area and access to dietary resources .
investigations spread over time and place support our observations and contribute to the idea of a continuing relationship between way of life , food quality and access , socioeconomic level , and dental caries evolution , a reliable predictor of the context of life . bearing this in mind , the direct , continuous impact of starch and sugar consumption on caries incidence is visible from the chalcolithic age to the present time , around the world and across civilizations .
 the majority of dental carie studies over the course of historical period underline mainly the prevalence evolution , the role of carbohydrates consumption and the impact of access to dietary resources . 
 the purpose of the present investigation was to compare population samples from two archaeological periods the chacolithic and middle age taking into account the geographical and socio economical situation . 
 the study concerned four archaelogical sites in south west france and population samples an inlander for the chalcolithic age , an inlander , an costal and urban for the middle age . 
 the materials studied included a total of 127 maxillaries , 103 mandibles and 3316 teeth . 
 data recorded allowed us to display that the chalcolithic population sample had the lowest carie percentage and the rural inlander population samples of middle age the highest ; in all cases molars were teeth most often affected . 
 these ones differences could be explained according to time period , carious lesions were usually less recorded in the chalcolithic age than the middle because of a lesser cultivation of cereals like in les treilles chacolithic population sample . in the middle age population samples , 
 the rural inland sample marsan showed the highest frequency of caries and ate more cereal than the coastal vilarnau and the poor urban st michel population samples , the first one ate fish and mediterranean vegetal and fruits and the second one met difficulties to food access , in both cases the consumption of carbohydrates was lesser than marsan population sample who lived in a geographical land convice to cereals cultivation . 
1. Introduction
2. Materials and Methods
3. Results and Discussion