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IntroductionThe generic term used to describe a developingtooth is “tooth germ”; the expression “tooth bud”should be avoided because of the overlap with thename of the first stage of tooth development [1].For an accurate definition of the tooth germthroughout its development, it is necessary to use adouble terminology that ensures a correlationbetween the functional phases and the morphologi-cal stages [1]. The phases of the development (ini-tiation, proliferation, differentiation and apposi-tion) follow one another dynamically and at timesthey partially overlap, the development being acontinuous process without clear demarcation lines[2]. The names of the stages are based on the mor-phology of the epithelial structure of the toothgerm: bud, cap, and bell. Consequently, the toothdevelopment process has the ensuing sequences:

(a) crown formation, (b) root formation and con-comitantly, (c) support tissue formation.

During tooth histo-morphogenesis changesoccur:

In the composition of the basement mem-brane (involving the metalloproteinases andtheir inhibitors) [3-5].In the signalling molecules mechanisms [6].In the localisation of cell surface elements(integrins [7,8], cadherins [9], signallingmolecules receptors [10]).

The regulation of the number of cells and theirarrangement is achieved through complex process-es including proliferation, apoptosis and plasticityin cell-cell and cell-matrix interactions [11,12].

Although similar studies have previously beenperformed by other groups, the study that is report-ed in this paper is the first on this topic designedand carried out in Romania.

Correlations Between Morphology and Evolution in the Structure ofthe Tooth Germ

Sergiu Daniel Sãvinescu1, Vlad Dãnilã2, Mioara Trandafirescu3, Irina-Draga Cãruntu4

1 Ph.D. Student. Department of Histology, Faculty of Medicine, Grigore T. Popa University of Medicine and Pharmacy, Iasi, andSpecialist in Forensic Medicine, Vaslui County Forensic Service, Romania. 2 Ph.D. Student and Specialist in OromaxillofacialSurgery, Faculty of Dental Medicine, Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania. 3 Ph.D., Assistant-Professor of Histology, Faculty of Medicine, Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania. 4 Ph.D.Professor of Histology, Faculty of Medicine, Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania.

AbstractAims: The aim of this study was the microscopic morphologic assessment of the development of deciduous tooth germsto investigate whether it correlated with the biologic age. Methods: The study material consisted of oral tissue from 20fetuses with a minimum gestational age of two months or newborns deceased at birth, cases autopsied in the VasluiCounty Forensic Service and in the Pathology Laboratory of the Municipal Hospital Barlad, Romania. Legal harvesting,manipulation and conservation conditions were respected; the collection of the specimens was performed only after eth-ical approval and with the written consent of the family concerned. The investigation methods consisted of routine his-tological examination of the specimens after they had been processed through special decalcification techniques andstained with hematoxylin-eosin and three special stains. Results: The microscopic evaluation of deciduous tooth germsin cap and bell stage allowed the characterisation of the three main components—enamel organ, dental papilla and den-tal sac—through the cellular details contributing to the development of the typical architecture of the tooth. Conclusions:In the cap and bell stage, the tooth germs had different morphologic features, without a direct correlation with the ges-tation age. The marked diversity in the shapes and sizes of the identified tooth germs supports the influence of geneticfactors and of the specific interactions with the micro-environment in tooth development.

Key Words: Tooth Germ, Development, Histology

Corresponding author: Professor Irina-Draga Cãruntu, Department of Histology, Faculty of Medicine, Grigore T. PopaUniversity, 16, University Street, 700115, Iaºi, Romania; e-mail: irinadragacaruntu@gmail.com

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AimThe aim of this study was the microscopic morpho-logic assessment of the development of deciduoustooth germs to investigate whether it correlatedwith the biologic age.

MethodsThe study material came from 20 human embryosand fetuses with a minimum gestational age of twomonths, resulting from medical or spontaneousabortions, and newborn babies that had died at birth.They were investigated in the Vaslui CountyForensic Service and Pathology Laboratory of theMunicipal Hospital Barlad, Romania. The gesta-tional age of each embryo and fetus was establishedfrom the relevant medical records. Specimens wereharvested, processed and stored according to theethical guidelines for these procedures. Collectionwas performed only with the written consent of thefamily concerned. The study was approved by theethics committees of the medical institutions men-tioned above, and of the Grigore T. Popa Universityof Medicine and Pharmacy, Iasi, Romania.

The embryos were fixed in 10% neutralbuffered formalin, fully embedded in paraffin, and4 µm-thick serial sections were cut. The cephalicextremities (fetuses aged from 10-16 weeks) andthe maxillas and mandibles (fetuses aged from 18-40 weeks) were carefully removed and dissected,fixed in 10% neutral buffered formalin, and decal-cified in 5% formic acid and 5 g sodium citrate.The decalcification time was variable (minimumthree days, maximum three weeks), depending onthe gestational age and on the degree of mineralisa-tion of the dental tissues. After demineralisation,the fragments were processed before being embed-ded in paraffin. Finally, cross-sections were cut toa thickness of 4 µm. All sections were stained withhaematoxylin and eosin (HE) for standard histolog-ical examination, as well as with special stains(trichrome Masson, trichrome Azan-Heidenhain,trichrome Szekelly, PAS). The special stains wereused in order to permit a clearer identification anddifferentiation of epithelial and mesenchymalstructures within the tooth germs.

Results

Identification of tooth germsThe study allowed a global assessment of eachcase. Excluding artefacts due to the difficulties ofthe harvesting and processing the human embryon-ic and fetal material (including the orientation ofthe specimens, and the particularities of the decal-

cification time), only tooth germs that were com-pletely sectioned and had preserved their structuralintegrity underwent morphological analysis. Inthese tooth germs, the histological stages of devel-opment reported in Tables 1 and 2 were seen.

Qualitative analysis of the identified toothgerms: defining histological elementsBud stageThe tooth germs at the bud stage presented a classi-cal pattern, including three types of cells:

Epithelial cells, which were compactlyorganised, maintaining the continuity withoral epithelial and forming the epithelialbud.Ectomesenchymal cells, which werearranged in a crowded manner around theepithelial bud and initiating the future den-tal papilla.Ectomesenchymal cells, which werelocalised more externally, as an accessorycondensation that ensures the developmentof the future dental follicle.

Cap stageThe tooth germs in cap stage had various shapesand sizes, without a direct correlation with the ges-tational age (Figures 1-3). The morphologicalstructure included the three main components: theenamel organ, the dental papilla and dental sac. Themicroscopic morphological evaluation of thesecomponents revealed cell structure details con-tributing to the establishment of the typical archi-tecture. In the enamel organ, the three epithelia—inner, outer and stellate reticulum—were identified(Figure 2). The outer enamel epithelium consistedof a single layer of small cells, most frequentlycuboidal with a curved spatial arrangement ratherthan a straight one. The inner enamel epitheliumwas characterised by the tall columnar shape of itscells. In the initial stages (incipient cap stage), thearrangement of the cells was rather crowded, sug-gesting a possible stratification; only later(advanced cap stage) did it become a single-layerstructure. The stellate reticulum, situated betweenthe inner and the outer ones, revealed different evo-lutionary aspects: in the initial stages, the extracel-lular matrix had a more mucoid consistency, thecells dissipated in the matrix and were without con-spicuous cytoplasmic expansions, and their identi-fication was supported by the presence of theirnuclei. During development, the extracellular

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Specimen Age (weeks) Maxillae: Number of tooth germs/stage of developmentI1 I2 C M1 M2

1 8th i.u. 2 / Bd 2 / Bd 2 / Bd 2 / Bd NO2 8th i.u. 2 / Bd 2 / Bd 1 / Bd 1 / Bd NO3 10th i.u. 1 / Cp1 1 / Cp1 1 / Cp1 1 / Cp1 NO4 10th i.u. 2 / Cp1 2 / Cp1 1 / Cp1 1 / Cp1 NO5 10th i.u. 1 / Cp1 1 / Cp1 NO NO NO6 14th i.u. 1 / B1 2 / Cp2 1 / Cp1 1 / Cp1 NO7 14th i.u. 2 / B1 2 / Cp2 1 / Cp1 1 / Cp1 NO8 14th i.u. 1 / B2 2 / Cp2 2 / Cp2 NO NO9 16th i.u. 2 / B2 2 / B1 1 / Cp2 1 / Cp2 NO10 18th i.u. 1 / B2 2 / B1 1 / B1 1 / Cp2 1 / Cp211 18th i.u. 2 / B2 1 / B2 1 / B1 1 / B1 NO12 22nd i.u. 1 / B2 1 / B2 1 / B2 1 / B2 1 / B113 22nd i.u. 2 / B2 1 / B2 NO 1 / B2 1 / B214 22nd i.u. 1 / B2 2 / B2 1 / B2 NO NO15 24th i.u. 2 / B2 1 / B2 1 / B2 1 / B2 1 / B216 24th i.u. NO 1 / B2 1 / B2 1 / B2 1 / B217 24th i.u. 2 / B2 2 / B2 2 / B2 1 / B2 1 / B218 24th i.u. 1 / B2 2 / B2 1 / B2 NO NO19 40th / birth 2 / B2 1 / B2 1 / B2 1 / B2 NO20 40th / birth 2 / B2 2 / B2 1 / B2 2 / B2 1 / B2

Table 1. Synopsis of the identified tooth germs in maxillae and the corresponding stage of development

*I1: central incisor, I2: lateral incisor, C: canine, M1: first molar, M2: second molar, Bd: bud stage, Cp1: incipient capstage, Cp2: advanced cap stage, B1: incipient bell stage, B2: advanced bell stage, NO: non-sectioned tooth germ(s)

Specimen Age (weeks) Mandible: Number of tooth germs/stage of developmentI1 I2 C M1 M2

1 8th i.u. 2 / Bd 1 / Bd 2 / Bd 2 / Bd NO2 8th i.u. 1 / Bd 2 / Bd 1 / Bd 1 / Bd NO3 10th i.u. 1 / Cp1 1 / Cp1 NO 1 / Cp1 NO4 10th i.u. 2 / Cp1 1 / Cp1 1 / Cp1 1 / Cp1 NO5 10th i.u. 2 / Cp1 2 / Cp1 1 / Cp1 1 / Cp1 1 / Cp16 14th i.u. 1 / Cp2 2 / Cp2 1 / Cp1 1 / Cp1 1 / Cp17 14th i.u. 1 / B1 2 / Cp2 2 / Cp2 NO NO8 14th i.u. 2 / B1 2 / Cp2 NO 1 / Cp2 NO9 16th i.u. 1 / B1 2 / Cp2, B1 1 / Cp2 1 / Cp2 NO10 18th i.u. 2 / B1 2 / B1 1 / Cp2 1 / Cp2 1 / Cp211 18th i.u. 1 / B2 1 / B2 NO 1 / Cp2 1 / Cp212 22nd i.u. 1 / B2 2 / B2 1 / B2 1 / B1 1 / B113 22nd i.u. 2 / B2 1 / B2 1 / B2 NO NO14 22nd i.u. 2 / B2 2 / B2 1 / B2 NO NO15 24th i.u. 1 / B2 1 / B2 1 / B2 1 / B2 1 / B216 24th i.u. NO 1 / B2 1 / B2 1 / B2 1 / B217 24th i.u. 2 / B2 2 / B2 2 / B2 1 / B2 1 / B218 24th i.u. 2 / B2 1 / B2 NO 1 / B2 1 / B219 40th / birth 1 / B2 1 / B2 1 / B2 1 / B2 1 / B220 40th / birth 2 / B2 1 / B2 1 / B2 2 / B2 1 / B2

Table 2. Synopsis of the identified tooth germs in mandibles and the corresponding stage of development

*I1: central incisor, I2: lateral incisor, C: canine, M1: first molar, M2: second molar, Bd: bud stage, Cp1: incipient capstage, Cp2: advanced cap stage, B1: incipient bell stage, B2: advanced bell stage, NO: non-sectioned tooth germ(s)

matrix became more transparent and clear and itscells achieved their stellate shape with multipleinterconnecting expansions defining an obviousaspect of a cell network. In the dental papilla, theabsence of homogenous features characteristic tothe stage of incipient cap was noted. The convexarea of the papilla, corresponding to the concavityof the enamel organ, showed distinct areas of celldensification typical of the advanced cap stage(Figure 3). The dental sac was evident, the ecto-mesenchyme was concentrically organised andsupported the other two components (Figure 3).

Bell stageThe tooth germs at the bell stage were of variousshapes and sizes, their diversity was independent ofthe gestational age, making a dynamic evaluationpossible for the three main components: enamel

organ, dental papilla and dental sac (Figures 4-9).The analysis of the enamel organ allowed the

observation of the multiple and successive changesin shape and size that determine the passage fromthe cap to the bell stage. For the outer epithelium ofthe enamel organ, the cells—mostly cuboidal withreduced cytoplasm and large conspicuous nucle-us—maintained the irregular curved arrangementfrom the cap stage along the concavity, except at itsupper pole where their arrangement was linear. Theappearance of the stellate reticulum of the enamelorgan was typically that of an interconnected cellnetwork, consisting of stellate cells with multidi-rectional processes. Between the stellate reticulumand the inner epithelium, the stratum intermediumof the enamel organ was identified; at this stage, itconsisted of one to four cell layers, which althoughextremely differently shaped, suggested a perpen-

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Figure 1. Developing oral cavity: tooth germs(central incisors) in cap stage, connected to the

oral epithelium (Masson, x 2).

Figure 3. Tooth germ (lateral incisor) inadvanced cap stage: obvious condensation of theectomesenchymal cells at the periphery of dental

papilla (HE, x 4).

Figure 2. Tooth germ in incipient cap stage (firstmolar): the enamel organ, the dental papilla, the

dental sac (HE, x 4).

Figure 4. Tooth germ (first molar) in incipientbell stage: first dentine layer, Hertwig epithelialsheath in different stages of development (mesial

vs. distal) (HE, x 4).

dicular disposition on the inner epithelium cells.The analysis of the inner epithelium revealed twodifferent aspects, allowing for the differentiation ofthe two stages: incipient bell (Figure 4) andadvanced bell (Figures 5 and 6). In the incipientbell stage, the inner epithelium consisted of colum-nar cells with a basophilic cytoplasm and, extreme-ly importantly, a central position of the nucleus.The advanced bell stage was defined by the pres-ence of dentine deposed by the odontoblasts in thedental pulp, with the consequent occurrence andlater apposition of the enamel layer (Figures 5 and6). In the inner epithelium of the enamel organ, theelongation of the columnar epithelial cells wasidentified: pre-ameloblasts and then ameloblasts,the characteristic morphologic feature being thechange in their polarity (Figure 7). The process was

conspicuous through the specific position of thenucleus and the reorganisation of the cytoplasmicdistribution. In the development of the bell stage,the thickness of the enamel layer was variable,clearly greater in the incisal areas and/or at the levelof the future cusps and smaller in the lateral areasof the future crown (Figures 8 and 9).

The structure characteristic for the enamelorgan included the transformation of Hertwig’sepithelial sheath (Figures 8 and 9), which con-tributed to the development of the epithelialdiaphragm, which was also identifiable in the spec-imens. The dental papilla showed transformationsdirectly correlated with those identified in theenamel organ. The mesenchymal cells clustered atthe periphery of the papilla at the end of the capstage and evolved morphologically and functional-

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Figure 5. Tooth germ (central incisor) inadvanced bell stage: the enamel organ, the dental

papilla, the dental sac (PAS, x 4).

Figure 7. Tooth germ (central incisor) in advanced bell stage: details for ameloblasts, enamel, dentine andodontoblasts (Masson, x 10(a), x 20(b)).

Figure 6. Tooth germ (central incisor) inadvanced bell stage: cusp tip presenting dentine,enamel, and the outer enamel epithelium posi-

tioned next to the stratum intermedium in areas ofmore advanced apposition (Masson, x 4).

ly. Consequently, in the incipient bell stage a con-tinuous cell layer consisting of tall cuboidal cellswith oval nuclei located at their basal pole—theodontoblasts—was present, covered by the first lineof dentine, with variable thickness according to thegestational age and, implicitly, to the age of thetooth germ (Figures 5-7). Under the odontoblasticlayer, the dental papilla converted into dental pulp,maintaining its clustering of mesenchymal cells,thus constructing the sub-odontoblastic layer withdifferentiation potential, under certain circum-stances, into odontoblasts. The central area of thedental pulp presented the typical features of a looseconnective tissue. The advanced bell stage wascharacterised by the changes from the periphery ofthe dental pulp, marking the development of theperipheral pulp with an architecture differing fromthat of the central pulp through the occurrence,under the odontoblastic layer, of the cell-free zone(zone of Weil) and of the cell-rich zone. The dentalsac was readily identifiable around the developingtooth germs; its structure was compact (Figures 8and 9).

DiscussionThe morphogenesis of the tooth and its supportingtissues is a component of the embryologic develop-ment of an individual and is in direct correlationwith the development of the head. For many years,researchers have studied the mechanisms involvedin the early development of the structures associat-ed with the oral cavity and specifically those build-ing the tooth and its supporting structures. Thesequential morphologic development of these

structures is already known, so current research(mainly experimental) focuses on a biologicapproach at a molecular and genetic level[4,6,9,10], opening broad perspectives for tissueengineering [13,14].

The development of the tooth takes place as aconsequence of the interrelations between the oralepithelium and its subjacent mesenchyme [2,15].The development process is essentially identical forall teeth, both deciduous and permanent. The odon-togenesis involves morphogenesis, epithelial histo-genesis and cell differentiation [2]. The regionaldevelopment of the teeth can be translated throughmodulation—space and time—involving the under-standing of the concepts of induction, competenceand differentiation [1]. Dental epithelial remodel-ling results from variations in the cell proliferationspeed, apoptosis and changes in the adherence andthe shape of the cell, leading to the placement of thecells with different functions [2]. Independent ofthe signalling molecules, which play a major role inthe induction or the modulation of specific stages,cell-cell and cell-matrix interactions regulate theplasticity/rigidity of certain areas in the enamelorgan [2].

Within this framework, for the first time inRomanian dental research, the current study hasdemonstrated the picture of the tooth developmentphases versus the biologic age, and, concomitantly,comments on the difficulties occurring during theinvestigation of tooth development.

The value of microscopic examination in thecharacterisation of the tooth development stagesThe entire deciduous dentition is initiated between

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Figure 8. Tooth germ (central incisor) inadvanced bell stage: the Hertwig epithelial sheath

(Masson, x 4).

Figure 9. Tooth germ (central incisor) inadvanced bell stage: transformation of the

Hertwig's epithelial sheath into the epithelialdiaphragm (PAS, x 4).

weeks 6 and 8 of embryonic development, the per-manent successional dentition between week 20 inutero and the tenth month after birth, and the per-manent molars between week 20 in utero (for firstmolars) and the fifth year of life (for third molars)[1]. In the current study, the ages of the embryosand fetuses enabled an investigation across manystages in the development of the deciduous denti-tion. However, the study focused on the identifica-tion of tooth germs in pre-eruptive stages.Depending on the biologic age, it was possible toobserve differences in the position of the develop-ing tooth germs on the inside of the maxillary bonein the areas equivalent to the dental alveoli. Thedifferences in the position of the tooth germsdepended on the development stage of the maxil-lary and constituted an indicator of the eruptiveposition. The tooth germs identified in the firsttrimester of pregnancy were of very small size andwere located at considerable distance from oneanother. The tooth germs identified in the secondand third trimesters of pregnancy were larger, as aresult of fast growth and, consequently, the spacebetween them was considerably reduced. However,as tooth formation occurs over a wide time span,the developmental stage of individual tooth germsis poorly related to gestation time.

The microscopic examination confirmed theasymmetrical growth characteristics of the compo-nents of the tooth germs and was representative ofthe proliferation stage, which leads to the cap stage.There was a transformation of the epithelial budwith a change in its shape from oval to round and,later on, to central curving leading to the appear-ance of the cap structure placed over a “ball” ofcondensed mesenchymal tissue.

The microscopic examination also allowedvisualisation, in the evolution from cap to bellstage, of the transformation of the cervical loop intoHertwig’s epithelial sheath by the expansion of theinner and outer epithelia of the enamel organ, andthe disappearance of the stratum intermedium andstellate reticulum.

The analysis of the structural details of thetooth germs leads to an evaluation of the changesoccurring in the ameloblasts, a morphological eventessential in the tooth development process. Thetooth germs of fetuses in the second and thirdtrimesters of pregnancy showed the elongation ofthe cells in the inner epithelium of the enamelorgan. Thus, in a first stage it was possible to iden-tify the central position of the oval nucleus, occu-

pying almost the entire cell cytoplasm. Later, anincrease in cell size, which causes the oval nucleusto become placed at the apical pole (towards thestratum intermedium), was noted. The tooth germspresenting the first dentine layer revealed prismaticameloblasts with a nucleus that had already migrat-ed to the basal pole (towards the dental papilla). Allthese modifications provided evidence of the mod-ifications in the polarity of the ameloblasts, anevent which precedes the occurrence of an apicalcytoplasmic differentiation (the Tomes process),conspicuous in the advanced bell stage and charac-terised by the deposition of the first enamel layer. Itshould be stressed that identification of the Tomesprocess, prominent in newly formed enamel, isextremely difficult due to the characteristics ofhard-tissue processing. However, because of thelarge number of tooth germs that were investigated,this was possible and the typical aspect of “saw-teeth” or “picket fence” created by the Tomesprocess at the junction between the ameloblasts andthe enamel in the process of formation wasobserved.

Concurrently with the appraisal of modifica-tions in the ameloblasts, it was also possible toobserve the transformation of the odontoblastsplaced at the periphery of the dental papilla, turninginto dental pulp. The first indication of this differ-entiation was identified in tooth germs at the incip-ient bell stage. It was the disappearance of the cell-deprived area between the papilla and the innerepithelium of the enamel organ while in theadvanced cap stage, the occupation of this territorywas achieved by the increase in size of the odonto-blast. The microscopic examination allowed identi-fication of tooth germs in the incipient bell stage atthe starting point from the area at the top of the den-tal papilla adjacent to the inner epithelium of theenamel organ, where the first layer of dentine aris-es. Other tooth germs revealed different stages inthe process of dentine deposition on the side of thefolding of the zone previously described and indi-cating the starting point of the development ofcusps, advancing downwards and laterally on thecusp slope up to the Hertwig epithelial sheath.Because of the differences in the thickness of thedentine layer, it was possible to separate the periph-eral outer dentine (mantle dentine), with a lowermineralisation degree, from the circumpulpal den-tine, which already contained dentinal tubules, theirslightly woven trajectory being a clear indicator ofthe arrangement of the odontoblasts in their cen-

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tripetal movement towards the inside of the dentalpulp.

During examination of the tooth germs, theexisting blood supply was also assessed in order toevaluate the differences between the cap and bellstages. The multiplication of the vascular elementsin the dental papilla was noted. They were simulta-neous with the passage from the cap to the bellstage. The important development of the blood sup-ply in the dental sac responsible for the nourishingsupport of the ameloblast and the entire enamelorgan—after differentiation of the ameloblasts andthe beginning of enamel formation, with the conse-quent interruption of contact with the dental pulp—was also noted.

These descriptive elements offer a picture ofthe microscopic morphology of tooth germ devel-opment. At an international level, the histologydescribed in this paper has previously been report-ed. However, it seems that it has previously beensupported by a relatively small number of imagesand they appear frequently, with due acknowledge-ment to their original authors, in a number of booksand other publications. This may be because of thedifficulties in accessing suitable material. Theauthors of this paper believe that the number ofembryos and fetuses examined and the number ofthe resulting images will make a contribution to thecurrent state of knowledge.

Difficulties in the tooth development researchTechnical considerationsThe histological processing of the tooth requires amandatory phase for demineralisation, because ofthe hard, inorganic material present in some struc-tures. Consequently, several technical problemscan occur. The choice of the best demineralisationsolution and the assessment/evaluation of the timeneeded for this procedure are dependent not onlyon the instructions available in books, but also onthe experience of each laboratory. We consider that

our technique leads to specimens of high quality,ensuring the visual support for our results.

Bioethical implicationsThe research on tooth development in humans isdifficult because of bioethical implications. The useof human material, represented by embryos andfetuses, is highly controversial [16] as a result ofmoral, ethical and religious considerations.However, studies based on cell lines or animalmodels cannot completely replace the humanmodel. In this context, the authors consider that thisstudy has been valuable and that the investigationof human material should be permitted as long asthe ethical principles of the research are respected.

ConclusionsIn the cap and bell stage, the tooth germs had dif-ferent morphologic features, without a direct corre-lation to gestational age. The marked diversity inthe shapes and sizes of the identified tooth germssupports the influence of genetic factors and of spe-cific interactions with the micro-environment intooth development.

Contribution of each authorSDS and IDC designed the study.SDS collected the specimens.VD performed the dissections of the fetuses,

before the fixation of the specimens.SDS and MT performed the histological exami-

nations and analysed the results. They produced thefirst draft of the manuscript.

IDC planned and conducted the study, participat-ed in the interpretation of the results, and draftedthe final version of the paper.

All authors approved its content.

Statement of conflict of interestsThe authors are aware of no conflicts of interest.

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