Indian Journal of Dental ResearchIndian Journal of Dental ResearchIndian Journal of Dental Research
Indian Journal of Dental Research   Login   |  Users online:

Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size         


Table of Contents   
Year : 2015  |  Volume : 26  |  Issue : 3  |  Page : 248-251
Dental fluorosis: A histological study using Light and Confocal microscopy

Department of Oral Pathology, SRM Dental College, Ramapuram, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India

Click here for correspondence address and email

Date of Submission13-Jan-2015
Date of Decision19-Feb-2015
Date of Acceptance03-Jun-2015
Date of Web Publication14-Aug-2015


Aims: To observe and characterize the histological features of fluorosed teeth under light and confocal microscope (CFM). Materials and Methods: A total of 25 fluorosed teeth and 5 normal teeth were collected from dentists across Dindigul, a known endemic area of fluorosis in South India. Ground sections of respective teeth were observed under light microscope and the sections were subsequently stained with acridine orange and studied under CFM. Results: Histological changes were observed in the ground sections of fluorosed teeth as compared with the normal teeth. Depending on the degree of fluorosis, the affected teeth showed various features of hypomineralization in enamel and dentin. Conclusions: Fluoride interacts with both mineral phases and organic macromolecules by strong ionic and hydrogen bonds resulting in incomplete crystal growth at prism peripheries. This presents as hypomineralization of enamel and dentin.

Keywords: Acridine orange, confocal microscopy, fluorosis, hypomineralization

How to cite this article:
Priyadharsini N, Malathi N, Tamizhchelvan H, Dineshkumar T. Dental fluorosis: A histological study using Light and Confocal microscopy. Indian J Dent Res 2015;26:248-51

How to cite this URL:
Priyadharsini N, Malathi N, Tamizhchelvan H, Dineshkumar T. Dental fluorosis: A histological study using Light and Confocal microscopy. Indian J Dent Res [serial online] 2015 [cited 2023 Sep 25];26:248-51. Available from:
Fluorine is the 13th most abundant element that is available in the crust of the earth and is an essential element for the calcification of bones and teeth. Fluoride when consumed under optimal level is found to have anticariogenic effect wherein when consumed in excess than the normal limits is known to cause dental and skeletal fluorosis. Fluorosis is an endemic problem affecting different parts of the country. Fluoride toxicity can be manifested either as dental fluorosis or skeletal fluorosis. As an early sign of dental fluorosis the teeth lose their shiny appearance with development of chalk-white spots. Later these white patches become yellow and sometimes brown or black and in severe cases, loss of enamel gives the teeth a corroded appearance. Detailed studies on enamel changes occurring in human dental fluorosis is a must to understand the pathogenic mechanisms of fluoride action on tooth structure resulting in mottling.[1] It is important to characterize histologically all signs of dental fluorosis, including the very early manifestations to find the histological signs of dental fluorosis. Since no detailed description is available in literature about the histological features of fluorosis, in an attempt to study the same, we collected extracted teeth with varying degrees of fluorosis from various dentists across Dindigul district, a known area of fluorosis endemicity and studied them under light microscope (LM) and confocal microscope (CFM). With these above facts, the present study was undertaken with an aim of characterizing the histological features of dental fluorosis and to observe the histological changes of fluorotic teeth under light and CFM.[/TAG:2]

   Materials and Methods Top

To study the histological features of fluorosed teeth, we conducted this study as a pilot study in Dindigul district in South India, a known area for endemic fluorosis. Fluorosed permanent teeth extracted for varied reasons from individuals of age group 35–40 years, living in Dindigul were collected from private dentists across the district. A total of 25 fluorotic teeth were collected and the degrees of fluorosis were classified using Dean's fluorosis index into severely fluorosed, moderately fluorosed, mildly fluorosed, very mildly fluorosed, and questionably fluorosed teeth with each group containing five teeth and were fixed in formalin. Five nonfluorotic teeth were used as control. Ground sections of all fluorotic and nonfluorotic teeth of thickness 100 µm were made using hard tissue microtome. Self-cure acrylic resin was used as the embedding medium. After making the sections of desired thickness, the sections were studied under LM for histological changes.

Subsequently the sections were stained with acridine orange solution and studied under CFM for a better understanding of the structural alterations.

Inclusion criteria

Extracted fluorosed teeth classified according to Dean's index.

Exclusion criteria

  • Teeth with dental caries
  • Teeth with any other extrinsic stains.

   Results Top

The coronal areas of ground sections of affected teeth were observed under light and CFM. When viewed under LM, teeth with questionable and very mild fluorosis did not present significant alterations histologically. Unlike unaffected teeth [Figure 1]a ground sections of fluorosed teeth showed crescent shaped hypomineralized areas in enamel along with disturbance in the pattern of incremental lines of retzius [Figure 1]b-[Figure 1]d which varied depending on the degree of fluorosis. Mildly fluorosed teeth showed mild disturbances [Figure 1]b whereas severely fluorosed teeth showed increased disturbances in the mineralization pattern along with chipping of surface enamel. Furthermore, moderately and severely fluorosed teeth showed dentinal disturbances. There were increased areas of interglobular dentin spaces indicating hypomineralization of dentin [Figure 1]c.
Figure 1: Light microscopic findings. (a) Normal nonfluorotic tooth showing the regular arrangement of enamel rods and dentinal tubules with no disturbance in the incremental lines of retzius, ×10. (b) Mildly fluorosed tooth showing disturbance in mineralization pattern, ×4. (c) Moderately fluorosed tooth showing increased interglobular dentin extending to the circumpulpal dentin, ×4. (d) Severely fluorotic tooth showing crescent shaped areas of subsurface hypomineralization along the striae of retzius, ×4

Click here to view

Under CFM the acridine orange stained sections of unaffected teeth showed regularly arranged well mineralized enamel rods and incremental lines of retzius along with the regular pattern of arrangement of dentinal tubules [Figure 2]a and [Figure 2]b. Fluorosed teeth showed disturbance in the pattern of arrangement of enamel rods [Figure 2]c and [Figure 2]d. Furthermore, severely fluorosed sections showed irregularly arranged and widened dentinal tubules [Figure 2]e. There was also disturbance observed in the mineralization pattern of dentinal tubules [Figure 2]f.
Figure 2: Confocal microscopy findings. (a and b) Unaffected tooth showing the regular arrangement of enamel rods and dentinal tubules. (c and d) Fluorosed enamel showing hypomineralized areas and altered incremental lines. (e) Fluorosed teeth showing irregularly arranged, hypomineralized, widened dentinal tubules. (f) Fluorosed teeth showing increased interglobular dentin

Click here to view

   Discussion Top

An important morphologic finding seen in fluorotic enamel is that of the presence of brownish discoloration in the surface enamel, which is proportional to the degree of fluorosis. This is because exposed porous fluorotic enamel may take up stain from the oral environment. Less advanced forms of fluorosis which did not show any surface damage also showed uptake of stain. Though this kind of posteruptive change does not occur in milder forms of fluorosis, this could probably be dependent on the dietary habits of the individuals.[2] The general morphology of fluorosed enamel does not differ from normal enamel but the tissue comprise areas of diffuse hypomineralization.[3]

Opacity is characteristic of fluorotic enamel, and this can result from incomplete apatite crystal growth.[4] The ground sections of fluorotic teeth made in our study showed areas of enamel opacity.

Subsurface hypomineralization of enamel is due to delay in removal of amelogenins at the early maturation stage of enamel formation.[5] The retention of amelogenin proteins by fluoride does not allow the enamel to mature and results in surface and subsurface porosities.[6] Inhibition of enzymatic degradation of amelogenins and their delayed removal from the developing enamel is the most important cause for the impaired crystal growth in fluorosis.[7] Fluoride, when incorporated into the crystal lattice, has a retarding effect on the degradation of amelogenin. The gradual arrest of delay in enamel maturation is the important pathogenic mechanism of enamel fluorosis.[8] This could probably be the reason for impaired degradation of amelogenin in fluorotic tissue.

With increasing severity of fluorosis, the subsurface enamel all along the tooth becomes increasingly porous (hypomineralized), and the lesion extends toward the inner enamel.[2] There is a disturbance in the arrangement of enamel rods which represents as disturbance in mineralization pattern and incremental lines of retzius in mildly fluorosed tooth [Figure 1]b. This is attributed to the irregular uptake and its distribution in fluorotic enamel.[9]

Though there was loss of enamel structure in the ground sections of moderately and severely fluorosed teeth used in our study, morphologically unaffected areas even in severely fluorosed teeth were histologically similar to unaffected teeth with no marked hypomineralization. This finding was similar to that observed by Thylstrup et al. using scanning electron microscopy.[10]

We also observed that in advanced stages of fluorosis the enamel becomes very porous that the outer layers break down and the exposed porous subsurface become discolored. Fejerskov et al. stated that there is extensive abrasion of the porous and soft exposed lesions in severe fluorotic enamel because when the well mineralized surface zone is fractured away, the exposed hypomineralized lesions are subjected to extensive modifications by the oral environment.[11]

It was found that the hypomineralized areas presented a crescent shaped appearance [Figure 1]c and [Figure 1]d. Furthermore, the hypomineralization appeared to be patchy in teeth with severe fluorosis [Figure 1]d. The mineral uptake occurs rather deep within the hypomineralized lesion in a crescent shaped fashion.[11]

Fejerskov et al. stated that in addition to the enamel, fluorosis also affects the underlying dentin. Histological examination of teeth from areas of high fluoride concentration showed more alterations in the enamel than that of dentin.[6]

In dentin though fluoride does not alter type I collagen which is the main extracellular component it does alter noncollagenous components. Fluoride is found to reduce the molecular size of dentin phosphoproteins and also lowers the phosphate content. The action of casein kinase II and alkaline phosphatase are also inhibited. The reduced phosphorylation reduces the mineral binding capacity and crystal initiation. The additional presence of dermatan and heparan sulfate reduces the size of glycosaminoglycans and makes them more anionic which while binding to mineral phase result in less mineral deposition.[12]

In the present study, dentin of moderately fluorosed tooth exhibited some amount of disturbances like increased interglobular spaces not only in the mantle dentin but also in the deeper areas which represents that there is marked hypomineralization in dentin as well [Figure 1]e. This is indicative of that fluoride affects the processes involved in biomineralization in general no matter if the crystal formation and growth occurs either in mesenchymal or ectodermally derived mineralized tissues.[12] Higher concentrations of fluoride like 5 ppm causes mottling and hypoplasia of the enamel and hypomineralized dentin, with increased interglobular spaces.[4]

Under CFM the sections of a non fluorosed normal tooth showed regular pattern of enamel rods and incremental lines of retzius [Figure 2]a and [Figure 2]b. Severely fluorosed tooth exhibited areas of hypomineralization in enamel and dentin [Figure 2]c and [Figure 2]d. There was disturbance in enamel rod pattern and arrangement of incremental lines of retzius in fluorotic tooth. There was widening of dentinal tubules [Figure 2]e and increase in interglobular dentin in fluorosed tooth [Figure 2]f which was well appreciated under CFM. This is in accordance with the fact that hypomineralized areas show increased intensity of fluorescence.[13]

   Conclusion Top

Fluoride interferes with the process responsible for the efficient removal of organic matrix components; as a result there is protein retention and disorganized enamel crystal formation resulting in hypomineralization of enamel as well as dentin.[14] From our study, we conclude that:

  • Histologically mild, moderate, and severe forms of fluorosed teeth showed subsurface hypomineralization, alteration in the striae of retzius
  • Moderately and severely fluorosed teeth showed hypomineralization of dentin and increased interglobular dentin not only in mantle dentin but in deeper areas as well
  • The subsurface hypomineralization occurs in a crescent shaped pattern indicating that mineral uptake occurs in crescent shaped fashion
  • Under CFM disturbance in the enamel rod arrangement, incremental lines of retzius and hypomineralization of enamel and dentin were well appreciated.

Since not many detailed studies correlating the histological features of fluorosis as observed under light and confocal microscopy are available in literature, we attempted at this pilot study for a better understanding of the histology of fluorosed tooth and the following observations were made. However, a much detailed study with increased sample size is to be done.

   References Top

Yanagisawa T, Takuma S, Fejerskov O. Ultrastructure and composition of enamel in human dental fluorosis. Adv Dent Res 1989;3:203-10.  Back to cited text no. 1
Fejerskov O, Larsen MJ, Richards A, Baelum V. Dental tissue effects of fluoride. Adv Dent Res 1994;8:15-31.  Back to cited text no. 2
Fejerskov O, Silverstone LM, Melsen B, Moller IJ. Histological features of fluorosed human dental enamel. Caries Res 1975;9:190-210.  Back to cited text no. 3
Avery JK. Oral Development and Histology. 2nd ed. Newyork: Thieme Medical Publishers, Inc.; 1994.  Back to cited text no. 4
Den Besten PK. Mechanism and timing of fluoride effects on developing enamel. J Public Health Dent 1999;59:247-51.  Back to cited text no. 5
Fejerskov O, Richards A, Denbesten P. Fluoride in Dentistry. 2nd ed. Munksgaard publishers, Copenhagen.1996. p. 275-90.  Back to cited text no. 6
Aoba T, Fejerskov O. Dental fluorosis: Chemistry and biology. Crit Rev Oral Biol Med 2002;13:155-70.  Back to cited text no. 7
Aoba T. The effect of fluoride on apatite structure and growth. Crit Rev Oral Biol Med 1997;8:136-53.  Back to cited text no. 8
Walton RE, Eisenmann DR. Ultrastructural examination of various stages of amelogenesis in the rat following parenteral fluoride administration. Arch Oral Biol 1974;19:171-82.  Back to cited text no. 9
Thylstrup A, Fejerskov O. A scanning electron microscopic and microradiographic study of pits in fluorosed human enamel. Scand J Dent Res 1979;87:105-14.  Back to cited text no. 10
Fejerskov O, Manji F, Baelum V. The nature and mechanisms of dental fluorosis in man. J Dent Res 1990;69:692-700.  Back to cited text no. 11
Robinson C, Connell S, Kirkham J, Brookes SJ, Shore RC, Smith AM. The effect of fluoride on the developing tooth. Caries Res 2004;38:268-76.  Back to cited text no. 12
Foreman PC. Fluorescent microstructure of mineralized dental tissues. Int Endod J 1988;21:251-6.  Back to cited text no. 13
Limeback H. Enamel formation and the effects of fluoride. Community Dent Oral Epidemiol 1994;22:144-7.  Back to cited text no. 14

Correspondence Address:
Nataraj Priyadharsini
Department of Oral Pathology, SRM Dental College, Ramapuram, Sri Ramachandra University, Porur, Chennai, Tamil Nadu
Login to access the Email id

Source of Support: Nil, Conflict of Interest: None

DOI: 10.4103/0970-9290.162896

Rights and Permissions


  [Figure 1], [Figure 2]

This article has been cited by
1 Effects of dimethyl sulfoxide pretreatment on the bonding properties of fluorotic dentin of different severity: An invitro study
Meifeng Zhang, Zhengfan Sun, Bo Zhang, Wei Peng, Ling Guo
The Journal of Prosthetic Dentistry. 2022;
[Pubmed] | [DOI]
E. V. Nikolishyna, N. M. Ilenko, I. A. Nikolyshyn
Bulletin of Problems Biology and Medicine. 2022; 2(2): 124
[Pubmed] | [DOI]
3 Disrupted Protein Expression and Altered Proteolytic Events in Hypophosphatemic Dentin Can Be Rescued by Dentin Matrix Protein 1
Elizabeth Guirado, Yinghua Chen, Ryan D. Ross, Youbin Zhang, Catherine Chaussain, Anne George
Frontiers in Physiology. 2020; 11
[Pubmed] | [DOI]
4 Conservative esthetic management of severe dental fluorosis with in-office power bleaching
AmbarW Raut, Vijay Mantri, PriyankaV Gedam, PoojaP Phabyani
Annals of African Medicine. 2020; 19(3): 211
[Pubmed] | [DOI]
5 Micromorphological analysis and bond strength comparison of two adhesives for different degrees of dental fluorosis
Shuangfeng Liu, Yanxia Zhu, Tana Gegen
Applied Adhesion Science. 2020; 8(1)
[Pubmed] | [DOI]
6 Dental fluorosis in the Vesuvius towns in AD 79: a multidisciplinary approach
Pierpaolo Petrone,Vincenzo Graziano,Chaturvedula Sastri,Thierry Sauvage,Marcello Mezzasalma,Mariano Paternoster,Fabio Maria Guarino
Annals of Human Biology. 2019; 46(5): 388
[Pubmed] | [DOI]
7 Dental fluorosis in the Vesuvius towns in AD 79: a multidisciplinary approach
Pierpaolo Petrone,Vincenzo Graziano,Chaturvedula Sastri,Thierry Sauvage,Marcello Mezzasalma,Mariano Paternoster,Fabio Maria Guarino
Annals of Human Biology. 2019; 46(5): 388
[Pubmed] | [DOI]
8 NaF Reduces KLK4 Gene Expression by Decreasing Foxo1 in LS8 Cells
Juedan Li,Peng Wang,Jianghong Gao,Xiuzhi Fei,Yan Liu,Jianping Ruan
Biological Trace Element Research. 2018; 186(2): 498
[Pubmed] | [DOI]
9 NaF Reduces KLK4 Gene Expression by Decreasing Foxo1 in LS8 Cells
Juedan Li,Peng Wang,Jianghong Gao,Xiuzhi Fei,Yan Liu,Jianping Ruan
Biological Trace Element Research. 2018; 186(2): 498
[Pubmed] | [DOI]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  

    Materials and Me...
    Materials and Me...
    Article Figures

 Article Access Statistics
    PDF Downloaded267    
    Comments [Add]    
    Cited by others 9    

Recommend this journal