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Laser Ther. 2016 October 1; 25(3): 201–208.
PMCID: PMC5108995

Comparative evaluation of microhardness and morphology of permanent tooth enamel surface after laser irradiation and fluoride treatment - An in vitro study


Background and aims: The aim of the study was to evaluate and compare the surface microhardness and surface morphology of permanent tooth enamel after Er,Cr:YSGG laser irradiation and Fluoride application.

Materials and methods: One hundred and twenty premolars extracted for orthodontic purpose were used in the study and randomly divided into 6 groups. Group A was not subjected to any treatment. Group B was subjected to Er,Cr:YSGG laser irradiation. Group C was subjected to Er,Cr:YSGG laser irradiation followed by application of 2% NaF gel for 4 minutes. Group D was subjected to laser irradiation and 1.23% APF gel for 4 minutes. Group E was subjected to 2% NaF gel pretreatment technique followed by laser irradiation. Group F was subjected to 1.23% APF gel pretreatment technique followed by laser irradiation. All the test groups were subjected to microhardness testing and scanning electron microscope evaluation at 500 X and 1500 X.

Results: All the treated groups showed an increase in microhardness value in comparison to the control group. The highest increase in microhardness was seen in Group F. Increase in microhardness values of Group B and Group D was not statistically significant as compared to Group A. Scanning Electron Micrographs showed few craters and fine porosities for Group A. These craters and porosities increased in size and often showed glass like appearance after laser irradiation.

Conclusions: It can be suggested by means of present study that Er,Cr:YSGG laser irradiation alone or in combination with fluoride gel is an effective tool to provide resistance against the caries. Significantly higher resistance (p< 0.05) was seen when APF gel was used prior to Er,Cr:YSGG laser irradiation and this combination can act as an efficient tool for prevention against dental caries.

Keywords: Er,Cr:YSGG laser; Fluoride; Caries prevention; Enamel microhardness


With shift of emphasis towards prevention of dental caries, a number of techniques have been introduced which have effectively reduced dental caries. 1, 2, 3) In the year 1965, Sognnaes and Stern demonstrated that irradiation of tooth surface using Ruby laser increases enamel resistance to demineralization. 4) Since then several investigations have been carried out on the effect of laser irradiation. Yamamoto and Sato (1980) 5) and Tooya (1982) 6) indicated that human enamel irradiated with Nd:YAG laser was more resistant to acid decalcification than a non — irradiated control. Nelson et al (1986) evaluated mineral profiles of artificial caries like lesions formed in intact human dental enamel and showed that infrared lasers caused clinically significant inhibition of lesion formation. 7) It has been proven that laser irradiation provides protection against both caries initiation and caries progression. Lasers have also been used in combination with fluoride for caries prevention and this technique is termed as laser-activated fluoride (LAF) therapy. 8, 9) Flaitz CM (1995) determined the combined effects of argon laser irradiation and acidulated phosphate fluoride treatment on caries like lesion formation in human enamel and concluded that both laser irradiation alone and in combination with acidulated phosphate fluoride decreased the lesion depth as compared to control. The values were significantly higher when the combination of argon laser and acidulated phosphate fluoride was used. 10) Santella MR (2004) assessed the caries preventive potential of diode laser (809 nm) treatment of the primary teeth enamel as compared to topical fluoride application and found topical fluoride application to be more effective in enhancing the resistance of primary teeth enamel as compared to diode laser. 11) Rechmann P (2013) used CO2 laser irradiation for prevention of occlusal pit and fissure caries lesion and concluded that CO2 laser irradiation markedly inhibits caries progression as compared to fluoride varnish alone over 12 months clinical trial. 12) Fornaini C (2014) investigated the effectiveness of demineralization reduction in enamel treated with sub ablative Er:YAG laser irradiation followed by fluoride varnish application and concluded that Sub-ablative Er:YAG laser irradiation before the acid etching did not reduce the shear bond whereas when associated with fluoride application it may play a role in caries prevention. 13) Esteves-Oliviera M (2012) screened CO2 laser (10.6 micron) parameters to increase enamel resistance to a continuous flow erosive challenge and concluded that a set of laser parameters was effective in reducing enamel mineral loss but was not recommended for use as it also caused surface cracking. 14) Although, the effect of laser irradiation on acid resistance of enamel is recognized, the studies on comparisons of acid resistance of enamel when irradiated with Er,Cr:YSGG lasers alone and in combination with topical fluoride application are scarce. Thus, aim of the present study was to evaluate and compare the microhardness and morphology of permanent tooth enamel surface after Er,Cr:YSGG Laser irradiation and Fluoride treatment.

Materials and methods

The present in vitro study was done after obtaining prior approval from the ethical committee of the institution. One hundred and twenty premolars extracted for orthodontic purpose were then disinfected and stored using OSHA recommendations. 15, 16, 17, 18) Each premolar was observed under magnifying glass and only those without any caries (active/arrested), cracks, mottling, demineralization and any restoration were selected for the study. In selected teeth, crown and root were entirely separated using diamond disc in a straight hand piece. Crowns were sliced vertically in mesio-distal direction to obtain bucco-lingual sections. Only the buccal enamel sections from each tooth were used for the study and stored in a plastic container. The enamel sections were then mounted on blocks made of self-cure acrylic. A 3mm x 3mm window was created on each sample surface. A pilot study was done with the aim of defining parameters for Er,Cr:YSGG laser for present study. 9, 19) Based on this pilot study, the following parameters were finalized for laser irradiation of the samples using Er,Cr:YSGG Laser (Waterlase iPlus 2011,Biolase,Germany) Power =0.5 Watt; Time = 10 seconds; Frequency = 20 Hz; Air =40 % and Water = 60%. The power density used was 5.55 W/cm2 with a fluence of 0.28 J/cm2. The samples were subdivided into 6 groups as shown in Table I.

Table 1:
Grouping of the samples

A force of 200g was applied for 10 seconds using Indentec Hardness Testing Machine (Zhv1-M, Zwick Roell,U.K). 20) The extent of indentation measured the length of diagonals which gave the Vicker's Hardness Number for the specimen. The procedure was repeated for two indentations and an average microhardness number for each tooth sample was obtained. The values were subjected to statistical analysis. Surface morphology was evaluated using Scanning Electron Microscope (TM3000, Hitachi, Japan). 21)

Data analysis was performed using Statistical Package for the Social Science-21 (SPSS-21). The data was analyzed on the basis of hardness values using Mann-Whitney test and Kruskal Wallis Test. p-values < 0.05 were considered significant.


I.) The values of Vicker's microhardness test obtained were obtained and Mann Whitney test was done to compare the hardness values at the two sites within each group. It was observed that there was no statistically significant difference in microhardness values at different sites (p > 0.05) on the same sample. The lowest value of microhardness was seen for Control group i.e. 257.1 Vicker's Hardness Number (VHN). All the groups had higher microhardness values in comparison to Control group. The highest microhardness value was seen for Group F i.e. APF gel application followed by Laser irradiation (302.1 VHN) as shown in Table 2.

Table 2:
Percentage increase in microhardness of all treatment groups as compared to control group.

II.) Wilcoxon's signed rank test was used to compare the microhardness variation of all the treated groups in comparison to the control group (Table 2). It was observed an increase of the microhardness value in all the groups in comparison to the control group (Table 2). It was observed that all the groups showed an increase in microhardness value in comparison to control. The highest increase in microhardness was seen in Group F. Increase in microhardness values of Group B and Group D was not statistically significant as compared to Group A.

Kruskal Wallis test (One way ANOVA of ranks) was used to compare the mean microhardness values of all the groups. (Table 3). p-values < 0.05 were considered significant.

Table 3:
Difference in microhardness amongst all the groups using Kruskal Wallis test.

III.) The Scanning electron micrographs obtained in the study for Group A showed the typical structure of normal enamel which was relatively smooth with shallow depressions and enamel prisms. In Group B both smooth and rough eroded surfaces were seen with crazing. At higher magnification eroded surface with irregular areas with rough surfaces could be appreciated. Slight melting could be observed and surface seemed to be degenerated thermally and presented a molten lava like appearance and as a whole an irregular structure with micro-holes. In Group C and Group D, the micrograph showed the surfaces peppered with cavitation and craters leading to an irregular undulated surface. The surface did not show any of the usual enamel prism ends as seen in control group. The surfaces possessed granular to globular irregularities with vitrified surface and occasional fine cracks, discontinuities and porosities which were small in size. The vitrified surface appeared to be homogenous and confluent. Only infrequent, loosely adherent surface granules and globules were present. In Group E and Group F, the surfaces showed irregular contours with numerous globular to granular irregularities. The surface was quite porous and had a prominent fractured pattern with the fine cracks. The surface coatings were present that mask the underlying enamel surfaces. The surface coatings had somewhat irregular contours with numerous granular-to-globular irregularities protruding from the surface coatings. The coatings were quite porous and had a prominent fracturing pattern with fine cracking of the surface coatings. Occasional signs of melting were also seen which could be because of thermal degeneration of enamel. (Fig 1)

Fig 1:
SEM images of all the groups


Hardness is a mechanical property which can be used to determine the degree of mineralization of tooth. It directly depends upon the mineral content, as well as on the crystalline arrangement of enamel prisms. As softening of enamel is a clinical feature of caries, microhardness measurement can also be used to assess the extent of protection afforded by laser. Hence, in the present study, microhardness test has been used because it is more accurate and less cumbersome method than others. 20)

The mean microhardness values obtained in the present study for Group A was 257.1 VHN. 22, 23, 24) The mean microhardness values obtained for Group B was 270.6 VHN. There was 5.5% increase in microhardness as compared to control and the value was not statistically significant (Table 2). This was in accordance with the values obtained in the study by Westerman et al (2003) 22) and Banda et al (2011) 23) where the values were 316 KHN and 320.3 VHN respectively. The percentage increase in microhardness was 5.43% and 8.47% respectively in two studies and the values were statistically not significant. In contrast, the study by Bedini et al (2010) showed that mean microhardness value after laser irradiation was 290.98 VHN and there was 7.9% reduction in microhardness. 24) This result was consistent with the result obtained by Laura et al (2010) who reported that the acid resistance of enamel due to subablative Er:YAG laser irradiation did not increase significantly compared to control. 25)

In Group C the value was 280.9 VHN with an increase of 9.25% in the value of microhardness as compared to the control group which was statistically significant. It has been suggested by Tagomori and Morioka (1989) that laser-modified enamel has an enhanced uptake of fluoride and that this fluoride uptake was greater when laser treatment was performed before the fluoride treatment. 26) This could decrease enamel demineralization by retention of fluoride. Fox et al (1992) hypothesized that thermal treatment with lasers converts the carbonated hydroxyapatite of tooth enamel to a less soluble mineral. 27) Similiarly, Meurman et al (1997) showed that it is possible to transform hydroxyapatite (HAP) crystals to fluorapatite (FAP) crystals instantaneously in the presence of fluoride using a CO2 laser. 28) In Group D the value was 258.7 VHN and there was 0.62% increase in the value of microhardness as compared to control which was statistically insignificant. In 2001, Hossain et al used an Er,Cr:YSGG laser to irradiate the enamel or dentin samples at 6 W (67.9 J/cm2) or 5 W (56.6 J/cm2) pulse energy, respectively, with or without water mist. The results showed that Er,Cr:YSCG laser irradiation with and without water mist appears to be effective for increasing acid resistance. 29) However, Bedini et al (2010) reported that laser enamel irradiation combined with fluoride (APF and/or fluoride varnish) did not present better results than in the control groups, and caries activity that led to cavities formation occurred after just 6 months, demonstrating no synergistic effect. 23) The conflicting result is most probably due to high number of variables such as type of laser, power, pulse frequency and duration of radiation involved in the lasing process.

In Group E there was 12.45% increase in the value of microhardness as compared to control which was statistically significant. It has been reported that, after a professional fluoride application, calcium fluoride (CaF2) is formed on enamel surface and fluoride is released to fluid phase. This effect promotes a consequent reduction of enamel demineralization. This effect promotes a consequent reduction of enamel demineralization. Also, a dose-response effect is observed between the concentration of CaF2, reservoirs on enamel and fluoride released, to “plaque fluid” and the subsequent inhibition of enamel demineralization. The findings of this study have shown that topical application of APF in primary teeth is effective in the demineralization process and caries control 30) In Group F the value was 302.1 VHN and there was 17.50% increase (highest) in the value of microhardness as compared to control which was statistically significant. The increase in microhardness of enamel results from the fusion of microparticles on its surface or melting, fusion and recrystallization of the enamel particles which creates a barrier on the tooth surface. 31) Thus, the ideal procedure is that laser be used before the lesion is established. The efficacy of fluoride in combination with laser to prevent demineralization is being extensively studied.

The surface morphology of enamel as viewed in Scanning electron microscope examination is in accordance with the study done by McCormack et al (1995) 32), Kantorowitz et al (1996), 33) Hicks et al (2003) 21) and Banda et al (2011). 23) Furthermore, the presence of extensive rugosities in all the area irradiated with laser was seen confirming the findings of Takahashi et al (1998). 34) Studies have also revealed melting and resolidification of the enamel surface of the human teeth irradiated by different lasers, based on the three-dimensional images by the scanning electron microscope. According to Shirazuka et al (1991) 35) and Pelino et al (1999) 36), the morphological alterations on the irradiated enamel surfaces make it more resistant with regard to demineralization. The surface coatings present may provide a reservoir of mineral phases during a cariogenic attack. 21, 36, 37) Surface layer contains higher amount of fluoride in the form of calcium fluoride obtained by reaction of fluoride gel with enamel surface making it more resistant to caries and increasing the microhardness.


Within the limitations of this study, it may be concluded that:

  1. Er,Cr:YSGG laser irradiation of enamel surface increases the surface microhardness thereby increasing the enamel resistance to caries.
  2. Er,Cr:YSGG laser irradiation followed by fluoride gel treatment increases the microhardness of the enamel surface. The microhardness value increased significantly when NaF gel was used while increase was statistically insignificant in case of APF gel.
  3. Fluoride gel treatment followed by laser irradiation leads to maximum increase in the microhardness of the enamel surface with highest increase in case of APF gel.
  4. There was marked change in surface topography after laser irradiation. The surface seemed more eroded with craters and cracks and synergistic application of fluoride gel leads to formation of globules and granules along with vitrified appearance of surface. This modified enamel surface has higher microhardness value and hence more resistance to cariogenic insult.
    Although laser irradiation along with fluoride gel treatment has shown promising in vitro results for prevention against caries, more in vivo researches are needed in this field.


1: Marsh P, Martin MV. (1999) Oral Microbiology. Edinburgh: Wright; 4(1):82-96.
2: De Moor RJ, Delme KI. (2009) Laser assisted cavity preparation and adhesion to Erbium-lased tooth structure: Part 1. Laser assisted cavity preaparation. J Adhes Dent; 11 (2):427-438. [PubMed]
3: Apel C, Meister J, Schmitt N, Graber HG, Gutknecht N. (2002) Calcium solubility of dental enamel following sub ablative Er:YAG and Er,Cr:YSGG laser irradiation in vitro. Laser Surg Med; 30 (6):337-341. [PubMed]
4: Stern RH, Sognnaes RF. (1965) Laser beam effect on dental hard tissues. J Dent Res; 43(3): 873-878.
5: Yamamoto H, Sato K. (1980) Prevention of dental caries by Nd:YAG laser irradiation. J Dent Res; 59(1): 2171-2177.
6: Tooya Y. (1982) Acousto-optically Q switched Nd:YAG laser resistance of human deciduous enamel to demineralization in vitro and in vivo. J Oral Bio; 24(5):442-452.
7: Nelson DGA, Shariati M, Glena R, Shields CP, Featherstone JDB. (1986) Effects of pulsed low energy infrared laser irradiation on artificial caries like lesion formation. Caries Res; 20(1):289-299. [PubMed]
8: Ghadimi S, Chiniforush N, Bouraima SA, Johari M. (2012) Clinical Approach of Laser Application in Different Aspects of Pediatric Dentistry. J Lasers Med Sci; 3(2):84-90.
9: Ana PA, Tabchoury CPM, Cury JA, Zezell DM. (2012) Effect of Er,Cr:YSGG Laser and Professional Fluoride Application on Enamel Demineralization and on Fluoride Retention. Caries Res; 46(3):441-451. [PubMed]
10: Flaitz CM, Hicks MJ, Westerman GH, Berg JH, Blankenau RJ, Powell GL. (1995) Argon laser irradiation and acidulated phosphate fluoride treatment in caries-like lesion formation in enamel: an in vitro study. Pediatr Dent; 17 (1):31-35. [PubMed]
11: Santaella MR, Braun A, Matson E, Frentzen M. (2004) Effect of diode laser and fluoride varnish on initial surface demineralization of primary dentition enamel: an in vitro study. Intl J Pediatr Dent; 14(3):199-203. [PubMed]
12: Rechmann P, Charland DA, Rechmann BMT, Le CQ, Featherstone JDB. (2013) In-vivo occlusal caries prevention by pulsed CO2-laser and fluoride varnish treatment—A clinical pilot study. Lasers Surg Med; 45(5):302-310. [PubMed]
13: Fornaini C, Brulat N, Milia G, Rockl A, Rocca JP. (2014) The use of sub-ablative Er:YAG laser irradiation in prevention of dental caries during orthodontic treatment. Laser Therapy; 23(3): 173-181. [PMC free article] [PubMed]
14: Esteves-Oliveira M, Yu H, Eduardo C, Meister J, Lampert F, Attin T, Wiegand A. (2012) Screening of CO2 Laser (10.6 micron) Parameters for Prevention of Enamel Erosion. Photomedicine and Laser Surgery;30(6):331-338. [PMC free article] [PubMed]
15: Lolayekar NV, Bhat VS, Bhat SS. Disinfiection methods of extracted human teeth. (2007) J Oral Health Comm Dent; 1(2):27-29.
16: CDC Gudelines for infection control in dental health care settings. MMWR 2003; 52(17);1-61. [PubMed]
17: Dominici JT, Eleazer PD, Clark JS, Staat RH, Scheetz JP. (2001) Disinfection/sterilization of extracted teeth for dental students use. J Dent Edu; 65:1278-1280. [PubMed]
18: OSHA Manual. OSHA Manuals for physicians, Dentists and Veterenarians. Avaialaible online at (last accessed on 19th September 2015).
19: Geraldo-Martins VR, Tanji EY, Wetter NU, Nogueira RD, Eduardo CP. (2005) Intrapulpal temperature during preparation with the Er:YAG laser: An in vitro study. Photomed Laser Surg; 23(2):182-186. [PubMed]
20: Guitterez-Salazar fMDP, Reyes-Gasga J. (2003) Microhardness and chemical composition of human tooth. Mat. Res; 6(3):367-373.
21: Hicks J, Flaitz C, Ellis R, Westerman GP, Owell L. (2003) Primary tooth enamel surface topography with in vitro argon laser irradiation alone and combined fluoride and argon laser treatment: scanning electron microscopic study. Pediatr Dent; 25(5):491-496. [PubMed]
22: Westerman GH, Randall WE, Latta MA, Powell GL. (2003) An in vitro study of enamel surface microhardness following argon laser irradiation and acidulated phosphate fluoride treatment. Pediatr Dent; 25(5):497-500. [PubMed]
23: Banda NR, Reddy GV, Shashikaran ND. (2011) Evaluation of primary tooth enamel surface morphology and microhardness after Nd:YAG laser irradiation and APF gel treatment- An in vitro study. J Clin Ped Dent; 35(4):377-382. [PubMed]
24: Bedini R, Manzon L, Fratto G, Pecci R. (2010) Microhardness and morphological changes induced by Nd:YAG laser on dental enamel: an invitro study. Caries Res; 46(2):168-172. [PubMed]
25: Rodríguez-Vilchis LE, Contreras-Bulnes R, Sánchez-Flores I, Samano EC. (2010) Acid resistance and structural changes of human dental enamel treated with Er:YAG laser. Photomed Laser Surg; 28(4):207-211. [PubMed]
26: Tagomori S, Morika T. (1989) Combined effects of laser and fluoride on acid resistance of human dental enamel. Caries Res; 23(5):225-231. [PubMed]
27: Fox JL, Yu D, Otsuka M, Higuchi WI, Wong J, Powell GL. (1992) Initial dissolution rate studies on dental enamel after CO2 laser irradiation. Dent Res; 71(3):1389-1398. [PubMed]
28: Meurman JH, Hemmerle J, Voegel JC, Rauhamaa-Makinen R, Luomanen M. (1997) Transformation of hydroxyapatite to fluorapatite by irradiation with high-energy CO2 laser. Caries Res; 31: 397-400. [PubMed]
29: Hossain M, Nakamura Y, Yamada Y, Kimura Y, Matsumoto N, Matsumoto K. (1999) Effects of Er,Cr:YSGG laser irradiation in human enamel and dentin: ablation and morphological studies. J Clin Laser Med Surg; 7(4), 155-159. [PubMed]
30: Tenuta LMA, Cerezetti RV, del bel Cury AA, Tabchoury CPM, Cury JA. (2008) Fluoride release from CaF2 and enamel demineralization. J Dent Res; 87(11):1032-1036 [PubMed]
31: Nelson DG, Wefel JS, Jongebloed WL, Featherstone JBD. (1987) Morphology, histology and crystallography of human dental enamel treated with pulsed low-energy infrared laser radiation. Caries Res; 21:411-426. [PubMed]
32: McCormack SM, Fried D, Featherstone JDB, Glena RE, Seka W. (1995) Scanning electron microscope observations of CO2 laser effects on dental enamel. J Dent Res; 74(10):1702-1708. [PubMed]
33: Kantorowitz Z, Featherstone J.D.B., Fried D. (1996) Argumentation of CO2 laser inhibition of in vitro caries by fluoride. J Dent Res; 75(1):25-29
34: Takahashi K., Kimura Y, Matsumoto K. (1998) Morphological and atomic analytical changes after CO2 laser irradiation emitted at 9.3mm on human dental hard tissues. J Clin Laser Med Surg; 16(3):167-173. [PubMed]
35: Shirazuka T, Kodaka K., Debari K, Matsumoto K. (1990) Acid resistance on human dental enamel by laser irradiation and fluoride treatment. J Dent Res; 70(4):350-355
36: Pelino JEP, Mello JB, Eduardo CP. (1999) In vitro study of the Nd:YAG laser effect on human dental enamel: optical and electron microscope analysis. J Clin Laser Med Surg.; 17(4):171-177. [PubMed]
37: Souza-Gabriel AE, Colucci V, Turssi CP, Serra MC, Corona SAM. (2010) Microhardness and SEM after CO2 laser irradiation or fluoride treatment in human and bovine enamel. Microsc Res Tech; 73(4):1030-1035. [PubMed]

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