Effect of Er: YAG or Nd:YAG Laser Exposure on Fluorosed and Non-Fluorosed Root Surfaces: An In Vitro Study

Abstract

Background and aims: Fluorosis affects tooth mineralization. The therapeutic benefit provided by lasers on fluorosed and non fluorosed cementum requires studying and comparing. The aim of this study was to evaluate and compare the root surface changes following Er:YAG or Nd:YAG laser irradiation on periodontally healthy fluorosed versus non-fluorosed teeth by scanning electron microscopy (SEM).

Materials and methods: A total of 76 periodontally healthy fluorosed (FH) and non-fluorosed (NFH) teeth specimens were included in this study. In one group, the experimental root specimens were irradiated using Er:YAG or with Nd:YAG laser in the other. A SEM evaluation was performed to assess the laser induced ultra structural changes in the root surface followed by statistical analysis using Fisher's exact test.

Results: It was observed that both FH and NFH groups were similarly affected by Nd:YAG or Er:YAG laser. However, the former caused more surface changes than the latter on melting of surface (p=0.12 for FH and p=0.08 for NFH), and Er:YAG laser caused more smear layer formation (p=0.51 for FH and p=0.16 for NFH).

Conclusion: Results suggest that undesirable morphological changes were observed almost similarly in FH and NFH groups using Er:YAG or Nd:YAG laser. Hence further in-vitro studies at lower energy settings followed by clinical trials are required in this aspect.

Introduction

Fluorine is a common element in the earth's crust. Fluoride ion has played a major role in dramatically reducing dental caries over past 40 years. Excessive systemic exposure to fluoride can lead to disturbances of bone homeostasis, enamel development (dental/enamel fluorosis) and mineralization. The severity of fluorosis on periodontal hard and soft tissues is dose dependent and also depends on timing and duration of fluoride exposure during development. The literature on fluoride and dental caries is well discussed in contrast to periodontal tissues. However a recent review by Dr. Vandana K L has explored an epidemiological association between fluorosis and periodontal disease, and also the influence of fluorosis on periodontal structures along with the comparison of influence of periodontal treatment on fluorosed and non fluorosed teeth. ¹⁾

Periodontitis, i.e., inflammation of the supportive structures of the teeth can be treated both by non -surgical and surgical therapy. Non- surgical therapy is gaining popularity due to its non invasive nature and patient compliance. Various procedures are included in non- surgical therapy and of recent interest is laser therapy. Previous research has shown laser induced deleterious thermal and chemical changes on human enamel and dentin. ^{2, 3)} However, there is paucity of research addressing the effect of laser irradiation on fluorosed teeth.

So far the laser induced beneficial effects have not been comparatively studied between fluorosed and non-fluorosed teeth. Fluorosis is known to bring mineralization changes (i.e., hypomineralisation) in dental hard tissues which may present a different response to laser treatment as compared to non-fluorosed teeth.

The use of lasers has been increasingly proposed for periodontal treatment in the hope for a more selective and atraumatic technique promoting periodontal healing. Geusic et al in 1964 developed Nd:YAG laser after discovery of the prototype of the neodymiumdoped: yttrium aluminum, garnet (Nd:YAG: 1.06 microns) laser in 1961 by Snitzer. ⁴⁾

Among all the lasers used in the field of dentistry, the Er:YAG laser has been reported to be the most promising laser for periodontal treatment. ⁵⁾ Its excellent ability to effectively ablate hard tissues and dental calculus without producing major thermal side-effects to adjacent tissue has been demonstrated in numerous studies. ^{1, 6, 7, 8)} The lack of thermal root surface changes is most likely caused by the special optical characteristics of the Er:YAG laser emitting radiation at a wavelength of 2.94 µm that peaks close to the absorption co-efficient of water (3µm). ⁹⁾ Scanning electron microscopy (SEM) observations from recent studies showed that the clinical use of an Er:YAG laser resulted in a smooth root surface morphology, even at higher energy settings. ^{2, 10, 11)}

The effects of Er:YAG or Nd:YAG laser on fluorosed and non fluorosed root surfaces have been reported previously, ¹⁾ but the comparative evaluation of the two lasers on fluorosed and non fluorosed root surfaces was not found in the literature.

The Medline search using the key words “laser”, “fluorosis”, “root surface” and “cementum”, has revealed few studies on the effect of lasers on fluorosed roots. Therefore, an initial attempt has been made in this in-vitro study to evaluate and compare the root surface changes following 2.94 µm Er:YAG and 1.06 µm Nd:YAG laser irradiation on periodontally healthy fluorosed and non-fluorosed teeth by SEM.

Materials and methods

In this study, fluorosed and non-fluorosed periodontally healthy teeth were included. The freshly extracted teeth were obtained from the Department of Oral and Maxillofacial Surgery, College of Dental Sciences, Davangere with patient's consent in accordance with the ethical guidelines of Institutional Review Board and Rajiv Gandhi University of Health Sciences, Karnataka, India. The study period was one year.

The periodontally healthy teeth were extracted non-traumatically due to orthodontic reasons. For fluorosed teeth, the fluorotic enamel stains were confirmed by the clinical examination and history of the subjects hailing from natural high water fluoride areas in and around Davangere (fluoride concentration >1.5–3ppm). Teeth with proximal caries extending to the cementum or with fillings extending beyond CEJ or with intrinsic stains caused by other reasons such as porphyria, erythroblastosis fetalis, tetracycline therapy, etc were excluded.

A total of 76 periodontally healthy fluorosed and non-fluorosed teeth specimens were included in this study. Equal numbers of tooth specimens were included in each group. The extracted teeth were immediately washed in running tap water and were stored in bottles containing 0.9% saline. The teeth specimens were bisected at the cemento-enamel junction to separate crown and root portion, using a sterile diamond disk running at low speed with sterile water coolant. This was followed by bisecting of the root bucco-lingually to get 2 halves. The detail description of each group is depicted in Table 1:

Table 1: Distribution of root specimens.

Laser	Fluorosis	Group	Test	Control
Er:YAG	Present	FH1	15	4
Er:YAG	Absent	NFH1	15	4
Nd:YAG	Present	FH2	15	4
Nd:YAG	Absent	NFH2	15	4

Laser treatment

Experimental (lased) specimens of group FH1 and NFH1 were irradiated with a 2.94µm Er:YAG laser (Fidelis Plus III, Fotona, Germany). Experimental (lased) specimens of group FH2 and NFH2 were irradiated with a 1.06 µm Nd:YAG laser (Fidelis Plus III, Fotona, Germany). All the irradiations were performed by the same operator.

The laser beam was guided onto the root surfaces under water irrigation with a R02 hand piece and a quartz fiber tip. Laser parameters were set at 100 mJ/pulse and 20 pulses/s (panel setting) for Nd:YAG and at 140 mJ/pulse and 10 pulses/s (panel setting) for Er:YAG laser irradiation. The treatment was performed from coronal to apical in parallel paths with an inclination of the fiber tip of approximately 15–20° to the root surface. The root samples were irradiated for 1 minute in a focused and contact mode.

SEM analysis:

Following irradiation, the specimens were placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for a minimum period of 24 hours. The specimens were then cleansed by means of three irrigation cycles with 10 ml of 0.1 M buffered phosphate solution. It was essential that the specimens were to be completely dry. The SEM works under a vacuum and for an image to be derived, the specimen must be dry, if not the specimen will simply collapse in the vacuumed chamber. They were submitted to a sequential dehydration process with 70% alcohol (10 minutes); 80% alcohol (10 minutes); 90% alcohol (10 minutes) and 100% alcohol (10 minutes) which replaced the water with alcohol and then the alcohol was slowly evaporated off leaving a dried specimen. Following dehydration, the specimens were mounted on SEM stubs. Mounted specimens were air-dried for 48 hours and sputter coated with 30 to 40 nm of gold. Finally, specimens were examined using a SEM (JEOL-JSM-840A, operating at an accelerating voltage of 20kV). Representative photomicrographs were obtained at 2500x magnifications for all the lased specimens and at 50x and 750x for control specimens. The photomicrographs were scored on a dichotomous basis, i.e., 0 (absent) and 1 (present) by a single, blinded examiner who had an experience in SEM interpretation (for over 30 years from University of Missouri-Kansas City, School of Dentistry); however, a special calibration of the examiner did not precede the study.

The SEM photomicrographs were assessed for following findings:

1. Melting of surface

2. Heat induced surface crazing/cracking

3. Smear layer

4. Open periodontal ligament (PDL) insertion sites into cementum

5. Closed PDL insertion sites into cementum

6. Exposure of the collagen tufts

7. Open dentinal tubules

Statistical analysis was done using Fisher's exact test to analyze the intergroup comparisons between fluorosed and non fluorosed root specimens treated with Er:YAG or Nd:YAG lasers.

Results

The results of the study are presented in Tables 2 to 7. Smear layer formation and melting of surfaces was observed in both surfaces treated with Er:YAG or Nd:YAGlaser (Tables 2 and 3). Heat induced crazing (Table 4), open and closed PDL insertion sites into cementum were appreciated in only those surfaces treated with Nd:YAG laser (Table 5). Exposure of collagen tufts (Table 6) and presence of open dentinal tubules (Table 7) were observed in surfaces treated with Er:YAG laser irradiation.

Table 2: Melting of surfaces following Er:YAG and Nd:YAG laser irradiation.

Magnification	Group	Melting of surfaces		FH1 Vs NFH1	FH2 Vs NFH2	FH1 Vs FH2	NFH1 Vs NFH2
		0	1
2500x	FH1	4	11 (73.3%)	P=0.42 (NS)	No diff	P=0.12 (NS)	P=0.08 (NS)
	NFH1	5	10 (66.7%)
	FH2	0	15 (100%)
	NFH2	0	13 (100%)

*NS = Non Significant

Table 7: Group wise data of open dentinal tubules following Er:YAG laser irradiation.

MAGNIFICATION	GROUPS	Opening of dentinal tubules		FH1 vs NFH1
		0	1
2500 X	FH1	3	12 (80%)	p=0.13, NS
	NFH1	2	13 (86.67%)

*NS = Non Significant

Table 3: Smear layer formation following Er:YAG and Nd:YAG laser irradiation.

MagNification	Group	Smear layer formation		FH1 Vs NFH1	FH2 Vs NFH2	FH1 Vs FH2	NFH1 Vs NFH2
		0	1
2500x	FH1	14	1 (6.67%)	P=0.17 (NS)	No diff	P=0.51 (NS)	P=0.16 (NS)
	NFH1	11	4 (26.7%)
	FH2	15(100%)	0
	NFH2	13 (100%)	0

*NS = Non Significant

Table 4: Heat induced crazing and cracking following Nd:YAG laser irradiation.

Magnification	FH2		NFH2		FH2 vs NFH2
	0	1	0	1
2500x	0	15 (100%)	0	13 (100%)	No difference

Table 5: Group wise data of open and closed PDL insertion sites into cementum-at 2500x following ND:YAG laser irradiation.

MAGNIFICATION	GROUPS	OPEN PDL INSERTION SITES INTO CEMENTUM		CLOSED PDL INSERTION SITES INTO CEMENTUM
		0	1	0	1
2500x	FH2	12 80%	3 20%	15 100%	0
	NFH2	13 100%	-	13 100%	0
		Groups	p-value	Groups	p-value
		FH2 Vs NFH2	X2=1.15 P=0.28,NS	FH2 Vs NFH2	NO DIFF

*NS = Non Significant

Table 6: Group wise data of exposure of collagen tufts following Er:YAG laser irradiation.

Magnification	Groups	YES	NO	p value
2500 X	FH1	4 (26.67%)	11	p = 0.46, NS
	NFH1	6 (40%)	9

*NS = Non Significant

In the present study, in case of control (untreated) fluorosed root specimens, the SEM observations at 50x and 750x magnification revealed an uneven smooth topography. In contrast, non-fluorosed control root specimens featured a rough topography with rounded elevations at 50x and 750x magnification.

In 2500x magnification, the fluorosed surfaces showed higher (73.3%) melting than the non fluorosed surfaces (66.67%) when irradiated with Er: YAG laser (p= 0.42) while all the fluorosed and non fluorosed surfaces treated with Nd:YAG laser showed melting of surfaces (Figures 1 and 2).

Figure 1:

Melting and heat induced crazing and cracking of fluorosed cementum at 500x (left) and 2500x (right) (after Nd:YAG laser irradiation)

Figure 2:

Melting and heat induced crazing and cracking of non-fluorosed cementum at 500x (left) and 2500x (right) (after Nd:YAG laser irradiation)

The smear layer was found in 6.67% and 26.7% of Er: YAG laser treated fluorosed and non-fluorosed root specimens respectively and was higher in the nonfluorosed group (p = 0.17) (Figure 3). None of the Nd:YAG lased root specimens demonstrated smear layer formation.

Figure 3:

PDL attachment sites obliterated by smear layer (arrows) at 2500x (after Er:YAG laser irradiation)

No heat induced surface crazing or cracking was observed in root surfaces treated with Er: YAG laser while all the fluorosed and non-fluorosed root surfaces treated with Nd:YAG laser showed heat induced crazing or cracking (Figures 1 and 2).

None of the non-fluorosed surfaces showed open PDL insertion sites into cementum and the fluorosed surfaces showed 80% open PDL insertion sites into cementum (p=0.28). On assessing the closed PDL insertion sites when irradiated with Nd:YAG laser, none of the surfaces showed closed PDL insertion sites into cementum.

The exposure of collagen tufts was only observed at 2500x magnification when irradiated with Er:YAG laser. In the fluorosed group, 26.67% root specimens showed exposure of collagen tufts, whereas in the nonfluorosed group 40% of root specimens showed exposure of collagen tufts (p= 0.46) (Figures 4 and 5).

Figure 4:

Exposure of collagen tufts and open dentinal tubules in fluorosed root specimen at 2500x (after Er:YAG laser irradiation)

Figure 5:

Exposure of collagen tufts and open dentinal tubules in non-fluorosed root specimen at 2500x (after Er:YAG laser irradiation)

The fluorosed and non-fluorosed groups showed 80% and 86.67% open dentinal tubules respectively following irradiation with Er:YAG laser (p= 0.13) (Figures 4 and 5).

Discussion

The root surface changes for control group in this study have been analyzed by using SEM at magnification of 50x and 750x similar to that used in studies by Israel et al. ¹³⁾ These lower magnifications were sufficient to observe the microscopic structure of a normal non lased root specimen unlike lased root specimens which required higher magnification for the changes to be observed.

The root surface changes for lased group in this study have been analyzed by using SEM at magnification of 2500x, which was similarly used in a study by Crespi et al. ¹¹⁾ Post Er: YAG and Nd:YAG laser irradiation on root surface, the subsequent root surface changes have been analyzed by a number of techniques like scanning electron microscope ¹⁾, light microscope ^{5, 7)} and stereomicroscope. ¹³⁾

Various hard tissue changes induced by fluorosis are hypomineralisation of enamel and dentin, hypercementosis, recession of alveolar crest, root resorption, hypermineralisation of cementum. The laser treatment effects on fluorosed root cementum have not been studied so far which is possibly hypomineralised similar to fluorosed dentin and enamel though the exact impact of fluorosis on cementum is not reported in literature. Hence the present study was designed to use those apparently beneficial laser parameters (tissue friendly and causing less thermal damage to the treated tissues) which differ for different laser types. For the present study we used the parameter of 100 mJ/pulse and 20 pulses/s for Nd:YAG, and 140 mJ/pulse and 10 pulses/s for Er:YAG laser irradiation. The comparative studies considered for discussion have utilized different laser parameters than the current study.

Within each laser group, the melting of root surface was similar in fluorosed and non-fluorosed root specimens (p>0.05). On comparison of Er: YAG versus Nd: YAG laser effects, the fluorosed and non-fluorosed healthy root specimens showed no significant difference (p>0.05). However, all the root specimens lased with Nd: YAG demonstrated melting of root surfaces which was relatively more (100% for both FH2 and NFH2) than those treated with Er:YAG laser (73.3% for FH1 and 66.7% for NFH1) because of the fact that the wavelength of Er:YAG laser was almost equivalent to absorption wavelength of water and hence there was better absorption of the laser energy by the water molecules in the root surfaces. ⁹⁾

In vitro studies on effect of Er:YAG laser on periodontally healthy root surfaces by Sasaki et al, ⁸⁾ Israel et al ¹³⁾ and Yamaguchi et al ¹⁴⁾ revealed no melting of the root surface using SEM observation.

Another in vitro study with Nd:YAG laser on periodontally healthy teeth by Morlock et al (with the sample size n=3 in each group, at the energy levels of 1.25W and 1.50W and 20pps for 20 seconds) which featured various surface changes like melting of surface, charring and crater formation as compared to untreated controls. ¹⁵⁾

The melting of root surfaces occurs at the energy levels 50 mJ or more, the melting temperature of hydroxyapatite (700°C) is reached, evaporation begins and steam forms, and micro explosions occur which eject the molten mineral phase from the crater's center to the wall and rim where it resolidifies thus there is melting of the surface. ¹⁵⁾ In Er:YAG laser, the tissue is explosively ablated hence increasing the diffusion process of the root surface without causing thermal damage while Nd:YAG laser removes only superficial part of ablated tissue leaving the remaining tissue chemically modified ,hence not favoring reattachment. ²⁾

Within each laser group, the smear layer formation was similar in fluorosed and non-fluorosed groups (p>0.05). However, previous in-vitro studies on periodontally healthy root surface by Sasaki et al, ⁸⁾ Israel et al ¹³⁾ and Yamaguchi et al ¹⁴⁾ reported absence of smear layer formation after Er:YAG irradiation, using SEM observation. The observation of absence of smear layer and melting of root surface in previous in-vitro studies with lower laser energy parameters on periodontally healthy teeth in case of Er:YAG lased surfaces were in contrast to the present study as higher laser energy parameters probably caused melting of root surface and subsequent smear layer formation. In our study smear layer was effectively removed at the energy levels 100mJ at 20pps when lased for 1min by Nd: YAG laser which was in accordance with the study conducted by Gaspirc et al. ²⁾

The surfaces treated with Er:YAG laser irradiation did not show any heat induced crazing or cracking as also found by Gaspirc et al. ²⁾ However, in case of heat induced crazing or cracking after Nd:YAG laser irradiation, both the fluorosed and non-fluorosed groups showed similar results. An in vitro study on periodontally healthy teeth, done by Wilder-Smith et al (with sample size of n= 25 in laser group, with energy densities ranging from 230.67 J/cm² to 1153.33 J/cm² at 1,2,3,4 or 5 minutes of exposure), the laser treated group at 1,2,3 minutes showed no laser damage, but small patches of laser damage like cracks, fissures were seen for 4 minutes group, even more damage was seen with 5 minutes group. ¹⁶⁾ These differences in the laser damage at various exposure time could be attributed to the fact that there was variability in the degree of susceptibility of different teeth and different parts of the same tooth to the laser. ¹⁷⁾ In general, the severity and extent of cracking observed probably related to increasing temperature associated with longer exposure times. These observations provide cause for concern regarding potential thermal, pulpal damage in the clinical situation. ³⁾

In case of open and closed PDL insertion sites in the cementum after Nd: YAG laser irradiation, a comparison between the fluorosed and non-fluorosed groups showed similar findings (p>0.05). Depending on the choice of laser parameter the effect on PDL insertion sites into cementum varied from total obliteration of insertion sites to little or no effect, the latter resulting in exposed and distinctly open sites. Interestingly, insertion sites that were open or partially obliterated were void of residual collagen tufts or remnants. The clinical significance of such an observation is unclear.

Obviously, those sites that are completely obliterated are essentially relevant to a surface topography generally associated with a smear layer. Several studies have demonstrated that mechanical debridement of root surfaces results in a smear layer containing remnants of dental calculus and contaminated root cementum, the resultant root surface inhibits fibroblast migration and attachment and may lead to healing by long junctional epithelium rather than fibrous attachment. ¹⁸⁾

In case of exposure of collagen tufts after Er:YAG laser irradiation in our study, the comparison between the fluorosed and non-fluorosed groups revealed similar findings (p>0.05). Also, in an in-vitro study on effect of Er:YAG laser on periodontally healthy root surface by Israel et al, ¹³⁾ an exposure of collagen bundles on the root surface was observed using SEM, which was in contrast to studies by Sasaki et al ¹¹⁾ and Yamaguchi et al ¹⁴⁾ which reported absence of exposure of collagen tufts after Er:YAG laser irradiation, using SEM observation. Thus, an inconsistency is observed in the previous studies on exposure of collagen tufts after Er:YAG laser irradiation, even when lower laser energy settings as compared to the present study, were used on periodontally healthy teeth. The Nd:YAG laser irradiated surfaces did not show open tubules because of the fact that permeability was declined due to denatured proteins which acted as barriers in dentinal tubules. ²⁾

The inter-group comparison of opening of dentinal tubules between the fluorosed and non-fluorosed groups treated with Er:YAG laser revealed similar findings (p>0.05). Also, previous in-vitro study on effect of Er:YAG laser irradiation on periodontally healthy root surface by Sasaki et al ⁸⁾ reported opening of dentinal tubules in contrast to studies by Israel et al ¹³⁾ and Yamaguchi et al ¹⁴⁾ which reported absence of opening of dentinal tubules after Er:YAG laser irradiation, using SEM observation. Thus, an inconsistency is observed in the previous studies on opening of dentinal tubules after Er:YAG laser irradiation, even when lower laser energy settings as compared to the present study, were used on periodontally healthy teeth.

A beneficial effect of acid resistance phenomenon has been observed in hypomineralised fluorosed teeth which also helps in prevention of caries in dental fluorosis patients. But on the other hand, the concentration of acid for root biomodification as well as the laser settings need to be modified separately for treating fluorosed and non fluorosed teeth. ¹⁾

Conclusion

To conclude the study in brief, the surface melting was found to be more in Er:YAG or Nd:YAG lased fluorosed healthy roots whereas the smear layer formation was found to be less in Er:YAG fluorosed than non-fluorosed group. The heat induced crazing was similar in Nd:YAG lased fluorosed and non-fluorosed groups. In Er:YAG lased group, the collagen tufts exposure and open dentinal tubules were found to be more in nonfluorosed group. The open and closed PDL fibers insertion sites were more in fluorosed healthy roots. The possible reason for response from fluorosed root surfaces could be due to hypomineralisation that requires to be analyzed. The possible shortcoming of the present study is the use of SEM for the evaluation of root surface changes as it does not detect subsurface damage. Therefore, further in-vitro studies using larger sample size, with different laser parameters (that can induce beneficial root changes for periodontal wound healing) and histological evaluation are required to better evaluate and understand the root surface changes following laser irradiation. Also, the biochemical and physical changes in periodontally healthy and diseased fluorosed root surface have to be further studied to understand the clinical implication of the results of the present study. Considering the 25 countries with endemic fluorosis globally, it is recommended to use laser parameters that are friendly to the hypomineralised fluorosed periodontal structures.