Effect of Exposure Times of Sodium Hypochlorite before Acid Etching on the Microshear Bond Strength to Fluorotic Enamel

INTRODUCTION

Because of successive exposures to high concentrations of fluoride during tooth development, dental fluorotic enamel is disturbed, which shows hypomineralized and porous [1]. Clinically, bilateral, diffuse (not sharply demarcated), opaque, and white lines following the perikymata were seen on the appearance of mild fluorotic enamel. The white patches may be formed by opacities. Severe fluorotic enamel may become entirely chalky-white, discolored from light to dark brown and/or pitted [1, 2]. According to biological aspects of dental fluorosis, TFI is used to be classified on a scale of one to nine: mild (TFI = 1-3), moderate (TFI = 4-5) and severe (TFI = 6-9) [1, 3, 4].

Although dental fluorosis might not affect our oral health seriously, it compromises the tooth’s aesthetics and brings potential psychosocial effects on many patients who experience social repercussions [5, 6]. Therefore, dentists have been proposed a broad range of therapies of various invasiveness to treat fluorotic enamel, including resin infiltration [7], microabrasion [8], dental veneers [1], external bleaching [9], crowns [10] or a combination of these methods [11, 12]. Because fluorotic enamel has high resistance in acid etching and the hypomineralization of enamel region has the porous nature, the shear adhesive strength of fluorotic enamel is lower than that of normal enamel [6]. Hence, how to increase the bonding strength of fluorotic enamel must be considered in most restorative procedures.

Some studies suggested that application with 5.25% NaOCl could increase enamel surface area before acid etching which is suitable for resin bonding [13, 14]. NaOCl has been known that it is efficient to remove organic matter at common temperature as a nonspecific proteolytic agent [13, 15]. 5.25% NaOCl solution has been applied to remove organic detritus which come from pulp canals in clinic. Additionally, some recent studies reported that NaOCl was applied to dissolve collagen before dentin bonding [16, 17]. The reason that NaOCl improves bonding strength is that it removes protein from surface of enamel [15, 18].

There are conclusions that good bonding results have been obtained on hypomineralized and normal enamel which applied 5.25% NaOCl [15, 18]. Besides, a recent study has found that treatment with 5.2%NaOCl before fluorotic enamel(TIF=4) acid etching increased orthodontic brackets’ bond strength [19]. However, most research showed that the duration of 5.25% NaOCl was 60s without giving a reason. To date, only Roberto et al evaluated that deproteinization with 5.25% NaOCl could increase μSBS before etching but processing time at 60s was more effective than 30s in enamel [14]. Whereas, there was a remarkably greater protein content by weight in fluorotic enamel compared with the normal [20]. We have to assume that extending pretreatment time of 5.25% NaOCl whether have an beneficial effect on bonding strength or not and whether there are differences in the processing time of 5.25% NaOCl between mild and moderate fluorotic enamel.

In consequence, this experiment was aimed at comparison between the μSBS of mild and moderate fluorotic enamel after pretreatment with 5.25%NaOCl at different times and make further exploration of enamel-etching pattern and ATR-FTIR. The null hypotheses tested that various exposure times with 5.25% NaOCl will make no difference to μSBS, etching pattern of enamel or ATR-FTIR of mild and moderate fluorotic enamel.

MATERIALS AND METHOD

The Human Ethics Committee that come from the School and Hospital of Stomatology, Southwest Medical University approved this study.

Forty-eight fluorotic molars were extracted from patients due to severe periodontal diseases after getting an informed consent which was taken to use the teeth in the research. According to teeth’s unique clinical feature, dimensions, morphology, appearance without any cracks or carious cavities, the fluorotic teeth were selected. Two examiners were trained and dental fluorosis was divided into two categories according to the TFI: mild dental fluorosis (ML-F) and moderate dental fluorosis (MD-F). Then, the periodontal film and dental calculus were removed and cleaned before teeth were stored at 4°C and in 1% thymol solution. Besides, teeth must be used within 6 months.

Gypsum was used to immobilize roots of teeth in mould and each dental crown was then cut it into four pieces which were in parallel with the long axis of teeth (buccal surface, lingual surface and two proximal surfaces), for 192 enamel surfaces in total: 168 enamel surfaces were used to evaluate μSBS, 16 were used for the etching pattern and 8 were used for ATR-FTIR analysis.

The samples in ML-F and MD-F group were respectively separated into 4 subgroups (n=21 per subgroup) based on application times (0s, 60s, 120s and 180s) of 5.25%NaOCl. Specimens were embedded in Polymethyl methacrylate through silicone mould (10 mm long × 10mm wide × 5mm high) (LELE, Shanghai, China). In order to acquire a smooth flat of enamel, using 600 grit wet silicon carbide sandpaper polished exposed enamel surfaces for 30 seconds. Each subgroup was treated as follows:

0s-group washed with water and then dried with oil-free compressed air for 10s [21]. (control group)

60s-group treated with 5.25% NaOCl by applying sterile cotton pellet for 60s, washed with water and then dried with oil-free compressed air for 10s.

120s-group treated with 5.25% NaOCl by applying sterile cotton pellet for 120s, washed with water and then dried with oil-free compressed air for 10s [13, 14].

180s-group treated with 5.25% NaOCl by applying sterile cotton pellet for 180s, washed with water and then dried with oil-free compressed air for 10s.

RESULTS

Microshear Bond Strength

As shown in table 1, the μSBS of each group is presented. The different severity of mild and moderate dental fluorosis had not significant effect on the μSBS when treated with 5.25%NaOCl at same time. However, the various duration of NaOCl had obviously different μSBS in mild or moderate fluorotic enamel. A significant difference was observed between 0s-group and 60s-group (p < 0.01). At the same time, 120s-group significantly increased the bond strengths compared to 0s-group as well (p < 0.01). Conversely, the μSBS at 180s was the lowest in all groups and showed a statistically significant decrease compared to 0s–group in ML-F group (p < 0.05). When 60s-group and 120s-group were compared, statistically significantly higher mean bond strengths were obtained to 120sgroup than to 60s-group (p < 0.05) in MD-F group. But in ML-F group, there was no difference between 60s-group and 120s-group (p > 0.05). Table 2 showed the fracture modes of each group and the majority of fracture modes was in Mode A. Nevertheless, Figure 1 showed the occurrence rate of facture in Mode A decreased as 5.25%NaOCl was applied.

Table 1.Microshear bond strengths in MPa (means ± standard deviations) of the different groups

Group	0s	60s	120s	180s
ML-F	25.55± 4.8a	28.40 ± 4.0b	29.31± 4.2b	22.98 ± 4.0c
MD-F	25.12 ± 3.6A	28.02 ± 3.8B	30.63 ± 3.9C	24.63± 3.9A
Note: For each line, different superscript letters indicate statistically significant differences between groups (two-way ANOVA; LSD test, p < 0.05). For each column, there is no statistical difference (p > 0.05).

Table 2.Number of specimens according to fracture mode* for all experimental groups

Group (facture mode)		ML-F			MD-F
Time	A	B	C	A	B	C
0s	25	2	6	25	1	7
60s	21	5	7	25	3	5
120s	24	2	7	24	4	5
180s	23	2	8	19	6	8
* A–adhesive; B–cohesive in enamel or resin; C–mixed;

Figure 1: Fracture modes (%) of ML-F and MD-F groups (Mode A: adhesive, Mode B: cohesive in enamel or resin, Mode C: mixed)

SEM observation

The representative SEM image of the enamel-etching pattern in each group are shown in Figure 2. Moderate dental fluorosis who compared the mild, after deproteinization with NaOCl and acid-etching, showed more dissolution of prisms cores in the same processing time. While more prism peripheries dissolve with the increase of NaOCl application time at the same degree of fluorotic enamel. An increase of irregularities and micro-porosities on the fluorotic enamel surface was visible compared to enamel with the 0s-groups. 180s-group revealed a more remarkable etching pattern with more underlying enamel compared with 60s or 120s. So deproteinization and acid-etching increased the porosity of enamel surface whose etching patterns showed more profound.

Figure 2: SEM images of various groups at a magnification of ×5,000. (ML-F: mild fluorotic enamel, MD-F: moderate fluorotic enamel). Mild and moderate fluorotic enamel treated with 5.25%NaOCl for different treatment times (0s, 60s, 120s, 180s). Fig. a, b: Enamel rods and inter-rod substance with a nearly uniform etching pattern but areas of hypomineralization of enamel can also be observed (white arrow). Fig. c, d, e and f: the inter-rod substances were removed and both type I etching (in which the enamel rod, or prism, head are dissolved) and type 2 etching (in which the enamel interprismatic substance is dissolved) patterns are evident (white arrow). Fig. g, h: barely visible enamel rods and irregular etching pattern were shown (white arrow).

Attenuated total reflection Fourier transform infrared (ATR-FTIR)

FTIR spectra showed major chemical groups in both mild and moderate fluorotic enamel (Figure 3): the phosphate ν₁, ν₃ stretching mode (900–1200 cm^-1), carbonate ν₃ stretching mode (1350–1520 cm^-1) and carbonate ν₂ deformational mode (845–890 cm^-1) from the mineral component, the amide Ⅰ bands (1544 cm^-1) and amide Ⅱ bands (1637 cm^-1) from the organic component [28, 29]. The spectra showed that mild and moderate fluorotic enamel were treated with different duration of NaOCl (Figure 4). Apatite is undissolving in NaOCl so the intensity of phosphate stretching vibration peak is constant [16, 17]. Therefore, we normalized the spectra to the phosphate v₃ (peak at 985/cm^-1) in Figure 3 and Figure 4.

As can be seen, the amide Ⅰ and amide Ⅱ bands are significantly larger in spectra of our moderate fluorotic enamel samples than the mild and so does carbonate ν₃ band (Figure 3). These spectra showed an obvious weakening of the peaks at 1,550, and 1,643/cm^-1 after NaOCl treatment (Figure 4). The presence of phosphate peak became more legible with prolonged time (Figure 4). With increased exposure time of NaOCl, the band at 1,643/cm^-1 showed weaker. But in 60s, 120s and 180s, the amide bands seemed to overlap in the ML-F group and the peaks at 1,643/cm^-1 in the MD-F group was relatively distinct. The differential spectra clearly indicate a decrease in the organic content with the application of NaOCl or an increase in the organic content with the severity of dental fluorosis.

Figure 3: A typical ATR spectra of mild fluorotic enamel (ML-F) and moderate fluorotic enamel (MD-F) without NaOCl treatment. The chemical components on enamel whose absorption peaks were between 700 and 2,000 cm^-1 are shown.

Figure 4: Representative ATR spectra of mild fluorotic enamel (ML-F) and moderate fluorotic enamel (MD-F) after 5.25%NaOCl treatments with different exposure times. The red, blue, green and black lines represent ATR spectra of enamel after NaOCl treatments for 0s, 60s, 120s, 180s groups, respectively.

DISCUSSION

The results of the present study indicated that the deproteinization of 5.25% NaOCl at 60s or 120s significantly increased bond strength than that at 0s in mild and moderate fluorosis. The optimum treatment time of 5.25% NaOCl was at 60s in ML-F group but 120s in MD-F group. There were obvious changes in bonding strength of fluorotic enamel after various duration of pre-treatment with NaOCl solution, so the null hypothesis that “various exposure times with 5.25% NaOCl will not influence the μSBS.” can be rejected. Besides, the longer the treatment time of NaOCl in 0 s to 180s was, the more profound etching patterns were under SEM. The spectra of enamel demonstrated that organic content decreased after NaOCl was applied. Hence, the hypothesis that ‘various exposure times with 5.25% NaOCl will not influence enamel-etching pattern and the ATR-FTIR of fluorotic enamel.’ was also rejected.

Besides, it is consistent with results from Mariana et al, in which the treatment of 5.2%NaOCl at 60s markedly increased bonding strength to fluorotic enamel [19]. we qualitatively analyzed the spectra on the amide bands and carbonate bands by ATR-FTIR and discovered that 5.25% NaOCl did indeed decrease protein content in enamel surface. This result is consistent with the findings of others studies [16, 17].

At the same time, what we found under SEM was consistent with Valencia et al that the center of the prism crystals was more easily attacked without NaOCl deproteinization, while the inter-rod substance was affected with NaOCl deproteinization [30]. Because inter-rod areas contain more organic material than enamel rods [30]. It was known that application with 5.25% NaOCl could be used to increase the bonding area between composite material and the tooth surface before acid etching [13, 21, 30, 31]. The type 1-2 etching pattern was increased when deproteinization with 5.25% NaOCl was used for enamel at 60s [14, 21, 30]. This could be used to explain application with 5.25% NaOCl can increase bond strengths for dental fluorosis.

In ML-F group, there is no difference in bond strength between 60s-group and 120s-group. However, in MD-F group, bond strengths of 120s-group were significantly increased than that of 60s-group. The results of spectrum showed that moderate fluorotic enamel surface had larger protein content than that mild (Figure 3). In addition, the amide bands which was in 60s and 120s seemed to overlap in the ML-F group but in the MD-F group, the band in 120s was distinctly weaker than that in 60s. Therefore, we speculated that MD-F needed a longer processing time of 5.25% NaOCl so that can remove more enamel surface protein. However, area integration of bands originating from carbonate and proteins was difficult to quantitative analysis due to broad or overlapping bands because processing time was too short.

However, the result indicated that treat time with 5.25% NaOCl before adhesive application was not the longer the better. Conversely, it has a specified time range. Time likes 60s or 120s would be beneficial but not 180s.The study found that the μSBS at 180s was the lowest in all groups and even showed a statistically significant decrease compared to 0s–group in ML-F group (p < 0.05). What’s more, SEM revealed that the 60s-group and 120s-group whose enamel rods and inter-rod substance were unambiguous and more uniform than 180s-group. On the contrary, 180s-group showed obscure enamel rods and anomalous etching pattern which was in a more pronounced etching pattern with more exposure of the porosity of enamel surface compared with 60s-group and 120s-group under SEM (Figure 3). The previous findings indicated that prolonged application of 5.25% NaOCl might increase porosity of enamel [19]. Thus we assumed that increased porosity which indicated less and less normal enamel rods structure led to bonding strength decreased.

In addition, many literatures suggest that NaOCl produces reactive free radicals which inhibit resin polymerization adequately [19, 32]. Because these residual free radicals compete with the propagating vinyl free radicals generated during light activation, it leads to premature chain termination and partial polymerization [2, 19]. Therefore, we speculate that the overlong application time of 5.25% NaOCl could have more free radicals on the increased enamel porosities which can’t be removed by rinsing completely and decreasing the conversion degree of the enamel-resin cement interface may result in a lowering of bond strength. Hence, we can go on with the experiment to explore in situ degree of conversion of adhesive after different time of 25% NaOCl application.

In summary, 5.25%NaOCl as an alternative deproteinization agent is beneficial for bonding to fluorotic enamel on the basis of the results. But the low surface energy of fluorotic enamel which has been verified more porous and hypomineralized impairs surface wetting and the surface with well outer mineralization is quite fragile in severe cases [19, 23, 33]. Further studies are needed to test the bonding strength involving thermocycling, resin penetration, in situ conversion degree of the resin cement and the mechanism of enamel surfaces after deproteinization by NaOCl.

CONCLUSION

The present study drew from these following conclusions. The maximal μSBS is acquired by using 5.25%NaOCl at 60s for mild fluorotic enamel but 120s for the moderate. The prolonged application time of 5.25%NaOCl enhanced enamel-etching pattern and minimize superficial enamel protein level. 5.25%NaOCl could be an alternative deproteinization agent for bonding dental adhesive to fluorotic enamel.