Enamel and Dentine Surface Morphology and chemistry

Scanning electron microscopy (SEM) has been used for qualitatively analysing the surface morphology of enamel and dentine specimens following bleaching. In addition profilometry has been used to measure the surface roughness.

Some authors have reported alterations of enamel surfaces, including shallow depression, increased porosity and slight erosion, associated with whitening treatments (Bitter, 1992; Bitter and Sander, 1993; Josey et al., 1996). In one study with two bleaching gels containing 16% and 35% carbamide peroxide, the authors concluded that the results indicated a need to warn patients of the potential for enamel alteration and its detrimental effect on tooth structure even if the long-term consequences have yet to be conclusively determined (Bitter 1998). It should be noted that studies have demonstrated that also soft drinks (e.g., Coca-Cola, Pepsi Cola) and fruit juices cause demineralisation and alteration of enamel (Grobler et al., 1990; Grando et al., 1996) which are comparable to those reported for whitening agents (McCracken and Haywood, 1996).

Shannon et al. (1993) subjected enamel slabs to different bleaching agents containing 10% carbamide peroxide for 15 hours a day for 2- and 4-week periods and evaluated by scanning electron microscopy. During the remaining 9 hours, the slabs were exposed to human saliva in vivo. Significant surface alterations in enamel topography were observed for slabs treated with the bleaching solutions for 4 weeks. Cubbon and Ore (1991) and Hammel (1998) have reported two clinical cases of serious adverse effects on enamel associated with whitening agents, both of which involved the use of “over-the-counter†products.

Attin et al. (1997) assessed effects of bleaching on enamel concurrent with fluoride remineralization. While bleaching produced a slight surface softening in their protocol, the group found that topical fluoride reversed this effect, promoting surface hardening through remineralization. Rothuijzen et al. [abstract](to be published) found that in vitro bleaching without intermittent remineralisation periods VivaStyle (10% carbamide peroxide; pH 5.2) rendered enamel vulnerable for subsequent demineralization, while bleaching with Opalescence (10% carbamide peroxide + 0.11% fluoride; pH 6.7) had a protective effect.

Numerous studies have indicated neglible changes in enamel surface texture associated with peroxide bleaching (McGuckin et al., 1992). When changes are observed, they are for the most part minor, involving the formation of shallow depressions or increased porosities. These are likely to be a side effect of the bleaching matrices. These changes are expected to be normalized through later prophylaxis or through salivary remineralization.

The majority of studies confirming the safety of bleaching systems are contrasted with a few investigations that have shown surface degradative changes associated with bleaching processes. Rotstein et al. (1996) reported that application of 35% carbamide peroxide produced etching and demineralisation on dental enamel surfaces and in subsurface areas.

From submission IV

The majority of the more recent studies that have used scanning electron microscopy or profilometry during the last years showed no significant changes in enamel surface morphology following bleaching even with one of the highest concentrations of hydrogen peroxide (35%) (Sulieman et al., 2004). Similarly, the lower levels of 6.5% hydrogen peroxide (Duschner et al., 2006) and 6.0% hydrogen peroxide (Duschner et al., 2006; Joiner et al., 2004; Nucci et al., 2004), and 10% carbamide peroxide (Nucci et al., 2004; Justino et al.,[abstract] 2004) were also shown to have no significant effects on enamel surface morphology following simulated 2 weeks product usages. This is contrasted with studies by Pinto et al. (2004), Cavalli et al. (2004a) and Yeh et al. (2005) who observed some changes in enamel morphology following bleaching with hydrogen peroxide or carbamide peroxide.

The differences between the positive and null effects on enamel may be due to differences in the in vitro protocols used and this is reflected in their differences with respect to replicating the in vivo environment. For example, Yeh et al. (2005) stored their samples between bleaching sessions in distilled water. Pinto et al. (2004) and Cavalli et al. (2004a) used artificial saliva consisting only of inorganic calcium and phosphate components, and were devoid of any organic components which could have the potential of forming a protective salivary pellicle. In the case of three studies which showed no effect of bleaching products on enamel surface morphology, (Duschner et al. 2006; Joiner et al. 2004; Justino et al. [abstract] 2004) human whole saliva was used as a key part of replicating the in vivo situation. Indeed, Justino et al. [abstract] (2004) demonstrated that any adverse effects evident for in vitro bleached and stored in water enamel specimens were not seen for similarly treated specimens placed on an intra-oral device and worn in the mouth.

In terms of changes in human enamel surface chemical composition, no differences were found between enamel treated with 30% hydrogen peroxide (120 hr treatment), 10% carbamide peroxide (6 h/day for 14 days) or water controls as measured by Raman spectroscopy (Park et al. 2004; Goo et al 2004). Similar results were obtained for electron spectroscopy for chemical analysis (ESCA) techniques used on human enamel treated with either 10% carbamide peroxide, 7% hydrogen peroxide or 12% hydrogen peroxide (7 h/d for 14 days) (Pugh et al. 2005).

COLIPA concluded that the majority of studies indicate that hydrogen peroxide and carbamide peroxide containing products have no significant deleterious effects on enamel and dentine surface morphology and that the contrasting studies that do show an effect, in general, have some limitations in the in vitro methodologies used which do not reflect the in vivo situation accurately.

Enamel and Dentine Surface Microhardness

Surface microhardness (SMH) measurement has been a frequently used technique for evaluating the effects of peroxide and bleaching products on enamel and dentine.

There are numerous published in vitro reports in the literature detailing the detrimental effects or lack of effects of peroxide-containing tooth whitening products on enamel microhardness (Seghi and Denry, 1992; Murchison et al., 1992), enamel resistance to abrasion (Seghi and Denry, 1992), dentin microhardness (Nathoo et al., 1994; Pecora et al., 1994), dentin roughening (Zalkind et al., 1996; Atrushkevich and Vasiukova, 1996), and restoration microhardness (Bailey and Swift, 1992; Nathoo et al., 1994). Results are dependent on the methodology used and the materials or products tested.

Several studies also reported minimal or no effects of whitening agents containing 10% carbamide peroxide on microhardness and mineral content of human enamel surfaces (Shannon et al., 1993; McCracken and Haywood, 1995, 1996; Nathoo et al., 1994; Murchison et al., 1992).

Crest Whitestrips gel containing up to 6.5% hydrogen peroxide was applied for up to 70 hours bleaching (five kits). Human tooth enamel specimens were cycled through a daily regime including salivary immersions and treatment with commercial tooth whitening gels containing hydrogen peroxide or carbamide peroxide. Following in vitro laboratory cycling, the teeth were cross-sectioned and remounted for observation of microhardness and ultrastructural characteristics in subsurface regions. It was concluded that the peroxide bleaching gels produced no changes in subsurface enamel and dentin ultrastructure or architecture. Tooth preparation – Human teeth were collected by dentists and periodontists in the course of their typical practice in the Cincinnati region. These teeth were collected as part of a longstanding Procter and Gamble program of tooth collection and preservation for their laboratory requirements. The results provided support for the clinical experience that vital tooth bleaching produces no effects on the structure or function of teeth (White et al., 2004a).

A 10% carbamide peroxide bleaching agent was evaluated against a placebo agent. Two hundred and forty dental fragments were randomly fixed on the vestibular surface of the first superior molars and second superior premolars of 30 volunteers. The results suggest that treatment with 10% carbamide peroxide bleaching materials for three weeks alters the enamel microhardness, although it does not seem to alter the dentin microhardness (Basting et al. 2001).

An in vitro study aimed to evaluate the effect of bleaching agents on dentin microhardness during and after bleaching was performed. Specimens were randomly assigned to seven groups using different bleaching agents as well as a placebo agent. The 42-day whitening treatment consisted of daily application of the agents to the dentin surface for 8 hours, followed by immersion in artificial saliva for 16 hours. After the bleaching treatment, specimens were kept immersed in artificial saliva for 14 days. Microhardness was measured at baseline as well as different times during bleaching and during the post-treatment period. It is concluded that throughout the bleaching treatment, depending on the agent applied, dentin showed a transitory decrease in microhardness values. In the post-treatment period, artificial saliva presented a remineralizing effect on the bleached surfaces (de Freitas et al. 2004).

Research has been carried out supporting the hard tissue safety of bleaching processes associated with strip bleaching gels (White et al., 2000). Studies included the assessment of strip gels containing different peroxide concentrations ranging from 5.3 to 16% hydrogen peroxide. Studies also included the application of gels for time periods up to 5x recommended consumer use. In a novel in vitro cycling protocol, bleach activity was first confirmed with image analysis colorimetry of enamel and dentin surfaces. Surface microhardness and texture assessments were complemented with analyses on cross sections samples. Hardness evaluations were then further complemented with ultrastructural observations realized through application of 3D confocal laser scanning microscopy image reconstructions, carried out on naturally wet specimens. Results illustrate the safety of the 6% hydrogen peroxide bleaching gels to both topically treated enamel and dentin and to surface regions of these specimens for all concentrations of hydrogen peroxide and all exposure regimens.

Sulieman et al. (2004) showed that 35% hydrogen peroxide treatment for 30 mins and Park et al. (2004) showed that 30% hydrogen peroxide treatment for 120 hours, both on human enamel, showed no significant reduction of SMH. Cycling experiments on enamel with 6-9.5% hydrogen peroxide where treatments were 30 mins, twice per day for 14 days simulated use (Teixeira et al. 2004), 6% and 6.5% hydrogen peroxide for 30 mins twice/day for 28 days (Duschner, 2006), 12% hydrogen peroxide for 7 h/day for 14 days (Pugh et al., 2005), 6% hydrogen peroxide for 20 mins, twice/day for 14 days (Joiner et al., 2004) all showed no reduction in SMH. A similar conclusion was obtained for similar simulated use cycling experiments when 10% carbamide peroxide (Justino et al., 2004; Unlu et al., 2004; Pugh et al., 2005; Leonard et al., 2005), 11% carbamide peroxide (Wong et al.,[abstract] 2006) and 15% carbamide peroxide (Unlu et al., 2004) were tested. Leonard et al.(2005), however, point out that when evaluating enamel microhardness, consumer available paint-on bleaching solutions may adversely affect enamel microhardness compared to a control and 10% carbamide peroxide dentist-prescribed, home-applied bleaching product.

Lewinstein et al. (2004) observed a reduction in SMH following 35% hydrogen peroxide or 35% carbamide peroxide treatments on human enamel which was reversed when treated with a 0.05% fluoride solution. Similarly, Basting et al.[abstract] (2005) also observed a slight reduction in SMH of human enamel following 8 h/day for 42 days of 10% carbamide peroxide treatments. In an in situ type study, Rodrigues et al. (2005) noted a slight reduction in SMH following in office 37% carbamide peroxide treatment (30 minutes x 2 on 3 days) plus at home use of 10% carbamide peroxide (6 h for 21 days). However, this was not significantly different from an equivalent series of placebo treatments and the authors considered the observed SMH reductions as clinically insignificant.

A reduction in enamel SMH was observed by Hairul Nizam et al (2005) following 24 h treatment with 30% hydrogen peroxide solution. Also two other studies observed a reduction in enamel SMH following bleaching with up to 35% hydrogen peroxide or 35% carbamide peroxide (Pinto et al., 2004; Attin et al., 2004). COLIPA points out that the conflicting data may be due to differences of the in vitro methods used. In particular, the two last studies used artificial saliva containing no organic components which could have formed a protective layer, there were no fluoride treatments to aid remineralisation and the study by Attin et al (2004) used bovine enamel which is known to have a three-fold faster rate of lesion progression compared to human enamel (see also Attin, 2006).

For dentine, no significant changes in SMH were reported in experiments involving 35% hydrogen peroxide for 30 minutes (Sulieman et al 2004) and 10% or 15% carbamide peroxide treatments for up to 28 hours (Unlu et al., 2004), or in cycling experiments using, 6% or 6.5% hydrogen peroxide treatments for 30 minutes twice/day for 28 days (Duschner et al., 2006) or 6% hydrogen peroxide for 20 minutes twice/day for 14 days (Joiner et al., 2004).

A transitory decrease in dentine SMH has been observed in some studies but recovered following a remineralisation period (Freitas et al., 2004a, 2004b) or 0.05% fluoride solution treatment (Lewinstein et al., 2004). Arcari et al. (2005) reported small reductions in dentine SMH (5.4%). A significant reduction in dentine SMH was observed for one 10% carbamide peroxide product (Basting et al., [abstract] 2005). In addition, the study by Hairul Nizam et al. (2005) showed a reduction in dentine SMH.

COLIPA concluded that overall, the majority of studies indicate that hydrogen peroxide and carbamide peroxide containing products have no significant deleterious effects on enamel and dentine SMH, even if one of the highest levels of hydrogen peroxide is used. The few contrasting studies that do show an effect, in general, again have some limitations in the in vitro methodologies used which do not reflect the in vivo situation accurately. Indeed, some studies demonstrated a transitory reduction in SMH which were recovered following a remineralisation period.

Subsurface Enamel and Dentine

Since hydrogen peroxide will diffuse through enamel towards the enamel-dentine junction, some studies have investigated the effects of bleach agents on subsurface enamel and dentine. This is typically accomplished by bleaching whole teeth or fragments and then cutting and polishing the specimens to reveal the internal subsurface enamel and dentine areas, followed by micro-hardness measurements.

Using the above approach, Teixeira et al. (2004) found no reductions in enamel subsurface microhardness following treatments with 6%-9.5% hydrogen peroxide (30 minutes x 2/day) or 10% carbamide peroxide (6 h/day) for 14 days in total. Similar results were found for both subsurface enamel and dentine following 6% hydrogen peroxide for 20 minutes x 2/day, for 14 days (Joiner and Thakker 2004) or for 14 hours and 70 hours total bleaching time with 5.3% hydrogen peroxide (White et al., 2004). In contrast, the study by Attin et al. (2005) showed some reduction in subsurface enamel but not subsurface dentine following bleaching protocols with up to 35% hydrogen peroxide or 35% carbamide peroxide. Again this contrast may be due to differences in the methodology.

An alternative approach to investigating the effects of bleaching on subsurface enamel, dentine and the enamel-dentine junction is to use confocal laser scanning microscopy which enables their ultrastructure to be investigated. Studies on bleached tooth specimens have demonstrated no changes in enamel and dentine ultrastructure (White et al., 2004; Duschner et al., 2006). On the other hand, Markovic et al. (2007) exposed teeth in vitro to either 10% or 16% carbamide peroxide for 4 hours per 7 days. The statistical analysis showed significantly higher microroughness for both groups of carbamide peroxide exposed enamel surfaces.

The ultimate tensile strength of subsurface enamel following treatment with up to 35% hydrogen peroxide and 37% carbamide peroxide (Silva et al.,[abstract] 2005) and 10 – 20% carbamide peroxide (Cavalli et al., 2004b) has been shown to be reduced compared to non bleached controls. Cavalli et al. (2004b) point out that the effects of bleaching agents on the mechanical properties of enamel have not been extensively studied. Although it is quite difficult to clinically associate enamel cracking or fractures with previous bleaching treatments, there is increasing evidence that enamel structural changes may occur due to exposure to such substances that may ultimately compromise its strength. There study showed that the ultimate tensile strength of enamel was significantly reduced when a routinely used bleaching regimen was followed and that the clinical implications must be further investigated.

Tam et al. (2007) have studied the effects of in vitro prolonged tooth bleaching on the fracture toughness of human dentin. Dentin from recently extracted molar teeth was directly or indirectly treated to simulate a prolonged at-home (10% carbamide peroxide or 3% hydrogen peroxide, 6 hours/day, 5 days/week for 8 weeks) or in-office (30% hydrogen peroxide, 1 hour/week for 8 weeks) bleaching regimen (N = 8/group). For direct bleach application, the treatment materials were applied onto dentin that was already prepared as compact tension specimens. For indirect bleach application, bleach was applied to the enamel of intact teeth prior to specimen preparation. There was a significant decrease in dentin fracture toughness after 8 weeks of direct bleach treatment. There were no significant differences between the bleach and control groups after 8 weeks of indirect bleach treatment (p = 0.19). The authors conclude that caution should be considered when using bleach for prolonged treatment times in clinical cases where there is dentin exposure such as occlusal attrition or gingival recession.

COLIPA concluded that the majority of relevant in vitro studies indicate that hydrogen peroxide and carbamide peroxide containing products have no significant deleterious effects on subsurface enamel and dentine microhardness or ultrastructure. Only one in vitro study showed a decrease in the subsurface microhardness of enamel.

Effects of Acid Challenges and Abrasion on Bleached Enamel/Dentine

Sulieman et al. (2004) found that pre-bleaching human enamel and dentine with 35% hydrogen peroxide for 30 minutes had no subsequent deleterious effect on enamel and dentine loss caused by citric acid erosive challenges or brushing with toothpaste, as measured by profilometry. Similarly, bleaching human enamel and dentine with 10%-22% carbamide peroxide for 2 h x 20 treatments did not increase their susceptibility to acid erosion or caries lesion formation as measured by quantitative light-induced fluorescence and transverse microradiography (Pretty et al., 2005).

Cia Worschech et al. (2006) studied how tooth bleaching abrasive dentifrices might change the outer superficial enamel. Human enamel slabs were exposed in vitro to a 10% carbamide peroxide bleaching agent at different times and submitted to different superficial cleaning treatments. Bleaching was performed on the enamel surface for six hours daily. After that, each slab received a cleaning surface treatment and was stored in artificial saliva. The study showed that the sole use of 10% carbamide peroxide did not alter the enamel surface roughness, but the cleaning treatments that employed the use of brushing with abrasive dentifrices resulted in a significant increase of enamel surface roughness.

The study by Wiegand et al. (2004) showed that bleaching with 35% or 38% hydrogen peroxide (15 min x 2/d, for 4 d) or 35% carbamide peroxide (1 h on 4 d) gave no significant increase in enamel wear caused by brushing with toothpaste. In the same study, they did show a significant increase in enamel wear following treatment protocols with 5.3% hydrogen peroxide, 10% carbamide peroxide and 15% carbamide peroxide. The authors conclude that bleaching treatment may result in increased tooth brushing abrasion. Acidic agents or long duration of bleaching seem to lead to an increased susceptibility to enamel loss by tooth brushing abrasion.

COLIPA concluded that in vitro studies indicate that hydrogen peroxide and carbamide peroxide containing products have no significant clinically relevant effects on subsequent enamel and dentine loss caused by acidic erosive challenges, toothpaste abrasion or caries lesion formation.

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