Effect of in-office bleaching agent on the surface roughness and microhardness of nanofilled and nanohybrid composite resins

Background: To compare the surface roughness and microhardness of Ceram.x® SphereTEC™ one and Filtek Z350 XT after in-office bleaching with Pola office. Methods: Twenty samples each of (10 mm diameter and 2 mm height) Ceram.x® SphereTEC™ one and Filtek Z350 XT were prepared. The samples were subjected to three bleaching sessions with 35% hydrogen peroxide (Pola office) with a seven-day interval between each session. Surface roughness and microhardness of the prepared samples prior to and after the bleaching regimen were measured using a profilometer and Vickers hardness tester, respectively. Results: A significant reduction (p <0.001) in the surface hardness of Filtek Z350 XT from 27.67 ± 2.10 to 17.83 ± 1.36 Vickers hardness number (VHN) was observed after the bleaching whereas no significant reduction in surface hardness was observed with Ceram.x® SphereTEC™ one. The adjusted mean (estimated marginal mean) microhardness after bleaching for Ceram.x ® SphereTEC™ one (35.79 ± 1.45) was significantly higher than Filtek Z350 XT (19.54 ± 1.45) (p < 0.001). However, in-office bleaching of these materials did not significantly alter their surface roughness. Conclusions: In office-bleaching with 35% hydrogen peroxide can reduce the microhardness of nanofilled composite. However, the surface roughness was not influenced by the bleaching procedure in both nanohybrid and nanofilled composite resin materials.


Introduction
Bleaching removes intrinsic and extrinsic stains from the dental tissues. 1,2 This procedure involves diffusion of bleaching agent which alters the structure of chromophore molecules present in enamel and dentin , thereby promoting tooth whitening. The outcome of tooth whitening depends on the concentration and the ability of the bleaching agent to reach the chromophore molecules coupled with duration and frequency of contact.
The most commonly used bleaching agents are hydrogen peroxide and carbamide peroxide. 3 These bleaching agents can be applied at-home and in-office and are considered to be effective and relatively safe when supervised by a dentist. 4 Although, bleaching is effective in improving esthetics, there has been a growing concern in the recent past on the effect of bleaching materials and techniques on existing restorative materials in the oral cavity. With greater demand for esthetics, there has been an increase in the use of direct esthetic restorative materials, especially dental composites. A dental composite restorative material mainly consists of a polymerizable resin matrix, reinforcing fillers, and a coupling agent that bonds resin with the fillers. The vast majority of dental composites are commercially available for clinical use mainly differ in terms of the resin matrix materials and the fillers used. The clinical performance of these materials significantly varies depending on the type, size, distribution, and concentration of fillers used in the composites.
Ceram.x ® SphereTEC™ one (Dentsply, Konstanz, Germany) is a light-curable nanohybrid composite consisting of nanometer and micrometer-sized fillers with a granulated filler technology (SphereTEC™). It consists of a blend of prepolymerized fillers of a size equivalent to 15 μm, non-agglomerated glass of 0.6 μm, and Ytterbium fluoride of 0.6 μm. It has distinctive handling characteristics, natural-looking gloss, and effortless polishing. Its resin matrix consists of a reformed version of the polysiloxane comprising matrix from the original Ceram.x ® mono+/duo+. It is combined with a well-established polyurethane methacrylate, bis-EMA, and TEGDMA to increase its mechanical strength.
Filtek™ Z350 XT (3M, ESPE, St. Paul, USA) is a universal nanocomposite consisting nanometer-sized filler particles in the composite matrix. The nanofillers consist of 20 nm silica and 4-11 nm zirconia, both in combination of nonagglomerated/non-aggregated and aggregated forms. The presence of nanofillers in agglomerated or clustered forms with a broad distribution in the size of the clusters permits higher filler loading as well as superior polishing ability and thus the esthetic characteristics. Both Ceram.x ® SphereTEC™ one and Filtek™ Z350 XT contain fillers in the nanometer range; however, their particle size and distribution is different.
Many studies have reported the action of bleaching agents on restorative materials. [5][6][7][8][9] The observed changes after bleaching of composite resin materials are alterations in smoothness, hardness and reduction in bond strength. 10,11 Despite extensive research, the observed changes among the composites seem to be varying which could be attributed to the differences in the concentration and type of bleaching agent used. In general, low concentrations of bleaching agent is used for longer times in home bleaching whereas in-office bleaching higher concentration of bleaching agent is used for shorter duration. In addition, compositional variations among dental composites such as type of resin matrix, ratio between resin matrix and filler may influence their susceptibility to bleaching. 9,12 In view of constant surge of newer dental composites with myriad of variation in the composition, it is essential to investigate the effect of bleaching on the properties of composites. In this regard, the present study aimed to compare the surface roughness and microhardness of nanofilled and nanohybrid composite restorative materials subjected to in-office bleaching.

Methods
Sample size was estimated based on the microhardness values reported by Sharafeddin and Jamalipour 5 which yielded an effect size of 1.42. With a power of 80% and 95% confidence interval, the sample size was estimated to be nine per group.
Twenty samples were prepared from each composite material; Ceram.x ® SphereTEC™ one (Dentsply, Sirona) and Filtek Z350 XT (3M ESPE, St. Paul, USA) using a customized stainless steel split mold of 10 mm diameter and 2 mm height. Table 1 summarizes information regarding the composition and manufacturers' details of composite resin materials.

REVISED Amendments from Version 1
We have revised the abstract, introduction with updated literature, details on sample size estimation and sample preparation, added additional details in table (t-value and Inter-group P-values), additional details in ANCOVA (f-value, degrees of freedom and 95% CI) and revised the conclusion. Limitations and scope for future research were also added in the end.
Any further responses from the reviewers can be found at the end of the article After the composite material was packed into the mold, mylar strip (SS White Co, Philadelphia, PA, USA) was used both on top and bottom surfaces to obtain a smooth surface on the composite. Subsequently, the composite material was cured for 20 seconds on both sides using a visible light curing unit (3M ESPE Elipar, St Paul, MN, USA) having a light intensity of 1200 wM/cm 2 .
The prepared discs were subjected to 0.1 ml of 35% hydrogen peroxide (Pola office, SDI Limited, Australia) for 15 mins followed by two additional applications in the same session. The discs were rinsed with distilled water for one minute between each application. The same protocol was repeated two times with one week interval between the applications. After the bleaching process, all the discs were washed and stored in distilled water at 37°C.
The surface hardness of the discs before and after the bleaching was measured using the Vickers hardness testing machine (MMT X7, Matsuzawa Company, Japan). The specimens were mounted on a platform of the device, and a load of 200 g was applied for 30 seconds. The load was removed after dwell time, and the length of the diagonal of the indentation was measured from which the area of the indentation was calculated. Three measurements of each sample were carried out. The surface hardness was calculated by dividing the load by the area of the indention and was reported as Vickers hardness number (VHN).
Surface roughness of the specimens pre-and post-bleaching was measured using a surface profilometer (Surtronics 3+, Taylor Hobson, UK). The samples were placed on a flat stable surface. The stylus of the profilometer was passed over the surface of the specimen to a distance of 0.8 mm. The experiment was carried out in triplicate on each disc, and average surface roughness, as Ra, was recorded in microns.

Statistical analysis
All the analyses were done using SPSS version 20 (RRID:SCR_019096). A p-value of < 0.05 was considered statistically significant. Normality was tested using the Kolmogorov Smirnov test. Comparison of mean surface roughness and microhardness before and after the bleaching was done using the Paired t-test. ANCOVA was used to evaluate the significant differences in the surface roughness and microhardness between the materials after adjusting the baseline values. Data for this study can be accessed at Mendeley Data. 13

Results
There was no significant difference in mean microhardness before and after bleaching (p = 0.954) in Ceram.x ® SphereTEC™ one. However, Filtek Z350 XT showed a significant reduction in the surface hardness after bleaching (p < 0.001). There were no significant differences in the mean surface roughness before and after bleaching in both the composite resin materials (p = 0.153 and 0.199), respectively (

Discussion
The main objective of the present study was to assess the effect of in-office bleaching on nanohybrid and nanofilled composites. As the bleaching process generally affects the surface characteristics of dental composites, both surface roughness and microhardness of Ceram.x ® SphereTEC™ one and Filtek Z350 XT were measured prior to and after bleaching using Pola office. The active ingredient of majority of bleaching agents is hydrogen peroxide and generally oxidizes the chromophores and improves the shade of the discolored tooth. Exposure of these bleaching materials can also potentially affect the existing restorative materials due to their strong oxidizing ability.
Some of the previous investigations have reported an increase in microhardness of composites after bleaching treatment with hydrogen peroxide. 14 In contrast, other research studies have indicated a reduction in surface hardness. 9 Our study did not show any significant changes in the microhardness and surface roughness concerning nanohybrid composite [Ceram.x ® SphereTEC™ one] which was in accordance with previous reports. 15,16 There was a significant reduction in microhardness of Filtek Z350 XT, whereas the surface roughness remained unaffected. These observations were in agreement with previous research. 17 An increase in the surface roughness of restorative materials will facilitate the plaque accumulation on the surface thus affecting the esthetics. 18 Similarly, a decrease in the surface hardness makes the material more vulnerable to wear during masticatory force application. 19 Hydrogen peroxide tends to cause oxidation, thereby facilitating the generation of free radicals. 20 The unreacted double bonds in the polymer resin are prone to oxidative cleavage by peroxides. The by-products of this reaction may bring about a reduction in microhardness. Moreover, the free radicals generated by the peroxides are capable of causing hydrolytic degradation of composite resin at the resin-filler interface, thereby paving the way for filler-matrix debonding, leading to microscopic cracks and thus increasing surface roughness. 21 Ceram.x ® SphereTEC™ has a high proportion of filler particles with advanced granulated filler technology. The nanohybrid composition with advanced filler technology ensures a higher filler loading and hence superior flexural strength, compressive strength, and low water sorption. 22 Higher filler loading and reduced resin matrix content reduces the chance of resin matrix oxidation by hydrogen peroxide, making them resilient to acidic bleaching agents. On the other hand, resin composite Filtek Z350 is a nanoparticulated composite compounded by BisGMA, UDMA, BisEMA, and minor proportions of TEGDMA. The overall inorganic filler loading in these composites is about 72% by weight, which is less than Ceram.x ® SphereTEC™ composites with an inorganic filler loading of 77-79% by weight. A low filler loading with a large resin matrix volume makes these composites more prone to oxidation or degradation by bleaching agents, hence a significant reduction in microhardness after bleaching. 23 Free radicals induced by peroxides may impact the resin-filler interface and cause a filler-matrix debonding. 24 The microhardness of the composites is highly influenced by the amount and type of the inorganic fillers. 25 Hence, a reduction in the surface microhardness for Filtek Z350 XT may be due to the inorganic filler loss on the surface. Ceram.x ® SphereTEC™ one has pre-polymerized filler particles of non-agglomerated barium glass and ytterbium fluoride and a resin matrix with highly dispersed methacrylic polysiloxane nanoparticles that are chemically similar to glass or ceramics. Such filler composition is more resistant to abrasion and inorganic filler loss at the surface. Hence no significant changes in microhardness and surface roughness were observed.
The results of the present study indicate that compositional variations influence the susceptibility of dental composites to bleaching. However, the present study selected only two types of dental composites. Additional studies on large number of composites and their types (microfilled, nanofilled, hybrid composites etc.) may provide more insights on the effect of bleaching on composites restorative materials.

Conclusions
Within the limitations of the present study, it can be concluded that in office-bleaching with 35% hydrogen peroxide can reduce the microhardness of nanofilled composite. However, the surface roughness is not affected in both nanohybrid and nanofilled composite resin materials. Hence, the effect of the bleaching agent on the existing composite resin restorations must be considered at the time of selection of the bleaching agent and the regimen for clinical use.

Open Peer Review
I have gone through the feedback provided by the authors and I agree with the information given.
In the introduction, although a clear description of the relevance of the study is provided together with a description of the composite materials, very little information is covered on bleaching materials and techniques and how these can specifically affect the material properties. For example, where this is attempted in the first two paragraphs, the information is scanty and supported by only two citations. Likewise, in the final paragraph, a general statement is made on the effect of the bleaching agents and suspected attributes regarding the material composition but no specific explanation and corresponding examples which would be helpful in justifying the need for this study. A clear distinction should be made between the nanofilled and the nanohybrid composite.
In the methods, the manufacture of Ceram.X SphereTec One in Table 1 is different from that stated in the introduction. The description of the specimens is also lacking important detail -how many specimens were prepared for each composite material? This should also be clarified in the abstract. What was the total sample size and was this calculated based on a determined statistical power?
What material was the split mould made of? Where was the mylar strip sourced from?
How much bleaching agent was applied on the discs? Was it the same quantity for all discs at all times? Were the discs dry at the initial bleaching application, before they were stored in distilled water?
For the hardness test, the length of indentation was measured but the calculation was derived from the area of indentation. This description should be clarified.
The description of the surface roughness measurement is also unclear, specifically, '...passed over the surface of the specimen perpendicularly to a distance of 0.8mm...' In the results, it would be useful to also report the t-values in Table 2 and F or relevant test values for ANCOVA, as well as the 95% CI. Table 2 shows paired t-test results within the same material, that is, before and after for the same material (within) not between the two materials.
A statement on the significant difference on microhardness between the two materials should also be captured in the abstract.
In the discussion, reference is made to a study on two nanohybrid composites in the first paragraph -this should be clarified as the title talks of a nanofilled and a nanohybrid.
The description of the bleaching agent in the discussion is different from that presented in the methodology -can this have any influence on the outcome observed?
Although clear arguments are presented in the discussion, some paragraphs lack supporting citations e.g only one in paragraph 4.
Were there any limitations in this study? Are the conclusions generalizable? Can the observations be accurately and undoubtedly attributed to the type of composite as implied in the conclusion? Do the authors believe that this study was able to show the effect of the bleaching agent on the composite due to the filler type and load?
Throughout the MS, some minor grammatical errors should also be addressed to improve clarity e.g In the title and objective, is the use of 'vital' correct considering that this was an in vitro study?; In the abstract conclusion, '...seems to vary due to variations...'; In the introduction, the second statement in the first paragraph, '..the majority of bleaching agents...' could improve clarity; in the second paragraph, third statement, '...ever-increasing demand...there has been an increase....'

If applicable, is the statistical analysis and its interpretation appropriate?
Yes Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Dental Biomaterials Science
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Query 1
In the introduction, Although a clear description of the relevance of the study is provided together with a description of the composite materials, very little information is covered on bleaching materials and techniques and how these can specifically affect the material properties. For example, where this is attempted in the first two paragraphs, the information is scanty and supported by only two citations. Likewise, in the final paragraph, a general statement is made on the effect of the bleaching agents and suspected attributes regarding the material composition but no specific explanation and corresponding examples which would be helpful in justifying the need for this study. A clear distinction should be made between the nanofilled and the nanohybrid composite.

Response 1
The introduction has been revised. Although a clear description of the relevance of the study is provided together with a description of the composite materials, very little information is covered on bleaching materials and techniques and how these can specifically affect the material properties-The most commonly used bleaching agents are hydrogen peroxide and carbamide peroxide. These bleaching agents can be applied at-home and in-office and are considered to be effective and relatively safe when supervised by a dentist. Many studies have reported the action of bleaching agents on restorative materials. The observed changes after bleaching of composite resin materials are alterations in smoothness, hardness and reduction in bond strength.
A clear distinction should be made between the nanofilled and the nanohybrid composite. Filtek™ Z350 XT (3M, ESPE, St. Paul, USA) is a universal nanocomposite consisting of nanometer-sized filler particles in the composite matrix.

Query 2
In the methods, the manufacture of Ceram.X SphereTec One in Table 1 is different from that stated in the introduction.

Response 2
Manufacturer details of Ceram X Sphertec One in Table 1 has been changed to Dentsply, Konstanz, Germany Query 3 The description of the specimens is also lacking important detail -how many specimens were prepared for each composite material? This should also be clarified in the abstract.
Response 3 20 samples each of (10 mm diameter and 2 mm height) Ceram.x® SphereTEC™ one and Filtek Z350 XT were prepared. It has been revised both in abstract and also in the main text.

Query 4
What was the total sample size and was this calculated based on a determined statistical power?

Response 4
Total sample size is 40. Sample size was estimated based on the microhardness values reported by Sharafeddin and Jamalipour which yielded an effect size of 1.42. With a power of 80% and 95% confidence interval, the sample size was estimated to be nine per group Query 5 What material was the split mould made of? Where was the mylar strip sourced from?
Response 5 Split mould was made of stainless steel. Manufacturing detail of mylar strip is (SS White Co, Philadelphia, PA, USA) and its been added to the main text Query 6 How much bleaching agent was applied on the discs? Was it the same quantity for all discs at all times? Were the discs dry at the initial bleaching application, before they were stored in distilled water?

Response 6
The prepared discs were subjected to 0.1 ml of 35% hydrogen peroxide (Pola office, SDI Limited, Australia) Same quantities were used for all the discs. Yes, the discs were dry at the initial bleaching application.

Query 7
For the hardness test, the length of indentation was measured but the calculation was derived from the area of indentation. This description should be clarified.

Response 7
Using the length of the diagonal of the indentation, area of the indentation [square-shaped indentation] was measured as follows: area of indentation = ½ x length of the diagonal 2

Query 8
The description of the surface roughness measurement is also unclear, specifically, '...passed over the surface of the specimen perpendicularly to a distance of 0.8mm...'

Response 8
The word perpendicular was used in the context that the stylus was moving over the surface We have removed the word "perpendicularly" from the text. Surface roughness of the specimens pre-and post-bleaching was measured using a surface profilometer (Surtronics 3+, Taylor Hobson, UK). The samples were placed on a flat stable surface. The stylus of the profilometer was passed over the surface of the specimen to a distance of 0.8 mm. The experiment was carried out in triplicate on each disc,and average surface roughness, as Ra, was recorded in microns Query 9 In the results, it would be useful to also report the t-values in Table 2 and F or relevant test values for ANCOVA, as well as the 95% CI.
Response 9 t-values ,F values and 95% CI have been added to the table 2 and text.
Query 10 Table 2 shows paired t-test results within the same material, that is, before and after for the same material (within) not between the two materials.
Response 10 It has been included in the Table 2 Query 11 A statement on the significant difference on microhardness between the two materials should also be captured in the abstract.

Query 12
In the discussion, reference is made to a study on two nanohybrid composites in the first paragraph -this should be clarified as the title talks of a nanofilled and a nanohybrid.
Response 12 It has been revised in the first paragraph of the discussion: The main objective of the present study was to assess the effect of in-office bleaching on nanohybrid and nanofilled composites Query 13 The description of the bleaching agent in the discussion is different from that presented in the methodology -can this have any influence on the outcome observed?

Response 13
The active ingredient of majority of bleaching agents is hydrogen peroxide and generally oxidizes the chromophores and improves the shade of the discolored tooth. It has been revised in the second paragraph of the discussion

Query 15
Were there any limitations in this study?

Response 15
The results of the present study indicate that compositional variations influence the susceptibility of dental composites to bleaching. However, the present study selected only two types of dental composites. Additional studies on large number of composites and their types (microfilled, nanofilled, hybrid composites etc) may provide more insights on the effect of bleaching on composites restorative materials.

Query 16
Are the conclusions generalizable? Can the observations be accurately and undoubtedly attributed to the type of composite as implied in the conclusion? Do the authors believe that this study was able to show the effect of the bleaching agent on the composite due to the filler type and load?

Response 16
From the results of the present study, we conclude that the variations in formulation of dental composites can influence their susceptibility to bleaching. In that sense, variations in filler size and loading would also alter the ratio between resin matrix to filler. Hence, the findings of the present study can be generalized to the extent that variations in composition changes their susceptibility to bleaching Query 17 Throughout the MS, some minor grammatical errors should also be addressed to improve clarity e.g In the title and objective, is the use of 'vital' correct considering that this was an in vitro study?; In the abstract conclusion, '...seems to vary due to variations...'; In the introduction, the second statement in the first paragraph, '..the majority of bleaching agents...' could improve clarity; in the second paragraph, third statement, '...ever-increasing demand...there has been an increase....'

Response 17
In the title and objective, is the use of 'vital' correct considering that this was an in vitro study?:Title has been rephrased to "Effect of in-office bleaching agent on the surface roughness and microhardness of nanofilled and nanohybrid composite resins" In the abstract conclusion, '...seems to vary due to variations..: Abstract has been revised to -In office-bleaching with 35% hydrogen peroxide can reduce the microhardness of nanofilled composite. However, the surface roughness was not influenced by the bleaching procedure in both nanohybrid and nanofilled composite resin materials.
In introduction-the majority of bleaching agents: Second statement in the first paragraph has been revised to :This procedure involves diffusion of bleaching agent which alters the structure of chromophore molecules present in enamel and dentin , thereby promoting tooth whitening ever-increasing demand...there has been an increase -third statement in the second paragraph has been revised to With greater demand for esthetics, there has been an increase in the use of direct esthetic restorative materials, especially dental composites with 10 x 2 mm were fabricated with Ceram.x® SphereTEC™ one and Filtek Z350 XT. The samples were subjected to two bleaching sessions with 35% hydrogen peroxide (Pola office) with a sevenday interval between each session. Surface roughness and microhardness of the prepared samples prior to and after the bleaching procedures were measured using a profilometer and Vickers hardness tester, respectively. This study reported a significant reduction (p <0.001) in the surface hardness of Filtek Z350 XT after the bleaching and no significant decrease in surface hardness with Ceram.x® SphereTEC™ one. However, in-office bleaching of these materials did not report a significant change in their surface roughness.
The detailed report is as follows.
This manuscript was well-written and adhered to the standards. The introduction was adequate and described the objectives of the study clearly. In the second paragraph, a few references should have been cited to substantiate the information presented.
○ The methodology was clearly described. The methodology can include information about the curing lamp specifications and the radiation/wavelength employed.

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Results described well based on the statistical analysis performed.

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The discussion is adequate and consistent with the results. The reasons for varying the surface hardness and roughness were discussed. The outcomes of the current study were compared with those of other comparable investigations. The limitations and the future scope of this study could have been emphasized.

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The conclusions were appropriate based on the results.

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The references were appropriate and mentioned the most recent studies.

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The data presented in the current study is useful to the scientific community and clinicians. This study reported a significant decrease in the surface hardness of the composites with the bleaching procedures. This decrease in hardness was dependent on the filler loading to resin ratio. Therefore, the data presented in this study is handy to clinicians for selecting an appropriate composite for their patient's needs.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? Yes