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Year : 2022  |  Volume : 25  |  Issue : 3  |  Page : 278-282
Microtensile resistance of an adhesive system modified with chitosan nanoparticles

1 Department of Dentistry, School of Biological and Health Sciences, Federal University of Jequitinhonha and Mucuri Valley, Diamantina, Brazil
2 Department of Dental Materials and Prosthesis, School of Dentistry, University of São Paulo, Ribeirão Preto, Brazil

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Date of Submission16-Dec-2021
Date of Decision26-Jan-2022
Date of Acceptance27-Jan-2022
Date of Web Publication13-Jun-2022


Aim: The aim of this study was to evaluate whether 2.5% chitosan applied after dentin acid etching or incorporated into the adhesive system interferes with the microtensile strength of the interface tooth-resin.
Subjects and Methods: Sixty healthy bovine teeth had the incisal thirds and the roots sectioned and were randomly allocated into three groups (n = 60). G1 (control): acid attack + Clearfil SE Bond + Z350 resin; G2: treatment with 2.5% chitosan + Clearfil SE Bond + Z350 resin; G3: treatment with 2.5% chitosan incorporated into dentin adhesive + Z350 resin. The stick-shaped specimens made in each group were randomly divided into two subgroups and subjected to degradation of the adhesive interface (with aging: specimens immersed in 2.5% sodium hypochlorite for 12 h and washed in distilled water for 1 h, and without aging). The groups were submitted to a microtensile strength test with a 0.5 mm/min speed until rupture. Statistical analysis of the data was performed according to normal distribution.
Results: Microtensile data were obtained by calculating the area of each toothpick, obtaining values in megapascal. No aging: G1 – 5251.69, G2 – 5.52 ± 1.46, and G3 – 3.91 ± 1.21. With aging: G1 – 3.45 ± 1.29, G2 – 2.75 ± 0.78, and G3 – 3.53 ± 1.33. Statistical analysis showed that the aging factor and adhesive modification decreased the bonding strength of the resin-dentin (P = 0.001). As well, the interaction between the analyzed factors showed statistically significant differences (P = 0.002).
Conclusions: Accelerated aging decreases the adhesion strength in G1, even when chitosan is previously applied to the adhesive system. However, when adding chitosan to the adhesive, adhesion remained similar after aging but lower when compared to the other groups.

Keywords: Aging; chitosan; dental cement; dentin; material strength

How to cite this article:
Botelho LP, Dias De Oliveira SG, Douglas De Oliveira DW, Galo R. Microtensile resistance of an adhesive system modified with chitosan nanoparticles. J Conserv Dent 2022;25:278-82

How to cite this URL:
Botelho LP, Dias De Oliveira SG, Douglas De Oliveira DW, Galo R. Microtensile resistance of an adhesive system modified with chitosan nanoparticles. J Conserv Dent [serial online] 2022 [cited 2022 Jul 4];25:278-82. Available from:

   Introduction Top

Adhesion is defined as the union of restorative materials to dental tissues. The tooth presents different tissues of distinct compositions, which makes the adhesive systems have different behaviors on their surfaces, both in enamel and dentin.[1] The dentin consists of organic and inorganic substances, as well as water, and the longevity of restorations is directly related to chemical and technological advances in the composition of these bonding agents.[2] However, dental treatment is often hampered by the presence of a larger area of the dentinal substrate that is about to be restored, making the adhesive procedure highly sensitive.[3] Failures in adhesive bonding can also occur due to degradation caused by the alteration of the adhesive matrix and the presence of bacteria when there is contact with saliva.[4]

When the enamel is lost, due to carious process or trauma, both dentin and collagen fibers are exposed to the oral environment, and initially, these fibers act as a mechanical barrier to mineral loss, associated with a buffering capacity.[5] This capacity may be explained due to the organic matrix acting as a protective membrane able to reduce demineralization, slowing the loss of minerals and diffusion of H+ (hydrogen ions) to the deeper dentin.[6]

In addition to mineral dissolution, the enzymatic degradation of collagen may interfere with the quality of the collagen network.[7] This reflects on the success of composite restorations, which are often first-choice materials because they are based on minimally invasive approaches.[8] The metalloproteinases (MMPs) can degrade the organic matrix of the dentin after demineralization caused by caries lesions or by the action of acids involved in restorative processes.[9] In addition, its action in the exposed collagen and the restorative union of the material and the dentin is currently recognized as an important mechanism of this interface degradation.[10]

Some substances have been used to strengthen and stabilize dentin collagen, improving the longevity of the adhesive restorations.[11] One of these substances is chitosan, a natural polysaccharide from the deacetylation of chitin, which is biocompatible, biodegradable, and nontoxic.[12] Studies evaluated the use of biopolymers, such as chitosan, to increase the number of cross-links between collagen fibers and to neutralize the MMPs.[13] However, the gradual increase in chitosan content may cause obliteration of the fibrillar spaces, negatively affecting the bonding of the restorative material to the dentin, which makes the concentration of chitosan an important factor for any subsequent application in adhesive dentistry.[13]

Thus, this study aimed to evaluate if the chitosan, applied at 2.5% after dentin acid etching or incorporated into the adhesive system, interferes with the microtensile strength of the interface of the tooth and the restorative material, in which the hypothesis is in the condition of improvement of the microtensile strength with the use of the modified adhesive system after modification.

   Subjects and Methods Top

Experimental design

Sixty healthy bovine incisors were cleaned and stored in distilled water at 4°C. The incisal thirds and the roots of the teeth were sectioned with a diamond disc (Struers A/S, Denmark), cooled with water in a metallographic cutter (Minitom, Struers A/S, Denmark). Then, the dentin surfaces were evaluated in a stereoscopic magnifying glass (magnification of ×40) (Carl Zeiss, Oberkochen, Germany), to ensure that the enamel has been completely removed. Then, the teeth were stored in distilled water at 4°C for 24 h.

Restorative procedures

The specimens were isolated with acid-resistant cosmetic nail polish (Colorama), leaving an area of approximately 5 mm × 6 mm of exposed dentin surface, and then, they were randomly divided into the following groups:

Group 1 (control) (n = 20): Clearfil SE Bond (Kuraray CO. LTDA, Umeda, Osaka, Japan) + composite resin Z350 XT, shade: A3.5 (3M Dental Products, St. Paul, MN, USA).

As suggested by the manufacturer, the two-step self-etch adhesive system was applied on the prepared surface. Then, the specimens were restored with composite resin with a resin plateau of approximately 3 mm in height, applying increments of 1 mm each and photopolymerizing for 20 s after each insertion (Photopolymerizer Ultralux, Dabi Atlante S/A Ind. Odontológicas, Ribeirão Preto, SP, Brazil), in continuous mode.

Group 2: Treatment with 2.5% chitosan + Clearfil SE Bond (Kuraray CO. LTDA, Umeda, Osaka, Japan) + composite resin Z350 XT, shade: A3.5 (3M Dental Products, St. Paul, MN, USA).

The chitosan solution was applied on the surface of the specimens with a brush for 5 s and the excess moisture was removed with absorbent paper.[14] For the production of chitosan 2.5 g (Sigma-Aldrich, Saint Louis, MO, USA) of low molecular weight chitosan (75%–85% of deacetylation) were weighed and slowly added to 100 ml of 1% acetic acid solution under magnetic agitation (Marconi Equip. Lab. Ltda, Piracicaba, SP, Brazil) for 20 min (enough time to solubilize the polysaccharide).[15] To avoid particle aggregation and to increase the pH of the solution, 1 mol/L of NaOH solution was added.[13] After the application of chitosan, the surface was prepared with the self-etching adhesive and restored with composite resin, as described in Group 1.

Group 3: Treatment with 2.5% chitosan incorporated to dentin adhesive + composite resin Z350 XT, shade: A3.5 (3M Dental Products, St. Paul, MN, USA).

The two-step self-etch adhesive containing chitosan was applied on the prepared surface. Firstly, chitosan was prepared by the emulsion and sonication method. For this, 1% (w/v) of solid lipid (illipe butter, Lipex 106, Polytechno) was heated at 60°C. Afterward, 20 mL of the aqueous phase with 0.5% (w/v) of stabilizer (Pluronic F68, Sigma-Aldrich, Germany) and cationic polymer chitosan (0.3%, w/v) of low molecular weight and with 75%–85% deacetylation (Sigma-Aldrich, São Paulo, Brazil) was added to the adhesive system. The experimental adhesive was used after the acid conditioning step, followed by the primer with 2.5% of chitosan. The specimens were restored with composite resin as described in Group 1.

For 24 h, all specimens were stored in distilled water at 37°C. Subsequently, they were cut into beams with a cross-sectional area of, approximately, 1 mm2, using a metallographic cutter (Minitom, Struers, Denmark) and diamond disc under refrigeration, 15 LC series 0.4 mm thick (Struers A/S, Copenhagen, Denmark). After this step, the three groups were divided into two subgroups (WA – with aging and NA – no aging).

For the aging process (WA), the specimens were immersed in 2.5% sodium hypochlorite solution (Neon Comercial Ltda., São Paulo, Brazil) for 12 h and then kept in distilled water for 1 h.

Microtensile test

The cross-sectional area of each beam was measured with a digital caliper (Mitutoyo, Tokyo, Japan). These beams were fixed by their extremities, by a cyanoacrylate-based glue (super bonder), and then subjected to the microtensile strength test (Ghiraldelli model). The tests were performed in a universal testing machine, with 0.5 mm/min speed, and 50 KgF load where the final values of the adhesive strength were expressed in megapascal, which was calculated based on the beams' cross-sectional areas. The pretest failures in the selected beams were included in the statistical analysis, and the strength value was considered zero.

After the microtensile test, the specimens were evaluated in a stereoscopic magnifying glass (Carl Zeiss, Oberkochen, Germany) with 40× magnification to analyze the fracture patterns by a calibrated operator. Fractures were classified as follows: adhesive fracture, cohesive fracture in dentin, cohesive fracture in resin, and mixed fracture.

Statistical analysis

Statistical analysis was performed by the statistical software SPSS for Windows, version 20.0 (SPSS Inc., Chicago, IL, USA). The data obtained were submitted to the Kolmogorov–Smirnov and Shapiro–Wilk normality tests. As the data showed normal distribution, the analysis of variance and Duncan's complementary test were used at a 5% significance level for two variation factors: aging and superficial treatment, and their interaction.

   Results Top

The mean and standard deviations of the microtensile strength are shown in [Table 1] (P = 0.045). The aging process had the lowest bonding strength values compared to groups that were not subjected to the aging process, except Group 3. Among the groups that received or did not receive the treatment with the MPP inhibitor, the group without chitosan presented higher and similar values compared to the group in which chitosan was applied before the self-etch adhesive system. However, the group where chitosan was manipulated as part of the adhesive system, showed lower and statistically significant values than the other nonaging groups, demonstrating maintenance of the resin/dentin bonding interface.
Table 1: Microtensile strength according to the group and aging (MPa)

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The fracture patterns were analyzed after the microtensile test, and the predominance of adhesive fractures (<80%) was observed in all studied groups [Figure 1].
Figure 1: Percentage of the types of fractures (Group 1 – self-etching adhesive system; Group 2 – chitosan solution and self-etching adhesive; Group 3 – chitosan added to the self-etching adhesive system. NA – no aging and WA – aging)

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   Discussion Top

In the context of adhesion of restorative materials to the dentin substrate, the inhibition potential of proteolytic activity is widely investigated, and the hypothesis was accepted in this study, in which the treatment of the dentin surface with an adhesive system modified by chitosan promoted improvements in mechanical strength over time.

The modification of the adhesive using chitosan was performed in a two-step self-etching system, as it presents good adhesion strength characteristics to the dentin and is not washable. Therefore, it promotes greater control of humidity and reduces the possibility of error in the surgical technique. In addition, this system provided more reliable and lasting bonding strength data when compared to other adhesives.[16]

The results obtained demonstrate values of microtensile strength on Group 2 (chitosan previously applied to the adhesive system) similar to the control group. However, when modifying the adhesive agent with the addition of chitosan, the microtensile strength values were statistically better. Similar results were found in other studies that used the modification of the adhesive system with the incorporation of chitosan.[17],[18] This incorporation has been studied in universal adhesive systems, also presenting higher microtensile strength values.[19]

The use of 2.5% chitosan concentration provides the formation of a calcium phosphate layer on the dentin, associated with an increase in the resistance of the dentin substrate itself against the MMP degradation.[20] However, it is likely that this concentration does not influence the wettability of the dentin substrate, which requires this biopolymer to be used to restore hydration of the collagen matrix.[14]

When checking the microtensile values after accelerated artificial aging, there was a reduction in microtensile strength in the control group, as reported by Deng et al.,[21] who demonstrated that the aging process significantly reduces microtensile strength values in the dentin-resin system. The values were also lower in the group that used chitosan as a pretreatment to the adhesive system. This fact can be explained by the hydrolytic attack of sodium hypochlorite, which promotes increased disintegration of the adhesive agent at the dentin-resin interface.[22] However, Baena et al.,[23] when verifying the use of chitosan as an additional primer, the immediate bonding strength of universal and self-etching adhesives was not significantly affected. Thus, the adhesives that used chitosan as pretreatment had higher microtensile strength values when compared to adhesive groups without pretreatment.[23]

It can be seen in the literature that, when samples are subjected to artificial aging, they decrease the bonding strength of groups of self-etching adhesives with and without the use of chitosan.[24] However, this work found that the adhesive system modified with chitosan did not influence the durability or bonding strength of the resin to the dentin with aging. It is suggested that the better dissolution of dentin constituents, especially the inorganic part, promotes better infiltration of the adhesive agent around the collagen, forming a more uniform hybrid layer that is resistant to chemical degradation by aging.[22] This result stands out as an advantage once the resistance values of the adhesive interface are maintained over time, suggesting greater durability of the resin-dentin bonding. Therefore, the incorporation of chitosan can improve the durability of dental restorations.[18] Corroborating these data, Curylofo-Zotti et al.[24] observed that 2.5% chitosan used as pretreatment to the adhesive system promoted greater resistance to microtensile after 6 months and 12 months of aging. This fact can be explained by the interaction between chitosan and collagen in demineralized dentin. This interaction results in an insoluble complex containing both polymers, thus increasing the stability of the resin-dentin interface.

The analysis of fracture patterns showed a predominance of adhesive fractures in all groups, with an increase in the number of mixed fractures in the nonaging group with pretreatment with chitosan and in the aging group with the modified adhesive system. This probably partly explains the microtensile values found in this study, as the adhesive applied after the chitosan had the highest values, demonstrating that the adhesive system provided a greater bonding strength, resulting in a more intact interface. However, the aging group in the adhesive system modified with chitosan did not provide the highest values of microtensile strength but showed an increase in mixed fractures. This demonstrates that aging did not interfere with resin adhesion to bovine dentin, and the adhesive system modified with 2.5% chitosan may be clinically promising since it also presents good responses in other properties.

   Conclusions Top

Thus, it may be concluded that the experimental adhesive system by the addition of chitosan resulted in the maintenance of the bonding strength after accelerated aging. It is likely to occur greater adhesive integrity, favoring longer restoration longevity.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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Correspondence Address:
Ms. Leticia Pena Botelho
Rua da Glória, n 187, Diamantina, MG
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcd.jcd_612_21

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