Is the Percentage of Collagen in Coronal Dentin Related to Microtensile Strength? An In Vitro Study

ABSTRACT

Objective:  To compare the percentage of collagen in the coronary dentin of human teeth across sexes, ethnicities, and age groups, and to correlate this percentage with the microtensile bond strength (μTBS) of an adhesive system.

Material and Methods:  Fifty-one sound extracted molars were selected from patients of different sexes and ethnicities with age variation between 16 and 51 years. The crown was separated from the root. The dentin of the coronal part was restored with a hybrid composite resin block. After 24 hours of storage in distilled water at 37 °C, each specimen was sectioned perpendicular to the bonding interface area to obtain beams with a cross-sectional area of approximately 0.9 mm². The beams were submitted to μTBS test until failure. The μTBS was expressed in MPa. The root portion containing 1.0 mm of the crown was processed histologically to assess the percentage of collagen. The following statistical tests were used: Kolmogorov-Smirnov, Mann-Whitney, Student's "t" test, Kruskal-Wallis, ANOVA, and the Pearson and Spearman correlation tests.

Results:  No-white and female individuals had the highest percentage of collagen when compared to white and male individuals (p<0.0001). The age group between 44 and 51 years had the highest percentage of collagen (p=0.0013). There were no significant differences in relation to µTBS regarding ethnicity, sex, and percentage of collagen.

Conclusion:  The percentage of collagen in coronal dentin is higher in the group between 44 and 51 years, in females, and in non-white individuals, and is not related to the microtensile bond strength.

Keywords:
Dentin-Bonding Agents; Collagen; Dentin; Tensile Strength

Introduction

Dental caries is the most common disease in the world and occurs when the ecological balance in oral microflora is interrupted by biological and environmental factors [¹]. Restorative dentistry allows for the restoration of dental function and aesthetics. Thus, the effective bond between restorative materials and dental structure is fundamental for the longevity of restorative procedures. The lack of satisfactory adhesion between dentin and restorative material leads to several problems, such as fracture, marginal infiltration, secondary caries, and postoperative sensitivity [²]. As a consequence of the degradation of the adhesive interface, recurrent failures of the tooth-composite interface have been considered as the main reason for replacing composite restorations [³].

Teeth are composed of enamel, cement, and dentin [⁴]. Dentin is a mineralized connective tissue consisting of approximately (volume) 50% inorganic matter, 30% organic matter that corresponds to the dentinal matrix, and 20% water [⁵]. Approximately 10% of the dentin matrix is composed of non-collagen proteins and lipids, while 90% is composed of type I collagen [⁶]. Type I collagen is a highly organized elastic structure with high tensile strength [⁷].

Collagen concentrations have been shown to vary according to ethnicity, sex, and age. Regarding ethnicity, African-American individuals have a higher incidence of collagenous deposition in the dermis when compared to Caucasian individuals [⁸]. Regarding sex, a study has demonstrated that estrogen is involved in the remodeling of the extracellular matrix and collagen, and is associated with the highest percentage of collagen in the female sex [⁹].

The total collagen content of the human skin surface shows an annual decline of approximately 1%. In addition, with advancing age, the collagen structure of the skin becomes irregular and unorganized, differentiating it from young skin, where collagen is abundant, organized, and highly regular [¹⁰]. In human dentin, age-related changes to collagen and apatite may alter dentin properties. Consequently, if the spatial distribution of dentin tubules and intertubular dentin affects mechanical properties, the compounding effect of age requires further investigation. Investigating in situ changes to dentin in human subjects poses ethical limitations; hence, most studies are laboratory-based or in vitro, and are conducted using donated teeth that have been extracted as part of dental management [¹¹].

Even due to the high mineral composition, the teeth are subject to demineralization associated with the decline in pH on the surface of the dental structure, which can cause lesions in the enamel and dentin [¹²]. The damaged areas can be replaced by restorative materials, which reestablish the function and aesthetics of the tooth. Thus, restorative procedures depend on the bond between materials and dental substrates [¹³].

As the dentinal tubules have spaces that contain fluids, which can prevent adequate bonding between dentin and restorative materials, the hybrid layer is the primary micromechanical bonding element. The hybrid layer is formed by the effective interrelation between the collagen fibrils and the adhesive, which penetrates the dentin and fills the interfibrillary microspaces [¹⁴]. The fact that dentin is a highly sensitive structure, and due to the presence of moisture, the bonding of restorative materials to dentin becomes highly challenging [¹⁵]. This is due to several intrinsic characteristics, including its organic composition, inhomogeneity, the humidity present in the dentinal tubules, and the presence of a smear layer [³,¹⁶]. Thus, adhesion of restorative materials to dentin becomes more difficult than adhesion to enamel [¹⁶].

It has already been demonstrated that the degradation of dentin collagen networks is closely related to flaws found in the dentin-adhesive interface [¹⁴]. Therefore, knowing the importance of collagen fibers for the adhesiveness of direct restorations with composite resin and that there are differences in the amount of collagen in relation to ethnicity and sex in other organs, the present study was justified. Furthermore, studies comparing the percentage of collagen in coronary dentin between ethnicities, sexes, and age groups, as well as those correlating the percentage of dentin collagen with the bond strength between the dentin substrate and composite resin, were not found.

As in other organs, differences in the percentage of collagen have already been observed between ethnicities, sexes, and ages. We believe that the null hypothesis is rejected and that female and non-white individuals have a higher percentage of collagen compared to male and white individuals. Thus, it is likely that there is a positive and significant correlation between the rate of dentin collagen formation and bond strength.

Thus, the present study aimed to compare the percentage of collagen in the coronary dentin of newly extracted human teeth between sexes, ethnicities, and age groups and to correlate the rate of collagen with the bond strength of an adhesive system.

Material and Methods

Study Design and Ethical Clearance

This in vitro experimental study was approved by the Human Research Ethics Committee (CAAE – 14423519.6.0000.5145).

Selection of Participants

Fifty-one extracted sound human third molars were obtained from 51 individuals between 16 and 51 years old at the Dental Clinic of the University of Uberaba from September 2019 to July 2020. These teeth were indicated for extraction due to orthodontic reasons. Prior to clinical procedures, data related to age, sex, ethnicity, and information on parafunctional habits or systemic diseases associated with collagen synthesis were recorded.

All eligible participants were informed of the study's nature and the potential risks and benefits of their participation by signing an informed consent form. When the patient was under 18 years of age, their legal guardian signed this consent form on their behalf.

The inclusion criteria were: sound third molars without restorations or wear. Teeth should be healthy because cavities and restorations can induce tertiary dentin formation that could alter the amount of dentin collagen. The exclusion criteria were: extracted teeth that presented any abnormality in size, lack of uniformity in enamel or dentin, or that had fractures, wear, a history of parafunctional habits, abfraction, cavities, or restorations. Teeth from individuals with systemic diseases associated with collagen synthesis, such as rheumatoid arthritis, progressive systemic sclerosis, systemic lupus erythematosus, dermatopolymyositis, mixed connective tissue disease, and Sjögren's syndrome, were excluded.

The 51 extracted teeth were divided into groups according to ethnicity, sex, and age of the individuals: White (W) (n=27); Non-White (NW) (n=24); Male (M) (n=20); Female (F) (n=31). The individuals were also divided into subgroups: Male White (MW) (n=14); Non-White Male (MNW) (n=6); Female White (FW) (n=13); Non-White Female (FNW) (n=18). According to age, individuals were grouped in the following intervals, in years: 16-22, 23-29, 37-43, 44-51.

After tooth extraction, remnants of cementum and the periodontal ligament were removed using curettes and running water. Then, a 1.0 mm cross-section was made above the cementum junction with a diamond disk (0.125 mm in diameter – Buehler Ltd., Lake Buff, IL, USA) mounted on a cutting machine (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA) to obtain two fragments: one fragment containing the coronary portion and another fragment containing the roots and 1.0 mm of the coronary portion.

Subsequently, the coronal portions of the teeth were kept in distilled water at 37 ºC. The root portions of the teeth, containing 1.0 mm of the coronal structure, were fixed in 10% buffered formaldehyde for later histological processing and analysis of the percentage of collagen.

Dental Crowns Demineralization and Histological Processing

After fixation in formaldehyde, the root portions containing 1.0 mm of the coronary structure were individually stored in sterile flasks containing 6% formic acid solution (60 mL of 6% formic acid + 940 mL of 10% buffered formaldehyde) for demineralization. The 6% formic acid solution was changed once a week for 15 weeks. Subsequently, this solution was changed every day for 60 days. After the complete demineralization of the dental structure, the crown segments destined for histological processing were dehydrated in alcohol, diaphanized in xylol, and included in paraffin. Then they were cut in a microtome (MRP 03 – Lupetec Tecnologia Aplicada, São Paulo, SP, Brazil) and sections 6 µm thick were obtained. These histological sections were placed on glass slides and stained with picrosirius red to analyze the percentage of collagen.

For staining with picrosirius red, the histological sections were washed in running water for five minutes. Then they were dewaxed with xylol, dehydrated in increasing concentrations of absolute alcohol, and then washed in running water for hydration. Subsequently, the slides containing the fragments were placed in the picrosirius red solution for five minutes and in the fuchsin G solution, also for five minutes. Finally, the cuts were quickly counterstained with Harris' hematoxylin and dipped in water to remove excess dyes. The slides were then assembled with Entelan.

Quantification of the Percentage of Collagen

The collagen morphometry was performed semi-automatically using a standard light microscope, Axio 4.1 (Carl Zeiss Microscopy GmbH, Berlin, Germany), coupled to an Axiocam image capture camera (Carl Zeiss Microscopy GmbH, Berlin, Germany) and a computer with the Axiovision 4.8 software installed (Carl Zeiss Microscopy GmbH, Berlin, Germany). The images viewed under the microscope were transmitted to the computer monitor. For this analysis, a 40X objective and a polarizing filter were used. In the polarized image, collagen exhibited birefringence with green, yellow, or red coloring, which was quantified semi-automatically [¹⁷].

To determine the total number of fields to be evaluated per case, the slide containing the largest fragment was selected. In this slide, all fields were evaluated, totaling 206 fields. Then, the accumulated average test was used to determine the number of fields to be evaluated in each case. With the aid of this test, it was possible to observe that the accumulated average stabilized with the evaluation of 100 fields. Thus, in each case, 100 histological fields were analyzed in the 40X objective.

Adhesion Procedures

In the fragments of the coronary portion, the ClearFil SE Bond adhesive system (Kuraray Noritake Dental Inc., Sakazu, Kurashiki, Okayama, Japan) was applied to the coronary dentin. The adhesive system consists of two bottles: one containing the acidic primer and another containing the adhesive, which must be used in conjunction with the self-etching technique. After drying the tooth surface, the primer was applied actively for 20 seconds, volatilizing slightly with a jet of air. The bond was then applied, the excess removed, and the material light-cured for 10 seconds, according to the manufacturer's instructions. Two layers of hybrid composite resin (Tetric N-Ceram, Empress Direct, Ivoclar Vivadent, Jagst) Germany), each with a thickness of 2 mm, were placed on the adhesive dentin surface of each tooth. Each layer of composite resin was photoactivated for 30 seconds using a light source unit (Bluephse, Ivoclar-Vivadente, Schaan, Liechtenstein), in low mode with an output density of 600 mW/cm². The static test condition was used. The static test was performed by storing the teeth in distilled water at 37 ºC for 24 hours.

Test of Bond Strength to Microtensile (µTBS)

Beams with an approximate transverse dimension of 0.9 mm x 0.9 mm were obtained from the most central area of each restored tooth using a water-cooled diamond blade in a low-speed saw machine (Isomet 1000, Buehler Ltd, Lake Bluff, IL, USA). All beams obtained per tooth were used for the µTBS test.

Each beam was attached to Geraldeli's device, a microtensile device, using cyanoacrylate glue [¹⁸]. This device was then coupled to a universal testing machine (EMIC DL 3000, São José dos Pinhais, PR, Brazil) equipped with a 10 kgf load cell, with a cross speed of 0.5 mm/min until the failure occurs. All beams were measured for thickness and width in the region of the adhesive interface before and after the test.

All analyses were performed by a single examiner in a blinded manner.

Statistical Analysis

Statistical analysis was performed using the GraphPad Prism 7 software (GraphPad, San Diego, California, USA). Statistical analysis for bond strength was performed using the tooth as an experimental unit. The average µTBS obtained from the sticks of each tooth was used to represent the bond strength of each tooth. For the statistical analysis of the percentage of collagen, all values of the 100 fields analyzed in each case were used.

The Kolmogorov-Smirnov test was used to assess normality. To compare variables with non-normal distribution between two groups, the Mann-Whitney test was performed, and for variables with normal distribution, Student's "t" test was performed, with the results expressed as mean and standard deviation. To compare variables with non-normal distributions between three or more groups, the Kruskal-Wallis test was used. For variables with normal distributions, the ANOVA test was employed. The correlation between the percentage of collagen and bond strength was performed using Pearson's correlation test. The correlation between the percentage of collagen and age was performed using the Spearman correlation test. The level of significance assumed was 5% (α=0.05).

Results

When assessing ethnicity, the percentage of collagen was significantly higher in non-white individuals (NW) when compared to white individuals (W) (p<0.0001) (Figure 1). When assessing sex, the percentage of collagen was significantly higher in females (F) compared to males (M) (p<0.0001) (Figure 2).

Figure 1
Percentage of collagen in groups of individuals according to ethnicity: Non-Whites (NW) and Whites (W). Normality test: Kolmogorov-Smirnov; Mann-Whitney test; p<0.0001. Results expressed in median (maximum – minimum) *indicates statistical difference.

Figure 2
Percentage of collagen in groups of individuals according to sex: Female (F) and Male (M). Normality test: Kolmogorov-Smirnov; Mann-Whitney test; p<0.0001. Results expressed in median (maximum – minimum) *indicates statistical difference.

When comparing all the MW, MNW, FW, and FNW subgroups, we observed a significantly higher percentage of collagen in the MNW, FW, and FNW subgroups when compared to the MW subgroup. There was a significant difference in the percentage of collagen being greater: in the MNW subgroup when compared to the FW subgroup; in the FNW subgroup when compared to the MNW subgroup, and in the FNW subgroup when compared to the FW subgroup (p<0.0001) (Figures 3 and 4).

Figure 3
Percentage of collagen between subgroups: Male White (MW); Non-White Male (MNW); Female White (FW); Non-White Female (FNW). Normality test: Kolmogorov-Smirnov; Kruskal-Wallis test; p<0.0001; Dunn’s multiple comparisons test. Results expressed in median (maximum – minimum) *indicates statistical difference.

Figure 4
Coronary dentin collagen in the subgroups according to sex: Female (F) and Male (M), and according to ethnicity: White (W) and Non-White (NW). A) Male White (MW), ordinary light microscopy; B) Male White (MW), polarized light; C) Non-White Male (MNW), ordinary light microscopy; D) Male Non-White (MNW), polarized light; E) Female White (FW), ordinary light microscopy; F) Female White (FW), polarized light; G) Non-White Female (FNW), ordinary light microscopy; H) Non-White Female (FNW), polarized light. Picrosirius red coloring, 40X.

Individuals aged 44 and 51 years presented a significantly higher percentage of collagen when compared to individuals aged 16 to 22 and 23 to 29 years (p=0.0013) (Figures 5 and 6). There was no significant difference in bond strength between the age groups (p=0.8596). There was a positive and non-significant correlation: a) between the percentage of collagen and age (p=0.9856) (Figure 7); b) between the percentage of collagen and the bond strength (p=0.2635) (Figure 8).

Figure 5
Percentage of collagen according to age groups: 16-22 years old, 23-29 years old, 37-43 years old, 44-51 years old. Normality test: Kolmogorov-Smirnov; Kruskal-Wallis test; p = 0.0013; Dunn’s multiple comparisons test. Results expressed in median (maximum – minimum) *indicates statistical difference.

Figure 6
Coronary dentin collagen in groups, according to age group. A) 16-22 years old; B) 23-29 years old; C) 37-43 years old; D) 44-51 years old. Picrosirius red coloring, polarized image, 40X.

Figure 7
Correlation between the mean of the collagen percentages and the ages of the 51 molars. Normality test: Kolmogorov-Smirnov; Spearman's correlation; (S= 0.002594); p=0.9856.

Figure 8
Correlation between the percentage of collagen and the maximum tension of the 51 molars. Normality test: Kolmogorov-Smirnov; Pearson's correlation (r= -0.1595); p=0.2635.

There was no significant difference in bond strength between ethnic groups (p=0.9338), sexes (p=0.9050), and among the subgroups (p=0.9093).

Discussion

In the present study, the null hypothesis was rejected because a significant difference in the percentage of collagen was observed among the sexes, ethnicities, and age groups. The null hypothesis for the bond strength was accepted, since there was no significant difference between the groups.

It is known that an inadequate diet [¹⁹] and unhealthy habits, such as smoking [²⁰], along with biological characteristics like age and sex, can impact the formation and degradation of collagen in various organs [¹⁹,²¹]. In the present study, a higher percentage of collagen was found in the coronary dentin of female individuals when compared to male individuals. Healthy premenopausal women accumulate significantly more collagen compared to men, suggesting greater collagen formation in females, likely due to variations in the inflammatory response associated with estrogen [¹⁹]. Thus, we suggest that the lower percentage of collagen found in the dentin of male individuals is associated with a lower amount of estrogen in men.

It has been shown that collagen in the skin declines with aging, with the crosslinking of collagen in human tendons also seeming to decrease with each decade of age. Likewise, postmenopausal women deposit significantly less collagen than premenopausal women [¹⁹]. In human dentin, age-related changes to collagen and apatite may alter dentin properties. Consequently, if the spatial distribution of dentin tubules and intertubular dentin affects mechanical properties, the compounding effect of age requires further investigation [¹¹]. Thus, increasing age can alter the balance between collagen formation and degradation, which leads to a reduction in the percentage of collagen with aging.

Additionally, another study demonstrated that during aging, type I collagen undergoes non-enzymatic post-translational changes, such as glycation [²²]. This glycation induces the generation of advanced glycated products (AGEs), which contribute to an increase in collagen fiber crosslinks with a consequent decrease in the diameter and length of collagen fibers [²²], which could also lead to a lower percentage of collagen with aging.

However, in the present study, we found a higher percentage of collagen in dentin with aging, perhaps due to the particularities of each tooth, since odontoblasts have the capacity to form dentin throughout life. Furthermore, it is known that with odontoblastic destruction after aggression or trauma, the mesenchymal cells of the pulp differentiate into odontoblasts and start depositing dentin [⁴], which would lead to an increase in dentin collagen with aging, as demonstrated in the present study.

In addition, with increasing age, sclerotic dentin is formed, which is characterized by the occlusion of dentinal tubules with mineralized material. It is believed that sclerotic dentin formation is a physiological response, and obliteration is achieved through the continuous deposition of peritubular dentin [²³]. Thus, as in the present study, a higher percentage of collagen was found with aging. We believe that this increase in collagen in dentin is associated with the physiological response of the dental organ resulting from the masticatory process over time.

African-American individuals have been shown to have greater collagenous deposition in the dermis and a more organized collagenous architecture compared to Caucasian individuals [⁸], which corroborates our results, as we observed a higher percentage of collagen in the coronary dentin of non-white individuals compared to white individuals. However, as our study was the first to compare the rate of dentin collagen associated with ethnicity, further studies are needed to clarify better the pathogenesis of ethnic differences in the percentage of dentin collagen.

When comparing all subgroups, the Male White subgroup had a significantly lower percentage of collagen compared to the other groups. The percentage of collagen was considerably higher in the subgroups: Male Non-White when compared to the Female White subgroup; in the Non-White Female when compared to the Non-White Male subgroup, and in the Non-White Female when compared to the White Female subgroup. Thus, we suggest that factors related to ethnicity and sex also prevailed in the subgroups.

Regarding bond strength, it is known that the adhesion of the restorative material to dentin is a challenge, since dentin is a tissue composed of approximately (volume) 50% inorganic matter, 30% organic matter (collagen), and 20% water [⁵]. In addition, most clinical substrates are covered by smear layers, which act as barriers against the penetration of adhesive molecules into dental substrates. Clinically, the removal or modification of the smear layer is crucial to create a satisfactory hybrid layer to ensure stable or high bond strength between resin and dentin [²⁴]. It is known that the maintenance of the smear layer interferes with the adhesion of some dental materials to dentin, at the same time that it can serve as a deposit of bacteria or their products, promoting the reinstatement of caries and pulp inflammation [²⁵]. Therefore, dentin adhesion depends on the formation of a hybrid layer, which is a structure composed of demineralized collagen fibrils reinforced by the resin matrix [²⁶].

The present study used a self-etching adhesive system, Clearfil SE Bond (Kuraray), which is considered the gold standard of adhesive systems [²⁷,²⁸,²⁹]. The basic composition of self-etching primers and self-etching adhesive systems is an aqueous solution of acid-functional monomers, with a relatively higher pH than that of phosphoric acid-based conditioning agents [³⁰]. The role of water is to provide the medium for the ionization and action of these acidic resin monomers. Self-etching adhesive systems also contain HEMA monomer (2-hydroxyethyl methacrylate) to increase the wettability of the dentin surface, as most acid monomers are poorly soluble in water. Bi or multifunctional monomers are added to provide resistance to crosslinking formed from the monomeric matrix [³¹].

Like self-etching adhesive systems, which do not require a separate acid conditioning and moisture control step, they are considered simplified adhesive materials. These adhesive systems offer several advantages over conventional conditioning and rinsing systems, including reduced postoperative sensitivity and a less sensitive technique. Another benefit of self-etching adhesive systems is that the infiltration of adhesive resin tends to co-occur with the self-etching process [³⁰].

The adhesion mechanism of self-etching adhesive systems has been intensively investigated, and two basic mechanisms have been described: micro-mechanical retention and chemical adhesion. The micromechanical bond contributes to providing resistance to mechanical stress, while the chemical interaction reduces hydrolytic degradation, maintaining the marginal sealing of restorations for longer [³²].

The present study showed that the percentage of collagen did not influence the results of adhesive strength in the different groups. Although no studies have been found on the relationship between the percentage of collagen and the adhesive strength of self-etching adhesive systems, the results of this study suggest that the quality of the hybrid layer would be more important than the amount of collagen available for hybridization. It has been documented that the quality of the hybrid layer is of paramount importance for the success and longevity of the restoration and that its degradation may occur due to the disorganization and solubilization of collagen fibrils, as well as by the hydrolysis and leaching of the adhesive in the interfibrillar spaces [³²].

However, the present study has limitations because, despite the use of controlled techniques, in vitro studies cannot fully reproduce the complex biological interactions that occur in the real oral environment. The absence of factors such as saliva, temperature variations, mechanical pressure, and humidity may reduce the applicability of the results to the clinical context.

Conclusion

The percentage of collagen in coronary dentin is significantly associated with sex, ethnicity, and age of individuals. Furthermore, the bond strength of the restorative material does not appear to depend on the percentage of collagen fibers in dentin. As this was the first study to associate the percentage of collagen with sex, age groups, ethnic groups, and bond strength, further studies are needed to understand better the pathogenesis of dentin collagen deposition in different groups.

Acknowledgements

We thank the Coordinator of the Dentistry Course at prof. Dr. Luis Henrique Borges, to students, professors, and other employees at Policlínica Getúlio Vargas, and also the technicians Marcelo Silveira Herméto and Juliano Carvalho da Silva for their collaborations.

• Financial Support
  This work was supported by the National Council for Scientific and Technological Development/CNPq (PQ-2018/Process number: 302867/2018-0); (PIBIC-UNIUBE 2020/002), CEFORES/UFTM, Minas Gerais Research Support Foundation/FAPEMIG, and the Coordination for the Improvement of Higher Education Personnel (CAPES).

Data Availability

The data used to support the findings of this study can be made available upon request to the corresponding author.

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Edited by

• Academic Editor:
  Alessandro Leite Cavalcanti

Publication Dates

• Publication in this collection
  28 Nov 2025
• Date of issue
  2026

History

• Received
  15 Dec 2024
• Reviewed
  20 Mar 2025
• Accepted
  25 Mar 2025