FT-Raman spectroscopy was used to study the human and bovine dentin and their interactions with adhesive systems. Ten human (H) molars and ten bovine (B) teeth were prepared exposing the dentin and then each specimen was divided into two parts. The resulted forty dentin segments were treated either with the total-etch one bottle adhesive (Prime & Bond 2.1, PB) or with the single-step self-etching adhesive (Xeno III, X) and divided into four groups: HPB (control), HX, BPB, and BX. Each group was analyzed by FT-Raman spectroscopy before and after the adhesive treatment. Six regions of the Raman spectrum were analyzed and the integrated areas of organic and inorganic peaks were calculated. Bovine untreated specimens showed higher peak area of
The characteristics of dental hard tissues are the main factor to be observed when analyzing the possibilities of the replacement of human teeth by animal teeth for
It has been reported that the adhesion to the superficial layer of dentin showed no significant differences between human and bovine dentin, and the dentin bond strength decreased with the depth of dentin because of the lower density of dentinal tubules in the bovine dentin [
Considering these factors, investigations on the chemical composition for hybridization of adhesive systems with bovine dentin are important to validate the studies to the human dentin. Contemporary adhesive systems interact with the dentin using two different approaches: either by completely removing the smear layer (etch-and-rinse technique) or by modifying it (self-etch technique) [
In selecting an adhesive system for the clinical use, it is very important to evaluate its bond strength and sealing ability [
Microscopy characterization can demonstrate the morphological relationship between the dentin and the adhesive layer, whereas Raman spectroscopy can elucidate the molecular interactions between the dentin and the adhesive monomers [
One of the distinct advantages of the Raman technique from other analytical techniques is the ability to record the molecular information of both collagen and mineral component of teeth without damaging the sample [
Based on previous studies on the application of adhesives using human or bovine dentin as a substrate, a more detailed characterization of the adhesive interaction with either human or bovine dentin components needs to be performed. Such characterization is important since detailed information on dentin structure is essential to understand the data from the investigations on dentin-adhesive materials [
Therefore, the purpose of this study was to investigate by FT-Raman spectroscopy whether there are differences in the inorganic and organic composition of human and bovine dentin before and after total-etch-and self-adhesive systems application.
Ethical approval of the study was granted by the Ethics Committee of the University of Vale do Paraíba (L087/2005/CEP). Ten extracted erupted noncarious human third molars and ten bovine teeth were used in this study. The human teeth were obtained from patients whose extractions were part of the dental treatment, and the bovine incisors teeth were obtained from bovine jaws. All specimens were stored in saline solution (Aster Produtos Médicos LTDA, Sorocaba, SP, Brazil) at 9°C until use. After the extraction, the remaining soft tissue was removed from the tooth surface with a dental scaler (7/8; Duflex, Rio de Janeiro, RJ, Brazil). The teeth were polished with a paste of pumice (S.S. White, Rio de Janeiro, RJ, Brazil) and filtered water using a Robinson brush (Viking-KG Sorensen, Barureri, SP, Brazil) in a low speed handpiece (KaVo do Brasil SA, Joinvile, SC, Brazil). After the cleaning procedure, the teeth were stored in 0.1% thymol aqueous solution at 9°C for one week long. To prepare the dentin specimens the teeth were washed for 24 h with filtered water to eliminate thymol residues [
The occlusal one-third of the human teeth crowns were sectioned perpendicularly to the long axis using a water-cooled low-speed diamond disc at 250 rpm with a 100 g load (Isomet 1000-BUEHLER, Lake Bluff, IL, USA). The dentin surface was grinded on wet 600-grit silicon carbide paper (Norton, São Paulo, SP, Brazil) at 150 rpm (Knuth Rotor-Struers, Brazil) for 1 min, under constant water cooling to produce a standardized smear layer [
Similarly, ten bovine teeth were cleaned after the extraction and stored in 0.1% thymol aqueous solution like the human teeth. The buccal enamel surface was removed using a water-cooled low-speed diamond disc at 250 rpm with a 100 g load and grinded on wet 600-grit silicon carbide paper at 150 rpm to expose the dentin layer. The dentin surface was polished for 1 min, under constant water cooling to produce a standard smear layer. The specimens were sectioned parallel to the long axis and the pulps were removed resulting into two parts. Ultrasonic cleaning (Maxiclean 1450, Merse, Campinas, SP, Brazil) with distillated water was performed for human and bovine teeth for 5 min in order to remove the excess of debris. The specimens were then stored in saline solution in a refrigerator at 9°C for one week.
The total of 20 human (H) dentin samples (~0.4 × 0.5 × 0.4 mm) and 20 bovine (B) teeth samples (~0.5 × 0.5 × 0.3 mm) were treated with Prime & Bond 2.1, PB (DENTSPLY De Trey GmbH, Konstanz, Germany), a total-etch one bottle adhesive or with Xeno III, X (DENTSPLY De Trey GmbH, Konstanz, Germany), a one-step self-etching adhesive, according to the experimental groups division: HPB (control), HX, BPB, and BX.
The chemical formulations of the two adhesives are listed in Table
Composition of the adhesives systems tested.
Adhesive | Composition |
---|---|
Xeno III | Liquid A: |
| |
Prime & Bond 2.1 | Etching: |
FT-Raman spectroscopy analyzed the top surface of dentin slabs before and after the treatments. One spectrum for each specimen was collected. The FT-Raman spectrometer (RFS 100/S-Bruker Inc., Karlsruhe, Germany) with a Ge diode detector cooled by liquid N2 was used to collect the data. The samples were excited by an air-cooled Nd : YAG laser (
For the qualitative and semiquantitative spectral analysis, the spectra were baseline corrected and then normalized to the 960 cm−1 peak [
The statistical analysis of the Raman results was performed by the one-way ANOVA at a 95% level of confidence. The Dunn’s Multiple Comparisons post hoc test was performed using the Instat software (GraphPad Software, San Diego, CA, USA) to assess the significance of the relative area evaluation of the treated dentin data. The Mann-Whitney test was also performed to study the significance between the average of twenty untreated bovine and the average of 20 untreated human spectra integrated areas.
The typical Raman spectra for the untreated human and bovine dentin in the regions of 300–1200 cm−1 and 1200–1800 cm−1 are shown in Figures
Comparison of FT-Raman spectra of mineral components from normal bovine dentin (BN) and normal human dentin (HN); human dentin treated with Xeno III (GI) and Prime & Bond 2.1 (GII) adhesives; bovine dentin treated with Xeno III (GIII) and Prime & Bond 2.1 adhesives (GIV).
Comparison of FT-Raman spectra of organic components from normal bovine dentin (BN) and normal human dentin (HN); human dentin treated with Xeno III (GI) and Prime & Bond 2.1 (GII) adhesives; bovine dentin treated with Xeno III (GIII) and Prime & Bond 2.1 adhesives (GIV).
Figure
FT-Raman spectra of Prime & Bond 2.1 (a) and Xeno III adhesives (b); dentin treated with Prime & Bond 2.1 (c) and Xeno III (d) adhesives.
Based on the calculated integrated areas of the Raman peaks, the results of the Mann-Whitney test showed statistical significant difference between the average of untreated human (
Mean values (standard deviation) of integrated areas obtained from Raman spectra: inorganic peaks (p1–p3) and organic peaks (p4–p6) of normal (N) and treated (T) dentin of experimental groups (
Groups |
p1 |
p2 |
p3 |
p4 |
p5 |
p6 |
---|---|---|---|---|---|---|
431 cm−1 | 590 cm−1 | 1071 cm−1 | 1245 cm−1 | 1452 cm−1 | 1665 cm−1 | |
HN | 3.49 (0.25)* | 2.99 (0.08)* | 3.83 (0.18)* | 2.04 (0.19)* | 2.70 (0.17)* | 2.56 (0.22)* |
BN | 3.83 (0.10)* | 2.78 (0.21)* | 3.46 (0.10)* | 1.60 (0.30)* | 2.06 (0.37)* | 2.02 (0.32)* |
HPB-N | 3.51 (0.13) | 2.80 (0.14) | 3.87 (0.16) | 2.04 (0.19) | 2.67 (0.21) | 2.59 (0.26) |
HPB-T | 3.53 (0.14) | 2.81 (0.09) | 3.76 (0.16) | 2.14 (0.19) | 2.85 (0.25) | 2.64 (0.19) |
HX-N | 3.47 (0.33) | 2.77 (0.26) | 3.79 (0.19) | 2.05 (0.21) | 2.74 (0.12) | 2.52 (0.17) |
HX-T | 3.35 (0.13) | 2.70 (0.10) | 3.74 (0.12) | 2.03 (0.25) | 2.77 (0.34) | 2.58 (0.22) |
BPB-N | 3.85 (0.10) | 2.98 (0.07) | 3.48 (0.10) | 1.66 (0.30) | 2.11 (0.41) | 2.08 (0.36) |
BPB-T | 3.74 (0.05) | 2.88 (0.07) | 3.41 (0.12) | 1.73 (0.24) | 2.25 (0.36) | 2.14 (0.37) |
BX-N | 3.81 (0.10) | 2.99 (0.08) | 3.45 (0.11) | 1.53 (0.29) | 2.00 (0.34) | 1.95 (0.27) |
BX-T | 3.64 (0.12) | 2.92 (0.12) | 3.41 (0.14) | 1.64 (0.27) | 2.09 (0.32) | 2.06 (0.28) |
Comparing the normal and treated spectra, it was observed that the adhesive treatment maintained or changed without statistical significance (
Statistical comparisons of the integrated areas of the Raman peaks between the adhesives and considering the same substrate (HPB versus HX; BPB versus BX) of treated dentin showed no significant statistical differences between the adhesives for both inorganic and organic contents (
Statistical results of the Dunn's Multiple Comparisons post-hoc test evaluation of integrated areas of the Raman peaks of the groups treated with adhesives (significant comparisons are in bold).
Group comparison | p1 | p2 | p3 | p4 | p5 | p6 |
---|---|---|---|---|---|---|
|
|
|
Amide III | CH2 | Amide I | |
431 cm−1 | 590 cm−1 | 1071 cm−1 | 1245 cm−1 | 1452 cm−1 | 1665 cm−1 | |
HX versus HPB |
ns
|
ns
|
ns
|
ns
|
ns
|
ns
|
BX versus BPB |
ns
|
ns
|
ns
|
ns
|
ns
|
ns
|
HX versus BX |
*
|
ns
|
**
|
ns
|
*
|
*
|
HPB versus BPB |
ns
|
ns
|
**
|
*
|
*
|
*
|
Comparisons of treated dentin between the two substrates and considering the same adhesive (HPB versus BPB; HX versus BX) showed statistical significant differences of the integrated areas of the Raman peaks, except for the peak at 590 cm−1 (
The integrated area of the p1 peak related to the
Despite previous histochemical, anatomical, morphological, and mechanical comparative studies considering human and bovine teeth [
FT-Raman analysis revealed that untreated human and bovine dentin had significant differences in the inorganic and organic contents in superficial dentin (Table
When analyzing the results of the Mann-Whitney test, to verify chemical differences between untreated human and bovine dentin, our study showed higher peak areas related to the inorganic (p2, p3) and organic contents (p4–p6) in human specimens than in bovine specimens (Table
The elevated organic content found in human untreated dentin could be another factor which influences the adhesion process since the exposed and stabilized collagen layer of dentin is a key factor to an adequate adhesion. Micromechanical retention is considered the most important mechanism for bonding resin to dentin. Such retention can occur when resin completely infiltrates dentin surfaces and creates a resin-reinforced dentin layer [
The statistical data presented in Table
The integrated area from the Raman peaks of human and bovine dentin after adhesives application, shows statistical significant differences between those two specimens (Tables
Regarding the analysis of adhesive interaction with dentin, some authors have found, by FT-Raman spectroscopy, piesces of evidence suggesting that ionic bonds and hydrogen bonding may form through the interaction of the ester function of adhesive ligand with the amide groups of collagen or through the formation of hydrogen bonds between the ligand and the collagen receptor [
In summary, we presented direct information regarding the differences in the chemical composition of the human and bovine dentin interaction with one single-step self-etching adhesive and one total-etch one bottle adhesive by using FT-Raman spectroscopy. The major contribution of this study was the chemical characterization of the differences between the mineral and organic components of untreated and adhesive-treated human and bovine dentin as substrates. Untreated bovine dentin showed lower organic content than the human dentin and this is a possible limitation for
The authors thank Walter Andrés Avendaño Jara for the MatLab analysis. This work was supported by FAPESP (01/14384-8) and CVPq (Grant nos. 302393/2003-0 and 302761/2009-8). The authors gratefully acknowledge the DENTSPLY De Trey GmbH Company, for donating the Xeno III adhesive and the phosphoric acid gel used in this study.