Specimen Preparation 

Three extracted unerupted human third molars and three extracted human molars with coronal caries were collected from the Oral Surgery Clinic at the University of Missouri-Kansas City (UMKC) School of Dentistry. The teeth were collected after the patient’s informed consent was obtained under a protocol approved by the UMKC adult health sciences institutional review board. Following extraction the teeth were placed in separate vials containing 0.9% normal saline and 0.002% sodium azide and stored at 4 °C. For healthy teeth, initial specimen preparation proceeded as follows: the occlusal one-third of the crown was sectioned perpendicular to the long axis of the tooth by means of a water-cooled low speed diamond saw (Buehler, Lake Bluff, IL). The exposed dentin surfaces were inspected with a microscope to ensure that no enamel remained. A uniform smear layer was created by abrading the exposed dentin surface with 600 grit SiC under water for 30 s.^{22 23}

The preparation of c-a dentin specimens followed the technique described by Nakajima etal.^{19 24} Characteristically, carious dentin is described as consisting of infected and affected layers. The affected layer is generally not removed during treatment. The carious material could be identified by visual inspection following staining with a caries detector solution. The occlusal surface was ground perpendicular to the long axis of the tooth until a flat surface composed of the carious lesion surrounded by normal dentin is exposed.

Prepared healthy dentin as well as prepared c-a dentin substrates were selected for treatment with a current commercial dentin adhesive Single Bond (3M/ESPE Dental Products, St. Paul, MN). The application of the adhesive followed the manufacturer’s instructions. The different dentin surfaces were etched for 15 s with 35% phosphoric acid. After acid etching, the teeth were rinsed with water for 10 s and blotted dry, leaving the dentin surface moist. Two consecutive coats of Single Bond were applied with a fully saturated brush. The surface was gently dried for 5 s and light cured for 20 s. These specimens were stored for a minimum of 24 h in H ₂ O at 37 °C before proceeding with the sectioning. The treated dentin surfaces were cut perpendicular to the bonded surface at 2 mm increments by means of a H ₂ O cooled low-speed diamond saw. The specimens were then cut at a depth of ∼2 mm below the interface to create a number of resin bonded beams or slabs. The final dimensions of these slabs were 10×2×2 mm.

Sectioning 

The rectangular beams were mounted on a methacrylate support with cyanoacrylate adhesive and 3-μm-thick sections were cut perpendicular to the adhesive-dentin face of the beam using a tungsten carbide knife mounted on a Leica Polycut S microtome (Leica, Deerfield IL). The knife edge and beams were moistened with distilled water while cutting. As each single section is cut, it is allowed to slide onto the knife, the microtome is stopped, and the thin section was collected on a 13 mm diameter, 2-mm-thick barium fluoride disk with the aid of a fine paintbrush. Most important for high quality image is the flatness of the sections at the end of procedure. For this reason, sections were covered by another BF ₂ disk to keep them flat. These sections were placed directly on the motorized stage for FTIR microspectroscopic and imaging analyses.

FTIR Microspectroscopic Imaging Analysis 

FTIR microspectroscopic and imaging analyses were completed using the Spectrum Spotlight FTIR imaging system (Perkin Elmer) with both single point and imaging mode. The liquid nitrogen cooled detector incorporates a narrow band mercury-cadmium-telluride (MCT) array detector and single point medium band MCT detector on a single substrate. It offers the wavelength range from 7800 down to 720 cm ⁻¹, supplying the mineral fingerprint detail missing from all other FTIR imaging systems. This system provides 25 and 6.25 μm pixel resolution, and could generate chemical image size up to 1.64 cm ². Images were scanned between 4000 and 720 cm ⁻¹ at 4 cm ⁻¹ spectral resolution, with two scans per pixel. Image size was ∼300 by ∼300 μm, using 6.25 μm pixel resolution. Approximately 2600 spectra were acquired in less than 5 min. An atmosphere correction was applied to the raw image, subtracting the contribution of atmosphere absorbance, i.e., water vapor and carbon dioxide. Images were created using Spectrum Spotlight software. In some cases, individual spectra were extracted from selected areas for more detailed analysis. The amide I band and the ν₁ν₃ PO ₄ domain between 1200 and 900 cm ⁻¹ were deconvoluted with Spectrum 3.3 software (Perkin Elmer). The deconvolution parameters were: gamma at 1.2 and no smoothing (length=0).

FTIR Spectra of Adhesive and Dentin 

The infrared spectra for Single Bond adhesive as well as healthy and carious dentin are presented in Figs. 1 and 2, respectively. The tentative assignments of the spectral features for adhesive and dentin are presented in Table 1. For Single Bond, the more intense bands occur at 1720 cm ⁻¹ (carbonyl), 1457 cm ⁻¹ (CH ₂ CH ₃) 1165 cm ⁻¹ (C–O stretch), and 832 cm ⁻¹ (C–C–O). These bands are associated with methacrylate monomers in adhesive liquid. Representative healthy and caries-affected dentin spectra are presented in Fig. 2. All spectra were recorded from 720 to 2000 cm ⁻¹, which spans the fingerprint region associated with collagen and mineral. The principal IR active bands associated with dentin have been well characterized. The most intense band at 900–1100 cm ⁻¹ range is associated with the inorganic component. Collagen features are present at 1650 cm ⁻¹ (amide I), 1550 cm ⁻¹ (amide II), 1450 cm ⁻¹ (CH), and 1240 cm ⁻¹ (amide III). The differences in spectra are clearly noticed in both the protein amide bands and ν₁ν₃ PO ₄ bands. The apparent feature is the mineral/collagen area ratio that is significantly smaller for caries-affected dentin as compared to healthy dentin.

Figure 1

Infrared spectrum of the single bond adhesive in the region 2000–720 cm ⁻¹.

Figure 2

Infrared spectra of healthy dentin (solid line) and caries-affected dentin (dotted line) in the region of 2000–720 cm ⁻¹.

Table 1

Changes in the spectral features associated with the collagen component are also shown in Figs. 2 and 3(a). Amide bands are thought to be representative of protein conformation. The amide I band is shifted from 1655 to 1638 cm ⁻¹ and amide II band also is shifted from 1553 to 1546 cm ⁻¹ (Fig. 2). In a comparison of this reported region in the deconvoluted spectra of healthy and c-a dentin [Fig. 3(a)], the relative intensity of this amide I band has changed dramatically and lost its asymmetrical structure in c-a dentin. The amide I $C==O$ stretching vibration) mode, with three bands at 1694, 1660, 1634 cm ⁻¹, is sensitive to the molecular conformation of the polypeptide chains. These spectral changes indicate that the ordered collagen structure became disorganized and denatured. The broadening and shift to low frequencies of this band could be caused by a loosening of the collagen fibril structure from water absorption.²⁵

Figure 3

Deconvoluted spectra of healthy dentin (solid line) and caries-affected dentin (dotted line). (a) In the region of 1710–1350 cm ⁻¹; (b) ν₁ν₃ PO ₄ domain between 1150 and 930 cm ⁻¹.

The ν₁ν₃ PO ₄ domain between 1150 and 930 cm ⁻¹ is explored to obtain data essentially on mineral crystallinity [Figs. 2 and 3(b)]. The ν₁ν₃ PO ₄ domain is shifted from 1040 cm ⁻¹ in the healthy dentin to 1014 cm ⁻¹ in the c-a dentin. In the c-a dentin spectrum, the greater broadness of the ν₁ν₃ PO ₄ domain band and shift to low frequencies indicate that the mineral structure and crystallinity alter dramatically (Fig. 2). The healthy and c-a dentin shows the different ν₁ν₃ PO ₄ deconvolution patterns [Fig. 3(b)]. The 1032 cm ⁻¹ is representative of stoichiometric apatites and the 1020 cm ⁻¹ of less crystalline apatites. The 1020/1030 cm ⁻¹ intensity ratio is frequently used for measuring degree of crystallinity. In the c-a dentin, the most intense band is shifted to 1011 cm ⁻¹, indicating the decreased crystallinity. The bands located between 1050 and 1100 cm ⁻¹ (1060, 1070, 1095, 1100 cm ⁻¹ due to different PO ₄ ³⁻ environments) also shows some differences between healthy and c-a dentin.

Two-Dimensional FTIR Imaging of the Dentin/ Adhesive Interface 

By combining spectroscopy with microscopy, molecular information can be obtained with great spatial resolution at the microscopic level. Specimens can be analyzed directly, in air, at room temperature and without destroying the specimen. In this study, the dentin/adhesive interface was analyzed by FTIR imaging. Figure 4 presents the visible image of the microtomed healthy dentin/adhesive interface slice. The two layers, i.e., dentin and adhesive are clearly visible, although lacking in detail. Figure 5(a) shows the total absorbance image of the above healthy dentin/adhesive interface collected from a 200×150 μm ² area. While there is no chemical information in the full absorbance image, it is a spatial representation of the energy impinging on the detectors. It is noted, however, that some additional features are observed, indicating stronger absorbance (less energy impinging the detectors) in the bottom half of the image. Inspection of Figs. 5(b)–5(d) reveals clear delineation between layers. Images of three spectral parameters (1721, 1658, and 1027 cm ⁻¹) provide information about the spatial distribution of the adhesive, collagen and mineral (apatite) components across the healthy d/a interface. Investigation of all of the images indicates that the interface is not a thin slice through the images. Its width appears to indicate an interaction between the adhesive and dentin layers, which is not obvious from observation of the visible image (Fig. 4). The interfacial width of 1027 cm ⁻¹ image appears to be bigger than that of 1721 cm ⁻¹ image. This indicates that the width of demineralized dentin layer is much deeper than that of the adhesive could penetrate, which is consistent with our previous micro-Raman results showing the gradient penetration of adhesive into the demineralized dentin layer.^{17 26}

Figure 4

Visible image of the microtomed slice of the dentin-adhesive interface. The regions associated with the adhesive and dentin are indicated on the figure.

Figure 5

Infrared spectroscopic images of the healthy dentin-adhesive interface (a) total absorbance image; (b) 1721 cm ⁻¹ image (carbonyl); (c) 1658 cm ⁻¹ image (amide I); (d) 1027 cm ⁻¹ image (phosphate).

Since these images contain thousands of very high quality spectra at a pixel resolution of 6.25 μm, the relative composition and other chemical information can be determined across the length and breadth of the d/a interface. As an example, a series of mapping spectra across the healthy dentin/adhesive interface [the position was represented by the dotted line in Fig. 5(d)] are shown in Fig. 6. The chemical characteristics of the two layers are apparent in the spectra. The three bottom spectra were acquired from pure adhesive, a methacrylate-based polymer. Vibrational bands associated with both adhesive and dentin components are noted in the fourth spectrum. The spectral intensity of the ν₁ν₃ PO4 domain increase gradually as a function of depth. The high spectral intensity of the ν₁ν₃ PO4 domain in the top spectrum indicates mineralized dentin. Spectral features associated with both adhesive (1721 cm ⁻¹) and collagen (1658 cm ⁻¹) appear in several spectra across the interface. This indicates that within the limits of resolution of this technique, the adhesive resin has penetrated into the dentin matrix, although the width of the interface layer could be overexaggerated due to the low spatial resolution of FTIR.

Figure 6

Mapping spectra across the healthy dentin-adhesive interface. These spectra are associated with the black dots noted across the dentin-adhesive interface in Fig. 5(d).

Representative microspectroscopic images across the caries-affected dentin/adhesive interface are shown in Fig. 7. Figure 7(a) shows the total absorbance image of the c-a dentin/adhesive interface collected from a 300×280 μm ² area. While there is no chemical information in the total absorbance image, it is noted, however, that a more complicated absorbance pattern is observed in this image. As shown in Figs. 7(b)–7(d), three images of spectral parameters (1721, 1658, and 1027 cm ⁻¹) were generated, representing the spatial distribution of the adhesive, collagen and mineral (apatite) components across the c-a d/a interface. It is noticed that the images of adhesive, collagen and mineral reveal very complex patterns in the interface. As compared to the healthy dentin/adhesive interface, the width of the interface is much bigger, and also the interface is not a uniform layer; it appears to intrude into either layer.

Figure 7

Infrared spectroscopic images of the caries-affected dentin-adhesive interface (a) total absorbance image; (b) 1721 cm ⁻¹ image (carbonyl); (c) 1658 cm ⁻¹ image (amide I); (d) 1027 cm ⁻¹ image (phosphate).

Due to the wide interface, two series of mapping spectra across the c-a dentin/adhesive interface are shown in Figs. 8(a) and 8(b) separately [the position was represented by the dotted lines 1 and 2 in Fig. 7(d), respectively]. As shown in Fig. 8(a), the amide I band is gradually shifted from 1656 to 1637 cm ⁻¹ across from healthy dentin to caries-affected dentin. This frequency shift indicates that the ordered collagen structure in healthy dentin changes to the disorganized and denatured structure in the c-a dentin. Similarly, the ν₁ν₃ PO ₄ domain becomes broad and is gradually shifted from 1038 to 1015 cm ⁻¹ across from the healthy dentin to the c-a dentin. The broadening of the ν₁ν₃ PO ₄ domain band and gradual shift to low frequencies indicate that the mineral structure undergoes dramatic changes when the dentin becomes caries affected. The relative intensity of the ν₁ν₃ PO ₄ domain also gradually decreased, indicating the caries-affected dentin has less mineral content as compared to the healthy dentin. A series of mapping spectra across the c-a dentin/adhesive interface are presented in Fig. 8(b). Spectral features associated with both adhesive (1721 cm ⁻¹) and collagen (1658 cm ⁻¹) are observed in several spectra across the interface. This indicates that the adhesive resin has penetrated into the demineralized dentin layer.

Figure 8

Mapping spectra across the caries-affected dentin-adhesive interface. (a) These spectra are associated with the black dots identified as 1 in the infrared spectroscopic image shown in Fig. 7(d). (b) These spectra are associated with the black dots identified as 2 in the infrared spectroscopic image shown in Fig. 7(d).

Representative microspectroscopic images collected from the adhesive region close to the healthy dentin and caries-affected dentin surface are presented in Figs. 9 and 10, respectively. Images were generated based on the band ratios of 1640 $\left(C==C\right)/1606$ (phenyl), which are used to measure the degree of double bond conversion (DC) in the light-cured adhesive resin. Red-yellow represents the highest relative intensity (lowest DC) while blue represents the lowest (highest DC). The image representing distribution of the degree of conversion in the adhesive layer on the top of the healthy dentin surface is shown in Figs. 9 and 10. Two spectra corresponding to the demarcations in the image were also shown. The DC of adhesive on the top of c-a dentin surface is lower as compared to that of the adhesive layer on healthy dentin surface. The DC of resin monomer differs substantially, and overall, the DC is lower in the region close to dentin surface. The differences in resin monomer conversion are likely due to outward water from dentin tubules as it interacts with the wet demineralized dentin matrix. Such survey maps of molecular information provide a reliable and powerful means of identifying resin monomer conversion distribution and flaws or defects in the pattern of adhesive/dentin interface.

Figure 9

Infrared spectroscopic image of the spatial distribution of double bond conversion in adhesive layer of healthy dentin-adhesive interface.

Figure 10

Infrared spectroscopic image of the spatial distribution of double bond conversion in the adhesive layer of caries affected dentin-adhesive interface.