Abstract
Objectives
We had previously discovered that the flexural and tensile strengths of human dentin were 2–2.4 times greater after being heated to 140 °C, and deduced that the generation of higher-density structures and therefore dehydration probably promoted the increased strength. Our test hypotheses were that intertubular dentin, which constitutes a major part of organic components, was selectively affected by heating, and such changes could happen without critical damages to the basic structure of dentin type I collagen.
Methods
Micro-mechanical changes of human dentin by heating at 140 °C were investigated by nano-indentation. Chemical changes in dentin collagen after heating were also investigated by X-ray diffraction study, a microscopic Fourier transform infrared (micro-FTIR) and a laser Raman spectroscopic analyses, and a cross-linking analysis by high-performance liquid chromatography.
Results
The results of nano-indentation showed that the micro-hardness of intertubular dentin increased after heating at 140 °C to 1.8 times more than unheated dentin; on the other hand, peritubular dentin was unchanged. Results of X-ray diffraction showed that the lateral packing of collagen molecules shrank from 13.6 ± 0.3 to 10.6 ± 0.1 Å after heating, but the shrinkage reversed to the original after rehydration for seven days. After heating, no substantial chemical changes in the collagen molecules were detected in tests by micro-FTIR or Raman analyses, or by cross-linking analysis.
Significance
These results suggest that intertubular dentin, which contains most of the type I collagen, was selectively affected by heating at 140 °C without critical damage to its collagen.
1
Introduction
In a previous significant clinical study , fracture was found to be the most frequent reason for tooth loss in persons involved in a long-term plaque control program. In this study, 62% of extractions were undertaken because of tooth fracture, compared with only 7% and 5% because of caries or periodontal disease, respectively. This research suggested that tooth fracture is a critical issue. Another etiological study on 227 vertical root fractures showed that 88% fracture happened in pulpless teeth . Therefore, effective methods to strengthen pulpless teeth could significantly help in preventing tooth fracture and thus be of potentially great assistance to clinicians in their front-line battle to promote oral general health.
In our previous research , we had discovered that the flexural and microtensile strengths of human dentin were 2–2.4 times greater after heating to either 110 °C or 140 °C. X-ray diffraction analyses indicated that dehydration probably generated higher-density structures and caused the shrinkage of the lateral packing of the collagen triple-helices from 14 Å to 11 Å. This was the probable cause of the greater strength of dentin after heating. As the next step in this series of studies, we investigated the effects of heating on the mechanical properties of human dentin by focusing on what happened when dentin was dehydrated and rehydrated . Several studies had reported that changes in the biomechanical properties of dentin after dehydration could be reversed by rehydration . We did discover that rehydration reverses the strength of heated dentin. Given how dentin reacted when dehydrated and then re-hydrated, we speculated whether strengthening effect of dentin by heating may be reversible without critical damage to its organic components, particularly type I collagen.
In human bone, which has a similar composition to dentin, the type I collagen network plays an important role in its toughness, and the toughness and strength of bone decreases with increasing denaturation of the collagen . Several studies have investigated how calcified and decalcified collagen in bone or dentin reacts to heating. Heat transforms the crystalline collagen triple helical structure into an amorphous random coil form . Some studies reported that the degree of mineralization significantly affected the denaturation temperature of type I collagen . Other studies showed that the calcified type I collagen molecules in bone broke down approximately at 150–175 °C , while the decalcified type I collagen molecules denature irreversibly at 35.9–60 °C . The denaturation temperature seemed to vary depending on testing conditions and origin of materials.
Armstrong et al. found the denatured temperature of human dentin collagen in mineralized versus demineralized teeth in relation to the extent of dehydration. They reported that the denaturation temperature of collagen in mineralized dentin was between 160 and 186 °C; but that of demineralized dentin exhibited a denaturation temperature of 65.6 °C that increased with dehydration to 176 °C. They suggested that presence of apatite crystallites in mineralized collagen or interpeptide bonding in demineralized collagen increased the denaturation temperature. Given that 176 °C is the denaturation temperature for dehydrated demineralized dentin, heating to 140 °C as shown in our previous study , can significantly strengthen human dentin without major damage to its collagen.
In this study, we focused on the performance of type I collagen in dentin, after the dentin had been heated at 140 °C to achieve the best strength. First, we tried to identify the exact changes in mechanical properties of heated dentin by conducting nano-indentation with combination to an atomic force microscope (AFM). This makes it possible to understand changes in the heated dentin at the microstructural level. Then, the thermal stability of dehydrated dentin collagen was investigated by various methods: an X-ray diffraction analysis, a microscopic Fourier transform infrared (micro-FTIR) spectroscopic analysis, a microscopic laser Raman spectroscopic analysis, and a cross-linking analysis by high-performance liquid chromatography (HPLC). Our test hypotheses were that intertubular dentin, which constitutes a major part of organic components, was selectively affected by heating, and such changes could happen without critical damages to the basic structure of dentin type I collagen.
2
Materials and methods
2.1
Preparation of dentin specimens
Human third molars free of caries were stored in Hanks’ balanced salt solution (HBSS) at 4 °C and used within three months of extraction. Teeth were obtained from patients aged from 22 to 67. Protocols were approved by the ethics committee of Osaka University. Disk and beam-shaped dentin specimens were prepared ( Fig. 1 ). Disk-shaped specimens with a thickness of approximately 1.5 mm were obtained from coronal central portions of the molars by sectioning perpendicular to the tooth axis. Beam-shaped specimens, measuring approximately 1.7 mm × 0.9 mm × 8.0 mm were obtained from the coronal central portions of the molars. Dentinal tubules in the beam-shaped specimens were organized to run perpendicular to the longitudinal surface along the specimen length.
2.2
Heat treatment and rehydration
The specimens prepared were treated according to the following protocols: control (wet) – soaked in HBSS; heat – heated from room temperature to 140 °C at a rate of 10 °C/min, since we had confirmed the mechanical properties of human dentin were strengthened greatest to that temperature ; rehydrated – heated, then soaked in HBSS at ambient temperature for seven days. Each experimental group contained 10 specimens.
2.3
Nano-indentation with an AFM
Each disk-shaped specimen was polished by using waterproof silicone carbide abrasive papers (#600, 1000, 1500, Buehler, Lake Bluff, IL) under running water with a grinding-polishing machine (Ecomet 3000, Buehler). Then, the specimen was cut in half. One was heated and the other was kept without heating as a control. Nano-indentation was conducted by using a pyramidal diamond tip with a diameter of 200 nm secured to a nano-indenter (Nano Indenter SA2, MTS Japan, Tokyo) at ambient temperature. The force for the indentation was 10 μN. The indentation was performed for 100 times per specimen in an area of 10 μm × 10 μm with frequency of every 1 μm. Then, the hardness and elastic moduli of the specimens with or without heating were calculated.
An image of the area of the indentation was taken using an AFM (Nanoscope Dimension 3100, Bruker AXS, NY, USA), and the distance between each indentation point and the center of the nearest dentinal tubule from the indentation was measured on the AFM image. Then, the correlation between their micro-hardness and elastic moduli and the distance from nearest dentinal tubule were analyzed. The micro-hardness and elastic moduli of dentin with and without heating were compared in relation to the microstructures of dentin by using ANOVA and Scheffe’s F test at a 95% level of confidence.
2.4
X-ray diffraction analysis
Each beam-shaped dentin specimen was demineralized by 0.5 M EDTA at a pH of 7.4 for 10 days. Complete demineralization was confirmed by the disappearance of Bragg diffraction rings originating from hydroxyapatite crystals. Each dentin specimen, whether wet, heated or rehydrated was prepared thus: glued onto a thin glass fiber, fixed to the goniometer head via a brass pin, and then were exposed to a monochromatic X-ray source (0.3 mm Ø, MoKα) from a rotating anode generator (50 kV, 250 mA, ultraX18, Rigaku, Tokyo, Japan). Diffraction patterns were recorded on a flat imaging plate (RAXIS-IV, Rigaku) by the normal-beam transmission technique. The lateral packing of collagen was examined by comparing diffraction patterns taken before and after the specimens were heated, and after rehydration.
2.5
Micro-FTIR spectroscopic analysis
IR measurements of collagen from the demineralized specimens, obtained by the same methods as described in the X-ray diffraction study, were conducted using an FTIR micro-spectrometer equipped with MCT detector (FTIR620 + IRT30, JASCO, Tokyo). A reference background spectrum ( T 0 ) was first measured adjacent to the specimen, and then the transmission IR spectrum of the specimen ( T ) was measured. The IR spectrum is described as IR absorbance (abs = − log 10 T / T 0 ) as a function of the wavenumber (cm −1 ). The spot size of the analysis was 200 μm × 200 μm. A total of 100 scans were accumulated at 4 cm −1 spectral resolution in a range from 4000 to 1000 cm −1 according to the analyzed spot size and IR signal intensities. All the IR spectral data were analyzed with Spectra Manager (JASCO).
In order to examine thermal changes in dentin collagen, heating experiments were also conducted. A heating stage (LK600, Linkam, Surrey, UK) was modified and used for the thermal degradation experiment. Each specimen was placed on an IR inactive CaF 2 disk with the thickness of 1 mm, which was mounted on the heating stage. When the specimen was heated from 40 to 140 °C at a rate of 10 °C per minute under an atmospheric condition, IR spectra were obtained every 10 °C. To investigate any irreversible thermal damages to collagen, the degraded specimens were measured after they had cooled to room temperature.
2.6
Micro-laser Raman spectroscopic analysis
Each demineralized beam-shaped specimen was heated to the target 140 °C. Raman spectra of a same specimen before and after heating were measured by laser Raman microscopy (RAMAN-11, Nanophoton, Osaka, Japan). The excitation source was a semiconductor laser operating at 785 nm with nominal power of 100 mW. The laser beam was focused and irradiated onto the specimen using an objective lens (5× NA = 0.3). The area of irradiation was 1.0 μm × 1.0 μm, and the accumulation time was 180 s. The excited Raman scattered light was collected with the same objective lens and guided to the spectrograph, which had a focal length of 500 mm. Raman light was then recorded with a thermoelectrically cooled CCD camera as the Raman spectrum, which contained chemical bond information. The Raman spectra obtained were normalized for the comparison, since the intensity of the raw spectra changes depending on the surface condition of dentin, such as the roughness and density of the collagen molecules. The normalization protocol was as follows: the background intensity caused by fluorescence was reduced according to the algorithm developed by methods previously reported ; then, the spectra were normalized with the peak intensity at 815 cm −1 , corresponding to the backbone vibration of collagen .
2.7
Characterization of enzymatic and non-enzymatic collagen cross-links
Quantification of the cross-links of the dentin collagen was conducted as reported previously . Disk-shaped dentin specimens were pulverized in liquid N 2 . Half were then heated, and the others were kept unheated as control. The powder was demineralized with 0.5 M EDTA in 50 mM Tris buffer (pH 7.4) for 96 h at 4 °C. The demineralized dentin residues were then sequentially suspended in potassium phosphate buffer (pH 7.6, ionic strength 0.15), and reduced at 37 °C with non-radioactive NaBH 4 (Sigma–Aldrich, St. Louise, MO, USA). The reduced specimens were hydrolyzed in 6 M hydrochloric acid at 110 °C for 24 h. The resulting hydrolysates were then analyzed for cross-links by using high-performance liquid chromatography (HPLC) (LC9, Shimadzu) fitted with a cation exchange column (Aa pack-Na, JASCO, Tokyo, Japan) linked to an on-line fluorescence flow monitor (RF10AXL, Shimadzu). The amounts of enzymatic cross-links, such as immature reducible and mature non-reducible cross-links, and non-enzymatic cross-links were calculated. Lysyl oxidase mediated reducible immature cross-links (dehydro-dihydroxylysinonorleucine; deH-DHLNL, dehydro-hydroxylysinonorleucine; deH-HLNL) were identified and quantified according to their reduced forms (DHLNL, HLNL). Reducible immature cross-links were detected by O-phthalaldehyde derivatization using a post-column method; and enzymatic non-reducible mature cross-links such as pyridinoline (Pyr), deoxypyridinoline (Dpyr), and non-enzymatic cross-links (pentosidine; Pen) were detected by natural fluorescence. The quantities of each cross-link were expressed as mol/mol collagen for enzymatic cross-links and mmol/mol collagen for non-enzymatic cross-links, and the values with and without heating were compared by Student’s t -test at a 95% level of confidence.
2
Materials and methods
2.1
Preparation of dentin specimens
Human third molars free of caries were stored in Hanks’ balanced salt solution (HBSS) at 4 °C and used within three months of extraction. Teeth were obtained from patients aged from 22 to 67. Protocols were approved by the ethics committee of Osaka University. Disk and beam-shaped dentin specimens were prepared ( Fig. 1 ). Disk-shaped specimens with a thickness of approximately 1.5 mm were obtained from coronal central portions of the molars by sectioning perpendicular to the tooth axis. Beam-shaped specimens, measuring approximately 1.7 mm × 0.9 mm × 8.0 mm were obtained from the coronal central portions of the molars. Dentinal tubules in the beam-shaped specimens were organized to run perpendicular to the longitudinal surface along the specimen length.
