archives of oral biology 59 (2014) 119–124
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ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Accumulation of advanced glycation end-products in human dentine Jiro Miura a,*, Kantaro Nishikawa b, Mizuho Kubo a, Shuichiro Fukushima b, Mamoru Hashimoto b, Fumio Takeshige a, Tsutomu Araki b a b
Division for Interdisciplinary Dentistry, Osaka University Dental Hospital, Japan Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, Japan
article info
abstract
Article history:
Cross-linking of collagen by Advanced Glycation End-products (AGEs) occurs by non-
Accepted 31 October 2013
enzymatic glycation (Maillard reaction). The purpose of this study was to examine whether AGEs are formed in human dentinal collagen, and to consider any possible influence of AGEs
Keywords:
on dentinal physiology. Mechanical characteristics, fluorescence spectra and immunohis-
Glycation
tochemical analyses of demineralized dentine sections from young subjects were compared
Human dentine
with those of aged ones. The same investigations were performed with young dentine
Ageing
artificially glycated by incubation in 0.1 M ribose solution. Indentation measurement indi-
Fluorescence
cated that the sections from aged dentine were mechanically harder than those from young
Collagen
dentine. The hardness of young dentine increased after incubation in ribose solution.
Browning
Fluorescence peak wavelength of the young dentine was shorter than that of the aged one, but shifted towards the peak wavelength of the aged one after incubation in ribose solution. These changes were considered to be due to accumulation of AGEs. Existence of AGEs in dentinal collagen was confirmed by immunohistochemical analysis. The obtained results suggest that AGEs accumulation occurs in dentinal collagen and is affected by both human age and physiological conditions such as glucose level in blood because dentinal collagen receives nourishment via dental pulp and tubules. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Ageing is an irreversible and physiological phenomenon that cannot be avoided in human beings. In aged persons, the teeth usually appear more brown than those in young subjects. This phenomenon is characteristic of ageing. Glycation of body protein is one of the most interesting processes in human ageing. Several studies have reported that the nonenzymatic reaction of blood glucose with body proteins leads to cumulative chemical modifications of tissue proteins throughout the body.1–4 This reaction, named Maillard reaction, is a biological process that occurs in tissues, finally resulting in formation of
advanced glycation end-products (AGEs). Maillard first noted in 1912 that amino acids heated in the presence of reducing sugars turned brown. During the formation of AGE, first the aldehyde group of reducing sugars binds to e-amino group of proteins without enzyme and forms Schiff base. The Schiff base becomes an Amadori product through the Amadori rearrangement. They are then converted non-reversibly into stable substances through oxidation, dehydration, and condensation. If oxidation accompanies glycation, the formed products are glycation products, for examples, pentosidine5 and N-carboxymethyllysine (CML).6–8 Thus, AGE is the collective term for the products of Maillard reaction. The free AGEs easily bind to collagen and act as a cross-link between collagen fibrils. Hence, accumulation
* Corresponding author at: Division for Interdisciplinary Dentistry, Osaka University Dental Hospital, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 2386; fax: +81 6 6879 2387. E-mail addresses: [email protected], [email protected] (J. Miura). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.10.012
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archives of oral biology 59 (2014) 119–124
of AGEs associated with cross-linking alters the mechanical characteristics of collagen-rich tissues. We assume that the Maillard reaction also occurs in dentinal collagen and as a result, AGEs accumulate near dental canals during one’s life. This phenomenon results in brownish discoloration and modification of the mechanical properties of dentine, because dentinal collagen receives nourishment through dentinal tubules via canals. However, there are few investigations concerning the Maillard reaction in vital tooth. The main purpose of this study was to determine whether AGEs produced by Maillard reaction accumulate in human dentine physiologically. In the present study, we performed mechanical indentation analysis, fluorescence spectroscopy, immunohistochemical staining and immuno-electron microscopy in demineralized dentine to detect AGEs formation.
the dentinal collagen was a few mm square, we have employed a conventional fluorescence spectrometer for liquid sample (Shimadzu RF-5300, Kyoto, Japan) instead of a microfluorometer. The fluorescence excitation wavelength was set around 370 nm according to the previous report by Kleter et al.,14 and fluorescence emission spectra between 390 nm and 700 nm were recorded. Because the spectrometer is designed for the measurement of a liquid sample in a quadratic prism-shaped glass cuvette, the sections were placed on the diagonal plane of the cuvette holder to examine the middle portion of the dentine layer. To eliminate unwanted stray light due to reflection and/or scattering on the sample surface, an additional UV cut filter was placed between the sample and the monochromator for fluorescence emission in the spectrometer.
2.4.
2.
Materials and methods
2.1.
Sample preparation
Six caries-free third molars of young and aged patients (young 18–26 yrs, aged 68–76 yrs), extracted as part of routine treatment at Osaka University Dental Hospital, were prepared for the experiment. The teeth were collected with the patients’ consent and preserved in Hank’s balanced salt solution (HBSS) at 4 8C till the experiments. The experimental protocol was approved by the Ethics committee of the Faculty of Dentistry, Osaka University. Samples were sectioned parallel to the tooth axis with an Isomet low-speed diamond wheel saw under water in order to obtain slabs of approximately 1.0 mm thickness. The sections were immersed in 4% paraformaldehyde and 0.1% glutaraldehyde (for immuno-electron microscopy), or 4% paraformaldehyde (for other measurements), then subsequently demineralized for four weeks with 10% EDTA in room temperature. For in vitro glycation, the demineralized sections were incubated in 0.1 M ribose solution in HBSS buffer at 37 8C for 6 weeks.9,10
2.2.
Mechanical test
Hardness of the demineralized sections was evaluated using a mechanical indentation tester (Shimadzu EZ-S) equipped with an indentation probe of diameter 1 mm. The probe indented up to 50 mm in depth from the sample surface to measure force–displacement characteristics. Based on the resultant force–displacement curve, the slope corresponding to a spring constant was calculated (note: unit of the slope is N/mm, the slope becomes steeper as the sample becomes harder). More than 25 points were probed in demineralized dentine to examine the regional heterogeneity of the hardness. The glycated young dentine samples were also subjected to the same mechanical trials.
2.3.
Fluorescence analysis
Autofluorescence of the sections from young and aged groups was measured. Because the measurement area of
Immunohistochemical staining
Demineralized dentine blocks were embedded in paraffin block after graded-ethanol dehydration, and sliced into 4 mmthick section samples using a microtome (Leica Microsystems GmbH, Wetzlar, Germany). Immunohistochemical staining was performed with an LSAB2 kit, according to the manufacturer’s instruction (DAKO, Glostrup, Denmark). The following primary antibody which recognizes the C-terminal region of CML was used: mouse anti-AGE monoclonal antibody (Clone No. 6D12; Transgenic Inc., Japan).11,12 Sections were lightly counterstained with haematoxylin. As negative controls, mouse serum IgG (Dako) was used as the primary antibody, and these gave uniformly negative results.
2.5.
Immuno-electron microscopy
Dentine blocks were embedded in LR Gold Resin System (Electron Microscopy Sciences, PA, USA) at 20 8C with an ultraviolet polymerizer system (DOSAKA EM CO. LTD., Kyoto Japan). After curing, samples were sectioned to 70 nm in thickness with a diamond knife (Nanotome Thick, Sakai Advanced Electron Microscope Research Center, Japan), and mounted on nickel grids (Nisshin EM Tokyo Japan) in a ultramicrotome (Ultrotome V, LKB, Sweden). The sections were immersed in Donkey serum (1:10) and incubated overnight at 4 8C with mouse monoclonal AGEs (1:100) primary antibody (Clone No. 6D12, Transgenic Inc., Japan). Secondary antibody, donkey anti-mouse IgG (18 nm colloidal gold; Jackson, PA, USA), was diluted (1:10) in 0.2% tween 20/ TBS plus 5% albumin. Specimens were fixed with 2% glutaraldehyde and then stained with 2% uranylacetate. A transmission electron microscope (H800, Hitachi, Tokyo, Japan) at 200 keV was employed for detection of AGE-bound colloidal golds.
2.6.
Statistical analysis
The values for the stiff were presented as mean SEM for statistical analysis. Obtained values were analyzed using oneway ANOVA and Tukey–Kramer tests with a significant level of p < 0.05.
archives of oral biology 59 (2014) 119–124
121
Fig. 1 – Colour and hardness of demineralized dentine. (a) and (b) show the distribution of the slope values in the young and aged dentine, respectively. The aged dentine is more brown and harder than the young dentine. Root portion was harder than the coronal one in both samples.
3.
Results
3.1.
Mechanical properties
Fig. 1(a) and (b) show distributions of the slope values in the young and aged dentine, respectively together with an overview of the dentinal collagen. As shown in these figures, the dentinal collagen in the aged was more brown and stiffer than that in the young. In particular, the root portion was stiffer than the coronal one in both dentins.
3.2.
Fluorescence analysis
Fig. 2 shows the fluorescence spectra of dentinal collagen. As shown in this figure, the fluorescence emission was distributed in the visible region, and the peak wavelengths of the young and aged dentine were 440 nm and 460 nm, respectively.
3.3. Immunohistochemical and immuno-electron microscopic analysis Young dentine showed AGE-negative immunoreaction in the coronal and root portion (Fig. 3(a)–(d)), while aged dentine showed AGE-positive reaction in both portions (Fig. 3(e)–(h)). In aged dentine, immunoreactions strongly appeared beside the predentin (Fig. 3(e) and (g)) and distributed along the dentinal tubules (Fig. 3(f) and (h)). In each section, there was no appreciable difference in the immunohistochemical staining between the coronal and root portions. In the images of immuno-electron microscopy (Fig. 3(i)–(l)), young dentine showed AGE-negative immunoreaction. On the contrary, AGEs labelled by colloidal golds (indicated as arrow heads) were found in the surrounding collagenous matrix in intertubular dentine of aged dentine (Fig. 3(i) and (j)).
3.4.
In vitro glycation
Fig. 4(a) shows comparison of stiffness between young, aged and glycated young dentine in the coronal and root portion. Data and p-values are analyzed using one-way ANOVA and Tukey–Kramer test (*p < 0.01, #p < 0.05 compared with young). After 6 weeks incubation in ribose solution, the young dentine became as stiff as that of aged control, and the fluorescence peak wavelength of the young dentine shifted towards that of the aged control (Fig. 2). The young dentine showed AGEpositive immunoreaction in intertubular dentine after incubation in ribose (Fig. 4(b)–(e)).
4. Fig. 2 – Fluorescence spectra of young and aged demineralized dentine before and after glycation.
Discussion
As described in the introduction, the main purpose of this research was to consider whether a Maillard reaction occurs
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archives of oral biology 59 (2014) 119–124
Fig. 3 – Immunohistochemical and immuno-electron microscopic images. (a)–(h): Localization of AGEs in the demineralized dentine with each age. Demineralized dentine sections were subjected to immunohistochemical staining with anti-AGEs antibody. (Black bar: 100 mm). (a): Young coronal dentine, (c): young root dentine, (b) and (d): high power view of the box in (a) and (c), respectively. (e): Aged coronal dentine, (g): aged root dentine, (f) and (h): high power view of the box in (e) and (g) (1000T), respectively. (i)-(l): Immuno-electron microscopic images of peritubular area. (i): Immuno-EM in young coronal dentine, (j): young root dentine, (k): aged coronal dentine, (l): aged root dentine. (P: pulp, DT: dentinal tubule, white bar: 500 nm, arrowheads: 18 nm dia. colloidal gold particles.)
and resultant AGEs accumulate in dentine. To confirm the effects of Maillard reaction on dentinal collagen, we performed mechanical tests, fluorescence measurement and immunohistochemical staining including both optical and electron microscopic analyses. The sections of young and aged demineralized dentine (age around 20 yrs and 70 yrs) were examined. Brownish discoloration was evident in the aged dentine (Fig. 1). The discoloration indicates possible accumulation of AGEs, and the resultant cross-linking is expected to reinforce the dentinal collagen mechanically. Results of the mechanical examination shown in Fig. 1 obviously support the formation of crosslinks. Though there was no appreciable heterogeneity in the distribution of colour density in the dentine, the hardness of the root portion was greater than that of the coronal portion in both young and aged dentine. This implies that localization of AGEs is influenced by metabolism in the dentine. One might speculate that residual minerals affect the mechanical property and cause heterogeneity in observed hardness values. Hence, we checked the hardness of the dentine section with an indentation tester every 2 days during the demineralization procedure, and confirmed that 2 weeks incubation in EDTA was sufficient for complete removal of hydroxyapatite. For in vitro glycation of dentinal collagen, the collagen was incubated in ribose solution instead of glucose solution that
corresponds to blood sugar, because reducibility of ribose is stronger than that of glucose,10 which shortens the incubation time. Reliability of the glycation was evaluated using the positive controls of primary antibody for CML, and we confirmed that in vitro glycation had progressed successfully. Our previous study, based on the measurement using a fluorescence microscope, had showed that fluorescence intensity increased without change in the peak wavelength in the ageing process.13 However, the present results shown in Fig. 2 indicate a red-shift of fluorescence peak by both ageing and in vitro glycation. Such an inconsistency in spectral shift may be due to different treatment of dentine; demineralized dentine was measured in the present study, while nondecalcified dentine was measured in the previous. Kleter reported that fluorescence profile of bovine dentinal collagen degraded with collagenase.14 He showed an increase in fluorescence intensity of the long wavelength component when dentine was exposed to glucose. Increase of the fluorescence component with the long wavelength region leads to red-shift of the observed fluorescence spectrum. Although we too found a red-shift of the fluorescence spectrum in the aged and glycated dentine, the resultant fluorescence intensity value was ambiguous because of the limitation of the employed spectrometer. To confirm the overall fluorescence spectral profile, we have to use an adequate fluorescence spectrophotometer. The combination
archives of oral biology 59 (2014) 119–124
123
Fig. 4 – Results of mechanical tests and immunohistochemical analysis of demineralized dentine before and after 6 weeks glycation in ribose solution. (a): Force–displacement characteristics of young, glycated young, and aged coronal and root dentine. The data are shown as mean W SEM (n = 3). P-values were determined by one-way ANOVA and Tukey–Kramer test (*p < 0.01, #p < 0.05). (b) and (d): Immunohistochemical images of young coronal and root dentine after 6 weeks glycation in ribose solution, (c) and (e): magnified image of the box in (b) and (d), respectively. Intertubular dentine was stained in each portion after the glycation, while peritubular dentine was stained before glycation (bar: 5 mm P: pulp, D: dentine).
results from the mechanical test and fluorometry for dentinal collagen strongly suggest the existence of AGEs in aged dentine. To confirm the suggestion, we next performed immunohistochemical staining. AGEs include many types of molecules, e.g., pentosidine is a marker for identification of osteoporosis in females and bone fracture in type 2 diabetes.15 CML is known to increase in human collagen with age, and age-adjusted concentrations of CML increase in skin collagen in diabetes.7 Ac¸il et al. focused on hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), and concluded that there was no relationship between the dentinal concentrations of these molecules and age.16 Though we stained only CML in the present study, we assume that there is an appreciable relationship between the dentinal concentration of AGEs and age. Because dentinal collagen does not regenerate within
one’s lifetime, the accumulated amount of AGE in dentinal collagen is a potential indicator of human age, making it applicable in forensic medicine. We observed the accumulation of AGEs in the collagen fibrils around dentinal tubules (Fig. 3(f) and (h)). These characteristics of localization indicated that Maillard reaction occurred at the contact region between dentinal collagen and the glucose rich tissue fluid. Collagen in which AGEs accumulate became harder and more fragile; therefore this accumulation with ageing may affect the mechanical properties of dentine such as dentinal crack propagation. Modifications of the collagenous matrix are structurally and mechanically important, and the breakage of cross-linking can result in severe tissue dysfunction.17 In recent years, several reports show that artificially formed
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archives of oral biology 59 (2014) 119–124
cross-linking in dentinal collagen reduces the activities of matrix metalloproteinase.18,19 Therefore, degradation of the hybrid layer between dentine and bonding agent was suppressed by cross-linking in dentinal collagen.20,21 Our report indicates that accumulation of AGEs with human ageing may also affect the bonding strength and long-term stability of dentine-bonding interfaces. Moreover, alteration of colour in dentinal collagen can be attributed to gradual accumulation of AGEs with ageing. This phenomenon will affect the concept of whitening discoloured tooth. To understand the homeostatic stability in dentine, we should further identify the types of AGE molecules observed in dentinal collagen.
Funding This work was supported by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant Numbers 22791831, 23240069, 23650260 and 25462957.
Competing interests The authors declare no potential conflicts of interest with respect to the authorship and publication of this article.
Ethical approval The experimental protocol was approved by the Ethics committee of the Faculty of Dentistry, Osaka University (H24-E25).
Acknowledgements This project was technically supported by the Research Center for Ultra-High-Voltage Electron Microscopy of Osaka University. The authors would like to thank Hirotaro Mori for his valuable suggestions and constant support, Yu Usami for technical support in the immunohistochemical analysis, and Keita Kondo for measuring the mechanical properties.
references
1. Vitek MP, Bhattacharya K, Glendening JM, Stopa E, Vlassara H, Bucala R, et al. Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc Natl Acad Sci U S A 1994;91:4766–70. 2. Raj DS, Choundhury D, Welbourne TC, Levi M. AGE: a nephrologist’s perspective. Am J Kidney Dis 2000;35:365–80. 3. Singh R, Barden A, Mori T, Beilin L. Advanced glycation endproducts: a review. Diabetologia 2001;44:129–46. 4. McIntyre NJ, Chesterton LJ, John SG, Jefferies HJ, Burton JO, Taal MW, et al. Tissue-advanced glycation end product concentration in dialysis patients. Clin J Am Soc Nephrol 2010;5:51–5.
5. Miyata T, Ueda Y, Shinzato T, Iida Y, Tanaka S, Kurokawa K, et al. Accumulation of albumin-linked and free-form pentosidine in the circulation of uraemic patients with endstage renal failure: renal implications in the pathology of pentosidine. J Am Soc Nephrol 1996;7:1198–206. 6. Fu MX, Requena JR, Jenkins AJ, Lyons TJ, Baynes JW, Thorpe SR. The advanced glycation end product, Ne(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem 1996;271:9982–6. 7. Requena JR, Ahmed MU, Fountain CW, Degenhardt TP, Reddy S, Perez C, et al. Carboxymethylethanolamine, a biomarker of phospholipid modification during the Maillard reaction in vivo. J Biol Chem 1997;272:17473–9. 8. Semba RD, Najjar SS, Sun K, Lakatta EG, Ferrucci L. Serum carboxymethyl–lysine, an advanced glycation end product, is associated with increased aortic pulse wave velocity in adults. Am J Hypertens 2009;22:74–9. 9. Roy R, Boskey A, Bonassar LJ. Processing of type I collagen gels using non-enzymatic glycation. J Biomed Mater Res A 2010;93:843–51. 10. Bailey AJ, Sims TJ, Avery NC, Halligan EP. Non-enzymic glycation of fibrous collagen: reaction products of glucose and ribose. Biochem J 1995;305:385–90. 11. Yoshida S, Yamada K, Hamaguchi K, Nishimura M, Hatakeyama E, Tsuchida H, et al. Immunohistochemical study of human advanced glycation end-products (AGE) and growth factors in cardiac tissue of patients on maintenance dialysis and kidney transplantation. Clin Nephrol 1998;49:273–80. 12. Ikeda K, Higashi T, Sano H, Jinnouchi Y, Yoshida M, Araki T, et al. N-carboxymethyl lysine protein adduct is a major immunological epitope in proteins modified with advanced glycation end products of the Maillard reaction. Biochemistry 1996;35:8075–83. 13. Matsumoto H, Kitamura S, Araki T. Autofluorescence in human dentine in relation to age, tooth type and temperature measured by nanosecond time-resolved fluorescence microscopy. Arch Oral Biol 1999;44:309–18. 14. Kleter GA, Damen JJM, Buijs MJ, Ten Cate JM. The Maillard reaction in demineralized dentin in vitro. Eur J Oral Sci 1997;105:278–84. 15. Tanaka S, Kuroda T, Saito M, Shiraki M. Urinary pentosidine improves risk classification using fracture risk assessment tools for postmenopausal woman. J Bone Miner Res 2011;26:2778–84. 16. Ac¸il Y, Springer ING, Prasse JG, Hedderich J, Jepsen S. Concentration of collagen cross-links in human dentin bears no relation to the individual age. Int J Legal Med 2002;116:340–3. 17. Knott L, Bailey AJ. Collagen cross-links in mineralizing tissues: a review of their chemistry, function, and clinical relevance. Bone 1998;22:181–7. 18. Breschi L, Mazzoni A, Ruggeri A, Cadenaro M, Di Lenarda R, De Stefano Dorigo E. Dental adhesion review: aging and stability of the bonded interface. Dent Mater 2008;24:90–101. 19. Breschi L, Martin P, Mazzoni A, Nato F, Carrilho M, Tjaderhane L, et al. Use of a specific MMP-inhibitor (galardin) for preservation of hybrid layer. Dent Mater 2010;26:571–8. 20. Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, et al. Collagen degradation by host-derived enzymes during aging. J Dent Res 2004;83:216–21. 21. Cova A, Breschi L, Nato F, Ruggeri Jr A, Carrilho M, Tja¨derhane L, et al. Effect of UVA-activated riboflavin on dentin bonding. J Dent Res 2011;90:1439–45.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Accumulation of advanced glycation end-products in human dentine Jiro Miura a,*, Kantaro Nishikawa b, Mizuho Kubo a, Shuichiro Fukushima b, Mamoru Hashimoto b, Fumio Takeshige a, Tsutomu Araki b a b
Division for Interdisciplinary Dentistry, Osaka University Dental Hospital, Japan Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, Japan
article info
abstract
Article history:
Cross-linking of collagen by Advanced Glycation End-products (AGEs) occurs by non-
Accepted 31 October 2013
enzymatic glycation (Maillard reaction). The purpose of this study was to examine whether AGEs are formed in human dentinal collagen, and to consider any possible influence of AGEs
Keywords:
on dentinal physiology. Mechanical characteristics, fluorescence spectra and immunohis-
Glycation
tochemical analyses of demineralized dentine sections from young subjects were compared
Human dentine
with those of aged ones. The same investigations were performed with young dentine
Ageing
artificially glycated by incubation in 0.1 M ribose solution. Indentation measurement indi-
Fluorescence
cated that the sections from aged dentine were mechanically harder than those from young
Collagen
dentine. The hardness of young dentine increased after incubation in ribose solution.
Browning
Fluorescence peak wavelength of the young dentine was shorter than that of the aged one, but shifted towards the peak wavelength of the aged one after incubation in ribose solution. These changes were considered to be due to accumulation of AGEs. Existence of AGEs in dentinal collagen was confirmed by immunohistochemical analysis. The obtained results suggest that AGEs accumulation occurs in dentinal collagen and is affected by both human age and physiological conditions such as glucose level in blood because dentinal collagen receives nourishment via dental pulp and tubules. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Ageing is an irreversible and physiological phenomenon that cannot be avoided in human beings. In aged persons, the teeth usually appear more brown than those in young subjects. This phenomenon is characteristic of ageing. Glycation of body protein is one of the most interesting processes in human ageing. Several studies have reported that the nonenzymatic reaction of blood glucose with body proteins leads to cumulative chemical modifications of tissue proteins throughout the body.1–4 This reaction, named Maillard reaction, is a biological process that occurs in tissues, finally resulting in formation of
advanced glycation end-products (AGEs). Maillard first noted in 1912 that amino acids heated in the presence of reducing sugars turned brown. During the formation of AGE, first the aldehyde group of reducing sugars binds to e-amino group of proteins without enzyme and forms Schiff base. The Schiff base becomes an Amadori product through the Amadori rearrangement. They are then converted non-reversibly into stable substances through oxidation, dehydration, and condensation. If oxidation accompanies glycation, the formed products are glycation products, for examples, pentosidine5 and N-carboxymethyllysine (CML).6–8 Thus, AGE is the collective term for the products of Maillard reaction. The free AGEs easily bind to collagen and act as a cross-link between collagen fibrils. Hence, accumulation
* Corresponding author at: Division for Interdisciplinary Dentistry, Osaka University Dental Hospital, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 2386; fax: +81 6 6879 2387. E-mail addresses: [email protected], [email protected] (J. Miura). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.10.012
120
archives of oral biology 59 (2014) 119–124
of AGEs associated with cross-linking alters the mechanical characteristics of collagen-rich tissues. We assume that the Maillard reaction also occurs in dentinal collagen and as a result, AGEs accumulate near dental canals during one’s life. This phenomenon results in brownish discoloration and modification of the mechanical properties of dentine, because dentinal collagen receives nourishment through dentinal tubules via canals. However, there are few investigations concerning the Maillard reaction in vital tooth. The main purpose of this study was to determine whether AGEs produced by Maillard reaction accumulate in human dentine physiologically. In the present study, we performed mechanical indentation analysis, fluorescence spectroscopy, immunohistochemical staining and immuno-electron microscopy in demineralized dentine to detect AGEs formation.
the dentinal collagen was a few mm square, we have employed a conventional fluorescence spectrometer for liquid sample (Shimadzu RF-5300, Kyoto, Japan) instead of a microfluorometer. The fluorescence excitation wavelength was set around 370 nm according to the previous report by Kleter et al.,14 and fluorescence emission spectra between 390 nm and 700 nm were recorded. Because the spectrometer is designed for the measurement of a liquid sample in a quadratic prism-shaped glass cuvette, the sections were placed on the diagonal plane of the cuvette holder to examine the middle portion of the dentine layer. To eliminate unwanted stray light due to reflection and/or scattering on the sample surface, an additional UV cut filter was placed between the sample and the monochromator for fluorescence emission in the spectrometer.
2.4.
2.
Materials and methods
2.1.
Sample preparation
Six caries-free third molars of young and aged patients (young 18–26 yrs, aged 68–76 yrs), extracted as part of routine treatment at Osaka University Dental Hospital, were prepared for the experiment. The teeth were collected with the patients’ consent and preserved in Hank’s balanced salt solution (HBSS) at 4 8C till the experiments. The experimental protocol was approved by the Ethics committee of the Faculty of Dentistry, Osaka University. Samples were sectioned parallel to the tooth axis with an Isomet low-speed diamond wheel saw under water in order to obtain slabs of approximately 1.0 mm thickness. The sections were immersed in 4% paraformaldehyde and 0.1% glutaraldehyde (for immuno-electron microscopy), or 4% paraformaldehyde (for other measurements), then subsequently demineralized for four weeks with 10% EDTA in room temperature. For in vitro glycation, the demineralized sections were incubated in 0.1 M ribose solution in HBSS buffer at 37 8C for 6 weeks.9,10
2.2.
Mechanical test
Hardness of the demineralized sections was evaluated using a mechanical indentation tester (Shimadzu EZ-S) equipped with an indentation probe of diameter 1 mm. The probe indented up to 50 mm in depth from the sample surface to measure force–displacement characteristics. Based on the resultant force–displacement curve, the slope corresponding to a spring constant was calculated (note: unit of the slope is N/mm, the slope becomes steeper as the sample becomes harder). More than 25 points were probed in demineralized dentine to examine the regional heterogeneity of the hardness. The glycated young dentine samples were also subjected to the same mechanical trials.
2.3.
Fluorescence analysis
Autofluorescence of the sections from young and aged groups was measured. Because the measurement area of
Immunohistochemical staining
Demineralized dentine blocks were embedded in paraffin block after graded-ethanol dehydration, and sliced into 4 mmthick section samples using a microtome (Leica Microsystems GmbH, Wetzlar, Germany). Immunohistochemical staining was performed with an LSAB2 kit, according to the manufacturer’s instruction (DAKO, Glostrup, Denmark). The following primary antibody which recognizes the C-terminal region of CML was used: mouse anti-AGE monoclonal antibody (Clone No. 6D12; Transgenic Inc., Japan).11,12 Sections were lightly counterstained with haematoxylin. As negative controls, mouse serum IgG (Dako) was used as the primary antibody, and these gave uniformly negative results.
2.5.
Immuno-electron microscopy
Dentine blocks were embedded in LR Gold Resin System (Electron Microscopy Sciences, PA, USA) at 20 8C with an ultraviolet polymerizer system (DOSAKA EM CO. LTD., Kyoto Japan). After curing, samples were sectioned to 70 nm in thickness with a diamond knife (Nanotome Thick, Sakai Advanced Electron Microscope Research Center, Japan), and mounted on nickel grids (Nisshin EM Tokyo Japan) in a ultramicrotome (Ultrotome V, LKB, Sweden). The sections were immersed in Donkey serum (1:10) and incubated overnight at 4 8C with mouse monoclonal AGEs (1:100) primary antibody (Clone No. 6D12, Transgenic Inc., Japan). Secondary antibody, donkey anti-mouse IgG (18 nm colloidal gold; Jackson, PA, USA), was diluted (1:10) in 0.2% tween 20/ TBS plus 5% albumin. Specimens were fixed with 2% glutaraldehyde and then stained with 2% uranylacetate. A transmission electron microscope (H800, Hitachi, Tokyo, Japan) at 200 keV was employed for detection of AGE-bound colloidal golds.
2.6.
Statistical analysis
The values for the stiff were presented as mean SEM for statistical analysis. Obtained values were analyzed using oneway ANOVA and Tukey–Kramer tests with a significant level of p < 0.05.
archives of oral biology 59 (2014) 119–124
121
Fig. 1 – Colour and hardness of demineralized dentine. (a) and (b) show the distribution of the slope values in the young and aged dentine, respectively. The aged dentine is more brown and harder than the young dentine. Root portion was harder than the coronal one in both samples.
3.
Results
3.1.
Mechanical properties
Fig. 1(a) and (b) show distributions of the slope values in the young and aged dentine, respectively together with an overview of the dentinal collagen. As shown in these figures, the dentinal collagen in the aged was more brown and stiffer than that in the young. In particular, the root portion was stiffer than the coronal one in both dentins.
3.2.
Fluorescence analysis
Fig. 2 shows the fluorescence spectra of dentinal collagen. As shown in this figure, the fluorescence emission was distributed in the visible region, and the peak wavelengths of the young and aged dentine were 440 nm and 460 nm, respectively.
3.3. Immunohistochemical and immuno-electron microscopic analysis Young dentine showed AGE-negative immunoreaction in the coronal and root portion (Fig. 3(a)–(d)), while aged dentine showed AGE-positive reaction in both portions (Fig. 3(e)–(h)). In aged dentine, immunoreactions strongly appeared beside the predentin (Fig. 3(e) and (g)) and distributed along the dentinal tubules (Fig. 3(f) and (h)). In each section, there was no appreciable difference in the immunohistochemical staining between the coronal and root portions. In the images of immuno-electron microscopy (Fig. 3(i)–(l)), young dentine showed AGE-negative immunoreaction. On the contrary, AGEs labelled by colloidal golds (indicated as arrow heads) were found in the surrounding collagenous matrix in intertubular dentine of aged dentine (Fig. 3(i) and (j)).
3.4.
In vitro glycation
Fig. 4(a) shows comparison of stiffness between young, aged and glycated young dentine in the coronal and root portion. Data and p-values are analyzed using one-way ANOVA and Tukey–Kramer test (*p < 0.01, #p < 0.05 compared with young). After 6 weeks incubation in ribose solution, the young dentine became as stiff as that of aged control, and the fluorescence peak wavelength of the young dentine shifted towards that of the aged control (Fig. 2). The young dentine showed AGEpositive immunoreaction in intertubular dentine after incubation in ribose (Fig. 4(b)–(e)).
4. Fig. 2 – Fluorescence spectra of young and aged demineralized dentine before and after glycation.
Discussion
As described in the introduction, the main purpose of this research was to consider whether a Maillard reaction occurs
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Fig. 3 – Immunohistochemical and immuno-electron microscopic images. (a)–(h): Localization of AGEs in the demineralized dentine with each age. Demineralized dentine sections were subjected to immunohistochemical staining with anti-AGEs antibody. (Black bar: 100 mm). (a): Young coronal dentine, (c): young root dentine, (b) and (d): high power view of the box in (a) and (c), respectively. (e): Aged coronal dentine, (g): aged root dentine, (f) and (h): high power view of the box in (e) and (g) (1000T), respectively. (i)-(l): Immuno-electron microscopic images of peritubular area. (i): Immuno-EM in young coronal dentine, (j): young root dentine, (k): aged coronal dentine, (l): aged root dentine. (P: pulp, DT: dentinal tubule, white bar: 500 nm, arrowheads: 18 nm dia. colloidal gold particles.)
and resultant AGEs accumulate in dentine. To confirm the effects of Maillard reaction on dentinal collagen, we performed mechanical tests, fluorescence measurement and immunohistochemical staining including both optical and electron microscopic analyses. The sections of young and aged demineralized dentine (age around 20 yrs and 70 yrs) were examined. Brownish discoloration was evident in the aged dentine (Fig. 1). The discoloration indicates possible accumulation of AGEs, and the resultant cross-linking is expected to reinforce the dentinal collagen mechanically. Results of the mechanical examination shown in Fig. 1 obviously support the formation of crosslinks. Though there was no appreciable heterogeneity in the distribution of colour density in the dentine, the hardness of the root portion was greater than that of the coronal portion in both young and aged dentine. This implies that localization of AGEs is influenced by metabolism in the dentine. One might speculate that residual minerals affect the mechanical property and cause heterogeneity in observed hardness values. Hence, we checked the hardness of the dentine section with an indentation tester every 2 days during the demineralization procedure, and confirmed that 2 weeks incubation in EDTA was sufficient for complete removal of hydroxyapatite. For in vitro glycation of dentinal collagen, the collagen was incubated in ribose solution instead of glucose solution that
corresponds to blood sugar, because reducibility of ribose is stronger than that of glucose,10 which shortens the incubation time. Reliability of the glycation was evaluated using the positive controls of primary antibody for CML, and we confirmed that in vitro glycation had progressed successfully. Our previous study, based on the measurement using a fluorescence microscope, had showed that fluorescence intensity increased without change in the peak wavelength in the ageing process.13 However, the present results shown in Fig. 2 indicate a red-shift of fluorescence peak by both ageing and in vitro glycation. Such an inconsistency in spectral shift may be due to different treatment of dentine; demineralized dentine was measured in the present study, while nondecalcified dentine was measured in the previous. Kleter reported that fluorescence profile of bovine dentinal collagen degraded with collagenase.14 He showed an increase in fluorescence intensity of the long wavelength component when dentine was exposed to glucose. Increase of the fluorescence component with the long wavelength region leads to red-shift of the observed fluorescence spectrum. Although we too found a red-shift of the fluorescence spectrum in the aged and glycated dentine, the resultant fluorescence intensity value was ambiguous because of the limitation of the employed spectrometer. To confirm the overall fluorescence spectral profile, we have to use an adequate fluorescence spectrophotometer. The combination
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Fig. 4 – Results of mechanical tests and immunohistochemical analysis of demineralized dentine before and after 6 weeks glycation in ribose solution. (a): Force–displacement characteristics of young, glycated young, and aged coronal and root dentine. The data are shown as mean W SEM (n = 3). P-values were determined by one-way ANOVA and Tukey–Kramer test (*p < 0.01, #p < 0.05). (b) and (d): Immunohistochemical images of young coronal and root dentine after 6 weeks glycation in ribose solution, (c) and (e): magnified image of the box in (b) and (d), respectively. Intertubular dentine was stained in each portion after the glycation, while peritubular dentine was stained before glycation (bar: 5 mm P: pulp, D: dentine).
results from the mechanical test and fluorometry for dentinal collagen strongly suggest the existence of AGEs in aged dentine. To confirm the suggestion, we next performed immunohistochemical staining. AGEs include many types of molecules, e.g., pentosidine is a marker for identification of osteoporosis in females and bone fracture in type 2 diabetes.15 CML is known to increase in human collagen with age, and age-adjusted concentrations of CML increase in skin collagen in diabetes.7 Ac¸il et al. focused on hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), and concluded that there was no relationship between the dentinal concentrations of these molecules and age.16 Though we stained only CML in the present study, we assume that there is an appreciable relationship between the dentinal concentration of AGEs and age. Because dentinal collagen does not regenerate within
one’s lifetime, the accumulated amount of AGE in dentinal collagen is a potential indicator of human age, making it applicable in forensic medicine. We observed the accumulation of AGEs in the collagen fibrils around dentinal tubules (Fig. 3(f) and (h)). These characteristics of localization indicated that Maillard reaction occurred at the contact region between dentinal collagen and the glucose rich tissue fluid. Collagen in which AGEs accumulate became harder and more fragile; therefore this accumulation with ageing may affect the mechanical properties of dentine such as dentinal crack propagation. Modifications of the collagenous matrix are structurally and mechanically important, and the breakage of cross-linking can result in severe tissue dysfunction.17 In recent years, several reports show that artificially formed
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cross-linking in dentinal collagen reduces the activities of matrix metalloproteinase.18,19 Therefore, degradation of the hybrid layer between dentine and bonding agent was suppressed by cross-linking in dentinal collagen.20,21 Our report indicates that accumulation of AGEs with human ageing may also affect the bonding strength and long-term stability of dentine-bonding interfaces. Moreover, alteration of colour in dentinal collagen can be attributed to gradual accumulation of AGEs with ageing. This phenomenon will affect the concept of whitening discoloured tooth. To understand the homeostatic stability in dentine, we should further identify the types of AGE molecules observed in dentinal collagen.
Funding This work was supported by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant Numbers 22791831, 23240069, 23650260 and 25462957.
Competing interests The authors declare no potential conflicts of interest with respect to the authorship and publication of this article.
Ethical approval The experimental protocol was approved by the Ethics committee of the Faculty of Dentistry, Osaka University (H24-E25).
Acknowledgements This project was technically supported by the Research Center for Ultra-High-Voltage Electron Microscopy of Osaka University. The authors would like to thank Hirotaro Mori for his valuable suggestions and constant support, Yu Usami for technical support in the immunohistochemical analysis, and Keita Kondo for measuring the mechanical properties.
references
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