- Email: [email protected]
Denaturation Temperatures of Dentin Matrices. I. Effect of Demineralization and Dehydration
Basic Research—Biology
Denaturation Temperatures of Dentin Matrices. I. Effect of Demineralization and Dehydration Steven R. Armstrong, DDS, PhD,* Julie L.P. Jessop, PhD,* Erik Winn, DDS,* Franklin R. Tay, BDSc (Hons), PhD,† and David H. Pashley, DMD, PhD† Abstract The denaturation temperature (Td) of dentin collagen in mineralized versus demineralized teeth was examined as a function of dentin age and the extent of dehydration. Using differential scanning calorimetry, Td of mineralized dentin was shown to be between 160°C to 186°C, depending on whether it was from young or old dentin that was hydrated or dehydrated, respectively. Demineralized dentin exhibited a Td of 65.6°C that increased with dehydration to 176°C. The presence of apatite crystallites or interpeptide bonding increased the Td of demineralized matrices. Interpeptide hydrogen bonding seems to stabilize collagen to thermal challenge. Water breaks interpeptide hydrogen bonds making collagen more susceptible to thermal denaturation. Rises in intracanal temperature are unlikely to cause extensive denaturation of mineralized root dentin walls. However, hydrated or partially dehydrated root canal walls that have been partially demineralized with chelating agents or mild acids may be susceptible to thermal denaturation. (J Endod 2006;32:638 – 641)
Key Words Collagen, dentin, differential scanning calorimetry, glass transition
*From the University of Iowa, Iowa City, Iowa; †Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, Georgia. Address requests for reprint to Dr. David H. Pashley, Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA 309121129. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2005.10.062
I
n endodontics, the use of poorly water-cooled high speed burs during access to root canals can burn mineralized dentin and turn its color from pale yellow to dark brown or black. During obturation of the root canals, intraradicular dentin that is in direct contact with heated instruments may be exposed to 170°C to 200°C during warm vertical compaction of thermoplastic root filling materials. There may be sufficient heat to thermally denature the adjacent mineralized dentin matrices causing a loss in mechanical strength. As dentin ages, it often becomes hypermineralized and sclerotic because of the deposition of mineral crystals within the dentinal tubules. Physiological sclerosis may occur in the apical third of root dentin (1). These extra minerals may make old mineralized dentin less hydrated than young dentin (2). During cleaning and shaping, root canals are exposed to chelating agents such as ethylenediamine tetra-acetic acid (EDTA), and mild acids such as doxycycline and/or citric acid that can partially demineralize the surface of intraradicular dentin and create 2 to 10 m thick layers of partially demineralized collagen (3–5). Unless a deproteinizing agent such as sodium hypochlorite is employed as the final rinse, these denuded collagen fibrils may remain hydrated to a variable extent, depending on the method employed for drying the irrigated root canals (6, 7). Presumably, the loss of apatite crystallites would make the demineralized dentin collagen matrix more susceptible to thermal denaturation. The effects of dehydration on the denaturation temperature (Td) of demineralized dentin collagen are unknown. However, for rat tail tendon that consists mostly of unmineralized type I collagen (8), Td is inversely related to the extent of hydration of collagen (8), up to a critical level when the intrafibrillar network is fully saturated with water (9). Thereafter, Td remains constant irrespective of how much excess water is added (9). Apparently, the thermal agitation of water is responsible for molecular disruptions of collagen molecules. Thus, thermal stability represents the resistance of the collagen molecules to unfolding as a result of heat treatment. This unfolding is believed to consist of two steps: the separation of the triple helices into individual helices, and the subsequent unfolding of these individual helices into random coils (10). The first step involves the disruption of water bridges (hydrogen bond) between the three polypeptide chains of the tropocollagen molecule (11). The second step involves the disruption of intrahelical hydrogen bonds of the ␣-chains (12). Previous reports are available on the thermal stability and denaturation temperatures of mineralized or demineralized collagen matrices were mostly performed on rat tails or turkey leg tendons (10, 13–17). Thus, the objective of this study was to examine, with the use of differential scanning calorimetry (DSC), the effect of dehydration on the denaturation temperatures of human dentin matrices. Our test hypothesis is that the denaturation temperatures of both mineralized and demineralized dentin matrices are determined, in part, by the amount of intrinsic hydrogen bonding between collagen peptides. In the presence of water, the strongest hydrogen bonding solvent known, little interpeptide hydrogen bonding can occur. At the other extreme, in completely dehydrated matrices, maximum hydrogen bonding can occur between adjacent collagen peptides. These should change the denaturation temperature of wet versus dry matrices.
Materials and Methods Extracted third molars were obtained from young patients (18 –23 yr) and mandibular incisors were obtained from old (⬎65 yr) patients, under a protocol approved by the Institutional Review Board of the University of Iowa.
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Basic Research—Biology TABLE 1. Denaturation temperature (Td) of human dentin Mineralized dentin
Demineralized dentin/hydration status Hydration of rat tail collagen (for comparison)*
Dentin Condition/Treatment
Td (°C)†
Hydrated young coronal dentin Dehydrated young coronal dentin Hydrated young root dentine Hydrated old root dentine Fully hydrated I Intermediate hydration II Low hydration III Very low hydration IV Fully hydrated I Intermediate hydration II Low hydration III Very low hydration IV
170.4 ⫾ 3.4b (4) 186.5 ⫾ 7.5b (4) 159.1 ⫾ 12.7a (3) 177.5 ⫾ 2.9b (3) 65.6 ⫾ 2.51d (5) 112.9 ⫾ 28.13c (7) 128.6 ⫾ 15.95c (10) 176.1 ⫾ 37.21b (3) 65.1 74.3 110.8 188.5
†Values are mean Td ⫾ SD in °C (number of specimens). Data identified by different superscript letters are significantly different (p ⬍ 0.05). *From reference 8, using regression lines to find mid-points of levels 2, 3, 4.
Mineralized Dentin Specimens Dentin disks 1-mm thick were cut from mid-coronal dentin or roots using an Isomet saw (Buehler Ltd., Lake Bluff, IL). These disks were cut into 3 mm ⫻ 3 mm dentin blocks using a medium diamond bur in a high speed dental handpiece with copious air-water spray. They were stored in artificial saliva (AS) containing 0.01% sodium azide (pH 7.0) to inhibit microbial growth. Demineralized Dentin Specimens Similarly prepared dentin disks were completely demineralized in 0.5 M EDTA (pH 7.4) with constant stirring for 14 days at 25°C, rinsed free of EDTA with water for 1 h, and then cut into 3 mm ⫻ 1 mm blocks with a razor blade and stored in AS containing sodium azide until used. Dehydration of Demineralized Dentin The demineralized dentin blocks were weighed wet and then placed in sealed jars containing anhydrous calcium sulfate (Drierite, Fisher Scientific, Chicago, IL) for 2 h to obtain dry weight. After rehydration in water, they were either used fully hydrated, or they were removed after varying times (minutes) from the container of Drierite and quickly sealed in tared high pressure DSC pans. All the blocks were subsequently weighed. This permitted gravimetric calculation of the water content of the matrix as a volume fraction (8). Measurement of denaturation temperature Specimens sealed in high pressure pans were placed in a differential scanning calorimeter (DSC-7, Perkin Elmer, Wellesley, MA) and scanned from 25°C to 200°C at a rate of 10°C/min. Subsequent cooling and reheating confirmed that the collagen denaturation was irreversible (9). The DSC was calibrated prior to use with iridium standards. Effects of age, location and dehydration on Td of mineralized dentin Beginning with fully hydrated young (18 –23 yr) coronal mineralized dentin, the Td was determined. In another group of mineralized dentin, the hydrated specimens were stored overnight at 121°C under a vacuum before being placed in DSC pans to test the effects of dehydration. Three specimens of fully hydrated young root dentin were used to determine the Td of root versus coronal dentin. This was repeated on fully hydrated root dentin from old patients (⬎65 yr). Effects of hydration of the Td of demineralized dentin Blocks of completely demineralized dentin from coronal dentin of young teeth were dried over anhydrous calcium sulfate to a constant dry weight. They were then fully rehydrated in water for 2 h and then stored JOE — Volume 32, Number 7, July 2006
in separate labeled containers. Each specimen was removed and then placed in a jar containing anhydrous calcium sulfate for 0 to 120 min to create a wide range of degrees of hydration. As soon as they were removed from the jar, they were sealed in tared high pressure DSC pans that were subsequently carefully weighed to calculate the water content that was expressed as the molar ratio of water to Glycine-X-Y tripeptide unit (8) or in water volume fraction. The specimens were then immediately scanned in the DSC. After cooling, specimens were rescanned to confirm that there was an irreversible phase transition (9). All pans were then pierced and heated to 125°C for 5 min to volatilize all solvents and reweighed for calculation of water volume fraction.
Statistical analysis Because of the broad range of the means, the data set was not normally distributed. Accordingly, the data were analyzed by one-way ANOVA on ranks. Multiple comparisons were done with Dunn’s method at the 95% confidence level.
Results The results of this study are summarized in Table 1 For mineralized dentin, Td for fully hydrated, young coronal dentin (i.e. 18 –23 yr-oldunerupted third molars) was 170.4 ⫾ 3.4°C (n ⫽ 4). When such dentin was dehydrated overnight, the Td rose significantly (p ⬍ 0.05) to 186.5 ⫾ 7.5°C (n ⫽ 4). Hydrated young root dentin had a Td of 159.1 ⫾ 12.7°C (n ⫽ 3) that was not significantly different from hydrated young coronal dentin. The Td of hydrated old (i.e. ⬎65 yr) root dentin (177.5 ⫾ 2.9°C) was not significantly different from hydrated young root dentin or hydrated young coronal dentin. When dentin specimens were completely demineralized in EDTA before scanning, the Td for fully hydrated young tooth specimens was only 65.6 ⫾ 2.5°C (n ⫽ 5). This Td value was significantly lower (p ⬍ 0.01) than that of fully hydrated mineralized dentin. When the state of hydration of young coronal dentin was varied according to the method of Miles and Ghelashvili (8), the Td of the specimens increased as the water content fell (Fig. 1). That is, when the molar ratio of water to collagen was in excess of 30, Td was constant at about 65.6°C (hydration level 1). As the water content fell (molar ratios between 6 –30, hydration level 2, Fig. 1), the Td increased to about 113°C. Further dehydration to molar ratios between 1 and 6, increased the Td to about 129°C (hydration level 3, Fig. 1). Prolonged dehydration to reduce the molar ratio below 1.0 gave a mean Td of 176°C (hydration level 4, Fig. 1, Table 1). Figure 2 shows the Td values of demineralized dentin matrices plotted as a function of the fractional volume of the fully hydrated state. When the data in hydration level 2 was plotted as least squares fits, the
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Basic Research—Biology
Figure 1. The effect of hydration, expressed as the molar ratio of water to collagen peptides, on thermal denaturation of the dentin matrix using the method of Miles and Ghelashvili (8): (1) fully hydrated state with adequate intrafibrillar and interfibrillar water with excess water (i.e. ratios above 30) having no effect on fiber arrangement; (2) intermediate hydration level with decreasing external but adequate intrafibrillar water to maintain fibril arrangement; (3) low hydration levels with progressive loss of hydrogen bonds (water bridges) to the triple helix; (4) very low hydration with one molecule or less of water per Gly-X-Y. This progressive loss of water leads to collapse of the matrix and the formation of interpeptide hydrogen bonding that stabilizes the collagen and therefore increases the denaturation temperature as measured by DSC.
y intercept of Td at a fractional water volume of zero yielded a theoretical Td of dry collagen of 163.3°C. This compares reasonably well with the Td values of 176.1 ⫾ 37.2°C for hydration level 4 for demineralized dentine matrices (Table 1). Table 1 also includes Td results obtained by Miles and Ghelashvili (8) using rat tail tendon for comparison with the results obtained in the current study using dentin matrices. The Td values given are water volume fraction mid points from regions 2, 3, and 4 calculated from published linear regressions (8). They are close to the values obtained with dentin matrices in the current study.
between the collagen fibrils are maximal. Water fills the interfibrillar spaces (20) and easily penetrates the collagen peptides where it hydrogen bonds to functional groups. In dry demineralized dentin (very low hydration state 4, Table 1, Td ⫽ 176.1°C), there is maximum interpeptide hydrogen bonding that stabilizes the collagen peptides (21, 22), and there is no solvent available to transmit the thermal energy from the pan to collagen to disrupt its structure. Thus, higher temperatures must be used to thermally agitate the weakest portion of the collagen matrix. To date, most of the studies that examined temperature rises associated with the use of endodontic heat sources have concentrated on increases in temperature along the external root surfaces (23–27). This is understandable, as potential damages to tooth-supporting structures are of immediate clinical concern. Temperature rise of the internal root surfaces up to 74.2°C above room temperature have been reported with the use of electrical heat sources (28, 29). As the Td of mineralized young and old root dentin varies between 159.1°C to 117.5°C, it is unlikely that the use of these heat sources will create gross denaturation of mineralized dentin collagen. However, prolonged direct contact of a System B heat source (SybronEndo, Orange CA) at 200°C to 250°C to paper-point blot-dried root canal walls that are not optimally coated with root canal sealers may result in localized denaturation of the mineralized root dentin. Ultrastructually, this may be manifested as the transformation of mineralized collagen to mineralized gelatin, that becomes apparent as the hard tissue is eventually demineralized and stained (Tay, unpublished results). Theoretically, these localized dehydrated/denatured regions may be more brittle (30) and may act as sites where crack initiation arises (31) within the endodontically-treated dentin. An even more pressing issue is the recent observation of demineralized collagen matrices on root canal walls (4, 5) that are irrigated with 17% EDTA or BioPure MTAD (DentsplyTulsa, Tulsa, OK), a new, acidic antibacterial endodontic irrigant that consists of doxycycline hyclate, citric acid, and a detergent (32, 33). As hydrated and briefly dehydrated demineralized dentin exhibited Td between 65.6°C to 112.9°C, thermal denaturation of demineralized root dentin collagen is
Discussion Within the limits of this study, it may be concluded that the presence of minerals increased the Td of demineralized dentin collagen matrices. This was shown by comparing the Td of young fully hydrated demineralized dentin (65.6 ⫾ 2.5°C) to that of young fully hydrated mineralized dentin (170.4 ⫾ 3.4°C). Additional increase in Td was achieved in mineralized dentin by dehydrating it. The results require acceptance of the test hypothesis. The differences in Td in demineralized dentin as a function of hydration were even larger in terms of percentage changes. That is, very dry demineralized dentin had a Td that was 260% higher than fully hydrated demineralized dentin (compare 176.1 to 65.6°C, Table). Even more remarkable is the observation that very dry demineralized dentin has a Td that is equivalent to the Td of fully hydrated, old mineralized root dentin (Table 1). Interpeptide hydrogen bonding within collagen fibrils seems to stabilize them against thermal challenge. We speculate that water breaks interpeptide hydrogen bonds making collagen more susceptible to thermal denaturation (18). The interfibrillar spaces between collagen fibrils are 20 to 30 nm wide in fully hydrated demineralized dentin (19), but can be virtually absent in air-dried matrices (20). In the presence of water, there is little interpeptide hydrogen bonding, and the spaces 640
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Figure 2. A linear relationship is demonstrated in the intermediate collagen hydration level (water volume fraction 0.32– 0.70). Using the reciprocal °K regression characteristics, y ⫽ 0.001096x ⫹ 0.002034, R2 ⫽ 0.5969, p ⫽ 0.0417, the midpoint of this region at 18 molar ratio or water volume fraction of 0.584 gives a Tmax ⫽ 101.9 °C. The regression was not significant in the lower hydration levels. This may be a result of a progressively collapsed structure as molecular rearrangement compensates for water bridging loss from the gaps that existed in a regular quarter-stagger fiber arrangement (8).
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Basic Research—Biology possible. As the denatured collagen is slowly digested by the release of host-derived matrix metalloproteinase (34, 35), leakage may appear in initially leakage-free root fillings. Such an issue is of clinical concern in the light of current interest in the application of dentin bonding technologies to endodontics, and will be reported in part 2 of this study.
Acknowledgments This work was supported, in part, by grants R01 DE 014911 and R01 DE 015306 (P.I.-D. H. Pashley) from the National Institute of Dental and Craniofacial Research and by R03 DE 14799-01 (P.I. S.R. Armstrong). The authors are grateful to Michelle Barnes for secretarial support.
References 1. Stanley HR, Pereira JC, Spiegel E, Broom C, Schultz M. The detection and prevalence of reactive and physiologic sclerotic dentin, reparative dentin and dead tracts beneath various types of dental lesions according to tooth surface and age. J Oral Pathol 1983;12:257– 89. 2. Toto PD, Kastelic EF, Duyvejonck KJ, Rapp GW. Effect of age on water content in human teeth. J Dent Res 1971;50:1284 –5. 3. Machnick TK, Torabinejad M, Munoz CA, Shabahang S. Effect of MTAD on the bond strength to enamel and dentin. J Endod 2003;29:818 –21. 4. Tay FR, Pashley DH, Loushine RJ, et al. Ultrastructure of smear layer-covered intraradicular dentin following irrigation with BioPure MTAD. J Endod (in press). 5. Tay FR, Hosoya Y, Loushine RJ, Pashley DH, Weller RN, Low DCY. Ultrastructure of intraradicular dentin following irrigation with BioPure MTAD. II. The consequence of obturation with an epoxy resin-based sealer. J Endod (in press). 6. Petschelt A. Drying of root canals. Dtsch Zahnarztl Z 1990;45:222– 6. 7. Hosoya N, Nomura M, Yoshikubo A, Arai T, Nakamura J, Cox CF. Effect of canal drying methods on the apical seal. J Endod 2000;26:292– 4. 8. Miles CA, Ghelashvili M. Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers. Biophy J 1999;76:3243–52. 9. Miles CA, Avery NC, Rodin VV, Bailey AJ. The increase in denaturation temperature following cross-linking of collagen is caused by dehydration of the fibres. J Mol Biol 2005;346:551– 6. 10. Rochdi A, Foucat L, Renou JP. Effect of thermal denaturation on water-collagen interactions: NMR relaxation and differential scanning calorimetry analysis. Biopolymers 1999;50:690 – 6. 11. Ramachandran GN, Chandrasekharan R. Interchain hydrogen bonds via bound water molecules in the collagen triple helix. Biopolymers 1968;6:1649 –58. 12. Luescher M, Rüegg M, Schindler P. Effect of hydration upon the thermal stability of tropocollagen and its dependence on the presence of neutral salts. Biopolymers 1974;13:2489 –2503. 13. Kopp J, Bonnet M, Renou JP. Effect of collagen crosslinking on collagen-water interactions (a DSC investigation). Matrix 1989;9:443–50.
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14. Bigi A, Ripamonti A, Cojazzi G, Pizzuto G, Roveri N, Koch MH. Structural analysis of turkey tendon collagen upon removal of the inorganic phase. Int J Biol Macromol 1991;13:110 – 4. 15. Kronick PL, Cooke P. Thermal stabilization of collagen fibers by calcification. Connect Tissue Res 1996;33:275– 82. 16. Friess W, Lee G. Basic thermoanalytical studies of insoluble collagen matrices. Biomaterials 1996;17:2289 –94. 17. Fukuda K, Nezu T, Terada Y. The effect of alcoholic compounds on the stability of type I collagen studied by differential scanning calorimetry. Dent Mater J 2000;19:221– 8. 18. Mogilner IG, Ruderman G, Grigera JR. Collagen stability, hydration and native state. J Mol Graph Model 2002;21:209 –13. 19. Pashley DH, Carvalho RM, Agee KA, Lee W-K, Tay FR, Callison TE. Effects of water- free polar solvents on tensile properties of demineralized dentin. Dent Mater 2003;19:347–52. 20. Carvalho RM, Mendonca JS, Santiago SL, et al. Effects of HEMA/solvent combinations on bond strength to dentin. J Dent Res 2003;82:597– 601. 21. Pashley DH, Agee K, Nakajima M, et al. Solvent-induced dimensional changes in EDTA-demineralized dentin matrix. J Biomed Mater Res 2001;56:273– 81. 22. Eddleston C, Hindle A, Rueggeberg FA, et al. Dimensional changes in demineralized dentin matrices following the use of HEMA-water vs. HEMA-alcohol primers. J Biomed Mater Res 2003;67A:900 –7. 23. Weller RN, Kimbrough WF, Anderson RW. Root surface temperatures produced during post space preparation. J Endod 1996;22:304 –7. 24. Lee FS, Van Cura JE, BeGole E. A comparison of root surface temperatures using different obturation heat sources. J Endod 1998;24:617–20. 25. Floren JW, Weller RN, Pashley DH, Kimbrough WF. Changes in root surface temperatures with in vitro use of the System B heat source. J Endod 1999;25:593–5. 26. Behnia A, McDonald NJ. In vitro infrared thermographic assessment of root surface temperatures generated by the Thermafil Plus system. J Endod 2001;27:203–5. 27. Lipski M. Root surface temperature rises in vitro during root canal obturation using hybrid and microseal techniques. J Endod 2005;31:297–300. 28. Jurcak JJ, Weller RN, Kulild JC, Donley DL. In vitro intracanal temperatures produced during warm lateral condensation of gutta-percha. J Endod 1992;18:1–3. 29. Sweatman TL, Baumgartner JC, Sakaguchi RL. Radicular temperatures associated with thermoplasticized gutta-percha. J Endod 2001;27:512–5. 30. Kishen A, Asundi A. Experimental investigation on the role of water in the mechanical behavior of structural dentine. J Biomed Mater Res A 2005;73:192–200. 31. Griffith AA. The phenomena of rupture and flow in solids. Phil Trans Roy Soc London A 1920;221:163–98. 32. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170 –5. 33. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. J Endod 2003;29:233–9. 34. Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, Ito S. Collagen degradation by host-derived enzymes during aging. J Dent Res 2004;83:216 –21. 35. Hebling J, Pashley DH, Tjäderhane L, Tay FR. Chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. J Dent Res 2005;84:741– 6.
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Denaturation Temperatures of Dentin Matrices. I. Effect of Demineralization and Dehydration Steven R. Armstrong, DDS, PhD,* Julie L.P. Jessop, PhD,* Erik Winn, DDS,* Franklin R. Tay, BDSc (Hons), PhD,† and David H. Pashley, DMD, PhD† Abstract The denaturation temperature (Td) of dentin collagen in mineralized versus demineralized teeth was examined as a function of dentin age and the extent of dehydration. Using differential scanning calorimetry, Td of mineralized dentin was shown to be between 160°C to 186°C, depending on whether it was from young or old dentin that was hydrated or dehydrated, respectively. Demineralized dentin exhibited a Td of 65.6°C that increased with dehydration to 176°C. The presence of apatite crystallites or interpeptide bonding increased the Td of demineralized matrices. Interpeptide hydrogen bonding seems to stabilize collagen to thermal challenge. Water breaks interpeptide hydrogen bonds making collagen more susceptible to thermal denaturation. Rises in intracanal temperature are unlikely to cause extensive denaturation of mineralized root dentin walls. However, hydrated or partially dehydrated root canal walls that have been partially demineralized with chelating agents or mild acids may be susceptible to thermal denaturation. (J Endod 2006;32:638 – 641)
Key Words Collagen, dentin, differential scanning calorimetry, glass transition
*From the University of Iowa, Iowa City, Iowa; †Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, Georgia. Address requests for reprint to Dr. David H. Pashley, Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA 309121129. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2005.10.062
I
n endodontics, the use of poorly water-cooled high speed burs during access to root canals can burn mineralized dentin and turn its color from pale yellow to dark brown or black. During obturation of the root canals, intraradicular dentin that is in direct contact with heated instruments may be exposed to 170°C to 200°C during warm vertical compaction of thermoplastic root filling materials. There may be sufficient heat to thermally denature the adjacent mineralized dentin matrices causing a loss in mechanical strength. As dentin ages, it often becomes hypermineralized and sclerotic because of the deposition of mineral crystals within the dentinal tubules. Physiological sclerosis may occur in the apical third of root dentin (1). These extra minerals may make old mineralized dentin less hydrated than young dentin (2). During cleaning and shaping, root canals are exposed to chelating agents such as ethylenediamine tetra-acetic acid (EDTA), and mild acids such as doxycycline and/or citric acid that can partially demineralize the surface of intraradicular dentin and create 2 to 10 m thick layers of partially demineralized collagen (3–5). Unless a deproteinizing agent such as sodium hypochlorite is employed as the final rinse, these denuded collagen fibrils may remain hydrated to a variable extent, depending on the method employed for drying the irrigated root canals (6, 7). Presumably, the loss of apatite crystallites would make the demineralized dentin collagen matrix more susceptible to thermal denaturation. The effects of dehydration on the denaturation temperature (Td) of demineralized dentin collagen are unknown. However, for rat tail tendon that consists mostly of unmineralized type I collagen (8), Td is inversely related to the extent of hydration of collagen (8), up to a critical level when the intrafibrillar network is fully saturated with water (9). Thereafter, Td remains constant irrespective of how much excess water is added (9). Apparently, the thermal agitation of water is responsible for molecular disruptions of collagen molecules. Thus, thermal stability represents the resistance of the collagen molecules to unfolding as a result of heat treatment. This unfolding is believed to consist of two steps: the separation of the triple helices into individual helices, and the subsequent unfolding of these individual helices into random coils (10). The first step involves the disruption of water bridges (hydrogen bond) between the three polypeptide chains of the tropocollagen molecule (11). The second step involves the disruption of intrahelical hydrogen bonds of the ␣-chains (12). Previous reports are available on the thermal stability and denaturation temperatures of mineralized or demineralized collagen matrices were mostly performed on rat tails or turkey leg tendons (10, 13–17). Thus, the objective of this study was to examine, with the use of differential scanning calorimetry (DSC), the effect of dehydration on the denaturation temperatures of human dentin matrices. Our test hypothesis is that the denaturation temperatures of both mineralized and demineralized dentin matrices are determined, in part, by the amount of intrinsic hydrogen bonding between collagen peptides. In the presence of water, the strongest hydrogen bonding solvent known, little interpeptide hydrogen bonding can occur. At the other extreme, in completely dehydrated matrices, maximum hydrogen bonding can occur between adjacent collagen peptides. These should change the denaturation temperature of wet versus dry matrices.
Materials and Methods Extracted third molars were obtained from young patients (18 –23 yr) and mandibular incisors were obtained from old (⬎65 yr) patients, under a protocol approved by the Institutional Review Board of the University of Iowa.
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Basic Research—Biology TABLE 1. Denaturation temperature (Td) of human dentin Mineralized dentin
Demineralized dentin/hydration status Hydration of rat tail collagen (for comparison)*
Dentin Condition/Treatment
Td (°C)†
Hydrated young coronal dentin Dehydrated young coronal dentin Hydrated young root dentine Hydrated old root dentine Fully hydrated I Intermediate hydration II Low hydration III Very low hydration IV Fully hydrated I Intermediate hydration II Low hydration III Very low hydration IV
170.4 ⫾ 3.4b (4) 186.5 ⫾ 7.5b (4) 159.1 ⫾ 12.7a (3) 177.5 ⫾ 2.9b (3) 65.6 ⫾ 2.51d (5) 112.9 ⫾ 28.13c (7) 128.6 ⫾ 15.95c (10) 176.1 ⫾ 37.21b (3) 65.1 74.3 110.8 188.5
†Values are mean Td ⫾ SD in °C (number of specimens). Data identified by different superscript letters are significantly different (p ⬍ 0.05). *From reference 8, using regression lines to find mid-points of levels 2, 3, 4.
Mineralized Dentin Specimens Dentin disks 1-mm thick were cut from mid-coronal dentin or roots using an Isomet saw (Buehler Ltd., Lake Bluff, IL). These disks were cut into 3 mm ⫻ 3 mm dentin blocks using a medium diamond bur in a high speed dental handpiece with copious air-water spray. They were stored in artificial saliva (AS) containing 0.01% sodium azide (pH 7.0) to inhibit microbial growth. Demineralized Dentin Specimens Similarly prepared dentin disks were completely demineralized in 0.5 M EDTA (pH 7.4) with constant stirring for 14 days at 25°C, rinsed free of EDTA with water for 1 h, and then cut into 3 mm ⫻ 1 mm blocks with a razor blade and stored in AS containing sodium azide until used. Dehydration of Demineralized Dentin The demineralized dentin blocks were weighed wet and then placed in sealed jars containing anhydrous calcium sulfate (Drierite, Fisher Scientific, Chicago, IL) for 2 h to obtain dry weight. After rehydration in water, they were either used fully hydrated, or they were removed after varying times (minutes) from the container of Drierite and quickly sealed in tared high pressure DSC pans. All the blocks were subsequently weighed. This permitted gravimetric calculation of the water content of the matrix as a volume fraction (8). Measurement of denaturation temperature Specimens sealed in high pressure pans were placed in a differential scanning calorimeter (DSC-7, Perkin Elmer, Wellesley, MA) and scanned from 25°C to 200°C at a rate of 10°C/min. Subsequent cooling and reheating confirmed that the collagen denaturation was irreversible (9). The DSC was calibrated prior to use with iridium standards. Effects of age, location and dehydration on Td of mineralized dentin Beginning with fully hydrated young (18 –23 yr) coronal mineralized dentin, the Td was determined. In another group of mineralized dentin, the hydrated specimens were stored overnight at 121°C under a vacuum before being placed in DSC pans to test the effects of dehydration. Three specimens of fully hydrated young root dentin were used to determine the Td of root versus coronal dentin. This was repeated on fully hydrated root dentin from old patients (⬎65 yr). Effects of hydration of the Td of demineralized dentin Blocks of completely demineralized dentin from coronal dentin of young teeth were dried over anhydrous calcium sulfate to a constant dry weight. They were then fully rehydrated in water for 2 h and then stored JOE — Volume 32, Number 7, July 2006
in separate labeled containers. Each specimen was removed and then placed in a jar containing anhydrous calcium sulfate for 0 to 120 min to create a wide range of degrees of hydration. As soon as they were removed from the jar, they were sealed in tared high pressure DSC pans that were subsequently carefully weighed to calculate the water content that was expressed as the molar ratio of water to Glycine-X-Y tripeptide unit (8) or in water volume fraction. The specimens were then immediately scanned in the DSC. After cooling, specimens were rescanned to confirm that there was an irreversible phase transition (9). All pans were then pierced and heated to 125°C for 5 min to volatilize all solvents and reweighed for calculation of water volume fraction.
Statistical analysis Because of the broad range of the means, the data set was not normally distributed. Accordingly, the data were analyzed by one-way ANOVA on ranks. Multiple comparisons were done with Dunn’s method at the 95% confidence level.
Results The results of this study are summarized in Table 1 For mineralized dentin, Td for fully hydrated, young coronal dentin (i.e. 18 –23 yr-oldunerupted third molars) was 170.4 ⫾ 3.4°C (n ⫽ 4). When such dentin was dehydrated overnight, the Td rose significantly (p ⬍ 0.05) to 186.5 ⫾ 7.5°C (n ⫽ 4). Hydrated young root dentin had a Td of 159.1 ⫾ 12.7°C (n ⫽ 3) that was not significantly different from hydrated young coronal dentin. The Td of hydrated old (i.e. ⬎65 yr) root dentin (177.5 ⫾ 2.9°C) was not significantly different from hydrated young root dentin or hydrated young coronal dentin. When dentin specimens were completely demineralized in EDTA before scanning, the Td for fully hydrated young tooth specimens was only 65.6 ⫾ 2.5°C (n ⫽ 5). This Td value was significantly lower (p ⬍ 0.01) than that of fully hydrated mineralized dentin. When the state of hydration of young coronal dentin was varied according to the method of Miles and Ghelashvili (8), the Td of the specimens increased as the water content fell (Fig. 1). That is, when the molar ratio of water to collagen was in excess of 30, Td was constant at about 65.6°C (hydration level 1). As the water content fell (molar ratios between 6 –30, hydration level 2, Fig. 1), the Td increased to about 113°C. Further dehydration to molar ratios between 1 and 6, increased the Td to about 129°C (hydration level 3, Fig. 1). Prolonged dehydration to reduce the molar ratio below 1.0 gave a mean Td of 176°C (hydration level 4, Fig. 1, Table 1). Figure 2 shows the Td values of demineralized dentin matrices plotted as a function of the fractional volume of the fully hydrated state. When the data in hydration level 2 was plotted as least squares fits, the
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Figure 1. The effect of hydration, expressed as the molar ratio of water to collagen peptides, on thermal denaturation of the dentin matrix using the method of Miles and Ghelashvili (8): (1) fully hydrated state with adequate intrafibrillar and interfibrillar water with excess water (i.e. ratios above 30) having no effect on fiber arrangement; (2) intermediate hydration level with decreasing external but adequate intrafibrillar water to maintain fibril arrangement; (3) low hydration levels with progressive loss of hydrogen bonds (water bridges) to the triple helix; (4) very low hydration with one molecule or less of water per Gly-X-Y. This progressive loss of water leads to collapse of the matrix and the formation of interpeptide hydrogen bonding that stabilizes the collagen and therefore increases the denaturation temperature as measured by DSC.
y intercept of Td at a fractional water volume of zero yielded a theoretical Td of dry collagen of 163.3°C. This compares reasonably well with the Td values of 176.1 ⫾ 37.2°C for hydration level 4 for demineralized dentine matrices (Table 1). Table 1 also includes Td results obtained by Miles and Ghelashvili (8) using rat tail tendon for comparison with the results obtained in the current study using dentin matrices. The Td values given are water volume fraction mid points from regions 2, 3, and 4 calculated from published linear regressions (8). They are close to the values obtained with dentin matrices in the current study.
between the collagen fibrils are maximal. Water fills the interfibrillar spaces (20) and easily penetrates the collagen peptides where it hydrogen bonds to functional groups. In dry demineralized dentin (very low hydration state 4, Table 1, Td ⫽ 176.1°C), there is maximum interpeptide hydrogen bonding that stabilizes the collagen peptides (21, 22), and there is no solvent available to transmit the thermal energy from the pan to collagen to disrupt its structure. Thus, higher temperatures must be used to thermally agitate the weakest portion of the collagen matrix. To date, most of the studies that examined temperature rises associated with the use of endodontic heat sources have concentrated on increases in temperature along the external root surfaces (23–27). This is understandable, as potential damages to tooth-supporting structures are of immediate clinical concern. Temperature rise of the internal root surfaces up to 74.2°C above room temperature have been reported with the use of electrical heat sources (28, 29). As the Td of mineralized young and old root dentin varies between 159.1°C to 117.5°C, it is unlikely that the use of these heat sources will create gross denaturation of mineralized dentin collagen. However, prolonged direct contact of a System B heat source (SybronEndo, Orange CA) at 200°C to 250°C to paper-point blot-dried root canal walls that are not optimally coated with root canal sealers may result in localized denaturation of the mineralized root dentin. Ultrastructually, this may be manifested as the transformation of mineralized collagen to mineralized gelatin, that becomes apparent as the hard tissue is eventually demineralized and stained (Tay, unpublished results). Theoretically, these localized dehydrated/denatured regions may be more brittle (30) and may act as sites where crack initiation arises (31) within the endodontically-treated dentin. An even more pressing issue is the recent observation of demineralized collagen matrices on root canal walls (4, 5) that are irrigated with 17% EDTA or BioPure MTAD (DentsplyTulsa, Tulsa, OK), a new, acidic antibacterial endodontic irrigant that consists of doxycycline hyclate, citric acid, and a detergent (32, 33). As hydrated and briefly dehydrated demineralized dentin exhibited Td between 65.6°C to 112.9°C, thermal denaturation of demineralized root dentin collagen is
Discussion Within the limits of this study, it may be concluded that the presence of minerals increased the Td of demineralized dentin collagen matrices. This was shown by comparing the Td of young fully hydrated demineralized dentin (65.6 ⫾ 2.5°C) to that of young fully hydrated mineralized dentin (170.4 ⫾ 3.4°C). Additional increase in Td was achieved in mineralized dentin by dehydrating it. The results require acceptance of the test hypothesis. The differences in Td in demineralized dentin as a function of hydration were even larger in terms of percentage changes. That is, very dry demineralized dentin had a Td that was 260% higher than fully hydrated demineralized dentin (compare 176.1 to 65.6°C, Table). Even more remarkable is the observation that very dry demineralized dentin has a Td that is equivalent to the Td of fully hydrated, old mineralized root dentin (Table 1). Interpeptide hydrogen bonding within collagen fibrils seems to stabilize them against thermal challenge. We speculate that water breaks interpeptide hydrogen bonds making collagen more susceptible to thermal denaturation (18). The interfibrillar spaces between collagen fibrils are 20 to 30 nm wide in fully hydrated demineralized dentin (19), but can be virtually absent in air-dried matrices (20). In the presence of water, there is little interpeptide hydrogen bonding, and the spaces 640
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Figure 2. A linear relationship is demonstrated in the intermediate collagen hydration level (water volume fraction 0.32– 0.70). Using the reciprocal °K regression characteristics, y ⫽ 0.001096x ⫹ 0.002034, R2 ⫽ 0.5969, p ⫽ 0.0417, the midpoint of this region at 18 molar ratio or water volume fraction of 0.584 gives a Tmax ⫽ 101.9 °C. The regression was not significant in the lower hydration levels. This may be a result of a progressively collapsed structure as molecular rearrangement compensates for water bridging loss from the gaps that existed in a regular quarter-stagger fiber arrangement (8).
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Basic Research—Biology possible. As the denatured collagen is slowly digested by the release of host-derived matrix metalloproteinase (34, 35), leakage may appear in initially leakage-free root fillings. Such an issue is of clinical concern in the light of current interest in the application of dentin bonding technologies to endodontics, and will be reported in part 2 of this study.
Acknowledgments This work was supported, in part, by grants R01 DE 014911 and R01 DE 015306 (P.I.-D. H. Pashley) from the National Institute of Dental and Craniofacial Research and by R03 DE 14799-01 (P.I. S.R. Armstrong). The authors are grateful to Michelle Barnes for secretarial support.
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14. Bigi A, Ripamonti A, Cojazzi G, Pizzuto G, Roveri N, Koch MH. Structural analysis of turkey tendon collagen upon removal of the inorganic phase. Int J Biol Macromol 1991;13:110 – 4. 15. Kronick PL, Cooke P. Thermal stabilization of collagen fibers by calcification. Connect Tissue Res 1996;33:275– 82. 16. Friess W, Lee G. Basic thermoanalytical studies of insoluble collagen matrices. Biomaterials 1996;17:2289 –94. 17. Fukuda K, Nezu T, Terada Y. The effect of alcoholic compounds on the stability of type I collagen studied by differential scanning calorimetry. Dent Mater J 2000;19:221– 8. 18. Mogilner IG, Ruderman G, Grigera JR. Collagen stability, hydration and native state. J Mol Graph Model 2002;21:209 –13. 19. Pashley DH, Carvalho RM, Agee KA, Lee W-K, Tay FR, Callison TE. Effects of water- free polar solvents on tensile properties of demineralized dentin. Dent Mater 2003;19:347–52. 20. Carvalho RM, Mendonca JS, Santiago SL, et al. Effects of HEMA/solvent combinations on bond strength to dentin. J Dent Res 2003;82:597– 601. 21. Pashley DH, Agee K, Nakajima M, et al. Solvent-induced dimensional changes in EDTA-demineralized dentin matrix. J Biomed Mater Res 2001;56:273– 81. 22. Eddleston C, Hindle A, Rueggeberg FA, et al. Dimensional changes in demineralized dentin matrices following the use of HEMA-water vs. HEMA-alcohol primers. J Biomed Mater Res 2003;67A:900 –7. 23. Weller RN, Kimbrough WF, Anderson RW. Root surface temperatures produced during post space preparation. J Endod 1996;22:304 –7. 24. Lee FS, Van Cura JE, BeGole E. A comparison of root surface temperatures using different obturation heat sources. J Endod 1998;24:617–20. 25. Floren JW, Weller RN, Pashley DH, Kimbrough WF. Changes in root surface temperatures with in vitro use of the System B heat source. J Endod 1999;25:593–5. 26. Behnia A, McDonald NJ. In vitro infrared thermographic assessment of root surface temperatures generated by the Thermafil Plus system. J Endod 2001;27:203–5. 27. Lipski M. Root surface temperature rises in vitro during root canal obturation using hybrid and microseal techniques. J Endod 2005;31:297–300. 28. Jurcak JJ, Weller RN, Kulild JC, Donley DL. In vitro intracanal temperatures produced during warm lateral condensation of gutta-percha. J Endod 1992;18:1–3. 29. Sweatman TL, Baumgartner JC, Sakaguchi RL. Radicular temperatures associated with thermoplasticized gutta-percha. J Endod 2001;27:512–5. 30. Kishen A, Asundi A. Experimental investigation on the role of water in the mechanical behavior of structural dentine. J Biomed Mater Res A 2005;73:192–200. 31. Griffith AA. The phenomena of rupture and flow in solids. Phil Trans Roy Soc London A 1920;221:163–98. 32. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170 –5. 33. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. J Endod 2003;29:233–9. 34. Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, Ito S. Collagen degradation by host-derived enzymes during aging. J Dent Res 2004;83:216 –21. 35. Hebling J, Pashley DH, Tjäderhane L, Tay FR. Chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. J Dent Res 2005;84:741– 6.
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