d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 901–906
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Effect of polyacrylic acid on dentin protease activities S. Ozcan a, R. Seseogullari-Dirihan b,c, M. Uctasli a, F.R. Tay d, D.H. Pashley d, A. Tezvergil-Mutluay c,e,∗ a
Department of Restorative Dentistry, Gazi University, Faculty of Dentistry, Ankara, Turkey Finnish Doctoral Program in Oral Sciences (FINDOS) University of Turku, Institute of Dentistry, Turku, Finland c Department of Restorative Dentistry and Cariology and Adhesive Dentistry Research Group, Institute of Dentistry, University of Turku, Turku, Finland d College of Dental Medicine, Georgia Regents University, Augusta, Georgia, USA e Turku University Hospital, TYKS, University of Turku, Turku, Finland b
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. This study tested whether treatment of demineralized dentin with polyacrylic acid
Received 13 November 2014
(PAA) has any activatory or inhibitory activity on dentin matrix metalloproteinases (MMP)s
Received in revised form
or cathepsin K (CAT-K).
9 April 2015
Methods. Dentin beams (1 mm × 2 mm × 6 mm; n = 10) were completely demineralized with
Accepted 28 April 2015
EDTA. After initial dry mass assessment, the beams were dipped into 37% phosphoric acid (PA), PA + 2% benzalkonium chloride (BAC), PA + 2% chlorhexidine digluconate (CHX), 10% PAA, PAA + BAC or PAA + CHX for 20 s. Demineralized beams without treatment served as
Keywords:
control. All beams were incubated in simulated body fluid (SBF) for 1 week and the dry mass
Polyacrylic acid
loss was evaluated. Aliquots of SBF were used to analyze solubilized telopeptide fragments
Dentin
using ICTP as indicator of MMP-mediated collagen degradation and CTX for CAT-K-mediated
Matrix metalloproteinase
degradation. Additional demineralized beams (n = 10) were used to measure the influence
Cysteine cathepsins
of different chemical treatments on total MMP activity of EDTA-demineralized dentin using
Degradation
generic MMP assay. Data were analyzed by ANOVA (˛ = 0.05). Results. Dry mass loss ranged from 6% (PA) to 2% for (PA-BAC) or (PAA-BAC) (p < 0.05). ICTP release of PAA-treated group was significantly higher (p < 0.05) than the control, and not significantly different from the PA group (p > 0.05). PA + CHX or PAA + CHX and PAA + BAC showed significantly lower ICTP than PA or PAA groups (p < 0.05). CAT-K activity increased significantly after 10% PAA treatment compared to control (p < 0.05) or to PA postreatment. Significance. Demineralized dentin treated with 10% polyacrylic acid activated CAT-K more than 37% phosphoric acid; 2% chlorhexidine digluconate seems to be a better inhibitor of MMPs and CAT-K than 2% benzalkonium chloride. © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗ Corresponding author at: Department of Restorative Dentistry and Cariology, Institute of Dentistry, University of Turku, Lemminkaisenkatu 2, 20520 Turku, Finland. Tel.: +358 2 3338340. E-mail address: arztez@utu.fi (A. Tezvergil-Mutluay).
http://dx.doi.org/10.1016/j.dental.2015.04.018 0109-5641/© 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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1.
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 901–906
Introduction
Although resin-enamel bonds are very stable over time [1], in vivo evidence indicates that resin–dentin bonds are prone to slow degradation in the oral environment [2–5]. The mechanism responsible for this degradation was first reported by Armstrong et al. [6], who published transmission electron microscopy images of normal vs. degraded hybrid layers in extracted human teeth that had been aged for four years in vitro. Those images showed the disappearance of collagen fibrils from hybrid layers. That same year, Pashley et al. [7] published results demonstrating that human dentin powder contained endogenous proteinases that could be inhibited by a variety of protease inhibitors, especially chlorhexidine, a potent antimicrobial agent known to inhibit matrix metalloproteinase (MMP)-2,-8 and -9 [8] and cysteine cathepsins [9]. More recently, cysteine cathepsins RNA was isolated from odontoblasts and pulp tissue [10] and both cathepsin B and cathepsin K (CAT-K) were identified in carious dentin suggesting the role of endogenous dentin proteases also in caries progression [11]. Therefore, an increasing amount of research is focused on how to inhibit or inactivate dentin proteases. During the application of etch-and-rinse adhesives on dentin, phosphoric acid (PA) is used to remove the smear layer and to demineralize dentin to a depth of 5–8 m [12]. This step enables infiltration of the dentin collagen matrix by adhesive resin monomers for micromechanical retention of resin composites. Unfortunately, both strong acids as well as mild acidic primers used at self-etch adhesives can activate the inactive proforms of endogenous dentin proteases [13–16]. Polyacrylic acid (PAA) etching is commonly employed in restorative dentistry to lightly etch dentin in preparation for placement of glass-ionomer cements. These cements contain aluminosilicate glass fillers that, when etched by PAA, release Al+++ , Ca++ and other ions that form a rigid, metallic salt bridge with polyacrylates [17]. Polyacrylic acids are polyanionic weak acids that may bind to dentin collagen matrices and endogenous proteases (e.g. MMPs and cathepsins). Although it is unlikely that a 10–15 s treatment of normal mineralized dentin will etch deep enough into mineralized normal dentin to activate the endogenous proteases of dentin, cariesaffected dentin is already partially demineralized. The authors previously reported that polyvinyl phosphonic acid, another linear polyanionic polymer, inhibits matrix-bound MMPs in dentin [18]. Hence, it was speculated that PAA may also have inhibitory effect on both MMPmediated and CAT-K mediated degradation of demineralized dentin. Accordingly, the objective of the present study was to evaluate the potential of PAA as an inhibitor of endogenous protease activities in ethylenediamine tetra-acetic acid (EDTA)-demineralized human dentin. The null hypotheses tested were: (1) PAA has no effect on the total MMP activity in demineralized dentin collagen matrices, and (2) combining PAA with other known inhibitors such as chlorhexidine or benzalkonium chloride has no effect on the loss of dry mass or release of MMP-mediated carboxyterminal cross-linked telepeptide (ICTP) or CAT-K mediated cross-linked C-terminal telopeptide (CTX) of type I collagen from EDTA-demineralized dentin over time.
2.
Materials and methods
2.1.
Specimen preparation
Eighty unerupted molars were obtained from 18 to 21-year-old patients under a protocol approved by the Human Assurance Committee of Georgia Regents University. The teeth were stored frozen until use. After thawing, the enamel and superficial dentin of each tooth were removed using an Isomet saw (Buehler Ltd., Lake Bluff, IL, USA) under water cooling. Dentin beams with dimensions 6 mm × 2 mm × 1 mm were sectioned from the mid-coronal dentin (160 beams). All beams were completely demineralized in 0.5 M EDTA (pH 7.4) for 30 days at 4 ◦ C with constant stirring. Three point flexure was used to confirm the absence of residual mineral. Mineralized beams have a modulus of elasticity of 16–19,000 MPa, while demineralized beams have a modulus of elasticity of 2–2.5 MPa [19]. Ten beams were assigned to each of 8 groups (n = 10).
2.2.
Total MMP activity of demineralized dentin
Generic colorimetric MMP assay was used to determine if acid pretreatments could inhibit dentin-derived endogenous MMPs, using 10 EDTA-demineralized beams for each group. After demineralization, the beams were individually incubated in 300 L of chromogenic thiopeptide substrate and assay buffer (Sensolyte Generic MMP assay; Anaspec, San Jose, CA, USA) in a 96-well plate for 60 min at 25 ◦ C. After 60 min, the beams were removed from the wells; the 96-well plate was placed in a microplate reader (Synergy HT; BioTek Instruments, Winooski, VT, USA) to measure the baseline total MMP activity of each beam at 412 nm [20]. The beams were rinsed free of MMP assay substrate and then distributed to different groups such that the mean baseline activity of each group was not statistically significant. The beams were dipped in the respective acid solutions (Table 1), rinsed and incubated in fresh chromogenic substrate and assay buffer in the 96-well plate for 60 min at 25 ◦ C. After 60 min of incubation, the activity was reassessed as described above. The total MMP activity was expressed as a percentage of the untreated baseline level to determine the percent inhibition or activation.
2.3.
Loss of dry mass
After demineralization, a set of beams (n = 10/group) was transferred to individually labeled polypropylene tubes and placed in a desiccator containing anhydrous calcium sulfate (Drierite, W.A. Hammond Drierite Co., Xenio, OH, USA). With the cap off, each beam in separate tubes was desiccated to a constant mass within 72 h. The initial dry mass was measured to the nearest 0.001 mg using an analytical balance (XP6 Microbalance, Mettler Toledo, Hightstown, NJ, USA). The beams were distributed to the 8 experimental groups such that the mean initial dry mass of each group was similar in all groups. After dry mass measurement, the beams were completely rehydrated in deionized water to recover their original dimensions [19,21]. The rehydrated dentin beams were then immersed in the respective acid solution for 20 s (Table 1). After acid treatment, the beams were dropped in 50 mL of buffered
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 901–906
Table 1 – Names and concentrations of the groups in the present study. Group designation
Demineralization Process
PA PA + BAC
PA + CHX
0.5 M EDTA
SELECT
(pH 7.4)
30 days at 4 ◦ C
CTRL PAA PAA + CHX
PAA + BAC
Applied chemicals 37% phosphoric acid (20 s) 37% phosphoric acid + 2% benzalkonium chloride (20 s) 37% phosphoric acid + 2% chlorhexidine (20 s) 35% phosphoric acid and 1% benzalkonium chloride gel (select HV, Bisco Inc.) (20 s) Control (no further treatment) 10% polyacrylic acid (20 s) 10% polyacrylic acid + 2% chlorhexidine (20 s) 10% polyacrylic acid + 2% benzalkonium chloride (20 s)
medium (pH 7.2) for 5 min with agitation to neutralize the acid conditioners, and blot-dried. The control group consisted of EDTA-demineralized dentin beams that were not exposed to any acid treatment. Each beam was then immersed in 0.5 mL of a calcium-and zinc-containing incubation medium in labeled polypropylene tubes. The medium contained 5 mM HEPES, 2.5 mM CaCl2 ·H2 O, and 0.05 mM ZnCl2 (pH 7.2). The sealed tubules were incubated in a shaker-water bath (60 cycles/min) at 37 ◦ C for one week. After removing the incubated beams and rinsing of the medium, loss of dry mass was reassessed as indirect measurement of the solubilization of insoluble collagen by matrix MMPs and CAT-K [18,19].
2.4.
2.5.
903
Statistical analyses
The percent loss of dry mass and the rate of release of ICTP and CTX (in ng telopeptide/mg dry dentin/unit time) from all groups were compared for normality (Kolmogorov–Smirnov test) and equality of variance (modified Levine test). When these statistical assumptions were valid, the data were analyzed using one-way analysis of variance. Post-hoc multiple comparisons were performed with the Holm–Sidak statistic. Statistical significance was preset at ˛ = 0.05.
3.
Results
3.1.
Total MMP activity assay
When demineralized beams were used as a source of MMP, the baseline activity of all beams after 60 min of incubation was not significant among the groups (p > 0.05). After the demineralized beams were treated with the respective acid solutions and further incubated for 60 min, significant differences were observed among the groups (p < 0.05; Fig. 1). The control beams showed 21% increase in total MMP activity during the second incubation period. All PA treated groups and the PAA group showed significant increases in the total MMP activity when compared to the control (p < 0.05). The group that was treated with PA alone for 20 s showed a 147% increase in MMP activity, whereas the groups with BAC or CHX treatment showed 90–92% increase in activity when compared to baseline activity level. Polyacrylic acid treatment alone showed only 51% increase in the baseline activity, whereas additional BAC or CHX treatment limited the increase in total activity to −37% or 8% respectively.
3.2.
Loss of dry mass
Loss of dry mass (Fig. 2) in the EDTA-demineralized control beams that were not exposed to any acid was 4.8 ± 0.4%. When
Solubilized telopeptides of collagen
Matrix degradation by MMPs was determined by measuring the amount of ICTP [22] after the week incubation period, using an ICTP ELISA kit (UniQ EIA, Orion Diagnostica, Finland). The only source of ICTP telopeptide fragments from collagen matrices is attributed to the telopeptidase activity of MMPs [22–24]. Matrix degradation by CAT-K was determined by measuring the amount of solubilized CTX in the incubation medium using the Serum CrossLaps ELISA Kit (Immunodiagnostic System, Farmington, UK). The only known source of CTX in dentin is CAT-K [23–25]. Although the optimum pH is 5.5 [25,26], release of CTX was performed at pH 7.2. Previous work showed that cathepsine K has it is still active, albeit at a low level at pH 7.4 [24]. After incubating in a shaker-water bath at 37 ◦ C, the entire 0.5 mL of medium was retrieved from the sealed tubes. Ten to twenty L aliquots of the incubation medium were used to measure solubilized ICTP and CTX telopeptide fragments.
Fig. 1 – Percent change in total MMP activity compared with untreated control specimens, using the Sensolyte Generic MMP assay kit. Groups identified with different letters are significantly different (p < 0.05).
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Fig. 2 – Loss of dry mass of demineralized dentin beams expressed as percent loss. For abbreviations please refer to Table 1. Groups identified with different letters are significantly different (p < 0.05).
the demineralized beams were treated for 20 s with 37% PA, the loss of dry mass was increased significantly (p < 0.05). When 37% PA treatment was combined with 2% BAC, 2% CHX, the dry mass loss significantly decreased (p < 0.05) compared to control, or 37% PA alone (p < 0.05). Immersion of the demineralized beams in 35% PA + 1%BAC (SELECT) also reduced mass loss significantly (p < 0.05) compared to 37% PA alone. When the demineralized dentin beams were treated with 10% PAA for 20 s, the loss of dry mass was significantly lower compared to control group and was only 54% of the PA-treated groups (Fig. 2). However, when the beams were treated with 10% PAA containing 2% CHX for 20 s there was no significant change in mass loss compared to PAA-treatment alone (p > 0.05). Similarly, when the PAA-treated beams were further treated with 2% BAC, there was slight reduction in dry mass loss that was not statistically significant when compared to the PAA group (p > 0.05).
Fig. 3 – The rate of ICTP telopeptide release from demineralized dentin beams. Values are ng telopeptide/mg dry dentin/week. Bar heights are mean values (n = 10); brackets indicate standard deviations. For abbreviations please refer to Table 1. Groups identified with different letters are significantly different (p < 0.05).
CTX release (pg/mg dry mass/week) from the incubation medium of the beams is shown in Fig. 4. Release of CTX was only 45.6 ± 3.1 pg/mg dry mass/week for the control group. Demineralized dentin beams treated with 37% PA produced a significant increase (p < 0.05) in CTX release which was 4.8fold more than the control, while those treated with 10% PAA produced a significantly higher release (p < 0.05) which was 8.5-fold more than the control. When PA-treated beams were post-treated with 2% CHX or 2% BAC, these two groups produced significant reduction in CTX release by 70–75% (p < 0.05). When dentin beams were
3.3. Inactivation of total endogenous proteases of dentin When the incubation medium of the beams was analyzed for ICTP release, the control group showed the lowest release (Fig. 3). The demineralized dentin beams treated with PA or PAA released significantly higher amount of ICTP compared to control (p < 0.05). When the demineralized dentin beams were treated with PA + 2% BAC, or SELECT HV, a high viscosity, 1% BAC-containing 35% PA etchant, there was small but insignificant reduction in ICTP release (p > 0.05) compared to 37% PA only. However, when the beams were post-treated with PA + 2% CHX, release of ICTP was reduced significantly (p < 0.05). Etching demineralized dentin beams with 10% PAA alone produced a significant (p < 0.05) increase in ICTP release compared to the control; the increase was only 66% of the ICTP that was released after 37% PA treatment. Post-treatment of the PAA-treated dentin beams with 2% CHX or 2% BAC significantly lowered the ICTP release (p < 0.05).
Fig. 4 – The rate of CTX telopeptide release from demineralized dentin beams. Values are pg telopeptide/mg dry dentin/week. Bar heights are mean values (n = 10); brackets indicate standard deviations. Abbreviations are defined in Table 1; Groups identified with different letters are significantly different (p < 0.05).
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 901–906
treated with Select HV for 20 s, release of CTX was significantly lower (p < 0.05) than the group treated with PA only. When PAA-treated beams were post-treated with 2% CHX, the CTX release fell 83%. In contrast, when PAA-treated beams were post-treated with 2% BAC, the CTX release fell only 22% (p < 0.05).
4.
Discussion
The present study was designed to determine whether polyacrylic acid has any inhibitory effect on endogenous MMPs and cathespin K in completely demineralized dentin matrices. Because PAA-treated demineralized dentin exhibited significant increases in total MMP activity, the results justify rejection of the first null hypothesis that PAA has no effect on the total MMP activity in demineralized dentin collagen matrices. While it is common practice to evaluate the mechanism of collagen degradation by means of recombinant enzymes, these observations may not be applicable to the matrix boundMMPs. Hence, demineralized dentin was used in lieu of recombinant enzymes as a source of MMPs to measure their activity using generic MMP assay. The EDTA employed in the present study to demineralize dentin is an excellent MMP-inhibitor, because it chelates Ca++ and Zn++ from mineralized dentin [27]. However, as soon as the dentin beams were rinsed free of EDTA and rehydrated in a calcium and zinc-containing media, there was an increase in their total MMP activity over the incubation period. This was confirmed by the observation that ICTP telopeptides were detected in the control group, indicating that the endogenous dentin MMPs were still active. In the absence of acids, endogenous MMP proforms in the control dentin beams may be activated by other biomolecules such as small integrinbinding N-linked glycoproteins (SIBLINGs). Bone sialoprotein, a SIBLING phosphoprotein, can activate MMP-2 proforms, while dentin matrix protein-1 (another SIBLING) can activate MMP-9 [28]. Activation of other MMP proforms may also be achieved via MMP-2 [29] or cysteine cathepsins [10]. Thus it is not necessary that acids must be present for activation of dentin proteases. The literature contains confusing results regarding the effects of PA etching of dentin on the activity of endogenous dentin proteases. Early qualitative studies of the collagenolytic activity of mineralized dentin powder reported that etching the powder for 15 s with 37% PA reduced its collagenolytic activity by 65% [7]. Similarly, when Nishitani et al. [14] measured the gelatinolytic activity before and after etching dentin with PA for 15 s, the authors reported reduction of gelatinolytic activity by 88%. Based on these studies, PA etching was suggested to denature and inactivate endogenous dentin proteases, and stabilize the demineralized collagen matrix over time. However, studies continued to be published showing degradation of resin–dentin bonds produced by etch-andrinse adhesives over time [5]. A recent study showed the activation of MMP proforms and related ICTP release from EDTA-demineralized beams [15], suggesting that PA does not denature dentin MMPs. It was speculated that MMPs were temporarily covered by CaHPO4 that solubilized to permit
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MMP activity. In the present study, immersion of EDTAdemineralized dentin into 37% PA increased the total MMP activity by about 147% compared to the baseline activity level, confirming the previous findings. When all calcium is removed from dentin by EDTA pretreatment, there are no calcium ions to react with the huge amount of phosphate in the phosphoric acid. Also, the amount of ICTP release increased significantly, suggesting that not all MMP proforms were in active form. When 2% BAC was mixed with 37% PA, it did not seem to be very effective as an MMP inhibitor. We speculate that cationic quaternary ammonium compounds (e.g. BAC) bind to fixed negative charges on collagen peptides, such as carboxylic acid functional groups in glutamic or aspartic acid. At pH 0.4 (the pH of 35–37% PA), all free carboxylic acid groups would be unionized and unable to bind cationic BAC. However, when 37% PA containing 2% CHX digluconate was used to treat EDTA-demineralized dentin beams, the MMPs appeared to be inhibited since the rate of release of ICTP was significantly (p < 0.05) lower than the PA- treated groups (Fig. 3). When demineralized dentin beams were post-treated with 10% PAA, the total MMP activity showed a 51% increase over the baseline level, which was significantly higher (p < 0.05) than the control group, but lower than the PA group. Similarly, the ICTP release was significantly higher than the control (Fig. 3), and lower than the PA group. This suggests that PAA is not an inhibitor of MMPs, but is an activating agent. When PAA + 2% CHX or 2% BAC was used to treat demineralized dentin beams, these 2 groups both inhibited MMPs compared to PAA treatment alone. We speculate that 10% PAA, with a pKa of 4.25, does not suppress the ionization of carboxylic acids, permitting more effective binding of BAC [30] and CHX [31] to MMPs. When the incubation medium of demineralized dentin beams was analyzed for CTX telopeptides, the activity of cathepsin K was very low (ca. 48 pg CTX/mg dry dentin matrix/week). The low activity levels might be partly related to the pH of the incubation medium (7.2) that was almost two orders above the optimum pH for cathepsin K (pH 5.5) [24,26]. However, when the demineralized dentin beams were treated with 37% PA, the rate of CTX release increased 4.8-fold, indicating that not all proforms of cathepsin K had been activated. When 2% BAC or 2% CHX were added to 37% PA, both inhibitors produced significant reduction in CTX release. The effectiveness PA + BAC or PA + CHX as a cathepsin inhibitor and its lack of effectiveness on MMPs suggest that the iso-electric pH of cathepsins may be lower than that of MMPs. This probably enables fixed negative charges from phosphoproteins to remain negative at low pH values. Within the limitations of the present study, it may be concluded that 10% polyacrylic acid activated cathepsin K more than 37% phosphoric acid. In addition, 2% chlorhexidine digluconate appears to be a better inhibitor of both MMPs and cathepsin K than 2% benzalkonium chloride. Future experiments should examine the uptake and binding of polyacrylic acid alone, and polyacrylic acid with chlorhexidine or benzalkonium chloride to collagen to understand the role played by these molecules in activation/inhibition of dentin-bound MMPs and cathepsin K.
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Acknowledgments This study was supported by grant #8126472 from the Academy of Finland to AT-M (PI) and by R01 DE015306 from the NIDCR to DHP, P.I. The authors do not have a financial interest in products, equipment, and companies cited in the manuscript.
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ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
Effect of polyacrylic acid on dentin protease activities S. Ozcan a, R. Seseogullari-Dirihan b,c, M. Uctasli a, F.R. Tay d, D.H. Pashley d, A. Tezvergil-Mutluay c,e,∗ a
Department of Restorative Dentistry, Gazi University, Faculty of Dentistry, Ankara, Turkey Finnish Doctoral Program in Oral Sciences (FINDOS) University of Turku, Institute of Dentistry, Turku, Finland c Department of Restorative Dentistry and Cariology and Adhesive Dentistry Research Group, Institute of Dentistry, University of Turku, Turku, Finland d College of Dental Medicine, Georgia Regents University, Augusta, Georgia, USA e Turku University Hospital, TYKS, University of Turku, Turku, Finland b
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. This study tested whether treatment of demineralized dentin with polyacrylic acid
Received 13 November 2014
(PAA) has any activatory or inhibitory activity on dentin matrix metalloproteinases (MMP)s
Received in revised form
or cathepsin K (CAT-K).
9 April 2015
Methods. Dentin beams (1 mm × 2 mm × 6 mm; n = 10) were completely demineralized with
Accepted 28 April 2015
EDTA. After initial dry mass assessment, the beams were dipped into 37% phosphoric acid (PA), PA + 2% benzalkonium chloride (BAC), PA + 2% chlorhexidine digluconate (CHX), 10% PAA, PAA + BAC or PAA + CHX for 20 s. Demineralized beams without treatment served as
Keywords:
control. All beams were incubated in simulated body fluid (SBF) for 1 week and the dry mass
Polyacrylic acid
loss was evaluated. Aliquots of SBF were used to analyze solubilized telopeptide fragments
Dentin
using ICTP as indicator of MMP-mediated collagen degradation and CTX for CAT-K-mediated
Matrix metalloproteinase
degradation. Additional demineralized beams (n = 10) were used to measure the influence
Cysteine cathepsins
of different chemical treatments on total MMP activity of EDTA-demineralized dentin using
Degradation
generic MMP assay. Data were analyzed by ANOVA (˛ = 0.05). Results. Dry mass loss ranged from 6% (PA) to 2% for (PA-BAC) or (PAA-BAC) (p < 0.05). ICTP release of PAA-treated group was significantly higher (p < 0.05) than the control, and not significantly different from the PA group (p > 0.05). PA + CHX or PAA + CHX and PAA + BAC showed significantly lower ICTP than PA or PAA groups (p < 0.05). CAT-K activity increased significantly after 10% PAA treatment compared to control (p < 0.05) or to PA postreatment. Significance. Demineralized dentin treated with 10% polyacrylic acid activated CAT-K more than 37% phosphoric acid; 2% chlorhexidine digluconate seems to be a better inhibitor of MMPs and CAT-K than 2% benzalkonium chloride. © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗ Corresponding author at: Department of Restorative Dentistry and Cariology, Institute of Dentistry, University of Turku, Lemminkaisenkatu 2, 20520 Turku, Finland. Tel.: +358 2 3338340. E-mail address: arztez@utu.fi (A. Tezvergil-Mutluay).
http://dx.doi.org/10.1016/j.dental.2015.04.018 0109-5641/© 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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1.
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Introduction
Although resin-enamel bonds are very stable over time [1], in vivo evidence indicates that resin–dentin bonds are prone to slow degradation in the oral environment [2–5]. The mechanism responsible for this degradation was first reported by Armstrong et al. [6], who published transmission electron microscopy images of normal vs. degraded hybrid layers in extracted human teeth that had been aged for four years in vitro. Those images showed the disappearance of collagen fibrils from hybrid layers. That same year, Pashley et al. [7] published results demonstrating that human dentin powder contained endogenous proteinases that could be inhibited by a variety of protease inhibitors, especially chlorhexidine, a potent antimicrobial agent known to inhibit matrix metalloproteinase (MMP)-2,-8 and -9 [8] and cysteine cathepsins [9]. More recently, cysteine cathepsins RNA was isolated from odontoblasts and pulp tissue [10] and both cathepsin B and cathepsin K (CAT-K) were identified in carious dentin suggesting the role of endogenous dentin proteases also in caries progression [11]. Therefore, an increasing amount of research is focused on how to inhibit or inactivate dentin proteases. During the application of etch-and-rinse adhesives on dentin, phosphoric acid (PA) is used to remove the smear layer and to demineralize dentin to a depth of 5–8 m [12]. This step enables infiltration of the dentin collagen matrix by adhesive resin monomers for micromechanical retention of resin composites. Unfortunately, both strong acids as well as mild acidic primers used at self-etch adhesives can activate the inactive proforms of endogenous dentin proteases [13–16]. Polyacrylic acid (PAA) etching is commonly employed in restorative dentistry to lightly etch dentin in preparation for placement of glass-ionomer cements. These cements contain aluminosilicate glass fillers that, when etched by PAA, release Al+++ , Ca++ and other ions that form a rigid, metallic salt bridge with polyacrylates [17]. Polyacrylic acids are polyanionic weak acids that may bind to dentin collagen matrices and endogenous proteases (e.g. MMPs and cathepsins). Although it is unlikely that a 10–15 s treatment of normal mineralized dentin will etch deep enough into mineralized normal dentin to activate the endogenous proteases of dentin, cariesaffected dentin is already partially demineralized. The authors previously reported that polyvinyl phosphonic acid, another linear polyanionic polymer, inhibits matrix-bound MMPs in dentin [18]. Hence, it was speculated that PAA may also have inhibitory effect on both MMPmediated and CAT-K mediated degradation of demineralized dentin. Accordingly, the objective of the present study was to evaluate the potential of PAA as an inhibitor of endogenous protease activities in ethylenediamine tetra-acetic acid (EDTA)-demineralized human dentin. The null hypotheses tested were: (1) PAA has no effect on the total MMP activity in demineralized dentin collagen matrices, and (2) combining PAA with other known inhibitors such as chlorhexidine or benzalkonium chloride has no effect on the loss of dry mass or release of MMP-mediated carboxyterminal cross-linked telepeptide (ICTP) or CAT-K mediated cross-linked C-terminal telopeptide (CTX) of type I collagen from EDTA-demineralized dentin over time.
2.
Materials and methods
2.1.
Specimen preparation
Eighty unerupted molars were obtained from 18 to 21-year-old patients under a protocol approved by the Human Assurance Committee of Georgia Regents University. The teeth were stored frozen until use. After thawing, the enamel and superficial dentin of each tooth were removed using an Isomet saw (Buehler Ltd., Lake Bluff, IL, USA) under water cooling. Dentin beams with dimensions 6 mm × 2 mm × 1 mm were sectioned from the mid-coronal dentin (160 beams). All beams were completely demineralized in 0.5 M EDTA (pH 7.4) for 30 days at 4 ◦ C with constant stirring. Three point flexure was used to confirm the absence of residual mineral. Mineralized beams have a modulus of elasticity of 16–19,000 MPa, while demineralized beams have a modulus of elasticity of 2–2.5 MPa [19]. Ten beams were assigned to each of 8 groups (n = 10).
2.2.
Total MMP activity of demineralized dentin
Generic colorimetric MMP assay was used to determine if acid pretreatments could inhibit dentin-derived endogenous MMPs, using 10 EDTA-demineralized beams for each group. After demineralization, the beams were individually incubated in 300 L of chromogenic thiopeptide substrate and assay buffer (Sensolyte Generic MMP assay; Anaspec, San Jose, CA, USA) in a 96-well plate for 60 min at 25 ◦ C. After 60 min, the beams were removed from the wells; the 96-well plate was placed in a microplate reader (Synergy HT; BioTek Instruments, Winooski, VT, USA) to measure the baseline total MMP activity of each beam at 412 nm [20]. The beams were rinsed free of MMP assay substrate and then distributed to different groups such that the mean baseline activity of each group was not statistically significant. The beams were dipped in the respective acid solutions (Table 1), rinsed and incubated in fresh chromogenic substrate and assay buffer in the 96-well plate for 60 min at 25 ◦ C. After 60 min of incubation, the activity was reassessed as described above. The total MMP activity was expressed as a percentage of the untreated baseline level to determine the percent inhibition or activation.
2.3.
Loss of dry mass
After demineralization, a set of beams (n = 10/group) was transferred to individually labeled polypropylene tubes and placed in a desiccator containing anhydrous calcium sulfate (Drierite, W.A. Hammond Drierite Co., Xenio, OH, USA). With the cap off, each beam in separate tubes was desiccated to a constant mass within 72 h. The initial dry mass was measured to the nearest 0.001 mg using an analytical balance (XP6 Microbalance, Mettler Toledo, Hightstown, NJ, USA). The beams were distributed to the 8 experimental groups such that the mean initial dry mass of each group was similar in all groups. After dry mass measurement, the beams were completely rehydrated in deionized water to recover their original dimensions [19,21]. The rehydrated dentin beams were then immersed in the respective acid solution for 20 s (Table 1). After acid treatment, the beams were dropped in 50 mL of buffered
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 901–906
Table 1 – Names and concentrations of the groups in the present study. Group designation
Demineralization Process
PA PA + BAC
PA + CHX
0.5 M EDTA
SELECT
(pH 7.4)
30 days at 4 ◦ C
CTRL PAA PAA + CHX
PAA + BAC
Applied chemicals 37% phosphoric acid (20 s) 37% phosphoric acid + 2% benzalkonium chloride (20 s) 37% phosphoric acid + 2% chlorhexidine (20 s) 35% phosphoric acid and 1% benzalkonium chloride gel (select HV, Bisco Inc.) (20 s) Control (no further treatment) 10% polyacrylic acid (20 s) 10% polyacrylic acid + 2% chlorhexidine (20 s) 10% polyacrylic acid + 2% benzalkonium chloride (20 s)
medium (pH 7.2) for 5 min with agitation to neutralize the acid conditioners, and blot-dried. The control group consisted of EDTA-demineralized dentin beams that were not exposed to any acid treatment. Each beam was then immersed in 0.5 mL of a calcium-and zinc-containing incubation medium in labeled polypropylene tubes. The medium contained 5 mM HEPES, 2.5 mM CaCl2 ·H2 O, and 0.05 mM ZnCl2 (pH 7.2). The sealed tubules were incubated in a shaker-water bath (60 cycles/min) at 37 ◦ C for one week. After removing the incubated beams and rinsing of the medium, loss of dry mass was reassessed as indirect measurement of the solubilization of insoluble collagen by matrix MMPs and CAT-K [18,19].
2.4.
2.5.
903
Statistical analyses
The percent loss of dry mass and the rate of release of ICTP and CTX (in ng telopeptide/mg dry dentin/unit time) from all groups were compared for normality (Kolmogorov–Smirnov test) and equality of variance (modified Levine test). When these statistical assumptions were valid, the data were analyzed using one-way analysis of variance. Post-hoc multiple comparisons were performed with the Holm–Sidak statistic. Statistical significance was preset at ˛ = 0.05.
3.
Results
3.1.
Total MMP activity assay
When demineralized beams were used as a source of MMP, the baseline activity of all beams after 60 min of incubation was not significant among the groups (p > 0.05). After the demineralized beams were treated with the respective acid solutions and further incubated for 60 min, significant differences were observed among the groups (p < 0.05; Fig. 1). The control beams showed 21% increase in total MMP activity during the second incubation period. All PA treated groups and the PAA group showed significant increases in the total MMP activity when compared to the control (p < 0.05). The group that was treated with PA alone for 20 s showed a 147% increase in MMP activity, whereas the groups with BAC or CHX treatment showed 90–92% increase in activity when compared to baseline activity level. Polyacrylic acid treatment alone showed only 51% increase in the baseline activity, whereas additional BAC or CHX treatment limited the increase in total activity to −37% or 8% respectively.
3.2.
Loss of dry mass
Loss of dry mass (Fig. 2) in the EDTA-demineralized control beams that were not exposed to any acid was 4.8 ± 0.4%. When
Solubilized telopeptides of collagen
Matrix degradation by MMPs was determined by measuring the amount of ICTP [22] after the week incubation period, using an ICTP ELISA kit (UniQ EIA, Orion Diagnostica, Finland). The only source of ICTP telopeptide fragments from collagen matrices is attributed to the telopeptidase activity of MMPs [22–24]. Matrix degradation by CAT-K was determined by measuring the amount of solubilized CTX in the incubation medium using the Serum CrossLaps ELISA Kit (Immunodiagnostic System, Farmington, UK). The only known source of CTX in dentin is CAT-K [23–25]. Although the optimum pH is 5.5 [25,26], release of CTX was performed at pH 7.2. Previous work showed that cathepsine K has it is still active, albeit at a low level at pH 7.4 [24]. After incubating in a shaker-water bath at 37 ◦ C, the entire 0.5 mL of medium was retrieved from the sealed tubes. Ten to twenty L aliquots of the incubation medium were used to measure solubilized ICTP and CTX telopeptide fragments.
Fig. 1 – Percent change in total MMP activity compared with untreated control specimens, using the Sensolyte Generic MMP assay kit. Groups identified with different letters are significantly different (p < 0.05).
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Fig. 2 – Loss of dry mass of demineralized dentin beams expressed as percent loss. For abbreviations please refer to Table 1. Groups identified with different letters are significantly different (p < 0.05).
the demineralized beams were treated for 20 s with 37% PA, the loss of dry mass was increased significantly (p < 0.05). When 37% PA treatment was combined with 2% BAC, 2% CHX, the dry mass loss significantly decreased (p < 0.05) compared to control, or 37% PA alone (p < 0.05). Immersion of the demineralized beams in 35% PA + 1%BAC (SELECT) also reduced mass loss significantly (p < 0.05) compared to 37% PA alone. When the demineralized dentin beams were treated with 10% PAA for 20 s, the loss of dry mass was significantly lower compared to control group and was only 54% of the PA-treated groups (Fig. 2). However, when the beams were treated with 10% PAA containing 2% CHX for 20 s there was no significant change in mass loss compared to PAA-treatment alone (p > 0.05). Similarly, when the PAA-treated beams were further treated with 2% BAC, there was slight reduction in dry mass loss that was not statistically significant when compared to the PAA group (p > 0.05).
Fig. 3 – The rate of ICTP telopeptide release from demineralized dentin beams. Values are ng telopeptide/mg dry dentin/week. Bar heights are mean values (n = 10); brackets indicate standard deviations. For abbreviations please refer to Table 1. Groups identified with different letters are significantly different (p < 0.05).
CTX release (pg/mg dry mass/week) from the incubation medium of the beams is shown in Fig. 4. Release of CTX was only 45.6 ± 3.1 pg/mg dry mass/week for the control group. Demineralized dentin beams treated with 37% PA produced a significant increase (p < 0.05) in CTX release which was 4.8fold more than the control, while those treated with 10% PAA produced a significantly higher release (p < 0.05) which was 8.5-fold more than the control. When PA-treated beams were post-treated with 2% CHX or 2% BAC, these two groups produced significant reduction in CTX release by 70–75% (p < 0.05). When dentin beams were
3.3. Inactivation of total endogenous proteases of dentin When the incubation medium of the beams was analyzed for ICTP release, the control group showed the lowest release (Fig. 3). The demineralized dentin beams treated with PA or PAA released significantly higher amount of ICTP compared to control (p < 0.05). When the demineralized dentin beams were treated with PA + 2% BAC, or SELECT HV, a high viscosity, 1% BAC-containing 35% PA etchant, there was small but insignificant reduction in ICTP release (p > 0.05) compared to 37% PA only. However, when the beams were post-treated with PA + 2% CHX, release of ICTP was reduced significantly (p < 0.05). Etching demineralized dentin beams with 10% PAA alone produced a significant (p < 0.05) increase in ICTP release compared to the control; the increase was only 66% of the ICTP that was released after 37% PA treatment. Post-treatment of the PAA-treated dentin beams with 2% CHX or 2% BAC significantly lowered the ICTP release (p < 0.05).
Fig. 4 – The rate of CTX telopeptide release from demineralized dentin beams. Values are pg telopeptide/mg dry dentin/week. Bar heights are mean values (n = 10); brackets indicate standard deviations. Abbreviations are defined in Table 1; Groups identified with different letters are significantly different (p < 0.05).
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 901–906
treated with Select HV for 20 s, release of CTX was significantly lower (p < 0.05) than the group treated with PA only. When PAA-treated beams were post-treated with 2% CHX, the CTX release fell 83%. In contrast, when PAA-treated beams were post-treated with 2% BAC, the CTX release fell only 22% (p < 0.05).
4.
Discussion
The present study was designed to determine whether polyacrylic acid has any inhibitory effect on endogenous MMPs and cathespin K in completely demineralized dentin matrices. Because PAA-treated demineralized dentin exhibited significant increases in total MMP activity, the results justify rejection of the first null hypothesis that PAA has no effect on the total MMP activity in demineralized dentin collagen matrices. While it is common practice to evaluate the mechanism of collagen degradation by means of recombinant enzymes, these observations may not be applicable to the matrix boundMMPs. Hence, demineralized dentin was used in lieu of recombinant enzymes as a source of MMPs to measure their activity using generic MMP assay. The EDTA employed in the present study to demineralize dentin is an excellent MMP-inhibitor, because it chelates Ca++ and Zn++ from mineralized dentin [27]. However, as soon as the dentin beams were rinsed free of EDTA and rehydrated in a calcium and zinc-containing media, there was an increase in their total MMP activity over the incubation period. This was confirmed by the observation that ICTP telopeptides were detected in the control group, indicating that the endogenous dentin MMPs were still active. In the absence of acids, endogenous MMP proforms in the control dentin beams may be activated by other biomolecules such as small integrinbinding N-linked glycoproteins (SIBLINGs). Bone sialoprotein, a SIBLING phosphoprotein, can activate MMP-2 proforms, while dentin matrix protein-1 (another SIBLING) can activate MMP-9 [28]. Activation of other MMP proforms may also be achieved via MMP-2 [29] or cysteine cathepsins [10]. Thus it is not necessary that acids must be present for activation of dentin proteases. The literature contains confusing results regarding the effects of PA etching of dentin on the activity of endogenous dentin proteases. Early qualitative studies of the collagenolytic activity of mineralized dentin powder reported that etching the powder for 15 s with 37% PA reduced its collagenolytic activity by 65% [7]. Similarly, when Nishitani et al. [14] measured the gelatinolytic activity before and after etching dentin with PA for 15 s, the authors reported reduction of gelatinolytic activity by 88%. Based on these studies, PA etching was suggested to denature and inactivate endogenous dentin proteases, and stabilize the demineralized collagen matrix over time. However, studies continued to be published showing degradation of resin–dentin bonds produced by etch-andrinse adhesives over time [5]. A recent study showed the activation of MMP proforms and related ICTP release from EDTA-demineralized beams [15], suggesting that PA does not denature dentin MMPs. It was speculated that MMPs were temporarily covered by CaHPO4 that solubilized to permit
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MMP activity. In the present study, immersion of EDTAdemineralized dentin into 37% PA increased the total MMP activity by about 147% compared to the baseline activity level, confirming the previous findings. When all calcium is removed from dentin by EDTA pretreatment, there are no calcium ions to react with the huge amount of phosphate in the phosphoric acid. Also, the amount of ICTP release increased significantly, suggesting that not all MMP proforms were in active form. When 2% BAC was mixed with 37% PA, it did not seem to be very effective as an MMP inhibitor. We speculate that cationic quaternary ammonium compounds (e.g. BAC) bind to fixed negative charges on collagen peptides, such as carboxylic acid functional groups in glutamic or aspartic acid. At pH 0.4 (the pH of 35–37% PA), all free carboxylic acid groups would be unionized and unable to bind cationic BAC. However, when 37% PA containing 2% CHX digluconate was used to treat EDTA-demineralized dentin beams, the MMPs appeared to be inhibited since the rate of release of ICTP was significantly (p < 0.05) lower than the PA- treated groups (Fig. 3). When demineralized dentin beams were post-treated with 10% PAA, the total MMP activity showed a 51% increase over the baseline level, which was significantly higher (p < 0.05) than the control group, but lower than the PA group. Similarly, the ICTP release was significantly higher than the control (Fig. 3), and lower than the PA group. This suggests that PAA is not an inhibitor of MMPs, but is an activating agent. When PAA + 2% CHX or 2% BAC was used to treat demineralized dentin beams, these 2 groups both inhibited MMPs compared to PAA treatment alone. We speculate that 10% PAA, with a pKa of 4.25, does not suppress the ionization of carboxylic acids, permitting more effective binding of BAC [30] and CHX [31] to MMPs. When the incubation medium of demineralized dentin beams was analyzed for CTX telopeptides, the activity of cathepsin K was very low (ca. 48 pg CTX/mg dry dentin matrix/week). The low activity levels might be partly related to the pH of the incubation medium (7.2) that was almost two orders above the optimum pH for cathepsin K (pH 5.5) [24,26]. However, when the demineralized dentin beams were treated with 37% PA, the rate of CTX release increased 4.8-fold, indicating that not all proforms of cathepsin K had been activated. When 2% BAC or 2% CHX were added to 37% PA, both inhibitors produced significant reduction in CTX release. The effectiveness PA + BAC or PA + CHX as a cathepsin inhibitor and its lack of effectiveness on MMPs suggest that the iso-electric pH of cathepsins may be lower than that of MMPs. This probably enables fixed negative charges from phosphoproteins to remain negative at low pH values. Within the limitations of the present study, it may be concluded that 10% polyacrylic acid activated cathepsin K more than 37% phosphoric acid. In addition, 2% chlorhexidine digluconate appears to be a better inhibitor of both MMPs and cathepsin K than 2% benzalkonium chloride. Future experiments should examine the uptake and binding of polyacrylic acid alone, and polyacrylic acid with chlorhexidine or benzalkonium chloride to collagen to understand the role played by these molecules in activation/inhibition of dentin-bound MMPs and cathepsin K.
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Acknowledgments This study was supported by grant #8126472 from the Academy of Finland to AT-M (PI) and by R01 DE015306 from the NIDCR to DHP, P.I. The authors do not have a financial interest in products, equipment, and companies cited in the manuscript.
references
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