Activation of gelatinolytic/collagenolytic activity in dentin by self-etching adhesives

Transcription

1 Eur J Oral Sci 2006; 114: Printed in Singapore. All rights reserved Activation of gelatinolytic/collagenolytic activity in dentin by self-etching adhesives Nishitani Y, Yoshiyama M, Wadgaonkar B, Breschi L, Mannello F, Mazzoni A, Carvalho RM, Tja derhane L, Tay FR, Pashley DH. Activation of gelatinolytic/collagenolytic activity in dentin by self-etching adhesives. Eur J Oral Sci 2006; 114: Ó 2006 The Authors. Journal compilation Ó 2006 Eur J Oral Sci Mild acids are known to activate dentin matrix metalloproteinase (MMPs). All selfetching dental adhesives are acidic (ph ) and may activate dentin MMPs. The purpose of this study was to compare the ability of several all-in-one adhesives to activate gelatinolytic and collagenolytic activities in powdered mineralized dentin. Powdered dentin made from human teeth was mixed with all-in-one adhesives (Clearfil Tri-S Bond, G-Bond, Adper Prompt L-Pop) or a self-etching primer (Clearfil SE Bond primer) for varying times and then the reaction was stopped by extracting the adhesives using acetone. Fresh untreated mineralized dentin powder had a gelatinolytic activity of 3.31 ± 0.39 relative fluorescent units (RFU) per mg dry weight (24 h) that increased, over storage time, to 87.5 RFU mg )1 (24 h) after 6 8 wk. When fresh powder was treated with acidic Tri-S Bond, the gelatinolytic activity increased from 3.24 ± 0.70 RFU mg )1 to > RFU mg )1 (24 h) after 20 min and then remained unchanged. Monomers with lower ph values produced less activity. There was a significant, direct correlation between gelatinolytic activity and ph, with Tri-S giving the highest activity. Coating dentin powder with Tri-S resin prevented fluorescent substrates from gaining access to the enzyme, even though it activated the enzyme. In conclusion, self-etch adhesives may activate latent MMP and increase the activity to near-maximum levels and contribute to the degradation of resin dentin bonds over time. Ó 2006 The Authors. Journal compilation Ó 2006 Eur J Oral Sci European Journal of Oral Sciences Yoshihiro Nishitani 1, Masahiro Yoshiyama 1, Bakul Wadgaonkar 2, Lorenzo Breschi 3, Ferdinando Mannello 4, Annalisa Mazzoni 5, Ricardo M. Carvalho 6, Leo Tjäderhane 7,8, Franklin R. Tay 2, David H. Pashley 2 1 Department of Operative Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 2 Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA; 3 Department of Surgical Sciences, UCO of Dental Sciences, University of Trieste, Trieste, Italy; 4 Institute of Histology and Laboratory Analysis, University of Urbino, Urbino, Italy; 5 Anatomical Sciences, University of Bologna, Bologna, Italy; 6 Department of Restorative Dentistry and Endodontics, Bauru School of Dentistry, University S¼o Paulo, Bauru, SP, Brazil; 7 Institute of Dentistry, University of Helsinki, Helsinki, Finland; 8 Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital (HUCH), Helsinki, Finland Dr David H. Pashley, Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA , USA Telefax: dpashley@mail.mcg.edu Key words: acrylics; collagen; dentin; matrix metalloproteinase Accepted for publication January 2006 It is well known that the dentin matrix contains matrix metalloproteinases (MMPs) that seem to be important in tooth development (1) and dentinal caries. Acids are also known to activate MMPs (2 5). Some of these MMPs (MMP-1, -8, -13) attack collagen, while others (MMP-2 and -9) attack gelatine. Dentin is known to contain MMP-2, MMP-20 and possibly MMP-8 (6, 7). Using fluorescein-labeled collagen, partially mineralized human dentin powder has been shown to have a low, but significant, collagenase activity, that seems to be responsible for the in vitro degradation of demineralized dentin over a 270-d time period (8) and may be responsible for the degradation of hybrid layers in vivo (9). However, the addition of four protease inhibitors to the incubation medium completely inhibited in vitro degradation of the dentin matrix over 270 d and inhibited the collagenolytic activity of mineralized dentin powder by 73% (8). In that same study, treatment of mineralized dentin powder with 37 weight percentage (wt%) phosphoric acid gel for 15 s, reduced the collagenolytic activity by 65% (8). It was thought that the low ph (10) of 37 wt% phosphoric acid gel (ph ¼ )0.17), while demineralizing the dentin powder and exposing more MMP activity, also denatured the enzymes. The residual collagenolytic activity was probably associated with the central portion of the powder that had not been demineralized in the 15-s etch. As the popularity of self-etching primers and all-in-one adhesives has increased, many all-in-one adhesives have been commercialized that have ph values of between 1 and 2. These acidic monomers may also demineralize dentin, but may not be sufficiently acidic to denature MMP activity. The purpose of this study was to determine whether self-etching adhesives increase the gelatinolytic and collagenolytic activities of human mineralized dentin powder. The hypothesis tested was that self-etching adhesives are sufficiently acidic to demineralize dentin and activate gelatinolytic and collagenolytic activities of dentin powder without denaturing the enzymes.

2 Self-etching adhesives activate dentin collagenases 161 Material and methods Assay for gelatinolytic/collagenolytic activity in dentin The functional gelatinolytic/collagenolytic activity of dentin was measured by means of the EnzChek assay kit, supplemented with type I bovine soluble skin collagen fluorescein conjugate (Cat. D-12060; Molecular Probes, Eugene, OR, USA). Extracted human teeth, ground free of enamel, pulpal soft tissue and cementum, were reduced to fine powder by freezing the dentin in liquid nitrogen and triturating it in a stainless steel mixer mill at )120 C (Model MM301; Retsch, Newtown, PA, USA) for 5 min at 30 Hz. The powder was then sieved through a 180-lm screen but retained on a 150-lm sieve and kept dry and frozen until used. The assay uses densely labeled fluorescein conjugated to gelatin. The fluorescein is covalently linked to the epsilonamino group of lysine on the gelatin peptides. Because the excitation and emission waves of fluorescein are so close to each other (495 vs. 515 nm, respectively), emitted fluorescent energy is absorbed by nearby fluorescein molecules, thereby quenching the fluorescence. However, when gelatin is cleaved by a peptidase such as gelatinase, the small, fluorescein-labeled peptide fragments become separated as they diffuse away from the substrate so that they are too far removed for any energy absorption. This allows increased fluorescence to occur in proportion to enzymatic degradation of the substrate. This mechanism of fluorogenic substrate fluorescence is sometimes called dye dye quenching. When the substrates are hydrolyzed, the cleavage products can be read in a 96-well fluorescent plate reader operated at an absorption maxima at 495 nm and a fluorescence emission maxima at 515 nm. Because the enzyme activity was very low, the reactions were run for 24 h at 37 C prior to performing fluorescence measurements. The assays were run at least twice with quadruplicate samples in each run. Activation of the enzyme by 4-amino-phenylmercuric acetate was not carried out, because preliminary studies found no difference in enzyme activity, with or without activation. We used a one-way analysis of variance (anova) to seek significant differences among untreated dentin powder, acidic-monomer etched powder, resin-coated powder, 2 wt% chlorhexidine-treated resin-coated powder, or powder acid-etched with 37 wt% phosphoric acid liquid for 15 s. Multiple comparisons were made by Tukey s test at a ¼ Experimental design Mineralized dentin powder (600 mg) was mixed by stirring, for various periods of time, with 0.7 ml of either 37 wt% phosphoric acid for 15 s or 0.7 ml of a self-etching all-in-one adhesive (Table 1). The acid etching was terminated by diluting the phosphoric acid liquid with 20 ml of 0.5 M HEPES-buffered saline (ph 7.4) over 2 min or by extracting the acid adhesive monomers from the dentin powder with 20 ml of 100% acetone. After brief (2 min) centrifugation to isolate the etched dentin powder, the phosphoric acid or self-etching acidic adhesive-treated powder was resuspended and rinsed in 20 ml of distilled water, recentrifuged and the water removed by aspiration to extract buffer salts or reaction products. After drying the dentin powder in an oven at 37 C for 8 h, the dry powder was weighed into aliquots of 80 mg that were transferred to individual wells in 96-well plates for measurement of gelatinolytic or collagenolytic activity using EnzChek kits. Aging of dentin powder Fresh vs. stored dentin powder was produced for each experiment after it became clear that mineralized dentin powder, stored at )10 C, exhibited a steady increase in gelatinolytic activity over time. This aged dentin powder was assayed weekly to determine how much enzyme activity could be associated with 80 mg of mineralized dentin powder. Resin-coated dentin powder The increase in gelatinolytic activity seen in all-in-one adhesive-treated dentin powder provided a convenient model for using to test the ability of adhesive resins to prevent access of soluble fluorescent gelatin substrate from the insoluble gelatinolytic enzyme in dentin powder. Resin coating of acidic monomer-activated dentin powder was accomplished by mixing 600 mg of freshly made mineralized dentin powder (that passed through a 180-lm diameter wire sieve but was retained on a 150-lm diameter sieve) with 0.7 ml of either Clearfil Tri-S Bond all-in-one adhesive or Clearfil SE Bond adhesive (both materials from Kuraray Medical, Tokyo, Japan) in subdued lighting. The paste was stirred, by hand, using a plastic spatula and then spread onto a glass slab for 20 s or 4 min, to permit further etching Table 1 Commercial adhesive systems tested Brand name Abbreviation ph Company Composition Adper Prompt L-Pop L-Pop M-ESPE, St Paul, MN, USA Phosphate methacrylates, water, ethanol, silanated colloidal silica, photoinitiator Clearfil SE Bond Primer SE Bond 1.76 Kuraray Medical, Tokyo, Japan Primer: MDP, HEMA, H 2 O, ethanol; Adhesives: MDP, HEMA, bisgma, CQ, silanated colloidal silica Clearfil S3 Tri-S 2.44 Kuraray Medical MDP, HEMA, bisgma, water, ethanol, CQ, silanated colloidal silica G-Bond G-Bond 2.25 GC Corporation, Tokyo, Japan 4-MET, phosphate acid, monomer, UDMA, silica, photoinitiator, water, acetone bisgma, 2,2-bis(4-2-hydroxy-3-methacryloyloxy-propoxphenyl)propane; CQ, camphorquinone; HEMA, 2-hydroxyethyl methacrylate; MDP, 10-methacryloyloxyde camethylene phosphoric acid; 4-MET, 4-methacryloyloxethyl trimellitic acid; UDMA, dimethylacryloyloxyethyl 2,2,4(3,3,5)-trimethylhexamethylene dicarbamate.

3 162 Nishitani et al. and evaporation of water and ethanol solvents from the paste, with a gentle air stream. This permitted the evaporation of volatile solvents. Spacers (0.4 mm thick) were placed on either end of the glass slab to ensure that the dentin powder-adhesive resin paste would be 0.4 mm thick when a Mylar film and second glass slab was pressed down on the evaporated paste to spread it into a film. Twenty seconds, or 5 min, after the initial mixing of the dentin powder with the adhesives, the sheet of resin-coated powder was light-cured using overlapping fields for 40 s, each at 600 mw cm 2. The sheet of resin-coated dentin powder was 7cm 4 cm. After light-curing through the glass slab, the top glass slab and Mylar film were removed, and the light-curing was repeated at a distance of 1 mm from the sheet. The sheet was then placed in a 37 C oven for 24 h. Then, the sheet was removed from the Mylar film and broken into large pieces that were placed in a mortar and hand-ground with a pestle into a fine powder. The powder was passed through a wire sieve with a mesh size of 300 lm but retained on a 225-lm sieve. This ensured that the dentin powder that was 180 lm in diameter, was coated with lm of resin all around the individual powder particles to give a diameter of 300 lm (Fig. 1). This resin-coated dentin powder was assayed for gelatinolytic or collagenolytic activity in a manner identical to that used for the original dentin powder. As the resin-coated powder had been acidetched by the acidic adhesive for 5 min, its activity was compared with similar batches of mineralized dentin powder that had been acid-etched by the same acidic adhesive for 20 s or 5 min, but had then been extracted with acetone to stop the acid-etching and remove all acidic adhesive monomers from the dentin powder. Some resin-coated powder was soaked in 2 wt% chlorhexidine digluconate (Cavity Cleanser; Bisco, Schaumburg, IL, USA) for 24 h. The long time was thought to be necessary for chlorhexidine to penetrate through the resin. The powder was then rinsed with water and dried. Weighed aliquots of chlorhexidine-treated resincoated dentin powder were assayed for residual gelatinolytic activity. Results Untreated freshly prepared mineralized dentin powder had collagenolytic and gelatinolytic activities of 1.71 ± 0.28 and 3.31 ± 0.34 relative fluorescence units (RFU) per mg of powder (24 h), respectively (Table 2). Untreated aged mineralized dentin powder increased both its collagenolytic and gelatinolytic activities over time (Fig. 2) until it was ± 3.3 and ± 2.71, respectively, RFU mg )1 (24 h), 7 wk after being prepared, even though it had only been unfrozen and refrozen 10 times. When the freshly prepared mineralized dentin powder was acid-etched with 37 wt% phosphoric acid liquid (ph 0.03) for 15 s, the activity fell from 3.31 ± 0.33 RFU mg )1 (24 h) to 0.76 ± 0.35 RFU mg )1 (24 h) (Table 2). Acid-etching freshly prepared mineralized powder with 10 wt% citric acid liquid (ph 1.8) or 1 N HCl (ph 0.75) for 15 s reduced the gelatinolytic activity of mineralized dentin powder from 3.31 ± 0.33 RFU mg )1 (24 h) to 1.76 ± 0.51 and 1.25 ± 0.30 RFU mg )1 (24 h), respectively (Table 2). When dentin powder from freshly extracted teeth was etched with the self-etching all-in-one adhesive Clearfil Tri-S Bond, the gelatinolytic activity increased linearly, with time, up to min, peaked at 20 min at RFU mg )1 (24 h) and then remained relatively constant over 120 min of further etching time (Fig. 3). When freshly prepared mineralized dentin powder was acid-etched with Clearfil Tri-S Bond (ph 2.44) for 5 min, the gelatinolytic activity increased from 3.24 ± 0.70 RFU mg )1 (24 h) to reach a plateau at ± 3.25 RFU mg )1 (24 h) (Fig. 4). Etching dentin powder with G-Bond (ph 2.25), another all-in-one adhesive, for 5 min, increased the gelatinolytic activity to ± 2.43 RFU mg )1 (24 h) after 5 min. Etching dentin powder with Clearfil SE Bond primer (ph 1.76) or Adper Prompt L-Pop (ph 0.88) for 5 min increased the activity of dentin powder to ± 1.20 and ± 3.94 RFU mg )1 (24 h), respectively (Fig. 4). Table 2 Gelatinolytic or collagenolytic activity of mineralized dentin powder Treatment Activity (RFU mg )1 ) (24 h) Gelatinolytic Collagenolytic Fig. 1. Schematic diagram of several possible orientations of resin-coated dentin particles. Note that resin thickness could not be controlled and was not uniform. Untreated fresh 3.31 ± 0.34 a 1.71 ± 0.28 A dentin powder (FDP) Aged dentin powder ± 3.31 b ± 2.71 B 37% phosphoric 0.76 ± 0.35 c acid gel, 15 s (FDP) 10% citric acid, 15 s, FDP 1.76 ± 0.51 c 1 N HCl, 15 s, FDP 1.25 ± 0.30 c Values represent the mean ± standard deviation (SD) (n ¼ 5 plates or 25 wells). Groups identified by different superscript letters are significantly different (P < 0.05). FDP, freshly made dentin powder was used in all acid treatments; RFU, relative fluorescent units.

4 Self-etching adhesives activate dentin collagenases 163 Fig. 2. Increase in gelatinolytic and collagenolytic activity of human mineralized dentin powder over storage time. The same pool of powder was thawed and refrozen periodically over a time-period of 50 d. RFU, relative fluorescent units. Fig. 4. Comparison of gelatinolytic activity of human mineralized dentin powder following no treatment (control) or etching for 15 s (phosphoric acid, PA) or 5 min for L-Pop, SE, GB or S3. GB, G-Bond; L-Pop, Adper Prompt L-Pop; S3, Clearfil Tri-S; SE, Clearfil SE Bond primer; RFU, relative fluorescent units. Values represent the mean ± standard deviation (SD), n ¼ 4 plates or 32 wells. Inset: gelatinolytic activity vs. ph of acidic agents. Table 3 Increase in gelatinolytic and collagenolytic activity of dentin powder by etching with Tri-S and decreased activity by resin-coating Treatment Activity (RFU mg )1 ) (24 h) Gelatinolytic Collagenolytic Fig. 3. Increase in gelatinolytic activity of freshly prepared human mineralized dentin powder that was treated with Clearfil Tris-S Bond, a self-etching adhesive, for 5, 10, 15, 20, 25, 30, 60 and 120 min prior to stopping the etching by extracting the adhesive from the powder with acetone. RFU, relative fluorescent units. Untreated FDP 3.24 ± 0.70 a 1.71 ± 0.28 A Tri-S 20 s etch of FDP ± 3.22 Tri-S 20 s etch of FDP ± % CH, 2 min Tri-S 20 s etch ± 0.27 resin coating Tri-S 5 min etch of FDP ± 3.25 b ± 2.43 B Tri-S 5 min etch of FDP ± 0.69 a 2.45 ± 0.24 C resin coating Tri-S 5 min etch of FDP ± 0.26 d resin coating/2% CH Tx* Untreated aged dentin powder ± 1.78 e Aged dentin powder + coating by SE Bond adhesive ± 0.82 f Values represent the mean ± standard deviation (SD) (n ¼ 5 plates). *After allowing Tri-S to etch for 20 s or 5 min, the resin was light-cured. After reducing the resin-coated dentin to powder, it was soaked in 2% chlorhexidine (CH) treatment for 24 h, rinsed with water and assayed. Groups identified by different superscript letters are significantly different (P < 0.05). FDP, freshly prepared dentin powder; RFU, relative fluorescent units. When the increase in gelatinolytic activity of dentin powder was plotted against the ph of the etchants, a significant (P < 0.05) positive linear relationship was obtained (R 2 ¼ 0.80, P < 0.05, Fig. 4). To determine if Tri-S adhesive could activate gelatinolytic activity in dentin powder in the 20-s application time recommended by the manufacturer, fresh dentin powder was etched for only 20 s (Table 3). This increased the activity from 3.28 ± 0.70 RFU mg )1 (24 h) to 34.9 ± 3.22 RFU mg )1 (24 h). When dentin powder that had been etched with Tri-S for 20 s was then treated with 0.2% chlorhexidine, the activity fell from 34.9 ± 3.22 RFU mg )1 (24 h) to 3.72 ± 0.27 RFU mg )1 (24 h). The addition of Tri-S (up to 80 ll per well) exhibited no intrinsic fluorescence. Addition of up to 80 ll of Tri-S to fluorescein-labeled gelatin substrate had no effect on its baseline fluorescence. That is, it neither increased nor quenched control fluorescence. Similarly, dentin powder without fluorescein-labeled substrates does not produce significant fluorescence. The ability of Clearfil Tri-S to increase collagenolytic activity of mineralized dentin powder was also shown when the 5-min etched dentin powder exhibited increased collagenolytic activity from 1.71 ± RFU mg )1 (24 h) in the untreated powder to ± 2.43 RFU mg )1 (24 h) in the treated powder. When we light-cured the Tri-S etched dentin powder (5 min), it

5 164 Nishitani et al. lowered the collagenolytic activity to 2.45 ± 0.24 RFU mg )1 (24 h) (Table 3). Coating the 20 s Tri-S activated powder by light-curing decreased the gelatinolytic activity, after 20 s, to 3.72 ± 0.27 RFU mg )1 (24 h). Coating freshly prepared mineralized dentin powder with Clearfil Tri-S Bond after allowing it to acid-etch dentin powder for 5 min, lowered the gelatinoltic activity of the dentin powder from ± 3.25 RFU mg )1 (24 h) to 1.83 ± 0.69 RFU mg )1 (24 h) (Table 3). Soaking Tri-S-coated dentin powder in 2 wt% chlorhexidine for 24 h, lowered the residual gelatinolytic activity of Tri-S-activated and -coated dentin powder from 1.83 ± 0.69 RFU mg )1 (24 h) to 0.20 ± 0.26 RFU mg )1 (24 h) (Table 3). The decrease in enzyme activity in resin-coated powder was caused, in part, by the resin having a density that was only half that of dentin. This is shown in Tables 4 and 5. The wt% of dentin in resin-coated dentin powder was only 42.3% (Table 4), while the vol% was 29.2% (Table 5). Thus, resin-coated dentin powder would be expected to have only 42.3% as much enzyme activity as original mineralized dentin powder (Table 6). However, the measured reduction in enzyme activity was 96% (Table 6). Discussion The results of this study support the test hypothesis that self-etching adhesives are sufficiently acidic to activate gelatinolytic and collagenolytic activities in mineralized dentin powder. Thirty-seven per cent phosphoric acid (ph 0.03) probably denatures the MMP activity as rapidly as it exposes more MMP enzymes during dentin demineralization, so that the net gelatinolytic activity is very low. The ph values of Tri-S and G-Bond are 2.44 and 2.25, respectively, while that of Prompt L-Pop is 0.88 (Fig. 4). A plot of gelatinolytic activity vs. ph of the acidic self-etching adhesives yields a highly significant (P < 0.05) positive relationship (R 2 ¼ 0.80, Fig. 4, insert) showing that ph values near zero denature whatever gelatinolytic activity is exposed by acid-etching. As a result of differences in buffering, the immersion of dentin powder in acidic etchants may overestimate the degree of clinical activation of MMPs that might accompany the application of etchants to solid surfaces (11). The fact that the instructions for bonding with these adhesives only recommends etching for s would also limit the increase in MMP activity that is possible during self-etching. While acetone extraction is thought to remove most of the acidic monomers from dentin Table 4 Weight of dentin in resin-coating powder wt% of dentin or resin wt% of dentin in resin Dry weight of dentin g 0.6 g 100 ¼ 42.3% Dry weight of resin g g Total weight of mixture g Table 5 Volume percent of dentin in resin-coating powder Volume of dentin vol% of dentin in resin *Volume of dentin cm cm ¼ 29.2% Volume of resin cm cm 3 Total volume of mixture cm 3 *Assumes that the density of dentin is 2.1 g cm )3. Table 6 Percentage reduction in enzyme activity by resin-coating Substance/treatment Gelatinolytic activity (RFU mg )1 ) (24 h) Observed reduction Expected reduction Tri-S activated FDP ± 3.25 same but resin-coated 1.83 ± % 42.3% FDP, freshly prepared dentin powder; RFU, relative fluorescent units. powder, residual monomers might continue to etch dentin and activate MMPs (12,13). However, it is also possible that once these acidic resins infiltrate the demineralized dentin and are light-cured, they effectively seal the activated MMPs from the fluorescein-labeled collagen or gelatin. As MMPs are hydrolases, they require water to hydrolyze peptide bonds in collagen. The importance of water in resin dentin interface degradation was demonstrated in a recent study in which mineral oil was used as a storage medium, replacing water. This resulted in no loss of adhesive-dentin bond strength with time, as observed with samples stored in artificial saliva (12). If the adhesive resins can seal the matrix from water, they may protect the collagen from the hydrolytic potential of endogenous MMPs (8,14). It is interesting that a non-solvated adhesive resin, Clearfil SE Bond adhesive, was as effective as Clearfil Tri-S Bond in preventing access of the soluble fluorescent collagen to the gelatinolytic enzyme activity in the dentin matrix. How long that sequestration lasts remains to be determined. It is important to point out that resin-coated dentin had a lower density than dentin powder alone. Table 4 shows that resin-coated dentin powder should have had only 42.3% of the enzyme activity of uncoated powder. However, the gelatinolytic and collagenolytic activity of TriS-coated powder fell by 96 and 86%, respectively (Table 3), indicating that the resins sealed dentin from the substrate in addition to ÔdilutingÕ the weight of the dentin powder. It is possible that resin coating of dentin powder may not block the endogenous collagenolytic activity of dentin MMPs on the collagen fibrils of the dentin matrix (8,12). While resin coating is successful in blocking the access of soluble fluorescent collagen (MW ¼ 350,000) to the enzyme, it may not prevent collagenolytic activity of MMPs bound to its own collagen fibril carrier. However, arguing against that pos-

6 Self-etching adhesives activate dentin collagenases 165 sibility is the report that the self-etching primer/adhesive, Clearfil SE Bond, showed little degradation or nanoleakage after 1 yr of function in humans (16). This is in sharp contrast to the severe loss of collagen staining in the etch-and-rinse adhesive Single Bond (3M-ESPE, St Paul, MN, USA) that was bonded to human primary teeth with 6 months of function (9). Although phosphoric acid etching decreases MMP activity (8), common simplified etch-and-rinse adhesives, including Single Bond, are able to reactivate gelatinolytic activity in phosphoric acid-etched, partially demineralized, dentin powder (A. Mazzoni et al. unpublished results). This may explain the degradation of hybrid layers observed in in vivo (9). Of great interest was the observation that the low level of gelatinolytic activity of mineralized dentin powder slowly increased over several months, from levels of 3.31 RFU mg )1 (24 h) to RFU mg )1 (24 h) (Table 2). This indicates that the enzyme is capable of autoactivation, probably induced by repeated freeze thaw cycles that are known to autoactivate purified MMPs (17). This result may also be partly caused by a progressive loss of inhibition by tissue inhibitors of matrix metalloproteinases (TIMPs) (18 20). In any case, it clearly demonstrates that the initial gelatinolytic activity of the mineralized dentin powder represents only about (3.31/ ¼ 0.03) 3% of its potential gelatinolytic activity. The highest gelatinolytic activity measured in this study, as a result of the use of acidic adhesives, was RFU mg )1 (24 h) produced by Clearfil Tri-S Bond after 20 min of etching (Fig. 4). If we assume that the maximum gelatinolytic activity of dentin powder is mg )1 (24 h) (i.e. the 50-d value in Fig. 2), then the highest activation by Clearfil Tri-S Bond was 90% of maximum (111.41/ ). This hypothesis has to be further validated. The increases in gelatinolytic activity reported in this study may underestimate the final increase in enzyme activity over time as a result of subsequent autoactivation of additional proenzymes over time. As most all-in-one adhesives contain hydrophilic resin monomers, they absorb water and exhibit relatively high solubility (21). Gradual loss of protecting resin might promote hydrolysis of dentin collagen over time. The in vivo stability of Clearfil SE Bond hybrid layers after 1 yr of function (16) may be a result of the fact that the acidic primer was covered with a separate neat, relatively hydrophobic, adhesive layer, a process known to lower water penetration into hybrid layers (22). However, selfetching primers leave collagen fibrils partially covered with residual apatite crystals. These crystals, and possible chemical interactions (23) of acidic monomers with residual dentin substrate, may provide more resistance to bond degradation than is possible in etch-and-rinse adhesives. Clearly, the durability of resin dentin bonds is more complex than previously thought. If relatively weak (i.e. ph 2.4) self-etching adhesives are used to etch dentin, they may activate MMPs in dentin far above baseline levels without denaturing the enzyme. This may not be a problem if the adhesive resins seal the exposed collagen very well. The alternative is to use them as all-in-one adhesives primers and cover them with hydrophobic adhesives that exhibit very low water sorption and solubility (21). Another alternative is to include MMP inhibitors, such as chlorhexidine (8,24), in all-in-one adhesives to inhibit the enzymes as they are exposed. How long the inhibitors would be effective remains to be determined. Acknowledgements This work was supported by grants R01 DE and R01 DE from the National Institute of Dental and Craniofacial Research, Bethesda, MD (P. I. D. Pashley), and by the Academy of Finland (grant , P. I. L. Tja derhane). The authors are grateful to the manufacturers for their donations of generous amounts of adhesives. The authors thank Mrs Michelle Barnes for her outstanding editorial assistance. References 1. Tjäderhane L, Palosaari H, Sulkala M, Wahlgren J, Salo T. The expression of matrix metalloproteinases (MMPs) in human odontoblasts. In: Ishikawa T, Takahashi K, Maeda T, Suda H, Shimono M, Inoue T, eds. Proceedings of the International Conference on Dentin/Pulp Complex Chicago: Quintessence Publishing, 2002; Davis GE. Identification of an abundant latent 94 kda gelatin-degrading metalloproteinase in human saliva which is activated by acid-exposure: implications for a role in digestion of collagenous proteins. Arch Biochem Biophys 1991; 286: Okada Y, Nada K, Kawamura K, Matsumoto T, Nakanishi I, Fujimoto N, Sato H, Seiki M. Localization of matrix metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase¼ gelatinase B) in osteoclasts: implications for bone resorption. Lab Invest 1995; 72: Tjäderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T. The activation and function of host matrix metalloproteinase in dentin matrix during breakdown in carious lesions. J Dent Res 1998; 77: Sulkala M, Wahlgren J, Larmas M, Sorsa T, Teronen O, Salo T, Tjäderhane L. The effects of MMP inhibitors on human salivary MMP activity and caries progression in rats. J Dent Res 2001; 80: Martin-De Las Heras S, Valenzuela A, Overall CM. The matrix metalloproteinase gelatinase A in human dentin. Arch Oral Biol 2000; 45: Sulkala M, Larmas M, Sorsa T, Salo T, Tjäderhane L. The location of matrix metalloproteinase-20 (MMP-20, Enamelysin) in mature human teeth. 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