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DISCOVERY! |
Department of Restorative Dentistry, Division of Oral Health Science, Hokkaido University Graduate School of Dental Medicine, North 13, West 7, Kita-ku, Sapporo 060-8586 Japan; sano{at}den.hokudai.ac.jp
Martin A. Taubman, Associate Editor
KEY WORDS: microtensile testing • nanoleakage • biodegradation • dentin bonding
INTRODUCTION
In the late 1980s, my research work was shifting from clinical cariology to dentin adhesion. At that time, total etching was widely performed in Japan, whereas etching of dentin was not recommended in the United States. This aversion to etching dentin was very interesting to me and was a strong driving force for me to enter the dentin bonding field. Some investigators reported that acid-etching of enamel decreased microleakage, but that acid-etching of dentin increased microleakage and produced pulpal inflammation.
Microleakage involves the penetration of tracer molecules, such as dyes, into 2- to 30-µm-wide gaps that develop between tooth structures and non-adhesive filling materials, including amalgam restorations, cements, and castings (Kidd, 1976). With the introduction of adhesive techniques (Nakabayashi et al., 1982) and materials to dentistry, the frequency of gap formation decreased, although gaps still form in high C-factor environments (Yoshikawa et al., 2001). I began testing the relationship between microleakage of dyes into gaps that developed between resin composites and those at cavity walls. At that time, all of the dentin adhesive systems, under light microscopy, showed mild to severe microleakage of dyes. It was a very interesting phenomenon, but it was difficult to understand how silver nitrate could stain adhesive interfaces in the absence of gaps. However, when we tested the microleakage of adhesive resins to dentin using silver nitrate as a tracer, we saw silver uptake into the resin-dentin interface in the absence of gap formation. Using cryo-SEM to increase spacial resolution (Sano et al., 1994a), we found that silver tracer sometimes penetrated the full thickness of the "hybrid layer" and even permeated around resin tags. The problem was solved during my 10-month sabbatical (1992 to 1993) at Professor David Pashley’s lab at the Medical College of Georgia. Professor Pashley was well-known for his series of studies on how water and other substances permeate across dentin. Fortunately, he had just begun research on the permeability properties of the smear layer, the layer of cutting debris left on enamel and dentin. He and Liwen Tao were bonding dentin adhesives to smear layers (Tagami et al., 1991). Dr. Junji Tagami had spent his sabbatical year with Professor Pashley in 1989 to evaluate how adhesives bonded to smear layers vs. acid-etched dentin, a procedure that removed the smear layers. Both Dr. Tagami and Professor Pashley were disappointed with the low resin-dentin bond strengths they obtained using either smear layers or acid-etched dentin as bonding substrates.
MICROTENSILE BOND-TESTING
When I arrived at Professor Pashley’s lab in October, 1992, he introduced me to Dr. Bernard Ciucchi, from the University of Geneva, who was also spending his sabbatical year at MCG. Bernard and I became a fine research team during my stay in Augusta. Professor Pashley wondered why resin bonds as low as 25 MPa caused dentin to fail cohesively. He suggested that we measure the cohesive tensile strength of mineralized and demineralized dentin (Sano et al., 1994b), and then resin-infiltrated demineralized dentin as a macro-model of hybrid layers (Sano et al., 1995a). We performed the testing using hourglass- and dumbbell-shaped specimens for measuring ultimate tensile strengths and revealed that the tensile strength of dentin was 60–100 MPa, which was far greater than the 25-MPa stresses that caused cohesive failures in resin-bonded dentin. We thought that this indicated that stress application to resin-bonded dentin was not uniform and, in fact, caused high stress concentrations.
That gave me the idea to test resin-dentin bond strengths using hourglass-shaped specimens (Fig. 1A
). We simply made resin composite build-ups on flat dentin surfaces. After soaking the bonded specimens in water for 24 hrs, we made vertical, serial sections through the build-ups to create slabs about 0.5 mm thick, the top half consisting of composite, and the bottom half composed of dentin. Our unique contribution to testing bond strength was to create hourglass-shaped specimens using a superfine diamond bur. We located the adhesive interface at the narrowest portion of the specimen, so that the highest stress would be concentrated at the resin-dentin interface. Then the specimens were glued to a jig for testing and pulled into tension until failure. The testing was successful, because all of the specimens failed adhesively, between the dentin and the adhesive. That is, rather than dentin failing cohesively, the joint failed adhesively, allowing us to calculate the true interfacial bond strength.
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To gain more insight into the important variables in measuring bond strength, we altered the surface area for adhesion, and measured the resulting apparent bond strength. We found an inverse logarithmic relationship between the bond strength and surface area for bonding. That is, as the bonding area was reduced, bond strengths increased to very high values.
At that time, I had to return to Japan. Just after returning, I was hospitalized because of a spontaneous pneumothorax of my left lung, so I had a lot of time to think about and write a first draft of the work I had done in Georgia. This was a unique opportunity for me. Generally, people working at universities are so busy doing research, treating patients in clinics, and giving lectures that they are too busy to think deeply about their research work (Smith, 2004). Fortunately, I had several quiet days to prepare a draft of our microtensile bond-testing manuscript. After finishing my draft and sending it to Professor Pashley, I had surgery to remove the bullae from my lung.
Soon thereafter, I received a faxed letter (no e-mail in 1993!) from my mentor, telling me that he had submitted the manuscript for the journal Dental Materials. The manuscript, which was well-received, was the first paper describing microtensile bond-testing (Sano et al., 1994c).
In that paper, we predicted future uses of microtensile bond-strength testing to evaluate the adhesive properties of resins to excavated carious (Nakajima et al., 1995) or sclerotic dentin (Yoshiyama et al., 1996), and testing the regional bond strength to various locations in tooth cavities (Pereira et al., 1999; Yoshikawa et al., 1999). Dr. Ricardo Carvalho, from the University of São Paulo, who had begun his two-year sabbatical at Professor Pashley’s laboratory, obtained similar results using resin-modified glass-ionomer cements (Sano et al., 1994c). He was responsible for the development of the squared-beam (non-trimming) model for microtensile testing, which was first presented at the IADR meeting in Hawaii, 1995. Still later, Dr. Y. Shono showed a similar relationship in resin-enamel bonds (Shono et al., 1997). This relationship between bond strength and specimen size is thought to be due to reductions in the number of intrinsic flaws or voids in resin-tooth interfaces as they are made smaller (Griffith, 1927).
The microtensile bond strength test is very useful and has rapidly spread around the world as the preferred method for testing the strength of resin-hard tissue bonds. Moreover, the testing method has contributed to improvements in the development of new adhesive systems, because the test can focus on the specific sites for the dentin adhesion.
We also proposed comparing the long-term stability of resin adhesion at various sites on the cavity wall, using the microtensile test on teeth extracted at various times after insertion of bonded restorations (Sano et al., 1999; Hashimoto et al., 2000, 2001, 2003; Takahashi et al., 2002; Koshiro et al., 2004).
NANOLEAKAGE
During my sabbatical, we also focused on the quality of the resin/dentin interface. The concept of hybrid layer formation, first proposed by Nakabayashi et al.(1982), was very exciting, and was thought to be responsible for the success of dentin bonding. Hybrid layers form when adhesive co-monomers infiltrate demineralized (i.e., acid-etched) dentin collagen fibrils. However, as mentioned in the INTRODUCTION, we observed silver nitrate tracer penetration into the hybrid layer without any gap formation (Fig.1B
). Tracer ions (silver) penetrated the full thickness of the hybrid layer in some areas, as well as into the adhesive resin. The phenomenon of tracer penetration of the hybrid layers was very interesting. We proposed that this type of leakage, which occurred within the hybrid layer in the absence of gap formation, should be called "nanoleakage", to distinguish it from microleakage, because the spaces that permitted the leakage were only around 20–100 nm wide (Sano et al., 1995b,c), compared with the 10- to 20-µm width of gaps causing microleakage. During the 1990s, some reviewers criticized the term "nanoleakage" as scientific slang. However, after the publication of numerous papers describing various aspects of nanoleakage by many researchers, "nanoleakage" is now regarded as a legitimate scientific term.
Although nanoleakage was shown to occur throughout the hybrid layer and/or adhesive resin, the clinical significance of nanoleakage was unclear. The spaces were too small to allow for bacterial penetration, but they were large enough for enzymes to enter. We hypothesized that nanoleakage revealed the location of defects at the resin-dentin interface, and could be the pathway for degradation of resin/dentin bonds over time.
Our original interpretation of nanoleakage was that silver occupied nanometer-sized spaces around naked collagen fibrils, where resin failed to infiltrate, or where residual water had not been displaced by adhesive resin (Sano et al., 1995c). Later TEM work demonstrated that water can pass from dentin, around resin tags, to form water-filled channels that project from the hybrid layer into the overlying adhesive (Tay et al., 2003; Hashimoto et al., 2004). As Tay et al.(2003) found, different types of nanoleakage occurred as specimens were aged in vitro. When these water-filled channels are stained with silver, they often look like microscopic trees (Fig. 2A). Dr. Tay called them "water trees" and suggested that they might act as potential sites for hydrolytic degradation of resin/dentin bonds. Thus far, all marketed products permitted some amount of nanoleakage and water-tree formation. Our goal was to minimize or diminish nanoleakage at the resin/dentin interface.
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In 1997, I moved from Tokyo Medical and Dental University to Hokkaido University, located in Northern Japan. Since Hokkaido was far from the center of Japan (Tokyo), and from cutting-edge techniques and information flow about adhesive research, I felt somewhat isolated from the latest advances in adhesion. I decided to shift my research focus again. Before I went to Hokkaido, most of my adhesion research was limited to short-term bonding performance of adhesive resins. In Hokkaido, I started to focus on the long-term durability of resin dentin bonds, in vivo and in vitro, that could provide an explanation for the clinical significance of nanoleakage.
I was still collaborating with my mentor, Professor Pashley, when I began my new life in Sapporo. Our first paper on the long-term durability of resin/dentin bonds in vivo was published in the Journal of Dental Research in 1999 (Sano et al., 1999). In that paper, we demonstrated that resinous materials were extracted from the resin-dentin interface over time, and that the hybrid layer became more porous in vivo. The following year, a very interesting paper was published by one of my students, Hashimoto et al.(2000). They placed resin composite restorations into primary teeth that were later recovered when the teeth were exfoliated. When the teeth were sectioned and examined by SEM, it was found that much of the hybrid layer had disappeared over 1–3 years of function. These investigators also reported loss of resinous materials from the resin-dentin interface. Moreover, they found loss of collagen fibrils within the hybrid layer.
The exact mechanism responsible for the degradation of the hybrid layer was not clear. However, we hypothesized that the biodegradation of hybrid layers involved a cascade of events in vivo (Fig. 2B). The first stage of biodegradation begins when dentin is acid-etched for removal of the smear layer, exposing the underlying collagen fibril matrix for hybrid layer formation. The second stage involves extraction of the resins that had infiltrated the dentin matrix via water-filled nanometer-sized voids within the hybrid layer. The third stage involves enzymatic attack of the exposed collagen fibrils, leading to depletion of collagen fibrils. If such biodegradation of resin-bonded dentin is to be avoided, complete penetration and curing of adhesive are essential. Additionally, we might need to block the adverse effects of enzymes (esterases and MMPs) at the resin/dentin interface.
Hashimoto recently completed his sabbatical year working with Professor Pashley. They found that mineralized dentin contains collagenolytic and gelatinolytic enzyme activities that are thought to be matrix metalloproteinases (Pashley et al., 2004). Although phosphoric acid-etching, used to remove the smear layer and expose collagen fibrils, lowers the collagenolytic activity (probably by partial denaturation of the enzymes), some residual activity remains. We believe that infiltration of adhesive resin around the collagen fibrils to which MMPs are bound lowers their enzymatic activity even more. However, if the resin is poorly infiltrated, or if the resin slowly hydrolyzes and leaches from the hybrid layer, the intrinsic MMP activity of the dentin matrix can be expressed and attack the collagen, causing it to solubilize (Hashimoto et al., 2001, 2003, 2004). This weakens the hybrid layer and shifts more functional stress to the remaining fibrils, causing them to defibrillate and enlarging the porosities within the hybrid layer. As the porosities within the hybrid layers merge, their size grows from nanometer-sized water-filled spaces to microsized water-filled spaces. Under occlusal function, resin composites may flex and permit compression of the water-filled voids in the degenerating hybrid layer. This may generate large fluid shear forces that accelerate loss of resin and collagen degradation products.
One of Hashimoto’s team’s recent research interests was to screen the effects of a variety of MMP inhibitors on intrinsic dentin collagenases and gelatinases, in an attempt to increase the durability of resin-dentin bonds. Their recent report, that chlorhexidine inhibits dentin-MMP activity, offers promise for increasing the durability of resin-dentin bonds (Hashimoto et al., 2005). If chlorhexidine can be used in primers, etchants, or as an additive to adhesive co-monomers, it may block the degradation cascade, thereby preserving hybrid layer structure and function.
So much has been learned about resin-dentin adhesion over the past 15 years, but I am certain that major improvements will be made in dentin adhesion over the next 15 years. My journey of discovery will continue, as I help young dental scientists develop to their full potential.
Received June 6, 2005; Last revision November 18, 2005; Accepted November 15, 2005
REFERENCES
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