The teeth used for in vitro bonding studies are principally obtained from humans and bovines. Teeth from both these sources are contaminated with bacteria so that the potential for the transmission of communicable diseases via blood-borne pathogens, particularly from human teeth, is a concern. It is important, therefore, that these teeth are decontaminated in a sterilizing medium before any bond-strength tests are done in the laboratory. The media in which they are stored after harvesting and the duration of storage, however, may influence the bond-strength results.

A variety of media that possess bacteriocidal and bacteriostatic properties have been used for storage purposes. These include chloramine, formalin, sodium hypochlorite, thymol, alcohol and glutaraldehyde. Autoclaving and ?-irradiation have also been used for decontamination. Distilled water, normal saline or freezing have also been used as methods of storage, although these are clearly unable to decontaminate teeth.

Our aims and objectives now were to examine the ScotchBond Multi-Purpose mediated shear bond strength of Z100 resin composite to dentine exposed on bovine incisors that had been freshly harvested (control group), stored for 2 months in a variety of decontaminating media, or decontaminated by irradiation. Storage in distilled water and freezing in distilled water were also included in this investigation.

Freshly extracted bovine incisor teeth obtained from the abattoir were used. In the laboratory the teeth were decoronated, the coronal pulps removed with a dental probe, and the crowns thoroughly washed free of blood and organic debris in running tap water. The crowns were then divided into groups of 10 and stored for 2 months, according to the method listed in Table 1, in a refrigerator at 4°C, except for two groups: the group that was used within 24 hr of harvesting was stored overnight in distilled water in a refrigerator at 4°C; the group subjected to freezing was placed in a container of distilled water and frozen at ?20°C until required. With the exception of the frozen teeth, the media were changed once a week for freshly prepared solutions.

After 24 hr in the case of fresh teeth, or 2 months of storage, the teeth were removed from their respective media, rinsed in running distilled water for 30 min and then embedded in autopolymerizing polymethyl methacrylate. Before embedding the pulp chamber of each tooth was filled with a water-soaked cellulose sponge. Polymethyl methacrylate was mixed in accordance with the manufacturers’instructions and poured into a mould of 2.5 cm dia. and 2.0 cm depth. During the embedding, we ensured that the labial surface projected above and parallel to the surface of the methacrylate and that the teeth were kept wet. The teeth were also kept wet by storing in distilled water at 4°C before surface preparation and resin application, which took place within 2 hr of embedding.

The superficial dentine surface was exposed by grinding away the enamel with water-irrigated No. 180 followed by No. 320 grit SiC paper on a grinding wheel. The dentine test surface was prepared with water-irrigated No. 600 grit SiC paper, as recommended by Pashley et al. (1988) and Titley et al. (1994). Care was taken to ensure that the test surface was limited to the superficial dentine layer, as recommended by Nakamichi et al. (1983). The teeth were kept moist by placing them in in distilled water at 4°C before resin application, which was done immediately after surface preparation.

The bonding resin system was the Scotchbond Multipurpose System and the resin composite was Z100 (3 M Co., MN, USA).

The prepared dentine surfaces of the embedded teeth were etched with a 10% maleic acid gel for 15 sec, washed under running distilled water for 60 sec, and primed with the Scotchbond Multipurpose System according to the manufacturers instructions, except that two coats of primer were applied in accordance with the development of a protocol and results obtained in this laboratory. At no time was the dentine surface allowed to become desiccated. Each embedded tooth was then placed on a bed of modelling clay between the vertical poles of a locating jig and the flat dentine surface oriented so that it was parallel to the horizontal plane. Gelatin cylinders 3 mm in height and 4.3 mm internal dia. were prepared from No. 5 gelatin capsules by removing the round end. The cylinders were placed on the primed dentine surfaces and held in place by an elastic band that had been stretched between the poles of the locating jig.

A thin layer of adhesive resin was applied to the dentine surface within the confines of the gelatin cylinder and photocured according to the manufacturers instructions (Visilux curing light; 3 M). The tooth and its attached gelatin cylinder were removed from the jig, the cylinder carefully filled with the Z100 resin composite, gently tamped with a plastic instrument to ensure good contact, and photocured from above for 60 sec, and then from the sides for a further 60 sec to ensure complete polymerization. The embedded teeth and their attached resin cylinders were stored in distilled water at 37°C for 24 hr before shear-bond strength testing.

The embedded specimens were clamped in an Instron Model 4301 test machine (Canton, MA, USA) so that the resin cylinder was at 90° to the vertical plane. A loop of prestretched stainless-steel wire was placed around the cylinder to contact the resin–tooth interface. With a cross-head speed of 0.5 cm/min and a reversible load cell of 50 kg, the specimens were shear-tested to failure. The failed specimens were each examined under a dissecting microscope at ×30 and the mode of failure recorded as being adhesive or adhesive/cohesive.

The means of the shear-bond strengths for the various storage media were compared for statistical significance by one-way ANOVA. Multiple comparisons were made with Bonferroni’s modification of the Student’s t-test. Fisher’s exact test was used to make a comparison between the modes of failure and the various storage conditions using the results for fresh teeth as the control.

The shear-bond strengths (in MPa) are listed in decreasing order of magnitude in Table 2. The different storage conditions resulted in significantly different shear-bond strengths (ANOVA, p<0.05). The highest bond strength was recorded with the fresh teeth (23.40 MPa) and the lowest after storage in glutaraldehyde (10.14 MPa). The Bonferroni groupings are displayed in Fig. 1, in which the means connected with the same line are not significantly different. In general, the results indicate that statistically significant higher shear-bond strengths were achieved using fresh teeth whereas teeth stored in thymol, methanol, and glutaraldehyde, or irradiated, had shear-bond strengths that were significantly lower. In descending order of shear-bond strength, teeth frozen in distilled water, or stored in neutral-buffered formalin, sodium hypochlorite, chloramine, homofix, and distilled water over 2 months at 4°C produce shear-bond strengths that were not statistically significantly different from one another. Fresh teeth and teeth frozen in distilled water had higher shear-bond strengths than with any other medium or method.

The mode of shear-test failure was recorded and these failures were shown to be either adhesive or a combination of adhesive–cohesive. Comparison of the mode of failure where fresh teeth were used as the control revealed that teeth stored in distilled water (p=0.01), thymol (p=0.08), glutaraldehyde (p=0.0007), or that were irradiated (p=0.03), showed patterns of failure that were statistically different from all other storage media or methods. It should also be noted that adhesive–cohesive failures were recorded at 100% for teeth that were either fresh or frozen, and 90% for teeth that were stored in neutral-buffered formalin or sodium hypochlorite.