Abstract
Objectives
This study aimed at evaluating the therapeutic bioactive effects on the bond strength of three experimental bonding agents containing modified Portland cement-based micro-fillers applied to acid-etched dentin and submitted to aging in simulated body fluid solution (SBS). Confocal laser (CLSM) and scanning electron microscopy (SEM) were also performed.
Methods
A type-I ordinary Portland cement was tailored using different compounds such as sodium–calcium–aluminum–magnesium silicate hydroxide (HOPC), aluminum–magnesium–carbonate hydroxide hydrates (HCPMM) and titanium oxide (HPCTO) to create three bioactive micro-fillers. A resin blend mainly constituted by Bis-GMA, PMDM and HEMA was used as control (RES-Ctr) or mixed with each micro-filler to create three experimental bonding agents: (i) Res-HOPC, (ii) Res-HCPMM and (iii) Res-HPCTO. The bonding agents were applied onto 37% H 3 PO 4 -etched dentin and light-cured for 30 s. After build-ups, they were prepared for micro-tensile bond strength (μTBS) and tested after 24 h or 6 months of SBS storage. SEM analysis was performed after de-bonding, while CLSM was used to evaluate the ultra-morphology/nanoleakage and the mineral deposition at the resin–dentin interface.
Results
High μTBS values were achieved in all groups after 24 h. Only Res-HOPC and Res-HCPMM showed stable μTBS after SBS storage (6 months). All the resin–dentin interfaces created using the bonding agents containing the bioactive micro-fillers tested in this study showed an evident reduction of nanoleakage and mineral deposition after SBS storage.
Conclusion
Resin bonding systems containing specifically tailored Portland cement micro-fillers may promote a therapeutic mineral deposition within the hybrid layer and increase the durability of the resin–dentin bond.
1
Introduction
The durability of resin–dentin interface represents one of the main concerns in adhesive dentistry as it is affected by severe degradation processes. Bond degradation occurs mainly via water sorption , hydrolysis of monomer methacrylates ester bonds caused by salivary esterases , and hydrolysis of collagen fibrils, which may be enhanced by activation of endogenous dentin matrix metalloproteinases (MMPs) . Regarding these different mechanisms of degradation, experimental strategies to preserve the hybrid layer such as ethanol-wet bonding and the use of MMP inhibitors have been proposed. Nevertheless, current attempts to extend the longevity of resin–dentin bonds via incorporation of more hydrolytically stable resin monomers and/or the use of matrix metalloproteinase inhibitors fail to address two fundamental issues: (1) replacement of the mineral phase within the demineralized dentin collagen; and (2) protection of the collagen from biodegradation through fossilization of MMPs .
The use of bioactive materials which promptly interact with dental hard tissues through therapeutic/protective effects may provide a feasible means to extend the longevity of resin–dentin interface . Experimental resin-based calcium-phosphate cements have been advocated as potential therapeutic restorative base-liner materials due to their ability to induce remineralization of caries-affected dentin . Nonetheless, alternative strategies are being developed in order to enhance calcium (Ca 2+ ), hydroxyl (OH − ), and phosphate (PO 4 −3 ) ions delivery within and beneath the resin–dentin hybrid layer. Calcium-silicate Portland-derived cements are able to release Ca 2+ and OH − , so creating favorable conditions for the remineralization of dental hard tissues (i.e. dentin and enamel) . These materials possess a bioactive activity since they are able to induce the formation of apatite-like crystals on their surface in a short induction period eliciting a positive response at the interface from the biological environment . However, the use of the Portland cements in operative dentistry is still debated due to clinical limitations related to their long setting time , high dissolution rate and “specific” mechanical properties . In contrast, the incorporation of resin specific monomers such as 2-hydroxyethyl methacrylate (HEMA), triethyleneglycol dimethacrylates (TEGDMA) and urethane dimethacrylates (UDMA) in silicate-based materials has been proposed to improve the mechanical properties, bond strength to dental tissues and reduce the setting time (light-curable systems) .
Since there is little information concerning the use of such “hybrid” resin-based photo-polymerizable dental adhesives, this study was purposed to assess the therapeutic/bioactive effects of three innovative bonding agents containing tailored Portland cement-based micro-fillers on the resin–dentin interface. This aim was accomplished by evaluating the micro-tensile bond strength (μTBS) after simulated body fluid solution (SBS) storage (24 h or 6 months). Fractography scanning electron microscopy (SEM) of the de-bonded specimens, ultra-morphology confocal microscopy (CLSM) and nanoleakage of the resin–dentin interface were also executed. The null hypotheses to be tested were that the inclusion of the tested micro-fillers within the composition of the experimental bonding agents induces: (i) no effect on the bond strength durability; and (ii) no mineral precipitation and nanoleakage reduction within the demineralized ‘poorly resin-infiltrated’ areas within the resin–dentin interface.
2
Materials and methods
2.1
Preparation of the experimental bioactive resin-base bonding agents
A type I ordinary Portland cement (82.5 wt%) (OPC: Italcementi Group, Cesena, Italy) mainly consisting of tri-calcium silicate (Alite: 3CaO × SiO 2 ), di-calcium silicate (Belite: 2CaO × SiO 2 ), tri-calcium aluminate (3CaO × Al 2 O 3 ) and gypsum (CaSO 4 × 2H 2 O) was mixed with 7.5 wt% of phyllosilicate consisting of sodium–calcium–aluminum–magnesium silicate hydroxide hydrate [(Na,Ca)(Al,Mg) 6 (Si 4 O 10 ) 3 (OH)·6H 2 O; Acros Organics, Fair Lawn, NJ, USA] in deionized water (ratio 2:1) to create the first experimental filler (HOPC). The second filler (HCPMM) was created by mixing 90 wt% of type I OPC, 7.5 wt% phyllosilicate and 2.5 wt% of hydrotalcite consisting of aluminum–magnesium–carbonate hydroxide hydrate [Mg 6 Al 2 (CO 3 )(OH) 16 ·4(H 2 O); Sigma–Aldrich, Gillingham, UK]. The third filler (HPCTO) used in this study was created by mixing OPC (80 wt%), phyllosilicate (7.5 wt%), hydrotalcite (2.5 wt%) and 10 wt% titanium oxide (TiO 2 : Sigma–Aldrich). The three modified Portland-based silicates were mixed with deionized water (ratio 2:1) and allowed to set in incubator at 37 °C for 24 h. Subsequently, they were ground in an agate jar and sieved to obtain <30 μm micro-filler particles.
A resin co-monomer blend was prepared as a typical three-step, etch-and-rinse bonding agent including a neat resin blend as bond and a 50 wt% ethanol–solvated resin mixture as primer (Res-Ctr – no filler). The neat resin blend was formulated by using 40 wt% of a hydrophobic cross-linking dimethacrylate 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl]-propane (Bis-GMA; Esstech, Essington, PA, USA) and 28.75 wt% of hydrophilic 2-hydroxyethyl methacrylate (HEMA; Sigma–Aldrich). An acidic functional monomer Bis(2-Methacryloyloxyethyl) Pyromellitate (PMDM; Esstech Essington) was also added (30 wt%) to the blend solution to obtain a dental bonding system with chemical affinity to the calcium present in the micro-fillers ( Fig. 1 ). The neat resin was made light-curable by adding 0.25 wt% camphoroquinone (CQ; Sigma–Aldrich), 0.5 wt% 2-ethyl-dimethyl-4-aminobenzoate (EDAB; Sigma–Aldrich) and 0.5% diphenyliodonium hexafluorophosphate (PIHF; Sigma–Aldrich).
The resin co-monomer blend was used as control filler-free or mixed with each micro-filler in order to formulate three experimental resin-base bonding agents (GB patent application no. 1118138.5 – filed on 20th October 2011): (i) Res-HOPC: 60 wt% of neat resin and 40 wt% of HOPC; (ii) Res-HCPMM: 60 wt% of neat resin and 40 wt% of HCPMM; and (iii) Res-HPCTO: 60 wt% of neat resin and 40 wt% of HPCTO filler ( Table 1 ). The hybrid calcium silicate-based bonding agents were prepared by mixing the neat resin and the fillers for 30 s on a glass plate to form a homogeneous paste prior the bonding procedures.
| Group | Primer | Bond | Bonding procedures |
|---|---|---|---|
| Res-Ctr pH (4.6) a |
20 wt% Bis-GMA 14.35 wt% HEMA 14.4 wt% PMDM 50 wt% ethanol |
40 wt% Bis-GMA 28.75 wt% HEMA 30 wt% PMDM 0.25 wt% camphoroquinone 0.5 wt% 2-ethyl-dimethyl-4-aminobenzoate 0.5% diphenyliodonium hexafluorophosphate |
(1) Dentin conditioning with 37% H 3 PO 4 for 15 s (2) Copious rinse with deionized water (3) Air-drying for 2 s (4) Application of a first layer of each experimental primer for 20 s (5) Air-drying for 5 s at maximum stream power (6) Application of a second layer of each experimental adhesive for 20 s (7) Gently air-drying for 2 s (8) Light-curing for 30 s (9) Resin composite application and light-curing |
| Res-HOPC pH (8.4) a |
20 wt% Bis-GMA 14.35 wt% HEMA 14.4 wt% PMDM 50 wt% ethanol |
24 wt% Bis-GMA 17.25 wt% HEMA 18 wt% PMDM 0.15 wt% camphoroquinone 0.3 wt% 2-ethyl-dimethyl-4-aminobenzoate 0.3 wt% diphenyliodonium hexafluorophosphate 40 wt% HOPC |
|
| Res-HCPMM pH (8.1) a |
20 wt% Bis-GMA 14.35 wt% HEMA 14.4 wt% PMDM 50 wt% ethanol |
24 wt% Bis-GMA 17.25 wt% HEMA 18 wt% PMDM 0.15 wt% camphoroquinone 0.3 wt% 2-ethyl-dimethyl-4-aminobenzoate 0.3 wt% diphenyliodonium hexafluorophosphate 40 wt% HCPMM |
|
| Res-HPCTO pH (8.3) a | 20 wt% Bis-GMA 14.35 wt% HEMA 14.4 wt% PMDM 50 wt% ethanol |
24 wt% Bis-GMA 17.25 wt% HEMA 18 wt% PMDM 0.15 wt% camphoroquinone 0.3 wt% 2-ethyl-dimethyl-4-aminobenzoate 0.3 wt% diphenyliodonium hexafluorophosphate 40 wt% HPCTO |
a Three discs for each experimental resin-base material (6 mm in diameter and 1 mm thick) and were light-cured for 30 s immersed in 25 ml of H 2 O (pH 6.7) at 37 °C and maintained for 30 days; the pH/alkalinizing activity was evaluated using a professional pH electrode (Mettler-Toledo, Leicester, UK) at room temperature (~24 °C).
2.2
Specimen preparation and bonding procedures
Caries-free human molars (age 20–40 years), extracted for periodontal reasons were used in this study. The treatment plan of any of the involved patients, who had given informed consent that their extracted teeth could be used for research purposes, was not altered by this investigation. This study was conducted in accordance with the ethical guidelines of the Research Ethics Committee (REC) for medical investigations.
The teeth were stored in deionized water (pH 7.1) at 4 °C and used within 1 month after extraction. A flat mid-coronal dentin surface was exposed using a hard tissue microtome (Accutom-50; Struers, Copenhagem, Denmark) equipped with a slow-speed, water-cooled diamond wafering saw (330-CA/RS-70300; Struers). A 180-grit silicon carbide (SiC) abrasive paper mounted on a water-cooled rotating polishing machine (Buehler Meta-Serv 3000; Grinder-Polisher, Düsseldorf, Germany) was used (30 s) to remove the diamond saw smear layer and to replace it with a standard and more clinically related smear layer . The specimens were divided into four groups ( n = 5/group) based on the tested materials ( Table 1 ).
The specimens were etched using a 37% phosphoric acid solution (H 3 PO 4 ; Aldrich Chemical) for 15 s followed by copious water rinse. The etched-dentin surfaces were air-dried for 2 s to remove the excess of water. The control (Res-Ctr) and experimental adhesives (Res-HOPC; Res-HCPMM; Res-HPCTO) were applied within a period of 20 s. The specimens were immediately light-cured for 30 s using a quartz–tungsten–halogen (QTH) system (>600 mW/cm 2 , Optilux VLC; Demetron, CT, USA). Five 1-mm-thick incremental build-up were performed using a resin composite (Filtek Z250; 3M-ESPE, St. Paul, MN, USA), light-activated for 20 s each step with a final curing of 60 s ( Fig. 2 ). The specimens were finally stored in SBS solutions (Oxoid, Basingstoke, Hampshire, UK) for 24 h and 6 months at 37 °C.
2.3
μTBS test and SEM analysis of the failed bonds
The specimens were sectioned perpendicular to the adhesive interface with a slow speed water-cooled diamond wafering blade (Accutom-50; Struers) mounted on a hard tissue microtome (Isomet 11/1180; Buehler). Subsequently, match-sticks with cross-sectional adhesive area of 0.9 mm 2 were created ( Fig. 2 ). As each tooth yielded 16 beams, there were a total of 80 match-sticks in each group. Half of these match-sticks ( n = 40) were tested after 24 h and the remaining half ( n = 40) after 6 months of static SBS storage (37 °C). Each resin–dentin match-stick was attached to a testing apparatus with a cyanoacrylate adhesive (Zapit; Dental Ventures, CA, USA). A tensile load was applied with a customized micro-tensile jig in a LAL300 linear actuator (SMAC Europe; Horsham, West Sussex, UK) with LAC-1 high speed controller single axis with built-in amplifier, that has a stroke length of 50 mm, peak force of 250 N, displacement resolution of 0.5 mm and crosshead speed of 1 mm/min . The load ( N ) at failure and the cross-sectional area of each failed beam (Digital micrometer Mitutoyo CD15; Mitutoyo, Kawasaki, Japan) permitted calculation of the μTBS in MPa. The μTBS (mean-MPa) data for each group were subjected to the repeated measures ANOVA and Tukey’s post hoc test for pair-wise comparisons ( α = 0.05). Fisher’s least significant difference (LSD) test was used to isolate and compare the significant differences ( P < 0.05) between the groups. Premature failures were included in the statistical analysis as zero values.
Modes of failure were classified as percentage of adhesive ( A ), mixed ( M ), or cohesive ( C ) when the failed bonds were examined at 30× using a stereoscopic microscope (Leica M205A; Leica Microsystems, Wetzlar, Germany). For each group, five representative de-bonded specimens, depicting the most frequent failure modes, were chosen for SEM ultra-morphology analysis of the fractured surfaces. They were dried overnight and mounted on aluminum stubs with carbon cement. They were sputter-coated with gold (SCD 004 Sputter Coater; Bal-Tec, Vaduz, Liechtenstein) and examined using an SEM (S3500; Hitachi, Wokingham, UK) with an accelerating voltage of 15 kV and a working distance of 25 mm at increasing magnifications from 60× to 5000×.
2.4
Dye-assisted CLSM evaluation
Three further dentin-bonded specimens were prepared as previously described for each group with the primer/bond resins doped with 0.05 wt% Rhodamine B (Rh-B: Sigma–Aldrich) and then serially sectioned across the adhesive interface to obtain resin–dentin slabs ( n = 12 per group) with a thickness of approx. 1 mm ( Fig. 2 ). The resin–dentin slabs were then allocated to two subgroups ( n = 6/group) based on the period of static storage in SBS (24 h or 6 months). Following each aging period, the specimens were coated with two layers of fast-setting nail varnish applied 1 mm away from the resin–dentin interfaces. Three specimens from each subgroup were immersed in 1 wt% aqueous fluorescein (Sigma–Aldrich) and the other three specimens in 0.5 wt% xylenol orange solution (XO: Sigma–Aldrich) for 24 h at 37 °C (pH 7.2). The latter is a calcium-chelator fluorophore commonly used in bone remineralization studies , due to its ability to form complexes with divalent calcium ions. The specimens were then treated in an ultrasonic water bath for 2 min and polished using ascending (#1200–4000) grit SiC abrasive papers (Versocit; Struers) on a water-cooled polishing device (Buehler Meta-Serv 3000 Grinder-Polisher; Buehler). A final ultrasonic treatment (5 min) concluded the specimen preparation for the confocal microscopy analysis which was performed using a confocal laser scanning microscope (DM-IRE2 CLSM; Leica, Heidelberg, Germany) equipped with a 63×/1.4 NA oil immersion lens. The fluorescein was excited at 488-nm, while XO at 514-nm using an argon laser. The ultra-morphology evaluation (resin-diffusion) was executed using a 568-nm krypton (rhodamine excitation) laser. CLSM images were obtained with a 1 μm z -step to optically section the specimens to a depth up to 20 μm below the surface . The z -axis scans of the interface surface were arbitrarily pseudo-colored by the same operator for better exposure and compiled into single projections using the Leica image-processing software (Leica). The configuration of the system was standardized and used at the same settings for the entire investigation. Each resin–dentin interface was completely investigated and then five optical images were randomly captured. Micrographs representing the most common features observed along the bonded interfaces were captured and recorded .
2
Materials and methods
2.1
Preparation of the experimental bioactive resin-base bonding agents
A type I ordinary Portland cement (82.5 wt%) (OPC: Italcementi Group, Cesena, Italy) mainly consisting of tri-calcium silicate (Alite: 3CaO × SiO 2 ), di-calcium silicate (Belite: 2CaO × SiO 2 ), tri-calcium aluminate (3CaO × Al 2 O 3 ) and gypsum (CaSO 4 × 2H 2 O) was mixed with 7.5 wt% of phyllosilicate consisting of sodium–calcium–aluminum–magnesium silicate hydroxide hydrate [(Na,Ca)(Al,Mg) 6 (Si 4 O 10 ) 3 (OH)·6H 2 O; Acros Organics, Fair Lawn, NJ, USA] in deionized water (ratio 2:1) to create the first experimental filler (HOPC). The second filler (HCPMM) was created by mixing 90 wt% of type I OPC, 7.5 wt% phyllosilicate and 2.5 wt% of hydrotalcite consisting of aluminum–magnesium–carbonate hydroxide hydrate [Mg 6 Al 2 (CO 3 )(OH) 16 ·4(H 2 O); Sigma–Aldrich, Gillingham, UK]. The third filler (HPCTO) used in this study was created by mixing OPC (80 wt%), phyllosilicate (7.5 wt%), hydrotalcite (2.5 wt%) and 10 wt% titanium oxide (TiO 2 : Sigma–Aldrich). The three modified Portland-based silicates were mixed with deionized water (ratio 2:1) and allowed to set in incubator at 37 °C for 24 h. Subsequently, they were ground in an agate jar and sieved to obtain <30 μm micro-filler particles.
A resin co-monomer blend was prepared as a typical three-step, etch-and-rinse bonding agent including a neat resin blend as bond and a 50 wt% ethanol–solvated resin mixture as primer (Res-Ctr – no filler). The neat resin blend was formulated by using 40 wt% of a hydrophobic cross-linking dimethacrylate 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl]-propane (Bis-GMA; Esstech, Essington, PA, USA) and 28.75 wt% of hydrophilic 2-hydroxyethyl methacrylate (HEMA; Sigma–Aldrich). An acidic functional monomer Bis(2-Methacryloyloxyethyl) Pyromellitate (PMDM; Esstech Essington) was also added (30 wt%) to the blend solution to obtain a dental bonding system with chemical affinity to the calcium present in the micro-fillers ( Fig. 1 ). The neat resin was made light-curable by adding 0.25 wt% camphoroquinone (CQ; Sigma–Aldrich), 0.5 wt% 2-ethyl-dimethyl-4-aminobenzoate (EDAB; Sigma–Aldrich) and 0.5% diphenyliodonium hexafluorophosphate (PIHF; Sigma–Aldrich).
The resin co-monomer blend was used as control filler-free or mixed with each micro-filler in order to formulate three experimental resin-base bonding agents (GB patent application no. 1118138.5 – filed on 20th October 2011): (i) Res-HOPC: 60 wt% of neat resin and 40 wt% of HOPC; (ii) Res-HCPMM: 60 wt% of neat resin and 40 wt% of HCPMM; and (iii) Res-HPCTO: 60 wt% of neat resin and 40 wt% of HPCTO filler ( Table 1 ). The hybrid calcium silicate-based bonding agents were prepared by mixing the neat resin and the fillers for 30 s on a glass plate to form a homogeneous paste prior the bonding procedures.
| Group | Primer | Bond | Bonding procedures |
|---|---|---|---|
| Res-Ctr pH (4.6) a |
20 wt% Bis-GMA 14.35 wt% HEMA 14.4 wt% PMDM 50 wt% ethanol |
40 wt% Bis-GMA 28.75 wt% HEMA 30 wt% PMDM 0.25 wt% camphoroquinone 0.5 wt% 2-ethyl-dimethyl-4-aminobenzoate 0.5% diphenyliodonium hexafluorophosphate |
(1) Dentin conditioning with 37% H 3 PO 4 for 15 s (2) Copious rinse with deionized water (3) Air-drying for 2 s (4) Application of a first layer of each experimental primer for 20 s (5) Air-drying for 5 s at maximum stream power (6) Application of a second layer of each experimental adhesive for 20 s (7) Gently air-drying for 2 s (8) Light-curing for 30 s (9) Resin composite application and light-curing |
| Res-HOPC pH (8.4) a |
20 wt% Bis-GMA 14.35 wt% HEMA 14.4 wt% PMDM 50 wt% ethanol |
24 wt% Bis-GMA 17.25 wt% HEMA 18 wt% PMDM 0.15 wt% camphoroquinone 0.3 wt% 2-ethyl-dimethyl-4-aminobenzoate 0.3 wt% diphenyliodonium hexafluorophosphate 40 wt% HOPC |
|
| Res-HCPMM pH (8.1) a |
20 wt% Bis-GMA 14.35 wt% HEMA 14.4 wt% PMDM 50 wt% ethanol |
24 wt% Bis-GMA 17.25 wt% HEMA 18 wt% PMDM 0.15 wt% camphoroquinone 0.3 wt% 2-ethyl-dimethyl-4-aminobenzoate 0.3 wt% diphenyliodonium hexafluorophosphate 40 wt% HCPMM |
|
| Res-HPCTO pH (8.3) a | 20 wt% Bis-GMA 14.35 wt% HEMA 14.4 wt% PMDM 50 wt% ethanol |
24 wt% Bis-GMA 17.25 wt% HEMA 18 wt% PMDM 0.15 wt% camphoroquinone 0.3 wt% 2-ethyl-dimethyl-4-aminobenzoate 0.3 wt% diphenyliodonium hexafluorophosphate 40 wt% HPCTO |
a Three discs for each experimental resin-base material (6 mm in diameter and 1 mm thick) and were light-cured for 30 s immersed in 25 ml of H 2 O (pH 6.7) at 37 °C and maintained for 30 days; the pH/alkalinizing activity was evaluated using a professional pH electrode (Mettler-Toledo, Leicester, UK) at room temperature (~24 °C).
2.2
Specimen preparation and bonding procedures
Caries-free human molars (age 20–40 years), extracted for periodontal reasons were used in this study. The treatment plan of any of the involved patients, who had given informed consent that their extracted teeth could be used for research purposes, was not altered by this investigation. This study was conducted in accordance with the ethical guidelines of the Research Ethics Committee (REC) for medical investigations.
The teeth were stored in deionized water (pH 7.1) at 4 °C and used within 1 month after extraction. A flat mid-coronal dentin surface was exposed using a hard tissue microtome (Accutom-50; Struers, Copenhagem, Denmark) equipped with a slow-speed, water-cooled diamond wafering saw (330-CA/RS-70300; Struers). A 180-grit silicon carbide (SiC) abrasive paper mounted on a water-cooled rotating polishing machine (Buehler Meta-Serv 3000; Grinder-Polisher, Düsseldorf, Germany) was used (30 s) to remove the diamond saw smear layer and to replace it with a standard and more clinically related smear layer . The specimens were divided into four groups ( n = 5/group) based on the tested materials ( Table 1 ).
The specimens were etched using a 37% phosphoric acid solution (H 3 PO 4 ; Aldrich Chemical) for 15 s followed by copious water rinse. The etched-dentin surfaces were air-dried for 2 s to remove the excess of water. The control (Res-Ctr) and experimental adhesives (Res-HOPC; Res-HCPMM; Res-HPCTO) were applied within a period of 20 s. The specimens were immediately light-cured for 30 s using a quartz–tungsten–halogen (QTH) system (>600 mW/cm 2 , Optilux VLC; Demetron, CT, USA). Five 1-mm-thick incremental build-up were performed using a resin composite (Filtek Z250; 3M-ESPE, St. Paul, MN, USA), light-activated for 20 s each step with a final curing of 60 s ( Fig. 2 ). The specimens were finally stored in SBS solutions (Oxoid, Basingstoke, Hampshire, UK) for 24 h and 6 months at 37 °C.
