1

Introduction

According to limited epidemiological studies, approximately ten percent of the general population suffers from perpetual xerostomia, and an estimated 30% of the population, 65 years and older, endures this condition . There are several possible etiologies for a patient experiencing xerostomia, which may be the result of medications, history of radiation therapy, or diagnosis of systemic disease such as Sjögren’s syndrome . The most common cause of xerostomia is medication-induced as a large population of elderly adults is treated by at least one medication that impairs salivary function . Typically, a patient suffering from xerostomia or salivary dysfunction may experience detrimental intraoral consequences such as oral discomfort, rampant and recurrent caries, increased risk of chronic infections, and desiccation of dental restorations—all of which can lead to a decrease in quality of life for the patient .

Selecting a restorative material with suitable physical, chemical, and clinical properties that matches to the xerostomic patients’ needs is not only imperative in terms of treating and managing patients’ dental disease but also is essential to their prognosis and the safeguarding of their oral health. For these patients, treatment using fluoride-releasing restorative materials may be advantageous due to their potential ability to inhibit caries progression, prevent secondary caries development, and promote mineralization as found in in vitro studies . Even though today’s fluoride-releasing restorative materials may have shortcomings (e.g., desiccation, hydrolysis, and dimensional instability) that limit their application, what makes these fluoride-releasing restorative materials attractive is their unique potential to leach and absorb (recharge) ions other than fluoride (e.g., CPP–ACP, CHX, or CPC) .

Apart from restoring a tooth from a diseased condition to a non-cariogenic state, the common intention behind placing these fluoride-releasing restorations is to modify the local environment, including that for adjacent teeth, since the effect of leachable components from a polyalkenoate acid-based restoration is well documented from in vitro studies . However, from a homeostasis perspective, in reality, the oral environment probably exerts a greater impact on these restorative materials than the ion-leaching influence from the restorations themselves. For example, the lack of salivary buffering in xerostomic patients may reduce normal plaque pH, which can alter the mechanical and surface properties of GIs accordingly .

GIs and RMGIs are sensitive to dimensional change when exposed to either a wet or dry environment. Previous studies have shown that conventional GIs and RMGIs are vulnerable to dehydration stress . Severe dehydration may produce loss of adhesion or debonding from tooth structure, shrinkage, and microleakage resulting in failure of the restoration or recurrent caries . Just as conventional GIs may undergo hygroscopic dimensional change when exposed to a moist environment, they may also contract under desiccated conditions and lose adhesion to tooth structure . Furthermore, the addition of resin to GIs as seen in RMGIs has not improved the susceptibility of the material to dehydration. In a study by Sidhu et al. when RMGI and conventional GI restorative materials were placed in Class V preparations of extracted human mandibular third molars and subjected to dehydration stress in vitro, after a period of time both materials demonstrated adhesive gap formation at the dentin interface . In an in vitro study by Watson, the strength of a RMGI material near dentin is weakened during dehydration resulting in shrinkage and cracking at the interface of the restoration with tooth structure . Clinically, the effect of dimensional change from dehydration of the material is manifested as debonding from tooth structure . Furthermore, compounded by cyclic fatiguing, small debonding disturbances have the potential to turn into marginal defects, which past clinical studies have shown that detectable defects in restoration margins have an increased risk of secondary caries formation.

Currently, the conventional GI’s acid-base chemistry continues to serve as a platform for future fluoride-releasing materials. An attractive goal is to create a restorative material that can not only offer the capability of fluoride release or uptake but can also assist in treating the localized microbes and mineralization by adding CPP–ACP, CHX, NaF or CPC into the conventional GI or RMGI. Secondary caries remains as one of the leading causes of replacement of restorations due to the colonization of bacterial biofilm at the tooth–restoration interface . Streptococcus mutans is the major contributing microorganism involved in the pathogenesis of dental caries in humans . Studies have shown that levels of S. mutans in dental plaque of conventional glass ionomers (GIs) and resin-modified glass ionomers (RMGIs) is lower compared to composite resin restorations . In addition, several in vitro studies have demonstrated that GI and RMGI, incorporated with agents like CPP–ACP, CHX, NaF, and CPC, can assist in hindering the formation of plaque biofilm, inhibiting demineralization, and preventing secondary caries in tooth structure adjacent to the restoration .

For example, CPP–ACP has been incorporated into GIs as a bioactive additive since ACP is a precursor to hydroxyapatite . CPP that contains the peptides with a continuous sequence of anionic amino acids (SerP–SerP–SerP–Glu–Glu), which maximizes the solubility of calcium phosphate through stabilization of ACP bound to the phosphopeptides, have shown to be anticariogenic . The reason for the mixing of CPP with the ACP component into the GI system is to facilitate increased efficiency of the transport of calcium and phosphate ions into the tooth. By increasing the CPP concentration in a leachable restorative material, this will alter the osmotic diffusion gradient and serve as a high concentration reservoir from which calcium and phosphate ions can be released into the enamel subsurface lesion . In an acidic oral environment, CPP–ACP has the ability to increase release of calcium, phosphate, and fluoride ions, which inhibits demineralization and promotes remineralization of enamel; a process in which the demineralized enamel crystalline voids receive a net mineral gain of calcium, phosphate, and fluoride ions .

CHX gluconate (0.12%), commonly found as topical antimicrobial mouth rinse, is considered the “gold standard” in its role as an antiplaque and antigingivitis agent . CHX is effective against both gram-positive and gram-negative bacteria and acts by increasing cell membrane permeability and causing the cell membrane to rupture . Known for its “substantivity”, CHX has been proven to be safe, but due to its side effects of staining, calculus formation, and taste alteration, it is better suited to be used on a short-term basis rather than long-term . CHX is commonly used as a pre-procedural mouth rinse to reduce the amount of bacteria in the mouth by approximately 90% as well as to improve wound healing prior to tooth extractions and after scaling and root planing or periodontal surgery . CHX has been evaluated as an additive to glass-ionomer materials .

Fluoride plays a significant role in dentistry and is influential in the treatment of incipient dental caries as well as prevention for future dental caries. Fluoride can be found as an active ingredient in several products such as mouth rinses, toothpastes, varnishes, gels, and glass ionomer restorative materials. Glass ionomers have the ability to release fluoride, whose release is based on a diffusion based process which has been reported to be beneficial to prevent the recurrence of secondary caries . Fluoride functions via three mechanisms including stimulation of remineralization, inhibition of demineralization, and antibacterial through microbial growth and metabolism inhibition with a concordant reduction of acid production. . In concentrations greater than 200 ppm, fluoride demonstrates bactericidal activity which is greater than that released from fluoride-releasing restorative materials . Furthermore, NaF causes inhibition of growth rate and growth levels of S. mutans , especially in an acidic environment .

CPC is a quaternary ammonium compound that is effective at preventing bacterial plaque accumulation thus inhibiting the development of gingivitis . CPC is regulated by the Food and Drug Administration (FDA) and is commonly sold as a mouth rinse and throat lozenges . CPC has the ability to destroy both gram-positive and gram-negative bacterial organisms with its bactericidal mechanism of action. In addition, it is effective against Candida albicans . Compared to CHX, CPC has fewer unwanted side effects; however, it is not as effective as CHX against plaque and gingivitis . CPC has been evaluated as an additive to glass-ionomer materials .

Despite the many advantageous properties of GI (e.g., chemical bonding to tooth, fluoride releasing, and appropriate thermal expansion), its limitations are more commonly linked to moisture or dehydration sensitivity. Practical understanding on whether strength and antimicrobial properties of a desiccated GI with surface crazing can be rejuvenated by imbibition of CPP–ACP, CHX, NaF, and CPC chemicals is critical to the bioactive functioning and long-term survival of any fluoride-releasing restoration. The results of this study should give evidence to clinicians treating xerostomic as well as other patients with existing GI or RMGI restorations or requiring placement of new GI or RMGI restorations whether a particular surface treatment may offer antimicrobial activity without hindering mechanical properties or perhaps improving mechanical properties of the restoration. Thus, our goal was to evaluate the effect of various surface treatments on the mechanical properties and antibacterial activity of desiccated GI and RMGI materials. This study tested two specific null hypotheses as follows:

• 1)

  There is no difference in the mechanical properties of desiccated GI and RMGI materials treated with CPP–ACP, CHX, NaF, or CPC compared to the untreated control.

• 2)

  There is no difference in the antimicrobial activity of desiccated GI and RMGI materials treated with CPP–ACP, CHX, NaF, or CPC compared to the untreated control.

2

Methods and materials

The protocol was approved by the Institutional Review Board at Wilford Hall Ambulatory Surgical Center JBSA-Lackland, Texas. See Tables 1 and 2 for a description of the materials used in this study. To ensure uniformity of fabrication and to minimize inter-operator differences, one provider created all samples.

Mechanical properties were evaluated using flexural strength, flexural modulus, and surface hardness testing. A total of 5 groups were created per chemical cure GI (Fuji IX, GC America, Alsip, IL, USA) and light cure RMGI (Ketac Nano, 3M/ESPE, St. Paul, MN, USA) restorative material. Ten specimens were prepared per group resulting in 100 total specimens. Fifty GI and fifty RMGI specimens were fabricated in molds and placed in the five different media. See Table 2 and Fig. 1 . To prepare each specimen for mechanical property testing, a (2 mm × 2 mm × 25 mm) aluminum mold (Sabri, Downers Grove, IL, USA) was lightly lubricated with a thin layer of silicone spray (WD-40, WD-40 Company, San Diego, CA, USA) to facilitate removal of specimens from the mold and then placed on a Mylar strip. The specimens were fabricated by inserting the GI restorative material into the mold ( n = 10). The top surface of the mold was covered with a second Mylar strip and glass slide to ensure that the end of the specimen was flat and parallel to the opposite surface of the mold. GIs were allowed to chemically cure for 6 min and RMGIs were light cured so that one side of the specimen was exposed to a light polymerization unit (Coltolux LED, Coltene/Whaledent Inc., Cuyahoga Falls, OH, USA) for 20 s each in three separate overlapping increments. Next, the mold was turned, and the opposite side of the specimen was exposed to the light in a similar manner. The adequacy of the light unit’s intensity was assessed to be at least 1000 mW/cm ² using a radiometer (LED Radiometer, SDS/Kerr, Orange, CA, USA) immediately prior to specimen preparation. The specimens were removed from the mold and lightly sanded with 600-grit sand paper (Imperial Wet or Dry, 3M, St. Paul, MN, USA) to remove flash and lubricant from the surface during preparation. The specimens were then stored in 100% humidity in a 37 °C oven (Model 20 GC, Quincy Lab, Chicago, IL, USA) for 24 h as the majority of the acid–base reaction takes approximately 1 day . At 24 h, all specimens were removed from 100% humidity and left in ambient room air at 23 °C and 40% relative humidity for 24 h for surface desiccation to occur and allow the formation of craze lines as described in a technique by Abduo and Swain . After desiccation, each of the five different surface treatments (i.e., four surface treatments and one untreated control) received 10 specimens of GI and 10 specimens of RMGI. See Table 2 . The specimens were immersed with 4 mL of each surface treatment and stored in 2 oz. plastic container jars (Thornton Plastics, Salt Lake City, UT, USA) in the laboratory oven at 37 °C. After 1 week, each specimen was tested using a universal testing machine (Model 5543, Instron, Canton, MA, USA) at a crosshead speed of 0.25 mm/min. Each specimen was placed on a three-point bending test device, which is constructed with a 20 mm span length between the supporting rods, and the central load was applied with a head diameter of 2 mm. The flexural strength was obtained using the expression, FS = 3 Fl /2 bd 2, where F is the loading force at the fracture point, l is the length of the support span (20 mm), b is the width, and d is the depth. Width and depth measurements were made using an electronic digital caliper (GA182, Grobet Vigor, Carlstadt, NJ, USA). The mean flexural strength and standard deviation were calculated for specimens from each of the two restorative materials that were stored in five different media over a one-week span. Flexural modulus was determined from the slope of the linear region of the load-deflection curve using the analytical software (Instron).

Flow chart of treatment groups.

Prior to testing, the fragments from the flexural strength test were returned to the original designated plastic container jars holding each surface treatment and microhardness testing was performed the next day. Knoop hardness testing (LM 300AT, Leco, St. Joseph, MI, USA) was performed with a 200 g load and a 10 s dwell time. Three hardness measurements were made for each specimen and averaged. A mean and standard deviation were determined per group. A two-way analysis of variance (ANOVA) and Tukey’s post hoc test was used to evaluate the effects of material type (2-levels) or storage media (5-levels) on each of the mechanical properties.

For antimicrobial activity testing, the number of groups and types of specimens were similar to mechanical testing. See Fig. 1 . Fifty GI and fifty RMGI specimens were fabricated in a (2 × 2 × 25 mm) beam-shaped mold ( n = 10) as before. The specimens were stored in 100% humidity at 37 °C for 24 h, placed in ambient room air at 23 °C and 40% relative humidity to desiccate for 24 h as before and then stored in one of the five media (i.e., four surface treatments and one untreated control) for one week as before. To determine antimicrobial activity, the specimens were covered with a bacterial suspension. Bacterial isolate S. mutans (ATCC 25175) was cultured on Trypticase Soy Agar with 5% sheep blood (TSAII, BBLTM221239/221262) and incubated at 35 ± 2 °C, 5% CO ₂ in an aerobic environment. An inoculation suspension of S. mutans was prepared by harvesting growth of the organism from TSA II and suspending it in sterile saline to a turbidity equal to a 0.5 McFarland turbidity standard (approx. 1.5 × 108 CFU/mL). A 1:100 dilution of the bacterial suspension was then prepared using Brain Heart Infusion broth (Bacto 237400). Each sample was transferred from its designated storage media with sterile forceps and placed in a vial with 2.0 mL of the bacterial suspension. Samples were incubated for 24 h at 35 ± 2 °C, 5% CO ₂ in an aerobic environment. After incubation, the bacterial suspension was removed and the samples were washed with 2 mL sterile water ×3. After the final wash, 2 mL of sterile saline was added to each sample and the samples were vigorously vortex mixed for 2 min. The saline solution was serially diluted 1:10 and plated on TSA II plates (102–106). Plates were incubated at 35 ± 2 °C, 5% CO2 in an aerobic environment for 3 days. The numbers of CFUs on the plates were counted. Lastly, CFU/mL recovered was calculated. A mean and standard deviation were determined per group. The data was analyzed with a Kruskall–Wallis and Mann–Whitney U non-parametric statistical tests per GI material.

2

Methods and materials

The protocol was approved by the Institutional Review Board at Wilford Hall Ambulatory Surgical Center JBSA-Lackland, Texas. See Tables 1 and 2 for a description of the materials used in this study. To ensure uniformity of fabrication and to minimize inter-operator differences, one provider created all samples.

Table 2

Surface treatment materials.

Material	Manufacturer
100% humidity in 37 °C oven	Model 20 GC, Quincy Lab, Chicago, IL
CPP–ACP (casein phosphopeptide–amorphous calcium phosphate)	Recaldent, GC America, Alsip, IL
5% NaF (sodium fluoride)	Duraflor, Medicom, Augusta, GA
0.12% CHX (chlorhexidine)	Colgate PerioGard, New York, NY
0.05% CPC cetylpyridinium chloride solution	Cepacol, Reckitt Benckiser Inc., Parsippany, NJ

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