Glass ionomer cement modified by a imidazolium salt: adding antifungal properties to a biomaterial

Abstract

We present the structural modification of a commercially available glass ionomer cement by inserting the imidazolium salt 1-n-hexadecyl-3-methylimidazolium chloride (C₁₆MImCl), composing a new biomaterial with antifungal biofilm activity. Test specimens were prepared using a commercial glass ionomer cement to which 10 ppm of cetylpyridinium chloride (reference ionic antifungal agent) or C₁₆MImCl were added. The feasibility and hypoallergenicity of the new biomaterial were assessed by microhardness plastic deformation and chorioallantoic membrane assays. Colony counting and scanning electron microscopy were used to evaluate the modified specimens’ antibiofilm activity against three multidrug-resistant Candida species. The modified glass ionomer cement presented a strong antibiofilm activity against Candida spp., without losing its original micromechanical and hypoallergenic properties, rendering it a promising candidate for further application in dentistry.

Introduction

A plethora of bacteria and fungi are able to alternate planktonic growth and community growth, commonly referred as biofilms. The use of orthodontic appliances favors the colonization by Candida albicans yeasts [1]. Moreover, Candida tropicalis, Candida parapsilosis, and Candida krusei were isolated from the saliva of individuals with or without clinical manifestations of candidiasis [2]. The importance of biofilm during orthodontic treatment, associated with dental plaque formation and periodontal diseases, is of concern [3, 4], considering that Candida spp. biofilms have a great resistance to antifungal drugs [5, 6].

Several strategies propose the development of biomaterials able of reducing the procession of adhesion, inactivating, and killing the guest biofilm forming agents [7–10]. Due to the biocompatibility with dental hard tissues, glass ionomer cements (GIC) have multiple applications in dentistry, such as restorative material and in orthodontic band fixation [8]. These compounds must present physical and chemical characteristics, especially concerning their physical structure maintenance, referred to as microhardness [11]. Moreover, they should allow fluoride exchange with the inner oral mucosa, inhibiting the dental plaque and biofilm formation by Streptococcus mutans, the main agent related to caries formation [9]. The GIC chemical structure allows the inclusion of different biologically active compounds such as cetylpyridinium chloride (CPC) and chlorhexidine (CHX) [7], or the substitution of a percentage of weight of the GIC for compounds such as magnesium oxide (MgO) or zinc oxide (ZnO) [12]. Nonetheless, CPC and CHX suffer the fate of most antimicrobial drugs, i.e., the reduction of their effectiveness due to microbial adaptation and resistance mechanism [13]. Thus, new effective drugs that are compatible to these materials are essential for the maintenance of the antimicrobial effect.

Imidazolium salts have been reported to be useful as additives for materials with antibiofilm surfaces. Moreover, the insertion of these salts into polylactic acid led to a promising material for biomedical applications [14], and they also presented a great performance when applied to mouthwash formulations, outperforming well-known commercial formulations [15]. Therefore, the aim of this study was to modify the structure of a commercially available GIC by inserting the 1-n-hexadecyl-3-methylimidazolium chloride (C₁₆MImCl) imidazolium salt to obtain a new biomaterial with antibiofilm activity.

Material and methods

Both the CPC (purity > 95%; Labsynth) and the C₁₆MImCl (purity > 98%; Iolitec) were acquired commercially and used without further purifications steps. Test specimens (TS) measuring 5 mm in diameter and 3 mm in height of the Ketac® Cem Easymix 3 M GIC were used for the tests. The TS were divided into three groups: TS₁, composed only of GIC (reference control); TS₂, GIC, and CPC at a 10-ppm (w/w) concentration added directly to the powder (reference drug); and TS₃, GIC, and C₁₆MImCl at a 10-ppm (w/w) concentration added directly to the powder. Immediately after the addition of CPC or C₁₆MImCl into the GIC powder, the compounds were placed in glass test tubes threaded with screw cap, homogenized by inversion in an ALB 260 hematological homogenizer for 2 h. The setting reactions of the TS were followed according to the GIC manufacturer’s instructions. Thus, the powder was mixed with acrylic acid polymer using a metal spatula (model 36), and left to complete the polymerization time. The TS were maintained in artificial saliva solution according to the methodology described by Hook et al. [8] for a period of 28 days.

For the plastic deformation evaluation, the TS were embedded into a flat epoxy base (Poxipol, Uruguay) and analyzed in a Shimadzu HMV-G 20ST Vickers microhardness tester with indentation loading of 200 g per 15 s. The test was performed for three times, and the results in Vickers (HV) were submitted to one-way ANOVA followed by the Tukey’s test.

The hypoallergenicity evaluation of the TS was performed using the Hens’ egg test on the chorioallantoic membrane (HET-CAM test), as described in details by Pippi et al. [16]. For the test, 0.3 ml of each compound at a concentration of 1 mg/ml was added to each egg. Solutions of 0.9% saline and 0.1 M NaOH (standardized with C₈H₅KO₄) were used as negative and positive controls, respectively. After 5 min, they were removed with isotonic saline solution and the degree of tissue change through hemolysis, cell lysis, and coagulation were evaluated. Irritation score results were given according to a formula Pippi et al. [16], where from 0 to 4.9 was denoted as nonirritant (or practically no irritation) and from 5 to 21 was denoted irritant (moderate/severe or extreme irritation).

The biofilm formation was evaluated using the methodology described by Bergamo et al. [15], with modifications. Three Candida glabrata (RL22, RL24, and RL25), three C. tropicalis (57A, 72A, and 72P), and three C. parapsilosis (RL11, RL20 and RL32) strains were used. Strains were resistant to ketoconazole and itraconazole and are deposited in the Mycology Collection of the Federal University of Rio Grande do Sul—UFRGS, Porto Alegre, Brazil. Yeasts were grown on Sabouraud dextrose agar (SDA) (HiMedia, India) for 24 h at 37 °C. Five young colonies were added to 2 ml of Tryptic Soy Broth (HiMedia) and incubated for 24 h at 35 °C to obtain the fungal inoculum. The TS were placed together with 200 μl of the inoculum and 1.8 ml of brain and heart infusion broth (HiMedia) into culture cell plate wells (Nest). After incubation for 72 h at 35 °C, TS were washed three times with 0.9% sterile saline, placed in test tubes with 2 mL of peptone water, and submitted to ultrasound at the frequency of 40 kHz per 10 min (Chemim, Brazil). Finally, serial dilutions corresponding to 10⁻¹, 10⁻², and 10⁻³ were performed, followed by 20 μl of each dilution incubated for 48 h at 35 °C on SDA plates for fungal cell counts (drop plate technique).

Antibiofilm activity of the TS was evaluated by scanning electron microscopy (SEM). Planktonic cells were removed from the TS by washing three times with 0.1 M cacodylate (Sigma-Aldrich) and then added 500 μl of 2.5% glutaraldehyde (Sigma-Aldrich) in 0.1 M sodium cacodylate (dimethylarsenate) for 10 min to fix the biofilm structure. Thereafter, samples were prepared according to Pippi et al. [16] and visualized in a scanning electron microscope (Carl Zeiss EVO® MA10, Germany), operating at 10 kV.

Results

Plastic deformation evaluation showed no significant difference among the three groups, with a mean value of 44.2 HV for TS₁, 43.5 HV for TS₂, and 43.1 HV for TS₃ (P > 0.05). A diamond shape central marking on the TS (Fig. 1a) represents the indentation area obtained with the biomaterial used in this study.

Fig. 1.

a Image of the indentation area in the TS, represented by the central diamond mark located in the vertical midline between the two parallel vertical markings. b Image of the HET-CAM test. The three powder compounds tested (GIC alone; GIC + CPC; GIC + C16MImCl) did not cause damage to the vascular structure

The hypoallergenicity evaluation (HET-CAM test) of the three TS showed no evidence of tissue alteration, considering that the eggs’ chorioallantoic membrane remained intact, with absence of hemolysis, cell lysis, and coagulation (Fig. 1b, c, and d).

GIC in its original composition did not present the capacity to inhibit the growth of any of the isolates tested (Table 1). C. glabrata, C. tropicalis, and C. parapsilosis were able to form biofilm on TS₁ (reference control) in the three dilutions used. Conversely, both TS₂ (CPC) and TS₃ (C₁₆MImCl) were able to inhibit surface biofilm growth for the three Candida species at almost all dilutions tested. This antibiofilm activity was confirmed by SEM. TS₁ surface was covered with fungal and ECM cells (Fig. 2a and b), whereas TS₂ (Fig. 2c and d) and TS₃ (Fig. 2e and f) surfaces showed complete biofilm inhibition, highlighting the absence of plaque growth.

Table 1.

Antibiofilm activity of test specimens (TS) composed of glass ionomer cement (GIC) modified or not by the addition of the imidazolium salts cetylpyridinium chloride (CPC) and 1-n-hexadecyl-3-methylimidazolium chloride (C₁₆MImCl)

Strains	TS1 (GIC)			TS2 (GIC + CPC)			TS3 (GIC + C16MImCl)
	10−1	10−2	10−3	10−1	10−2	10−3	10−1	10−2	10−3
C. glabrata (RL22)	+  +  +	+  +  +	+  +  +	-	-	-	-	-	-
C. glabrata (RL24)	+  +  +	+  +  +	+  +  +	-	-	-	+	-	-
C. glabrata (RL25)	+  +  +	+  +  +	+  +  +	-	-	-	-	-	-
C. tropicalis (57A)	+  +  +	+  +  +	+  +  +	-	-	-	-	-	-
C. tropicalis (72A)	+  +  +	+  +  +	+  +  +	-	-	-	-	-	-
C. tropicalis (72P)	+  +  +	+  +  +	+  +  +	+	-	-	-	-	-
C. parapsilosis (RL11)	+  +  +	+  +  +	+  +  +	-	-	-	+	-	-
C. parapsilosis (RL20)	+  +  +	+  +  +	+  +  +	-	-	-	-	-	-
C. parapsilosis (RL32)	+  +  +	+  +  +	+  +  +	-	-	-	-	-	-

-, negative growth; + , one colony growth; +  + , 2–5 colonies growth; +  +  + , > 05 colonies growth

Fig. 2.

Scanning electron microscopy showing the test specimens surface evaluated against the C. tropicalis 72A isolate. a, b Unmodified GIC, with biofilm development on its surface, with 20 µm and 10 µm scale bars, respectively. c, d GIC + 10 ppm CPC, without biofilm development on its surface, with 20 µm and 10 µm scale bars, respectively. e, f GIC + 10 ppm C₁₆MImCl, with no biofilm development on its surface, with 20 µm and 10 µm scale bars, respectively

Discussion

Herein, we evaluated the modification of a GIC type widely used in odontology, considering the formation of a new biomaterial with fungal antibiofilm activity. The CPC or C₁₆MImCl incorporation into the glass powder and subsequent polymerization reaction did not modify the microhardness of the TS. This initial step is of utmost importance, as the inclusion of additives can compromise the physical–mechanical properties of GIC. Several studies already demonstrated the effectiveness of the insertion of compounds with antimicrobial activities [17]. For example, El-Tatari et al. [18] demonstrated that the insertion of Salvadora persica extract in the proportion of 4% (w/w) into the GIC inhibited C. albicans. Bertolini et al. [19] observed reduction on the C. albicans biofilm formation but also on the GIC microhardness. This can be explained possibly due to the difference in the brand and the type of constitution of the GIC.

Data from HET-CAM test reinforce previous findings on the biocompatibility of conventional ionomeric materials with human cells and tissues. Moreover, previous studies have already indicated that there is no cytotoxicity, genotoxicity, or mutagenicity directly related to effective inhibitory doses of imidazolium salts [11, 14, 20].

Few commercial GIC brands have presented modifications for achieving antimicrobial activity; however, they did not present inhibitory effect against C. albicans biofilm [21, 22]. The potential risk of C. albicans and non-Candida albicans infections in the oral cavity is demonstrated by the isolates ability to form biofilm similar to those of invasive candidiasis [1, 14, 23]. The fact that both CPC and C₁₆MImCl are organic salts that present the same anion (Cl) and bear a positively charged pyridinium and imidazolium ring, respectively, indicates an important role of the cationic head in inhibiting Candida spp. [15]. CPC is a quaternary ammonium salt that causes de-structuring of the biological composition through the hydrogen bond of the molecule with components of the cell wall. This causes an imbalance between the intra- and extracellular medium, with deregulation of the cytoplasmic content, resulting in cell death [13, 24]. Studies indicate the effectiveness of the antibacterial and antifungal action of CPC in biomaterials, dental products, and mouthwash formulations [15, 24], including against fluconazole-resistant Candida dubliniensis [13]. We have previously demonstrated that the C₁₆MImCl mechanism of action is related to interference in membrane regeneration capacity by decreasing the amount of available sterol in the fungal cell, causing depletion of intracellular components [21]. The amphipathicity of the cell wall presents a favorable physicochemical interaction with the imidazolium salt, allowing effective binding and inhibitory action on biofilm formation. In line with these results, Bergamo et al. [25] tested the activity of C₁₆MImCl against planktonic cells and biofilms formed by non-resistant and multidrug-resistant C. tropicalis isolates. A good biofilm penetration was observed, breaking the fungal protection and characterizing an antifungal effect even at very low concentrations. The same antibiofilm activity was observed by inserting the compound into polymeric structures such as resin bases and catheters [21].

In conclusion, addition of low concentrations of the imidazolium salts CPC or C₁₆MImCl to GIC formulation resulted in high antibiofilm activity against Candida spp., without losing GIC original micromechanical and hypoallergenic properties. Considering the demand for biomaterials with antibiofilm activity, this new biomaterial could potentially improve the classical ionomer cement applicable in many areas of dentistry.

Author contribution

AE and AMF made the conception of the work. AE wrote the draft paper with input from AMF. Writing reviews were done by RAZ. RKD obtained and curated the data. VB made the inferences and results analysis. Material preparation, data collection, and analysis were performed by AE, JZAM, and WL.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All the authors are in consent with the inclusion of their names in this manuscript and with the submission for publication.

Conflict of interest

The authors declare no competing interests.