Incorporation of amoxicillin-loaded microspheres in mineral trioxide aggregate cement: an in vitro study

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Restor Dent Endod. 2020;45.e50

Publication date (electronic) : 2020 October 07

doi : https://doi.org/10.5395/rde.2020.45.e50

Fábio Rocha Bohns¹, Vicente Castelo Branco Leitune¹, Isadora Martini Garcia¹, Bruna Genari^1,2, Nélio Bairros Dornelles¹, Silvia Stanisçuaski Guterres³, Fabrício Aulo Ogliari⁴, Mary Anne Sampaio de Melo^5,6, Fabrício Mezzomo Collares¹

¹Department of Dental Materials, School of Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.

²Department of Orthodontics and Biomaterials, Centro Universitário UDF, Brasília, DF, Brazil.

³Cosmetology Laboratory, School of Pharmaceutical Sciences, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.

⁴Yller Biomaterials, Pelotas, RS, Brazil.

⁵Program in Biomedical Sciences, University of Maryland School of Dentistry, Baltimore, MD, USA.

⁶Division of Operative Dentistry, Department of General Dentistry, University of Maryland School of Dentistry, Baltimore, MD, USA.

Correspondence to Mary Anne Sampaio de Melo, DDS, MSc, PhD. Associate Professor, Division of Operative Dentistry, Department of General Dentistry, University of Maryland School of Dentistry, 650 W Baltimore St, Baltimore, MD 21201, USA. mmelo@umaryland.edu

Correspondence to Fabrício Mezzomo Collares, DDS, MSc, PhD. Associate Professor, Department of Dental Materials, School of Dentistry, Federal University of Rio Grande do Sul, Rua Ramiro Barcelos, 2492 Rio Branco, Porto Alegre, RS 90035-003, Brazil. fabricio.collares@ufrgs.br

Received 2020 March 08; Revised 2020 May 11; Accepted 2020 May 26.

Abstract

INTRODUCTION

Bacterial pathogens can reach the dental pulp, compromising the inner section of a tooth that contains the nerve tissue and blood vessels [1]. As a result of carious lesions or tooth fractures, bacteria gain entry into the pulp of the tooth and can be sheltered into dentinal tubules and root canal [2]. These microorganisms and their byproducts are responsible for initiating/maintaining a periapical inflammatory process of the pulp tissue that often become necrotic [3]. As the infection progresses, bacteria spread down the root, and through the apical foramen, reach the apex of the tooth root [4]. In cases of tooth infections in young patients, the affected tooth can be immature with incompletely formed roots. The treatment that met the standard of care for infected immature teeth relies on the use of a specific reparative material to neutralize the bacteria and to stimulate the root end closure process by forming a mineralized apical barrier [56].

The failure to induce root-end closure (apexification) is often caused by the persistence of microorganisms in the root canal system [7]. Mineral trioxide aggregate (MTA)-cements are Portland-like cement compounded by tricalcium silicate, tricalcium aluminate, tricalcium oxide, silicate oxide, and bismuth oxide [8]. This class of material has outstanding bioactivity with substantial calcium ions release and satisfactory performance as sealing material for root canal treatment [9]. Regrettably, most sealing materials, including MTA-based root canal sealers, lack of substantial antibacterial properties [10]. Some of them that were found to have bacteriostatic behavior, only retain this property for few days or lose this quality after the setting of the material [1112]. As these materials have intimate contact with the root canal structures and tissues, it would be beneficial if sealing materials retained their antibacterial properties.

Polymer-based drug delivery systems provide a robust technology platform to enable sustained, spatiotemporally controlled drug release [13]. Microspheres have long been used in drug delivery applications because of their managed release capabilities [14]. Microsphere-based strategies have attracted attention in Dentistry because these materials may provide controlled release of bioactive molecules to promote disinfection and consequent healing of surrounds tissues [15]. Microspheres are organic or inorganic, spherical, free-flowing particles ranging from 1 to 1,000 μm in diameter that may carry drugs or bioactive molecules [16]. In medicine, they have been extensively used in drug delivery/targeting applications mainly because of their ability to enhance the efficacy of the associated drug by providing both a large surface area–to–volume ratio for drug release and spatial and temporal control over release [17].

Poly-methacrylic acid-co-methyl methacrylates are copolymers of methacrylate and methacrylic acid, and its use to delayed drug delivery are well reported [18]. This class of synthetic polymers, as known as Eudragits, presents variations in their proportion of neutral, alkaline, or acid groups, resulting in different physicochemical properties, including controlled release [19]. Overall, β-lactam antibiotics are used as the first option for the treatment of endodontic infections [20]. Amoxicillin is a broad-spectrum antibiotic with lower rates of bacterial resistance and the risk of side effects in endodontics [21]. The addition of the antibiotics system with a delayed-release profile to MTA could be a promising approach to improve the treatment outcomes (Figure 1).

Figure 1

Illustrative scheme of a possible approach for the proposed bioactive dental cement with amoxicillin-loaded polymeric microspheres.

Based on it, the present study investigated a simple and effective approach for the incorporation of amoxicillin-loaded polymeric microspheres into a modified formulation of bioactive cement and evaluated the resulting drug release profile and the effect on clinically relevant physical and mechanical properties.

MATERIALS AND METHODS

Amoxicillin microspheres preparation

Microspheres loaded with amoxicillin (MS_AMOX) were prepared with poly(methacrylic acid-co-methyl methacrylate [Eudragit S100]), and trihydrate amoxicillin following a previous study [22]. Briefly, the polymer was first dissolved in acetone (200 mL), and subsequently, the amoxicillin was added under magnetic stirring at 25°C. The resultant solution was spray-dried using a Mini-Spray Dryer B-290 (Buchi, Flawi, Switzerland) coupled to a dehumidifier (B-292, Buchi) using the following parameters: feed pump rate of 5.0 mL min⁻¹, 100% aspiration, 0.7 mm nozzle, atomization air at 819 Lh⁻¹, and inlet temperature of 60°C with a resulting outlet temperature of approximately 40°C. Figure 2 summarizes the synthesis route for the microspheres. Morphological characteristics of microspheres of amoxicillin were evaluated using scanning electron microscopy (SEM; JSM 6060, Jeol, Tokyo, Japan) with a voltage of 15 kW.

Figure 2

Schematic demonstration of the synthesis route for microsphere preparation by spray-dry method.

Composition of mineral trioxide aggregate formulations

Synthetized MS_AMOX presented a mean size of 2.664 μm, d10 of 1.369 μm, d50 of 2.430 nm, and d90 of 4.315 μm according to a previous study [22]. To prepare the groups of MTA, MS_AMOX was mixed with the MTA at the weight percentages of 0%, 2.5%, and 5%. They were selected following favorable results of pilot tests. The MTA (WMTA Angelus, Angelus, Londrina, PR, Brazil) was used as parent material for the MS_AMOX's incorporation. Preliminary studies were performed to tune the optimal liquid/powder ratio able to reach an acceptable homogenization of 5.0 wt% of MS_AMOX. The resulting homogeneity was achieved at a liquid/powder ratio of 1.16 mL/g. Based on these screening results, 2 formulations were used as a control: 1) cement mixed with deionized water using the manufacturer's instruction at a liquid/powder ratio of 0.30 mL/g, (Control_{ratio 0.3}) and 2) cement mixed with deionized water at a liquid/powder ratio of 1.16 mL/g (Control_{ratio 1.16}). The experimental cements were prepared by mixing the tested MS_AMOX concentrations with deionized water at a liquid/powder ratio of 1.16. Table 1 describes the chemical compositions of formulations assessed in this study.

Table 1

Description of the composition from each group formulated in this study

Diametral tensile strength

The samples (n = 3) for diametral tensile strength (DTS) tests were prepared with cylindrical silicone molds measuring 9 mm in diameter and 1.5 mm in height. Each group was mixed and poured into the molds; the excess of the material was removed, and a polyester strip was put over the samples to secure a flat surface. All samples were allowed to set in an incubator at 37°C for 7 days. DTS of the samples was conducted on a universal testing machine Shimadzu (EZ-SX, Shimadzu, Japan) at a loading rate of 1 mm/min. DTS of the specimens were calculated using the relationship:

DTS = 2Pπbw

Where: P is the maximum load (N), b is the diameter (mm), and w is the thickness (mm) of the measured specimen.

RESULTS

Figure 3 shows SEM images of the microspheres fabricated by the spray-dry method. Figure 3A shows Spherical-shaped particles with a diameter ranging from 2 µm to 5 µm. The microspheres with its mashed shell indicate that the microspheres were not solid capsules but consist of a single internal cavity, which was occupied by amoxicillin. The non-spherical appearance of some of the particles can be due to the applied cutting force. The microspheres present irregular morphology and various size distribution. Some of these microspheres were also seen to be interconnected (arrow; Figure 3B). This is thought to have occurred as a result of liquid residue remaining between the microspheres and polymeric interconnection between the particles. The acetone subsequently acted as a solvent on minimal quantities of the shell surface, which is clearly indicated in Figure 3C.

Figure 3

Scanning electron microscopy images of amoxicillin-loaded microspheres.

The amoxicillin release profile of MS_AMOX mixed with the reparative cement at the weight percentages of 5% is shown in Table 2. The release profile demonstrated that the MS_AMOX effectively retarded the drug release after 7 days of storage and showed an approximately 17% of drug release after 96 hours of evaluation.

Table 2

Amoxicillin release profile of 5% microspheres loaded with amoxicillin

Figure 4 displays the initial setting time of the reparative cement with and without MS_AMOX. One way ANOVA and Tukey tests showed that the modification of power: liquid ratio of MS_AMOX significantly prolonged the initial setting time from 805 seconds to 1,433 seconds. When adding MS_AMOX at 2.5%, only slightly prolonged the initial setting time of 83 seconds was noted concerning Control_{ratio 1.16}. Increasing the concentration of MS_AMOX revealed an initial setting time of 1,417 seconds, similar to the Control_{ratio 1.16}. All the experimental formulations were statistically different from the Control_{ratio 0.3} (p < 0.05).

Figure 4

Setting times of tested cement formulations as the microsphere concentration increases. Values followed by the same letters are not significantly different (p > 0.05).

The means and standard deviations of the compressive strength of the groups are shown in Figure 5. One way ANOVA and Tukey tests indicated statistically significant different values among groups. The compressive strength values of the Control_{ratio 1.16} were significantly lower than the Control_{ratio 0.3} (p < 0.05). Overall, the incorporation of MS_AMOX has improved the compressive strength results. MS_AMOX at 2.5% and MS_AMOX at 5% has shown an increase of 90.8% and 92.8%, respectively, in relation to Control_{ratio 1.16}. Comparing to the Control_{ratio 0.3}, the MS_AMOX at 5% has statistically similar.

Figure 5

Diametral tensile strength (DTS) of the tested cement formulations according to microsphere concentration. Values followed by the same letters are not significantly different (p > 0.05).

DISCUSSION

In the present study, MS_AMOX were successfully synthesized and incorporated in a tuned formulation to grant delays in the amoxicillin release profile for the first time. With this approach, the prolonged antimicrobial effect during extended periods could increase the killing of remaining bacteria, which improve the healing tissue integrity and cellular function. Therefore, recent interests and significant efforts in preventing and actively treating tooth infections by directly targeting infection causative agents through direct application of antimicrobial agents either alone or loaded into dental materials. These have the advantage of overcoming challenges such as poor, locally delivered antibiotics [25]. Single-visit apexification using calcium silicate cements has been successfully used for the endodontic treatment of teeth with necrotic pulps and open apices [26].

Here, MS_AMOX effectively delayed the drug release when incorporated in the regenerative cement. At the measuring time of 96 hours, the amoxicillin release profile showed 16.68% of drug release but showed no release at previous measurements (Table 2). Similar results have been obtained by other researchers when using of acrylate-based polymers as a delayed carrier [26]. The microspheres obtained are monolithic particles retained in a porous or solid polymer matrix, differently from microcapsules that consist of a solid or liquid drug container, surrounded by a polymeric membrane [2728]. The primary process of drug release from microspheres relays on diffusion, biodegradation, desorption of the adsorbed drug from the surface [14].

MS_AMOX can be prepared by solid-state reaction, co-precipitation, sol-gel method, and spray-dry routes [16]. In the current study, a facile method obtained the loaded microspheres using a Eudragit S100. The used copolymer solubilizes at pH 7. However, the alkaline medium induced by Ca²⁺ released from MTA-cements may delay its degradation, retarding the release of the drug [2930]. The MTA's ability to increase the local pH makes this material very favorable as a vehicle for polymeric microspheres.

The setting behavior of MTA is driven by many variables such as the fineness of the particles, the water/powder ratio, temperature, humidity of application site^, and the addition of substances [31]. As a Portland cement, MTA absorbs water. The water trapped in the cement mix and saturated by calcium hydroxide is then released to the environment [32]. High water content and the presence of amoxicillin microspheres may have affected the hydration mechanism of the used MTA-base cement, increasing the setting times of experimental groups. Fridland and Rosado [32] found that the physical properties of MTA changed when mixed with different water-to-powder proportions [33]. However, the amount of water incorporated in the mix is limited by its loss of consistency in the presence of excessive liquid. The modified MTA-base cement had a higher liquid-to-powder ratio in comparison with MTA, which needs more mixing liquid to achieve desired consistency to work. The targeted formulation of MS_AMOX at 5% presented suitable handing properties at the water-to-powder ratio of 1.16.

The liquid-to-powder ratio can also determine the mechanical properties of MTA-base cement. A higher liquid-to-powder ratio gives rise to an increasing degree of water pore volume, porosity, and solubility, which consequently can influence the mechanical characteristics of the cement. In our study, the incorporation of MS_AMOX has improved the compressive strength results. As amoxicillin has hydrophilic characteristics, its addition to the mixture may have contributed to absorbing water, remaining less unreacted water in the cement bulk, significantly increasing the strength of the targeted formulation of MS_AMOX at 5% [34]. Also, the smooth and round shape of microspheres works as a distributor of stress, which may contribute to the significant increase in mechanical property. The MS_AMOX at 5% presenting adequate strength can get the benefit of the humid environment because of the inherent hydrophilic nature. Further studies to investigate the in vivo behavior of MTA-base cement with synthesized MS_AMOX at 5% seem relevant.

CONCLUSIONS

In this work, we have tuned the formulation of a bioactive cement with polymeric microspheres to impart delayed release of antibiotics as a potential drug delivery system to treat local tooth infections. The resulting synthesis is facile, reproducible, and highly reliable. The incorporation of polymeric microspheres at the 5 wt% conveys delayed-release without significant detrimental effects of the physical and mechanical behavior of the material. The possibility of slowing the release of the antibiotics delivered at local sites of infection could find potential applications within dental materials that are in close contact with dental tissues.

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