Biological properties of experimental dental alginate modified for self-disinfection using green nanotechnology

Many materials are available nowadays to take primary and secondary impressions for patients in dental clinics [1]. The choice of an impression material for a particular situation is mainly dependent on the treatment protocol and the operator’s preference. Meanwhile, hydrocolloids and elastomeric polymers are the most used impression materials for various dental treatments [2]. Hydrocolloid dental materials include both agar–agar and alginates, which are viscous liquids, present in a sol state or a gelatinous consistency.

Alginates are salts of alginic acid, a polysaccharide extracted from the cell walls of brown algae. They belong to the Phaeophyceae family, which is widespread, especially in the colder oceans of the Northern Hemisphere [3]. Dental alginates are irreversible elastic hydrocolloids that were developed in the 1940s when the agar impression material was limited [4]. Irreversible alginates consist of salts of alginic acid, calcium sulfate as a reactor, zinc oxide, potassium titanium fluoride, diatomaceous earth, and coloring or flavoring agents [5]. Alginate is provided in the form of a powder to be mixed with water and set by a chemical reaction that cross-links the carbohydrate polymer, forming a hydrogel [5].

Alginates are one of the most frequently and routinely used dental materials in every dental practice, especially at the first dental visit for a pre-treatment evaluation. Their common usage is due to their cost-effectiveness, ease of use, and fast setting time that can be even controlled by temperature [6]. Disadvantages include less accurate reproduction of details as compared with elastomeric impression materials, poor dimensional stability, and inadequate retention of non-perforated trays [7]. Moreover, hydrocolloids are hydrophilic by nature; therefore, they swell if immersed in water or disinfectant; and thus; their dimensional stability upon disinfection and storage is problematic [8].

Dental impressions present a source of cross-infection to the dentist and dental technicians since they are exposed to blood and saliva inside the oral cavity; hence, disinfection is mandatory for impression materials [9]. On the other hand, distortion can be a problem if disinfection guidelines are not strictly followed. Disinfectant sprays are used for alginate impressions, but they do produce air bubbles in the cast, thereby affecting accuracy [10]. Although immersion in disinfectants like 1% sodium hypochlorite or 2% glutaraldehyde can result in dimensional changes of only 0.1%, still the quality of the impression surface may be greatly compromised if the recommended period and other factors are not strictly controlled [11, 12].

Attempts to incorporate disinfectants into the alginate powder or mixing water were found to be an effective way of disinfection with minimal adverse effects on dimensional accuracy and surface details [13, 14]. Follow-up studies of irreversible hydrocolloid impression materials pre-impregnated with disinfectants have shown that this technique saves time, is active against oral pathogens, and demonstrates greater dimensional stability than spray and immersion techniques [15].

The antibacterial activity of metal and metal oxide nanoparticles is extensively studied in medicine [16, 17]. Silver and its compounds have been used as antimicrobial agents for various medical purposes. Silver ions are effective against bacteria, viruses, and fungi besides causing no harm to humans at low concentrations [18]. Materials with at least one external dimension of 1–100 nm are defined as nanomaterials or nanoparticles (NPs), and they have attracted increasing interest in recent years, especially in dentistry [19]. Silver nanoparticles have demonstrated unique and significantly different physical and chemical properties compared to their macroscopic counterparts. The smaller the nanoparticles, the greater the surface-to-volume ratio, dispersion, and antimicrobial efficacy [19].

Conventional approaches for the production of nanoparticles (NPs) are typically expensive, toxic, complicated, and non-ecological [20]. Green nanotechnology is a recent approach, which utilizes microorganisms, plants, or their extracts as reducing and capping agents in the synthesis of AgNPs. Plants play an important role in the biosynthesis of NPs and their major advantage is that they are easily available, safe, and contain a variety of metabolites that can contribute to the reduction of silver ions [20]. Green nanoparticles exhibit distinct characteristics compared to those generated through physical and chemical means. The green process employs a bottom-up approach to create magnetic nanoparticles using biological constituents like plant extracts or bacteria to replace the costly chemical-reducing agents. The eco-friendly transformation of microparticles into NPs through green reduction is environmentally favorable, sustainable, devoid of chemicals, cost-effective, and scalable. Additionally, green synthesis leads to the recovery and recycling of valuable metal salts like gold (Au) and silver (Ag) from waste streams [21, 22]. Boswellia sacra (B. sacra) is a tree in the genus Boswellia from which frankincense oleo gum resin is collected. It is native to Oman, Yemen, and Somalia. B. sacra finest sorts are presented under the local names Houjri, Najdi, and Sahli or Shaebi, based on the region of cultivation in Oman. Houjri is the first-grade, most expensive resin that is growing in the north of the Samhan Mountains [23]. Resin essential oils contain several pharmacologically active compounds (monoterpenes, sesquiterpenes monoterpenes, sequiterpinols, and ketones) that have antimicrobial activity against important human pathogens, both bacterial and fungal organisms, such as Staphylococcus aureus, Escherichia coli, Proteus vulgaris, and Candida albicans [23].

To the best of our knowledge, Boswellia sacra (B. sacra) plant extract has never been used for the green synthesis of silver nanoparticles and has not been incorporated before or used for the disinfection of any hydrocolloid impression material in dentistry. Therefore, in our previous and present investigations, we aimed to replace the water used for the preparation of alginate with a prepared aqueous solution of B. sacra added to silver nitrate in a given concentration to biosynthesize nanoparticles for enhanced antimicrobial activity. Moreover, a 0.2% silver nitrate solution and a 0.2% CHX solution were used for the preparation of two other antimicrobial-modified groups for comparison.

Our former results which assessed our modification on alginate physical and mechanical properties showed that detail reproduction and accuracy of alginate were not negatively impacted by the different self-disinfection modifications. Moreover, elastic recovery was improved by the addition of CHX, AgNO₃, and BS + AgNPs. Additionally, it was found that all groups reported tear strength values that were within the acceptable range, with CHX and BS + AgNPs showing significantly higher tear strength values compared to the control group [24].

Therefore, based on the fact that the green synthesis of metal nanoparticles using the Boswellia sacra extract did not alter the functional performance of alginate, this study aimed to chemically characterize B. sacra extract and to confirm the production of the green-synthesized nanoparticles using color change, UV–Vis spectroscopy, and scanning electron microscopy (SEM). Moreover, the effect of the modifications was tested along with control alginate against six microbial strains to confirm its efficacy.

Alginate impressions are regularly contaminated with patients’ blood and saliva, thus acting as a potential medium for the transfer of infectious microorganisms and viruses between patients, operators, and dental auxiliaries [9]. The presence of numerous microbes, including streptococci, staphylococci, Candida, Pseudomonas aeruginosa, and MRSA on alginate impressions and gypsum casts has been documented and can be a cause of infections in dental clinics [31]. Since alginates undergo syneresis and imbibition according to the surrounding conditions, post-setting disinfection by spraying or immersion often compromises the accuracy of alginate impressions [32].

In the present study, a color change was observed over time after mixing BS extract and silver nitrate solution. The color change resulted from the excitation of surface plasmon resonance (collective movement of free electrons in the silver when light falls on it) due to the reduction of Ag²⁺ ions to Ag 0 by biomolecules such as phenols, flavonoids, ketones, tannins, and proteins present in BS extract [33]. These phytochemicals contain hydroxyl and ketone groups that induce the reduction of Ag ions to form appropriate nuclei that grow during the development phase into spherical AgNPs [34].

Furthermore, UV–Vis spectrophotometry confirmed the formation of AgNPs by yielding a bell-shaped spectrum after the different time intervals. The broad plasmon band may be due to the presence of plant metabolites in the solution, which may adsorb the light in this spectrophotometric range as well. The peak was observed at 440 nm, and an absorbance between 400 and 460 nm is always characteristic for the formation of silver nanoparticles [35]. It is worth mentioning that spherical nanoparticles show only a single SPR band and the number of peaks increases with an increasing range of particle shapes [36, 37]. SEM magnified images of samples have shown that the particles were mostly spherical with a size distribution in the range of 50 to 100 nm. Agglomerated AgNPs were also present, which may be a sign of sedimentation [38].

The antimicrobial activity of the modified groups was tested using agar diffusion assays against different microbial strains including four Gram-positive bacteria (S. aureus, methicillin-sensitive and resistant, S. mutans, and M. luteus), one Gram-negative bacterium (E. coli) and a yeast (C. albicans). The three modified groups were significantly more active than the unmodified alginate against all microbial strains. Results showed that the BS+AgNPs group differed insignificantly from AgNO₃ only against all strains except S. aureus. On the other hand, the antimicrobial activity of BS+AgNPs was comparable to the CHX group against C. albicans and MRSA. CHX showed significantly higher activity than all other groups against S. mutans, S. aureus, E. coli, and M. luteus. The unmodified alginate also showed a reproducible weak antibacterial activity against both S. aureus strains and M. luteus, which might be due to the presence of zinc ions in the alginate powder.

The results are in agreement with several studies, which reported that the incorporation of disinfecting agents such as silver nanoparticles, quaternary ammonium compounds, chlorhexidine, iodine, bisguanidine compounds, and ammonium chloride into the impression materials eliminates the need for separate disinfection of the impression after removal from the mouth [39, 40]. Our results showed also that BS+AgNPs were efficient against an MRSA which is an antibiotic-resistant strain and thus might have the potential to be used in medicine. Profound antimicrobial activities of green-synthesized AgNPs were previously reported by Vanlalveni et al. [41], Akhtar et al [42], and Tahmasebi et al. [43].

The antimicrobial activity of BS+AgNPs could be due to the synergistic action of both the plant extract and AgNPs. Different phytochemical constituents were identified by gas chromatography/mass spectrometry in this and a previous study [44]. Terpenoids (e.g., boswellic acids, cymene-7-ol, L-pinocarveol, carveol, and cis-sesquisabinene hydrate) were detected in considerable amounts in the B. sacra extract. Although the antibacterial mode of action of terpenes remains mostly unknown, it has been reported that most terpenoids act by partitioning into the membrane, increasing its permeability and dissipating proton motive force [45].

Carveol, for example, has been shown to affect the membrane integrity in E. coli and S. aureus and induce leakage of potassium from S. aureus cells [46]. Moreover, alkaloids (e.g., quinidine), sterols (e.g., stigmasterol), flavonoids (e.g., vitexin), phenols (e.g., cis-p-Mentha-2, 8-dien-1-ol), and saponins (e.g., squalane) were detected. Many of these secondary plant compounds have long-established antimicrobial activities, e.g., saponins damage the bacterial cell membrane [47], stigmasterol is bacteriostatic for MRSA [48], and vitexin inhibits biofilm formation of P. aeruginosa and alters the surface properties of S. aureus) [49].

Silver in a nanometre scale of less than 100 nm is toxic to a wide range of microorganisms [50]. Although the exact mechanism of silver nanoparticles’ antibacterial effects has not been completely clarified, various antibacterial actions have been proposed [51]. Silver nanoparticles can penetrate bacterial cell walls, damage the cytoplasmic membrane, and even result in cell lysis [50]. There is also an influence of the particle size and shape on the release of silver ions since AgNPs with spherical or quasi-spherical format are more susceptible to silver release, due to their larger surface area [51]. Moreover, Gram-negative bacteria are more susceptible to silver nanoparticles than Gram-positive strains. The cell walls of Gram-positive bacteria are composed of a thick peptidoglycan layer of linear polysaccharide chains cross-linked by short peptides, thus forming a more rigid structure leading to difficult penetration of the AgNPs compared to the Gram-negative bacteria, where the cell wall possesses thinner peptidoglycan layers [52, 53].

Silver nitrate, which is a suspension of sub-microscopic silver ions, can significantly reduce the duration and severity of many bacterial infections [54]. Silver is an inert metal but it is biologically active in an aqueous environment in which it is present in an ionic soluble state (Ag⁺) [55]. One of the main advantages of silver is its oligo dynamic effect, of having high microbicidal capacity in water at a very low concentration (one part per million) [55]. One of the important mechanisms of Ag⁺ toxicity is the ability of silver ions to interact with the bacterial inner membrane and impair its integrity. Additionally, silver ions target the SH groups on proteins, disrupt their disulfide bonds, and inactivate dehydratases by breaking down 4Fe-4S clusters [56]. A recent study showed that in S. aureus, silver ions target proteins involved in glycolysis, pentose phosphate cycle, and defense against ROS [57].

The toxicity of silver nitrate is dosage-dependent; oral ingestion of more than 2 g of silver nitrate can be fatal [54]. Silver nitrate rapidly reacts with chloride yielding extremely non-soluble silver chloride that causes a fatal electrolyte imbalance. However, the dosage of silver nitrate used in this study (0.2%) is very low, and for the BS+AgNPs group, it was even lower. One milliliter of 0.2% silver nitrate solution contains 0.002 g silver nitrate. A 38-ml solution that is used to make a full arch impression contains 0.08 mg silver nitrate and is equivalent to 0.33% of a fatal dose. In addition, it should be taken into consideration that silver nitrate was not used in a free form or ingested, but instead, it went through a chemical reaction and is enclosed in a gel that is used topically inside the mouth and not in a liquid form.

Chlorhexidine in the present study was used as it has a proven antimicrobial activity against various microbial strains [58]. The selected concentration of CHX (0.2%) is believed to be effective for oral disinfection and plaque inhibition with no serious side effects [58, 59]. The positively charged chlorhexidine works actively against bacteria by binding to the negatively charged sites on the bacterial cell wall causing destabilization of the cytoplasmic membrane and may affect membrane proteins [60, 61]. The bacterial uptake of chlorhexidine is very fast, typically functioning within 20 s. For C. albicans (fungus), the mechanism of action is almost the same in that the fungus uptakes chlorhexidine rapidly and damages the integrity of the cell wall and the plasma membrane resulting in leakage and cell death [62].

It is important to note that impression materials that come into contact with contaminated saliva and blood can pose a substantial risk of cross-contamination, not only with bacteria and fungi but also with highly contagious viruses like hepatitis B, hepatitis C, herpes, and HIV. As a result, there are plans to conduct further research to assess the effectiveness of the green-modified alginate against these viruses.

Within the context of this study, it can be concluded that the usage of Boswellia sacra plant extract was effective for the biosynthesis of AgNPs in a simple, inexpensive, and ecologically friendly way at room temperature. The analysis of the BS extract confirmed the presence of different organic compounds that can act as reducing and stabilizing agents for the biosynthesis of AgNPs. Furthermore, the antimicrobial activity of dental alginate was enhanced by the incorporation of 0.2% CHX, 0.2% AgNO₃, and green-synthesized AgNPs against all tested microbial strains. Future work involving testing the effect of the three used antimicrobial agents on the dimensional accuracy, detail reproduction, tear strength, elastic recovery, setting time, and flow of alginate is in progress.