Photodynamic Therapy in the Management of MDR Candida spp. Infection Associated with Palatal Expander: In Vitro Evaluation

Abstract

The aim of this work is to evaluate the effectiveness of antimicrobial photodynamic therapy (aPDT) against oral MDR (multi-drug-resistant) Candida spp. infections related to orthodontic treatment with palatal expanders through in vitro study. Methods: PDT protocol: Curcumin + H₂O₂ was used as a photosensitizer activated by a 460 nm diode LED lamp, with an 8 mm blunt tip for 2 min in each spot of interest. In vitro simulation: A palatal expander sterile device was inserted into a custom-designed orthodontic bioreactor, realized with 10 mL of Sabouraud dextrose broth plus 10% human saliva and infected with an MDR C. albicans clinical isolate CA95 strain to reproduce an oral palatal expander infection. After 48 h of incubation at 37 °C, the device was treated with the PDT protocol. Two samples before and 5 min after the PDT process were taken and used to contaminate a Petri dish with a Sabouraud field to evaluate Candida spp. CFUs (colony-forming units). Results: A nearly 99% reduction in C. albicans colonies in the palatal expander biofilm was found after PDT. Conclusion: The data showed the effectiveness of using aPDT to treat palatal infection; however, specific patient oral micro-environment reproduction (Ph values, salivary flow, mucosal adhesion of photosensitizer) must be further analyzed.

1. Introduction

The use of palatal expanders (PEs), as well as all fixed orthodontic appliances, raises the incidence of tissue stress lesions and related bacterial and fungal infections [1]. The palatal expander must be rigid and carry out its action thanks to a screw intrinsic to the device placed centrally between the two hemiarches and generally anchored to the first molars or first deciduous molars by bands. A PE is indicated in the treatment of upper transverse skeletal insufficiencies; the device is capable of generating forces close to 10 kg on the anchoring structures; therefore, rigidity represents a fundamental requirement due to the resistance to these separations of the maxillae offered not so much by the suture but from the basal portions of the bone [2]. Recent studies by Carli et al., 2023, evaluated the prevalence of interceptive orthodontic treatments in schoolchildren [3]. Among 2199 subjects, in the case of tissues that correspond with orthodontic expanders, there has frequently been documentation of an inflammatory response of the mucosa due to mechanical traumatic injuries and/or accompanied by an increase in local or disseminated dysbiosis. In fact, in general, the existence of retentive areas around the parts of fixed appliances affixed to the teeth appears to be the primary cause of the elevated build-up of dental plaque and the inflammatory general response [4]. Furthermore, following fixed orthodontic treatment, a notable decrease in periodontal attachment has been shown [5,6]. In order to prevent these side effects, patients who have fixed equipment should adhere to a very strict oral hygiene regimen [7]. Initially, clinical changes that occur in oral mucosa (gingival and palatal) during PE treatment are related to mucosal hyperplasia, in which, in some cases, the histological analysis performed to have a correct diagnosis confirmed the mechanical nature of the lesion, thus corroborating the link between the expander’s insertion and the development of palatal mucosal hyperplasia [8]. In these conditions, in overstressed tissues, an associated infection with pathogenic bacteria or fungi is highly likely. In this context, palatal infection appears to be a multifactorial phenomenon, mainly due to the following: (i) PE positioning strategy; (ii) patient oral microbial condition; (iii) patient hygiene rules; and (iv) laboratory monitoring for infection and inflammation level. In terms of laboratory diagnosis for mucosal infection related to PE, many authors reported bacterial and especially fungal infections. The most common fungal genera associated with PEs belong to Candida spp.; a recent article published by Brzezińska-Zając et al., 2023, reported C. albicans and C. parapsilosis as the predominant yeast strains on removable appliances in young patients [9]; the same authors identified new Candida strains, isolated at the end of the follow-up, namely C. dubliniensis, C. kefyr, and C. krusei, which are associated with C. albicans [9]; other publications reported C. tropicalis, C. guilliermondii, and Candida krusei as species associated with C. albicans [10,11], which represents the main strain found on orthodontic appliances (more than 50%) [10].

• Orthodontic appliances and impact on oral microbiota

As described before, the biological impact of orthodontic appliances is related to the mechanical interactions between these devices and gingival and palatal tissue cells. In fact, an intensive mechanical force and/or an inappropriate manufacturing material could be responsible for a chronic inflammation around the involved tissues [11]. This biological “host–device” disequilibrium is able to interfere with the largest and most complex oral microbiota. Considering the study by Kado et al. on Japanese patients [12], which utilized 16S rRNA meta-analysis sequencing, the use of an orthodontic appliance causes dynamic shifts in the composition of the oral microbiome. Bacteroidetes and TM7 bacteria significantly increased with time in both plaque and saliva, while Proteobacteria and Actinobacteria decreased, according to taxonomic analysis at the phylum level, in which anaerobic and facultative anaerobes were found in much higher numbers in both saliva and plaque. A rise in the relative abundance of obligatory anaerobes, such as periodontal pathogens, i.e., Porhyromonas gingivalis, was observed. In summary, this dysbiosis level observed with fixed orthodontic appliances could probably be a stage in between a healthy and periodontitis microbiome transition. This result is comparable with other authors who described an increasing risk for periodontal diseases and caries in orthodontic patients [13].

• Oral microbiota differences in fixed orthodontic appliances and aligners

There are differences in oral microbial population between patients with fixed orthodontic treatment with brackets and aligners. Several factors can influence the oral microbiota in orthodontic patients, such as plaque accumulation, metal corrosion (brackets), host immunity, hormone levels (age of the patient), and tooth movement [14]. In fact, orthodontic appliances, both fixed and removable, promote the retention of food particles and dental plaque, making it more difficult to maintain oral hygiene and increasing the percentage of oral infections, white spot lesions (WSLs), and bad breath. Researchers have demonstrated that levels of S. mutans, Lactobacilli, and Candida albicans increased six months after the insertion of orthodontic appliances, with higher values in the fixed appliance group of patients than in the removable appliance group [14]. Aggregatibacter actinomycetemcomitans in the subgingival sites is increased 3–6 months after the insertion of the fixed appliance compared to untreated patients. Recently, researchers observed that metal brackets with helical ligatures retained more plaque among fixed orthodontic appliances, which was responsible for the worsening of bleeding parameters on probing, and found that the plaque index is higher compared to steel ligatures [15]. No important differences are observed in the average counts of P. gingivalis, S. mutans, and other periodontal and cariogenic bacteria between aesthetic ceramic brackets and metal ones [16]. Patients with aligners compared to those with fixed appliances showed a similar increase in the total bacterial load, but subjects with fixed appliances had a significant increase in the amounts of cariogenic species in their saliva [17].

• Clinical approach against oral infections due to palatal expanders

Certain changes in the oral environment are brought about by orthodontic therapy, especially when fixed appliances are used. These changes include a decrease in pH and an increase in the buildup of dental biofilm. Oral hygiene is hampered by the uneven surfaces of brackets, bands, wires, and other orthodontic components, and the effectiveness of salivary and muscle movements as mechanical self-cleaning agents is restricted. The presence of inflammatory components and altered salivary parameters, such as pH, could elevate the mass of pathobionts and cariogenic bacteria [18,19]. While a normal treatment regimen could be performed with a mouthwash [20], a problem could increase if the infection is due to a multi-drug-resistant (MDR) strain [21]. Pellissari et al., in a study of subjects wearing a fixed orthodontic appliance, among the genera Streptococcus, Staphylococcus, Sphingomonas, Enterobacter, and Candida, found that 74% of clinical isolates showed antimicrobial resistance [21]. In this context, a raised problem is the presence of MDR strains of Candida spp.; these strains have become resistant to first-line antifungal agents. The alternative treatment options against these fungal infections are limited because of their high toxicity [22]. For this reason, the research has shifted toward testing alternative medicines due to the growing aspect of drug resistance and the rise in oral infections caused by non-albicans species that are less sensitive to traditional antifungals, and the promising new therapeutic protocols include (i) plant products that can be used in conjunction with phytotherapy [23,24]; (ii) topical probiotics for balancing the oral microbiota in cases of dysbiosis [25], as well as ozone therapy (ozone-based gel or ozonized water) [26,27]; and (iii) antimicrobial photodynamic therapy (aPDT).

• Photodynamic Therapy (PDT) in orthodontics

PDT is a potent substitute for drug administration that exhibits highly selective antifungal action without causing any side effects [28,29]. PDT represents a therapeutic approach that involves the presence of three elements: a photosensitizer that binds specifically to microbial cells; a laser or non-laser light, with a specific wavelength for each type of photosensitizer (which corresponds to its maximum absorption spectrum); and the bioavailability of oxygen. These three elements react to determine the formation of free radicals and reactive oxygen species that are released inside the target cells to which the photosensitizer has adhered. For this reason, this therapy is considered safe for host cells and precise in controlling microbial infections, such as oral resistant fungal infections [30,31,32].

The use of PDT in patients undergoing orthodontic therapy is widely documented in the literature through clinical trials [33]. Although it has demonstrated a certain effectiveness in lowering the periodontal bacterial load, in modulating gingival hyperplasia, and in preventing white spots, no study has ever proposed it as a tool for decontaminating the oral tissues beneath a palatal expander [33].

The most used parameters of PDT in orthodontics are methylene blue with a concentration of 0.0005% as a photosensitizer and then irradiation with light at 670 nm, J/cm², 150 mW, and curcumin as photosensitizer activated by light source with a wavelength of 450 nm, fluence of 96 J/cm², and fluence rate of 165 mW/cm² [22,33].

In vitro studies carried out almost exclusively on brackets report the use of curcumin enriched with nanoparticles and activated by lights at 405 nm and a fluence rate of 150 mW/cm² and the use of riboflavin and rose bengal activated with lights at 375 nm and 3 mW/cm², reporting a certain efficacy against S. mutans [34,35].

So far, we have not found any work that has evaluated the effectiveness of PDT on refractory candidiasis in patients undergoing orthodontic treatment.

The objective of this work is to use an in vitro model to evaluate the effectiveness of PDT, carried out with a curcumin-based photosensitizer, in the eradication of MDR Candida spp. infections, with a reconstruction experiment against C. albicans in vitro biofilm.

2. Materials and Methods

2.1. Photodynamic Therapy Treatment Procedure

We clarify that this is an entirely in vitro study with no human or animal subjects; hence, IRB approval was not required. An in vitro photodynamic therapy session was realized with a commercial curcumin-based photosensitizer with 3% hydrogen peroxide activated with FlashMax^® P7 blue light (CMS Dental, Copenhagen, Denmark). This commercial photosensitizer was already evaluated in previous research with the same concentration provided by the Parent Company (CMS Dental, Copenhagen, Denmark), in which different light irradiations were used to activate it [29]. The authors reported a good result, in terms of antifungal activity, by using the photosensitizer already mentioned, activated with a λ = 460 nm diode LED light supporting 7 watts of power. In this work, we used a 2.5 mL syringe of the previously mentioned photosensitizer, supplied by the Parent Company, in which it was released on the surface to be treated, so as to cover the areas of interest. The illumination was realized with the previously cited LED light, with an output power of 4000 mW/cm² (CMS Dental, Copenhagen, Denmark), using a blunt tip with a diameter of 8 mm, for 2 min, at a distance of 1 cm from the surface, as suggested by the Parent Company. This type of LED lamp has 2 buttons that activate the light for 3 or 20 s, and its power, energy, and fluence are standard and cannot be adjusted by the operator. The photosensitizer was then removed.

2.2. In Vitro Experiment

An orthodontic bioreactor (OB) represents a dynamic structure able to reproduce some oral parameters such as saliva flow and temperature. As shown in Figure 1, a sterile glass Petri dish, Ø = 90 mm, containing a pure 15% agarose gel in the plate bottom able to support the palatal expander, was selected for this study. This orthodontic device was immersed in 10 mL of Sabouraud dextrose broth (Microbiol, Uta, Italy) plus 10% human saliva. The plate was then inoculated with an MDR C. albicans clinical isolate CA95 strain, which was already used in our previously published work with aPDT [29].

The MDR Candida strain was grown in dextrose Sabouraud broth to mid-log phase (OD550 = 0.125 or 0.5 McFarland), equal to about 1.5 × 10⁷ CFUs/mL, and was used as a 100× inoculum in the orthodontic bioreactor. This apparatus was incubated for 48 h at 37 °C. The bioreactor was subjected to vertical oscillations by alternating motion by using a tilting laboratory shaker (LMS, Rome, Italy). The bioreactor oscillation time (ωt) needed to obtain a salivary flow of about ≃ 0.3–0.5 mL/min was calculated per cm² of Petri dish area, following the formula ωt = [A × 0.5/V].

Here,

✔

ωt = medium oscillations min per cm², regulated by tilting apparatus.

✔

A = occupied cross-sectional area of the ø 90 mm Petri dish in cm².

✔

V = the available volume of the liquid medium (6.5 mL).

In these conditions, a flow within the already mentioned range was obtained by setting ωt_s from 3 to 5 oscillations/min.

At the experiment endpoint (48 h), the liquid medium was discarded, and the PE was treated with the photodynamic procedure as described before. Two sampling sets were performed: before and 5 min after the PDT treatment. In every case, the sampling was executed by brushing the points indicated in Figure 1 for 60 s for each one. The brush material collected was put in a sterile Eppendorf tube containing 500 µL of sterile saline NaCl 0.9% solution after a mild pipetting on the biofilm zone, and 100 µL of this solution was inoculated on the surface of a plate containing Sabouraud dextrose agar after different 1/10 dilution series, as provided by the CLSI (https://clsi.org) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST (http://www.eucast.org accessed on 1 April 2025). The colony-forming units (CFUs) were counted after 48 h of 37 °C incubation in air. This experiment was executed in triplicate. In brief, the final CFU titer was calculated using the formula [CFUf = CFUpXdil], where CFUf = the final titer, CFUp = the number of colonies in the examined plate, dil = sample dilution factor. A comparison between CFU values found in the infected Petri dishes with samples taken before and after PDT was performed to express the effectiveness of the treatment.

2.3. Irradiation Experimental Setup

An irradiation setup was necessary to normalize the holding of the light source and the positioning of the sample being irradiated. In this work, we used the system shown in Figure 2. The PDT treatment was performed inside a Class II Biosafety Cabinet to avoid microbial contamination. It was critical to use a homemade metal support that keeps a steady distance between the light emitter and the palatal expander with the C. albicans biofilm. The irradiation procedure was started after the complete discarding of the liquid medium previously described.

The main parameters were

• Output power: 4000 mW/cm².
• Wavelength range from 460 to 470 nm.
• Irradiation cone (about 1 cm) for each treatment point.
• The distance from the PFT apparatus to the palatal expander: 2 cm.
• Time of exposition for each treatment point = 10 s.
• Total irradiation time/prosthesis = 3.5 min.

2.4. Statistical Analysis

Each experiment was executed in triplicate, and quantitative data were expressed as mean ± SD. We employed Fisher tests to assess the importance of distinct analytical groups: palatal expander (PE) biofilm without treatment and PE biofilm following antibacterial photodynamic therapy treatment, at both To and T1. p values were calculated by using a social science statistic program, available at https://www.socscistatistics.com/ (accessed on 1 April 2025) [36,37].

3. Results

In this work, we have chosen to perform in vitro evaluation by using a bioreactor (Figure 1) that is capable of reproducing, in part, some physical and biochemical conditions operating in the mouth. After the step of infection using a multi-drug-resistant strain of C. albicans, photodynamic treatment was executed with the same conditions previously described [29]. An evaluation by cultural procedure, executed after 48 h, comparing the number of CFUs of Petri dishes contaminated with a brush taken before and after PDT, revealed a dramatic decrease in viable Candida cells of about 99%, as reported in Figure 3. These data reveal that the differences are statistically significant (p < 0.01).

4. Discussion

Generally, in the oral cavity, Candida infections are treated with antifungal medications such as nystatin, clotrimazole, amphotericin B, and miconazole. In recent decades, the increase in antimicrobial resistance to conventional antimycotics has become a major health problem that requires the development of new drugs [22,38,39]. Therefore, some researchers have experimented with alternative therapies, one of which is photodynamic therapy (PDT). In fact, until now, no mechanism of drug resistance linked to PDT has been described in the literature. Antimicrobial photodynamic therapy (aPDT) is an innovative treatment that uses a combination of light-sensitive compounds called photosensitizers (PTs) and light of a specific wavelength to produce reactive oxygen species (ROS) that can selectively kill microbial cells, including bacteria, fungi, and viruses. Curcumin, a bright yellow chemical produced by Curcuma longa plants, activated with blue lights composed of 420–480 nm and 20–120 J/cm², represents one of the most used products used recently against microbial infections, including Candida infections [40,41,42]. In this in vitro work, we confirm the real effectiveness of this therapeutic treatment against Candida infections; in fact, we have obtained a reduction in C. albicans colonies on the in vitro palatal surfaces of about 99.9%. For the first time in the literature, PDT effectiveness was evaluated against a particular resistant Candida infection (MDR). On the palatal surface of the patient with a PE, not only are Candida spp. present but also the oral microbiota could interfere with fungal infection. The mechanism of action of the aPDT process against salivary microbiota in a patient who wears an orthodontic palatal expander is shown in Figure 4.

In a recent work [29], the same commercial product containing curcumin/H₂O₂ 3% was used in an in vitro study as a photosensitizer and evaluated in a PDT treatment that used two different light sources: traditional irradiation with 7 W light at λ = 460 nm and a polarized light source of 25 W with a wavelength range of λ = 380–3400 nm. In this work, the authors found a crucial, strongly time-dependent fungistatic effect of PDT performed with the curcumin-based photosensitizer and the two lights described [29].

The commercial photosensitizer used in this work contains concentrations of curcumin and hydrogen peroxide already tested by the manufacturer for use on patients. The effect is synergistic between curcumin, 3% hydrogen peroxide, and a light source (the same wavelength used by the manufacturer). If we were to remove even one of these elements, the results would be very different. The effect of the natural product (curcumin) as a single photosensitizer is well documented in the scientific literature, while the idea of increasing the activity of another natural photosensitizer with hydrogen peroxide as a catalyst—a chemical element that can increase the formation of ROS—has recently been successfully proposed [43].

The authors improved the antifungal activity of lactoferrin by mixing it with hydrogen peroxide. They also tested the activity of hydrogen peroxide alone on the same MDR Candida species, demonstrating, in any case, significantly lower activity than the combined use of lactoferrin and hydrogen peroxide, both photoactivated and non-photoactivated. We can therefore deduce that the antifungal activity is not solely due to 3% hydrogen peroxide, even in our study, but we cannot determine whether curcumin, hydrogen peroxide, or the light source has a predominant effect on the others [43]. Since the PDT protocol, already validated for in vivo use, requires all three, we prefer to limit ourselves to observing a very interesting synergistic effect, despite all the limitations of the evaluation performed: the CFUs were evaluated, not the activity on the biofilm of MDR Candida species, and the in vitro evaluation with a bioreactor could not predict other patient-related variables.

Curcumin and riboflavin as photosensitizers activated by blue light in the aPDT procedure have the potential for reducing the bacterial count in periodontal infection, especially against several species of microorganisms, including A. actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, with very low concentrations of photosensitizers and activated for only 30–60 s [42]. A reduction in these periodontal bacteria can also influence the success in the eradication of MDR Candida spp.; it is also important to find an effective PDT protocol against these bacteria [14,15]. A randomized clinical study has evaluated the effectiveness of a curcumin-based photosensitizer in PDT against most common oral pathogens with irradiation parameters of 455 ± 30 nm wavelength, 400 mW average optical power, 5 min application, illumination area of 0.6 cm², 600 mW/cm²; intensity, and 200 J/cm² fluence [44]. The parameters used in this work to activate the curcumin-based photosensitizer are in line with those used in previous scientific works.

Compared to laser light at the same wavelength, LED light determines a greater effect also by virtue of higher powers and a slight photothermal effect, well tolerated by tissues when used in vivo, which enhances the activation of the photosensitizer itself. This is also the reason why an LED at that wavelength was chosen instead of a laser, as already chosen by other researchers [45]. Other researchers recently found interesting results using laser devices with very similar wavelengths against E. faecalis and Candida spp. [46,47]. Although the cytotoxic effect of LED light may be greater, in vivo laser light could also act as a photobiomodulator, promoting faster tissue healing [45,46,47].

Toluidine Blue (TBO) solution was the most common type of photosensitizer in PDT against the infection of Candida spp. In a recent review on the subject, the studies selected reported the antifungal effectiveness of aPDT with TBO against C. albicans and non-albicans Candida, but are overall in vitro studies without clinical observations and without comparison with bacterial load reduction [48]. Furthermore, TBO is toxic if accidentally ingested, while a photosensitizer such as a derivative of Curcuma Longa is very safe, overall, in pediatric patients [29,40,48]. The efficacy of photodynamic therapy is strongly dependent on many variable parameters. One of the basic factors is the light source. In photodynamic therapy, it is also extremely important that the maximum absorption of the photosensitizer used is aligned with the wavelength emitted, so the effectiveness could be influenced by different parameters [48,49].

In previous works in the presence of mobile appliances in the oral cavity, an increased count of Candida albicans, a common fungal opportunistic pathogen found in the oral cavity of 62% of preschool children and 71% of school children, was shown [50]. In the presence of a fixed appliance, an equal increase in the presence of this fungus has been demonstrated, often in conjunction with an increase in the load of Streptococcus mutans, a bacterium responsible for caries and white spot phenomena [49]. Candida non-albicans species, such as C. parapsilosis, C. glabrata, and C. tropicalis, seem to be linked to certain diseases and present in a high percentage of the population in recent decades [49]. A fact not to be overlooked is that some Candida non-albicans species are used in the production of certain foods. They are also used as an adjuvant in cheese production or as starters for coffee, cocoa, vegetables, and meat and for fermentation of beer and wine. Several Candida strains used as culture starters have been associated with some types of fungal infections, although they are generally considered less virulent than C. albicans [49]. The intensity of Candida infection depends on the response of the immune system. While it may seem that the patient’s diet is linked to the presence of these Candida species [51], we have no studies on the type of Candida spp. on the palatal expanders. If it is true that some authors reported that candidiasis in pediatric patients wearing palatal orthodontic appliances is very similar to that developed in adult patients wearing removable prostheses [51], it is also true that the phenomenon has never been well studied from a microbiological and molecular biology point of view. The young age of the patients, the type of material with which the palatal expanders are made (predominantly metal and not resin), and the geometry of what actually adheres to the palate could determine a greater proliferation of different fungal species compared to what we are used to observing. Finally, the use of photodynamic therapy as a tool against fungal and bacterial infections under palatal orthodontic devices has never been proposed. Although other approaches have been documented in the literature for treating the problem, photodynamic therapy may be more beneficial.

In comparison to PDT, topical ozone therapy has the function of activating the blood microcirculation, contributing to better oxygenation of the tissues, and modulating the inflammatory process, and its antibacterial mechanism, also based on the production of free radicals, is less selective [26,27]. The possibility that ozone increases the bioavailability of oxygen led some researchers to test ozonized water (0.060 mg/L), in an in vitro study, as a photosensitizer in photodynamic therapy, activated by blue lights, very close to the maximum absorption spectrum, vs. S. mutans, with good results [52].

Probiotics and paraprobiotics have been shown to be effective and safe in managing an oral ecosystem. If administered after photodynamic therapy, with the aim of enhancing the stability and microbiological balance achieved after PDT, they could be interesting strategies to be used safely even in pediatric age, in patients with orthodontic appliances [53,54]. However, they require a certain compliance on the part of the patient in taking the tablets constantly, an objective that is not always easy, especially in pediatric age.

• Study limitations and future perspectives

This study describes an in vitro approach for evaluating new photodynamic protocols. It should be considered a phase 1 study in photodynamic therapy for oral prosthetic infections. It could be a model for evaluating new photosensitizers and new wavelengths. We believe the bioreactor structure should be further improved, using new denture supports other than agarose. Alternatively, a standardized in vitro system that closely mimics the oral cavity could serve as a valid production control system for pharmaceutical companies producing photodynamic devices.

5. Conclusions

Patients undergoing orthodontic therapy with a palatal expander (PE), often pediatric, are potentially at risk of developing oral candidiasis. In this in vitro study, which simulated a fungal infection with PE-related MDR Candida spp., PDT realized with a curcumin-based photosensitizer and 460 nm LED light was shown to be an effective method of oral decontamination. This preliminary in vitro study, however, has some limitations: (1) it does not take into account salivary flow at the palatal level, linked to the presence of minor salivary glands that could influence the density of the photosensitizer, an element very complex to reproduce in a bioreactor; (2) the pH during the photodynamic process was not evaluated, an element that could influence the activation of the photosensitizer.

Alternatively, and not simple to realize, it could be intriguing to build a prosthetic infection model on cell cultures, such as gum lines or other epithelial lines, to evaluate the host cell response to the prosthesis and to the fungal infection, thus representing in vitro the cross-kingdom communication cited in the latest scientific works.