Annali di Stomatologia | 2026; 17(3): 537-549

ISSN 1971-1441 | DOI: 10.59987/ads/2026.3.537-549

Articles

Morphological, microbiological and clinical evaluation of different mouthwashes for periodontal and peri-implant tissue oral hygiene maintenance: do we really need them?

Francesca De Angelis¹, Sara Bernardi², Pierangelo Bellio³, Lorenza Fagnani³, Maurizio D’Amario², Diana Torge⁴, Stefano Di Carlo¹, Edoardo Brauner¹, Nicola Pranno¹

¹Department of Oral and Maxillofacial Sciences, Sapienza University of Rome, Rome, Italy

²Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy

³Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy

⁴Department of Life Science, Health, and Health Professions, Link Campus University, Rome, Italy

^*Corresponding author: Francesca De Angelis - francesca.deangelis@uniroma1.it

Article History

Received: June 3, 2026

Accepted: July 22, 2026

Published: July 30, 2026

Abstract

Aim

The success of dental prosthetic rehabilitation relies not only on resistant crowns, but also on the maintenance of periodontal and peri-implant tissue health, which oral hygiene procedures support by keeping microbial load and tissue inflammation low. Chlorhexidine (CHX)-based mouthwashes effectively control oral microbial load, but their prolonged use is associated with side effects, prompting research into alternatives such as those based on umbelliferone. This study aimed to compare, in vitro and clinically, the efficacy of two different mouthwashes in reducing microbial load and their associated side effects.

Methods

Thirteen patients undergoing prosthetic rehabilitation on natural and implant abutments were divided into three subgroups: a test group prescribed an umbelliferone-based mouthwash, a positive control group prescribed a chlorhexidine mouthwash, and a negative control group following standard oral hygiene only. Clinical evaluation assessed periodontal status and pigmentation of resin temporary crowns. In vitro evaluation included morphological observation of temporary crowns using optical and electron microscopy for natural-tooth rehabilitations and microbial evaluation using the Agar Snyder test for implant rehabilitations.

Results

Clinical assessment revealed few side effects in both mouthwash groups. Morphological evaluation showed low microbial biofilm organization in both mouthwash groups, with slight qualitative differences between them, while the negative control group displayed an organized matrix network. Microbial analysis showed no microbial metabolic activity in either mouthwash group, with no differences between them.

Conclusion

Within the limits of this preliminary study, the umbelliferone-based mouthwash appeared to reduce the microbial load comparably to chlorhexidine, with numerically fewer recorded events, suggesting it may be a promising alternative for maintaining oral hygiene in patients undergoing prosthetic rehabilitation. Further studies with larger samples are needed to confirm these findings. Dental hygienists and dentists may play an important role in guiding patients toward correct adherence to daily oral hygiene routines.

Keywords: oral health; mouthwashes; microscopy; microscopy electron scanning; microbiology; prosthodontics; chlorhexidine; umbelliferones; biofilms.

1. Introduction

The combination of resistant crowns and periodontal/peri-implant tissue health is a fundamental elements that determine the overall success of dental prosthetic rehabilitation. Oral hygiene plays a regulatory role in this balance by managing oral microbial load and limiting inflammatory dynamics [1]. The oral cavity is the most external section of the gastrointestinal system, performing sensory functions, mechanical food processing, and a barrier function that protects the distal gastrointestinal tract from pathogens [1]. A significant part of oral physiology is its microbiome, which forms a biofilm on dental surfaces [1]. The oral cavity is therefore a complex microbial ecosystem, characterized by a unique community of microorganisms — the oral microbiota — that changes throughout life depending on diet, genetic predisposition, medical conditions, applied therapies, and hygienic habits [2]. Bacteria, fungi, viruses, archaea, and protozoa predominantly colonize the oral cavity [2,3], from hard tissues (teeth) to mucosal tissues (lips, buccal mucosa, tongue, palate, gingival mucosa) [2,4], forming microecological communities essential for maintaining the balance of the host oral ecosystem and overall oral health [2]. Because the oral cavity is exposed to the external environment, pathogens can easily colonize it, promoting medical conditions usually characterized by an underlying inflammatory state [1]. Disturbance of this microbiological balance may be associated with poor oral hygiene, compromised immune response, smoking, or chemo-or radiotherapy, potentially leading to oral mucositis, periodontitis, or other painful conditions that negatively affect quality of life [1,5].

The onset and progression of prevalent oral diseases such as caries and periodontitis are often associated with complex, dynamic interactions between oral bacteria and the host oral environment [6–8]. Bacterial species within the oral biofilm can spread through the bloodstream, with negative systemic effects [1]. Biofilm — well-organized microbial communities embedded in a matrix of extracellular polymeric substances (EPS) — adhere to tooth surfaces and oral soft tissues, promoting inflammation and contributing to caries and periodontitis [6,9]. For this reason, considerable attention has been devoted to developing oral hygiene devices effective in maintaining optimal oral health, including toothbrushes, which physically disrupt and dislodge biofilms from tooth surfaces [6,10], and toothpastes, whose abrasive nature and chemical components help remove biofilms [6,11]. Mouthwashes, characterized by varied and often natural compositions, also contribute significantly to oral hygiene. Antiseptic molecules are applied in mouthwash formulations to limit inflammation, counteract microbial proliferation, and manage post-operative interventions on the oral cavity or following prosthetic implants, which can damage mucous membranes and tissues [12]. Modern implantology techniques use implant prostheses made of metal pins (usually titanium) designed to integrate with the maxillary or mandibular bone, on which a definitive ceramic or ceramic-composite crown is mounted [12]. A high level of implant osseointegration is fundamental for effective, predictable bone regeneration [13]. The porous nature of crown materials and the implant placement procedure inevitably carry a risk of inflammatory and infectious complications, including edema and intense pain [14]; managing the post-implant stage is therefore essential to avoid bacterial infections leading to abscesses and sepsis, underscoring the importance of home hygiene for prophylactic purposes [14]. A mouthwash is an antiseptic liquid solution used to clean the oral cavity and freshen breath [2], often enriched with antibacterial ingredients and essential oils to reduce plaque and bad breath [2,15] progressively. Dentists commonly prescribe mouthwashes with anti-inflammatory and, above all, antibacterial action, typically delivered through chlorhexidine (1,1′-hexamethylene bis [5-(p-chlorophenyl) biguanide] di-D-gluconate; CHX) [16]. CHX is a chemical disinfectant with broad antiseptic activity against Gram-positive and Gram-negative bacteria, yeasts, and viruses [16], showing antimicrobial activity against both planktonic and biofilm forms of intracanal bacteria [17] by modifying the protein architecture of the bacterial cell membrane, while also showing low toxicity to vital tissues [17]. In dentistry, CHX is frequently used to reduce oral bacterial load after surgical, implantological, and periodontal procedures, as well as for inflammatory issues, abscesses, and plaque control [17]. CHX is a broad-spectrum, biocompatible antimicrobial agent with potential clinical benefits for some oral diseases [18–19]; however, recent evidence highlights several challenges. Long-term application of this synthetic biguanide may cause yellow-brownish staining of tooth surfaces and prosthetic materials [20]; while natural dental pigmentation can be restored by discontinuing CHX use, the porous nature of prosthetic implants can make this staining permanent. Among the most frequent side effects associated with CHX use are rare sensitization and oral mucosal desquamation, dysgeusia, and impaired taste perception [20]; glossitis, xerostomia, and alteration of the resident bacterial flora are also associated with long-term CHX mouthwash use [17,20]. A valid alternative to CHX mouthwashes is represented by oral topical solutions characterized by anti-inflammatory, antibacterial, and anti-edematous properties, with re-epithelializing and film-forming potential [21], suitable for extended use without significant side effects. One such oral topical solution is composed as follows:

10% Calcium Hydroxide (Ca(OH)₂): reduces bacterial counts in the oral cavity, with significant effects on apical periodontitis, dental traumatology, and intracanal dressings. This compound is widely used due to its direct effect on Gram-positive and Gram-negative bacteria (from Streptococcus to Lactobacillus, Actinomycetes, and Staphylococcus), often involved in peri-radicular lesions [22]. The notable biocompatibility and antimicrobial properties of Ca(OH)₂ are primarily attributed to the sustained release of hydroxyl ions (OH-). These ions act as highly reactive free radicals with strong oxidizing properties, inducing lethal damage to bacterial membranes and proteins [23].
Umbelliferone (UMB): an aromatic compound and one of the most common plant-based coumarins, present as a secondary metabolite in the flowers, fruits, and roots of almost all higher plants, mainly from the Umbelliferae/Apiaceae family [24]. UMB displays anti-adhesive activity against microorganisms (E. coli, E. faecalis, St. aureus), a feature useful for reducing plaque development on periodontal pockets and prosthetic implants [24–25], and also displays antioxidant properties [25].
Oligomeric proanthocyanidin (OPC): characterized by anti-inflammatory properties, OPC easily penetrates the lipid membrane of the cell, exerting antioxidant functions with significant anti-inflammatory effects [26]. Among its biological potentials, OPC can alleviate metabolic disorders, ameliorate inflammation, and improve dental biostability [26]. Several studies attribute to OPC specific antioxidant, anti-inflammatory, anti-aging, antibacterial, and antineoplastic activities [26].
Hyaluronic Acid (low molecular weight: 200–400 kDa): the most abundant glycosaminoglycan (GAG) of the extracellular matrix of soft periodontal tissues, essential for promoting regeneration of damaged tissues. It is widely used in periodontal regeneration due to its hygroscopicity, viscoelasticity, biocompatibility, and anti-edematous action [27]. This natural, non-sulfated GAG is present in the extracellular matrix of connective tissue, synovial fluid, embryonic mesenchyme, vitreous humor, skin, and other organs and tissues, playing a significant role in the wound-healing process [28] and is often applied in the treatment of inflammation [28].
Fluoride: prevents caries and provides beneficial effects in treating dental hypersensitivity [29], strengthening tooth enamel, lowering acid levels, and helping rebuild minerals [30]. Fluoride is considered the main component for the prevention and non-invasive control of dental caries, reducing caries risk and reversing early caries lesions, as recent evidence confirms [31–32].

Several recent studies have evaluated the clinical efficacy and long-term effects of CHX-free oral solutions in different clinical contexts [21]. The development of microscopical techniques, which has revolutionized morphological sciences by providing new levels of magnification and resolution for exploring biological and non-biological samples [33], represents a promising tool for understanding the structural and ultrastructural features of the oral microbial ecosystem; when combined with cellular, molecular, and microbiological data, microscopic evidence allows researchers to explore the profile of predominant oral microbes. For these reasons, the aim of this study was to perform a clinical and morphological comparison between CHX mouthwashes and CHX-free oral solutions, evaluating their clinical efficacy and side effects. Selected parameters were: the presence of bacterial species on prosthetic implants and in the oral cavity, pigmentation of the prosthetic surface, burning sensation in the oral cavity, bacterial resistance, and dysgeusia in patients undergoing different prosthetic implant procedures. The primary endpoint of this study was to evaluate improvement in pigmentation parameters of prosthetic surfaces; secondary aims included assessing pathogenic bacterial species, reducing the most frequent side effects commonly associated with CHX mouthwash use, and improving tissue regeneration over long-term application of the examined oral solution.

2. Materials and Methods

2.1 Study Design

This was a prospective, longitudinal, controlled pilot clinical study including both in vitro and in vivo assessments of the antiseptic and clinical efficacy of an umbelliferone-based mouthwash. Temporary resin provisional crowns on natural and implant abutments were analyzed according to the assigned study arm. Patients were recruited at the Implant Prosthodontics Unit, Umberto I University Hospital of Rome, Italy.

2.2 Population, Inclusion and Exclusion Criteria

Clinical evaluation results are reported for 13 patients who completed follow-up, of whom 4 were assigned to Arm A, 5 to Arm B, and 4 to Arm C. Although the sample size was limited, this pilot study aimed to generate preliminary data for larger randomized clinical trials. Inclusion criteria were good implant capacity and dental implant stability (>35 Ncm). Exclusion criteria were detection of strong bacterial or fungal infection at T0, implants with non-optimal capacity, bruxism, incorrect masticatory occlusal plane, and presence of oral cavity cancers.

Enrolled patients were divided into three groups: Group A, subjected to a solution of 10% Ca(OH)₂, 10 ppm of UMB, 0.5% of OPC, 0.3% hyaluronic acid, and 200 ppm fluoride; Group B (CHX mouthwash), a solution composed of 0.2 g CHX gluconate, polyoxyethylene hydrogenated castor oil, non-crystallizable sorbitol (70%), mint essence, ethylic alcohol (96%), and 20 cc purified water; and Group C, a negative control not exposed to any mouthwash treatment (20 cc).

Follow-ups were performed at T0, T1 (15 days), and T2 (30 days). A numerical value was associated with the initial color of the implant and natural teeth to record any chromatic changes during follow-up. After implant placement, Group A patients were recommended to use the Ca(OH)₂/UMB/OPC/hyaluronic acid/fluoride solution after dental hygiene (10 mL oral rinses, three times daily) for 15 days (T1) and 30 days (T2), avoiding food or drink for at least one hour after rinsing. Group B patients were recommended to perform three oral rinses daily with the CHX mouthwash for 15 days (T1) and 30 days (T2), rinsing after each daily dental cleaning. Group C followed standard oral hygiene procedures (medium-bristled toothbrush, fluoride toothpaste, brushing and/or dental floss).

Patients were randomly assigned to study groups using a computer-generated randomization list. The operator performing clinical evaluations was blinded to the type of mouthwash prescribed.

2.3 Follow-Up Evaluations

After clinical examination, the following parameters were evaluated: inflammation and gingival edema, pain, alteration of tooth and implant color recorded at T0, burning sensation in the oral mucosa, dysgeusia, white gums, desquamation, and ulceration. Scoring was binary (yes/no). The T2 follow-up assessed the same parameters as T1.

2.4 Agar Snyder Test

Resin provisional crowns were unscrewed at T1 and T2 follow-ups, immersed with sterile tweezers in PBS, sonicated at 30 kHz for 5 minutes, and screwed back into place. The sonicated biofilm in PBS was stored at –20°C for subsequent use in the Agar Snyder Test, performed as follows: the agar Snyder medium was dissolved in distilled water and sterilized by autoclave (medium setup); the prepared medium was inoculated with a sample of saliva or tooth surface material to obtain a uniform suspension (inoculation); the inoculated medium was incubated at 37°C for 72 hours (incubation); finally, the medium was observed for acid formation, indicated by a color change from green to yellow. A yellow color change indicates acid production and increased caries susceptibility, while a green color indicates low acid production and lower caries susceptibility.

2.5 In Vitro Analysis: Morphological Analysis

Morphological examination was performed to assess the secondary endpoint of the study by analyzing biofilm derived from control and test groups exposed to mouthwashes. Light microscopy (LM; stereomicroscope, Zeiss Axio Zoom V16) and scanning electron microscopy (SEM; Zeiss GeminiSEM 500), with standard preparative protocols, were applied to morphologically characterize bacterial residues on surfaces exposed and not exposed to mouthwashes.

2.6 Morphological Analysis: Scanning Electron Microscopy

SEM morphological analyses were performed at T2 on p resin provisional crowns placed on natural teeth to evaluate, from a morphological standpoint, biofilm on resin provisional crowns exposed and not exposed to tested and non-tested mouthwashes. Resin provisional crowns were removed, placed in a sterile test tube, and immersed in 0.1 M glutaraldehyde. Samples were rinsed in PBS and dehydrated through an ascending ethanol series (50%–70%–90%–95%–100%), then immersed in hexamethyldisilane (HMDS; Sigma-Aldrich S.r.l., Milan, Italy). Samples were air-dried by HMDS evaporation, mounted on metal stubs, and observed with a stereomicroscope at different magnifications. Finally, samples were gold-coated and observed by SEM at different magnifications using secondary electron probes.

2.7 Statistical Analysis

Each clinical sign was recorded as the number of affected patients per arm at each follow-up. Between-arm comparisons for each endpoint were performed using Fisher’s exact test (Freeman–Halton extension for the three-arm comparison). Endpoints with no events in any arm were not subjected to inferential testing and are reported descriptively. Given the exploratory nature and limited sample size of this pilot study, results are reported primarily in descriptive terms. A two-sided p < 0.05 was considered statistically significant. Analyses were performed with GraphPad Prism 9.5.1 (GraphPad Software, San Diego, CA, USA).

3. Results

3.1 Morphological Assessments

Observation under an optical microscope allowed qualitative assessment of residual plaque on the surfaces of the resin provisional crowns. Arm C elements showed visibly greater plaque and cement residue accumulation than Arm A and Arm B elements (Figure 1).

**Figure 1.** Light microscopy of resin provisional crowns. (A,C) Arm C (negative control), 8x and 56x magnification: opaque resin crown surface with plaque/cement residues. (B,D) Arm A, 8x and 56x magnification: surface free of opaque layers, with visible resin porosity. (E-G) Arm B, 8x, 56x, and 32x fluorescence reconstruction: mixed plaque-related opacity and translucent areas showing resin porosity.

SEM observation confirmed the microbial nature of the areas highlighted by optical observation: the highest presence of microbial biofilm was found in Arm C samples, a low presence of microbes on Arm A sample surfaces, and a medium presence of microorganisms on Arm B crown surfaces (Figure 2).

**Figure 2.** SEM analysis of resin provisional crowns. (A,B) Arm C (negative control), 1000x and 5000x magnification: biofilm matrix covering the resin crown surface, with numerous cocci visible at higher magnification. (C,D) Arm A, 1000x and 5000x magnification: absence of an organized microbial matrix, with isolated microbial colonies. (E,F) Arm B, 1000x and 5000x magnification: occasional organized biofilm areas and lower microbial presence elsewhere.

3.2 Microbiological Assessment

Regarding the Agar Snyder Test results, salivary samples from three patients in Arm A (T1 n=3, T2 n=3), three patients in Arm B (T1 n=3, T2 n=3), and three patients in Arm C (T0 n=1, T1 n=2, T2 n=2) were analyzed; at T0, only the Arm C patient sample was kept as a baseline reference for comparison. Negative-control reference samples showed Snyder agar medium acidification as early as 24 h (inset in Figure 3A). In contrast, positive controls and test samples at both follow-ups showed no Snyder agar medium acidification at 24, 48, or 72 h (Figure 3).

**Figure 3.** Snyder agar test after 24, 48, and 72 h. (A) At 24 h, treated samples retained green coloration, indicating absence of acidification and low acidogenic activity; the inset shows reference tubes, including a negative control with gas production/acidification. (B,C) At 48 and 72 h, no relevant color change was observed in treated samples compared with 24 h.

3.3 Clinical Assessment

Clinical evaluation results are reported for the 13 recruited patients with resin provisional crowns on implants enrolled in the study, of whom 4 were assigned to Arm A, 5 to Arm B, and 4 to Arm C. Arm A patients did not show particular variation between the two follow-ups in terms of pain, chromatic alteration recorded at T0 of the provisional, burning oral mucosa, dysgeusia, pale gingiva, presence of phlogosis, or possible ulceration; one patient reported discomfort related to gingival edema and ulceration (Table 1). Arm B patients presented a picture generally similar to Arm A patients. No chromatic alteration was recorded in any arm at any time point. One patient in Arm B reported discomfort at T2 related to edema, and two patients presented ulceration at T2. Arm C patients did not show substantial variation from T0 to T2 (Table 1).

Table 1. Clinical signs reported per study arm at baseline (T0), 15-day follow-up (T1), and 30-day follow-up (T2). Values are numbers of patients with the event. Between-arm comparisons at T2 were performed with Fisher’s exact test, Freeman-Halton extension. n.t., not tested because no events were recorded. No endpoint differed significantly among arms.

Clinical endpoint	Arm A (n = 4)			Arm B (n = 5)			Arm C (n = 4)			p value (T2)
Clinical endpoint	T0	T1	T2	T0	T1	T2	T0	T1	T2	p value (T2)
Gingival inflammation / edema	0	0	1	0	0	1	1	1	1	1.000
Pain reported	0	0	0	0	0	0	1	1	1	0.615
Chromatic alteration	0	0	0	0	0	0	0	0	0	n.t.
Oral mucosa burning	0	0	0	0	0	0	0	0	0	n.t.
Dysgeusia	0	0	0	0	0	0	0	0	0	n.t.
Gingival blanching	0	0	0	0	0	0	0	0	0	n.t.
Ulceration	0	0	1	0	0	2	0	0	1	1.000

3.4 Statistics

At the final follow-up, Fisher’s exact test showed no statistically significant differences among the three arms for any clinical endpoint with recorded events (gingival inflammation/edema, p = 1.000; pain, p = 0.615; ulceration, p = 1.000). The remaining endpoints (chromatic alteration, oral burning, dysgeusia, gingival blanching) showed no events in any arm at any time point and were reported descriptively (Table 1). Mild, isolated events (gingival edema and ulceration) emerged only at T2 and resolved without clinical consequences.

4. Discussion

Oral microbial biofilm is an ever-present, natural phenomenon on every surface within the oral cavity. Once teeth are cleaned, it takes approximately six hours for the initial biofilm to begin re-forming, while biofilm on dental implants requires somewhat longer to establish [34]. Within this biofilm environment, microorganisms coexist in a fragile equilibrium; when this balance is disrupted — dysbiosis — various oral health issues such as caries, periodontitis, and peri-implantitis may emerge, potentially leading to loss of dental and periodontal tissue [35].

Correct oral hygiene procedures and related professional home care products are crucial to ensuring the success of implant and prosthodontic therapies. The primary objective of this study was to compare, in vitro and in vivo, the efficacy and incidence of side effects of two different mouthwashes, aiming to identify a valid alternative to chlorhexidine-based products, whose long-term use is associated with several adverse effects widely reported in the literature [17].

4.1 Morphological and Microbiological Findings

The morphological results of this study confirmed that the mouthwash containing umbelliferone and calcium hydroxide possesses notable antibacterial properties, in agreement with previous findings from Cai et al., Lee et al., and Monte et al. [36–38]. Interestingly, although the microbial load observed on the surface of resin provisional crowns in patients using the umbelliferone-based mouthwash (Group A) was slightly higher than in patients using the chlorhexidine-based mouthwash (Group B), the nature of the biofilm differed significantly between the two groups: the biofilm on Group A restorations appeared disrupted and disorganized, whereas in Group B, when present, the biofilm maintained a more structured and organized architecture. An organized biofilm structure enables microbial communities to communicate and cooperate through dynamic interactions, forming a protective extracellular polymeric matrix that enhances resistance to external challenges, including antimicrobial agents and host immune responses; a mature, organized biofilm is therefore considered a potential pathogenic factor, particularly under conditions favoring opportunistic microorganism proliferation. These observations are consistent with Bowen et al. [39], who demonstrated that dental caries pathogenesis involves not only specific risk factors (such as a sugar-rich diet) but also synergistic interactions among organized colonies of opportunistic pathogens that outcompete commensal flora and create a pathogenic microenvironment via extracellular matrix production.

In light of these findings, the ability of the umbelliferone- and calcium-hydroxide-based mouthwash to disrupt biofilm architecture may represent a significant clinical advantage over chlorhexidine-based formulations, especially for long-term use, where biofilm management may be critical.

Microbiological findings were consistent with morphological observations, showing no significant metabolic microbial activity in either group, as assessed by the Snyder test. It should be noted, however, that the Snyder test primarily measures global acidogenicity, providing only an indirect estimate of bacterial metabolic activity rather than a quantitative assessment of microbial load or species composition [40].

These results align with the ecological hypothesis of oral biofilm behavior, which posits that controlling bacterial acid production — rather than pursuing complete eradication — is key to maintaining oral health and preventing biofilm-associated diseases [41]. Preclinical studies have shown that umbelliferone exhibits antibiofilm properties mainly by interfering with bacterial adhesion and extracellular matrix formation rather than through a broad bactericidal effect [42], which could explain the observed absence of acidification despite the persistence of some viable cells.

Moreover, consistent evidence indicates that mouthwashes are effective as adjuncts to mechanical plaque control but should not replace professional and home-based hygiene procedures [43–44]. The observed low metabolic activity in both treated groups may therefore reflect a synergistic effect between the umbelliferone-based mouthwash and regular professional dental hygiene maintenance, supporting host-biofilm balance.

4.2 Clinical Assessments

Regarding adverse effects, both mouthwashes were generally well tolerated, with only mild mucosal inflammation and ulceration reported in four patients overall. This outcome was somewhat expected in the chlorhexidine group (Group B), as oral ulceration is a well-documented side effect of chlorhexidine-based rinses [45]. In the single case observed in Group A, the ulceration was likely unrelated to the mouthwash itself and may instead have been associated with other factors, such as poorly adapted resin provisional crowns, mechanical trauma, or individual predisposition to aphthous ulceration. Previous experimental studies have likewise reported a very low incidence of mucosal irritation or cytotoxicity associated with umbelliferone-based formulations [42].

An unexpected finding was the absence of staining or discoloration on resin provisional crowns among patients using the 0.2% chlorhexidine mouthwash, contrasting with previous clinical evidence describing brown staining as one of the most frequent and aesthetically concerning side effects of chlorhexidine use [46–47]. A plausible explanation for this discrepancy is the limited sample size of this subgroup, which may not have been large enough to reliably detect this effect.

Finally, it is worth reflecting on findings from the control group (Group C), in which patients did not use any antimicrobial mouthwash. Despite visibly greater plaque accumulation on resin provisional crowns, no significant clinical changes were observed between T0 and T2. These results suggest that, under certain conditions, adjunctive antiseptic mouthwash use may not be strictly necessary if patients maintain high compliance with effective mechanical plaque control at home, consistent with current evidence emphasizing that mechanical biofilm removal — via toothbrushing, dental floss, and interdental cleaning — remains the corner-stone of periodontal and peri-implant disease prevention, while mouthrinses serve primarily as supportive agents [48–49].

4.3 Limitations of the Study

Limitations of this study include the small sample size and the in vitro assessment methodologies, with morphological evaluation being qualitative in nature and microbiological evaluation providing only an indirect measurement of microorganism metabolic activity.

Nonetheless, these data can be used to inform larger future clinical studies; total morphological assessment could be performed using fluorescence microscopy, and Snyder test data could lead to studies employing culture-based and molecular methods (qPCR, 16S rRNA sequencing) to better characterize microbial composition and load [33].

5. Conclusions

Within the limitations of this pilot study, the umbelliferone-based mouthwash showed comparable clinical and microbiological findings to chlorhexidine and may represent a promising adjunctive option for prosthetic and peri-implant maintenance. Larger randomized clinical trials with quantitative microbiological analyses are required before definitive conclusions can be drawn.

Author Contributions

Conceptualization, F.D.A., S.B., P.B. and S.D.C.; methodology, F.D.A., S.B., P.B. and L.F.; validation, S.B., M.D.A. and N.P.; formal analysis, review and editing, S.B. and P.B.; investigation, E.B.; resources, F.D.A. and S.B.; data curation, S.B.; writing — original draft preparation, D.T. and S.B.; writing — review and editing, F.D.A.; supervision, F.D.A.; project administration, F.D.A.; funding acquisition, F.D.A. All authors have read and agreed to the published version of the manuscript.

Funding

The authors gratefully acknowledge BIO-STILOGIT PHARMACEUTICALS S.R.L. (Via Lilliano e Meoli, 78) for kindly providing the oral solution used in this study for the treatment protocol administered to the study groups. The company had no role in the study design, data collection, analysis, interpretation of results, or writing of the manuscript.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of the Department of Oral and Maxillofacial Sciences, Sapienza University of Rome (Protocol Code n. 0000776, Date of Approval: 20/04/2026).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Data will be available from the corresponding author upon reasonable request.

Acknowledgments

Conflicts of Interest

The authors declare no conflicts of interest. The BIO-STILOGIT PHARMACEUTICALS S.R.L. company had no role in the study design, data collection, analysis, interpretation of results, or writing of the manuscript.

Abbreviations

Ca(OH)₂: Calcium hydroxide
CHX: Chlorhexidine
EPS: Extracellular polymeric substances
GAG: Glycosaminoglycan
HMDS: Hexamethyldisilane
LM: Light microscopy
OPC: Oligomeric proanthocyanidin
SEM: Scanning electron microscopy
UMB: Umbelliferone

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