📄 medicine

Development of an In Vitro Denture Stomatitis Model Simulating Candida albicans Biofilm Formation and Detachment Under Daily Antiseptic Immersion

This study developed a reproducible in vitro model using standardized acrylic resin to demonstrate that daily immersion in 0.5% sodium hypochlorite or 0.12% chlorhexidine gluconate significantly reduces *Candida albicans* biofilm loads compared to water controls, providing a validated framework for evaluating denture-cleansing agents prior to clinical application.

Original authors: Pornpot Jiangkongkho, Thatawee Khemwong

Published 2026-07-22

📖 4 min read☕ Coffee break read

CC BY 4.0

Original authors: Pornpot Jiangkongkho, Thatawee Khemwong

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

Imagine your mouth as a bustling, microscopic city. Inside this city, there are tiny buildings made of hard plastic called dentures. Sometimes, invisible tenants called Candida albicans (a type of fungus) decide to move in. At first, they are just polite guests, but if they stay too long without being cleaned, they throw a wild party. They build a sticky, tough fortress called a "biofilm" that clings to the denture surface. This fortress is so strong that normal rinsing can't wash it away, and it can irritate the soft skin (mucosa) underneath, causing a painful red rash known as denture stomatitis. The big question for scientists and dentists is: How do we break down this sticky fortress without hurting the patient? To answer this, researchers need a way to test cleaning products in a controlled setting, like a miniature laboratory city, before trying them on real people.

This study, conducted by researchers at Naresuan University in Thailand, built exactly that kind of miniature laboratory city. They wanted to see how well two common cleaning agents—0.5% sodium hypochlorite (a type of bleach) and 0.12% chlorhexidine gluconate (a common mouthwash ingredient)—could knock down a Candida albicans party on a piece of denture plastic. To make their test fair and realistic, they followed strict international rules (ISO 20795:2013) to create smooth, 10x10x3 mm³ blocks of heat-polymerized acrylic resin, polishing them until they were as smooth as a glass marble (roughness below 0.2 µm) so the fungus wouldn't get a foothold just because the surface was bumpy.

The researchers set up a 10-day experiment where they let the fungus grow on these plastic blocks for different amounts of time: 24 hours, 48 hours, or 72 hours. These timeframes represented the fungus just starting to arrive, getting comfortable, and building a mature, tough fortress. Once the "parties" were formed, they treated the blocks daily with either the bleach, the mouthwash, or just plain deionized water (as a control group). To see how many fungus cells were left, they used a special sonic bath (a machine that uses sound waves at 35,000 Hz) to shake the fungus off the plastic, and then used a chemical test called an MTS assay to count how many living cells remained.

The results showed that both cleaning agents were much better than plain water at reducing the number of living fungus cells. When the fungus had only been there for 24 or 48 hours, the treatments caused a significant drop in the number of survivors. The data suggested a clear pattern: the longer you kept treating the denture with these agents, the fewer fungus cells were left. However, the story got a little tricky with the 72-hour group. When the fungus had been allowed to build its fortress for a full 72 hours, the difference between the cleaning agents and the plain water wasn't as statistically clear, though the water group still had surprisingly low loads by the end.

The researchers used math to connect the dots, finding that the length of time the fungus was allowed to grow before treatment, the type of cleaner used, and the number of days the treatment lasted were all linked to how many fungus cells survived. They found that for the 24-hour and 48-hour groups, the cleaning agents worked significantly better than water. The study suggests that this new, standardized way of testing—using smooth plastic blocks and a 10-day timeline—could be a reliable tool for dentists to figure out which cleaning products work best before they recommend them to patients. While the study didn't test a mix of different germs (it only used Candida albicans), it successfully created a repeatable model to help understand how to break down these sticky fungal fortresses.

Technical Summary: Development of an In Vitro Denture Stomatitis Model Simulating Candida albicans Biofilm Formation and Detachment Under Daily Antiseptic Immersion

Problem Statement
Denture stomatitis (DS) is a prevalent chronic inflammatory condition affecting denture wearers, frequently attributed to persistent polymicrobial biofilms on prosthetic surfaces. Candida albicans is the primary pathogen, capable of transitioning from a commensal to an invasive pathogen by adhering to acrylic surfaces and forming drug-resistant biofilms. While chemical disinfection using agents like 0.5% sodium hypochlorite and 0.12% chlorhexidine gluconate is a standard clinical intervention, evaluating their efficacy is hindered by a lack of standardized in vitro methodologies. Existing protocols often vary significantly in sample dimensions, incubation times, and endpoints, making it difficult to compare results or translate them reliably to clinical practice. Furthermore, no widely adopted protocol exists for studying biofilm development on solid polymethyl methacrylate (PMMA) denture base coupons that adhere to the ISO 20795:2013 standard.

Methodology
The study developed a reproducible in vitro framework to simulate DS conditions and evaluate the efficacy of daily antiseptic immersion.

Substrate Preparation: 216 heat-polymerized acrylic resin specimens (Triplex™ hot) were fabricated according to ISO 20795-1:2013. Samples were cut to 10×10×3 mm³ and polished to a surface roughness below 0.2 µm to standardize adherence conditions.
Biofilm Induction: Specimens were inoculated with C. albicans (ATCC 90028) in Sabouraud dextrose broth. Colonization periods were set at 24, 48, and 72 hours to simulate early, intermediate, and mature biofilm phases.
Treatment Protocol: Following colonization, samples underwent daily immersion for 10 days in one of three solutions: 0.5% sodium hypochlorite, 0.12% chlorhexidine gluconate (combined with 0.5% cetylpyridinium chloride), or deionized water (control).
Quantification: C. albicans loads were measured at baseline and on Days 1, 3, 5, 7, and 10. Detachment was achieved via sonication (35 kHz, 5 minutes) to isolate chemical efficacy from physical disruption. Viable cell counts were quantified using an MTS cell viability assay, calibrated against a standard curve generated from spectrophotometric absorbance (490 nm).
Statistical Analysis: Data were analyzed using non-parametric tests (Kruskal-Wallis, Mann-Whitney U) and multiple linear regression with bootstrapping to examine relationships between treatment duration, cleansing agent type, and pre-treatment colonization time.

Key Results

Standardization: A robust linear standard curve was established ( $y = 2.444x + 5.390$ , $R^2 = 0.969$ ) to convert absorbance values to log10 C. albicans loads.
Efficacy of Disinfectants: Compared to the deionized water control, both 0.5% sodium hypochlorite and 0.12% chlorhexidine gluconate resulted in significant reductions in C. albicans loads across the 24-hour and 48-hour colonization groups.
Temporal Dynamics:
- In the 24-hour group, sodium hypochlorite showed a significant decrease on Day 1, followed by a rebound on Day 3, and a continuous significant decrease by Day 10. Chlorhexidine showed a similar trend with pronounced effects toward the end of the treatment period.
- In the 48-hour group, both agents demonstrated significant reductions from baseline across all measured days, with chlorhexidine showing significant differences between Day 1 and Days 7 and 10.
- In the 72-hour group, no significant differences were observed between the two disinfectant groups across treatment dates; however, the deionized water control showed a distinct decrease in load relative to the other groups, though overall loads remained low.
Regression Analysis: Linear regression confirmed significant relationships among treatment duration, cleansing agent type, and C. albicans incubation time for the 24-hour and 48-hour groups ( $P=0.05$ ). Treatment duration and cleansing agent type were significant predictors of residual C. albicans load.

Significance and Claims
The authors claim to have established a reproducible in vitro framework that characterizes biofilm dynamics and evaluates the antiseptic efficacy of denture-cleansing agents under clinically relevant conditions. The study validates that 24-hour and 48-hour incubation periods are suitable models for simulating C. albicans colonization on PMMA surfaces. The framework allows for the quantitative assessment of residual fungal loads following standardized chemical interventions, facilitating the development of evidence-based strategies for managing denture stomatitis. The authors position this model as a necessary step prior to in vivo validation, offering a standardized method to compare novel antifungal interventions against established chemical cleaners.

Limitations Noted
The paper explicitly acknowledges that the study relies on a monomicrobial model (C. albicans in isolation), which does not capture the complex multi-species interactions and inter-kingdom signaling found in natural denture biofilms. Additionally, the authors note that the framework requires further validation and repeatability testing due to funding and capacity constraints. Future applications suggested by the authors include expanding the model to dual-species biofilms and evaluating newer antimicrobial agents or functionalized resins.

Technical Summary: Development of an In Vitro Denture Stomatitis Model Simulating Candida albicans Biofilm Formation and Detachment Under Daily Antiseptic Immersion

More like this