An In Vitro Comparative Evaluation of Conventional and Novel Thymus vulgaris Derived Herbal Disinfectant Solutions against Pathogenic Biofilm on Maxillofacial Silicones and Its Impact on Color Stability

INTRODUCTION

Maxillofacial silicones represent specialized materials employed in facial prosthetics to enhance or restore the appearance of individuals who have undergone facial surgery, experienced facial trauma, or were born with facial abnormalities. These materials play a significant role in enhancing the overall quality of life for these individuals.¹ They are commonly used with adhesives, implant-retained bars, and magnets being the most common retention methods.² However, the use of these prostheses can lead to complications if not properly disinfected, with Whear et al., reporting an infection rate of 16%.³ Disinfection methods such as chlorhexidine solution and liquid soap can affect the color stability of the silicone, with pre-colored silicone showing higher stability than hand-colored silicone.⁴ Furthermore, silicones used for maxillofacial prostheses can harbor microorganisms, with silicone being more vulnerable to microbial adhesion due to its larger porosities.⁵ Therefore, proper disinfection and maintenance of maxillofacial silicone prostheses are crucial to prevent complications.

It is imperative to sterilize and disinfect maxillofacial silicones to ensure cleanliness, sterility, and patient safety. This process serves to eliminate bacteria, viruses, and various other types of microorganisms that might inhabit the silicone surface.⁶ Effective cleaning procedures not only reduce the risk of infections but also extend the lifespan of prostheses and help maintain their aesthetic appeal. Therefore, the implementation of proper disinfection protocols for maxillofacial silicones is of paramount importance.⁴

A significant concern arises from the proliferation of pathogenic biofilms on maxillofacial silicones. Microorganisms such as Candida (C.) albicans and Staphylococcus (S.) aureus play a role in infections associated with these biofilms. Specifically, C. albicans exhibits adhesion to silicone surfaces and undergoes morphological changes that promote the development of biofilms. On the other hand, S. aureus employs virulence factors in the formation of biofilms, leading to increased resistance to antimicrobial treatments and immune responses. A comprehensive understanding of these mechanisms is imperative for the development of effective strategies to both prevent and address biofilm-related infections on maxillofacial silicones.^7,8

Conventional hygiene practices for maxillofacial silicones involve rinsing with warm water, applying mild soap or a non-abrasive cleanser, and gentle scrubbing with a soft brush or sponge. Despite these methods, limitations arise, as mild soap may struggle to remove persistent stains or biofilm, potentially heightening infection susceptibility. Hard-to-reach areas may not be effectively cleaned with soft-bristled brushes, and air drying or cloth drying could leave residual moisture, fostering microbial growth. Given these challenges, specialized antimicrobial agents or disinfectants become imperative for thorough silicone disinfection.⁹

Thymus vulgaris, commonly known as thyme, is a perennial herb renowned for its versatile pharmacological properties and culinary applications. This herb contains various bioactive compounds, including phenolic compounds, flavonoids, and terpenoids which contribute to its noteworthy antioxidant, antimicrobial, and anti-inflammatory attributes. Thymol and carvacrol are the major phenolic compounds present in T. vulgaris.¹⁰ Flavonoids such as luteolin, apigenin, and naringenin are also present.¹¹ It is also rich in terpenoids, including p-cymene, γ-terpinene, and thymol along with Rhamnogalacturonan I-type polysaccharides.^10,12

Thyme demonstrates robust antimicrobial effects against bacteria, fungi, and even viruses, including drug-resistant strains. Its antioxidant activity serves to safeguard cells from oxidative damage, consequently diminishing the risk of chronic diseases. Moreover, thyme possesses anti-inflammatory properties and bolsters the immune response.^13,14 Furthermore, extracts derived from T. vulgaris have exhibited promising anticancer potential by inducing cell death, restraining tumor growth, and suppressing cancer cell proliferation across various cancer types. It stands as a valuable herb with substantial medicinal properties. Notably, Thymus essential oil has demonstrated superior antimicrobial and antifungal qualities in vitro, surpassing the performance of chemical solutions for root canal irrigation. In a separate in vitro investigation, T. vulgaris essential oil exhibited heightened antifungal effectiveness when compared to chlorhexidine for disinfecting removable orthodontic appliances. These appliances, with their more hydrophobic surface, tend to increase salivary Candida carriers, particularly of the albicans species, by facilitating Candida binding through hydrophobic interactions.¹⁵ This study aims to delve into the therapeutic potential of T. vulgaris and its standardized extracts as alternative methods for disinfecting maxillofacial silicones.¹⁶

The preservation of color stability in maxillofacial silicones holds significant importance in maintaining the enduring aesthetic appeal of facial prostheses. These silicones are meticulously designed to harmonize with natural skin tones, thus creating a seamless and lifelike appearance. Their formulation incorporates specific pigments and additives to counteract fading or discoloration induced by various factors such as UV radiation, heat, and moisture.¹⁷

This study introduces a novel approach to managing biofilms on maxillofacial silicones by employing previously unexplored herbal disinfectant solutions derived from T. vulgaris. The primary objective of this investigation is to assess the antimicrobial efficacy of these herbal solutions and their impact on color stability in comparison to conventional disinfectants when applied to maxillofacial silicone materials.

MATERIALS AND METHODS

The present study was conducted as an in vitro investigation and adhered to the guidelines of good laboratory practice (GLP). It received approval from the Institutional Review Board (IRB No: EC:1364/2021). To create the experimental specimens, 100 disk-shaped samples were fashioned, each measuring 9 mm in diameter and 4 mm in thickness. These specimens were crafted using MDX4-4210 room-temperature vulcanization elastomer enclosed within a metallic matrix. Notably, MDX4-4210 is a silicone material commonly used in maxillofacial applications.

During the fabrication process, intrinsic pigments were incorporated into the silicone to achieve the desired coloration. The silicone elastomer was prepared following the manufacturer’s instructions, which entailed mixing the base and catalyst at a ratio of 10:1. The resulting mixture was applied to the matrix and allowed to polymerize at room temperature for a period of three days. Subsequently, the specimens were delicately removed, any irregularities were trimmed, and sterilization was carried out using ethylene oxide. No additional finishing or polishing procedures were conducted to preserve a smooth surface conducive to cell attachment.

The standard fresh strains of S. aureus – MTCC 12598 and C. albicans – MTCC 2091, were procured from Dr Prabhakar Kore Basic Science Research Center, Belagavi, India. The specimens were divided into two distinct groups, with 35 specimens each. One group was intentionally contaminated with S. aureus, while the other group was contaminated with C. albicans. To assess cell viability, the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was used. Subsequently, biofilms of C. albicans and S. aureus developed on the surfaces of these silicone specimens. The next step involved placing these contaminated specimens into two separate, pre-sterilized 24-well microtiter plates. To each well, 2 µL of standardized cell suspensions were added, along with 980 mL of Sabouraud medium for C. albicans and Brain-heart infusion medium for S. aureus. Subsequently, the microtiter plates were incubated under aerobic conditions in an orbital shaker at 37ºC, with an agitation speed of 75 rpm, for a duration of 72 hours. This incubation period allowed for the formation of biofilms on the surface of the silicone material.

The sample size for the five groups was determined through a power and effect size analysis using Cohen’s statistical power analysis.¹⁸ The estimation of power and effect size relied on data obtained from the pilot study, with a fixed p-value of 0.05 (alpha = 0.05, power = 80%). The assumed effect size was set at 0.5. Consequently, the calculated sample size for each group was determined to be 35. Following the formation of biofilms, each group of 35 specimens for both microorganisms was further subdivided into five distinct groups. In the control group (Group I), the specimens were immersed in a saline solution for a duration of 10 minutes. In the experimental groups, various disinfection methods were used. This included submerging the specimens in 4% chlorhexidine solution (Group II), soap water solution (Group III), and T. vulgaris solutions at concentrations of 5% (Group IVa) and 10% (Group IVb), all for a 10 minute duration.¹⁹ Commercially available thyme oil was procured and authenticated from Surajbala Exports Private Limited, New Delhi (HSN Code: 3301). The essential oil was made into a disinfectant solution at KLE Pharmacy College, Belagavi. Previous studies established the minimum inhibitory concentration (MIC) of T. vulgaris essential oil specifically for S. aureus and C. albicans.^14,20,21 Considering these findings, the concentrations for T. vulgaris disinfectant were established at 5 and 10%. The contents of the disinfectants included thyme oil as the active ingredient, Tween 80 and Span 60 were the emulsifying agents, and water was used as the solvent. The composition of the T. vulgaris disinfectant solutions at concentrations of 5 and 10% is detailed in Table 1.

To evaluate the influence of disinfection protocols on biofilm viability, the MTT reduction assay was employed, using L929 mouse fibroblast cell lines sourced from the National Centre for Cell Science in Pune. This assay quantifies the conversion of MTT into ‘Formazan blue’ by viable cells. Following the formation of biofilms, the specimens were rinsed with phosphate buffered saline (PBS) and transferred to new wells containing PBS enriched with glucose and MTT. The plates were then incubated in darkness at 37°C for a duration of 3 hours. After incubation, 300 µL from each well was transferred to a fresh plate, and 100 µL of DMSO was added to dissolve the Formazan crystals. Optical density (OD) measurements were conducted at 492 nm using an ELISA reader. The impact of the disinfection solutions on biofilm metabolic activity was determined by calculating the percentage of cell viability relative to the control group, which represented 100% viability. Optical density data were transformed into numerical values that represented cell viability, with the equation used being % of cell viability = [OD (test specimen) × 100%]/[OD (control specimen)].

In the second part of the study, 30 disk-shaped specimens were manufactured using MDX4-4210 silicone elastomer, employing the same procedure as previously described for specimen fabrication. These specimens shared the same dimensions, measuring 9 mm in diameter and 4 mm in thickness. The evaluation was centered on assessing the color stability of conventional disinfectants applied to the silicone elastomer. Color assessment was carried out at two specific time points: initially, at the commencement of the disinfection procedure, and subsequently, after a 30-day duration of disinfection. The color analysis was conducted with the utilization of the Spectrophotometer X-Rite Europe software. Color changes were quantified utilizing the CIE Lab × color system, employing the formula ΔE = [(ΔL)² + (Δa)² + (Δb)²]½.¹³ The ΔE values obtained were categorized into three clinically relevant intervals: ΔE <1 (indicating undetectable color alteration), 1 < ΔE < 3.3 (representing color changes clinically considered acceptable), and ΔE > 3.3 (indicating color changes clinically regarded as unacceptable.

RESULTS

The comparison of mean absorbance value after the MTT assay for C. albicans and S. aureus biofilms is presented in Table 2.

DISCUSSION

Silicone is a widely favored material for maxillofacial prostheses due to its comfort and flexibility. However, the porous nature of these prostheses, along with moisture accumulation and the acidic pH of the skin, especially in individuals with limited dexterity, creates a favorable environment for the growth of bacteria and fungi.²² In order to manage and prevent infections in individuals using maxillofacial prostheses, strict adherence to proper hygiene practices and thorough disinfection of the prosthesis is essential.²³ The current study aimed to assess the effectiveness of T. vulgaris solution, a herbal disinfectant for maxillofacial prostheses, and its impact on color stability, addressing the critical need for improved disinfection methods in this context.

In this study, the antifungal activity of five disinfectants against C. albicans was assessed using the MTT assay. Soap water exhibited the highest antifungal action, followed by the 5% and 10% concentrations of T. vulgaris solution, respectively. Treatment with these solutions resulted in a significant reduction in C. albicans viability, on par with the effect of soap water. While soap water demonstrated the highest antifungal activity, the efficacy of T. vulgaris was nearly as potent and even surpassed that of chlorhexidine. These findings indicate that T. vulgaris solution holds promise as an effective disinfectant for maxillofacial prostheses, particularly in combating C. albicans infections. These results are consistent with those of Naseri et al., where a 2% T. vulgaris solution exhibited favorable antimicrobial activity against C. albicans.¹⁵ Additionally, Jafri and Ahmad demonstrated that T. vulgaris is effective in treating biofilms associated with C. albicans, supporting the findings of this study.²⁴ It was also found to be effective against removable orthodontic appliances infected with C. albicans as reported by Kavianirad et al.²⁵

A similar trend was observed in this study against S. aureus. Soap water exhibited the highest antibacterial activity, followed by T. vulgaris solutions at 5 and 10%, respectively. The cell viability of the herbal disinfectants was comparable to that of soap water and superior to chlorhexidine. Notably, chlorhexidine demonstrated stronger antimicrobial activity against S. aureus compared to C. albicans. These results indicate that the T. vulgaris disinfectant possesses significant antibacterial effects against S. aureus, on par with soap water and superior to chlorhexidine. These findings align with those reported by Guiotti et al. and Galgano et al.^18,26 It was found to be effective in treating S. aureus, including methicillin-resistant S. aureus. It has been found to inhibit biofilm formation, and disrupt preformed biofilms.²⁷ It also demonstrated synergistic effects when combined with other antimicrobial compounds, enhancing its antibacterial activity.²⁸ These findings suggest that T. vulgaris can be effective in treating S. aureus infections.

Saline solution, being a neutral and isotonic substance without inherent antimicrobial properties, serves as a baseline for assessing disinfectant effects. Its similarity to body fluid sodium levels ensures its suitability for biological research.²⁹ In contrast, a 4% chlorhexidine solution, known for its biocompatibility and comprehensive disinfection capabilities, has consistently proven effective in disinfecting dental impression materials and prostheses using both spray and immersion methods.³⁰

Color stability is recognized as a pivotal characteristic when assessing the functionality of a facial prosthesis from the patient’s perspective. It is a common parameter used to evaluate the longevity and aesthetic appeal of materials. In a study by Cantor et al., methods for assessing the aesthetics and color matching of prosthetic facial materials with human skin using reflectance spectrophotometry were explored.³¹ In the present study, the chlorhexidine group exhibited the least color alterations among the tested disinfectants, followed by T. vulgaris at a 10% concentration. These findings were in line with a study conducted by Chamaria et al.,¹⁹ although they contradicted the results of Chotprasert et al.⁴ Conversely, other disinfectants demonstrated substantial color alterations after a 30-day period, including T. vulgaris at a 5% concentration and the soap water group.

The observed color changes in the maxillofacial silicones indicate that conventional disinfectants such as soap may have a mild adverse effect on the aesthetic quality of the prostheses over time.⁹ The specific concentration of soap for disinfecting maxillofacial silicones is not explicitly detailed in the provided studies. However, the study by Goiato et al. recommends the use of neutral soap for cleansing maxillofacial prostheses. It’s important to note that the process of disinfecting specimens using neutral soap involves applying digital friction, which may gradually remove the superficial layer of silicone.³² Therefore, careful consideration must be given to selecting a cleaning solution that can achieve both disinfection and preservation of the prosthetic material’s aesthetic quality simultaneously.

Numerous environmental factors, including sunlight, humidity, air pollutants, facial secretions, and disinfection methods, can impact the color stability of silicone prostheses. Research conducted by Goiato et al. and Mancuso et al. underscores how external factors and chemical cleansers can lead to discoloration and the alteration of material properties.^32,33 The influence of UV light exposure, as documented in studies by Lemon et al., Haug et al., Eleni et al., and Hatamleh et al. has been observed to induce significant changes in coloration. These changes are attributed to processes like cross-linking and interaction with fatty acids.^34–37 Additionally, research by Hatamleh et al. and Polyzois et al. has shown that exposure to simulated sebum solutions can result in color changes and the decomposition of elastomeric materials.^37,38

Despite soap water’s superior antimicrobial efficacy, it did not exhibit higher color stability on maxillofacial silicones in this study. In contrast, the herbal disinfectant proved more effective in preserving color while still providing adequate antimicrobial protection. When choosing chemical disinfectants, it is crucial to consider both their antimicrobial properties and compatibility with preserving the material’s physical characteristics. The minimal impact of the T. vulgaris disinfectant (10%) on color stability is promising, indicating that antimicrobial treatment with Thymus does not compromise the aesthetic quality of maxillofacial silicones. Herbal disinfectants, with their bioactive phytochemical composition, demonstrate potent antimicrobial activity against pathogens, offering a more sustainable and cost-effective solution compared to synthetic counterparts. Conducting clinical trials with patients having maxillofacial defects could provide a more comprehensive and scientifically rigorous assessment of the efficacy of T. vulgaris disinfectant.

CONCLUSION

This study highlights T. vulgaris as a promising herbal disinfectant against microbial biofilms on maxillofacial silicones, particularly at 5 and 10% concentrations, though not surpassing the efficacy of conventional soap water. This suggests T. vulgaris is a potential alternative in clinical settings, emphasizing its compatibility with silicone materials. Additionally, the study underscores the importance of color stability in maxillofacial prosthetics, with Chlorhexidine and 10% T. vulgaris effectively preserving color, aiding practitioners in selecting disinfection agents that ensure both microbial control and visual integrity.

ACKNOWLEDGMENT

The authors would like to thank KLE’s Dr Prabhakar Kore Basic Science Research Center for providing resources for the study.

ORCID

Meekha Peter https://orcid.org/0000-0001-7509-4070

Hema Kanathila https://orcid.org/0000-0002-5876-1377

Mahantesh Bembalagi https://orcid.org/0000-0002-2809-7313

Varkey Nadakkavukaran Santhosh https://orcid.org/0000-0001-9197-2646

Rhea Vas https://orcid.org/0000-0003-4401-5696

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