Optimization of Facial Silicone Elastomers Antimicrobial Activity: In-Vitro Study View PDF

Maxillofacial prostheses are an efficient treatment option when maxillofacial defects resulting from ablative surgery, trauma, or congenital anomalies cannot be surgically reconstructed. Silicone elastomers, mainly polydimethylsilioxane (PDMS) have been used for over 50 years in constructing maxillofacial prostheses for individuals with facial defects [1]. Such prostheses play integral role in improving patient’s quality of life. However, they usually last for 18 to 24 months as they deteriorate in structure, colour fade and become stained requiring their remake [2].

The silicones are porous in nature [3], making those favourable sites for bacterial colonization causing prosthesis staining and infections [4]. A black discoloration is usually evident on the prosthesis’s inside surfaces after they have been worn for a period of time (Figure 1).

Microbiologic techniques and SEM studies revealed that a fungus belonging to the genus Penicillium is associated with such discolored areas. Furthermore, disk diffusion tests determined that the antifungal agent clotrimazole, when incorporated into silicone samples, was effective in inhibiting it’s in vitro growth [5,6]. Titanium oxide (TiO₂) has been advocated as an antibacterial agent based on its photocatalytic properties proposing its use for preventing bacterial contamination and disinfection [7]. Other oxides of similar anti-fungal activities were ZnO, SiO₂ and Al₂O₃ [8]. But there is contradictory results of their effects requiring further evidence.

For example, ZnO which is a bio-safe material that possesses photo-oxidizing and photocatalysis impacts on chemical and biological species. It exhibits attractive antibacterial properties due to increased specific surface area as the reduced particle size leading to enhanced particle surface reactivity but research still inconclusive about its antibacterial effect [9]. CHX is well known for its antibacterial and antifungal activities and have been commonly used in different fileds of dentistry [10]. It affects the colonization rate of oral bacteria on the enamel surface by coating the surface of the bacteria which results in prolonged and persistent antimicrobial action.

The use of the above mentioned inserts in maxillofacial silicone prostheses is limited. Thus, this study aimed to investigate the effect of Zinc Oxide nano particles (ZnO-NP) and Chlorohexidine Diacetate Salt (CHX) inserts on the antimicrobial activity of maxillofacial silicone elastomer at three difference concentrations (1%, 3%, and 5%).

Preparing Silicone Samples

Commonly used maxillofacial silicone elastomer (M511, Technovent, Newport, Wales) was mixed according to manufacturer instructions with ZnO nano particles (ZnO-NP) (BET= 67 m²/g) and Chlorohexidine Diacetate Salt (CHX) at different concentrations: 1%, 3% and 5% (by weight). The mixture was manually mixed for 5 sec and placed in a Speed Mixer at 2000 rpm (revolution per minute) for 60 sec (Speedmixer, Dac 150, Hauchild Engineering, Germany). Then it was packed inside disc-shaped steel moulds (40 mm diameter and 0.5 mm height) and cured for 1 hour at 100?. Once curd, smaller silicone discs (10 mm diameter) were produced. Sixty specimen per insert were made available for the three concentrations. Each concentration included 20 specimen which were divided equally between two inoculants; Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans). Eighty specimens of silicone without insert were made and divided into 4 groups acting as negative control (NC) and 4 groups acting as double negative control (DNC). All groups are present in table 1.

Table 1: Test groups presented.

Abbreviations: DNC: Double negative control where silicone discs contained no inoculum and the discs were inoculated with distilled water only; NC: Negative control where silicone discs contained no inoculum and the discs were inoculated with 200 µl of suspension of either microorganisms. S. aureus: Stapylococcus aureus; C. albicans: Candida albicans. 

Preparation of the Microbial Test Inoculum

S. aureus ATCC 25923 and C. albicans ATCC 10231 were obtained from the microbiology laboratory, Faculty of Medicine, Mutah University. Microorganisms were retrieved from the -70ºC and streaked using sterile inoculating loop onto nutrient and hypertonic Dextrose Agar (SDA) (BBL Microbiology Systems, Becton Dickinson, Company, Sparks, MD, U.S.A.) to obtain single colonies. Plates were incubated at 36? ± 1 overnight. Growing S. aureus colonies were confirmed by their typical colony morphology, Gram’s staining, catalase production and tube coagulase test. C. albicans was confirmed by colony appearance, gram stain and germ tube test. Ten colonies of each growing organism were suspended separately in 5 ml brain heart infusion and incubated at 36? ± 1 overnight. The culture suspension of S. aureus and C. albicans was utilized to calculate the neat colony forming units for each organism using serial dilution method.

Testing Silicone Discs

Testing the Sterility of the Silicone Discs: Sterility of silicone discs was carried out by washing them using either 70% ethanol, distilled water or nothing. The discs were then incubated at 36? ± 1 overnight. After that, the uppermost face of each disc was then cultured using sterile forceps by placing onto nutrient agar for 15s then removed. Agars were then incubated at 36? ± 1 overnight before being examined for any growth of microorganisms.

Antimicrobial Inhibitory Effect of Silicone Discs: Antimicrobial activity of the silicone discs were assessed as previously described [11]. S. aureus and C. albicans suspensions were adjusted to 5x10⁶ colony forming units/ml (cfu/ml). For S. aureus, the first test group was silicone discs that contains 1%, 3% or 5% chlorhexidine in triplicates. The second test group was silicone discs with 1%, 3% or 5 % ZnO-NP in triplicates. The silicone samples were aseptically placed in a sterile petri dish using sterile forceps with their release liner facing up.

The upper surface of each disc was inoculated with 200µl of S. aureus suspension (10⁶ cfu) using a sterile pipette. One of the silicone discs with 0% concentration was inoculated with 200 µl sterile distilled water and considered as double negative control (DNC), since it has no CHX or ZnO-NP and no bacteria was added. Another 0% disc was inoculated with 200 µl S. aureus suspension and considered as a negative control (NC), since it has no CHX or ZnO-NP but with bacteria added. To prevent inoculum leakage, drying and to ensure proper contact to the discs, the inoculated silicone samples were covered with a sterile cover film.

The discs were incubated at 36? ± 1 overnight. Thereafter, excess inoculum was removed using filter paper and the bacterial-exposed face of each disc was cultured by placing onto nutrient agar for 15s then removed. Plates were then incubated at 36? ± 1 for 18-24 hours and observed for bacterial growth. Experimental procedure for testing discs against C. albicans using SDA was the same as described above for S. aureus.    

The demonstration of inhibitory effect of CHX and ZnO-NP variable concentrations is shown in Figure 2. There was a significant decrease in colony forming units of S. aureus and C. albicans when tested against 1%, 3%, and 5% concentrations of CHX and ZnO-NP.

Table 2 shows that both inserts reduced bacterial and fungal number of colony forming units compared to the negative control with CHX showing an overall better antimicrobial activity than ZnO-NP regardless of their concentration. However, the complete inhibition of S. aureus and C. albicans growth was zero number of colonies (100% growth inhibition) and only obtained using the 5% concentrations of CHX compared to other CHX and ZnO-NP concentrations.

Table 2. Silicone discs impregnated with different concentrations of CHX and ZnO, and tested against S .aureus and C. albicans

Abbreviations: CFUs: Colony Forming Units; CHX: Chlorhexidine; DNC: Double Negative Control; NC: Negative Control; ZnO: Zinc Oxide.

No colonies were detected at the DNC disc as expected since this control was inoculated with sterile distilled water only. For the NC disc, numerous uncountable colonies were detected as expected. Images of silicone samples without any insert were captured using Micro Computer Topography (u-CT) and optical microscope (X40 magnification). The images show pores trapped as a result of the manual mixing of the silicone elastomer as shown in Figure 3.

Facial silicone prostheses communicating with the oral cavity may be contaminated with oral microflora. These microorganisms can grow over a wide temperature range (40 to 45^oC). Human skin especially around craniofacial percutaneous implants shows a microflora consisting of Staphylococcus aureus Streptococcus species, bacilli and Candida parapsilosis, that cause peri-abutment infection [5,6]. They are associated with reported infections of medical devices [12], especially maxillofacial silicone prostheses [4,13]. This study aimed to investigate the effect of two different inserts (ZnO-NP and CHX) at three different concentrations (1%, 3%, and 5%) on the antibacterial and antifungal activities of maxillofacial silicone elastomer. There was significant effect of insert and concentration on bacterial and fungal growth. However, the complete growth inhibition was only observed at 5% CHX concentration, this inhibition was maintained at 18 and 24 hours of incubation which indicates strong bactericidal effect of 5% CHX. The CHX and ZnO-NP were suggested to have such antibacterial activity [14,15,16]. Different concentrations were tested against C. albicans and S. aureus as they are common organisms that may lead to prosthesis infection [11,12,14]. Both inserts decreased bacterial and fungal growth with CHX showing an overall better antimicrobial activity than ZnO-NP regardless of their concentration.

CHX impregnated in medical devices kills organisms and protects against microbial colonization and subsequently biofilm development by disrupting the pathogen’s cell membrane [10]. It markedly decreased the growth of both organisms at 1% and 3% concentrations, and completely inhibited the growth at 5% concentration. Its antimicrobial effect is novel and attributed to its broad-spectrum biocide effect against fungi and bacteria. It has both bacteriostatic (inhibits bacterial growth) and bactericidal (kills bacteria) mechanisms of action, depending on its concentration [17].

Taking into consideration that CHX has been used safely in human [14]; incorporating it in silicone would have potential clinical applications in medical devices such facial prosthetics, indwelling catheters, needleless connectors and antimicrobial dressings [18]. A study found that CHX antimicrobial activity can last for 48 hours on the skin when used topically and is not affected by the presence of blood or other body fluids [18]. Hence, it is anticipated that facial prosthetics impregnated with CHX can be worn for long hours without any CHX inferior effect on the skin.

ZnO-NP were previously found to have antimicrobial effect which is enhanced by the surface area to volume ratio of the particles [15]. However, in our study, the antimicrobial effect of ZnO-NP against S. aureus and C. albican was not as effective as CHX. That was evidenced by the remaining microbial growth the silicone discs with ZnO-NP of the three different concentrations. Therefore, the current study limited in vitro antimicrobial benefits of using zinc oxide in silicone discs.

ZnO-NP antibacterial activity is directly correlated with their concentration and size. Larger surface area and higher concentration are accountable for ZnO-NP antibacterial activity [19]. This was evident in current study as samples impregnated with 5% ZnO had the least growth of microbes. However, size effect was found to be inconclusive as some studies indicated no effect [20], while others reported increased antibacterial activity with decreased size [21]. Other factors such as material aging and presence of surface defects may affect the antibacterial activity of ZnO-NP [9,22]. For example, some studies suggested that freshly mixed zinc has shown better antibacterial effect than aging mixtures, probably because increased zinc is released from fresh material and such effect becomes less over time [22,23]. Also, the insolubility nature of the ZnO-NPs was suggested to hinder the diffusion of enough antibacterial amount of Zn to the disc surface environment leading to a decreased visible antibacterial effect [24]. Toxicity mechanism of ZnO-NP differs among studies due to variances in test conditions and mechanisms being investigated [9].

Facial silicone prostheses are porous in nature. Clinically, pigments and colourants are added to the external surfaces of the prostheses for better camouflage effect and skin-tone matching of surrounding tissues. Patients are advised not to use any corrosive solvents, household soaps, or industrial detergents but tap water with mild soap in cleaning their prosthesis [2,25, and 26]. While such protocol can maintain the colour and properties of the silicone prosthesis, however, it is insufficient to kill all bacterial growth as patients notice black staining present inside the fitting surfaces of the prostheses over time. This is likely fungal and bacterial growth inside the pores. Manually mixed silicones have significantly high percentage of pores (0.97-2.15%) and pore volume (0.64-1.43 mm³) when compared to mechanical mixing under vacuum [3]. Such pores, especially on prosthesis fitting surface are favourable sites for microbial colonization and cannot be reached by the brush during normal prosthesis cleaning [4]. Hence it is advocated to use anti-microbial silicone-cleaning solution, however it can inferiorly affect silicone’s tear strength [25]. A side from this, maxillofacial silicone composition is polydimethylsiloxane chains with no antimicrobial ingredients [1]. Thus, incorporating anti-microbial agent can be the ideal solution. But more studies need to be carried out to determine its influence on silicone’s prosthetic materials properties and color.

This study complemented the ongoing in vitro work on silicone disinfection, however its results is restricted to baseline, which is considered a limitation. It needs to be widened to other types of bacteria that exist on silicone prostheses surface.

Within the limitations of this study Chlorohexidine diacetate salt exhibited antibacterial activity against some oral pathogenic strains better than ZnO nano particles. It has the potential to completely eliminate bacteria that adhere to maxillofacial elastomer when mixed at 5% (by weight), thus, contributing to the disinfection of silicone-based materials and the destruction of the source of infections caused by Candida albicans or Staphylococcus aureus.

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