Discussion
This study investigated the potential use of CA, a natural substance renowned for its broad-spectrum antimicrobial properties, in the disinfection of alginate dental impression materials. The effect of CA disinfection was comprehensively verified from the following three aspects: physical properties, antimicrobial effect, and influence on the accuracy of gypsum model. The study yielded three significant findings. Firstly, the elastic recovery, compressive strain, fluidity, and initial setting time of alginate impression material mixed with 10 mg/mL CA were all within the standard range. Secondly, this study demonstrated that CA had a significant bactericidal effect on the microorganisms on the surface of alginate-stained bacterial impressions, and thirty minutes after mixing and spraying disinfection, no viable bacteria was detected on the four bacteria-stained alginate impressions. Lastly, there was no statistically significant difference between the CA intervention group, the traditional chemical disinfectant intervention group, and the distilled water mixing group on the accuracy of the plaster model made by the impression. These research findings provide guidance for the clinical application of alginate impression disinfection and establish the groundwork for the potential use of CA as a disinfectant for alginate impressions.

In our previous study on the disinfection effect of CA sprayed on silicone rubber impressions, CA was found to have a significant disinfection effect on pathogenic bacteria. However, another study found that after the same disinfection procedure, the microbial load on the surface of alginate impressions was higher than that of silicone rubber impressions ( Divya Dharshini, Somasundaram & Muralidhara, 2020 ; Demajo et al., 2016 ). This may be because the water absorption properties of alginate materials result in a lower amount of disinfectant remaining on the impression’s surface after spraying ( Ginjupalli et al., 2018 ), making disinfection procedures for alginate impressions more demanding than those for other types of impressions. In recent years, many scholars have studied the self-disinfection method of adding bacteriostatic agents directly to alginate impression materials ( Ginjupalli et al., 2016 ; Hussian & Jassim, 2015 ). Self-disinfection not only ensures the disinfection of the entire impression material but also prevents the infiltration of alginate caused by prolonged immersion disinfection ( Ahmed, Jawad & Nahidh, 2020 ). Self-disinfection also allows for long-term contact of the disinfectant with microorganisms. This paper studied CA as a self-disinfectant for alginate impressions because of its anti-inflammatory, antimicrobial, safe, and pollution-free properties.

The complexity of the oral structure requires impression materials to have corresponding properties, including good elasticity, plasticity, fluidity, and appropriate solidification time, which are reflected in the physical properties of elastic recovery, strain-in-compression, fluidity, and initial setting time ( Dilip, Gupta & Geiger, 2023 ). The fluidity of alginate impression materials mixed with 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, or 30 mg/mL CA solutions was explored in pre-experiments, which showed that alginate impression material mixed with more than 20 mg/mL CA was too mobile to meet the required standards. Based on this finding, this study explored the physical properties of alginate impression materials mixed with CA concentrations of 10 mg/mL, 15 mg/mL, and 20 mg/mL.

The elastic recovery of impression materials is crucial for achieving an accurate reproduction of the oral cavity. Elasticity refers to the ability of the material to recover after solidification, ensuring that the impression does not undergo permanent deformation, affecting dimensional accuracy, upon removal from the mouth ( Wezgowiec et al., 2022 ). ISO 21563 ( Standardization, 2021 ) specifies a minimum elastic recovery value of 95% for alginate impression materials.

Strain-in-compression is an important indicator that assesses the flexibility or rigidity of a material. It provides insights into several aspects of the impression, such as if it can be easily removed from the mouth without causing damage to oral tissues and if the cured plaster model can be successfully removed from the impression without any breakage. Strain-in-compression also evaluates the rigidity of the impression’s weak peripheral areas, ensuring they can withstand the pressure exerted during mold creation without deforming. According to ISO 21563 ( Standardization, 2021 ) standards, the strain-in-compression of alginate impression materials should range between 5% and 20%. In this study, varying concentrations of CA solutions led to statistically significant differences in the elastic recovery and strain-in-compression of the mixed alginate impression materials ( P < 0.01). This finding aligns with the studies conducted by Singer & Bourauel, (2023) and Dreesen et al. (2013) , who found that modifications in filler content and mixing techniques can result in changes to the properties of elastic recovery and strain-in-compression. The research results of this study show that the elastic recovery, strain-in-compression, and average values of alginate dental impressions prepared using different concentrations of CA solution were all within the standard range.

The initial setting time of alginate impression materials refers to the time required from the start of mixing until the material attains the necessary elasticity and strength for separating and removing the impression. Initial setting time serves as an indicator of the curing speed of dental impression materials ( Ramer, Gerhardt & McNally, 1993 ). An excessively long initial setting time can cause discomfort to the patient, while an overly short initial setting time may not provide enough time for the dentist to perform the procedure ( Cervino et al., 2018 ). The T-6505 Japanese Industrial Standard ( Kitamura & Kawai, 2015 ) specifies an initial setting time range of 1–5 min for alginate impression materials. In this experiment, only the alginate dental impressions mixed with 10 mg/mL CA solution had an initial setting time that fell within the 1–5-minute range. There was a positive correlation between the primary coagulation time and the concentration of CA solutions ( P < 0.01). The solidification time of alginate material is determined by how fast the replacement reaction between calcium sulfate and sodium alginate occurs after mixing with water. Factors affecting the initial coagulation time include the amount of retardant used, the ratio of alginate to binder, and temperature ( Cervino et al., 2018 ). The initial setting time of alginate material is greatly affected by CA concentration, which may be due to the interaction between calcium alginate and CA ( Demircan & Oral, 2023 ).

It is essential for the fluidity of the impression material to fall within a specific range. According to ANSI/ADA Specification No. 18-1992 ( Dental Materials, 1992 ), the average diameter of the solidification test piece for alginate impression materials should be between 27 mm and 36 mm. In this study, the concentration of the CA solution affected the fluidity of the alginate impression material ( P < 0.01), with the fluidity of the impression material increasing alongside CA concentration. The average diameter of the solidification plate for CA self-disinfecting alginate impression materials ranged from 28.86 mm to 32.60 mm, thus meeting the relevant standards.

Based on the results of the experiment on physical properties, a 10 mg/mL CA solution was chosen as a suitable self-disinfecting solution for alginate impression materials. Previous studies have demonstrated the effectiveness of CA spraying at concentrations of 100 mg/mL, 150 mg/mL, and 200 mg/mL for disinfecting silicone rubber stain impressions. However, since this study focused on mixing disinfection, CA concentrations of 50 mg/mL, 55 mg/mL, and 60 mg/mL were chosen for spray disinfection. In the experimental group, the CA solution mixing protocol was combined with spray disinfection of infected impressions with 50 mg/mL, 55 mg/mL, or 60 mg/mL CA solutions. The control groups consisted of only a 10 mg/mL CA solution for mixing disinfection and only a 60 mg/mL CA solution for spraying disinfection. Maciel et al. (2023) performed an experiment using the inhibition zone method to measure the antimicrobial effect of a 2% chlorhexidine solution as an impression disinfectant. However, this method only demonstrated the killing effect of the disinfectant on the corresponding bacteria without providing insight into the disinfection effect of the procedure on the impression’s surface. In this experiment, contaminated impressions were disinfected and sampled, thereby maximizing the assessment of the disinfectant’s antimicrobial effect on the corresponding bacteria under clinical disinfection procedures.

In recent years, the antimicrobial effect of CA has been confirmed by many researchers. This effect occurs mainly through direct targeting of the bacterial cell wall and membrane, leading to irreversible permeability damage ( Feng et al., 2023 ; Liu et al., 2019 ). The results of this experiment also showed the powerful antimicrobial effect of CA. As intervention time increased, the colony count of each group decreased significantly. The growth of Escherichia coli , Staphylococcus aureus and Candida albicans on the impression surface was completely inhibited after 30 min of intervention in the experimental group. Escherichia coli , is a gram-negative bacterium, which can cause gastrointestinal infection or urinary tract infection and impact other local tissues and organs of humans and animals under certain conditions ( Yang, Lan & Xie, 2022 ). Staphylococcus aureus , a gram-positive coccus, can cause a variety of diseases including skin infection, respiratory infection, and bacteremia ( Ahmad-Mansour et al., 2021 ; Plumet et al., 2022 ) and is one of the main causes of nosocomial infections ( Konstantinovski et al., 2021 ). Candida albicans is an opportunistic pathogenic fungus, which can cause oral fungal infections in people with low immunity ( Eichelberger et al., 2023 ) and is also an indicator strain for killing fungi in laboratory tests. It has been reported that pathogens such as Candida albicans and Staphylococcus aureus can still be detected on oral impressions and plaster models after disinfection ( Egusa et al., 2008 ). A study on medical microfluidic devices also found that CA inhibited the growth of Escherichia coli on polydimethylsiloxane (PDMS) surfaces ( Ren et al., 2015 ). The findings of this study align with the results of Yang, Lan & Xie (2022) , who demonstrated that CA influences the antioxidant system and energy metabolism of Staphylococcus aureus by significantly reducing its ATPase and CAT activity. A study by Yun & Lee (2017) also discovered that CA induces apoptosis in Candida albicans by blocking K ion channels. This study showed that 10 mg/mL CA mixing combined with 55 mg/mL or 60 mg/mL CA spraying disinfection for 20 min can completely inhibit the growth of Streptococcus pneumoniae on the impression surface. This may be because Streptococcus pneumoniae is more sensitive to CA ( Lou et al., 2011 ). However, the results of the control groups were less positive than those of the experimental groups; a significant number of live bacteria (1.6–2.77 lgCFU/mL) remained on the control group impressions even after 30 min of disinfection, indicating a poor disinfection effect. Even when the impression was sprayed with a high concentration CA solution for 30 min, viable bacteria still persisted on the impression surface, which may be attributed to the physical properties of alginate impression materials ( Hardan et al., 2022 ). During the initial solidification stage, hydrocolloid impression materials exhibit continuous percolation, followed by condensation. This percolation and condensation process leads to the transfer of microorganisms from inside the material to the impression surface ( Al Shikh & Milosevic, 2020 ). The results of this study show that CA has a similar disinfection effect to traditional chemical disinfectants. In addition, because of CA’s natural extraction, its use as an alginate impression disinfectant can help eliminate potential health damage caused by chemical disinfectants ( Pimpley et al., 2020 ; Tajik et al., 2017 ).

The ANSI/ADA’s Specification No. 18-1992 and the International Organization for Standardization’s 21563:2021, which both specifically focus on dental alginate, do not provide specific requirements or limits on dimensional change values ( Dental Materials, 1992 ; Standardization, 2021 ). Currently, there are two primary methods for evaluating the impact of disinfectants on impression dimensional accuracy. One method involves measuring linear changes at specific sites on standard impression materials or plaster models ( Babiker, Khalifa & Alhajj, 2018 ; Singer et al., 2023 ). The other method uses model scanning technology, which has seen rapid development in recent years, to analyze three-dimensional deviations in the impression materials using software ( Reich et al., 2023 ), thus reducing systematic errors caused by manual measurements ( Parize et al., 2022 ). Due to the absence of standard feature points in the dentition, the “best fit alignment” algorithm was employed to align the two digital models in this study. The distilled water mixing group was used as a blank control to eliminate the influence of any systematic errors that may occur during the impression-making process. The systematic analysis results demonstrated that chlorine spray disinfection at a concentration of 2,000 mg/L and CA intervention had little effect on the dimensional accuracy of alginate dental impressions. Moreover, the difference in dimensional accuracy between these disinfection methods and the distilled water mixing group was not statistically significant. These findings align with studies conducted by Vrbova et al. (2020) , Babiker, Khalifa & Alhajj (2018) and Singer & Bourauel, (2023) , who modified alginate materials with hydrogen peroxide and silver ion powder and observed no changes in the dimensional accuracy of the tested groups.