Materials & Methods
Ethical approval and informed consent were not required for this study since it did not involve human subjects. This study performed in vitro laboratory testing of dental alginate impression material (V514450, Dentsply, Charlotte, NC, USA). To prepare the experimental materials, a CA solution (CJS 20220620, XI’AN FOREVIEW Bio-tech Co., Ltd., China), at concentrations of 10 mg/mL, 15 mg/mL, and 20 mg/mL, along with dental alginate impression powder, was mixed into a uniform paste using an automatic alginate mixer (GX300, Xianyang Holy Medical Co, Ltd, Shaanxi, China). The liquid to powder ratio used was 23 mL:10 g, as specified in the instructions of the dental alginate impression material. The prepared paste material was immediately used for subsequent experiments.

To evaluate the elastic recovery and strain-in-compression, cylindrical samples were prepared using a split-mold specimen with fixation ring following the ISO 21563 standard ( Standardization, 2021 ). For this procedure, the mold was placed on a glass plate and the impression mixture was poured into the mold. The glass plate with the mold was then placed in a constant temperature water bath at 35 °C for two minutes and 30 s to ensure proper setting of the alginate material. The final alginate specimens had a diameter of 12.5 mm and a height of 20 mm.

Immediately after the specimen was separated from the forming assembly, it was placed on the base of the well-tuned universal material testing instrument (AGS-10KN, SHIMADZU, Kyoto, Japan). The initial height of the sample (h_1) was then recorded. To assess elastic recovery, a deforming force was gradually released over a five-second period. After the deformation force was completely removed from the specimens, the sample height (h_2) was measured again after 40 s.

The elastic recovery, represented as a percentage (K), was evaluated by calculating the change in sample height before and after the deformation force, using the following Eq. (1) : (1) K = 100 − 100 ∗ h _ 1 − h _ 2 / h _ 0 .

Here, h_0 denotes the measured height (in mm) of the split mold used in the experiment.

To evaluate strain-in-compression, five samples per material were tested. An initial force of 1.2 ± 0.1 N was applied to the specimen, and the sample height was recorded after 30 s (h_1). The pressure was then increased to 12.2 ± 0.1 N for 10 s, and the sample height (h_2) was recorded again after 30 s under the total load. The percentage of strain-in-compression, E, was calculated by measuring the change in sample height before and after the application of pressure, using the following Eq. (2) : (2) E = 100 ∗ h _ 1 − h _ 2 / h _ 0 2 . Here, h_0 represents the height (in mm) of the split mold used for preparing the samples.

The initial setting time test involved the preparation of cylindrical samples measuring 16 mm in length and 30 mm in diameter using a rigid ring mold, in accordance with the ISO 21563 standard ( Standardization, 2021 ). The mold was placed on a flat glass plate measuring 50 mm by 50 mm, and the prepared impression mixture material was injected into the mold.

To determine the initial setting time, one end of the test rod (measuring 100 mm in length and 6 mm in diameter) was briefly brought into contact with the unset specimen material. Any excess material on the rod was carefully removed before repeating the contact/withdrawal and rod clearing steps at intervals of 10 s. This process was repeated until the test rod cleanly separated from the material, and the time at which this separation occurred was recorded as the initial setting time.

The experimental method used for testing the fluidity of the material followed the guidelines set by the ANSI/ADA Specification No. 18 for irreversible hydrocolloid impression material ( Dental Materials, 1992 ). To begin the test, an open syringe was used to quickly measure and dispense 0.5 mL of the impression mixture into the center of a clean glass plate. After mixing the material for 60 s, another clean glass plate was gently placed on top of the mixture with a vertical light pressure of 1.5 kg. After applying the pressure for five seconds, the load was released, and the diameter of the solidified specimen was measured at five different locations using a digital vernier caliper. The average of these measurements was then taken as the measurement value for the fluidity of the impression material mix.

Based on the results obtained from the physical performance test experiment, a solution of 10 mg/mL CA was selected as an autologous disinfectant for the alginate printing material. Four pathogenic bacteria, namely Escherichia coli ATCC 25922 (provided by the Oral Biology Laboratory of the Key Laboratory of the Ministry of Education), Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231, and Streptococcus pneumoniae ATCC 49619 (all provided by the Jiangxi Clinical Laboratory Center), were cultured, isolated, and chosen for further testing.

For the experiment, Escherichia coli and Staphylococcus aureus were inoculated on nutrient agar medium, with Streptococcus pneumoniae cultured on blood agar medium and Candida albicans cultured in Sabouraud’s agar medium. Bacterial suspensions were prepared at a concentration of 10 7 cfu/mL. Standard dentition model (Nobel BIOCARE, Sweden) was disinfected with ultraviolet lamp (ZYW-170Z, Zhong Yi, China) for 1 h before impression preparation. The alginate material was mixed with the CA solution (10 mg/mL) to prepare a total of 244 pairs of impressions on a standardized maxillary dentition model, four disinfection methods, four bacteria, three time periods, five replicates per group, required a total of 240 CA-mixed impressions, and four blank impressions are required to observe the effect of bacterial contamination. An additional 64 pairs of impressions were prepared using distilled water, four strains, three time periods, five replicates per group, required a total of 60 impressions mixed with distilled water, also required four blank impressions. Resulting in a total of 308 pairs.

The impressions were divided into four groups based on the four different types of bacteria, and 300 µL of bacterial suspension was inoculated onto the surface of each impression in a clean laboratory setting. After five minutes of bacterial inoculation, the impressions mixed with CA solutions were sprayed with distilled water, then with a CA solution at a concentration of either 50 mg/mL, 55 mg/mL, or 60 mg/mL. The impressions mixed with distilled water were sprayed with a 60 mg/mL CA solution. Each impression was sprayed with 40 mL of solution. The remaining six pairs (three pairs of distilled water mix and three pairs of CA mix) were not disinfected, and only samples were taken to observe the effect of infection. At 10 min, 20 min, and 30 min, sterile cotton swabs were used to sample the same area of each impression with consistent applied force. The sampled cotton swabs were collected in test tubes containing five mL of sterile phosphate-buffered saline (PBS) under sterile conditions and vigorously shaken using an IKA MS3 Basic Vortex (IKA, STAUFEN, Germany) for three minutes. Following this, 200 µL of the PBS containing the sampled material was inoculated onto the appropriate agar plates (blood agar for Streptococcus pneumoniae and Sabouraud’s agar for Candida albicans ). The plates were then incubated at 36 ± 1 °C for 48 h to allow for the recovery of any contaminating microorganisms. In the case of Streptococcus pneumoniae , incubation occurred in a 36 ± 1 °C carbon dioxide incubator for 48 h. Lastly, the colony-forming units (CFU) on each plate were counted and recorded.

Based on the experimental results obtained from the physical property detection and bacteriostatic effect detection, a 10 mg/mL CA solution was chosen as an appropriate self-disinfectant for the alginate impression material, while a 60 mg/mL CA solution was selected as the spray disinfectant. For this experiment, a total of 36 pairs of impressions were prepared: 24 pairs using distilled water and 12 pairs using a 10 mg/mL CA solution. The impressions prepared with CA solutions were further sterilized by spraying them with a 60 mg/mL CA solution. Another 12 pairs of impressions were made with distilled water and underwent no further treatment, while the remaining 12 pairs were sterilized by spraying them with a disinfectant containing 2,000 mg/L of available chlorine. The impressions were then poured with gypsum product (E.50102, HERAEUS, China) after 30 min of disinfection, and the molds were released after two hours. The standard model was first scanned using the SHINING3D dental desktop scanner (AutoScan-DS-EX Pro(H), SHINING 3D, China). Subsequently, the plaster models were scanned in 3D and the digital models were output in standard triangular language (STL) format, with each one being named and saved. Geomagic Quality 13.0 software was used for data analysis, digital model trimming, and preservation of dental morphological data. The digitized superanhydrite models were compared with the standard model using the “best fitting alignment” algorithm based on dental surface and subsequently assessed in a 3D comparison. The root mean square (RMS) was employed as a parameter to measure the deviation between the gypsum model and the standard model, while a 3D chromatogram of the deviation was generated and saved for visual analysis.

A statistical analysis of the obtained results was conducted using GraphPad Prism 9.1.2 software (GraphPad Software, San Diego, CA, USA). A minimum of n ≥ 5 measurements were performed for each group, and the results were presented as mean ± standard deviation. Parametric one-way ANOVA with post hoc Tukey HSD test or nonparametric Kruskal–Wallis with post hoc Dunn’s test was used to assess differences between the disinfection methods and the non-disinfected control. Statistical significance was determined at p < 0.05. For the comparison of colony counts at different time points among the five groups, a repeated measures ANOVA analysis was performed using SPSS software. Mean results for each procedure were compared using pairwise tests, with a post hoc Bonferroni adjustment for multiple comparisons at a confidence level of 95%.