Bacterial adhesion and surface properties of computer-aided design-computer-aided manufacturing restorative materials

Abstract

Purpose: This study aimed to evaluate the surface properties and bacterial adhesion of computer-aided design-computer-aided manufacturing (CAD-CAM) restorative materials.

Methods: Four CAD-CAM resin-based blocks (Vita Enamic, Shofu block HC, Cerasmart [CS] and Lava Ultimate [LU]) and a leucite-reinforced glass ceramic block (IPS Empress CAD) were used in the present study. Specimens prepared with dimensions of 10 × 10 × 1 mm were polished. Surface characteristics were assessed with hydrophobicity and surface free energy (SFE) analysis. Surface roughness was measured using a profilometer, and elemental and topographic evaluations were performed with SEM-EDX analysis. After being kept in artificial saliva for 1 h, Streptococcus mutans (S. mutans) and Streptococcus mitis (S. mitis) were incubated separately in 5% CO₂ atmosphere at 37°C for 24 h. The adhered bacteria were counted as ×10⁸ CFU/mL.

Results: Surface roughness, contact angle and SFE measurement values were found to be in the range of 0.144-0.264 Ra, 28.362°-70.074° and 39.65-63.62 mN/m, respectively. The highest adhered amount of S. mutans was found in CS and the lowest in LU, while there was no significant difference between the amounts of adhered S. mitis.

Conclusion: Despite differences in the surface properties of the materials used for the study, the materials exhibited identical properties with respect to bacterial adhesion.

Introduction

Computer-aided design-computer-aided manufacturing (CAD-CAM) technology is widely used in indirect restorations due to its advantages such as less time spent chairside and ease of application [1,2]. With the development of CAD-CAM technology, the materials used for the production of CAD-CAM restorations are also progressing. Recent advancements in millable materials have expanded the options available for various applications. These materials include wax, poly(methyl methacrylate) (PMMA), composite resins, high-performance polymers, metals, and ceramics; these materials offer enhanced properties and performance, making them suitable for a wide range of uses [3].

Complex biofilms known as human dental plaque are found on both tooth tissues and restoration materials. The oral biofilm has the potential to host several microorganisms that contribute to the progression of various disease conditions, including secondary caries, the demineralization of marginal enamel and dentin, and periodontal disease [4]. Following the establishment of an acquired salivary pellicle, the surfaces in question undergo initial colonization by streptococci [5]. After 24 h, the plaque mostly contains gram-positive rod bacteria such as streptococci and actinomyces [5,6,7]. Caries-associated mutans streptococcus (MS) can also be detected in early plaque [7]. MS is a group of bacteria associated with dental caries, including species such as Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sobrinus), Streptococcus cricetus (S. cricetus), Streptococcus rattus (S. rattus), Streptococcus downei (S. downei), and Streptococcus macacae (S. macacae) [8]. S. mutans and S. sobrinus are the mainly detected species in the human oral cavity. When sucrose is present, MS creates extracellular polysaccharides and uses adhesins to cling to salivary pellicle components [9]. Although successful restorations have been produced with CAD-CAM applications, it is known that the surface properties of the materials are extremely important for the longevity of restorations in the oral environment where microbial density is high. Relationships between the quantity of adherent bacteria, surface roughness, surface free energy and charge, and hydrophobicity of substratum have all been reported in vitro [10].

Although there have been numerous studies on the effect of the material surface, different studies have shown that various restorative materials can have antibacterial activity as well as induce the growth of various bacteria [11]. In general, despite numerous studies on oral biofilm morphology and development [11,12,13]. only limited information is available on bacterial adhesion on the surface of new restorative materials, especially those manufactured for CAD-CAM technology. Numerous studies evaluating the mechanical, physical, and additional properties of CAD-CAM materials with a wide range of materials and proven clinical use have been published in the literature. However, few studies have evaluated the relationship between surface properties of materials and bacterial adhesion. Therefore, the aim of this study was to examine the surface roughness, surface free energy and contact angle of five different CAD-CAM materials and to evaluate the bacterial adhesion that may develop on these materials. The null hypothesis of this study is that there will be no difference in bacterial adhesion to the CAD-CAM materials tested.

Materials and Methods

Specimen preparation

Four resin-based composite CAD-CAM blocks; Vita Enamic (VE) (VITA Zahnfabrik, Bad Säckingen, Germany), Shofu block HC (SB) (Shofu Inc, Kyoto, Japan), Lava Ultimate (LU) (3M ESPE, St. Paul, MN, USA), Cerasmart (CS) (GC Europe, Leuven, Belgium) and one leucite-reinforced glass ceramic block, IPS Empress CAD (IPS) (Ivoclar Vivadent AG, Schaan, Liechtenstein) were included in the present study. The manufacturer and materials information are presented in Table 1. Using a low-speed diamond saw (Isomet Low Speed Saw, Buehler, Lake Bluff, IL, USA), 20 specimens (10 × 10 × 1 mm) were prepared for each product, and then specimens were polished for a total of 240 s using Sof-Lex (3M ESPE Dental Products, St Paul, MN, USA) Al₂O₃ polishing discs; Coarse (100 µm), Medium (29 µm), Fine (14 µm), and Super Fine (8 µm), respectively. After polishing with abrasive, the specimens were polished with felt discs (Super-Snap Buff, Shofu Inc.) and diamond polishing paste (Diamond Polish, Ultradent Products, South Jordan, UT, USA). Then all specimens were ultrasonically cleaned and dried with air.

 

Table 1 The compositions and manufacturers of resin-based CAD-CAM blocks tested in present study

Materials	Codes	Manufacturer	Content			Batch No
			organic phase	inorganic filler
IPS Empress CAD	IPS	Ivoclar Vivadent AG, Schaan, Liechtenstein	SiO2 (60-65%), Al2O3 (16-20%), K2O (10-14.%), Na2O (3.5-6.5%), other oxides (0.5-7%), pigments (0.2-1%)			T15791
Vita Enamic	VE	VITA Zahnfabrik, Bad Säckingen, Germany	UDMA, TEGDMA	glass-ceramic sintered network	86 wt%	38630
Shofu block HC	SB	Shofu Inc, Kyoto, Japan	UDMA, TEGDMA	SiO2, zirconium silicate	61 wt%	111501
Lava Ultimate	LU	3M ESPE, St.Paul, MN, USA	Bis-GMA, UDMA, Bis-EMA, TEGDMA	SiO2 (20 nm), ZrO2 (4-11 nm), ZrO2/SiO2 clusters	79 wt%	N538336
Cerasmart	CS	GC Europe, Leuven, Belgium	Bis-MEPP, UDMA, other DMA	SiO2 (20 nm), barium glass (300 nm)	71 wt%	160411

 

Surface roughness measurement

A tactile profilometer (Surftest SJ 201, Mitutoyo, Tokyo, Japan) with a 0.25-mm cutoff value was used to determine the surface roughness of all specimens. The resolution of the profilometer was 0.01 mm with the transverse length of 4.0 mm and the diamond recording pin stylus diameter of 5 µm. To determine a roughness profile, the constant measuring speed of 0.5 mm/s was used and roughness measurements were performed on three sites of each specimen and the arithmetic average of the data was taken. The data obtained belongs to one specimen. These measurements were performed for all specimens. (n = 10). The measured roughness parameters were recorded as Ra value (arithmetical average value of all absolute distances of the roughness profile).

Contact angle and surface free energy determination

The hydrophobicity and surface free energy (SFE) measurement was evaluated by using an automated contact angle and surface tension measurement device (KVS, Attension Theta, Biolin Scientific UK, Manchester, UK). Five specimens for all groups were prepared as described above. Following that the contact angle and SFE of all tested specimens were evaluated by using distilled water, dimethyl sulfoxide, and ethylene glycol. For contact angle (°) measurements, the specimens were placed on the platform of the devices and droplets of fluids were dropped. The volume of the droplet was measured using One Attension (2.6 version) software (Biolin Scientific, Gothenburg, Sweden) and the software automatically calculated the contact angle on both sides of the droplets. The average of the right and left contact angle values of each droplet represents the contact angle of the sample. A total of 5 contact angle values were calculated for each group. For SFE measurements, the total SFE (γ^tot), dispersive (γ^d), and polar (γ^p) components for each specimen were calculated according to the previous report [14].

Scanning electron microscope (SEM) and energy distribution X-ray spectroscopy (EDX) analysis

The scanning electron microscope (SEM) and energy distribution X-ray spectroscopy (EDX) analysis was performed using Field Emission SEM (FEI QUANTA 400F, FEI Company, Hillsboro, OR, USA). Three specimens for each material were subjected to SEM followed by EDX analysis. After coating the specimens with an Au-Pd layer using the sputter coater device (Polaron, East Sussex, England), represented images at ×2,000 and ×20,000 magnifications were obtained, and elemental analysis of specimens was performed.

Assessment of bacterial adhesion

After surface roughness measurements, specimens were cleaned in an ultrasonic cleaner for 15 min and then sterilized in 122°C for 15 min in an autoclave (Tomy Model SX-700 E Autoclave, Katsushika, Tokyo, Japan) before assessment of bacterial adhesion. To produce a pellicle layer covering all specimens surface, artificial saliva with mucin was prepared according to the previous formulation [15]; 8.4 mg NaF, 2,560 mg NaCl, 332.97 mg CaCl₂, 250 mg MgCl₂ (6H₂O), 189.48 mg KCl, 0.1 mL H₃PO₄ for 2 L of artificial saliva. After the mixing of artificial saliva, the pH was adjusted to 6.7-7.0 by adding 0.1M NaOH into solution and 140 mg of Type II mucin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was added to each 100 mL of artificial saliva. All specimens were incubated in artificial saliva with mucin (5 mL) in a petri dish and left for 1 h at 37°C in a 5% CO₂ atmosphere to produce a pellicle layer. The specimens were washed with 5 mL of saline and transferred to sterilized petri dishes. Ten specimens were divided into two subgroups to evaluate adhesion of S. mutans and S. mitis (n = 5). S. mutans strain (ATCC 25175) and S. mitis strain (obtained from the microbiology laboratory of the institute) were used as test bacteria in this study. The cultivation medium consisted of 5% sheep blood agar (BBL, BD Diagnostic, Sparks, MD, USA). Bacteria grown in cultures were transferred to tubes containing 5 mL brain-heart infusion (BHI) (BBL, BD Diagnostic) and were incubated at 37°C in a 5% CO₂ atmosphere for 24 h. At the end of the incubation, tubes were centrifuged for 5 min, followed by adding 5 mL of phosphate-buffered saline (PBS) into each tube, and centrifuging was repeated for 15 s. Bacterial suspension was prepared as 10⁹ CFU /mL and 200 µL of bacterial suspension was added to the surface of each specimen. After 15 min, BHI with 5% glucose was added to each petri dish to cover all specimens, and dishes were kept in the incubator (CO₂ Water Jacketed Incubator, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in a 5% CO₂ for 24 h. Following 24 h incubation, each specimen was placed into a tube containing 2 mL of PBS and mixed with a centrifuge (MX-S Vortex Mixer, Scilogex LLC, Rocky Hill, CT, USA) for 60 s to separate the free bacteria. Subsequently, 200 µL of BHI broth was added to each well of ninety-six-well microplates, and 20 µL of washing solution from the vortexed samples was added to the microplates. The BHI broth was incubated at 37°C in 5% CO₂ atmosphere for 24 h. At the end of incubation, the optical densities of the microplates were determined at 630 nm in an automatic microplate reader (Readwell Touch Automatic ELISA Plate Analyzer, Robonik Pvt., Ltd., Thane, India). Bacteria counts were indicated as ×10⁸ CFU/mL. Two additional specimens per bacteria were prepared and analyzed using a confocal laser scanning microscope (CLSM). Selected specimens were gently removed from the flow cells, rinsed twice with sterile PBS to remove non-adherent cells, and stained using the Live/Dead BacLight Viability Kit (Invitrogen L-13152, Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions and then observed using a CLSM (Zeiss LSM 510-Meta, Carl Zeiss Microscopy GmbH, Jena, Germany) immediately after the staining procedures.

Statistical analysis

Data were analyzed using statistical software (IBM SPSS Statistics, v20; IBM Corp., Armonk, NY, USA) at P ≦ 0.05. The surface roughness, contact angle values and count of adhering S. mutans and S.mitis showed normal and homogeneous distribution according to the Kolmogorov-Smirnov and Shapiro-Wilk’s test; accordingly, one-way ANOVA and Tukey HSD multiple comparison tests were used to evaluate the differences between the groups. A software program (G*Power 3.1; Universitat Düsseldorf, Düsseldorf, Germany) was used to perform the power analysis.

Results

Surface roughness

Mean surface roughness values and standard deviations are shown in Fig. 1. While the highest Ra values were seen in IPS (0.264 µm), the lowest Ra value was measured in LU (0.144 µm) (P < 0.05). However, there was no statistically significant difference between LU and CS (0.164 µm) and between IPS and VE (0.22 µm). The SB which has 0.197 µm Ra value was not statistically different from VE and CS.

Fig. 1 Mean and standard deviations of surface roughness values (different letters indicate statistical differences between groups) (P < 0.05)

Contact angle and surface free energy

The mean contact angle values and images are presented in Fig. 2. The lowest water contact angle (CAW) values were seen in IPS (28.36). While the highest CAW value was obtained in CS (70.07), there was no statistical difference between CS, SB (67.49) and LU (57.34). The CAW of VE (37.34) was statistically different from all other materials (P < 0.05).

According to the SFE values presented in Fig. 3, IPS had the highest γ^tot value (63.61). This was followed by VE with γ^tot value of 55.80. The lowest γ^tot was also found in CS, but the γ^tot values of LU (41.87) and SB (40.67) were close values to each other. Also, γ^tot occurs in disperse (γ^d) and polar (γ^p) components. However, the effects of these components change according to the material. γ^d data of CS (γ^d 28.49) and SB (γ^d 28.75) provide a greater contribution to γ^tot. This situation was different for IPS (γ^p 56.16), VE (γ^p 46.00) and LU (γ^p 24.38). The effect of polar components was greater in these materials.

Fig. 2 Mean and standard deviations of contact angle values (different letters indicate statistical differences between groups) (P < 0.05)

Fig. 3 Mean of surface free energy values

SEM and EDX

SEM images of all CAD-CAM materials at ×2,000 and ×20,000 magnification are shown in Fig. 4. SEM images allowed good observation of the surface morphology of the specimens. Scratches from polishing were seen especially on IPS, SB and LU. In the polished samples, the IPS with the roughest surface exhibited a more irregular surface with indentations as evidenced in the images. A network structure consisting of macro, micro and nano filler particles was also seen in VE. It also showed a smoother surface in LU and CS images, which had the least roughness. However, on the SB surface, there were distinct hole-like round gaps. Large filler particles are clearly observed in SB and LU.

The differences obtained in the elemental analysis of CAD-CAM materials are presented in Table 2. According to elemental analysis, there were important differences in the contents of CAD-CAM materials. Silicon (Si) was available in all material groups. IPS and VE contained aluminum (Al), sodium (Na) and potassium (K). Barium (Ba) was only found in CS. However, attention was drawn to the fact that LU (19.6) contained high levels of zirconium (Zr).

The organic resin matrix content was represented indirectly by carbon measurement (ref). IPS (7.7) had the lowest C content and there was a distinct difference between other materials. The C content of other materials, respectively, was CS (26.6), LU (27.9), VE (30.0), and SB had the highest value (34.6).

Fig. 4 Scanning electron microscopy (SEM) images of CAD-CAM blocks at magnifications of ×2,000 and ×20,000

Table 2 Mean composition (wt%) of the tested CAD-CAM blocks by EDX

	C	O	Al	Si	Ba	Na	K	Zr
IPS	7.7	33.6	10.0	33.7	-	3.5	11.5	-
VE	30.0	29.8	8.7	21.8	-	4.2	4.4	1.2
SB	34.6	31.7	-	33.7	-	-	-	-
LU	27.9	27.1	-	25.4	-	-	-	19.6
CS	26.6	30.8	-	36.2	6.3	-	-	-

 

Bacterial adhesion

The CFU values and standard deviations of S. mutans and S. mitis on the surface of CAD-CAM materials are shown in Fig. 5. There was a statistically significant difference between the LU with the lowest S. mutans CFU value and the CS with the highest CFU value (P < 0.05). However, there was no statistical difference between the other groups. There was no statistically significant difference between the S. mitis CFU values of all CAD-CAM groups.

In Fig. 6, CLSM images of the CAD-CAM materials can be seen. The red color represents dead bacteria and the green color also represents living bacteria. Living bacteria are more intensely observed in all material groups. Parallel to the CFU data, S. mitis adhesion was seen more intensely in all materials than in S. mutans.

Fig. 5 Mean and standard deviations of S.mutans and S.mitis CFU/mL values (different letters indicate statistical differences between groups [P < 0.05])

Fig. 6 The CLSM images of S.mutans and S.mitis adhesions. Viable bacterial cells are shown in green, whereas dead cells are shown in red.

Discussion

The deposition of biofilm, also known as bacterial plaque, on the surface of restorative materials has been found to promote the development of secondary caries and periodontal inflammation [16]. This phenomenon is a significant factor that influences the durability and lifespan of restorations. It is important to have restorative materials with a low sensitivity to bacterial adherence on their surfaces. It has been demonstrated that differences in microbial adhesion between different materials are related to their chemical composition and surface properties [17]. The adherence of microorganisms to oral surfaces is significantly influenced by the roughness of the substratum surface and its surface free energy [18]. In particular, substrates characterized by a high SFE, indicating a hydrophilic surface, tend to have a greater amount of biofilm formation compared to substrates with a low SFE, indicating a hydrophobic surface. Both SFE and roughness have an impact on microbial adhesion and biofilm formation [19]. However, the influence of roughness appears to be more significant in terms of the accumulation and composition of biofilm [15]. On the other hand, the effect of SFE becomes more pronounced when comparing surfaces that have a comparable level of roughness [20]. A significant amount of research indicates that, in vivo, smooth surfaces exhibit a lower propensity for biofilm formation compared to rough surfaces [21]. According to a series of split-mouth examinations, it can be concluded that the presence of a surface roughness above a threshold of 0.2 µm and/or an elevation in surface free-energy promotes the development of biofilm on restorative materials. According to Teughels et al. [22], the determining factor in the interaction between surface attributes is surface roughness. The surface roughness, contact angle and surface free energy of the CAD-CAM materials used in the present study were compared and the results showed that there was a difference between used materials, and thus, the null hypothesis was rejected. In a literature review, the relationship between the initial surface roughness of intraoral hard materials and plaque accumulation was examined and it was suggested that the threshold surface roughness for in vivo bacterial retention is a Ra above 0.2 µm [18]. In the present study, the Ra of specimens were far below the threshold level of 0.2 µm except for IPS and VE. However, in the present study, no differences in bacterial adhesion between the materials were detected in the analyses that could be associated with surface roughness. When analyzing the results of bacterial adhesion examinations, it is important to highlight that the attachment of S. mitis is not influenced by variations in surface roughness. The group classified as LU had the lowest surface roughness and the lowest S. mutans CFU value. Nevertheless, despite similar roughness levels between the LU and CS groups, it was noted that the adherence of S. mutans was more prominent in the CS group. The adherence of S. mutans has been demonstrated to be influenced only by the surface roughness of LU.

Many studies have reported that the exact effects of surface roughness on how bacteria adhere to and make biofilms depending on the type and number of bacteria cells as well as other factors in the environment [17,23]. A study conducted by Lin et al. [24] found that altering the roughness of ceramic surfaces within the range of 0.2 to 2 µm did not provide any significant impact on the production of biofilm by S. mutans. Possible reasons for the various results are the types of materials used, the degree of roughness, the bacteria used, and the conditions under which they were grown. It is noteworthy to mention that the entirety of the oral surfaces is covered by a salivary pellicle, which may reach a thickness of up to 1,000 nm [25]. This pellicle has the potential to modify nanotopography [26], thus exerting a significant influence on surface roughness. Moreover, the pellicle contains bacterially produced enzymes that could synthesize exopolysaccharides within their immediate environment in the presence of sucrose [27]. The alteration of topography can be induced by glucans that are generated within the local environment, concurrently offering binding sites for pathogenic organisms like S. mutans [23], Therefore, in order to regulate the formation of biofilms, the roughness of materials must either counteract the impact of saliva or indirectly modify bacterial adherence by changing the characteristics, quantity, and/or structure of adsorbed saliva/microbial proteins. Reducing or increasing the hydrophobicity of a surface can generally result in the promotion or inhibition of bacterial adhesion. Studies found supragingival biofilms developed less on hydrophobic surfaces than hydrophilic ones [10,28]. The correlation between the adhesion of bacterial species and surface hydrophobicity, however, varies. The potential impact of salivary coating on the interaction between dental materials and oral bacteria is significantly influenced by the hydrophobicity of the bacteria and the surfaces of the teeth. In the present study, it was shown that there was no significant correlation between surface hydrophobicity and bacterial adhesion.

Based on previous studies, it has been reported that the average SFE values for S. mutans, S. sanguinis, and C. albicans are 48.4, 47.7, and 40.1 mN/m, respectively [29]. It is important to note that these values demonstrate variation between different strains. Minagi et al. [30] have reported that there appears to be a positive correlation between the SFE of a material and its probability of adherence by microorganisms. Gram-negative bacterial cells predominantly have higher SFE (35 to 65 mN/m), while some gram-positive bacterial cells have high (35 to 65 mN/m) or low (0 to 25 mN/m) values [29]. The available evidence indicates that the occurrence of polysaccharides on the cellular surface of gram-positive bacteria, specifically S. mutans and S. sanguinis, has a tendency to enhance the hydrophilicity of the bacterial cell. According to the results of the present study, S. mutans adhered more to CS with higher SFE, but no correlation between the materials was found to show this difference. On the other hand, it has also been reported that with the increase of the hydrophobic property of the surface, stronger bacterial adhesion is achieved by removing the water between the water-soluble bacteria and the surface [31,32]. Despite having the lowest surface roughness and SFE, the higher S. mutans CFU value in the CS group can be explained by the fact that S. mutans is hydrophobic [33]. It has been reported that the surface is considered hydrophobic when the water contact angle value is greater than 65° [34]. As a result of electrostatic forces between the surface and bacteria, S. mutans tend to bind more to hydrophobic surfaces [19,35]. In this study, only the IPS and VE groups had total free surface energies (γ^tot) greater than 50 mN/m and the Ra values of these two materials were higher than the clinical threshold value (0.2 µm) even after standard surface polishing. These results suggest that SFE and surface roughness may be interrelated, but the differences in SFE and Ra values do not affect bacterial adhesion when compared to other materials.

In the present study, EDX analysis was performed together with SEM images to test the hypothesis whether there is a relationship between biofilm formation and material content. SEM images of IPS, a leucite-reinforced glass-ceramic material, show irregularity of the surface, and Si, Na, K and Al were detected in the EDX analysis. In the SEM findings of VE, the surface has an irregular structure like IPS, and Si, Na, K and Al elements detected in ceramic materials were also observed in the EDX analysis. The surface roughness values of these two materials are similar to each other, and the SEM images and EDX findings are also similar. CFU values of S. mutans bacteria were also found similar in VE and IPS groups. The CFU values of S. mutans were also found to be similar in the findings of this study. The highest biofilm formation was observed only in the CS group with high Si content and Ba element in its structure. The lowest biofilm formation was observed in the LU group with the highest Zr element. In the CLSM images, it is seen that S. mutans exhibited the lowest intensity painted areas in the LU group (Fig. 6). In an in vivo study evaluating biofilm formation in various dental ceramic materials, low bacterial adhesion was observed, especially in zirconia [36]. They evaluated zirconia as a promising material due to its low biofilm formation.

Another issue that should be emphasized is that all surfaces in the oral cavity that come into contact with saliva are covered with pellicle [37]. Pellicle formation can mask the physico-chemical surface properties of dental materials. This may affect surface roughness and bacterial adhesion [38]. In this study, membrane formation was achieved with mucin-containing artificial saliva to mimic intraoral conditions.

Adhesion of the salivary pellicle layer to the tooth surface is the first step for oral bacterial colonization. Oral bacteria adhere to host-derived receptors in the salivary pellicle. In the present study, tested materials were coated with artificial saliva and mucin to simulate the oral environment. The in vitro model adopted in this study evaluated the adhesion of S. mutans and S. mitis at neutral pH and under laboratory conditions; and the presence of essential salivary proteins such as lysozyme, agglutinin, and mucins, which have been found to be involved in bacterial attachment to surfaces in the oral environment in vivo, were not considered. The presence of acquired pellicles in oral conditions containing host- and bacterially derived proteins, which are essential for the development of bacterial adhesion and biofilm formation, was simulated in vitro in this study. However, besides the surface properties of the materials, other factors such as personal dietary intake and the complex oral microbiome also influence biofilm formation. Therefore, new materials for dental applications need to be evaluated under conditions that mimic the oral cavity for bacterial adhesion, and further studies will disclose how changes in the surface properties of materials affect pellicle formation and composition, and how the pellicle modifies the surface properties of different dental materials. More importantly, how the interaction between surface properties and pellicle formation affects the bacterial adhesion strength as well as the mechanical stability and detachment of biofilms needs further elucidation. A limitation of this in vitro study is that only Streptococcus bacterial species were used. Therefore, the study conditions do not fully reflect the range of oral microbial flora. The surface properties of the materials and bacterial adhesion were quantitatively evaluated. In addition, morphological examinations by confocal laser scanning microscopy showed bacteria adhering to the specimen surface but did not reflect bacterial biofilm. Therefore, further studies are needed to clarify the qualitative differences in the surface properties of materials and bacterial adhesion. Further in vitro and perhaps in vivo studies are needed to evaluate the effects of newly developed materials on biofilm development, subsequent oral bacterial dental plaque formation and survival of plaque bacteria.

Within the limitations of the present study, the following results were found:

1. LU, which had the lowest surface roughness, showed the least S. mutans adhesion and S. mutans adhesion of only LU was affected by surface roughness.

2. S. mitis adhesion was not affected by materials and their surface properties.

3. In general, there was a correlation between the surface roughness values and the SFE of the CAD-CAM materials.

Abbreviations

Al₂O₃: aluminum oxide; Bis-EMA: ethoxylated bisphenol A dimethacrylate; Bis-GMA: bisphenol A diglycidylmethacrylate; Bis-MEPP: bisphenole A ethoxylate dimethacrylate; CAD-CAM: computer-aided design and computer-aided manufacturing; CFU: colony-forming unit; CLSM: confocal laser scanning microscope; DMA: dimethacrylate; K₂O: Potassium oxide; Na₂O: sodium oxide; PBS: phosphate-buffered saline; PMMA: poly(methyl methacrylate); SEM-EDX: scanning electron microscopy / energy distribution X-ray spectroscopy; SFE: surface free energy; SiO₂: silica, TEGDMA: triethylene glycol dimethacrylate; UDMA: urethane dimethacrylate; ZrO₂: zirconia

Ethics Statements

Not applicable

Conflicts of Interest

Çağatay Barutçugil declares that he has no conflict of interest. Deniz Tayfun declares that he has no conflict of interest. Nurgül Çetin Tuncer declares that she has no conflict of interest. Ayşe Dündar declares that he has no conflict of interest.

Funding

This study was supported by the Scientific Research Projects Management Unit from Akdeniz University with the project number TDH-2017-3003.

Author Contributions

ÇB: conceptualization, resources, formal analysis, writing, review, editing, supervision; DT: methodology, investigation; NÇT: original draft writing, editing; AD: writing, review, editing, supervision

ORCID iD

¹⁾ÇB: [e-mail], https://orcid.org/0000-0002-5321-2299

²⁾DT: [e-mail], https://orcid.org/0000-0001-7702-2943

¹⁾NÇT: [e-mail], https://orcid.org/0000-0002-9318-9441

¹⁾AD*: [e-mail], https://orcid.org/0000-0001-6373-6267

Data Availability Statements

All authors gave approval for the data in this study to be available.

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