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Article

Cytotoxicity and Microbiological Properties of Ceramic CAD/CAM Materials Subjected to Surface Treatment with Nanometric Copper Layer

by
Aleksandra Piszko
1,*,
Wojciech Grzebieluch
2,
Paweł J. Piszko
1,3,
Agnieszka Rusak
4,
Magdalena Pajączkowska
5,
Joanna Nowicka
5,
Magdalena Kobielarz
6,
Marcin Mikulewicz
7 and
Maciej Dobrzyński
1,*
1
Department of Pediatric Dentistry and Preclinical Dentistry, Wroclaw Medical University, Krakowska 26, 50-425 Wroclaw, Poland
2
Laboratory for Digital Dentistry, Department of Conservative Dentistry with Endodontics, Wroclaw Medical University, Krakowska 26, 50-425 Wroclaw, Poland
3
Department of Polymer Engineering and Technology, Faculty of Chemistry, Wroclaw University of Science and Technology (WUST), Wyb. Wyspiańskiego 27, 50-370 Wroclaw, Poland
4
Division of Histology and Embryology, Department of Human Morphology and Embryology, Wroclaw Medical University, T. Chalubinskiego 6a St., 50-368 Wrocław, Poland
5
Department of Microbiology, Faculty of Medicine, Wroclaw Medical University, T. Chalubinskiego 4, 50-368 Wroclaw, Poland
6
Department of Mechanics, Material and Biomedical Engineering, Faculty of Mechanical Engineering, Wroclaw University of Science and Technology (WUST), Wyb. Wyspiańskiego 27, 50-370 Wroclaw, Poland
7
Department of Facial Developmental Defects, Wroclaw Medical University, Krakowska 26, 50-425 Wroclaw, Poland
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2024, 14(20), 9224; https://doi.org/10.3390/app14209224
Submission received: 7 August 2024 / Revised: 24 September 2024 / Accepted: 4 October 2024 / Published: 11 October 2024
(This article belongs to the Section Applied Dentistry and Oral Sciences)
Browse Figures
Versions Notes

Abstract

:
The aim of this study is to present the characteristics and a comparison of four different commercial materials dedicated to the CAD/CAM technique in dentistry, all of which can be classified as ceramic materials. Its purpose is also to evaluate the impact of surface treatment on the cytotoxicity and microbiological properties of the materials. The CAD/CAM technique has a perpetually growing role in modern reconstructive dentistry. It requires a material’s possession of peculiar characteristics, such as mechanical resistance, durability, functionality (similar to natural tissues), good aesthetics and biocompatibility. To critically evaluate a biomaterial, both manufacturer claims and in vitro tests should be considered. Further steps of evaluation may include animal tests and clinical trials. There are certain attributes of biomaterials that may be modified by surface treatment that can be crucial to the clinical success of the material. The evaluated materials were Vita Suprinity (VITA-Zahnfabrik, Germany), Vita Mark II (VITA-Zahnfabrik, Germany), Celtra Duo (Dentsply Sirona, USA) and Empress Cad (Ivoclar Vivadent, Liechtenstein). They are available in the form of prefabricated blocks of various diameters and are popular among operators performing clinical procedures using CAD/CAM. Standardized blocks of each material were prepared. Half of them had their surface polished. Further, half of all the samples were covered by a nano-copper layer. The samples were evaluated for cytotoxicity, presented on a 0–4 scale, adhesion susceptibility and potential of forming a biofilm on their surface. Physicochemical properties such as the water contact angle (WCA) were evaluated for the tested materials. The influence of copper coating on cytotoxicity cannot be unequivocally stated or denied. Surface polishing did not affect the materials’ cytotoxicity, but it increased the WCA of all materials and, therefore, their hydrophobicity. Different degrees of adhesion ability and biofilm formation were dependent on the species of microorganisms and properties of the dental materials.

1. Introduction

CAD/CAM, which stands for computer-aided design and computer-aided manufacturing is a technique gaining significant popularity in many fields, among which is modern reconstructive dentistry. On the market, there is a wide variety of materials, sold as prefabricated blocks, that may be milled and used in the temporary or definitive reconstruction of hard tissues in patients’ oral cavities. In general, materials for CAD/CAM can be divided into major groups, such as metal, polymer, ceramic, or composite materials [1]. Materials are constantly evaluated and enhanced to meet the high demands of modern dentistry. Among the favourable characteristics of dental materials for indirect restorations, we may list mechanical resistance, durability and functionality similar to natural tissues, as well as good aesthetics and biocompatibility [2,3]. To critically evaluate a biomaterial, many aspects should be appraised considering the material’s purpose. Its marketing claims must be considered, and in vitro and animal tests should be performed [4]. In our paper, we focused on several crucial aspects of the in vitro evaluation of a new material. One of them is biocompatibility, defined as “the ability of a material to elicit an appropriate biological response in a given application” [5]. This is especially important when the material remains in contact with patients’ tissues for a long time, such as in reconstructive dentistry [4]. Materials whose purpose involves contact with human cells should be validated according to standards such as EN ISO 10993-5 (Biological evaluation of medical devices—Part 5: Tests for in vitro cytotoxicity) [6]. Cytotoxicity was evaluated considering the cell morphological changes described in this standard, according to which a material can be regarded as cytotoxic when it presents at a second or higher grade in the set scale. CAD/CAM restorations frequently come into contact not only with hard tissues of the tooth such as the enamel or dentin but also with soft gingival connective tissue, in which its major population is fibroblasts [7]. Testing the adhesion of fibroblast cells to a material allows a prediction of its potential interaction with a tissue.
Dental caries is regarded as a complex transmissible disease affecting biofilm development [8]. The risk for caries includes many factors—physical, biological, environmental, behavioural and lifestyle-related—among which we can distinguish levels of colonization of the oral cavity by cariogenic bacteria [9]. In the human oral microbiota, over 700 different species of bacteria coexist [10]. Both natural tissues and restorative and prosthetic materials in the oral cavity are covered by an acquired pellicle followed by bacterial colonization. Bacteria tend to bind to the pellicle in the course of the first 4 h after cleaning the teeth. Both Gram-positive (Streptococcus mutans, S. salivarius, S. motis and Lactobacillus spp.) and Gram-negative (Actinobacillus spp., Campylobacter spp., Fusobacterium nucleatum and Porphyromonas gingivalis) bacteria colonize the oral cavity [11]. The multispecies biofilm of the oral cavity is the main factor in the development of tooth decay, periodontitis, peri-implantitis and stomatitis [12,13,14,15,16]. Due to the formation of biofilms on biomaterials, especially in marginal gaps, secondary caries may develop [14,17]. Restorative material are desired to be precise and stable in their form to reduce the risk of microleakage or of aforementioned marginal gaps and secondary caries as a result [18]. Modern restorative materials should play a role in the limitation of the level of attachment of microorganisms inside the oral cavity. A number of studies have noticed that the colonization of materials by their biofilm depends on different factors, such as free energy or hydrophilic/hydrophobic properties [19,20]. To evaluate the hydrophilic/hydrophobic character of the tested materials, the water contact angle was measured and taken into interpretation.
Materials dedicated for direct or indirect restorations of tooth tissues that show potential in cariogenic bacteria reduction are still searched for. There are studies that show the antimicrobial potential of nanomaterials based on metals. Metals or their oxides may interfere with bacterial metabolism and prevent biofilm formation [21,22,23,24]. While there are many studies showing the antimicrobial potential of copper addition to dental materials, there are not many showing its cytotoxicity [25,26]. Copper has an impact on bacterial metabolism by disrupting their nucleic acids and key enzymes. Together with silver ions, which are more expensive, copper gained popularity in caries prevention in recent years [22]. To evaluate the influence of copper on the surface of CAD/CAM materials on biofilm growth, half of the samples were covered by 8 nm of a chemically pure and inert homogenous copper layer.

2. Materials and Methods

2.1. Material and Sample Preparation

The juxtaposition of the evaluated materials is presented in Table 1. Vita Suprinity (VS) is a lithium silicate glass-ceramic reinforced with zirconium oxide (8–12 wt%), which makes it resistant to loads according to the manufacturer. Vita Zahnfabric declares this material to be long-lasting in the restoration of patients’ oral cavities and characterized by good aesthetics, as well as being biocompatible according to ISO standards [27,28]. Vita Mark II (VM2) is a feldspar ceramic that the manufacturer declares to have high aesthetic value, and to be resistant and durable over a long period of time. As the perk of the material, an abrasion behaviour very similar to natural enamel may be listed. The specific cytotoxicity or any biocompatibility tests regarding this material provided by the manufacturer could not be found [29]. Celtra Duo (CD) is a zirconia-reinforced lithium silicate ceramic material. It is declared to be a biocompatible ceramic with translucent properties. It is also claimed to have a high aesthetic value and to have the properties of a high-strength glass-ceramic [30]. Empress Cad (EC) is a leucite-based glass-ceramic with elasticity at the level of 62 and other desired physical properties declared by its manufacturer [1,31].
Blocks of the CAD/CAM materials were cut into 4 × 4 × 2 mm samples using a low-speed, water-cooled Miracut 151 (Metcon, Bursa, Turkey) diamond saw. Subsequently, half of the samples intended for cytotoxicity and adhesion tests were polished on one side (wall 4 × 4 mm). For this purpose, a Strong 204 straight handpiece was used (Strongdril, Fuzhou, China) (Figure 1A,B) and a 2-step polishing procedure was performed. SoftCut polishers (SHOFU INC., Kyoto, Japan) were used in the 1st step, and CeraMaster (SHOFU INC., Kyoto, Japan) polishers were used in the 2nd step (Figure 2B). During the polishing process, each sample was placed into a 3D-printed holder (Figure 2A). All samples were polished by the same operator using equal conditions. All the samples were degreased by isopropyl alcohol. Half of the samples, both polished (P-) and non-polished (NP-), were covered by a nanolayer of copper. All the samples were sterilized in a steam autoclave at 134 °C and 202.6 kPa.

2.2. Copper Coating

Half of the P- material specimens and half of the NP- ones were sputtered with cooper in a vacuum using a Leica EM ACE200 coating system. The copper target (70-CU5410) was used during two-stage coating processes, i.e., a 4 nm layer was created in directional mode, and the next 4 nm layer was created in diffuse mode. Finally, all samples were covered by an 8 nm chemically pure and inert homogenous Cu layer. The thickness of the Cu layer was verified using scanning electron microscopy on Phenom ProX (Thermo Fisher Scientific, Eindhoven, The Netherlands). The average thickness of the Cu layer was 7.93 + 0.47 nm, independent of the type of coated material.

2.3. Cytotoxicity and Adhesion

2.3.1. Cell Lines

The cytotoxicity test was performed on a line of Balb/3T3 healthy mouse fibroblasts (ATCC, American Type Culture Collection ATCC, Old Town Manassas, VA, USA)—the models were used for the in vitro evaluation of the biomaterials [36,37,38]. The Balb/3T3 cells were cultured in DMEM with the addition of 4.5 g/L glucose and 25 mM of HEPES (Lonza, Basel, Switzerland), as well as an addition of 1% L-glutamine with streptomycin and penicillin present in the mixture (Sigma-Aldrich, St. Louis, MO, USA) and 10% fetal bovine serum (FBS, Sigma-Aldrich). The cellular adhesion test of the evaluated materials was performed on NHDF healthy human dermal fibroblasts (Lonza, Basel, Switzerland). The NHDF cells were cultured in a medium with supplements from an FGMTM bullet kit (Lonza). Cell culture was conducted in standard conditions (37 °C, 5% CO2 and constant air humidity) in a HERA cell 150i incubator (Thermo Scientific, Waltham, MA, USA).

2.3.2. Cytotoxicity Testing—Direct Contact

Balb/3T3 cells were trypsinized using 0.25% Trypsine–EDTA (Sigma-Aldrich), resuspended in culture medium and plated in a plate with 6 wells (TPP, Trasadingen, Switzerland) at 1.5 × 105 cells per well. After 24 h, the tested material was placed on the wells. After 24 h of incubation with the material, the cell morphology was assessed under the disc, in its vicinity and in the rest of the well (CKX53 inverted phase-contrast microscope, Olympus, Tokyo, Japan). The control in the research was a culture carried out in a full medium and in standard conditions that had no contact with the tested material. The cytotoxicity of the analyzed materials was assessed according to the criteria describing changes in cell morphology presented in the EN ISO 10993-5 standard (Biological evaluation of medical devices—Part 5: Tests for in vitro cytotoxicity) [36,37,38]. The degree of cytotoxicity and changes in the cells’ morphology were evaluated on a scale of 0–4 [6] as follows: 0—no toxicity, when no changes are visible under or near the tested material; 1—weak degree of reactivity, when individual cells have degenerated under the sample; 2—moderate degree of reactivity, when the zone of changed cells is limited to the surface under the sample; 3—medium degree of reactivity, when the zone of changed cells is limited to 1 cm around the sample; 4—strong degree of reactivity, where the zone of changed cells exceeds the 1 cm limit around the sample. According to the above standard, a cytotoxic material has a grade of at least 2 [6].

2.3.3. Cellular Adhesion to the Surface of the Material

NHDF cells were trypsinized using the TrypLE™ Express Enzyme (GibcoTM Thermo Fisher Scientific, Waltham, MA USA), suspended in culture medium and plated in a 6-well plate (TPP, Trasadingen, Switzerland) at 6.0 × 104 cells/well. Prior to commencing the culture, the evaluated material was placed in each well. The cells were then cultured for 72 h and 5 days in a gentle swinging motion, which enabled the assessment of the adhesive properties of the materials on the NHDF cells. A laboratory pendulum stirrer set to “gentle motion” mode was used to obtain the gentle swinging mode of operation. After the incubation of the cells with the material, cell adhesion was assessed using an Eclipse80i fluorescence microscope (Nikon Corporation, Tokyo, Japan) and a DAPI mixture with a concentration of 0.1 µg/mL (Thermo Fisher, Waltham, MA USA) and propidium iodide (0.5 mg/mL) (Roche, Mannheim, Germany). Cell adhesion to the polished and unpolished surfaces of the material was assessed in accordance with a previously established protocol [36].

2.4. Adhesion Abilities of Strains

The following reference strains were utilized in the performed experiments: Streptococcus mutans (ATCC 25175), Lactobacillus rhamnosus (ATCC 9595), Candida albicans (ATCC 90028) and Candida albicans (ATCC 10231).
Suspensions of microorganisms were prepared from fresh cultures of the analyzed strains with a density of 0.5 on the McFarland scale (1.5 × 106 CFU/mL) for fungi and 1.0 (3 × 108 CFU/mL) for bacteria. To obtain suspensions of the strains, 2 mL of liquid Sabouraud Dextrose Broth (Biomaxima, Lublin, Poland) medium with the addition of 5% sucrose; 2 mL of liquid medium Brain Heart Infusion Broth—BHI—(Biomaxima, Lublin, Polska) with the addition of 5% sucrose; and 2 mL medium De Man–Rogosa–Sharpe Broth—MRS—with the addition of 5% sucrose (Biomaxima, Lublin, Poland) were used, respectively, for C. albicans, S. mutans and L. rhamnosus. Sterile materials were introduced into the prepared suspensions of microorganisms. After the incubation period (S. mutans—37 °C, 48 h in 5% CO2; L. rhamnosus—37 °C, 48 h in anaerobic conditions; C. albicans—37 °C, 48 h in aerobic conditions), the materials were rinsed 3 times in 2 mL of NaCl and shaken in 1 mL of 0.5% saponin solution (Sigma-Aldrich, Poznan, Poland) for 1 min. The resulting suspension of microorganisms, desorbed from the surface of the material, was inoculated quantitatively on solid substrates appropriate for the given microorganism (BHI Agar, MRS Agar, Sabouraud Agar—Biomaxima, Lublin, Poland). After the incubation period (S. mutans—BHI agar, 37 °C, 48 h, in 5% CO2; L. rhamnosus—MRS agar, 37 °C, 48 h, in anaerobic conditions; C. albicans—Sabouraud agar, 37 °C, 48 h, in aerobic conditions), the grown colonies were counted and the number of colony-forming units per 1 millilitre of suspension was assessed (CFU/mL). The CFU/mL value was counted according to Equation (1):
CFU/mL = average number of colonies × reciprocal dilution × 10
The study was carried out in 2 repetitions. One material was used for one replicate for each strain species.

2.5. Water Contact Angle

WCA measurement was performed utilizing a PG-X contact angle goniometer (Testing Machines, Inc., New Castle, DE, USA). Each specimen was measured 5 times. The mean value of the contact angle was calculated, as well the standard deviation as the error.

3. Results

3.1. Direct Contact—Cytotoxicity Evaluation

For the NP-EC material, the cells after contact with the tested material showed a correct morphology, and no differences were observed in relation to the control culture (cells without contact with the material) at the edge of the sample or further away from the sample. Slight changes in Balb/3T3 cell morphology were observed under the test material (Figure 3). The tests showed a weak cytotoxicity of the material (first degree on the cytotoxicity scale). The conducted tests showed a lack of cytotoxicity of the evaluated material because according to the PN-EN ISO 10993-5 standard [6], the cytotoxic material must have at least a second degree in the assessment. The results for the other materials are included in Table 2 and imaging for the remaining materials is presented in the Supplementary Material (Figures S1–S11).

3.2. Adhesion of Cells to the Surface of the Material

The obtained results show that among tested materials, none have a weak adhesion affinity for human NHDFs (Figure 4 and Figure 5).

3.3. Adhesion Abilities of Strains

The results of the performed microbiological assessment of the polished CAD/CAM materials with and without copper coating are presented in Table 3 and graphically in Figure 6. In total, 28 and 31 strains of C. albicans adhered to all the material surfaces, whereas no adhesion was observed for VM2, VM2 + Cu or EC + Cu during testing with S. mutans or for VS and EC against L. rhamnosus.

3.4. Water Contact Angle

The hydrophilic/hydrophobic character of the evaluated CAD/CAM dental materials was assessed by the determination of the contact angle between their surface and water droplets (Figure 7). Commonly, when the angle is over 90°, the material is considered hydrophobic, and if the value is below 90°, it is regarded as hydrophilic [39]. In all measured specimens, both before and after surface treatment, the WCA was below 90°. The WCA was utilized to assess the effect of polishing on the surface properties of the materials.
Before polishing, the evaluated lithium silicate ceramic (VS and CD) exhibited lower contact angle values (58.6° and 43.2°, respectively) than the Feldspar leucite-reinforced glass-ceramic (EC, 82.8°). Polishing reduced the WCA in all samples’ contact angles, specifically, VS by 36.2% to 37.4°; VM2 by 41.8% to 42.1°; CD by 23.0% to 33.2°; and EC by 56.8% to 35.8°. Images of the representative water droplet angle for all measured samples are presented in Figure S12 in the Supplementary Material.

4. Discussion

The CAD/CAM technique has a growing role in reconstructive dentistry. A wide range of constantly evolving materials is used to obtain desired properties for restorations [40]. These include good aesthetics, biocompatibility, marginal adaptation and sufficient mechanical resistance. This method is gaining popularity among dentists around the world; however, not many studies on surface treatment can be found. Surface modifications can be either physical—changing topography or morphology— or chemical, such as by layer deposition. The aim of surface modification is to create a specific chemical and physical environment that offers a favourable cellular response in the surrounding tissues [41].
The study by Siddanna et al. demonstrated statistically significant differences in the surface roughness of both composite and ceramic materials for CAD/CAM technology implicated by surface polishing [42]. A series of experiments were conducted by researchers to assess the efficacy of different polishing systems. These included spiral polishers (Diacomp FeatherLite/Brasseler), rubber cup polishers (Enhance/Dentsply Caulk) and brush–paste polishers (Diashine/VH Technologies). Surface roughness in the form of arithmetic mean height (Sa) and squared mean height (Sq) was evaluated using a confocal laser microscope. The authors came to the conclusion that the finished surfaces exhibited a markedly reduced level of roughness in comparison to the milled surfaces for all materials. Generally, the brush–paste polishing technique and the use of spiral polishers yielded the lowest surface roughness values for their evaluated CAD/CAM materials.
The application of different processes and surface treatments has been found to have a significant impact on the maximum contact stresses of the lithium disilicate glass-ceramic CAD/CAM material [43]. The findings indicate that surfaces obtained by CAD/CAM milling, polishing and sintering exhibited increased contact stresses and the least fatigue damage, thereby demonstrating superior fatigue performance compared to the other processed ceramic surfaces. The authors emphasize the impact of surface treatment on the subsequent mechanical damage to the material.
Biological response may be influenced by surface characteristics, including topography and chemistry. In this study, we can observe higher WCA values for polished materials. However, the precise degree of hydrophilicity that optimizes biological and clinical outcomes remains uncertain. The review by Gittens et al. indicates that recent studies suggest that increased surface hydrophilicity may facilitate the integration of hard and soft tissue with dental implants, potentially accelerating healing and promoting early osseointegration [44]. The values of the physicochemical parameters of modern biomaterials can be compared to those observed in natural tissues. The mean WCA value for human enamel is dependent upon the presence of an acquired pellicle, with values ranging from 47.2 to 60.2 [45].
Possible complications after the implantation of biomaterials, dental implants or restorative materials include the risk of developing infections. The implanted biomaterial is susceptible to microbial colonization, which may lead to the formation of biofilm structures. Biofilms can lead to tissue destruction and failure of the therapeutic process, as well as carrying the risk of systemic infection [46]. Oral microorganisms have strong adhesive abilities. That is why it is so important to create materials or alter them to make them less susceptible to the adhesion of microorganisms and biofilm formation [47]. This study assessed the adhesion and biofilm formation abilities of S. mutans, L. rhamnosus and C. albicans. To assess the adhesive ability, a qualitative method (calculation of the CFU/mL value) was used. The highest CFU/mL values—3.9 × 103 and 4.15 × 103—were denoted for C. albicans ATCC 90028, respectively, for VM2 and EC. Higher CFU/mL values were observed for fungi, and lower values for bacteria. The surface that was most susceptible to the adhesion of L. rhamnosus and both strains of C. albicans was VM2 + Cu. S. mutans did not adhere to VM2, VM2 + Cu or EC + Cu—VM2 was denoted with the highest and EC with the second highest WCA values. L. rhamnosus did not adhere to VS and EC.
In their evaluation of CAD/CAM materials, Dobrzyński et al. [11] observed that non-polished surfaces exhibited a greater susceptibility to the adhesion of C. albicans ATCC 90028 than polished ones. In accordance with this cited article, the presented study demonstrated that the C. albicans ATCC 90028 strain exhibited a strong adhesion to non-polished surfaces. Furthermore, Özarslan et al. [48] investigated the impact of polishing methods on surface properties. With the same conclusion as the presented article, the referenced study concluded that the adhesion of C. albicans ATCC 90028 to rough (non-polished) surfaces is higher than to polished ones. Vulović et al. evaluated the adhesion of Streptococcus oralis and C. albicans to CAD/CAM dental material surfaces. The authors concluded that the higher the roughness and contact angle are, the more favourable the adhesion to the material surface for both the analyzed microbial strains [49]. During the evaluation of strains F. nucleatum, C. albicans, S. oralis, V. parvula and P. gingivalis in terms of their adhesion to rough surfaces, the same authors [50] assessed the contact angle. Their study concluded that materials exhibiting higher contact angle values and greater hydrophobic properties were likely to exhibit increased microbial adhesion. VM2 before modification with Cu possessed the highest contact angle. Both the strains of C. albicans and L. rhamnosus exhibited the highest adhesion among all the evaluated materials, which is in line with the referred study. However, no adhesion in S. mutans was observed.
Nanoparticles of copper find an application in dentistry as a microorganism-inhibiting component [51]. It has been established that copper is capable of damaging microbial cells thanks to its ability to generate reactive oxygen species and to replace or bind with native cofactors in metalloproteins [52]. Moreover, copper may destroy cell walls and cell membranes and react with proteins and DNA [51]. As summarized in the review by Agnihotri et al., there are studies proving the toxic effect of copper and its compounds on some kinds of human tissues, such as brain, lung or spleen tissue [53]. At the same time, there are studies that prove no or a weak cytotoxic effect of copper-containing materials on human fibroblast-like cells [54] or osteoblasts [55]. The cellular effect of copper-containing nanoparticles may depend on their different aspects, such as size or morphology, as well as on environmental conditions and chemical factors [51]. This fact may explain the slight differences in the cytotoxicity of coated and non-coated materials. In our study, two materials (NP-VM2 + Cu and NP-EC + Cu) showed moderate changes in cell cultures, described as grade 2 according to ISO norms [6]. The other evaluated materials (CD and VS) coated with copper showed no or weak changes in the cell cultures. Furthermore, our studies show that human NHDFs adhere to the surfaces of the evaluated materials, confirming the known properties of CAD/CAM materials.

5. Conclusions

Based on the obtained results, different degrees of adhesion ability and biofilm formation were depended on the species of microorganisms and the properties of the dental materials. S. mutans and L. rhamnosus turned out to be the strains that adhered to the surface of the tested materials the least. S. mutans did not adhere to VM2 or VM2 + Cu, unlike C. albicans ATCC 90028, which formed a strong biofilm on both the surface of VITA MARK II and its copper counterpart. Candida albicans ATCC 90028, Candida albicans ATCC 10231 and L. rhamnosus adhered better to VM2 + Cu compared to the same material without copper. Interestingly, the addition of copper did not result in a significant reduction in bacterial or fungal presence compared to the non-copper materials. The conducted tests showed that the majority of the materials are not cytotoxic according to an ISO norm [6]. Two materials coated with copper nanoparticles exhibited moderate cytotoxicity (VM2 and EC). The influence of copper on cytotoxicity remains inconclusive, neither confirmed nor refuted. Surface polishing did not alter the cytotoxicity of the materials. The tests carried out on human NHDFs showed the adhesive properties of the materials’ surfaces. All of the evaluated materials can be described as hydrophilic, as they present a WCA below 90°. The values of the WCA are higher for polished samples, which indicates that they are more hydrophobic.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/app14209224/s1, Figure S1: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for NP-VS; Figure S2: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for NP-VM2; Figure S3: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for NP-CD; Figure S4: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for NP-VS + Cu; Figure S5: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for NP-VM2 + Cu; Figure S6: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for NP-CD + Cu.; Figure S7: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for NP-EC + Cu.; Figure S8: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for P-VS.; Figure S9: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for P-VM2.; Figure S10: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for P-CD.; Figure S11: (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for P-EC.; Figure S12: Representative images of water droplets during WCA measurement. Polished samples: VS (A), VM2 (B), CD (C), EC (D). Non-polished samples: VS (E), VM2 (F), CD (G), EC (H).

Author Contributions

Conceptualization, A.P., W.G., M.K. and M.D.; methodology, W.G., P.J.P., A.R., M.P. and M.K.; software, A.P. and P.J.P.; validation, A.P., P.J.P., A.R., M.P., M.M. and M.D.; formal analysis, A.P. and P.J.P.; investigation, A.P., W.G., P.J.P., A.R., M.P., J.N. and M.K.; resources, M.M. and M.D.; data curation, A.P., P.J.P., A.R., M.P. and J.N.; writing—original draft preparation, A.P., W.G., P.J.P., A.R., M.P., J.N. and M.K.; writing—review and editing, A.P. and P.J.P.; visualization, P.J.P.; supervision, W.G., M.M. and M.D.; project administration, M.M. and M.D.; funding acquisition, M.M. and M.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a subsidy from Wroclaw Medical University, number SUBZ.B180.24.058.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article or Supplementary Material.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 09224 g001
Figure 1. Strong 2004 motor (A), handpiece for Strong 2004 motor (B), and prefabricated block and 4 × 4 × 2 mm cut block of Vita Mark II (C).
Figure 1. Strong 2004 motor (A), handpiece for Strong 2004 motor (B), and prefabricated block and 4 × 4 × 2 mm cut block of Vita Mark II (C).
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Applsci 14 09224 g002
Figure 2. Three-dimensionally printed holder used for finishing and polishing (A), and SoftCut and CeraMaster polishers (SHOFU INC., Kyoto, Japan) (B).
Figure 2. Three-dimensionally printed holder used for finishing and polishing (A), and SoftCut and CeraMaster polishers (SHOFU INC., Kyoto, Japan) (B).
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Figure 3. (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for NP-EC.
Figure 3. (A) Direct contact of Balb/3T3 fibroblasts with the test material at the edge of the sample, (B) under the sample and (C) further from the sample after 24 h; (D) control culture without contact with the test material. Ampl. 100× for NP-EC.
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Figure 4. NHDF adhesion to the tested material after 72 h (A–D) and after 5 days (E–H). Ampl. 100×—NP-VS (A,E); NP-VM2 (B,F); NP-CD (C,G); NP-EC (D,H).
Figure 4. NHDF adhesion to the tested material after 72 h (A–D) and after 5 days (E–H). Ampl. 100×—NP-VS (A,E); NP-VM2 (B,F); NP-CD (C,G); NP-EC (D,H).
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Figure 5. NHDF adhesion to the tested material after 72 h (A–D) and after 5 days (E–H). Ampl. 100×—P-VS (A,E); P-VM2 (B,F); P-CD (C,G); P-EC (D,H).
Figure 5. NHDF adhesion to the tested material after 72 h (A–D) and after 5 days (E–H). Ampl. 100×—P-VS (A,E); P-VM2 (B,F); P-CD (C,G); P-EC (D,H).
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Figure 6. Adhesion abilities of different strains to dental CAD/CAM materials and the same materials coated with Cu. Results presented as CFU/mL (mean value + SD) in the logarithmic scale.
Figure 6. Adhesion abilities of different strains to dental CAD/CAM materials and the same materials coated with Cu. Results presented as CFU/mL (mean value + SD) in the logarithmic scale.
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Figure 7. Water contact angle of polished and non-polished CAD/CAM blocks.
Figure 7. Water contact angle of polished and non-polished CAD/CAM blocks.
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Table 1. Description of materials selected for the study.
Table 1. Description of materials selected for the study.
Material (Abbr.)Sample PhotographyManufacturerClassificationCompositionSeries Number (lot)Reference
Vita Suprinity (VS)Applsci 14 09224 i001VITA-ZahnfabrikLithium Silicate CeramicsSiO2 (56–64 wt%), Li2O (15–21 wt%), K2O (1–4 wt%), P2O5 (3–8 wt%), Al2O3 (1–4 wt%), ZrO2 (8–12 wt%), CeO2 (0–4 wt%), La2O3 (0.1 wt%), pigments (0–6 wt%)41781[32]
Vita Mark II (VM2)Applsci 14 09224 i002VITA-ZahnfabrikFeldspar CeramicsSiO2 (56.0–64.0 wt%), Al2O3 (20.0–23.0 wt%), Na2O (6.0–9.0 wt%), K2O (6.0–8.0 wt%), CaO (0.3–0.6 wt%), TiO2 (<0.1 wt%)80560[33]
Celtra Duo (CD)Applsci 14 09224 i003Dentsply SironaLithium Silicate CeramicsSiO2 (58.0 wt%), P2O5 (5.0 wt%), Al2O3 (1.9 wt%), Li2O (18.5 wt%), ZrO2 (10.1 wt%), Tb2O3 (1.0 wt%), CeO2 (2.0 wt%)18030920[34]
Empress Cad (EC)Applsci 14 09224 i004Ivoclar VivadentLeucite-Reinforced Glass-CeramicsSiO2 (60.0–65.0 wt%), Al2O3 (16.0–20.0 wt%), K2O (10.0–14.0 wt%), Na2O (3.5–6.5 wt%), other oxides (0.5–7.0 wt%), pigments (0.2–1.0 wt%)S50510[35]
Table 2. Evaluation of cytotoxicity of materials selected for the study.
Table 2. Evaluation of cytotoxicity of materials selected for the study.
Evaluated
Material		Description of Morphological Changes in Cell CultureEvaluation of Changes in Cell CultureCytotoxicity
NP-VSno changes in the environment of the material and under the material; slight inhibition of cell growth under the samplenone0
NP-VM2no changes in the environment of the material and under the material; slight inhibition of cell growth under the samplenone0
NP-CDindividual cells have degenerated or distorted under the materiallow1
NP-ECindividual cells have degenerated or distorted under the materiallow1
NP-VS + Cuindividual cells have degenerated or distorted under the materiallow1
NP-VM2 + Cuthe zone of changed cells is limited to the surface under the materialmoderate2
NP-CD + Cuno changes in the environment of the material and under the material; slight inhibition of cell growth under the samplenone0
NP-EC + Cuthe zone of changed cells is limited to the surface under the materialmoderate2
P-VSno changes in the environment of the material and under the material; slight inhibition of cell growth under the samplenone0
P-VM2no changes in the environment of the material and under the material; slight inhibition of cell growth under the samplenone0
P-CDindividual cells have degenerated or distorted under the materiallow1
P-ECindividual cells have degenerated or distorted under the materiallow1
Table 3. Adhesion abilities of different strains to dental CAD/CAM materials and the same materials coated with Cu. Results presented as CFU/mL (mean value ± SD).
Table 3. Adhesion abilities of different strains to dental CAD/CAM materials and the same materials coated with Cu. Results presented as CFU/mL (mean value ± SD).
MaterialVSVS + CuVM2VM2 + CuCDCD + CuECEC + Cu
C. albicans ATCC 90028
[CFU/mL]		4.1 × 102 ± 9.9 × 1013.5 × 103 ± 2.8 × 1033.9 × 103 ± 1.6 × 1036.6 × 103 ± 6.8 × 1031.3 × 103 ± 7.1 × 1012.7 × 103 ± 7.8 × 1024.2 × 103 ± 1.2 × 1032.4 × 103 ± 1.4 × 103
C. albicans ATCC 10231
[CFU/mL]		1.0 × 103 ± 1.3 × 1032.0 × 102 ± 9.2 × 1011.2 × 103 ± 9.2 × 1024.2 × 103 ± 4.6 × 1037.0 × 102 ± 03.9 × 103 ± 1.6 × 1031.1 × 103 ± 5.1 × 1023.4 × 103 ± 2.4 × 103
S. mutans
ATCC 25175
[CFU/mL]		3.5 × 102 ± 7.1 × 1016.0 × 102 ± 8.5 × 102002.0 × 102 ± 01.5 × 102 ± 7.1 × 1015.0 × 101 ± 7.1 × 1010
L. rhamnosus ATCC 9595
[CFU/mL]		05.0 × 101 ± 7.1 × 1014.0 × 102 ± 2.8 × 1023.2 × 103 ± 1.4 × 1032.0 × 102 ± 07.5 × 102 ± 6.4 × 10203.8 × 102 ± 1.4 × 102
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Piszko, A.; Grzebieluch, W.; Piszko, P.J.; Rusak, A.; Pajączkowska, M.; Nowicka, J.; Kobielarz, M.; Mikulewicz, M.; Dobrzyński, M. Cytotoxicity and Microbiological Properties of Ceramic CAD/CAM Materials Subjected to Surface Treatment with Nanometric Copper Layer. Appl. Sci. 2024, 14, 9224. https://doi.org/10.3390/app14209224

AMA Style

Piszko A, Grzebieluch W, Piszko PJ, Rusak A, Pajączkowska M, Nowicka J, Kobielarz M, Mikulewicz M, Dobrzyński M. Cytotoxicity and Microbiological Properties of Ceramic CAD/CAM Materials Subjected to Surface Treatment with Nanometric Copper Layer. Applied Sciences. 2024; 14(20):9224. https://doi.org/10.3390/app14209224

Chicago/Turabian Style

Piszko, Aleksandra, Wojciech Grzebieluch, Paweł J. Piszko, Agnieszka Rusak, Magdalena Pajączkowska, Joanna Nowicka, Magdalena Kobielarz, Marcin Mikulewicz, and Maciej Dobrzyński. 2024. "Cytotoxicity and Microbiological Properties of Ceramic CAD/CAM Materials Subjected to Surface Treatment with Nanometric Copper Layer" Applied Sciences 14, no. 20: 9224. https://doi.org/10.3390/app14209224

APA Style

Piszko, A., Grzebieluch, W., Piszko, P. J., Rusak, A., Pajączkowska, M., Nowicka, J., Kobielarz, M., Mikulewicz, M., & Dobrzyński, M. (2024). Cytotoxicity and Microbiological Properties of Ceramic CAD/CAM Materials Subjected to Surface Treatment with Nanometric Copper Layer. Applied Sciences, 14(20), 9224. https://doi.org/10.3390/app14209224

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