The Effects of Thermocycling on the Physical Properties and Biocompatibilities of Various CAD/CAM Restorative Materials

The purpose of this study is to evaluate the changes in physical properties and biocompatibilities caused by thermocycling of CAD/CAM restorative materials (lithium disilicate, zirconia reinforced lithium silicate, polymer-infiltrated ceramic network, resin nanoceramic, highly translucent zirconia). A total of 225 specimens were prepared (12.0 × 10.0 × 1.5 mm) and divided into three groups subjected to water storage at 37 °C for 24 h (control group), 10,000 cycles in distilled water at 5–55 °C (first aged group), and 22,000 cycles in distilled water at 5–55 °C (second aged group) [(n= 15, each]). The nanoindentation hardness and Young’s modulus (nanoindenter), surface roughness (atomic force microscopy (AFM)), surface texture (scanning electron microscopy (FE-SEM)), elemental concentration (energy dispersive spectroscopy (EDS)) and contact angle were evaluated. The morphology, proliferation and adhesion of cultured human gingival fibroblasts (HGFs) were analyzed. The data were analyzed using one-way ANOVA and Tukey’s test (p < 0.05). The results showed that the nanoindentation hardness and Young’s modulus were decreased after thermocycling aging. Cell viability and proliferation of the material decreased with aging except for the highly translucent zirconia. Zirconia-reinforced lithium silicate exhibited significantly lower cell viability compared to other materials. The surface roughnesses of all groups increased with aging. Cell viability and Cell adhesion were influenced by various factors, including the surface chemical composition, hydrophilicity, surface roughness, and topography.


Introduction
Ceramics exhibit high wear resistance, aesthetics, and excellent color stability, as well as a high degree of biocompatibility [1]. However, they also accelerate the wear cause by opposing dentition and they are prone to fracture due to their brittleness [2]. In contrast, resin composites have the appropriate esthetics, easy milling processes, easy repairability in The effect of surface roughness on attachment and proliferation is a subject of controversy, with certain studies suggesting that smooth surfaces are more favorable, while others indicate the opposite [28,29]. Therefore, this in vitro study investigated the physical and surface properties as well as the biocompatibilities of materials under thermal cycling aging, which is one of the aging processes. The null hypothesis presented in this study is that the physical properties of various CAD/CAM restorative materials do not differ before and after aging, the biocompatibility of various CAD/CAM restorative materials does not differ before and after aging, and the surface properties of these restoration materials are no difference before and after aging, and that the surface characteristics are not associated with cell adhesion.  Table 1.

Aging Procedure
Five samples of each material were randomly selected. The first aged group was subjected to thermal cycling (TW-D813, Taewon tech, Bucheon-si, Republic of Korea) (Figure 1b) in distilled water at 5-55 °C for 10,000 cycles (5 °C for 30 s; 55 °C for 30 s; rest period; 10 s), which corresponded to approximately 1 year in the clinical setting. Another group (second aged) underwent thermal cycling for 22,000 cycles under the same conditions, which corresponded to approximately 3 years in a clinical environment [30,31]. The control group was stored in distilled water at 37 °C for 24 h.

Nanoindentation Hardness and Young's Modulus (Elastic Modulus)
The nanoindentation hardness and Young's modulus (Elastic modulus) were measured with a Nanoindenter (Nanoindenter XP MTS, Chicago, IL, USA). The Berkovich diamond tip roundness was 40 nm, the essential load was 45 mN, the strain rate was 0.05%/s, the maximum penetration depth was 2 µm, and each specimen was measured 3 times.

Aging Procedure
Five samples of each material were randomly selected. The first aged group was subjected to thermal cycling (TW-D813, Taewon tech, Bucheon-si, Republic of Korea) (Figure 1b) in distilled water at 5-55 • C for 10,000 cycles (5 • C for 30 s; 55 • C for 30 s; rest period; 10 s), which corresponded to approximately 1 year in the clinical setting. Another group (second aged) underwent thermal cycling for 22,000 cycles under the same conditions, which corresponded to approximately 3 years in a clinical environment [30,31]. The control group was stored in distilled water at 37 • C for 24 h.

Measurements of Physical Properties Nanoindentation Hardness and Young's Modulus (Elastic Modulus)
The nanoindentation hardness and Young's modulus (Elastic modulus) were measured with a Nanoindenter (Nanoindenter ® XP MTS, Chicago, IL, USA). The Berkovich diamond tip roundness was 40 nm, the essential load was 45 mN, the strain rate was 0.05%/s, the maximum penetration depth was 2 µm, and each specimen was measured 3 times.

Energy Dispersive X-ray Spectroscopy (EDS)
Energy dispersive X-ray spectroscopy (SU8230; Hitachi High Technologies, Tokyo, Japan) with a measurement voltage of 15 kV was used to observe differences in the surface components before and after aging.

Field-Emission Scanning Electron Microscopy (FE-SEM)
Energy dispersive X-ray spectroscopy (SU8230; Hitachi High Technologies, Tokyo, Japan) with a measurement voltage of 15 kV at 1000 magnifications was used to observe differences in the surface components before and after aging.

Atomic Force Microscopy (AFM)
To analyze the surface roughnesses (R a , R q ), atomic force microscopy (XE-100;Park Systems, Suwon, Republic of Korea) was used for 3 measurements per specimen. The scan sizes were 10 × 10 µm 2 , the resolution was 256 × 256 pixels, and the scan rate was 0.8 Hz. And the non-contact mode Kelvin probe force microscopy equipped with a Pt/Cr-coated silicon tip (tip radius < 25 nm, force constant 3 N m −1 , and a resonance frequency of 75 kHz) was employed to measure the surface charge potentials.

Contact Angle Measurements
The surface wettabilities of the samples were characterized by water contact angle measurements with a contact angle analyzer (Phoenix 300; SEO, Suwon, Republic of Korea). The sessile drop method was used for contact angle measurements. Images of the drops were captured with a video camera as soon as they landed on the surfaces. In each case, three water droplets were measured for each test specimen. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the images and measure the contact angles. The average angle of the three droplets was obtained.

CCK-8 Assay
The Cell Counting Kit-8 (CCK-8, CK04-20, Dojindo, Japan) assay was used to assess the activity of the mitochondrial dehydrogenase in the living cells, which is a marker of cell viability. First, three samples per group were placed in 12-well untreated plates, seeded with cells at a density of 5.0 × 10 4 per well, and incubated for 24 h. After 24 h of incubation, the samples were washed with PBS and then incubated for 2 h at 37 • C in 1 mL of culture medium with 100 µL of CCK solution added to each well. The optical density was measured at 450 nm with a microplate reader (Scientific Varioskan LUX Multimode Microplate Reader, Thermo Fisher Scientific, Waltham, MA, USA).
Finally, a fluorescence microscope (Olympus IX71, Olympus, Tokyo, Japan) was used to collect the fluorescence images. The images were quantified with ImageJ software to determine the F-actin intensity, cell area, Talin 1 intensity and Vinculin intensity.

Nanoindentation Hardness
The means and standard deviations of the nanoindentation hardness before and after aging are presented for all materials Figure 2a, Supplementary Table S1. In all groups except for zirconia, the nanoindentation hardness of both of the aged groups was significantly lower than that of the control group. The nanoindentation hardness was found to be the lowest in the Group S, while the Group Z exhibited the highest values among all the groups Pharmaceutics 2023, 15, 2122 7 of 20 before and after aging (p < 0.05). The values of Groups M, C, E, and S exhibited significant decreases after the 2nd aging (p < 0.05).

Young's Modulus (Elastic Modulus)
The mean and standard deviations for the Young's moduli determined for each material before and after aging are presented in Figure 2b, Supplementary Table S2. Group S exhibited the lowest value among all the tested materials before and after aging (p < 0.05). There were no significant differences in Young's moduli of Group M and Group C (p > 0.05), while significant differences were observed for all other materials both before and after aging. The values of all material groups exhibited significant decreases after aging (p < 0.05). Group M showed a decrease between the 1st age group and the 2nd aged group.

Energy Dispersive X-ray Spectroscopy (EDS)
In comparing the surface compositions of all materials before and after aging, there were several elements that showed statistically significant differences between the materials, as described in detail in Table 3.
Group M was mainly composed of C, O, Al, Si, P, and K, and the carbon and potassium components showed significant decreases as the aging process progressed. In Group C, while the carbon (C) and oxygen (O) levels increased in the 2nd aged group compared to the 1st aged group, the zirconium (Zr) content decreased significantly. Group E was mainly composed of C,O, Al, Si, Na, and C was the base of the polymer. In the 2nd aged group, the carbon (C) and oxygen (O) contents showed significant increases compared to the control group. Group S was mainly composed of C, O, Al, Si, and Ba. There was little

Young's Modulus (Elastic Modulus)
The mean and standard deviations for the Young's moduli determined for each material before and after aging are presented in Figure 2b, Supplementary Table S2. Group S exhibited the lowest value among all the tested materials before and after aging (p < 0.05). There were no significant differences in Young's moduli of Group M and Group C (p > 0.05), while significant differences were observed for all other materials both before and after aging. The values of all material groups exhibited significant decreases after aging (p < 0.05). Group M showed a decrease between the 1st age group and the 2nd aged group.

Energy Dispersive X-ray Spectroscopy (EDS)
In comparing the surface compositions of all materials before and after aging, there were several elements that showed statistically significant differences between the materials, as described in detail in Table 3.
Group M was mainly composed of C, O, Al, Si, P, and K, and the carbon and potassium components showed significant decreases as the aging process progressed. In Group C, while the carbon (C) and oxygen (O) levels increased in the 2nd aged group compared to the 1st aged group, the zirconium (Zr) content decreased significantly. Group E was mainly composed of C, O, Al, Si, Na, and C was the base of the polymer. In the 2nd aged group, the carbon (C) and oxygen (O) contents showed significant increases compared to the control group. Group S was mainly composed of C, O, Al, Si, and Ba. There was little difference in the elemental composition after aging. Group Z, which is a type of zirconia, showed an increase in the carbon (C) content as the aging process progressed, while the zirconium (Zr) content decreased in contrast. FE-SEM images showed that the topographic patterns differed among all groups ( Figure 3). The surface of the control group was relatively smooth, whereas the aged group showed micro irregularities, porosity, and defects. In Group M, rectangular shaped lithium disilicate particles were well observed, but some irregular shape was observed after aging.
In Group E, plate-like ceramic supports were well observed in all groups and remained after aging, Group S exhibited uniformly sized ceramic fillers dispersed in all groups, and no significant changes were observed even after aging. And Polycrystalline particles were observed in Group Z, and changes in crystal morphology were observed after aging. Representative AFM images for all groups are shown in Figure 4. In the control group of all materials, the grains exhibited a smooth surface with a dense network of interlocking

Atomic Force Microscopy (AFM)
Representative AFM images for all groups are shown in Figure 4. In the control group of all materials, the grains exhibited a smooth surface with a dense network of interlocking acicular branches, and the spaces between them were filled with a compact amorphous phase, such as the glassy matrix or ground mass. After aging, the surface texture showed localized surface uplifts on the grains. Surface roughness and microcracks primarily progressed along the grain boundaries.  The mean and standard deviation for the surface roughness (R a and R q ) measured before and after aging for each group, as well as the statistical analyses, are presented in Figure 5a,b and Supplementary Table S3. Both before and after aging, Group S showed the highest Ra and Rq values, while Group Z showed the lowest Ra and Rq values. In all materials, the Ra and Rq values increased relative to that of the control group as aging progressed. In Group M, Rq and Sq showed significant increases in the 2nd aged group compared to the control.

Contact Angle Measurements
The means, standard deviations, and statistical analyses of the contact angle strengths are presented in Figure 5c,d and Supplementary Table S4. In all groups, the contact angles were significantly lower for the 2nd aged group than for the control group (p < 0.05). Group Z exhibited the highest contact angles before and after aging, followed by Groups C, S, E, and M. In both Groups C and Z, the contact angles decreased significantly as the aging time increased (p < 0.05). Groups E and S showed similar contact angle values before and after aging.

Cell Properties
The following are the experimental results of the biocompatibility analysis of various CAD/CAM materials. The HGF cells were cultured on specimens to analyze cell viability through Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay and LIVE/DEAD assay, and cell spreading and focal adhesion through immunofluorescence staining (Figure 6a).
3.3.1. Cell Viability Figure 6 shows the cell viability results after incubation of HGFs in five types of materials for 24 h. The cell viability was evaluated by measuring the cell viability of HGFs

Contact Angle Measurements
The means, standard deviations, and statistical analyses of the contact angle strengths are presented in Figure 5c,d and Supplementary Table S4. In all groups, the contact angles were significantly lower for the 2nd aged group than for the control group (p < 0.05). Group Z exhibited the highest contact angles before and after aging, followed by Groups C, S, E, and M. In both Groups C and Z, the contact angles decreased significantly as the aging time increased (p < 0.05). Groups E and S showed similar contact angle values before and after aging.

Cell Properties
The following are the experimental results of the biocompatibility analysis of various CAD/CAM materials. The HGF cells were cultured on specimens to analyze cell viability through Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay and LIVE/DEAD assay, and cell spreading and focal adhesion through immunofluorescence staining (Figure 6a).
To confirm cell viability in culture conditions, LIVE/DEAD staining was performed and confirmed by fluorescence microscopy images (Figure 6c). Live cells are stained green and dead cells are stained red, with dead cells marked with white arrows. The percentage of dead cells in all groups was low, which means that most of the cells were able to survive on the surface, which indicates that all samples exhibited good biocompatibility for HGFs. Also, as shown in the image, cell viability decreased with aging, except for the group Z.   Figure 6 shows the cell viability results after incubation of HGFs in five types of materials for 24 h. The cell viability was evaluated by measuring the cell viability of HGFs upon aging by thermocycling for each material using the CCK-8 assay, and then re-evaluated by fluorescence microscopy images using the LIVE/DEAD assay.

Cell Viability
The group M and the group C showed no significant difference in cell viability between the control and 1st aged groups, but there was a tendency for HGF cell viability to decrease in the 2nd aged group (Figure 6b). The group E and group S also showed a decrease in HGFs viability in all aged groups compared to the control. However, the group Z increased the viability of HGFs in the aged group compared to the control group.
To confirm cell viability in culture conditions, LIVE/DEAD staining was performed and confirmed by fluorescence microscopy images (Figure 6c). Live cells are stained green and dead cells are stained red, with dead cells marked with white arrows. The percentage of dead cells in all groups was low, which means that most of the cells were able to survive on the surface, which indicates that all samples exhibited good biocompatibility for HGFs. Also, as shown in the image, cell viability decreased with aging, except for the group Z.

Immunocytochemistry of Cell Spreading and Focal Adhesion
Cell size and spreading were assessed F-actin cytoskeleton stained with phalloidin (red) and nuclear stained with SYTOX™ (green) followed by fluorescence microscopy images (Figure 7a). As shown in the images, the cells cultured in the control and aged groups of each material look different in cell morphology, either wider or thinner, but all are well spread. To assess the size and spread of the cells, the cell area was quantified using Image J software. In all materials, there was no significant difference in cell area with aging. We also quantified and analyzed the intensity of actin, which is important for cell structure and movement. All material groups except the group E showed lower f-actin intensity in all aged groups compared to the control group. This suggests that the ability of cells to adhere or growth may be reduced in the aged groups. On the other hand, the group E showed higher f-actin intensity in the aged group compared to the control group. This indicates that the cells in the aged group are able to adhere and growth more effectively to the material surface.
cein-AM and dead cells were stained red with ethidium homodimer-1. white arrow indicating dead cells. Data are presented as mean ± SEM (scale bar, 100 µm).

Immunocytochemistry of Cell Spreading and Focal Adhesion
Cell size and spreading were assessed F-actin cytoskeleton stained with phalloidin (red) and nuclear stained with SYTOX™ (green) followed by fluorescence microscopy images ( Figure 7a). As shown in the images, the cells cultured in the control and aged groups of each material look different in cell morphology, either wider or thinner, but all are well spread. To assess the size and spread of the cells, the cell area was quantified using Image J software. In all materials, there was no significant difference in cell area with aging. We also quantified and analyzed the intensity of actin, which is important for cell structure and movement. All material groups except the group E showed lower f-actin intensity in all aged groups compared to the control group. This suggests that the ability of cells to adhere or growth may be reduced in the aged groups. On the other hand, the group E showed higher f-actin intensity in the aged group compared to the control group. This indicates that the cells in the aged group are able to adhere and growth more effectively to the material surface.
To further investigate the cell adhesion of HGF to the materials, we analyzed the focal adhesion markers Talin1 and Vinculin by staining after 4 h of HGFs incubation on the five materials (Figure 7b). The quantification of Talin1 and Vinculin showed that the intensity of focal adhesion related proteins decreased in the aged groups of the group M, group C and group Z compared to the control group. This suggests that cell adhesion and cell motility may have decreased in the aged groups. However, in the group C and group E, the intensity of focal adhesion related proteins decreased in the aged group compared to the control group. This suggests that the formation or stability of focal adhesions may have increased with aging.  Representative immunofluorescence microscopy images of cytoskeletal organization (F-actin filament) stained with phalloidin (red), and nuclei stained with SYTOX™ (green) of HGF cells cultured for 24 h in all groups. The merged images are shown (scale bar, 50 µm). Graph shows quantification of cell spreading area and intensity using Image J software. Data are presented as mean ± SEM (n = 30). Statistical significance was determined using one-way ANOVA. * denotes a significant difference at p < 0.05. (b) Focal adhesion. Representative immunofluorescence microscopy mages with the focal adhesion markers indicating Vinculin (red) and Talin 1 (green) for HGF cells cultured for 4 h in all groups. The merged images are shown (scale bar, 50 µm). Graph shows quantification of focal adhesion intensity using Image J software. Data are presented as mean ± SEM (n = 30). Statistical significance was determined using one-way ANOVA. * denotes a significant difference at p < 0.05.
To further investigate the cell adhesion of HGF to the materials, we analyzed the focal adhesion markers Talin1 and Vinculin by staining after 4 h of HGFs incubation on the five materials (Figure 7b). The quantification of Talin1 and Vinculin showed that the intensity of focal adhesion related proteins decreased in the aged groups of the group M, group C and group Z compared to the control group. This suggests that cell adhesion and cell motility may have decreased in the aged groups. However, in the group C and group E, the intensity of focal adhesion related proteins decreased in the aged group compared to the control group. This suggests that the formation or stability of focal adhesions may have increased with aging.

Discussion
Dental restorative materials undergo chemical, biological, physical, and thermal changes due to the complex oral environment [32]. Changes in oral temperature can lead to decreases in the strengths of restorations and accelerate the progression of cracks [33,34]. In previous studies, various accelerated aging procedures have been applied to conventional CAD/CAM restorative materials, and thermal cycling is one of the most commonly used methods for aging dental restorative materials [35]. Therefore, in this study, the physical properties, surface properties, and biological properties of various hybrid CAD/CAM restorations before and after thermal cycling aging were compared.
The data suggested that the physical properties of the tested materials exhibited gradual declines after thermocycling; thus, the first null hypothesis suggesting that the physical properties of the different CAD/CAM restorations would not be affected by thermal cycling aging is rejected. All CAD/CAM ceramic materials except for high translucence zirconia (Group Z) showed statistically significant decreases in hardness and elastic moduli with increasing thermocycling. This occurred because thermocycling involved cyclic temperature changes, which induced thermal stresses within the material. These thermal stresses can lead to microstructural changes, such as grain boundary cracking or phase transformations, and these weaken the material and result in decreased hardness and elastic modulus. Additionally, the second null hypothesis suggesting that the material type would not affect the properties of different CAD/CAM restorations is also rejected. The nanoindentation hardness and Young's modulus values were highest for the Lava plus zirconia (Group Z), followed by IPS e.max CAD (Group M) and then the hybrid CAD/CAM materials. Zirconia was less affected by thermocycling due to its low thermal expansion coefficient, structural stability, and high crystal structure density. These characteristics allowed the zirconia to maintain its mechanical properties more effectively during thermocycling conditions. IPS e.max CAD (Group M) is a glass-ceramic material, which means it contained both glass and crystalline phases. The presence of crystalline particles in the material contributed to the higher hardness and elastic modulus values compared to the purely resin-based hybrid CAD/CAM ceramics such as the resin nanoceramic (RNC) and polymerinfiltrated ceramic network (PICN). Among the resin-based hybrid CAD/CAM restorations, Cerasmart (Group S) showed significantly lower values even after thermal cycling, followed by Vita Enamic (Group E). This was consistent with previous studies. Alamoush et al. [36] evaluated the physical properties of various CAD/CAM ceramics and found that the specimens fabricated from RNC recorded the lowest nanoindentation hardness values and elastic moduli, followed by those from PICN. This was explained by the fact that PICN is composed of two continuous interpenetrating networks: a ceramic network and a polymer network, which differentiates it from other resin composite CAD/CAM blocks that are typically made up of a resin matrix with dispersed ceramic fillers.
The EDS and SEM analyses revealed significant alterations in the surface microstructures and compositions of the tested materials after thermal cycling aging. The initially homogeneous structures of the materials were found to deteriorate, and they exhibited noticeable surface uplifts, microcracks, and irregular defects and slight damage after aging. These changes were attributed to reductions in the particle sizes caused by aging, which in turn affected the elemental compositions of the materials. Vita Enamic (Group E) and Cerasmart (Group S) exhibited clear alterations in their surface microstructures, whereas IPS e.max CAD (Group M) and Celtra Duo (Group C) exhibited relatively slight alterations. As a result, thermal cycling had a greater impact on polymer-based materials.
The surface roughnesses had increased for all groups after the aging process. This was consistent with a previous study showing that simulated accelerated aging led to increases in the surface roughnesses of ceramic materials, which was attributed to the crystalline components, and the difference in the dissolution rates may have led to the increased surface roughnesses [37]. Among the materials, Cerasmart (Group S) showed the highest Ra value. The results of this study were consistent with previous findings indicating that when water is absorbed by the resin matrix, it causes expansion and plasticization of the resin, which lead to hydrolysis of the silane coupling agent and eventual loss of the surface fillers. Additionally, the absorbed water can diffuse into the interface between the ceramic and polymeric phases, potentially creating microcracks on the surface and further increasing the surface roughness of Cerasmart (Group S) compared to the other tested materials [38]. Lava Plus zirconia (Group Z) is a fully stabilized zirconia with a 5% yttria content, and it showed the lowest surface roughness value. The addition of yttria to the zirconia structure stabilized the crystal structure and made it more resistant to changes in the surface roughness even under different conditions, such as thermal cycling.
In this study, the contact angles for all materials tended to decrease after thermocycling. After thermocycling, the resin matrix of the CAD/CAM ceramic was expected to be more hydrophilic [39] (decreased WCA) due to water sorption during thermocycling. Sturz et al. [40] suggested that changes in the WCA may be attributed to the chemical composition of the material, particularly differences in the matrix composition and filler fraction, as well as potential surface inhomogeneities caused by the fillers.
In terms of biocompatibility, the ceramic restorations are in prolonged contact with oral soft tissues, especially with the marginal keratinized gingiva. Thus, biocompatibility is a critical characteristic that should be carefully evaluated prior to clinical use. In vivo, the formation of a mucosal seal around the prosthetic restoration is critical for success, and this process is highly dependent on the attachment of HGFs in the connective tissues to the substrate surface [41]. For cell viability, as revealed by the LIVE & DEAD assay, most cells remained viable on all surfaces, suggesting that all samples had excellent biocompatibilities toward HGFs. Except for the Lava plus zirconia (Group Z), the cell viability decreased with increasing aging time for all materials, as shown by the results of the CCK-8 assay. The vital reaction to dental restoration is affected by the surface topography and surface physiochemistry of the material used. The surface features of a material have a significant impact on the way cells interact with it. First, the surface wettability of a biomaterial, as determined with contact angle measurements, has been shown to have an impact on protein adsorption onto the surface of the material. Generally, a hydrophilic surface inhibits cell adhesion by preventing protein adsorption, while a hydrophobic surface promotes cell adhesion by inducing protein adsorption. Second, surface roughness has a significant impact on protein adsorption and subsequent cell behavior [42,43]. As shown by the results of the cell viability analysis, the present study revealed that surface roughness and wettability had significant effects on the initial stages of host tissue-biomaterial integration, and particularly on cell adhesion and proliferation. The impact of surface roughness on the attachment and proliferation of HGFs is a subject of debate, with some studies suggesting that smooth surfaces are more beneficial, while others report the opposite [44][45][46][47].
In this study, all of the materials exhibited increased surface roughness after thermal cycling aging, which resulted in reduced performance of the HGFs in terms of adhesion, proliferation, and migration. Therefore, based on the results of this study, it can be inferred that HGFs exhibit higher proliferation rates on smooth surfaces. Unlike other ceramic materials, zirconia showed a slight increase in cell viability in the aged group compared to the control group. This was assumed to arise because the surface of zirconia was very smooth, with a very low R a value for surface roughness, even in the aged group, which may have led to a higher fibroblast survival rate [48]. In our study, Celtra Duo (Group C) exhibited a significantly lower cell viability than the other materials. It can be assumed that the absence of a crystallization process in the zirconia-reinforced lithium silicate (ZLS) may have resulted in an unstable crystalline structure of the material, which negatively impacted the cell viability [49]. Additionally, cytoskeletal organization provides the fundamental framework of the cell and maintains the cell shape, promotes cell maturation, and facilitates cell migration [50]. In the present study, it was observed that cell spreading of the HGFs was faster on smoother surfaces in the control group compared to rougher surfaces in the aged group, as shown by the F-actin intensities. Previous studies suggested that cells on smooth surfaces underwent spreading and developed robust actin cytoskeletons to achieve mechanical stability on smooth surfaces, whereas on rough surfaces, the topographical features of the surface were used by the cells for stabilization [51,52]. However, in this study, there were no differences in the cell areas of the control group and aged group after 24 h of seeding. The actin binding protein Talin1 is closely related to cell adhesion and is the key regulator of focal adhesion. Talin1 interacts with actin to form a connection with integrin, the main adhesion protein in cells, which helps cells attach to their surrounding matrix and spread [53]. Vinculin is involved in the formation of intercellular adhesions, cell spreading and movement, and regulates the structural stability of cells [54]. Talin1 and Vinculin proteins are important in the formation of cell adhesions, which are essential for the adhesion and proliferation of HGF on material surfaces. Consequently, Talin1 and Vinculin proteins are vital in the establishment of secure soft tissue seals around restoration materials. In Celtra Duo (Group C) and Vita Enamic (Group E), the fluorescence intensity of talin1 and vinculin proteins per cell was higher in aged samples, indicating stronger expression, whereas in the other groups, weaker signals were observed in aged samples. This suggested that various factors influenced cell adhesion, and based on these results, it can be inferred that this is attributable to changes in the chemical composition or surface topology after aging rather than the surface roughness. It is widely recognized that cell adhesion and growth on a material surface are affected by factors such as the chemical composition, roughness, and topography.
Considering the physical property perspective, the Lava plus zirconia biomaterial exhibited a surface hardness and elastic modulus that were more than double those of glass ceramics such as IPS e.max CAD and hybrid CAD/CAM ceramics such as Celtra Duo, Vita Enamic, and Cerasmart. This characteristic enables utilization of zirconia in a wide range of applications in dentistry, including long bridges and implant frameworks, where it effectively replaces the traditional metal materials. On the other hand, CAD/CAM zirconia blocks are predominantly made of partially sintered zirconia, as seen for the material used in this study (Lava plus zirconia), because fully sintered zirconia blocks are too strong and difficult to machine. However, partially sintered zirconia undergoes significant shrinkage during the sintering process, which can affect the accuracy of the final restoration. Hybrid CAD/CAM ceramics offer elastic moduli and surface hardnesses similar to those of natural teeth, and they exhibit excellent milling capabilities that enable faster fabrication of restorations and cause less wear on the milling equipment than zirconia. Since they already possess the desired final strengths, there is no need for additional sintering after milling, which eliminates inaccuracies due to shrinkage. Moreover, the time required for digital scanning, milling, polishing, and bonding is shorter, and the fabrication process is simpler than that for zirconia, which is advantageous [55,56]. Finally, we confirmed in vitro that the ceramic material used in this experiment was not cytotoxic. This demonstrated that the ceramic material used in this study shows promise in improving the esthetic outcomes of dental implants and in tightening the peri-implant junction.
From a biological perspective, the ceramic materials used in this study demonstrated that the HGFs prefer smooth surfaces, which underscores the recommendation for surface grinding and polishing based on faster cellular responses. In the case of implant placements, the marginal gingiva is injured, which initiates a regenerative process. Therefore, the HGFs migrate from the wound edges to the wound area, generate a collagen matrix and facilitate attachment of the keratinocytes. By modulating the surface characteristics, such as by polishing or grinding the implant material, tissue adhesion can be finely regulated, which enables the induction of tissue regeneration at the appropriate time.
The limitations of this study include the aging procedures. This artificial aging technique predicts the long-term degradation caused by aging the ceramic. However, in the oral environment, dental restorations are exposed to different stimuli, including mechanical aging, thermocycling, and chemical aging. Therefore, to reflect the clinical situation, more studies are required to assess the changes occurring in the physical properties, surface topography and cellular properties of dental ceramics under various aging conditions. Due to the in vitro nature of this study, further studies will be needed to define the technical protocols for the surface ceramic treatments and to understand the biological and mechanical advantages of these procedures. Furthermore, our study was limited to analyzing the expression patterns of cell adhesion proteins, and further research is needed to investigate the gene expression levels in more detail. Additionally, further research is needed on bacterial adhesion to CAD/CAM ceramics to reduce the risk of periprosthetic infections, as bacterial adhesion and biofilm formation on prosthetic surfaces are key steps in the pathogenesis of these infections.

Conclusions
The results of this study demonstrated that i.
The accelerated aging procedure induced changes in the physical properties of the CAD/CAM materials. The nanoindentation hardnesses and Young's moduli were decreased after thermocycling aging. Lithium disilicate and hybrid CAD/CAM restorative ceramics tend to be lower than those of highly translucent zirconia before and after aging. ii. Cell viability and proliferation of the material decreased with aging except for the highly translucent zirconia materials. Significant differences were observed in the viabilities of the HGFs on the CAD/CAM materials. Zirconia-reinforced lithium silicate exhibited significantly lower cell viability than the other materials. iii. The surface roughness increased with aging for all groups, and the resin nanoceramic showed the highest roughness, while the highly translucent zirconia exhibited the smoothest surface among the materials. After aging, changes were observed in the surface microstructures, compositions, and hydrophilicities. iv. Cell adhesion and growth on a material surface are influenced by various factors, such as the surface chemical composition, hydrophilicity, roughness, and topography.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pharmaceutics15082122/s1, Table S1: Mean and SD values of nanoindentation hardnesses (GPa); Table S2: Means and standard deviations of the Young's moduli (GPa); Table S3: Mean ± SD values and statistical analyses of surface roughnesses (R a and R q ); Table S4: Means and standard deviations of the contact angles (degrees).