*2.3. Cell Viability and Adhesion Behavior of the Ni-Fe-TiO<sup>2</sup> Nanocomposites*

The cell viability of L929 of the Ni-Fe-TiO<sup>2</sup> nanocomposites for 24 h is shown in Figure 6a. Obviously, the Ni-Fe-TiO<sup>2</sup> nanocomposites exhibited a cell survival rate of more than 70%. It is considered an acute cytotoxic potential when the cell viability of the sample is less than <70% of the blank, according to ISO 109993-5. No statistically significant difference (*n* = 5) between investigated Ni-Fe-TiO<sup>2</sup> nanocomposites. Following cell seeding on the Ni-Fe-TiO<sup>2</sup> nanocomposites, morphology and cell adhesion in Ni-Fe-TiO<sup>2</sup> nanocomposites were observed through FE-SEM as illustrated in Figure 6b. It was found that all Ni-Fe-TiO<sup>2</sup> nanocomposites showed numerous elongated filopodia after 3 days of cell seeding. The filopodia of cells not only adhered flat, but also tightly grabbed the surface structure (as indicated by arrows). The cell viability and response features demonstrated all Ni-Fe-TiO<sup>2</sup> nanocomposites possessed good biocompatibility.

*Inorganics* **2022**, *10*, x FOR PEER REVIEW 5 of 11

**Figure 4.** The FE-SEM micrographs of a car-like shape model of the Ni-Fe-5 wt.% TiO2 nanocomposite fabricated through an optimal UV-LIGA method: (**a**) car-like cavity image and (**b**) car-like shape image. **Figure 4.** The FE-SEM micrographs of a car-like shape model of the Ni-Fe-5 wt.% TiO<sup>2</sup> nanocomposite fabricated through an optimal UV-LIGA method: (**a**) car-like cavity image and (**b**) car-like shape image. **Figure 4.** The FE-SEM micrographs of a car-like shape model of the Ni-Fe-5 wt.% TiO2 nanocomposite fabricated through an optimal UV-LIGA method: (**a**) car-like cavity image and (**b**) car-like shape image.

posited micro-scale car-like shape model exhibits a smooth surface and structural integrity. *2.3. Cell Viability and Adhesion Behavior of the Ni-Fe-TiO2 Nanocomposites*  **Figure 5.** A higher magnification FE-SEM micrograph of the car-like shape model. The electrodeposited micro-scale car-like shape model exhibits a smooth surface and structural integrity. **Figure 5.** A higher magnification FE-SEM micrograph of the car-like shape model. The electrodeposited micro-scale car-like shape model exhibits a smooth surface and structural integrity. *Inorganics* **2022**, *10*, x FOR PEER REVIEW 6 of 11

**Figure 5.** A higher magnification FE-SEM micrograph of the car-like shape model. The electrode-

**Figure 6.** *Cont.*

**3. Discussion** 

**Th i ti t d l**

**Figure 6.** (**a**) Cell viability of L929 of the Ni-Fe-TiO2 nanocomposites for 24 h and cell morphologies of the Ni-Fe-TiO2 nanocomposites after culturing with L929 cells for 3 days: (**b**) Ni-Fe-5 wt.% TiO2, (**c**) Ni-Fe-10 wt.% TiO2, and (**d**) Ni-Fe-20 wt.% TiO2. The filopodia (as indicated by arrows) of cells

In this study, it was found that the electrodeposited nanocomposite produced better smooth sidewall and surface as well as structural integrity, revealing that the Ni-Fe alloy with TiO2 nanoparticles promoted formability. To explore possible reinforcement factors in nanocomposite materials, we compared results from different groups of nanocomposites including a traditional matrix of polycrystalline Ni containing reinforced nanoparticles of Al2O3 [30]. Oberle et al. [31] reported that with large hardness and mechanical strength enhancement in the composite matrix, the volume fraction of the co-deposited

not only adhered flat, but also tightly grabbed the surface structure.

**Ni-Fe-5 wt.% TiO2**

**3DPP-1 3DPP-2 3DPP-3**

**Ni-Fe-10 wt.% TiO2**

**Ni-Fe-20 wt.% TiO2**

**Figure 6.** (**a**) Cell viability of L929 of the Ni-Fe-TiO2 nanocomposites for 24 h and cell morphologies of the Ni-Fe-TiO2 nanocomposites after culturing with L929 cells for 3 days: (**b**) Ni-Fe-5 wt.% TiO2, (**c**) Ni-Fe-10 wt.% TiO2, and (**d**) Ni-Fe-20 wt.% TiO2. The filopodia (as indicated by arrows) of cells not only adhered flat, but also tightly grabbed the surface structure. **Figure 6.** (**a**) Cell viability of L929 of the Ni-Fe-TiO<sup>2</sup> nanocomposites for 24 h and cell morphologies of the Ni-Fe-TiO<sup>2</sup> nanocomposites after culturing with L929 cells for 3 days: (**b**) Ni-Fe-5 wt.% TiO<sup>2</sup> , (**c**) Ni-Fe-10 wt.% TiO<sup>2</sup> , and (**d**) Ni-Fe-20 wt.% TiO<sup>2</sup> . The filopodia (as indicated by arrows) of cells not only adhered flat, but also tightly grabbed the surface structure.

#### **3. Discussion 3. Discussion**

**(a)**

**Cell viability (%)**

**Control (medium only)**

**Blank (medium only)**

**0**

**20**

**40**

**60**

**80**

**100**

In this study, it was found that the electrodeposited nanocomposite produced better smooth sidewall and surface as well as structural integrity, revealing that the Ni-Fe alloy with TiO2 nanoparticles promoted formability. To explore possible reinforcement factors in nanocomposite materials, we compared results from different groups of nanocomposites including a traditional matrix of polycrystalline Ni containing reinforced nanoparticles of Al2O3 [30]. Oberle et al. [31] reported that with large hardness and mechanical strength enhancement in the composite matrix, the volume fraction of the co-deposited In this study, it was found that the electrodeposited nanocomposite produced better smooth sidewall and surface as well as structural integrity, revealing that the Ni-Fe alloy with TiO<sup>2</sup> nanoparticles promoted formability. To explore possible reinforcement factors in nanocomposite materials, we compared results from different groups of nanocomposites including a traditional matrix of polycrystalline Ni containing reinforced nanoparticles of Al2O<sup>3</sup> [30]. Oberle et al. [31] reported that with large hardness and mechanical strength enhancement in the composite matrix, the volume fraction of the co-deposited oxide reinforcements with 50 nm and 300 nm particle sizes is relatively low (i.e., 1–2% volume). Muller et al. [32] also indicated that there was a hardness and mechanical strength increased in the co-deposited Ni nanocomposite matrix containing at least 23 vol.% of Al2O<sup>3</sup> (average 14 nm in diameter), and grain size of Ni is over 50 nm. However, the fabricated Ni-Fe with reinforced TiO<sup>2</sup> nanoparticles nanocomposite exhibited a similar microstructure texture with a grain size of ~50 nm. The grain size decreased when TiO<sup>2</sup> nanoparticles were added, which indicated that the nanocrystallization effect would occur due to the reactions of energetic ions in the solution during electrodeposition [22,33].

For a small matrix, the grain size was only meaningful within a limited range when used in nanocrystalline range dislocation models. The concept of the original dislocation model of the Hall-Petch relationship was based on that grain boundaries were played as barriers to dislocation movement, so a pile-up of dislocation formed at grain boundaries. As the length of pile-up with a range of 10~100 nm, the models of pile-up become doubtful. For example, the nanocrystalline range of dislocations in a pile-up rapidly reduced when the size of the grain decreased [34]. Likewise, the typical mechanism of the Orowan type was unlikely to work in the investigated nanocomposite material, since the particles of reinforcing were one order of magnitude larger than the average grain size of the matrix at least, leading to a structure in which one hard particle was surrounded by numerous differently oriented grains in the matrix of Ni-Fe. Accordingly, the nanocomposite materials with higher mechanical strength were mainly owing to the nanocrystalline Ni matrix with

the presence of a reinforced second phase [35]. It is believed that the mechanical properties can be promoted for the Ni-Fe-TiO<sup>2</sup> nanocomposite.

Cytotoxicity testing is designed to assess the general toxicity of biomaterials and medical devices. The test consists of extracting the device in a cell culture medium and then exposing the extract to L929 mouse fibroblasts. ISO 10993-5 specification indicates reasonable cell viability of 70% under the MTT assay. Cell viability near or below 70% may be highly toxic to cells. In the present study, cell viability results showed that the investigated Ni-Fe-TiO<sup>2</sup> nanocomposites had high cell viability of over 90% after culturing with L929 cells for 24 h. A high cell viability rate reveals that the investigated material possesses better cell proliferation behavior and excellent biocompatibility [36,37]. In addition, it was found that there was no statistically significant difference between investigated Ni-Fe-TiO<sup>2</sup> nanocomposites. These findings demonstrated the electrodeposited Ni-Fe-TiO<sup>2</sup> nanocomposites with different concentrations of TiO<sup>2</sup> nanoparticles did not influence the proliferation and adhesion behaviors of L929 cells. Nanostructured TiO<sup>2</sup> has broad potential applications due to its nanosized features, low toxicity, and good biocompatibility [38]. Mohammadi et al. [39] also indicated that the mechanical strength of calcium phosphate cement can be enhanced with TiO<sup>2</sup> nanoparticles addition in the short-term. As a result, it is believed that the electrodeposited Ni-Fe-TiO<sup>2</sup> nanocomposites can not only enhance cell viability but can also improve mechanical properties to obtain the desired results. As discussed above, the optimal Ni-Fe-TiO<sup>2</sup> nanocomposite can be fabricated through the UV-LIGA approach. The Ni-Fe-TiO<sup>2</sup> is a potential nanocomposite material that could be unitized as an endodontic file for dental applications. However, further studies should be performed to provide additional information concerning the mechanical properties including micro-hardness, tensile strength, torsional strength, and cyclic fatigue in the presence of electrodeposited Ni-Fe-TiO<sup>2</sup> nanocomposites.

#### **4. Materials and Methods**

#### *4.1. Materials Preparation*

In this study, a modified Watts bath solution [40] was adopted as a plating solution and TiO<sup>2</sup> nanoparticles with an average diameter of 40 nm (Merk Taiwan, Taipei, Taiwan) were used as reinforced material. The pH value was controlled at a range of 2.5–3.5. Ni carbonate and hydrochloric/sulphuric acid (ratio 1:9) were used for pH value adjustment. Before fabrication, the TiO<sup>2</sup> nanoparticles powder was slowly added to the modified Watts bath solution with continuous mixing to avoid the TiO<sup>2</sup> nanoparticles agglomeration. Subsequently, the TiO<sup>2</sup> slurry with different concentrations of 5 wt.%, 15 wt.%, and 20 wt.% was added to the solution of bulk in the final bath, respectively. Hereafter, the pulsed electrodeposition process was performed with a galvanostat/potentiostat electrochemical instrument (EG & G, Princeton Applied Research 263A, Artisan Technology Group, Champaign, IL, USA), which could control the direct or pulsed electrodepositions. The parameter of pulse plating of duty cycle (pulse on time divided by pulse on time plus pulse off time) was set in direct current between 30% and 100%. The peak current densities were up to 10 A/dm<sup>2</sup> . The stirring rate was calculated by means of a mechanical impeller to maintain the TiO<sup>2</sup> particulate in suspension. The time of plating was set constantly at 750 rpm for 3 h. The plating solution contained in the plating cell was kept in a water bath at 60 ◦C. The plating cell anode was made by the electrolytic Ni (purity 99.99%) containing Ti basket. The cathode was made by the Ti substrate (1 cm × 2 cm). Finally, the electrodeposits were mechanically removed from the Ti substrate (cathode side) to analyze surface and microstructural characterizations.

#### *4.2. Surface Characterization*

Surface morphology was studied by a JEOL JSM-6500F field emission scanning electron microscope (FE-SEM, Tokyo, Japan) equipped with a high-energy dispersive X-ray spectroscope (EDS; INCA, Oxford Instruments, Abingdon, UK). The operating voltage was kept at 20 kV. The samples were observed and analyzed under different magnifications.

#### *4.3. Microstructure Identification*

The model 657 dimple grinder (Gatan Inc., Pleasanton, CA, USA) and model 691 precision ion polishing system (Gatan Inc., Pleasanton, CA, USA) were used to prepare the TEM specimen with an electron transparent area. Hereafter, a model JEOL-2100 highresolution transmission electron microscope (TEM; JEOL Ltd., Tokyo, Japan) was used to research crystallinity and phase identification with an accelerating voltage of 200 kV.

#### *4.4. Cytotoxicity Assay*

In this study, the L929 RM60091 mouse fibroblast cell line (Bioresource Collection, and Research Center, Hsinchu, Taiwan) was used for cytotoxicity evaluation. The cells were seeded in culture dishes at a density of 5 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per 100 µL in α-Minimum Essential Medium (MEM; Level Biotechnology, New Taipei City, Taiwan). Cells from passage 2 were harvested at 80% confluence and used for further 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. The extracts of the investigated samples were placed in an orbital shaker maintained at 37 ◦C for 24 h with a mass to volume extraction ratio of 0.2 g/mL, which was followed by filtering and sealing in sterile bottles. L929 cells at a density of 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well were cultured in MEM and seeded on the 24-well culture plates. After obtaining a confluent monolayer, the medium was replaced by 0.1 mL sample extracts and incubated for 24 h at 37 ◦C in an atmosphere of 5% CO<sup>2</sup> (*n* = 5). Afterward, a 10 µL MTT assay kit (R&D system, Minneapolis, MN, USA) was added to each well and incubated for 2 h. The optical density value of each plate was read at 570 nm through the ELx800 microplate reader (BioTek, Winooski, VT, USA). According to ISO 10993-5 specification, the cell viability (%) in the short-term culturing (24 h) experiment was adopted to assess the material's acute cytotoxicity response.

#### *4.5. Cell Morphology Observation*

The morphology of L929 cells was observed after 3 days of culture. The adhered L929 cells were washed with PBS, placed in a fixative consisting of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h at 4 ◦C, rinsed in deionized water, and dehydrated in serial of ethanol solutions for 15 min each concentration. Hereafter, dehydrated samples were soaked in hexamethyldisilazane, sputter coated with platinum, and analyzed with JEOL-6500F FE-SEM at 25 kV under different magnifications.

#### *4.6. Statistical Analysis*

The experimental results with multiple readings are presented as mean ± standard deviation. Data were analyzed through the variance from the Student's *t*-test (Excel 2016 version, Microsoft Corporation, Redmond, WA, USA). P values ≤ 0.05 were considered statistically significant.

#### **5. Conclusions**

The present work fabricated a potential nanocomposite material consisting of anatase TiO<sup>2</sup> nanoparticles contained in the nanocrystalline matrix of Ni-Fe using electrodeposition from a modified Watts bath. The size of grain in the nanocrystalline Ni-Fe matrix decreased with the addition of the TiO<sup>2</sup> nanoparticles. The Ni-Fe-TiO<sup>2</sup> nanocomposite exhibited a smooth surface and structural integrity. The TiO<sup>2</sup> nanoparticles doped in the Ni-Fe alloy can facilitate formability. The electrodeposited Ni-Fe-TiO<sup>2</sup> nanocomposites with different concentrations of TiO<sup>2</sup> nanoparticles did not influence the proliferation and adhesion behaviors of cells. Therefore, the electrodeposited Ni-Fe-TiO<sup>2</sup> nanocomposite is a promising endodontic file material for dental applications.

**Author Contributions:** Writing—original draft, C.-W.C.; Investigation, C.-W.C. and C.-H.T. (Chen-Han Tsou); Data curation, C.-C.H. and C.-H.T. (Chi-Hsun Tsai); Methodology, B.-H.H.; Supervision, T.S.; Resources, Y.-C.C.; Validation, K.-S.H.; Writing—review & editing, W.-C.L. and C.-M.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors would like to thank the Zuoying Branch of Kaohsiung Armed Forces General Hospital and Taipei Medical University Hospital for financially supporting this research under contract no. KAFGH-ZY-A-110015 and 111-D-TMUH-003.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

