The Effect of Selenium Nanoparticles on the Osteogenic Differentiation of MC3T3-E1 Cells

Reactive oxygen species (ROS) regulate various functions of cells, including cell death, viability, and differentiation, and nanoparticles influence ROS depending on their size and shape. Selenium is known to regulate various physiological functions, such as cell differentiations and anti-inflammatory functions, and plays an important role in the regulation of ROS as an antioxidant. This study aims to investigate the effect of selenium nanoparticles (SeNPs) on the differentiation of osteogenic MC3T3-E1 cells. After fabrication of SeNPs with a size of 25.3 ± 2.6 nm, and confirmation of its oxidase-like activity, SeNPs were added to MC3T3-E1 cells with or without H2O2: 5~20 μg/mL SeNPs recovered cells damaged by 200 μM H2O2 via the intracellular ROS downregulating role of SeNPs, revealed by the ROS staining assay. The increase in osteogenic maturation with SeNPs was gradually investigated by expression of osteogenic genes at 3 and 7 days, Alkaline phosphatase activity staining at 14 days, and Alizarin red S staining at 28 days. Therefore, the role of SeNPs in regulating ROS and their therapeutic effects on the differentiation of MC3T3-E1 cells were determined, leading to possible applications for bone treatment.


Introduction
Bone remodeling is mediated by osteoblasts and osteoclasts, and osteoblasts are the main cells in bone formation and are responsible for the synthesis, secretion, and mineralization of the bone matrix, which accounts for 4-6% of cell content, forming bone [1,2]. If the balance between osteoblasts and osteoclasts is abnormal, bone diseases such as osteoporosis and inflammatory bone erosion occur, threatening human health [3]. During bone regeneration, reactive oxygen species (ROS) are inevitably produced [4].

Synthesis of Selenium Nanoparticles
Selenium nanoparticles (SeNPs) were selectively synthesized by the reduction of an aqueous solution of sodium selenite with ascorbic acid. In a typical SeNP synthesis procedure, 100 mM sodium selenite aqueous solution was mixed with 100 mM ascorbic acid, and the solution pH was adjusted to 7.1 using NaOH or HCl under rigorous magnetic stirring conditions. The resulting solution color changed to red and was centrifuged at 15,000 rpm for 30 min and washed with distilled water. The final solution was lyophilized to collect the SeNPs.

Peroxidase Activity of SeNPs
The oxidase-like activity of the SeNPs was determined as previously reported [53]. Briefly, we first performed the catalytic oxidation of the peroxidase substrate 3,3 ,5,5tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H 2 O 2 ). For the experiment, varying concentrations of SeNPs (10,20,40, and 80 µg/mL) were dispersed in a mixture containing 350 µL of acetate buffer (0.2 M), 300 µL of TMB (0.2 M), and finally 100 µL of H 2 O 2 (30%, 2 M). After uniform mixing, the mixture was incubated in the dark for approximately 30 min. Later, the supernatant was collected by centrifugation, and UVvisible absorption was performed in the range of 400-800 nm to confirm the oxidase-like activity of the SeNPs.

Cell Viability
Cell viability was measured using a cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan) assay. Briefly, MC3T3-E1 cells were seeded in a 96-well plate (Costar, Corning Incorporated, USA) at 1 × 10 4 cells per well, and after 24 h, cells were treated with SeNPs (10-320 µg/mL). After 30 min of treatment with SeNPs, high oxidative stress conditions were enabled by treatment with aqueous solution of H 2 O 2 in different concentrations (0, 100, 200, and 300 µM) for 24 h. Then, medium containing 10% CCK-8 solution was replaced in each well of the plate, followed by incubation for 2 h at 37 • C. After that, the absorbance of each well at 450 nm was recorded using a microplate reader (Thermo Fisher Varioskan TM LUX, Waltham, MA, USA). Cell viability (%) was quantified by the average absorbance of the selenium treatment group/average absorbance of the control group × 100%. Cell survival was also examined by live/dead staining (0.5 µM calcein AM and 2 µM ethidium homodimer-1 solutions, Thermo Fisher, USA), and images were taken using an optical microscope (IX71, Olympus, Tokyo, Japan).

ROS Staining
High oxidative stress conditions were enabled by pretreatment with H 2 O 2 for 4 h. ROS levels were measured using the Image-iT LIVE Green Reactive Oxygen Species Detection Kit (Invitrogen, Carlsbad, CA, USA). The cells were gently washed with Hanks' Balanced Salt solution (HBSS)/Ca/Mg twice and labeled with 25 µM of 6-carboxy-2 ,7dichlorodihydrofluorescein diacetate (carboxy-H 2 DCFDA) which is general oxidative stress indicator to cover the adherent cells for 30 min at 37 • C. The labeled cells were gently washed three times and observed using a microscope.

Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
MC3T3-E1 cells were cultured with or without SeNPs and H 2 O 2 for 7 days. Total RNA was isolated using an RNA preparation kit (Geneall, Songpa-gu, Seoul, Korea) according to the manufacturer's instructions, and the RNA concentration was measured using a Nanodrop (Thermo Fisher, USA). First-strand cDNA was developed using an RNA reverse transcription (RT) kit (Bioneer, Daeduk-gu, Daejeon, Korea) according to the manufacturer's instructions. PCR was performed using target gene expression levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels. The delta cycle threshold (Ct) method was used to calculate relative levels of expression. Primer sequence is as follows: Alkaline phosphatase activity(ALP) forward 5 -ACA CCT TGA CTG TGG TTA CT-3 , reverse 5 -CCT TGT AGC CAG GCC CGT TA-3 , Osterix forward 5 -CCC TTC TCA AGC ACC AAT GG-3 , reverse 5 -AGG GTG GGT AGT CAT TTG CA-3 , GAPDH forward 5 -GAG CAT CTC CCT CAC AAT TT-3 , reverse 5 -GGG TGC AGC GAA CTT TAT-3 .

Alkaline Phosphatase Activity (ALP)
MC3T3-E1 cells were seeded in 24-well plates at a density of 6 × 10 4 cells/well, and after 24 h, cells were treated with SeNPs (5-20 µg/mL). After 30 min of treatment with SeNPs, H 2 O 2 was treated to maintain high oxidative stress conditions and osteogenic differentiation medium was treated for 14 days. ALP staining was conducted by using a staining kit (Sigma-Aldrich, USA). The cells were washed with phosphate buffer solution (PBS, Tech and Innovation, Chuncheon, Korea) twice and fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. The fixed cells were washed with PBS three times and stained with ALP solution dissolved in distilled water for 1 h at 37 • C. Stained cells were washed with PBS three times and observed using an optical microscope. After staining, quantification was performed using ImageJ software (Bethesda, MD, USA).

Alizarin Red S Staining (ARS)
Alizarin red S (ARS, Sigma-Aldrich, USA) staining was performed to determine MC3T3-E1 mineralization. MC3T3-E1 cells were seeded in 24-well plates at a density of 6 × 10 4 cells/well in osteogenic differentiation medium for 28 days. The cells were washed twice with PBS and then fixed with 4% PFA for 30 min. The fixed cells were also washed 3 times with PBS and subsequently stained with 40 mM ARS solution for 10 min. The remaining solution was washed out 5 times with distilled water and observed using a microscope. The stained calcium deposits were dissolved in 10% (w/v) cetylpyridinium chloride (CPC, Sigma-Aldrich, MO, USA) solution on a rocking shaker. After 1 h, the eluted solutions were transferred to 96-well plates and measured at 562 nm using a microplate reader.

Statistical Analysis
Statistical significance between groups was evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons tests or two-way ANOVA followed by Dunnett's multiple comparisons tests. GraphPad Prism 8 software (San Diego, CA, USA) was used.

Characterization of SeNPs
The morphology and shape of the sol-gel synthesized SeNPs are shown in the transmission electron microscopy (TEM) image in Figure 1a. The SeNPs were spherical in shape with a size of 25.3 ± 2.6 nm, and the particle size distribution was analyzed by ImageJ software. Figure 1b shows the particle size distribution histogram from 18 to 32 nm. The XRD pattern of SeNPs is shown in Figure 1c and exhibits two broad peaks at 2θ = 20-30 • and 45-55 • , demonstrating the crystalline and amorphous nature of the particles [54]. The FTIR spectra of the SeNPs showed four main peaks, as shown in Figure 1d. The sharp and intense peak at 2919.5 cm −1 corresponds to the -CH group, the peak at 1592.53 cm −1 corresponds to the -COO group, and the peaks at 1112.05 and 557.68 cm −1 correspond to the -CO and Se-O groups, respectively [55,56]. Furthermore, the surface charge of the SeNPs was estimated to be −13.9 mVas, as shown in Figure 1e, which is mainly due to the presence of -COO, -CO, and -OH groups on the surface, as verified by FTIR analysis. Higher zeta potentials, either negative or positive, provide stable particle colloidal suspensions. The optical properties of the SeNPs were analyzed by UV-visible spectroscopy. The absorption spectra of the SeNPs are depicted in Figure 1f. The SeNPs exhibit a broad absorption peak at 321.4 nm [57].

Oxidase-Like Activity
The oxidase-like activity of the SeNPs was performed using the TMB assay. Figure

Oxidase-Like Activity
The oxidase-like activity of the SeNPs was performed using the TMB assay. Figure 2a shows the optical image of the TMB and SeNPs with concentration from 10 to 80 µg/mL in TMB and produces typical light blue color after 30 min of reaction. The changing of color of SeNPs in TMB to blue in the presence of H 2 O 2 is the confirmation of oxidase-like activity of SeNPs. After confirmation of oxidase-like activity of SeNPs, we performed the UV-visible absorption for the quantification, and the maximum absorption peak of the oxidized TMB products was observed at 652 nm. The UV-visible absorption spectrum of the TMB solutions before (Reference) and after reaction (0 µg/mL) and SeNPs in TMB solutions after reaction of 30 min is shown in Figure 2a. The oxidized products with TMB solution in the presence of SeNPs shows higher absorption peak intensity at 652 nm, which was quantified. The quantitative analysis of the absorption values of the TMB and SeNPs in TMB solution are shown in Figure 2b. The intensity of the absorption peak at 652 nm increases with increasing the SeNPs concentrations, which confirmed the oxidation of TMB and intrinsic oxidase-like activity of the prepared SeNPs. Moreover, it is common to increase the oxidase activity with increasing the concentration of the nanoparticles, as reported in other reports [58]. It has also been reported that the smaller the nanoparticles' size, the higher the oxidase activity because the smaller size would expose more active sites due to the high surface-to-volume ratio [59]. Overall, our results suggest that 40 µg/mL concentration of SeNPs is the optimal concentration to achieve effective oxidase activity.

Effect of Selenium Nanoparticles on Cell Viability in MC3T3-E1-Induced ROS Conditions
Thirty-two milligrams of SeNPs were quantified and dissolved in PBS. After that, the selenium stock was diluted to the highest concentration of 320 μg/mL and used to treat MC3T3-E1 cells, followed by serial dilution by 1/2. As a result of treatment with SeNPs,

Effect of Selenium Nanoparticles on Cell Viability in MC3T3-E1-Induced ROS Conditions
Thirty-two milligrams of SeNPs were quantified and dissolved in PBS. After that, the selenium stock was diluted to the highest concentration of 320 µg/mL and used to treat MC3T3-E1 cells, followed by serial dilution by 1/2. As a result of treatment with SeNPs, MC3T3-E1 cell viability was shown at a concentration of 40 µg/mL or less, confirmed by Live/Dead staining ( Figure 3a) and CCK-8 assay (Figure 3b), respectively. In the case of treatment of MC3T3-E1 cells with hydrogen peroxide, Live/Dead staining images showed that cells survived in H 2 O 2 concentrations of 400 µM or less (Figure 3c). As a result of treatment with SeNPs and H 2 O 2 together, cell viability increased compared to the untreated group when selenium was applied, and the SeNP-treated group showed an approximately 17.24-39.58% increase in cell viability compared to the SeNP-untreated group, when 300 µM H 2 O 2 was treated (Figure 3d). This confirmed the cell viability at a relatively higher selenium concentration compared to previous studies that treated MC3T3-E1 cells with selenium at 0-800 ng/mL [60].  . The statistical significance of (b) was calculated using one-way analysis of variance (ANOVA), and (d) was calculated using two-way ANOVA followed by a two-sided Dunnett's multiple comparison test compared to control (CTL) (scale bar = 350 μm). * Represents p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, # is compared with SeNPs and H2O2-untreated groups. # Represents p < 0.05, #### p < 0.0001; n = 4.

ROS Staining
Because ROS generation is mostly governed by mitochondria, loss of the mitochondrial membrane triggers ROS generation, and increased ROS production leads to further mitochondrial disruption. We thus examined whether SeNP treatment affected ROS pro- The statistical significance of (b) was calculated using one-way analysis of variance (ANOVA), and (d) was calculated using two-way ANOVA followed by a two-sided Dunnett's multiple comparison test compared to control (CTL) (scale bar = 350 µm). * Represents p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, # is compared with SeNPs and H 2 O 2 -untreated groups. # Represents p < 0.05, #### p < 0.0001; n = 4.

ROS Staining
Because ROS generation is mostly governed by mitochondria, loss of the mitochondrial membrane triggers ROS generation, and increased ROS production leads to further mitochondrial disruption. We thus examined whether SeNP treatment affected ROS production. To measure ROS production, we used 5-(and-6)-carboxy-29,79-dichlorodihydrofluorescein diacetate (carboxy H 2 DCFDA) after SeNP and 400 µM H 2 O 2 treatment. As shown in Figure 4a, control cells showed a large number of cells stained with fluorescence. In contrast, the cells treated with 5 µg/mL SeNPs showed weak fluorescence, indicating that 5 µg/mL SeNPs efficiently controlled ROS. Furthermore, it has been suggested that ROS may affect several cellular activities. Additionally, Figure 4b shows the results obtained by analyzing the intensity and positive area of the staining value using ImageJ. As a result of the intensity analysis of fluorescently stained cells, the SeNP-treated group showed lower intensity than the untreated group, and the highest decrease was observed at 5 µg/mL SeNPs. This result suggests that selenium nanoparticles can be involved in various activities of cells by regulating ROS, in addition to previous studies showing that SeNPs act as antioxidants [61,62].  The fluorescence intensity of cells and ROS-positive areas was measured using ImageJ. Statistical significance was calculated using one-way ANOVA followed by a two-sided Dunnett post hoc test compared to CTL (scale bar = 350 μm). **** Represents p < 0.0001; n = 5.

Effect of Selenium Nanoparticles on the Expression of Osteogenic Genes Determined by qRT-PCR
qRT-PCR was used to investigate the expression levels of osterix, one of the major osteoblast transcription factors in bone formation [63], and ALP, one of the most reliable markers for osteogenic differentiation produced by osteogenic cells [64][65][66], to determine the effect of SeNPs on the expression levels of MC3T3-E1 cells. Figure 5a shows that treatment with SeNPs for 3 days resulted in an increase in the expression of the osteogenic genes analyzed. For osterix, the value of the negative control group was 1.00 ± 0.05, the positive control was 1.19 ± 0.06, SeNPs at 5 μg/mL was 1.47 ± 0.03, SeNPs at 10 μg/mL was The fluorescence intensity of cells and ROS-positive areas was measured using ImageJ. Statistical significance was calculated using one-way ANOVA followed by a two-sided Dunnett post hoc test compared to CTL (scale bar = 350 µm). **** Represents p < 0.0001; n = 5.

Effect of Selenium Nanoparticles on the Expression of Osteogenic Genes Determined by qRT-PCR
qRT-PCR was used to investigate the expression levels of osterix, one of the major osteoblast transcription factors in bone formation [63], and ALP, one of the most reliable markers for osteogenic differentiation produced by osteogenic cells [64][65][66], to determine the effect of SeNPs on the expression levels of MC3T3-E1 cells. Figure 5a shows that treatment with SeNPs for 3 days resulted in an increase in the expression of the osteogenic genes analyzed. For osterix, the value of the negative control group was 1.00 ± 0.05, the positive control was 1.19 ± 0.06, SeNPs at 5 µg/mL was 1.47 ± 0.03, SeNPs at 10 µg/mL was 1.47 ± 0.13, and SeNPs at 20 µg/mL was 1.51 ± 0.03. In the case of ALP, the value of the negative control group was 1.00 ± 0.01, the positive control was 2.41 ± 0.04, SeNPs at 5 µg/mL was 3.40 ± 0.09, SeNPs at 10 µg/mL was 3.48 ± 0.04, and SeNPs at 20 µg/mL was 3.33 ± 0.07. Figure 5b shows the results after treatment with SeNPs for 7 days. In the case of osterix, the value of the negative control group was 1.00 ± 0.14, the positive control was 1.32 ± 0.02, SeNPs at 5 µg/mL was 1.60 ± 0.13, SeNPs at 10 µg/mL was 1.45 ± 0.04, and SeNPs at 20 µg/mL was 1.05 ± 0.01. According to the ALP expression results, the value of the negative control group was 1.00 ± 0.01, the positive control was 2.19 ± 0.66, SeNPs at 5 µg/mL was 2.86 ± 0.08, SeNPs at 10 µg/mL was 2.75 ± 0.05, and SeNPs at 20 µg/mL was 2.55 ± 0.15. In the group treated with selenium, the activities of osterix and ALP were higher than those in the group not treated with selenium. (b) Results of qRT-PCR analysis on the seventh day after treatment with osteogenic differentiation media. The selenium-treated group showed higher osterix and ALP activity than the selenium-treated group, and the highest gene expression was confirmed at 5 μg/mL. Statistical significance was calculated using one-way ANOVA followed by a two-sided Dunnett post hoc test compared to CTL (N.C: Negative control, P.C: Positive control). * Represents p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3.

Effect of Selenium Nanoparticles on Cell Differentiation
To assess whether SeNPs were effective for the differentiation of MC3T3-E1 cells, ALP activity was measured at 14 days. Figure 6a shows the ALP staining data of MC3T3-E1 cells treated with SeNPs for 14 days. ALP staining was performed for 14 days after cells

Osterix (Day 3) ALP (Day 3)
Osterix (Day 7) ALP (Day 7) Figure 5. The effect of SeNPs on the expression of osteogenic genes through qRT-PCR. The relative expression levels of target genes normalized to GAPDH were calculated using the delta cycle threshold (Ct) method. The figure shows the relative expression of multiple genes relative to gene expression in the negative control treatment cells. (a) Results of qRT-PCR analysis of osteogenic markers on the third day after treatment with osteogenic differentiation media. In the group treated with selenium, the activities of osterix and ALP were higher than those in the group not treated with selenium. (b) Results of qRT-PCR analysis on the seventh day after treatment with osteogenic differentiation media. The selenium-treated group showed higher osterix and ALP activity than the selenium-treated group, and the highest gene expression was confirmed at 5 µg/mL. Statistical significance was calculated using one-way ANOVA followed by a two-sided Dunnett post hoc test compared to CTL (N.C: Negative control, P.C: Positive control). * Represents p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3.

Effect of Selenium Nanoparticles on Cell Differentiation
To assess whether SeNPs were effective for the differentiation of MC3T3-E1 cells, ALP activity was measured at 14 days. Figure 6a shows the ALP staining data of MC3T3-E1 cells treated with SeNPs for 14 days. ALP staining was performed for 14 days after cells were treated with osteogenic differentiation media. The results of ALP staining in 5-10 µg/mL SeNP-treated cells were higher than those in positive control cells. In addition, even at 50 µM H 2 O 2 , the staining was higher in the 5-10 µg/mL SeNP-treated group than in the SeNP-untreated group. Figure 6b showed quantification of ALP staining. Both H 2 O 2 -treated and untreated groups showed high ALP activity at SeNP concentrations of 5-10 µg/mL. Based on these results, it was confirmed that SeNPs express early osteogenic markers such as ALP in the H 2 O 2 environment. As a result of ALP staining, higher ALP activity was confirmed in H 2 O 2 untreated group. Therefore, the mineralization of MC3T3-E1 cells were investigated through ARS staining in the H 2 O 2 -untreated group. Another osteoblast differentiation marker, ARS staining, was used to evaluate the level of differentiation. The effects of SeNPs on the development of calcium deposits in MC3T3-E1 cells were analyzed by ARS staining. Figure 6c shows that SeNPs promoted the mineralization function of MC3T3-E1 cells. Cells treated with osteogenic differentiation media (ODM) and 5-10 µg/mL SeNPs showed higher staining than the group treated only with ODM, and the group treated with 50 µM H 2 O 2 showed lower staining than the selenium-treated group. Figure 6d shows a graph quantified by adding CPC solution to the stained well after ARS staining. In the 5-10 µg/mL SeNP-treated group showed higher mineralization than the SeNPs untreated group. Through these results, it was confirmed that SeNPs at a concentration of 5-10 µg/mL promote the differentiation of MC3T3-E1 cells. In addition to previous studies reporting improved cell adhesion and osteoblast function [36], SeNPs protect against ROS-induced damage and are related to cell growth and differentiation regulation [67][68][69], confirming that they also affect the differentiation of cells. were analyzed by ARS staining. Figure 6c shows that SeNPs promoted the mineralization function of MC3T3-E1 cells. Cells treated with osteogenic differentiation media (ODM) and 5-10 μg/mL SeNPs showed higher staining than the group treated only with ODM, and the group treated with 50 μM H2O2 showed lower staining than the selenium-treated group. Figure 6d shows a graph quantified by adding CPC solution to the stained well after ARS staining. In the 5-10 μg/mL SeNP-treated group showed higher mineralization than the SeNPs untreated group. Through these results, it was confirmed that SeNPs at a concentration of 5-10 μg/mL promote the differentiation of MC3T3-E1 cells. In addition to previous studies reporting improved cell adhesion and osteoblast function [36], SeNPs protect against ROS-induced damage and are related to cell growth and differentiation regulation [67][68][69], confirming that they also affect the differentiation of cells.

Conclusions
The purpose of this study was to investigate the ROS regulation ability and efficiency of SeNPs in MC3T3-E1 cells for ROS produced during the cell differentiation (Figure 7). In this study, as a result of analyzing the cell viability after treatment of MC3T3-E1 cells with SeNP and H2O2 together, cell viability was confirmed at a SeNP concentration of 20 μg/mL or less and H2O2 concentration of 200 μM or less. In addition, it was confirmed that The formation of calcium deposits is indicated by ARS staining. After treatment with different concentrations of SeNPs, cells were stained with ARS 28 days after treatment with osteogenic differentiation medium. Higher calcium deposition was observed in the group treated with 5 µg/mL selenium and 10 µg/mL selenium without H 2 O 2 . (d) Results of quantification of ARS staining using CPC extraction. Statistical significance was calculated using one-way ANOVA followed by a two-sided Dunnett post hoc test compared to CTL. * Represents p < 0.05, *** p < 0.001, **** p < 0.0001.

Conclusions
The purpose of this study was to investigate the ROS regulation ability and efficiency of SeNPs in MC3T3-E1 cells for ROS produced during the cell differentiation (Figure 7). In this study, as a result of analyzing the cell viability after treatment of MC3T3-E1 cells with SeNP and H 2 O 2 together, cell viability was confirmed at a SeNP concentration of 20 µg/mL or less and H 2 O 2 concentration of 200 µM or less. In addition, it was confirmed that SeNP can control ROS and protect cells by treating H 2 O 2 . As a result of qRT-PCR to investigate the expression level of the osteogenic differentiation marker, it was confirmed that gene expression increased in the SeNP (5-10 µg/mL) treatment group compared to the control group 3 and 7 days after treatment with the osteogenic differentiation medium. We analyzed the effect of SeNP on the differentiation of MC3T3-E1 cells at high free radical concentrations. As a result of ALP staining 14 days after the osteogenic differentiation medium treatment, it was confirmed that ALP activity was higher in the 5-10 µg/mL SeNP-treated group and the untreated group. As a result of ALP activity analysis, higher ALP activity was confirmed in the H 2 O 2 untreated group than in the treated group, and the degree of calcification of MC3T3-E1 cells according to SeNP treatment proceeded only in the H 2 O 2 untreated group. ARS staining was performed 28 days after treatment with bone differentiation medium. In the H 2 O 2 untreated group, the SeNPtreated group showed higher staining at a concentration of 5-10 µg/mL compared to the untreated group. Based on these results, the treatment of SeNPs seems to have an effect on early differentiation but not on late differentiation. These results suggest that SeNPs may affect the differentiation of MC3T3-E1 cells. In addition, previous studies have shown that SeNP protects LPS-treated MC3T3-E1 cells from death through regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and improves cell adhesion and osteoblast function. However, in this paper, we confirmed the potential as a bone therapy through studies on whether SeNP, which has not been studied previously, modulates ROS and affects the differentiation of MC3T3-E1 cells. We analyzed the effect of SeNP on the differentiation of MC3T3-E1 cells at high free radical concentrations. As a result of ALP staining 14 days after the osteogenic differentiation medium treatment, it was confirmed that ALP activity was higher in the 5-10 μg/mL SeNP-treated group and the untreated group. As a result of ALP activity analysis, higher ALP activity was confirmed in the H2O2 untreated group than in the treated group, and the degree of calcification of MC3T3-E1 cells according to SeNP treatment proceeded only in the H2O2 untreated group. ARS staining was performed 28 days after treatment with bone differentiation medium. In the H2O2 untreated group, the SeNP-treated group showed higher staining at a concentration of 5-10 μg/mL compared to the untreated group. Based on these results, the treatment of SeNPs seems to have an effect on early differentiation but not on late differentiation. These results suggest that SeNPs may affect the differentiation of MC3T3-E1 cells. In addition, previous studies have shown that SeNP protects LPS-treated MC3T3-E1 cells from death through regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and improves cell adhesion and osteoblast function. However, in this paper, we confirmed the potential as a bone therapy through studies on whether SeNP, which has not been studied previously, modulates ROS and affects the differentiation of MC3T3-E1 cells.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.