Silver Nanoparticles Alone or in Combination with Calcium Hydroxide Modulate the Viability, Attachment, Migration, and Osteogenic Differentiation of Human Mesenchymal Stem Cells

This study aimed to evaluate the effect of silver nanoparticles (AgNPs) alone or in combination with calcium hydroxide (Ca(OH)2) on the proliferation, viability, attachment, migration, and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Different concentrations of AgNPs alone or mixed with Ca(OH)2 were prepared. Cell proliferation was measured using AlamarBlue, and hMSCs attachment to dentin disks was evaluated using scanning electron microscopy. Live–dead imaging was performed to assess apoptosis. Wound healing ability was determined using the scratch-migration assay. To evaluate osteogenic differentiation, the expression of Runt-related transcription factor (RUNX2), Transforming growth factor beta-1 (TGF-β1), Alkaline Phosphatase (ALP), and Osteocalcin (OCN) were measured using real-time reverse transcriptase polymerase chain reaction. ALP staining and activity were also performed as indicators of osteogenic differentiation. AgNPs alone seemed to favor cell attachment. Lower concentrations of AgNPs enhanced cell proliferation. AgNP groups showed markedly less apoptosis. None of the medicaments had adverse effects on wound closure. The expression of TGF-β1 was significantly upregulated in all groups, and OCN was highly expressed in the AgNP groups. AgNPs 0.06% showed the most enhanced ALP gene expression levels, activity, and marked cytochemical staining. In conclusion, AgNPs positively affect hMSCs, making them a potential biomaterial for various clinical applications.


Introduction
Treatment of immature necrotic teeth is considered one the most challenging procedures in Endodontics. Long-term calcium hydroxide (Ca(OH) 2 ) apexification was the original proposed treatment modality for immature necrotic teeth [1][2][3]. Due to the increase in root fracture and the need for high patient compliance, apexification later evolved into a one-step method with mineral trioxide aggregate (MTA) [4][5][6][7][8]. Even though both methods have comparable success rates, neither can achieve continued root development or restoration of pulp tissues [9,10].
This led to a paradigm shift towards endodontic regeneration. Regenerative endodontic procedures (REPs) are defined as: biologically based procedures designed to physiologically replace damaged tooth structures, including dentin and root structures, as well as cells of the pulp-dentin complex [11,12]. The primary goal of endodontic treatment is elimination of disease, but REPs also aim to gain root thickness and length, and restore the functions of the pulp-dentin complex [13,14]. In addition to the classic tissue engineering triad (stem cells, scaffolds, and growth factors), REPs have been found to rely on a new fourth pillar: root canal disinfection [15,16].
The nature of dental pulp (endodontic) pathosis was proven to be microorganismmediated [17,18]. Therefore, root canal disinfection is a fundamental step in endodontics, especially in REPs. A recent systematic review showed that 79% of failed regeneration cases presented with persistent infection [16]. Given the complex microbiota of the root canal system, many intracanal medicaments have been proposed for endodontic disinfection, such as calcium hydroxide [Ca(OH) 2 ] and triple antibiotic paste [TAP]. Despite the clinical efficacy of these medicaments, many limitations have been reported; including toxicity, discoloration, and antibiotic resistance [19][20][21][22][23].
The potential toxicity of AgNPs is a concern. However, this is dependent on many factors, and the mechanism is poorly understood [59,60]. An in vitro and in vivo study reported that smaller AgNPs caused more cytotoxicity, oxidative stress and pulmonary inflammation than larger AgNPs [61]. In contrast, another investigation reported that AgNP size did not play a role in the viability of the tumor cells studied [62]. Moreover, AgNPs in higher concentrations ranging from 0.1-5% suppressed neural outgrowth and reduced cell viability when evaluated on rat cortical cell cultures [63]. The differences in AgNP size, dose, concentration, duration of action, and the cell lines studied might explain the variations in the literature in regard to the cytotoxicity of AgNPs [59]. Nevertheless, it is crucial to identify an adequate concentration of AgNPs, with or without Ca(OH) 2 , which can eradicate microorganisms without hindering the healing process or harming the surrounding stem cells. Therefore, this study aimed to evaluate the effect of AgNPs alone or in combination with Ca(OH) 2 on the proliferation, viability, attachment, migration, and osteogenic differentiation of hMSCs.

Results
For UV-Vis, an absorption band at around 190-1100 nm wavelength was used. The sample presented the characteristic surface plasmon of silver nanoparticles, and showed three peaks: two narrow bands at 307 nm and 322 nm, and a broader band at 386 nm. The interband transitions of electrons caused an absorption that was detected over a broad range (approximately 250 nm-325 nm) ( Figure 1). The intensity of the localized surface plasmon resonance (LSPR) band continued to increase, shifted to a shorter wavelength, and narrowed down with further UV irradiation. Compositional analysis of the elements was conducted using EDS to conf istence and purity of AgNPs. A noticeable difference in the percentages of c oxygen (O), and silver (Ag) elements is shown in Figure 2A. Generally, around optical absorption peak of silver appeared. Some particles got oxidized and agg by forming silver oxide, as indicated by the presence of oxygen, but the quantit small. SEM imaging showed crystalline particles that were spherical, semi-i shape, and well distributed ( Figure 2B).  Compositional analysis of the elements was conducted using EDS to confirm the existence and purity of AgNPs. A noticeable difference in the percentages of carbon (C), oxygen (O), and silver (Ag) elements is shown in Figure 2A. Generally, around 3 keV, the optical absorption peak of silver appeared. Some particles got oxidized and agglomerated by forming silver oxide, as indicated by the presence of oxygen, but the quantity was very small. SEM imaging showed crystalline particles that were spherical, semi-identical in shape, and well distributed ( Figure 2B). Compositional analysis of the elements was conducted using EDS to confirm the existence and purity of AgNPs. A noticeable difference in the percentages of carbon (C), oxygen (O), and silver (Ag) elements is shown in Figure 2A. Generally, around 3 keV, the optical absorption peak of silver appeared. Some particles got oxidized and agglomerated by forming silver oxide, as indicated by the presence of oxygen, but the quantity was very small. SEM imaging showed crystalline particles that were spherical, semi-identical in shape, and well distributed ( Figure 2B). FTIR helps analyze multi-component systems and provides necessary information pertaining the material's phase composition and type of interactions between various compounds and polymers. In this study, FTIR was employed to detect the purity of AgNPs. The FTIR spectrum ( Figure 3) showed the fingerprint zones at the 500-400 cm −1 , which are identical to the vibration frequency of Ag-O ionic bond groups. FTIR helps analyze multi-component systems and provides necessary information pertaining the material's phase composition and type of interactions between various compounds and polymers. In this study, FTIR was employed to detect the purity of AgNPs. The FTIR spectrum ( Figure 3) showed the fingerprint zones at the 500-400 cm −1 , which are identical to the vibration frequency of Ag-O ionic bond groups.

AgNPs Favor Cell Attachment
Representative SEM images are shown in Figure 4. HMSCs were grown on dentin disks immersed in the test medicaments for either 7 or 14 days. Calcium hydroxide and TAP samples showed altered cell morphology (rounder) and more debris, while AgNPs alone favored cell attachment. The combination groups (Ca(OH)2 + AgNPs) exhibited elongated cells that appeared adhered to dentin. For all groups, day 14 samples showed a higher number of cells that are more widely distributed than day 7 samples.
Lower Concentrations of AgNPs Enhance Cell Proliferation: AlamarBlue results are shown in Figure 5.

AgNPs Favor Cell Attachment
Representative SEM images are shown in Figure 4. HMSCs were grown on dentin disks immersed in the test medicaments for either 7 or 14 days. Calcium hydroxide and TAP samples showed altered cell morphology (rounder) and more debris, while AgNPs alone favored cell attachment. The combination groups (Ca(OH) 2 + AgNPs) exhibited elongated cells that appeared adhered to dentin. For all groups, day 14 samples showed a higher number of cells that are more widely distributed than day 7 samples.  Lower Concentrations of AgNPs Enhance Cell Proliferation: AlamarBlue results are shown in Figure 5. All groups exhibited a trend towards increased cell proliferation on day 5, except for [AgNPs 0.06% + Ca(OH) 2 ], Ca(OH) 2, and TAP. The highest concentration of the combination groups [AgNPs 0.06% + Ca(OH) 2 ] showed a gradual reduction in cell proliferation in a similar manner to Ca(OH) 2 and TAP (p < 0.05). The [AgNPs 0.03% + Ca(OH) 2 ] group had significantly higher cell proliferation across all time points when compared with treatment controls Ca(OH) 2

AgNPs Induce Less Apoptosis
Representative images from the acridine orange/ethidium bromide (AO/EtBr) staining on days 1 and 2 post-treatment are shown in Figure 6, and the calculated percentages of dead cells on day 2 are shown in Figure 7. On day 2, increased cell proliferation was evident in all groups. In addition, cells treated with TAP exhibited more apoptotic bodies

AgNPs Induce Less Apoptosis
Representative images from the acridine orange/ethidium bromide (AO/EtBr) staining on days 1 and 2 post-treatment are shown in Figure 6, and the calculated percentages of dead cells on day 2 are shown in Figure 7. On day 2, increased cell proliferation was evident in all groups. In addition, cells treated with TAP exhibited more apoptotic bodies (condensed chromatin) and a higher number of necrotic cells (red dots) than the remaining groups (Figures 6 and 7). However, these changes were mainly observed on Day 2 ( Figures 6 and 7).    AgNPs Have No Adverse Effects on Wound Closure: Wound closure was quantified, and the results are shown in Figure 8A. Resu cate that the wound healing percentages significantly increased with time for each and none of the medicaments decreased the migration ability at any time. On th hand, TAP exhibited the least migration ability among all groups. This differen significant compared with the control and 3 experimental groups, as shown in Fig  The combination group AgNPs 0.03%+ Ca(OH)2 appeared to have the highest per of wound closure among all groups. However, this was only significant when com with TAP. Representative images are shown in Figure 8B.
All Medicaments Enhanced Osteogenic Marker Expression: For data simplification, the gene expressions of the test medicaments only (Ag combination and alone) are presented in Figure 9. On day 10 of osteoblastic diff tion, the expression of TGF-β1 was significantly upregulated in all groups compar their levels on day 5 and the negative control. On day 10, the most potent comb groups [(AgNPs 0.06% and 0.04%+ Ca(OH)2)] showed the highest expression of T with a 22 (p < 0.05) and 12.7-fold increase, respectively. Conversely, the two lowe centrations of AgNPs (in combination or alone) resulted in expression levels com AgNPs Have No Adverse Effects on Wound Closure: Wound closure was quantified, and the results are shown in Figure 8A. Results indicate that the wound healing percentages significantly increased with time for each group, and none of the medicaments decreased the migration ability at any time. On the other hand, TAP exhibited the least migration ability among all groups. This difference was significant compared with the control and 3 experimental groups, as shown in Figure 8A. The combination group AgNPs 0.03%+ Ca(OH) 2 appeared to have the highest percentage of wound closure among all groups. However, this was only significant when compared with TAP. Representative images are shown in Figure 8B.
All Medicaments Enhanced Osteogenic Marker Expression: For data simplification, the gene expressions of the test medicaments only (AgNPs in combination and alone) are presented in Figure 9. On day 10 of osteoblastic differentiation, the expression of TGF-β1 was significantly upregulated in all groups compared with their levels on day 5 and the negative control. On day 10, the most potent combination groups [(AgNPs 0.06% and 0.04%+ Ca(OH) 2 )] showed the highest expression of TGF-β1, with a 22 (p < 0.05) and 12.7-fold increase, respectively. Conversely, the two lowest concentrations of AgNPs (in combination or alone) resulted in expression levels comparable to the positive control.  All groups significantly upregulated ALP on days 5 and 10 compared with the negative control. On day 10, the expression of ALP was significantly elevated in three groups [AgNPs 0.06%, AgNPs 0.04% and AgNPs 0.06% %+ Ca(OH)2] (p < 0.05). The remaining medicaments exhibited similar expression levels to the positive control.
AgNPs at 0.06% Enhance Alkaline Phosphatase Activity and Staining: All the tested molecules upregulated the ALP activity with time ( Figure 10A), concordant with an increase in the intensity of the ALP cytochemical staining ( Figure 10B). Day 5 levels of ALP activity were similar in all groups. However, on day 10, AgNPs 0.06% showed the highest ALP activity, and this increase significantly differed from all remaining groups. The Runx2 expression was significantly upregulated on day 5 compared with the negative control (p < 0.05). The mixture of AgNPs + Ca(OH) 2 showed higher levels compared with the AgNPs alone and the positive control. The AgNPs 0.03% + Ca(OH) 2 showed the highest expression levels of Runx2 on both day 5 and day 10, compared with the rest of the groups. All groups exhibited a down-regulated expression of Runx2 with time (p < 0.05).
On day 10, the expression of OCN showed the most dramatic increase in the AgNPs 0.06% (1.5-fold) and AgNPs 0.04% (2.84-fold) groups when compared with their levels on day 5 (p < 0.05). These two groups also upregulated the day 10 expression of OCN significantly more than all the other groups (p < 0.05). The OCN expression levels on day 10 were not very different when comparing the combination groups (Ca(OH) 2 and AgNPs) to the positive control.
All groups significantly upregulated ALP on days 5 and 10 compared with the negative control. On day 10, the expression of ALP was significantly elevated in three groups [AgNPs 0.06%, AgNPs 0.04% and AgNPs 0.06% + Ca(OH) 2 ] (p < 0.05). The remaining medicaments exhibited similar expression levels to the positive control.
AgNPs at 0.06% Enhance Alkaline Phosphatase Activity and Staining: All the tested molecules upregulated the ALP activity with time ( Figure 10A), concordant with an increase in the intensity of the ALP cytochemical staining ( Figure 10B). Day 5 levels of ALP activity were similar in all groups. However, on day 10, AgNPs 0.06% showed the highest ALP activity, and this increase significantly differed from all remaining groups.

Discussion
Microorganisms, especially in biofilm form, are the primary culprits of endodontic pathosis [17,18,64,65]. Biofilm microorganisms are highly resistant to disinfecting agents used in endodontic treatment; therefore, combined agents were recommended to enhance their activities and to balance their deficiencies against persistent infections [66][67][68]. Accordingly, the combination of AgNPs with Ca(OH) 2 has been investigated and showed a synergistic effect compared with the use of Ca(OH) 2 alone [44,47,48,57]. The ideal intracanal medicament must be biocompatible and ensures the survival of the surrounding stem cells [69]. In this study, we investigated the effect of previously optimized concentrations of AgNPs, alone and in combination with Ca(OH) 2 , on the proliferation, viability, attachment, migration, and differentiation of human bone marrow mesenchymal stem cells [48].
As a model for human bone marrow stem cells (hBMSCs), a telomerized hMSC line (hMSC-TERT) was employed in this study. This cell line was generated by overexpression of the human telomerase reverse transcriptase gene (hTERT). It manifests all the characteristics and genetic markers of primary hBMSCs, including multipotency, high differentiation capability, and unlimited self-renewal [70,71]. Bone marrow stem cells have similar features to dental pulp stem cells, and have exhibited multipotency and strong differentiation abilities, making them a possible source for the regeneration of dental tissues [72][73][74][75].
The toxicity of AgNPs is frequently cited as a concern and depends on many factors, including the AgNP concentration [59]. The cytotoxicity of AgNPs is directly proportional to their concentration, and several concentrations of AgNPs were tested in the literature. 0.01% AgNPs is the lowest concentration that exhibited good antibacterial properties in published studies [42,57,76,77] with no significant changes in tooth color [78]. Furthermore, the concentrations of tested AgNPs range up to 0.15%, which has been used as an additive in endodontic sealers and showed improved antibacterial effects with minimal discoloration [53,79].
When used as an irrigant at concentrations up to 0.025%, AgNPs demonstrated low cytotoxicity and genotoxicity against L929 fibroblasts [80]. Additionally, using L929 fibroblasts, Takamiya et al. evaluated the cytotoxicity of silver nanoparticles synthesized with ammonia and polyvinyl pyrrolidone at different concentrations ranging from 0.00001-0.01% [81]. They also assessed the reaction of subcutaneous connective tissue of Wistar rats to AgNPs and reported that AgNPs were not cytotoxic at 0.0025% or lower. [81]. An in-vivo study concluded that 0.0047% nanosilver dispersions were biocompatible compared with 2.5% NaOCl solution when tested on fibrous connective tissues of rats [55]. In the present study, optimized concentrations were used [48]. The AlamarBlue results presented herein showed that all groups exhibited a trend towards increased cell proliferation on day 5 ( Figure 5) and that the lowest concentrations had significantly higher cell proliferation, which agrees with the previously mentioned studies.
Cell attachment and morphology were also examined using SEM. AgNPs alone seemed to favor cell attachment, and the combination groups showed elongated cells adhering to the dentin surface. Cells treated with Ca(OH) 2 and TAP had altered morphology, consistent with the AlamarBlue results, where these two groups exhibited less viable cells. The detrimental effect of TAP on cell attachment is in agreement with a previous study [82].
The size and shape of the nanoparticles are other essential features that affect their toxicity [59,[83][84][85][86]. For example, Liu et al. reported that 5 nm AgNPs were more cytotoxic than 20 nm AgNPs when tested against four cell lines [83]. On the other hand, another study reported that spherical-shaped particles did not induce adverse effects on cells [84]. Therefore, the present study utilized spherical silver nanoparticles with a 20 nm size.
Cell migration is a critical step in pulp wound healing, and TGF-β1 plays a pivotal role in this process. TGF-β1 is released early in the healing stages and acts in the recruitment of inflammatory cells and the expression of essential proteins that form the extracellular matrix [87]. In this study, the gene expression of TGF-β1 on day 10 was significantly upregulated in all groups compared with their levels on day 5 and the negative control ( Figure 9). This was also in accordance with the wound-healing assay, where the migration ability was enhanced in cells treated with AgNPs, especially combined with Ca(OH) 2 ( Figure 8). The results agreed with previous reports, which observed that AgNPs and Ca(OH) 2 increased migration rate and wound healing [85,[88][89][90].
In addition to its role in wound healing, TGF-β1 is a crucial osteogenic marker. It regulates osteoblastogenesis and bone formation and increases osteoprogenitor cells through chemotaxis [86,91]. TGF-β1 is also a known inducer of odontoblastogenesis and mineralization of dental tissues [92][93][94]. The upregulation of TGF-β1 gene expression highlights the potential favorable effect of the tested medicaments on the osteogenic differentiation and mineralization.
Runx2 is considered the master-switch of osteoblastic differentiation. It is highly expressed in preosteoblasts, reaches the peak level in immature osteoblasts, and is reduced with osteoblastic maturation [95]. The results in this experiment exhibited the classic behavior of Runx2, where its expression levels were initially upregulated, but later decreased at 10 days. Moreover, groups containing Ca(OH) 2 showed higher levels of Runx2 expression, which is consistent with previous reports [96,97].
Other assays were used to evaluate the effect of the tested medicaments on osteogenic differentiation. The expression of ALP and OCN was quantified using RT-PCR (Figure 8), and the ALP activity was also quantitatively and qualitatively assessed (Figure 9). ALP is one of the earliest osteoblastic differentiation markers, while OCN is a relatively late marker [98,99]. The q-RT-PCR indicated that AgNPs significantly upregulated the expression of ALP and OCN after 10 days of osteoblastic differentiation and that AgNPs 0.06% had the highest expression levels ( Figure 5). Consistently, AgNPs 0.06% showed the most increased ALP activity ( Figure 6) and intense cytochemical staining (Figure 7). This is in agreement with previous studies [100][101][102][103]. However, one study reported that AgNPs decreased ALP and OCN expression, which could be attributed to the differences in cell lines and periods [104].

Materials and Methods
The study protocol was reviewed and approved by the Institutional Review Board (IRB No. E-20-5025) and the College of Dentistry Research Center (CDRC No. PR 0116).

Characterization of AgNPs
Silver nanoparticles (20 nm) were obtained as a powder from Zhengzhou Dongyao Nano Materials Company. Their size, shape, size, distribution, and aggregation were all examined using ultraviolet-visible (UV-Vis) spectroscopy (PerkinElmer, Norwalk, CT, USA), scanning electron microscopy (JSM-7610F-Field Emission Scanning Electron Microscope-JEOL-Japan), and the Nicolet 6700 Fourier-transform infrared (FTIR) Spectrometer (Thermo Fisher Scientific, Madison, WI, USA). Moreover, the existence and purity of the AgNPs were confirmed by compositional analysis of the elements using energy dispersive spectroscopy (EDS).

Medicament Selection and Preparation
The concentrations of AgNPs were selected based on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of a previous study [48], in which the chosen medicaments exhibited desirable antibacterial effects.
To prepare the silver nanoparticle medicaments, 0.5% carboxymethyl cellulose (CMC) was dispersed in distilled water with a magnetic stirrer at 120 rpm until a smooth consis-tency was obtained. Next, AgNPs (size = 20 nm) (Zhengzhou Dongyao Nano Materials Co., Ltd., Zhengzhou, China) were diffused in the prepared gel with gentle stirring for 1 h. After that, the samples were placed in a sonicator for 2 h. Finally, the medicaments were refrigerated (4 • C) for 4 h to achieve homogeneous gels without entrapped air. The manufacturer already characterized the physical properties of these AgNPs.
Preparation of TAP (Conc. 1 mg/mL) was performed by dissolving Ciprofloxacin, Minocycline, and Metronidazole (Xi'an Sgonek Biological Technology Co., Ltd., Shaanxi, China), at a ratio of 1:1:1 in 1 mL distilled water. Then, stirring was continued using a magnetic stirrer until completely dissolved.

Cell Culture and Dentin Disk Preparation
As a model for human bone marrow stem cells (hBMSCs), A telomerized human mesenchymal stem cell line (hMSC-TERT) was employed in this study [70]. This cell line was obtained from the stem cell unit, anatomy department, College of Medicine, King Saud University and was generated by overexpression of the human telomerase reverse transcriptase gene (hTERT) [70,71]. Cells were cultured in basal medium (Dulbecco's Modified Eagle medium, DMEM), which contains 4mM L-glutamine, 4500 mg/L D-glucose, and 110 mg/L 10% sodium pyruvate, and supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids and 1% penicillin-streptomycin (All acquired from Thermo-Fisher Scientific Life Sciences, Waltham, MA, USA). Cells were kept in a 5% CO 2 incubator at 37 • C and 95% humidity, and the medium was changed every 2 days. After reaching 80-90% confluency, cells were treated with 0.025% trypsin (Gibco), sub-cultured in flasks, or used in experiments. An automated cell counter (TC10 TM , Bio-Rad, Hercules, CA, USA) was used to determine the cell count before seeding.
For the attachment assay, dentin disks with 1 mm thickness were prepared from extracted human teeth following a previous protocol [20,48]. Briefly, human teeth were freshly extracted and stored in thymol. Teeth with cracks, root resorption, previous root canal treatment, and calcified canals were excluded. A surgical blade was used to remove gingival tissues. Then, a precision saw at 1000 rpm (IsoMet 1000 Precision Saw, IsoMet, Buehler, IL, USA) was used to section the teeth after embedding them in ortho-resin. Disks with 1 mm thickness were obtained, washed with 17% EDTA in an ultrasonic bath, and rinsed with sterile saline. Lastly, they were sterilized using gamma radiation at 25 kilos Gray [106].
For SEM, cells were cultured on treated dentin disks. For the remaining assays, medicaments were applied to cell culture inserts (Falcon ® , Corning, NY, USA) with a 0.4 µm pore size. This allowed the medicaments to diffuse into the media without direct contact between the cells and any insolubilized particles [107].

Cell Morphology and Attachment using Scanning Electron Microscopy (SEM)
Dentin disks were utilized in this assay to observe the cellular morphology and attachment to dentin. The disks were placed into 24-well culture plates and randomly allocated to one of the groups. Each sample was immersed in 0.2 mL of the assigned material and was incubated for 7 or 14 days at 37 • C in a humidified atmosphere of 5% CO 2 . After each time period, specimens were washed twice with PBS for 5 min and then with 17% EDTA for 1 min [82]. To prepare for cell seeding, the disks were placed in new culture plates containing basal medium (DMEM) and incubated for 24 h [82]. Then, cells were seeded on the treated dentin disks and cultured for 3 days.

Cell Proliferation Assay
Cell proliferation was evaluated using the Alamar Blue assay. At days 1, 3, and 5 of treatment, 10% Alamar blue substrate (Thermo Fisher Scientific Life Sciences, Waltham, MA, USA) was added to each well. Cells were incubated for 3 h in the dark, and the fluorescence (Ex 530 nm/Em 590 nm) was measured using a microplate reader (BioTek Synergy II, BioTek Inc., Winooski, VT, USA).

Live-Dead Imaging
Cell apoptosis was evaluated using the acridine orange/ethidium bromide (AO/EB) method, a fluorescence-based staining technique described previously [108]. At 24 and 48 h of treatment, cells were washed twice with PBS and stained with a dual fluorescent dye containing 100 µg/mL AO and 100 µg/mL EB (Sigma ® , St. Louis, MO, USA) for 1 min. Images were then taken and assessed using a fluorescence microscope (Eclipse Ti, Nikon Imaging Inc., Tokyo, Japan). The differences in uptake of the dual dye indicate viable (green) and non-viable (red) cells. Percentage of dead to live cells was quantified as described previously [108]. Data are representative of three replicas for each experimental condition, where more than 4 images were captured per condition.

Scratch Migration (Wound healing) Assay
Cells (0.1 × 10 6 /well) were seeded in 12-well plates for 24 h to achieve confluency. A sterile ruler was used to reference the center, and an artificial scratch wound was produced in each well using a sterile 200 µL pipette tip. Each well was washed three times with PBS to remove debris. Medicaments were applied, and the wound closure was assessed at 12, 24, and 36 h using a light microscope (Carl Zeiss Canada, North York, ON, Canada). Images were obtained using Zen software (Carl Zeiss Canada, North York, Ontario, ON, Canada). Then, wound healing area percentages were calculated using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA) [109] at three periods: 0 to 12 h, 12 to 24 h, and 24 to 36 h, as previously described [110]. Data are representative of 3 replicas for each experimental condition, where 3 images were captured per condition.

Quantitative Reverse Transcription Real-Time PCR (qRT-PCR)
On days 5 and 10 of osteoblastic differentiation, total RNA was extracted from cell pellets using the innuPREP RNA Mini Kit (Analytic-Jena, Jena, Germany). The concentration and quality of the extracted RNA were measured using the NanoDrop TM 2000 spectrophotometer (ThermoFisher Scientific), and complementary DNA (cDNA) was synthesized using the high-capacity cDNA reverse transcription kit (ThermoFisher Scientific) according to the manufacturer's instructions. Expression levels of Transforming growth factor beta 1 (TGF-β1), Runt-related transcription factor (Runx2), Alkaline Phosphatase (ALP), and Osteocalcin (OCN) were evaluated using qRT-PCR. Each PCR was carried out in triplicate on a ViiA TM 7 Real-Time PCR System (ThermoFisher Scientific), using a Fast-SYBR Green master mix (ThermoFisher Scientific). Data were normalized to the housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase GAPDH), and the relative gene expression was calculated using the delta-delta CT method. Primer sequences used in this study are listed in Table 1. Table 1. List of primer sequences used in qRT-PCR.

Primer
Forward Reverse ALP activity was quantified using the ALP Activity Colorimetric Assay Kit (BioVision, Inc., Milpitas, CA, USA) with some modifications. The Cells were cultured in 24-well plates. On days 5 and 10 of osteoblastic differentiation, the wells were rinsed with PBS and fixed with a fixative solution (3.7% formaldehyde in 90% ethanol) for 30 s. The fixative was removed and replaced with a p-nitro-phenyl phosphate solution, and the cells were incubated in the dark for 1 h at room temperature. The optical density for each well was measured at 405 nm using a microplate reader (SpectraMax ® M5, Molecular Devices, San Jose, CA, USA). ALP enzyme activity was normalized to the cell count.

ALP Staining
On days 5 and 10 of osteoblastic differentiation, cells were washed with PBS and fixed using an acetone/citrate buffer fixative for 5 min at 25 • C. The stain was prepared by dissolving 2 mg Naphthol-AS-TR-phosphate (Sigma) in 10 mL H2O and 10 mg Fast Red (Sigma) in 12 mL 0.1 Tris buffer. Then, the fixative was replaced with the Naphthol/Fast red stain (1:1), and cells were stained for 1 h at room temperature. Cells were then rinsed 3 times with water and counterstained using Mayer's hematoxylin solution for 3-4 min at room temperature. Specimens were washed with water, kept in PBS, and observed using a light microscope.

Statistical Analysis
All numerical data are presented as the mean ± SEM. Statistical analysis was performed using one and two-way analysis of variance [ANOVA] with GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA), and significance was set at (p < 0.05). Tukey's post hoc was used for multiple comparisons.

Conclusions
In conclusion, compared with Ca(OH) 2 and TAP, AgNPs have positive effects on cell proliferation, viability, attachment, migration, and osteoblastic differentiation potential. These results highlight the potential application of AgNPs combined with Ca(OH) 2 or alone as a biomaterial for various clinical applications, such as intracanal medicaments in root canal treatments and endodontic regeneration. However, given the limitations of in vitro studies, further in vivo investigations are needed to deem these medicaments ready for clinical application.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

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