Zirconia Nanoparticles Trigger ROS‑Mediated Cytotoxicity and Suppress Osteogenic Differentiation in 3T3‑E1 Osteoblasts

Abstract

Zirconia (ZrO₂) nanoparticles are increasingly employed in biosensors, cancer therapy, implants, and dentistry because of their high mechanical strength and low reported toxicity. However, as usage expands, so does potential human exposure, making a clear toxicological profile essential. Titanium dioxide (TiO₂) nanoparticles, known to be weakly toxic, served as a benchmark in this study. We examined the cytotoxicity of TiO₂ and ZrO₂ NPs in osteoblast‑like 3T3‑E1 cells, focusing on reactive oxygen species (ROS) generation, apoptosis, morphological changes, and osteogenic differentiation. Both NPs induced ROS in a concentration‑dependent manner, leading to apoptosis and altered cell morphology at high concentrations. High doses also impaired osteogenic differentiation, with ZrO₂ NPs displaying more pronounced toxicity than TiO₂ NPs. These findings highlight the need for careful dose management when considering ZrO₂ nanoparticles in biomedical applications.

Introduction

Engineered nanoparticles (NPs) have permeated diverse sectors, from electronics to medicine. Zirconia (ZrO₂) NPs, prized for their mechanical robustness, are now common in biosensors, cancer therapeutics, implants, and dental materials [1, 2]. Despite their advantages, the expanding use of ZrO₂ NPs raises legitimate concerns about potential health and environmental risks. Existing toxicological data are sparse and sometimes contradictory: some studies report excellent biocompatibility compared to other oxides such as ferric oxide, TiO₂, or ZnO [3–6], while others have detected mild cytotoxicity or cellular damage [7–12].

Previous work has also demonstrated that nanoparticles can enhance osteogenic differentiation in osteoblasts [14–17]. Nevertheless, the safety profile of ZrO₂ NPs, particularly regarding bone cell function, remains unclear. This study evaluates the cytotoxic effects of ZrO₂ NPs on 3T3‑E1 cells, using TiO₂ NPs as a well‑characterized control. Outcomes assessed include cell viability, ROS production, apoptosis, morphology, and osteogenic markers.

Materials and Methods

Materials Preparation and Characterization

TiO₂ (CAS 637262) and ZrO₂ (CAS 544760) NPs were purchased from Sigma‑Aldrich and characterized by transmission electron microscopy (TEM), zeta potential, and dynamic light scattering (DLS). TEM provided morphology and primary particle size, while DLS assessed agglomeration in complete culture medium. Prior to cell exposure, NPs were sonicated for 30 min and diluted to the desired concentrations in α‑MEM.

3T3‑E1 Cell Culture

3T3‑E1 cells were maintained in α‑MEM supplemented with 10% FBS and 1% antibiotic/antimycotic, at 37 °C, 5% CO₂. Medium was refreshed every other day.

Cell Proliferation Assay

Cell viability was quantified using the CCK‑8 assay. Cells (5 × 10³ per well) were exposed to TiO₂ or ZrO₂ NPs (0–150 μg/mL) for 24 or 48 h, with or without 10 mM N‑acetyl‑l‑cysteine (NAC). After 2 h incubation with CCK‑8, reagents were transferred to a fresh plate to avoid NP interference, and absorbance was measured at 450 nm.

Annexin V Apoptosis Analysis

After 48 h exposure, cells were harvested, stained with FITC‑Annexin V and propidium iodide, and analyzed by flow cytometry to quantify early and late apoptosis.

ROS Generation Analysis

Intracellular ROS were measured using DCFH‑DA after 48 h exposure to NPs (0–100 μg/mL). Fluorescence was detected by flow cytometry.

Confocal Microscopy

At 24 h, cells on coverslips were fixed, permeabilized, and stained for α‑tubulin (green), F‑actin (red), and nuclei (blue). Imaging was performed with an Olympus FV10i confocal microscope.

Mineralization Induction Detection

Cells were cultured for 7, 14, or 21 days with 10 or 100 μg/mL NPs, then fixed and stained with alizarin red S to visualize mineralized nodules.

RNA Extraction and RT‑PCR

Total RNA was isolated, reverse‑transcribed, and quantified by SYBR‑green RT‑PCR. Osteogenic genes (RUNX2, COL1α1, ALP, OPN, OCN, BSP) were examined at days 3, 7, 14, and 21.

Statistical Analysis

Data are expressed as mean ± SEM. ANOVA with Bonferroni or Dunnett’s T3 post‑hoc tests evaluated significance (p < 0.05).

Results

Nanoparticle Characterization

TEM revealed TiO₂ and ZrO₂ NPs as rod‑shaped spheres with primary diameters of 25.4 ± 2.8 nm and 31.9 ± 1.9 nm, respectively. DLS showed hydrodynamic sizes of 81.2 nm (TiO₂) and 93.1 nm (ZrO₂), indicating moderate agglomeration in medium. Zeta potentials were +32.9 mV (TiO₂) and +42.4 mV (ZrO₂), suggesting good colloidal stability (Fig. 1).

Figure 1. (a) TEM images of TiO₂ and ZrO₂ NPs. (b) DLS size distribution. (c) Distribution of NPs on 3T3‑E1 cells after 1 h exposure.

Cytotoxicity Assessment

CCK‑8 assays revealed no significant toxicity at 10 μg/mL for either NP. Viability decreased with concentrations above 20 μg/mL, reaching <50% at 150 μg/mL after 48 h (Fig. 2). NAC partially restored viability, confirming ROS involvement.

Figure 2. Cell viability after exposure to TiO₂ (a) and ZrO₂ (b) NPs, with and without NAC.

ROS Generation

Both NPs induced ROS in a concentration‑dependent manner, peaking at 100 μg/mL after 48 h. ZrO₂ NPs produced higher ROS levels at lower concentrations than TiO₂. NAC effectively suppressed ROS (Fig. 3).

Figure 3. ROS levels after NP exposure.

Apoptosis and Necrosis

Flow cytometry indicated dose‑dependent apoptosis. At 50 μg/mL, late apoptotic/necrotic cells rose to 43% (ZrO₂) and 29% (TiO₂). At 100 μg/mL, early apoptosis was more pronounced with TiO₂ (34%) compared to ZrO₂ (17%) (Fig. 4).

Figure 4. Apoptosis and necrosis profiles after NP exposure.

Morphological Alterations

Confocal imaging revealed minimal changes at 10 μg/mL. At 100 μg/mL, cells shrank, exhibited rounded morphology, and displayed disrupted actin filaments and microtubules. Quantitative analysis confirmed reduced cell area (Fig. 5).

Figure 5. Cell area changes after 24 h exposure.

Figure 6. Cytoskeletal organization after NP treatment.

Mineralization

Alizarin red staining showed no significant mineralization at 10 μg/mL across 14–21 days. At 100 μg/mL, mineralized nodules were reduced and less defined, indicating impaired osteogenic capacity (Fig. 7).

Figure 7. Alizarin red staining of mineralized nodules.

Gene Expression

RT‑PCR revealed upregulation of RUNX2, COL1α1, and ALP at 10 μg/mL, peaking at day 3. High‑dose (100 μg/mL) exposure downregulated these genes by day 7. Late‑phase markers (OPN, OCN, BSP) were increased at 10 μg/mL but suppressed at 100 μg/mL (Fig. 8).

Figure 8. Osteogenic gene expression over time.

Discussion

Our data confirm that ZrO₂ NPs are biocompatible at low concentrations but become cytotoxic above 20 μg/mL, primarily through ROS‑mediated apoptosis and cytoskeletal disruption. Compared to TiO₂, ZrO₂ exerts stronger oxidative stress and faster apoptosis at equivalent doses. The observed ROS production likely originates from nanoparticle interaction with mitochondrial membranes, consistent with prior reports of organelle damage by ZrO₂ [12].

The impairment of osteogenic differentiation at high NP doses aligns with reduced mineralization and downregulation of key osteogenic genes. These findings underscore the importance of dose optimization for any ZrO₂‑based biomaterial, especially in load‑bearing implants where osteointegration is critical.

Conclusion

Zirconia nanoparticles are generally safe for bone cells at low concentrations but exhibit dose‑dependent cytotoxicity, ROS generation, apoptosis, and suppression of osteogenesis at higher levels. These insights are essential for the rational design of ZrO₂‑containing biomaterials.

Abbreviations

Ag:

Silver

ALP:

Alkaline phosphatase

Col1α1:

Collagen 1α1

DLS:

Dynamic light scattering

FBS:

Fetal bovine serum

NAC:

N-acetyl‑l‑cysteine

NPs:

Nanoparticles

OC:

Osteocalcin

OD:

Optical density

OPN:

Osteopontin

PBS:

Phosphate‑buffered saline

ROS:

Reactive oxygen species

RUNX2:

Runt‑related transcription factor 2

Si:

Silica

TEM:

Transmission electron microscope

TiO₂:

Titanium dioxide

ZrO₂:

Zirconia

α-MEM:

Minimum essential medium‑alpha