Hyaluronic Acid Loaded with Cerium Oxide Nanoparticles as Antioxidant in Hydrogen Peroxide Induced Chondrocytes Injury: An In Vitro Osteoarthritis Model

Osteoarthritis (OA) is the most common joint disease type and is accompanied by varying degrees of functional limitation. Both hyaluronic acid (HA) joint injections and pain relievers are efficient treatments for early-stage osteoarthritis. However, for the decomposition by hyaluronidase and free radicals in the knee joint, HA injection treatment has limited effect time. The cerium oxide nanoparticles (CeO2) is a long time free radical scavenger. CeO2 combined with HA expected, may extend the HA decomposition time and have a positive effect on osteoarthritis therapy. In this study, CeO2 was successfully synthesized using the hydrothermal method with a particle size of about 120 nm, which possessed excellent dispersibility in the culture medium. The in vitro OA model was established by cell treated with H2O2 for 30 min. Our study found that the inhibition of chondrocyte proliferation dose-dependently increased with H2O2 concentration but was significantly decreased by supplementation of cerium oxide nanoparticles. COL2a1 and ACAN gene expression in chondrocytes was significantly decreased after H2O2 treatment; however, the tendency was changed after cerium oxide nanoparticles treatment, which suggested that damaged chondrocytes were protected against oxidative stress. These findings suggest that cerium oxide nanoparticles are potential therapeutic applications in the early stage of OA.


Introduction
Osteoarthritis (OA), the most common form of arthritis, has long been considered a complex metabolic disease disorder which leads to focal damage to articular cartilage at the weight-bearing areas [1]. This slowly progressive, disabling joint disorder can significantly impair life quality (QOL) and affects nearly 34% of those ages 65 and older [2][3][4]. Initially, increased pressure on the joint, which leads to the cartilage matrix's fragility, was considered to be the primary pathological process. As a result of the progress in molecular biology in the 1990s, scientists discovered that many soluble mediators could increase the synthesis of matrix metalloproteinases by chondrocytes and led to the inflammatory process. Recent data have shown that OA is a much more complex metabolic syndrome induced by the inflammatory mediators released by cartilage, subchondral bone, and synovium [5].

Morphology and Composition of CeO 2 Nanoparticles
The morphology and composition of CeO 2 nanoparticles were analyzed. The SEM images showed that cubic crystals cerium oxide nanoparticles agglomerated, with a particle size from 30 nm to 60 nm ( Figure 1a). The chemical composition of the synthesized cerium oxide nanoparticles was analyzed by SEM-accessorized EDS, which indicated the composition was mainly cerium and oxygen (Figure 1b). The TEM images showed that the grain size of cerium oxide nanoparticle was around 10 nm, and the exposing surface was unstable, where three crystal planes could be found by the lattice spacing, namely (111) (0.3127 nm), (100) (0.2711 nm), and (110) (0.1971 nm). (Figure 1c). The particle size distribution of cerium oxide nanoparticles in cell culture medium was 131.1 ± 0.7 nm (PDI = 0.104), which was measured by Zetasizer ( Figure 1d).

Crystal Phase Identification
CeO2 nanoparticles were scanned from 10-80°, after drying, polishing, and evenly spreading on XRD sample stage. Figure 2 shows the X-ray pattern of the synthesized cerium oxide nanoparticles; where the characteristic diffraction peaks on 2 theta of (111), (200), (220), (311), (222), (400), (331), and (420), respectively, are fully matched to standard cerium oxide pattern of JCPDS No. 340394. The crystal structure was identified as cerium oxide without the second phase to be traced in the pattern. From the results, we could tell that the crystal structure of synthesized cerium oxide nanoparticles was the same as that of CeO2 prepared by a conventional method.

Crystal Phase Identification
CeO 2 nanoparticles were scanned from 10-80 • , after drying, polishing, and evenly spreading on XRD sample stage. Figure 2 shows the X-ray pattern of the synthesized cerium oxide nanoparticles; where the characteristic diffraction peaks on 2 theta of (111), (200), (220), (311), (222), (400), (331), and (420), respectively, are fully matched to standard cerium oxide pattern of JCPDS No. 340394. The crystal structure was identified as cerium oxide without the second phase to be traced in the pattern. From the results, we could tell that the crystal structure of synthesized cerium oxide nanoparticles was the same as that of CeO 2 prepared by a conventional method.

Crystal Phase Identification
CeO2 nanoparticles were scanned from 10-80°, after drying, polishing, and evenly spreading on XRD sample stage. Figure 2 shows the X-ray pattern of the synthesized cerium oxide nanoparticles; where the characteristic diffraction peaks on 2 theta of (111), (200), (220), (311), (222), (400), (331), and (420), respectively, are fully matched to standard cerium oxide pattern of JCPDS No. 340394. The crystal structure was identified as cerium oxide without the second phase to be traced in the pattern. From the results, we could tell that the crystal structure of synthesized cerium oxide nanoparticles was the same as that of CeO2 prepared by a conventional method.

Determination of the Effect of Oxidative Stress on Chondrocytes
In the presence of 0.3 mM H 2 O 2 oxidative stress for 30 min, there was no significant cytotoxicity, but chondrocytes' viability was significantly affected (Figure 3a,b). The results of live/dead staining also showed that at the concentration of 0.3 mM H 2 O 2 , the treated chondrocytes did not show any significant evidence of cell death or cell damage, whereas the slightly excessive amount of H 2 O 2 (1 mM) was harmful to the chondrocytes (Figure 3c). In a further study, 0.3 mM H 2 O 2 was selected to be the concentration as the inductive agent for the chondrocytes apoptosis [25]. Similar to above, the expression of COL1A1, COL2A1, and aggrecan (ACAN) genes was downregulated in the presence of H 2 O 2 oxidative stress; this phenomenon was even more evident at 1.0 mM H 2 O 2 oxidative stress ( Figure 4).

Determination of the Effect of Oxidative Stress on Chondrocytes
In the presence of 0.3 mM H2O2 oxidative stress for 30 min, there was no significant cytotoxicity, but chondrocytes' viability was significantly affected (Figure 3a,b). The results of live/dead staining also showed that at the concentration of 0.3 mM H2O2, the treated chondrocytes did not show any significant evidence of cell death or cell damage, whereas the slightly excessive amount of H2O2 (1 mM) was harmful to the chondrocytes (Figure 3c). In a further study, 0.3 mM H2O2 was selected to be the concentration as the inductive agent for the chondrocytes apoptosis [25]. Similar to above, the expression of COL1A1, COL2A1, and aggrecan (ACAN) genes was downregulated in the presence of H2O2 oxidative stress; this phenomenon was even more evident at 1.0 mM H2O2 oxidative stress ( Figure 4).

Biocompatibility of Cerium Oxide Nanoparticles
Our results indicated that the viability of chondrocytes was inhibited and their cytotoxicity getting increased as the concentration of CeO2 nanoparticles increased ( Figure 5). At the lower concentration of CeO2 (less than 0.02 μg/mL), it showed no cytotoxic effect and was more biocompatible to chondrocytes ( Figure 5).

Biocompatibility of Cerium Oxide Nanoparticles
Our results indicated that the viability of chondrocytes was inhibited and their cytotoxicity getting increased as the concentration of CeO 2 nanoparticles increased ( Figure 5). At the lower concentration of CeO 2 (less than 0.02 µg/mL), it showed no cytotoxic effect and was more biocompatible to chondrocytes ( Figure 5).

Biocompatibility of Cerium Oxide Nanoparticles
Our results indicated that the viability of chondrocytes was inhibited and their cytotoxicity getting increased as the concentration of CeO2 nanoparticles increased ( Figure 5). At the lower concentration of CeO2 (less than 0.02 μg/mL), it showed no cytotoxic effect and was more biocompatible to chondrocytes ( Figure 5).

Cell Apoptosis and Gene Assay
In flow cytometry, the results for detection of cell apoptosis rate by Annexin V/PI apoptosis assay are divided into the following four quadrants: Annexin V-/PI-(Q3) which represent living cell, Annexin V+/PI-(Q4) which represent early apoptotic cell, Annexin V+/PI+(Q2) which characterize late stage apoptotic cells, and Annexin V-/PI+(Q1) which identify cells with permeabilized membranes only. As unstained controls, normal cell was used to gate for a negative cell population. The percentage of each quadrant of figures of flow cytometry are summarized in Table 1. The results indicated that 0.3 mM H 2 O 2 could permeate chondrocytes membranes as that induced in chondrocytes of early OA, 0.02 ng/mL CeO 2 nanoparticles and 1% HA were biocompatible, and the combination of 0.02 ng/mL CeO 2 nanoparticles/1% HA could protect chondrocytes from the harmful effect induced by 0.3 mM H 2 O 2 ( Figure 6).

Cell Apoptosis and Gene Assay
In flow cytometry, the results for detection of cell apoptosis rate by Annexin V/PI apoptosis assay are divided into the following four quadrants: Annexin V-/PI-(Q3) which represent living cell, Annexin V+/PI-(Q4) which represent early apoptotic cell, Annexin V+/PI+(Q2) which characterize late stage apoptotic cells, and Annexin V-/PI+(Q1) which identify cells with permeabilized membranes only. As unstained controls, normal cell was used to gate for a negative cell population. The percentage of each quadrant of figures of flow cytometry are summarized in Table 1. The results indicated that 0.3 mM H2O2 could permeate chondrocytes membranes as that induced in chondrocytes of early OA, 0.02 ng/mL CeO2 nanoparticles and 1% HA were biocompatible, and the combination of 0.02 ng/mL CeO2 nanoparticles/1% HA could protect chondrocytes from the harmful effect induced by 0.3 mM H2O2 ( Figure 6).

Effect of CeO2 Nanoparticles-Loaded Hyaluronic Acid Treatment When Chondrocytes under Oxidative Stress
As mentioned above, in the presence of H2O2 oxidative stress, the expression of COL1A1, COL2A1, and aggrecan (ACAN) genes were all downregulated. HA's presence can scavenger the H2O2 oxidative stress-induced gene effect on chondrocytes, while the CeO2 cannot scavenger the H2O2 induced COL2A1 gene effect on chondrocytes. The presence of HA + CeO2 can scavenger the H2O2 oxidative stress on chondrocytes, and further enhance HA's scavenger effect on the H2O2 oxidative stress induced COL1A1 and COL2A1 gene expression on chondrocytes (Figure 7).

Effect of CeO2 Nanoparticles-Loaded Hyaluronic Acid Treatment Glycosaminoglycan (GAG) Synthesis
Consistent with the gene expression patterns of ACAN, COL1A1, and COL2A1, H2O2 reduced production of sulfated proteoglycan as determined by alcian blue staining. In the H2O2-treated samples, cellularity was relatively sparse with destructed cell membrane as compared with the control samples. With the treatment of HA and CeO2, the effects of H2O2-treatment (both cellularity and cell membrane destruction) seem to be reversed. The accumulation of sulfated proteoglycan was recovered and was most obvious when the cells were pretreated with both HA and CeO2 (Figure 8). In the H2O2-treated samples, cellularity was relatively sparse with destructed cell membrane; whereas with the treatment of HA, CeO2 can reverse the H2O2-treatment effects. The accumulation of sulfated proteoglycan was most obvious when the cells were pretreated with both HA and CeO2.

Discussion
Osteoarthritis (OA) is a degenerative disease of articular cartilage induced by various factors. Although OA's pathogenesis remains to be fully elucidated, it has been generally recognized that overexpression of reactive oxygen species (ROS) plays a vital role in the degeneration of articular cartilage [26]. Submillimolar concentrations of H2O2 can induce inhibition of the extracellular matrix (ECM) synthesis, chondrocyte apoptosis, lipid peroxidation, and inflammatory cytokines overproduction, and lead to the matrix metalloproteinase (MMPs) formation [5][6][7]. Effective diseasemodifying OA therapies could lead to potentially transformative therapy. H2O2-induced oxidative stress helps study the occurrence and development of OA and evaluate the therapeutic strategies [27]. In this study, primary cultured chondrocytes treated with H2O2 to partly mimic their physiological conditions under oxidative stress were used as a model to examine the protective effects of cerium oxide and HA.
The particle size of synthesized cerium oxide nanoparticles was 131.1 ± 0.7 nm. Because the TEM images indicated that the grain size of CeO2 nanoparticles was around 10 nm, and the exposing surface was unstable, the larger nanoparticles may be due to the aggregation of the smaller ones [28]. H2O2 can be endogenously produced in OA's pathogenesis and can induce chondrocytes injury [29]. This study found that inhibition of chondrocyte proliferation and chondrocytes-related gene expression was dose-dependent with H2O2 concentration. H2O2-mediated oxidative stress can enhance ROS and lipid peroxidation levels in chondrocytes. Previous studies have shown that chondrocyte apoptosis induced by oxidative stress was responsible for the development of OA [26,30,31]; lipid peroxidation could be induced by ROS and caused significant tissue damage in degenerative osteoarthritis [32][33][34][35].
Hyaluronic acid (HA), present in the healthy joint's synovial fluid, has a protective effect against the invasion of PMN cells. In inflamed joints, the HA concentration decreases by depolymerization,

Discussion
Osteoarthritis (OA) is a degenerative disease of articular cartilage induced by various factors. Although OA's pathogenesis remains to be fully elucidated, it has been generally recognized that overexpression of reactive oxygen species (ROS) plays a vital role in the degeneration of articular cartilage [26]. Submillimolar concentrations of H 2 O 2 can induce inhibition of the extracellular matrix (ECM) synthesis, chondrocyte apoptosis, lipid peroxidation, and inflammatory cytokines overproduction, and lead to the matrix metalloproteinase (MMPs) formation [5][6][7]. Effective diseasemodifying OA therapies could lead to potentially transformative therapy. H 2 O 2 -induced oxidative stress helps study the occurrence and development of OA and evaluate the therapeutic strategies [27]. In this study, primary cultured chondrocytes treated with H 2 O 2 to partly mimic their physiological conditions under oxidative stress were used as a model to examine the protective effects of cerium oxide and HA.
The particle size of synthesized cerium oxide nanoparticles was 131.1 ± 0.7 nm. Because the TEM images indicated that the grain size of CeO 2 nanoparticles was around 10 nm, and the exposing surface was unstable, the larger nanoparticles may be due to the aggregation of the smaller ones [28]. H 2 O 2 can be endogenously produced in OA's pathogenesis and can induce chondrocytes injury [29]. This study found that inhibition of chondrocyte proliferation and chondrocytes-related gene expression was dose-dependent with H 2 O 2 concentration. H 2 O 2 -mediated oxidative stress can enhance ROS and lipid peroxidation levels in chondrocytes. Previous studies have shown that chondrocyte apoptosis induced by oxidative stress was responsible for the development of OA [26,30,31]; lipid peroxidation could be induced by ROS and caused significant tissue damage in degenerative osteoarthritis [32][33][34][35].
Hyaluronic acid (HA), present in the healthy joint's synovial fluid, has a protective effect against the invasion of PMN cells. In inflamed joints, the HA concentration decreases by depolymerization, the intraarticular application of high molecular weight HA might be an essential therapeutic regimen to restore the natural barrier against PMN migration and to interrupt the inflammatory cascade [36]. Cerium oxide nanoparticles, widely applied in our life [37], have recently come into consideration for biomedical use due to their potent antioxidant properties and have been proposed as a treatment for oxidative stress-associated chronic diseases [38][39][40]. Cerium oxide nanoparticles present the mimetic activity of superoxide dismutase. Mimicking natural antioxidant enzymes such as superoxide dismutase and catalase, the switching between CeO 2 and CeO 2 -x during redox reactions makes CeO 2 nanoparticles a lucrative catalytic nanoparticle. It is able to inactivate excess reactive oxygen species (ROS) which is correlated with a large number of pathologies [41]. CeO 2 nanoparticles can scavenge most reactive oxygen species and nitrogen species via an auto-regenerative mechanism. In such circumstances, a minimum dose can exhibit catalytic activity for a longer duration [42].
Chondrocyte ECM mainly contains type 2 collagen and aggrecan. Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. A previous study has demonstrated that H 2 O 2 can induce chondrocytes apoptosis and caspase-3 activation in rat chondrocytes [43]. In the study, both HA and CeO 2 nanoparticles could lower chondrocytes apoptosis induced by H 2 O 2 . We also observed that COL2A1 and ACAN gene expression in chondrocytes was significantly decreased after H 2 O 2 treatment. Both hyaluronic acid (HA) and CeO 2 can scavenger H 2 O 2 oxidative stress on chondrocytes, while the presence of HA + CeO 2 can further enhance the scavenger effect of HA on the H 2 O 2 oxidative stress induced COL1A1 and COL2A1 gene expression on chondrocytes. Inconsistent with the gene expression patterns of ACAN, COL1A1, and COL2A1, H 2 O 2 treatment can significantly reduce the production of sulfated proteoglycan as determined by alcian blue staining; while the treatment of HA + CeO 2 can reverse the H 2 O 2 -treatment effects. The accumulation of sulfated proteoglycan was most apparent when the cells were pretreated with both HA and CeO 2 . The results demonstrated that cartilage degeneration was significantly improved after CeO 2 nanoparticles treatment and this tendency suggested that cerium oxide nanoparticles can protect damaged chondrocytes against oxidative stress. The results of this study indicate that cerium oxide nanoparticles can attenuate the progression of OA through suppression of H 2 O 2 -mediated injury.

Synthesize of Cerium Oxide Nanoparticles
CeO 2 nanoparticles were synthesized according to the following manner [44]. In separate burettes, 0.02 M solution of cerium(III) nitrate hexahydrate was prepared by dissolving 2.17 g, Ce(NO 3 ) 3 ·6H 2 O in 250 mL distilled water and 0.03 M of K 2 CO 3 solution was prepared by dissolving 1.036 g, K 2 CO 3 in 250 mL distilled water. By adding drop by drop the aqueous solution of cerium(III) nitrate hexahydrate (50 mL) and potassium carbonate (20 mL) to a well stirred water (100 mL), a white precursor, cerium(III) carbonate, was precipitated. During the precipitation method, the constant Ph = 6 was maintained. The product was aged for 2.5 h (at 220 • C) without any washing and purification and finally calcined for 3 h (at 600 • C). The resulting CeO 2 nanoparticles were dried for 2 h (at 65 • C), and then cooled to room temperature.

Morphology and Composition of Cerium Oxide Nanoparticles
Scanning electron microscopy (SEM) was used to examine the microstructure of the CeO 2 nanoparticles. The specimens were mounted onto an adhesive copper stub, and then gold sputtered. SEM analyses were performed using a JSM-7600F (JEOL, Tokyo, Japan) electron microscope with a current and voltage of 20 mA and 10 kV, respectively. Additionally, electron dispersive spectrophotometry (EDS) were used to determine the composition of the synthesized particles. According to the Nanogenotox protocol [45], the 0.5% absolute ethanol pre-wetted CeO 2 nanoparticles were dispersed at 2.56 mg/mL, in 0.05% bovine serum albumin (BSA), in double-distilled water by 16 min sonication. Then, the sonicated CeO 2 nanoparticles were dispersed in DMEM high glucose with 10% FBS for the transmission electron microscopy (TEM) examination. A small drop of the stock suspension was pipetted onto a TEM grid and allowed to dry at room temperature and the observed by transmission electron microscopy (TEM) (JEM-2011, JEOL instrument, Tokyo, Japan) at a voltage of 200 kV.

Particle Size Identification
At an incident angle of 90 • , the particle sizes of the CeO 2 nanoparticles were measured by dynamic light scattering (DLS) at 25 • C; while the zeta potential was determined by DLS associated with electrophoretic mobility at pH 7.4. Then, 5 mg CeO 2 nanoparticles were dispersed in 5 mL de-ionized water for measurements of mean size. Then, the obtained dispersion was vortexed for 30 s with 5 repeats. After complete homogenization, the samples were placed in a cuvette for measurements in a Zeta-sizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK).

Crystal Structure Identification
X-ray diffractometry (XRD, TTRAX III, Rigaku, TX, USA) was used to determine the crystal structure of the synthesized CeO 2 nanoparticles [46]. By using a Ni filter with a potential of 30 kV and current of 15 mA, the synthesized CeO 2 nanoparticles were collected and mounted onto the sample holder of the X-ray powder diffractometer under Cu KαI radiation (λ = 0.15406 nm). In the range from 20 • to 80 • , each specimen was scanned at a speed of 2 • /min. The patterns were analyzed using a model auto-matched to the international center for the diffraction database using Jade 6.0 software. JCPDS Card No. 340394 was used as the standard pattern.

Isolation of Chondrocytes
Cartilage from the knees of bovine was minced into small pieces, then sequentially digested in 0.25% trypsin for 30 min and placed on 2 mg/mL collagenase II-containing medium for 4-5 h, at 37 • C. The solution was washed using phosphate-buffered saline (PBS) and filtered through a 200 µm cell strainer. The cells were collected by centrifugation, and then cultured in DMEM/F12 medium containing 10% FBS in a humidified atmosphere (at 37 • C, 5% CO 2 ). Chondrocytes at passage 2 were selected for the subsequent processes.

Determination of Experimental Concentrations of H 2 O 2
Chondrocytes exposed to H 2 O 2 were used as the experimental oxidative stress model [25]. At the preparation of 1 × 10 5 /mL single-cell suspension, the cultured chondrocytes were seeded into 96-well plate at 10 4 cells in each well. When chondrocytes adhered to the wall, the cells were starved for 24 h by adding 100 µL serum-free culture medium. Then, chondrocytes were treated with H 2 O 2 (0, 0.1, 0.3, 1, 3, and 10 mM) for 30 min. Then, chondrocytes would be evaluated by lactate dehydrogenase (LDH) assays, water-soluble tetrazolium (WST-1) assays, and live/dead staining. Finally, cell apoptosis assay, gene expression, and alcian blue staining were used to evaluate the capacity of CeO 2 nanoparticles and CeO 2 /HA.

Cell Viability
The biocompatibility of the CeO 2 nanoparticles was evaluated using the water-soluble tetrazolium (WST-1) assay (Sigma, St. Louis, MO, USA) [46]. The biocompatibility of the as-prepared composite was tested according to the ISO 10993-5 standard [47]. An extract medium was prepared by adding 0.2 g/mL of the CeO 2 nanoparticles to high-glucose DMEM (Sigma, St. Louis, MO, USA), followed by incubation at 37 • C, for 24 h. At a cell density of 5 × 10 3 cells/well, chondrocytes were seeded in 96-well plates and incubated at 37 • C, for 1 day. Then, the culture medium was replaced with the extract medium, and samples and cells were incubated for 1 to 3 days. Before the assay, 10 µL WST-1 reagent was added into each well for 4 h incubation; then, the plate was placed in a spectrophotometric plate reader (ELISA reader, Tecan Sunrise, Hombrechtikon, Switzerland) and read at the 450 nm absorbance (with a reference filter at 600 nm) to determine the amount of Formazan formed. The percentage of cell viability was calculated by the following Equation (1):

Cytotoxicity
The CytoTox 96 Assay Kit (Promega Corporation, Madison, WI, USA) for measuring the extracellular lactate dehydrogenase (LDH) content was used to evaluate the chondrocytes cytotoxicity. Briefly, after transferring the suspension medium to a new enzymatic assay plate, the LDH substrate solution was added for 30 min incubation; then, the stop solution was added, and the absorbance at 490 nm was measured on a spectrophotometric plate reader (ELISA reader, Tecan Sunrise, Hombrechtikon, Switzerland). The percentage of cytotoxicity was calculated by the following Equation (2)

Detection of Cell Apoptosis Rate by Flow Cytometry
The FITC Annexin V Apoptosis Detection Kit (Thermo Fisher Scientific Inc., MA 02451, USA) was used to quantify the percentage of cells undergoing apoptosis, according to the manufacturer's instructions. Briefly, chondrocytes were harvested after treatment, washed twice with cold PBS, then re-suspended in 100 µL of binding buffer (containing 5 µL FITC Annexin V and 5 µL propidium iodide (PI)), incubated (at 25 • C) for 15 min in the dark), then 400 µL binding buffer was added, the cells were analyzed with a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA).
The primers (Biotools Co., Ltd., Taipei, Taiwan) are shown in Table 2. For total RNA extraction, Qiazol (Qiagen, Valencia, CA, USA) was used, according to the manufacturer's protocol. For the first-strand cDNA synthesis, random hexamers (Vivantis Inc., Oceanalde, CA, USA) and reverse transcriptase (Vivantis Cat No: RTPL12) were used with the following PCR parameters: 95 • C for denaturation (3 min), 40 cycles of 95 • C for 20 s, 60 • C for annealing (30 s), and 72 • C for elongation (30 s). TOOLS 2X SYBR qPCR Mix (Biotools Co., Ltd., Taipei, Taiwan) was applied for real-time RT-PCR using a CFX Connect Real-Time PCR Detection System (BioRed, CA, USA). The expression of the target genes was calculated by using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control.

Alcian Blue Staining for Mucopolysaccharides
The deparaffinize slides were rehydrated with distilled water, stained with alcian blue solution (pH 2.5) for 30 min, washed by running tap water for 2 min, rinsed in distilled water, then counterstained with nuclear fast red solution, and mounted for later observation.

Statistical Analysis
All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by using one-way ANOVA and the post hoc comparisons used was Bonferroni test. Statistically significance was defined at p-value less than 0.05. All analyses were performed by using SPSS version 16.0 software (SPSS Inc., Chicago, IL, USA).

Conclusions
In this study, the particle size of synthesized cerium oxide nanoparticles was in the 131.1 ± 0.7 nm range, and the TEM images indicated that the grain size of CeO 2 nanoparticles was around 10 nm. H 2 O 2 can be endogenously produced in OA's pathogenesis and can induce chondrocytes injury, and chondrocytes-related gene expression was dose-dependent with H 2 O 2 concentration. Our findings suggest that CeO 2 nanoparticles can prevent H 2 O 2 -induced chondrocytes injury through its antioxidant effects in vitro and reduced cartilage damage. Cerium oxide nanoparticles present the mimetic activity of superoxide dismutase. It was mimicking natural antioxidant enzymes such as superoxide dismutase and catalase, which could inactivate the excess of ROS correlated with a large number of pathologies. In vitro OA model, the results demonstrated that cartilage degeneration was significantly improved after CeO 2 nanoparticles treatment and this tendency suggested that cerium oxide nanoparticles can protect damaged chondrocytes against oxidative stress. These results support the potential therapeutic applications of CeO 2 nanoparticles as a supplementation in human OA treatment.
Funding: This research received no external funding.