Anti-osteoarthritis effects of injectable hyaluronic acid hydrogel loaded with platelet lysate and cerium oxide nanoparticle by regulating TNF-α: An in vitro model

2.1 Materials

Cerium (III) nitrate hexahydrate (Ce (NO₃)₃·6H₂O, Hyaluronic acid sodium salt (HA; Mw 1.3 × 10⁶ Da), 1, 4-butanediol diglycidyl ether (BDDE), potassium carbonate, and NaOH were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2 Synthesis and characterization of CeO₂-NPs

The CeO₂-NPs were synthesized based on previous reports with minor modification. Briefly, 1.085 g, Ce (NO₃)₃·6H₂O was dissolved in 125 mL double distilled water to obtain a 0.02 M solution. Simultaneously, 518 mg K₂CO₃ was dissolved in 250 mL double distilled water to obtain 0.03 M solution. Then, 50 mL of the cerium solution was added dropwise to 20 mL of potassium carbonate under vigorous stirring. The pH of the reaction mixture was set at 6.0 during the synthesis. The aging process was conducted at 220 °C for 150 min without any further washing steps. Finally, the aged NPs were calcined at 600 °C for 180 min. The synthesized CeO₂-NPs were characterized using Scanning electron microscopy (SEM, JSM-7600F. JEOL, Tokyo, Japan) at the accelerating voltage of 20 kV. CeO₂-NPs were mounted on the adhesive copper stub and sputter-coated with electroconductive element (gold). Using TEM instrument (ZEISS CEM 902 A) the morphology of CeO₂-NPs were evaluated. A small droplet of NPs dispersion was dried on a TEM grid and visualized at a voltage of 200 kV. The hydrodynamic size and Zeta potential of the synthesized CeO₂-NPs were investigated using a Zeta-sizer Nano ZS (Malvern Instruments Ltd, UK) at 25 ^◦C. Using a Thermo Scientific Genesys 6 UV–Vis spectrophotometer the UV–Vis spectra of the synthesized CeO₂-NPs was recorded. The analysis was conducted using quartz cuvettes of 1 cm in length in the range of wavelengths between 200 and 900 nm. The FTIR spectra of the synthesized CeO₂-NPs was obtained using a VERTEX 70 V (Bruker, Germany) spectrometer at room temperature over the wavenumber range of 4000–––400 cm⁻¹. Using the DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay kit the radical scavenging potential of the synthesized CeO₂-NPs was evaluated. According to the previous report (Soren et al., 2015), different concentrations of CeO₂-NPs (10, 20, 40, 100, 200 μg/mL) were dispersed in one mL of methanol and incubated with a reaction mixture contained DPPH (76 μM). The reaction mixture was allowed to incubate for 60 min at room temperature, and the absorption was measured at 517 nm using a microplate reader.

2.3 Platelet lysate preparation

Platelet lysate (PL) was obtained from rat plasma platelet concentrates following a two-step procedure, adapted from previous studies. In this modified method, the anti-coagulated blood (in 3 % v/v sodium citrate) underwent a 10-minute centrifugation at 210 × g at 4 °C to yield a yellowish fraction containing plasma with buffy coat, which was further centrifuged at 4 °C for 5 min at 210 × g. Subsequently, a series of three freeze–thaw cycles between −80 °C and 37 °C resulted in the lysis of concentrated platelets, and the remaining platelet residue was then removed by centrifugation at 2,000 × g for 10 min.

2.4 Fabrication of injectable hyaluronic acid hydrogel

The hydrogel was prepared using a solution impregnation based on previous protocol with minor revision. For pure hydrogel synthesis, HA was dissolved in 0.1 M NaOH solution and stirred for 24 h at room temperature to obtain a homogenous solution of 2 % (w/v) HA. The obtained polymeric solution was blended with BDDE solution (1 % w/v) containing 0.1 M NaOH and gently shacked for one h and incubated at 30 °C for 12 h to complete the cross-linking. The hydrogel was rinsed with ultra-pure water three times to eliminate any unreacted residual substances. We followed a similar protocol to produce the HA hydrogel loaded with PA and CeO2 NPs, with the exception of the initial step. In this case, we introduced CeO2 NPs (5 % w/v) and PL (5 %w/v) into the starting solution, after which we added HA and proceeded with the aforementioned protocol.

2.5 Characterization of hydrogel

The hydrogels' morphology was examined using the SEM imaging technique. Following lyophilization with a freeze-dryer (Thermo Heto PowerDry LL3000, USA), the hydrogels were cross-sectioned with a thin blade, coated with gold, and then observed using an SEM microscope. Furthermore, the semi-quantitative elemental analysis and distribution of CeO₂ nanoparticles within the hydrogel were assessed using EDX analysis. This experiment was performed using the SEM apparatus integrated with an EDX detector following gold coating.. The surface functional groups of the synthesized CeO₂ NPs and the fabricated nanocomposites was evaluated using the FTIR spectroscopy (VERTEX 70 V spectrometer, Bruker, Germany) at room temperature over the wavenumber range of 4000–––400 cm⁻¹. The XRD pattern of the fabricated hydrogels was obtained using a PANalytical Empyrean diffractometer (PANalytical, USA). The experiment was conducted on lyophilized samples after pressing them into flat samples and mounting then onto a quartz sample holder. The using a Cu Kα radiation (45 kV and 40 mA) and a step size of 0.026^◦. The injectability of the prepared hydrogel was assessed by examining its ability to be loaded into a syringe. The flow of the hydrogel from the syringe under a constant piston pressure served as an indication of its injectability. To evaluate the water absorption capacity of the hydrogels, they were immersed in PBS and the resulting weight change was measured. The hydrogel samples were then freeze-dried, weighed, immersed into a solution, incubated for 48 h, and their wet weights were measured at preselected times. The obtained values were used to calculate the water absorption percentage for the formulation. $$\mathrm{W}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\left(\frac{\mathit{wetweight}-initialdryweight}{\mathit{initialdryweight}}\right)\times 100$$

2.6 Biological evaluations

The cytocompatibility of the fabricated hydrogels was assessed using the MTT assay kit. The samples in their wet form underwent sterilization through 20 min of UV irradiation, followed by a 20-minute incubation in 70 % ethanol and three washes with autoclaved PBS (pH: 7.4). Subsequently, the sterilized hydrogels were incubated with 10^5 cells per well in 96-well plates, each containing 100 μl of cell culture medium (DMEM)/FBS (10 %)/Pen-Strep. The cells were then incubated for 48 and 72 h under standard cell culture conditions. For the 72-hour incubation group, the cell culture medium was refreshed once. At the end of the designated incubation period, MTT solution was added to the cells and incubated for 4 h in the dark. Following this, a DMSO solution was added to the cells, and the absorbance of the medium was measured at 590 nm using a microplate reader. Wells without hydrogels served as the control group. The percentage of cell viability was calculated using the following equation:. $$Cellviablity\left(\%\right)=(\frac{\mathit{ODtest}-ODBackground}{\mathit{ODcontrol}-ODBackground})\times 100$$

Real time PCR (qPCR) assay was applied to evaluate the expression of target genes under treatment. The cells were treated with TNF-α (10 ng/ml) for 6 h to induce OA. The cells without any treatments were considered as healthy cells, the cells treated with only TNF-α as the control group, and the cells treated with TNF-α plus hydrogel as the treatment. TRIzol reagent was applied to extract total RNA and the quality of the product was evaluated by NanoDrop2000 spectrophotometer. Then, cDNA was synthesized by the reverse transcription. The applied reaction mixture (20 μl) comprising PCR Forward Primer (0.4 μl), template cDNA (1 μl), SYBR® Premix Ex Taq II (Tli RnaseH Plus) (10 μl), PCR Reverse Primer (0.4 μl) and ddH₂O (8.2 μl). The qPCR reaction was conducted using the following condition: initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 10 sec (40 cycles), and annealing and extension at 60 °C for 30 sec. The reference gene was β-Actin and the relative mRNA expression was measured using the 2^-ΔΔCT method. The experiment was conducted using an ABI QuantStudioTM 7 Flex Real-Time PCR System.

Concentrations of Col2, Aggrecan, Col10, Mmp13 (Cloud Clone, USA) and Adamts, Adamts9 (MyBioSource, USA) in the cell culture supernatant were determined using commercially available ELISA kits according to the instructions provided by the manufacturer. A standard curve was calculated for each assay with detection limits based on protocol assay. Values are presented in ng/mL.

2.7 Data analysis

Using the statistical analyzing software (SPSS,16.0, SPSS Inc., Chicago, IL, USA) the obtained data (carried out at least in triplicate) were analyzed and expressed as mean ± SD. The p-value less than 0.05. was considered as statistically significance of the results.

3.1 CeO₂-NPs properties

The hydrodynamic and actual size, surface zeta potential, and morphology of the synthesized CeO₂- NPs were characterized using DLS, Zetasizer, and SEM/TEM imaging, respectively. The results revealed that the synthesized CeO₂- NPs have a zeta potential of 11 ± 2 mV (Fig. 1A) and a hydrodynamic size of 460 ± 20 nm (Fig. 1B). The morphology of the synthesized CeO₂ NPs was observed through SEM (Fig. 1C) and TEM (Fig. 1D) imaging, showing a spherical and uniform morphology. These findings are consistent with those reported in other studies.. Lin et al. (Lin, 2020) synthesized nanoceria and observed cubic crystals CeO₂-NPs agglomerates under SEM imaging. They reported a hydrodynamic size of 131 ± 0.7 nm with the PDI value of 0.104. in another study, Marzi et al. (De Marzi et al., 2013) synthesized ceria NPs observed spherical agglomerates of NPs under SEM imaging. Tumkur rt al. (Tumkur et al., 2021) used a hydroxide-mediated method to synthesize cerium oxide nanoparticles and observed clusters of uniformly distributed particles through SEM imaging.

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Fig. 1

Characteristics of CeO₂-NPs. (A) Zetapotential, (B) hydrodynamic size, (C) SEM image, and (D) TEM image of CeO₂-NPs.

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The FTIR spectroscopy was conducted to evaluate the functional groups of the synthesized CeO₂-NPs and the results are presented in Fig. 2A. The characteristic peaks of HA can be seen around 1600 cm⁻¹ (related to antisymmetric stretching vibration of COO⁻), 1405 cm⁻¹ (related to symmetric stretching vibration of COO⁻), 3427 cm⁻¹ (related to the plethora of hydroxyl groups, O–H stretching), and 2922 cm⁻¹ (related to the aliphatic symmetric C–H group stretching) (Culica et al., 2020; Safat et al., 2021). In the FTIR spectrum of the synthesized CeO₂-NPs the peak located at around 3420 cm⁻¹ can be attributed to the –OH stretching vibrations of the hydroxyl groups. Moreover, the peaks related to the –CH₃ and –CH₂ asymmetric stretching vibrations can be seen at around 2850 cm⁻¹ and 2920 cm⁻¹. The peak located at around 450 cm⁻¹ can be attributed to CeO₂ stretching vibration (Zamiri et al., 2015). The FTIR spectrum of the hydrogel nanocomposite exhibited the characteristic peaks of HA, indicating the entrapment of CeO₂-NPs into the hydrogel.

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Fig. 2

FTIR spectrum (A) and UV–Vis spectrum (B) of CeO₂-NPs.

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The UV–Vis spectrum of the synthesized CeO₂-NPs was recorded and the results are presented in Fig. 2B. The characteristic absorption peak of Ce⁺⁴ and Ce³⁺ of CeO₂-NPs can be seen around 325 and 200 nm. According the results, it can be concluded that the synthesized CeO₂-NPs consist of Ce⁺⁴ and Ce³⁺ ions. It is reported that the presence of Ce³⁺ ions has direct and vital roles in radical scavenging activity of CeO₂-NPs (Dunnick et al., 2015; Nurhasanah et al., 2018).

The antioxidant activity of the synthesized CeO₂-NPs was assessed using the DPPH assay kit. DPPH is a stable free radical and antioxidant substance neutralize this free radical via donation of hydrogen or electron and form the stable DPPH molecule (Nurhasanah, 2018). The results of the experiment revealed a dose-dependent radical scavenging activity of the synthesized CeO₂-NPs (Fig. 3).

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Fig. 3

Radical scavenging activity of CeO₂-NPs, measured using the DPPH assay kit.

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3.2 Hydrogel nanocomposite properties

The hydrogel was created through the interaction of BDDE with HA polymer, which led to the formation of covalent ether linkages between adjacent HA-HA polymeric chains. Furthermore, the interconnected HA-HA network structure and the abundant surface OH groups are believed to facilitate macromolecular entanglements and hydrogen bonding. The transformation from sol to gel and the injectability of the resulting hydrogel are illustrated in Fig. 4A-D. The results (Fig. 4C) demonstrate the consistent flow of the hydrogel through the syringe.

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Fig. 4

Macroscopic features of the hydrogel. (A) Polymeric solution, (B) the formed hydrogel, and (C) syringeability of the hydrogel.

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3.3 SEM and EDX results

In addition to macroscopic characteristics, microscopic properties such as internal structure, porosity, and pore architecture are essential and decisive for the intended application. We utilized SEM imaging to examine the internal structure and pore architecture of pure and CeO₂-NPs-loaded hydrogels, and the findings are depicted in Fig. 5. It is evident that the fabricated hydrogels possess a porous structure with interconnected pores. Furthermore, the incorporation of CeO₂-NPs into the HA hydrogel (Fig. 5B) did not alter the structure of the HA hydrogel (Fig. 5A). Numerous studies have underscored the use of porous hydrogels for osteoarthritis treatment. (Zhao et al., 2022). The porous nature of the hydrogels can be applied to deliver various therapeutic agents. It was observed that porous hydrogels can provide slow and sustained release of the therapeutic agents into the synovial cavity and subsequently, effectively promote cell proliferation, tissue formation, and repairing cartilage (Jeuken et al., 2016; Réeff et al., 2013). The distribution of CeO₂-NPs within the HA hydrogel was evaluated using the semi-quantitative EDX analysis. The results (Fig. 5C) showed that the synthesized CeO₂-NPs are homogenously dispersed within the matrix of the HA hydrogel.

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Fig. 5

SEM image of (A) pure HA hydrogel, (B) CeO₂ NPs-loaded hydrogel, and (C) EDX analysis results.

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3.4 XRD results

The crystallinity of the synthesized CeO₂-NPs and fabricated hydrogel was evaluated using the XRD analysis. The XRD pattern of CeO₂-NPs (Fig. 6) showed that the CeO₂-NPs exhibited the characteristics peaks of CeO₂ NPs. The peaks and indicating lattice planes are as follow: 2θ = 28.5°: (1 1 1), 2θ = 33.9°: (2 0 0), 2θ = 47.8°: (2 2 0), 2θ = 56.2°: (3 1 1), 2θ = 58.5°: (2 2 2), and 2θ = 69.1°: (4 0 0). The obtained XRD pattern is match with JCPDS No. 340,394 (standard cerium oxide pattern) (Lin, 2020; Kumar et al., 2010; Farahmandjou et al., 2016). Moreover, the wide peak at the HA/CeO₂-NPs pattern located at 22° to 25° can be associated to the reflection at (1 1 0) and (2 0 0) diffraction planes of HA structure (Fig. 6), indicating the semi-crystalline nature of fabricated HA hydrogel (Sundarrajan et al., 2012; Hezma et al., 2023). In this pattern (HA/CeO₂-NPs) the characteristic peaks of CeO₂-NPs can be observed, indicating that the incorporation of CeO₂-NPs into the hydrogel structure did not altered the crystallinity of the synthesized CeO₂-NPs. Moreover, using the Debye-Scherrer equation we found that the synthesized NPs have an average crystallite size (D) of around 8 nm. $$D=\frac{k\lambda }{\beta \cos \theta }$$

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Fig. 6

XRD patterns of the synthesized CeO₂-NPs and HA/CeO₂-NPs hydrogel.

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3.5 Swelling results

The water absorption capacity and water content are essential indicators for biomaterials, especially in the context of osteoarthritis treatment. Hydrogels with high water absorption and retention capacity have garnered significant attention for their potential to alleviate symptoms of OA. We conducted an assessment of the water absorption and retention capacity of the prepared hydrogels, and the results (Fig. 7) indicate that the fabricated hydrogels swelled by 800 % within the initial 10 h of the experiment. Furthermore, the hydrogels retained the absorbed water for approximately 25 h before the retention decreased. These findings suggest that the structure of the hydrogel remains stable and intact within the first 24 h, after which the cross-linking of the biopolymer is broken down through hydrolysis, leading to desorption. Additionally, we observed that the inclusion of CeO₂-NPs in the HA hydrogel reduced its water absorption and retention potential. This observation may be attributed to interactions between the NPs and the hydrophilic groups of the HA polymer, which occupied these groups and hindered the absorption of water through these functional groups.. Zhang et al. (Zhang et al., 2010) also reported the same trend, they fabricated pH-sensitive alginate/hydroxyapatite nanocomposite beads via the in situ generation of hydroxyapatite during the sol–gel transition process of alginate. They observed that increasing the concentration of hydroxyapatite decreased the swelling capacity of the hydrogel.

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Fig. 7

Water absorption and retention of the fabricated hydrogels.

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3.6 Cell viability results

The viability of the cells under incubation with the hydrogels was evaluated using the MTT assay kit. The cells were incubated with the hydrogels for 24 and 72 and the results (Fig. 8) showed that the fabricated hydrogels did not induce any adverse effect on the cells growth. Moreover, CeO₂-NPs-loaded HA hydrogels enhanced cell growth after 72 h cell seeding. These observations imply that HA hydrogel is not cytotoxic and incorporation of CeO₂-NPs improved proliferative effects of the HA hydrogels. Results of other studies are controversial, some of them showed toxicity of CeO₂-NPs and some other showed cytocompatibility. Lin et al. (Lin, 2020) reported that the synthesized CeO₂-NPs were biocompatible to chondrocytes at low concentration (lower than 0.02 µg/mL), but at the higher concentration induce dose-dependent toxicity. Tumkur et al. (Tumkur, 2021) reported that hydroxide-mediated CeO₂-NPs did not induce cell death even at higher concentrations (100 μg per 100 μl), evaluated by MTT and Live/Dead assays. Arya et al. (Arya et al., 2014) reported that CeO₂- NPs not only failed to induce toxic effects on primary cortical cells, but also prevented apoptotic cell loss during treatment with hydrogen peroxide (H₂O₂, 50 µM). They suggested that the observed protective effects could be linked to the preservation or restoration of major redox equivalents, such as the NADH/NAD⁺ ratio, mitochondrial membrane potential, and cellular ATP.

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Fig. 8

Cell viability results of the fabricated hydrogels at 24 and 72 h after cell sedding, measured by the MTT assay.

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3.7 Molecular evaluation result

qPCR assay was applied to reveal the regulative effects of the fabricated hydrogel on gene expressions of chondrocytes. The cells were treated with TNF-α to induce OA condition. The results (Fig. 9) showed that the expression of Col2 and Aggrecan genes was downregulated under treatment with TNF-α (Control group) while expressions of Col10, Mmp13, Adamts5, and Adamts9 upregulated under treatment with TNF-α (Control group), compared with healthy cells (P < 0.05). On the other hand, the results (Fig. 9) showed that the treatment using the hydrogel significantly reversed the effect of TNF-α (the OA condition) and restored the expression of the target genes.

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Fig. 9

Relative mRNA expressions of Col2, Aggrecan, Col10, Mmp13, Adamts, Adamts9 genes in chondrocytes treated with TNF-α (Control) and TNF-α plus hydrogel. Values are shown as mean ± SD. *P < 0.05.

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ELISA results confirmed this potent antioxidant activity of hydrogel and also increased the growth of chondrocytes over the culturing period. Fig. 10 shows that concentration of Col2, Aggrecan, Col10, Mmp13, Adamts5, Adamts9 in the cell culture supernatant. ELISA detected similarly a significant higher level of Col2 and Aggrecan expression for treatment group compared to control (OA group), while a low level in Col10, Mmp13, Adamts5, Adamts9 antigen-concentration was found in this group.

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Fig. 10

Concentration (ng/ml) of Col2, Aggrecan, Col10, Mmp13, Adamts5, Adamts9 in the cell culture treated with TNF-α (Control) and TNF-α plus hydrogel. Values are shown as mean ± SD. *P < 0.05.

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