Nano-enzyme hydrogels for cartilage repair effectiveness based on ternary strategy therapy

Abstract

Designing artificial nano-enzymes for scavenging reactive oxygen species (ROS) in chondrocytes (CHOs) is considered the most feasible pathway for the treatment of osteoarthritis (OA). However, the accumulation of ROS due to the amount of nano-enzymatic catalytic site exposure and insufficient oxygen supply seriously threatens the clinical application of this therapy. Although metal–organic framework (MOF) immobilization of artificial nano-enzymes to enhance active site exposure has been extensively studied, artificial nano-enzymes/MOFs for ROS scavenging in OA treatment are still lacking. In this study, a biocompatible lubricating hydrogel-loaded iron-doped zeolitic imidazolate framework-8 (Fe/ZIF-8/Gel) centrase was engineered to scavenge endogenous overexpressed ROS synergistically generating dissolved oxygen and enhancing sustained lubrication for CHOs as a ternary artificial nano-enzyme. This property enabled the nano-enzymatic hydrogels to mitigate OA hypoxia and inhibit oxidative stress damage successfully. Ternary strategy-based therapies show excellent cartilage repair in vivo. The experimental results suggest that nano-enzyme-enhanced lubricating hydrogels are a potentially effective OA treatment and a novel strategy.

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1. Introduction

Osteoarthritis (OA) is globally known to be a highly prevalent degenerative disease of cartilage and is recognized by the medical community as one of today's major public health problems.^1,2 According to preliminary statistics, more than 250 million people worldwide suffer from this disease each year and end up with up to ∼41% disability.^2,3 A recent study reported that with the rapid growth of the aging population, nearly 25% of adults are expected to have OA by 2030.⁴ Despite the high prevalence and disability caused by OA, there is a lack of effective methods to target the management of this widespread disease.⁵ Surgery is the only option for patients with advanced disease. There is still no treatment for early-stage patients to reverse or slow the progression of OA.⁶ Recently, scavenging the ROS generated by oxidative stress in chondrocytes (CHOs) has been recognized as the most promising approach for the treatment of OA.^2,7–10 However, the biggest bottleneck in clinical practice is how to achieve sustained and efficient ROS removal, reducing damage to CHOs to rebuild cartilage structure and function.

In vivo, ROS levels are mainly regulated by superoxide dismutase (SOD) and catalase (CAT).¹¹ However, the expression of these biological enzymes in OA joint tissues is significantly decreased and leads to ROS accumulation, accelerating the progression of OA in normal articular cartilage.¹² Therefore, ROS scavenging by supplementation with exogenous bio-enzymes may be a potential therapeutic strategy for OA treatment.^4,13–16 However, defects such as rapid inactivation, uneven distribution, rapid depletion, and insufficient retention of the biological enzymes at the lesion site limit the clinical application of this therapy in OA.^4,17,18 Drug platforms based on the construction of transported bio-enzymes such as liposomes,^19–21 hollow silica nanospheres,^22,23 polymersomes,^24,25etc. through well-improved pharmacokinetics, biodistribution, and solubility have been achieved. However, surface conjugation or immobilization of these carriers leading to rapid inactivation of the enzyme, poor stability, and difficulties in application and storage remains a problem.⁴ Besides, lower enzyme loading, high burst release, and difficulties in surface functionalization further limit their application in OA.^18,26 In conclusion, it is virtually impossible to achieve long-term sustained treatment of OA with a regimen based on the scavenging of ROS by biological enzymes.

Encouragingly, artificial nano-enzymes with ROS scavenging ability have been considered as the most promising avenue for the treatment of OA in recent years due to their catalytic efficiency and enzymatic reaction kinetics that are similar to biological enzymes.^27–29 For example, nanomaterials based on MoS₂,^30,31 polydopamine (PDA),³² platinum particles,^12,28 MnO₂,^33,34 Prussian blue,^35,36 and CeO₂³⁷ have demonstrated excellent abilities in inhibiting inflammation and scavenging ROS with highly effective OA treatment. Unfortunately, these nano-enzymes not only involve complex preparation procedures and high costs, but also suffer from severe loss of exposed active centers due to unavoidable agglomeration during synthesis, storage, and application, resulting in a large gap between the catalytic activity and kinetics of artificial nano-enzymes and those of biological enzymes.^38–40 Although recently reported single-atom nano-enzymes can drastically disperse and expose metal active centers, providing a solution for improving catalytic activity, most of these nano-enzymes must rely on additional photothermal effects to achieve ROS scavenging.³⁸ This time-consuming and labor-intensive process not only causes inconvenience in treating OA, but the prolonged exposure to near-infrared radiation and elevated temperatures may also be potentially harmful to the patient.⁴¹ Metal–organic frameworks (MOFs) are considered ideal carriers for the immobilization and loading of drugs and various nanocatalytic materials due to their high surface area and porous structure.^42–45 However, recent attempts at artificial nano-enzymes/MOF engineering in phase studies have generally focused on cancer therapy, and biosensing and antimicrobial activities.^46–48 Besides, the expensive prices, low natural reserves, and large biotoxicity of the heavy, precious, and rare metals used in the current design of nano-enzymes greatly limit their application in biomedical engineering.¹² It is urgent and challenging to find highly safe and abundant nano-enzymatic/MOF-engineered materials to expand the application for OA therapy.

Furthermore, normal metabolism and nutrition of cartilage depend on the maintenance of synovial and lymphatic fluids.⁴⁹ The flow of these fluids results in the rapid clearance of nano-enzymes from the OA joint cavity which cannot sustainably clear ROS.⁵⁰ Friction from degeneration of articular cartilage is thought to be at the root of the development of OA.⁵¹ Therefore, the development of OA is highly correlated with lubrication at the articular cartilage interface, and the simultaneous maintenance of joint hyperlubrication while using artificial nano-enzymes is crucial and a prerequisite for the inhibition of OA.⁵² Hypoxia of CHOs is another important characteristic pathological change in OA and it promotes synovial inflammation.⁵³ Hypoxia also decreases mitochondrial respiration, generates ROS and increases oxidative damage, which further induces OA inflammation.⁵⁴ Therefore, a ternary synergistic regimen of decreasing ROS levels while increasing the oxygen content of the cartilage environment and maintaining lubrication at the articular cartilage interface may be an effective strategy for the treatment of OA. To the best of our knowledge, this ternary synergy scheme has not been reported.

Here, we innovatively proposed nano-enzyme-enhanced lubricating hydrogels as a ternary strategy for hydrogen peroxide (H₂O₂)-driven synergistic oxygen production to treat OA (Fig. 1). This lubricating hydrogel complex inspired by biological metabolism is made of natural pigskin gelatin (Gel) and catalase-mimicking nano-enzymes (Fe/ZIF-8), and has properties such as injectability and high ROS scavenging activity. Zn and Fe used in nanozymes obtained using a simple and rapid room temperature coordination reaction (3 h) have great crustal abundance and are essential trace elements for the human body, and have strong biocompatibility (Fig. 1a). The catalytic nano-enzymes can mimic SOD and CAT bio-enzymatic activities, and the resulting hydrogel system (Fe/ZIF-8/Gel) can efficiently decompose endogenous H₂O₂ and synergistically produce O₂ (Fig. 1b). In vitro experiments demonstrated that the nanoenzyme-enhanced hydrogels successfully alleviated OA hypoxia and inhibited oxidative damage. Physical parameters such as the high water content and chemical composition of hydrogels as well as their adjustable mechanical strength allow them to well mimic the temporal and spatial complexity required for CHO growth and provide continuous lubricity.⁵⁵ The three-dimensional (3D) spatial structure and large specific surface area of the hydrogel effectively attenuated the clearance of the nano-enzymes in the OA joint cavity. The 3D spatial structure and large specific surface area of the hydrogel effectively reduced the clearance rate of nano-enzymes in the OA joint cavity, providing a suitable 3D microenvironment for ROS reduction and cartilage repair in CHOs. To further investigate the effectiveness of the ternary strategy therapy in vivo, a nano-enzyme-enhanced lubricating hydrogel was injected into a large cartilage injury in animal models of OA. The results showed excellent cartilage repair. These findings suggest that lubricating hydrogels based on a ternary strategy has great promise for treating OA.

Fig. 1 Schematic synthesis of Fe/ZIF-8/Gel nano-enzyme hydrogels and the principle of scavenging endogenous ROS inspired by metabolic processes to drive O₂ generators and treat OA. (a) Schematic diagram of the Fe/ZIF-8/Gel nano-enzyme hydrogel synthesis process; (b) the Fe/ZIF-8/Gel nano-enzymatic hydrogels alleviate OA through a ternary strategy of scavenging ROS, driving O₂ production, and providing lubrication to the bone and joint.

2. Experimental

2.1. Reagents and apparatus

All chemicals are of analytical grade and do not require further purification for use. Zinc nitrate hexahydrate (Zn(NO₃)₃·6H₂O) (Z111703, Aladdin), 2-methylimidazole (77272A, Adamas), ferric nitrate nine hydrate (Fe(NO₃)₃·9H₂O) (82876F, Adamas), potassium hexacyanoferrate(III) (K₃[Fe(CN)]₆·3H₂O) (22174A, Adamas), tetrapotassium hexacyanoferrate (K₄[Fe(CN)]₆) (22721, HAdamas), methacryloyl gelatin (Gel) (EFL-GM-60) (Engineering For Life), hydrophilic 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) (32437E, Adamas), diphenylpropylphenylhydrazine (DPPH) (33386D, Adamas), lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP) (3321898BA, Adamas), methanol (CH₃OH) (75851V, Adamas), H₂O₂ (82427D, Adamas), anhydrous ethanol (CH₃CH₂OH) (G73537AC, Greagent), ultrapure water (UP, 18.25 MΩ cm⁻¹), and phosphate buffered salt solution (PBS, 0.01 M, pH = 7.2–7.4).

Drum-air electric thermostatic drying oven and ultrasonic cleaning instrument were purchased from Wuxi Yibaofan Environmental Test Equipment Co., Ltd (Wuxi, China) and Li Fang Hua Cheng (Chengdu, China), respectively. A thermostatic bath was purchased from Xinzhi. The glassy carbon electrode (GCE) (d = 5 mm), Ag/AgCl electrode, and platinum wire electrode were purchased from Tianjin Incole Union Technology Co., Ltd. An electronic balance was purchased from Tuoliduo. A refrigerated centrifuge was purchased from Zhongkezhongjia. A CHI760E electrochemical workstation was purchased from Chen Hua Co. (Shanghai, China). Scanning electron microscopy (SEM, JSM-7610FPlus), transmission electron microscopy (TEM, JEM-2100plus), grazing incidence X-ray diffractometer (GIXRD, SmartLab 9 kW), energy-dispersive spectrometry (EDS, ULTIM MAX 40), Raman spectroscopy (Thermo DXR2xi), Fourier transforms attenuated total reflectance infrared spectroscopy (ATR-FTIR, PerkinElmer UATR two), inductively coupled plasma spectroscopy/mass spectrometry (ICP-AES/MS, Aglient-7800(MS)), and X-ray photoelectron spectroscopy (XPS, Thermo Scientific scalable Xi+) were used to characterize the morphological structure, elemental composition, and valence state of the modified electrode surfaces.

2.2. Synthesis of Fe/ZIF-8 nano-enzymes

Metal–organometallic frameworks were synthesized using a simple room-temperature coordination reaction. Briefly, 2.57 g of Zn(NO₃)₂·6H₂O and 1.74 g of Fe(NO₃)₃·9H₂O were added to 18 mL of CH₃OH and dissolved with stirring. Then 5.68 g of 2-methylimidazole was dissolved in 82 mL of CH₃OH. Finally, the two solutions were quickly mixed and stirred for 24 h at room-temperature. The precipitate was collected by centrifugation and washed three times with CH₃OH, and the resulting product was dried overnight in an oven at 60 °C to obtain a light yellow powder labeled Fe/ZIF-8. ZIF-8 was prepared according to the same procedure without the addition of Fe(NO₃)₃·9H₂O.

2.3. Preparation of Fe/ZIF-8/Gel hydrogels

First, a standard solution of LAP initiator at a concentration of 0.25% (w/v) was prepared. A 5 wt% hydrogel precursor was prepared by dissolving the purchased gel in 0.25% (w/v) LAP at 60–70 °C. Then, different masses of Fe/ZIF-8 (10 μg, 50 μg, 100 μg, 200 μg, and 400 μg) were added to 2 mL of 5 wt% hydrogel precursor and stirred well. Finally, hydrogels containing different concentrations of Fe/ZIF-8 (Fe/ZIF-8/Gel) were prepared under ultraviolet illumination for 5 min. Pure gel was prepared without the addition of Fe/ZIF-8 following the same procedure.

2.4. Cell culture and processing

The chondrocytes (CHOs) used in this study were obtained from Sprague Dawley (SD) rats and extracted according to established procedures. Briefly, articular cartilage tissue from each knee was aseptically processed and incubated in type II collagenase (0.25%; Sigma-Aldrich) for 2 h at 37 °C. Filtration, centrifugation (1000 rpm min⁻¹, 5 min), and collection of the supernatant were carried out. The supernatant was then resuspended in a 10 cm diameter Petri dish and incubated in 8 mL of Dulbecco's modified Eagle's medium (DMEM) (low sugar, Gibco) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) and incubated at 37 °C and 5% CO₂. The medium was changed every 2 days until the cells reached ∼80% concentration.

2.5. In vitro cell viability studies

To explore the biocompatibility of the prepared Fe/ZIF-8/Gel, CHOs (at a density of 5000 cells per well) were cultured in 96-well plates and then treated with Fe/ZIF-8 hydrogel (100 μg mL⁻¹) (200 μL) or PBS (200 μL) processing. After 24 h of incubation, the cells were treated with Cell Counting Kit-8 (CCK8) (C8022, Adamas life) assay kit volume and tested for cell viability at 450 nm with a microplate spectrophotometer (Thermo Fisher, USA). Cell viability was also assessed using the calcein-AM/PI kit (56496-20X50UG, Sigma-Aldrich) according to the manufacturer's instructions, and the cell morphology and number were observed using a high-throughput multiparameter cell dynamic analysis system (PE/Opera Phenix Plus). To simulate the oxidative environment of OA, CHOs were cultured in 96-well plates in different groups (PBS, ZIF-8/Gel, and Fe/ZIF-8/Gel) according to the same procedure, and H₂O₂ (10 μL, 100 μM) was added. After 24 h of incubation, cell proliferation, and viability were determined using the CCK-8 assay kit and calcineurin-AM/PI staining.

2.6. Assessment of intracellular ROS (H₂O₂) consumption and O₂ production

The ability of Fe/ZIF-8/Gel hydrogels to scavenge intracellular H₂O₂ was verified using the H₂O₂ probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, CHOs (5.0 × 10⁴ cells per well) were cultured in 96-well plates and then incubated with ZIF-8/Gel or Fe/ZIF-8/Gel in a ROS environment (concentration of 100 μM H₂O₂). For comparison, CHOs were incubated with PBS with or without H₂O₂ following the same procedure. After 3 days of culture, 10 μM DCFH-DA was added to all experimental groups, and the intracellular ROS levels were observed using confocal microscopy. A Ru(dpp)₃Cl₂ probe was used to verify the intracellular O₂ generation capacity of Fe/ZIF-8/Gel hydrogels. Briefly, after Ru(dpp)₃Cl₂ (10 μg mL⁻¹) was added to the different groups and incubated at 37 °C for 3 days, the O₂ levels of all treatment groups were observed using fluorescence confocal microscopy.⁵⁶

2.7. Evaluation of SOD-like enzyme activities in Fe/ZIF-8/Gel hydrogels

The SOD activities of ZIF-8/Gel and Fe/ZIF-8/Gel (100 μg mL⁻¹) were assayed by total superoxide dismutase. Assay Kit with NBT (S0109, Beyotime, China) by following the instructions of the manufacturer. Briefly, CHO cells were collected and washed twice with 4 °C 0.01 M PBS (pH = 7.2–7.4). The CHO cells were collected by centrifugation (2000 rpm min⁻¹, 5 min) and homogenized in a 4 °C PBS ice bath with a glass homogenizer. The homogenate was then centrifuged at 4 °C (2000 rpm min⁻¹, 5 min) and the supernatant was taken as the sample to be tested. The appropriate volume of NBT/enzyme working solution was configured according to the volume of 160 μL per reaction (SOD assay buffer: NBT: enzyme solution = 158 : 1 : 1) and stored at 4 °C. 40 μL of the startup working solution was prepared at a ratio of reaction startup solution (40X): SOD = 1 : 39 and stored at 4 °C. Adding 200 μL of the mixture to a 96-well plate at a ratio of sample: reaction initiation work solution: NBT/enzyme work solution = 20 : 20 : 160 and incubating for 30 min at 37 °C. Finally, using the microplate spectrophotometer the absorbance of the mixture was measured at 560 nm. The SOD activities of the blank control, PBS, and PBS + H₂O₂ groups were tested according to the same procedure and instructions. Finally, the SOD activity of each group of samples was calculated according to the formula in the instructions.

2.8. Assessment of intracellular inflammatory factors

The CHOs were processed according to the same procedure as in section 2.6. The supernatants of different experimental groups were collected after 3 days of incubation and the expression level of interleukin 6 (IL-6) in the supernatants was detected using an ELISA kit (Meinian, China).

2.9. Evaluation of the mitochondrial protective capacity of Fe/ZIF-8/Gel hydrogels

The CHOs were processed according to the same procedure as in section 2.6. After 3 days of incubation, the membrane potential and calcium content of mitochondria were measured using a JC-1 kit (Beyotime Biotech, China) and Fluo-4 fluorescent probe (Beyotime Biotech, China), respectively, and the images were collected using fluorescence confocal microscopy.

2.10. In vivo therapeutic OA performance evaluation of the Fe/ZIF-8/Gel hydrogel

The experiments were performed using SD rats weighing 200–250 g (Sichuan University Animal Center, Chengdu, China). The rats were placed in a climate-controlled room, under the light/dark cycle of 12 h/12 h with free access to food and water. All animal experiments in this work were reviewed, approved, and supervised by the Institutional Animal Care and Use Committee of Sichuan University. During all the experiments, the animals were deeply anesthetized with 2 wt% sodium pentobarbital. Using an anterior cruciate ligament transection (ACLT) procedure to establish the OA model.²

All SD rats were randomly and equally divided into 4 groups (n = 3). The interventions were as follows: (1) Sham group: 100 μL of saline only; (2) Control group: OA SD rats + 100 μL saline; Experimental group: (3) OA SD rats + 100 μL ZIF-8/Gel (100 μg mL⁻¹); (4) OA SD rat + 100 μL Fe/ZIF-8/Gel (100 μg mL⁻¹). Four groups of SD rats were injected weekly until 4 or 8 weeks. After the rats were sacrificed, the knee joints and major organs were collected for further experiments and the knee joints were imaged and evaluated by macroscopic scoring. The expression level of MMP13 in synovial fluid was detected using an ELISA kit and assayed at 450 nm absorbance using a microplate detector (Synergy H1, BioTek).

2.11. Tissue staining of joint sections

The collected knee samples were fixed in 4% paraformaldehyde and decalcified with 10% EDTA (Beyotime, China). The joints were then embedded and sectioned. The joint sections were stained with Hematoxylin–eosin (H&E) (Solarbio, China), Safranin O/Fast Green (Solarbio, China), and Toluidine blue (Solarbio, China), respectively. The severity of cartilage degeneration was then assessed using the Osteoarthritis Research Society International (OARSI) score. Besides, immunohistochemical staining was used to detect the expression of matrix metalloproteinase 13 (MMP13) and collagen type II (COL2). Briefly, after dewaxing and dehydration, the sections were incubated with 3 vt% H₂O₂ for 10 min and heated in a water bath containing EDTA (Beyotime, China) for 5 min. After occlusion with 5% BSA for 30 min at 37 °C anti-MMP13 and anti-COL2 (dilution 1 : 200) were added to the sections and left overnight at 4 °C. After rinsing with PBS, the sections were incubated with a secondary antibody (Beyotime, China). Finally, they were stained with DAB (Boster, China) and Mayer's hematoxylin (Slarbio, China) and observed with fluorescence confocal microscopy.

2.12. In vivo cytotoxicity assessment

The major organs (heart, liver, spleen, lung, and kidney) were embedded and sectioned, stained with H&E, and visualized using fluorescence confocal microscopy.

2.13. Statistical analysis

The differences between various groups were analyzed by one-way analysis of variance (ANOVA) or Dunnett's T3. The levels of significance were set at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001 (*symbol compared with the sham group and # symbol compared between groups). Data are presented as means ± standard deviations (mean ± SD). All statistical analyses and graphing were performed using Origin 2018 64Bit and GraphPad.Prism.9.5.

3. Results and discussion

3.1. Design and characterization of Fe/ZIF-8/Gel

The ZIF-8 with a dodecahedral structure was synthesized through a simple room-temperature coordination reaction of Zn²⁺ and 2-methylimidazole. The synthesized ZIF-8 has a typical ∼200 nm center size, is relatively uniform, has a smooth surface with rounded surroundings, and appears to be relatively regular and complete (Fig. 2a and c). Here, 2-methylimidazole was chosen as a ligand because of its mild conditions, shorter time, and simplicity in coordination with Zn²⁺. Besides, the relatively homogeneous size and regular structure facilitate the loading of other metal elements (such as Cu²⁺) and expose the active catalytic sites as large as possible.⁵⁷ Numerous studies have shown that iron ions can rapidly catalyze the decomposition of ROS, especially H₂O₂, and have good biocompatibility.^58–61 To alleviate ROS-induced OA, Fe-doped ZIF-8 (Fe/ZIF-8) was prepared by adding Fe(NO₃)·9H₂O to the coordination process (Fig. 1a). The obtained Fe/ZIF-8 inherits the original polyhedral structure of ZIF-8, while its center size increases to ∼600 nm (Fig. 2b and d). The increase in center size may be because adding Fe³⁺ leads to partial deprotonation of the 2-methylimidazole ligand, which allows the ZIF-8 crystals to grow in all directions and produces larger, better-grown crystals.⁶² Energy dispersive spectroscopy (EDS) of ZIF-8 showed a very homogeneous distribution of the elements Zn, C, N, and O, which can depict the unique dodecahedral profile of ZIF-8 (Fig. 2e). Besides, the uniform distribution of Fe in ZIF-8 ensures efficient catalytic decomposition of H₂O₂ to generate O₂, thus rapidly reducing oxidative stress and hypoxia and effectively alleviating OA (Fig. 2f). To achieve targeted delivery and slow release of Fe/ZIF-8 nanoparticles, we used methacryloyl Gel hydrogel with a hydrophilic 3D network as a carrier to accomplish long-term catalysis of H₂O₂ by injecting it into the lesion site. To observe the internal structure of Fe/ZIF-8/Gel, the gelatinized composites were rapidly frozen in liquid nitrogen, lyophilized overnight, and characterized. The Fe/ZIF-8/Gel hydrogel showed an irregular porous structure, which facilitated the adhesion and growth of CHO cells (Fig. 2g). Besides, the large specific surface area of the porous structure provides abundant sites for the attachment of Fe/ZIF-8 and the catalytic decomposition of H₂O₂. SEM showed that Fe/ZIF-8 was uniformly dispersed in the gel hydrogel (Fig. 2h). The unique rheological properties of gel hydrogels also allow for excellent cartilage protection and lubrication within the joint cavity, lubrication that is essential for inhibiting OA and relieving pain caused by friction in patients during exercise.⁶³ Rheological measurements were performed to assess the mechanical properties of the gel hydrogels, and swept-frequency measurements of the gel hydrogels at 0.5% strain showed that the storage modulus (G′) was about an order of magnitude higher than the loss modulus (G′′) in the region of the measured frequency, indicating successful gelation (Fig. 2i).⁶⁴ The consistently smaller viscosity of the hydrogel with increasing shear rate verifies the good shear thinning properties and injectability of the gel (Fig. 2j).⁶⁵ In fact, the hydrogel can be injected smoothly through a 26-gauge needle to form the specified pattern (e.g., “DH”) and can still be injected in a liquid environment (e.g., PBS) (Fig. 3a–d).

Fig. 2 Synthesis and characterization of the Fe/ZIF-8/Gel nano-enzyme hydrogel. (a) and (b) The SEM images of (a) ZIF-8 and (b) Fe/ZIF-8 (scale bar: 500 nm). (c) and (d) The TEM images of (c) ZIF-8 and (d) Fe/ZIF-8 (scale bar: 100 nm). (e) and (f) Elemental mapping images of (e) ZIF-8 (scale bar: 50 nm) and (f) Fe/ZIF-8 (scale bar: 200 nm). (g) and (h) SEM images of (g) Gel hydrogel (scale bar: 500 μm) and (h) Fe/ZIF-8/Gel (scale bar: 50 μm). (i) Rheological behavior of the Gel in the frequency sweep mode (frequency range from 0.1–10 Hz). (j) Variation in viscosity of the Gel with shear rate from 0.01–1000 s⁻¹.

Fig. 3 Characterization of the Fe/ZIF-8/Gel nano-enzyme hydrogel. (a)–(c) The formation of a Gel from a liquid state to a hydrogel (scale bar: 1 cm). (d) Injectability of the Fe/ZIF-8/Gel nano-enzymatic hydrogel (scale bar: 1 cm). (e) XRD image of ZIF/8 and Fe/ZIF-8. (f) Full-scan XPS image and elemental contents of ZIF/8 and Fe/ZIF-8. (g)–(i) Deconvolution energy spectra of (g) C 1s, (h) O 1s, and (i) Zn 2p for ZIF/8 and Fe/ZIF-8.

The phase composition of ZIF-8 was investigated by X-ray diffraction (XRD). The diffraction peaks located at 7.3°, 10.4°, 12.7°, 14.7°, 16.4°, 18.0°, 24.5, and 26.6° are narrow and sharp, indicating that ZIF-8 is well crystallized (Fig. 3e). These spikes can be attributed to the (011), (002), (112), (022), (013), (222), (114), (233), (1134), and (044) lattice planes of ZIF-8, belonging to the cubic space group (I4̄₃m) (JPCDS No. 89-3739).^66,67 The average microcrystalline sizes of ZIF-8 and Fe/ZIF-8 were estimated to be ∼190 nm and ∼587 nm, respectively, using the Debye–Scherrer equation (D_hkl = kλ/β cos θ), and the peak widths of the strongest characteristic diffraction peaks (011) crystal planes, which indicated that the results estimated by XRD were in general agreement with the TEM measurements.⁶⁸ Apparently, although the sizes of ZIF-8 and Fe/ZIF-8 are different, they have similar crystalline properties, indicating that Fe doping does not affect the crystallization process (Fig. 3e), which can maintain the unique framework structure of ZIF-8 and help to achieve Fe immobilization and efficient catalysis of H₂O₂. The chemical state and surface group composition of ZIF-8 and Fe/ZIF-8 nanoparticles were investigated by X-ray photoelectron spectroscopy (XPS). The full-scan XPS spectrum of ZIF-8 contains peaks for C 1s (∼284 eV), N (∼284 eV), O 1s (∼532 eV), and Zn 2p (1022 - 1045 eV) (Fig. 3f). The presence of Fe signals in the full spectrum of Fe/ZIF-8 indicates that iron has been successfully doped into the polyhedra. The C 1s of both ZIF-8 and Fe/ZIF-8 were deconvoluted into three subpeaks, C–C/C=C, =C–OH, and N–C=N, which are closely related to their ligands (Fig. 3g).⁶⁹ To investigate the effect of Fe doping on ZIF-8, the high-resolution spectra of ZIF-8 and Fe/ZIF-8 O 1s and Zn 2p were deconvolved, respectively (Fig. 3h and i). The peaks of the high-resolution spectrum of ZIF-8 O 1s were fitted to 531.8 and 533.3 eV, corresponding to the C–O and –OH bonds, respectively⁷⁰ (Fig. 3h). In contrast, the fitted peak of O 1s in Fe/ZIF-8 was shifted by 0.3 eV toward lower binding energy (Fig. 3h). This phenomenon can also be observed in the deconvolution spectra of Zn 2p, where Fe doping leads to a decrease in the binding energy of Zn 2p in ZIF-8 from 1021.8 and 1044.6 eV to 1019.8 and 1042.7 eV compared to ZIF-8⁷¹ (Fig. 3i). This was attributed to the fact that the doping of Fe³⁺ significantly improved the electrical conductivity of the composites and effectively modulated the electron cloud density of ZIF-8,⁷² resulting in the electron transfer being accelerated, which facilitated its rapid catalysis and decomposition of H₂O₂in vivo. This process was visually verified by cyclic voltammetry curves (CV) and electrochemical impedance spectroscopy (EIS) (Fig. S1, ESI†). The response current of the redox peak of Fe/ZIF-8 was significantly higher than that of ZIF-8, while the semicircle diameter of its EIS was substantially reduced, which proved that the electron transfer ability of Fe/ZIF-8 was significantly higher than that of ZIF-8, and the scavenging of ROS could be realized rapidly (Fig. S1, ESI†). Besides, the high-resolution spectra of Fe in Fe/ZIF-8 were fitted to Fe^(III), indicating that the doping process does not change the valence state of Fe, which is essential to ensure the efficient catalysis of H₂O₂ (Fig. S2, ESI†).

3.2. Evaluation of the Fe/ZIF-8/Gel in vitro catalyzed ROS decomposition performance

H₂O₂ is one of the most common ROS in tissues.⁷³ The exchange of substances during metabolism is recognized as a determining factor in the regulation of physiological functions.⁷⁴ Inspired by metabolic processes, nano-enzyme-enhanced self-protecting hydrogels were designed as H₂O₂-driven O₂ generators (Fig. 1b and 4a). The Fe/ZIF-8/Gel catalytic ability to decompose H₂O₂ was proportional to the content of Fe/ZIF-8 nano-enzymes, indicating that the excellent catalytic ability of the hydrogel was mainly attributed to the nano-enzymes (Fig. 4b). Besides, Fe/ZIF-8/Gel showed an H₂O₂ scavenging capacity comparable to that of natural catalase in neutral PBS (pH 7.4) (Fig. 4c). In acidic PBS (pH 6.5), the catalytic activity of Fe/ZIF-8/Gel remained almost unchanged, whereas catalase activity was significantly reduced (Fig. 4c). Significant bubble production can be observed in acidic PBS containing H₂O₂ and Fe/ZIF-8/Gel, which is attributed to the catalase-like activity of Fe/ZIF-8/Gel that catalyzes the decomposition of H₂O₂ to produce O₂ (Fig. 4a, d and Fig. S3, ESI†). Notably, ZIF-8/Gel also catalyzes the decomposition of H₂O₂ to produce O₂, because Zn²⁺ can scavenge H₂O₂.⁷⁵ However, the ability of the ZIF-8/Gel to catalyze the decomposition of H₂O₂ to produce O₂ was significantly lower than that of Fe/ZIF-8/Gel (Fig. S4, ESI†). More importantly, Fe/ZIF-8/Gel can durably catalyze H₂O₂. Even after repeated additions of H₂O₂, the hydrogels continued to exhibit excellent catalytic activity without showing a significant delay (Fig. 4e). Besides, Fe/ZIF-8/Gel was able to continuously produce O₂ in the presence of H₂O₂, showing its excellent catalytic persistence (Fig. 4f). To verify the advantages of the ortho-dodecahedral structure of Fe/ZIF-8 for H₂O₂ scavenging, we prepared Fe-MOF according to the method reported in the literature and investigated the scavenging effect of the nano-enzymatic hydrogel with the same concentration (100 μg mL⁻¹) on H₂O₂ scavenging.⁷⁶ The synthesized Fe-MOF has an irregular bulk structure with a size of ∼2 μM (Fig. S5, ESI†). Due to the particular spatial structure and large specific surface area of ZIF-8 favoring the loading and exposure of Fe³⁺, it exhibits a better H₂O₂ scavenging effect than Fe-MOF (Fig. S6, ESI†). The Michaelis–Menten kinetics of Fe/ZIF-8/Gel were evaluated by the fast and slow production of O₂ catalyzed by Fe/ZIF-8/Gel at different concentrations of H₂O₂ (pH 6.5) (Fig. 4d and g). The maximum velocity (V_max) of the reaction catalyzed by Fe/ZIF-8/Gel nano-enzymes was 6.857 × 10⁻⁶ M s⁻¹, and the Mie constant (K_m) was calculated to be 47.241 mM, which was higher than that of the K_m value of catalase (25 mM),⁷⁷ indicating that Fe/ZIF-8/Gel has high catalytic activity in acidic environments, which is difficult for other catalytic ROS nanocatalysts (such as Mn₃O₄) to achieve (Table S1, ESI†). Such nano-enzymes that maintain high catalytic activity under acidic conditions are ideally suited for the treatment and palliation of OA, as the local synovial fluid at the patient's lesion site due to the presence of ROS may often be maintained in an acidic environment as low as pH 6.5, leading to little efficacy of current mainstream development of ROS scavenging strategies based on exogenous bio-enzymes due to enzyme inactivation.²² Moreover, the H₂O₂-driven Fe/ZIF-8/Gel oxygen generator showed excellent concentration dependence and responsiveness, and its oxygen production and oxygen production rate was essentially proportional to the H₂O₂ concentration (Fig. 4d). Briefly, when the concentration of H₂O₂ is doubled, the rate and mass of O₂ production are doubled. This suggests that Fe/ZIF-8/Gel can dynamically and intelligently track and quickly remove ROS from OA very well. In other words, the catalytic H₂O₂ and oxygen production capacity of Fe/ZIF-8/Gel does not change depending on the concentration of ROS. This excellent performance in catalyzing H₂O₂ decomposition was also verified by time–current (i–t) curves (Fig. 4h). After the baseline was stabilized and different concentrations of H₂O₂ were added successively, Fe/ZIF-8/Gel consistently showed excellent catalytic ability and all of them obtained obvious current response platforms. Interestingly, Fe/ZIF-8/Gel can linearly catalyze the decomposition of H₂O₂ in the range of 10 μM–1.1 mM in the class of 10 μM, with a calibration curve regression equation of I_p (μA) = 0.02377C_H2O2 + 0.4106 (R² = 0.9941) (Fig. 4i), and the lower limit of catalysis is 1.2 μM. There was essentially no time delay in the response of Fe/ZIF-8/Gel to H₂O₂, indicating that Fe/ZIF-8/Gel nanohydrogels have an extremely fast response rate (Fig. 4j). It is important to note that to the best of our knowledge, this is the first report developed with direct evidence that nano-enzymatic hydrogels can accomplish the catalytic decomposition of physiological level concentrations of H₂O₂ (50 μM).⁷⁸ This wide range of catalytic ability ensures that Fe/ZIF-8/Gel is fully capable of treating OA due to ROS.

Fig. 4 Catalytic-like activity of Fe/ZIF-8/Gel nano-enzyme hydrogel and characterization of ROS(H₂O₂)-driven O₂ generation. (a) The generation of O₂ bubbles in acidic PBS (pH 6.5) containing H₂O₂ (25 mM) and Fe/ZIF-8/Gel nano-enzyme hydrogel was observed using light microscopy (scale bar: 100 μm). (b) UV-visible absorption spectra of nano-enzymatic hydrogels containing different concentrations of Fe/ZIF-8 upon the addition of Ti(SO₄)₂ solution (concentration of H₂O₂ was 1.0 mM). (c) H₂O₂ inhibition rates of Fe/ZIF-8/Gel nano-enzyme hydrogel and catalase in PBS at different pH values. (d) Time course of O₂ production by Fe/ZIF-8/Gel nano-enzyme hydrogels in acidic PBS (pH 6.5) with different concentrations of H₂O₂ (the inset represents the principle of Fe/ZIF-8/Gel nano-enzyme hydrogel driving H₂O₂ to produce O₂). (e) Repetitive catalytic H₂O₂ consumptionability of the Fe/ZIF-8/Gel nano-enzyme hydrogel with repetitive addition of H₂O₂ (1.0 mM). (f) Continuous catalytic O₂ generation ability of Fe/ZIF-8/Gel nano-enzyme hydrogel with repetitive addition of H₂O₂ (0.1 mM). (g) Michaelis–Menten kinetics of Fe/ZIF-8/Gel nano-enzyme hydrogel calculated from panel (d). (h) i–t Current response curves of the Fe/ZIF-8/Gel nano-enzyme hydrogel to the continuous addition of H₂O₂ (the inset illustrates the mechanism of electrocatalytic H₂O₂ generation of O₂) and (i) the corresponding calibration curves. (j) Fe/ZIF-8/Gel nano-enzyme hydrogel fast drive curve for oxygen production from H₂O₂. In all experiments, the Fe/ZIF-8/Gel nano-enzyme hydrogel concentration was 100 μg mL⁻¹. These data are presented as mean values ± SD (n = 3).

OA is a synovial tissue-associated inflammation that results from an over-activated immune response against self-antigens.⁷⁹ Therefore, the ROS scavenging ability of Fe/ZIF-8/Gel against hydrophilic 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) radicals and hydrophobic diphenylpicrylphenylhydrazyl (DPPH) radicals was further verified (Fig. 5a and b). The scavenging of PTIO and DPPH radicals by Fe/ZIF-8/Gel hydrogels increased with increasing Fe/ZIF-8 nano-enzyme content (Fig. 5c). This suggests that Fe/ZIF-8 nanoparticles can significantly improve the ability to scavenge ROS, which is attributed to the fact that iron atoms can rapidly perform electron transfer and catalyze free radical decomposition, which helps to eliminate oxidative stress in the OA lesion site and promote tissue repair. Similar to the catalyzed H₂O₂ decomposition, the Fe/ZIF-8/Gel hydrogel maintained a high scavenging capacity for these radicals under acidic conditions (Fig. 5b).

Fig. 5 In vitro behavioral analysis of CHOs protected by the Fe/ZIF-8/Gel nano-enzyme hydrogel. (a) and (b) Scavenging of PTIO and DPPH radicals by Fe/ZIF-8/Gel nano-enzyme hydrogels at different pH values. (c) Scavenging rates of PTIO and DPPH radicals by nano-enzymatic hydrogels containing different concentrations of Fe/ZIF-8. (d) Calcein-AM/PI staining of CHOs after different treatment groups (scale bar: 200 μm). (e) Cell proliferation of CHOs in different groups on the 1st, 3rd, and 5th days. (f) Cell survival in different treatment groups after 5 days of co-culture of CHOs and Fe/ZIF-8/Gel nano-enzyme hydrogels. (g) ROS scavenging ability validated by a ROS probe (DCFHDA) after different treatments. Green fluorescence from DCFH-DA indicates the presence of ROS (scale bar: 50 μm). (h) Intracellular O₂ generation assay monitored by an O₂ probe [Ru(dpp)₃Cl₂]. Red fluorescence from Ru(dpp)₃Cl₂ is quenched by O₂ (scale bar: 100 μm). (i) MDA activity of CHOs after different treatments. (j) SOD activity of CHOs after different treatments. (k) The JC-1 probe verified mitochondria's membrane potential recovery ability after different treatments. JC-1 emitted green fluorescence to indicate the membrane damage of mitochondria (scale bar: 20 μm). These data are presented as mean values ± SD (n = 3). * and # indicate significant differences between Fe/ZIF-8/Gel and Gel and ZIF-8/Gel, respectively.

3.3. In vitro behavioral analysis of CHO protected by hydrogel

The CHO apoptosis directly contributes to articular cartilage damage in OA and causes many other cartilage diseases.⁸⁰ The morphological and functional changes in CHO cells can be an important marker for determining the progression of OA.⁷⁹ Oxidative stress caused by elevated ROS in the joints can severely accelerate CHO senescence.⁸¹ To address these limitations, we creatively designed a Fe/ZIF-8/Gel nano-enzymatic hydrogel as an H₂O₂-driven O₂ generator for the co-culture of CHO. Before in vivo application, the biocompatibility of Fe/ZIF-8/Gel nano-enzymatic hydrogels was investigated by live/dead staining and CCK-8 assay. In the PBS group, ZIF-8/Gel and Fe/ZIF-8/Gel groups, CHO had good cell survival, and no significant differences were observed between the groups (Fig. S7–S9, ESI†). Besides, CHOs were uniformly distributed in the Fe/ZIF-8/Gel nano-enzyme hydrogel matrix with intact morphology and no obvious damage (Fig. S7, ESI†). The Fe/ZIF-8/Gel nanoenzymatic hydrogel also had a high biosafety profile, as it was non-invasive to CHO cells (Fig. S8 and S9, ESI†). These results confirm that the developed and designed Fe/ZIF-8/Gel nano-enzymatic hydrogels have good biocompatibility and no significant cytotoxicity against CHO.

The good biocompatibility prompted us to further investigate the protective effect of Fe/ZIF-8/Gel nano-enzymatic hydrogels on CHO viability in the pathological microenvironment of OA. The OA pathological microenvironment enriched with H₂O₂ was simulated by adding 0.1 mM H₂O₂ co-culture to CHOs. Normal growth conditions for CHO growth were also simulated using PBS instead of H₂O₂. Oxidative stress injury resulted in significant CHO cell death when CHO was co-incubated with PBS + H₂O₂ and ZIF-8/Gel + H₂O₂ groups (Fig. 5d). However, the cells in the Fe/ZIF-8/Gel + H₂O₂ group were essentially identical to normal, with only a slight reduction in survival (Fig. 5e). These results also applied to the detection of CCK-8. The cell viability of CHO cells in the PBS + H₂O₂ and ZIF-8/Gel + H₂O₂ groups was significantly decreased. In contrast, the Fe/ZIF-8/Gel + H₂O₂ group effectively mitigated the damaging effect of H₂O₂ on the cells. The cell viability after 5 days of co-culturing was 32.05%, 83.12%, and 97.09% for the PBS + H₂O₂, ZIF-8/Gel + H₂O₂, and the Fe/ZIF-8/Gel + H₂O₂ groups, respectively (Fig. 5f).

3.4. In vitro evaluation of antioxidant and anti-inflammatory properties of Fe/ZIF-8/Gel hydrogels

To visually verify the performance of Fe/ZIF-8/Gel hydrogels for ROS scavenging, the CHOs were incubated in different treatment groups and stained with the ROS indicator 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA).⁵⁵ The green fluorescence intensity of the co-cultured CHOs in the Fe/ZIF-8/Gel + H₂O₂ group decreased by 3.14-fold and 1.92-fold, respectively, compared with that in the PBS + H₂O₂ and ZIF-8/Gel + H₂O₂ groups, suggesting that Fe/ZIF-8/Gel nano-enzymatic hydrogels significantly alleviated the intracellular oxidative stress (Fig. 5g and Fig. S10, ESI†). Previous studies have shown that ROS catabolism and O₂ generation help protect CHOs from oxidative damage.⁵⁴ Afterward, the in vitro O₂-generating capacity was monitored with a characteristic indicator of O₂ [Ru(dpp)₃Cl₂]. The COHs cultured in Fe/ZIF-8/Gel + H₂O₂ exhibited significant red fluorescence quenching due to elevated intracellular O₂, which fully demonstrated the high driving oxygen-producing capacity of Fe/ZIF-8/Gel + H₂O₂ nano-enzymatic hydrogels in scavenging endogenous ROS (Fig. 5h and Fig. S11, ESI†). Malondialdehyde (MDA) is an important end product of lipid peroxidation and can visually reflect the degree of oxidative damage to CHOs.⁵⁵ The MDA content of CHOs treated with Fe/ZIF-8/Gel was significantly lower than that of the PBS + H₂O₂ and ZIF-8/Gel + H₂O₂ groups and was comparable to that of the PBS group throughout the incubation period. In contrast, the MDA content of CHOs in the PBS + H₂O₂ group and the ZIF-8/Gel + H₂O₂ group was significantly elevated on days 3 and 5 (Fig. 5i). Increasing SOD activity improves the survival and proliferation of H₂O₂-injured cells.⁸² SOD also acts as the most important antioxidant protein in the body and is involved in repairing H₂O₂-induced cellular damage and improving survival. Therefore, we evaluated the SOD activity of Fe/ZIF-8/Gel nano-enzymatic hydrogels for CHO activation in an H₂O₂-rich OA pathology microenvironment. The SOD activity of CHOs was significantly decreased in the PBS + H₂O₂ group and the ZIF-8/Gel + H₂O₂ group (Fig. 5j), because H₂O₂ greatly inhibited the SOD activity. However, it is noteworthy that the antioxidant function of Fe/ZIF-8/Gel nanoenzymatic hydrogels significantly mitigated these deleterious effects. These findings emphasize that Fe/ZIF-8/Gel nano-enzymatic hydrogels may reduce the detrimental effects of H₂O₂ on its survival and activity by maintaining the intrinsic SOD activity of CHO and inhibiting the production of intracellular MDA, which may be beneficial for the suppression of inflammation.

Mitochondria are the main organelles in cells that produce ROS, and scavenging ROS is a viable treatment for OA.² Because excessive ROS causes mitochondrial dysfunction and disrupts homeostasis in CHO cells, leading to changes in membrane potential (Δψ_m) and abnormal calcium regulation. These dysfunctions, in turn, further induce a “vicious cycle” of ROS and promote the development of OA.⁸³In vivo, scavenging excess ROS in mitochondria promotes the recovery of mitochondrial membrane potential, oxidative respiratory chain, and mitochondrial autophagy, and repairs the CHO damage. First, the effect of Fe/ZIF-8/Gel nano-enzyme hydrogels on the oxidative respiratory chain associated with mitochondrial dysfunction was examined. The Δψ_m of different treatment groups was first tested with the JC-1 probe, and the PBS + H₂O₂ group and the ZIF-8/Gel + H₂O₂ group exhibited reduced JC-1 aggregate (red)/JC-1 monomer (green) ratios due to the mitochondrial dysfunction significantly leading to the reduction of mitochondrial membrane potential. This suggests that H₂O₂ stimulation disrupts mitochondrial membranes in CHOs, leading to the disappearance of JC-1 aggregates and the appearance of large numbers of JC-1 monomers (Fig. 5k and Fig. S12, ESI†). The Fe/ZIF-8/Gel + H₂O₂ group effectively scavenged ROS, inhibited H₂O₂-induced ROS damage to mitochondria, and showed negligible potential changes, which indicated that the H₂O₂-damaged mitochondrial membranes were completely restored, and the red/green ratios measured by the JC-1 probe were significantly higher than that of the other H₂O₂-treated groups (Fig. S13, ESI†).

Continued oxidative damage to OA exacerbates excessive inflammation. A cascade of inflammatory responses plays a key role in the pathologic microenvironment of OA, and pro-inflammatory cytokines or proteins, such as IL-6 and MMP13, are abundantly expressed in the joint cavity of OA.⁸⁴ Since Fe/ZIF-8/Gel nano-enzyme hydrogels have antioxidant capacity, they also have significant anti-inflammatory effects. ELISA confirmed that sustained oxidative stress induced by H₂O₂ treatment (0.1 mM) significantly increased the expression of IL-6 and MMP13 in CHOs, whereas Fe/ZIF-8/Gel nano-enzymatic hydrogels inhibited the expression of these cytokines even under H₂O₂-rich conditions (Fig. S14 and S15, ESI†).

3.5. In vivo performance evaluation of Fe/ZIF-8/Gel hydrogel-mediated therapeutic OA

In vitro, we confirmed the excellent ROS scavenging (Fig. 5d), antioxidant (Fig. 5i and j), and anti-inflammatory (Fig. 5e and f) abilities of the designed Fe/ZIF-8/Gel, and exhibited acceptable biocompatibility. These excellent properties prompted us to demonstrate further the possibility of Fe/ZIF-8/Gel nano-enzymatic hydrogels for the in situ treatment of OA in living animals. To evaluate the therapeutic effect of OA in vivo, ACLT surgery was used to construct a rat model of OA, and the therapeutic effect of Fe/ZIF-8/Gel nano-enzymatic hydrogel on OA was investigated (Fig. 6a). Twenty-eight days after surgery, knee samples were collected for macroscopic examination of the knee joint by macroscopic photography and radiography. The cartilage surface of the SD rats in the ACLT-treated group was severely damaged, showing the typical OA features of erosion and osteomalacia, indicating the successful establishment of an OA model (Fig. 6b). Besides, the joint space width in the OA model group was significantly smaller than that in the sham-operated group. There was significant erosive peeling of the articular surface (Fig. 6b). The cartilage of SD rats treated with Fe/ZIF-8/Gel nanoenzyme hydrogel was observed macroscopically in the knee joint to be smooth and shiny, behaving similarly to normal cartilage, suggesting that it could alleviate OA degeneration without cartilage damage (Fig. 6b). Besides, cartilage degeneration and joint space width were significantly suppressed in SD rats in the Fe/ZIF-8/Gel nano-enzymatic hydrogel treatment group. Macroscopic scoring, which is a criterion for assessing cartilage damage, was further performed. After 4 and 8 weeks of treatment, the OA joint scores obtained with Fe/ZIF-8/Gel nano-enzymatic hydrogel were significantly lower than those of the OA group (1.59 ± 0.18 and 1.54 ± 0.16) and were 4.5 and 2.1 times lower than those of the surgical group, and the surgical + ZIF-8/Gel group, respectively, which indirectly revealed the relief of OA (Fig. 6c and Fig. S16, ESI†). The articular cartilage damage grading criteria (ICRS) also indicated that Fe/ZIF-8/Gel nano-enzymatic hydrogel had good OA treatment results (Fig. 6h). Morphologic and pathologic examinations demonstrated that Fe/ZIF-8/Gel treatment avoided cartilage damage (e.g., surface fibrosis and loss of proteoglycans) and prevented severe cartilage erosion throughout the uncalcified area (Fig. 6d–g). The articular cartilage morphology and structure were significantly improved and the cartilage surface was almost intact when compared with the OA and ZIF-8/Gel groups (Fig. 6e and f). Besides, the Fe/ZIF-8/Gel nano-enzymatic hydrogel well reduced the synovial mucosal cell layer and retention cell density without significant inflammatory infiltration (Fig. 6d). In conclusion, the Fe/ZIF-8/Gel nano-enzymatic hydrogel has a better therapeutic effect on the ACLT-induced OA SD rat model.

Fig. 6 In vivo performance of Fe/ZIF-8/Gel nano-enzyme hydrogel-mediated therapy for OA. (a) Schematic diagram of the procedure for the treatment of OA in vivo using Fe/ZIF-8/Gel nano-enzyme hydrogel. (b) Representative micro-CT images of different treatment groups after 4 and 8 weeks of treatment. (c) The macroscopic scores of the knee joint after 4 and 8 weeks of treatment in different treatment groups. (d)–(f) Optical images of knee joints stained with (d) H&E, (e) safranin O/fast green, and (f) toluidine blue staining of joint tissues after 4 weeks and 8 weeks of treatment in different treatment groups (scale bar: 1 mm). (g) MMP13 and COL2 sections of joint tissues after 8 weeks of treatment in different treatment groups (scale bar: 50 μm). (h) The ICRS scores of macroscopic observations of rat knee joints in different treatment groups. (i) The MMP13 expression in joint fluid detected by ELISA. (j) The OARSI scores of macroscopic observations of rat knee joints in different treatment groups. These data are presented as mean values ± SD (n = 3). * Indicates significant differences between the ZIF-8/Gel and Fe/ZIF-8/Gel and Sham groups.

Besides, cartilage destruction and related inflammatory changes were detected by H&E staining in different treatment groups at 4 and 8 weeks. Compared with the operated group, the Fe/ZIF-8/Gel nano-enzymatic hydrogel well reduced the synovial mucosal cell layer and retention cell density without significant inflammatory infiltration, but the therapeutic effect of the ZIF-8/Gel treatment group was not obvious (Fig. 6d and Fig. S17, ESI†). This may be because the Fe/ZIF-8/Gel nano-enzymatic hydrogel was significantly effective in reducing cartilage destruction and inflammatory cell recruitment caused by the surgery. To further evaluate the therapeutic effect of Fe/ZIF-8/Gel nano-enzymatic hydrogel on OA, safranin O/fast green staining was also performed. Thinning and destruction of the cartilage surface can be observed in the surgical group. However, after treatment with Fe/ZIF-8/Gel nano-enzymatic hydrogel, the thickness of the cartilage and the integrity of the cartilage surface were maintained, with a smooth surface and regular arrangement of chondrocytes (Fig. 6e and Fig. S18, ESI†). In patients with OA, ROS-mediated oxidative stress leads to the differentiation of cartilage into hypertrophic CHOs, resulting in difficulty in forming stable articular cartilage.⁸⁵ Toluidine blue staining demonstrated that Fe/ZIF-8/Gel was effective in reducing hypertrophic differentiation of cartilage, exhibiting a morphology essentially identical to that of the sham-operated group after 4 and 8 weeks of treatment (Fig. 6f and Fig. S19, ESI†). Research has shown that COL2 is involved in the formation of the extracellular matrix of CHO and is essential for maintaining the overall integrity of cartilage.⁸⁶ In the surgical group, COL2 expression was significantly decreased, and after treatment with Fe/ZIF-8/Gel nano-enzymatic hydrogel, COL2 expression was significantly increased, which was basically similar to that in the sham-surgical group (Fig. 6g). Besides, to verify the anti-inflammatory effect of Fe/ZIF-8/Gel nano-enzymatic hydrogel, MMP13 immunohistochemical staining and ELISA were performed on knee cartilage and synovial fluid. Little to no brown staining was observed on cartilage in the sham-operated group. In contrast, brown staining on cartilage was significantly increased in the surgical group, indicating stronger expression of MMP13 (Fig. 6g and Fig. S20, ESI†). The results of ELISA were similar to the trend of immunohistochemistry, which also showed that the Fe/ZIF-8/Gel nano-enzymatic hydrogel group had the lowest expression levels of inflammatory factors (Fig. 6i). These experimental results confirmed that Fe/ZIF-8/Gel nano-enzymatic hydrogel could effectively remove excessive ROS from CHOs and generate O₂, alleviate the microenvironment of OA, and reduce the expression of inflammatory factors, thereby promoting cartilage healing for the treatment of OA. This was also reflected by the OARSI score, which was negatively correlated with OA lesion progression, with the Fe/ZF-8/Gel nano-enzymatic hydrogel contributing the most to ROS clearance, and thus significantly reducing the OARSI score close to that of the sham-operated group, confirming the robust efficacy of OA (Fig. 6j).

3.6. In vivo biosafety assessment of Fe/ZF-8/Gel nano-enzymatic hydrogels

To further evaluate the biosafety of Fe/ZF-8/Gel nano-enzymatic hydrogels in vivo, the H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) of rats in different treatment groups at 8 weeks was performed. The Fe/ZF-8/Gel nano-enzymatic hydrogel was similar to that of the sham-operated group, with no significant inflammatory lesions or tissue necrosis observed in major organs (Fig. 7). Meanwhile, the Fe content in the internal organs of rats in the Fe/ZF-8/Gel nano-enzymatic hydrogel group was evaluated using AAS (Fig. S21, ESI†), which was basically consistent with that of normal organs.^87,88 It was shown that Fe/ZF-8/Gel nano-enzymatic hydrogels were fully metabolized after 8 weeks of treatment in rats and had no significant toxic side effects on the major organs of rats. Further hemolysis tests showed that the Fe/ZF-8/Gel nano-enzyme hydrogel had good blood biocompatibility (Fig. S22, ESI†). The above results confirmed that the Fe/ZF-8/Gel nano-enzyme hydrogel has good biosafety and blood compatibility in vivo with less toxic side effects.

Fig. 7 The H&E images of major organs of SD rats in different treatment groups.

4. Conclusion

In conclusion, we successfully constructed a ternary artificial nano-enzymatic hydrogel Fe/ZIF-8/Gel nano-enzyme for scavenging overexpressed ROS in CHOs synergistically generating dissolved oxygen and providing sustained lubrication and achieved good results in repairing and protecting OA cartilage. The scavenging of ROS by Fe/ZIF-8/Gel nano-enzyme hydrogel synergistically generates the release of dissolved oxygen that can mitigate OA by restoring mitochondrial function and reducing inflammatory factors to protect CHOs from oxidative stress-induced apoptosis. The nano-enzyme hydrocoagulation can minimize the removal of nano-enzymes in the OA joint cavity and provide a suitable 3D microenvironment for cartilage repair. The excellent biocompatibility ensures that nano-enzymatic hypercoagulation has a high potential for clinical translation, but also for the treatment of other diseases caused by excessive ROS (e.g. inflammatory bowel disease, atherosclerosis, etc.). However, we used a conventional culture method to validate the ROS scavenging effect of Fe/ZIF-8/Gel nanoenzymes on CHOs. In the future, the 3D culture modeling of spherical aggregates should be used to better and more intuitively understand the biological functions of nano-enzymatic hydrogels and to better simulate the real in vivo microenvironment. Although Fe/ZIF-8/Gel nanoenzymes showed acceptable results for the treatment of OA in SD rats, the effectiveness of OA treatment should also be verified by primates with closer evolutionary affinities to humans.

Author contributions

Wei Deng: conceptualization, data curation, formal analysis, writing – original draft preparation and editing. Yue Zhou: conceptualization, data curation, formal analysis, writing – original draft preparation and Editing. Qinlin Wan: conceptualization, investigation. Lei Li: investigation, formal analysis, writing – original draft preparation. Hui Deng: supervision, formal analysis. Yong Yin: supervision, investigation. Qingsong Zhou: investigation, supervision. Qiujiang Li: methodology. Duo Cheng: conceptualization, investigation. Xuefeng Hu: resources, supervision. Yunbing Wang: conceptualization, resources, supervision, writing – reviewing and editing. Ganjun Feng: conceptualization, resources, methodology, supervision, funding acquisition, writing – reviewing, and editing.

Conflicts of interest

There is no conflict of interest for any of the authors.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 82072434, 82272546), the Sichuan Science and Technology Program (2023NSFSC0659), the Sichuan Provincial Medical Association Special Research Fund (2021SAT05, 2019HR18), the Clinical Scientific Research Fund Project of Chengdu Medical College and the Third Affiliated Hospital of Chengdu Medical College (23LHPDZYB20), the West China Nursing Discipline Development Special Fund Project, Sichuan University (HXHL21005), and the Chengdu Medical University Science and Technology Foundation (CYZYB23-05). We sincerely appreciate Huaiqiang Sun and Shenglan You from Animal Imaging Core Facilities, West China Hospital, Sichuan University for their assistance and guidance. We gratefully acknowledge the technical assistance of Core Facilities of West China Hospital (Li Chai, Yi Li, Xing Xu, Hongying Chen, Yan Wang, Jian Yang, and Mengli Zhu). We also appreciate eceshi (https://www.eceshi.com) for the SEM, TEM, XPS, and XRD analysis.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb00307a

‡ These authors contributed equally to this work.

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