Copper-incorporated hydrogels loaded with curcumin microspheres for the repair of bone defects

Abstract

Addressing bone defects continues to be an exceedingly difficult challenge owing to local immune dysregulation, vascular injury, and disorders of osteogenesis. No entirely satisfactory resolution has been achieved. In this work, through loading curcumin/poly (lactic-co-glycolic acid) microspheres constructed via a double emulsion technique, a copper-incorporated carboxymethyl chitosan/sodium alginate hydrogel (CA-Cur@Cu hydrogel) was developed. The CA-Cur@Cu hydrogels can sustain the release of curcumin for a long time. This sustained release demonstrates effective antioxidant and anti-inflammatory properties, inducing M2 polarization of macrophages and enhancing the bone immune microenvironment. Furthermore, Cu²⁺ released from the CA-Cur@Cu hydrogels induces angiogenesis and osteogenesis, which synergize with anti-inflammatory properties of curcumin to accelerate the repair of bone defects. Additionally, the CA-Cur@Cu hydrogels exhibit remarkable antibacterial activity, effectively inhibiting clinical bacterial infections. Overall, this study successfully constructed a multifunctional hydrogel integrating antioxidant, anti-inflammatory, pro-angiogenic, and osteogenic properties, providing a new perspective and potential solution for the treatment of bone defects.

Graphical abstract

Curcumin PLGA microspheres and copper ions endow the hydrogels with anti-oxidative stress capabilities, angiogenesis, and osteogenesis. The CA-Cur@Cu hydrogels can effectively clear excessive reactive oxygen species (ROS) in the microenvironment through the sustained release of curcumin, thereby exerting a significant anti-inflammatory effect and promoting macrophage polarization toward the M2 phenotype, which optimizes the osteogenic microenvironment. Additionally, the sustained-release Cu²⁺ ions from the hydrogels possess unique properties for promoting angiogenesis and osteogenesis, and synergistically with curcumin facilitate the repair of bone defects.

1. Introduction

With the acceleration of the global population aging process, the number of patients suffering from bone defects due to various diseases, including osteoporosis (OP), tumor resection, and trauma, has been increasing annually [1,2]. Presently, bone defect treatments include options such as autologous bone grafting, allogeneic bone grafting, and artificial bone substitutes. Nevertheless, all these approaches come with intrinsic limitations that hinder their more extensive use. Despite the significant advancements made in the field of medical treatment and bone repair materials [[3], [4], [5], [6]], successfully managing bone defects remains a major challenge for patients and orthopedic surgeons around the world [7].

The reasons for inadequate bone regeneration are complex, involving immune dysfunction and vascular damage [8]. Local inflammation and excessive reactive oxygen species (ROS) disrupt the balance of the bone immune microenvironment. Simultaneously, bone defects are accompanied by significant blood supply disruption, which collectively contribute to the challenges in repairing bone defects. Consequently, an ideal bone repair biomaterial must not only exhibit strong osteogenic potential but also demonstrate superior angiogenic and immunomodulatory capabilities.

Copper, a vital trace element in the human body, is crucial for bone development. Studies have shown that copper ions can stimulate various signaling pathways that are closely associated with osteogenesis, including MAPK, Wnt/β-catenin, and BMP-Smad pathways [9]. This activation significantly boosts the osteogenic differentiation potential of osteoblasts. More importantly, copper ions (Cu²⁺) can mimic a hypoxic environment and activate the HIF-1 signaling pathway by inhibiting the degradation of hypoxia-inducible factor 1α (HIF-1α) [10,11]. This mechanism further enhances the expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS), markedly improving angiogenesis capability [12,13]. Furthermore, copper is a well-recognized antibacterial metal element and a highly effective antibacterial agent with a broad spectrum of activity [[14], [15], [16]]. Consequently, incorporating copper ions into biomaterials can not only enhance osteogenic differentiation but also stimulate angiogenesis, thus ensuring efficient bone tissue repair. Moreover, the superior antibacterial properties of copper ions can prevent implant-related infections, which play a crucial role in successful bone repair.

However, a single repair component alone is insufficient to achieve adequate bone repair effects. Additionally, a significant challenge in bone defect repair is the presence of local inflammation and elevated reactive oxygen species (ROS) [17,18]. Inflammation and excessive ROS can not only promote osteoclast differentiation but also inhibit osteoblast activity, thereby hindering the bone repair process [[19], [20], [21], [22], [23], [24]]. Consequently, an ideal bone repair biomaterial must not only support osteogenesis and angiogenesis but also possess robust anti-inflammatory and immunomodulatory capabilities.

In recent years, immunomodulatory strategies aimed at enhancing bone tissue regeneration have garnered significant attention in the field of biomaterials [[25], [26], [27]]. These strategies primarily involve the use of nanomaterials, pharmaceutical agents, bioactive factors, and exosomes [25,[28], [29], [30]]. Although these approaches have demonstrated efficacy in modulating the bone immune microenvironment to facilitate tissue repair, several limitations remain noteworthy. For instance, the sudden release of substances may lead to toxicity; rapid drug metabolism hinders sustained therapeutic effects; bioactive factors may become inactivated; and low bioavailability and limited production efficiency reduce effectiveness. Therefore, the development of biomaterials capable of achieving stable and long-term immunomodulation is of critical importance for effective bone defect repair.

Curcumin is an acidic polyphenolic compound extracted from Curcuma longa, known for its potent anti-inflammatory and antioxidant properties [31,32]. Additionally, curcumin has been reported to exhibit osteogenic effects. Xiong et al. demonstrated that curcumin promoted the osteogenic differentiation of periodontal ligament stem cells [33]. Sarkar et al. further confirmed that curcumin enhanced the activity and proliferation of osteoblasts [34]. However, due to its poor water solubility, chemical instability and low bioavailability in vivo, the biological activity of curcumin is difficult to be fully exerted. To address these challenges, numerous studies have encapsulated curcumin into controlled-release systems, such as polymer particles, lipid nanoparticles, and liposomes. Poly(lactic-co-glycolic acid) (PLGA) microspheres stand out as one of the most promising drug carriers due to their exceptional stability, low toxicity, and high encapsulation efficiency for hydrophobic compounds [35,36].

Hydrogels are three-dimensional network structures formed by the cross-linking of hydrophilic polymer chains, exhibiting a broad spectrum of adjustable physical and chemical properties. They can effectively encapsulate various nanomaterials, small molecule compounds, and metal ions, making them highly attractive for applications in bone tissue engineering [[37], [38], [39]]. Among these materials, carboxymethyl chitosan (CMC), a natural cationic polymer, not only demonstrates excellent biodegradability but also exhibits significant anti-tumor activity, antibacterial properties, and strong adsorption capacity for metal ions [40]. Alginate (Alg), a natural anionic polymer, is renowned for its superior water absorption, biocompatibility, and biodegradability, which makes it particularly suitable for bone repair filling materials. When carboxymethyl chitosan and alginate are cross-linked to form a hydrogel, the resulting material serves as an ideal matrix for bone repair biomaterials. By constructing a well-defined hydrogel network structure and incorporating bioactive components, the efficiency of bone defect repair can be significantly enhanced.

In this study, by incorporating Cur/PLGA microspheres into a carboxymethyl chitosan/alginic acid salt solution, a composite hydrogel (CA-Cur@Cu hydrogel) was fabricated via copper ion cross-linking. This innovative CA-Cur@Cu hydrogel is capable of not only slowly releasing curcumin to scavenge excessive reactive oxygen species (ROS) in the microenvironment, thereby exerting significant anti-inflammatory effects and promoting macrophage polarization toward the M2 phenotype to optimize the osteogenic microenvironment, but also releasing Cu²⁺ to promote angiogenesis and osteogenesis, synergistically working with curcumin to repair bone defects (Fig. 1). This study confirmed via material characterization that the CA-Cur@Cu hydrogels exhibited a loose and porous structure, stable sustained release of curcumin, and significant antioxidant activity. The biocompatibility, osteogenic capacity, angiogenic activity, immunomodulatory function, and antibacterial properties of the composite hydrogels were further evaluated in vitro. Additionally, a rat cranial defect model was constructed to validate the repair efficacy of the hydrogels. Consequently, this study successfully developed a multifunctional hydrogel that integrates antioxidant, pro-angiogenic, osteogenic, and antibacterial capabilities, offering a novel and promising strategy for treating bone defects.

Fig. 1.

Schematic diagram of the preparation of CA-Cur@Cu hydrogels and the mechanism of CA-Cur@Cu hydrogels for repairing bone defects. The CA-Cur@Cu hydrogels can effectively clear excessive reactive oxygen species (ROS) in the microenvironment through the sustained release of curcumin, thereby exerting a significant anti-inflammatory effect and promoting macrophage polarization toward the M2 phenotype, which optimizes the osteogenic microenvironment. Additionally, the sustained-release Cu²⁺ ions from the hydrogels possess unique properties for promoting angiogenesis and osteogenesis, and synergistically with curcumin facilitate the repair of bone defects.

2. Methods

2.1. Materials preparation

Preparation of Cur/PLGA microspheres: Cur/PLGA microspheres were prepared by the double emulsion method. Briefly, 100 mg of PLGA (LA:GA = 75:25, molecular weight 50,000) was dissolved in 1 mL of dichloromethane (Macklin, China). Then, 16 mg of curcumin (Macklin, China; AR) dissolved in 100 μL of DMSO was added to the PLGA solution. The mixture was vortexed for 30 s and slowly added to 25 mL of polyvinyl alcohol solution (PVA, Macklin, China; AR; 0.5 %, w/v). Stirring at room temperature for 4 h ensured complete volatilization of dichloromethane. The mixture was centrifuged at 4000 rpm for 5 min, washed three times with deionized water, and freeze-dried under vacuum. The resulting Cur/PLGA microspheres were stored at −20 °C.

Preparation of CA/Cur-Cu composite hydrogels: 300 mg of carboxymethyl chitosan (CMC, Macklin; AR) and 300 mg of sodium alginate (Alg, Macklin; AR) were dissolved in 10 mL of deionized water to form a CMC-Alg (CA) solution. Then, 40 mg of Cur/PLGA microspheres were added and stirred for 30 min to disperse uniformly. The mixture was placed in a 24-well plate, crosslinked with a 3 wt% calcium chloride solution containing 100 μM copper ions, and washed three times with deionized water to obtain the CA/Cur-Cu hydrogels. For the CA/Cur hydrogels, the same procedures were followed but without copper ions in the crosslinking solution.

2.2. Characterization

The surface morphologies of Cur/PLGA microspheres and CA-Cur@Cu hydrogels were observed using SEM (ZEISS, Germany). FT-IR analysis was performed to examine CMC, Alg, CA, and CA@Cu.

2.3. Mechanical properties of hydrogels

Hydrogels (CA, CA-Cur, CA-Cur@Cu; dimensions: 6 mm × 6 mm × 4 mm) were tested using a universal testing machine (model Z050, Zwick/Roell) at a compression rate of 1 mm/min. The compressive modulus and strength were calculated.

2.4. Hydrogels degradation

The CA, CA-Cur, and CA-Cur@Cu hydrogels were immersed in PBS and incubated at 37 °C. The samples were removed from the PBS at specific time points and weighed to determine the weight loss. Meanwhile, the pH value of the PBS was measured using a pH meter, and the zeta potential was analyzed using dynamic light scattering (Zetasizer Nano S90, Malvern Panalytical, UK). Weight loss (%) was calculated as follows: (Wo − Ws)/Wo × 100 %, where Wo is the initial hydrogel weight and Ws is the weight at each specific time point.

2.5. Swelling ratio

The CA, CA-Cur, and CA-Cur@Cu hydrogels were freeze-dried for 72 h, and the weight of the freeze-dried hydrogels (Wd) was measured. Then, the three types of hydrogels were immersed in phosphate buffer solution (PBS), incubated at 37 °C for 24 h, and the wet weight of the hydrogels (Ww) was measured again. The swelling ratio was calculated as: Swelling ratio = (Ww - Wd)/Wd × 100 %.

2.6. Encapsulation efficiency and in vitro release kinetics of Cur/PLGA microspheres

Determination of the encapsulation efficiency of curcumin in microspheres: 1 mg of Cur/PLGA microspheres was dissolved in 10 mL of dichloromethane at room temperature. Absorbance was measured at 420 nm, and curcumin concentration was calculated using the standard curve to determine curcumin content. Encapsulation efficiency was calculated as: Encapsulation efficiency = (Actual drug loading/Theoretical drug loading) × 100 %.

Sustained release determination of curcumin: 1 mg of Cur microspheres and 250 mg of CA/Cur hydrogel were immersed in 10 mL of PBS, incubated at 37 °C and 100 rpm. Supernatant was collected at set times, absorbance was measured at 420 nm, and curcumin content was calculated. Fresh PBS was added after each collection.

2.7. In vitro antioxidant activity of Cur/PLGA microspheres

The in vitro antioxidant activity of Cur/PLGA microspheres was evaluated using H₂O₂, DPPH, and ABTS scavenging experiments. Working solutions were prepared following the instructions from the H₂O₂ kit (Beyotime, China), DPPH kit (DOJINDO, Japan), and ABTS kit (Jiancheng). Cur/PLGA microspheres (final concentrations of 0.25, 0.5, and 1 mg/mL) were mixed with the respective working solutions and incubated at room temperature in the dark for 30 min. Absorbance was measured using a microplate reader according to the kit instructions.

2.8. In vitro study

2.8.1. Extraction and culture of periosteum-derived stem cells

Animal experiments were approved by the Ethics Committee of Shenzhen Hospital of Southern Medical University. Periosteum-derived mesenchymal stem cells (PDSCs) were isolated from the skulls of 4-week-old SD rats and were routinely cultured and passaged. Third-generation cells were cryopreserved for subsequent in vitro studies. The culture medium for PDSCs was composed of DMEM/F12 supplemented with 10 % fetal bovine serum (FBS) and 1 % streptomycin/penicillin (10,000 U/mL). Osteogenic induction medium included β-glycerophosphate (10 mmol/L), ascorbic acid (50 μmol/L), and dexamethasone (10⁻⁷ mol/L). Human umbilical vein endothelial cells (HUVECs) and mouse mononuclear macrophage leukemia cells (RAW 264.7) were cultured in DMEM with 10 % FBS and 1 % streptomycin/penicillin. Hydrogels were sterilized by UV radiation overnight and co-cultured with cells at 37 °C, 5 % CO₂, with medium changes every 3 days. Each group had three biological replicates.

2.8.2. Biocompatibility of CA-Cur@Cu composite hydrogels

The control, CA-Cur, and CA-Cur@Cu groups were established. PDSCs were seeded at 3 × 10⁴ cells/well in 24-well plates and co-cultured with the hydrogels. On days 1, 3, and 5, cell activity was measured using a CCK-8 kit (DOJINDO, Japan), and live cells were stained with a live cell staining kit (BestBio, China). Cell growth was then observed under a fluorescence microscope.

2.8.3. Evaluation of in vitro osteogenic activity

To evaluate the in vitro osteogenic performance of the CA-Cur@Cu hydrogels, this study used alkaline phosphatase (ALP) staining and ALP activity determination, ARS staining, and analysis of osteogenesis-related gene expression. Three groups were set up: control, CA-Cur, and CA-Cur@Cu. PDSCs (3 × 10⁴/well) were seeded in 24-well plates and co-cultured with the hydrogels.

Detection and staining of ALP activity: On day 14, ALP activity was detected using the ALP activity detection kit (Solarbio) and stained with the ALP staining kit (Solarbio). Staining results were observed under an inverted microscope (LEICA DMi8, Germany).

Alizarin red S (ARS) staining: On day 14, cells were fixed with 4 % paraformaldehyde for 30 min. The fixative was discarded, and the cells were washed three times with PBS and stained with 0.2 % ARS solution (Solarbio) for 20 min. Subsequently, 10 % cetylpyridinium chloride solution was added to each well and incubated overnight. The solution was transferred to an EP tube, centrifuged at 13,000 rpm for 15 min, and the absorbance was measured at 570 nm to quantify ARS staining.

Osteogenesis-related gene detection: At 7 and 14 days, total RNA was extracted using TRIzol reagent (AG), converted to cDNA with a reverse transcription kit (AG), and analyzed by SYBR Green qPCR (AG). Expression levels of OPN, COL-1, and Runx2 were quantified and normalized to GAPDH. Primer sequences are showed in Table S1.

2.8.4. In vitro angiogenesis

The in vitro angiogenic ability of the CA-Cur@Cu hydrogels was evaluated using CD31 immunofluorescence staining, tube formation assays, and detection of angiogenesis-related genes. Three groups were established: control, CA-Cur, and CA-Cu@Cur. HUVECs (3 × 10⁴/well) were seeded in 24-well plates and co-cultured with the hydrogels.

CD31 immunofluorescence staining: After 3 days of co-culture, cells were fixed with 4 % paraformaldehyde for 30 min and permeabilized with 0.1 % Triton X-100, followed by blocking. Then they were incubated with the primary antibody (CD31, rabbit anti-mouse, Affinity Biosciences, USA) and the secondary antibody (Goat anti-rabbit IgG, Alexa Fluor 488, Affinity Biosciences, USA). Nuclei were stained with DAPI, and images were acquired using an inverted fluorescence microscope (Leica DMi8, Germany).

Tube formation assays: HUVECs were co-cultured with various hydrogels for 3 days. Matrigel (Corning, USA) was spread on 24-well plates. HUVECs were seeded on the Matrigel at a density of 10⁵ cells per well. After 8 h, images were captured using an optical microscope, and tube formation was analyzed using ImageJ.

Detection of angiogenesis-related genes: After 3 days of culture, total RNA was extracted using TRIzol reagent and converted into cDNA with a reverse transcription kit. The expression levels of bFGF, HIF-α, CD31, and VEGF were analyzed by SYBR Green qPCR, with GAPDH as the internal reference. Primer sequences are listed in Table S2.

2.8.5. In vitro antioxidant stress

The control group, the CA-Cur group, and the CA-Cur@Cu group were established. PDSCs were seeded at a density of 5 × 10⁴ cells per well in 12-well plates. After 6 h, the medium was replaced with fresh medium containing 100 μM H₂O₂, and the hydrogels were added for co-culture for an additional 24 h. The DCFH-DA kit (Beyotime) was used for staining to detect intracellular ROS levels, and images were captured and recorded using an inverted fluorescence microscope (Leica, DMi8, Germany).

2.8.6. In vitro immunomodulation

The in vitro immunomodulatory activity of the CA-Cur@Cu hydrogels was evaluated using immunofluorescence staining and detection of inflammation-related genes. Four groups were set: Normal, control, CA-Cur, and CA-Cur@Cu. RAW 264.7 cells were seeded in 24-well plates at 5 × 10⁴ cells/well. After 6 h, the Control, CA-Cur, and CA-Cur@Cu groups were treated with medium containing 100 ng/mL LPS and co-cultured with the hydrogels.

Immunofluorescence staining: Cells were fixed with 4 % paraformaldehyde for 30 min after 24 h of culture, permeabilized with 0.1 % Triton X-100, and blocked. Primary antibodies (CD86, CD206, rabbit anti-mouse, Affinity Biosciences, USA) and secondary antibodies (Goat anti-rabbit IgG, Alexa Fluor 488/594, Affinity Biosciences, USA) were applied sequentially. Nuclei were stained with DAPI, and images were captured using an inverted fluorescence microscope (Leica DMi8, Germany).

Inflammation-related gene detection: Cells were cultured for 24 h, and total RNA was extracted using TRIzol reagent. The RNA was reverse-transcribed into cDNA, and the expression levels of IL-1β, TNF-α, IL-10, and Arg-1 were analyzed by SYBR Green qPCR. GAPDH served as the internal reference gene. Primer sequences are listed in Table S3.

2.9. Antibacterial performance

Staphylococcus aureus (S. aureus, ATCC 25923) and Escherichia coli (E. coli, ATCC 25922) were used to assess the antibacterial efficacy of the CA-Cur@Cu hydrogels. The control group, the CA-Cur group, and the CA-Cur@Cu group were set up. A bacterial suspension (10⁶ CFU/well) was added to a 24-well plate and co-incubated with the respective hydrogel groups for 24 h. Subsequently, 100 μL of the co-culture medium was sampled and uniformly spread onto agar plates. Following incubation at 37 °C for 24 h, the number of viable colonies was quantified. Additionally, the absorbance of the co-culture medium at 600 nm was measured to further evaluate bacterial growth inhibition.

2.10. In vivo study

Sprague Dawley (SD) rats (specific pathogen-free, 8 weeks old, 220–240 g, male) were used for the animal experiments. This study was approved by the Animal Ethics Committee of Shenzhen Hospital of Southern Medical University (No. 2025-0225). The rats were randomly divided into three groups: the control group, the CA-Cur group, and the CA-Cur@Cu group. Rat cranial defect model construction: Rats were anesthetized via intraperitoneal injection of pentobarbital sodium (1.5 %, 0.3 mL/100 g). Following anesthesia, the head skin was disinfected with iodophor, and a surgical drape was applied. A midline incision was made on the scalp, the periosteum was carefully separated, and the parietal bone was exposed. Using a 5-mm-diameter electric drill, a circular defect with a diameter of 5 mm was created in the parietal bone while physiological saline was continuously dripped to maintain cooling during drilling. Subsequently, a 5-mm-diameter hydrogel was implanted into the defect. The incision was then sutured. To prevent infection, penicillin was administered intramuscularly for the first 3 days post-surgery. At 8 weeks post-operation, the rats were euthanized, their skulls were harvested and then fixed in 4 % paraformaldehyde for further analysis.

The skulls of rats in each group were scanned using Micro-CT (Imaging 100, Raycision Medical Technology Co., Ltd., China). The scanning parameters were set as follows: layer thickness of 15 μm, voltage of 45 kV, and current of 435 μA. Following the acquisition of images, three-dimensional reconstruction was performed, and the data were analyzed for trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), bone volume fraction (BV/TV), and trabecular separation (Tb.Sp).

For histological analysis, the specimens were fixed in 4 % paraformaldehyde for 24 h and then decalcified. Routine sections were prepared for hematoxylin-eosin (HE) staining, Masson staining, and osteocalcin (OCN) immunohistochemical staining to comprehensively evaluate the bone repair efficacy of the CA-Cur@Cu hydrogels.

Furthermore, the major organs of rats in different groups were collected at 8 weeks and stained with H&E to assess the biocompatibility of CA-Cur@Cu hydrogels in vivo.

2.11. Statistical analysis

All statistical evaluations were performed using GraphPad Prism 8.0. Data are expressed as the mean ± standard deviation. Comparisons among multiple groups were assessed through one-way ANOVA, with post-hoc pairwise comparisons carried out using Tukey's test. Statistical significance was defined as a p-value less than 0.05.

3. Results and discussion

3.1. Characterization

In this study, curcumin was encapsulated in PLGA microspheres to achieve long-term sustained release of curcumin, thereby enhancing the promotion of bone defect repair. Fig. 2A shows the preparation process of the CA-Cur@Cu hydrogels. Scanning electron microscopy (SEM) analysis (Fig. 2B) revealed that the Cur/PLGA microspheres exhibited a spherical shape with a smooth surface. and the diameters of most microspheres were within the range of 40–50 μm (Fig. S1). CA@Cu hydrogels and CA-Cur@Cu hydrogels were successfully fabricated via the ion cross-linking method. Both types of hydrogels possessed a loose, porous, and interconnected structure (Fig. 2B), which facilitated uniform cell seeding, nutrient exchange, vascular ingrowth, and ultimately promoted osteogenic differentiation and bone repair [41,42]. In the CA-Cur@Cu hydrogels, Cur/PLGA microspheres were uniformly dispersed within the hydrogel pores, confirming the successful preparation of the CA-Cur@Cu hydrogels.

Fig. 2.

Preparation and characterization of CA-Cur@Cu hydrogels. A) Schematic illustration of the preparation process for the CA-Cur@Cu hydrogels. B) SEM images of Cur microspheres, CA@Cu, and CA-Cur@Cu hydrogels, with local magnified images below. C) FTIR spectra analysis. D) Compression modulus of hydrogels. E) Compression strength of hydrogels.

The FTIR spectra of the hydrogels and their components are presented in Fig. 2C. Both hydrogels exhibited characteristic peaks corresponding to Alg and CMC. Specifically, the characteristic peaks of CMC and Alg at 1618 cm⁻¹ and 1616 cm⁻¹ were replaced by new peaks at 1627 cm⁻¹ for the CA hydrogel and 1635 cm⁻¹ for the CA@Cu hydrogel, respectively. This indicates that the CA hydrogel was cross-linked by Ca²⁺, while the CA@Cu hydrogel was cross-linked by both Ca²⁺ and Cu²⁺. These findings collectively confirm the successful preparation of the CA@Cu hydrogel via the ion cross-linking method.

3.2. Mechanical properties of hydrogels

The mechanical properties of the hydrogels were assessed through compression tests (Fig. 2D and E). The compressive moduli of CA, CA-Cur, and CA-Cur@Cu hydrogels were 0.274 ± 0.008 MPa, 0.294 ± 0.005 MPa, and 0.308 ± 0.015 MPa, respectively. The compressive strengths of CA, CA-Cur, and CA-Cur@Cu hydrogels were 0.204 ± 0.005 MPa, 0.223 ± 0.006 MPa, and 0.238 ± 0.010 MPa, respectively. The above results demonstrate that the CA-Cur@Cu hydrogel exhibits appropriate mechanical properties, thereby enabling it to provide effective mechanical support during bone defect repair.

3.3. Hydrogels degradation

The degradation performance of biomaterials plays a crucial role in bone defect repair. Biomaterials with poor degradability may cause prolonged space-occupying effects at the defect site, thereby hindering the natural bone repair process. As shown in Fig. S2, the three hydrogels exhibited similar degradation patterns, characterized by an initial slow phase followed by a faster degradation rate. After 28 days, the degradation rate reached approximately 70 %. During this process, the pH of the solution slightly decreased but remained generally close to neutral. Concurrently, the zeta potential showed a slight upward trend, although it remained around 0 mV throughout. These findings demonstrate that the CA-Cur@Cu hydrogels possess favorable biodegradability. Their degradation does not generate significant acidic byproducts, which can help avoid adverse changes to the surrounding tissue environment and minimize potential irritation to the host organism.

The CA-Cur@Cu hydrogels exhibited a slow degradation rate in the initial stage, followed by an accelerated degradation phase in the later stage. This degradation behavior allows the hydrogels to provide sufficient mechanical support during the early phase of tissue repair, thereby promoting osteogenic differentiation and the formation of new bone tissue. As the healing process advances, the degradation of the hydrogels increases, entering a more active phase. This dynamic degradation profile aligns well with the natural growth pattern of bone tissue, enabling a synchronized match between material degradation and the maturation of newly formed bone tissue. Consequently, a dynamic equilibrium between "material degradation" and "tissue regeneration" is achieved. As a result, the CA-Cur@Cu hydrogels not only maintain mechanical stability at the repair site but also minimize adverse effects associated with prolonged residue. This characteristic significantly reduces the long-term space-occupying effect of the hydrogels in vivo, allowing for gradual metabolism or absorption as the bone tissue matures, thereby minimizing interference with the regenerating bone tissue.

3.4. Swelling ratio

CA, CA-Cur, and CA-Cur@Cu hydrogels exhibit a relatively high swelling ratio (Fig. 3A), suggesting that all three hydrogels possess excellent water absorption capacity and hydrophilicity. This can be attributed to the well-distributed hydrophilic groups and porous network structure within the hydrogels, which endow them with superior water retention capabilities. Such characteristics not only enable the effective absorption of excessive exudate at bone defect sites but also provide a favorable moist microenvironment for cell adhesion, proliferation, and migration.

Fig. 3.

Swelling ratio, in vitro release kinetics, and in vitro antioxidant properties of the CA-Cur@Cu hydrogels. A) Swelling behavior of the hydrogels. B) In vitro release kinetics of curcumin. C) Assessment of the in vitro antioxidant capabilities of the CA-Cur@Cu hydrogels.

3.5. Encapsulation efficiency of Cur/PLGA microspheres and in vitro release kinetics of curcumin

The encapsulation efficiency of the Cur/PLGA microspheres prepared in this study was determined to be 78.82 ± 5.81 %. The in vitro release kinetics of curcumin (Fig. 3B) demonstrated that the cumulative release of Cur/PLGA microspheres reached 17 % on the first day, followed by a gradual decrease in the release rate. By day 40, the sustained-release amount had reached 80 %. In contrast, the sustained-release curve of curcumin in the CA-Cur@Cu composite hydrogels exhibited a relatively flat profile. On the first day, the sustained-release amount was 8 %, which was lower than that of the pure Cur/PLGA microspheres. Subsequently, the release rate slowed down, resulting in a cumulative release of 61 % by day 40. Previous studies have demonstrated that PLGA microspheres exhibit a burst release phenomenon, and the sustained-release rate decreases and tends to stabilize. The initial burst release may be caused by the rapid diffusion of surface-bound drugs, and then the gradual degradation of PLGA leads to the gradual sustained release of internal drugs [[43], [44], [45], [46]]. This also explains the explosive release of simple Cur/PLGA microspheres on the first day, followed by a decrease in the release amount and a tendency to stabilize. Interestingly, when Cur microspheres are incorporated into a composite hydrogel, the burst release rate is significantly reduced, and the overall sustained-release rate becomes slower. This could be due to the encapsulation of Cur microspheres within the network structure of the hydrogels, which introduces an additional diffusion barrier, slowing down the sustained-release rate of curcumin and further achieving its long-term sustained release.

3.6. Antioxidant activity

Excessive reactive oxygen species (ROS) in the antioxidant-active microenvironment can induce DNA damage, protein degradation, and lipid peroxidation, leading to oxidative stress damage in cells [47]. Oxidative stress has been shown to impair the bone regeneration process by promoting osteoclastogenesis, reducing osteoblast activity, and inducing osteocyte apoptosis [19,47]. Therefore, it is highly significant for biomaterials to possess antioxidant properties for bone defect repair. To assess the antioxidant capacity of Cur/PLGA microspheres, H₂O₂, DPPH, and ABTS scavenging assays were employed to comprehensively evaluate their performance. The results indicate that Cur/PLGA microspheres exhibit excellent scavenging efficiency for H₂O₂, DPPH, and ABTS (Fig. 3C). In contrast, pure PLGA microspheres lack scavenging ability. Curcumin is well-known for its potent antioxidant effects [31,32]. Consequently, Cur/PLGA microspheres demonstrate a robust ROS scavenging capability. By incorporating Cur/PLGA microspheres into the hydrogels, CA-Cur@Cu hydrogels are endowed with superior antioxidant activity, which facilitates the reduction of excessive ROS in the microenvironment and promotes the establishment of an optimal osteogenic microenvironment.

3.7. Biocompatibility

The biocompatibility of the CA-Cur@Cu hydrogels was comprehensively evaluated using live cell staining and CCK-8 assays. Live cell staining (Fig. 4A) revealed that PDSCs maintained a long spindle shape with extended pseudopodia. On day 1, no significant difference in cell numbers was observed among the three groups. However, on days 3 and 5, the cell numbers progressively increased, with the CA-Cur@Cu group exhibiting the highest cell count, followed by the CA-Cur group, and the control group having the lowest count. The CCK-8 results (Fig. 4B) corroborated the findings from live cell staining. Collectively, these results demonstrate that the CA-Cur@Cu hydrogels exhibit excellent biocompatibility and effectively promote cell proliferation.

Fig. 4.

Biocompatibility of CA-Cur@Cu hydrogels. A) Live cell staining images on days 1, 3, and 5; B) Quantitative analysis of cell viability by CCK-8 assay.

3.8. In vitro osteogenesis

The ability of the CA-Cur@Cu hydrogels to promote osteogenic differentiation of PDSCs was comprehensively evaluated using alkaline phosphatase (ALP) staining, ALP activity assays, alizarin red (ARS) staining, and detection of osteogenesis-related genes (Runx2, COL-1, and OPN).

Alkaline phosphatase (ALP) activity serves as a critical marker for assessing osteogenic performance. During bone formation, ALP plays an essential role by catalyzing the hydrolysis of phosphate esters and providing the necessary inorganic phosphorus for mineralization. Consequently, the level of ALP activity directly reflects the degree of osteogenic activity. As illustrated in Fig. 5A, the CA-Cur@Cu group exhibited the highest number of blue nodules with the darkest coloration, indicating the strongest ALP activity, followed by the CA-Cur group, while the control group demonstrated the lowest activity. This trend was further confirmed by quantitative analysis (Fig. 5C) and ALP activity measurements (Fig. 5D).

Fig. 5.

In vitro osteogenesis evaluation of CA-Cur@Cu hydrogels. A) Alkaline phosphatase staining. B) Alizarin red staining. C) Quantitative analysis of alkaline phosphatase staining). D) ALP activity detection. E, F) Quantitative analysis of the alizarin red staining presented in B). G) qPCR detection of osteogenesis-related genes (COL-1, OPN, Runx2). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Alizarin red staining is a widely recognized method for assessing osteogenic differentiation and mineralization capabilities. Alizarin red can specifically bind to calcium salts and produce red precipitates. Consequently, the activity of osteogenic mineralization can be evaluated by the number and density of stained nodules. As illustrated in Fig. 5B, on day 14, the CA-Cur@Cu group exhibited a significantly higher number of mature red-stained nodules, indicating robust osteogenic mineralization activity. Conversely, both the CA-Cur and control groups displayed fewer and less dense red-stained mineralized nodules, reflecting diminished osteogenic potential. These observations were further supported by ARS quantitative analysis (Fig. 5E and F).

COL-1, Runx2, and OPN are widely recognized as key indicators in the detection of osteogenesis-related genes. These genes are crucial for bone formation, development, and repair processes. As illustrated in Fig. 5G, on days 7 and 14, the expression trends of COL-1, Runx2, and OPN were consistent. Specifically, the highest expression levels were observed in the CA-Cur@Cu group, followed by the CA-Cur group, with the lowest levels detected in the control group. Additionally, the expression levels exhibited a time-dependent increase.

The CA-Cur@Cu hydrogels have demonstrated remarkable osteogenic performance in the above experiments, primarily due to the synergistic effects of copper ions and curcumin. Research indicates that copper ions enhance the proliferation, adhesion, and migration of mesenchymal stem cells (MSCs), while promoting alkaline phosphatase (ALP) activity and upregulating osteogenic-related genes, thereby positively regulating osteogenic differentiation [[48], [49], [50]]. Wang et al. [51] incorporated copper into 3D-printed titanium alloys, achieving sustained copper ion release that promoted ALP activity and osteogenic mineralization of bone marrow mesenchymal stem cells. Similarly, a study reported a composite hydrogel loaded with copper-doped mesoporous silica nanospheres (Cu-MSNs) for bone repair, demonstrating that copper significantly accelerated bone regeneration [52]. Additionally, curcumin has been confirmed to enhance osteoblast activity and proliferation, further promoting osteogenic differentiation [34]. Consequently, the CA-Cur@Cu hydrogels synergistically enhance osteogenic differentiation through the sustained release of copper ions and curcumin.

3.9. In vitro angiogenesis

Bone contains an extensive vascular network, which plays a vital role in maintaining bone health and function. When bone injury occurs, it is invariably accompanied by the disruption of local blood supply. The loss of this blood supply not only compromises nutrient delivery to the damaged area but also potentially impedes the transport of growth factors, thereby prolonging the repair process of bone defects [53]. Consequently, the reconstruction of the local vascular network is important for repairing bone defects, and biomaterials with pro-angiogenic properties are especially critical. In this study, CD31 immunofluorescence staining, tube formation assay, and detection of angiogenesis-related genes were employed to comprehensively evaluate the angiogenic activity of the CA-Cur@Cu hydrogels.

The results of immunofluorescence staining (Fig. 6A) revealed that CD31 was significantly expressed in the CA-Cur@Cu group, whereas the expression of CD31 in both the CA-Cur group and the control group was notably weak. The quantitative analysis of fluorescence intensity (Fig. 6B) confirmed these staining results. The tube formation assay and corresponding quantitative analysis (Fig. 6C–E) demonstrated that the CA-Cur@Cu group exhibited the strongest tube formation-promoting activity, significantly enhancing tubular structure formation, increasing the total length of blood vessels, and promoting a greater number of vascular connections. In contrast, neither the control group nor the CA-Cur group showed notable angiogenesis-promoting effects. Furthermore, the expression levels of angiogenesis-related genes, including bFGF, HIF-α, CD31, and VEGF, were markedly higher in the CA-Cur@Cu group compared to those in the CA-Cur group and the control group (Fig. 6F–I). No significant difference was observed between the CA-Cur group and the control group. The enhanced angiogenic activity of the CA-Cur@Cu group can be primarily attributed to its sustained release of copper ions. Numerous studies have validated the pro-angiogenic effects of copper. Yang et al. [54] demonstrated that doping Cu²⁺ into hydroxyapatite scaffolds effectively promoted vascularized osteogenesis by enhancing the migration and tube formation capabilities of HUVECs. This observation was reinforced in a subsequent study on a novel copper-containing phenolic nanozyme that facilitated diabetic wound healing [55]. Additionally, it has been verified that Cu²⁺ activates the HIF-α signaling pathway, induces VEGF secretion, and thereby promotes angiogenesis [56].

Fig. 6.

In vitro angiogenesis of CA-Cur@Cu hydrogels. A) CD31 immunofluorescence staining. B) Quantitative analysis of A). C) Tube formation assay. D, E) Quantitative analysis of C). F-I) qPCR detection of angiogenesis-related genes.

3.10. In vitro antioxidant stress

To evaluate the anti-oxidative stress activity of the CA-Cur@Cu hydrogels, an oxidative stress microenvironment was created by adding H₂O₂ to the culture medium. After 24 h, the intracellular ROS levels were assessed using DCFH-DA staining. As shown in Fig. S3A, strong green fluorescence, indicative of ROS accumulation, was observed in the control group, whereas minimal fluorescence was detected in both the CA-Cur and CA-Cur@Cu groups. Quantitative analysis of DCFH-DA fluorescence intensity (Fig. S3B) corroborated the qualitative staining results. These findings indicated that the CA-Cur@Cu hydrogels effectively scavenge ROS within the cellular microenvironment and mitigate intracellular oxidative stress, thereby creating conditions conducive to osteogenic differentiation.

3.11. In vitro immunomodulation

It has been confirmed that the immune response plays a critical role in the osteogenesis process [57,58]. Macrophages, as the primary immune cells, are responsible for initiating and maintaining the inflammatory response. Under microenvironmental stimulation, macrophages can polarize into two distinct phenotypes: M1 and M2. M1 macrophages secrete pro-inflammatory cytokines, thereby promoting inflammation persistence, whereas M2 macrophages release anti-inflammatory factors to facilitate tissue repair [25,[59], [60], [61]]. Biomaterials with immunomodulatory properties are expected to significantly enhance osteogenic repair. The immunomodulatory effects of the CA-Cur@Cu hydrogels were comprehensively evaluated using CD86 and CD206 immunofluorescence staining, as well as through the detection of inflammation-related gene expression (IL-1β, TNF-α, IL-10, Arg-1). Specifically, CD86, IL-1β, and TNF-α serve as characteristic markers of M1 macrophages, while CD206, IL-10, and Arg-1 are typical indicators of M2 macrophages.

Immunofluorescence staining (Fig. 7A) revealed that, compared with the normal group, under LPS stimulation, CD86 expression was significantly upregulated in the control group, whereas CD206 expression remained negligible. In contrast, in both the CA-Cur and CA-Cur@Cu groups, CD86 expression was markedly reduced, while CD206 expression was significantly enhanced. Fluorescence quantification analysis (Fig. 7B and C) supported this trend. Furthermore, the detection of inflammation-related gene expression (Fig. 7D–G) demonstrated that, compared with the normal group, pro-inflammatory genes (IL-1β and TNF-α) were significantly upregulated in the control group. However, this upregulation was markedly attenuated in both the CA-Cur and CA-Cur@Cu groups. Conversely, anti-inflammatory genes (IL-10 and Arg-1) were significantly downregulated in the control group but were markedly upregulated in the CA-Cur and CA-Cur@Cu groups. These findings suggest that both CA-Cur and CA-Cur@Cu hydrogels effectively promote M2 polarization of macrophages while inhibiting M1 polarization, thereby exhibiting superior immunomodulatory effects. Consistent with these results, numerous studies have reported that biomaterials capable of inducing M2 polarization can accelerate bone regeneration. Li et al. [62] developed an electrospun naringin-loaded microsphere/sucrose acetate isobutyrate system that successfully repaired osteoporotic bone defects by promoting M2 polarization of macrophages. Another study utilized a 3D-printed scaffold loaded with BMP-4 to induce M2 polarization, secrete anti-inflammatory factors, and improve the bone immune microenvironment, ultimately achieving successful repair of diabetic bone defects [63]. Similarly, Zhu et al. fabricated a multifunctional hydrogel platform that scavenged excessive ROS in the microenvironment, thereby promoting M2 polarization and reducing inflammation, leading to remodeling of the bone immune environment and rapid bone repair [24].

Fig. 7.

Immunomodulatory effects of CA-Cur@Cu hydrogels. A) Immunofluorescence staining for CD86 and CD206 markers. B) Quantitative analysis of CD86 immunofluorescence intensity. C) Quantitative analysis of CD206 immunofluorescence intensity. D-G) qPCR analysis of inflammation-related gene expression levels.

3.12. Antibacterial properties

The presence of implants can elevate the risk of infection and facilitate the formation of bacterial biofilms. Implant-related infections are among the most frequent and severe complications in biomaterial-associated surgeries, with an incidence rate that accounts for 25.6 % of all medical device-related infections in the United States [64]. Consequently, enhancing the antibacterial properties of biomaterials may effectively reduce or prevent implant-related infections.

The antibacterial performance of the CA-Cur@Cu hydrogels was evaluated using the spread plate method and bacterial activity detection. The spread plate method (Fig. 8A–C) revealed that for S. aureus, the colony counts in the control, CA-Cur, and CA-Cur@Cu groups were 246.00 ± 4.00, 24.33 ± 14.15, and 1.67 ± 1.53, respectively. For E. coli, the counts were 396.30 ± 14.29 in the control group, 38.00 ± 8.89 in the CA-Cur group, and 0.00 ± 0.00 in the CA-Cur@Cu group. Bacterial activity detection (Fig. 8D and E) indicated that the absorbance value of the CA-Cur@Cu group was the lowest, followed by the CA-Cur group, with the control group exhibiting the highest value. These results demonstrate that the CA-Cur hydrogel possesses good antibacterial properties, while the CA-Cur@Cu hydrogel exhibits superior antibacterial performance.

Fig. 8.

Antibacterial properties of CA-Cur@Cu hydrogels. A) Results of the spread plate method. B, C) Quantitative analysis of A). D, E) Detection of bacterial activity.

The antibacterial performance of the CA-Cur@Cu hydrogels is attributed to its multifaceted composition, including carboxymethyl chitosan, curcumin, and Cu²⁺. CMC and its hydrogels are renowned for their antibacterial properties, which have been extensively validated in numerous studies [[65], [66], [67]]. The positively charged amino groups on the CMC molecules can interact with negatively charged bacterial cell membranes, causing membrane disruption and subsequent bacterial cell death. Furthermore, CMC induces the production of reactive oxygen species (ROS) in bacteria, leading to damage of cellular structures such as proteins, lipids, and DNA. Additionally, CMC interferes with bacterial cell wall synthesis and enzyme metabolism, thereby inhibiting bacterial growth [68]. Curcumin, known for its potent antioxidant and anti-inflammatory properties, also exhibits antibacterial activity. Its mechanisms include inhibiting the key enzyme FtsZ involved in bacterial cell division, disrupting bacterial cell membranes through its amphiphilic nature, and importantly, preventing the formation of bacterial biofilms [[69], [70], [71]]. Copper, a well-recognized antibacterial metal element with a broad spectrum of activity [[14], [15], [16]], has been confirmed to exhibit significant antibacterial effects against various microorganisms, including bacteria and fungi [[72], [73], [74], [75], [76]]. Consequently, the synergistic action of these multiple antibacterial components in the CA-Cur@Cu hydrogels confers them with a robust antibacterial effect, enabling effective prevention of infections during surgery and playing a crucial role in mitigating biomaterial-related implant infections.

3.13. In vivo study

A skull defect model with a 5-mm diameter was established, and a 5-mm hydrogel was implanted at the bone defect site. The rats were randomly divided into three groups: the control group, the CA-Cur group, and the CA-Cur@Cu group. At the 8th week post-operation, the rats were euthanized, and their skulls were harvested. The repair efficacy of the CA-Cur@Cu hydrogels was comprehensively assessed using Micro-CT analysis and histological evaluation (HE staining, Masson staining, and OCN immunohistochemical staining).

Micro-CT analysis (Fig. 9A–C) revealed that in the control group, there was minimal new bone formation at the defect site, with sparse attachment to the edges of the defect. In comparison, the CA-Cur group exhibited an increased amount of new bone, covering approximately half of the defect area, which indicates the good osteogenic properties of the CA-Cur hydrogels. Notably, the CA-Cur@Cu group demonstrated the highest level of new bone formation among the three groups, with new bone extending from the edge to the center of the defect, thus showing excellent bone repair-promoting ability. Quantitative analysis of new bone formation (Fig. 9D) was conducted. Consistent with the Micro-CT findings, the bone analysis parameters, including BMD, BV/TV, Tb.Th, and Tb.N, were significantly higher in the CA-Cur@Cu group compared to the CA-Cur group, with the control group having the lowest values. The trabecular separation (Tb.Sp) was lowest in the CA-Cur@Cu group, indicating that the CA-Cur@Cu group exhibited the strongest capacity for promoting bone repair.

Fig. 9.

Micro-CT results of CA-Cur@Cu for in vivo bone defect repair. A, B) Three-dimensional reconstructions from Micro-CT imaging. C) Coronal cross-sectional images. D) Quantitative analysis of Micro-CT data.

HE staining (Fig. 10A) revealed that the defect in the control group was predominantly filled with fibrous tissue, with only a small amount of new bone formation adhering to the edges. In the CA-Cur group, the defect was filled with both fibrous and new bone tissue, and the extent of new bone formation was significantly greater than that in the control group. Notably, in the CA-Cur@Cu group, the defect was predominantly occupied by new bone tissue, which bridged across the defect, indicating robust osteogenic activity. This observation was further corroborated by quantitative analysis of new bone tissue (Fig. 10D). Moreover, HE staining of the major organs in rats demonstrated that the CA-Cur@Cu hydrogels exhibited no significant in vivo toxicity (Fig. S4).

Fig. 10.

Histological analysis of in vivo bone defect repair using CA-Cur@Cu hydrogels. A) H&E staining. B) Masson staining. C) OCN immunohistochemical staining. D) Quantitative analysis of A). E) Quantitative analysis of B). F) Quantitative analysis of C).

Masson staining can specifically stain collagen tissue and newly formed bone tissue blue, with the extent and intensity of the blue color reflecting osteogenic activity. As illustrated in Fig. 10B, fewer blue-stained collagen fibers were observed at the defect site in the control group, indicating relatively lower osteogenic activity. In contrast, the CA-Cur group showed visible blue-stained collagen fibers and new bone formation at the defect site. Notably, in the CA-Cur@Cu group, a substantial amount of blue-stained new bone tissue was evident, with a darker blue color and a broader distribution, suggesting the highest level of osteogenic activity among the groups. The quantitative analysis of collagen tissue (Fig. 10E) supported these staining observations.

OCN is a widely recognized osteogenic marker that can effectively evaluate the osteogenic activity of tissues. Immunohistochemical analysis of OCN (Fig. 10C) revealed that the CA-Cur@Cu group exhibited the largest positive staining area and the darkest brown color, followed by the CA-Cur group, while the control group showed the smallest positive area. These findings were further confirmed by quantitative analysis (Fig. 10F).

Collectively, these results demonstrate that the CA-Cur@Cu hydrogels retain an outstanding capacity to promote osteogenic repair in an in vivo bone defect animal model. These effects can be attributed to the sustained release of curcumin from the hydrogels, which confers antioxidant and anti-inflammatory properties, thereby enhancing the osteogenic microenvironment. Additionally, the synergistic release of Cu²⁺ promotes angiogenesis and osteogenesis, ultimately facilitating effective bone defect repair.

The repair of bone defects is a complex, multifaceted process that involves multiple stages, including immunomodulation, angiogenesis, and bone regeneration. An ideal bone repair biomaterial should be capable of meeting the diverse requirements in the treatment of bone defects. Previous studies have demonstrated that copper ions exert significant pro-angiogenic and osteogenic effects [[48], [49], [50],[54], [55], [56]], thereby effectively promoting bone tissue repair. However, a single repair component alone is insufficient to achieve adequate bone repair effects. Moreover, a critical challenge in bone defect repair is the presence of local inflammatory responses and the excessive accumulation of reactive oxygen species (ROS) [17,18]. These pathological conditions not only promote osteoclast differentiation but also inhibit osteoblast activity, thereby substantially delaying or impairing the bone repair process [[19], [20], [21], [22], [23], [24]]. Therefore, incorporating curcumin, which possesses immunomodulatory properties, can effectively compensate for the deficiencies of copper ions in this regard. However, studies have demonstrated that curcumin exhibits limitations such as poor water solubility, low chemical stability, and limited bioavailability [77,78], which hinder its ability to exert a sustained and stable therapeutic effect in vivo. To overcome these challenges, this study employed PLGA microspheres to encapsulate curcumin, which were further embedded within a hydrogel network to enable sustained and controlled release of curcumin. By integrating the angiogenic and osteogenic properties of copper ions with the antioxidant and immunomodulatory effects of curcumin, the system can more effectively address the multifaceted requirements of bone defect repair.

Overall, the in vitro and in vivo research results of this study confirmed that the CA-Cur@Cu hydrogels exhibit excellent biocompatibility, effectively improve the bone immune microenvironment, promote angiogenesis, and enhance osteogenesis performance. Moreover, the CA-Cur@Cu hydrogels demonstrated remarkable antibacterial effects, which can prevent infections caused by common pathogenic bacteria during bone repair, thereby reducing or avoiding implant-related infections.

4. Conclusion

In this study, a multifunctional CA-Cur@Cu hydrogel was successfully fabricated by doping with Cur/PLGA microspheres and cross-linking using copper ions. The integration of Cur/PLGA microspheres and copper ions conferred the CA-Cur@Cu hydrogels with diverse functionalities, including antioxidant and immunomodulatory capabilities, pro-angiogenic activity, and osteogenic properties. Notably, the CA-Cur@Cu hydrogels enable sustained long-term release of curcumin, thereby exerting potent antioxidant and anti-inflammatory effects, promoting M2 macrophage polarization, and enhancing the bone immune microenvironment. Simultaneously, the hydrogels facilitate the slow release of Cu²⁺ ions, which induces angiogenesis and osteogenesis while synergistically interacting with the anti-inflammatory properties of curcumin to collectively promote bone repair and achieve optimal therapeutic outcomes. Furthermore, the robust antibacterial performance of the CA-Cur@Cu hydrogels significantly reduces the risk of implant-associated infections, offering critical support for infection prevention in clinical orthopedic applications. In conclusion, the multifunctional CA-Cur@Cu hydrogel, which integrates immunomodulatory, pro-angiogenic, osteoinductive, and antibacterial properties, can not only be applied to the repair of simple bone defects, but also to infected bone defects, diabetic bone defects, and other types of tissue injuries. Therefore, this multifunctional hydrogel provides novel insights and practical solutions for the treatment of complex tissue injuries and exhibits extensive clinical application potential.

CRediT authorship contribution statement

Wencan Lu: Writing – original draft, Data curation, Conceptualization. Weida Zhuang: Supervision, Methodology, Investigation. Wenhua Li: Writing – review & editing, Supervision, Data curation, Conceptualization. Hongxun Sang: Supervision, Project administration, Investigation, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

The data that has been used is confidential.