A bioactive Cu-grafted gel coating with micro–nano structures for simultaneous enhancement of bone regeneration and infection resistance

Abstract

Prosthetic joint infection (PJI) remains a significant challenge in clinical applications. It not only impedes the recovery of bone tissue at the site of bone defect but also leads to multiple debridements, long-lasting antibiotic treatment and even secondary replacement. Titanium alloy Ti6Al4V (TC4) is widely used in orthopedic implants due to its excellent mechanical properties and biocompatibility; however, it lacks inherent antibacterial and osteoinductive functions. In this study, a composite coating based on polyvinyl alcohol (PVA) with tissue repair and antibacterial properties was applied on the surface of TC4. A PVA gel coating functionalized with terpyridine and catechol groups (PVA–TP–CA) was synthesized and subsequently complexed with copper (Cu) ions. The differential binding affinities of TP and CA groups to Cu enabled a sustained and controlled release of metal ions. Furthermore, a micro–nano surface structure was fabricated on TC4 using femtosecond laser technology to achieve a micro–nano structure interface and enhanced bonding strength. Biological evaluations demonstrated that the modified surface significantly improved the antibacterial, angiogenic, and osteogenic properties of TC4. These findings indicate that this multifunctional composite coating holds great promise for surface modification of orthopedic implants, offering an effective strategy for preventing PJI while promoting bone regeneration.

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1. Introduction

With the increasing number of orthopedic implants in recent years, orthopedic implant infections have become a major clinical concern.¹ Taking artificial joints as an example, approximately 2 million total joint replacement surgeries are performed annually in the USA, which is projected to reach approximately 4 million by 2030.² In China, around one million joint replacement surgeries are performed annually, with an annual growth rate of 25%.³ Total knee arthroplasty (TKA) is one of the most common and reliable surgical treatment options for treating knee conditions such as rheumatoid arthritis, osteoarthritis, and osteonecrosis.⁴ As the life expectancy increases, the demand for TKA and the corresponding costs are estimated to increase significantly.⁵ PJI is one of the most catastrophic complications associated with TKA, accounting for approximately 25% of TKA failures.⁶ Acute PJI most commonly occurs within 3–4 weeks postoperatively,⁷ making this period critical for preventing implant-associated infections.

Debridement, antibiotics, and implant retention are the recommended treatments for all acute PJI, but their efficacy in patients with late acute PJI is not well described.⁸ Compared with postoperative treatment, preventive strategies have demonstrated superior clinical efficacy. Current clinical practice primarily involves patient-specific risk assessment and perioperative systemic antibiotic prophylaxis to prevent PJI.⁹ However, the former is associated with numerous confounding variables and clinical uncertainty, while the latter carries the risk of inducing antibiotic-resistant bacterial strains.⁴ These limitations have driven growing interest in the development of novel preventive biomaterials. In addition to infection, TKA can also result in vascular damage, which further complicates healing and implant integration. Therefore, given the challenges of impaired bone regeneration, insufficient neovascularization, and the relatively long incubation period of PJI, there is an urgent need for advanced implant materials that can address these complications simultaneously. Currently, metals such as TC4 are commonly used as primary materials for TKA in clinical practice.^10,11 These materials exhibit excellent biocompatibility, suitable mechanical strength, fracture toughness, and corrosion resistance. They provide robust support and enhance structural stability in large bone defect areas during TKA;¹² however, they lack inherent antibacterial, angiogenic, and osteogenic properties.¹³ Therefore, developing a suitable implantable biomaterial based on TC4 that can prevent PJI has greater potential for clinical translation.

Current surface modification techniques have become effective strategies to promote bone formation and enhance antibacterial performance. During the healing process, antibacterial-functionalized surfaces can effectively inhibit bacterial proliferation, thereby reducing the rates of postoperative infections. Most antibacterial functionalizations on titanium alloy surfaces involve structural modifications and surface coatings. Structural modification can directly change the original structure or chemical composition of the material surface, affecting the interaction between the material and bacteria.^14,15 Surface coating, such as gel coating, on orthopedic implants is also an effective surface modification strategy for promoting bone formation and enhancing antibacterial performance.^16–19 The construction of functional gel coatings facilitates the effective integration of the substrate's mechanical properties with the coating's functional advantages and modulates the electrochemical microenvironment at the composite surface, thereby enhancing the biofunctional performance of the implant in physiological environments. Through the modification of gel coatings, the adjustment of physicochemical properties, such as the release rate of bioactive functional components and the elastic modulus, on the surface of medical metals can be achieved, further optimizing the biological functions of biomedical metals.^20,21 Among the polymers, PVA is a biodegradable polymer with low toxicity and is considered non-harmful.²² PVA has been widely used in drug delivery systems, tissue engineering scaffolds, and other biomedical applications, offering novel solutions for disease treatment.^23,24 Additionally, PVA is an FDA-approved biodegradable biomaterial. It can enhance the antibacterial properties of materials through acylation reactions, grafting of different functional groups, and incorporation of antibacterial agents.^25–27 However, the single antibacterial mechanism of structural modification and the insufficient bonding strength between the coating and the substrate have always been challenging issues in the field of surface modification. Therefore, there is an urgent need to develop a gel coating based on TC4 with high bonding strength, long-term antibacterial efficacy along with osteogenic and angiogenic properties to improve the outcomes of TKA and effectively prevent PJI.

Osteointegration plays a crucial role in the success of clinical implants. To enhance osteointegration, various biofunctional substances, such as enzymes, peptides, and growth factors, have been incorporated into biomaterials. However, ensuring the preservation of their high bioactivity during the fabrication process has proven to be a significant challenge. Cu is a broad-spectrum inorganic antibacterial agent, one of the essential elements for the human body, and is more stable than antibiotics.²⁸ Cu has good biocompatibility among metal elements with antibacterial properties.^29,30 Cu also plays a critical role in stimulating angiogenesis, though its direct molecular pathways in bone regeneration remain unclear. Importantly, osteogenesis and angiogenesis are closely coupled biological processes.³¹ Vascular endothelial cells can release osteogenic factors via paracrine signaling and establish microvascular networks that supply essential nutrients and a regenerative microenvironment for bone formation.³² Thus, vascular network regeneration serves as a fundamental prerequisite for bone regeneration. Moreover, studies have shown that metal elements in gel coating can enhance the mechanical properties of the material surface, thereby promoting the expression of osteogenesis-related proteins.³³ Terpyridine and catechol groups were used on PVA substrates to graft Cu simultaneously through metal–ligand coordination bonds.^34,35 These coordination bonds allow biomaterials to retain the inherent physicochemical properties of the metal ions.^36,37 The strength of the coordination bonds can vary from strong to weak, depending on the ligand type and the properties of the metal ions.³⁸ The binding strength between Cu²⁺ and one of the two functional groups is represented by the stability constant (K). The lower limit of the logarithm of the binding constant between terpyridine and Cu²⁺ is 8, while the logarithm of the binding constant between catechol and Cu²⁺ is around 6.^39,40 This suggests that the stability of Cu²⁺ bound to terpyridine is higher, and the binding strength between Cu²⁺ and the two functional groups differs, resulting in different release rates. A long-term antibacterial effect can be achieved by attaining long-lasting release of Cu²⁺.

The adhesion between the coating and substrate is a critical challenge that affects both long-term stability and biofunctional performance. Therefore, femtosecond laser processing is used to construct micro–nano structures on the surface of TC4, enhancing the bonding strength between the coating and the substrate by increasing the number of binding sites. Femtosecond laser processing offers unique advantages,^41,42 including minimal thermal effects, high processing precision, and strong three-dimensional processing capability.⁴³ This technique allows for precise control of the surface modification area and enables the direct fabrication of micro–nano multi-level structures.^44,45 The bonding strength of the coating is an important evaluation standard of composite materials during implantation.

We fabricated a micro–nano structure on the surface of TC4 and applied a PVA gel coating (PVA–TP–CA/Cu) incorporating terpyridine and catechol groups complexed with Cu onto the substrate surface to obtain PVA–TP–CA/Cu@TC4 (Fig. 1(A)). The micro–nano structure constructed by femtosecond laser processing can enhance the bonding strength between the coating and TC4. Furthermore, the differing binding affinities of the terpyridine and catechol groups for Cu²⁺ enabled a sustained release of Cu over time. In the present study, PVA–TP–CA/Cu@TC4 exhibited enhanced antibacterial properties by disrupting the bacterial membrane potential and promoting protein leakages through Cu²⁺. It also demonstrated good biocompatibility, promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs). When it is implanted into the distal femur of rats to simulate TKA, PVA–TP–CA/Cu@TC4 achieved superior vascularization and osteogenic induction. In short, our research offers a novel approach to preventing PJI after TKA.

Fig. 1 (A) Synthetic process of PVA–TP–CA/Cu@TC4, (B) antibacterial mechanism, and (C) the bone-promoting mechanism of PVA–TP–CA/Cu@TC4 implants.

2. Experimental

2.1 Synthesis of PVA–TP–CA

0.5 g of PVA (Aladdin, Shanghai, China) and 0.07 g succinic anhydride (Macklin, Shanghai, China) were dissolved in a mixture of 60 mL dimethyl sulfoxide (DMSO, Aladdin, Shanghai, China) and 0.2 mL of triethylamine (Aladdin, Shanghai, China) at 95 °C for 30 min. The mixed solution was transferred to room temperature, gently stirred overnight, and then dialyzed in ultrapure water for 3 days. The resulting product, PVA–COOH, was obtained by freeze-drying. Next, 0.5 g of PVA–COOH, 0.139 g of 2-([2,2′:6′,2′′-terpyridin]-4′-yloxy) ethan-1-amine (TP, Extension, Changchun, China), 0.087 g 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, Macklin, Shanghai, China), and 0.056 g 4-(dimethylamino) pyridine (DMAP, Heowns, Tianjin, China) were dissolved in a mixture of 60 mL DMSO at 95 °C for 30 min. Then, the mixture solution was stirred overnight at room temperature and dialyzed in ultrapure water for 3 days. Finally, freeze-drying yielded PVA–TP–COOH. To prepare PVA–TP–CA, 0.3 g of PVA–TP–COOH, 0.78 g of EDCI, and 0.78 g of dimethylformamide (DMF, Rhawn, Shanghai, China) and 2-(N-morpholino)ethanesulfonic acid (MES, pH 4.8, 0.1 M, Leagene, Beijing, China) buffer (60 mL, 1v/1v) were mixed. Then, the mixture solution was stirred overnight at room temperature and dialyzed in ultrapure water for 3 days. Freeze-drying produced PVA–TP–CA. Finally, PVA–TP–CA was dissolved in ultrapure water at a concentration of 3 mg mL⁻¹. The solution was mixed with 3-(N-morpholino)-propanesulfonic acid (MOPs, pH 8.5, Leagene, Beijing, China) buffer at a 1 : 2 ratio.

2.2 Synthesis of PVA–TP–CA/Cu@TC4

TC4 thin slices with a size of 10 mm × 10 mm × 1 mm were polished sequentially using 600#, 1000#, 2000#, and 5000# SiC sandpaper. The polished samples were then cleaned with 75% ethanol and ultrapure water, respectively, followed by drying for further use. A Femtosecond Laser System (Oscillator PumpSource-Verdi-V6, Oscillator-Mira 900, Amplifier-Legend Elite, Coherent Incorporated, USA) was used to irradiate the surface of the samples. Subsequently, the femtosecond-laser-treated titanium alloy (FSL) slices were cleaned and then immersed in a mixed solution of PVA–TP–CA solution and MOP buffer for 2, 4, and 12 h to prepare PVA–TP–CA@TC4. The resulting PVA–TP–CA@TC4 was further immersed in the CuCl₂ aqueous solution for 1 h to obtain PVA–TP–CA/Cu@TC4.

2.3 Characterization of PVA–TP–CA/Cu@TC4

The surface morphology of TC4, FSL, PVA–TP–CA@TC4 and PVA–TP–CA/Cu@TC4 was analyzed using scanning electron microscopy (SEM, Regulus 8100, HITACHI, Japan) and atomic force microscopy (AFM, MFP-30, Oxinst, UK). The hydrophilicity was measured using a contact angle device (JC2000DM, Powere, China) with a 2 μL droplet of distilled water applied to the sample. The element composition and chemical valence sites of PVA–TP–CA/Cu@TC4 were identified using an X-ray photoelectron spectrometer (XPS, Escalab, Thermo Fisher, USA). The composite materials (PVA–TP–CA/Cu@TC4) were immersed in 5 mL of phosphate-buffered saline (PBS, pH 7.4) under static incubation at 37 °C. The samples were collected at predetermined time points (1, 3, 6, 9, and 12 days), and the release of Cu was quantified using inductively coupled plasma mass spectrometry (ICP-MS, Optima 8300, PerkinElmer, USA). The potentiodynamic polarization curve measurements were conducted in PBS buffer using an Electrochemical Workstation (CHI660, Shiruisi, China).

2.4 Antibacterial activity assessments

Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) strains used in this work were obtained from Guangdong Microbial Culture Collection Center.

First, the samples were placed into a standard 24-well culture plate. The corresponding bacterial suspension (500 μL, 1 × 10⁷ CFU per mL) was then added to the samples, followed by incubation for 24 h to assess their antibacterial properties. After incubation, the bacteria adhering to the samples were separated. Subsequently, 100 μL of sequentially diluted bacterial suspensions were spread on nutrient agar plates, and the colonies were counted after overnight incubation. The zeta potential of the bacteria was measured using a Nanoparticle Size and Zeta Potential Analyzer (ZS920, ZiMeng, Shanghai). To evaluate the bacterial membrane potential, DiBAC₄(3) (MedChemExpress, Shanghai, China), a membrane potential-sensitive fluorescent dye that binds to depolarized cells to enhance fluorescence, was used. DiBAC₄(3) (1 μM) was added to the bacterial suspension (1 × 10⁷ CFU per mL) and incubated for 5 min at 37 °C. Afterward, the bacterial suspension was exposed to the samples for 12 h. The fluorescence intensity was measured at excitation and emission wavelengths of 490 nm and 505 nm, respectively.

2.5 Angiogenesis in vitro

HUVECs (Pricella Biotechnology, Wuhan, China) were grown in Endothelial Cell Medium (ScienCell, USA). The samples with a size of 10 mm × 10 mm × 1 mm were incubated for 24 h at 37 °C in 1 mL medium to prepare the sample extracts.

For the tube formation assay, HUVECs (3 × 10⁴ per well) were cultivated in Matrigel-coated plates containing the sample extracts and cultured for 3 and 6 h. The formed tubular structures were quantitatively analyzed by counting the number of nodes and meshes using microscopy and ImageJ software.

2.6 Biocompatibility and cell adhesion

BMSCs (Pricella Biotechnology, Wuhan, China) were grown in complete medium including α-minimum essential medium (α-MEM, Gibco, USA), 10% fetal bovine serum (FBS, Gibco, USA), and 1% penicillin/streptomycin (Solarbio, Beijing, China).

First, 1.5 × 10⁴ BMSCs were cultured on the samples with a size of 10 mm × 10 mm × 1 mm for 24 h and 72 h. The cytoactivity of the BMSCs was analyzed using a CCK-8 assay (Beyotime Biotechnology, Shanghai, China). In addition, the cell morphology at 24 h was analyzed by staining with Actin-Tracker Red-Rhodamine (Beyotime Biotechnology, Shanghai, China) and DAPI (Beyotime Biotechnology, Shanghai, China), and the images were captured using a fluorescence microscope (DMi8, Leica, China).

2.7 Osteogenesis in vitro

BMSCs were seeded (5000 cells per well) onto the samples with a size of 10 mm × 10 mm × 1 mm in 24-well plates. The cells were stained and analyzed for osteogenic markers using anti-Runx2, anti-BMP-2, or anti-COL-I antibodies (Beyotime Biotechnology, Shanghai, China). The average fluorescence intensity of a single cell was quantitatively analyzed using ImageJ software.

2.8 Evaluation of new bone formation and angiogenesis

Sprague–Dawley (N = 24) rats weighing between 230 and 350 g were randomly divided into four groups and used in this study. First, the distal femurs were exposed, and 1.6 mm × 3 mm defects were created. Next, the four sets of samples were implanted into the defect. Then, the skin and muscles were sutured and disinfected. In order to test the effect on PJI prevention after TKA, we hereby build the animal model without bacteria injection. During the postoperative feeding process, all four groups of rats were not injected with antibiotics to verify the effectiveness of PVA–TP–CA/Cu@TC4 in preventing PJI after TKA. Finally, the animals were euthanized in the sixth week, and the femurs were removed and placed in a 4% polyformaldehyde solution. All the animal experiments complied with the guidelines of Tianjin Medical Experimental Animal Care, and animal protocols were approved by the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) Co., Ltd (protocol number: YSY-DWLL-2024530).

The femurs fixed in 4% polyformaldehyde for 48 h were imaged using the Micro-CT system (Quantum GX2, PerkinElmer, USA) and a 3D reconstruction was performed. Then, the bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) were calculated.

Hematoxylin and eosin (H&E) staining and Masson's trichrome (Masson) staining. The femurs were dehydrated in a series of ethanol (70%, 90%, 95%, 100%), embedded, and cut into 300 μm thick sections using an Interlocked Diamond Saw (300CP, EXAKT, Germany). The sections were then polished to 30 μm thick slices using a Grinder (400S, EXAKT, Germany) for H&E and Masson staining.

Immunohistochemical staining. The femurs were decalcified in 10% EDTA solution for 1 month and dehydrated in ethanol and dimethylbenzene after embedding in paraffin. Thin sections were cut by a Microtome (HistoCore Multicut, Leica, Germany) in the sagittal plane along the implantation direction. The antibodies used for staining included anti-BMP-2 (Boster, Wuhan, China), anti-OCN (Proteintech, Wuhan, China), and anti-CD31 (Abcam, Shanghai, China).

2.9 Statistical analysis

All experiments were performed with at least 3 independent replications and analyzed using Origin 2024 and SPSS 27.0 software. The results are expressed as mean ± standard deviations (SD). Statistical analyses were assessed using one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls (SNK) post hoc test, where * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001 and were considered statistically significant.

3. Results and discussion

3.1 Characterization of PVA–TP–CA/Cu@TC4

In order to increase the bonding strength between the coating and the TC4, femtosecond laser processing was performed on the TC4. Based on ISO 13779.2, the bonding strength between PVA–TP–CA and TC4 is 29.04 MPa, while the bonding strength between PVA–TP–CA and TC4 treated using a femtosecond laser (FSL) is 38.8 MPa. Moreover, a supplementary experiment was conducted to evaluate the bonding strength according to the ASTM D3359 standard.⁴⁶ The peeled part of the TC4 surface coating extends beyond the scratch region, while the FSL surface coating exhibits serrated peeling (Fig. S1, ESI†).

The surface morphology of PVA–TP–CA@TC4 and PVA–TP–CA/Cu@TC4 was analyzed using SEM (Fig. 2(A) and (B)). The micro–nano structure obtained by femtosecond laser treatment can still be maintained after the gel coating process. To further validate this observation, AFM was used to analyze the surface morphology of TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4. The AFM results showed that the PVA–TP–CA@TC4 and PVA–TP–CA/Cu@TC4 retained most of the micro–nano structures obtained by femtosecond laser processing (Fig. 2(C)). Moreover, the application of the gel coating resulted in a decrease in the Young's modulus of the materials. The Young's modulus of PVA–TP–CA/Cu@TC4 is 32 GPa tested by nano-indentation, which is comparable with the Young's modulus of human bones.^47,48 To confirm the grafting of Cu onto the terpyridine and catechol groups, the elemental composition of PVA–TP–CA/Cu@TC4 was analyzed (Fig. 2(D)). The results demonstrated that Cu was uniformly distributed on the surface of the material. XPS results further verified that Cu exists in its ionic form, grafted to the two functional groups (Fig. 2(E) and Fig. S3, ESI†). Furthermore, the ratio of copper grafted to terpyridine to that grafted to catechol is approximately 1 : 1.2. To determine the optimal concentration of Cu grafting, the cell compatibility and antibacterial performance of PVA–TP–CA/Cu@TC4 prepared via the dip coating process of soaking in CuCl₂ solutions with different concentrations were evaluated (Fig. S2, ESI†). The results showed that optimal cell compatibility and antibacterial performance were achieved at a CuCl₂ soaking concentration of 0.5 mg mL⁻¹. Additionally, the release of Cu from PVA–TP–CA/Cu@TC4 was analyzed using ICP-MS. A slow and sustained release of Cu was observed over 12 days, with the amount of released Cu not exceeding the inhibitory concentration that BMSCs could tolerate (Fig. 2(F)). The release profile exhibits a pronounced gradient release pattern, consistent with XPS evidence confirming successful Cu coordination with both terpyridine and catechol groups. The sustained release of Cu over 12 days indicates the long-term stability of PVA–TP–CA/Cu@TC4 under a PBS buffer. Moreover, a previous study demonstrated that the coordination between catechol and Cu is highly stable, enabling sustained Cu release for more than 40 days.⁴⁹ To further evaluate the material's stability, electrochemical corrosion experiments were conducted under a PBS buffer. The gel coating on the metal surface enhanced the material's corrosion resistance, and the addition of Cu further improved this property. These results suggest that PVA–TP–CA/Cu@TC4 exhibits a stable performance.

Fig. 2 Characterization of TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4. SEM images of (A) PVA–TP–CA@TC4 and (B) PVA–TP–CA/Cu@TC4. (C) The topographies of TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4 detected by AFM. (D) The element mappings of PVA–TP–CA/Cu@TC4. (E) XPS survey spectrum of the PVA–TP–CA/Cu coating. (F) The cumulative release behavior of Cu from PVA–TP–CA/Cu@TC4. (G) The potentiodynamic polarization curves revealing the corrosion behavior of PVA–TP–CA/Cu@TC4. (H) The electrochemical mechanism of corrosion resistance of PVA–TP–CA/Cu@TC4.

3.2 Antibacterial evaluation

S. aureus and E. coli are the most common bacteria associated with infections following orthopedic implant surgery, and they can significantly hinder the process of angiogenesis and bone regeneration. Implant related infections can lead to a range of serious clinical complications, including the need for secondary surgeries or even amputations.^50–52

To investigate the antibacterial properties of TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4, S. aureus and E. coli were co-cultured with the materials, and the colony conditions were observed and quantified. The statistical analysis confirmed that the S. aureus amount decreased from 6.4 × 10⁵ to 82 CFU per mL, and the E. coli amount decreased from 1.7 × 10⁶ CFU per mL⁻¹ to below the detection limit (Fig. 3(A)–(C)). Moreover, the release profile showed that Cu was continuously released for at least 12 days, indicating that PVA–TP–CA/Cu@TC4 can provide a sustained antibacterial effect over this period. It can effectively prevent PJI during the acute susceptible period. The Cu-containing composite gel coating offers significant advantages over conventional antibacterial materials by enabling controlled Cu release. This stabilized ion release kinetics extends the duration of antimicrobial activity while maintaining an optimal balance between antibacterial efficacy and biocompatibility, presenting a promising strategy for advanced biomaterial surface engineering.

Fig. 3 Antibacterial evaluation. (A) Bacterial colonies were observed after the co-culture of TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4 with S. aureus and E. coli, respectively. Statistical analysis of the colonies of (B) S. aureus and (C) E. coli in different groups. (D) Zeta potential and (E) fluorometric assay for the membrane potential of S. aureus and E. coli induced by treatment with TC4 and PVA–TP–CA/Cu@TC4. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. n = 3.

Further investigations were conducted to explore the mechanisms underlying the antibacterial activity of the materials. It is widely accepted that Cu²⁺ interacts with negatively charged bacterial membranes, disrupting their integrity and leading to bacterial death. To further investigate this, the zeta potential and bacterial membrane potential of the co-cultured bacterial solution were tested (Fig. 3(D) and (E)). We observed an increase in the zeta potential of bacteria following co-culture, suggesting that the material influenced the bacterial potential. Additionally, the reduction in fluorescence intensity of the bacterial suspension indicated the dissipation of membrane potential and damage to membrane integrity. This effect is attributed to the interaction between the positively charged Cu²⁺ and the negatively charged bacterial membrane. Alterations in surface potential compromise membrane stability and permeability, facilitating further Cu²⁺ penetration into the bacterial cytoplasm. This, in turn, leads to intracellular protein leakage and ultimately results in bacterial inactivation.

3.3 In vitro angiogenic activity

When bones are injured, not only is the integrity of the damaged area of the bone compromised, but there is also a significant interruption in the blood vessels within the bone. However, after the implantation of conventional implants, the spontaneously formed neovascularization of the host is often insufficient to ensure proper integration of the implants. This highlights the need for new methods to accelerate neovascularization and enhance implant integration.^53,54

The blood vessels can provide nutrients, oxygen, and signaling molecules to promote the generation of new bone.^55,56 In this study, the material extracts were prepared, and HUVECs were cultured for 3 and 6 h to observe the tubular structure formed by the cells (Fig. 4(A)). The ability of the material extracts to promote angiogenesis was evaluated by analyzing the number of nodes and meshes in HUVECs (Fig. 4(B) and (C)). The results indicated that PVA–TP–CA/Cu@TC4 significantly enhances angiogenesis.

Fig. 4 Angiogenic activity of HUVECs cultured in extract medium of TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4 in vitro. (A) Optical images of tubule formation assay on HUVECs treated with TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4 extract medium; scale bar, 200 μm. Quantification of angiogenic (B) mesh number and (C) node number. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. n = 3.

3.4 In vitro osteogenic activity

First, the biocompatibility of the materials was evaluated. As shown in Fig. 5(C), a large number of live BMSCs were detected on the surface of PVA–TP–CA/Cu@TC4 on both the first and third days, with no significant difference compared to the group of TC4. Subsequently, the adhesion of BMSCs to the surface of the composite gel coating was evaluated (Fig. 5(A) and (C)). Cells in all four groups of materials exhibited typical spindle-shaped morphology of BMSCs. The spreading area of cells on the PVA–TP–CA/Cu@TC4 surfaces was significantly larger compared to that on PVA–TP–CA@TC4 (Fig. 5(C)). Next, we evaluated the expression of specific proteins in BMSCs cultured on different material surfaces to assess osteogenic differentiation (Fig. 6(A)–(F)). The expression levels of intracellular Runx2, BMP-2, and COL-I in the PVA–TP–CA/Cu@TC4-treated group were obviously higher than those in other groups. The experiment results indicated that PVA–TP–CA/Cu@TC4 significantly promotes osteogenic differentiation.

Fig. 5 The cell behavior on the surface of TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4 in vitro. (A) Fluorescence images of BMSCs cultured on the substrates for 24 h; scale bar, 125 μm. Cells were stained with actin filaments (red) and cell nuclei (blue) in this study. (B) The cytoactivity of BMSCs cultured on TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4 after 1 and 3 days. (C) Quantification of cell spreading area. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. n = 3.

Fig. 6 Osteogenic assay for BMSCs cultured on TC4, FSL, PVA–TP–CA@TC4, and PVA–TP–CA/Cu@TC4 in vitro. Representative immunofluorescence images showing the expression of (A) Runx2, (B) BMP-2, and (C) COL-I; scale bar, 125 μm. The corresponding quantitative analysis of fluorescence intensity of (D) Runx2, (E) BMP-2, and (F) COL-I. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. n = 3.

3.5 In vivo osteogenic and angiogenic activities

To investigate the osteogenic, angiogenic, and PJI prevention properties of PVA–TP–CA/Cu@TC4 in vivo, a series of animal experiments were conducted without the administration of antibiotics, thereby allowing for effective verification of the material's ability to prevent bacterial infections during bone tissue repair. First, micro-CT was used to observe osteogenesis around the implants after six weeks. Three-dimensional (3D) reconstructions revealed newly formed bone tissue in each group (Fig. 7(A)). The corresponding quantitative analysis demonstrated that PVA–TP–CA/Cu@TC4 significantly promoted new bone formation compared to the other groups (Fig. 7(B) and (C)). To investigate in situ bone formation, we conducted hard tissue sectioning followed by H&E and Masson staining. The PVA–TP–CA/Cu@TC4 implant exhibited tight integration with the surrounding bone tissue and evident collagen deposition (Fig. 7(D) and (E)).

Fig. 7 Evaluation of bone regeneration in vivo. (A) 3D-reconstructed images of micro-CT of bone regeneration (cyan for the implant, white for bone tissue). (B) and (C) Quantification of the new bone regeneration around the implants. The images of (D) H&E and (E) Masson staining; scale bar, 150 μm. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. n = 3.

Next, to further confirm endogenous bone generation, in situ analyses of specific osteogenic markers, including BMP-2 and osteocalcin (OCN), were performed. The bone tissue surrounding the PVA–TP–CA/Cu@TC4 showed a more complete structure and higher protein expression levels (Fig. 8(A) and (B)). The expression of BMP-2 in vivo aligned with cellular experiments in vitro (Fig. 8(D)). Notably, the expression of OCN in the bone tissue of the PVA–TP–CA/Cu@TC4-treated group was significantly higher than that in other groups (Fig. 8(E)). To further assess the in vivo angiogenic activity of PVA–TP–CA/Cu@TC4, sections surrounding the implant were analyzed for the expression of the angiogenic marker platelet endothelial cell adhesion molecule-1 (CD31).⁵⁷ Increased vascularity was observed in the bone tissue adjacent to the PVA–TP–CA/Cu@TC4, with this group exhibiting the highest vascular density (Fig. 8(C) and (F)). Based on the above experiments, we concluded that the PVA–TP–CA/Cu@TC4 significantly enhanced angiogenesis and osteogenesis in the rat bone defect model.

Fig. 8 Immunohistochemistry analysis in the rat bone defect model. Images of (A) BMP-2, (B) OCN, and (C) CD31 (the red arrows represent neovascularization); scale bar, 100 μm. Quantification of (D) BMP-2, (E) OCN, and (F) CD31 positive expression area. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. n = 3.

The surface properties of titanium play a critical role in determining the rate of osseointegration and the overall success of implants. In this study, a femtosecond laser-induced micro–nano structure was fabricated on the surface of TC4, followed by the application of a Cu-loaded gel coating. The micro–nano structure between the matrix and the coating significantly enhanced the bonding strength. Recent studies have highlighted that the controlled release of metal ions from material surfaces is essential for balancing cytotoxicity and antibacterial efficacy, as well as for regulating complex biological responses.⁵⁸ Terpyridine and catechol groups with different binding strengths to Cu were used in this study to achieve controlled slow release of Cu.

In summary, compared with TC4, PVA–TP–CA/Cu@TC4 exhibits excellent antibacterial, angiogenic, and osteogenic activities. On the one hand, Cu²⁺ inhibits bacterial growth by disrupting their membrane potential. On the other hand, the surface properties and chemical composition of PVA–TP–CA/Cu@TC4 promote angiogenesis and osteogenic differentiation. Based on experimental results and previous studies, we found that the incorporation of Cu into the gel coating increased the Young's modulus of the material from 28 GPa to 32 GPa, thereby promoting the expression of Yes-associated protein (YAP) and increasing cell adhesion³³ (Fig. 9). Furthermore, we constructed the micro–nano structure between the coating and TC4 using femtosecond laser processing, which enhanced the bonding strength and provided a more stable connection. Compared to TC4, PVA–TP–CA/Cu@TC4 not only prevents PJI by its antibacterial property, but also improves angiogenesis, addresses vascular damage associated with PJI, and enhances the osseointegration. In short, PVA–TP–CA/Cu@TC4 with its antibacterial, angiogenic and osteogenic properties, holds significant potential, as a novel material for artificial joint applications.

Fig. 9 The surface structure and chemical composition of PVA–TP–CA/Cu@TC4 promote osteogenic differentiation.

4. Conclusions

We developed a universal coating for various orthopedic implants, functionalized with antibacterial, angiogenic, and osteogenic properties. To enhance the adhesion strength of the coating, a micro–nano structure was constructed on the titanium alloy surface using femtosecond laser technology. This approach constructs the micro–nano structure, increasing the number of binding sites and ensuring a more stable connection between the coating and the titanium alloy substrate. Additionally, Cu was grafted into the coating, enabling sustained and long-term release. PVA–TP–CA/Cu@TC4 demonstrates good biocompatibility and excellent antibacterial properties while also promoting the osteogenic differentiation of BMSCs and angiogenesis in HUVECs. The experiments in vivo further reveal that PVA–TP–CA/Cu@TC4 accelerates the formation of new bone and blood vessels in a rat femoral bone defect model. These results underscore the potential of PVA–TP–CA/Cu@TC4 as an effective strategy for preventing PJI after TKA while simultaneously enhancing vascularization and bone regeneration. In short, we have developed a multifunctional coating capable of modifying orthopedic implants and enhancing their bioactivity at the implant–tissue interface.

Author contributions

Hu Ying: data curation formal analysis, methodology, writing – original draft. Li Mingjun: conceptualization, funding acquisition, investigation, methodology, project administration, resources, supervision, writing – review & editing. Xu Xun: project administration, supervision, writing – review & editing. Ma Nan: conceptualization, supervision, writing – review & editing. Luo Jiahao: data curation, methodology. Wu Xiaoxuan: data curation, formal analysis. Ping Qixiang: data curation, methodology. Lin Xiao: conceptualization, supervision. Zhang Tingbin: data curation, methodology. Liang Chunyong: conceptualization, funding acquisition, project administration, supervision, writing – review & editing. Yang Lei: conceptualization, funding acquisition, project administration, supervision, writing – review & editing.

Data availability

Data are available upon request from the authors.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

The authors would like to thank the National Natural Science Foundation of China (52201296, 82025025, U21A2055, and 82472108), the Hebei Natural Science Foundation (C2022202001), the Science and Technology Project of Hebei Education Department (BJK2022026), the Hebei Provincial Department of Human Resources and Social Security (C20220317), and the “Chunhui Plan” Cooperative Research Project Foundation of Ministry of Education of China (HZKY20220260).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00211g

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