1. Introduction
Polyetheretherketone (PEEK), a high-performance semi-crystalline polymer, is valued as a leading contender in bone-implant materials owing to its superior biocompatibility, chemical stability, and intrinsic radiolucency, as well as its elastic modulus that matches that of human cortical bone
[1],
[2]. However, its biological inertness obstructs its integration with natural bone tissue and consequently stifles normal osteogenesis
[3],
[4]. Furthermore, the inert nature of PEEK permits bacterial colonization at the implant–bone interface, leading to infections and subsequent deterioration of peri-implant tissues
[5],
[6]. These risks could lead to implant loosening or even total failure, potentially necessitating extended medical interventions or repeat surgeries
[7]. Therefore, the versatile PEEK needs to be designed in such a way as to gain excellent antibacterial and osteogenic properties; this will shift PEEK’s role in bone repair from passive to active and thereby significantly extend the lifespan of PEEK implants and widen their scope in clinical applications.
In order to endow implants with antibacterial properties, various surface-modification methods based on the surface integration of antibiotics, antibacterial nanoparticles, antibacterial peptides, and topography structures have been reported. However, such antibacterial methods are highly dependent upon the intrinsic antibacterial activity of the loaded agents
[8],
[9]. These approaches frequently necessitate much more time for bacteria disintegration, and it is difficult to provide on-demand, cyclic antibacterial action. In contrast, photothermal therapy (PTT), which converts light energy into thermal energy to thermally ablate bacteria
in situ, is remotely controllable, noninvasive, deep-tissue penetrating, and histocompatible; thus, it is a promising new option for the treatment of bacterial infections
[10],
[11],
[12]. Interestingly, studies have shown that local hyperthermia can upregulate the expression of proteins such as heat shock protein and bone morphogenetic protein, increase the generation of mineralized crystals, and accelerate the process of bone repair
[13]. Photothermal transfer agents (PTAs) play a decisive role in photothermal performance; the currently used PTAs for antimicrobial implants include carbonaceous nanomaterials, narrow bandgap semiconductors, plasmonic nanomaterials, and MXenes
[14]. Among them, black phosphorus (BP) nanosheets are new two-dimensional (2D) narrow bandgap semiconductors that can kill bacteria through the photothermal effect, such as via cell wall disruption and protein denaturation
[15],
[16]. In addition, BP nanosheets exhibit a very high surface area/volume ratio due to their unique puckered structure, making them suitable carriers for the efficient loading of drug molecules or biomolecules
[17],
[18]. Moreover, the degradation of BP produces nontoxic phosphate, which accelerates mineralization at the site of bone defects by trapping calcium ions and accelerating cellular differentiation and signal transduction
[19]. Despite their excellent antibacterial and bone-repair potential, BP nanosheets are highly biodegradable in complex physiological environments
[20]. Therefore, they often require modification in biological applications. It has been reported that the use of polydopamine (PDA) can enhance the stability and photothermal properties of bare BP nanosheets
[21],
[22]. A multifunctional coating of PDA acts as “biological glue” to stabilize BP nanosheets, and composite nanoparticles formed by the self-assembly of BP and PDA have the dual advantages of high specific surface area and viscosity, providing good conditions for the subsequent loading of functional molecules
[19],
[23].
In an infected microenvironment, bone regeneration and repair are extremely challenging due to severe destruction of the local osteogenic environment. Therefore, in addition to possessing anti-infective properties, the ability of bone-implant materials to reshape the peri-implant microenvironment is crucial. Recently, various attempts have been made to fabricate multifunctional coatings for the simultaneous anti-infection and effective osseointegration of PEEK implants
[24],
[25],
[26],
[27]. Studies have pointed out that recruiting endogenous bone marrow mesenchymal stem cells (BMSCs) to the implant-bone interface is a promising approach to achieve osseointegration
[28],
[29]. Moreover, early targeted recruitment of BMSCs into the defect area is a prerequisite for subsequent osteogenic differentiation
[30],
[31]. In addition, the higher adhesion efficiency of BMSCs can reduce the bacterial infection rate through “surface competition”
[32]. E7, a short peptide with seven amino acids, has been found through phage display technology to have a specific affinity to BMSCs
[33]. It can promote the adhesion and migration of BMSCs for
in situ bone regeneration by upregulating the secretion of chemokines
[29],
[34]. Moreover, compared with functional proteins, short peptides are more resistant to local environmental changes such as pH and thermal changes. At the same time, short peptides are easily prepared and low in cost, making them more suitable for the surface modification of materials
[35].
In our previous study, we successfully modified E7 peptide on the surface of a silk protein scaffold. The
in vivo and
in vitro results demonstrated that the E7-modified scaffold recruited more BMSCs in the defect area and promoted cell proliferation and osteogenic differentiation on the scaffold surface, thereby promoting early rapid bone repair
[36]. Hence, E7 has great potential for enhancing the recruitment of BMSCs and bone formation. By introducing E7 peptides onto the surface of inert PEEK, we hope to improve early osteogenesis on the implant surface and achieve an osseointegration-friendly microenvironment.
Based on these considerations, we designed and fabricated the new versatile nano-coated PEEK implant material (sulfonated PEEK (sPEEK)/BP/E7) reported herein, which first surface-sulfonated PEEK and sequentially assembled BP/PDA and E7. Among these components, the sPEEK acquired a three-dimensional (3D) network structure on the implant surface, which promoted cell proliferation and the expression of osteogenic-related genes
[37]. Under near-infrared (NIR) irradiation, BP/PDA served as a PTA for efficient, noninvasive, and controlled photothermal antibacterial treatment around the implants. E7 can play a key role in the osseointegration and osteogenesis of implants by recruiting stem cells. Meanwhile, as the multifunctional coating degrades, the degradation products of BP attract free calcium ions, forming calcium phosphate deposition, and promote local mineralization. Overall, we envisaged that this sPEEK/BP/E7 implant could provide controlled photothermal antibacterial effects and permit the PEEK-implant
in situ recruitment of BMSCs for osseointegration and bone regeneration. Both osteogenic and antibacterial evaluations
in vitro and
in vivo were carried out to verify the biological safety and effectiveness of our modified PEEK implant.
2. Materials and methods
2.1. Preparation of sPEEK/BP/E7
Medical-grade PEEK (450 G; Victrex, UK) of different sizes (discs: ϕ10 or 20 mm × 1 mm; rods: ϕ2 mm × 8 mm) was used in this study. First, sPEEK with a uniform 3D porous nanostructured surface network was constructed by immersing PEEK into 97–99 wt% sulfuric acid (Sigma, USA) under magnetic stirring at 25 °C for 5 min. To remove residual sulfuric acid and other residues, the samples underwent hydrothermal treatment at 120 °C for 6 h in a 100-mL Teflon-lined autoclave and were then cooled to 25 °C. Second, BP nanosheets (0.5 mg∙mL−1; HWRK Chemical Co., Ltd., China) were mixed with PDA (2 mg∙mL−1; Aladdin, China) in Tris-HCl (pH 8.5), and sPEEK was then immersed in the mixed BP/PDA solution for 12 h, followed by ultrasonic cleaning in distilled water thrice. Subsequently, the BP/PDA-decorated sPEEK (sPEEK/BP) was soaked in 200 μg∙mL−1 of E7 (purity: 95.64%; Scilight-Peptide Beijing, China) solution at 4 °C for 24 h to graft the E7 peptide and then transferred to a vacuum air pump for vacuum drying. After rinsing in deionized water to remove unattached E7, the final samples (sPEEK/BP/E7) were obtained by air drying.
2.2. Material characterization
The surface morphologies and elemental distribution of all samples were examined using field-emission scanning electron microscope (FE-SEM; Zeiss, Germany) and energy-dispersive spectrometry (EDS; Phenom ProX, the Netherlands). Hydrophilicity was evaluated by measuring the water contact angle (WCA) using a contact-angle meter (Dataphysics, Germany). The distribution of E7 on the samples was visualized using rhodamine-labeled E7 and observed with a confocal laser scanning microscope (CLSM; Leica, Germany). Moreover, to evaluate the release of E7, E7 peptide concentrations in a phosphate-buffered saline (PBS) environment at 37 °C were quantified on days 1, 3, 5, 7, 14, 21, and 28 using a bicinchoninic acid (BCA) protein assay kit (Abcam, UK).
2.3. Photothermal effect of modified samples
The photothermal abilities of the modified samples (sPEEK, sPEEK/BP, and sPEEK/BP/E7) were characterized using an infrared thermal imaging system (FLIR, USA). In brief, different samples (ϕ10 mm × 1 mm) placed in an 24-well plate were immersed in 1 mL of PBS and irradiated with a NIR 808-nm laser (0.5 W∙cm−2) for 500 s. The temperature and thermal images of the samples were recorded every 20 s by the infrared thermal imaging system. Similarly, we investigated the variation in photothermal conversion efficiencies under 808-nm laser irradiation at different power densities (0.3, 0.4, and 0.5 W∙cm−2). In addition, the photothermal change of the samples in a dried condition was detected under 808-nm laser irradiation. We further evaluated the photothermal performance of sPEEK/BP/E7 rods implanted in rat femurs to simulate heat transfer in vivo. Implantation details are described in Section 2.7.
2.4. Cell activity
2.4.1. Cell cultivation
The BMSCs used in this study were derived from the femoral bone marrow of 4-week-old Sprague–Dawley (SD) rats. All surgical procedures were approved by the Animal Ethics Committee of Shanghai Ninth People’s Hospital. The cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco) and maintained at 37 °C in a 5% CO2 atmosphere. The cell medium was refreshed every three days. BMSCs at passage 2–3 were used in the following experiments.
2.4.2. BMSC proliferation
Cell proliferation on various PEEK samples was assayed using a 5-ethynyl-2′-deoxyuridine (EdU; Thermo Fisher Scientific, USA) staining and cell counting kit (CCK-8; Dojindo, Japan) in accordance with the manufacturer’s instructions. For EdU staining, following a 3-d incubation with the samples in the 24-well plates (3 × 104 cells∙well−1), all samples were incubated with EdU medium for 2 h in the dark. Subsequently, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, China). The cells were then visualized and photographed using a fluorescence microscope (Zeiss). In the CCK-8 assay, the culture medium was mixed with 10% CCK-8 reagent after 1, 3, 5, and 7 d of culturing. After a 1-h incubation, 100 μL of the supernatant was transferred to a 96-well plate, and the optical density (OD) of the solution was measured at λ = 450 nm using an enzyme labeling instrument (TECAN Spark, Switzerland).
2.4.3. BMSC adhesion
For the cell adhesion investigation, the expression of adhesion-related proteins was measured. In brief, the expression levels of vinculin and integrin β1 were evaluated by means of immunofluorescence staining. BMSCs (5 × 104 cells∙well−1) cultured on various sample surfaces in the 24-well plates for 12 h were fixed with 4% paraformaldehyde and treated with mouse monoclonal anti-vinculin or anti-integrin β1 primary antibody (Servicebio Technology Co., Ltd., China), followed by Alexa Fluor 488-conjugated secondary antibody (Abclonal, USA). Subsequently, the cells were stained with TRITC-phalloidin (Abclonal) and DAPI. Finally, the stained cells were observed using a CLSM.
2.5. In vitro osteogenicity
2.5.1. BMSC recruitment
The recruiting effect of various samples on the BMSCs was assessed using a Transwell assay. In brief, various samples were placed in 24-well plates, while BMSCs (5 × 104 cells∙well−1) were seeded in the upper chamber of the Transwell system, which was then positioned in the well plates. After 24-h incubation in a serum-free medium, non-migrated cells on the membrane were wiped off. Cells that had migrated to the underside of the membrane were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet solution for 10 min. Subsequently, the number and morphology of the cells were observed under a light microscope (Zeiss) and analyzed via ImageJ software (Rawak Software, Germany) to evaluate their migration ability.
2.5.2. Alkaline phosphatase (ALP) assays
Alkaline phosphatase staining was carried out after cultivating BMSCs (5 × 104 cells∙well−1) on various sample surfaces in 24-well plates for 7 d. The BMSCs were fixed with 4% paraformaldehyde and subsequently washed with PBS. ALP activity was detected using Nitro Blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (NBT/BCIP) staining (Beyotime) in accordance with the manufacturer’s instructions; the staining results were observed using an inverted microscope (Nikon, Japan). The ALP protein was quantitatively measured using an ALP assay kit (Beyotime) and recorded as the OD value at 405 nm with the total protein concentration, determined using a BCA protein assay kit (Thermo, USA) for normalization.
2.5.3. Alizarin red S (ARS) assays
Calcium deposition was investigated after cultivating BMSCs (5 × 104 cells∙well−1) on samples in 24-well plates for 14 d. The cells were fixed and stained with 1% alizarin red S (Sigma) for 10 min, after which calcium deposition was observed under an inverted microscope. Next, the stained samples were immersed in 10% cetylpyridinium chloride (Sigma) solution, and the OD values were quantitatively detected at 590 nm. Total protein concentration was used for normalization.
2.5.4. Immunofluorescence staining
Bone-specific proteins, including osterix, Runx-2, and osteocalcin (OCN), were analyzed through immunofluorescence staining (primary antibody from Servicebio Technology Co., Ltd.), employing the method described in Section 2.4.3. For detecting osterix and Runx-2, the BMSCs (5 × 104 cells∙well−1) were cultured on sample surfaces in 24-well plates for 3 d; for detecting OCN, the culture duration was extended to 7 d. Osterix was labeled using Alexa Fluor 594-modified secondary antibody (Abclonal), while Runx-2 and OCN were labeled with Alexa Fluor 488-modified secondary antibody (Abclonal).
2.5.5. Real-time polymerase chain reaction
The expression levels of osteogenic genes were further detected. Following cell culture (2 × 105 cells∙well−1) on sPEEK, sPEEK/BP, and sPEEK/BP/E7 surfaces (20 mm in diameter) in 6-well plates for 3 and 7 d, the total messenger RNA (mRNA) of the BMSCs on different samples was isolated via TRIzol (Invitrogen, USA) and transformed into complementary DNA (cDNA) with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), in accordance with the manufacturer’s guidelines. Real-time polymerase chain reaction (RT-PCR) analysis of Runx-2, ALP (3 d), OCN, and collagen I (Col Ⅰ) (7 d) was conducted using an ABI 7500 RT-PCR machine (Thermo Fisher Scientific) with SYBR Green (Roche, Switzerland). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the house-keeping gene for normalization. Quantitative analysis was then performed using the 2−ΔΔCt method.
2.6. In vitro antibacterial activity
2.6.1. Bacterial culture and treatment
Staphylococcus aureus (S. aureus; ATCC 25923) and Escherichia coli (E. coli; ATCC 25922) were used for the antibacterial assay. Both bacterial strains were cultured in Mueller–Hinton Broth (MHB; Thermo Fisher Scientific). To evaluate the samples’ antibacterial ability after NIR irradiation, 500 μL of bacterial suspension (1 × 106 colony-forming units (CFU)∙mL−1) was co-incubated with sPEEK, sPEEK/BP, and sPEEK/BP/E7 substrates (10 mm in diameter) in a 24-well plate and treated with 808-nm NIR irradiation (0.5 W∙cm−2) for 8 min. The culture plate was then cultured further at 37 °C for 12 h for the surviving bacteria to grow and colonize the surface of the material.
2.6.2. Bacterial spread plate test
The in vitro antibacterial rate of various samples after NIR irradiation was evaluated by means of the spread plate method. After placement in a bacterial incubator for 12 h, the samples were gently rinsed with sterile PBS to remove unattached bacteria. Subsequently, adherent bacteria were dislodged using ultrasonication in PBS for 5 min. After dilution, the bacterial suspensions were spread on agar plates and incubated at 37 °C for 18 h. The number of active CFUs was imaged and counted.
2.6.3. Live/dead bacterial staining
Bacterial viability and membrane integrity on the samples’ surfaces were detected by means of live/dead bacteria staining (Invitrogen). The bacteria on the substrates were incubated with SYTO9 and propidium iodide (PI) stains after co-culturing for 12 h. Under CLSM, the live bacteria fluoresced green, while the dead bacteria, indicative of compromised cell membranes, appeared red.
2.6.4. Biological transmission electron microscopy (TEM) analysis
The integrities of the S. aureus and E. coil cultured on the samples’ surfaces were examined using biological TEM. After incubation, bacteria adhering to the samples were separated via ultrasonic vibration and collected through centrifugation. These bacteria were then fixed with 2.5% glutaraldehyde at 4 °C for 1 h, followed by dehydration with a series of graded ethanol solutions. Finally, they were embedded in resin and sectioned into ultrathin slices, about 100-nm thick, for observation via biological TEM (Philips EM 201, USA).
2.7. In vivo study
2.7.1. Implantation surgery and postoperative procedures
In vivo experiments were performed after approval by the Animal Ethics Committee (SH9H-2024-A617-SB). Before surgery, various PEEK rods were uniformly coated with S. aureus inoculum (2 × 103 CFUs) to construct bacterially infected implants. These implants were then placed in a bacterial incubator at 37 °C for 4 h to facilitate bacterial adherence.
A rat femoral condylar defect model was established for in vivo experiments. The rats used in this study were 8-week-old SD rats weighing approximately 200 g each. For the in vivo photothermal antibacterial study, the left and right femurs of six rats were implanted with sPEEK implants and sPEEK/BP/E7 implants, respectively. For the osteogenesis study, the rats were divided into three groups (six rats per group): the sPEEK group, the sPEEK/BP group, and the sPEEK/BP/E7 group. All surgeries were performed under aseptic conditions. After anesthesia via intraperitoneal injection with sodium pentobarbital (40 mg∙kg−1), the femurs near the knee joint of the rats were exposed through surgical incision. A 2-mm-diameter surgical drill was used to create an approximately 8-mm-long narrow channel in the condyle, aligned with the femur’s long axis, penetrating into the marrow. The surgical site was intermittently irrigated with cold sterile physiological saline to prevent thermal injury. Subsequently, different implants were carefully inserted into the created defects using sterile forceps. The soft tissues and skin were then sutured. Both the right and left femurs of each rat received implants. All animals were given corresponding analgesic and soothing measures after surgery.
One day after surgery, the implant sites were irradiated with an 808-nm laser at 0.5 W∙cm−2 for 10 min, with images and temperatures captured using an infrared thermal imaging system. Additionally, for the rats used for osteogenesis studies, at 2 and 4 weeks after surgery, the rats received intraperitoneal injections of 30 mg∙kg−1 ARS (Sigma) and 20 mg∙kg−1 calcein (Sigma), respectively, to mark new bone formation.
2.7.2. Antibacterial evaluation in vivo
One week after surgery, six rats were sacrificed via excessive injection of sodium pentobarbital, and their femurs were harvested. To ascertain the bacterial load on the various implants, three rods from each group were sonicated for 10 min in 1 mL of PBS to isolate all adherent bacteria. These dilutions were plated onto bacterial culture plates and incubated at 37 °C for 18 h; after that, the colonies were counted. In addition, the remaining rods were immersed in 5 mL of MHB medium (one rod per tube, n = 3) and incubated at 37 °C overnight. Subsequently, the turbidity of the bacterial cultures was recorded using digital photographs.
The remaining femurs were gently obtained and fixed in formaldehyde solution for 2 d. Subsequently, these samples underwent gradual dehydration in ethanol solutions of increasing concentrations (50%, 70%, 90%, 100%, and 100%), followed by xylene infiltration and paraffin embedding. Finally, the paraffin-embedded sections were stained using hematoxylin and eosin (H&E), and the infection status in the tissues surrounding the implants was evaluated by observing the inflammation in these sections.
2.7.3. Micro-computed tomography (micro-CT) analysis
Eight weeks after surgery, the rats for the osteogenesis studies were euthanized. To study the osseointegration at the bone–implant interface, high-resolution images of the newly formed bone around the implants were acquired using a micro-CT scanner (Bruker, USA). Subsequently, 3D reconstruction was performed using accessory analysis software, allowing for the isolation of the bone tissue surrounding the implant from the background. Furthermore, the medullary cavity area with a radius of 1 mm from the implant surface was selected, and bone volume/total volume (BV/TV), bone mineral density (BMD), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were analyzed.
2.7.4. Histopathological evaluation
After micro-CT analysis, Van Gieson (VG)’s picrofuchsin staining was performed. More specifically, the femurs with various PEEK implants were fixed in 4% paraformaldehyde, dehydrated with progressive ethanol solutions, infiltrated with xylene, and ultimately embedded in polymethylmethacrylate. Undecalcified sections were prepared using a microtome (Leica), cutting along or perpendicular to the long axis of the implant with the implant at the center. These sections were then ground and polished to a thickness of 40 µm. The chelating fluorescent dyes ARS (red) and calcein (green) were visualized under CLSM at excitation/emission wavelengths of 543/617 nm and 488/517 nm, respectively. Finally, VG staining images were obtained using a light microscope, and new bone area and bone–implant contact were assessed.
In addition, to evaluate the biosafety of the sPEEK/BP/E7 under NIR irradiation, major organs such as the heart, liver, spleen, lungs, and kidneys underwent histological evaluation eight weeks after surgery. Following the embedding and sectioning of these organs, as per the aforementioned method, H&E staining and microscopic observation were performed.
2.8. Statistical analysis
Data from semi-quantitative and quantitative experiments are expressed as mean ± standard deviation, and the results from all experiments (at least three times for each experiment) were statistically analyzed using GraphPad Prism (9.0). The results of the in vivo antibacterial evaluation comparing the sPEEK group and the sPEEK/BP/E7 group were analyzed using Student’s t test. One-way analysis of variance (ANOVA) was used to analyze other results comparing the sPEEK, sPEEK/BP, and sPEEK/BP/E7. Statistical significance was assessed at a p value of less than 0.05.
3. Results and discussion
3.1. Characterization of sPEEK/BP/E7
The novel versatile implant material (sPEEK/BP/E7) expected to have osteogenic and antibacterial properties was fabricated by constructing a multifunctional drug- or functional molecule-delivery system on the surface of inert PEEK, as shown in
Fig. 1.
The surface morphology of the different coatings was observed under FE-SEM (
Fig. 2(a)). A micro-/nano-scale 3D porous network structure was formed on the surface of the sPEEK, which would facilitate cell anchoring and tissue growth
[3],
[38]; this pore structure became more complex, with an increased pore number and reduced pore size (∼500 nm), after layer-by-layer modification using BP/PDA and E7. The elemental composition and distribution on the surface of the sPEEK/BP/E7 were detected using EDS. As shown in
Figs. 2(b) and
(c), the elements carbon (C), oxygen (O), sulfur (S), nitrogen (N), and phosphorus (P) were uniformly distributed on the surface of the sPEEK/BP/E7, where the phosphorus peak and the relative increase in N originate from the loading of the BP nanosheets and short peptides, indicating uniform deposition of the BP nanosheets. This result can mainly be attributed to the active sites on the surface of the BP nanosheets and the unique bonding properties of PDA, permitting interaction with various polymers through π−π stacking and electrostatic interactions
[13],
[26],
[39]. In addition, the fluorescence image after E7 labeling (
Fig. 2(d)) showed that the short peptide E7 (red fluorescence) was successfully and uniformly immobilized on the sPEEK/BP surface, with the process relying on a Michael addition or Schiff base reaction derived from the BP/PDA
[40].
The surface hydrophilicity of the different samples was also evaluated. sPEEK exhibited hydrophobicity, with a WCA of more than 90° (99.85° ± 9.57°) (
Fig. 2(e)). After modification with BP/PDA, the hydrophilicity of the sPEEK/BP increased (WCA of 53.7°) due to the hydrophilicity of the BP/PDA
[13],
[41]. After subsequent conjugation with E7 peptide, the surface hydrophilicity was further enhanced, with a WCA of 40.3°. Hence, the sPEEK/BP/E7 samples showed improved wettability compared with the sPEEK substrates, which would facilitate bone-related cell adhesion and osteogenic response by increasing the affinity between the cells and biomaterials
[42].
The effective loading of functional factors on the surface of the implant can enhance its biological activity. Therefore, we further investigated the sustained release of E7 peptide on the modified surface (
Fig. 2(f)). Initial release of E7 peptide was evident, which coincided with the fact that the cell migration capacity was the highest at an early stage during osteogenesis
[43],
[44]. The E7 peptide in sPEEK/BP/E7 realized long-lasting and slow release, which was attributed to the threefold effect of the 3D network structure of the surface, the high specific surface area of the BP nanosheets, and the potent adhesion of PDA.
Subsequently, the photothermal properties of the different samples were evaluated. The temperature changes in the sPEEK, sPEEK/BP, and sPEEK/BP/E7 in PBS after 808-nm laser (0.5 W∙cm
−2) irradiation are shown in
Fig. 1(g). After 500 s of irradiation, the surface temperature of sPEEK increased from 26.8 to 39.5 °C, while that of sPEEK/BP and sPEEK/BP/E7 rapidly increased to 51.4 and 51.2 °C, respectively, indicating that these two surface coatings had a significant photothermal effect. Moreover, when the laser power was varied from 0.3 to 0.4 or 0.5 W∙cm
−2, the surface temperature of sPEEK/BP/E7 increased to 44.8, 48.6, and 51.3 °C, respectively (
Fig. 2(h)). This power-dependent photothermal conversion capability is consistent with previous reports of BP nanosheets and is advantageous for achieving temperature control
[45]. This time dependence and power dependence of the temperature change suggested that the photothermal effect of this novel coating was controllable. Infrared thermal images in a dry environment showed that the temperature of sPEEK/BP/E7 increased to 45.5, 62.7, and 70.1 °C after 1, 3, and 5 min of irradiation, respectively (
Fig. 2(i)). In a wet PBS environment, the temperature of sPEEK/BP/E7 increased to 38.4, 46.4, and 50.8 °C after 1, 3, and 5 min of irradiation, respectively (
Fig. 2(j)). The homogeneous surface temperature of the sPEEK/BP/E7 sample in the air environment indirectly demonstrated that the coating on the sample surface was well-distributed. These results revealed that the presence and homogeneous distribution of the BP/PDA coating played a key role in endowing the sPEEK/BP/E7 sample with a good photothermal effect under NIR irradiation.
3.2. Cell activity in vitro
To demonstrate the potential application of the bone–implant materials, we explored the cytocompatibility of the novel nano-coatings.
Fig. 3(a) shows the EdU staining after 3 d of culturing BMSCs on sPEEK, sPEEK/BP, and sPEEK/BP/E7 plates. The EdU (red) was doped into DNA replicating within the cell, thereby revealing the cells that were proliferating, while the nuclei were stained blue by DAPI. The sPEEK/BP and sPEEK/BP/E7—especially the substrates modified with E7—had not only a high number of cells on their surfaces but also a high number of cells in a proliferative state. Changes in the cell proliferation on different sample surfaces over time were then quantified using a CCK-8 assay (
Fig. 3(b)). The number of cells gradually increased with time. There were more cells on the surface of sPEEK/BP than on the surface of sPEEK at 3, 5, and 7 d, which is consistent with previous literature reporting that BP/PDA promotes the proliferation of BMSCs
[19]. sPEEK/BP/E7 showed the highest cell viability, and it has previously been reported that E7 peptide helps maintain the viability of BMSCs
[34].
To evaluate the role of adhesion-related proteins in the BMSCs cultured on various samples, immunostaining specific to integrin β1 and vinculin was performed. As shown in
Fig. 3(c), vinculin (green) was only located at the focal adhesion sites, where it overlapped with actin filaments (red) terminating in extending filopodium-like structures. As exhibited, the BMSCs adhering to BP- or BP/E7-modified surfaces with more extended cytoskeletons expressed much more vinculin. Moreover, the integrin β1 of the BMSCs co-cultured with sPEEK/BP and sPEEK/BP/E7 (
Fig. 3(d), green) was more widely distributed along with the actin cytoskeleton (
Fig. 3(d), red) at 12 h compared with that of the cells cultured on the sPEEK plate. The microstructural promotion of BMSC adhesion might occur through the facilitation of serum protein absorption. Good cell adhesion activity—especially the upregulation of integrin β1—plays an important role in stimulating the integrin/FAK signaling pathways; this can further activate many downstream pathways, such as MAPK/Erk and PI3K/Akt, which have a close relationship with both angiogenic and osteogenic differentiation
[46].
3.3. Osteogenesis in vitro
Recruiting endogenous BMSCs and capturing them for
in situ bone regeneration after implantation is an effective repair strategy. Our previous study demonstrated that the use of chemokines
in situ helps recruit more stem cells to the damaged area and promotes the proliferation of cells that help repair tissue defects
[36]. In this study, the migration-inducing effect of different samples on BMSCs was assessed by means of a Transwell migration test. The number of cells spanning the pore in the sPEEK/BP/E7 group was found to be significantly greater than that in the sPEEK and sPEEK/BP groups (
Figs. 4(a) and
(b)), confirming the high affinity and recruitment of E7 to BMSCs. E7 has been reported to promote autocrine secretion of the endogenous chemokine SDF-1α in mesenchymal stem cells, thereby inducing cell migration
[29],
[47]. Moreover, this
in situ recruitment of stem cells avoids the tedious process of
in vitro seed cell culture and immunogenic diseases
[48], so E7 is a very useful component for recruiting BMSCs.
We then further validated the osteogenic induction effect of the modified surface. First, ALP was detected as an early marker of osteogenic differentiation (
Figs. 4(c) and
(d))
[49]. After incubating the cells on the samples’ surfaces for 7 d, significant ALP staining appeared on all of the samples. Notably, in the sPEEK/BP/E7 group, most cells exhibited strong positive ALP expression. In contrast, the other two groups displayed relatively fewer cells with strong positive expression. This observation is consistent with the semi-quantitative ALP data presented, suggesting that BP/PDA and E7 favor early osteogenic differentiation.
Mineralization of the extracellular matrix (ECM) on different surfaces was further assessed by means of ARS staining after 14 d of culture. The results (
Figs. 4(e) and
(f)) showed that sPEEK/BP/E7 exhibited the highest ECM mineralization level, followed by the sPEEK/BP group. These findings suggest that E7 plays an important role in the enhancement of cellular matrix mineralization, which has a synergistic effect with BP/PDA. The osteogenic outcomes of sPEEK/BP/E7 may be attributed to the degradation products of the BP nanosheets and the osteoinductive properties of E7. The BP nanosheets degrade into phosphate ions, which can capture calcium ions and thereby facilitate bone tissue mineralization. Moreover, E7 has been reported to have certain osteogenic properties, and the good cell adhesion of sPEEK/BP/E7 facilitates osteogenic differentiation
[35],
[50],
[51].
The results of the immunofluorescence staining revealed the expression of osteogenesis-related proteins in the cells. Runx2 can increase the expression levels of osteogenesis-related osterix, OCN, and Col I
[52]. Osterix is a zinc finger-containing transcription factor necessary for osteogenic differentiation and is an early marker of osteogenic differentiation
[53]. After 3 d of co-culture, sPEEK/BP increased the expression of Runx2 and osterix in minor quantities compared with the control group, while the loading of E7 increased the expression dramatically (
Fig. 5(a)), indicating that sPEEK/BP/E7 has excellent osteogenic induction ability. In the later stages of osteogenic differentiation, OCN activity signaled mineral deposition, and the BMSCs on the sPEEK/BP/E7 surface exhibited a significant upregulation of OCN at 7 d (
Fig. 5(b)). Quantitative fluorescence intensity showed significant difference between groups. (
Figs. 5(c) and
(d)).
RT-PCR was used to evaluate osteogenesis at the genetic level; the results are shown in
Figs. 5(e–h).
Runx2 and
ALP are associated with early osteogenic differentiation. At day 3, the difference between the three groups was statistically significant, with the highest mRNA levels in the sPEEK/BP/E7 group. After 7 d, the expression levels of osteogenic markers (
Col I and
OCN) were significantly upregulated in the sPEEK/BP/E7 group and markedly higher than those in the sPEEK/BP and sPEEK groups, suggesting that BP/PDA and E7 synergistically created better osteogenic conditions. The osteogenic effect of E7 in combination with the superb cell adhesion and phosphate environment of sPEEK/BP/E7 likely accounted for the significantly higher expression levels of osteogenic bone markers observed in the sPEEK/BP/E7 group. These findings, which were consistent with the protein expression results, clearly revealed the ability of sPEEK/BP/E7 to induce the differentiation of BMSCs into osteoblasts. This strategy of synergistic stem cell recruitment and cell osteogenic differentiation endows the implant surface with a superior osteogenic ability
[54].
3.4. Photothermal antibacterial activity in vitro
Bacterial infection in the surgical area is a common complication after implant placement. The antibacterial activity of different samples was evaluated under NIR irradiation. The effect of different coatings of
S. aureus and
E. coli was first investigated using the plate-spreading method. As shown in
Figs. 6(a) and
(b), a large number of
S. aureus cells could be eluted from the surface of sPEEK due to its lack of photothermal action. In contrast, no
S. aureus adhered to the sPEEK/BP and sPEEK/BP/E7 surfaces after NIR irradiation. The loading of BP/PDA, compared with sPEEK, resulted in 100% inhibition efficiency against
S. aureus. As for
E. coli (
Figs. 6(c) and
(d)), in comparison with sPEEK, sPEEK/BP and sPEEK/BP/E7 inhibited
E. coli by 92.7% and 93.1%, respectively, after irradiation with NIR light for 5 min. These results indicate that sPEEK/BP and sPEEK/BP/E7 have excellent photothermal antibacterial effects.
To understand the potential antibacterial mechanisms of sPEEK/BP and sPEEK/BP/E7, the integrity of the membranes of
S. aureus and
E. coli was examined via live/dead (green/red) fluorescence staining (
Figs. 6(e) and
(f)). After NIR irradiation, almost all the bacteria were green and grew well in the sPEEK group, whereas a large amount of red staining was observed in the sPEEK/BP and sPEEK/BP/E7 groups. The red fluorescent stain PI only penetrates into damaged bacterial membranes
[55], indicating that NIR irradiation had led to the rupture of bacterial membranes in the sPEEK/BP and sPEEK/BP/E7 groups, resulting in significant bacterial death on the surface of the samples. To further explore the antibacterial effects of the sPEEK/BP and sPEEK/BP/E7 samples, biological TEM was employed to observe morphological alterations in
S. aureus and
E. coli. As depicted in
Fig. 6(g), the bacteria treated with sPEEK presented robust, visible walls and membranes, enclosing a densely colored cytoplasm. In contrast, after interacting with sPEEK/BP/E7 and after NIR irradiation, significant disruption was noted in the bacterial walls and membranes. In addition, a sparser and brighter cytoplasm was evident, suggesting cytoplasmic leakage. These observations indicate the active role of the BP/PDA-modified surfaces in the destruction of bacterial walls and membranes. This result was consistent with the results of the plate-spreading experiments and live/dead staining, suggesting that sPEEK/BP/E7 is effective for the controllable destruction of bacteria using NIR light.
3.5. Antibacterial activity in vivo
Encouraged by the excellent antibacterial activity
in vitro, we further evaluated the antibacterial performance of sPEEK/BP/E7
in vivo. As
S. aureus is one of the most common causes of peri-implant infection
[48], we implanted various
S. aureus-contaminated implants into the femoral condyles of rats to construct an implant-associated infection model. First, we evaluated the photothermal conversion ability of sPEEK/BP/E7 after implantation
in vivo: The real-time temperature was recorded with a thermal imaging instrument during irradiation of the implant site with an 808-nm laser for 8 min. As shown in
Figs. 7(a) and
(b), the temperature of sPEEK increased from 32 to 39.3 °C, while the surrounding temperature of sPEEK/BP/E7 showed a greater increase, eventually stabilizing at about 51.3 °C. Although the thermal conversion speed of sPEEK/BP/E7
in vivo was slightly lower than that
in vitro, it was still able to reach the effective temperature for sterilization
[56].
After being implanted and treated with NIR irradiation, the implants were removed and incubated at 37 °C in MHB for 12 h to assess the amount of live bacteria that remained on the modified surface. As shown in
Fig. 7(c), the medium of the sPEEK group was turbid, indicating that there was a large amount of residual bacterial growth on it. In contrast, the media containing sPEEK/BP and sPEEK/BP/E7 were transparent and clear, meaning that the number of bacteria on the surface of the implants was significantly reduced after treatment with BP nanosheets. The amount of bacterial adhesion of the different implants
in vivo was further quantitatively evaluated by means of sonication and dilution treatment. The results showed that the antibacterial rate of sPEEK/BP and sPEEK/BP/E7 reached almost 100% (
Fig. 7(d)), which was comparable to the
in vitro level. These results suggest that
S. aureus can be controllably removed from implant surfaces
in vivo by means of remote NIR irradiation.
Furthermore, the H&E images (
Fig. 7(e)) revealed a significant reduction in inflammatory cell infiltration around the sPEEK/BP/E7, in comparison with the numerous inflammatory cells observed near the sPEEK implant. These findings imply that the photothermal effects of BP/PDA may suppress intense inflammatory responses by enhancing the
in vivo antimicrobial efficacy of implants.
3.6. Osteogenesis and safety assessment in vivo
The femoral condylar infection implant model was also used to assess the
in vivo osseointegration performance. The femurs of the rats were removed after 8 weeks of implantation, and all implants were seen to be still present in the condyle. Micro-CT scanning showed that the implant extended through the condylar cortex to the cancellous bone (
Fig. 8(a)). The 3D reconstruction images showed that, compared with sPEEK, there was obvious new bone formation surrounding the implant in the sPEEK/BP and sPEEK/BP/E7 groups—especially in sPEEK/BP/E7, where it completely encircled the implant surface.
Figs. 8(b–e) depicted the further quantitative analysis results from the micro-CT. BV/TV, Tb.N, BMD, and Tb.Th were significantly higher in the sPEEK/BP and sPEEK/BP/E7 groups than in the sPEEK group, and these parameters were significantly higher in the sPEEK/BP/E7 group than in the sPEEK/BP group.
VG-stained tissue sections were further prepared for microscopic observation of the implant surface and quantitative analysis of new bone formation and bone–implant contact (
Figs. 8(f–h)). There was only a small portion of discontinuous new bone around the sPEEK (21.67% ± 1.21%), and a significant space existed between the bone and the implant. In contrast, the BP/PDA loading in the sPEEK/BP group promoted new bone formation; the new bone area (45.48% ± 2.68%) was significantly higher than that around sPEEK, and more new bone (77.33% ± 1.89%) was formed in the sPEEK/BP/E7 group. In addition, the percentage of bone–implant contact showed a similar trend, where sPEEK/BP/E7 had 89.55% ± 2.73% contact with bone, significantly higher than that of sPEEK/BP (40.53% ± 1.89%) and sPEEK (12.07% ± 1.62%). These results were consistent with the recruiting and osteogenic effects of sPEEK/BP/E7 on BMCSs in the
in vitro assays, and this high bone-implant contact suggested superior osseointegration.
New bone formation was recorded at different time points by injecting red and green fluorescent dyes at 2 and 4 weeks; the results are shown in
Fig. 8(i). There was no significant new bone formation in the sPEEK group even at 4 weeks. The fluorescence area was significantly greater in the sPEEK/BP/E7 group than in the sPEEK/BP and sPEEK groups at 2 or 4 weeks (
Fig. 8(j)). Overall, our developed sPEEK/BP/E7 promoted new bone formation and mineralization at the bone–implant interface in the early (2 weeks), middle (4 weeks), and late (8 weeks) stages.
Finally, the major organs of the abovementioned three groups of rats—including the major metabolic organs, the liver, kidney, and lungs—were harvested to evaluate the
in vivo safety of the sPEEK/BP/E7 coating. H&E staining (
Fig. 9) revealed no microscopic differences in these organs between sPEEK and sPEEK/BP/E7, and no enrichment of nanoparticles was found in any of the groups. These findings demonstrated that the sPEEK/BP/E7 coating did not have metabolic toxicity; however, for further safety, a future
in vivo evaluation in humans is still needed.
4. Conclusions
In this study, a multifunctional sPEEK/BP/E7 implant was successfully constructed by means of the layer-by-layer assembly of BP/PDA and E7 on sPEEK. The key findings regarding this newly constructed “functionally integrated” implant-modified surface are as follows:
(1) Photothermal effect and photothermal antibacterial activity. The BP, in conjunction with PDA, features photothermal properties that are controllable via NIR light. This capability provides the implant with remote, noninvasive, and on-demand photothermal antibacterial activity, which is critical for preventing or treating implant-related infections.
(2) BMSC recruitment and osteogenesis. Benefiting from the affinity of E7 for BMSCs and the phosphorus content of BP, the sPEEK/BP/E7 implant effectively enhances the recruitment, adhesion, proliferation, and osteogenic differentiation of BMSCs. Consequently, the novel implant demonstrates superior osseointegration and bone-regeneration capabilities.
The development of the sPEEK/BP/E7 implant can thus offer substantial clinical benefits and improved patient outcomes. By incorporating both biochemical and photothermal cues, this innovative implant not only enhances osseointegration but also significantly lowers the risk of postoperative infections. Such dual functionality can improve patient prognosis by increasing the success rates of implants and decreasing the incidence of complications, leading to considerable savings in healthcare costs and treatment durations. Although the mechanisms of the multifunctional coating in infectious bone repair and its long-term therapeutic effects require further research, the integrated “all-in-one functional” approach represented by the sPEEK/BP/E7 implant is a forward-looking solution for implant designs, providing a new idea for the application of PEEK-based materials in the field of bone repair.
Acknowledgments
The authors acknowledge the support by the Fundamental Research Funds for the Central Universities (YG2024QNB16), the National Natural Science Foundation of China (82270953 and 82201115), Shanghai Rising-Star Program (21QA1405400), the Natural Science Foundation of Shanghai (22ZR1436400), the Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZLCX20212400), and the Opening Research fund from Shanghai Key Laboratory of Stomatology, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine (2022SKLS-KFKT008).
Compliance with ethics guidelines
Xiao Wang, Shuning Zhang, Ai Zhu, Lingyan Cao, Long Xu, Junjie Wang, Fei Zheng, Xiangkai Zhang, Hongyan Chen, and Xinquan Jiang declare that they have no conflict of interest or financial conflicts to disclose.
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