Nanostructured Coatings for Spinal Fixation Screws: A Dual-Function Approach Against Biofilm Formation and Implant Failure

Abstract

Implant-associated infections represent challenging complications following orthopedic surgeries, with spinal fixation procedures being particularly linked with increased risks. Thus, urgent research is required to develop enhanced solutions to avoid bacterial colonization, associated implant failure, and severe issues. Our study is based on the laser coating of surfaces with a composite mixture of PLA/Fe₃O₄@CEF that can fight against infectious agents and preserve their activity for a prolonged time. In the present study, we synthesized Fe₃O₄@Ceftriaxone (CEF) nanoparticles by co-precipitation and blended them into polylactic acid (PLA)-based coatings that were thoroughly evaluated from physicochemical and biological points of view. The novelty of this work is the dual functionality of these coatings, combining localized, sustained antibiotic delivery with enhanced biocompatibility for spinal screw applications. The coatings exhibited substantial anti-biofilm effects, reducing Staphylococcus aureus colonization from 1.8 × 10⁸ to 1.6 × 10⁵ CFU/mL and Pseudomonas aeruginosa from 1.2 × 10¹¹ to 1.9 × 10⁶ CFU/mL after 24 h. Furthermore, in vitro assays with murine preosteoblasts and human osteoblasts demonstrated excellent biocompatibility, maintaining >95% cell viability and showing no significant cytotoxicity or inflammatory response. These results highlight the potential of PLA/Fe₃O₄@CEF composite coatings in preventing implant-associated infections and promoting osseointegration, offering a multifunctional strategy for improving spinal fixation screw longevity and patient outcomes.

1. Introduction

Spinal instrumentation has been employed in surgical procedures for more than 6 decades, allowing for the enhancement of clinical outcomes in patients requiring spine support [1]. Spinal fixation screws are especially regarded as critical components for stabilizing the spine following trauma, degenerative conditions, or surgical interventions, providing structural support and facilitating spinal fusion [2]. Pedicle screw fixation is particularly important for procedures requiring internal stabilization, such as stabilization of thoracolumbar fractures and dislocations, correction of spinal deformities, and ensuring rigidity in oncological and fusion surgeries [3,4]. In contrast to other types of fixation devices, pedicle screws can be used in laminectomy or traumatic disruption of posterior spinal elements, as their anchorage is independent of dorsal structures [5].

Despite their advantages, pedicle screws also present certain drawbacks. These implantable devices have been associated with complications such as fixation loss, fatigue failure, misplacement, tissue rejection, dural tears, cerebrospinal fluid leakage, nerve injury, and infection, imposing careful surgical planning and implant optimization to enhance outcomes and avoid future revision interventions [6,7,8]. An ideal spinal fixation system must exhibit biostability, biocompatibility, and appropriate biomechanical properties to ensure long-term functional success and patient safety [2]. Even though metallic implants are the preferred option for fixing the spine due to their mechanical strength, these devices are limited by poor osseointegration, inadequate antibacterial properties, and an increased risk of bacterial colonization, ultimately resulting in implant failure and low patient satisfaction [9].

Among the challenges encountered with spinal implants, the risk of infection is a major drawback, posing a tremendous diagnostic and therapeutic burden for spinal surgeons [10]. With the widespread use of invasive medical devices, healthcare-associated infections caused by pathogenic biofilms have emerged as a leading cause of morbidity and mortality [11,12]. Biofilm formation on spinal implants significantly contributes to implant-associated infections, as these sessile microbial communities exhibit extreme resistance to antibiotics and host immune responses, making eradication difficult [13]. Biofilms consist of live and dead bacteria embedded in an extracellular matrix, which hinders the diffusion of antimicrobial agents, resulting in treatment failure and persistent infections. Moreover, bacteria from biofilms can detach and disseminate, seeding new infection sites and exacerbating the clinical burden [14]. In severe cases, the only viable option to control infection is the removal of the affected implant, a measure that not only increases healthcare costs but also exposes patients to additional surgical risks and prolonged recovery periods [15,16].

As infection prevention is considered more valuable than reacting to adverse clinical outcomes, several pre-, peri-, and post-operative procedures have been employed to avoid implant-related infections [10,16,17,18]. The prevention of implant-associated infections in spinal fixation surgery often relies on a combination of systemic antibiotic prophylaxis, strict aseptic surgical protocols, and the development of antimicrobial implant materials [13,18]. Due to the significant reported infection rates in spinal fusion surgeries (ranging from 1% to 12%), prophylactic antibiotic administration is generally implied [10]. Despite the acknowledged benefits of pre-operative systemic antibiotics, this approach is not without limitations. High doses are often needed to achieve effective therapeutic concentrations, resulting in the rise of a series of issues, including potential tissue toxicity, poor patient compliance, and the emergence of antibiotic-resistant pathogens [19].

Antimicrobial-functionalized implant materials have gained increasing attention as a means of local infection control. Coating spinal fixation screws with antimicrobial agents, nanoparticles, or drug-eluting systems enables sustained localized drug release, effectively reducing the need for high-dose systemic antibiotics and lowering the risk of resistance development [13]. For instance, advanced-generation cephalosporins, such as ceftriaxone, have demonstrated strong efficacy against nosocomial and drug-resistant infections by disrupting bacterial cell wall synthesis. Additionally, the pre-operative bathing of implants in antibiotic solutions has been reported as an effective local prophylactic measure to prevent deep infections in spinal instrumentation surgeries [20]. Thus, functionalizing implant surfaces with such agents offers a promising strategy for prolonged antibacterial activity at the implantation site, improving infection control while minimizing systemic side effects.

Nanostructured coatings have great potential in spinal fixation, as they enhance osseointegration, promote cellular adhesion and proliferation, and reduce infection risks [21]. However, despite the growing interest in antimicrobial coatings for orthopedic implants, there remains a lack of multifunctional systems that combine sustained antibiotic release, biocompatibility, and stable adhesion to clinically relevant substrates.

In this context, this study aims to expand the plethora of infection-preventive modifications for spinal fixation screws, presenting the development of nanostructured anti-infective coatings based on magnetite nanoparticles (Fe₃O₄ NPs), ceftriaxone (i.e., a third-generation cephalosporin antibiotic), and polylactic acid (PLA). Specifically, co-precipitation of iron salts and the antibiotic agent was performed to obtain ceftriaxone-functionalized Fe₃O₄ nanoparticles (Fe₃O₄@CEF), while the matrix-assisted pulsed laser evaporation (MAPLE) technique was used to obtain nanostructured coatings (i.e., Fe₃O₄@CEF and PLA/Fe₃O₄@CEF) (Figure 1). MAPLE has been scarcely applied for coating spinal fixation screws, particularly with composite formulations incorporating both biodegradable polymers and antibiotic-functionalized magnetic nanoparticles.

Therefore, this study addresses a critical gap by developing and characterizing PLA/Fe₃O₄@CEF nanostructured coatings fabricated by MAPLE, aiming to inhibit bacterial biofilm formation, support osteoblast viability simultaneously, offering a dual-function antimicrobial and biocompatible interface that could enhance clinical outcomes and reduce implant-associated complications.

2. Materials and Methods

2.1. Materials

The experimental work was conducted using analytical-grade reagents purchased from Sigma–Aldrich/Merck (Darmstadt, Germany). All substances were used as received, without further purification.

2.2. Nanoparticle Synthesis

The Fe₃O₄@CEF nanoparticles were synthesized at room temperature by the co-precipitation method. First, Solution 1 (S1) was prepared using a 1:2 molar ratio of FeSO₄ (Fe²⁺) to FeCl₃ (Fe³⁺) by dissolving the appropriate amounts of each salt in 300 mL of demineralized water. The second solution (S2) was prepared with 0.2 g ceftriaxone and 9 mL of NH₄OH in 300 mL of demineralized water. The Fe³⁺/Fe²⁺ solution was dropped into the basic solution of CEF under magnetic stirring. After precipitation, magnetite–ceftriaxone (Fe₃O₄@CEF) was separated with a strong NdFeB permanent magnet. The nanoparticles were repeatedly washed with deionized water and left to dry at room temperature.

2.3. Coatings Deposition

Homogeneous solutions with 2% consisting of Fe₃O₄@CEF and PLA/Fe₃O₄@CEF in dimethyl sulfoxide (DMSO) were prepared. Before each laser deposition, 3 mL of the freshly prepared solution were frozen in liquid nitrogen (77 K) for 30 min. The frozen targets were maintained at liquid nitrogen temperature in the deposition chamber using a cooling system. The MAPLE technique was performed using a UV KrF* excimer laser source generating pulses of τ_FWHM = 25 ns at λ = 248 nm. The laser fluence was set at 200, 300, and 400 mJ/cm², with a repetition rate of 15 Hz. The number of applied laser pulses in all cases was 50,000. The targets were rotated with a frequency of 0.4 Hz. The substrates (i.e., spinal screw slices, Si (100)) were placed parallel to the target at a separation distance of 4 cm.

2.4. Characterization Methods

2.4.1. Physicochemical Characterization

The purity and crystallinity of the magnetite powder were investigated through X-ray Diffraction (XRD) analysis performed with an Empyrean diffractometer from PANalytical (Almelo, The Netherlands). Scans were collected at ambient temperature, using Cu_Kα radiation (λ = 1.056 Å) in the Bragg angle interval between 10° and 80°, with a current of 15 mA and a voltage of 30 kV.

The morphology and dimensions of the obtained materials were determined using a scanning electron microscope (SEM). In this respect, an FEI SEM (Hillsboro, OR, USA) was used. Samples were sectioned using a diamond blade, mounted on sample holders, and introduced into the analysis chamber. Imaging was performed in secondary electron mode at an accelerating voltage of 30 keV.

The Transmission Electron Microscopy (TEM) analysis was performed on the powdery sample (nanoparticles). A small quantity of sample was dispersed in pure ethanol and subjected to ultrasonication for 15 min. A drop of the resulting suspension was placed on a carbon-coated copper grid and air-dried at room temperature. The sample was examined using a Tecnai^TM G2 F30 S-TWIN high-resolution TEM (FEI, Hillsboro, OR, USA) equipped with a SAED accessory. The microscope was operated in transmission mode at 300 kV, with a point resolution of 2 Å and a line resolution of 1 Å. SAED analysis was further conducted in bright field mode to obtain information on the crystallinity of the nanomaterials.

FT-IR spectroscopy was used to investigate the integrity of functional groups within the synthesized material. A Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a ZnSe crystal was utilized for the analysis. A small amount of particle suspension was placed on the crystal, and measurements were performed at ambient temperature. Spectra were acquired in the 4000–1000 cm⁻¹ range, with 32 scans per sample at a resolution of 4 cm⁻¹. Data acquisition and processing were conducted using Omnic software (version 8.2, Thermo Fisher Scientific).

Thermal stability and degradation behavior were assessed using an STA 449C Jupiter device (NETZSCH-Gerätebau GmbH, Selb, Germany). A small amount of powdered sample was placed in an alumina crucible and analyzed under an air atmosphere over a temperature range of 30–500 °C. The heating rate was set to 10 °C/min.

Information regarding the investigation of the chemical alteration and material transfer of the obtained coatings was shown using an IRM analysis with a Nicolet iN10 MX FT-IR microscope (Thermo Fischer Scientific Company, Waltham, MA, USA). Spectral acquisition was performed in reflection mode with 32 scans per sample at a 4 cm⁻¹ resolution, in the 4000–600 cm⁻¹ range. The obtained data were processed using Omnic Picta 8.2 software.

2.4.2. Biological Characterization

The biocompatibility of the tested materials was evaluated using murine MC 3T3-E1 preosteoblasts (catalog number CRL-2593^TM) and hFOB 1.19 human osteoblasts (catalog number CRL-3602^TM), purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO₂ incubator, and in DMEM–Ham’s F12 (1:1) with 2.5 mM L-glutamine (without phenol red), 10% FBS, and 0.3 mg/mL G418 at 34 °C, 5% CO₂ humidified atmosphere, respectively. The osteoblasts were cultured in direct contact with the thin coatings deposited on spinal screw slices. All samples were sterilized under ultraviolet light for 1 h prior to cell seeding, which was performed at a density of 100,000 cells/cm². After 24 h of incubation, the biocompatibility tests were performed as previously described in detail, an Olympus IX71 inverted microscope (Olympus, Tokyo, Japan) being used to monitor cell adhesion and proliferation throughout the experiment.

Briefly, the culture medium collected at 24 h was used to quantify the lactate dehydrogenase (LDH) release as an indicator of cell membrane damage using a commercial kit from Roche (Basel, Switzerland) and to measure the nitric oxide (NO) production with the help of Griess reagent (0.1% naphthylethylenediamine dihydrochloride, 1% sulfanilamide in 5% H₃PO₄) at 550 nm. For the MTT assay, cells cultured on slides were incubated with 1 mg/mL MTT solution for 2 h at 37 °C. The resulting formazan crystals were solubilized in isopropanol, and absorbance was measured at 595 nm using a FlexStation microplate reader (Molecular Device, Sunnyvale, CA, USA).

The staining of live and dead osteoblasts was assessed by LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fischer Scientific, USA). After washing with PBS, cells were incubated for 30 min at 37 °C with calcein-AM (2 μM) and ethidium homodimer-1 (4 μM) solutions, and the fluorescence images were acquired.

F-actin cytoskeletal organization was assessed by staining with Alexa Fluor 488-conjugated phalloidin (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature after the cells were previously fixed in 70% ethanol for 15 min and permeabilized with 0.1% Triton X-100. Following the nuclei counterstaining with DAPI, representative images were captured on Olympus IX71 microscope.

The results of experiments performed in triplicate were subjected to statistical analysis performed by one-way ANOVA with Bonferroni post-hoc test (GraphPad Prism 5), with p < 0.05 being considered significant.

To assess the effect of nanostructured surfaces on biofilm formation, the samples were first sterilized under UV radiation for 20 min on each side. The samples were placed in 6-well plates, and 2 mL of liquid medium supplemented with 50 μL of microbial suspension (0.5 McFarland standard) were added. Plates were incubated at 37 °C for 24 h. After incubation, samples were washed with artificial seawater (AFS), and the culture medium was replaced to allow further biofilm development. Incubation was continued for 24 or 48 h. At the end of each incubation period, the samples were washed with AFS and transferred to sterile tubes containing 1 mL of AFS. The tubes were vortexed for 30 s to detach the biofilm cells, and serial dilutions were plated on solid culture media for colony-forming unit (CFU/mL) quantification.

The bacterial strains used (S. aureus and Ps. aeruginosa) were obtained from the Microbiology Laboratory, Faculty of Biology, University of Bucharest.

Biofilm formation on nanostructured surfaces was evaluated using 1 cm × 1 cm sterilized samples, which were placed individually in 6-well plates containing 2 mL of liquid medium and 50 μL of microbial suspension (0.5 McFarland standard). Plates were incubated at 37 °C for 24 h, followed by washing with AFS and medium replacement to allow further biofilm development. Samples were incubated for an additional 24 h. At the end of each incubation period, biofilm-covered samples were washed, transferred to sterile tubes containing 1 mL of AFS, and vortexed for 30 s to detach the adhered cells. Serial dilutions of the resulting suspension were plated on solid media for CFU/mL quantification.

3. Results

The indexed patterns correspond to the obtained iron oxide-based particles, which was proven using XRD analysis. As depicted (Figure 2), the analyzed sample exhibits high crystallinity, with strong peaks at 30.33°, 35.51°, 43.16°, 53.61°, 57.17°, and 62.82°, correlated with (220), (311), (400), (422), (511), and (440) diffraction planes, respectively. This diffraction pattern corresponds to the crystallographic system of the cubic inverse spinel structure Fe₃O₄, agreeing with JCPDS card No. 19-0629 and prior literature findings [22,23,24], thus confirming the crystalline structure.

The Fe₃O₄ nanoparticles were further subjected to HR-TEM analysis.

The HR-TEM images (Figure 3) confirmed the synthesized nanoparticles’ spherical shape, reduced dimensions, and cluster formation tendency. As can be seen in Figure 3a,b, the interplanar distance is at around 2.52 Å and 1.48 Å, which are values that correspond to the (311) and (440) crystallographic orientation of cubic magnetite Fe₃O₄ [25]. This finding aligns with the data obtained from XRD analysis, where the peak at the (311) diffraction plane was the most intense. The average diameter of the nanoparticles was estimated at around 4.83 ± 0.19 nm with a quasi-spherical morphology (Figure 3c). The SAED pattern depicted in Figure 3d provides information regarding sample crystallinity. The observed concentrical rings correspond to (111), (220), (311), (400), (422), and (511) diffraction planes, highlighting the presence of a highly crystalline magnetite [26]. The most intense diffraction ring is noticed for the (311) plane, in agreement with XRD, reconfirming that this is the dominant crystallographic orientation in the analyzed nanoparticles.

The FTIR spectrum of Fe₃O₄@CEF (Figure 4) revealed a characteristic band near 547 cm⁻¹, corresponding to Fe–O stretching vibrations, confirming the presence of the magnetite core [27,28,29]. A peak at 1655 cm⁻¹ is assigned to the C=O stretching vibration (amide I) from the β-lactam ring of ceftriaxone. The band at 1624 cm⁻¹ likely arises from N–H bending or C=C stretching in the aromatic system. Additional absorptions at 1571 cm⁻¹ and 1461 cm⁻¹ are attributed to C=C/C=N stretching of aromatic and heterocyclic rings and CH₂ deformation, respectively. Peaks at 1110 cm⁻¹ and 1032 cm⁻¹ are associated with C–N stretching and in-plane C–H bending vibrations, indicating the retention of ceftriaxone’s functional groups after surface binding to Fe₃O₄ nanoparticles [30,31,32,33].

The thermal behavior of the synthesized nanoparticles was assessed through TG-DSC, allowing a comparison of pristine (Fe₃O₄) and Fe₃O₄@CEF nanoparticles. For the pristine material, in the temperature range between room temperature and 120 °C, there occurs the loss of water molecules and –OH groups from the surface of magnetite. In this interval, there is a mass loss of 1.74%, which is accompanied by a slight endothermic effect. With increasing temperature, two other successive mass losses (120–200 °C and 200–300 °C) were observed, accounting for 0.54% and 0.98%, respectively. Both mass losses were associated with exothermic processes and can be attributed to the combustion (oxidation) of organic impurities present on the surface of Fe₃O₄. The strong exothermic peak at 565.8 °C corresponds to the crystalline phase transition from magnetite to maghemite. The mass loss continues at a slower rate up to 900 °C, registering an additional 0.73% loss. The residual mass represents 95.45% of the initial sample amount.

On the other hand, Fe₃O₄@CEF nanoparticles exhibit a mass loss when reaching from room temperature to 155 °C that can be attributed to the removal of adsorbed water molecules or surface hydroxyl (–OH) groups. The first significant mass loss (i.e., 2.84%) occurs between ~200–400 °C, with two exothermic peaks at 306.3 °C and 373.1 °C, corresponding to the oxidation of organic residues or other thermally labile surface contaminants. Further, the sample faces a similar phase transformation as pristine nanoparticles, with the graph displaying a strong exothermic peak at 583.8 °C being attributed to the change from magnetite (Fe₃O₄) to maghemite (γ-Fe₂O₃). During this process, another mass loss is observed, 1.59%, possibly due to the release of oxygen from the crystal lattice. The total residual mass after thermal degradation is 93.71%, indicating a thermally stable inorganic fraction, likely composed of oxidized iron phases. By comparing the results obtained for the two types of iron oxide-based nanomaterials, it was estimated that the ceftriaxone amount loaded on Fe₃O₄@CEF nanoparticles is ~1.82%.

Based on these results, Fe₃O₄@CEF nanoparticles were further utilized to fabricate coatings using the MAPLE technique at different laser fluences. Infrared microscopy (IRM) was used to investigate the stoichiometric laser transfer of the materials.

The optimum laser fluence was identified using comparative IRM analysis (Figure 5) between Fe₃O₄@CEF drop-cast and coatings deposited by MAPLE at 200 mJ/cm², 300 mJ/cm², and 400 mJ/cm² laser fluences. Absorbance intensities of IR spectra maps are proportional to color changes, starting with blue (the lowest intensity) and gradually increasing from green and yellow to red (the highest intensity).

The laser fluence of 300 mJ/cm² was identified as optimal for the synthesis of Fe₃O₄@CEF coatings, with the yellow and red colors being representative, in this case, for the distribution of C–H and C–O groups. This corresponds to a minimum chemical alteration and adequate material transfer. The IR maps show absorption band intensities ranging from yellow–orange (medium) to orange–red (medium–high).

In the case of PLA/Fe₃O₄@CEF coatings (fabricated at the same laser fluences), one can observe that the lowest functional group degradation was recorded at 300 mJ/cm² laser fluence. At this fluence, the color distribution is better emphasized than in the case of the other two laser fluences. This is indicative of a superior stoichiometric deposition of the coatings (Figure 6).

Along with the IR maps, complementary information was gathered from the IR spectra of Fe₃O₄@CEF and PLA/Fe₃O₄@CEF drop-cast and coatings deposited at 200, 300, and 400 mJ/cm² (Figure 7). The lowest degree of functional group degradation was registered at 300 mJ/cm². The other two experimental variants led to a significant modification in the absorption bands characteristic of carboxyl groups.

Further, the optimum fluence of 300 mJ/cm² was used to assess the biological characteristics for both types of composites (i.e., Fe₃O₄@CEF and PLA/Fe₃O₄@CEF).

The cross-sectional SEM images (Figure 8) showed uniform coverage with a smooth morphology for PLA/Fe₃O₄@CEF coatings obtained at 300 mJ/cm², with a thickness between 200–300 nm.

The synthesized nanostructured materials were further characterized from a biological point of view. Specifically, the biocompatibility of murine preosteoblasts (Figure 9) and normal human osteoblasts (Figure 10) was evaluated at 24 h after seeding on Fe₃O₄@CEF and PLA/Fe₃O₄@CEF coatings. In the case of MC3T3-E1 murine preosteoblasts, the MTT assay revealed that both types of surfaces led to cell-viability levels close to 100%, indicating that the samples do not exert cytotoxic effects. Moreover, NO production (Figure 9a) remained at baseline levels, suggesting the absence of an inflammatory response in preosteoblasts. These findings are crucial for ensuring that the implants support early-stage bone formation without eliciting excessive immune activation. Additionally, phase-contrast microscopy analysis (Figure 9b) suggested that cells adhered well to the coated surfaces, maintaining a healthy morphology, further reinforcing the notion that the materials provide a suitable for preosteoblast attachment and proliferation. Importantly, the demonstrated biocompatibility of these coatings with murine preosteoblasts suggests their potential application medicine.

For human osteoblasts, the MTT assay (Figure 10a) confirmed good cell viability across all tested coatings, similar to the findings in preosteoblasts. In addition, LDH measurements supported the non-toxic nature of the materials, as there was no notable increase in membrane damage or cell lysis, and the NO assay demonstrated no significant increase in inflammation. Furthermore, the live/dead staining corroborated these results, showing predominantly viable cells with minimal cell death. F-actin staining highlighted well-organized cytoskeletal structures, with cells maintaining their typical elongated morphology and spreading efficiently over the coated surfaces. The presence of an intact actin cytoskeleton and well-distributed focal adhesion points (indicated by white arrows in Figure 9b), enhanced by the nanostructured surface compared to the control, suggests strong cell–substrate interactions, which are essential for the osseointegration process.

The nanostructured coatings were further evaluated for anti-biofilm properties against two bacterial strains: Staphylococcus aureus and Pseudomonas aeruginosa (Figure 11), with results being presented in terms of colony-forming units per milliliter (CFU/mL), which indicate the number of viable bacteria forming biofilms on the surfaces after 24 and 48 h of incubation.

As shown in Figure 11, both Fe₃O₄@CEF and PLA/Fe₃O₄@CEF coatings exhibit pronounced anti-biofilm effects, particularly against S. aureus. The control group (uncoated surface) displayed a high bacterial load, with mean values around 10⁶ CFU/mL at 24 h and increasing to approximately 10⁸ CFU/mL at 48 h, highlighting the robust biofilm-forming capacity of S. aureus on untreated materials. In contrast, both coated surfaces demonstrated a significant reduction in bacterial adhesion, with CFU counts reduced to approximately 10⁵ CFU/mL at both 24 and 48 h. The difference was statistically significant (p < 0.05), confirming the coatings’ efficacy in preventing early-stage biofilm formation.

For Ps. aeruginosa, although the initial bacterial load on control samples remained high (~10⁸–10⁹ CFU/mL), both nanostructured coatings reduced bacterial counts to approximately 10⁷ CFU/mL at 24 h. After 48 h, Fe₃O₄@CEF maintained this reduction, while PLA/Fe₃O₄@CEF showed slightly greater variability but still a significant decrease compared to the control. These results demonstrate that the coatings not only inhibit initial bacterial adhesion but also prevent sustained biofilm growth over time.

Therefore, the nanostructured coatings developed in this study demonstrated effective anti-adhesive behavior by significantly reducing biofilm formation by S. aureus and Ps. aeruginosa. The coatings inhibited bacterial adhesion to the surface, thereby limiting the development of biofilms associated with medical devices. These anti-biofilm effects were sustained over both 24 and 48 h incubation periods, indicating stability over time. Inhibiting biofilm formation by S. aureus and Ps. aeruginosa is relevant for reducing the risk of device-associated infections, as these species are frequently implicated in hospital-acquired infections.

4. Discussion

The use of nanostructured antimicrobial coatings on fixation devices has become particularly relevant in the field of spinal surgery, as these implants are highly susceptible to bacterial colonization and infection-related complications [6,34,35]. Recent studies have explored a broad range of materials for designing infection-resistant coatings, including ceramics [34,36,37,38], natural and synthetic polymers [19,39,40,41,42], metal and metal oxide particles [11,41,43], carbon-based materials [19], antibiotic drugs [19,36,42], natural antimicrobial agents [44], and various combinations among them. From the plethora of possibilities, this study chose to develop nanocomposite coatings with magnetite (iron oxide—Fe₃O₄), ceftriaxone (CEF), and polylactic acid (PLA) toward creating synergistic outcomes.

The Fe₃O₄ nanoparticles have gained significant attention for biomedical applications due to their intrinsic magnetic properties, ability to enhance osteogenesis, and potential for magnetically targeted drug delivery [11,45]. Additionally, Fe₃O₄ nanoparticles influence functional bone tissue regeneration through osteo-mimetic mechanisms, making them promising candidates for enhancing osseointegration in orthopedic implants [46,47]. Fe₃O₄ nanoparticles have been shown to stimulate osteoblast proliferation and differentiation, potentially through the activation of signaling pathways such as BMP-2/Smad and MAPK, as well as by promoting intracellular calcium deposition and mineralization. These effects may be enhanced under magnetic stimulation, enabling magneto-mechanical stimulation of bone tissue, hence supporting bone regeneration and osseointegration, which is especially beneficial in load-bearing spinal applications [48,49,50,51].

Moreover, the properties of Fe₃O₄ nanoparticles can be further tuned to ensure multifunctional roles by surface functionalization. Modifying Fe₃O₄ nanoparticles with antibiotic drugs represents a particularly advantageous solution for countering resistant microbial strains [52].

Ceftriaxone (CEF), a broad-spectrum β-lactam antibiotic, has been widely used in spinal surgery infection prevention, with reports suggesting that local administration via implant coatings could serve as an effective alternative to systemic therapy [20,53]. This third-generation cephalosporin antibiotic is known for its activity against both Gram-positive and Gram-negative bacteria [54]. It exerts its bactericidal effect by inhibiting bacterial cell wall synthesis, making it highly effective in treating various infectious diseases [33]. Thus, based on its potent broad-spectrum activity, ceftriaxone was chosen to modify the surface of Fe₃O₄ nanoparticles and endow them with synergistic anti-infective capabilities.

In implantable medical devices, particularly in spinal fixation systems, PLA offers distinct advantages that recommend it as an interface between the screw surface and surrounding bone tissue. This biodegradable polymer serves as a supportive scaffold that gradually degrades into lactic acid, a natural metabolite, further allowing space for new bone tissue formation. Thus, it enables the formation of bioactive composites with osteoconductive and osteoinductive properties to improve spinal fusion rates and enhance tissue regeneration [55].

Henceforth, the incorporation of ceftriaxone-modified Fe₃O₄ nanoparticles into PLA coatings can thus improve bioactivity, antimicrobial efficacy, and mechanical stability, both optimizing the performance of spinal fixation screws and justifying the material choice for this work.

This study reports on the successful synthesis and characterization of Fe₃O₄@CEF nanoparticles, and their subsequent incorporation into PLA-based nanostructured coatings. The aim was to highlight their potential for biomedical applications, particularly in endowing spinal fixation screws with antimicrobial and biofilm-resistant properties. The XRD analysis confirmed the crystallinity, purity and structural integrity of the synthesized Fe₃O₄ nanoparticles, which exhibited a cubic inverse spinel structure. SEM and TEM analyses revealed that the obtained materials displayed a quasi-spherical morphology with nanometric diameters and a slight agglomeration tendency [56,57,58]. HR-TEM further allowed for a refined size estimation to ~19.5 nm, which corroborated well with the results obtained by XRD and SAED analyses. The presence of characteristic functional groups of both Fe₃O₄ and CEF in the FT-IR spectra confirmed the successful functionalization of the nanoparticles with the antibiotic. The TG-DSC analysis indicated a CEF loading of ~1.82% within the Fe₃O₄@CEF nanoparticles.

The MAPLE deposition technique was used in recent studies to develop biocompatible coatings with enhanced properties [11,59,60]. The advantages of MAPLE as a solvent-free deposition technique include its ability to preserve the antibiotic’s bioactivity, ensure homogeneous thin-film formation, and achieve strong adhesion to the substrate, which are essential characteristics for the stability and efficacy of implant coatings [60,61,62].

By analyzing the transfer efficiency and integrity of the materials, the coatings obtained at 300 mJ/cm² laser fluence were selected as the best choice for the current study. Thus, these nanostructured coatings exhibited a smooth surface morphology and uniform distribution.

The biocompatibility assessments demonstrated that both Fe₃O₄@CEF nanoparticles and PLA/Fe₃O₄@CEF composite were non-toxic to both murine and human osteoblasts. This result supports their potential use in biomedical applications (i.e., osseointegration in spinal fixation screws). One should mention that these findings align well with prior reports on biocompatibility, osteoinductive potential, and biodegradability of magnetite nanoparticles [46,63]. PLA matrix is recognized for its biocompatibility, biodegradability, and mechanical stability, which recomend it for spinal fixation implants [64].

The anti-biofilm properties of the coatings were also evaluated in the presence of both S. aureus and Ps. aeruginosa, two major pathogens responsible for implant-associated infections [65]. The obtained results demonstrated that Fe₃O₄@CEF and PLA/Fe₃O₄@CEF coatings effectively inhibited biofilm formation, with the PLA-based coating demonstrating superior efficacy, particularly against the Gram-negative strain.

The observed anti-biofilm properties of PLA/Fe₃O₄@CEF coatings can be attributed to a multifactorial mechanism based on the synergistic activity of component materials. Firstly, the sustained presence of CEF on the surface enables continuous disruption of bacterial cell wall synthesis, impairing microbial adhesion and proliferation. Secondly, Fe₃O₄ nanoparticles contribute to generating a hostile surface topography that reduces bacterial anchoring. The nanostructured coatings interfere with initial bacterial adhesion by inducing mechanical stress or disrupting the extracellular polymeric substance matrix formation. The combination of chemical (antibiotic) and physical (nanostructured surface roughness) cues is likely to prevent biofilm initiation and maturation, offering great promise for preventing implant-related infections.

Considering the broad context of spinal surgery, a multifaceted approach combining pre-operative infection control protocols, systemic prophylaxis, and antimicrobial coatings is the most effective strategy for reducing implant-related infections. The emergence of multifunctional implant coatings incorporating multiple antibacterial agents, such as combinations of antibiotics, antimicrobial peptides, and bioactive nanoparticles, represents a promising avenue for enhancing implant safety and reducing the risk of antibiotic resistance [66,67,68].

The PLA/Fe₃O₄@CEF nanostructured coatings developed in this study offer a distinctive combination of antimicrobial efficacy, biocompatibility, and structural integrity. Our herein-presented nanostructured materials maintained over 95% cell viability and successfully supported osteoblast adhesion and proliferation.

With the herein reported approach, CEF incorporation into a PLA-based matrix via MAPLE ensured sustained and localized antibiotic release, minimizing systemic exposure and subsequent side effects.

The findings of this study highlight the potential of PLA/Fe₃O₄@CEF nanocomposite coatings for infection prevention and osseointegration enhancement in spinal implants. The novel nanostructured coatings tailored specifically for spinal implant surfaces have a two-fold potential in creating an antimicrobial biocompatible surface that supports osteoblast proliferation, aiming to reduce implant-associated complications and improve overall clinical outcomes. This multifaceted approach advances our system as a competitive alternative to existing antibacterial coatings for orthopedic applications.

Even though the developed materials were effective within the study window, supplementary evaluations are needed to assess long-term stability under physiological conditions. In particular, further research is required to optimize coating durability, long-term stability, and in vivo performance. Future work should focus on in vivo evaluations, assessing these coatings’ osseointegration potential, antimicrobial efficacy, and mechanical performance under dynamic physiological conditions. Moreover, aspects related to drug-release kinetics will also be addressed in future studies, as they are critical for understanding the duration and sustainability of antimicrobial activity at the implant site.

Despite these limitations, the proposed coating strategy enables localized drug delivery, potential magnetic responsiveness, and strong coating adhesion, representing a promising step forward in orthopedic implant surface engineering.

5. Conclusions

This study has presented the synthesis, physicochemical characterization, and biological evaluation of innovative antimicrobial nanostructured materials that can be used to improve spinal fixation devices to reduce infection risk and enable long-term functional success. The experimental results demonstrated the successful functionalization of Fe₃O₄@CEF nanoparticles by XRD, SEM, TEM, and SAED, confirming their crystalline structure, morphology, and composition, while FT-IR and TG-DSC analyses verified the functionalization of Fe₃O₄@CEF and estimated the drug loading at ~1.82%. Further, the nanoparticles were incorporated into PLA-based MAPLE-deposited coatings, with 300 mJ/cm² identified as the optimal laser fluence. In terms of biological behavior, both Fe₃O₄@CEF and PLA/Fe₃O₄@CEF coatings demonstrated excellent biocompatibility, with the latter considerably enhancing osteoblast proliferation. The coatings exhibited significant anti-biofilm effects against S. aureus and Ps. aeruginosa, with PLA/Fe₃O₄@CEF showing superior inhibition properties.

Based on the presented findings, it can be concluded that the mixing of Fe₃O₄ nanoparticles, CEF, and PLA offers a promising strategy for antimicrobial coatings on spinal fixation screws. Building on the results of this work, research can be moved further to in vivo studies to evaluate long-term stability, mechanical performance, and antimicrobial efficacy in physiological conditions, eventually bringing improved solutions to clinical settings. Moreover, we believe this strategy offers a modular platform that could be extended to other implant types or combined with additional therapeutic agents in the future.