Hierarchical Peptide Functionalized Titania Nanotubes: Improving Stability, Promoting Osteogenesis, and Reducing Infection Risk

ABSTRACT

In this study, a hierarchical surface functionalization strategy was developed that combines engineering of titania nanotubes (TiNTs) on the titanium surface, followed by dual‐mode peptide functionalization. Fmoc‐FF‐OH dipeptide was first covalently anchored onto the TiNT surface, then a secondary layer of the self‐assembling FF dipeptide was physically deposited. This technique harnesses the strength of covalent bonds and the extensive surface coverage provided by self‐assembling peptides to create a long‐lasting, bioactive, and functional surface that promotes superior biocompatibility. The nanotubular topography and peptide‐mediated biochemical cues recreate an extracellular matrix–like microenvironment, supporting stem cell adhesion, proliferation, and osteogenic differentiation. Additionally, the self‐assembling peptide significantly reduced bacterial adhesion compared to unmodified titanium, demonstrating a marked reduction in bacterial adhesion and early biofilm formation in vitro. Overall, this multifunctional approach combines nanotopographical engineering with peptide‐based bioactivity to enhance implant osseointegration and prevent implant‐associated infections, offering a promising strategy for next‐generation titanium biomedical devices.

The study demonstrates a dual‐level modification of titania nanotube surfaces through the covalent grafting of Fmoc‐FF dipeptides, followed by a secondary self‐assembled layer of FF dipeptide. This hierarchical strategy significantly enhances coating density and structural integrity without compromising the nano‐topographical and biochemical cues essential for cell signaling. The resulting surfaces retain their bioactivity under physiological conditions.

1. Introduction

The long‐term success of orthopedic implants depends critically on their ability to achieve stable osseointegration while preventing bacterial colonization [1, 2, 3]. Titanium and its alloys remain the materials of choice for load‐bearing biomedical devices due to their high mechanical strength, excellent corrosion resistance, and greater biocompatibility, which collectively contribute to their stability in physiological environments [1, 2, 3, 4]. However, the intrinsic bio‐inertness of titanium often limits direct cellular interactions at the bone implant interface, thereby motivating the need for surface engineering approaches to enhance biological integration and reduce the risk of implant‐associated infections [1, 5, 6] Two main strategies have been generally employed to address these limitations: (i) biofunctionalization with biomolecules, and (ii) nanostructuring of the titanium oxide layer on the titanium surface [2, 3, 7, 8, 9].

Biofunctionalization can be achieved through either non‐covalent physical adsorption or covalent coupling of bioactive agents. Physical deposition provides greater surface coverage but is generally weakly bound due to the limited number of van der Waals binding sites. While covalent immobilization offers more durable anchoring, it often suffers from a relatively low density [8, 10, 11]. In parallel, engineering titania nanostructures on the titanium surface, such as nanotubes, nanopillars, and nanopores, provides a topographic effect to tune implant surfaces [11, 12, 13]. Among these, titania nanotubes (TiNTs) are particularly attractive due to their high surface area, tunable nanoscale dimensions, and ordered morphology, which mimic structural aspects of the extracellular matrix (ECM).^3,14 Fabrication of TiNTs on titanium surfaces promotes osteoblast adhesion, proliferation, and differentiation, thereby supporting osseointegration [5, 14]. The bioactivity of TiNT surfaces is due to their nano‐topographies, while remaining chemically inert and lacking biochemical signaling capacity. Therefore, they showed moderate activity at a very early stage. Their intrinsic antibacterial efficacy alone is often insufficient for preventing long‐term infections [3, 8, 14, 15, 16].

Therefore, this study proposes a hierarchical functionalization strategy that integrates covalent coupling with subsequent supramolecular self‐assembly on titania nanotube surfaces to achieve a dense, bioactive, and stable functionalized surface. In this context, multifunctional self‐assembling short peptides emerge as promising molecular building blocks that can impart both dynamic bioactivity and chemical stability to inert inorganic surfaces [17, 18, 19]. The short peptides have been widely explored for various biomedical applications, including drug delivery, tissue engineering, and antimicrobial therapies, due to their unique ability to form ordered nanostructures through non‐covalent interactions such as hydrogen bonding, π–π stacking, hydrophobic interactions, and van der Waals forces [19, 20, 21, 22] This self‐assembly process enables the creation of nanoscale architectures that mimic aspects of the extracellular matrix (ECM). With their tunable chemistry, inherent biocompatibility, and responsiveness to physiological stimuli, self‐assembling peptides are ideal candidates for engineering multifunctional surfaces that integrate structural guidance with biochemical signaling [19, 20].

The diphenylalanine (Phe‐Phe, FF) motif and its N‐fluorenylmethoxycarbonyl‐protected derivative (Fmoc‐FF) are well‐known self‐assembling dipeptides. Driven by π–π stacking and hydrogen bonding, these peptides form ordered nanostructures, fibrils, and hydrogels under physiological conditions [23]. Such assemblies mimic the ECM, providing a supportive microenvironment for cellular adhesion, proliferation, and osteogenic differentiation [23]. In addition, FF‐based nanomaterials exhibit inherent antimicrobial properties by disrupting bacterial membranes and preventing biofilm formation, while maintaining excellent cytocompatibility [23, 24, 25, 26, 27].

This study builds on these considerations by developing nanostructures and a hierarchical dual‐mode surface functionalization strategy that integrates nano‐topographic and biochemical cues on titanium implants. Initially, titanium nanotubes (TiNTs) are fabricated on the titanium surface to impart nanoscale structural guidance. Subsequently, Fmoc‐FF peptides are covalently immobilized onto the nanotubular surface, establishing a stable biochemical interface. Finally, a secondary layer of self‐assembling FF dipeptide is applied, increasing biomolecular density and enhancing bioactivity while maintaining environmental responsiveness under physiological conditions. In our previous work, we established a stable covalent Fmoc‐FF conjugation on TiNTs, thereby reducing bacterial adhesion while preserving nanotopography [26]. The covalently bonded peptide layer increases the number of non‐covalent binding sites on TiNTs, enabling an additional layer and providing greater stability than direct physical deposition. Therefore, this study hypothesizes that this combined covalent and noncovalent peptide immobilization strategy will produce a stable, high‐density biofunctional surface (Scheme 1) that mimics ECM features, promotes osteogenic differentiation of host cells, and exhibits antimicrobial activity, thereby addressing two significant challenges facing titanium‐based implants.

SCHEME 1.

A hierarchical dual‐mode surface‐functionalization strategy for titanium implants is illustrated.

2. Results and Discussions

2.1. Synthesis and Characterization of TiNTs and Fmoc‐FF ‐TiNTs

TiNTs were synthesized using electrochemical anodization followed by annealing, a well‐established laboratory procedure (Figure 1A); see Materials and Methods for a detailed procedure [28, 29]. SEM images revealed the formation of TiNT arrays on the titanium surface. These nanotubes have diameters of 120–160 nm and lengths of 1.4–1.7 µm. The nanotubular surfaces were functionalized with (3‐aminopropyl) triethoxysilane (APTES), introducing amine groups [26, 30, 31]. The aminated surfaces were then treated with Fmoc‐diphenylalanine (Fmoc‐FF) using N‐hydroxysuccinimide (NHS) and 1‐ethyl‐3‐ (3‐dimethylaminopropyl) carbodiimide (EDC) for amide coupling (Figure 1B); see Materials and Methods for a detailed procedure [26, 32] To confirm the synthesis of nanotubes and the subsequent conjugation of Fmoc‐FF onto TiNT, the process was validated by SEM, FT‐IR, XPS survey scans, and EDS analysis.

FIGURE 1.

Fabrication and Analytical Confirmation of Peptide‐Functionalized Titanium Nanotube (TiNT) Surfaces. (A) illustrates the setup for the TiNT fabrication process, which involves anodization followed by annealing. (B) Reaction scheme of covalent conjugation of peptides. (C) SEM image of the synthesized TiNTs surface. (D) SEM image of peptide (Fmoc‐FF) functionalized TiNTs surface. (E) presents EDS color map images of Fmoc‐FF‐TiNT surface (Ti‐Red, O‐Cyan, C‐Green, Si‐Magenta, N‐Yellow), with the electronic image shown in the inset (See Figure S1 for individual EDS color maps). (F) compares the FT‐IR spectra and (G) the XPS spectra of the unmodified (blue) and modified (green) TiNT surfaces. (H–K) High‐resolution XPS spectra and deconvolution fits for C1s, N1s, O1s, and Si2p for Fmoc‐TiNT surfaces.

SEM images confirmed the formation of vertically oriented titania nanotubes on the titanium surface (Figure 1C). FT‑IR revealed the characteristic broad band for titanium oxide_, ranging from 880 to 520 cm⁻¹ with peaks at ∼620 and 606 cm⁻¹ for the stretching and a shoulder at ∼796 cm⁻¹ (Figure 1F, red box). After peptide functionalization, the nature of the band changes to exhibit multiple peaks, indicating the presence of multiple groups, including the Ti─O, Si─O─Ti, and organic linkages [26, 28]. The emergence of amidic N–H (∼3349 cm⁻¹), aromatic C─H (∼3025 cm⁻¹), and carbonyl C═O (∼1662 cm⁻¹) bands confirmed the covalent bonding of Fmoc‑FF [26, 33]. Further supporting these results, the surfaces were analyzed with XPS (Figure 1G), which revealed that all samples exhibited Ti 2p (∼459.9 and 464.8 eV) and O1s (∼530.4 eV) peaks, along with a C 1s peak (∼284.8 eV) (environmental carbon used as a reference) [26, 34]. Peptide‐modified surfaces displayed additional signals for N 1s (∼400.2 eV) and distinct Si 2p (∼102.1 eV) and Si 2s (∼154.1 eV) peaks, which were absent in pristine TiNT [26, 28].

High‐resolution XPS of covalently conjugated Fmoc‐FF Titania surfaces C1s, N1s, O1s, Si2p, and Ti2p peaks (Figure 1H–K and Figure S8), and their deconvolution results further support the covalent conjugation of Fmoc‐FF (Table S2). The observed atomic % for C1s, N1s, O1s, Si2p, and Ti2p are 34, 5, 43, 2, and 16, respectively. The C1s peak deconvolution revealing subpeaks at 284.8 eV for C─C/C─H bonds and 284.2 eV for aromatic carbons indicates conjugation in organic compounds. C1s deconvolution reveals C─N (286.48 eV, 12.4%) and C═O (288.44 eV, 11.4%) peaks characteristic of peptide amide. N 1s peaks at 400.0 eV (amide) and 401.3 eV (protonated amine) further support the presence of peptides. Further, the O1s peptide carbonyl (532.45 eV, 19.0%) emerges alongside Ti‐O (71.8%) [26]. Furthermore, SEM imaging confirmed the preservation of nanotubular morphology after functionalization (Figure 1D), and EDS elemental color mapping demonstrated homogeneous distribution of Ti, O, C, N, and Si (Figure 1E and Figure S1). Collectively, these results confirmed stable peptide conjugation without compromising the structural features of the nanotube arrays.

2.2. Self‐Assembly of FF‐Dipeptide on Fmoc‐FF‐TiNT Surface

The FF dipeptide is well known for its ability to self‐assemble into robust, amyloid‐like fibers and rods in aqueous environments [35]. To investigate the effect of the surfaces on the self‐assembly of FF dipeptide, 10 µL aliquots of a 1 mg/mL FF solution were drop‐cast onto peptide‐modified and unmodified surfaces: planar Ti, TiNTs, and Fmoc‐FF‐TiNTs, and subsequently analyzed using SEM. SEM imaging revealed the formation of long, straight fibers on all three surfaces, consistently indicating that the rapid self‐assembly kinetics of FF in water dominated any surface‐specific effects (Figure 2A, 1st column). However, the surface‐dependent differences were observed when FF was dissolved in ethanol and aqueous ethanol. In the case of an ethanolic solution, FF still produced fibrous assemblies on planar Ti, whereas the TiNT surface did not display any fiber formation (Figure 2A, 3^rd column) [35]. This indicates that the droplets cast over the nanotubular surface rapidly spread out the nanotubular domains, likely dispersing the peptide solution before self‐assembly can occur, resulting in peptide deposition onto the nanotube walls rather than the formation of robust fibers. The plainer Ti lacks this nanoroughness, and therefore FF has enough time to self‐assemble into fibers. Meanwhile, an ethanol‐water mixture (80%) exhibits a balance between self‐assembly rate and spreading across the inter‐nanotubular gaps, resulting in interconnected fiber networks between nanotubes. However, the straight fiber's structure was obtained over the Ti surface (Figure 2A, 2nd column). The FF nanofiber on covalently conjugated TiNT surfaces, Fmoc‐FF‐TiNT, appears to be more closely connected through the nanotube walls than on the plain TiNT surface, indicating the covalent layer of peptide facilitates strong binding of FF fibers through multiple noncovalent interactions such as π‐π stacking, hydrogen bonding, and van der Waals force, which are absent on unmodified TiNT surfaces (Figure 2a,b) [35].

FIGURE 2.

Self‐Assembly and Morphology of FF Peptide on Modified Titanium Surfaces: Panel A: Solvent Effects. SEM images show that the solvent composition (water, 80% ethanol‐water, and ethanol) determines the deposition pattern of dipeptide FF (1 mg/mL). Long fibers form on the Ti surface regardless of solvent, and on all three surfaces in pure water. On the TiNT substrates, ethanol restricts deposition to the nanotube walls, whereas an 80% ethanol‐water mixture facilitates the spreading and interlinking of FF fibers across the gaps between nanotubes. Panel B: Concentration Effects. SEM images of FF at different concentrations (2, 3, and 4 mg/mL) on the Fmoc‐FF‐TiNT surface. The 2 and 3 mg/mL concentrations result in a dense, hydrogel‐like filling of FF across the TINT surface, whereas 4mg/mL shows a multi‐morphology. SEM images highlighting the diverse multi‐morphological structures observed for the 4 mg/mL FF peptide assembly on the Fmoc‐FF‐TiNT surface. (a and b) Zoomed images of FF dipeptide at concentrations of 1 mg/mL and 2 mg/mL deposited on Fmoc‐FF‐TiNT surfaces, revealing a hydrogel‐like network of nanofibers that form interconnected structures spanning the spaces between the nanotubes.

Furthermore, the concentration‐based investigation of the Fmoc‐FF‐TiNT surface revealed that increasing concentrations to 2 and 3 mg/mL led to a denser filling of the self‐assembled peptide, resulting in a hydrogel‐like structure within the inter‐nanotubular space (Figure 2B). A further increase in concentration resulted in multiple morphological outcomes. A 4 mg/mL FF concentration resulted in a wide range of morphologies on the nanotubular surfaces, including overfilling of the nanotubular surface, fiber structure within nanotube gaps, and overgrowth of the nanotubes (Figure 2B). This indicates that at high concentrations, FF peptides rapidly self‐assemble in solution before spreading, thereby limiting ordered interaction with the surface. Critically, the SEM analysis confirmed the sustained presence of the peptide material on the surface throughout the entire 4‐week incubation, thereby demonstrating the functionalization's significantly longer‐term stability.

2.3. Stability of Self‐Assembled FF Functionalization

The binding stability of the self‐assembled FF (diphenylalanine) dipeptide on Ti, TiNT, and Fmoc‐FF‐TiNT surfaces was rigorously evaluated over 28 days under physiological conditions (37°C in PBS). Three methods were employed for stability evaluation: SEM imaging, XPS spectroscopy, and peptide release kinetics measured by absorbance at 255 nm (Figure 3A). A 2 mg/mL FF solution, which yielded a dense, hydrogel‐like structure within the nanotubular surface, was selected for the study (Figure 3A, day 0). Initial assessment by SEM showed that the homogeneous peptide layer remained largely intact on the Fmoc‐FF‐TiNT surface through Day 1. Subsequent incubation, however, led to structural evolution by Days 2 and 7, with the appearance of peptide layers on the surface, suggesting that hydrogel swelling pushes the peptide up. From Day 14 onward, SEM images showed that the peptide film began to form interconnected fibrous structures. This transformation reflects the inherent FF self‐assembly process, resulting in long fiber structures. However, smaller peptide fibers were also observed bridging the nanotubes (Figure 3A, day 28).

FIGURE 3.

Evaluating the Long‐Term Stability and Release Profile of Self‐Assembled FF on Fmoc‐FF‐TiNTs: Panel (A) SEM images track the surface evolution of FF (2mg/mL) solution deposited on Fmoc‐FF‐TiNT surfaces after incubation in PBS at 37°C for up to 28 days. (B) XPS spectra characterize the chemical composition of these surfaces over time. (C) The kinetics of FF release from different substrates (Ti, TiNT, and Fmoc‐FF‐TiNT) are compared. Error bars represent mean ± SD (n = 5, independent biological replicates). (D) Bar graph represents the % FF binding capacity on the various surfaces, which is quantified. Statistical significance (^* p < 0.05) was determined by an ANOVA with Tukey post‐hoc multiple comparison; ^**** p < 0.0001, ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05.

The SEM analysis confirmed the sustained presence of the peptide on the surface throughout the 4‐week incubation, demonstrating the significant stability of the self‐assembled FF dipeptide on functionalized surfaces over 28 days of incubation in PBS at 37 °C. In the case of an unmodified TiNT surface, SEM images showed depositions of FF on day 0 and day 1, but no appreciable peptide deposition was observed after 7 days (Figure S2A). In contrast, for a plain Ti surface, no peptide deposition was observed even after a single day of incubation (Figure S2C); however, the peptide layer detached rapidly during immersion in PBS (as confirmed by manual observation). These samples were also investigated using XPS. The presence of nitrogen and silicon peaks throughout the samples from day 0 to day 28 also supports the presence of a peptide on the Fmoc‐FF‐TiNT surface (Figure 3B). In contrast, for the unmodified TiNT surface, the nitrogen peak appeared on day 7 and subsequently disappeared (Figure S2B). The SEM and XPS analyses collectively indicated prolonged peptide retention for up to 28 days in PBS on Fmoc‐FF‐TiNT surfaces relative to Ti and TiNT surfaces.

Furthermore, the binding/loading capacity and stability of the FF dipeptide on different surfaces were quantitatively assessed using FF release kinetics, leveraging the characteristic absorbance of the peptide solution at 255 nm (Figure 3C,D). The percentage of bonded FF was determined by comparing the amount of FF released upon rinsing to the maximum amount initially drop‐cast on a surface. This analysis revealed that the covalently conjugated Fmoc‐FF‐TiNT surface exhibited a significantly higher maximum binding capacity (57±2%) compared to non‐functionalized TiNTs (21±1%) and plain Ti (13±0.2%) surfaces (Figure 3D and Table S1). This enhanced binding capacity of Fmoc‐FF‐TiNT is perhaps due to the peptide's self‐assembling properties, which provide more non‐covalent binding sites for subsequent peptide layers. Furthermore, stability evaluated over 28 days by the FF release profile showed a stark difference across surfaces: the peptide rapidly detached from the planar Ti surface within 4–6 h, whereas for the TiNT surfaces, it took 8–10 days (Figure 3C,D). In contrast, the release profile from the Fmoc‐FF‐TiNT surface was noticeably slow and incomplete; the peptide was not fully released even after 28 days of incubation. Therefore, the release kinetics indicate the superior retention of FF peptide on the Fmoc‐FF‐TiNT surface. These findings, obtained through XPS analysis, SEM imaging, and UV–V is absorption spectroscopy, collectively support the retention of hierarchical peptide‐functionalized TiNT surfaces after 28 days of incubation in PBS under static conditions and provide a promising platform for durable bioactive coating design. Future studies should examine more complex physiological conditions, such as enzymatic degradation, mechanical loading, and shear resistance, to better approximate implant environments.

2.4. Cell Viability and Cytotoxicity of Modified Surfaces

The biocompatibility of the modified titanium surfaces was systematically evaluated in comparison to the plain Ti control through LDH cytotoxicity and CellTiter Blue cell viability assays, as well as cell adhesion and proliferation using human adipose‐derived stem cells (hADSCs) (Figure 4). The LDH assay, which measures enzyme release from damaged or lysed cells, demonstrated that all tested surfaces, including modified and non‐modified surfaces, exhibited LDH levels comparable to those of the negative control and significantly lower than those of the positive control. This observation confirms that neither the peptide‐modified nor the non‐modified surfaces induced cytotoxic effects (Figure 4B). Complementarily, the CellTiter Blue assay, which assesses metabolic activity as an indicator of viable cells by quantifying resazurin reduction, showed a progressive increase in cellular viability and proliferation from day 1 through day 7 on all surfaces (Figure 4C). This trend highlights the ability of both modified and unmodified surfaces to support sustained cell growth and metabolic function over time.

FIGURE 4.

Adhesion and proliferation of hADSCs on various surfaces. Panel (A) Representative fluorescence microscopy images showing hADSCs after 1, 4, and 7 days of culture; (B) Cytotoxicity assessment based on lactate dehydrogenase (LDH) release assay; and (C) Cell viability measured at days 1, 4, and 7 using the CellTiter Blue metabolic activity assay. (D) Quantification of cells adhered and proliferation by day 1, 4, and day 7, corresponding to panel A. Panel (E) Fluorescence and SEM images of ADSCs morphology, cell‐cell, and cell‐surface interactions on different surfaces (Ti, TiNT, Fmoc‐FF‐TiNT, TiNT+FF, and Fmoc‐FF‐TiNT+FF) after 24 h of culture represent the Initial Cell Response. Error bars represent mean ± SD (n = 9, independent biological replicates), and statistical significance (p‐value) was determined by an ANOVA two‐way analysis and Tukey post hoc using appropriate multiple‐comparison; ^**** p < 0.0001, ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05.

2.5. Cellular Responses of Modified Ti Surfaces

Cell behavior on implant surfaces is crucial because it directly influences the integration, stability, and success of biomedical devices within the body. The initial interaction between cells and the implant surface is influenced by physical and chemical surface properties, including roughness, topography, wettability, and surface energy [36, 37]. Therefore, a surface equipped with nano‐ or micro‐structures, together with biochemical activity, is crucial for the integration of implants with the surrounding tissue. The early cellular responses of surface stem cells are crucial for osseointegration, in which the implant directly bonds to bone tissue, thereby bypassing interference from soft tissue. Implants that encourage favorable cellular behavior, characterized by robust adhesion, efficient proliferation, and maintenance of healthy cell morphology, support enhanced osseointegration and accelerate tissue healing [36].

To evaluate these responses, hADSCs were cultured on modified and unmodified titanium surfaces and analyzed at 1, 4, and 7 days using fluorescence microscopy and SEM. A 2 mg/mL FF solution, which yielded a dense, hydrogel‐like structure within the nanotubular surface, was selected for the study. Fluorescence imaging at day 1 showed that ADSC densities on peptide‐functionalized TiNT surfaces (TiNT+FF, Fmoc‐FF‐TiNT, and Fmoc‐FF‐TiNT+FF) were comparable to those on unmodified Ti, while the bare TiNT samples demonstrated fewer attached cells (Figure 4A). By day 4, cell growth and proliferation were similar across all surfaces; however, the cells on the peptide‐conjugated surface appeared interconnected or communicating with each other. Intercellular communication facilitates the exchange of growth factors, cytokines, and other signaling molecules that encourage sustained proliferation and prevent apoptosis. On day 7, confluency increased, covering more than 80% of the surface area.

Detailed imaging at day 1 also revealed morphology‐dependent interactions, as shown by fluorescence and SEM imaging, which revealed distinct differences in cell‐surface interactions (Figure 4E and Figure S3). ADSCs on plain Ti appeared well‐spread and flattened, a typical adhesion pattern. In contrast, the nanotubular topography of TiNT surfaces induced cytoskeletal rearrangements, leading cells to adopt an elongated morphology consistent with previous reports [14, 16]. The elongated shapes contribute to enhanced bone integration through various mechanisms. Interestingly, on peptide‐conjugated TiNT surfaces, both covalently (Fmoc‐FF‐TiNT) and non‐covalently (TiNT+FF) bound peptides demonstrate even more pronounced effects, showing multiple elongated cells aligned together, suggesting enhanced intercellular communication and alignment [14, 15, 16]. Importantly, the TiNT surface that was first covalently functionalized with Fmoc‐FF and then coated with an additional non‐covalent layer of FF dipeptide (Fmoc‐FF‐TiNT+FF) displayed distinctive cellular self‐assembly behavior. Quantification of the aggregate cell count showed that FF‐deposited surfaces had a greater number of elongated cells aligned together (Figure S11). On this dual‐modified surface, ADSCs organized into cylindrical aggregates, a phenomenon likely resulting from the synergistic effects of the peptide biochemical cues and the underlying nano‐topographic features.

2.6. Evaluation of Osteogenic Differentiation of ADSCs on Functionalized Surfaces

The ability of various modified titanium surfaces to support the osteogenic differentiation of ADSCs was thoroughly investigated. Following an initial 7‐day culture period, the cells were exposed to osteogenic induction media, which consisted of growth medium supplemented with dexamethasone, β‐glycerophosphate, and ascorbic acid, to stimulate their maturation into osteoblasts [38, 39] The progression of ADSCs toward the osteogenic lineage was monitored at weeks 1 and 3 post‐induction by immunofluorescent detection of osteocalcin, a late‐stage non‐collagenous protein highly specific to osteoblasts and typically secreted during the mineralization phase of bone formation. Additionally, functional assays targeting osteoblast activity were performed, including measurements of alkaline phosphatase (ALP) activity and calcium accumulation. ALP plays a vital role by hydrolyzing phosphate‐containing compounds, thereby increasing local phosphate availability and facilitating subsequent mineralization. Calcium deposition represents the final, mature stage of osteogenesis, during which the mineralized bone matrix (predominantly calcium phosphate crystals) is formed [39].

Immunofluorescence microscopy demonstrated that, following one week of osteogenic induction, ADSCs cultured on all tested surfaces began to express osteocalcin, as evidenced by the appearance of green, fluorescent aggregates (Figure 5A). This early secretion of osteocalcin is indicative of the initial commitment of ADSCs toward the osteoblastic lineage. By week 3 of induction, a marked increase in osteocalcin expression was observed across all surfaces, signifying progressive maturation of the cells into osteoblasts (Figure 5B). Quantitative analysis of osteocalcin release per unit area revealed a distinct hierarchy among the tested substrates: the Fmoc‐FF‐TiNT+FF surface exhibited the highest osteocalcin levels, followed by Fmoc‐FF‐TiNT, TiNT+FF, TiNT, and, lastly, the unmodified Ti surface (Figure 5D). These findings underline the critical role of nanotubular topography in supporting osteogenic differentiation, while also highlighting the synergistic effect of FF dipeptide modification in further enhancing osteogenic commitment. The robust and stable self‐assembly of FF dipeptides on the Fmoc‐FF‐TiNT surface, facilitated by the additional binding interface on both the Fmoc‐FF and TiNT, appears to provide sustained biochemical signaling, thereby promoting consistent osteogenic differentiation beyond that observed on TiNT alone.

FIGURE 5.

Adipose‐Derived Stem Cell (ADSC) Behavior and Osteogenesis: Panels (A,B) Representative fluorescence images depicting osteogenic differentiation at 1 and 3 weeks, respectively, highlighting Osteocalcin (green), nuclei (blue), and Actin fibers (red). Panel (C) Representative SEM Images of cells after week 3 of differentiation. The spherulite‐like structures on the surface are marked by red arrows, and the circle may be mineralized calcium salts. (D) The bar graph represents the quantification of Osteocalcin expression, calculated from the green fluorescence intensity in panels B and C with respect to area. (E) The bar graph represents total protein expression. (F) Alkaline Phosphatase (ALP) activity after 1 and 3 weeks. (G) Quantification of calcium (Ca) deposition after 1 and 3 weeks of differentiation. Error bars represent mean ± SD (n = 9, independent biological replicates), and statistical significance (p‐value) was determined by an ANOVA two‐way analysis and Tukey post‐hoc using appropriate multiple‐comparison; ^**** p < 0.0001, ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05.

The total protein assay showed that overall protein expression remained comparable across all tested surfaces, with no significant differences observed initially. Notably, a marked increase in total protein content was detected after week 3 of osteogenic differentiation, specifically on the dual‐functionalized Fmoc‐FF‐TiNT+FF surface (Figure 5E). This enhanced protein expression signifies greater cellular activity, improved cell adhesion, and increased matrix production, all of which are essential for effective osseointegration and bone tissue formation on implant biomaterials [40]. The ALP activity assessment revealed slightly elevated values on peptide‐modified TiNT surfaces after week 1 of induction. By the third week, these differences became more pronounced, with ALP activity increasing progressively in order: Ti < TiNT < TiNT+FF < Fmoc‐FF‐TiNT < Fmoc‐FF‐TiNT+FF (Figure 5F and Figure S10). The ALP activity is a hallmark of osteoblast differentiation that promotes mineralization by hydrolyzing phosphate substrates and thereby promoting bone matrix formation [41]. The increase in ALP activity observed on the Fmoc‐FF‐TiNT+FF surface supports the expression of bone‐specific proteins and their mineralization. Calcium deposition analysis reveals late‐stage matrix mineralization, exhibiting a slight increase over the Fmoc‐FF‐TiNT+FF surface and similar trends across all surfaces at both one and three weeks, with a general increase in calcium content from week one to week three (Figure 5G). This temporal increase is consistent with the expected progression of osteogenic maturation and matrix mineralization during differentiation.

The phosphate produced by ALP activity initiates the mineralization of calcium into hydroxyapatite, which helps form hard bone tissue [42, 43, 44]. SEM imaging after three weeks of osteogenic induction revealed spherulite‐like material deposits and dense cells on all tested surfaces, to varying degrees. These materials suggest that differentiated osteoblasts undergo active extracellular matrix mineralization [43, 44]. Such spherulitic mineralization is a hallmark of mature bone tissue formation, reflecting the successful progression of ADSCs through the osteogenic lineage [42, 43, 44]. These spherical aggregates exhibited a distribution with the highest abundance on the Fmoc‐FF‐TiNT+FF surface, followed by Fmoc‐FF‐TiNT, TiNT+FF, TiNT, and the lowest density on the plain Ti substrate. These biochemical markers, with higher ALP activity and osteocalcin secretion on the Fmoc‐FF‐TiNT+FF surface, suggest a more functionally mature osteoblastic phenotype capable of producing a well‐organized and biologically relevant mineralized matrix. Collectively, these results demonstrate that the combination of nanotubular topography and FF dipeptide functionalization synergistically enhances osteogenic differentiation and extracellular matrix mineralization. These findings have significant implications for the design of advanced biomaterial implants intended to promote efficient stem cell‐driven bone regeneration.

2.7. Antibacterial Evaluation of Functionalized Surfaces

Bacterial infection remains a significant clinical challenge in the application of titanium‐based biomedical implants [45]. The onset of infection typically occurs when bacterial cells initially adhere to the implant surface, subsequently leading to the development of complex polymicrobial biofilms [2, 8]. These biofilms exhibit remarkable resistance to conventional antimicrobial treatments, thereby complicating therapeutic interventions. Their intricate three‐dimensional structure and heterogeneous microbial community contribute to elevated antibiotic tolerance, which promotes persistent inflammation and increases the likelihood of implant malfunction or failure [2, 8, 27]. The antibacterial and antibiofilm activities of the engineered surfaces were rigorously evaluated using both Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), two of the most clinically significant pathogens implicated in implant‐associated infections [45, 46, 47]. S. aureus, a Gram‐positive bacterium, is the predominant cause of orthopedic and medical device‐related infections due to its strong surface adhesion, robust biofilm formation, and ability to evade host immune responses [46]. P. aeruginosa, a Gram‐negative bacterium, is equally important in the context of implant infections, particularly in immunocompromised patients, owing to its high intrinsic antibiotic resistance, persistent biofilm‐forming capacity, and adaptability to diverse environments [47].

A two‐time‐point assay was conducted at 6 and 24 h to assess bacterial adhesion dynamics, with live/dead fluorescence microscopy used for visualization (Figure 6 and Figures S4–S7). SYTO 9 staining identified live bacteria (green), while propidium iodide (PI) marked dead bacteria (red). Fluorescence images of S. aureus revealed that the plain Ti surface exhibited the highest bacterial adherence at both time points, initiating biofilm formation by 24 h (Figure 6A), as evidenced by the development of dense bacterial aggregates and an extracellular matrix (Figure 6B). Notably, TiNT surfaces exhibited reduced bacterial adherence at 6 h (Figure S4), but this increased by 24 h, suggesting a time‐dependent colonization pattern. Both Ti and TiNT surfaces also displayed dead bacteria, likely due to contact‐mediated antibacterial effects on the initial adherent layer; however, the overall bacterial growth remained high, and the contact‐killing effect was insufficient to significantly reduce total bacterial burden (Figure 6A,B and Figure S5) [2, 48]. In contrast, the peptide‐modified TiNT surfaces (TiNT+FF, Fmoc‐FF‐TiNT, and Fmoc‐FF‐TiNT+FF) showed significantly less bacterial adherence and consistently inhibited bacterial growth by 24 h. SEM Images further confirmed the reduction of bacterial adhesion. Quantification of bacterial adhesion was performed by calculating the area covered by bacteria from fluorescence images taken at 6 and 24 h. This further supports the significant inhibition of S. aureus on the peptide‐modified TiNT surfaces, including TiNT+FF, Fmoc‐FF‐TiNT, and Fmoc‐FF‐TiNT+FF (Figure 6H,I).

FIGURE 6.

Evaluation of Bacterial Adhesion and Antibacterial Activity: Panel (A) Live/Dead Fluorescence Microscopy Images of S. aureus after 24 h. Panel (B) Corresponding SEM images of adhered S. aureus. Panel C: Live/Dead Fluorescence Microscopy Images of P. aeruginosa after 24 h. Panel (D) Corresponding SEM images of adhered P. aeruginosa. (E–G). Zoomed SEM images highlighting the disruption of bacterial cells on TiNT surfaces coated with self‐assembling FF dipeptide. (H–K) Quantification of the area covered by the adhesion of bacteria on different surfaces measured from fluorescence images taken at 6 and 24 h. (H) S. aureus at 6 h, (I) S. aureus 24 h, (J) P. aeruginosa at 6 h, and (K) P. aeruginosa at 24 h. Error bars represent mean ± SD (n = 9, independent biological replicates), and statistical significance (p‐value) was determined by an ANOVA one‐way analysis (for each live and dead) and Tukey post‐hoc testing using appropriate multiple‐comparison; ^**** p < 0.0001, ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05.

Similar trends were observed for P. aeruginosa; the highest bacterial growth was observed on the plain Ti surface at both 6 and 24 h, followed by the TiNT surfaces (Figure 6C,D and Figures S6 and S7). However, more dead than live colonies were observed on the TiNT surface, resulting in a significant reduction in the growth of live P. aeruginosa. However, peptide‐modified TiNT surfaces, including TiNT+FF, Fmoc‐FF‐TiNT, and Fmoc‐FF‐TiNT+FF, showed the least bacterial adhesion, with most bacteria dead. SEM imaging provided further insight into the antibacterial mechanisms. On peptide‐modified surfaces, particularly TiNT+FF and Fmoc‐FF‐TiNT+FF, P. aeruginosa cells displayed morphological disruption, with many appearing as empty bacterial shells, suggesting potential structural damage and reduced bacterial viability [24]. These observations confirm that the combination of nanotubular topography and the inherent antibacterial properties of self‐assembling FF dipeptide effectively prevents biofilm formation and shows antibacterial activity [24, 25, 27]. Overall, these observations are consistent with established TiNT contact killing via nanomechanical membrane rupture [11] and FF peptide membrane disruption [24]. Consequently, the synergistic combination of nanotopography and peptide bioactivity reduced bacterial adhesion and prevented biofilm formation on dual‐modified surfaces.

Overall, the hierarchical functionalization strategy introduces distinct quantitative advantages over the unmodified and covalent‐only systems. Regarding peptide loading capacity, unmodified TiNTs exhibited a binding capacity of 21±1%, whereas the covalently anchored Fmoc‐FF‐TiNT surface significantly increased this capacity to 57±2%. Retention duration was correspondingly enhanced; physically deposited peptide on TiNT detached within 8–10 days, while the Fmoc‐FF‐TiNT interface retained the peptide for over 28 days. Biological performance was also progressively enhanced across the systems. Osteocalcin secretion and alkaline phosphatase (ALP) activity increased sequentially from TiNT to Fmoc‐FF‐TiNT and peaked on the dual‐functionalized Fmoc‐FF‐TiNT+FF surface, which also displayed the highest marked increase in total protein content. Similarly, while TiNT surfaces showed an initial reduction in bacterial adherence at 6 h, followed by increased colonization by 24 h, both Fmoc‐FF‐TiNT and Fmoc‐FF‐TiNT+FF maintained significant inhibition of bacterial growth and adhesion by 24 h.

3. Conclusion

In this study, an engineered, robust peptide‐functionalized TiNT surface is developed to address major challenges in implant biomaterials, including long‐term stability, scalability, biocompatibility, Osteogenesis, and bacterial infection prevention. Covalent conjugation of Fmoc‐FF peptides equips TiNTs with multiple binding sites. This enables the strong binding of a physically deposited secondary layer of self‐assembling peptides through noncovalent forces (such as π‐π stacking, H‐bonding, and van der Waals forces), which are absent on unmodified TiNT surfaces. These hierarchical functionalizations demonstrated prolonged peptide retention over 28 days in PBS and enhanced stability relative to Ti and TiNT under the test conditions. Due to their inherent biocompatibility, peptide‐functionalized TiNTs exhibit enhanced biocompatibility, promoting the viability and proliferation of human adipose‐derived stem cells without any cytotoxic effects in vitro. The synergistic effect of the nanotubular topography, combined with the biochemical cues of FF dipeptide fibers and hydrogel‐like networks, significantly enhances stem cell adhesion, proliferation, and osteogenic differentiation by providing a favorable microenvironment for cellular attachment and signaling. Furthermore, the intrinsic antibacterial properties of the FF nanostructures impart potent activity against key implant‐associated pathogens, including gram‐positive Staphylococcus aureus and gram‐negative Pseudomonas aeruginosa, thereby markedly reducing bacterial adhesion and biofilm formation. Taken together, these multifunctional characteristics, which integrate durable surface chemistry, superior biocompatibility, osteoinductive capacity, and antimicrobial efficacy, highlight the promise of hierarchical peptide Functionalization on TiNTs as advanced materials for next‐generation biomedical implants.

Author Contributions

RS conceptualized and designed the study, performed all the necessary experiments, analyzed the data, and wrote the first draft under the supervision of KCP. This draft was reviewed with KCP, who provided feedback and recommendations to enhance the quality and clarity of the written work.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll74888‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.