Synthesis and Characterization of Bioactive Coatings with Bone Regeneration Potential and Anti-Resorptive Effect

Abstract

Bioactive coatings are of great interest for orthopedic applications, as they combine mechanical stability with biological functionality. In this study, stainless steel discs were coated with 45S5 bioactive glass doped with 1.0 wt% samarium by spin coating, followed by surface functionalization with benfotiamine through spraying. This strategy integrates three components: a metallic substrate as a stable and inexpensive support, a bioactive glass layer with well-known osteogenic potential, and a superficial organic layer of benfotiamine, a lipid-soluble analog of vitamin B1 with higher bioavailability. Samarium doping was selected based on previously reported antimicrobial potential against clinically relevant staphylococci, while the rationale for benfotiamine functionalization derives from literature describing vitamin B1 derivatives with anti-resorptive and osteogenic activity. The coatings were characterized by scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR) microscopy. Bioactivity was assessed by immersion in simulated body fluid (SBF), where phosphate bands indicated the formation of calcium phosphate phases (CaPs). Wettability tests showed a reduced contact angle after benfotiamine functionalization. Cytocompatibility was evaluated by LDH and MTT assays with MC3T3-E1 cells, suggesting overall biocompatibility and enhanced cell viability after 7 days for the benfotiamine-functionalized coatings. The present findings support a simple and cost-effective route to multifunctional coatings with potential relevance for future orthopedic applications.

1. Introduction

Bioactive materials, particularly 45S5 bioactive glass, have been extensively studied for their ability to form a direct bond with bone tissue, thereby stimulating the osseointegration process [1,2,3]. Such materials are widely applied in biomedical fields, especially in orthopedic and dental implantology [4,5,6].

In parallel, the addition of benfotiamine, a fat-soluble form of vitamin B1, depicted in Figure 1 has been investigated in the literature as a factor that may influence cellular behavior in the bone environment. Reports indicate that vitamin B1 can reduce oxidative stress and inflammation, promote osteoblast proliferation, and inhibit osteoclast activity, thereby potentially preventing bone resorption [7,8,9,10,11]. Benfotiamine, as a lipid-soluble analog of vitamin B1 with improved stability and bioavailability, has therefore been considered as a promising candidate to enhance the biological performance of bioactive coatings. Pharmacokinetic studies have shown substantially higher bioavailability of thiamine derivatives from benfotiamine compared to thiamine hydrochloride, and enhanced tissue penetration has also been documented [12,13,14].

The deposition of bioactive materials as thin films onto metallic substrates such as stainless steel is significant for biomedical applications. The spin-coating technique allows for the fabrication of uniform and controlled layers and has been successfully used in similar contexts [15,16,17].

In a previous study conducted by our group, 45S5 bioactive glass doped with samarium was reported to exhibit a moderate antimicrobial effect, particularly against Staphylococcus aureus and Staphylococcus epidermidis, pathogens frequently involved in implant-associated infections [18]. These findings, obtained in earlier work, support the potential relevance of Sm-doped systems for orthopedic applications, although the biological response may vary depending on the microbial strain.

This work aims to obtain and characterize bioactive coatings based on 45S5 glass doped with 1.0 wt% samarium and functionalized with benfotiamine, and spin-coated onto stainless steel supports. Considering the previously demonstrated antimicrobial effect of Sm-doped bioactive glass [18] and the anti-resorptive effect of vitamin B1 derivatives [10], benfotiamine was added to enhance the biological activity of the coatings. This study investigates the morphology, chemical composition, and behavior in simulated body fluid (SBF), as well as the cytocompatibility of the coatings, evaluated by MTT and LDH assays.

2. Materials and Methods

2.1. Materials

The experiments were conducted using a range of analytical-grade reagents. Tetraethyl orthosilicate (TEOS, 99.9%) was purchased from VWR (Radnor, PA, USA). Merck (Darmstadt, Germany) supplied triethyl phosphate (TEP, 99%), nitric acid (65%), tris(hydroxymethyl)aminomethane (TRIS, 99%), acetone (99.8%), methanol (99.9%), and hydrochloric acid (37%). Sigma-Aldrich (St. Louis, MO, USA) provided calcium nitrate tetrahydrate (99%), samarium nitrate hexahydrate (99.9%), sodium chloride (99%), and calcium chloride dihydrate (99%). From SILAL (Bucharest, Romania), the following compounds were sourced: sodium nitrate (99.5%), potassium chloride (99%), sodium bicarbonate (99%), potassium phosphate dibasic (99%), and sodium sulfate (99%). Magnesium chloride hexahydrate (99%) was obtained from Fluka (Charlotte, NC, USA). Benfotiamine was synthesized according to the procedure described in our previously published work [19].

2.2. Bioactive Coatings Preparation—45S5 + 1.0 wt% Sm + Benfotiamine

The bioactive coatings were obtained in several successive steps: surface preparation of the stainless steel supports, synthesis of the sol–gel solution of 45S5 bioactive glass doped with 1.0 wt% samarium, deposition by the spin-coating technique, thermal treatment of the deposited layer, and finally, benfotiamine deposition.

The supports consisted of circular discs made of AISI 304L stainless steel, with a diameter of 25 mm. The surfaces were sanded with 400-grit abrasive paper, degreased by ultrasonic cleaning in acetone, and then immersed for 3 min in 0.1 M HCl solution to remove surface oxides [17]. The samples were subsequently rinsed with demineralized water and isopropyl alcohol and dried at room temperature.

The 45S5 bioactive glass doped with 1.0 wt% samarium was synthesized in sol form, following a previously described procedure [18]. First, 37.5 g of demineralized water and 1 mL of 2 N nitric acid were placed in a laboratory beaker and heated to 40 °C. Subsequently, 19.5 g of tetraethoxysilane (TEOS) was gradually added over 2.5 h under continuous stirring. After this step, the solution was stirred for an additional 30 min, after which 1.925 g of triethyl phosphate (TEP) was added over 10 min, followed by another 30 min of stirring. Then, 12.9 g of calcium nitrate tetrahydrate was slowly added, followed by 8.4 g of sodium nitrate, and finally 0.375 g of samarium nitrate hexahydrate. The resulting sol was continuously stirred for 3 days at room temperature (22–24 °C).

The deposition of the bioactive glass layer was performed using the spin-coating technique by applying 1 mL of sol onto each disc, followed by spinning for 45 s at 2500 rpm. Each sample received two successive layers. The coated samples were then dried at 130 °C for 20 h and subsequently heat-treated at 800 °C for 3 h. The total mass of bioactive glass deposited per sample was approximately 10 mg.

Benfotiamine deposition was performed by spraying a concentrated solution directly onto the surface of the bioactive glass layer. The solution was prepared by dissolving 0.5 g of benfotiamine (anhydrous form) in a mixture of 15 mL methanol and 10 mL demineralized water, heated to 60–70 °C, corresponding to a final concentration of ~20 mg/mL. After complete dissolution, the solution was cooled to 40 °C and used immediately. Spraying was carried out in several successive thin layers using a fine manual sprayer (non-professional device, spray distance ~20 cm), with drying at 40 °C after each layer and gravimetric monitoring of the sample mass. The spraying was repeated until ~1 mg of benfotiamine was deposited per disc, typically requiring 3–4 passes. After deposition, the samples were dried at room temperature until a constant mass was achieved. Each disc therefore contained approximately 10 mg of Sm-doped bioactive glass and ~1 mg of benfotiamine. Uniformity of the deposited benfotiamine layer was indirectly confirmed by SEM and FTIR microscopy. Although coating thickness and surface roughness were not directly measured (e.g., by cross-sectional SEM or profilometry), thickness could be indirectly approximated considering the deposited mass, the surface area of the disc, and density values of bioactive glass coatings reported in the literature [18,20,21].

2.3. Bioactivity Evaluation and Surface Characterization

The bioactivity of the prepared samples was evaluated through in vitro immersion tests in simulated body fluid (SBF) over periods ranging from 1 to 7 days at 36 ± 1 °C. Tests were performed on both Sm-doped 45S5 coatings and their benfotiamine-functionalized counterparts. Each 25 mm stainless steel disc was immersed in a sealed polypropylene container containing 30 mL of SBF. The samples were weighed before and after immersion to determine mass changes. Following the immersion period, the samples were rinsed sequentially with demineralized water and acetone, then dried at 36 °C for 24 h. The pH of the SBF solution was periodically monitored throughout the immersion. The composition of the SBF solution is shown in

Table 1. Ionic concentrations of simulated body fluid (SBF) and human blood plasma.

Ions	Na+	K+	Ca2+	Mg2+	HCO3−	Cl−	HPO42−	SO42−
SBF	142	5.0	2.5	1.5	4.2	148	1.0	0.5
Human blood plasma	142	5.0	2.5	1.5	27.0	103	1.0	0.5

, matching the ionic concentrations of human blood plasma as described by Kokubo and Takadama [22].

Surface characterization was performed using Fourier-transform infrared (FT-IR) microscopy with a Nicolet iN10MX spectrometer (Thermo Scientific, Madison, WI, USA). The detector was cooled with liquid nitrogen, and measurements were conducted in reflectance mode. Spectra were collected in the 4000–600 cm⁻¹ range with a resolution of 4 cm⁻¹, from randomly selected areas on each sample surface. Data were processed in absorbance mode, with baseline correction applied in the OMNIC Picta software (version 9.1.0.24 Thermo Scientific), to reduce noise and improve spectral interpretation. Spectra were analyzed qualitatively.

Surface morphology was examined using scanning electron microscopy (SEM) with a QUANTA INSPECT F50 instrument (Thermo Fisher Scientific, Eindhoven, The Netherlands), equipped with a field-emission gun, which provides a resolution of 1.2 nm. Images were acquired at an accelerating voltage of 30 kV, with a spot size of 3.5, a working distance of ~23 mm, and magnifications of 1000×, 5000×, and 20,000×. Elemental composition was assessed using the attached energy-dispersive X-ray spectrometer (EDS, MnK resolution 133 eV; Thermo Fisher Scientific, Eindhoven, The Netherlands). Spectra were collected in area mode from representative regions of each sample.

Contact angle measurements were performed using the static sessile drop method with a Krüss Mobile Surface Analyzer (Krüss, Hamburg, Germany) at 20 °C, employing demineralized water as the test liquid. The droplet volume was approximately 4 µL, applied by automated dosing, and the contact angle was calculated using the ellipse (Tangent–1) fitting method provided by the Krüss ADVANCE software (version 1.19.2.26301, Krüss, Hamburg, Germany). For each sample, three measurements were performed at different surface locations, and the average contact angle was reported.

2.4. Cytocompatibility Assessment of Metal Composites

Quantitative and qualitative biocompatibility assays were performed to evaluate the biological response of metal composites coated with 45S5 1.0 wt% enriched Sm bioactive glasses, supplemented or not with benfotiamine. Before experiments, the tested materials which covered the entire well bottom were sterilized by exposure to UV for 6 h on each side. Murine pre-osteoblast cells, MC3T3-E1 (code CRL-2593, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% glutaMAX, 1% sodium pyruvate, and 1% antibiotic antimycotic solution and maintained under standard culture conditions (37 °C, 5% CO₂, humidity). Pre-osteoblasts were seeded at a density of 2 × 10⁴ cells/cm² directly onto coated stainless steel discs to ensure uniform contact with the surface, and incubated for 7 days. Biocompatibility assays, including quantitative (MTT, LDH) and qualitative (Live/Dead fluorescence analysis) testing, were performed 2 and 7 days post-seeding. During quantitative biocompatibility studies, the tested composites were compared to a background control (BC), namely metal composites coated with 45S5 1.0 wt% enriched Sm bioactive glasses, supplemented or not with benfotiamine exposed directly to cell culture media (in the absence of the cellular component). In addition, a tissue culture plastic (TCP) control was considered in this study. The final results were carried out in percentages based on their absorbance.

2.5. MTT Assay

Cell viability of pre-osteoblasts was evaluated using the spectrophotometric 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay (MTT, Sigma-Aldrich, Steinheim, Germany). Cells were incubated with MTT solution for 4 h at 37 °C. The solution was prepared according to the manufacturer’s instructions in PBS at a concentration of 1 mg/mL. Following formazan crystallization, isopropanol was used to dissolve the resulting crystals to a final violet solution. The solution’s optical density was measured at a wavelength of 550 nm using a FlexStation 3 Spectrophotometer (Molecular Devices, San Jose, CA, USA) to determine the number of metabolically active cells.

2.6. LDH Assay

The cytotoxic effect of both composites was evaluated using the “In Vitro Toxicology Assay kit, Lactic Dehydrogenase Based” TOX7 kit (Sigma/Merck, Steinheim, Germany). Following the manufacturer’s instructions, culture medium was collected and mixed with the previously prepared LDH reaction mix and transferred to a 96-well plate. The samples were incubated in the dark at room temperature for 15–20 min. The levels of lactate dehydrogenase enzyme released in culture medium by apoptotic cells were measured spectrophotometrically at a wavelength of 490 nm using a FlexStation 3 Spectrophotometer (Molecular Devices, San Jose, CA, USA). Apart from the BC and TCP controls we also considered a positive control represented by a pre-osteoblast MC3T3-E1 biosystem, which was treated with 2% Triton-X100 (Sigma/Merck, Steinheim, Germany) to ensure 100% of cell cytotoxicity.

2.7. Live/Dead Assay

The proliferation and viability of pre-osteoblasts in contact with composites were qualitatively measured using the Live/Dead Kit (ThermoFisher Scientific, Waltham, MA, USA). Live cells (green) were labelled with calcein acetoxymethyl (AM), whereas nuclei of dead cells (red) were labelled with ethidium homodimer-1 (EthD-1). The staining solution was prepared following the manufacturer’s instructions, and a volume of 500 µL was added to each sample and incubated for 1 h in the dark at room temperature. Visualization of live and dead cells was performed using an Olympus IX73 (Olympus, Tokyo, Japan) microscope equipped with a HamamatsuORCA-03G camera (A3472-06, Hamamatsu, Japan). The obtained images were acquired using CellSens Dimension software (v1.11, Olympus).

2.8. Statistical Analysis

All cytocompatibility tests were performed in triplicate (n = 3). Quantification of fluorescence intensity was performed using ImageJ software (version 1.54p, National Institutes of Health, Bethesda, MD, USA), analysis was performed using a minimum of 10 frame images per condition. Data were analyzed using GraphPad Prism Software 3.0 (GraphPad Software Inc., San Diego, CA, USA) and further assessed by applying the one-way ANOVA method and the Bonferroni post-test, taking a p-value < 0.05 as statistically significant.

3. Results and Discussion

3.1. Surface Characterization

3.1.1. SEM

The surfaces obtained after deposition of the bioactive glass, and again after deposition of benfotiamine, were analyzed by scanning electron microscopy (SEM). The corresponding images, obtained at different magnifications, are presented in Figure 2.

After the deposition of Sm-doped 45S5 bioactive glass, a relatively homogeneous and continuous coating was observed, without macroscopic cracks or evident microcracks, indicating good adhesion to the stainless steel substrate and a uniform distribution of the material achieved by the spin coating technique. At 1000× magnification (Figure 2a), the surface displayed a smooth texture, with uniformly distributed particles and without major agglomerations. Fine lines resulting from the mechanical preparation of the stainless steel substrate with abrasive paper were also visible. At 5000× magnification (Figure 2b), a granular morphology with a dense network of fused particles was observed, typical of partially crystallized glass. Numerous cubic crystals with well-defined edges were visible across the surface, embedded within a compact matrix. At higher magnification of 20,000× (Figure 2c), the cubic crystals were clearly identified, with sizes ranging from a few hundred nanometers and surrounded by amorphous regions, suggesting the coexistence of vitreous and crystalline phases. The compact appearance and absence of major discontinuities indicate the formation of a stable layer, suitable for biomedical applications.

The images obtained after benfotiamine deposition (Figure 2d–f) revealed a modified morphology, influenced by the presence of organic crystals. At 1000× magnification (Figure 2d), the surface appeared rougher compared to the non-functionalized coating, with crystalline structures distributed across the entire surface. Areas with higher local density alternated with more dispersed regions, while no cracks or defects were observed. At 5000× magnification (Figure 2e), well-defined needle-like crystals, characteristic of benfotiamine, were observed on the surface. These structures were present both individually and grouped in small aggregates, while the underlying glass matrix maintained its compact aspect. At 20,000× magnification (Figure 2f), the acicular crystals were confirmed to be located on the superficial layer, above the inorganic matrix. Their random orientation and variable sizes created a complex three-dimensional surface morphology, without altering the integrity of the inorganic coating.

The thickness of the coating was evaluated based on gravimetric data, by correlating the deposited mass (~10 mg per 25 mm disc, corresponding to ~2.0 mg/cm²) with density values reported for bioactive glass coatings (2.0–2.7 g/cm³). Considering the compact, pore-free morphology revealed by SEM, the average thickness of the coating was estimated to be in the range of 7.5–10 μm. The additional benfotiamine layer (~1 mg per disc) represents a very thin coverage, which modifies only the superficial morphology without significantly altering the overall thickness of the coating.

3.1.2. Energy-Dispersive X-Ray Spectroscopy (EDS) of the Coatings

In order to confirm the presence of the characteristic elements of the bioactive glass, to demonstrate the incorporation of samarium into the coating composition, and to monitor the chemical changes associated with CaPs formation after immersion in SBF, energy-dispersive X-ray spectroscopy (EDS) analysis was performed. Representative spectra of the coatings, recorded both before immersion and after 7 days in SBF, are shown in Figure 3 while the semi-quantitative data are listed in

Table 2. Semi-quantitative EDS results (normalized atomic %) of Sm-doped 45S5 coatings, with and without benfotiamine, before and after SBF immersion.

Sample	Na %at	Ca %at	Si %at	P %at	Sm %at	Ca/P Ratio
45S5 + 1.0% Sm (initial)	25.7	12.1	56.3	5.1	0.8	2.35
45S5 + 1.0% Sm (1 day in SBF)	3.6	16.4	70.2	8.8	1.0	1.87
45S5 + 1.0% Sm (3 days in SBF)	nd	14.1	64.7	19.9	1.3	0.71
45S5 + 1.0% Sm (7 days in SBF)	7.2	12.9	58.5	19.7	1.6	0.66
45S5 + 1.0% Sm + Benfotiamine (initial)	19.3	17.2	56.8	6.1	0.6	2.83
45S5 + 1.0% Sm + Benfotiamine (1 day in SBF)	nd	17.6	70.8	10.7	0.9	1.65
45S5 + 1.0% Sm + Benfotiamine (3 days in SBF)	12.6	9.8	60.4	13.8	3.3	0.71
45S5 + 1.0% Sm + Benfotiamine (7 days in SBF)	8.1	10.1	67.4	13.2	1.2	0.77

nd = not detected (below detection limit of the EDS technique). Values were normalized by excluding substrate contributions (Fe, Cr, Ni). Results represent single-point measurements and are therefore indicative and semi-quantitative.

.

The EDS spectra confirmed the presence of the main elements characteristic of 45S5 bioactive glass—O, Na, Si, P, and Ca—together with samarium as a dopant. The Sm signal was observed in the region of 5.6–7.3 keV, where it partially overlaps with Fe, Cr, and Ni peaks originating from the stainless-steel substrate; nevertheless, the detection of Sm supports its incorporation into the glass matrix. Although benfotiamine contains C, N, and S in its molecular structure, these elements were not detected in the present EDS measurements. This can be explained by the inherent limitations of EDS in detecting low-Z elements but also because of the low content of benfotiamine. The presence of benfotiamine was instead confirmed by complementary techniques: FTIR spectroscopy, which revealed its characteristic vibrational bands, and SEM analysis, which showed needle-like crystalline structures absent in the initial bioactive glass coatings.

After immersion in SBF for 7 days, the spectra showed increased relative intensities of Ca and P, consistent with the precipitation of CaPs on the coating surface. This trend aligns with the known bioactive behavior of 45S5-based materials and further supports the FTIR evidence of CaPs formation. The semi-quantitative results obtained by EDS are summarized in Table 2, providing a clearer picture of the compositional changes occurring during immersion.

The data presented in Table 2 include only the elements relevant to the bioactive glass composition (Na, Si, P, Ca, and Sm). Signals of Fe, Cr, and Ni, originating from the stainless-steel substrate, were excluded from the normalization and are not reported in Table 1. Oxygen was not considered, since its quantification by EDS is notoriously less reliable due to absorption effects and detector limitations for low-energy X-ray lines. By normalizing the atomic percentages of the selected cations to their sum, the trends of Na depletion and Ca–P enrichment during SBF immersion could be more clearly assessed.

According to the data in Table 2, sodium decreased sharply during the initial days of immersion for both types of coatings. In the non-functionalized sample, Na was almost completely depleted by day 3, with a slight reappearance at day 7, while in the benfotiamine-functionalized sample it disappeared already after 1 day and partially reappeared thereafter. This behavior reflects the rapid Na⁺ ↔ H₃O⁺ ionic exchange from the surface but, after a while, the Na⁺ is slightly released from the deeper structure as well as redeposition from the solution.

Calcium concentrations showed only minor variations, such as less soluble ion in SBF media. In the non-functionalized sample, a slight increase was observed at day 1, followed by a return to values close to the initial level, whereas in the benfotiamine-functionalized sample the Ca content remained similar to the initial value after 1 day and decreased thereafter.

Silicon increased in the initial stage (day 1), as the more mobile ions (such as Na and partially Ca) were rapidly released, leading to a relative enrichment of the layer in Si and Sm. Subsequently, the Si content gradually decreased, a phenomenon that can be attributed to the partial dissolution of the silicate network and to the precipitation of CaPs, which altered the relative proportions of the detected elements.

Phosphorus increased continuously during the first three days and then stabilized for both types of coatings, consistent with the progressive precipitation of CaPs at the surface, an indicator of their bioactive behavior. The benfotiamine-functionalized sample showed a slightly higher phosphorus content initially (≈20% relative increase compared to the non-functionalized coating), likely related to the intrinsic phosphate group of benfotiamine. However, at later immersion times the phosphorus level was higher in the non-functionalized sample, suggesting differences in the kinetics of CaPs deposition. This observation should be interpreted with caution, since EDS is only semi-quantitative, especially for thin films on metallic substrates.

Samarium was detected both in the initial samples and after SBF immersion. In both cases, its relative concentration appeared to increase slightly with immersion time, which can be explained by its lower mobility compared with Na and Ca. As Na and Ca are released, Sm remains in the glass matrix and may appear enriched at the surface. This result suggests that Sm is less prone to rapid release under the tested conditions, although this interpretation should be treated with caution, since EDS provides only semi-quantitative information. Further studies (e.g., solution analysis by ICP-MS) would be required to confirm the actual release behavior of Sm.

Ca/P ratios for both coatings remained below 1.67 throughout immersion because some phosphate is also adsorbed from the SBF (the samples were washed gently with water after immersion to not further alter the surface characteristics). The benfotiamine-functionalized coating showed a lower Ca/P at early times—consistent with its higher P—whereas at later times the non-functionalized sample exhibited higher P and correspondingly low Ca/P. These values likely reflect P-rich CaPs and thin-film EDS constraints (even after 3 days), rather than fully matured apatite stoichiometry. Future work will aim to clarify this mechanism by monitoring the evolution of Ca²⁺ and phosphate concentrations in SBF, since in the present case the bioglass surface appears to release phosphate more rapidly than it deposits Ca²⁺, which could explain the low Ca/P ratio.

Overall, the EDS results confirm the characteristic behavior of 45S5 glass, with rapid sodium release, partial dissolution of the silicate network, and progressive formation of CaPs. Samarium was consistently detected throughout the immersion period, suggesting that it remains relatively stable within the coating under the tested conditions. Comparison between the non-functionalized and benfotiamine-functionalized samples revealed similar general trends, but with subtle differences in Si, Ca, and P, which may be related to the influence of functionalization on ion release and CaPs deposition. These results suggest that functionalization with benfotiamine does not negatively affect the bioactive behavior of the 45S5 glass, while inducing only minor compositional differences during immersion. Nonetheless, the EDS data do not provide direct confirmation of hydroxyapatite, but only indicate early CaPs precipitation on the surface.

3.1.3. FTIR Microscopy

To determine the surface composition, FTIR microscopy analysis was conducted. Figure 4 shows the images obtained after performing the coating with bioactive glass. From the optical image (Figure 4a), it can be observed that a uniform surface, with a finely granulated texture, that is characteristic of bioactive glass coatings, was obtained. In addition, the coating is free of obvious cracks or discontinuities, which indicates good adhesion and homogeneous surface coverage.

The image from Figure 4b represents the FTIR transmittance map recorded at ~1010 cm⁻¹, corresponding to the Si–O–Si asymmetric stretching band. It indicates slight variations in surface absorption, reflecting possible minor fluctuations in the thickness of the glass layer. The dominant colors (green, yellow) suggest a relatively uniform distribution of the chemical composition, with some areas with lower absorption (blue) or higher absorption (red), depending on the color scale adjacent to the image.

The spectrum in Figure 4c presents three partially overlapping but distinguishable bands, characteristic of the silicate lattice in SiO₂–based bioactive glass. A broad band in the region of ~1010 cm⁻¹ is attributed to the asymmetric stretching vibrations of Si–O–Si bridging oxygens, while the band at ~950 cm⁻¹ corresponds to Si–O stretching vibrations of non-bridging oxygens (NBO). The signal near ~880 cm⁻¹ is assigned to the symmetric stretching of Si–O–Si groups [23,24,25,26]. Thus, Figure 4 represents the characterization of the surface coated with 1.0 wt% Sm-doped 45S5 bioactive glass. The optical image shows a continuous and homogeneous coating. The FTIR transmittance map indicates a uniform distribution of the silicate network-specific signal, and the point spectrum confirms the presence of Si–O–Si bonds and the absence of organic components, consistent with the inorganic composition of the layer.

The surface obtained after benfotiamine deposition was analyzed using FTIR microscopy, and the results are presented in Figure 5a. A uniformly coated surface with a slightly granulated texture, free of visible defects, can be observed. The FTIR absorbance maps from Figure 5b and Figure 5c corresponds to the characteristic peaks at 1010 cm⁻¹ and 1665 cm⁻¹, respectively.

The band at 1010 cm⁻¹ is associated with the vibrations of the Si–O–Si bonds in the bioactive glass lattice [27], while the band at 1665 cm⁻¹ is attributed to the C=N and/or C=O groups present in the benfotiamine structure [28]. This selection allows a visual comparison between the distribution of inorganic (bioactive glass) and organic (benfotiamine) components on the coated surface.

The colors in the FTIR maps represent absorbance levels: red areas indicate higher absorbance, corresponding to regions with a locally thicker layer, while blue areas indicate lower absorbance, corresponding to a thinner layer. These chromatic variations reflect slight non-uniformities in layer thickness, resulting from the spray functionalization method.

The FTIR spectrum recorded on the benfotiamine-functionalized sample is shown in Figure 5d. As already discussed for the Sm-doped glass coating, the intense band around ~1010 cm⁻¹ and the feature near ~950 cm⁻¹ are characteristic of the silicate network, corresponding to Si–O–Si asymmetric stretching vibrations (bridging oxygens) and Si–O stretching of non-bridging oxygens (NBO), respectively. In addition, the signal near ~894 cm⁻¹ can be attributed to the symmetric stretching of Si–O–Si groups. These bands are typical for 45S5 bioactive glass, whose composition is predominantly silicon-based, with phosphate present in smaller amounts.

In the 1650–1670 cm⁻¹ region, several additional peaks are observed, attributed to C=O and/or C=N stretching vibrations within the benfotiamine structure. Other characteristic peaks associated with benfotiamine are also present in the 1400–1300 cm⁻¹ region (C–H deformations and C–N bonds), at 1260 and 1200 cm⁻¹ (C–O, S–C and C–N stretching), and in the 3000–3500 cm⁻¹ range (O–H vibrations, likely from surface moisture or residual hydroxyl groups). These organic-specific signals confirm the presence of the benfotiamine layer, and their detailed assignments are summarized in

Table 3. Characteristic FTIR bands of Sm-doped 45S5 bioactive glass coatings functionalized with benfotiamine.

Wavenumber (cm−1)	Assignment (concise)
3000–3500 (broad)	ν(O–H)
1665	C=O/C=N (benfotiamine)
1620	C=N/C=C (heterocyclic/aromatic)
1530–1515	C=C aromatic/C=N
1442–1407	δ(CH2/CH3) ± C=C (aromatic)
1338	ν(C–N) ± δ(CH3)
1260	ν(C–O)/ν(S–C)/ν(C–N)
1200	ν(C–O)/ν(C–N)
1010	ν_as(Si–O–Si)
950	ν(Si–O−) (non-bridging oxygen, NBO)
896	ν_sym(Si–O–Si)
685	γ(C–H) (aromatic) ± ν(C–S)

[28].

The spectrum supports the presence of a bilayered coating, consisting of a continuous inorganic matrix (Sm-doped glass) and a superficial organic layer (benfotiamine), both of which are detectable in the IR spectrum and consistent with morphological observations.

It should be noted that the adhesion strength of the coatings was not assessed in the present study. As adhesion represents a critical parameter for biomedical coatings, this aspect will be investigated in future work.

3.1.4. Bioactivity Assessment

To evaluate the bioactivity of the obtained coatings, the samples were immersed in simulated body fluid (SBF) for 1, 3, and 7 days at 36 ± 1 °C. The variation in pH and the weight loss during immersion are shown in Figure 6 and Figure 7, comparing Sm-doped coatings with and without benfotiamine functionalization.

As shown in Figure 6, the pH of the SBF solution increases slightly during the first 3 days, after which it stabilized. This trend was similar for both functionalized and non-functionalized coatings, with only minor differences. The initial increase is consistent with the well-known ion exchange between the glass surface and the SBF solution, involving the release of Na⁺ and Ca²⁺ ions and the concomitant consumption of H⁺ ions, leading to a moderate increase in alkalinity. Subsequently, Ca²⁺ and PO₄³⁻ ions precipitate on the coating surface, stabilizing the pH as dissolution and reprecipitation processes approach dynamic equilibrium.

As shown in Figure 7, the coatings exhibited an initial mass loss during the first day, followed by a slower evolution between 3 and 7 days. Both functionalized and non-functionalized coatings displayed comparable dissolution kinetics, characterized by an initial rapid ion release stage, followed by stabilization. This behavior suggests that the dissolution process tends to slow down after the early stages of immersion. The slightly higher mass loss observed for the functionalized coatings during the first day can most likely be attributed to the concurrent release of benfotiamine, in addition to the dissolution of the glass matrix.

These measurements were interpreted as indicative dissolution trends rather than quantitative proof of bioactivity, given the limited number of replicates. Nevertheless, the observed behavior is consistent with previous reports on 45S5-based coatings [29,30]. Together with the phosphate bands identified in FTIR spectra, these findings support the ability of the coatings to interact dynamically with the simulated physiological environment.

The surfaces of the samples after immersion in SBF were analyzed by FTIR microscopy, focusing on the band at 1060 cm⁻¹, characteristic of phosphate groups (PO₄³⁻), indicative of the formation of CaPs. The evolution of the surface was characterized after 1, 3 and 7 days, and the results are presented in Figure 8.

The optical images (Figure 8a,d,g) indicate a progressive surface modification upon contact with SBF. After one day (a), the surface retains a granular appearance, but with larger granules. After 3 days (d), a decrease in the size of the granules is observed, while at 7 days (g) the surface acquires a more uniform appearance, as a result of the formation of a mineral layer.

The FTIR absorbance maps at 1060 cm⁻¹ (Figure 8b,e,h) reflect the distribution of phosphate groups on the surface, providing an indirect picture of the formation of CaPs. The colors on the map indicate the level of absorbance, where red corresponds to a higher absorption (thicker layer of phosphate compound), and blue to a lower absorption. In all three cases, the local variation in absorbance values is relatively small, which suggests a uniform deposition of the reaction product over the entire analyzed area. The presence of phosphate groups is noted from the first day, and the signal intensity remains well-defined even after 3 and 7 days, which confirms the progressive formation of a phosphate-rich layer.

The FTIR spectra recorded at the points marked in red on the analyzed surfaces confirm these observations (Figure 8c,f,i).

After 1 day (c), a decrease in the intensities of the characteristic peaks of bioactive glass at 1010, 950 and 896 cm⁻¹ and an intensification of the 1060 cm⁻¹ peak due to phosphate groups is observed [31,32]. A new band appears at 794 cm⁻¹, which was not present in the initial spectrum. This band is attributed to Si–O–Si bending vibrations within the silicate network, reflecting structural rearrangements of the glass matrix during immersion in SBF [33]. The intensity of the 1650–1670 cm⁻¹ and 1400–1500 cm⁻¹ bands characteristic of benfotiamine decreases, which suggests the release of benfotiamine.

After 3 days (f), the band at 1060 cm⁻¹ becomes pronounced, and those at 1650–1670 cm⁻¹ and 1400–1500 cm⁻¹ are attenuated, suggesting almost complete release of benfotiamine and formation of the phosphate layer. After 7 days (i), the spectrum is dominated by an intense and clearly defined band at 1060 cm⁻¹, specific for PO₄³⁻ groups, consistent with the formation of CaPs, while the signals associated with benfotiamine are almost absent, indicating its complete or almost complete release.

These results suggest a bioactive behavior of the coatings, characterized by the gradual formation of CaPs on the surface in contact with SBF. In parallel, benfotiamine appears to be released during immersion, most likely from superficial crystalline deposits, a phenomenon that could contribute to a local therapeutic effect. Overall, the observed behavior supports the potential of these composite coatings for applications in bone regeneration, where both the stimulation of osteointegration and the reduction in bone resorption are desired.

3.1.5. Hydrophilicity Assessment via Contact Angle Measurements

The hydrophilicity of the samples was evaluated by measuring the static contact angle of water droplets on the surface, using the sessile drop method at 20 °C. The results, presented in

Table 4. Contact angle measurements for bioactive glass coatings and bioactive glass coated with benfotiamine.

Sample	Value 1 (°)	Value 2 (°)	Value 3 (°)	Mean (°)	Standard Deviation (°)
45S5 + 1.0 wt% Sm	73.00	66.52	63.86	67.79	4.70
45S5 + 1.0 wt% Sm + Benfotiamine	30.71	24.41	36.27	30.46	5.94

, show a clear difference between the samples. The 45S5 bioactive glass coating doped with 1.0 wt% Sm exhibited a contact angle of 67.79° ± 4.70°, indicating a moderately hydrophilic surface. After benfotiamine functionalization, the average contact angle decreased significantly to 30.46° ± 5.94°, revealing a substantial increase in surface hydrophilicity.

This enhanced hydrophilic behavior may improve the initial protein adsorption and cell adhesion on the implant surface, facilitating faster integration with bone tissue. The reduction in contact angle suggests that the benfotiamine coating favourably modifies the surface energy, potentially contributing to improved biological performance of the composite material.

3.2. In Vitro Cytocompatibility Assessment of Stainless Steel Discs Coated with 45S5 Sm-Doped Bioactive Glass, Functionalized or Not with Benfotiamine

To evaluate the effect of benfotiamine on cell behavior and response, biocompatibility assessment was carried out at 2 and 7 days of cell culture in standard conditions. The control group consisted of stainless steel discs coated with 45S5 1.0 wt% Sm-dopedbioactive glass, while the experimental group consisted of stainless steel discs coated with 45S5 1.0 wt% Sm-doped bioactive glass and further functionalized with benfotiamine.

The cytotoxic potential of both 45S5 Sm-doped bioactive glass coatings was tested by performing the LDH assay. This quantitative test allows measurement of the amount of LDH released from damaged cells in the culture medium. Data obtained after 2 days of culture reported an overall low cytotoxicity, with no significant differences between the two tested coatings (Figure 9a). After 7 days of culture, LDH levels showed a slight increase compared to those measured at 2 days post-seeding; however, the difference was not statistically significant and may be attributed to the high proliferation rate. The obtained data indicated that the benfotiamine-functionalized and non-functionalized Sm-doped bioactive glass coatings exhibit an overall low cytotoxicity and may be considered biocompatible.

To further investigate the biocompatibility of the tested composites, cell viability assessment using the MTT assay was performed. Both metal composites exhibited overall similar cellular viability after 2 days of in vitro culture without significant differences. Interestingly, after one week, a significant increase in the viability of MC3T3-E1 cells in contact with the benfotiamine-functionalized coatings was observed (p < 0.05, Figure 9b) compared to the non-functionalized controls. These results indicate that the addition of benfotiamine generally favors pre-osteoblasts proliferation and growth, suggesting that this compound may potentially serve as a beneficial agent in future osteogenic studies. The enhanced proliferation observed for the benfotiamine-functionalized coatings may be linked to the known stimulatory effects of vitamin B1 derivatives on osteoblasts. Given the higher stability and bioavailability of benfotiamine compared to vitamin B1, such functionalization could provide additional benefits, although further dedicated studies are needed to confirm this mechanism.

The Live/Dead staining, coupled with fluorescence microscopy, allowed the qualitative visualization of cellular distribution, morphology, and viability after 2 and 7 days of cell culture in contact with the tested coatings. After 2 days of incubation, fluorescence microscopy images showed an increased cellular growth and viability on bothSm-doped bioactive glass coatings, indicating that the metal composites coated with 45S5 bioactive glasses doped with 1.0 wt% Sm, with or without benfotiamine, support cellular proliferation and viability. We previously demonstrated that the incorporation of 1.0 wt% Sm into bioactive glasses resulted in an increased cell proliferation [18]. Moreover, after 7 days of in vitro cell culture, a higher cellular density and proliferation rate were observed in both composites, with the highest density reported at the level of benfotiamine-supplemented composites, compared to the control (Figure 9c). In addition, cells exposed to benfotiamine were observed to form aggregates after 7 days of culture. These results were further demonstrated by calculating the percentage of area covered by green fluorescence. After 7 days of culture, a statistically significant increase in fluorescence intensity was observed in cells in contact with 45S5 Sm-doped bioactive glass supplemented with benfotiamine, compared to coatings without benfotiamine (p < 0.05, Figure 9d). These data align with the MTT and LDH results. Therefore, the addition of benfotiamine to Sm-doped 45S5 bioactive glass coatings on stainless steel discs could potentially support cell viability and enhance their applicability in bone regeneration.

4. Conclusions

This study presents the development of multifunctional bioactive coatings composed of 45S5 bioactive glass doped with 1.0 wt% samarium and functionalized with benfotiamine. The coatings were successfully deposited onto stainless steel substrates using the spin coating method, resulting in homogeneous and adherent layers.

Surface analyses revealed a compact and partially crystalline morphology for the Sm-doped bioglass, while benfotiamine functionalization led to the formation of characteristic needle-like crystals. FTIR analysis confirmed the presence of both inorganic and organic phases. Contact angle measurements showed a significant increase in hydrophilicity after benfotiamine application, which is favorable for biological integration.

In vitro bioactivity tests in SBF showed trends consistent with the deposition of calcium phosphate phases (CaPs), which are generally associated with bioactive behavior and bone-bonding potential. Cytocompatibility assays revealed low cytotoxicity and enhanced osteoblast proliferation in the presence of benfotiamine, suggesting a dual role of the coatings in promoting osteointegration and potentially contributing to anti-resorptive effects.

These findings indicate that benfotiamine-functionalized, Sm-doped bioactive glass coatings are promising candidates for future applications in orthopedic and dental implants, where both regeneration and long-term stability are essential. The paper was designed to have an initial proof of concept followed by a series of publications treating additional research, such as an evaluation of how the coatings’ adhesion correlated with the surface preparation and deposition procedure, as well as the control of the benfotiamine release (including by developing a more dense or porous coating). Further in vitro and in vivo evaluations will be realized.