Synergistic Integration of Cobalt Ferrite and Carvacrol in a Chitosan Scaffold: Multimodal Antimicrobial Activity and Magnetic Responsiveness

Abstract

This study developed multifunctional chitosan–hydroxyapatite (CH–HAp) scaffolds incorporating cobalt ferrite (CoFe₂O₄, CFO) nanoparticles and carvacrol to combine bone regeneration potential with magnetic responsiveness and antimicrobial activity. Scaffolds containing 5 wt% CFO and 10–30 wt% carvacrol (free or Tween 80-emulsified) were fabricated via freeze-drying. The inclusion of CFO provided ferrimagnetic behavior, while carvacrol reduced chitosan crystallinity and increased scaffold porosity. Formulations with 30 wt% carvacrol demonstrated the strongest antimicrobial effect, showing inhibition halos against Staphylococcus aureus, Escherichia coli, Candida albicans, and Candida glabrata. The scaffold combining emulsified carvacrol and CFO exhibited a highly porous (≈90%) structure, preserved magnetic response, and mild cytotoxicity toward L929 fibroblasts, indicating cytocompatibility. The synergistic integration of CFO and carvacrol in a CH–HAp matrix yielded a multifunctional platform that simultaneously provides structural support, magnetic responsiveness, and antimicrobial performance, showing great promise for advanced bone tissue engineering applications.

1. Introduction

Bone Tissue Engineering (BTE) aims to develop biomaterial systems capable of supporting efficient bone regeneration while ensuring structural stability, biocompatibility, and infection prevention, which are among the main factors associated with implant failure [1,2]. Three-dimensional scaffolds combining CH and Hap have been widely explored due to their bioactivity, osteoconductivity, and chemical similarity to the natural bone matrix [3,4,5]. Recent studies have shown that CH–HAp hybrid scaffolds can be functionalized with a variety of bioactive agents, enhancing their regenerative and antimicrobial potential [6,7,8,9,10]. Despite these advances, important challenges remain, particularly those related to microbial contamination and the need for combined therapeutic strategies within BTE.

Carvacrol, a phenolic monoterpene found in essential oils, exhibits strong antimicrobial, antifungal, and anti-inflammatory activities [11,12]. However, its high hydrophobicity and volatility hinder its stable incorporation into hydrophilic polymer matrices. For this reason, emulsifiers such as Tween 80 (T80) are often required to improve their dispersion and retention in polymeric systems [13,14]. In parallel, CFO have attracted significant interest in the biomedical field due to their high coercivity, chemical stability, and efficient response to external magnetic fields [15,16,17,18]. While both components are well-studied individually, their synergistic integration into CH–HAp scaffolds has not been systematically explored.

Here, we develop and characterize multifunctional CH–HAp scaffolds functionalized with CFO and carvacrol, the latter incorporated in both free and emulsified forms. We evaluate the effects of these components on the physicochemical, magnetic, structural, and biological properties of the scaffolds, focusing on their antimicrobial efficacy, cytocompatibility, and potential as a magnetically responsive platform for bone tissue engineering.

2. Experimental Section

2.1. Materials

Carvacrol (purity ≥ 98%), chitosan (75–85% deacetylation, 50–150 kDa), hydroxyapatite (purity ≥ 97%, particle size < 200 nm), Tween 80 (≥58% oleic acid content), glacial acetic acid (purity ≥ 99.8%), and phosphate-buffered saline (PBS, pH 7.4) were obtained from Sigma-Aldrich^® (Merck Group, Darmstadt, Germany). Ammonium hydroxide (28–30%) was sourced from Neon^® (Suzano, SP, Brazil). Cobalt ferrite (CoFe₂O₄, CFO) nanoparticles were synthesized via combustion reaction at the Laboratory for the Synthesis of Ceramic Materials (LabSMaC, Campina Grande, Brazil). All materials were used as received.

2.2. Preparation of CH–HAp/CFO/Carvacrol Scaffolds

Scaffolds were prepared using a polymer–ceramic matrix composed of CH (1.5% w/v in 1% acetic acid) and HAp (30% w/w relative to the mass of chitosan), following an adapted methodology from previous studies [19]. The CH–HAp base dispersion (total batch volume: 100 mL) was homogenized by mechanical stirring at 350 ± 10 rpm for 1 h at room temperature (25 ± 2 °C).

The experimental design (Table 1) included three independent factors:

(i)

CFO presence (0 or 5 wt% relative to CH)

(ii)

Carvacrol concentration (10, 20, or 30 wt% relative to CH)

(iii)

Carvacrol incorporation method (free or emulsified with T80 at 1:1 w/w ratio)

The proportions of CFO constituting the scaffolds were determined according to the method by Farzaneh et al. [20], and the HAp composition followed the procedure described by Ponciano et al. [21].

Table 1. Composition of scaffolds according to the presence of CFO, carvacrol concentration, and carvacrol incorporation form (free or emulsified).

	Scaffold	CFO (% w/w)	Carvacrol (% w/w)	Carvacrol Form
Group 1	S1	0	10	Free
	S2	0	20	Free
	S3	0	30	Free
Group 2	S4	5	10	Free
	S5	5	20	Free
	S6	5	30	Free
Group 3	S7	0	10	Emulsified
	S8	0	20	Emulsified
	S9	0	30	Emulsified
Group 4	S10	5	10	Emulsified
	S11	5	20	Emulsified
	S12	5	30	Emulsified

Table 1. Composition of scaffolds according to the presence of CFO, carvacrol concentration, and carvacrol incorporation form (free or emulsified).

	Scaffold	CFO (% w/w)	Carvacrol (% w/w)	Carvacrol Form
Group 1	S1	0	10	Free
	S2	0	20	Free
	S3	0	30	Free
Group 2	S4	5	10	Free
	S5	5	20	Free
	S6	5	30	Free
Group 3	S7	0	10	Emulsified
	S8	0	20	Emulsified
	S9	0	30	Emulsified
Group 4	S10	5	10	Emulsified
	S11	5	20	Emulsified
	S12	5	30	Emulsified

Each formulation was prepared individually, with reagents weighed separately to ensure reproducibility and uniformity within each experimental group.

Group 1—CH–HAp containing free carvacrol: Free carvacrol (10–30 wt%) was added dropwise to CH–HAp dispersion with continuous stirring (1 h).

Group 2—CH–HAp + CFO + free carvacrol: CFO (5 wt%) was first dispersed in CH–HAp (30 min stirring), followed by free carvacrol addition (10–30 wt%) and further mixing (1 h).

Group 3—CH–HAp + emulsified carvacrol (without CFO): An aqueous surfactant phase was first prepared by dispersing T80 in deionized water under magnetic stirring for 30 min. Carvacrol was then added dropwise to this aqueous T80 solution (1:1 w/w carvacrol:T80), followed by 30 min of stirring and 10 min of ultrasonication, forming a stable oil-in-water nanoemulsion. This preformed nanoemulsion was then incorporated into the CH–HAp dispersion under continuous mixing.

Group 4—CH–HAp + CFO + emulsified carvacrol: CFO (5 wt%) was first dispersed in the CH–HAp matrix (30 min stirring). The preformed nanoemulsion (T80 dispersed in water + carvacrol incorporated afterward) was then added dropwise to the CH–HAp–CFO dispersion, followed by 1 h of homogenization.

All formulations underwent identical post-processing: freezing at −48 °C (24 h), freeze-drying at −56 °C (72 h), neutralization in ammonium hydroxide vapor (24 h), and secondary freeze-drying.

Figure 1 presents a schematic illustration of the scaffold preparation process.

2.3. Physicochemical and Biological Characterization

All scaffold formulations (S1–S12) and precursor materials were characterized using the following techniques. Based on preliminary antimicrobial screening, scaffolds S3, S6, S9, and S12 were selected for detailed morphological, porosity, and cytotoxicity analysis.

FT-IR analysis: Spectra were acquired using a Bruker Vertex 70 spectrometer (Bruker Optics, Ettlingen, Germany) with ATR accessory. Measurements were taken in the 4000–400 cm⁻¹ range (4 cm⁻¹ resolution, 64 scans).

XRD analysis: Patterns were obtained using a Bruker D2 PHASER diffractometer (Bruker AXS, Karlsruhe, Germany) with Cu Kα radiation (λ = 1.5406 Å, 30 kV, 10 mA) over 2θ = 10–60° (0.02° step size, 2° min⁻¹ scan rate). Crystallite sizes were estimated using the Scherrer equation [21].

Magnetic measurements: Hysteresis loops were measured at room temperature using a vibrating sample magnetometer (MicroSense EZ9, MicroSense LLC, Lowell, MA, USA) with maximum applied field of 13,700 G. Magnetization values were normalized to the total mass of each scaffold (0.0063–0.0245 g).

Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) determination: Antimicrobial evaluation included two complementary assays: broth microdilution and agar diffusion. Different microbial panels were used depending on the assay. The MIC and MBC values were determined exclusively for standard strains from the American Type Culture Collection (ATCC) Staphylococcus aureus MRSA (ATCC 43300) and Escherichia coli (ATCC 25922), following the protocol by Ellof et al. [22] and the Clinical and Laboratory Standards Institute M07 and M27 protocols (CLSI M07/M27) [23]. Sterile 96-well plates received 190 μL of tryptic soy broth (TSB), CFO powder at 10% (w/v), and carvacrol at 100 µg·mL⁻¹. Serial dilutions of CFO were performed to achieve final concentrations ranging from 100 × 10³ μg·mL⁻¹ to 390.62 μg·mL⁻¹. A bacterial suspension standardized to 1.5 × 10⁸ CFU mL⁻¹ was added (10 μL per well), followed by incubation at 35 ± 2 °C for 24 h. After incubation, 20 μL of TTC (2%) was added for viability detection. MBC values were determined by transferring samples from inhibited wells to Mueller–Hinton agar and incubating at 37 ± 1 °C for 24 h. The MBC corresponded to the lowest concentration producing no visible growth [24].

Antimicrobial assays: Agar diffusion assays were performed for all scaffold formulations (S1–S12) using a broader microbial panel, including: Staphylococcus aureus (ATCC:77193), methicillin-resistant Staphylococcus aureus (MRSA, ATCC:43300), Escherichia coli (ATCC:25922), Pseudomonas aeruginosa (ATCC:27853), Candida albicans (ATCC:14053), and Candida glabrata (ATCC:90030). The inoculum was prepared in TSB and incubated for 24 h at 35 °C. The resulting bacterial suspensions were standardized via spectrophotometry at 580 nm (25% transmittance), achieving a final concentration of 1 × 10⁸ CFU·mL⁻¹. For each strain, 50 µL of inoculum was mixed with 4.95 mL of molten Mueller–Hinton agar (double-layer technique). Scaffold discs (8 mm) and positive controls (cefazolin for Gram-positive and gentamicin for Gram-negative bacteria) were placed on the agar surface and incubated at 35 °C for 24 h. For fungal assays, inocula were prepared in Sabouraud dextrose broth, incubated for 48 h at 25 °C, standardized, and tested using the same double-layer method. Plates were incubated at 25 °C for 48 h. Inhibition zones were measured in triplicate. Based on preliminary screening, samples S2, S3, S6, S9, and S12 displayed measurable antimicrobial activity and were selected for extended characterization.

SEM analysis: Surface morphology was examined using a VEGA3 TESCAN microscope at 20 kV accelerating voltage under high vacuum (<10⁻⁴ Torr). Images were captured at 100× and 2000× magnification using a secondary electron detector.

Apparent porosity (AP): Porosity was determined by ethanol displacement method using Equation (1):

$$\mathrm{A}\mathrm{P} \left(\mathrm{\%}\right)={\displaystyle \frac{{\mathrm{V}}_1-{\mathrm{V}}_3}{{\mathrm{V}}_2-{\mathrm{V}}_3}}\times 100\mathrm{\%}$$

(1)

where ${\mathrm{V}}_1$, ${\mathrm{V}}_2$, and ${\mathrm{V}}_3$ represent the ethanol volume before immersion, after immersion, and after scaffold removal, respectively. Measurements were performed in triplicate.

Cytotoxic activity: Cytotoxicity according to ISO 10993-5:2009 [25] using the agar diffusion method and L929 mouse fibroblasts (ATCC NCTC clone 929, Cell Bank of Rio de Janeiro, Rio de Janeiro, Brazil). Cells were cultured in RPMI 1640 medium (Gibco-Invitrogen Corporation, Grand Island, NE, USA) at 37 °C with 5% CO₂ until reaching 80% confluence. Trypsinization was performed with 0.25% trypsin (Gibco^®, Life Technologies, Grand Island, NY, USA), and cell density was quantified using an automated cell counter (Interwoven-Thermo Fisher, Waltham, MA, USA). Suspensions of 1.0 × 10⁵ cells·mL⁻¹ were distributed into 6-well plates (4 mL per well) and incubated under the same conditions for 24 h. After cell adhesion, 2× concentrated MEM medium (Gibco^®-Invitrogen Corporation, Grand Island, NE, USA) and agar solution with neutral red (Sigma-Aldrich, St. Louis, MO, USA) were added. Scaffolds, as well as the latex and filter paper, were sectioned into 1 cm² pieces and inserted into the wells, with a positive control (toxic latex sheet) and a negative control (quantitative filter paper with a pore size of 0.45 µm, obtained from Química Moderna, Barueri, SP, Brazil). The plates were incubated for 24 h, and the clear zones were quantified (ISO 10993-5). Cell lysis was evaluated using a NIKON ECLIPSE TS100 inverted digital microscope (Minato, Tokyo, Japan).

3. Results and Discussion

3.1. Chemical Interactions and Structural Properties (FT-IR/XRD)

FT-IR analysis confirmed the presence of characteristic functional groups from all components in the composite scaffolds. Figure 2a shows the reference spectra of precursor materials, with leading absorption bands summarized in

Table 2. Main FT-IR absorption bands and their assignments for precursor materials, compared with literature data.

Material	Wavenumber (cm−1)	Assignment	Reference Values (cm−1)	Vibration Type/Functional Group	Reference
Chitosan (CH)	3288	O–H and N–H stretching	3290–3360	Hydrogen-bonded hydroxyl and amine groups	[26,27,28]
	2918, 2874	C–H stretching	2920–2850	Aliphatic CH2 groups
	1652, 1590	Amide I (C=O), Amide II (N–H)	1650–1590	N-acetyl groups
	1020	C–O–C stretching	1020–1070	Polysaccharide backbone
Hydroxyapatite (HAp)	3293	O–H stretching	3300–3570	Structural hydroxyl	[29,30]
	1154	ν3 (PO43− asymmetric stretch)	1090–1150	Phosphate group
	888, 698, 556	ν4 (PO43− bending)	870–560	Phosphate group
Cobalt ferrite (CFO)	526, 434	Fe–O stretching	540–420	Tetrahedral and octahedral Fe–O vibrations	[31]
Tween 80	3484	O–H stretching	3400–3500	Hydroxyl group	[32,33]
	2920	C–H stretching	2910–2850	Aliphatic chain
	1730	C=O stretching	1730–1745	Ester group
	1096	C–O–C stretching	1080–1120	Polyoxyethylene ether group
Carvacrol	3362	O–H stretching	3350–3400	Phenolic hydroxyl	[34]
	2958	C–H stretching	2960–2850	Aliphatic chain
	1249	C–O stretching	1230–1270	Phenolic group
	800–900	C–H out-of-plane bending	800–890	Aromatic ring substitution

The composite scaffolds (Figure 2b) exhibited spectral features corresponding to all incorporated components, providing qualitative verification of their successful integration. Characteristic HAp phosphate bands (ν₃ at ~1020 cm⁻¹ and ν₄ at 560–600 cm⁻¹) remained evident across all formulations, confirming the preservation of the calcium phosphate phase. Scaffolds containing magnetic nanoparticles showed detectable bands in the 525–433 cm⁻¹ region associated with CFO, indicating successful physical incorporation without chemical modification.

.

Spectral regions including the broad O-H/N-H stretching (~3300 cm⁻¹) and C-H stretching (2960–2870 cm⁻¹) showed overlapping contributions from CH, carvacrol, and T80 components. Variations in relative intensity were observed in the amide I region (~1650 cm⁻¹) among different formulations.

To further evaluate structural preservation and phase composition, XRD analysis was performed. Figure 3 shows the diffractograms of the precursor materials (CH, HAp, CFO) and the scaffolds (S1–S12), compared with the CH-HAp control.

The XRD pattern of CH revealed two characteristic crystalline regions at 2θ ≈ 9.8° and 20.1°, consistent with the known organization of ordered domains formed through intra and interchain packing of the semicrystalline polysaccharide [35]. The CFO pattern exhibited a single-phase inverse spinel structure, with diffraction peaks at approximately 2θ = 18.4°, 30.2°, 35.6°, 43.24°, and 57°, corresponding to the (111), (220), (311), (400), and (511) planes of the cubic spinel phase (space group Fd-3m, No. 227). These reflections are consistent with the reference pattern from the JCPDS 01-076-7254 database, indicating 66% crystallinity and an average crystallite size of 31 nm. The HAp exhibited the characteristic reflections of the hexagonal phase (ICDD 00-064-0738), including peaks at 2θ ≈ 25.9°, 28.9°, 31.86°, 32.1°, and 32.9°, corresponding to the (002), (102), (210), (211), and (112) planes.

The CH-HAp control scaffold displayed the expected combination of CH’s semicrystalline region (around 20°) and HAp reflections (25–35°), indicating that scaffold processing did not induce detectable phase changes. Across the composite scaffolds (S1–S12), the XRD patterns consistently showed the characteristic reflections of HAp and, when applicable, CFO, indicating that both phases were preserved after incorporation into the polymeric matrix.

Scaffolds containing carvacrol (Groups 1 and 3) displayed a reduction in the intensity of CH characteristic reflections and a broadening of the diffuse halo in the 20–35° region. This effect, which increased with higher carvacrol content, reflects a decrease in the ordering of semicrystalline domains. Additionally, scaffolds incorporating CFO (Groups 2 and 4) retained the magnetic phase structure without alteration. The overlapping contributions of CH, HAp, and CFO reflect the hybrid nature of the composite and the coexistence of multiple crystalline and amorphous domains.

Altogether, the XRD results indicate the preservation of the crystalline phases of HAp and CFO, expected modifications in the semicrystalline organization of CH due to carvacrol, and the absence of new crystalline phases after scaffold processing.

3.2. Magnetic Properties

Figure 4 illustrates the magnetization curves of the precursors.

Based on the magnetization curves illustrated in Figure 4a,b, CH, HAp, carvacrol, and T80 exhibited diamagnetic behavior, evidenced by their negative magnetic susceptibility and the absence of a magnetic hysteresis loop [36]. In contrast, CFO nanoparticles displayed typical ferrimagnetic behavior, with a well-defined magnetic hysteresis loop (Figure 4b). The CFO particles exhibited saturation magnetization (M_s) of 55.9 emu·g⁻¹, remanence (M_r) of 24.22 emu·g⁻¹, and coercivity (H_c) of 1127.9 Oe, values consistent with cobalt ferrite nanoparticles synthesized via combustion reactions reported by Santos et al. [37] and E. Leal et al. [38]. These findings align with previously reported magnetic properties for ferrimagnetic materials, particularly CFO [39].

Figure 5 shows the magnetization curves for the scaffolds (S1–S12). Scaffolds without CFO (S1–S3 and S7–S9) maintained the intrinsic diamagnetism of the polymeric matrix (Figure 5a,c). Conversely, incorporated with 5 wt% CFO (S4–S6 and S10–S12) exhibited clear ferrimagnetic signatures, characteristic of the magnetic nanoparticles incorporated in the formulations (Figure 5b,d). The persistence of ferrimagnetism contrasts with findings by Cevallos et al. [40], who observed suppression of ferrimagnetic behavior in polyvinylpyrrolidone (PVP) fibers containing CFO. In the present work, the CH–HAp matrix successfully supported the retention of CFO’s magnetic properties.

Table 3. Magnetic parameters of scaffolds S4, S5, S6, S10, S11, and S12.

Scaffold	Ms (emu·g−1)	Mr (emu·g−1)	Hc (Oe)
S4	2.5	1.16	1199
S5	3.11	1.26	1479
S6	1.6	1.18	1722
S10	2.23	1	1193
S11	2.56	1.2	745
S12	2.45	1.1	715

summarizes the magnetic parameters of the magnetic scaffolds. Although all formulations contained identical CFO loading (5 wt%), the magnetic response varied significantly, reflecting differences in nanoparticle distribution and agglomeration within the matrix [41].

The highest M_s value was observed for scaffold S5 (3.11 emu·g⁻¹), formulated with 20% free carvacrol. In contrast, scaffold S6, containing 30% free carvacrol, showed the lowest M_s (1.60 emu·g⁻¹), representing a reduction of approximately 50%. This decrease suggests that higher free-carvacrol content may reduce the homogeneous dispersion of CFO particles, promoting agglomeration and decreasing the net magnetic response.

In contrast, scaffolds containing emulsified carvacrol (Group 4, S10–S12) displayed more uniform M_s values (2.23–2.56 emu·g⁻¹). This uniformity is likely attributable to T80, which acts as a surfactant to enhance nanoparticle dispersion. The literature suggests that magnetic nanoparticles in T80 solutions can form stabilized core–shell structures, reducing agglomeration [42]. Roacho-Péres et al. also showed that T80 coatings decrease the aggregate size of magnetite nanoparticles [43].

Coercivity values also varied widely, ranging from 715 Oe (S12) to 1722 Oe (S6). The high Hc in S6 indicates stronger magnetic-domain coupling, likely associated with greater nanoparticle clustering. Conversely, the lower Hc values in S11 and S12 reflect more efficient CFO dispersion facilitated by T80, which minimizes interparticle interactions [44].

3.3. Antimicrobial Evaluation

Table 4. MIC and MBC values for CFO nanoparticles and carvacrol against S. aureus MRSA and E. coli bacteria.

Samples	Microrganisms MIC/MBC (μg·mL−1)
	S. aureus MRSA ATCC (43300)		E. coli ATCC (25922)
	MIC	MBC	MIC	MBC
CFO	6.250	-	12.500	12.500
Carvacrol	48.8	48.8	48.8	48.8
Cefalexin	-	-	3.125	50.000

summarizes the MIC and MBC values obtained for CFO nanoparticles and for carvacrol against S. aureus MRSA and E. coli. Values represent the mean of three independent experiments (n = 3).

Both carvacrol and CFO exhibited measurable antimicrobial activity. CFO displayed a potent antibacterial effect, with MIC values of 6.25 µg·mL⁻¹ (S. aureus MRSA) and 12.5 µg·mL⁻¹ (E. coli). The high sensitivity of MRSA ATCC 43300 to CFO is significant, given MRSA strains, suggesting that combustion-synthesized CFO holds potential as an alternative antimicrobial agent [15,45].

Carvacrol also showed bactericidal activity, with MIC and MBC values of 48.8 µg·mL⁻¹ for both organisms. These results indicate greater potency against MRSA ATCC 43300 compared to findings by Bakhtiari et al. [46], who reported significantly higher MIC (250 µg·mL⁻¹) and MBC (500 µg·mL⁻¹) values for carvacrol against MRSA ATCC 33592. Such variations are likely due to differences among strains and assay conditions.

In addition to the MIC/MBC values obtained in this study, carvacrol is widely recognized as a broad-spectrum antimicrobial agent. Previous reports demonstrate its effectiveness against a wide range of clinically relevant microorganisms, including Gram-positive bacteria (Staphylococcus aureus ATCC 25923, ATCC 6538P, ATCC 29213, ATCC BAA-1707, ATCC 43300; S. epidermidis ATCC 12228; Micrococcus luteus ATCC 10240; Bacillus subtilis ATCC 6633; B. cereus ATCC 10876; Enterococcus faecalis ATCC 29212), Gram-negative bacteria (Escherichia coli ATCC 25922; Salmonella typhimurium ATCC 14028; Bordetella bronchiseptica ATCC 4617; Proteus mirabilis ATCC 12453; Pseudomonas aeruginosa ATCC 27852; Klebsiella pneumoniae), and several fungal species (Candida albicans ATCC 10231, ATCC 2091; C. auris CDC B11903; C. glabrata ATCC 90030; C. krusei ATCC 14243; C. parapsilosis ATCC 22019; C. lusitaniae ATCC 34449; C. tropicalis ATCC 1369) [47,48].

Table 5. Antimicrobial activity of scaffolds S1–S12 evaluated by the agar diffusion assay.

Microrganisms
Scaffold	S. aureus	E. coli	P. aeruginosa	C. albicans	C. glabrata
	Average Inhibition Halo (mm)
S1	-	-	-	-	-
S2	11.66 ± 1.69	10.33 ± 0.47	-	-	-
S3	12.66 ± 2.05	12.66 ± 2.05	-	-	-
S4	-	-	-	-	-
S5	-	9.00 ± 1.63	-	-	-
S6	16.00 ± 0.81	10.66 ± 0.94	-	-	-
S7	-	-	-	-	-
S8	-	-	-	-	-
S9	10.33 ± 0.47	9.66 ± 2.05	-	20.33 ± 0.47	14.33 ± 0.47
S10	-	-	-	-	-
S11	-	-	-	-	-
S12	10.00 ± 0	10.00 ± 0	-	14.00 ± 0.81	13.00 ± 0
Standard	Cefazolin	Gentamicin	Gentamicin	Nistatin	Nistatin
	23.00 ± 0	21.00 ± 0	21.00 ± 0	21.00 ± 0.81	20.00 ± 0.81

describe the antimicrobial activity of scaffolds S1–S12 evaluated by the agar diffusion method.

The antimicrobial behavior of the scaffolds reflected their composition, carvacrol loading, incorporation method, and the intrinsic antimicrobial potency of the isolated active agents.

Scaffolds containing 10% carvacrol, whether in free form (S1, S4) or nanoemulsified with T80 (S7, S10), did not produce inhibition halos against any of the tested microorganisms. This absence of activity indicates that a 10% loading was insufficient to release carvacrol at concentrations that reach the MIC (48.8 µg·mL⁻¹) within the agar medium, a limitation often observed in controlled-release matrices where drug loading is below the threshold for effective diffusion [49,50].

In contrast, scaffolds formulated with 20% and 30% free carvacrol (S2, S3, S5, S6) exhibited marked antibacterial activity. The inhibition zones for the 30% formulations were larger, suggesting that higher loading enabled release concentrations that approached the MBC, resulting in a pronounced bactericidal effect.

Distinct behavior was observed in formulations containing nanoemulsified carvacrol. Scaffolds with 10% and 20% nanoemulsified carvacrol (S7, S8) produced no detectable antimicrobial activity, whereas those containing 30% (S9, S12) demonstrated apparent antibacterial and antifungal effects. This delayed onset is characteristic of nanoemulsion-loaded systems; T80 encapsulates carvacrol within its hydrophobic core, thereby restricting rapid release. Only at higher loadings does sufficient carvacrol overcome this barrier to diffuse at effective concentrations [51,52].

Among all formulations, scaffolds S6 and S12 displayed the most significant antimicrobial activity (both containing 30% carvacrol and 5 wt% CFO). The literature suggests that ferrimagnetic nanoparticles can enhance antimicrobial performance through mechanisms such as ROS generation [53] or local microdisturbances that increase membrane permeability [54,55]. Although no external magnetic field was applied, these intrinsic effects may contribute to the enhanced performance of the magnetic scaffolds.

Regarding antifungal activity, only scaffolds containing 30% nanoemulsified carvacrol (S9 and S12) exhibited inhibitory effects against C. albicans and C. glabrata. This aligns with reports that nanoemulsions improve antifungal efficacy by enhancing solubility and facilitating membrane fusion and intracellular release [12,56,57,58]. The superior performance of the nanoemulsified system is likely attributed to two factors: the higher concentration of carvacrol (30%) required to overcome the intrinsic resistance of yeast cells, and the high specific surface area and mobility of the nanodroplets, which facilitate mass transfer and fusion with the fungal membrane [59,60]. Peculiarly, the absence of inhibition zones in scaffolds containing 30% free carvacrol underscores the critical role of the nanoemulsion system. While free carvacrol failed to permeate the complex fungal structure efficiently, the T80-stabilized nanocarriers successfully enhanced bioavailability and localized action, thereby inhibiting fungal growth.

3.4. Morphology and Porosoty

To correlate microstructural features with antimicrobial performance, selective screening was performed. Scaffolds exhibiting the highest activity at maximum carvacrol concentration (S3, S6, S9, and S12) were selected for detailed characterization. Figure 6 presents the surface morphology of the selected scaffolds.

The microstructure of scaffold S3 (30% free carvacrol) (Figure 6a,b) revealed a heterogeneous matrix and irregular pore distribution. The polymeric walls exhibited local collapse and discontinuities. At higher magnification, disorganized fibrous structures and granular domains were observed adhering to pore walls. These features likely correspond to phase-separated carvacrol regions formed during solvent removal, suggesting that direct incorporation leads to an uncontrolled distribution within the CH-HAp matrix.

In contrast, scaffold S6 (CFO + 30% free carvacrol, Figure 6c,d) presented a markedly more organized architecture. The incorporation of the CFO contributed to matrix reinforcement, resulting in thicker, continuous polymeric walls. Well-defined spherical agglomerates, consistent with CFO nanoparticles, were observed embedded within the matrix. These particles appeared well-distributed, conferring structural stability and mitigating pore collapse. The coexistence of lamellar structures and globular domains indicates partial incorporation of carvacrol within the CFO-reinforced matrix.

The morphology of S9 (30% emulsified carvacrol; Figure 6e,f) showed a highly porous, less-dense matrix with thin, interconnected walls. The use of T80 promoted finer dispersion of carvacrol droplets, yielding a more homogeneous pore distribution compared to free-carvacrol formulations. High-magnification images reveal globular vesicular domains partially integrated into the polymer network. The smoother topography indicates improved interfacial compatibility between the polymeric and oily phases.

Finally, scaffold S12 (CFO + 30% emulsified carvacrol, Figure 6g,h) exhibited the most compact and structurally organized morphology among the evaluated formulations. The combination of emulsification and magnetic nanoparticles resulted in a scaffold with minimal phase separation and enhanced structural stability. This densification effect aligns with findings by Heidari et al., who observed that the incorporation of iron oxide nanoparticles into chitosan/hydroxyapatite scaffolds qualitatively reduced matrix porosity, promoting a denser, more mechanically robust structure [61].

Figure 7 presents the apparent porosity of scaffolds S3, S6, S9, and S12. The results reveal distinct trends associated with the method of carvacrol incorporation and the presence of cobalt ferrite.

Scaffold S3 (30% free carvacrol) exhibited the lowest porosity (81.9 ± 4.17%) and the highest variability among samples. This behavior is consistent with the heterogeneous microstructure observed in SEM analysis, where the direct incorporation of carvacrol without emulsifying agents promoted the formation of irregular hydrophobic domains and local wall collapse during drying. These structural defects likely obstructed the pore network, reducing the total void volume.

In contrast, for scaffold S6 (CFO + 30% free carvacrol), porosity increased markedly to 88.5 ± 0.50%, accompanied by a decrease in variability. The presence of CFO appeared to contribute to greater matrix stability; the rigid nanoparticles likely reinforced the polymer walls, preventing shrinkage during lyophilization. This reinforcement is reflected in the SEM images, which show a cohesive structure with fewer collapsed regions and a uniform distribution of interconnected pores.

Scaffold S9 (30% emulsified carvacrol) showed even higher porosity 89.5 ± 0.47%). The emulsification process enabled a homogeneous distribution of carvacrol throughout the polymeric matrix, reducing phase separation. As evidenced by SEM, this resulted in thin, continuous pore walls and a smoother topography. The high porosity indicates that the nanoemulsion effectively mitigated the morphological disruptions commonly observed in scaffolds containing free hydrophobic agents.

The highest porosity value was observed for scaffold S12 (90.5 ± 2.25%), which combined emulsified carvacrol and CFO. SEM analysis revealed an organized architecture with uniform pores, well-distributed CFO particles, and minimal surface collapse. This result suggests a synergistic effect: T80 promotes the efficient dispersion of carvacrol, while CFO reinforces the polymeric network. Consequently, this combination yielded a highly porous and interconnected structure, as recently highlighted by Apăvăloaiei et al. [62]. Their study emphasizes that interconnected pores are essential to stimulate osteoblast adhesion, nutrient flow, and vascularization. Furthermore, they note that pore sizes in the 50–150 µm range are particularly effective for fibrovascular growth and cell migration, features consistent with the optimized morphology observed in this work.

3.5. Biocompatibility and Trade-Offs

Figure 8 illustrates the cytotoxic behavior of the scaffolds against L929 fibroblasts evaluated via the agar diffusion method.

The results, summarized in

Table 6. Results of the agar diffusion cytotoxicity test according to ISO 10993-5:2009.

Tested Material	Discoloration Grade	Cell Lysis Grade	Interpretation
Positive Control	3	4	Severe
Negative Control	0	0	Non-cytotoxic
Blank	-	-	-
S3	2	2	Mild
S6	2	2	Mild
S9	2	2	Mild
S12	2	2	Mild

, indicate that all scaffolds exhibited only mild cytotoxicity (Grade 2), characterized by decolorization zones smaller than 0.45 cm. According to ISO 10993-5:2009 standards, materials falling within this range are interpreted as having “mild” reactivity, bordering the limit of biological acceptance for implantable devices but considered distinct from “moderate” or “severe” toxicity.

This outcome is significant considering the high concentration of carvacrol incorporated into the matrices. Carvacrol is a phenolic compound with known intrinsic cytotoxicity; Ranjitkar et al. reported that concentrations above 100 µg·mL⁻¹ reduced the viability of both tumor and non-tumor cell lines by more than 50%, demonstrating limited selectivity [63].

In contrast, the present results show that, when incorporated into CH-HAp scaffolds, carvacrol’s cytotoxic effects are markedly attenuated. This mitigation is likely related to the polymeric matrix acting as a barrier, reducing the burst release and controlling cellular exposure to the bioactive agent. This “protective” effect aligns with findings by Akhlaq et al., who demonstrated that nanostructuring carvacrol into chitosan-based particles reduced toxicity towards non-tumoral cells, achieving selectivity indices greater than 2 [64].

Complementarily, Farto-Vaamonde et al. observed that PLA scaffolds loaded with carvacrol maintained tissue viability even during the initial release phase, suggesting that the scaffold architecture modulates the interaction between the drug and the surrounding tissue [65]. Therefore, the magnetic scaffolds developed in this work demonstrate a favorable balance: they effectively inhibit resistant bacteria and fungi while maintaining mild cytotoxicity. This suggests that the controlled incorporation of bioactive compounds can ensure antimicrobial efficacy without compromising biomedical safety.

4. Conclusions

In this study, multicomponent magnetic and antimicrobial scaffolds were successfully developed by integrating CFO nanoparticles and carvacrol into a CH–HAp matrix. Both components were effectively incorporated and directly influenced the structural, magnetic, and biological properties of the scaffolds. Carvacrol, especially at 30 wt%, acted as a plasticizer, reducing CH crystallinity and promoting a more amorphous structure, whereas 5 wt% CFO imparted ferrimagnetic behavior and enabled measurable magnetization. T80 improved carvacrol stabilization and dispersion, enhancing the homogeneity of the polymeric network and facilitating a more uniform distribution of CFO. Among all formulations, those with higher carvacrol content and the combination of emulsified carvacrol with CFO demonstrated the most promising multifunctional performance. These scaffolds exhibited broad-spectrum antimicrobial activity against S. aureus, E. coli, C. albicans, and C. glabrata, while maintaining only mild cytotoxicity (discoloration halos < 0.45 cm), in accordance with ISO 10993-5 standards. Of particular significance, the scaffold containing emulsified carvacrol and CFO achieved the best balance of porosity (≈90%), surface organization, magnetic response, and biological safety. Future studies should focus on in vivo evaluation, drug-release kinetics, mechanical characterization, and intermediate carvacrol concentrations to optimize the balance between antimicrobial activity and cytocompatibility.