Triggered by light and magnetism: smart foam PLLA/HAP/Fe3O4 scaffolds for heat-controlled biomedical applications

1 Triggered by Light and Magnetism: Smart Foam PLLA/HAP/Fe3O4 Scaffolds for HeatControlled Biomedical Applications Emilia Zachanowicz1*, Anna Tomaszewska2, Magdalena Kulpa-Greszta2, Piotr Krzemiński3, Jean-Marie Nedelec4, Dominika Zákutná5, Štefan Hricov6, Aleksandra Nurzyńska7, Anna Belcarz-Romaniuk7 and Robert Pązik2* 1Polymer Engineering and Technology Division, Wroclaw University of Technology, 50-370 Wrocław, Poland 2Faculty of Biotechnology, Collegium Medicum, University of Rzeszow, Pigonia 1, 35-310 Rzeszow, Poland 3Institute of Materials Engineering, Faculty of Exact and Technical Sciences, University of Rzeszow, Pigonia 1, 35-100 Rzeszow, Poland 4Université Clermont Auvergne, Clermont Auvergne INP, CNRS, ICCF, Clermont-Ferrand, F-63000, France 5Department of Inorganic Chemistry, Charles University, Hlavova 2030, Prague 2 128 40, Czechia 6Department of Condensed Matter Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, 121 16 Prague 2, Czechia 7Chair and Department of Biochemistry and Biotechnology, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland *Corresponding authors: Emilia Zachanowicz (WUT), [email protected], Robert Pązik (UR), [email protected] Abstract The ternary composite foam materials containing PLLA, HAP (20 nm), and morphologically controlled Fe3O4 nanoparticles (80 nm) were fabricated using the thermally induced phase separation (TIPS) technique over a broad concentration range of the magnetic component (1 – 30 wt%). The foam scaffolds were highly porous (>95%) and lightweight, with a high capacity for soaking in Ringer’s solution. The foam density varied with the inorganic component content, ranging from 0.02 to 0.079 g/mL, while the mean pore size was approximately 330 µm. The magnetic behavior of Fe3O4 nanocubes and foam composites was characterized. The presence of the inorganic filler caused a shift towards a lower decomposition temperature of PLLA. The energy conversion of both dry and Ringer’s solution-soaked foams was studied in detail, demonstrating that the fabricated ternary composites are highly temperature-responsive under the influence of an alternating magnetic field (AMF), nearinfrared (NIR) laser radiation (808, 880, and 1122 nm), and the synergistic effect of both external stimuli. This synergy resulted in faster heating and a higher maximum temperature (Tmax ≈ 80°C). Biological characterization and heating ability analysis enabled the selection of the most reliable foam, which contained 15% magnetic filler, based on its appropriate microstructure, sufficient biocompatibility, and ability to reach biologically relevant temperatures under AMF exposure and the combined action of NIR and AMF. The fabricated materials exhibit high potential for biomedical applications as well as other areas requiring temperature-controlled stimulation of various processes. Keywords: ferrites, multifunctional nanomaterials, foams, magnetic hybrids, energy conversion. 1. Introduction 2 Composites are considered among the most promising and modern future materials as they allow for the flexible integration of different compounds and combine physicochemical properties of their components within the same construct1–3. An example of such materials includes hybrids composed of naturally derived polymers (chitosan, collagen, glucan, polylactide, etc.), which upon integration with inorganic substances of the apatite family, i.e., hydroxyapatites, fluorapatites, carbonated apatites, calcium and/or strontium phosphates, etc. enable the development of advanced bone substitute scaffolds for regenerative medicine4–10. The ability to use various apatite compounds and their different properties in biological media allows control over new bone tissue formation, which is one of their key advantages11–13. Polymeric materials are often used as a matrix that maintains the appropriate microstructure of the bone replacement material, serving as a scaffold for newly colonizing cells. In the case of bone tissue, apatites combined with polymers facilitate achieving the desired properties of implants by stimulation of remodeling processes, integration with bone tissue as well as affect mechanical properties and degradability1. An important aspect of composite materials is their microstructure, which significantly determines whether the scaffold can be applied to compact or cancellous (spongy) bone. Spongy bones are commonly found at the ends of long bones, within flat bones, and inside short bones and are characterized by a highly porous structure with pore sizes ranging from 150 to 800 µm14. Polymers and their composites containing inorganic materials with a foam-like microstructure represent an innovative solution for modern implants whereas their microstructure, porosity and other critical parameters can be controlled during the fabrication process15–17. The PLLA is a well-known biodegradable polymer widely used in tissue engineering due to its favorable mechanical properties and high biocompatibility. PLLA can be processed into various structures, such as scaffolds and films, that support cell growth and tissue formation18. Calcium hydroxyapatite (HAP) is a key component of bone tissue, making it an ideal material for bone regeneration and repair. Its biocompatibility and osteoconductive properties are well-documented, and support cell adhesion and proliferation in tissue engineering applications19. On the other hand, a combination of PLLA with HAP can lead to an increase in the mechanical strength and bioactivity of the resulting composite materials, making them suitable for load-bearing applications in bone regeneration20. There are several techniques for the fabrication of the hybrid organic-inorganic composite towards highly porous materials, 3 namely, solvent casting and thermally induced phase separation (TIPS), microsphere sintering, selective polymer extraction, gas foaming, 3-D printing, and many more21,22. A very interesting idea is to use magnetic compounds as a functional additive to polymeric or inorganic scaffolds, such as ferrites (MFe2O4, M - Co2+, Mn2+, Ni2+, Fe2+, Zn2+)2,3 as a functional additive to polymeric or inorganic scaffolds. The main advantage of magnetic nanoparticles is their ability to generate heat under the action of external stimuli, i.e., alternating magnetic field (AMF) or near-infrared light, which makes them very useful in magneto-photothermal therapies2,23–25. The mechanisms of heat generation vary strongly depending on the type of stimulation. In the case of the AMF, three main contributions to heat induction were identified (1) hysteresis losses, (2) generation of eddy currents, and (3) residual losses that include Néel’s internal and Brownian external losses26. Meanwhile, temperature effects associated with NIR light interaction are predominantly caused by non-radiative processes that lead to energy dissipation (lattice vibrations)25,27. Both forms of magnetic nanoparticles stimulation (AMF and NIR) allow the achievement of relevant temperature ranges that affect biological processes (<43°C) or can lead to cancer cell elimination (>43°C)27– 30. Literature review reports mainly on binary organic-inorganic composites of PLLA/Fe3O431– 39, HAP/Fe3O440, 41 ceramic materials or HAP embedded iron apatite42 for enhancing cellular activity, cell adhesion, biocompatibility, mechanical properties, and only a few are devoted to the temperature effects, mainly focused on the interaction with the AMF. In the context of the ternary hybrids PLLA/HAP/Fe3O4 of any form, there is no data on their responsiveness to AMF and NIR. Some results can be found for other type of polymer like collagen/HAP/Fe3O443 (AMF only), chitosan/HAP/Fe3O444 (no AMF and NIR), PLA/ENR/Fe3O436 (ENR – epoxidized rubber) shape memory triggered by AMF and NIR, PLLA/PGA/Fe3O438 (PGA – polyglycolic acid, AMF only), polycaprolactone/HAP/HAP:Fe42 (AMF only), cellulose/HAP/Fe3O445 (no AMF and NIR). Therefore, the engineering of new, multifunctional composite materials is necessary and can open attractive directions for possible practical applications. In this work poly(L-lactic acid) polymer (PLLA) with calcium hydroxyapatite (Ca10(PO4)6(OH)2 nanoparticles foams were incorporated with morphologically controlled cubic nanomagnetite (80 nm Fe3O4) particles to achieve highly AMF and NIR temperature responsive composites characterized by the porous microstructure for potential use in biomedicine related field. 4 2. Experimental 2.1. Synthesis procedure of magnetic foams preparation The synthesis of magnetic foams with incorporated magnetite cubic nanoparticles, calcium hydroxyapatite, and polylactide polymer was carried out in three distinct steps: (I) preparation of morphology-controlled Fe3O4 cubic nanoparticles, (II) synthesis of nano-sized Ca₁₀(PO₄)₆(OH)₂, and (III) integration of the inorganic components into the polylactide matrix to fabricate magnetic and light-responsive functional foam hybrids. (I) Synthesis of magneto and light temperature-responsive Fe3O4 nanocubes. For the preparation of magnetite cubic particles, the well-known thermal decomposition method reported by Kim et al.46 was adopted with minor modifications. In a typical procedure, 2 mmol of the iron(III) complex (iron(III) acetylacetonate, Fe(acac)₃, 99.7%, Thermo Fisher Scientific, Germany) was mixed with 10 mL of dibenzyl ether (BE, 98%, Sigma Aldrich, Poland) in a three-neck glass flask until the substrate was completely dissolved. Subsequently, 4 mmol (1.4 mL) of oleic acid (OA, 90%, Sigma Aldrich, Poland) was added to the reaction mixture. All operations involving the iron source and reaction solution were performed under an inert N₂ atmosphere (99.999%, Linde, Poland) using a glove box (GS Glove Box Systemtechnik GmbH P10R250T2, Germany) equipped with an automatic gas control unit. The flask containing all necessary chemicals was then assembled into a reaction setup comprising a mechanical stirrer, a temperature controller with a Pt-100 sensor (LTR 2500, Juchheim, Germany), a heating mantle, an Allihn’s condenser column, and N₂ support gas line. In addition, the N₂ was passed through laboratory scrubbers containing mineral oil and molecular sieves (separately). After that, a solution was degassed with N2 for 60 min at room temperature. Subsequently, the reaction mixture temperature was increased to 285°C and left for an additional 30 min under constant stirring and a protective N2 atmosphere. The final mother liquor with visible black precipitate was cooled down to room temperature and further separated by centrifugation and washing cycles with the solvent mixture containing hexane/acetone/ethanol (1:1:1 v/v ratio, all delivered by Chempur, Poland, pure for analysis). The resulting magnetic particles were resuspended in ethanol and stored in a laboratory refrigerator. Nanoparticle concentration in a suspension was determined using a microbalance technique with a Radwag MYA 5.4Y scale by filling up an aluminum foil crucible 5 with 50 µL of particle dispersion and solvent evaporation until dry mass. Three repetitions were done, and an average mass value was calculated. (II) Preparation of calcium nanohydroxyapatite polymer filler For the fabrication of Ca₁₀(PO₄)₆(OH)₂ inorganic nanoparticles, a well-known precipitation technique proposed by Rodriguez-Lorenzo et al.47 was employed, with modified process parameters to obtain smaller particles. Briefly, to prepare approximately 100 g of hydroxyapatite, 1 mol (236.15 g) of Ca(NO₃)₂·4H₂O (99%, Sigma Aldrich, Poland) and 0.6 mol (79.23 g) of (NH₄)₂HPO₄ (99%, Sigma Aldrich, Poland) were used, maintaining a Ca:P ratio of 1.66. Both salts were separately dissolved in deionized water in glass beakers. The phosphatecontaining solution was then slowly added to the calcium nitrate solution, leading to the rapid precipitation of a white powder. Vigorous stirring, preferably mechanical for large batches, was essential to prevent powder adhesion and dense material formation. During mixing, a 25% NH₄OH solution (99%, Avantor, Poland) was added in 20 mL portions to keep a strongly basic pH above 9. This step was crucial for transforming the initially precipitated apatite into a hydroxyapatite structure. Maintaining the pH above 9 was particularly important, as a neutral pH could lead to the formation of a TCP/HAP mixture48. The presence of biphasic apatite can be desirable in certain cases to adjust phosphate resorption rates for bone integration processes in apatite-based implants49. The whole mixing stage lasted approximately 2 h, with continuous pH monitoring. The product was then separated via centrifugation and washed with Milli-Q water to purify it and remove ammonium nitrate byproduct, which accounted for nearly half of the total mixture. Purification was critical for safety reasons, preventing the risk of explosion and NOx contamination during thermal treatment. However, the separated NH₄NO₃ could be collected and reused as a key ingredient in plant fertilizers. Finally, the resulting white powder was dried at 60°C for 24 h and annealed at 500°C for 3 h. Due to significant particle aggregation caused by sintering, a final milling step was performed to enhance sample uniformity. The primary advantage of this protocol lies in scalability, enabling large-scale hydroxyapatite production. (III) Fabrication of functional foam hybrid materials The integration of inorganic components, magneto-light-responsive Fe3O4 cubic nanoparticles, and bioresorbable HAP nanoparticles into the PLLA matrix was carried out by using the TIPS technique supported with SLP (salt-leaching process). Further details on this 6 approach can be found elsewhere50. However, for this study, the process parameters were significantly modified. Specifically, PLLA (Resomer L210s, Evonik, Germany), along with the previously prepared HAP and cubic Fe3O4 nanoparticles, served as substrates. In the standard procedure, PLLA and HAP in a 50:50 weight ratio were suspended in 1,4-dioxane (99%, Sigma Aldrich, Poland) at 75°C under mechanical stirring overnight, ensuring the formation of a homogeneous mixture. Subsequently, different amounts of Fe3O4 in dioxane were added to achieve magnetic particle concentrations ranging from 1 to 30 wt%. The final solution was mixed vigorously with a stirrer and placed on a shaker to eliminate air bubbles. Sodium chloride (NaCl, Stanlab, Poland) with an average particle size of 500 µm was used as a porogen. After mixing, the blend was dispensed into a 24-wells plate (0.5 mL/well) and frozen at -40°C for 24 h. This was followed by freeze-drying at -50°C under a vacuum of 10 Pa for an additional 24 h to form porous foams. In the final step, foams, including the reference sample without Fe3O4, were subjected to SLP, which was repeated multiple times with fresh water exchange to ensure NaCl removal. Washing effectiveness was confirmed by the reaction of the AgNO3 solution with the supernatant to verify the absence of Cl⁻ anions. Final foam composites were dried at 40°C for 24 h and used for physicochemical and biological characterization. 2.3. Characterization of physicochemical properties of fabricated materials The X-ray powder diffraction technique (XRD) was employed to study the structural properties of the stock Fe3O4 magnetite and HAP particles as well as fabricated foams. All diffraction patterns were recorded using a Bruker D8 Advanced diffractometer with an X-ray source copper lamp (Kα1 1.54060 Å) within a 2Θ range of 15-65°. In addition, a Ni filter was utilized to filter out Kα2 associated reflections. In the case of highly X-ray-absorbing Fe3O4 particles and foams with incorporated magnetite, the data curation relied on background correction (Diffrac.Eva software (V.2)), normalization, and signal smoothing (Origin Pro 9, Origin Lab). The final results were compared with the reference cards from the ICDD database (International Centre for Diffraction Data). Sample preparation involved the evaporation of part of the ethanol suspension of Fe3O4 to a dry powder. No special treatment for HAP and foam measurements was used. Magnetite and calcium hydroxyapatite particle size, morphology, and distribution were determined by means of transmission electron microscopy (TEM) with a Tecnai Osiris X-FEG HRTEM microscope (FEI Company, USA) operating at 200 kV. Sample preparation required 7 placing a droplet of the ethanol-based nanoparticle suspensions of all inorganic materials (0.25 mg/ml) on a carbon-coated 200 mesh copper grids (EM Resolutions, United Kingdom) and slow drying overnight at room temperature under dust protection. In the case of the foam hybrids, microstructure imaging was carried out using a Tescan Vega 3 (Tescan Group, Czech Republic) scanning electron microscope. The sample was deposited to the carbon tape, mounted on an aluminum stage, and placed in the microscope chamber. Due to the risk of polymer melting, a low-voltage regime was used (max 5 kV). The size distribution analysis of Fe3O4, HAP particles, and pore diameter was performed using freeware ImageJ software (ver. 1.46r). The density and porosity of foams were measured using a hydrostatic balance Shimadzu AX 220 (Shimadzu Ltd., Japan). The porosity was calculated with the following formula: Φ= [1 −  ], (1) where Φp is porosity, ρsc represents scaffold density, and ρb is the bulk density of polymer. Both ρsc and ρb (1.24 g/mL) were measured by the buoyancy method. Fourier transform infrared spectroscopy (FTIR) was employed to analyze the structural features of the fabricated foams. Thermo Scientific Nicolet iZ10 spectrometer equipped with an attenuated total reflection (ATR) accessory. Spectra were recorded over a range of 2000 - 500 cm-1. Before measurement, a standard calibration procedure was performed to correct for air humidity. No special sample treatment was required. Samples were directly placed on the diamond crystal and pressed against the hot-spot using a bolt. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were performed using a TGA/DSC1 Mettler Toledo system under N2 atmosphere. For each foam sample, 5 mg of material was taken and heated from 50°C to 550°C at a heating rate of 20°C/min. Data from the TGA characterization were processed with STARe software and plotted in Origin 9.0. Composite glass transition and crystallization temperatures were determined utilizing a DSC apparatus coupled with a TC 100 intercooler under an inert N2 atmosphere (30 mL/min). For this purpose, approximately 6 mg of foam was placed inside an aluminum pan and heated from 0°C to 200°C at a rate of 5°C/min. Afterward, sample was cooled down to 0°C, and a reheating scan was performed to eliminate the foam’s thermal history and evaluate thermal transitions. 8 Magnetic measurements were performed using a Quantum Design MPMS7XL superconducting quantum interference device (SQUID) magnetometer. Zero-field-cooled (ZFC) and field-cooled (FC) magnetization curves were recorded during warming from 3 K to 350 K under an applied magnetic field of 10 mT. Isothermal magnetization measurements were conducted at 3 K and 300 K with magnetic fields ranging from –5 T to +5 T. To prevent physical rotation of the magnetic nanoparticles during measurements, the samples were immobilized within gelatin capsules using a small amount of adhesive. The organic content, originating from surfactant residues and the PLLA matrix, was quantified by thermogravimetric analysis (TGA) for each sample, and the magnetization data were subsequently corrected based on the inorganic (Fe₃O₄) nanoparticle mass. Heat generation ability of magnetite containing foams as well as reference PLLA/HAP sample, was measured using two external stimuli, namely alternating magnetic field (AMF) and near-infrared laser radiation (NIR, 808, 880, and 1122 nm) under separate and synergy mode. G2 D5 Series Multimode 1500 W driver (nanoScale Biomagnetics, Spain) equipped with a thermally insulated S32 coil was used (polystyrene box). In the case of laser stimulation, 808 nm, 880 nm, and 1122 nm continuous laser modules (power stability no worse than 1%) equipped with 400 µm optical fibers (CNI, China) were utilized. Lasers were calibrated with an Ophir StarLite power meter using a beam track thermal sensor 10 A-PPS (Ophir, Israel) with a measurable laser power range of 20 mW - 10 W, with laser beam size detection accuracy of 5%. The magnetic field frequency and intensity were chosen between 145 - 396 kHz and 22 – 32 kA/m, while laser output power for all three wavelengths was between 100 - 600 mW. All measurements were done on dry and Ringer’s solution soaked foams (2 h at room temperature), mimicking conditions at possible implantation sites. Temperature effects were recorded with a FLIR T660 scientific thermovision camera (FLIR, USA) and analyzed with dedicated ResearchIR software (FLIR, USA). The final data presentation was performed in Origin 9.0 software. 2.5. Cell culture experiments The normal human fetal osteoblast cell line (hFOB 1.19, American Type Culture Collection (ATCC), USA) was used to assess cytotoxicity through both indirect and direct methods, as well as to evaluate cell proliferation. Cells were cultured in a 1:1 mixture of DMEM/Ham’s F12 medium without phenol red (Gibco Thermo Fisher Scientific, USA), supplemented with 10% 9 fetal bovine serum (Pan-Biotech GmbH, Germany), 100 U/mL penicillin, 100 μg/mL streptomycin, and 300 μg/mL G418 (Sigma-Aldrich, Poland). The cells were maintained at 34°C in a 5% CO2 atmosphere. 2.5.1. Evaluation of cytotoxicity by indirect method Cytotoxicity was assessed using the MTT assay, in accordance with ISO 10993-5, using extracts of biomaterials prepared according to ISO 10993-12. In summary, extracts were obtained by incubating 25 mg of the composites in 1 mL of culture medium, which was dictated by the high capacity of the materials to absorb fluids, at 37°C for 24 h. Concurrently, a negative control for cytotoxicity was prepared. Cells were seeded in 96-well plates at a density of 1×105 cells/mL and cultured at 34°C for 24 h. Subsequently, the medium was replaced with 100 µL of the respective foam extracts, and the cells were incubated for an additional 24 h. After incubation, an MTT colorimetric assay (Sigma-Aldrich, Poland) was performed to evaluate cell viability and metabolic activity. Briefly, 25 µL of MTT solution (5 mg MTT/1 mL PBS) was added to each well and incubated for 3 h at 34°C to allow mitochondrial dehydrogenase enzymes in viable cells to reduce MTT to formazan. As a result of this reduction, formazan accumulated as crystals within the living cells. After the specified incubation period, 100 µL of 10% SDS solution in 0.01M HCl was added to each well to dissolve the formazan crystals. After 12 h, the absorbance of the referred to solubilized formazan concentration was measured using a plate reader at a wavelength of 570 nm. The amount of formazan is proportional to the number of viable cells. The results of the MTT assay were expressed as a percentage of the optical density (OD) value obtained relative to the negative control for cytotoxicity. Cytotoxicity was also assessed qualitatively with the Live/Dead Double Staining Kit (Sigma-Aldrich, Poland) following the manufacturer's protocol. The hFOB 1.19 cells treated with biomaterials extracts were stained with fluorescent dyes and observed utilizing a confocal laser scanning microscope (CLSM, Olympus Fluoview equipped with FV1000, Japan) 2.5.2. Evaluation of cytotoxicity by direct method In order to assess cytotoxicity using the direct method, hFOB 1.19 cells were seeded directly onto the materials at a density of 5×104 cells/mL. After 48 hours of culture, the cells were stained with the Live/Dead Double Staining Kit (Sigma-Aldrich, Poland), following the manufacturer's protocol. Subsequently, the samples were visualized with a confocal laser scanning microscope (CLSM, Olympus Fluoview equipped with FV1000, Poland). 16 a narrower temperature range. This suggests that magnetite nanoparticles may act as catalysts or oxidants, accelerating PLLA depolymerization at lower temperatures. Figure 6. TGA (left panel) and DTA curves (right panel) of the chosen PLLA/HAP/Fe3O4 foams as a function of magnetite concentration. Figure 7. The 1st heating DSC curves (a), the cooling DSC curves (b), and the 2nd heating DSC curves of PLLA/HAP and PLLA/HAP/Fe3O4 foams. 17 Analysis of the DSC results (Fig. 7) shows three distinct thermal effects, namely glass transition (Tg) within a temperature range of 61 - 69°C, cold crystallization around 100°C, and a melting temperature (Tm) between 178 - 185°C. During the 1st heating, a slight decrease in glass transition temperature was found, followed by an increase in magnetite concentration. A similar effect was noticed in the melting process, whose temperature peak for binary PLLA/HAP foam was at 185°C and decreased to 181°C for the composite with 20% Fe3O4. Upon integrating the magnetic particles into the PLLA/HAP, a noticeable promotion of nucleation in the crystallization process was observed. This phenomenon is particularly evident during heating and cooling cycles. It manifests as an increase in the endothermic and exothermic peaks. During the cooling, one can note that the exotherm peak is monomodal with a maximum of around 100°C for all foams. We did not observe a bimodal shape of the exothermic peak (two Tc temperatures) and their shift towards higher temperatures (up to 119°C) upon HAP amount increase as reported by Szustakiewicz50. In this study, the Tc remained stable. We believe that this is probably due to the different character of the interaction of magnetite particles. They did not significantly alter the crystallization of the PLLA polymer, like HAP. A similar observation was reported by Laraba et al.52 with graphene oxide (GO) as a filler of the PLLA matrix. However, GO shifted the melting temperature toward higher values. Interestingly, in the plot representing the 2nd heating, one can distinguish an exothermic peak with a maximum of around 161°C. In accordance with literature data, this behavior is associated with the phase transition of α’ to α form of PLLA, meaning that composite foams consist of a mixture of both PLLA structures57. The former one was not detected by XRD, probably due to its low content and the instrument's sensitivity. Moreover, the rise of concentration of magnetic particles led to an increase in the exothermic peak surface area, indicating the effect of Fe3O4 on the formation of a greater amount of the α crystalline phase. ZFC and FC magnetization curves (Fig. 8) exhibit a bifurcation above 350 K, indicating that all samples remain in a magnetically blocked state throughout the entire measured temperature range58. This behavior is consistent with the critical size for single-domain Fe3O4 nanoparticles (approximately 91 nm)59,60. The ZFC curves display a pronounced drop in magnetization around 100 K, which is attributed to the Verwey transition61 - a phenomenon characteristic of magnetite and absent in maghemite. No significant shift in the Verwey transition temperature was detected with increasing Fe3O4 nanocubes concentration in the 18 PLLA matrix, suggesting that interparticle interactions remain relatively weak and do not significantly influence the transition behavior. Isothermal magnetization measurements at 3 K reveal moderate coercivity (μ0Hc ≈ 0.04 T, or 400 Oe)46 across all samples (Fig. 8). The saturation magnetization values (83, 97, 87, and 94 Am²kg⁻¹ for 1%, 5%, 10%, and 15% Fe3O4 loading, respectively) remain close to that of bulk magnetite (Ms ≈ 90 Am²kg⁻¹), indicating the high crystallinity and preservation of the magnetic properties of the Fe3O4 nanocubes. At room temperature, the isothermal magnetization curves exhibit very low coercivity, further supporting the presence of a blocked magnetic state for the Fe3O4 nanocubes under ambient conditions. Figure 8. ZFC/FC magnetization curves (a) of Fe3O4 nanocubes and PLLA/HAP/Fe3O4 foams (dashed line indicates the Verwey transition, Tv) and their isothermal magnetization (b) at 300 K (inset: zoomed 3 K isothermal magnetization). 3. 2. Heating ability of the ternary PLLA/HAP/Fe3O4 foam composites under AMF and NIR stimulation The effectiveness of PLLA/HAP/Fe3O4 foam in converting energy into heat under an alternating magnetic field (AMF) and near-infrared light (NIR) was evaluated for both separate and synergic action (dual mode) of external stimuli. All measurements were conducted as a function of Fe3O4 concentration (1 - 30%) and optimized for the magnetic field frequency (149 - 496 kHz), intensity (22 - 32 kA/m, corresponding to 27.6 - 40.2 mT), laser wavelengths (808, 880, 1122 nm), and laser power (100 - 600 mW) for dry and Ringer’s solution soaked materials 19 (Figs. 9 - 13). A binary PLLA/HAP hybrid foam was used as the reference material. Optimization of the external stimulation parameters was performed on a PLLA/HAP/Fe3O4 sample containing 8% Fe3O4, which resulted in obtaining temperatures exceeding 43°C crucial for potential biological applications. Figure 9. Heating curves of the PLLA/HAP/Fe3O4 8% dry foam measured for (a) AMF stimulation, (b) 808 nm laser, (c) 880 nm laser, and (d) 1122 nm laser as a function of the AMF parameters and laser power (separate modes). The complete characterization of stock Fe3O4 nanoparticles and their heat-generation capabilities was the subject of our previous study on magneto-plasmonic heterostructures62. The Fe3O4 concentration dependence in PLLA/HAP/Fe3O4 was measured for Ringer’s solutionsoaked foams by using the following AMF parameters 336 kHz, 27 kA/m (Fig. 10). For dual 20 mode (synergy) heating experiments were conducted at 336 kHZ, 27kA/m, and 600 mW for three wavelengths 808, 880 and 1122 nm covering the Ist and the IInd optical biological window (Fig. 13). In all cases, the cooling stage was recorded for both dry and soaked composites. The optimization of the alternating field parameters and laser power was performed on a dry PLLA/HAP/Fe3O4 foam containing 8% magnetite cubic particles in order to choose the most reliable conditions for contactless heat generation. As shown in Fig. 9a, the best performance was achieved for the 336 kHz and 27 kA/m (33.9 mT) with a maximum temperature of 54.1°C. The 145 kHz and 32 kA/m (40.2 mT) also gave a high temperature of around 50°C for dry composite. In the case of the laser light, three wavelengths were tested 808, 880, and 1122 nm (see Fig 9b - 9d) for the 100, 200, and 300 mW laser power, respectively. A higher power was not applied since the Tmax of 93°C was achieved. Therefore, further increasing this parameter might lead to polymer melting and microstructure degradation, which will be seen as a detrimental effect. The variation in the Tmax (47 - 98°C) for lasers was attributed to the laser light spot size affecting heating ability. All used wavelengths have a capacity for significant heat induction due to the Fe3O4 efficient absorption that covers a broad spectral range63. Table 2. AMF and NIR conversion parameters, i.e., heating speed dT/dt and maximum temperature (Tmax) for the PLLA/HAP/Fe3O4 8% dry foam. Sample Parameters (AMF and NIR) dT/dt (°C/s) Tmax (°C) AMF PLLA/HAP/Fe3O4 8% 145 kHz, 32 kA/m 1.91 50.3 PLLA/HAP/Fe3O4 8% 336 kHz, 27 kA/m 2.49 54.1 PLLA/HAP/Fe3O4 8% 496 kHz, 22 kA/m 1.59 43.5 Laser 808 nm PLLA/HAP/Fe3O4 8% 100 mW 6.05 50.5 PLLA/HAP/Fe3O4 8% 200 mW 18.17 76.5 PLLA/HAP/Fe3O4 8% 300 mW 25.27 97.6 Laser 880 nm PLLA/HAP/Fe3O4 8% 100 mW 4.32 47.7 PLLA/HAP/Fe3O4 8% 200 mW 9.58 60.2 PLLA/HAP/Fe3O4 8% 300 mW 15.43 78.3 Laser 1122 nm PLLA/HAP/Fe3O4 8%% 100 mW 5.10 50.8 PLLA/HAP/Fe3O4 8% 200 mW 9.17 73.8 PLLA/HAP/Fe3O4 8% 300 mW 16.32 93.4 A comparison of the heating speed (Table 2) revealed a difference in the effectiveness of the heat generation mechanisms over one order of magnitude. For the AMF stimulation, the dT/dt 21 is between 1.59 – 2.49°C/s, while laser stimulation led to the values within the range of 5.125.2°C/s, showing its superior role over AMF heat induction. However, it has to be stressed that laser light utilization in biomedical applications will be problematic due to tissue penetration limitations, while AMF overcomes this drawback. Therefore, laser light will be limited to on-skin and shallow treatments. For deeper-tissue procedures, more invasive techniques such as laparoscopy must be used64,65. Although, foams are primarily designed for bone tissue replacement, other potential applications, such as wound-healing composites, remain viable. It is of great importance to measure the ability of heat induction in an environment that resembles the implantation site. Therefore, the PLLA/HAP/Fe3O4 foams were soaked for 2 h in the Ringer’s solution (Table 3), and further characterization of energy conversion was performed (Fig. 10 – 13). The interesting feature of all foams is their ability to water-based solution loading of ≈ 440 - 500%, except for PLLA/HAP/Fe3O4 above 20%, when a water loading drops below 400% due to the microstructure degradation. Figure 10 presents the heating curves recorded for the different concentrations of magnetic components (1 - 30%) under stimulation with the optimal AMF (336 kHz, 27 kA/m) parameters. Table 3. Mass change of the PLLA/HAP/Fe3O4 foams before and after soaking with Ringer’s solution. Sample Foam mass (g) Ringer’s solution soaked foam (g) Water (g) Water loading (%) PLLA/HAP/Fe3O4 1% 0.0318 0.1612 0.1294 506.9 PLLA/HAP/Fe3O4 5% 0.0229 0.1230 0.1001 537.1 PLLA/HAP/Fe3O4 10% 0.0382 0.1694 0.1311 443.2 PLLA/HAP/Fe3O4 15% 0.0373 0.1988 0.1615 533.0 PLLA/HAP/Fe3O4 20% 0.0465 0.1902 0.1437 409.2 PLLA/HAP/Fe3O4 30% 0.0592 0.1803 0.1211 304.6 It is very clear that the presence of a high water loading (Ringer’s solution) dramatically affects the heating ability due to the high specific heat capacity of water (4.184 J/g°C). In general, values of the heating speed dT/dt (Table 4) decreased strongly if directly compared to the dT/dt obtained for the PLLA/HAP/Fe3O4 8% composite (dry foam - 2.49°C/s, soaked foam - 0.07°C/s). It also caused a two-fold decrease of the Tmax from 54.1°C to 27.8°C. Since the AMF is a key stimulant for deep-tissue penetration, the bottom limit for samples that will allow achieving a biologically relevant temperature was a PLLA/HAP/Fe3O4 with 15% of magnetite (43.8°C with dT/dt of 0.52°C/s). In the case of the higher-doped foams, one can see a progressive increase in the Tmax (up to 74°C) and heating speed (0.52 to 1.39°C/s). However, as mentioned earlier, the degradation of the foam microstructure was already noticed above 22 20% of the Fe3O4. Thus, only samples with a maximum of 15% of magnetite were considered for further biological evaluation. Figure 10. Heating curves of the magnetite concentration dependence of the soaked PLLA/HAP/Fe3O4 foams under the action of the AMF (336 kHz, 27 kA/m). Table 4. AMF conversion parameters, i.e., heating speed dT/dt and maximum temperature (Tmax) for the PLLA/HAP/Fe3O4 Ringer’s solution soaked foams – concentration dependence. Sample dT/dt (°C/s) Tmax (°C) AMF 336 kHz. 27 kA/m PLLA/HAP/Fe3O4 1% 0.01 22.2 PLLA/HAP/Fe3O4 2% 0.01 22.9 PLLA/HAP/Fe3O4 5% 0.025 23.7 PLLA/HAP/Fe3O4 8% 0.07 27.8 PLLA/HAP/Fe3O4 10% 0.22 35.5 PLLA/HAP/Fe3O4 15% 0.52 43.8 PLLA/HAP/Fe3O4 20% 1.28 58.8 PLLA/HAP/Fe3O4 30% 1.39 74.7 To evaluate the effect of laser light stimulation, measuring the dependence of the laser power within the 300 - 600 mW range was necessary. It was anticipated that the presence of water molecules in soaked foams would significantly affect heating efficiency across all three wavelengths (Fig. 11). To ensure a proper comparison, heat induction was analysed using PLLA/HAP/Fe3O4 foams containing 8% magnetite. The final data are summarized in Table 5. It is evident that the high heat capacity of the solvent strongly influences energy conversion. This phenomenon was reflected in a drastic change of dT/dt values, which ranged from 0.36 23 to 2.32°C/s for soaked composites (dry 4.32 - 25.27°C/s, Tmax 43 - 97°C), along with a reduced Tmax of 33 - 45°C at 8% Fe3O4 doping. Figure 11. Heating curves of the PLLA/HAP/Fe3O4 8% soaked foam measured for (a) 808 nm, (b) 880 nm, and (c) 1122 nm laser modules as a function of the laser power 300 - 600 mW. Table 5. NIR conversion parameters, i.e., heating speed dT/dt and maximum temperature (Tmax) for the PLLA/HAP/Fe3O4 8% soaked foam. Sample Parameters (NIR) dT/dt (°C/s) Tmax (°C) Laser 808 nm PLLA/HAP/Fe3O4 8% 300 mW 0.64 34.5 PLLA/HAP/Fe3O4 8% 450 mW 1.59 40.8 PLLA/HAP/Fe3O4 8% 600 mW 2.32 45.2 Laser 880 nm PLLA/HAP/Fe3O4 8% 300 mW 0.46 33.2 PLLA/HAP/Fe3O4 8% 450 mW 0.42 34.7 PLLA/HAP/Fe3O4 8% 600 mW 1.04 40.2 Laser 1122 nm PLLA/HAP/Fe3O4 8%% 300 mW 0.36 33.7 PLLA/HAP/Fe3O4 8% 450 mW 0.39 38.6 PLLA/HAP/Fe3O4 8% 600 mW 1.35 43.1 Notably, NIR stimulation is significantly more effective than AMF in terms of both heating speed and maximum temperature. This feature is crucial to avoid the induction of cell thermotolerance in hyperthermia applications66. The direct comparison of the dry and soaked foams during the action of the NIR light (808, 880, and 1122 nm) was shown in Fig. 12. The concentration dependence of cubic magnetite nanoparticles in PLLA/HAP/Fe3O4 foams was measured under synergy mode. It involved, referred to here as dual mode, a combined action 24 of AMF and NIR laser light (AMF: 336 kHz, 27 kA/m; NIR: 600 mW at 808, 880, and 1122 nm) for soaked ternary composites (Fig. 13). For the PLLA/HAP/Fe3O4 sample with 8% magnetite, the maximum temperature (Tmax) reached was 49°C (AMF: 27.8°C, NIR808: 45°C, NIR880: 40°C, NIR1122: 43°C), demonstrating that the synergistic interaction of both stimuli leads to a further temperature increase. As shown, the Tmax obtained for all foams under dual mode was significantly higher than that achieved with individual stimulation. The highest recorded Tmax was observed for PLLA/HAP/Fe3O4 foams containing 30% magnetite, reaching 84°C (NIR808), 80°C (NIR880), and 82°C (NIR1122). However, foams with 20% and 30% Fe3O4 content exhibited a loss of their microstructure, making them unsuitable for further biological characterization. Figure 14 shows a comparison of different stimulation modes for PLLA/HAP/Fe3O4 with 15% magnetite, highlighting striking differences between the various energy conversion mechanisms. Notably, this sample could reach biologically relevant temperature ranges using AMF, NIR, and their synergistic combination. Figure 12. Comparison of the dT/dt and Tmax values for the dry, and Ringer’s solution-soaked foams with 8% of Fe3O4 under NIR stimulation (open symbols - dry, full-colored - soaked foams). 25 Figure 13. Heating curves of the magnetite concentration dependence under the action of both stimuli (AMF 336 kHz, 27 kA/m, 600 mW, 808, 880, and 1122 nm lasers). Figure 14. Comparison of the heating generation using different stimuli AMF, and NIR separately and under synergy mode (dual) for different laser wavelengths (808, 880, 1122 nm) on soaked PLLA/HAP/Fe3O4 15% foam. 3.3. 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