Composite magnetic 3D-printing filament fabrication protocol opens new perspectives in magnetic hyperthermia

Three-dimensional (3D) printing technology has emerged as a promising tool for meticulously fabricated scaffolds with high precision and accuracy, resulting in intricately detailed biomimetic 3D structures. Producing magnetic scaffolds with the aid of additive processes, known as 3D printing, reveals multitude and state-of-the-art areas of application such as tissue engineering, bone repair and regeneration, drug delivery and magnetic hyperthermia. A crucial first step is the development of innovative polymeric composite magnetic materials. The current work presents a fabrication protocol of 3D printed polymer-bonded magnets using the Fused Deposition Modeling 3D printing method. Polymer-bonded magnets are defined as composites with permanent-magnet powder embedded in a polymer binder matrix. By using a low-cost mixing extruder, four (4) different filament types of 1.75 mm were fabricated using commercial magnetite magnetic nanoparticles mixed with a pure polylactic acid powder (PLA) and a ferromagnetic PLA (Iron particles included) filaments. The powder mixture of the basic filaments was compounded mixed with the nanoparticles (NPs), and extruded to fabricate the 3D printing filament, which is subsequently characterized structurally and magnetically before the printing process. Magnetic polymer scaffolds are finally printed using composite filaments of different concentration in magnetite. Our results demonstrate that the heating efficiency (expressed in W g−1) of the 3D printed magnetic polymer scaffolds (ranging from 2 to 5.5 W g−1 at magnetic field intensity of 30 mT and field frequency of 365 kHz) can be tuned by choosing either a magnetic or a non-magnetic filament mixed with an amount of magnetite NPs in different concentrations of 10 or 20 wt%. Our work opens up new perspectives for future research, such as the fabrication of complex structures with suitable ferromagnetic custom-made filaments adjusting the mixing of different filaments for the construction of scaffolds aimed at improving the accuracy of magnetic hyperthermia treatment.

Three-dimensional (3D) printing technology has emerged as a promising tool for meticulously fabricated scaffolds with high precision and accuracy, resulting in intricately detailed biomimetic 3D structures. Producing magnetic scaffolds with the aid of additive processes, known as 3D printing, reveals multitude and state-of-the-art areas of application such as tissue engineering, bone repair and regeneration, drug delivery and magnetic hyperthermia. A crucial first step is the development of innovative polymeric composite magnetic materials. The current work presents a fabrication protocol of 3D printed polymer-bonded magnets using the Fused Deposition Modeling 3D printing method. Polymer-bonded magnets are defined as composites with permanent-magnet powder embedded in a polymer binder matrix. By using a low-cost mixing extruder, four (4) different filament types of 1.75 mm were fabricated using commercial magnetite magnetic nanoparticles mixed with a pure polylactic acid powder (PLA) and a ferromagnetic PLA (Iron particles included) filaments. The powder mixture of the basic filaments was compounded mixed with the nanoparticles (NPs), and extruded to fabricate the 3D printing filament, which is subsequently characterized structurally and magnetically before the printing process. Magnetic polymer scaffolds are finally printed using composite filaments of different concentration in magnetite. Our results demonstrate that the heating efficiency (expressed in W g −1 ) of the 3D printed magnetic polymer scaffolds (ranging from 2 to 5.5 W g −1 at magnetic field intensity of 30 mT and field frequency of 365 kHz) can be tuned by choosing either a magnetic or a non-magnetic filament mixed with an amount of magnetite NPs in different concentrations of 10 or 20 wt%. Our work opens up new perspectives for future research, such as the fabrication of complex structures with suitable ferromagnetic custom-made filaments adjusting the mixing of different filaments for the construction of scaffolds aimed at improving the accuracy of magnetic hyperthermia treatment. * Author to whom any correspondence should be addressed.
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Introduction
There have been more than two thousand years since magnetism has been associated with medicine. As early as 624-547 BC, it was the Greek scientist and astronomer Thales of Miletus who is considered the first one who made a connection between humans and magnets, believing that the soul produced motion and concluding that, as a magnet also produces motion in that it moves iron, it must also possess a soul [1]. In the middle of the 20th century, it was Gilchrist and his colleagues treated lymphatic nodes and metastases by injecting metallic particles heated by an external magnetic field [2]. Recently, magnetic nanoparticles (MNPs) have been increasingly linked with a plethora of biomedical applications [3] concerning not only therapy, with their use as heating agents in magnetic hyperthermia [4] and as specific agents in drug delivery [5] but also as diagnostic tools in MRI, serving as contrast agents [6].
In recent years, the need to create multifunctional materials, that can be used in multiple applications not only in the biomedical field, as has been already mentioned, previously, but also for household and automotive use as well as for audio, video, and computer industries, has led the scientific community to focus on the magnetic composite materials that are especially attractive by the respective manufacturing industries [7,8]. These materials combine properties of conventional polymers and magnetic materials (ferromagnetic and/or ferrimagnetic particles embedded or mixed in a polymer matrix) and lately are classified as 'magneto-polymeric materials' [9].
Meanwhile, there have been 30 years since threedimensional (3D) printing technology was invented. To improve understanding and communication among persons working in diverse industries, the ISO (International Organization for Standardization) has classified seven types of additive manufacturing (AM) processes [10]: UV-Vat Photopolymerization, Material Jetting, Binder Jetting, Material Extrusion, Powder Bed Fusion, Sheet Lamination and Directed Energy Deposition. A vat of liquid photopolymer resin on a platform is lowered in UV-Vat Photopolymerization method (also known as stereolithography) [11], and a laser beam draws a shape, forming a layer of photopolymerization that hardens. This liquid method is suitable with silicone materials and is quite precise, but it must be reinforced with structural support given by other materials, which frequently necessitates considerable post-processing time. It is mostly employed in the manufacture of complex medical gadgets and high-end recreational items. The print head, which is placed above the platform, drops micro-droplets that are positioned with charged deflection plates in the Material Jetting process [12]. This technology, similar to an inkjet printer, may employ a variety of materials, including silicones, polymers, and waxes. It takes a long time to build realistic models, prototypes, or, increasingly, extremely precise working components in small quantities since the reservoir must be often replenished. To bond the layers, the Binder Jetting process employs powderbased materials such as polymers, ceramics, and metals that are deposited by a roller and a liquid. While this procedure is quick and adaptable (changing powder ratio and colors), it has a significant post-processing time and is not structurally sound. It is utilized in industrial applications (including aircraft), dentistry and medical equipment, luxury applications, etc. For this work's purposes, the Material Extrusion Method (mostly known as fused deposition modeling (FDM) Method) is followed [13,14]. This approach is commonly employed in personal 3D printers, with material flowing through a heated nozzle and depositing in a continuous stream. Melting or spontaneous adhesion is used to connect the material from one layer to the next. Material extrusion has evolved beyond its domestic usage, and its polymer, plastic, and silicone materials give strong structural support for some industrial applications, notably in the automobile sector. Powder Bed Fusion method [15], like binder jetting, employs a powdered material deposited with a roller or blade but is fused by a thermal energy source given by an electron beam or laser. Powder bed fusion necessitates a pre-heated, near-vacuum chamber. It can work with metal and polymer materials to create sturdy support structures that are excellent for prototypes and visual models. It is, however, a time-consuming procedure that is utilized to produce crucial components such as jet engine parts. The Sheet Lamination technique has two variations: Ultrasonic additive manufacturing [16], which uses metal components tied together using ultrasonic welding, and laminated object manufacturing [17], which uses paper materials glued together with an adhesive. Layers of various materials are applied and bonded before the form is carved using a knife or laser beam. This procedure is low-cost and quick, but the initial precision is imprecise, necessitating post-processing. It is mostly used to create prototypes. Lastly, the Directed Energy Deposition technique [18] is fairly sophisticated, employing four or five coordinated axis arms to deposit metal powder or wires, ceramics or polymer materials melted by an electron beam or laser, around a stationary object, achieving excellent precision and a variety of finishings. This method is used to both produce and repair components. Until now, AM was associated with various biological uses, which inspired novel printing techniques to emerge and had been constantly improved upon to suit different unmet clinical needs [19]. This cost-efficient technique exploits biocompatible materials and is used to develop novel model implants to provide a greater understanding of human anatomy and diseases, and can be used for organ transplants, surgical planning, and the manufacturing of advanced drug delivery systems [20,21]. In addition, nowadays, 3D-printed medical devices and implants can be designed and customized for each patient to provide a more tailored treatment approach. These highly cost-effective, smaller-scale production, personalized devices are manufactured preoperationally and are benefited for their efficiency, accuracy, and the personalized uniqueness they provide [22].
In the last few years, although the 'marriage' of 3D technology and the production of magnetic composites has already begun [23][24][25], with a lot of groups working on the design and the fabrication of 3D-printed magnetic composite scaffolds [26][27][28][29][30], for biomedical use [31], and more specifically for bone tissue engineering, for targeted drug delivery [32] and magnetic hyperthermia [33,34], either with or without the mixing of MNPs, the need for establishing a composite magnetic 3D-printing filament fabrication protocol seems more demanding than ever. Many groups investigated combinations of magnetic materials [35] with polymer matrixes [36][37][38][39][40][41] to finally construct a respective filament to be used as a source to create 3D-printed polymer magnetic scaffolds [42], but none of them studied the influence of the filament fabrication protocol steps on the final application of the magnetic scaffolds. In our recently published work [43], we analyzed the methodology to create a reproducible and accurate protocol for assessing the heating efficiency of magnetic scaffolds serving as bone implants for deep-seated hyperthermia tumor treatment. Here, by taking advantage of our previous work, we present how a magnetic polymer filament fabrication protocol can be linked with the heating efficiency of the final 3D-printed magnetic polymer scaffolds. A detailed structural and magnetic analysis of the magnetic polymer filaments along with the 3Dprinted magnetic polymer scaffolds' specific absorption rate results, that give the fingertip of their magnetic hyperthermia efficacy, demonstrate how magnetite MNPs can be used as a tuning guide to fabricate suitable magnetic polymer scaffolds for a successful magnetic hyperthermia treatment.

Materials
For the purposes of this work a typical polylactide (EasyFil polylactic acid powder (PLA) filament of 1.75 mm in diameter equipped by FormFutura) thermoplastic filament, commercial magnetite NPs provided by Alfa Aesar (consisting of spherical shape MNPs with a diameter of 50-100 nm) as well as magnetic filament provided by Protopasta (PP) [44], known as PP Magnetic Iron PLA, which is a compound of Natureworks 4043D PLA and finely ground iron powder, are used as reference filaments and as a feedstock material to prepare the composite filaments. The choice of the PLA, as the thermoplastic material used in our filaments, is made as it is one of the most popular thermoplastics in the field of 3D printing industry, available almost in any 3D printing laboratory and for its remarkable features; presents a relatively low melting point between 170 • C and 180 • C and a low viscosity approximately 4.0 dl g −1 at 0.1% (w v −1 ) in chloroform (25 • C). Its biodegradable behavior makes it suitable for biomedical applications as well. Finally, it does not release toxic pollutants, as is the case with acrylonitrile butadiene styrene (ABS), which is presented as a key advantage over other thermoplastic materials.
Magnetite NPs, being by far the most employed iron oxide NPs in biomedical applications, have been chosen in this work as the magnetic compound of the composite filaments. The two different MNPs contents that are used to synthesize the composite filaments are 10 and 20 wt% relative to the PLA thermoplastic material.

Filament fabrication
The flow chart in figure 1 provides an overview of this work's protocol suggestion, beginning with the production of composite filaments and progressing through the 3D printing of magnetic scaffolds and finally to their heating evaluation. The manufacture of composite filaments can be divided into four main stages, as illustrated in figure 2: (a) commercial filament pulverization, (b) materials mixing, (c) composite material drying and (d) extrusion of the composite filament.
For the fabrication and more specifically for the extrusion process of the composite filaments, a low-cost and highly efficient single screw extruder (figure 2(c)), named Filament Extruder VT110 and provided by the 3D-Tech company, was used.
The main process followed for the filament extraction is illustrated in figure 2. After cutting the PLA filament into pieces, a grinder is used to make the PLA granules. The main part of the process is to convert the desired composite into granules (∼5 mm in diameter as shown in figure 2(a)) to create the PLA pellets or the PLA ground material (figure 2(b)). The latter is inserted as feedstock material into the single screw extruder, as it is shown in figure 2(c). To have better control of PLA filament grinding results, the material temperature is kept below 0 • C. Regarding the extrusion process, the rotation speed of the extruder is set at 10 rpm. An automatic filament winder (automatic filament winder is also equipped together with the Extruder VT110 by the 3D-Tech company) is adapted onto the extruder, providing feedback to the extruder and the rotational speed of the spool (this controls the filament diameter: the faster the spool rotates, the thinner the filament). The main process for the filament extraction: (a) after cutting PLA filament into pieces, a grinder is used to make PLA granules. The (b) mixed material is used for feedstock of (c) the single screw extruder to finally produce (d) the resulting magnetic filament. The speed of the spool is determined by a closed-loop control system. The feedback is given by the laser micrometer that measures the filament's diameter as it exits the extruder, thus, controlling at the same time the rheological behavior of the composite filaments by tuning the extrusion flow rate. After extruding the filament, to achieve better flow results and a more consistent diameter, the filament is ground again and the whole process is repeated at least three times. Following filament extrusion, the filaments were placed in a desiccator at 70 • C for 12 h to maintain zero humidity conditions. The extruded synthetic filaments (with a 1.75 ± 0.01 mm filament diameter as shown in figure 2(d)) are composed of Fe 3 O 4 and thermoplastic material either with (PP material) or without (PLA material) iron particles, named as Fe@PP and Fe@PLA, respectively. Fe@PP and Fe@PLA composite filaments are fabricated using two different concentrations of magnetite in thermoplastic material, 10 and 20 wt%, named as Fe@PP10, Fe@PLA10 and Fe@PP20, Fe@PLA20, respectively. The nomenclature used in this work is summarized in table 1.
It should be mentioned here that the presence of MNPs in composite filaments influences their fluid dynamic behavior and, more specifically, their extrusion temperature. In order to achieve the optimal filament extrusion conditions (when the filament is not extruded properly then it is too soft, mechanically unstable and brittle), we adjust the extrusion temperature by +5 • C for every 10 wt% of MNP filament addition. This means that for the reference filaments of PLA and PP, the extrusion temperature is set at 200 • C, for the composite filaments with 10 wt% of NPs content (Fe@PLA10 and Fe@PP10) the extrusion temperature is set at 205 o C and last, for the filaments with the highest MNP content of 20 wt% (Fe@PLA20 and Fe@PP20), the extrusion temperature reaches 210 • C.
2.2.1. 3D printing scaffolds. The produced 3D printing filaments are used to construct the magnetic scaffolds. A low-cost and easy-to-operate FDM printer, LK4 Longer, was used to 3D print the magnetic scaffolds. Scaffolds' models were printed using a custom-made software written in MATLAB [45,46], which is currently under development as the first image-guided slicing software named 'Reform' from the startup company 'Morphé' [47]. Our recently published work [43] was a guide here to choose the scaffolds dimensions. In that work, a standardization protocol with certain experimental steps was proposed for an accurate evaluation of the heating efficiency of the 3D-printed magnetic scaffolds bone phantoms. Based on that, the shape investigated comprised of cylindrical (height 16 mm diameter 19 mm) structure. Morphé's under-development slicing software differs from other conventional 3D printing software. The goal of this software is to deposit material over the entire printing surface. In 'Reform' the infill density parameter (which is the crucial parameter that one must consider using a conventional slicing software) is replaced by the extrusion flow rate parameter. This parameter defines the material extrusion flow rate per a predefined voxel, and thus the amount of a melted filament deposited on each layer during 3D printing. This parameter essentially replaces the infill density parameter in commercial slicing software such as Ultimaker Cura [48]. 3D printing scaffolds' dimensions and printing parameters kept the same for all fabricated scaffolds examined in this work. Finally, six scaffolds were printed in this study. Four for each magnetic filament we extruded as well as two reference scaffolds printed with commercial PP and PLA filaments, respectively.

Structural and magnetic characterization
For the structural and magnetic characterization analysis of the filaments used in this work, the following characterization techniques are used: the phase identification of the filaments are examined by SIEMENS D500 x-ray diffractometer using the Ka line of Cu as a radiation source. The data were collected in the range of diffraction angle 2θ = 10-80 • at 8 • min −1 . The micromorphology of the filaments is characterized by scanning electron microscopy (SEM): A JEOL JSM-6390LV scanning microscope is used to study the topography of the surface as well as of the cross-section of the filaments. Magnetic properties of the scaffolds are investigated by vibrating sample magnetometry (VSM; 1.2H/CF/HT Oxford Instruments VSM) in a magnetic field range of ±1 T.

Phantom preparation
In our previous work [43], agarose gel was used not only as a tissue-mimicking phantom but as a heat diffusion medium between the scaffolds' pores as well. Based on that protocol, to prepare the magnetic scaffolds for magnetic hyperthermia experiments, scaffolds were placed in an agarose matrix. Gels are made by heating up agarose at 80 • C in an appropriate buffer followed by a freeze-thaw cycle of the aqueous agarose solution. Phantoms were prepared by mixing the scaffolds with distilled water (50 ml in agarose concentration of 2 g l −1 ) at room temperature. A magnetic stirrer was used to achieve uniform distribution. Gels were fabricated in small open glass vessels where the magnetic scaffolds were placed.

Magnetic hyperthermia
The Easyheat AC field induction heating system, operating at the power of 2.4 kW and provided by Ambrell, was used for the magnetic hyperthermia measurements of this work. An eightturn water-cooled induction copper coil is connected to the head of the induction system. Two chillers in a row are used, in a closed water-cooling system, to keep the coil temperature at ∼17 • C with a flow rate of 2.8 l min −1 and input pressure of a minimum 2.8 bar, during alternating magnetic field (AMF) application. All magnetic hyperthermia measurements were done under the magnetic field of 24 kA m −1 at the frequency of 365 kHz while the scaffolds in agarose matrix were centered in the eight-turn induction heating coil with optical fiber positioned in the center of each scaffold. All samples' initial temperature (before turning the magnetic field on) was set at 17 • C, while coil cooling water temperature was kept stable at 17 • C during the whole experimental procedure. Due to optical fiber's temperature sensitivity (not able to withstand high temperatures, over 80 • C), AC magnetic field was turned on for 100 s for each sample measurement, while samples' cooling was also applied at 17 • C for approximately 2 min.

Results and discussion
The structural and magnetic profiles of the commercial PP filament are characterized using x-ray diffraction (XRD), SEM and VSM characterization techniques as shown in figures 3-5. XRD peaks of PP 3D printing magnetic filament demonstrate the existence of iron particles in PP magnetic filament is given in figure 3. The surface along with the cross-section morphology of the commercial PP filament is shown in figure 4(d), emphasizing that the iron particles are homogeneously dispersed into the whole area. Energy dispersive x-ray analysis (EDX) analysis gives an estimation of the amount of iron in the filament, it ranges between 50-80 wt%. Magnetic hysteresis loop of PP 3D printing magnetic filament presented in figure 5 shows the ferromagnetic behavior of the commercial filament  This value resides within the range estimated by EDX since the saturation magnetization value is a macroscopic quantity of the whole specimen in contrast to EDX analysis which depends strongly on the regional spectrum that is chosen to be analyzed. Coercive field and saturation magnetization are also measured at 12.5 mT and 126 A m 2 kg −1 respectively, outlining the iron content i.e. the ferromagnetic nature and the potential exploitation in bioapplications [49]. Figure 3 represents the XRD spectra of the reference filaments of PP and polylactide (PLA), as shown in red and purple color, respectively, of the reference magnetite NPs (Fe 3 O 4 ), as shown in dark yellow color, as well as of the composite filaments Fe@PP10, FePP20, Fe@PLA10 and Fe@PLA20 shown in orange, light-purple, blue and green color, respectively. The labeling of the strongest crystal peaks is shown according to the reference sheets PDF: #19-0629 and PDF: #06-696 for magnetite and iron, respectively. It can be clearly seen from figure 3 that magnetite main diffraction peaks at (220), (311), (400), (422), (511), and (440) shown with dotted pink lines, are revealed not only in crystalline magnetite NP powder (shown with dark yellow color in figure 3) but also in composite filaments with magnetite NPs (Fe@PP10, FePP20, Fe@PLA10, and Fe@PLA20). Additionally, the two main diffraction peaks of Fe crystal at (011) and (002) crystal planes are observed at commercial PP filament as well as at the respective composite filaments Fe@PP10 and Fe@@PP20, shown in figure 3 with red, orange and light purple color, respectively.
The surface morphology of the commercial PLA and iron PP filaments together with their respective composite filaments Fe@PLA10, Fe@PLA20, Fe@PP10, and Fe@PP20 are shown in figure 4, emphasizing, in the case of Fe composite filaments (figures 4(b)-(f)), that the iron particles are homogeneously dispersed within filament volume. Additionally, magnetite MNPs dispersed in PLA matrix, in the case of composite filaments, seem to form clusters of a few µm in diameter (figures 4(b), (c), (e) and (f)). It can be also noticed that iron and iron oxide particle clusters of a few µm are formed in both cases for commercial and composite PP filaments as well as for composite filaments Fe@PLA10, Fe@PLA20. The existence of iron microparticles in PP filaments (both commercial and composites, as shown in figures 4(d)-(f)) has an impact on their surface morphology compared with the respective PLA filaments (both commercial and composites, as shown in figures 4(a)-(c)). It must be noted here that no degradation effects were observed after material reprocessing. It has been observed that even after the first melt-extrusion cycle the filaments remain stable with quite precise dimensions (1.75 ± 0.01 mm in diameter) and were able to be inserted in 3D-printed head. It is worth mentioning that the brittleness of each filament was reduced after each cycle, making the filament more stable for printing. As it has been elsewhere [50] analyzed before, the final filament's homogeneity can be further improved by rheological studies. More specifically, in Ahmad et al work, the rheological and morphological properties of oil palm fiber-reinforced ABS composites are used as a feedstock material for FDM. In our work, the three parallel and symmetrically engraved prominent notches that one can see in figure 4(a) and (d) are essential for the proper filament extrusion mechanism, carried out by the FDM extrusion head.
The magnetic hysteresis loops of the magnetic composite filaments containing Fe 3 O 4 NPs and Fe particles as well as of the commercial PP filament and magnetite NPs are shown in figure 5, where their coercivity can be observed in the close-up image in the inset of figure 5. It can be clearly seen that the existence of pure iron microparticles in the PP filament is the reason why its saturation magnetization (M s ) presents the highest value (126 A m 2 kg −1 ) among the other samples in figure 5. The respective magnetic hysteresis loops of the composite filaments Fe@PP10 and Fe@PP20, colored orange and light purple in figure 5, respectively, indicate that the presence of magnetite NPs in PP filament has an impact on the respective saturation magnetization value. More specifically, the higher the wt% MNPs content in the filament the stronger the M s reduction. Since M s of bulk iron (M s = 240 A m 2 kg −1 ) is significantly higher (more than 2.5 times) than M s of bulk magnetite (M s = 90 A m 2 kg −1 ), such an impact on the respective M s values of the composite filaments Fe@PP10 (M s = 105 A m 2 kg −1 ) and Fe@PP20 (M s = 91 A m 2 kg −1 ), can be expected and is clearly presented in figure 5. On the other hand, the injection of magnetite MNPs into the nonmagnetic PLA filament has a positive effect on the respective composite filament's M s . Hysteresis loops of Fe@PLA10 and Fe@PLA20 filaments presented with blue and green color in figure 5, respectively, demonstrate that the composite filament's magnetization can be controlled by the amount of MNPs dispersed into the filament. 10 and 20 wt% of MNPs at Fe@PLA10 and Fe@PLA20 filaments increase the filaments' M s values to 9 and 17 A m 2 kg −1 , respectively. It is also apparent from the magnetic hysteresis loop of magnetite MNPs, presented with dark yellow color in figure 5, that the M s value (96 A m 2 kg −1 ) reaches the M s value of the bulk magnetite (96 A m 2 kg −1 ), indicating the high crystallinity of the magnetite-MNPs, a fact that also results from their sharp XRDs peaks shown in the respective diffraction pattern in figure 3. Regarding the coercivity values (H c ), presented in the inset of figure 5, one can notice that upper and lower H c correspond to magnetite MNPs and PP filament, 13 and 4 mT, respectively, with coercivity values of the composite filaments varying between the aforementioned limits. It should be noted that H c , as well as M s , are crucial filaments' magnetic properties that have a significant impact on the heating efficiency of the corresponding magnetic scaffolds fabricated by the respective filaments and finally on their potential use as magnetic hyperthermia agents. Finally, looking at the remanence magnetization (M r ) values in the inset of figure 5, it can be noticed that this can be controlled by the amount of magnetite MNPs in the filaments, since pure magnetite MNPs M r value reaches 13 A m 2 kg −1 , where the composite filaments show M r values lower than 4 emu g −1 . All the samples' magnetic properties are summarized in table 2.
Our results are in very good agreement with the recently published of Kania et al work [37] and their suggestion to fabricate magnetic filaments made from nanocomposite materials by material extrusion. In that work, similarly to us, 3D-printed magnetic polymer composites were synthesized by mixing magnetite NPs with a polymeric matrix (polyethylene glycol, polyvinyl butyral and silicone gel). Similar to our results, their composite filaments presented comparable to our M s values, showing that magnetization is enhanced with increasing magnetite NP concentration. More specifically, for the filaments with 20 wt% of magnetite they show M s ranging between 20-30 A m 2 kg −1 while the coercive field remains similar to our filaments' H c levels (less than 10 mT). M r values follow the same trend as in Kania et al work [37]. M r of the final filaments were between 2-3 A m 2 kg −1 for the filaments with 20 wt% of magnetite.

Heating evaluation of composite magnetic scaffolds
In our previous work [43], the difference between MNPs and magnetic scaffolds heating evaluation was highlighted and corroborated. The terms SLP and SAR were discussed in detail, emphasizing their differences in calculating the heating performance of MNPs and magnetic scaffolds, respectively. To conclude the most appropriate term for the magnetic scaffolds heating evaluation is SAR. Since heat is diffused in the scaffold's surrounding medium (agarose in our case, tissue in the case of in vivo experiments), a simple formula of SAR, as given in equation (1), can give accurate results of the scaffold's heating profile. The following formula represents the correlation between the rise in the phantom temperature and SAR [51]: where, ∆T ∆t −1 ( • C s −1 ) is the initial heating rate, and c (J kg −1 o C −1 ) is the specific heat capacity of the agarosegel phantom that was set to be 4184 J kg −1 • C −1 . Based on that proposed protocol, the heating evaluation of all scaffolds prepared in this work began with their hyperthermia heating curves, as presented in figure 6. The temperature of the 3D-printed magnetic scaffolds in agarose matrix is rapidly increased after the application of AC field at the magnetic field amplitude of 30 mT and at the frequency of 365 kHz. On the contrary, temperature remains unchanged for the scaffold printed by the non-magnetic PLA filament under the same AC conditions (black curve in figure 6). Upon a first reading of figure 6, 10 or 20 wt% of magnetite in scaffolds gives them a magnetic profile suitable for magnetic hyperthermia. This is strongly demonstrated by the pink hyperthermia zone, shown in figure 6, that all magnetic scaffold samples exceed in very few seconds (less than a minute) after the AC magnetic field is on. In our recently published work [52], we showed that among the different heating evaluation methods used in magnetic hyperthermia laboratories worldwide, the modified law of cooling is the most accurate one to follow, limiting the SLP uncertainty to values under 5%-compared to other conventional methods like 'initial slope' and 'Box-Lucas'. The modified law of cooling is also used here to quantify figure 6 hyperthermia curve results, giving the SAR values of all scaffolds depicted in figure 7. It should be noticed that all magnetic scaffolds studied in this work show significantly high SAR values comparable with those mentioned in our previous study [43]. Additionally, in Zhang et al work [42], 3D printed mesoporous bioactive glass/polycaprolactone (Fe 3 O 4 /MBG/PCL) composite scaffolds containing Fe 3 O 4 NPs in wt% concentrations between 0-9.3 were tested in magnetic hyperthermia at AC frequency of 409 kHz and AMF amplitude of 14.3 kA m 1 . For similar AC induction heating conditions followed in this work, magnetic composite scaffolds at in Zhang et al study [42] showed a positive correlation between heating rate and Fe 3 O 4 content in the scaffolds. In that work, SAR values ranged from 0 to 4.7 W g 1 , for magnetic scaffolds without or with 9.3 wt% magnetite NPs, respectively. Heating performance of the composite scaffolds fabricated with 3D printing in this work show comparable to Zhang et al work results, regarding the SAR value level as well as the trend their heating performance follows with respect to the magnetite composition in the scaffold.
More specifically, magnetic scaffold printed with PP filament (reference scaffold PP shown with red color in figures 6 and 7) shows the best heating performance among all magnetic scaffolds. The respective thermal efficiency of the composite magnetic scaffolds Fe@PP10 and Fe@PP20, colored with orange and light-purple color, in figures 6 and 7, respectively, is slightly reduced compared with the ones of PP. Additionally, the more the amount of MNPs dispersed in the filament the higher the reduction in the heating performance. On the other hand, the heating performance of the composite magnetic scaffolds Fe@PLA10 and Fe@PLA20, shown in blue and green color in figures 6 and 7, respectively, indicates that SAR values of composite scaffolds, printed with a non-magnetic PLA material, are more enhanced for higher magnetite NP percentages.
To summarize, these results indicate that the heating efficiency of a composite magnetic scaffold can be controlled by choosing either a magnetic (like PP) or a non-magnetic (like PLA) filament mixed with an amount of magnetite NPs. These heating efficiency results come in very good agreement with the magnetic characterization findings. More specifically, we previously showed in figure 5 that the magnetic profile of all scaffolds can be tuned by the amount of magnetite NPs dispersed in the filament. Starting with a non-magnetic material like PLA, the filament's M s can be increased by filling the filament with magnetite NPs. On the contrary, magnetic iron filament's M s can be easily reduced by increasing the percentage of magnetite MNPs in the filament. The impact of the MNPs' existence in the filament on its final magnetic properties strongly affects the heating efficiency of the final magnetic scaffolds printed by the respective filament. Until now, magnetic filament fabrication protocols [36][37][38][39][40][53][54][55] have been referred to as the specific steps we take to construct the filament without considering final application feedback. In our work, we propose a protocol to produce magnetic filaments for magnetic hyperthermia use. These filaments are used to fabricate 3D printed magnetic scaffolds, with structural and magnetic properties influencing not only the filament's stability but also the final heating result. The magnetic scaffold's heating evaluation is a part of the filament's construction process. The optimal percentage of NPs dispersed in the filament is adjusted by the filaments' magnetic and structural properties as well as by the final 3D printed scaffolds thermal effect. Additionally, after extruding the filament and in order to improve the filament's homogeneity, we propose three melt-extrusion cycles, applied to enhance the NPs distribution homogeneity in the filaments. This work opens up new avenues for future research, such as the fabrication of complex structures with suitable ferromagnetic custom-made filaments controlling the extrusion rate per voxel and the mixing of different filaments for the fabrication of scaffolds aimed at improving the accuracy of magnetic hyperthermia treatment [46,47].

Conclusions
This work proposes a detailed protocol for magnetic polymer filament fabrication with tunable magnetic features and consequently magnetic heating. By following very precise steps, magnetite MNPs were mixed with a pure (PLA) or/and with a magnetic polymer (PLA with iron particles) matrix in different concentrations 10 and 20 wt%. The impact of the MNPs' presence in the filament was examined not only on the magnetic and structural properties of the filament but also on the fabrication process of the magnetic polymer scaffolds as well. Finally, magnetic polymer scaffolds 3D printed at specific dimensions, to mimic bone implants, and their heating efficiency was examined in AC magnetic hyperthermia. Our results demonstrated that the magnetite NPs may play a key role in the magnetic polymer filament fabrication as they can act as tuning factors on their final magnetic, structural and thermal properties. Furthermore, our results highlight the significance of controlling the MNP dispersion homogeneity during filament extrusion procedure, which can be accomplished by controlling the flow rate in the filament outflow, step that will further reduce the uncertainty and probability of failure during 3D printing while also increasing the accuracy in magnetic hyperthermia. Based on the findings of this work, future studies will focus on the fabrication of complex structures using suitable ferromagnetic custom-made filaments that control the extrusion rate per voxel and the mixing of different filaments for the fabrication of scaffolds aimed at improving the accuracy of magnetic hyperthermia treatment.

Data availability statement
All data that support the findings of this study are included within the article (and any supplementary files).