Co0.5Mn0.5Fe2O4@PMMA Nanoparticles Promotes Preosteoblast Differentiation through Activation of OPN-BGLAP2-DMP1 Axis and Modulates Osteoclastogenesis under Magnetic Field Conditions

The prevalence of osteoporosis in recent years is rapidly increasing. For this reason, there is an urgent need to develop bone substitutes and composites able to enhance the regeneration of damaged tissues which meet the patients’ needs. In the case of osteoporosis, personalized, tailored materials should enhance the impaired healing process and restore the balance between osteoblast and osteoclast activity. In this study, we fabricated a novel hybrid material (Co0.5Mn0.5Fe2O4@PMMA) and investigated its properties and potential utility in the treatment of osteoporosis. The material structure was investigated with X-ray diffraction, Fourier-transform infrared spectroscopy with attenuated total reflectance, FTIR-ATR, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and selected area (electron) diffraction (SAED). Then, the biological properties of the material were investigated with pre-osteoblast (MC3T3-E1) and pre-osteoclasts (4B12) and in the presence or absence of magnetic field, using RT-qPCR and RT-PCR. During the studies, we established that the impact of the new hybrids on the pre-osteoblasts and pre-osteoclasts could be modified by the presence of the magnetic field, which could influence on the PMMA covered by magnetic nanoparticles impact on the expression of genes related to the apoptosis, cells differentiation, adhesion, microRNAs or regulating the inflammatory processes in both murine cell lines. In summary, the Co0.5Mn0.5Fe2O4@PMMA hybrid may represent a novel approach for material optimization and may be a way forward in the fabrication of scaffolds with enhanced bioactivity that benefits osteoporotic patients.


Introduction
Osteoporosis (OP) is to the most frequent bone disease which can occur at any age stage, although women are more predisposed than men [1]. OP is common in societies all over the world due to progressive aging. The disease is characterized by reduced bone strength, mineral density and biomechanical properties, which together trigger bone fractures. According to the NIH Consensus Development Panel on Osteoporosis For that reason, in the presented study, we developed Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA nanohybrids that were cultured with both pre-osteoblasts and osteoclasts under normal and static magnetic field conditions. In this study, we have found that under normal and magnetic field condition, Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA improves pre-osteoblast activity and induces the expression of OPN-BGLAP2-DMP1 axis by activation of integrins, while inhibiting osteoclastogenesis.
In summary, by utilizing Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA nanohybrids modulating of osteoblasts/osteoclast activity might occur, therefore becoming an interesting approach for developing a strategy for future osteoporotic related fracture and bone regeneration.

Materials and Methods
2.1. In Situ Synthesis of the Binary Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA Hybrids The magnetic field responsive stock colloidal suspension of the Co 0.5 Mn 0.5 Fe 2 O 4 nanoparticles was prepared via microwave driven non-hydrolytic approach described in detail elsewhere [14]. The magnetic characterization of Co 0.5 Mn 0.5 Fe 2 O 4 was presented by our group previously interested reader is advised to follow that article [14]. In short, the following metal acetylacetonates were taken 1 mmol (257 mg) of Co(acac) 2 (99.9%, Alfa Aesar, Kandel, Germany), 1 mmol (253 mg) of Mn(acac) 2 (99.9% Alfa Aesar, Kandel Germany), 4 mmol (1413 mg) of Fe(acac) 3 (99.99%, Alfa Aesar, Kandel Germany) and subsequently dissolved in 70 mL of acetophenone (99% Sigma Aldrich, Poznań, Poland, used without further purification). All handling with the metal complexes has been done under an inert atmosphere of N 2 using an acrylic glove box (GS Glove Box Systemtechnik GMBH P10R250T2, Sömmerda, Germany). The reaction mixture was directly transferred into the Teflon vessel, secured and placed inside of the Ertec ® Magnum V2 microwave reactor (Ertec, Wrocław, Poland). The process was carried out under autogenous pressure of 15 atm, at 200 • C for 60 min. Afterward, Co 0.5 Mn 0.5 Fe 2 O 4 nanoparticles were separated from the solvent through washing, centrifuging cycles and re-suspended in 30 mL of de-ionized water. The final concentration of the Co 0.5 Mn 0.5 Fe 2 O 4 particles was measured using the micro-scale technique. In the case of the binary Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA hybrids, the in situ polymerization protocol was adopted without any changes as described previously [15]. The main chemicals were methyl methacrylate (MMA) monomer (99% Sigma Aldrich, Poznań, Poland), potassium peroxydisulfate as a polymerization initiator (≥99.0%, KPS, Sigma Aldrich, Poznań, Poland), benzethonium chloride (≥96.0%, Hyamine ® 1622, Sigma Aldrich, Poznań, Poland), as well as stock Co 0.5 Mn 0.5 Fe 2 O 4 particles. The MMA monomer was purified from the hydroquinone (inhibitor) through washing with 10% water solution of NaOH (≥97.0% Sigma Aldrich, Poznań, Poland) and, finally, dried with MgSO 4 (≥97.0% Sigma Aldrich, Poznań, Poland) prior usage. Briefly, 3 mL (4 mmol) of hyamine containing aqueous solution was added to the 6 mL of the Co 0.5 Mn 0.5 Fe 2 O 4 (concentrated stock suspension containing 100 mg particles) and mixed with 13 mL of de-ionized water. An ultrasound bath was used to homogenize dispersion for 20 min. After that, the mixture was transferred to a four-neck flask equipped with a mechanical stirrer, gas inlet (N 2 ), dropping funnels and Pt-100 sensor for temperature control. The MMA (in proportion of 80% to 20% of particles) was slowly injected and a KPS initiator was added. The reaction vessel was heated up to 80 • C for 3 h under constant stirring and nitrogen blanket. The binary hybrids were separated using a laboratory magnet and carefully dried under a vacuum.

Characterization of Basic Physicochemical Properties of PMMA@Co 0.5 Mn 0.5 Fe 2 O 4 Hybrids
Structure identification of Co 0.5 Mn 0.5 Fe 2 O 4 nanoparticles and Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA hybrid materials was carried out employing X-ray powder diffraction technique using a PANalytical X'Pert PRO X-ray diffractometer (Cu-K α1 = 1.54060 Å, nickel filtering, Malvern, UK) recording XRD patterns at the range of 2Q = 10-75 • and their direct comparison with the reference standards from the ICDD database (International Centre for Diffraction Data). The morphology and particle size were discussed based on the transmission electron microscopy (TEM) in the case of the nanoparticles whereas hybrid materials were subjected to the scanning electron microscopy (SEM) technique to prevent hybrids from unwanted deterioration induced by the high energy electron beam. Therefore, a Philips CM-20 Super Twin microscope (Philips, Amsterdam, The Netherlands), operated at 200 kV was used for the TEM characterization while hybrids samples were imaged with a Nova Nano-SEM 230 microscope (FEI Company, ThermoFisher Scienitific, Waltham, MA, USA). The measurements of the FTIR-ATR spectra were performed on a Nicolet iZ10 spectrometer (Thermo Fischer Scientific, Waltham, MA, USA) equipped in diamond ATR accessory covering the spectral range between 4000-500 cm −1 at room temperature. Magnetic characterization of the Co 1−x Mn x Fe 2 O 4 colloids was presented by us previously [16]; thus, the interested reader is advised to follow that article for details. The hybrid particle mean size and distribution were estimated by using free-image processing software ImageJ v.1.46 [17] through analysis of SEM images by taking into consideration of 100 hybrid particles (longest diameter was measured due to elongated shape of objects).

Cell Lines
The mouse pre-osteoblast mouse cell line (MC3T3-E1-subclone 4) was obtained from the European Collection of Authenticated Cell Cultures (EACC, Sigma-Aldrich, Munich, Germany), while the mouse pre-osteoclast cell line (4B12) was a kind gift from the Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry (Professor S. Amano) [18]. The MC3T3-E1 cell line was maintained in Minimum Essential Medium Alpha (MEM-α, Gibco, Waltham, MA, USA) without ascorbic acid supplemented with 10% Fetal Bovine Serum (FBS, Merck, KGaA, Darmstadt, Germany) and 1% of standard antibiotics (Merck KGaA, Darmstadt, Germany). The 4B12 cell line was also cultured in MEM-α with the addition of 30% CSCM (calvaria-derived stromal cell conditioned media), 10% of FBS and 1% of antibiotics in standard conditions.

Magnetic Field (MF)
The cells were exposed to the magnetic field using the static magnetic field (SMF) stimulation system designed in Wroclaw University of Science and Technology. This system is appropriate to produce a uniform SMF through action of two parallel magnets with opposite polarity. Plates with samples were placed in the central core, as shown in Figure 1. In this place, the MF strength was equaled 0.3T. The daily exposure of cells on the MF was 15 min.
subjected to the scanning electron microscopy (SEM) technique to prevent hybrid unwanted deterioration induced by the high energy electron beam. Therefore, a CM-20 Super Twin microscope (Philips, Amsterdam, The Netherlands), operated kV was used for the TEM characterization while hybrids samples were imaged Nova Nano-SEM 230 microscope (FEI Company, ThermoFisher Scienitific, Waltham USA). The measurements of the FTIR-ATR spectra were performed on a Nicolet iZ1 trometer (Thermo Fischer Scientific, Waltham, MA, USA) equipped in diamond A cessory covering the spectral range between 4000-500 cm −1 at room temperature. M characterization of the Co1−xMnxFe2O4 colloids was presented by us previously [16 the interested reader is advised to follow that article for details. The hybrid particl size and distribution were estimated by using free-image processing software v.1.46 [17] through analysis of SEM images by taking into consideration of 100 particles (longest diameter was measured due to elongated shape of objects).

Cell Lines
The mouse pre-osteoblast mouse cell line (MC3T3-E1-subclone 4) was obtaine the European Collection of Authenticated Cell Cultures (EACC, Sigma-Aldrich, M Germany), while the mouse pre-osteoclast cell line (4B12) was a kind gift from the D ment of Oral Biology and Tissue Engineering, Meikai University School of Dentistr fessor S. Amano) [18]. The MC3T3-E1 cell line was maintained in Minimum Essent dium Alpha (MEM-α, Gibco, Waltham, MA, USA) without ascorbic acid supplem with 10% Fetal Bovine Serum (FBS, Merck, KGaA, Darmstadt, Germany) and 1% of ard antibiotics (Merck KGaA, Darmstadt, Germany). The 4B12 cell line was also cu in MEM-α with the addition of 30% CSCM (calvaria-derived stromal cell condition dia), 10% of FBS and 1% of antibiotics in standard conditions.

Magnetic Field (MF)
The cells were exposed to the magnetic field using the static magnetic field stimulation system designed in Wroclaw University of Science and Technology. Th tem is appropriate to produce a uniform SMF through action of two parallel magne opposite polarity. Plates with samples were placed in the central core, as shown in 1. In this place, the MF strength was equaled 0.3T. The daily exposure of cells on was 15 min.

Cell Proliferation Assay
In order to determine the viability of cells after treating them with PMMA, Co 0.5 Mn 0.5 Fe 2 O 4 and their combination (Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA) in a ratio of 80/20 and at a final con-centration of 90.8 µg/mL, after 24, 48 and 72 h in the magnetic field conditions, TOX-8 kit (Merck KGaA, Darmstadt, Germany) was performed according to the manufacturer's protocol. The absorbance in the appropriate wells was evaluated by 96-well microplate reader (Epoch; Biotek Instruments, Winnoski, VT, USA) equipped with Gen5 software version 2.0 [19]. The measurements were taken at 600 nm and 690 nm as the reference lengths. Each experiment was performed at least three times independently.

Morphology and Mitochondria Status Analysis
The mitochondria, actin filaments and the nucleus of treated/untreated pre-osteoblasts and pre-osteoclasts were stained as described previously [20]. Briefly, mitochondria were stained using MitoRed dye, the F-actin filaments with Phalloidin-Atto 488 and the cells nuclei with 4 ,6-diamidino-2-phenylindole DAPI (all from Life Technologies, Carslbad, CA, USA).
Briefly, the cells after incubation with the PMMA and its combination, were incubated for 30 min with MitoRed solution at concentration 1:1000 at 37 • C and fixed with 4% PFA (POCh, Gliwice, Poland). Then, the cells were stained with Phalloidin-Atto 488 for 45 min at RT and then stained with DAPI. Visualization was made by a confocal microscope (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany) at 0.5 µm steps up to a final depth of 25 µm. Images were captured at magnification 630× and analyzed using Fiji New ImageJ with Colour Pixel Counter plugin version 1.52 developed by Wayne Rasband from NIH, USA. Each photograph was taken at least three times independently.

Gene Expression Analysis
The gene expression analysis was performed using qPCR technique. Briefly, cells seeded at the plastic plates at the density of 1 × 10 4 /well in the appropriate medium with the addition of PMMA or its modification and placed in the magnetic field were collected after 24 h and suspended in the Extrazol (BLIRT DNA, Gdańsk, Poland). The total RNA was obtained using acid guanidinium thiocyanate-phenol-chloroform extraction method described by Chomczynski and Sacchi [21] using the reagents from Merck KGaA, Darmstadt, Germany. The total of RNA quality and quantity were determined using a spectrophotometer (Epoch, Biotek Instruments, Winnoski, VT, USA).
The process of digestion of gDNA and cDNA synthesis was performed using Takara PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Bio Europe, Goteborg, Sweden) according to the manufacturer protocol. Both processes were performed using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA).
Each cDNA template was amplified by the quantitative reverse transcription polymerase chain reaction, using SensiFAST™ SYBR No-ROX Kit (Bioline, London, UK) in total volume of 10 µL (for a single reaction-1 µL of cDNA and 500 nM of each primer, according to the protocol). The sequences of the specific primers obtained from Merck KGaA, are listed in Table 1. The qRT-PCR reactions were performed using a CFX Connect Real-Time PCR Detection System (CFX Connect Optics Module, Bio-Rad, Hercules, CA, USA) equipped with the software BioRad CFX Maestro and the transcript levels were normalized to Gaph as a standard control (house-keeping gene).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 5 Software and the statistical significance was marked with asterisk (*). The p value less than 0.05 (p < 0.05) are marked with one asterisk (*), while p value less than 0.01 (p < 0.01) with two asterisks (**) and, finally, the p values less than 0.001 (p < 0.001) with three asterisks (***). The analysis of the diffraction patterns ( Figure 2A) leads to the conclusion that the structure of the Co 0.5 Mn 0.5 Fe 2 O 4 can be ascribed to the spinel-type materials as supported by reference card no. ICDD 10-0319 as well as 22-1086. A detailed analysis of the structural properties of the same nanoparticles with a broader concentration range of Mn doping was a subject of our previous article [15] where it was proven that the final compound formed solid solution of respective elements in appropriate ratio. The results of the formation of the hybrid material were confirmed by the FTIR-ATR spectra analysis ( Figure 2B). One can see a range of the PMMA characteristic vibration modes which are present either in the case of hybrid Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA and reference polymer sample prepared in the same way as composite. The difference in peak positions and their structure reflects the interaction of the PMMA with the Co 0.5 Mn 0.5 Fe 2 O 4 surface [22]. The average crystallite size was calculated from the peak broadening with help of Scherrer's formula: where k stands for constant value set at 0.9, λ is the wavelength of the Cu lamp (1.54060 Å), β 0 means apparatus broadening; β is full width at half maximum (FWHM) and θ corresponds with the peak maximum taken for the calculations [22] and compared with the size extracted from the TEM image ( Figure 2C). We noted that there is a very good match between both size estimation methods. The mean crystallite size calculated from the broadening of the (220) crystallographic plane reflection was around 7 nm, whereas the particle size obtained from the TEM imaging is 8 ± 2 nm. Analysis of the SEM micrographs leads to the observation that the hybrid materials have elongated shapes, sample morphology shows sufficient homogeneity, while the size of the objects has been estimated to range from 120 to 200 nm. formed solid solution of respective elements in appropriate ratio. The results of the formation of the hybrid material were confirmed by the FTIR-ATR spectra analysis ( Figure  2B). One can see a range of the PMMA characteristic vibration modes which are present either in the case of hybrid Co0.5Mn0.5Fe2O4@PMMA and reference polymer sample prepared in the same way as composite. The difference in peak positions and their structure reflects the interaction of the PMMA with the Co0.5Mn0.5Fe2O4 surface [22]. The average crystallite size was calculated from the peak broadening with help of Scherrer's formula: where k stands for constant value set at 0.9,  is the wavelength of the Cu lamp (1.54060 Å), β0 means apparatus broadening; β is full width at half maximum (FWHM) and θ corresponds with the peak maximum taken for the calculations [22] and compared with the size extracted from the TEM image ( Figure 2C). We noted that there is a very good match between both size estimation methods. The mean crystallite size calculated from the broadening of the (220) crystallographic plane reflection was around 7 nm, whereas the particle size obtained from the TEM imaging is 8 ± 2 nm. Analysis of the SEM micrographs leads to the observation that the hybrid materials have elongated shapes, sample morphology shows sufficient homogeneity, while the size of the objects has been estimated to range from 120 to 200 nm.  All reflections were indexed accordingly; the 14 2θ broad peak corresponds to the amorphous PMMA shell.
The Co 0.5 Mn 0.5 Fe 2 O 4 sample was taken as a core material due to its best magnetic properties within the whole concentration range of Mn 2+ studied in Ref. [14]. The sample and the sample after covering it with the PMMA shell (to improve biocompatibility) assure the best response upon action of static magnetic field. The effect of PMMA and Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA modification was evaluated on the pre-osteoblasts in the presence of magnetic field and the results showed that applying of magnetic fields decrease the viability of MC3T3-E1 after 48 h ( Figure 3B).
Materials 2021, 14, x FOR PEER REVIEW 8 o All reflections were indexed accordingly; the 14 2θ broad peak corresponds to t amorphous PMMA shell.
The Co0.5Mn0.5Fe2O4 sample was taken as a core material due to its best magne properties within the whole concentration range of Mn 2+ studied in Ref. [14]. The sam and the sample after covering it with the PMMA shell (to improve biocompatibility) sure the best response upon action of static magnetic field.

Anti-Proliferative Effect of PMMA Modified by Co0.5Mn0.5Fe2O4 in Ratio 80/20 towards P Osteoblasts and Pre-Osteoclasts in the Presence of Magnetic Field.
The effect of PMMA and Co0.5Mn0.5Fe2O4@PMMA modification was evaluated on t pre-osteoblasts in the presence of magnetic field and the results showed that applying magnetic fields decrease the viability of MC3T3-E1 after 48 h ( Figure 3B). In turn, Co0.5Mn0.5Fe2O4@PMMA increases the viability in the MF(+) condition in co parison to control cells ( Figure 3C).
In the case of pre-osteoclasts, we observed statistically significant increase of viabil of cells treated with the PMMA in the presence of MF(+) after 48 h ( Figure 3E) and af 72 h, independently of the MF application ( Figure 3F)

Morphology and Mitochondria Network Development Related to PMMA and PMMA@Co0.5Mn0.5Fe2O4 towards Osteoblasts and Osteoclasts in the Presence of Magnetic Fie
The impact of the PMMA and its modification on the morphology and mitochond network rearrangement in the magnetic field on the pre-osteoblasts and pre-osteocla was determined using MitoRed and F-actin staining.
Our studies showed that PMMA alone Co0.5Mn0.5Fe2O4@PMMA limited the grow of cytoskeleton in the pre-osteoblasts in the MF(−) condition ( Figure 4A(iii)). Moreov the comparison between MF(+) and MF(−) revealed that pre-osteoblasts cultured in t MF(+) condition showed weak cytoskeleton development than these in the MF(−) (Figu In turn, Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA increases the viability in the MF(+) condition in comparison to control cells ( Figure 3C).
In the case of pre-osteoclasts, we observed statistically significant increase of viability of cells treated with the PMMA in the presence of MF(+) after 48 h ( Figure 3E) and after 72 h, independently of the MF application ( Figure 3F

towards Osteoblasts and Osteoclasts in the Presence of Magnetic Field
The impact of the PMMA and its modification on the morphology and mitochondria network rearrangement in the magnetic field on the pre-osteoblasts and pre-osteoclasts was determined using MitoRed and F-actin staining.
Our studies showed that PMMA alone Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA limited the growth of cytoskeleton in the pre-osteoblasts in the MF(−) condition ( Figure 4A(iii)). Moreover, the comparison between MF(+) and MF(−) revealed that pre-osteoblasts cultured in the MF(+) condition showed weak cytoskeleton development than these in the MF(−) ( Figure 4A(iii,iiii)). From the other side, the mitochondrial network was less developed in case of PMMA combination in the MF(−) condition in comparison to control cells ( Figure 4A(i)).  The opposite effect was reported in the pre-osteoclasts treated by PMMA and its combination in both magnetic conditions, where the clearly visible mitochondrial network and cytoskeleton development were reported ( Figure 4B(i-iiii)).

The Impact of PMMA Modified by Co0.5Mn0.5Fe2O4 in Ratio 80/20 on the Expression of Genes Related to the Apoptosis towards Pre-Osteoblasts and Pre-Osteoclasts in the Presence of Magnetic Field
The impact of the PMMA and its modification on the apoptosis of pre-osteoblasts and pre-osteoclasts was determined using qPCR. The expression of p21, p53, Casp9, Bad, Bax and Bcl-2 was analyzed after 24 h of incubation of the cells with the addition of PMMA and Co0.5Mn0.5Fe2O4@PMMA in ratio 80/20 in the MF condition.
Our studies showed that PMMA alone increasing the expression of p53 gene as compared to control in the MF(−) condition ( Figure 5B). Moreover, the effect of the magnetic field application was observed after pre-osteoblasts culturing with PMMA. The addition of PMMA caused increase of the expression of the p21, Casp9, Bad and Bax in the MF(+) as compared to MF(−) ( Figure 5A,C-E). The impact of the PMMA and its modification on the apoptosis of pre-osteoblasts and pre-osteoclasts was determined using qPCR. The expression of p21, p53, Casp9, Bad, Bax and Bcl-2 was analyzed after 24 h of incubation of the cells with the addition of PMMA and Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA in ratio 80/20 in the MF condition.
Our studies showed that PMMA alone increasing the expression of p53 gene as compared to control in the MF(−) condition ( Figure 5B). Moreover, the effect of the magnetic field application was observed after pre-osteoblasts culturing with PMMA. The addition of PMMA caused increase of the expression of the p21, Casp9, Bad and Bax in the MF(+) as compared to MF(−) ( Figure 5A,C-E).
In turn, the expression of the p53 gene after 24 h exposition to Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA was increased in the MF(−) condition, compared to control ( Figure 5B). In parallel, the expression of the Casp9 and Bcl-2 were increased after PMMA@Co 0. 5   In turn, the expression of the p53 gene after 24 h exposition to Co0.5Mn0.5Fe2O4@PMMA was increased in the MF(−) condition, compared to control (Figure 5B). In parallel, the expression of the Casp9 and Bcl-2 were increased after PMMA@Co0.5Mn0.5Fe2O4 incubation in the MF(−), in comparison to MF(+) (Figure 5C,F).
The effect of PMMA alone on the pre-osteoclasts expression of genes associated with apoptosis was similar in the MF(+) and MF(−) conditions. The significant decrease in the expression of p21 and Bad in the MF(−) condition, as well as the decrease of the expression of p21 in the MF(+) condition, was observed ( Figure 6A,D).  In turn, the expression of the p53 gene after 24 h exposition to Co0.5Mn0.5Fe2O4@PMMA was increased in the MF(−) condition, compared to control (Figure 5B). In parallel, the expression of the Casp9 and Bcl-2 were increased after PMMA@Co0.5Mn0.5Fe2O4 incubation in the MF(−), in comparison to MF(+) (Figure 5C,F).
The effect of PMMA alone on the pre-osteoclasts expression of genes associated with apoptosis was similar in the MF(+) and MF(−) conditions. The significant decrease in the expression of p21 and Bad in the MF(−) condition, as well as the decrease of the expression of p21 in the MF(+) condition, was observed ( Figure 6A,D).  The opposite effect was observed after pre-osteoclasts treating with Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA in ratio 80/20 in the MF(−) condition, where we observed the increase of the expression of the p21, Casp9 and Bax ( Figure 6A,C,E), while after the applied magnetic field, the significant decrease of p53 and Bax was reported ( Figure 6B,E). Interestingly, applying of magnetic field decreased the expression of p21, Casp9 and Bax after treating pre-osteoclasts with modified PMMA (Figure 6A,C,E). Our studies revealed the impact of the PMMA on the expression of genes related to process of osteogenesis and osteoclastogenesis in the case of the exposition of them to the magnetic field. Interesting observation was noticed in case of the expression of Alp gene after pre-osteoblasts incubation with PMMA. From one side, the expression of the Alp was decreased independently of MF application, but from the other side, its expression was lower in the MF(−) than in MF(+) condition ( Figure 7B). Moreover, PMMA caused the statistical significant decrease of the Col1A1 expression in the MF(+) condition in comparison to control ( Figure 7C).
The opposite effect was observed after pre-osteoclasts treating with Co0.5Mn0.5Fe2O4@PMMA in ratio 80/20 in the MF(−) condition, where we observed the increase of the expression of the p21, Casp9 and Bax ( Figure 6A,C,E), while after the applied magnetic field, the significant decrease of p53 and Bax was reported ( Figure 6B,E). Interestingly, applying of magnetic field decreased the expression of p21, Casp9 and Bax after treating pre-osteoclasts with modified PMMA (Figure 6A,C,E).

The Impact of PMMA Modified by Co0.5Mn0.5Fe2O4 in Ratio 80/20 on the Expression of Genes and Proteins Related to Osteogenesis/Osteoclastogenesiss towards Pre-Osteoblasts and Pre-Osteoclasts in the Presence of Magnetic Field
Our studies revealed the impact of the PMMA on the expression of genes related to process of osteogenesis and osteoclastogenesis in the case of the exposition of them to the magnetic field. Interesting observation was noticed in case of the expression of Alp gene after pre-osteoblasts incubation with PMMA. From one side, the expression of the Alp was decreased independently of MF application, but from the other side, its expression was lower in the MF(−) than in MF(+) condition ( Figure 7B). Moreover, PMMA caused the statistical significant decrease of the Col1A1 expression in the MF(+) condition in comparison to control ( Figure 7C). The impact of the magnetic field application was also observed in the case of Bglap 2 and Dmp1 expression after PMMA addition to culture. In the MF(−) condition, the expression of Bglap 2 was increased as compared to control, while after comparison Bglap2 expression between MF(−) and MF(+), we noticed the increase expression after PMMA in the MF(−) than in MF(+) ( Figure 7E). Moreover, the expression of Dmp1 in the MF(+) was higher than in the MF(−) condition ( Figure 7F). The impact of the magnetic field application was also observed in the case of Bglap 2 and Dmp1 expression after PMMA addition to culture. In the MF(−) condition, the expression of Bglap 2 was increased as compared to control, while after comparison Bglap2 expression between MF(−) and MF(+), we noticed the increase expression after PMMA in the MF(−) than in MF(+) ( Figure 7E). Moreover, the expression of Dmp1 in the MF(+) was higher than in the MF(−) condition ( Figure 7F).
In turn, the combination of the Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA increased the expression of Alp in the pre-osteoblasts in MF(+) as compared to MF(−) condition ( Figure 7B), while Opn and Bglap 2 was increased as compared to the control only in the case of the MF(−) condition ( Figure 7D,E). Additionally, the combination of the Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA in a ratio of 80/20 increases the expression of the Col1A1 as compared to control in both magnetic conditions ( Figure 7C).
Interestingly, the expression of the Dmp1 was increased in the MF(−) condition as compared to control, while in the MF(+) was decreased. Moreover, the expression of the Dmp1 in the MF(−) condition was increased as compared to MF(+) ( Figure 7F).
The impact of the PMMA and its combination in the magnetic field condition on the pre-osteoclasts was determined based on the expression of the Mmp9, PU.1, Itgav and c-fos.
Our studies revealed the enhanced expression of the Mmp9 after PMMA incubation of pre-osteoclasts as compared to control cells independently of the magnetic field conditions ( Figure 8A). In addition, we reported that presence of the magnetic field is associated with decreasing expression of Mmp9 ( Figure 8A). Additionally, it was noticed that in both magnetic conditions the expression of PU.1 and Itgav was decreased as compared to control ( Figure 8B,C).

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12 of 20 of 80/20 increases the expression of the Col1A1 as compared to control in both magnetic conditions ( Figure 7C). Interestingly, the expression of the Dmp1 was increased in the MF(−) condition as compared to control, while in the MF(+) was decreased. Moreover, the expression of the Dmp1 in the MF(−) condition was increased as compared to MF(+) ( Figure 7F).
The impact of the PMMA and its combination in the magnetic field condition on the pre-osteoclasts was determined based on the expression of the Mmp9, PU.1, Itgav and cfos.
Our studies revealed the enhanced expression of the Mmp9 after PMMA incubation of pre-osteoclasts as compared to control cells independently of the magnetic field conditions ( Figure 8A). In addition, we reported that presence of the magnetic field is associated with decreasing expression of Mmp9 ( Figure 8A). Additionally, it was noticed that in both magnetic conditions the expression of PU.1 and Itgav was decreased as compared to control ( Figure 8B,C).

The Impact of PMMA Modified by Co0.5Mn0.5Fe2O4 in Ratio 80/20 on the Expression of Genes Related to Integrins towards Pre-Osteoblasts and Pre-Osteoclasts in the Presence of Magnetic Field
Finally, we established the impact of the PMMA and its modification on the integrins expression in mouse pre-osteoblasts and pre-osteoclasts cell lines. In the case of  Figure 9B-D).  In the case of the pre-osteoclasts, PMMA alone decreased the expression of the INTb1 in the presence of the magnetic field and also INTa6 in both magnetic conditions ( Figure  9E,F). Moreover, the combination of the PMMA and Co0.5Mn0.5Fe2O4 decrease the expression of the INTa6 in the magnetic field condition ( Figure 9F). Interestingly, the expression of the INTb1 was increased in the pre-osteoclasts in the MF(+) condition after culturing them in the presence of the PMMA combination.

The Impact of PMMA Modified by Co0.5Mn0.5Fe2O4 in Ratio 80/20 on the Expression of Genes Related to Inflammation Process towards Pre-Osteoblasts and Pre-Osteoclasts in the Presence of Magnetic Field
Interestingly, the results associated with the inflammation profile showed that the new modification of PMMA could decrease the expression of Il-6 independently of the presence of magnetic field in the pre-osteoblasts, while increasing in the pre-osteoclasts ( Figure 10A,C). The opposite, also independently of the presence of MF, Co0.5Mn0.5Fe2O4 @PMMA in a ratio of 80/20 caused the decrease of the Tgfb in MC3T3-E1 cell line and enhanced the expression of Tnfa (Figure 10B,D).

The Impact of PMMA Modified by Co0.5Mn0.5Fe2O4 in Ratio 80/20 on the Expression of Genes Related to microRNA Involved in the Process of Osteoblastogenesis and Osteoclatogenesis towards Pre-Osteoblasts and Pre-Osteoclasts in the Presence of Magnetic Field
Additional studies revealed statistically significant decrease of the expression of miR-17-5p, miR-21-5p, miR-124-3p and miR-145-5p in comparison to control only in case of MF absence, while the expression of miR-7a-5p, miR-203a and miR-223a was decreased independently of the magnetic field presence in the MC3T3-E1 cell line ( Figure 11A-G). Figure 11. The impact of PMMA and its modification on the microRNA profile associated with osteoblastogenesis and osteoclastogenesis expression of miR-7a-5p (A), miR-17-5p (B), miR-21-5p  (C), miR-124-3p (D), miR-145-5p (E), miR-203a (F) and miR-223a (G) in the magnetic field condition towards mouse pre-osteoblasts (MC3T3-E1 cell line. Statistical differences are indicated by * p < Additional studies revealed statistically significant decrease of the expression of miR-17-5p, miR-21-5p, miR-124-3p and miR-145-5p in comparison to control only in case of MF absence, while the expression of miR-7a-5p, miR-203a and miR-223a was decreased independently of the magnetic field presence in the MC3T3-E1 cell line ( Figure 11A-G).

The Impact of PMMA Modified by Co0.5Mn0.5Fe2O4 in Ratio 80/20 on the Expression of Genes Related to microRNA Involved in the Process of Osteoblastogenesis and Osteoclatogenesis towards Pre-Osteoblasts and Pre-Osteoclasts in the Presence of Magnetic Field
Additional studies revealed statistically significant decrease of the expression of miR-17-5p, miR-21-5p, miR-124-3p and miR-145-5p in comparison to control only in case of MF absence, while the expression of miR-7a-5p, miR-203a and miR-223a was decreased independently of the magnetic field presence in the MC3T3-E1 cell line ( Figure 11A-G).  Comparing the results between the presence and absence of the magnetic field, only in the case of miR-7a-5p was the slightly decreased of expression of this miR observed under magnetic field condition ( Figure 11A).
x FOR PEER REVIEW 15 of 20 Comparing the results between the presence and absence of the magnetic field, only in the case of miR-7a-5p was the slightly decreased of expression of this miR observed under magnetic field condition ( Figure 11A).

Discussion
The effectiveness of bone implants tailored to osteoporotic patients depends on both stimulation of osteoblast proliferation and differentiation, as well as inhibition of osteoclasts activity. Poly(methyl methacrylate) (PMMA) has become one of the attractive and frequently used polymers in the synthesis of bone cements since its first biomedical application in 1937. In order to enhance its bioactivity, PMMA can be combined with a wide range of chemicals in order to synthetize nanoparticles with improved osteoinductive properties. One of the potential candidates for small molecule additives are bioinorganic ions which represents an inexpensive and stable alternative to peptides, nucleic acids and growth factors. In the presented study, we doped PMMA with Co, Mn and Fe2O4 and analyzed in vitro the osteogenic and osteoclastogenic properties of composite. Furthermore, we have investigated whether exposition of fabricated biomaterials to magnetic field enhance their bioactive properties. Previous studies have confirmed that cobalt ions

Discussion
The effectiveness of bone implants tailored to osteoporotic patients depends on both stimulation of osteoblast proliferation and differentiation, as well as inhibition of osteoclasts activity. Poly(methyl methacrylate) (PMMA) has become one of the attractive and frequently used polymers in the synthesis of bone cements since its first biomedical application in 1937. In order to enhance its bioactivity, PMMA can be combined with a wide range of chemicals in order to synthetize nanoparticles with improved osteoinductive properties. One of the potential candidates for small molecule additives are bioinorganic ions which represents an inexpensive and stable alternative to peptides, nucleic acids and growth factors. In the presented study, we doped PMMA with Co, Mn and Fe 2 O 4 and analyzed in vitro the osteogenic and osteoclastogenic properties of composite. Furthermore, we have investigated whether exposition of fabricated biomaterials to magnetic field enhance their bioactive properties. Previous studies have confirmed that cobalt ions and MF are both able to enhance angiogenesis and bone tissue regeneration, which supports their application in the fabrication of nanomaterials for bone tissue engineering.
For that reason, in order to confirm the utility of bone-filling material, its impact on the cells surrounding the microenvironment of damaged tissue should be investigated carefully. Biocompatibility results revealed that pure Co decreased proliferation of MC3T3-E1 cell line in comparison to control group; however, that effect was ameliorated in PMMA + Co group. Previous studies revealed that a concentration of cobalt ions <10 ppm enhance proliferation of bone cells [23,24]. In the presented study, we employed the MC3T3-E1 cell line as it has behaviors similar to primary calvarial osteoblasts and for the evaluation of biocompatibility of implants is preferable to osteosarcoma cell line, since it better reflects physiological condition. A similar phenomenon was observed for 4B12 cells, in which addition of Co resulted in decreased growth kinetics.
In the presented study, we demonstrated that Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA hybrid and Co 0.5 Mn 0.5 Fe 2 O 4 alone exert a pro-ostegoenic and anit-osteoclastogenic effects. The biological activity of the materials has been demonstrated by the analysis of gene expression related to bone cells metabolism under normal and MF condition. Interestingly, hybrid material showed the better cell response for the expression of Runx2, Alp, Col1a1, while Co 0.5 Mn 0.5 Fe 2 O 4 enhanced expression of Opn, Bglap2, Dmp1. Observed phenomenon can be explained at least by two different ways. Enhanced differentiation of MC3T3-E1 cells may by directly activated by each hybrid components. Modification of PMMA surface with different inorganic compounds was proved to enhance osteoblasts adhesion and response. Recently, it was shown by Phakatkar et al. [25] that novel PMMA bone cement nanocomposites containing magnesium phosphate nanosheets and hydroxyapatite nanofibers possess antibacterial attributes with enhanced cytocompatibility and mechanical properties. Another group showed that incorporation of hydroxyapatite into PMMA increases the biological response to the cement from tissue around the implant site [20]. The authors revealed that, after the transplantation of material in vivo, its surface is immediately covered by the cells with initiate the osteointegration. They proved that the incorporation of hydroxyapatite improves the attachment of extracellular matrix (ECM) protein in comparison to PMMA only. In the same analogy, enhanced osteogenesis on the PMMA hybrid observed in the presented study may results from its modification with inorganic ions. Fan et al. [23] have shown that cobalt chloride (CoCl 2 -treated bone progenitor cells induced higher degree of vascularization and enhanced osteogenesis. Furthermore, cobalt-substituted hydroxyapatite (COHA) effectively promotes bone cell growth, reduces the inflammatory response and is an antibacterial agent [26].
Another possible mechanism is related to the presence of iron oxide in fabricated materials. Recently, iron oxide nanoparticles (IONPs) have been widely studied in the areas of bone regenerative medicine. It was shown that IONPs incorporated to gelatin sponge scaffold enhance bone formation in vivo and is visible in MRI imaging without using of external magnetic field [27]. On the other hand, nanocomposites of iron oxide and hydroxyapatite were characterized by superparamagnetic and biocompatible properties [28]. Vlad et al. [27] have shown that incorporation of iron oxide nanoparticles into the powder phase of an alpha-tricalcium phosphate-based cement improved injectability of apatitic bone cement for vertebroplasty. The application of IONPs in bone tissue engineering was also investigated in our own studies. We have shown previously that α-Fe 2 O 3 /γ-Fe 2 O 3 nanocomposite exerts dual action as they enhance osteogenic differentiation while reduce the activity of osteoclast [20]. We also found that polyurethane/poly(lactic) acid sponges doped with iron oxide nanoparticles under magnetic field enhance osteogenesis of adipose derived mesenchymal stem cells by enhanced expression of osteopontin and collagen type I.
An additional mechanism for better cell response may result from the application of external magnetic field (MF), which is therapeutic agent per se and further enhance the bioactive properties of materials doped with magnetic responsive particles. MF potential to affect osteoblast behavior on different biomaterials have been proved in multiple studies including our own. Based on the obtained data, MF represent a potential tool to improve the clinical outcome of selected regenerative therapies not only in orthopedics but also in dentistry. However, due to discrepancies between some research works, MF should be more thoroughly investigated by proper clinical trials. Herein, we have shown that application of MF enhances the cellular response of scaffold doped with Fe 2 O 4 . It stands with our and other previous research which showed that MF application enhance osteogenesis, modulates progenitor stem cells fate and diminish osteoclasts activity.
It also should be mentioned that fabricated, hybrid material, due to the incorporation of Co and iron oxides, represents a potent MRI contrasting agent. In diagnostics, MRI is applied to differ between healthy and tumor tissue and to visualize the location of lesions. However, metal implants can interfere with the MRI, causing misinterpretation of the obtained results. In previous experiments, it was confirmed that COHAC can be used as a T2 contrast localization agent and does not cause image interference [29,30].
In the next step, we investigated how fabricated nanocomposites affects the expression of the integrins as they are involved in the regulation of multiple cell functions, including their interactions with matrix, proliferation and differentiation. It was shown that cell movement on the material surface is possible through the formation of cytoskeletal projections called filopodia, which in turn stimulates the activation of integrins [18,31]. Transmembrane receptors in a great amount can be found in cells forming focal contacts (adhesion plaques), which are directly responsible for the adhesion of cells to material surface. Integrins bound to selected ECM components, e.g., collagen, fibronectin, osteopontin and through signal transduction modulates the fate of bone forming cells and, thus, are directly involved in the regeneration process. Their activation stimulates cells to migration, movement, adhesion and, finally, differentiation. The findings in this study show that both Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA and Co 0.5 Mn 0.5 Fe 2 O 4 in control and MF condition modulate the integrins expression. We found that the abovementioned materials enhanced the mRNA levels of INTa6, INTa1 and INTa3 in relation to pure PMMA.
Here, we also proved that the new biomaterials could influence on the action of miR-NAs involved in the processes of bone remodeling and can modulate the pro-inflammatory response that is important in the case of the potential future use of such biomaterials in the OP treatments.

Conclusions
In the presented paper, we fabricated a Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA hybrid and investigated its physicochemical and biological properties. The bioactivity of scaffolds was determined using in vitro osteoblast and osteoclasts system. The hybrid material showed better cellular response in comparison to pure PMMA under control and magnetic field condition. This can be explained by the presence of bioactive, inorganic ions-Fe 2 O 4 and Co, which are known to enhance bone regeneration. Therefore, a newly developed Co 0.5 Mn 0.5 Fe 2 O 4 @PMMA might be useful for a bone substitute or filler.
The material for bone regeneration should be characterized by activation of preosteoblasts, which induce deposition of ECM proteins and leads to its mineralization. Yet, while taking into consideration its application in osteoporotic patients, novel, smart biomaterials should be incorporated with particles that not only stimulate bone forming cells, but at the same time inhibit the overactivity of osteoclasts. The enhanced activity of bone resorbing cells disrupts tissue homeostasis, contributing to bone mass loss and altered bone microstructures, which make it prone to fractures. Thus, in order to re-establish normal bone repair, bone grafts should be tailored to meet the patients' need and restore the balance between cells in affected tissue. Here, we provide a proof of evidence that the modification of PMMA with Co 0.5 Mn 0.5 Fe 2 O may represent a novel approach for the material optimization and may be the way forward in the fabrication of scaffold with enhanced bioactivity that benefits osteoporotic patients.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ongoing studies.

Acknowledgments:
The authors are grateful of Shigeru Amano from the Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry for sharing with the 4B12 cell line.

Conflicts of Interest:
The authors declare no conflict of interests.