Hydroxyapatite Based Composites for Bone Regeneration

doi:10.21203/rs.3.rs-1942868/v1

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Hydroxyapatite Based Composites for Bone Regeneration

https://doi.org/10.21203/rs.3.rs-1942868/v1

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Aim of study:

Preparation and characterization of the new nanocomposite, magneto-hydroxyapatite-alendronate (mag-HA-Ald np), for bone tissue scaffolds formation.

Material and methods

Iron oxide nanoparticles (Fe₃O₄ np) were added to aqueous solutions of Ca(NO₃)₂.4H₂O and (NH₄)₂HPO₄ for obtaining mag-HA np. The target nanocomposites, mag-HA-Ald np, were synthesized by using aqueous solution of alendronate (Ald), with different ratios. The structure of the prepared nanocomposites were investigated based on spectral (FTIR, XRD, SEM) and EDX analysis. The cytotoxicity of the nanocomposites were performed using MTT assay, to detect the effect of magnetic field application on the cell viability; two culture methods were applied (with and without magnetic field exposure).

Results

The spectral, FTIR, XRD, SEM as well as EDX measurements confirmed the synthesis of nanocomposite, magneto-hydroxyapatite-alendronate (mag-HA-Ald np) with different ratios. MTT assay results suggested the positive correlation between Ald and iron oxide content with cell viability. In addition, Magnetic field application significantly decreases the cell viability in all groups in comparison to non- magnetic field application.

Hydroxyapatite

Alendronate

Spectral

EDX

Cytotoxicity

Scaffold

Preparation and characterization of the new nanocomposites, mag-HA np & The target nanocomposites (mag-HA-Ald np) for bone tissue scaffolds formation.

The cytotoxicity of the nanocomposites was performed using MTT assay
The effect of magnetic field application on the cell viability were tested
Magnetic field application significantly decreases the cell viability

Bone grafting is considered a traditional approach to treat bone defects. It is a surgical technique that substitutes missed bone with a bone that is either from patient himself (autograft), another person of the same species (allograft), bone of different species (xenograft). Because of the many disadvantages associated with bone grafts, such as risks of immune reactions and transmission of diseases or infections, scientists have long searched to overcome existing transplant drawbacks by modeling biocompatible scaffolds that could regenerate new bone. These synthetic scaffolds should fulfill some critical requirements. In short, they should be biocompatible, chemically inert, biodegradable, mechanically strong, osteoconductive, osteoinductive and also must be able to be osteointegrated at the defect site [Moussa and Dym 2020; Christy et al. 2020). On this basis, ceramics especially hydroxyapatite (HA; Fig. 1i) are the most investigated materials in bone regeneration field. The inorganic portion of HA mimics the natural bone matrix and this is necessary to encourage the integration with the living bone tissue. HA possesses exceptional biocompatibility and bioactivity properties with respect to bone cells and tissues, probably due to its similarity with the hard tissues of the body. HA np properties (surface grain size, pore size, wettability, biodegradability) could control protein interactions, and therefore, modulating subsequent enhanced osteoblast adhesion and long term functionality (Fathy et al. 2017; Fathy et al. 2018). Luckily, hydroxyapatite can be simply prepared or extracted from natural sources (Calabrese et al. 2020).

Living bone constantly undergoes a coupled resorptive-formative process, i.e., equilibrium between osteoblastic bone formation and osteoclastic bone resorption. The disruption of this equilibrium causes disease circumstances such as osteoporosis, metabolic bone disorder, which are most prevalent among the elderly, post-menopausal women and children suffering from malnutrition (Granito et al. 2018). The exaggerated activity of osteoclasts (OC) leads to destructions in the skeleton and reductions of bone mass, hence, treatment of osteoporotic bone defects or fractures with traditional hydroxyapatite-based materials faces challenges and difficulties (Yu and Wang 2016). Many strategies have been established to control the equilibrium between osteoblasts and osteoclasts, including the addition of growth factors that can promote bone formation by regulating gene expression related osteogenesis, but their application is restricted due to their high price and limited half-life in the human body (Suárez-González et al. 2012).

Bisphosphonates, {Bps; containing P-C-P units; Fig. 1ii} are a class of drugs that prevent the loss of bone mass. They are applied for the treatment of calcium disorders and bone metabolism. They cause an early reduction in bone resorption and a later increase in bone formation through their strong binding to the bone mineral hydroxyapatite that inhibits bone resorption and crystal dissolution and many other diseases, such as hypercalcemia, cancer, multiple myeloma, metastatic bone disease and Paget disease (Vasikaran 2001; Larsson and Fazzalari 2014).

Bps are selectively absorbed by osteoclasts in bone matrix and can inhibit osteoclasts precursors differentiation and mature osteoclasts resorptive activity. Bps may cause apoptosis of mature osteoclasts as well as many morphological changes in osteoclasts such as loss of their ruffled borders (Baylink et al. 1993). Many studies have also shown that Bps can encourage osteoinduction and osteoblasts activity especially when integrated with calcium phosphate nanocrystals. The effect of Bps is varying from one to another based on the substituent lengths and the atoms (R₁ and R₂) attached to the central carbon atom of the P-C-P groups (Fulfaro et al. 1998). For most current Bps, R₁ is a OH⁻ group that boosts HA affinity through calcium binding, while R₂ is the main determinant of antiresorptive ability (Ri et al. 2018).

Magnetic iron oxide nanoparticles (MNPs) are already well known in nanomedicine. They are highly corrosion resistant and safe for in-vivo applications; including magnetic resonance imaging (MRI) contrasting agents, magnetic-guided drugs or gene delivery, cell targeting, and cancer therapy (hyperthermia) (Hsu and Chang 2010). Nowadays, great attention has been given to the interactions between magnetic iron oxide nanoparticles (MNPs) and cells, showing that MNPs could enhance the growth of the cells due to their capability to reduce intracellular H₂O₂ through intrinsic peroxidase-like potency. In addition, the magnetic force produced by magnetic field, along with MNPs, may affect the surrounding environment of the materials, causing a number of changes in the behavior of the cells and accelerating the progression of the cell cycle. Furthermore, some studies proved that the incorporation of MNPs in the scaffold enhances the microstructure and the surface area of the scaffold. Therefore, the ability to absorb protein and biological behavior could be significantly enhanced (Huang et al. 2009). Although hydroxyapatite (HA), is an important inorganic biomineral constituent of bone, teeth, and phosphate mineral rocks, very few literatures exist showing the application of hydroxyapatite functionalized with magnetic nano particles.

We have published the synthesis and design of Pd (II) complexes based on alendronate, with anticancer activities against the human prostate (DU-145) and breast (MDA-MB231) cancer cell lines (Fathy et al. 2018). Consequently, these studies have encouraged us to synthesise new nanocomposites, magneto-hydroxyapatite-alendronate (mag-HA-Ald np), for bone tissue scaffolds formation. Their structures were investigated based on variety of physico-chemical measurements. In addition, their cytotoxicity was performed using MTT assay, to detect the effect of magnetic field application on the cell viability.

Chemicals and apparatus

All manipulations were performed under aerobic conditions using materials and solvents as received. Chemicals and solvents were obtained from sigma and merk. Calcium nitrate, Diammonium hydrogen phosphate and Alendronate sodium trihydrate were purchased from Alfa Aesar (USA), Iron oxide nanoparticles were purchased from Nano Gate Company (Egypt).

Ultrasonication was performed on Digital ultrasonic bath CD-4830, 35 kHZ, China. The centerfuge apparatus used is Safety centrifuge Thermo Fisher Scientific Company, United States. FT-IR spectra were measured on a Matson 5000 FT-IR spectrometer. X-Ray powder diffraction patterns were were assesed using X-ray diffractometer (XRD, PANalytical X'Pert PRO, Netherlands, The Electron Microscope Unit, Mansoura University- Egypt) equipped with mono-chromatized Cu-Kα radiation (λ=1.542 Å, 50 kV and 40 mA). Sample analysis was covered the 2θ range from 4^° to 80^° at a scan rate of 0.02^° /sec (Sabry and Salih 2020). The size of the crystallites was calculated using the XRD data to determine the Full-Width Half Maximum (FWHM). Then, by applying Scherrer formula; {D, K, λ, B and θ are the crystallite size (nm), Scherer constant (0.9), the wavelength of X-rays (1.54056 A°), the line broadening at FWHM in radians, and the Bragg^ˈs angle in degrees, respectively) (Jakab et al. 2020). EDX measurements were performed using energy dispersive x-ray unit (EDX, JEOLJSM-5500LV SEM, JEOL Ltd, Japan) (Bauer et al. 2021). SEM measurements were carried out using a Hitachi S-3000 VP-SEM Variable Pressure-SEM.

Preparation of Nanocomposites (mag-HA-Ald np)

The nanocomopsites were prepared by chemical wet precipitation method.

Magnetohydroxyapatite nanoparticles (mag-HA np)

Iron oxide nanoparticles (Fe₃O₄np; 0.5 – 1.5%) were added to an aqueous solution of hydrated Ca(NO₃)₂.4H₂O (2.36 g, 10 mmol; 10 mL). The reaction mixture was mixed in for 30 min at 40^°C. Subsequently with continuous sonication, (NH₄)₂HPO₄ (0.78 g, 8 mmol) in water (25 mL) was added dropwise with vigorous sonication over the period of 30 min.

The pH of the reaction mixture was adjusted to ~10-11 by using ammonia solution and stand for 48 hs at room temperature. The supernatant was discarded, and the precipitate was washed repeatedly with distilled water, centrifuged for 5 min., and finally dried at 50^oC for 24 hs. The sediment was manually pulverized using a mortar and pestle for fine powder formation (Ferraris et al. 2020).

Magnetohydroxyapatite alendronate nanoparticles (mag-HA-Ald np)

An aqueous solution of sodium alendronate (0.25 g, 0.78 mmol; 15 mL) was added dropwise to mag-HA np powders (0.5 g) under continuous sonication. The obtained mixture could precipitate for 48 hs at room temperature. The obtained nanocomposite was isolated via centrifugation and washed by dist. water and dried at 50^oC for. 24 hs.

Notes

Eight mag-HA-Ald np composites were prepared with different weight ratios of mag-HAnp and Ald (Table 1).
All prepared HA and mag-HA np nanocomposites were taken in such an amount that Ca / P molar ratio was maintained at ~ 1.67, which decreased to 1.15 upon adding Ald.

Biological characterization

Cytotoxicity assay

In vitro cytotoxicity test is one of the indicators of the evaluation of compatibility of biomedical nanocomposite (mag-HA-Ald np). In this study, assessments of cell viability of MSCs were carried out using the MTT assay, an indirect quantitative colorimetric assay method for determination of cell growth and proliferation. It based on the conversion of the yellow tetrazolium bromide to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells (Li et al. 2015). Firstly, disks of 5 mm diameter and 2 mm thickness were prepared by compressing the nanocomposite powders into cylindrical mold. The disks were sterilized under UV-lamp for 3 hs, washed 3 times with phosphate buffer saline, PSB (10 min for each wash). The disks were placed in 96-well plates then rat bone marrow mesenchymal stem cells (MSCs) were seeded over them (5 × 10³ cells in each well; the control was cells without any samples’ disks).

Two culture methods were applied with and without magnetic field-exposed groups.

With magnetic field-exposed group a magnet disk (10 cm diameter and 3 cm thickness)) was used to produce the magnetic field. The magnet was placed above the well plates (0.75 mm thickness) to expose the cultures to a continuous magnetic field during the entire test period. After culturing for 24 hs at 37^°C, 40 μl MTT solution was added onto each well and incubated for 4 hs at 37^°C. After incubation, 200 μl Dimethyl sulfoxide solution was added to each well and incubated for 15 minutes to dissolve the insoluble formazan. Absorbance of the resulting purple formazan produced by active cells was recorded at 570 nm using media as a blank by ELISA. The percent relative cell viability was calculated using the following equation (Xia et al. 2018).

Statistical analysis

At least three repeated tests were performed in all the experiment and the results were presented as means ± standard deviation (SD). One-way analysis of variance (ANOVA) and Tukey’s post hoc tests were used to compare between groups, while values with and without magnetic field were compared using Paired (dependent) test. A value of p < 0.05 was considered of statistical significance.

Characterization of nanocomposites

HA np (Ca/P molar ration 1.65 ± 0.1 and surface Ca/P atomic ratio 1.30 ± 0.05) has high affinity of interaction of positively charged Ca²⁺ with alendronate phosphate (PO₃^2-)-richer surface groups (Sobhi et al. 2020).

FTIR spectroscopy is one of the classical methods for structure determination of small molecules and is a valuable for investigation the structure and molecular mechanism of mag-HA np and Ald interaction (Cheng et al. 2022).

The FTIR spectrum of NaHAld shows bands at 3350 and 3246 cm^-1assigned to n_as(NH₂) and n_s(NH₂), respectively (Fathy et al. 2018). The n_as(P-C-P) and n_s(P-C-P) are observed in the range of 800-655 cm^-1(in Raman at 797 and 665 cm^-1), and the multiple absorption bands in the range of 3600–3300 cm^-1 are attributed to n(OH) stretching of P–OH and C–OH groups. The n_as(P-O) and n_s(P-O) stretching vibrations are observed at 1057 and (1021, 920) cm^-1 (in Raman at 1055 and (1020, 919 cm^-1), respectively, while the stretching and bending vibrations of P=O and P-OH groups giving rise to the bands in the fingerprint region below 1320 cm^-1. The bands at 1234 and 1183 cm^-1 (in Raman at 1233 and 1173 cm^-1) due to n(P=O) and n(P-O) stretches, respectively (Fathy et al. 2017; Fathy et al. 2018).

In the synthesized nanocomposites (Figs.2-4), the bands near 1037 and (988, 602) cm^-1 attributed to {n_as(P-O) and n_s(P-O)} and {n_as(P-C-P) and n_s(P-C-P)} stretching vibrations, respectively, were shifted to lower wavenumber. This feature indicates the participation of P-O in H-bond formation with HA (Neamtu et al. 2017; Doca et al. 2020). The bands observed in 3537-3487 cm⁻¹ region, are due to n(OH) stretching of (P–OH, C–OH groups) and hydrated water (Mir et al. 2012). Moreover, the charactarestic band near 565 cm⁻¹ is related to n(Fe-O) vibrations for Fe₃O₄, confirmed the presence of the isolated nanocomposites in the magneto form (Mondal et al. 2017). This band is overlapped with Asymmetric bending vibration of PO₄^3- of HA, which observed at 569 cm^-1.

Moreover, free HA shows bands at 3568, 1032 and 569 cm^-1, that owning to n(OH), n_as(O-P-O of PO₄^3-) and d_as(PO₄^3-) vibrations, respectively. In the composites, the bands near 1132, 1037 and 988 due to n_as(PO₄^3-), showed slight shifts to 1095, 1033 and 960 cm⁻¹, while those associated to d_as(PO₄^3-) vibration became much weaker. The broadening of the IR composites bands in the 1200–900 and 700–500 cm⁻¹ regions, indicates the formation of interlinked bonds into the layer between hydroxyapatite PO₄^-3and P-OH/P-O bonds specific to Ald (Putra et al. 2019).

The XRD patterns of the synthesized HA np (Fig. 5i) shows diffraction peaks with d-spacing values of 2q 26.10 and 32.89, 39.72 and 50.0^o which are assigned to (210), (211), (130) and (213) planes, respectively, due to d-spacing values matching with the hexagonal system with primitive lattice, in good agreement with the reported data of crystalline hydroxylapatite (Arunseshan et al. 2013). The crystallite size and crystallinity of HA (88 Å and 4.38%) have high similarities with biological apatite (103 Å and 4.35%), which have high similarities (Feng et al. 2017).

On the other hand, the XRD patterns of mag-HA np composite (Fig. 5ii) shows sharper peaks than those of HA, indicating higher crystallinity (Arunseshan et al. 2013). The peaks observed at 2q 25.91, 32.89, 39.71 and 49.99° are closely mimicking to those of HA. The XRD patterns exhibit peaks at 2q 29.99, 35.00, 44.82, 53.00 and 64.88^o corresponding to (220), (311), (400), (422) and (511), respectively, which marked with Fe₃O₄nanoparticles (Loh et al. 2008; Gong et al. 2003; Liao et al. 2001). The X-ray diffraction patterns of mag-HA–Ald nanocomposites (Fig. 5iii) show that the presence of Ald does not change the lattice constants of the HA phase (Neamtu et al. 2017).

The average crystallite size for fabricated HA np, mag-HA np and mag-HA-Ald np composites (Fig. 6) is determined by using the Debye-Scherrer equation (eq. 2) (Monshi et al. 2012).

L= Kλ /(β. Cosθ) (2)

K is the shape factor, L = crystallite average size, λ is the wavelength of Cu Kα radiation, β Full-Width Half Maximum (FWHM) of reflection (in radians) placed at 2 θ and θ is the reflection angle. Average crystallite size calculated in degrees.

The addition of Ald greatly decreased the composites crystal size. This is due to the interaction between Ald^- anion and Ca(II) ions of HA, which would not significantly alter the crystal structure of HA. However, Ald^- anion affects the crystal dimensions and leads to smaller composite crystals’ size compared to pure HA (Boanini et al. 2007; Boanini et al. 2012).

The morphology of the nanocomposites, mag-HA np, mag-HA-Ald np and HA-Ald np, were investigated in SEM analysis (Fig. 7). The SEM image of mag-HA np exhibits homogeneous dispersed spherical nanoparticles. In case of mag-HA np, white spots are clearly visible in the SEM micrographs and are likely to be representative of Fe₃O₄ (Zeng et al. 2012). The results indicate that both the HA np and mag-HA np scaffolds exhibit similar nano- and nanoporosity and good interconnectivity, indicating that the scaffold structure mimicked the morphology of natural spongy bone (Yuan et al. 2006). The matrix of mag-HA-Ald np is characterized by nanopores which average size is ~ 21-24 nm. Ald particles decorate the surface of mag-HA np and the size of particles were gradually decreased by decreasing Fe₃O₄ content in the nanocomposites (Puppi et al 2011).

The mag-HA np and mag-HA-Ald np nanocomposite was characterized using energy dispersive X-ray (EDX) analysis to identify the elemental components in the nanocomposites. The elemental composition of HA np, mag-HA np and mag-HA-Ald np were obtained by EDX analysis (Table 2). HA np has 4.38 (C), 46.34 (O), 18.65 (P), 30.63 (Ca) wt% with Ca/P 1.64, while in case of mag-HA np, 10.19 (C), 35.11 (O), 13.94 (P),22.54 (Ca), 0.22 (Fe) wt% with Ca/P 1.62. Upon adding Ald, both P and Ca wt% decreased as well as the Ca/P with the appearance of Na. For example, G7 (ratio of mag: HA: Ald is 1:100:40) has 24.52 (C), 48.17 (O), 1.38 (Na), 11.96 (P), 13.72 (Ca), 0.25 (Fe) wt% with Ca/P 1.15.

Biological characterization

Cytotoxicity assay

In bone diseases, magnetic therapy has been considered a promising alternative in disease treatments (Zeng et al. 2012). The magnetic fields may stimulate the proliferation and differentiation of osteoblasts, promote the expression of growth factors such as bone morphological protein, increase osteointegration, and accelerate new bone formation (Santini et al. 2003; Jansen et al. 2010). Moreover, magnetic fields have also been reported to be beneficial to increase the bone density and calcium content, the integration of bone and implants, and enhance the healing of bone fractures (Zhang et al. 2006; Taylor et al. 2006). In the biomedical field, magnetic nanoparticles (mag nps) have shown great interest based on their magnetic properties, i. e., they become superparamagnetic at diameters < 20 nm (Sun et al. 2004).

MSCs were chosen, as they are the most prevalent cells used in bone regeneration. They are characterized by high proliferation capacity besides their capability to withstand the preparation conditions necessary for in vitro expansion. Furthermore, many studies have shown that in vivo, MSCs can form various mesenchymal tissues including bone (Ressler et al.2022). MTT assay is the most common method applied to evaluate the cytotoxicity. This test is more accurate and time saving than other conventional hemocytometer counting methods. Cell viability was evaluated using the MTT microculture tetrazolium assay based on the ability of mitochondrial enzymes to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into purple formazan crystals, which is directly proportional to the quantity of dehydrogenase enzyme (an indicator of the cell vitality), i.e., the inhibition in the metabolic activity is considered as a first sign for cell damage and decreased cell vitality (Oh and Hong 2022).

The toxicity of HA to vascular smooth muscle cells has been discussed in many research articles (Chen et al. 2018). It is related to the size and morphology. On the uptake and function of human umbilical vein endothelial cells, nano HA was found to be more conducive to cellular uptake and exerts greater damage to normal cell function than micro HA (Shi et al. 2017). The inhibitory effect and the mechanism of nano HA of different sizes (60, 170, 290 nm) on cancer cells (Bel-7402) have been reported (Han et al. 2014). As the concentration was 0.56 g L^-1, the inhibition rates were found 75, 40 and 20%, respectively. Moreover, the anti-tumor activity and apoptosis-inducing ability of 26 and 45 nm HA were found to be greater than those of 78 and 175 nm in case of HepG2 cells (Yuan et al. 2010).

Fig.8 shows the obtained results of MTT assay for the prepared nanocomposites and Tables 3, 4 list the statistics and comparison of cell proliferation of the nanocomposites (G1 – G8) via ANOVA test.

Without magnetic field, the highest mean value was recorded for G5 (84.77±0.75) and G6 (84.67±0.73) groups, followed by G7 and G8 groups, then G4 group, and finally G2, G1 and G3 (61.7±0.93) groups. On the other hand, With magnetic field, the highest mean value was recorded for G7 (61.27±0.9), followed by G8 and G5 groups, then G6, G4, G3, G2 groups and least value recorded for G1 group (50.04±1.82).

There is a positive correlation between Fe₃O₄ content and cell proliferation. This feature is coming from the superior topographical features and the increase in the surface roughness of Fe₃O₄ containing nanocomposites (mag-HA np & mag-HA-Ald np) than free Fe₃O₄ (Heidari et al. 2020). In addition, the chemical composition of the scaffolds that could effectively regulate cell proliferation. Iron is an essential element that has a necessary role in cell metabolism, and incorporating it into scaffolds could enhance cell growth, and thus the magneto nanocomposites can control the trans-membrane flux of some ions, especially Ca⁺²which can adjust factors related to cell proliferation and growth (Han et al. 2021). In addition, the chemical composition of the scaffolds that could effectively regulate cell proliferation.

Based on the obtained results, the presence of Ald significantly increases cell proliferation. The proliferative effect of bisphosphonates was due to their stimulation of fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) gene expression have been reported (Lee et al. 2021). These genes are well recognized for stimulating proliferation and differentiation of mesenchymal progenitor cells. These data are in agreement with those demonstrated that Ald has an effective role in the proliferation and maturation of primary human osteoblast cells.

Moreover, the application of magnetic field decreases the cell viability in all samples due to its thermal effect which could cause depolarization, excitation, and breakdown of cell membrane and show obvious side effect on the cells (Chen et al. 2017), while the non-thermal effects of magnetic field due to the direct interactions of magnetic field with biological cells. The magnetic field could trigger DNA damage, cell ultrastructure alteration, cell cycle arrest and cell proliferation inhibition (Xu et al. 2021; Ashdown et al. 2020; Lee et al. 2015). These results are in contrast with that obtained by Gonçalves AI et al (Gonçalves et al. 2016), they concluded that the magnetic field stimulus promoted the proliferation of the cells in both magnetite-containing and magnetite-free scaffolds, suggesting that the cellular behavior is favored by magnetic forces. In addition, magnetite-containing scaffolds exhibit significant higher cell proliferation values in comparison to magnetite-free scaffolds which highlighted the synergistic effect of combining iron oxide nanoparticles with magnetic actuation (Gonçalves et al. 2016).

Note: For each group, the mean value of cell proliferation was greater without magnetic field compared to those with magnetic field (p = 0.00).

New composites combine hydroxyapatite, alendronate and Fe₃O₄ nanoparticles were successfully synthesized which may potentially be used as a bone tissue scaffold.
These composites have been characterized based on spectral (FTIR, XRD, SEM) and EDX measurements
According to MTT assay results, the prepared composites are considered non –toxic and a positive correlation between bisphosphonate and iron oxide content and cell viability was suggested.
Magnetic field application is not recommended as it significantly decreases the cell viability in all groups.

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Funding & Acknowledgements

This research is funded by the High Ministry of Education, Post 2014 and BRIDGES-Development-3 Project, Scientific Research Academy, Ministry of Research, Egypt.
The work reported has been carried out by Marwa S. Ismail for her Ph D degree. Authors are grateful to the Egyptian High Ministry of Education.

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Dental Biomaterials Department, Faculty of Dentistry, Mansoura University, Mansoura, Egypt

Marwa S. Ismail, Hagag A. Mohammed, Manal F. Osman, Noha A. Elwassefy

Chemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt

Sahar Ibrahim Mostafa

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Correspondence to Sahar Ibrahim Mostafa

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Table 1: The ratios of the prepared nanocomposites mag-HA np and Ald

Groups	*Fe₃O₄ (mag) np*	HA	Ald
G1	0	100	0
G2	0.5	100	0
G3	1	100	0
G4	1.5	100	0
G5	0	100	40
G6	0.5	100	40
G7	1	100	40
G8	1.5	100	40

Table 2: EDX analysis of prepared nanocomposites and their Ca/P ratios

Composite	Element	Mass%	Ca/P
(G1)	C	4.38	1.64
	O	46.34
	P	18.65
	Ca	30.63
(G2)	C	10.19	1.62
	O	53.11
	P	13.94
	Ca	22.54
	Fe	0.22
(G3)	C	14.14	1.67
	O	49.78
	P	13.31
	Ca	22.29
	Fe	0.48
(G4)	C	12.66	1.62
	O	52.02
	P	13.18
	Ca	21.44
	Fe	0.70
(G5) (G6)	C	35.56	1.15 1.15
	O	43.95
	Na P Ca C O	1.01 9.02 10.46 41.05 41.55
	Na	0.69
	P	7.72
	Ca	8.86
	Fe	0.13
(G7)	C	24.52	1.15
	O	48.17
	Na	1.38
	P	11.96
	Ca	13.72
	Fe	0.25
(G8)	C	24.26	1.15
	O	48.17
	Na	1.38
	P	11.96
	Ca	13.72
	Fe	0.51

Table 3: Crystal size (nm) of the prepared nanocomposites

Groups	Crystal size (nm)	Groups	Crystal size (nm)
G1	68 nm	G5	21.6 nm
G2	67.8 nm	G6	21 nm
G3	69 nm	G7	22.5 nm
G4	71 nm	G8	24 nm

Table 4: Descriptive statistics and comparison of cell proliferation of the prepared nanocomposites (ANOVA test)

Cell proliferation		Mean	SD	F	P
Without magnetic field	G1	61.79^d	.87
	G2	62.29^d	1.00	800.3	.000*
	G3	61.70^d	.93
	G4	69.65^c	.74
	G5	84.77^a	.75
	G6	84.67^a	.73
	G7	73.60^b	.79
	G8	73.58^b	.75
With magnetic field	G1	50.04^e	1.82
	G2	51.54 ^z,e	1.37	61.21	.000*
	G3	53.41^z	1.24
	G4	53.59^z	1.20
	G5	59.32^x,y	1.71
	G6	57.62^y	.93
	G7	61.27^x	.90
	G8	60.05^x	1.14

Significance level p ≤ 0.05, *significant; within the same comparison, means sharing the same superscript letter are not significantly different.

Table 5: Comparison data of cell proliferation of the prepared nanocomposites with and without magnetic field

(Paired test)

Groups		Mean	SD	t	P
G1	Without magnetic field	61.79	.87	10.720	0.00*
G1	With magnetic field	50.04	1.82
G2	Without magnetic field	62.29	1.00	22.172	0.00*
G2	With magnetic field	51.54	1.37
G3	Without magnetic field	61.70	.93	10.279	0.00*
G3	With magnetic field	53.41	1.24
G4	Without magnetic field	69.65	.74	20.209	0.00*
G4	With magnetic field	53.59	1.20
G5	Without magnetic field	84.77	.75	64.132	0.00*
G5	With magnetic field	59.32	1.71
G6	Without magnetic field	84.67	.73	40.240	0.00*
G6	With magnetic field	57.62	.93
G7	Without magnetic field	73.60	.79	259.747	0.00*
G7	With magnetic field	61.27	.90
G8	Without magnetic field	73.58	.75	17.584	0.00*
G8	With magnetic field	60.05	1.14

Significance level p ≤ 0.05, *significant

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