Synthesis, Characterization, and in Vitro Biological Evaluation of

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Tissue Engineering and Regenerative Medicine

Synthesis, characterization and in vitro biological evaluation of copper-containing magnetic bioactive glasses for hyperthermia in bone defect treatment Razieh Koohkan, Tabassom Hooshmand, davod mohebbikalhori, Mohammadreza Tahriri, and Mohammad Taha Marefati ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b01030 • Publication Date (Web): 13 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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ACS Biomaterials Science & Engineering

Synthesis, characterization and in vitro biological evaluation of coppercontaining magnetic bioactive glasses for hyperthermia in bone defect treatment Razieh Koohkana, Tabassom Hooshmanda*, Davod Mohebbi-Kalhorib, Mohammadreza Tahriric, Mohammad Taha Marefatid

a

Department of Dental Biomaterials, School of Dentistry/Research Center for Science and Technology in Medicine, Tehran University of Medical Sciences, North-Kargar street, 14146, Tehran, Iran. b

Chemical Engineering Department, Faculty of Engineering, University of Sistan and Baluchestan, Daneshgah street, Zahedan, Iran. c

School of Dentistry, Marquette University, Milwaukee, WI 53233, USA.

d

School of Metallurgy and Materials Engineering, University of Tehran, North-Kargar street, 14395, Tehran, Iran

*Corresponding author: Dr. Tabassom Hooshmand (DDS, Ph.D) Department of Dental Biomaterials, School of Dentistry/Research Center for Science and Technology in Medicine, Tehran University of Medical Sciences, North-Kargar street, 14146, Tehran, Iran. E.mail: [email protected] Tel: +98-9123495663

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Abstract Hyperthermia treatment induced by magnetic mesoporous glasses has been applied as a potential therapeutic approach for bone defects due to malignant tumors. The objective of this study was to synthesize and characterize the structural and biological properties of magnetic bioactive glasses (BGs) for producing multifunctional materials. The effect of the addition of copper (Cu) to the bioactive glass composition was also evaluated. Fe BG and FeCu BG as magnetic mesoporous BGs, and Cu BG as mesoporous BG were synthesized and dried by template sol-gel method. Then, the synthesized bioglasses were characterized and analyzed using Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Energy-dispersive electron disperse spectroscopy (EDS), Brunauer-Emmett-Teller (BET) and Vibrating sample magnetometer (VSM). In addition, the antibacterial behavior, cytotoxicity assay (MTT test), proliferation assay of HUVEC cell assay, and bioactivity (ALP activity test) of the synthesized BGs were evaluated. The characterization results exhibited that the synthesized powders formed mesoporous glasses with nanoparticle morphology, good surface area and magnetic properties. The synthesized BGs also demonstrated suitable biological behavior. The magnetic saturation of bioactive glasses was increased by the addition of copper oxide. A two-phase structure was observed for the magnetic glasses compared to the coppercontaining glasses and thus, making them suitable for drug delivery systems. The antibacterial behavior was found to be better for the Cu BG and Fe BG compared to the FeCu BG. However, the least amount of cytotoxicity was observed for the Fe BG and FeCu BG, compared to the Cu BG. Also, the Fe-containing BGs compared with the control group showed a lack of HUVEC cell proliferation and angiogenesis motivation. From the ALP assay, higher bioactivity for the


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magnetic bioglasses in the presence of mesenchymal cells was found. From the results of this in vitro study, the FeCu BG could be considered as a new generation of magnetic glasses for inducing hyperthermia in treatment of bone defects due to malignant tumors. However, further in vitro and in vivo studies are required to confirm their applications in healing of bone defects and tissue engineering.

Keywords: Bioactive glass; Magnetic; Mesoporous; Bone defect; Hyperthermia; Fe; Cu


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Introduction For years, problems such as osteoporosis, bone loss, jawbone deterioration and bone injuries due to tumor ablation have caused significant clinical complications in terms of restoration and bone reconstruction, particularly in the head and neck area 1. Practical solutions in this field may 2

include bone graft, auto graft, allergenic grafts, and xenogeneic grafts

. Despite the

osteoconductive and osteoinductive abilities of this type of graft, access to these resources are often difficult 3. Problems include the lack of access to an appropriate source for grafting, limitation of bone resection for wide restoration, rejection of grafted bone, pain and patient discomfort after surgery. Therefore, the production and application of the bioactive compounds will be more feasible for the purpose of restoration and reconstruction of bone. Tissue engineering deals with biological substances that restores, replaces or reconstructs the injured or lost tissues

4-7

. Recently, bioactive glasses/glass ceramics, as a new class of biomaterials, have

been widely used in bone regeneration and drug delivery applications due to their appropriate properties that can increase specific surface area and bond with living tissue

8-9

. The proteins

absorbed on the amorphous calcium phosphate (ACP) or carbonate hydroxyapatite (CHA) layer which forms on the surface of bioactive material could cause gene expression and stimulation of osteoblast cells 10. The bioactivity characteristics and behavior of glasses depends on the composition and amount of elements present. Researchers are attempting to control different glass properties and behavior by changing the type and amount of elements in the structure of bioactive glasses

11

. Today, the role of bioactive glass (BG) in creating different

characteristics, such as angiogenesis capacity and chronic wound healing, has been demonstrated

for

the

copper

doped

borate

bioactive


glasses 12.

In

addition,

the

4

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osteogenesis capacity, anti-inflammatory and antibacterial activity were reported for the Zn-containing bioactive glasses magnetic

ions,

Fe+3,

significantly

13-14

. It has been shown that the propagation of

improved

the

mitochondrial

activity

and

gene

expression of BMSCs cells for bone formation, along with reconstructing the bone defects

15

. Iron combined with composite scaffold of glasses creates a magnetic

bioactivity structure that can be utilized for bone tumor treatment with hyperthermia. The magnetic bioactive glass composition in the magnetic core has been widely used to treat magnetic hyperthermia of cancer tumors. Magnetic biomaterials generate heat under a magnetic field in the region of the patient's cancer tissue, and cancer cells are destroyed at a higher temperature (>43 °C), while normal cells survive at this temperature. Furthermore, the composite particles can be used as a drug delivery system for magnetic hyperthermia treatment

16-17

. Copper has shown angiogenesis

behavior when is incorporated in the glass composition

12

. These glasses have been

used for the treatment of malignant tissues, and thus, the angiogenesis behavior in these glasses should be considered. In our previous work, the use of copper in the glass structure did not inhibit its anti-bacterial properties and resulted in a low risk toxic bioglass

18

.

The aim of this study was to synthesize and characterize the structural, magnetic and in vitro biological properties of the synthesized BGs based on SiO2-CaO-Fe2O3-P2O5 with the addition of copper oxide (CuO) to develop a multifunctional behavior such as hyperthermia treatment and antibacterial applications.


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Experiment Materials The chemical composition as raw materials of the following were used: tetraethyl orthosilicate (TEOS, 98%; Merck), triethylphosphate (TEP, Merck, 99.8%), calcium nitrate tetrahydrate (99%;

Merck),

ferric

nitrate

nonahydrate

(Fe(NO3)3.9H2O;

Merck),

copper

nitrate

(Cu(NO3)2.3H2O, 99.5%; Merck), HNO3 (65%; 2 M; Merck), absolute ethanol (Merck) and a non-ionic block copolymer P123 (5800, Sigma- Aldrich, CAS Number 9003-11-6 (ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO–PPO–PEO), EO20PO70EO20).

Methods Synthesis of BGs In this study, three mesoporous BG systems with a mass ratio of (i) and (ii) SiO2 (68%), CaO (23%), P2O5 (4%), X (5%) (X= Fe2O3 or CuO) and (iii) SiO2 (68%), CaO (18%), P2O5 (4%), CuO (5%), Fe2O3 (5%), were synthesized by sol-gel method using the evaporation-induced self-assembly (EISA) process for drying, with P123 surfactant used as co-templates. The details of synthesis of the BGs have been detailed in other studies

18-19

. BGs were prepared by dissolving P123 (12 g) in 180 ml of ethanol and 25

ml of distilled water, under stirring at 40 °C until the solution became clear. Then, 2.7 ml of 2 M HNO3 and tetraethylortosilicate were added to the solution for 40 min to complete the hydrolysis reaction of TEOS by acid. TEP, calcium nitrate tetrahydrate, copper nitratetrihydrate and ferric nitrate nonahydrate were added sequentially through an interval of 40 min to the clear solution. The final pH was 3.5 and the mixture was stirred at 30 °C for 24 h. The resulting solution was poured into a petri dish then


6

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transferred into a drying oven at 38 °C for 7 days. Finally, the dried gel was dried and calcined in a nitrogen/air oven at 650 °C with a heating rate of 2.8 °С.min-1, starting at 25 °С to eliminate the organic substances and residual nitrate. First, nitrogen gas was used to decline the oxidation probability of the metal elements. Second, at the final 105 min, the air was utilized for better removal of polymer from the glass structure. The calcined powders were then collected for analytical analyses.

Characterization of BGs The calcination temperature and thermal behavior of the sample were determined by Thermogravimetry and differential thermal analysis (TG-DTA; STA PT-1000, Linseis, Germany) on dried gels at a heating rate of 10 °C.min-1, from 25 °C to 900 °C in air. The structure of prepared glasses was analyzed by X-ray diffraction (XRD; utilizing INEL Equinox 3000, France), with Cu-Kα radiation at 40 KV and 40 mA. Fourier transform Infrared spectroscopy (FT-IR; Nicolet iS10 spectrometer, USA) was used to investigate the functional groups of molecules, chemical composition of the samples, and to ensure the formation of the synthesized BGs. The samples, before and after soaking in SBF solution, were analyzed at the wave number range of 400–4000 cm–1. Magnetic

characterization

of

the

BGs

was

confirmed

using

a

Vibrating

sample

magnetometer (VSM, PAR-155) with maximum applied field up to 2 kOe at room temperature.

To

study

the

geometries,

composition

and

microscopic

structure

of

calcined BGs, the powders were analyzed using Scanning electron microscopy (SEM) and Electron disperse spectroscopy (EDS) (SEM; TESCAN MIRA3 LMH Schottky, Czech Republic, 15.0 KV). The nanostructure was investigated using Transmission


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electron microscopy (TEM; Zeiss-EM10C, Germany, 80 KV). The powder specific surface area and porosity characterization were evaluated by Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH), respectively, with a PHSCHINA, PHS1020 device. These methods were based on the adsorption–desorption of low-temperature nitrogen gas. For measuring the bioactivity of the synthesized BGs, first, the SBF solution was prepared according to the method proposed by Kukubo et al.

20

. Then, 0.3

mg of the synthesized BGs were dissolved in 200 ml of SBF solution and incubated at 37 °C. After the duration of 1, 7, and 14 days, the samples were separated from the solution by centrifuge and Whitman membrane filter (50 mm diameter/size of 40) and then dried. The releasing ion exchange into the biologic solution was analyzed by Inductively Coupled Plasma (ICP). The dried samples were also analyzed using SEM and EDS to investigate the formation of apatite layer deposition. The cell metabolic activity and cell proliferation were measured over time, utilizing the MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide)

assay

with

direct

method. This is a colorimetric assay for assessing the cell metabolic activity. The bioactivity of glasses was evaluated by Alkaline Phosphatase Activity (ALP). Horse Mesenchymal Stem Cells-adipose (HMSC-ad) were exposed to the synthesized BGs to evaluate the cytotoxicity and bioactivity properties, which were achieved by MTT and ALP assay, respectively

21-22

. The angiogenesis behavior was examined by measuring

the proliferation of the HUVEC (Human Umbilical Vein Endothelial Cells/C554). The MTT test was performed directly on the BGs, according to the study done by Labbaf et al.

21

. For this purpose, the number of evaluated cell was 6000 cells per well and the

medium was Ham’s F12+DMEM (1/1V) and FBS10% at NCBI.


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The

antibacterial

activity

inhibitory concentration assessment

on

the

of

(MIC)

standard

the and

BGs the

was

investigated

minimal

gram-negative

E.

by

bactericidal coli

measuring

minimum

concentration

ATTC25922

14

.

The

(MBC) initial

suspension was obtained by mixing of sterile powder and nutrient broth culture solution through a concentration of 100 mg.mL-1 in the sterile tubes. The nutrient broth without the BG powder was selected as the control group. Broth Microdilution method was used to obtain MIC in 96-well microtiter plate. Briefly, a solution of prepared suspensions of the samples, at various concentrations by dilution method (12.5, 25, 50, and 100 mg.mL-1), in a mixture of Muller Hinton Broth medium with 50/50 v/v ratio was added to the wells. Then, these wells were inoculated by a bacterial suspension according to McFarland standard of 0.5 at (1.5×108 CFU/mL) value. The MIC of bacteria was chosen according to the lowest concentration well lacking turbidity (colonies of bacteria) after 24 h of incubation at 37 °C. To confirm complimentary, the MIC value was certified using ELISA reader at a wavelength between 450 and 620 nm. To determine the minimal bactericidal concentration, all wells lacking turbidity or bacterial colony were initially inoculated on Mueller-Hinton Agar plates separately. Then, the lowest concentration of the plates lacking bacterial colonies was reported as MBC after 24 h of incubation at 37 °C. To obtain reliable data, each test was performed twice using a constant concentration on three samples.

Statistical evaluation The data for the MTT, ALP, and HUVEC cell proliferation were analyzed by One-way ANOVA followed by Tukey HSD post hoc test. The data for the antibacterial


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evaluation were not statistically analyzed because the antibacterial analysis was only performed twice using a constant concentration.

Results and Discussion Thermal analysis Fig. 1.A shows the TG-DTA curves of the dried gels containing copper. Three exothermic peak temperatures of this sample at 178, 294 and 365 °C were observed in the DTA curve during heating up to 700 °C. To explore the nature of these exothermic events, the pure initial nitrates and the P123 surfactant were also analyzed by the DTATG apparatus in a similar manner. However, their graphs have not been presented here for summarization. The results showed that the iron nitrate and calcium nitrate decomposed after melting in a single step via endothermic reactions below 200 ºC

23-25

.

However, the endothermic decomposition reaction of copper nitrate happened after its melting in two steps at 183 ºC and 265 ºC. Similar results have been also reported by other researchers

23-24

. Moreover, the surfactant burned mainly in the temperature range

between 178 ºC to 350 ºC. Nevertheless, it is interesting to note that for the synthesized gels, the removal of nitrates occurred through the exothermic reactions. It is due to this fact that the surfactant is mainly composed of glycol which is used as a fuel in the solution combustion reaction of metal nitrates to fabricate metal oxides

26,27

. In other

words, the reaction between P123 and metal nitrates is considerably exothermic

26-29

. It

seems that this reaction overlapped with the endothermic decomposition reaction of nitrates and the sum of these reactions appeared as exothermic events in the DTA curves of the gels. Therefore, the first exothermic peak with a considerable mass loss of


10

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48% was due to the elimination of nitrate group through its reaction with the surfactant, the evaporation of volatile components

26-29

, the decomposition of un-dissolved copper

nitride to copper oxide 24, and the extrication of water and CO2 by-products 28,30. The second weight loss (about 5.5%) could be attributed to the omission of remaining calcium nitrate and burning of remaining surfactant through an exothermic reaction. However, no significant weight loss was recorded during the third exothermic event. Therefore, as reported by other studies

28, 31

, this event may be related to the formation

of crystalline phases. Similar exothermic peaks were observed in the DTA/DTG curves of the prepared Fe-containing gel, as shown in Fig. 1.B. However, the first peak temperature in the Fe-containing gels was slightly lower than that of the Cu-containing gel. Since the only difference between the gels was their copper and iron nitrates contents, this shift could be related to the presence of these nitrates. In the other words, because of the lower melting point of iron nitrate than the copper nitrate, the decomposition temperature of iron nitrate was less than the copper nitrate during heating 24, 26,27 . The TG-DTA curves of the copper-iron containing gel are shown in Fig. 1.C. Three consecutive exothermic events were observed in the DTA curve, along with three sudden weight losses during these exothermic events. Comparing these curves with those presented in Figs. 1.A and B, it can be concluded that in addition to the evaporation of volatile components, the first and the second exothermic events at 158 °C and 202 °C in Fig. 1.C were related to the decomposition of iron nitrate and copper nitrate through their reaction with the P123 surfactant, respectively

24, 26, 29, 32

. Also, the

third broad exothermic event was related to the combustion of remaining surfactant and


11


omission of remaining calcium nitrate

26,29

Page 12 of 52

. Above 500 °C, no considerable weight loss

was observed in any of the samples. Thus, it can be concluded that the glasses were formed during calcinations of the gels above 500 °C. Hence, regarding the TG-DTA results, the majority of cytotoxic components were removed, which might be effective on the biologic environment. Based on these results, the calcination temperature was selected as 650 °C. In a similar work on the magnetic glass and glass ceramics done by Baino et al.

33

flowing) on

, the effect of different processing conditions (calcination in air vs. argon the formation of magnetic crystalline phases

was

investigated.

The

endothermic peaks were observed in some of the TG-DTA curves, however, the surfactant was not used in the synthesis method of bioglasses. In addition, the crystallization of magnetic phase occurred around 550-570 °C. In this study, the weak exothermic peak observed at 365 °C in Fig. 1.B might be related to the initial formation of magnetic phases as found by the XRD (Fig. 2 in the next section). Also, because of P123 surfactant burning during the thermal analysis, the formation of these phases might have occurred at lower temperatures.

XRD analysis Fig. 2 shows the XRD patterns of the Fe, Cu, and FeCu BGs. The presence of broad peaks in these spectra showed that the calcinated samples were mainly composed of an amorphous

phase.

There

were

peaks

related

to

the

crystalline

of

Hedenbergite

(CaFeO6Si2, JCPDS No. 96-900-0339, and 22-1 reflection) and minor concentrations of Hematite (Fe2O3, JCPDS No. 96-101-1241 and 211 reflection) as crystalline phases


12

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observed in the Fe BG. The XRD patterns of FeCu BG exhibited crystalline phases with

diffraction peaks of Hematite and Magnetite (Fe3O4, JCPDs No. 96-900-2317)

corresponding to the (10-1) and (311) reflections, respectively 34. The presence of Magnetite, Hematite and Hedenbergite crystallization would lead to the saturation of magnetization values

35

.

XRD patterns of all samples displayed a

broad peak between 15-35 °C. These results revealed that the dominant phase in the synthesized samples was glass.

FTIR analysis The FTIR spectra of Fe, Cu, and FeCu synthesized BGs are shown in Fig. 3. Most absorption bands around 460 (455-470) cm−1 and 800 (795-805) cm−1 were related to the Si–O–Si bending vibration mode and symmetric stretching of Si–O–Si band, respectively. The absorption band located at 1070 (1000-12000) cm−1 was attributed to Si–O–Si asymmetric stretching in SiO4 group

36-37

. The weak absorption peak at 1238

cm−1 corresponded to the presence of PO4-3 groups in the glass and was not related to the formation of apatite-like phase .The peaks around 1639 cm−1 -1648cm−1 confirmed the bending vibration modes of water (O-H) groups

37

. The band observed at 569 cm−1

in Fe BG and FeCu BG was related to metal-oxygen (metal-O) bond, which could confirm the formation of magnetic iron oxide (Fe-O) or (Cu-O)

38

. Additionally, the

absorption bands at 3550cm−1 were attributed to –OH group and confirmed the formation of silanol groups on the surface of glasses groups

39

.

Finally, the IR data

confirmed the possibility of formation of silica framework (glass).


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Page 14 of 52

SEM/EDS observations FE-SEM images of Fe, Cu, and FeCu synthesized BGs are shown in Figs. 4. A homogeneous structure including agglomerated nanoparticles for the Cu BG can be observed. Microscopic examination of the Fe BG and FeCu BG demonstrated the twophase structure, including the spherical cavities in a homogeneous background of glasses (Figs. 4.D and G). The spherical cavities, including agglomerated particles, can be observed in the interior of the cavities (Figs. 4.E, F, H, and I), while a homogeneous structure is obvious at the periphery of the cavities (Figs. 4.D and G). Figs. 4.G, H, and I are related to the Fe BG sample, which has a similar structure to FeCu BG. However, the amount of spherical cavities in Fe BG was much more than FeCu BG (Figs. 4.D and G). Furthermore, the agglomerated nanoparticles set inside the cavities of Fe BG were separated from the wall, and the agglomerated nanoparticles were encompassed as a ball-liked shape inside the cavities (Fig. 4.H). While, the nanoparticles had almost no distance from the inside walls of the cavities in the FeCu BG sample (Fig. 4.E). The Cu BG did not clearly show the two-phase structure (Fig. 4.A). A finding obtained by EDS confirmed the presence of elements (Si, Ca, P, O, Fe, and Cu) in the structure of glasses. Although, EDS test is not a purely quantitative analysis, it could be inferred that there was a difference between the weight percent of iron inside and outside of the cavities in the EDS diagram of FeCu BG and Fe BG (Figs. 5.B, C, D, and E). The content of Fe inside the cavities (spherical pellets) (Figs. 5.B and D) was higher than that of the outside in both glasses containing iron. Also, in all samples, there was a higher presence of oxygen and Si peaks compared to the other element peaks, confirming the formation of glass or glass ceramic 40.


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Figs. 4.A, D, and G show that the spherical cavities in Fe Cu BG were created, with a maximum for the Fe BG samples. Further studies are required to assess the observed structures for the synthesized bioglasses in this study. According to another studies

41-42

, the probable reason for these changes could be due

to the nucleation tendency and crystal growth for iron oxide ions in the silicate glass structure,

leading

to

a

two-phase

structure

containing

spherical

cavities.

These

spherical cavities are like the crystal phases which could be formed in a glass silicate network through the nucleation and sediment of ions elements during the synthesis process. In addition, other studies have reported that both types of Fe ions, ferrous (Fe2+) and ferric (Fe3+) ions can exist in the silicate network of glasses containing Fe

41-42

. In this

study, the presence of both Fe ions was also proved by the formation of Magnetite and Hematite phases by XRD (Figs. 4). In studies done on the Fe-containing glasses

41, 43

, the iron atom/ions were able to

change their valence in the glass matrix. Thus, the regions of local energy difference were

created

by

transferring

of

valence

electrons

between

the

ions

and

thus,

accelerating the nuclei formation. The grain and particle were changed from tiny and very fine to aggregates of prismatic particle microstructures of glasses 41, 43. The solubility of ferric ion is less than that of Cu2+ and Fe2+ due to the smaller atomic radius and higher valence for Fe3+. For the same reason, the solubility of Cu2+ is less than that of Fe2+. Hence, the replacement of Fe3+ with Cu2+ in the FeCu BG can be due to the low ferric solubility and the primary penetration of copper ion in the silicate


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Page 16 of 52

network during its synthesis, as the copper nitrate was added to the solution prior to the iron nitrate. Likely, the copper–iron reaction can occur by the following equation 44:

Fe3+ + Cu+ ⇌ Fe2+ + Cu2+

Eq. 1

According to the above explanations, the concentration of ferric (Fe3+) ions in the silicate network of the FeCu BG decreased during synthesis process due to the nucleation and crystal growth by ferric ions in the presence of copper ions. Therefore, the electron valence difference between the iron ions (Fe2+ and Fe3+) could be changed by the reduction of (Fe3+) ions concentration in the FeCu BG structure compared to Fe BG. Consequently, this event leads to the declination of the spherical cavities and nuclei formation in FeCu BG microstructures (Figs. 4.D and E). On the other hand, due to the lower solubility of Cu2+ compared to Fe2+ ions, ferrous cannot effectively induce nucleation in the FeCu BG structure. As a result, the number of spherical cavities was less formed and the crystalline growth was increased by the sedimentation of ions in the FeCu silicate network compared with that of Fe BG (Figs. 4.D and G). Therefore, there was no distance between the spherical agglomerated particles with the homogeny area in the FeCu BG structure due to the increment of crystallization inside the cavities surface on the nuclei in the FeCu BG structure compared with Fe BG (Figs. 4.H and E). However, this distance with surfactant was created within the solution stage, which then disappeared during calcination stage. EDS results also confirmed the presence of higher amount of iron inside the cavities. On the other hand, copper could act as a glass modifier

45

such that Cu displaces the

ions in the silicate structure of FeCu BG. Thus, the nucleation of Fe ions would occur


16

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with distances for the FeCu BG. Because both magnetic glasses had the same percentage of Fe (%5), more crystallization growth and less nucleation was found for the FeCu BG compared to the Fe BG (Figs. 4.D and G).

TEM observations Nanostructure formation in the samples was confirmed using TEM images, with their size in nano-aspect and under 100 nm

46

. In addition, all the mentioned samples showed mesoporous

structure formation. Regular mesoporous structure formed in Cu BG (Figs. 6.A and B), while both regular and irregular mesoporous structure were observed in the TEM images (Fig. 6.D, F and I). These results were explained by the two phase structure observed for FeCu BG and Fe BG as shown in SEM images. The regular mesoporous structure in FeCu BG was related to the formation of homogenous structure placed outside of cavities (Fig. 6.D). The irregular mesoporous structure for the above-mentioned glasses containing iron was attributed to the formation of non-homogenous structure which were observed in SEM images (Figs. 4.E and H) and placed inside the cavities, as shown in Figs. 6.F, G, and H. While, a regular mesoporous structure was not observed for the Fe BG due to the high numbers of spherical cavities (Fig. 6.G and H). Also, Fig. 6.F was most likely an image of the spherical-shape mass, which included a porous nanoparticle set with irregular mesoporous shape.

Textural characteristics The

surface

area

of

Fe

BG,

Cu

BG

and

FeCu

BG

was

evaluated

by

N2

adsorption/desorption isotherms, as shown in Fig. 7A. The isotherm curves of Fe BG, Cu BG and FeCu BG exhibited a type IV isotherm that was associated with the


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mesoporous materials. Type H1 hysteresis loop was shown for FeCu BG and Cu BG, which is often consistent with the mesoporous structure of compacts or agglomerates of approximately uniform spheres in fairly regular array47. H4 hysteresis loop is shown for Fe BG in Fig. 7A, which is indicative of the presence of narrow slit-like pores 47. The textural characteristics obtained from the BJH assay and BET results for the three bioglasses are provided in Table 1. It should be noted that the addition of copper to the magnetic glass structure improved the surface area in the present study. However, in comparison to the glasses in other studies48 , the three samples showed a relatively large specific area of 169.467, 203.492, and 284.822 m2.g-1 for the Fe BG, Cu BG, and FeCu BG, respectively. In a similar study done by Zhang et al.48 , the specific spatial surface areas (SSA) for the mesoporous bioglasses with 3, 6, and 9% of Fe were 121, 114, and 84 m2.g-1, respectively. Furthermore, due to the use of surfactant during synthesis of bioglasses in the present study, FeCu BG, Fe BG and Cu BG showed higher specific areas compared with the magnetic bioglasses that were synthesized without surfactant by Baino et al. 33. Baino et al. 33 have reported the specific areas around 59.7 and 41.5 m2.g-1 for the 60SiO238CaO-2Fe2O3 mesoporous according

and

60SiO2-30CaO-10Fe2O3,

respectively.

In

addition,

the

materials are characterized by pore size in the range of 2 to 50 nm to

the

IUPAC

definition

(International

Union

of

Pure

and

Applied

Chemistry) 49. Fig. 7B demonstrated that the diameter pore sizes were in the range of 2-5 nm and 2-7 nm for the Fe-containing glasses and Cu BG, respectively. The distribution of pore size for the Fe BG and FeCu BG was uniform. As shown in Table 1, the average pore


18

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diameter of the samples was 2.519, 2.5, and 2.41 nm for the FeCu BG, Fe BG, and Cu BG, respectively.

Bioactivity evaluation of synthesized BGs XRD analysis XRD pattern of Fe BG, Cu BG, and FeCu BG showed the characteristic peaks of hydroxyl apatite, in accordance with the JCPDS No. 96-901-1092 (Fig. 8). The presence of a diffraction peak around 31-34 degrees (121), the minor peaks at 25 degrees (002), 46.9 degrees (221), and other minor peaks (130), (123), and (502) could indicate the deposition of amorphous hydroxyl apatite layer on the surfaces of the samples 50.

FTIR analysis For the bioactivity evaluation of treated glasses by SBF, the functional group band related to HCA (hydroxyl carbonate apatite) or ACP formation could be observed in the FTIR spectra for Fe BG, FeCu BG, and Cu BG (Fig. 9). The band at 460 cm-1 was shifted to a higher band at 467 cm-1 after 7 days of treatment with SBF. The band at 1079 cm−1 was shifted to a range of 1024-1093 cm−1, which specifically corresponded to the phosphate groups simultaneously. Before soaking, all the glasses displayed a double peak at 1421 cm-1, as shown in Fig. 3. When the immersion time for samples was increased to 7 days, the intensity of peaks was also increased. The bimodal peaks observed around the wavenumbers of 1415-1460 cm−1 were related to the CO3-2 (C=O) group

37, 39, 50

. The peaks around 1640-1648 cm−1 and 3420 cm-1 confirmed the bending

vibration modes of water and (hydroxyl) O-H groups, respectively 37-38.


19


Page 20 of 52

It has been reported that the absorption bands around 3550 cm−1 are attributed to –OH group, confirming the formation of the surface silanol groups

39

. Additionally, the

characteristic bands for the presence of a carbonated hydroxyl apatite (HCA) layer appeared at, 568-608, and 1024-1093 cm−1, corresponding to PO4-3 groups (P-O). It can be concluded that these results confirmed the formation of HCA

or ACP amorphous

calcium phosphate 37, 39, 50.

ICP analysis and pH measurements Leaching and exchanged ion of filtered solutions from the treated glasses (in duration of 1, 7 and 14 days) were analyzed by Inductively Coupled Plasma (ICP). On the first day, all three samples of the synthesized glasses showed an increase in the calcium ion concentration (Fig. 10.A). Cu BG had the highest value of releasing calcium ions, with the maximum calcium ions release occurring in the first two days, as shown in Fig. 10.A, while for the Fe BG and FeCu BG was at the seventh day. Meanwhile, for the Cu BG and Fe BG, the calcium ion concentration was reduced after 3 and 8 days, respectively. Fig. 10.B shows that the phosphorus absorption was high within the first three days. The maximum phosphorus ions absorption occurred in both Cu BG and Fe BG surfaces during the first day. However, the phosphorus ions released from the FeCu BG to SBF solution during the first two days and then, the absorption value of phosphorus ions increased in the following days. Fig. 10.C showed that Cu BG and FeCu BG had the maximum and minimum copper ions releasing into SBF solution, respectively. Fig. 10.D showed that the silicon ions releasing from the FeCu BG and Cu BG into the SBF solution reached the maximum during the first day and then remained constant. As a result, the increase of Ca and decrease of P ion concentrations, along with increased pH in the SBF solution, could be attributed to the sedimentation of the HCA or ACP layer on the surface of the bioactive glasses


20

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51-53

. Fe releasing was lower than the level worthy to be reported. This is most likely due to the

strong bond of Fe compared with the weak band of Ca and Cu found in the glass structure, which resulted in a more permanence of Fe in the glass structure, and thus, increased magnetic saturation and super-paramagnetic behavior of glass for the hyperthermia treatment. The pH of samples was in the range of 7 to 7.9 in SBF (Fig. 11). The low release of Fe and average pH could provide a desirable condition for growing and cohesion of cells onto the glasses

53

. In other studies

16, 53

, the releasing of Ca and Si ions decreased with the iron oxide

substitution in the glass structure. This phenomenon was observed in both types of glass containing iron, especially in Fe BG compared to FeCu BG. It is worth mentioning that the Ca releasing in FeCu BG was higher than Fe BG, which was due to the presence of Cu in FeCu BG. In fact, , it has been shown

45

that Cu could act as a modifier and the CaO (modifier) was

replaced by CuO in the Cu-containing silicate–phosphate glasses and therefore, this could have resulted in higher amount of Ca releasing in FeCu BG compared to Fe BG. The release of silicon ion in Fe BG reached the maximum on the seventh day, followed by a decrease in its releasing into the SBF solution Fig. 11.D. In addition, the pH diagram followed the pattern of silicon ions releasing into SBF solution up to the seventh day, which was more significant for Fe BG at this time, as shown in Fig. 11. In the Fe BG, the increase in pH was slow due to the low speed of calcium releasing (Fig.10.A and Fig. 11). Therefore, breaking the SiO2 in the SBF solutions occurred with low speed. Decrease of the Si concentration might have occurred due to the Si– OH group formation on the surface of the glasses after 7 days.


21


Page 22 of 52

SEM/EDS observations The SEM micrographs of three bioactive glasses (Fe BG, Cu BG, and FeCu BG) showed a sediment formation on the glasses surface after 7 days. Fig. 12 clearly showed this sediment formation, however, HCA formation speed could not be judged in SEM images. The broad peak and higher weight percentage of Ca, P, and O could confirm the semi-apatite or ACP layer formation on the surface of bioactive glasses in EDS graphs

54

(Fig. 13). These results were

coordinated with what was observed by FTIR and ICP analyses on the seventh day. The Ca to P ratios are shown in Fig. 13. The Ca/P ratios in the precipitation for the FeCu BG, Fe BG, and Cu BG were 1.393 (14.06/10.09), 1.59 (12.41/7.76), and 1.43 (15.22/10.64), respectively. The Ca to P ratio ranges from 1.5 in amorphous calcium phosphate (ACP) to 1.67 in hydroxyl apatite (HA) 55 . The Fe BG showed the closest Ca/P ratio to that of the ACP or HA. The low Ca/P ratio observed for the Cu-containing glasses in this study could be due to the high affinity of Cu ion for bonding to the P ion and formation of the Ca19Cu2(PO4)14 and thus, high amount of P remains in the deposition on the bioglass surfaces. Therefore, it can be concluded that the amount of deposition on the glass surfaces after soaking in SBS is not a reliable criteria for determining the bioactivity of glasses and the ALP evaluation must be also performed.

VSM analysis Fig. 14 shows the VSM graphs for all three synthesized BGs, including a narrow hysteresis loop and very high coactivity (Hc) for the FeCu BG sample compared to Fe BG. The magnetic properties were determined under a magnetic field of 20,000 Oe for the synthesized BGs (Fe BG and FeCu BG), as shown in Fig. 14. The saturation magnetization of FeCu BG (1.043 emu/g) was almost five times the Fe BG sample


22

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(0.236 emu/g). The remanence magnetization in Fe BG was much higher than that of FeCu BG, which was 86.26 emu/g and 31.259 emu/g, respectively. The superparamagnetic behaviors were justifiable for FeCu BG regarding the obtained data in Fig. 14. According to a previous study (Fe3O4),

Hematite (Fe2O3),

and

56

, the magnetic phases such as Magnetite

Hedenbergite (CaFeO6Si2) could

improve

the

paramagnetic behavior. It appeared that the addition of Cu to the glass composition resulted in better entrance of iron oxide into the glass structure in this study, and therefore,

a

better

formation

of

crystalline

phase

of

Magnetite,

Hematite,

and

Hedenbergite as shown in the XRD diagram (Fig. 2). If the size of iron oxide particles was

similar

to

that

as

quantum

dot,

the

magnetic

behavior

transforms

from

ferromagnetic into super-paramagnetic status. The reason for the higher paramagnetic property observed for the FeCu BG might be related to synthesis stages compared to Fe BG. In the synthesis stages, copper nitrate salt and then iron nitrate salt were added into the synthesizing solution. Because of this, copper was replaced with iron. In fact, copper simplifies iron replacement in the glass composition. Finally, the magnetite phase formation was simplified during synthesis and calcination processes in the FeCu BG. According to the ICP results, the lack of Fe ions released in SBE solution (as soaking samples were separated) could be attributed to the Magnetite, Hematite and Hedenbergite phase formation in the FeCu BG and Fe BG addition,

enclosing

of

spherical

agglomerated

16

as shown in Fig. 2. In

nanoparticles with

high

iron

content

inside the cavities in the glasses microstructures (SEM images in Figs. 4.D and G) could also contribute to the lack of iron releasing.


23


Page 24 of 52

MTT assay The cytotoxicity test was performed by the

Horse Mesenchymal Stem Cells-adipose

(HMSC-ad) with two specific concentrations according to the method reported by Labbaf et al.

21

. Fig. 15 presents the effect of synthesized glasses on the cellular growth

proliferation rate of mesenchymal stem cell and statistically difference between the mean values using one-way ANOVA analysis. No significant cell proliferation under the low concentration (200 µg.mL-1) in the samples compared to the control group within the first day was found (Fig. 15). The control group showed more proliferation of the samples under the high concentration (400 µg.mL-1). However, there was no significant cell proliferation difference between the glass samples and control group for either concentration. The ICP results showed that the maximum ion exchange between the glasses and biologic environment happened on the first day, while the minimum amount occurred for the Fe-BG. Furthermore, this sample contained no copper. Therefore, it could be concluded that the cells were involved in less shock due to the ion exchange volume for the Fe BG glass compared with that of the two other glasses on the first day. All the synthesized BGs showed a significant proliferation under both concentrations within the third day compared to the control group, while Fe BG and Cu BG samples performed more effectively under the high concentration. No difference was observed within the third and seventh days in respect to the amount of cell proliferation for both Fe BG and Cu BG samples under the two concentrations. However, the cells in FeCu BG showed significant proliferation compared to the third day. The cell growth difference was significant compared to the control group for the samples containing Fe.


24

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Since all the three glasses had metal compositions, releasing of ions could act as a shock for the cells, which occurred for the Cu sample within the first day based on the ICP results. The Cu ion concentration was increased more in the Cu BG medium compared to the FeCu BG within the seven days, which resulted in less cellar growth for the Cu BG. On the other hand, Si concentration has been considered as an effective factor to promote the biological activity and cell growth

57

. Based on the ICP results in

this study, lower amounts of Si existed in the medium of Fe-glass culture compared with that of FeCu BG. Therefore, the amount of cellar growth in the Fe-glass was less than that of FeCu BG on the third and seventh days.

ALP activity The in vitro bioactivity was assessed using alkaline phosphatase activity assay (ALP), and consequently, the differentiation was performed from mesenchymal stem cells to osteoblast cells

22

. The 45S5 glass was selected as the control positive. Fig. 16 shows

the activity of alkaline phosphatase enzyme evaluated through exposure of Horse Mesenchymal Stem Cells-adipose (HMSC-ad) to synthesized glasses and statistically difference between the mean values using one-way ANOVA analysis. On the first day, both concentrations of ALP level for Fe BG and FeCu BG showed no significant increase compared to the control group. However, the ALP level was increased in Feglass compared to 45S5. At this time, the ALP level in the copper glass was slightly less than that of control groups for both concentrations. On the third day, the ALP levels increased in all the glasses at a concentration of 200 µg.mL-1 compared to the control group. However, this increase was significant only for the 45S5 BG. All of the


25


Page 26 of 52

samples, except FeCu BG at the concentration 400 µg.mL-1, showed a significant increase in the alkaline phosphatase level. On the seventh day, there was a significant increase of ALP activity for the 45S5, Fe BG, and FeCu BG samples at the two concentrations compared to the control group. However, this difference for Cu BG was only found to be significant at a concentration of 400 µg.mL-1. On the third day, the cellular differentiation and bioactivity rates were significantly higher for the Fe BG at the concentration of 400 µg.mL-1 compared to that of Cu BG and FeCu BG. However, significant cellular differentiation and bioactivity rates were observed in the FeCu BG for both concentrations at the seventh day. It is worth mentioning that the ICP results showed that the maximum range of releasing Ca, Cu and Si ions occurred within the first two days. However, only Fe BG with a concentration of 400 µg.mL-1 had high ALP level compared to other glasses. Therefore, the lack of copper in Fe BG composition and less ion interchange resulted in the increased ALP level on the first day. At the same time, the FeCu BG and Cu BG not only released Ca ions and absorbed phosphorus ions, but also released Cu ions into the environment containing cells. Leaching Cu ion into the medium probably led to decreased cellular differentiation rate and amount of alkaline phosphatase protein. However, the amount of Cu ions decreased due to the changing medium during the other days. Therefore, it can be concluded that decreasing the amount of Cu resulted in an increased alkaline phosphatase protein level in the medium. Based on the ICP results, the primary sediment of calcium phosphate (CaP) layer on the glass surface as well as the completion of this layer as time passes, led to the increased ALP activity on the third day and later. Cu BG sample showed less ALP


26

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level due to the higher Cu-releasing in the medium compared to that of other groups. A high-chemical affinity of copper to phosphorous resulted in the formation of P–O–Cu bonds

45

, which as a result led to the decreased formation of an amorphous hydroxyl

apatite or ACP layer due to the competition between calcium and copper for bonding with phosphorus.

Proliferation assay of HUVEC cell

The results of the growth and proliferation of HUVCs when exposed to the BGs are shown in Fig. 17.

A statistically significant reduction in HUVEC cell proliferation was

observed for both concentrations of 100 and 200 µg.mL-1 by the first day. The magnetic glasses showed less growth at the concentration of 100 µg.mL-1 compared with the control group. While, a significant reduction in the cellar growth was only observed for the Fe-glass under the concentration of 200 µg.mL-1 by the third day. A significant reduction in HUVEC growth was observed on the seventh day compared with the control group in both concentrations. The overall results of the cellar proliferation test showed that the glasses containing Cu had better cellar growth compared with the Fe-glasses. However, none of the three glasses could lead to a more motivation in HUVEC growth, which is a vain cell subset. This is important because these glasses are designed for use in cancer cell treatment. However, angiogenesis is an obstacle with treating cancer cells. Contrary to the results by another study 12 which improved angiogenesis by Cu-containing glasses was reported, a significant reduction in the HUVEC cell proliferation and angiogenesis by the addition of Cu in the magnetic glasses was observed in the present study. Therefore, we believe that the lack


27


Page 28 of 52

of angiogenesis motivation by these samples, especially for the Cu containing sample, can

be

considered

their

main

advantage

in

treatment

of

cancer

cells.

Because

angiogenesis could result in the metastasis of malignant tumors 58.

Antibacterial evaluation Table 2 shows that the bacterium inhibitory concentrations of Cu BG, Fe BG, and FeCu BG during 24 h were 25, 50, and 100 mg.mL-1, respectively. The bactericidal concentrations for the Cu BG, as well as Fe BG and FeCu BG were 50, 50, and 100 mg.mL-1, respectively. It is noticeable that the results were obtained using three samples and two experiment repetitions. The antibacterial behavior of Cu-containing bioactive glasses has been also shown in the previous studies 18, 59. Finally, in the present study, the synthesized FeCu BG showed better magnetic behavior and surface area compared with the similar types in other studies

16, 48, 60

. The reason for the

improvement of magnetic behavior was the use of Cu in the glass composition. To our knowledge, this study is the first that showed the presence of Cu in the magnetic bioglasses has led to a better Fe replacement in the glass composition and also improved magnetic saturation and paramagnetic property. Therefore, Fe toxic side effects in the magnetic glasses might be reduced significantly by the addition of Cu to the glass composition as less Fe is required. In other words, more magnetic saturation by less amount of Fe in the bioglass structure could be obtained. Moreover, the presence of Cu in the structure of magnetic glasses would result in a more release of Ca and thus, improved bioactivity similar to that of observed for the FeCu BG in this study. Furthermore, the mesoporous and two-phase structure in the Fe-containing glasses can provide their use in drug delivery systems. Due to the complications of chemotherapy in cancer


28

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treatment, it is hoped that future studies on hyperthermia treatments could lead to a reduction in the side effects of chemotherapy.

Conclusions In the present study, the synthesized Fe-containing BGs had relatively glass-ceramic structures, while the Cu BG had a glass structure. The bioactivity behavior of glasses was confirmed by the semi-apatite layer formation and ALP assay, suggesting that they could be appropriate candidates for bone tissue regeneration. All the synthesized BGs were biocompatible and the FeCu BG had super-paramagnetic property. This study showed that the addition of Cu along with Fe in the composition of BGs could lead to the improved super-paramagnetic behaviors with reduced amount of Fe required, and also with no angiogenesis property. From the results of this in vitro study, the copper-containing magnetic bioglasses as a new generation of magnetic glasses might be considered as an appropriate candidate for inducing hyperthermia in treatment of bone defects due to malignant tumors. However, further in vitro and in vivo studies are required to confirm their applications in healing of bone defects and tissue engineering.

Acknowledgement This study was supported and funded by a grant (Grant No. 94-01-69-27257) from the Deputy of Research of Tehran University of Medical Sciences.

Conflict of Interest The authors declare no competing financial interest.


29


Page 30 of 52

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Page 37 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. The textural characteristics obtained from the BJH assay and BET results for the synthesized bioglasses.

Samples

SSA* (BET) (m2.g-1)

Average pore diameter (nm)

Cu BG

203.49

2.41

FeCu BG

284.82

2.52

Fe BG

169.47

2.50

* Specific spatial surface area

Table 2. The antibacterial results for the synthesized bioglasses on E. coli ATTC25922.

-1

-1

Samples

MIC* (mg.mL )

MBC** (mg.mL )

Cu BG

25

50

Fe BG

50

50

100

100

FeCu BG

* Minimum Inhibitory Concentration ** Minimal Bactericidal Concentration


37


Page 38 of 52

Figure 1. The TG-DTA curves of the mesoporous bioactive glasses; (A) Cu BG, (B) Fe BG, and (C) FeCu BG

Figure 2. The XRD patterns of Cu, Fe, and FeCu BGs before soaking in SBF solution.


38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 - - - - - - - - - - - - - - - - 800

ACS Paragon Plus Environment OH- - - - - - - - - - -

- - - - - - - - - - - - 3550

OH-- - - - - - - - - - - - - - - - - - - - - - - - 1639 - 1648

Si-0-Si - - - - - - - - - - - - - - - 1070 P-O - - - - - - - - - - - - - - - - - - - - - - - - 1238

Si-O-Si

Si-O-Si - - - - - - - - - - - - - - - - - -455 - 470 Metal-O -- - - - - - - - - - - - - - - - - - - - -569

Transmittance (%)

Page 39 of 52 ACS Biomaterials Science & Engineering

FeCu BG Fe BG Cu BG

500 1000 1500 2000 2500 3000 3500 Wavenumbers (cm¯¹)

Figure 3. The FTIR spectra for Fe, Cu, and FeCu BGs before soaking in SBF solution.

39


Page 40 of 52

Figure 4. SEM micrographs of Cu BG (A, B and C), FeCu BG (D, E and F), and Fe BG (G, H and I).


40

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Figure 5. EDS patterns of Cu BG (A), Fe BG (B and C); (B) inside of cavities and (C) outside of cavities, FeCu BG (D and E); (D) inside of cavities and (E) outside of cavities.


41


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Figure 6. TEM micrographs of Cu BG (A, B and C), FeCu BG (D, E and F), and Fe BG (G, H and I).


42

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25 0 Cu B G F eCu B G Fe BG

V a/cm 3 (STP ) g-¹

20 0

15 0

10 0

50

A 0 0

0.2

0.4

0 .6

0 .8

1

ρ /ρ ₒ

45 Cu B G

40

F e Cu B G

35

Fe BG

30

d V ρ /d r ρ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25 20 15 10 5

B

0 0

10

20

30

40

50

60

70

80

r ρ /Å

Figure 7. BET (A) and BJH (B) patterns of Fe BG, FeCu BG, and Cu BG.


43


Page 44 of 52

Figure 8. XRD patterns of FeCu BG, Fe BG, and Cu BG after soaking in SBF solution.

Figure 9. FTIR spectra for FeCu BG, Fe BG, and Cu BG after soaking in SBF solution.


44

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Figure 10. ICP results for FeCu BG, Fe BG, and Cu BG after of soaking in SBF solution, (A) Ca ion, (B) P ion, (C) Cu ion and (D) Si ion.


45


7.95 7.90 7.85 7.80

pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 52

7.75 7.70 Cu BG

7.65

FeCu BG

7.60

Fe BG

7.55 0

1

2

3

4

5

6

7

8

9 10 11 12 13 14

Time (day)

Figure 11. pH diagram for FeCu BG, Fe BG, and Cu BG after of soaking in SBF solution.


46

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Figure 12. SEM micrographs of Cu BG (A, B and C), FeCu BG (D, E and F), and Fe BG (G, H and I) after soaking in SBF solution.


47


Figure 13. EDX patterns for Cu BG (A), FeCu BG (B), and Fe BG (C) after soaking in SBF solution.

Magnetization (emu/g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 52

1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 -0.8 -1.0 -1.2

FeCu BG Fe BG Cu BG

-25000

-15000

-5000

5000

15000

25000

Magnetic Field ( Oe) Figure 14. VSM graphs for the Cu BG, FeCu BG and Fe BG.


48

Page 49 of 52

2 1.8 1.6

**

1.4

**

Control FeCu BG **

Fe BG

** ** **

*

Cu BG

*

*

*

*

1.2

OD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 0.8 0.6 0.4 0.2 0 low/1d

high/1d

low/3d

high/3d

low/7d

high/7d

Concentration (µg.ml-1 )/Culture time (day) Figure 15. MTT assay of the synthesized bioactive glasses, along with negative and positive controls with two concentrations (low: 200 µg.mL-1 and high: 400 µg.mL-1) for 1, 3 and 7 days [The mean difference compared to control group is significant at the 0.05 level (*) and 0.001 (**)].


49


60 Control

50

FeCu BG

40

**

**

** **

# **

Fe BG

**

Cu BG ## *

45S5

OD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 52

##

30 **

20 10

*

* * #

**

##

#

0 low.1d

high.1d

low.3d

Concentration

high.3d

(µg.ml-1).Culture

low.7d

high.7d

time (day)

Figure 16. ALP activity of the synthesized bioactive glasses, along with negative and positive controls with two concentrations (low: 200 µg.mL-1and high: 400 µg.mL-1) for 1, 3 and 7 days [The mean difference compared to control group is significant at the 0.05 level (*) and 0.001 (**). The mean difference compared to 45S5 group is significant at the 0.05 level (#) and 0.001 (##)].


50

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Figure 17. HUVEC proliferation under exposure to the synthesized bioactive glasses with controls at two concentrations (low: 100 µg.mL-1 and high: 200 µg.mL-1) for 1, 3 and 7 days. [The mean difference compared to control group is significant at the 0.05 level (*) and 0.001 (**)].


51


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"For Table of Contents Use Only"

Table of Contents Graphic

Synthesis, characterization and in vitro biological evaluation of coppercontaining magnetic bioactive glasses for hyperthermia in bone defect treatment Razieh Koohkan, Tabassom Hooshmand, Davod Mohebbi-Kalhori, Mohammadreza Tahriri, Mohammad Taha Marefati


52

Synthesis, Characterization, and in Vitro Biological Evaluation of

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