Synthesis and characterization of magnesium-lanthanum dual doped bioactive glasses

Journal Pre-proof Synthesis and characterization of magnesium-lanthanum dual doped bioactive glasses Hossein Jodati, Bahadır Güner, Zafer Evis, Dilek Keskin, Ayşen Tezcaner PII:

S0272-8842(20)30051-1

DOI:

https://doi.org/10.1016/j.ceramint.2020.01.050

Reference:

CERI 23982

To appear in:

Ceramics International

Received Date: 9 October 2019 Revised Date:

17 December 2019

Accepted Date: 6 January 2020

Please cite this article as: H. Jodati, Bahadı. Güner, Z. Evis, D. Keskin, Ayş. Tezcaner, Synthesis and characterization of magnesium-lanthanum dual doped bioactive glasses, Ceramics International (2020), doi: https://doi.org/10.1016/j.ceramint.2020.01.050. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Synthesis and Characterization of Magnesium-Lanthanum Dual Doped Bioactive Glasses Hossein Jodatia, Bahadır Günera, Zafer Evisa,b, Dilek Keskina,b, and Ayşen Tezcaner a,b,* a

Department of Biomedical Engineering, Middle East Technical University, Ankara, 06800, Turkey

b

Department of Engineering Sciences, Middle East Technical University, Ankara, 06800, Turkey

Abstract Dual ion-doped bioactive glass has drawn high attention during recent years because of wellimproved therapeutic characteristics and mechanical properties. In this study, 5 different iondoped (1wt.% Lanthanum with 0, 1, 2.5, 5, and 10 wt.% Magnesium) 58S bioactive glasses (BGs) were synthesized. The physicochemical results revealed that Lanthanum (La) and Magnesium (Mg) ion doping has been successfully done. Mechanical characterization results depicted that La-doped and Mg-La double doped samples with Mg less than 2.5 wt.% had higher diametral tensile strength compared to BG and other co-doped samples. However, Vickers hardness values were not affected by ion dopings. Apatite formation and bioactivity were also improved in double ion-doped samples, especially in those with the higher magnesium content. Cell culture studies showed that synthesized BGs were not cytotoxic. Especially, groups having 2.5 to 5 wt.% Mg showed the highest cell viabilities at day 7. Results indicated that Mg- La dual doped bioactive glasses possess improved properties compared to BG, and thus, have potential for bone regeneration applications.

Keywords: bioactive glass; Magnesium-Lanthanum; dual doped; bone

*Corresponding author. Email address: [email protected] (A. Tezcaner)

1. Introduction Application of bioceramics, such as bioactive glasses, provided major progress in the success of bone repair during the past 40 years [1–4]. After favourable outcomes of bioactive glasses in dental bone regeneration [5–8], they were investigated as orthopaedic particulate bone grafts in many studies and clinical trials [9,10]. Nowadays, application of bioactive glasses is a very fast-growing field in bone regeneration explorations, especially their blends with polymers, ceramics, or hydrogels [11,12]. However, clinical products made of bioactive glasses have not stretched to the highest potential in the matter of bone regeneration due to poor mechanical strength. The first bioactive glass synthesized by Dr Larry Hench in the late 1960s and was later named as Bioglass  45S5 by the University of Florida. BGs have no adverse effects on cells or tissues and have never been identified as cytotoxic, carcinogenic, mutagenic or antigenic materials [13]. BGs are amorphous solids that elicit a specific biological reaction when the material interacts with living tissues. Not only fibrous tissue doesn’t warp them once implanted, but also BGs develop a strong bond with surrounding host tissues and trigger cell proliferation, as well as gene response [14,15]. Although most of the biomaterials that have been used over decades for bone regeneration possess bioactivity and osteoconductive properties, bioactive glasses are the only materials that also have osteogenic property, which triggers proliferation as well as differentiation of osteoprogenitor cells [13]. Nevertheless, high solubility and poor mechanical properties such as low toughness and fracture resistance are the disadvantages of BGs [13,16]. Incorporation of various elements into the composition, ion doping, is one of the well-known methods to improve therapeutic characteristics, mechanical properties, and/or biochemical behaviour of BGs. Bioactivity of glasses is significantly affected by their surface composition and reactivity. Therefore, BGs with modified reactivates and compositions may provide more suitable materials for physical or chemical needs of human body parts. Influences of several metals and other additive ions, especially the abundant ions in human bone, magnesium, strontium [17], fluoride [18], potassium [19], and zinc [20] on biomechanical or biological behaviour of BGs have been studied by different research groups during past years [21]. Magnesium (Mg) is one of the most important mineral elements of bone matrix [22,23]. It is taking part in more than 300 chemical reactions and is the co-factor of several enzymes in the human body. Mg has also been reported to make a positive influence on wound healing, bone fracture hindrance, and bone density [24]. Presence and functional roles of Mg in bone tissue 1

suggest that it may be involved in the development and maintenance of bone [14]. Mg takes action as a network former or modifier when it is doped [25,26]. Mg-doped BG could act as an appropriate substrate on which human osteoblasts proliferate and perform their osteogenic activities [27,28]. Enhanced surface reactivity, which has a role on bioactivity, has been observed in Mg-doped BGs [13,14,22,23]. However, addition of network former oxides and neglecting the structural role of MgO were mentioned as reasons for lower bioactivity of Mgdoped BGs in-vitro [25,29]. Lanthanum (La) is a rare earth element that has a chemical affinity like calcium (Ca) element. It exists in interior or surface of bone [30]. Lanthanum carbonate has been addressed as a possible substitute to Ca-based phosphate binders for treating end-stage renal disease (ESRD) patients [30]. It was reported that La-doped BGs indicated were non-toxic up to 10 mol% [31,32]. Antibacterial effect of La-doped nanoparticles has been shown in a couple of studies [33–35].

Composites

of

hydroxyapatite

(HA)

and

La

exhibited

better

biocompatibility, as well as higher bioactivity in simulated body fluid (SBF) [36,37]. Positive effect of La ion on the improvement of proliferation and osteogenic differentiation of mesenchymal cells has been reported in the literature [38–41]. Concerning mechanical properties, some properties, like elastic modulus and hardness of silicate containing glasses were improved when doped with La [42] besides the physicochemical properties like thermal stability or glass transition temperature [43]. In this study, Mg-La dual-doped BGs were synthesized and characterized (microstructural characterization, bioactivity, mechanical properties and biological response of cells) for the first time in literature. By studying the characteristics of dual-doped BGs, it is hoped that these novel materials can serve as a new biomaterial for further biomedical applications.

2. Materials and Methods 2.1 Synthesis of Pure and Doped BGs All 58S BGs with composition of 58SiO2-38CaO-4P2O4 (wt.%) were synthesized by quick alkali-mediated sol-gel method [44]. Analytical grade precursor materials were prepared according to specified nominal composition values stated in Table 1. To synthesize BG, 21.26 ml tetraethyl orthosilicate (TEOS, Merck, USA) and 50 ml ethanol were stirred in a beaker for 10 minutes. Then, 2.8 ml nitric acid (2M HNO3, Merck, USA) and 13.9 ml

2

distilled water were added into the solution and it was stirred for 30 minutes at room temperature. 2.15 ml triethyl phosphate (TEP, Merck, USA) was added into the obtained acid silica sol and was stirred for 20 minutes. Then, 14.04g calcium nitrate tetrahydrate (Ca[NO3]2.4H2O, Sigma, USA) was added to the solution. For synthesis of doped BGs, La (III) oxide (La2O3, Merck, USA) and magnesium nitrate hexahydrate (Mg[NO3]2.6H2O, Merck, USA) were added at the required amount with the calcium source. When added compounds were completely dissolved, the sol was gelled with the addition of 1 M Ammonia (NH3, Merck, USA) while stirring continuously. The obtained gel was dried at 60 oC for 24 hours after muddling in order to eliminate possible residual ethanol and water, and the dried gel was calcined at 600 o C for 2 hours.

Table 1. The nominal composition of BGs (weight ratio). Sample BG

SiO% 58

Si% 27.09

CaO% 38

Ca% 27.17

P2O% 4

P% 1.74

Mg% 0

Mg% 0

La2O3% 0

La% 0

1LaBG

57

26.62

38

27.17

4

1.74

0

0

1

0.86

1Mg1LaBG

57

26.62

37

26.45

4

1.74

1

0.6

1

0.86

2.5Mg1LaBG

57

26.62

35.5

25.38

4

1.74

2.5

1.51

1

0.86

5Mg1LaBG

57

26.62

33

23.59

4

1.74

5

3.00

1

0.86

10Mg1LaBG

57

26.62

28

20.02

4

1.74

10

6.03

1

0.86

2.2. Microstructural characterization of BG powders X-ray diffraction patterns of pure and doped BG powders were achieved using Rigaku Ultima-IV operating at 30 mA and 40 kV utilizing CuKα radiation with step sizes of 0.028 and a counting time of 1 second. All diffractograms were collected between 10-90 degrees of 2θ. Fourier transform infrared spectroscopy (FTIR, Bruker IFS 66/S, FRA 106/S, HYPERION 1000, RAMANSCOPE II) analysis was performed to examine the functional groups of BGs. The range of infrared spectra was between 4000-400 cm-1. Chemical compositions of pure and doped BGs were determined using Inductively Coupled Plasma Mass Spectrometry (Perkin Elmer DRC II model ICP-MS) by dissolving particles in 2% NO3 solution for analysis. 2.3. Mechanical Characterization

3

BG powders were dried at 60°C overnight and were calcined at 600°C for 2 hours in order to purge all impurities. The BG powders were passed through sieve with 212 µm orifice and then were pressed in the shape of discs (12mm in diameter) via automatic hydraulic cold press device (Carver, USA) under the imposed force of 22 kN for 3 minutes before mechanical analysis. Diametral tests were conducted on the discs on the discs of BGs using Univert Biomaterial Mechanical Testing Device (Cell scale, Canada). Prepared discs were subjected to compression force diametrically. In the course of compression, the maximum tensile force is created across surface diameters of discs perpendicular to the compressive load direction. Equation 1 was employed to calculate the tensile strength of the samples [45,46]: =

. .

(1) Where S is tensile strength, F is failure force, t is disk thickness, and D is sample’s diameter. One way ANOVA (p=0.05) and Tukey’s post hoc tests were used for statistical analysis (n=4).

Density and porosity of materials were measured with respect to Archimedes principle through using formulas (2) and (3).

Density =





×















(2)

Wet weight of the samples was measured after removing excess water by filtrate paper, and the porosity of the samples was calculated using the formula (3) [47].

Porosity =























(3)

Theoretical densities of the pure and doped BGs were calculated using equation (4), where w and

are weights and densities respectively [48]. Relative densities were quantified by

means of dividing real density to theoretical density values.

4

Theoretical

!"

Density= '

#$ "

%&

- #$ 1 .( 0"' %&)( * ( ! *", #$ %&

!)

+



/

(4)

Micro-indentation measurements of pure and doped BG samples were accomplished using a micro-hardness tester (SHIMADZU, Japan). Three discs for each group were prepared and each disc was subjected to Vicker’s indentation test under 1.961 N force for 20 seconds. The indentation size was measured by a light microscope. The Vickers hardness number was calculated using the formula below [49]: HV=0.001854P/d2

(5)

where P is applied force, d is indented diagonal length. All the discs were examined through single indent mode with 10 numbers of indents for each disk, which was chosen at random spots. One-way analysis of variance (ANOVA) with Game-Howell post hoc test for multiple comparisons was executed on data (n=30, p=0.05).

2.4. In vitro Bioactivity Test Bioactivity of pure and doped BGs was studied by immersing discs in simulated body fluid (SBF) [50] at 37oC for 1, 3, 7, and 14 days. Incubated discs were removed from SBF, washed with distilled water, and then were dried at room temperature. Scanning electron microscope (SEM QUANTA 400F Field Emission) examination was employed to investigate the morphology of formed apatite on the disc surface. Moreover, changes in the concentration of ions in the SBF was determined using ICP-MS analysis. 2.5. In-vitro Cytotoxicity Analysis In vitro cytotoxicity evaluation of pure and doped BGs was carried out using Saos-2 cells (human osteosarcoma cell line). Discs were sterilized by incubation in 70% ethanol for 4 hours and UV irradiation for 1 hour on both surfaces before cell seeding. Saos-2 cells were grown in high glucose Dulbecco’s modified Eagle medium (DMEM) boosted with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin. The initial cell seeding density on discs was 2x104 cells/disc and discs of each group (n=4) were incubated at predetermined time points inside the incubator (Panasonic, MCO-18AC, Kadoma, Japan) at 37°C under 5% CO2 5

humidified environment. Viability of cells on discs was evaluated by Alamar Blue® cell viability assay (Invitrogen, USA) using supplier’s protocol after 1, 4 and 7 days of incubation. Increased in cell viability with time was interpreted as cell proliferation. At each incubation time point, the media were removed from the wells and discs were washed with PBS. After that, PBS was replaced with Alamar Blue solution (10% v/v) prepared with DMEM without phenol red. Discs were incubated at 37°C for 4 hours in dark. At the end of incubation time, the solutions were transferred to a new 48 well plate for absorbance reading at 570 and 600nm wavelengths. Cell free discs of each group were used as blank control and TCPS with cells was used as a positive control. 3. Results and Discussion 3.1. Microstructural Investigation of BG Powders XRD analysis (Fig. 1) showed the glassy nature of synthesized BGs powders. All samples were in the amorphous phase since just weak wide-spreading band has been observed. Similar XRD pattern was also observed in another study, in which nano 58S BG powder had been synthesized using quick alkali-mediated sol-gel method [44]. XRD patterns of calcium silicates have diffraction with a board and asymmetric maxima at 2θ=32.0 [51,52]. Observed negligible sharper peaks at 2θ= 32.16° on the board maxima patterns of pure BG and 1LaBG were assigned to partially crystalized calcium silicate with a formula of Ca2SiO2 (JCPDS# 20-0237).

The phase of amorphous calcium silicate could turn into crystalline phase

especially at a temperature around 800 or above [53]. However, similar peaks have been reported even at a lower temperature in a study conducted by Meiszterics et al. [54]. In addition to high temperatures, keeping samples below the liquidus temperature for a long time, cooling slowly in methods that use melting approach for glass synthesis, or re-heating of synthesized glasses to the temperature below their melting temperature, were mentioned in literature as reasons for crystallization of glasses [55]. Reheating BGs in order to remove moisture from powders might be the reason for those crystalline peaks in this study like classical behaviour of amorphous glasses [56].

Fig. 1. XRD pattern of synthesized pure and five doped BG groups.

FTIR spectra of pure and Mg-La co-doped BGs are illustrated in Fig. 2. Major absorption bands at 1044 23 4 and 470 23

4

were assigned to Si-O-Si symmetric and asymmetric

stretching in BGs [57,58]. Absorption band at 790 23

4

belongs to Si-O stretching of 6

nonbridging oxygen atoms, and also Si-O symmetric stretching of bridging oxygen atoms

among tetrahedrons. The vibration band 1375 23 4 was attributed to CO32- [23,59]. The absorption band at 570 cm-1 was dedicated to PO43- [57]. Those shoulders at 958 cm-1 (Fig. 2) related to BG and 1LaBG graphs are assigned to the Si–O–Ca [44] and are in agreement with observed partially crystalline calcium silicate in XRD results (Fig. 1). These shoulders shift to lower wavenumbers near 925 cm-1 in other groups, which are attributed to absorption band

of Si-O of non-bridging oxygen atoms [60–62]. Moreover, absorption bands at 173023 4 in the spectra of FTIR of all BGs showed the hygroscopic characteristic of the silicate glass and were assigned to OH- group bending vibration mode [53].

Fig. 2. FTIR spectra of pure and doped BGs.

In Table 2, ICP-MS results of pure and Mg-La doped BGs are listed. Presence of La and Mg in doped materials indicated successful doping of La and Mg ions. Mg and La were doped in place of Ca [63–65]. However, in all groups, amounts of Ca, Si, La, Mg, and P of powders were lower than Theoretical amounts (Table 1). A slight deviation from theoretical amounts is inevitable in the synthesis of BG powders [43]. Table 2. Weight ratio of elements in the chemical composition of pure and Mg-La doped BG powders determined with ICP-MS. Sample

Si wt.% Ca wt.%

P wt.%

Mg wt.%

La wt.%

BG

18.9

26.1

1.03

0

0

1LaBG

19.6

26.3

0.82

0

0.69

1Mg1LaBG

18.8

25.2

0.83

0.57

0.7

2.5Mg1LaBG

18.9

22.4

0.97

1.50

0.73

5Mg1LaBG

19.2

20.2

1.00

2.96

0.74

10Mg1LaBG

19.1

15.7

0.91

5.9

0.72

Measured, theoretical and relative density values, as well as porosity ratios, are given in Table 3. There was no significant difference between the relative density of pure BG and other groups except 10Mg1LaBG samples. Relative density of 1LaBG and 5Mg1LaBG

7

followed the same pattern as pure BG group. BGs with 1 and 2.5 % Mg content had similar densities. Higher relative density of doped BGs compared to pure BG was reported in previous studies [66]. Such higher values were explained with an increase in the densification of La and/or Mg containing samples [67]. However, in the present study, doping of ions had no effect on relative densities and only in samples with 10% Mg content the relative density decreased. Compared to non-doped BGs there exist studies, in which doped BG had lower

[68].

density Table 3. Densities and porosity values of pure and doped BGs (n=3). Sample

Density(g/cm3)

Theoretical

Relative

Porosity

density

density

%

(g/ccm3)

%

BG

2.42±0.16

2.86

84.64

90.35±0.70

1LaBG

2.41±0.16

2.88

83.66

89.38±0.78

1Mg1LaBG

2.27±0.13

2.88

78.75

88.46±1.00

2.5Mg1LaBG

2.31±0.01

2.88

80.49

87.39±0.83

5Mg1LaBG

2.38±0.09

2.89

82.23

91.45±0.34

10Mg1LaBG

1.95±0.27

2.90

67.14

88.54±1.42

3.2. Mechanical Characterization Diametral tensile strength results of pure and doped BGs are shown in Fig. 3. A significant difference was observed in tensile strengths of groups. Tensile strengths of 1LaBG and 1Mg1LaBG were statistically higher than values of pure and other groups (p<0.05). However, there was no significant difference between 1LaBG and 1Mg1LaBG groups (p>0.05). When Mg content was 5wt.% and more the diametral tensile strength of samples decreased and strength values were even lower than pure BG. Minimum tensile strength was observed for the sample with 10 wt.% Mg. Theoretically, doped materials show greater mechanical strength compared to pure materials because of the strengthening impression of the inorganic fillers [61,66,67]. Results of 1LaBG and 1Mg1LaBG confirmed this theory owing to La doping. Similar results for La-doped materials were reported in the literature [37]. However, above a specific Mg-doping value, the strength values reduced dramatically. Inorganic ions aggregation and resultant microcrack formation were addressed as the reason for such decrease [69]. Thus, an increase in depth and number of microcracks could be 8

addressed as reasons for the lesser tensile strength of BG with 5 or 10 wt. % of magnesium. Also, an increase in the amount of MgO might have contributed to lower strength values in these samples. Moreover, porosity and relative density are biomaterial’s parameters, which play a role in mechanical properties and bioactivity of scaffolds [70,71]. It has been reported that improvement in mechanical properties (compressive or tensile strength) of materials was observed for materials with higher relative density [72]. Thus, lower relative density of 10Mg1LaBG (Table 3) could also be a reason for decreased tensile strength. Nonetheless, effect of porosity on tensile strength could be neglected since porosity of all samples were about 90%.

Fig. 3. Mean diametral tensile strength values of pure and doped BGs (n=4, p<0.05).

Micro-hardness test results are given in Fig. 4. No significant difference in hardness was observed between pure BG and doped BGs. Hardness value of 1LaBG samples was similar to the values of 5Mg1LaBG and 10Mg1LaBG groups. Doped and pure BGs samples had similar porosity values which could be the reason for very small difference observed between hardness of doped groups [66].

Fig. 4 Vickers micro-hardness values for pure and doped BGs (n=30, p<0.05). There is no significant difference between groups.

3.3. Bioactivity Test In-vitro SBF bioactivity test was widely known protocol to inspect the possibility of apatite formation on the surface of tested material in order to evaluate its ability for bonding with hard tissues. The concentrations of Ca, Si, and Mg in the SBF, as well as pH changes of SBF, are depicted for different soaking times in Fig. 5 (a) – (d). One way to measure glass network dissolution is ion release profile of Si. According to Figure 5(a) remarkable release of Si ion in all BGs groups was observed 7 days of incubation in SBF at 37°C. Relatively slower release of Si ion was observed until day 14, which could be interpreted as reaching equilibrium at day 14 [69]. Apatite formation, which leads to diffusion barrier formation, or insoluble Si salts precipitation could also be the reason for the reduction in Si ion release [38,70]. Moreover, decrease in Si precipitation rate was more noticeable in 10Mg1LaBG, which could be due to decrease in BG solubility.Compact structure of 10Mg1LaBG, due to 9

the smaller ionic radius of Mg ion, might have prevented easy filtration of SBF solution into its structure compared to pure BG [71,73]. Almost the same release pattern was observed for Ca2+ ion (Fig. 5-b). Change in Mg2+ ion concentration during 14 days of incubation in SBF solution is shown in Fig. 5-c. Decrease in Mg2+ ion concentration of SBF was addressed to the formation of calcium phosphate apatites, containing Mg. Mg2+ and Ca2+ ions showed different precipitations dynamics; brisk precipitation of Ca2+ at initial days of immersion was followed by gradual precipitation of Mg2+ [71,73]. The amount of La3+ in all SBF samples was below 0.004µg/L due to the small amount of doped La or its fast precipitation. In all groups, pH of SBF increased gradually during 14 days (Fig. 5-d). Ion exchange between H+ ions in the SBF and alkaline ions on the surface of BGs, such as Ca2+, could be the reason for the increase observed in pH of SBF [13,61,73,74]. Fig. 5. Changes in concentration a) Si, b) Ca, and c) Mg ions, and, d) pH change in SBF during 14 days of incubation. Surface morphology of pure and doped BGs after 1 and 14 days of incubation in SBF were demonstrated in Fig. 6. After 1 day incubation in SBF, almost no apatite particle was observed on BGs surfaces. Apatite formation was observed on the surfaces of all BGs after 14 days of incubation. Formation of apatite happens in five steps. First, alkali earth metal ion such as Ca2+ exchanges ions with H+ of SBF. Second, Si(OH)4 forms and leads to occurring of dissolution. Thirdly, silica-rich layer starts to establish on the surface, through polycondensation of silanols. As the last step, incorporation of PO43- and Ca2+ results in the formation

of

amorphous calcium-phosphate

(Ca-P)

layer.

Eventually,

apatite

structures crystallize from Ca-P rich layer [13]. Higher deposition of apatite-like particles were observed on surfaces of pure BG and 10Mg1LaBG samples (Fig. 6-A, and F), which was in agreement with almost similar ion release profile of these groups (Fig. 5). Mg content increase to 10 wt.%, resulted in more apatite formation compared to other doped BGs , which could be related to the change in surface reactivity [70]. Fig. 6. SEM images of BGs; left (day 1) right (day 14). A) BG, B) 1LaBG, C) 1Mg1LaBG, D) 2.5Mg1LaBG, E) 5Mg1LaBG, F) 10Mg1LaBG. White arrows show apatite particles on the surface of BGs. 3.4. Cell Viability Test Cell viability on 2.5% and 10% Mg-doped BG discs was statistically higher than that observed on TCPS (p<0.05) after 1 day (Fig. 7). This finding suggested that increment in 10

magnesium content in discs might have augmented Saos-2 cell attachment onto the disc surface by enabling integrin binding [75]. However, other Mg containing groups, (5% magnesium doped BG) did not show a significant increase which might be due to higher variability in cell viability. This group also had statistically higher porosity (Table 3) compared to 2.5% and 10Mg1LaBG discs. Thus, variation in the attachment observed in the 5Mg1LaBG group might be caused by leakage of cells through pores of the discs to the plate surface during the cell seeding process. For this reason, cells were just washed out the surface of the discs with higher porosity, before they settled in. Moreover, a similar variation was detected in pure BG group which also had higher porosity than 2.5Mg1LaBG. Increase in reduction percentage was observed for all groups during 7 days of culturing. Such increase showed that cells proliferated on the discs through the incubation period. Highest cell viability was observed on 2.5Mg1La and 5Mg1La containing BG discs on day 7. Lowest cell viability was observed in 10% Mg doped and pure BG groups. Although cell viability is this group was lower than TCPS, cell viability was still above 85% relative to TCPS. Overall, results showed that pure doped BGs were not cytotoxic [76]. Observed lower cell viability in 10Mg1LaBG compared to TCPS could be related with pH change. Previous studies reported that Mg related cytotoxicity is related to the formation of Mg hydroxide that causes an increase in pH [77]. However, change in pH of SBF for all groups was similar (Fig 5-D). Therefore, the lowest cell viability in this group might be related with earlier differentiation of the cells leading to a decrease in cell proliferation [78]. There are many studies reporting that Mg enhances osteoblast growth, cell adhesion, and bone formation [57,79].

Fig. 7. Viability of Saos-2 cells on pure and doped BG discs during 7 days of incubation (n=4).

4. Conclusion In this study, Mg and La elements were doped to BG for the first time in the literature. The Mg content was increased in four steps from 1 to 10 wt. %; while La quantity was kept constant (1 wt.%). Physicochemical characterization of microwave-mediated synthesized powders including XRD, FTIR, and ICP-MS demonstrated that ion doping was achieved. From a mechanical point of view, La doping has increased the diametral strength of BG when doped alone or with a low amount of Mg element (less than 2.5 wt.%). However, Mg had negative effect on diametral strength when doped at high percentage in BG. Increased 11

bioactivity and apatite formation on samples’ surface were a common feature of all doped BG groups. La seemed to have less effect on bioactivity; while, groups with higher Mg content had higher bioactivity. All groups, including doped and pure BGs were cytocompatible. Additionally, BG groups that were doped with La and an optimum amount of Mg ( 2.5-5 wt.% ) had positive effect on cell proliferation. Overall, it could be concluded that Mg-La doped BGs with Mg content 2.5 wt.% is a biomaterial that holds potential for use as regenerative bone substitutes.

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List of Figures Fig. 1. XRD pattern of synthesized pure and five doped BG groups. Fig. 2. FTIR spectra of pure and doped BGs. Fig. 3. Mean diametral tensile strength values of pure and doped BGs (n=4, p<0.05). Fig. 4 Vickers micro-hardness values for pure and doped BGs (n=30, p<0.05). There is no significant difference between groups. Fig. 5. Changes in concentration a) Si, b) Ca, and c) Mg ions, and, d) pH change in SBF during 14 days of incubation. Fig. 6. SEM images of BGs; left (day 1) right (day 14). A) BG, B) 1LaBG, C) 1Mg1LaBG, D) 2.5Mg1LaBG, E) 5Mg1LaBG, F) 10Mg1LaBG. White arrows show apatite particles on the surface of BGs. Fig. 7. Viability of Saos-2 cells on pure and doped BG discs during 7 days of incubation (n=4).

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List of Tables Table 1. The nominal composition of BGs (weight ratio). Table 2. Weight ratio of elements in the chemical composition of pure and Mg-La doped BG powders determined with ICP-MS. Table 3. Densities and porosity values of pure and doped BGs (n=3).

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There is no conflict of interest for this study.