Cytocompatibility studies of titania-doped calcium borosilicate bioactive glasses in-vitro

Accepted Manuscript Cytocompatibility studies of titania-doped calcium borosilicate bioactive glasses in-vitro

Rajkumar Samudrala, P. Abdul Azeem, Vasudeva Rao Penugurti, M. Bramanandam PII: DOI: Reference:

S0928-4931(16)31871-9 doi: 10.1016/j.msec.2017.03.245 MSC 7757

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

23 October 2016 26 January 2017 25 March 2017

Please cite this article as: Rajkumar Samudrala, P. Abdul Azeem, Vasudeva Rao Penugurti, M. Bramanandam , Cytocompatibility studies of titania-doped calcium borosilicate bioactive glasses in-vitro. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/ j.msec.2017.03.245

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT

Cytocompatibility studies of titania-doped calcium borosilicate bioactive glasses In-vitro Rajkumar Samudralaa, P. Abdul Azeema*, Vasudeva Rao Penugurtib, M. Bramanandamb, Department of Physics, National Institute of Technology Warangal,

PT

Warangal-506004, India a

RI

Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad,

AC

CE

PT E

D

MA

NU

SC

India b

Corresponding author: P. Abdul Azeem Email: [email protected], [email protected] Contact Number: + 91-870-2462578 Fax: +91-870-2459547

1

ACCEPTED MANUSCRIPT Abstract The present study aims to elucidate the applications of Titania (TiO2) doped calcium borosilicate glass as a biocompatible material in regenerative orthopedic applications. In this context, we have examined the bioactivity of various concentrations of TiO2 doped glasses with the help of simulated body fluid (SBF). Cytocompatibility, cell proliferation, and protein expression studies revealed the potential candidature of TiO2

PT

doped glasses on osteoblast cell lines (MG-63). We hypothesized that TiO2 doped calcium borosilicate glasses are most cytocompatible material for bone implants.

RI

Glasses with composition 31B2O3-20SiO2-24.5Na2O-(24.5-x) CaO-xTiO2 x=0,0.5,1,2

SC

have been prepared by the conventional melt-quenching technique. After immersion of glasses in the SBF, formation of hydroxyapatite layer on the surface was confirmed by

NU

X-ray Diffractometer (XRD), Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS) analysis. Significant change in the pH of the body fluid was observed with the addition of titania.

MA

Degradation test was performed as per the ISO 10993. The results showed that partial substitution of TiO2 with CaO negatively influenced bioactivity; it decreased with

D

increase in concentration of TiO2. Vickers hardness tester was used to measure the Micro hardness values of the prepared glasses. With the increasing of TiO2 content, the

PT E

micro hardness of the glass samples was increased from 545 Hv to 576 Hv. Cytocompatibility has been evaluated with MG-63 cells with MTT assay. Further, we observed that there was no change in expression of cyclin levels even after the

CE

incorporation of titania. The antibacterial properties were examined against E. coli and S. aureus. Strong antibacterial efficacy was observed for 2% TiO2 in the system. Hence

AC

it can be concluded that titania-doped borosilicate glasses may be used as potential materials in bone tissue engineering. Keywords: Biocompatible material, Bioactivity, Cell viability, Cytocompatibility, Proliferation, Human osteoblast cells

2

ACCEPTED MANUSCRIPT 1. Introduction ‘Bioceramics’ are biocompatible materials, which find use in several clinical applications. Bioceramics can be produced in crystalline and amorphous forms and they are generally classified into two groups based on their chemical composition; calcium phosphates

PT

and others, including yttria -stabilized tetragonal zirconia, alumina ceramics, silicate and phosphate families of glasses and glass-ceramics[1]. Bioactive glasses are considered as

RI

potential materials for bone substitution, as they can form a direct bond with the living bone

SC

without formation of bond with surrounding fibrous tissue. One of the essential conditions for the bioactive material is that it should form a biologically active apatite called

NU

hydroxylapatite layer (HAp) on its surface through which the material could form a bond

MA

with both soft tissues as well as hard tissues[2]. 45S5 is the best implant material, not only for dental and orthopedic applications but also, for ossicular prostheses, endosseous ridge

D

maintenance, and other applications[1,3].

PT E

Boron plays a vital role in bone formation and depends on its concentration in the composition. The highest concentrations of boron are found in bone, nails, and hair [4]. Day

CE

et al. [5–9] have extensively studied the use of borate glasses in biomedical applications. Recent studies have demonstrated that the potential bioactivity of borate glasses comes from

AC

their lower chemical durability, faster degradation rate and almost complete conversion to hydroxyapatite (HAp) than the widely studied 45S5 bioglass when placed in SBF. Studies have shown that some borate glasses have the ability to support the growth and differentiation of human mesenchymal stem cells, and to promote bone formation more rapidly than silicate based 45S5 glass [10,11]. S. M. Wiederhorn et al.[10] reported that higher amount of borate content weakens the glass structure. Glass structure was strengthened by incorporation of silica as former and CaO as modifier respectively[12].

3

ACCEPTED MANUSCRIPT Titanium has gained importance recently for its broad range of applications in the biomedical field[13]; it is one of the earliest transition metals to be investigated for antitumor properties; also it is found to elicit favorable cell response and is concluded to be one of the best materials suited for biological requirements[14]. Hence, titanium is widely used as a biomaterial for several dental and orthopedic clinical purposes. In the present work, titanium

PT

dioxide (TiO2) was incorporated into borosilicate glass to produce compositions with

RI

controlled degradation rate and enhanced biological response which is a suitable material for

SC

bone tissue engineering applications. Numerous studies were focused on TiO2 doped glasses and glass ceramics[15–21]. Devi et al.[22] reported titania-doped phosphate glasses and

NU

glass-ceramics are bioactive. Though the 2 mol% of TiO2 content controlled the solubility and improved the density of glass, however poor bioactivity was reported. Mohini et al. [23]

MA

studied the in-vitro bioactivity of TiO2 doped borosilicate glasses (up to 10 mol%) and showed that higher concentration of TiO2 did not affect the bioactivity of the glasses.

D

Titanium is acting as both active as well as inert material depending on the host composition.

PT E

Nowadays, clinical interest is aimed at attaining increased biocompatibility and better mechanical surface properties for the development of the biomaterial for dental as well

CE

as orthopedic applications. Hence, there is a great need to modify the biomaterial surfaces to

AC

produce novel material which meets the clinical demands. So far a tailor-made composition of standard bioglass as a potential tool for orthopedic and dental applications has not yet been fully elucidated. In this context, the present study aims at producing different TiO2 doped borosilicate bioglasses and also to investigate their bioactivity, cytocompatibility using cell proliferation assays, protein expression studies.

4

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Media and chemicals All chemicals used in this work were of analytical grade and without further purification. Silica (SiO2, 99.99%) taken from Umicore thin film coating quality, B2O3 (99.9%), CaO (99.9%), TiO2 (99.9%) have been purchased from Sigma-Aldrich. Na2CO3

PT

with 99.9% purity was purchased from Sisco Research Laboratories.

RI

DMEM (Gibco, 11965092), Trypan blue (Himedia, RM 100125), MTT 3-(4,5-

SC

Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (Himedia, TC191), NP – 40 (Sigma, I8896), 1X PBS(PH 7.4) (Gibco, 10010023), TBST FBS (Gibco, 10270106), Anti-anti

NU

(Gibco, 15240062), Sodium pyruvate (Gibco, 11360070), and Trypsin-EDTA ( Gibco, 25200056) were purchased from Invitrogen. Ponceau stain was from Himedia, RM977, and

MA

NP – 40 detergent was purchased from Sigma-Aldrich.

Reagent A and Reagent B for protein estimation (BIO-RAD Catalog no: 5000113) were purchased from Bio-Rad, PVDF membrane was from GE healthcare (Cat.

D

No.10600002)

PT E

Cyclin A, Cyclin B1 antibodies were from Santacruz, β- Tubulin antibody was of Sigma-Aldrich, Mouse, and Rabbit HRP conjugated secondary antibodies were from GE Healthcare, Alexa Fluor 588, and DAPI were from Invitrogen Life tech. β- Actin was from

AC

2.2 Cell line

CE

Cell signaling Technology, USA.

MG 63 Human osteoblast cells were purchased from National Centre for Cell Science (NCCS), Pune, India. The expression of various features by these cells makes them handy in investigating the osteoblast response on different biomaterials as they are derived from osteosarcoma cells[14][24].

5

ACCEPTED MANUSCRIPT 2.3 Preparation of calcium borosilicate glass The glasses with the composition 31B2O3-20SiO2-24.5Na2O-(24.5-x) CaO-xTiO2 (mol %) (x=0,0.5,1,2) were prepared by a melt quenching technique, as described earlier[12]. Appropriate amounts of analytical grade oxide powders with 99.9% purity were heated in a platinum crucible in an electric furnace for 3h under the temperature range 1000oC -1200oC.

PT

The melts were cast into pre-heated stainless steel moulds, annealed at 350oC for two hours.

RI

For convenience, these glass systems are labelled T0, T0.5, T1 and T2 according to the TiO2

SC

content in the glass matrix as shown in Table 1. The resulting glasses were grounded and sieved to obtain a fine glass powder.

NU

2.4 Preparation of SBF

MA

In order to investigate the in-vitro bioactivity of a material, SBF is widely used for its mimicking nature with human blood plasma. Simulated body fluid was prepared by using

D

standard procedure developed by Kokubo et al[25] in polypropylene bottles. The experiment

PT E

was performed in a static condition. powder to SBF solution ratio was used as 0.1g of glass powder in 50ml at 37oC [26].

CE

2.5 Characterization

AC

The amorphous nature of glasses was confirmed by X-ray diffraction (XRD) analysis (Model: PANALYTICAL XPERT POWDER); The radiation source was CuKα with the scanning angle ranging from 20 oC and 60oC. The measurement was done with the step size of 0.02°C with the time per step being 50 sec. IR absorption spectra were measured to identify the functional groups in the glass specimens before and after immersion into SBF solution to confirm the formation of apatite layer. The spectra were recorded using KBr pelleted samples with a resolution of 4 cm−1 in a wavelength range of 4000- 400 cm−1 using FTIR transmittance spectrometer (model S 100; PerkinElmer). pH measurements were done 6

ACCEPTED MANUSCRIPT with Thermo Scientific (ORION pH 7000). The surface morphologies and elemental compositions of the glasses were examined using SEM-EDS (Carl Zeiss EVO 18 and Oxford). Confocal microscopy images of the samples has been obtain with Carl Zeiss, NLO 710 Germany. The hardness of the glass samples were carried out by using Vickers hardness tester (HMV-2000 SHIMADZU). The applied load was 100 g and loading time was 15 s.

PT

Each sample was measured ten times, and the mean value of the test results was taken.

RI

2.6 Degradation study:

SC

Degradation test was performed as per the ISO 10993 “Biological evaluation of biomedical devices – Part 14: Identification and quantification of degradation products from

NU

ceramics”. These tests were performed at 37 °C in SBF at pH 7.4, weight loss as a function of

MA

immersion time were measured up to 21 days. A ratio of 0.50 g powders to 50.0 mL solution was used, and the SBF was replaced for every three days. Weight changes were measured by

D

separating the powders from SBF, washing with deionized water, and dried at 95 °C.

PT E

2.7 Cytotoxicity and Cell proliferation assay with MTT The cytocompatibility of the prepared glasses has been assessed using MTT

CE

Assay[27]. For this, 5x103 MG-63 cells were added to each well of 96-well plate. The cell counting was done by Trypan blue staining assay using Hemocytometer. Cells were treated

AC

with each of the four compounds within a range of concentrations from 50 µg/mL to 1000 µg/mL. The work was done in triplicates. After 48 hours, 20 μL (5mg/mL) of MTT [3-(4,5Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] reagent was added to each well and incubated for four hours. After that medium with MTT was removed and 100 μL of acidified isopropanol (Isopropanol with 4mM HCl and 0.1% NP40) was added to each well and incubated for half an hour. The absorbance (OD) of the cells was measured at 570 nm with

7

ACCEPTED MANUSCRIPT reference 650 nm using plate reader (BioRad, USA). The percentage of cell viability was calculated using the formula given below, % Cell Viability = [OD]test/[OD]control After the above steps, the proliferation of cells in the presence of respective

PT

compounds at a concentration of 1000 μg/mL for a time period of seven days was monitored using MTT Assay. For this, to each well containing 5x103 cells, each of the respective

RI

compounds was added at a concentration of 1000 μg/mL. The absorption values were

SC

measured after 1, 3, and 7 days.

NU

2.8 Immuno Blotting

MG-63 cells were treated with the respective compounds (T0, T0.5, T1, T2) of 1000

MA

μg/mL for 48 hrs. Following the treatment, cells were washed with 1X PBS (PH 7.4) and then lysed with NP-40 Lysis buffer (150 mM NaCl, 50 mM Tris-HCl of pH 8.0 , 1% NP 40

D

with 1X Protease Inhibitors, 1mM NaF, 1 mM PMSF, 1mM Na3VO4). Protein estimation was

PT E

done by RC-DC assay (BioRad, USA) and then an equal amount of protein was resolved on 12% SDS-PAGE and then transferred to nitrocellulose membrane. After that non-specific

CE

protein was blocked with 5% non-fat milk, and then membrane was incubated with primary antibody overnight (Cyclin A # sc751 with 1:500 dilutions, Cyclin B1 # sc-250 with 1:500

AC

(Santacruz Biotech, USA), β-Actin # CST 4967 with 1:1000 (Cell signalling Technology, USA). Later unbound antibody was removed with TBST washings for 10 min each for three times. Rabbit-HRP secondary antibody for Cyclin A and β- Actin and mouse HRP Secondary antibody for Cyclin B1 was added for one hour at room temperature. Unbound antibody was removed with TBST buffer washings thrice for 10 min each. Blots were developed with Amersham ECL Prime Western Blotting Detection Reagents (GE Healthcare Product code: RPN2232) and images were captured using Chemidac (BioRad, USA)[28].

8

ACCEPTED MANUSCRIPT 2.9 Immunofluorescence and Confocal Imaging MG – 63 cells were cultured in 35 mm dishes along with a sterilised coverslips by using DMEM and 10% FBS. Cells incubated for overnight for adherence, treatment was done with respective compounds of 1000 μg/mL except for control plate. After 1 and 7days of

PT

treatment, plates were removed from the incubator and washed with PBS. After that cells were fixed with 4% Paraformaldehyde for 20 min at Room temperature and then

RI

permeabilized with cold Acetone – methanol (1:3) mixture (It should be kept in -20 oC at

SC

least 30 min before adding) for 30 min. Washings were done with TBS for 5 min each for 3 times. Blocking was done with 3% BSA for one hour at room temperature and Primary

NU

antibody (β- Tubulin) was added with 1: 100 dilutions for overnight at 4 oC. Next day washings were done with TBS, TBST, and TBS for 15 min each. After that secondary

MA

antibody (Alexa flour 588) was added for one hour at room temperature and then washings were done with TBS, TBST, and TBS for 15 minutes each. Coverslip was removed from

PT E

D

culture plate and was allowed to dry for five minutes and fixed on a glass slide with a prolonged gold antifade reagent with DAPI and incubated for 16 - 24 hrs in dark. Nail polish was applied around the cover slip which prevents the air from entering the slide. Later the

CE

images were taken with confocal microscopy.

AC

2.10 Anti bacterial activity test: Stock solutions were prepared using UV sterilized TiO2 [T0 ,T0.5 ,T1 ,T2] bioglass particles at 0 (control), and 10 mg ml−1 in NB. These solutions were incubated for 72 h at 37 °C under aerobic conditions in shaker (with a shaking speed of 50 rpm) incubators and then filtered using a 0.45 micron syringe filter. One ml of filter sterilized stock solution was neutralised to achieve a pH of 7.3 using HCl and inoculated with the test microorganisms at 106 CFU. The remainder of the stock solution was used to measure the pH. The inoculated samples were incubated at 37 °C under static and 9

ACCEPTED MANUSCRIPT shaker (100 rpm) conditions. After 24 and 96 h of incubation, the samples were removed, serial dilutions performed and 100 μl aliquots dispensed onto NA plates, which were incubated overnight at 37 °C. Following incubation, the total viable CFU count was determined, and growth reductions calculated.

PT

2.11 Statistical Analysis All the experiments were performed in triplicates. The results obtained from the

RI

cytocompatibility assays (cytotoxicity and cell proliferation) were submitted to a Two-way

SC

Analysis of Variance (2-way ANOVA).The data were represented as means ± standard deviation for all experiments and analyzed using two-way analysis of variance with a student

NU

t- test. A p-value < 0.05 was considered statistically significant. The resulting data was

MA

statistically analysed using origin 8.0

3. Results and Discussion

D

The XRD patterns of all glasses before soaking in SBF (a) after soaking in SBF (b &

PT E

c) for three and seven days respectively were shown in Fig.1. The XRD analysis of prepared glasses shows no sharp diffraction peaks which confirm the amorphous character of the

CE

glasses (Figure 1a). Fig.1b & 1c shows the crystalline phases on the glass surfaces after immersion in SBF for three and seven days. The observed diffraction peaks were at 2 =

AC

25.87o, 31.77o and 46.71o, related to (002), (211) and (222) crystalline planes respectively. On increasing titania content, the peak intensity is reduced in all the glass samples. The XRD pattern of the immersed samples (Fig. 1) matches with the peaks of hydroxyapatite [Ca10(PO4)6(OH)2] according to the standard JCPDS card number 09-0432 [29–31]. As reported earlier, the base glass (T0) acts as a bioactive material[12]. The hydroxyapatite layer formation can be observed on all glass samples but there is a decrease in peak intensity with increase in TiO2 content up to three days of incubation in SBF. Saturation of hydroxyapatite

10

ACCEPTED MANUSCRIPT layer formation in all glasses with the increase of immersion time (seven days) can be observed from Fig.1c. The room temperature FT-IR transmittance spectra of all investigated glasses before and after soaking in SBF are shown in Fig.2a and b respectively. The wavenumber 468 cm−1 corresponds to rocking vibrations of Si-O-Si bridges [32,33], characteristic wave numbers

PT

710-720 cm-1 related to B-O-B bond bending vibrations of borate network[34]. While, the

RI

peak observed at 930-1197 cm−1 is due to stretching vibration of tetrahedral (BO4)− units and

SC

band at 1200-1500 cm−1 B–O stretching vibrations of trigonal BO3 units respectively [35] . With increasing titania content additional shoulder peaks were noticed in all titania-doped

NU

glass samples before in vitro studies. From Fig.2b it is observed that the new peak values were positioned at ~561, ~609, ~875, ~ 960, ~1024, ~ 1422 and ~1639 cm-1 in all glasses

MA

after seven days of immersion in SBF which confirms the formation of hydroxyapatite layer. In all glasses, the double peak at ~561, ~609 cm-1 can be observed which is due to P-O

D

bending vibrations in PO4 tetrahedral and a characteristic band of HAp phase [36–38]. The

PT E

small shoulder around 875cm-1 is related to the stretching vibration of SiO44- units (Q0) and to A-type carbonate-substituted hydroxyapatite (CHA) phase [35,39]. The band at 1024cm-1

CE

indicates P-O bending vibrations in PO4 tetrahedral and a characteristic band of the formation

AC

of apatite layer[38,40] . The peak observed at 1639cm-1 is the absorption band of water molecule [41]

The SEM micrograph confirms bioactivity of the glasses by the formation of a white globular precipitate on the surface of the glasses. The SEM-EDS micrograph of T2 glass before and after in-vitro studies is shown in Fig. 3. It was observed that before immersion T2 glass shows rod-like morphology, with the width of the order of approximately 100nm which may be due to the precursor TiO2 nanoparticles. EDS results showed that there is no phosphate content in the glass (T2). The prolonged exposure of samples in SBF for three days 11

ACCEPTED MANUSCRIPT and seven days, there was an enormous increase in the white precipitate formation, confirmed by Ca/P atomic ratio 1.64 (± 0.03) and 1.66 (± 0.02) respectively, which is nearly equal to bone composition (1.67)[42,43]. The pH changes of the SBF solutions, in which different glasses were soaked, were

PT

measured in order to check their bioactivity. Bioglasses usually degrade in phosphate buffered salines and SBF because of the solubility of constituent elements in the host (base)

RI

glass composition. It was found that pH value of un-doped (T0) sample increases rapidly

SC

within 7 days, after which the pH value increment slowdown (shown in Fig. 4). pH of the body fluid increased from 7.4 to 8.4 due to the rapid dissolution of B2O3 content and

NU

exchange of alkali (Ca++, Na+) ions and hydrogen (H+, OH−) ions in the undoped glass composition. From previous studies, higher pH of the solution accelerates apatite

MA

formation[44]. The pH value decreases in the bulk solution for all glasses except for the undoped sample (T0). The reduction in bioactivity is indicated by decrease in ionic reactivity of

PT E

D

the bodies in physiological fluid with progressive addition of TiO2 in the system. The decrement in pH values were observed with the addition of TiO2 to the host glass as shown in Fig. 4. The incorporation of TiO2 decreases the degradation rate followed by slow

CE

dissolution. This shows that the chemical durability of the system increases due to addition of

AC

transition metal oxide [45].

Fig. 5. shows weight loss as a function of immersion time. Weight loss of undoped borosilicate glass and TiO2 doped glasses are showing same trend with the immersion time as depicted in Fig. 5. However, weight loss is higher in undoped borosilicate glass as compared to TiO2 doped glasses due to rapid dissolution of constituents in the base glass (T0). It was observed that all glasses showed nearly same trend, initially within the first week the weight loss increased at a rapid rate afterwards slowed down. This initial sudden increase in weight loss is due to series of rapid chemical reaction occurred when powder of material soaked in 12

ACCEPTED MANUSCRIPT physiological fluid [46]. After seven days of immersion weight losses are 43.25%, 41%, 35.48%, and 29.17% for T0, T0.5, T1, and T2 respectively. Chemical durability is increased with addition concentration of TiO2. Eventually, the dissolution rate is decreases with addition of TiO2 to the glass. A strong covalent Ti–O bond formed than Ca–O bond, when Ti4+ ions substitute for Ca2+ ions, because the electrostatic field strength of Ti4+ is larger than that of Ca2+

PT

[19][44][47].This clearly demonstrating that the addition of TiO2 to base glass lowers the

RI

degradation rate. The incorporation of TiO2 leads to reduction in the weight loss of prepared glasses, which has also been observed with pH measurements (fig. 4) and suppression of

SC

peaks in XRD as shown in fig 1b.

NU

The measured micro hardness values are shown in Table 1. The hardness values are in the range 545-576 Hv. The hardness of TiO2 doped glasses is increased with increasing TiO2

MA

concentration. Among the all glasses 2 mol% TiO2 doped glass showing better micro

D

hardness.

PT E

The viability of MG-63 cells at different dosages (1000 to 50 μg/mL) with varied TiO2 doped glasses was analysed using MTT assay. Base glassT0 and TiO2 doped glasses T0.5, T1, T2 are treated with MG-63 cells to know the non-toxic concentrations with MTT

CE

Assay. MTT forms formazan crystals with the live cells and these crystals when dissolved in

AC

acidified isopropanol give a colour which can be measured spectrophotometrically. The amount of absorbance is equal to the cell viability. The results of the test are shown in Fig. 6. As can be seen from the graph, the cell viability is affected by the dosages (50 μg/mL - 1000 μg/mL) of the respective glasses, but not by the content of the titania. Even though the cell viability decreases with the increase in the dosage, the change was not significant (as nearly 70-80% of the cells are viable). According to ISO 10993 - 5: 2009 [48] standards, it can be confirmed that the prepared samples were non toxic in nature, because the cell viability is greater than 70%. 13

ACCEPTED MANUSCRIPT In view of the results from MTT assay, the concentration of compounds (T0, T0.5, T1, T2) was fixed to 1000 μg/ml for further in vitro studies. The cytotoxic nature of the compounds on cell cycle progression was characterized by measuring the levels of cyclins. As cyclins are the key regulators of cell cycle progression; alterations in the levels of these proteins will lead to decreased cell proliferation and also the death of the cells. For this,

PT

immunoblotting with cyclin antibodies was performed. This data is summarized in the Fig. 7.

RI

These observations clearly demonstrate that the compounds exerted no impact on the cyclin

SC

levels even at the maximum concentration tested (viz., 1000 μg/ mL) and thus they are not cytotoxic.

NU

Further, MTT assay was also employed to quantify the effects of the test compounds on cell proliferation for over seven days. The results (Fig. 8) suggest that the cells treated

MA

with the test compounds did not demonstrate enhanced proliferative ability in comparison to

D

the untreated cells.

PT E

An ideal biomaterial should not affect the viability as well as the morphology of the seeded cells. Further studies highlight the effect of all the four compounds on the morphology of MG-63 cells. To investigate this, cells were treated with and without compounds for 1-7

CE

days and the cell morphology was checked by probing β–Tubulin which is a cytoskeletal

AC

marker by immunofluorescence approach and nuclear stain with DAPI. The results are shown in the Fig. 9. From these results, it can be deduced that the cells were healthy and there was no significant difference in morphology of the compound-treated to -untreated cells. This implies that these compounds do not have the cell proliferative capacity but at the same time were not cytotoxic.

14

ACCEPTED MANUSCRIPT Figure 10 shows the antibacterial effect of undoped and TiO2 doped borosilicate bioglass particles over a 96 h period under static incubation condition. The result shows that particulate TiO2 Bioglass showed considerable broad-spectrum antibacterial properties [49][50][51], as growth inhibition was demonstrated for E. coli and S. aureus. In the present study bioglass particles at the concentration of 2% TiO2 for 10 mg ml-l exhibited a strong

PT

antibacterial effect against E. coli and S. aureus. [50]. Under the static conditions T2 bioglass

RI

particles inhibited S.aureus and E.coli growth within 96 hours of incubation, with 4 log

SC

reduction and 3 log reduction respectively. The remaining bioglasses [T0, T0.5, T1] did not posses strong antibacterial effect against both E. coli and S. aureus. The mechanism of

NU

bacterial inhibition of Ti+4 is not yet fully elucidated. Though results are encouraging, the results obtained in the present study are not sufficient for the complete understanding of

MA

antibacterial effect. More experiments should be performed in the near future to fully understand the anti-bacterial nature of the titanium oxide doped glasses.

PT E

D

5. Conclusions

We have successfully synthesized novel TiO2 doped calcium borosilicate glasses using conventional melting and quenching technique. The experimental results of XRD, FT-

CE

IR and SEM-EDS have demonstrated that TiO2 doped calcium borosilicate glasses are

AC

bioactive materials. The incorporation of TiO2 decreases the degradation rate followed by slow dissolution. As Ti4+ having stronger field strength as compared to Si4+ & Ca2+ so its replacement became the cause for reduction in degradation that in turn improved the chemical stability. Biological properties cytocompatibility, cell proliferation studies clearly reveal the non-toxic nature or cytocompatible nature of the prepared glass samples. Strong antibacterial efficacy was observed for 2% TiO2 in the system against E. coli and S. aureus. From these findings, TiO2 doped calcium borosilicate glasses are considered as useful materials for orthopedic and dental applications. 15

ACCEPTED MANUSCRIPT Acknowledgements The authors wish to express their thanks to the Department of Science and Technology & MHRD, New Delhi, Government of India for the financial support (Order No: SR/S2/CMP/0090/2010) and sincere gratitude to the Director NIT Warangal, for providing the required

PT

facilities in the institute for carrying out research work. References:

L.L. Hench, An introduction to bioceramics, Second edition, Imperial college press,

RI

[1]

[2]

SC

London 2013.

M.A. Sainz, P. Pena, S. Serena, A. Caballero, Acta Biomaterialia Influence of design on bioactivity of novel CaSiO3 – CaMg (SiO3)2 bioceramics : In vitro simulated body

J.R. Jones, Review of bioactive glass: From Hench to hybrids, Acta Biomaterialia. 9

MA

[3]

NU

fluid test and thermodynamic simulation, 6 (2010) 2797–2807.

(2013) 4457–4486.

R.E. Newnham, Essentiality of Boron for Healthy Bones and Joints, (1994) 83–85.

[5]

R.F. Brown, D.E. Day, T.E. Day, S. Jung, M.N. Rahaman, Q. Fu, Growth and

D

[4]

PT E

differentiation of osteoblastic cells on 13-93 bioactive glass fibers and scaffolds., Acta Biomaterialia. 4 (2008) 387–96. X. Zhang, W. Jia, Y. Gu, W. Xiao, X. Liu, D. Wang, C. Zhang, W. Huang, M.N.

CE

[6]

Rahaman, D.E. Day, N. Zhou, Teicoplanin-loaded borate bioactive glass implants for treating chronic bone infection in a rabbit tibia osteomyelitis model., Biomaterials. 31

[7]

AC

(2010) 5865–74.

Y. Gu, W. Huang, M.N. Rahaman, D.E. Day, Bone regeneration in rat calvarial defects implanted with fibrous scaffolds composed of a mixture of silicate and borate bioactive glasses., Acta Biomaterialia. 9 (2013) 9126–36.

[8]

D. Zhao, W. Huang, M.N. Rahaman, D.E. Day, D. Wang, Mechanism for converting Al2O3-containing borate glass to hydroxyapatite in aqueous phosphate solution., Acta Biomaterialia. 5 (2009) 1265–73.

[9]

W.-T. Jia, X. Zhang, S.-H. Luo, X. Liu, W.-H. Huang, M.N. Rahaman, D.E. Day, C.-

16

ACCEPTED MANUSCRIPT Q. Zhang, Z.-P. Xie, J.-Q. Wang, Novel borate glass/chitosan composite as a delivery vehicle for teicoplanin in the treatment of chronic osteomyelitis., Acta Biomaterialia. 6 (2010) 812–9. [10] S.M. Wiederhorn, Y.-H. Chae, C.G. Simon, J. Cahn, Y. Deng, D. Day, Cell adhesion to borate glasses by colloidal probe microscopy., Acta Biomaterialia. 7 (2011) 2256– 2263.

PT

[11] W. Huang, D.E. Day, K. Kittiratanapiboon, M.N. Rahaman, Kinetics and mechanisms of the conversion of silicate (45S5), borate, and borosilicate glasses to hydroxyapatite

RI

in dilute phosphate solutions, Journal of Materials Science: Materials in Medicine. 17

SC

(2006) 583–596.

[12] R. Samudrala, G.V.N. Reddy, B. Manavathi, P.A. Azeem, Synthesis , characterization

NU

and cytocompatibility of ZrO2 doped borosilicate bioglasses, Journal of NonCrystalline Solids. 447 (2016) 150–155.

MA

[13] S. Rafique, M. Idrees, A. Nasim, H. Akbar, A. Athar, Transition metal complexes as potential therapeutic agents, Molecular Biology. 5 (2010) 38–45. [14] E. a. Abou Neel, J.C. Knowles, Physical and biocompatibility studies of novel titanium

D

dioxide doped phosphate-based glasses for bone tissue engineering applications,

PT E

Journal of Materials Science: Materials in Medicine. 19 (2008) 377–386. [15] S.M. Naga, A.M. El-Kady, H.F. El-Maghraby, M. Awaad, R. Detsch, A.R. Boccaccini, Novel porous Al2O3-SiO2-TiO2 bone grafting materials: Formation and

CE

characterization., Journal of Biomaterials Applications. 28 (2014) 813–824.

AC

[16] C. Ma, A. Nagai, Y. Yamazaki, T. Toyama, Y. Tsutsumi, T. Hanawa, W. Wang, K. Yamashita, Electrically polarized micro-arc oxidized TiO2 coatings with enhanced surface hydrophilicity., Acta Biomaterialia. 8 (2012) 860–5. [17] B.D. Boyan, T.W. Hummert, D.D. Dean, Z. Schwartz, Role of material surfaces in regulating bone and cartilage cell response., Biomaterials. 17 (1996) 137–46. [18] A. Kiani, J. V Hanna, S.P. King, G.J. Rees, M.E. Smith, N. Roohpour, V. Salih, J.C. Knowles, Structural characterization and physical properties of P2O5-CaO-Na2O-TiO2 glasses by Fourier transform infrared, Raman and solid-state magic angle spinning nuclear magnetic resonance spectroscopies., Acta Biomaterialia. 8 (2012)

17

ACCEPTED MANUSCRIPT [19] A. V. Gayathri Devi, V. Rajendran, N. Rajendran, Structure, solubility and bioactivity in TiO2-doped phosphate-based bioglasses and glass-ceramics, Materials Chemistry and Physics. 124 (2010) 312–318. [20] V. Rajendran, A. V. Gayathri Devi, M. Azooz, F.H. El-Batal, Physicochemical studies of phosphate based P2O5–Na2O–CaO–TiO2 glasses for biomedical applications, Journal of Non-Crystalline Solids. 353 (2007) 77–84.

PT

[21] X. Du, W. He, X. Zhang, J. Ma, C. Wang, C. Li, Y. Yue, Low temperature biosynthesis of Li2O-MgO-P2O5-TiO2 nanocrystalline glass with mesoporous structure

RI

exhibiting fast lithium ion conduction, Materials Science and Engineering C. 33 (2013)

SC

1592–1600.

[22] A.V. Gayathri Devi, V. Rajendran, N. Rajendran, Structure, solubility and bioactivity

and Physics. 124 (2010) 312–318.

NU

in TiO2-doped phosphate-based bioglasses and glass–ceramics, Materials Chemistry

MA

[23] G. Jagan Mohini, G. Sahaya Baskaran, V. Ravi Kumar, M. Piasecki, N. Veeraiah, Bioactivity studies on TiO2-bearing Na2O–CaO–SiO2–B2O3 glasses, Materials Science and Engineering: C. 57 (2015) 240–248.

D

[24] E.A. Abou Neel, T. Mizoguchi, M. Ito, M. Bitar, V. Salih, J.C. Knowles, In vitro

PT E

bioactivity and gene expression by cells cultured on titanium dioxide doped phosphatebased glasses, Biomaterials. 28 (2007) 2967–2977. [25] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity?,

CE

Biomaterials. 27 (2006) 2907–2915.

AC

[26] A. Goel, R.R. Rajagopal, J.M.F. Ferreira, Influence of strontium on structure, sintering and biodegradation behaviour of CaO-MgO-SrO-SiO2-P2O5-CaF2 glasses., Acta Biomaterialia. 7 (2011) 4071–4080. [27] N. Siddiqui, K. Pramanik, E. Jabbari, Osteogenic differentiation of human mesenchymal stem cells in freeze-gelled chitosan/nano ??-tricalcium phosphate porous scaffolds crosslinked with genipin, Materials Science and Engineering C. 54 (2015) 76–83. [28] S. Bugide, D. David, a Nair, N. Kannan, V.S.K. Samanthapudi, J. Prabhakar, B. Manavathi, Hematopoietic PBX-interacting protein (HPIP) is over expressed in breast infiltrative ductal carcinoma and regulates cell adhesion and migration through 18

ACCEPTED MANUSCRIPT modulation of focal adhesion dynamics, Oncogene. (2014) 1–12. [29] L. Ji, W. Wang, D. Jin, S. Zhou, X. Song, In vitro bioactivity and mechanical properties of bioactive glass nanoparticles / polycaprolactone composites, Materials Science & Engineering C. 46 (2014) 1–9. [30] G. Kaur, G. Pickrell, G. Kimsawatde, D. Homa, H. a Allbee, N. Sriranganathan, Synthesis, cytotoxicity, and hydroxyapatite formation in 27-Tris-SBF for sol-gel based

PT

CaO-P2O5-SiO2-B2O3-ZnO bioactive glasses., Scientific Reports. 4 (2014) 4392.

RI

[31] R. Govindan and E. K.Girija, Drug loaded phosphate glass/hydroxyapatite nanocomposite for orthopedic applications, Journal of Materials Chemistry B. 2 (2014)

SC

5468–5477.

[32] A.M. Abdelghany, H. Kamal, Spectroscopic investigation of synergetic bioactivity

International. 40 (2014) 8003–8011.

NU

behavior of some ternary borate glasses containing fluoride anions, Ceramics

MA

[33] P. Ducheyne, S. Radin, M. Heughebaert, J.C. Heughebaert, Calcium phosphate ceramic coatings on porous titanium: effect of structure and composition on electrophoretic deposition, vacuum sintering and in vitro dissolution, Biomaterials. 11

PT E

D

(1990) 244–254.

[34] E.I. Kamitsos, G.D. Chryssikos, Borate glass structure by Raman and infrared spectroscopies, Journal of Molecular Structure. 247 (1991) 1–16.

CE

[35] S. Agathopoulos, D.U. Tulyaganov, J.M.G. Ventura, S. Kannan, A. Saranti, M.A. Karakassides, J.M.F. Ferreira, Structural analysis and devitrification of glasses based

AC

on the CaO-MgO-SiO2 system with B2O3, Na2O, CaF2 and P2O5 additives, Journal of Non-Crystalline Solids. 352 (2006) 322–328. [36] S. Srinivasan, R. Jayasree, K.P. Chennazhi, S. V Nair, R. Jayakumar, Biocompatible alginate / nano bioactive glass ceramic composite scaffolds for periodontal tissue regeneration, Carbohydrate Polymers. 87 (2012) 274–283. [37] K. Taek Jung, Y.-H. Chu, S. Haam, Y. Gun Shul, Synthesis of mesoporous silica fiber using spinning method, Journal of Non-Crystalline Solids. 298 (2002) 193–201. 38]

I. Rehman, W. Bonfield, Characterization of hydroxyapatite and carbonated apatite by photo acoustic FTIR spectroscopy, Journal of Materials Science: Materials in

19

ACCEPTED MANUSCRIPT Medicine. 8 (1997) 1–4. [39] Z. Hong, A. Liu, L. Chen, X. Chen, X. Jing, Preparation of bioactive glass ceramic nanoparticles by combination of sol-gel and coprecipitation method, Journal of NonCrystalline Solids. 355 (2009) 368–372. [40] C.Y. Kim, a. E. Clark, L.L. Hench, Early stages of calcium-phosphate layer formation

PT

in bioglasses, Journal of Non-Crystalline Solids. 113 (1989) 195–202. [41] R. Lucacel Ciceo, D.L. Trandafir, T. Radu, O. Ponta, V. Simon, Synthesis,

RI

characterisation and in vitro evaluation of sol-gel derived SiO2-P2O5-CaO-B2O3 bioactive system, Ceramics International. 40 (2014) 9517–9524.

mechanical properties, 24 (2003) 2161–2175.

SC

[42] T. Kokubo, H. Kim, M. Kawashita, Novel bioactive materials with different

NU

[43] T.M. Coelho, E.S. Nogueira, a. Steimacher, a. N. Medina, W.R. Weinand, W.M. Lima, M.L. Baesso, A. C. Bento, Characterization of natural nanostructured

MA

hydroxyapatite obtained from the bones of Brazilian river fish, Journal of Applied Physics. 100 (2006).

D

[44] S. Ni, R. Du, S. Ni, The influence of Na and Ti on the in vitro degradation and

PT E

bioactivity in 58S sol-gel bioactive glass, Advances in Materials Science and Engineering. 2012 (2012)1-7.

[45] O. Peitl, E. Dutra Zanotto, L.L. Hench, Highly bioactive P2O5–Na2O–CaO–SiO2 glass-

CE

ceramics, Journal of Non-Crystalline Solids. 292 (2001) 115–126. [46] L.L. Hench, The story of Bioglass, J Mater Sci: Mater Med 17 (2006) 967–978

AC

[47] M. Navarro, M. Ginebra, J. Cle, S. Mart, Physicochemical Degradation of TitaniaStabilized Soluble Phosphate Glasses for Medical Applications, Journal of Ameican Ceramic Society. 52 (2003) 5–12. [48] ISO/EN 10993-5. Biological evaluation of medical devices – Part 5. Tests for cytotoxicity, in vitro methods: 8.2 tests on extract. [49] S. Begum, W.E. Johnson, T. Worthington, R.A. Martin, The influence of pH and fluid dynamics on the antibacterial efficacy of 45S5 Bioglass, Biomedical Materials. 11 (2016) 15006.

20

ACCEPTED MANUSCRIPT [50] M. Riaz, R. Zia, F. Saleemi, T. Hussain, F. Bashir, H. Ikhram, Effect of Ti+4 on in vitro bioactivity and antibacterial activity of silicate glass-ceramics, Materials Science & Engineering C. 69 (2016) 1058–1067. [51] S. Hu, J. Chang, M. Liu, C. Ning, Study on antibacterial effect of 45S5 Bioglass??, Journal of Materials Science: Materials in Medicine. 20 (2009) 281–286.

PT

Figure Captions: Table 1. The composition of glasses with various concentrations of TiO2 (mol %) and

RI

Microhardness.

SC

Figure 1. XRD Spectra of Glasses before (a) and after (b), (c) in-vitro studies. (a) zero days of immersion in SBF, (b) three days of immersion in SBF (c) seven days of immersion in

NU

SBF

MA

Figure 2. FTIR Spectra of Glasses before (a) and after (b) in-vitro studies. (a) zero days of immersion in SBF, (b) seven days of immersion in SBF Figure 3. SEM-EDS micrographs of (2 mol %) TiO2 glasses before (a) and after [(b), (c), (d)]

PT E

D

in-vitro studies. (a) zero days of immersion in SBF (b) one day of immersion in SBF (c) three days of immersion in SBF (d) seven days of immersion in SBF Figure 4. pH variation of glasses up to 7 days of immersion period.

AC

immersion time.

CE

Figure 5. Weight loss (%) of undoped (T0) and TiO2 (T0.5, T1, T2) glasses as a function of

Figure 6. Cell Viability of undoped (T0) and TiO2 (T0.5, T1, T2) glasses with MG-63 cells using MTT assay.

Figure 7. Immunoblot analysis of the compounds: MG-63 cells were treated with 1000 μg/mL of all compounds and cyclins were analyzed to see the effect on cell cycle progression of the compounds.

21

ACCEPTED MANUSCRIPT Figure 8. Cell Proliferation of TiO2 glasses with MG-63 cells using MTT assay. The proliferation was not significant with the TiO2 glass compounds compared to the normal untreated cells. Figure 9. Confocal laser scanning spectroscopy images MG-63 cells treated with T0 and T2. Figure 10. The Anti-microbial properties of undoped and TiO2 doped borosilicate glass

AC

CE

PT E

D

MA

NU

SC

RI

PT

particles on the viability of (a) E coli and (b) S aureus, under different incubation time.

22

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

Fig. 1

23

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

Fig. 2

24

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

Fig. 3

25

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Fig. 4

26

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Fig. 5

27

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Fig. 6

28

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Fig. 7

29

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

Fig. 8

30

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Fig. 9

31

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

Fig. 10

32

ACCEPTED MANUSCRIPT Table 1

Sample

Composition (mol %)

Micro hardness

SiO2

Na2CO3

CaO

TiO2

(Hv)

T0

31

20

24.5

24.5

0

545 (±20)

T0.5

31

20

24.5

24

0.5

558 (±22)

T1

31

20

24.5

23.5

1

564 (±20)

T2

31

20

24.5

22.5

2

576 (±21)

AC

CE

PT E

D

MA

NU

SC

RI

PT

B 2O 3

33

ACCEPTED MANUSCRIPT Highlights

CE

PT E

D

MA

NU

SC

RI

PT

A tailor-made composition of TiO2 doped borosilicate glasses were synthesized TiO2 doped borosilicate glasses are bioactive and cytocompatible. Degradation rate is reduced by the addition of TiO2 content. Immunoblotting studies demonstrated that no significant change in cyclin levels.

AC

1. 2. 3. 4.

34