Materials Science and Engineering C 49 (2015) 549–559

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Influence of barium substitution on bioactivity, thermal and physico-mechanical properties of bioactive glass Sampath Kumar Arepalli ⁎, Himanshu Tripathi, Vikash Kumar Vyas, Shubham Jain, Shyam Kumar Suman, Ram Pyare, S.P. Singh ⁎ Department of Ceramic Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005, India

a r t i c l e

i n f o

Article history: Received 9 June 2014 Received in revised form 11 December 2014 Accepted 9 January 2015 Available online 12 January 2015 Keywords: Bioactive glass Barium oxide Physico-mechanical properties Thermal behavior

a b s t r a c t Barium with low concentration in the glasses acts as a muscle stimulant and is found in human teeth. We have made a primary study by substituting barium in the bioactive glass. The chemical composition containing (46.1 − X) SiO2−–24.3 Na2O–26.9 CaO–2.6 P2O5, where X = 0, 0.4, 0.8, 1.2 and 1.6 mol% of BaO was chosen and melted in an electric furnace at 1400 ± 5 °C. The glasses were characterized to determine their use in biomedical applications. The nucleation and crystallization regimes were determined by DTA and the controlled crystallization was carried out by suitable heat treatment. The crystalline phase formed was identified by using XRD technique. Bioactivity of these glasses was assessed by immersion in simulated body fluid (SBF) for various time periods. The formation of hydroxy carbonate apatite (HCA) layer was identified by FTIR spectrometry, scanning electron microscope (SEM) and XRD which showed the presence of HCA as the main phase in all tested bioactive glass samples. Flexural strength and densities of bioactive glasses have been measured and found to increase with increasing the barium content. The human blood compatibility of the samples was evaluated and found to be pertinent. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Bioactive glasses degrade in physiological solutions, forming a hydroxyl-carbonate apatite (HCA) layer on surface of the bioactive glass, which are an intimate bond between the glass and living bone [1,2]. Bioactive glasses can be produced by the traditional melting method which is regarded as simple and suitable for mass production [3]. The bioactive glass systems containing SiO2–Na2O–CaO–P2O5 have shown higher bioactivity in comparison to hydroxy apatite [4–6]. The bioactive glasses were able to bind to bone and to promote bone formation, which are also of interest for use as bone grafts and implant coatings [7–9]. The 45S5 bioactive glass is a very successful biomaterial for clinical applications and many researchers have studied with an incorporation of some ions such as Li, Zn, Ti, K, Zr, Mg, Fe, and Sr in the base bioactive glass because of their unique effect on osteoblastic cell proliferation of different ions in the base bioactive glasses [10–13]. It was reported that in 45S5 bioactive glass, SrO can easily be substituted for calcium oxide due to their similar ionic charge and radius [14,15]. Donnell and Hill [16] discussed the difference in atomic weight of Sr = 87.62 and Ca = 40, while Sr2+ containing bioactive glasses were shown to combine with bone regenerative properties of bioactive glasses [16]. The 89Sr ⁎ Corresponding authors. E-mail addresses: [email protected] (S.K. Arepalli), [email protected] (S.P. Singh).

http://dx.doi.org/10.1016/j.msec.2015.01.049 0928-4931/© 2015 Elsevier B.V. All rights reserved.

1488 keV beta radiation has been used for the treatment of pain in the bone due to metastases from prostatic cancer [17]. It is well known that the general formula for apatite is [M10(XO4) 6Y2], where M represents a bivalent cation, X represents a trivalent anion and Y represents a monovalent anion [18]. The divalent cations (Mg2 +), (Sr2 +) and (Ba2 +) with a similar charge of (Ca2 +) calcium can readily be substituted in the lattice of hydroxyapatite. Barium is one of the alkaline earth metals like calcium. It has been reported that low doses of barium in glasses act as a muscle stimulant [19]. Austin et al. [20] confirmed that the barium distributions in the teeth of human children and found early life dietary transitions in primates. Kaur et al. [21] studied the bioactivity in the system of barium–zinc-borosilicate glass containing 30 mol% of barium in their compositions. Leenakul et al. [22] substituted BaO–Fe2O3 in 45S5 bioactive glass and showed the improvement in bioactivity. Though, the barium is radioactive but the lower concentration in the amorphous state is worth to investigate its role which is due to be studied. There is a continuous demand for bone implant formulations such as bioactive glasses which have better osteoblastic properties. One of the approaches to improve bone stimulating properties of these biomaterials is incorporation of bone stimulator ions into their chemical compositions. Moreover, there are limited studies on barium containing bioactive glass for bone substitutes. This makes Ba2+ substitution in bioactive glass an interesting group of materials to study and hence we carried out the preliminary screening tests as a biomaterial.

550

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559

2. Materials and methods 2.1. Preparation of bioactive glasses The chemicals were used in this experiment are analytical reagent grade (all from Loba Chemie, Mumbai, India) such as quartz, sodium carbonate, calcium carbonate, barium carbonate and ammonium dihydrogen orthophosphate as a source of SiO2, Na2O, CaO, BaO and P2O5, respectively with a purity of 98–99.9%. All were introduced in the form of their respective anhydrous state. The weighed batches were mixed thoroughly for 30 min and melted in pure alumina crucibles to get the desired bioactive glasses as given above in Table 1. The melting was carried out in an electric furnace at 1400 ± 5 °C for 2 h in air as furnace atmosphere and homogenized melts were poured on preheated aluminum sheet. The prepared bioactive glass samples were directly transferred to a regulated muffle furnace at 500 °C for annealing. Further, the glass samples were cooled gradually under a controlled rate of 10 °C per minute to room temperature after annealing for 1 h at 500 °C. In order to explore the possibility for contamination of aluminum in the glass samples melted in 99.9% pure alumina crucibles at 1400 °C for 2 h, the XRF (Thermo Scientific, USA) and the chemical analysis of the samples were done for aluminum content. The chemical analysis of the glass samples for determination of aluminum by Al(OH)3 precipitation method did not show any presence of Al3 + ion in the glass [23]. Further, the XRF analysis also did not show any appreciable contamination of aluminum in the glass melts prepared at above temperature in a small duration of 2 h. Since the glass samples have been melted in alumina crucibles as such the problem of contamination of silicon in the melts does not arise. Hence the results obtained for the present investigation on SiO2–CaO–Na2O–P2O5–BaO bioactive glasses have not been affected by contamination of aluminum and silicon from the crucibles.

2.3.2. Heat-treatment system Sometimes the bioactive glasses are used as coating materials on metal implants and composites therefore they are subjected to heattreatment. Hence it is better to know their crystalline phases present. The prepared bioactive glass samples were heat-treated in two-step system, firstly nucleation temperature for the formation of nuclei sites and after holding for the specific time, it was then further heated to reach the second selected crystal growth temperature after holding for the specific time. The samples were left to cool inside the muffle furnace to room temperature at a cooling rate of 10 °C per min. 2.3.3. Powder X-ray diffraction analysis In order to identity the crystalline phase present in the bioactive glasses, glass-ceramics and SBF treated glass samples were ground to 75 μm and the fine powders were subjected to X-ray diffraction analysis (XRD) using RIGAKU-Miniflex II diffractometer adopted Cu-Kα radiation (λ = 1.5405 Å) with a tube voltage of 40 kV and current of 35 mA in a 2θ range between 20° and 80°. During measurement the step size and speed were set to 0.02° and 1° per min, respectively and were followed in the present investigation. The JCPDS-International Centre for diffraction Data Cards were used as a reference. 2.3.4. Structural analysis of bioactive glasses The functional groups of bioactive glasses were investigated at room temperature in the frequency range of 4000–400 cm−1 using a Fourier transform infrared (FTIR) spectrometer (VARIAN scimitar 1000, USA). The fine bioactive glass powdered samples were mixed with spectroscopic grade KBr in the ratio of 1 part of sample with 99 parts of KBr. The mixtures were subjected to an evocable die at load of 10 MPa to produce clear homogeneous discs. The discs were immediately put in the instrument for FTIR spectral transmission measurements and the spectra of samples were recorded.

2.2. Preparation of SBF To carry out in vitro studies, we prepared simulated body fluid according to Kokubo [24] that has inorganic ion concentrations similar to those of human body fluid in order to reproduce formation of apatite on bioactive materials in vitro. The SBF solution was prepared at 37 °C by dissolving reagent grade NaCl, KCl, NaHCO3, MgCl2·6H2O, CaCl2 and KH2PO4 into double distilled water and it was buffered at pH = 7.4 with TRIS (trishydroxy methyl aminomethane) and 1 N HCl.

2.3. Characterization of samples 2.3.1. Thermal behavior In order to identify the thermal behavior of the barium substituted bioactive glasses, the differential thermal analysis (SETARAM Instrumentation, France) was carried out on powdered samples in air up to 1000 °C using powdered alumina as a reference material with the heating rate of 10 °C min− 1. The glass nucleation and crystallization temperatures were obtained from the DTA results which were used for proper heat treatment for converting glass to their corresponding glass–ceramic.

Table 1 Chemical composition of the bioactive glasses (mol%). Constituents

Ba-0

Ba-1

Ba-2

Ba-3

Ba-4

SiO2 Na2O CaO P2O5 BaO

46.1 24.3 26.9 2.6 0.0

45.4 24.5 27.1 2.6 0.4

44.6 24.6 27.3 2.6 0.8

43.9 24.8 27.4 2.7 1.2

43.1 25.0 27.6 2.7 1.6

2.3.5. In vitro bioactivity study of bioactive glass The bioactivity of the prepared bioactive glass samples were examined through in vitro test. The test was performed by immersing 1 g of each samples in 10 mL of SBF solution contained in a small polyurethane container and incubated at 37 °C in a static condition for time periods of 1, 3, 7, 14 and 30 days. After soaking, the samples were filtered, rinsed with double distilled water and dried in an electric air oven at 100 °C for 1 h. The formation of hydroxy carbonate apatite layer (HCA) on the surface of the bioactive glass samples were determined using FTIR, XRD and SEM techniques. 2.3.6. pH behavior The stages of formation of hydroxyl carbonate apatite layer on the surface of the samples were checked by pH behavior of the SBF solution containing bioactive glasses. Each 1 g of powdered bioactive glass samples was soaked in 10 mL of SBF solution at 37 °C for 7 days and the pH of the leached solution was measured continuously using Universal Biomicroprocessor pH meter at room temperature. The pH values were recorded timely after definite intervals of time. The instrument was calibrated each time with standard buffer solutions of pH 4.0 and 7.0. 2.3.7. Surface morphology of bioactive glass sample by SEM The surface morphology of samples was analyzed before and after SBF treatment using a scanning electron microscope (SEM) (Inspect S50, FEI). The bioactive glass samples were cut into required dimensions and immersed into SBF for 14 days at 37 °C. Further, the samples were removed, washed with double distilled water and dried at 100 °C for 1 h and they were coated with gold by sputter coating instrument before their examination with SEM.

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559

551

2.3.8. Toxicity on blood (hemolysis assay) Hemolysis assay was performed on the bioactive glass samples (Ba0, Ba-1, Ba-2, Ba-3 and Ba-4) with acid citrate dextrose (ACD) human blood using a spectrophotometer. The ACD solution was prepared by mixing 0.544 g of anhydrous citric acid, 1.65 g of trisodium citrate dehydrate and 1.84 g of dextrose monohydrate to 75 mL of double distilled water. The ACD blood (5 mL) solution was prepared by adding 4.5 mL of fresh human blood to 0.5 mL of ACD. Equal weights of bioactive glass samples were taken in different test tubes. 10 mL of phosphate buffer solution (PBS) and 10 mL of water was added to it. Control experiment contained only 10 mL PBS solution. All these test tubes were kept in desiccators for 30 min at 37 °C and 0.2 mL of ACD blood was added to each test tube, mixed gently and the test tubes were kept for 1 h in incubator at 37 °C. The test tubes were centrifuged for 15 min. The supernatant is carefully taken from test tube without disturbing the precipitate. Optical density (OD) of each 1 mL supernatant, positive and negative controls was determined at 545 nm using a Shimadzu (UV-1700), UV–vis spectrophotometer. The percentage of hemolysis was calculated as follows by Eq. (1): Hemolysis ð%Þ OD of the test sample−OD of the negative control  100: ¼ OD of the positive sample−OD of the negative control

ð1Þ

2.3.9. Density and flexural strength of bioactive glasses The densities of annealed bioactive glass samples were determined by Archimedes' principle with water as the immersion liquid. The flexural strength was measured using three point bending test. The bioactive glass samples were prepared into rectangular shape and they were ground and polished for required dimensions (60 mm length × 10 mm width × 5 mm thickness) using grinding and polishing machine. The test was performed at room temperature using a Universal Testing Machine (AGS 10kND, SHIMADZU, Japan) having cross-head speed at 0.5 mm/min and flexural strength was calculated using Eq. (2). Three samples were tested for each composition and the results were averaged.

Flexural strength ¼

3PL 2BD2

Fig. 1. DTA curve of bioactive glass samples (Ba-0, Ba-1, Ba-2, Ba-3 and Ba-4).

ð2Þ

where, P is the breaking load (Newton), L is the length (mm), B is the breadth (mm) and D is the thickness (mm) of the sample. 3. Results and discussion 3.1. Differential thermal analysis curves of bioactive glasses The thermal behavior curves of the bioactive glasses are shown in Fig. 1 and the results dictate that the incorporation of barium in the base bioactive glass reduced both the nucleation temperature from 614 to 542 °C and the crystallization temperature from 760 to 680 °C. Further, it could be observed that the higher amount of modifiers in the composition has facilitated in lowering Tg point and the viscosity of the glass melt [25]. On substitution of BaO for SiO2 content in the composition causes the shift of exothermic peaks to lower temperatures as compared to that of the base glass. Therefore, a lower energy is required to promote crystallization in the glass. This may be attributed due to the presence of larger Ba2+ ions in the system which increases interference in glass network. The modifiers occupied the interstitial positions in the glass structure and thereby weaken oxygen bond strength [26]. During the process of thermal treatment, the high concentration of barium in the glassy system might have allowed an early nucleation by the nucleating agents like P2O5 in the glass composition.

Fig. 2. XRD pattern of bioactive glass samples sintered at different temperatures as given in Table 2 (Ba-0, Ba-1, Ba-2, Ba-3 and Ba-4).

552

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559

3.2. Phase analysis of heat treated bioactive glasses Fig. 2 shows the XRD patterns of bioactive glass samples after controlled thermal treatment as given in Table 2. All the bioactive glass samples show specific crystalline phases with small inconsistency in the intensity of the peaks depending on the composition and the heattreatment schedule. The major diffraction sharp peak identified at 2θ = 33.56° (hkl = 204) corresponds to Na2Ca2Si3O9 (sodium calcium silicate) and the intensities of the diffraction peaks were matched with the standard PDF#: 22-1455. The earlier studies on sintered 45S5 bioactive glass showed the same crystalline phase [27]. It is well known that the Na2O–CaO–P2O5–SiO2 system has the tendency to form the sodium–calcium-silicate phase as the main phase which was also confirmed earlier by earlier workers [1,28]. The secondary peak observed after barium substitution in the samples Ba-2, Ba-3 and Ba-4 at about 21.1° corresponding to Ca (PO3)2 (calcium phosphate) PDF#: 50-0584. The intensity of the secondary peak increased with an increase in the concentration of barium oxide. It is well known that the addition of a few percent of P2O5 to silicate glass compositions promotes the volume nucleation and formation of glass–ceramic as indicated by Mastelaro et al. [29]. It can be understood from XRD spectra that on addition of barium up to 1.6 mol% in the base bioactive glass system has not affected on the main crystalline phase within the specified heat treatment conditions. 3.3. Structural analysis of bioactive glasses by FTIR spectrometry Fig. 3 shows the Fourier transform infrared transmittance spectra of the bioactive glass samples recorded in the frequency range of 400– 4000 cm−1 on the FTIR spectrometer. The FTIR spectra were clearly presented in Figs. 3, 5–9 and the vibrational bands have been marked by the vertical dotted lines which were prepared similar to transmission mode spectra as given by earlier workers [40]. The parent bioactive glass (Ba0) revealed the sharp bands successively at about 450, 740, 1025, 1420, 2350 and 3420 cm−1 indicating various functional groups. The transmittance spectral bands of bioactive glasses have confirmed the main characteristic of SiO4 tetrahedral silicate network and it is attributed due to the presence of SiO2 as a major constituent [30]. The resultant FTIR spectra at around 450 cm−1 is associated with a Si–O–Si symmetric bending mode and the band at 740 cm−1 corresponded to Si–O–Si symmetric stretching of non-bridging oxygen atoms between SiO4 tetrahedral. The major broad band at about 1025 cm−1 can be attributed due to Si–O–Si asymmetric stretching. The minor sharp peak at 1420 cm−1 is attributed due to stretching mode of C–O vibration of CO3 groups. The infrared frequencies and related functional groups were also reported by ElBatal et al. [31] in their bioglass ceramic systems. The FTIR spectral bands of Ba-1, Ba-2, Ba-3 and Ba-4 samples have clearly shown the similar behavior like Ba-0 with small change in the band intensities (Fig. 3). The barium substituted bioactive glasses up to 1.6 mol% did not show any major changes in the FTIR transmission spectral characteristics.

Fig. 3. FTIR transmittance spectra of bioactive glass samples (Ba-0, Ba-1, Ba-2, Ba-3 and Ba4).

were recorded in all the samples within first 3 days as compared to the initial pH of the solution (pH = 7.4). The pH values on third day were measured as pH = 9.38, 9.36, 9.44, 10.29 and 9.92 for the samples Ba-0, Ba-1, Ba-2, Ba-3 and Ba-4 respectively under physiological conditions. The increase in pH can be explained as due to the fast release of Na+ and Ca2+ ions through the exchange with H+ or H3O+ ions into the solution. The H+ ions being replaced with cations thereby resulted in an increase in hydroxyl concentration of the solution which led to attack in silicate glass network and formation of silanols. The dissolution rate as well as pH increment decreased after 4 days due to the decrease of Na+ and Ca2+ ionic concentration from sample surface. The reason for this decrease in the pH can be considered due to the precipitation of Ca2+ ions from the solution to form calcium phosphates and carbonates, Greenspan and others showed the same behavior and changes in

3.4. pH assessment The pH behavior of the SBF solution after immersion of the bioactive glass samples can be clearly seen from Fig. 4. The maximum pH values Table 2 Heat treatment temperatures used for nucleation and crystal growth of bioactive glasses. Sample No

Nucleation temperature (°C)

Soaking time (hrs)

Crystallization temperature (°C)

Soaking time (hrs)

Ba-0 Ba-1 Ba-2 Ba-3 Ba-4

614 591 586 558 542

4 4 4 4 4

760 726 717 695 681

3 3 3 3 3

Fig. 4. pH behavior after immersion of the bioactive glass samples in SBF.

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559

553

pH after in vitro dissolution of the samples for various time periods [32–34]. Moreover, the sample numbers Ba-3 and Ba-4 with higher barium content were found to posses the highest rate of dissolution and hence the maximum pH values were recorded as compared with base glass sample No. Ba-0. The incorporation of barium oxide into 45S5 bioactive glass resulted in an increase in the pH of SBF. Their high degradation rate leads to a higher pH value and favored an early development of hydroxy carbonate apatite layer on the sample surface with more crystallinity as evident from SEM image (Fig. 11). The formation of apatite in SBF is strongly pH dependent and the most interesting aspect is that the cross linking of the collagen chains and the subsequent precipitation of hydroxy apatite on bone formation are pH dependent. Hence it requires a high pH at the bone formation site. The barium substituted bioactive glasses have been proposed as potential materials for bone tissue regeneration. 3.5. In vitro bioactivity of bioactive glasses by FTIR spectrometry Figs. 5–9 show the FTIR transmittance spectral bands of the bioactive glasses before and after immersion in SBF for different time periods such as 1, 3, 7, 14, and 30 days. In general, it is well known that a decrease in the intensity of transmittance bands indicates an increase in molecular concentration of species formed at surface of the bioactive glass treated with SBF solution with an increase in soaking time. The present results obtained by FTIR spectrometry were also in good agreement with the earlier studies made by previous workers [30,34,35]. Fig. 5 shows the infrared spectral bands of Ba-0 sample before and after immersion in SBF. The new bands were found to appear after 1 day immersion in SBF at wavenumbers 578 and 661 cm−1 which correspond to (phosphate) P–O bending. The bands corresponding to the frequencies of 883 and 1602 cm−1 are associated with (carbonate) C– O stretching mode and a minor peak at around 2908 cm−1 as well as

Fig. 5. FTIR transmittance spectra of the bioactive glass sample Ba-0 before and after immersion in SBF for different time periods 1, 3, 7, 14, and 30 days.

Fig. 6. FTIR transmittance spectra of the bioactive glass sample Ba-1 before and after immersion in SBF for different time periods 1, 3, 7, 14, and 30 days.

broad band at about 3380 cm−1 can be assigned due to the presence of (hydroxyl) O\H groups on the surface. The prolonged period of the sample in SBF shows the similar behavior with small decrease in the intensities of the bands which resulted favorably due to the formation of hydroxyl carbonate apatite (HCA) layer.

Fig. 7. FTIR transmittance spectra of the bioactive glass sample Ba-2 before and after immersion in SBF for different time periods 1, 3, 7, 14, and 30 days.

554

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559

Fig. 8. FTIR transmittance spectra of the bioactive glass sample Ba-3 before and after immersion in SBF for different time periods 1, 3, 7, 14, and 30 days.

Fig. 6 shows the IR spectral bands of Ba-1 sample before and after treatment with SBF. The new bands were revealed after 1 day treatment with SBF at 586 and 659 cm−1 which are attributed due to (phosphate) P–O bending. The bands at 879 and 1618 cm− 1 are assigned due to

(carbonate) C–O stretching mode of vibration and a minor peak at around 2914 cm−1 as well as broad band at about 3414 cm− 1 was due to the presence of (hydroxyl) O–H groups on the surface. The prolonged period in SBF shows the similar behavior with small decrease in the intensities of the bands which resulted due to the formation of HCA layer on the sample surface. Fig. 7 shows the bands of Ba-2 sample before and after immersion in SBF. The new bands appeared after immersion in SBF for 1 day at 551 and 632 cm−1 which are related to (phosphate) P–O bending. The bands at 872 and 1573 cm−1 correspond to (carbonate) C–O stretching mode and a minor peak at around 2859 cm−1 as well as broad band at about 3359 cm−1 are assigned as usual due to formation of (hydroxyl) O–H groups on the surface of the sample. The prolonged period of the treatment of sample in SBF shows the like behavior with small decrease in the intensities of the bands which resulted in hydroxyl carbonated apatite layer formation on the sample. Fig. 8 shows the bands of Ba-3 sample before and after immersion in SBF. The new bands recorded after immersion in SBF for 1 day at 567 and 640 cm−1 and they are associated with (phosphate) P–O bending. The bands at 860 and 1596 cm−1 correspond to (carbonate) C–O stretching mode and a minor peak at around 2902 cm−1 as well as broad band at about 3402 cm−1 are attributed due to the presence of (hydroxyl) O–H groups on the surface of the bioactive glass sample. The prolonged period of the sample in SBF shows the similar behavior with small decrease in the intensities of the bands and this dictated for the hydroxyl carbonated apatite layer formation. Fig. 9 shows the bands of Ba-4 sample before and after it was soaked in SBF. The new bands were recorded after 1 day soaking in SBF at 535 and 616 cm− 1 which are associated with (phosphate) P–O bending. The FTIR bands obtained at 844 and 1562 cm−1 correspond to (carbonate) C–O stretching mode of vibration and a minor peak at around 2880 cm−1 as well as broad the band at about 3350 cm−1 was attributed due to (hydroxyl) O–H groups on the surface of the sample. The prolonged period of the sample in SBF shows the similar behavior with small decrease in the intensities of the bands which favored for the formation of hydroxyl carbonated apatite layer. It may be noted from FTIR spectra as given in Figs. 5–9 that the barium substitution in base bioactive glass at the cost of silica did not affect in apatite formation. 3.6. In vitro bioactivity of bioactive glasses by X-ray diffractometry

Fig. 9. FTIR transmittance spectra of the bioactive glass sample Ba-4 before and after immersion in SBF for different time periods 1, 3, 7, 14, and 30 days.

Fig. 10 shows the XRD pattern of the bioactive glass samples (Ba-0, Ba-1, Ba-2, Ba-3 and Ba-4) before and after immersion in SBF for 14 days under physiological condition. It is evident from the XRD data that the prepared bioactive glasses had shown homogeneity and amorphous state which is an indicative of the internal disorder and glassy nature before immersion in SBF. Notably, the amorphous scattering of a broad hump at ≈ 32° found in the base bioactive glass and the broad peak became more intense as the concentration of BaO increases in the glasses. This difference might be due to the effect of barium contracting the silicate network. Fig. 10 also contains the XRD pattern of hydroxyapatite (HA) powder [36] for comparison of crystalline HA layer formation on the surface of the SBF treated bioactive glass samples. In light of this, the XRD patterns of the SBF treated samples have clearly shown the formation of crystalline HA on their surfaces after immersion in SBF for 14 days. The XRD peak located at 2θ at around 31.8° corresponds to (211) crystalline phase regarded as hydroxyapatite [Ca10(PO4)6(OH)2] and the diffraction peaks were matched with the standard PDF#: 74-0565 [37,38]. However, all the samples after immersion in SBF showed the calcite phase at 2θ at around 29.46° (PDF No. 85-1108) in addition to HA, whereas the Ba-0 sample has shown the calcite as the major phase. In the Ba-0 bioactive glass sample formation of calcite phase has hindered the formation of HA layer on the surface of the bioactive glass. Tulyaganov et al. [39] had also observed that a decreasing intensity of

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559

Fig. 10. XRD pattern of bioactive glass samples Ba-0, Ba-1, Ba-2, Ba-3 and Ba-4 before and after SBF treatment for 14 days and hydroxyapatite.

555

556

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559

HA peaks was accompanied by the formation of relatively high intensity calcium carbonate (calcite) peaks, which suggested that the early deposition of HA layer was gradually suppressed by freshly precipitated calcite in 45S5 Bioglass®. Kansal et al. [40] had also shown the calcite formation in all their SBF treated glasses including 45S5 Bioglass® which masked the precipitation of crystalline HA layer on the surface of the samples. The present trends are well supported by earlier results [39,40]. Further, it is mentioned herewith that calcite is a biodegradable material which exhibits strong bonding to the bone. It is interesting to note as evident from XRD patterns of bioactive glasses that an increase in barium content in the glass samples has decreased the tendency of calcite formation and simultaneously resulted in the formation of crystalline HA layer on the surface of the bioactive glasses (Fig. 10). The investigation was carried out here for the qualitative characterization of HCA phase identification in the barium substituted bioactive glasses. The diffraction pattern of all the tested samples Ba-0, Ba-1, Ba2, Ba-3 and Ba-4 demonstrated the same crystalline phases as hydroxyapatite and calcite. The intensity of the diffraction pattern is the only difference which was observed and this difference is attributed due to the different amounts of phases formed on each sample. 3.7. Surface morphology of bioactive glass samples by SEM Figs. 11 and 12 show surface morphology of all the bioactive glass samples (Ba-1, Ba-2, Ba-3 and Ba-4) by their SEM images before and after the immersion in the simulated body fluid for 14 days at 37 °C. The changes in the surfaces of the glass samples after immersion in SBF can be easily seen from the SEM images (Fig. 12A–D) with respect to the images of samples before SBF treatment as presented in Fig. 11A–D. The micrographs as given in Fig. 12 exhibit the layer of polycrystalline ball-like particles formed on surface of the samples after SBF treatment, which encourages the growth of the same crystallinity [41]. The developed crystals on the surface of the bioactive glasses is assumed to be hydroxy carbonate apatite, since the HCA layer is round in shape and varies in intensity from one sample to another [31]. It is interesting to note that with the increase in barium concentration the extent of HCA crystals was found to increase progressively as evident from the SEM

pictures of the samples presented in Fig. 12A–D. It is worth to note that the barium substituted bioactive glasses can also produce HCA like structure on their surfaces in SBF in vitro. The formation of apatite layer is also supported by the results obtained from XRD and FTIR spectrometry after immersion of the bioactive glass samples in SBF. 3.8. Hemolysis assay Hemolysis is the breakage of the red blood cell (RBC) membrane causing the release of the hemoglobin and other internal components into the surrounding fluids. As mentioned earlier, the hemolysis phenomenon of blood is directly related to bio-incompatibility. Hemolysis takes place when the red blood cells come in contact with materials and it is important to check this implant material before use. It has been well described that the permissible limit of hemolysis for biomedical implants should be less in all the cases [42]. The percentage of hemolysis of ACD blood was calculated on the heparinized film of bioactive glass samples and found as Ba-0 = 8.7%, Ba-1 = 6.5%, Ba2 = 4.2%, Ba-3 = 3.1% and Ba-4 = 5.4% with an increase in barium concentration as shown in Fig. 13. The base bioactive glass (Ba-0) is reported herewith as biocompatible with 8.7% hemolysis in it. Interestingly, all the barium contained bioactive glasses showed even lower hemolysis which is an indication for their better biocompatibility. However, the glass sample No. Ba-3 having 3.1% hemolysis was found to exhibit better biocompatibility as compared to other bioactive glasses. It may thus be concluded that biocompatibility was found to be improved in all the bioactive glasses due to the presence of barium oxide content and the sample with 1.2 mol% BaO has a higher potential as a biomaterials. 3.9. Density of bioactive glasses Fig. 14 shows the variation of densities of bioactive glasses with BaO/ SiO2 ratio in the glass samples (Ba-0, Ba-1, Ba-2, Ba-3 and Ba-4). It is observed that the densities of the samples were found to increase with increasing barium content in the glass from 2.70 to 2.81 g/cm3. It may be due to partial replacement of SiO2 with BaO which is attributed due to

Fig. 11. Scanning electron micrographs of bioactive glasses Ba-1, Ba-2 Ba-3 and Ba-4 before immersion in SBF (A, B, C and D) respectively.

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559

557

Fig. 12. Scanning electron micrographs of bioactive glasses Ba-1, Ba-2 Ba-3 and Ba-4 after immersion in SBF for 14 days (A, B, C and D) respectively.

the replacement of a smaller ion (Si4+) with a bigger (Ba2+) ion in the glass. In other words the lighter molar mass of SiO2 (60.08 g/mol) has been replaced by the heavier BaO (153.3 g/mol) in the bioactive glass samples. 3.10. Flexural strength of bioactive glasses Fig. 15 shows the flexural strength of bioactive glasses determined to investigate the effect of barium substitution on the mechanical properties by change in BaO/SiO2 ratio in the bioactive glass sample nos. Ba-0, Ba-1, Ba-2, Ba-3 and Ba-4. The flexural strength of the samples was found to increase with increasing barium concentration in the glass from 47.38 to 65.39 MPa. On substitution of barium for silica in the

Fig. 13. Change in hemolysis with BaO/SiO2 ratio in bioactive glass samples.

composition acts as sintering additive which facilitated for early melting with more homogeneity in the glass, this would make the glass more rigid. The another main reason for the superior fracture strength can be explained by the Ba2 + ion which possesses a larger ionic radius than Si4 + ion and occupies more space to make the structure more dense in the glass network [43]. This might have resulted in higher mechanical properties. It can be clearly seen that the result of Ba-4 sample possessed highest mechanical strength as compared to Ba-0 sample because of the presence of barium oxide only in the glass composition at the cost of SiO2. The results obtained are comparable and within the range of human cortical bone [44].

Fig. 14. Change in density with BaO/SiO2 ratio in bioactive glass samples.

558

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559

References

Fig. 15. Change in flexural strength with BaO/SiO2 ratio in bioactive glass samples.

4. Summary The main spirit of the research work is to enhance the properties of parent bioactive glass (45S5) without major alteration of the base glass composition. The bioactive glass system SiO2–Na2O–CaO–P2O5 with small substitution of barium oxide at the cost of silica has been successfully prepared and the salient features of the work are as follows: 1. The present bioactive glasses with barium oxide substitution show the decrease in glass nucleation and crystallization temperatures. 2. Thermally treated base bioactive glass and barium substituted glasses show the main crystalline phases as Na2Ca2Si3O9. 3. The XRD analysis of the bioactive glass before immersion in SBF dictated the amorphous nature of the glass. 4. The FTIR spectrometry confirmed the presence of SiO4 tetrahedral in the silicate glass network. 5. FTIR transmission spectra, pH behavior, XRD and SEM images indicated the formation of hydroxyl carbonate apatite layer on the surface of the barium containing bioactive glasses after immersion in simulated body fluid. 6. The density and flexural strength were enhanced with an increase in barium content in the base bioactive glass. 7. The hemolysis of the bioactive glasses had been improved by substitution of barium oxide, whereas the sample with 1.2 mol% BaO has a higher potential. It can be concluded from the results that the barium oxide can easily be substituted in base bioactive glass to the extent of experimental work. All the barium substituted bioactive glasses had shown improved properties, but the sample with 1.2 mol% of barium oxide demonstrated better result on the hemolysis. The prepared bioactive glasses are promising for bone substitutes in biomedical applications.

Acknowledgments The authors, gratefully acknowledge the Head of the Department of Ceramic Engineering, IIT (BHU) and the honorable Director, Indian Institute of Technology (BHU) Varanasi, India for providing necessary facilities for the present research work. The author, Sampath Kumar Arepalli is also very much grateful to the University Grants Commission, New Delhi, India (F.14-2(SC)/2010(SA-III)) for providing the Rajiv Gandhi National Fellowship for the research work.

[1] L.L. Hench, Bioceramics, J. Am. Ceram. Soc. 81 (7) (1998) 1705–1728. [2] L.L. Hench, Bioceramics: from concept to clinic, J. Am. Ceram. Soc. 74 (7) (1991) 1487–1510. [3] P. Sepulveda, J.R. Jones, L.L. Hench, Characterization of melt-derived 45S5 and sol– gel-derived 58S bioactive glasses, 58 [6]2001. 734–740. [4] Clerk, L.L. Hench, The influence of surface chemistry on implant interface, J. Biomed. Mater. Res. 10 (2) (1976) 161–174. [5] T. Kasuga, Y. Abe, Calcium phosphate invert glasses with soda and titania, J. NonCryst. Solids 243 (1) (1999) 70–74. [6] P. Saravanapavan, J.R. Jones, R.S. Pryce, L.L. Hench, Bioactivity of gel–glass powders in the CaO–SiO2 system: a comparison with ternary (CaO–P2O5–SiO2) and quaternary glasses (SiO2–CaO–P2O5–Na2O), J. Biomed. Mater. Res. 66 (1) (2003) 110–119. [7] J. Moura, L.N. Teixeira, C. Ravagnani, O. Peitl, E.D. Zanotto, M.M. Beloti, et al., In vitro osteogenesis on a highly bioactive glass ceramic (biosilicate), J. Biomed. Mater. Res. 82 (3) (2007) 545–557. [8] N. Lotfibakhshaiesh, D.S. Brauer, R.G. Hill, Bioactive glass engineered coatings for Ti6Al4V alloys: influence of strontium substitution for calcium on sintering behaviour, J. Non-Cryst. Solids 44 (2010) 2583–2590. [9] L.L. Hench, Anderson, Bioactive glass coatings, in: L.L. Hench, J. Wilson (Eds.), An Introduction to Bioceramics, World Scientific, Singapore, 1993, pp. 239–259. [10] F.H. ElBatal, A. ElKheshen, Preparation and characterization of some substituted bioglasses and their ceramic derivatives from the system SiO2–Na2O–CaO–P2O5 and effect of gamma irradiation, Mater. Chem. Phys. 110 (2–3) (2008) 352–362. [11] S. Kumar, P. Vinatier, A. Levasseur, K.J. Rao, Investigations of structure and transport in lithium and silver borophosphate glasses, J. Solid State Chem. 177 (4–5) (2004) 1723–1737. [12] A. Oki, B. Parveen, S. Hossain, S. Adeniji, H. Donahue, Preparation and in vitro bioactivity of zinc containing sol–gel-derived bioglass materials, J. Biomed. Mater. Res. A 69 (2) (2004) 216–221. [13] A. Saboori, M. Sheikhi, F. Moztarzadeh, M. Rabiee, S. Hesaraki, M. Tahriri, N. Nezafati, Sol–gel preparation, characterisation and in vitro bioactivity of Mg containing bioactive glass, Adv. Appl. Ceram. 108 (3) (2009) 155–161. [14] M.D. O'Donnell, P.L. Candarlioglu, C.a. Miller, E. Gentleman, M.M. Stevens, Materials characterisation and cytotoxic assessment of strontium-substituted bioactive glasses for bone regeneration, J. Mater. Chem. 20 (40) (2010) 8934–8941. [15] M.D. O'Donnell, Y. Fredholm, A. de Rouffignac, R.G. Hill, Structural analysis of a series of strontium-substituted apatites, Acta Biomater. 4 (5) (2008) 1455–1464. [16] M.D. O'Donnell, R.G. Hill, Influence of strontium and the importance of glass chemistry and structure when designing bioactive glasses for bone regeneration, Acta Biomater. 6 (7) (2010) 2382–2385. [17] S. Pors Nielsen, The biological role of strontium, Bone 35 (3) (2004) 583–588. [18] M. Mathew, S. Takagi, Structures of biological minerals in dental research, J. Res. Natl. Inst. Stand. Technol. 106 (6) (2001) 1035–1044. [19] Wellington Moore, Comparative metabolism of barium-133 and calcium-45 by embryonic bone grown in vitro embryonic, Radiat. Res. 21 (3) (2014) 376–382. [20] C. Austin, T.M. Smith, A. Bradman, K. Hinde, R. Joannes-Boyau, D. Bishop, D.J. Hare, P. Doble, et al., Barium distributions in teeth reveal early-life dietary transitions in primates, Nature 498 (7453) (2013) 216–219. [21] G. Kaur, P. Sharma, V. Kumar, K. Singh, Assessment of in vitro bioactivity of SiO2– BaO–ZnO–B2O3–Al2O3 glasses: an optico-analytical approach, Mater. Sci. Eng. C 32 (7) (2012) 1941–1947. [22] W. Leenakul, P. Kantha, N. Pisitpipathsin, G. Rujijanagul, S. Eitssayeam, K. Pengpat, Structural and magnetic properties of SiO2–CaO–Na2O–P2O5 containing BaO–Fe2O3 glass–ceramics, J. Magn. Magn. Mater. 325 (2013) 102–106. [23] A.I. Vogel, A Text-book of Quantitative Inorganic Analysis, Third edition The English Language Book Society, London, 1969. 472–473. [24] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27 (15) (2006) 2907–2915. [25] P. Sooksaen, S. Suttiruengwong, K. Oniem, K. Ngamlamiad, J. Atireklapwarodom, Fabrication of porous bioactive glass–ceramics via decomposition of natural fibres, J. Met. Mater. Miner. 18 (2) (2008) 85–91. [26] S.M. Salman, S.N. Salama, H.a. Abo-Mosallam, The role of strontium and potassium on crystallization and bioactivity of Na2O–CaO–P2O5–SiO2 glasses, Ceram. Int. 38 (1) (2012) 55–63. [27] O. Bretcanu, X. Chatzistavrou, K. Paraskevopoulos, R. Conradt, I. Thompson, A.R. Boccaccini, Sintering and crystallisation of 45S5 Bioglass® powder, J. Eur. Ceram. Soc. 29 (16) (2009) 3299–3306. [28] O. Peitl Filho, G.P. LaTorre, L.L. Hench, Effect of crystallization on apatite-layer formation of bioactive glass 45S5, J. Biomed. Mater. Res. 30 (4) (1996) 509–514. [29] V.R. Mastelaro, E.D. Zanotto, N. Lequeux, R. Cortes, Relationship between shortrange order and ease of nucleation in Na2Ca2Si3O9, CaSiO3 and PbSiO3 glasses, J. Non-Cryst. Solids 262 (22) (2000) 191–199. [30] C.Y. Kim, A.E. Clark, L.L. Hench, Early stages of calcium-phosphate layer formation in bioglasses, J. Non-Cryst. Solids 113 (2) (1989) 195–202. [31] H. ElBatal, M. Azooz, E.M. Khalil, A. Soltan Monem, Y. Hamdy, Characterization of some bioglass–ceramics, Mater. Chem. Phys. 80 (3) (2003) 599–609. [32] Marta Giulia Cerruti, Characterization of Bioactive Glasses. Effect of the Immersion in Solutions That Simulate Body FluidsPhD thesis University of Turin, Torino, Italy, 2004. [33] M. Cerruti, D. Greenspan, K. Powers, Effect of pH and ionic strength on the reactivity of Bioglass 45S5, Biomaterials 26 (14) (2005) 1665–1674. [34] M.R. Filguei ras, G. LaTorre, L.L. Hench, Solution effects on the surface reactions of three bioactive glass compositions, J. Biomed. Mater. Res. 27 (12) (1993) 1485–1493.

S.K. Arepalli et al. / Materials Science and Engineering C 49 (2015) 549–559 [35] I. Rehman, M. Karsh, L.L. Hench, W. Bonfield, Analysis of apatite layers on glass–ceramic particulate using FTIR and FT-Raman spectroscopy, J. Biomed. Mater. Res. 50 (2) (2000) 97–100. [36] M.H. Fathi, A. Hanifi, Evaluation and characterization of nanostructure hydroxyapatite powder prepared by simple sol–gel method, Mater. Lett. 61 (2007) 3978–3983. [37] Qizhi Z. Chen, Ian D. Thompson, Aldo R. Boccaccini, 45S5 bioglasss-derived glass–ceramic scaffolds for bone tissue engineering, Biomaterials 27 (11) (2006) 2414–2425. [38] Q.-Z. Chen, G. a Thouas, Fabrication and characterization of sol–gel derived 45S5 Bioglass®–ceramic scaffolds, Acta Biomater. 7 (10) (2011) 3616–3626. [39] D.U. Tulyaganov, M.E. Makhkamov, A. Urazbaev, A. Goel, J.M.F. Ferreira, Synthesis, processing and characterization of a bioactive glass composition for bone regeneration, Ceram. Int. 39 (3) (2013) 2519–2526.

559

[40] I. Kansal, A. Goel, D.U. Tulyaganov, L.F. Santos, J.M.F. Ferreira, Structure, surface reactivity and physico-chemical degradation of fluoride containing phospho-silicate glasses, J. Mater. Chem. 21 (22) (2011) 8074–8084. [41] T. Kokubo, H.-M. Kim, M. Kawashita, Novel bioactive materials with different mechanical properties, Biomaterials 24 (13) (2003) 2161–2175. [42] D. Shim, D.S. Wechsler, T.R. Lloyd, R.H. Beekman, Hemolysis following coil embolization of a patent ductus arteriosus, Catheter. Cardiovasc. Diagn. 39 (3) (1996) 287–290. [43] S.J. Watts, R.G. Hill, M.D. O'Donnell, R.V. Law, Influence of magnesia on the structure and properties of bioactive glasses, J. Non-Cryst. Solids 356 (9–10) (2010) 517–524. [44] K. Rezwan, Q.Z. Chen, J.J. Blaker, A.R. Boccaccini, Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering, Biomaterials 27 (18) (2006) 3413–3431.