Journal of Non-Crystalline Solids 406 (2014) 88–94

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Journal of Non-Crystalline Solids

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Evaluation of zinc and magnesium doped 45S5 mesoporous bioactiveglass system for the growth of hydroxyl apatite layer

Vikas Anand, K.J. Singh ⁎, Kulwinder KaurDepartment of Physics, Guru Nanak Dev University, Amritsar 143005, India

⁎ Corresponding author.E-mail address: kanwarjitsingh@yahoo.com (K.J. Singh

http://dx.doi.org/10.1016/j.jnoncrysol.2014.09.0500022-3093/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 July 2014Received in revised form 21 September 2014Accepted 28 September 2014Available online xxxx

Keywords:Mesoporous bioactive glass;Tris SBF;Apatite layer

Bioactive glass samples of xZnO(22.4 − x)Na2O·46.1SiO2·26.9CaO.2·6P2O5·2MgO system have been preparedby using the sol gel technique. Investigations of structural and bioactive properties of these samples have beenundertaken by using X-ray diffraction, field emission scanning electron microscopy, Raman and energy-dispersive X-ray spectroscopy, Brunauer, Emmett and Teller technique, thermogravimetry, differential thermalanalysis and derivative thermogravimetrymethods and pH studies. X-ray diffraction spectra of prepared samplesindicate the formation of hydroxyl apatite layer after 7 and 14 days during in vitro analysis. Observed peaks ofRaman spectra confirm the growth of hydroxyl apatite layer during in vitro analysis. The formation of apatitephase is responsible for change in themorphology of samples which has been studied by field emission scanningelectronmicroscopy. Ca/P ratio and ion dissolution rate have been evaluated from energy dispersive X-ray spec-troscopy results. The observed Ca/P ratio further confirms the growth of apatite layer with time during in vitroanalysis. The effect of the addition of zinc on surface area and mesoporous nature of all the samples has beeninvestigated by the Brunauer, Emmett and Teller technique. All glass samples have been found to be thermallystable from thermogravimetry, differential thermal analysis and derivative thermogravimetry techniques. pHstudies indicate the non-acidic nature of all the prepared glass samples. Drug release properties of the glass sam-ples have been investigated by using gentamycin as an antibiotic. Prepared samples have shown excellent drugrelease properties which can be related to the porous nature of the samples. Due to slow dissolution rate, gooddrug delivery properties and formation of apatite phase within 7 to 14 days, our glass samples can be promisingcandidates for bone regeneration applications.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Fracture in the bonemay cause a serious problemwhen its impact ison a large scale. Sometimes, fracture can lead to the loss of the bone andthere can be many other incidents like cancer therapies, accidents, andage factors (old age) responsible for the elimination of the bone fromthe human body. In a survey, it has been estimated that in womenand men (specially over the age of 50 years), the lifetime risk of devel-oping a fracture is 40% and 13% respectively [1]. Replacement of dam-aged bone is a major problem in orthopedics surgery and in order tomeet this purpose, bioinert metallic implants are used. These implantsact like a foreign particle for host body and sometimes, they may getrejection. Moreover, metallic implantsmay not stimulate the osteoblas-tic and osteoconductive phenomena in the host body. Due to metallicnature, these implants are temperature sensitive and start to cause thepain on surrounding tissues under hot and cold conditions. To overcomeall these problems, a special kind of glass has been developedwhich hasthe ability to bondwith bone and soft tissues [2]. This glass is popularly

).

known as ‘bioactive glass’. Bioactive glasses are one of the categories ofbio-materials and they can be potential materials for applications inorthopedics and surgery. Bioactive glasses are bioresorbable and theyprovide friendly environment for the growth of bone (apatite layer)and soft tissues. Therefore, these can be good alternatives of metallicimplants. First, bio-active glass was prepared by using the conventionalmelt-quenching technique by Larry Hench. This glass was termed as45S5 Bioglass®. Lower Ca/P ratios do not bond to bone [3]. Bioactiveglasses can also be prepared by the sol–gel technique.

Recently, borate glasses have also been investigated for biomedical ap-plications by removing silica from the bioactive glass compositions [4,5].Silica free glass samples with the composition (45B2O3, 24.5Na2O,24.5CaO, 6P2O5) were prepared with additional 2% of fluoride cationswhich were added separately. It has been observed that dissolution rateis better for LiF as compared to other fluorides (ZnF2, CaF2 and NaF) [4].Oxide glass samples of the B2O3, Na2O, CaO, K2O and LiO constituentshave also been prepared in the laboratory [5]. It has been noticed thatdissolution rate proceeds rapidly than as observed in silicate Hench'sbioglass (45S5). It has been inferred that the corrosion mechanisms ofborate glasses in aqueous environments, generally undergo hydration,hydrolysis, and ion exchange reactions.

Table 2Ion concentration of Tris SBF and Human plasma.

89V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

In order to check the bioactivity of the samples, the following is theconventional procedure. Samples are soaked in the TRIS Simulated BodyFluid (SBF). SBF has concentration of ions equal to human blood plasma.The creation of apatite layer on the samples (from few hours up to fewdays) can confirm the bioactive nature of prepared samples. It has beenfound that formation rate of apatite layer improves at higher concentra-tion of calcium ions when the phosphate ions remain in the range of 4–6 mol% in the composition. When the composition of phosphate ions isincreased from 6mol% onwards, it shows a negative effect in the rate offormation of apatite layer.

Applications of bioactive glass in human body depend upon its bio-activity, structural and dissolution properties. Bioactivity of glass pro-vides the information regarding the growth of apatite layer, whereas,structural and dissolution rate properties inform about the strength ofbioactive glass. 45S5 has been themost successful bioactive glass. More-over, 45S5 bio-glass cannot be used as a good scaffold owing to highdissolution rate. In this work, authors havemade an attempt to improvethe dissolution rate of 45S5 bio-glass alongwith constant Ca and P ratio.All glass samples have been prepared in the laboratory by the sol geltechnique.

2. Materials and method

Glass system xZnO(22.4 − x)Na2O·46.1SiO2·26.9CaO.2·6P2O5·2MgO was prepared in the laboratory by using the sol gel method.Details of the chemical composition of the glass samples are provided inTable 1. The solutions to obtain glasses were prepared from the stoichio-metric amounts of tetraethyl orthosilicate (TEOS), triethyl phosphate(TEP), Ca(NO3)2·4H2O, NaNO3, Mg(NO3)·6H2O and Zn(NO3)·4H2O (ARgrade). 1 M HNO3 was used as the catalyst for hydrolysis process.TEOS was added into 1 M HNO3 solution (TEOS and H2O molar ratioequal to eight) and the mixture was stirred up to 1 h for completehydrolysis. TEP, calcium nitrate tetra hydrate and magnesium nitratehexa hydrate were dissolved in 1 M HNO3 solution and stirred up tomore than 40 min. Both solutions were mixed under vigorous stirringand sodium nitrate was added into the solution. After 1 h of vigorousstirring, transparent solution was obtained. Solution was kept in an airtight beaker for 5 days for aging. Gel was heated up to 60 °C for 12 h,120 °C for 12 h and the product had been calcinated up to 700 °C for4 h. Prepared samples had been crushed in agar and mortar for 1 h.

X-raydiffraction (XRD) study has beenundertaken by usingBRUKERD8 FOCUS XRDmachine. Raman studies of prepared samples have beenperformed by the RENISHAW IN VIA REFLEX MICRO RAMAN SPEC-TROMETER with 785 nm laser beam of exposure time of 40 s with therange of 300 to 1500 cm−1. Field emission scanning electron microsco-py (FESEM) study has been carried out by ZEISS SUPERA 55. In order toget FESEM images, samples have been filtered from SBF and washedwith acetone and DI water four times. Moisture has been removedfrom samples by drying them up to 60 °C. Platinum coating has beenused to make the samples conductive. Gold coating of samples hasbeen avoided due to overlapping of gold and phosphorus peaks duringenergy dispersive X-ray (EDX) analysis. Brunauer–Emmett–Teller(BET) analysis has been undertaken by micrometrics ASAP 2020.Thermogravimetry, differential thermal analysis and derivativethermogravimetry (TG–DTA–DTG) techniques have been used to inves-tigate the thermal behavior of prepared bioactive glasses by EXSTAR TG/DTA 6300 instrument up to 1400 °Cwith the increase in temperature of10 °C per minute.

Table 1Composition (in mol%) of samples.

Sample code ZnO Na2O SiO2 CaO P2O5 MgO

BG-1 6 16.4 46.1 26.9 2.6 2BG-2 8 14.4 46.1 26.9 2.6 2BG-3 10 12.4 46.1 26.9 2.6 2

3. Assessment of in vitro bioactivity

Bioactivity nature of samples had been evaluated with the help of27-Tris SBF solution. SBF solutionwas prepared as per the recipe report-ed elsewhere [6]. 3mg of powder samplewas soaked in 30ml of 27-TrisSBF solution for 7 and 14 days under 37 °C temperature. After every12 h, sample was replaced by a fresh TRIS SBF solution. pH of Tris SBFwas measured after regular interval of time to check the ion exchangeprocess in between the surface of sample and Tris SBF solution. Ion con-centration of Tris SBF and human blood plasma is provided in Table 2.The results of in vitro bioactivity were analyzed with the help of XRD,Raman, SEM and EDX studies.

4. Results and discussion

4.1. XRD studies

XRD patterns of the samples are provided in Fig. 1. All samples showamorphous nature of material before soaking into SBF. As shown in theFig., sharp peaks of Calcium Hydroxide Phosphate [Hydroxylapatite(HAp)] (JCPDS No. 00-074-0566) are observed after 7 days. Intensityof peaks has increased when the number of days is increased to14 days. Along with HAp layer, calcium carbonate and calcium phos-phate peaks are also observed in all three XRD patterns. The formationof HAp layer on the surface of prepared glass samples and shifting ofamorphous to crystalline behavior after soaking of glass samples inSBF solution with the passage of time indicate the bioactive behaviorof prepared samples. Sometimes, there may be slight shifting of peaksof HAp due to the small substitution of calciummagnesium in biologicalapatite [7].

4.2. Raman spectroscopy

It is shown in the Raman spectra that before the in vitro analysis, onesharp peak appears around 1070 cm−1 indicating the presence of car-bonate ions (Fig. 2). Same peak splits into two small peaks after 7 and14 days during in vitro test which indicate the presence of phosphateions. Peaks around 1089–1091 and 1052–1055 cm−1 in the Ramanspectra corresponds to the asymmetric stretching (ν3) of the P\Obond in phosphate. The peak around 963–965 cm−1 corresponds tothe symmetric stretching mode (ν1) of the P\O bond of the phosphategroup. This peak is generally observed around 963 cm−1 in hydroxylap-atite [8]. The minor shift from 963 cm−1 may be due to minor incorpo-ration of magnesium or zinc ions into the HAp lattice. The resultantcompressive stresses in the lattice may lead to this difference in peakposition [9]. Raman data support the inferences of XRD studies interms of observation of bioactive nature of the glass samples.

4.3. FESEM and EDX studies

Morphology of samples has been confirmed by FESEM before andafter the immersion of samples in SBF solution. FESEM micrographshave observed that after 7 days, the content of calcium and phosphate

S. no. Ions Ion conc. (in 27-Tris SBF) Ion conc. (in human plasma)

1 Na+ 142 1422 K+ 5 53 Mg2+ 1.5 1.54 Ca2+ 2.5 2.55 HPO4

2− 1 16 HCO3

− 27 277 Cl− 125 1038 SO4

2− 0.5 0.5

Fig. 1. XRD pattern of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples before and after thein vitro analysis.

Fig. 2. Raman peaks of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples before and after thein vitro analysis.

90 V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

starts to increase which indicates that the apatite layer (calcium phos-phate) starts to generate on the surface of samples (shown in Fig. 3)After 14 days, growth of layer has been observed to increase and almostthewhole surface of the sample has been found to be covered by the ap-atite layer. This growth phenomenon is common in all three preparedsamples but the growth rate of the layer is high at the surface of BG-1and low at the surface of BG-3. It may be due to two reasons:(i) Increase in ZnO content (BG-3 sample). The presence of ZnO orMgO in the samples may have slowed down the deposition rate ofHAp. This observation is consistent with the results of Li et al. [10] andDu and Chang [11]; and (ii) lesser surface area for growth of apatitelayer (BG-3 sample, Table 4).

Due to small surface area in BG-3 thanBG-1, the rate of growth of ap-atite layermay have slowed down. It is reported by Li et al. that leaching

of ions is one of the reasons to slow down the rate of apatite layer butwhen zinc is added in the presence ofmagnesiumwith the replacementsodium content, there is a decrease in the leaching of Mg, Zn and siliconions as shown in EDX (Fig. 4). This can be explained as follows. BothMgand Zn ions have a dual character to behave like a network former ornetwork modifier. If they act like a former then Si\O\Si bond isreplaced by Si\O\Mg or Si\O\Zn bond and as a result, structurebecomes weak because the newly formed bond has less strength ascompared to the old one. But in case, Mg and Zn behave like modifiersthey give strength to the structure which reduces the solubility ofglass in SBF [12].

Enhancement in growth of apatite layer may be due to the presenceof magnesium ions along with zinc ions. Magnesium being a major ele-ment in the bone, may increase the growth of apatite layer. Moreover,magnesium plays a vital role in providing the apoptosis control to cell.EDX study of the prepared samples has been undertaken before andafter 7 and 14 days of immersion in SBF. In EDX study, it has been

Fig. 3. SEM images of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples before and after in vitro study.

91V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

observed that there is an increase in the concentration of calcium andphosphorus with time due to the formation of apatite layer on thesurface of the sample but the dissolution of silicon ions is very slow inall three samples. Due to this low solubility rate, these samples mayshow good scaffold properties. Exchange of ions is also confirmed byEDX graphs (Fig. 4). The graphs show the change of calcium and phos-phorus ions from Tris SBF and the growth of apatite layer on the surfaceof glass has been established within seven days.

At the end of 14th day, the concentration of calcium and phosphorusions increases due to the formation of calcium phosphate phases on thesurface of glass. Phase formation has also been confirmed with theweight (%) data of EDX (Table 3). The results indicate that Ca/P ratioof samples changes before and after in vitro analysis. Ca/P ratio hasbeen reported as 1.67 for the stoichiometric composition of HAp [13].Synthetic calcium phosphate materials have also been prepared andstudied extensively in vitro and in vivo. Somematerials have been called“hydroxylapatite”with Ca/P ratios ranging from 1.3 up to 2.0 [14]. Dur-ing the EDX analysis for our glass samples, authors have found that after14 days, Ca/P ratio lies in the range of 1.68–1.77 for all the glass samplesprepared in the laboratory. These values are very close to Ca/P ratio(1.67) for stoichiometric composition of HAp and also, lie in the rangeof already reported values of Ca/P ratio (1.3–2.0) for hydroxyl apatite.These results confirm the growth of HAp on the surface of the glass sam-ples. Moreover, these results also support the inferences of XRD andRaman studies. No major change in the concentration of Si, Mg and Znions has been observed even after 14 days of samples in Tris SBFsolution.

4.4. Brunauer–Emmett–Teller studies

BET study has been undertaken to find out the surface area andporous nature of prepared samples (Fig. 5). N2 adsorption–desorptionphenomena have been studied to obtain the desired results. BET surfacearea is calculated by using all adsorption data points from .01 to 1.0(total points 46) in the relative pressure (P/Po). In order to calculatethe pore size, the Barrett–Joyner–Halenda (BJH) desorption method(relative pressure from 1.0 to .12, total 16 points) has been preferredover the adsorption method because desorption is carried out at a lowrelative pressure which is useful for thermodynamic equilibrium [15].Passage of time indicates the bioactive behavior of prepared samples.Sometimes, there may be slight shifting of peaks of HAp due to smallsubstitution of calcium magnesium in biological apatite [7]. Surfacearea, pore volume and BJH desorption average pore volume of preparedsamples are provided in Table 4. As shown in the table, pore volume andpore size of samples decrease with the increase of the amount of Zn.This is due to the role of Zn as the network modifier to increase thestrength of glass samples.

Fig. 5 shows the N2 physisorption i.e. adsorption and desorptionisotherms and pore size distribution of prepared bioactive samples.The pore size of samples (Table 4) comes in the range of type II(2–50 nm). According to International Union of Pure and AppliedChemistry (IUPAC) classification of adsorption isotherm, type II de-scribes the presence of mesoporous and microspores which exhibitsa hysteresis loop and a variation point at lower pressure in a material[16].

Fig. 4. EDX results of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples before and after in vitro study.

92 V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

4.5. TG/DTA/DTG studies

Graphical representation of thermal behavior of samples is givenin Fig. 6. There is no sharp change in the mass of samples. For BG-1,BG-2 and BG-3, there is 5.60%, 5.24% and 2.6% loss of weight up to300 °C respectively which is due to the evaporation of adsorbedwater and ethanol molecules. BG-1 has high porosity which may bea reason of highest weight loss among all three samples. After that,from 300 to 700 °C, the weight loss is 2.81%, 1.86% and 2.09% forBG-1, BG-2 and BG-3 respectively. This may be due to the decompo-sition of precursor and removal of nitrate from samples. Further,samples are heated up to 1400 °C with 0.57%, 0.72%, and 0.79%weight loss of BG-1, BG-2 and BG-3 respectively. Weight loss from(700 to 1400 °C) may be due to the decomposition of carbonate

Table 3Weight percentage of the elements of samples from EDX data. EDX results are average (Avg.)

Element Standard used BG-1 (wt.%) BG-2 (w

Before After 14 days Before

Avg. ±Er%x10 Avg. ±Er%x10 Avg.

C CaCO3 14.6 0.02 16.44 0.024 14.45O SiO2 48.59 0.03 47.02 0.032 49.72Na Albite 12.97 0.025 8.48 0.031 8.48Mg MgO 2.78 0.031 3.28 0.034 3.28Si SiO2 14.65 0.032 14.12 0.031 14.12P GaP 1.96 0.031 2.51 0.032 2.68Ca Wollastonite 2.11 0.021 4.21 0.041 3.13Zn Zn 2.34 0.021 4.14 0.01 4.14Ca/P ratio 1.07 1.68 1.17

and formation of crystalline phases in the sample. All three samplesshow a stable thermal response of up to 1400 °C.

4.6. pH studies

pH study of samples gives an idea about ion exchange process in be-tween the surface of sample and Tris SBF solution. Variation of pH ofsamples in Tris SBF is shown in Fig. 7. A sudden rise in the pH of solutionindicates the exchange of ions between sample and Tris SBF whichstarted in the first hour. Total variation of pH of solution is from 7.4 upto 8.74 within 180 h. The pH of solution changes in a narrow range of1.34which shows that samples are chemically stable in Tris SBF solutionand do not show any acidic nature in Tris SBF.

of 5 measurements and the observed error (Er) percentage is also presented.

t.%) BG-3 (wt.%)

After 14 days Before After 14 days

±Er%x10 Avg. ±Er%x10 Avg. ±Er%x10 Avg. ±Er%x10

0.031 16.44 0.01 9.62 0.031 13.01 0.0390.034 49.11 0.04 48.35 0.041 49.01 0.0210.023 6.48 0.028 2.63 0.031 1.13 0.0410.025 3.28 0.032 0.54 0.029 0.41 0.0310.027 13.12 0.031 23.51 0.028 18.47 0.0210.041 3.12 0.049 4.54 0.05 4.98 0.0430.048 5.31 0.042 4.92 0.031 8.8 0.0290.014 3.14 0.021 5.89 0.018 4.23 0.022

1.7 1.08 1.77

Fig. 5. (a) Adsorption desorption curves and (b) pore volume of bioactive samples. Lines have been drawn as a guide to the eye.

93V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

4.7. In vitro drug release studies

In vitro drug release of sample has been studied with the help ofgentamicin. 2 g of prepared sample has been immersed in 40 ml ofgentamicin solution (10mgml−1). Sample has been kept in the solutionup to 24 h. After filtering the powder and drying at 40 °C up to 48 h,in vitro release of gentamicin from the drug-loaded bioactive glass iscarried out in an incubator at 37 °C. 2 g of powder is dipped in the20 ml of Tris SBF under 37 °C. Gentamicin release was determined byUV analysis. The release mediumwas withdrawn at the predeterminedtime intervals and replaced with fresh SBF solution each time. Duringthe drug release mechanism, all three samples show a quick release inthe first hour and then there is a decrease in the rate of release of drugin SBF (shown in Fig. 8). All the three samples show similar drug releasebehavior as reported bymesoporous channel [17] This study shows thatprepared samples have a good response in drug delivery phenomenaand it is due to their mesoporous behavior.

4.8. Statistical calculations

Data for pH and drug delivery studies is the average of 3 times rep-lication of experiment. Error bars have been used in the correspondingfigures. EDX measurements have been undertaken 5 times. The ob-served maximum deviation is 0.5%.

5. Conclusions

XRD peaks at 25.8°, 28.9°, 31.7°, 32.7°, 32.19°, 32.8°, and 39.78° indi-cate the presence of HAp phase on the surface of samples during in vitroanalysis. Growth of HAp phase has been confirmed by HAp(P\O bond)peak at 963 cm−1 in Raman spectra. FESEM images further confirm thefindings of XRD and Raman investigations. Observed Ca/P ratio (1.68,1.70 and 1.77) by the EDX technique during in vitro analysis also con-firms the formation of HAp phase. Low dissolution rate of silicon, mag-nesium and zinc ions has been observed by the EDX analysis. From BETstudy, it can be inferred that the increase in the content of zinc leads tothe decrease in the surface area and pore size which can be related tothe network modifier behavior of zinc. These results are consistentwith the findings of the TGA–DTA–DTG techniquewhich shows a stable

Table 4BET surface area, pore volume and pore size of bioactive samples.

Sample code BET surface area (m2/g) Pore volume (cm3/g A°) Pore size (nm)

BG-1 14.9 7.5 × 10−4 30BG-2 13.8 3.8 × 10−4 21BG-3 9.4 1.4 × 10−4 18

thermal behavior of samples having high content of zinc. Drug releaseproperties concluded that glass sample 6ZnO·16.4Na2O·46.1SiO2·26.9CaO·2.6P2O5·2MgO is better due its higher surface area and poresize. Porosity, surface area and chemical composition of the bioactiveglasses have been found to play a significant role in controlling the bio-active behavior of the glass samples.

Fig. 6. TG–DTA–DTG response of (a) BG-1, (b) BG-2 and (c) BG-3 glass samples.

Fig. 7.pHvariation of samples during in vitro analysis. Lines have beendrawn as a guide tothe eye.

Fig. 8. Gentamicin release by glass samples after regular time intervals. Lines have beendrawn as a guide to the eye.

94 V. Anand et al. / Journal of Non-Crystalline Solids 406 (2014) 88–94

Acknowledgments

The authors Vikas Anand and Kulwinder Kaur are grateful to thefinancial assistance provided by the UGC, New Delhi (India) throughJRF (NET)[F.17-74/2008 (SA-I)] and DST, New Delhi (India) through IN-SPIRE program [IF-120620] respectively.

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