CERAMICSINTERNATIONAL

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http://dx.doi.org0272-8842/& 20

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(2016) 3638–3651

Ceramics International 42 www.elsevier.com/locate/ceramint

B2O3–MgO–SiO2–Na2O–CaO–P2O5–ZnO bioactive system for boneregeneration applications

Vikas Ananda, K.J. Singha,n, Kulwinder Kaura, Harpreet Kaurb, Daljit Singh Arorab

aDepartment of Physics, Guru Nanak Dev University, Amritsar 143005, IndiabDepartment of Microbiology, Guru Nanak Dev University, Amritsar 143005,India

Received 22 August 2015; received in revised form 22 October 2015; accepted 4 November 2015Available online 12 November 2015

Abstract

Bioactive samples of composition x �B2O3 � (2xþ2)MgO.(22.4� (2xþ2))Na2O.(46.1�x) SiO2.26.9CaO � 2.6P2O5 � 2ZnO (x varying from 0 to4) have been prepared in the laboratory by the sol–gel technique. Structural information has been drawn from X-ray Diffraction, FourierTransform Infrared spectroscopy, Field Emission Scanning Electron Microscopy, Energy Dispersive X-ray and Atomic Absorption Spectroscopy.By using the Brunauer, Emmett and Teller technique, it has been found that the samples are mesoporus with pore size varying from 19 to 42 nm.Detailed analysis of degradation behavior of the materials has been undertaken. Gentamycin has been tested as an antibiotic to study their drugrelease properties. Swelling, antimicrobial and cell culture studies have also been conducted. Attempt has been made to search for suitablechemical composition for the purpose of developing effective implant material for bone regeneration applications.& 2015 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Keywords: Bioceramic; SBF; Hydroxylapatite

1. Introduction

Hard tissue replacement or regeneration are the only clinicalsolutions available for the treatment of broken bone [1]. Due tobone formation ability, many bioactive glasses or ceramicshave been tested for hard tissue engineering [2]. Severalbioactive systems have been reported but most of them sufferfrom several drawbacks like abrupt dissolution rate, slowbioactivity, poor mechanical strength and lack of some specialproperties, for example, antimicrobial, cell proliferation andosteogenesis, which restrict their use as implant materials forclinical applications. It has been reported that dissolution rate,bioactivity and mechanical properties of the bioactive samplescan be improved by addition of different phases includinghydroxylapatite (HAp) and whitlockite [3–5]. Growth of HApand whitlockite layers on the bioactive samples are keyindicators for applications of samples as implant materialsfor bone regeneration applications. HAp (Ca10(PO4)6(OH)2)

/10.1016/j.ceramint.2015.11.02915 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

g author. Tel.: +91183 2452190.ss: kanwarjitsingh@yahoo.com (K.J. Singh).

has similar chemical composition to human bone. Mechanicalstrength, dissolution rate and osteoblast properties of HAp arealso comparable to human bone. Moreover, HAp can makestrong and stable bond with old bone and the surroundingtissues. Whitlockite is a term for the mineral or syntheticmaterial in which Mg2þ and HPO4

2� ions substitute in β-TriCalcium Phosphate (TCP). Its solubility is lower than pure β-TCP which indicate the enhanced stability of the lattice [6]. Itoccurs in various pathological calcifications and also, as amajor constituent of human dental calculus [7].Significant efforts have been made by researchers to

enhance the properties of bioactive samples by doping severalelements like, Ag, Mg, Sr, Zn etc [8–12]. Each dopant canalter the characteristics which further leads to change in theproperties of bioactive sample. Zinc, magnesium and boron asconstituents of bioactive system have special place due to thefollowing observations. It has been observed that partialsubstitution of zinc with the replacement of sodium in 45S5bioglasss improves the bond formation activity by increasingcell proliferation and differentiation and increased amount ofzinc leads to formation of bone structure [13,14]. Moreover,

Table 1Composition in mol% and pore size in nm. Pore size is the average of3 measurements and the observed standard deviation (SD) is presented.

Samplecode

B2O3 MgO Na2O SiO2 CaO P2O5 ZnO Pore Size(nm)7SD.

MZB-0 0 2 20.4 46.1 26.9 2.6 2 4271MZB-1 1 4 18.4 45.1 26.9 2.6 2 3472MZB-2 2 6 16.4 44.1 26.9 2.6 2 2571MZB-3 3 8 14.4 43.1 26.9 2.6 2 1971MZB-4 4 10 12.4 42.1 26.9 2.6 2 1972

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zinc also provide DNA replication [15], bone re-sorption [16]and antibacterial properties [16,17]. Zinc helps in the bondformation in the implant and accelerate the recovery of patient[14]. Magnesium is one of the most abundant cations in thehuman body. As a bone constituent, it covers 50–60% weightof bone [18,19]. Mg is essential for many enzyme reactions.Mg can directly stimulate the osteoblast proliferation [20].MgO also incorporate the synthesis of HAp layer as reportedby Vallet-Regi et al. [1] and Balamurgan et al. [8,21].Magnesium is naturally present in human bone and essentialfor the human metabolism [18,19]. It can stimulate the growthof new bone and tissues [20,22]. Borate can also be used inbioactive systems as a dopant. Borate is a very interestingmaterial for doping because it can easily change its coordina-tion number from three to four and hence, it can form thevariable structural units [23]. Recently, silica free borateglasses [24] have also been investigated for biomedicalapplications. It has been inferred that the corrosion mechan-isms of borate glasses in aqueous environments, generallyundergo hydration, hydrolysis, and ion exchange reactions.Most of studies reported earlier provide information about theeffect of doping of individual ZnO, MgO and B2O3 on theproperties of bioactive ceramics. Four out of five samplesreported in this article contain all the three compounds. Themain aim of presented work is to investigate the effect ofborate and magnesium oxide in the presence of zinc oxide onHAp and whitlockite layer formation and biological properties.

2. Materials and methods

2.1. Preparation of bioactive samples

Bioactive ceramics of the system x �B2O3(2xþ2)MgO �(22.4�2xþ2)Na2O � (46.1�x) SiO2 � 26.9CaO � 2.6P2O5 �2ZnO have been prepared in the laboratory by using thesol–gel method. Tetraethyl orthosilicate (TEOS), triethylphosphate (TEP), calcium nitrate tetra hydrate, sodiumnitrate, magnesium nitrate hexahydrate, zinc nitrate tetrahydrate and boric acid (AR grade) have been used assource materials for SiO2, P2O5, CaO, Na2O, MgO, ZnOand B2O3 respectively. 1 M HNO3 was used as the catalystfor hydrolysis process. TEOS was added into 1 M HNO3

solution (TEOS and H2O molar ratio equal to eight) and themixture was stirred up to one hour for complete hydrolysis.TEP,calcium nitrate tetra hydrate and magnesium nitratehexahydrate were dissolved in 1 M HNO3 solution andstirred up to 40 min. Both solutions were mixed undervigorous stirring condition. Sodium nitrate was added intothe solution followed by boric acid. After one hour ofvigorous stirring, transparent solution was obtained. Solu-tion was kept in air tight beaker for 3 days for aging. Gelwas heated up to 60 1C for 12 h and 120 1C for 12 h. Thesamples were calcinated up to 700 1C for 8 h to attaincrystalline nature. Prepared samples had been crushed inagar and mortar for one hour. Chemical composition of theprepared samples is provided in Table 1.

2.2. Assessment of in vitro bioactivity

In vitro bioactive nature of samples has been evaluated withthe help of simulated body fluid (SBF) solution. SBF solutionhas been prepared as per the recipe reported elsewhere [25].One gram of powder sample was soaked in 50 ml of SBFsolution under 37 1C. After every 12 h, old SBF was replacedwith fresh SBF solution.

2.3. Characterization techniques

X-ray diffraction (XRD) study has been undertaken by usingBruker D8 focus XRD machine. Fourier Transform InfraredSpectroscopy (FTIR) investigations of the samples have beenundertaken in transmittance mode by using Perkin ElmerSpectrometer(C92035),Germany. Field emission scanningelectron microscopy (FESEM) and energy dispersive X-ray(EDX) studies have been carried out by ZEISS SUPERA 55.In order to get FESEM images, samples have been filteredfrom SBF and washed with acetone and DI water four times.Moisture has been removed from samples by drying them upto 60 1C. Platinum coating has been used to make the samplesconductive. EDX study has been undertaken without coatingof samples. Differential thermal analysis technique has beenused to investigate the thermal behavior of the samples byEXSTAR TG/DTA 6300 instrument up to 1400 1C with theincrease in temperature of 10 1C min�1. Atomic AbsorptionSpectroscopy (AAS) study has been undertaken by using AAS240FS Agilent Atomic Absorption Spectrometer to check theconcentration of ions in decant SBF and citric buffer.Biological properties of samples have been studied with thehelp of swelling, drug release, cytotoxicity and cell culturestudies. Labsystem Multiskan EX ELISA and Biorad 680-XR,Japan reader with 570 and 590 nm wavelengths of UV–visiblerange have been used for biological studies.

3. Results and discussion

3.1. Apatite forming ability of the system

Apatite formation ability of samples have been checkedbefore and after immersion in the SBF solution. During theseinvestigations, structural changes, morphology and Ca/P ratioshave been investigated with the help of XRD, FTIR, FESEM

Fig. 1. XRD spectra of sample before and after in vitro analysis of samples (□ Calcite, ∎ Whitlockite�Magnesium phosphate, ▾ Hydroxylapatite, △Sodiumcalcium silicate).

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and EDX studies. The results obtained have been correlatedwith the bioactive nature of the samples.

3.1.1. XRD studiesXRD patterns of the samples are provided in Fig. 1.

Sharp peaks of sodium calcium silicate (JCPDF-78-1650)(in Fig. 1(a)) whitlockite (JCPDF-70-2064) (in Fig. 1(b)–(e)) along with the appearance of sodium nitrate (Fig. 1(b)), magnesium phosphate (Fig. 1(e)) and calcite (Fig. 1

(b)–(e)) indicates the crystalline behavior samples. It hasbeen noticed that peaks of whitlockite phase becomesharper with the addition of magnesium. Magnesiumcontaining whitlockite is one of the abundant bio-mineralin the bone [26,27]. However, its importance and role hasnot been fully identified. Along with calcium ion, magne-sium ions are also present in the human bone [26,28].Dentin contains 26% to 58% of whitlockite mineral byweight (Fig. 2).

Fig. 2. FTIR spectra of sample before and after in vitro analysis.

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It is difficult to synthesize the whitlockite phase in pure formand there are only limited reports available for the synthesis ofwhitlockite in aqueous solutions [28–30]. It is also reportedthat whitlockite phase is stable only in acidic pH(4.5–5) [31].

In the light of this situation, it is imperative that when oursamples (containing whitlockite phase) come in contact withhigh pH SBF solution (pH�7.4), it leads to dissolving of thewhitlockite phase and formation of HAp phase (JCPDF no.

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07-0747) takes place. During immersion of samples in SBF,structural changes have been observed after 2, 7 and 14 days.Within 2 days, it has been seen that due to exchange of ionsbetween samples and SBF, the pH of solution rose up to 8.4. Ithas also been seen that calcite phase (in Fig. 1(b)–(e)) start toform with the exchange of calcium ions between sample andSBF solution. After few hours, calcium carbonate dissociateinto calcium and carbonate ions leading to higher concentra-tion of Ca2þ ions in the solution which can be used for theformation of apatite layer. Reaction may occur as per thefollowing equation;

CaCO3þHþ Ca2þ þHCO�3 ð1Þ

After every 12 h, old SBF has been replaced with fresh SBFsolution to maintain the ions concentration and pH of solution.Within the 7days, stable HAp phase start to grow on thesurface of samples. Peak at 31.91 in the XRD spectra indicatesthe formation HAp phase after 7 days (Fig. 1(a)–(e)). Growthof hydroxylapatite has not been observed after 24 h.

DTA spectra of the dried gels indicate the glass transitiontemperatures of samples as 611, 641, 691, 751 and 82 1C forMZB-0. MZB-1, MBZ-2, MBZ-3 and MBZ-4 samples. Inorder to confirm the observations of glass transition tempera-ture from DTA data, XRD spectra of the dried gels have beenundertaken at 50 1C which confirms the amorphous nature.Broad humps of crystallization temperatures have beenobserved at 3961, 3601, 3421, 3331 and 317 1C for MZB-0.MZB-1, MBZ-2, MBZ-3 and MBZ-4 samples. Broad humpsmay be due to the formation of multiple crystalline phases.

3.1.2. FTIR studiesSome characteristic peaks have been observed at 470 cm�1,

near about 710 cm�1 and 1080 cm�1 which can be assignedto Si–O–Si bending, Si–O–Si bond and Si–O–Si asymmetricalstretching. Vibration of P¼O has been observed near1220 cm�1 along with C–O bond at 1420–1470 cm�1. SomeBO3 vibrations at 1380 cm�1 indicate the presence of boron inthe samples. Peaks at 960-975 cm�1 show the presence of B–O–M (M may be any metal). Two small humps at 876 cm�1

and 920 cm�1 are attributed to the vibrations of SiO44� and

Si2O74� bonds.. Humps at 619 cm�1 and near 986-1000 cm �1

represent the Mg band with influence of γ4PO4 vibrations.When the samples are immersed into SBF and analyzed after7 days and 14 days, some distinctive changes thus observedare provided below;

� Two sharp peaks at 601 and 558 cm�1 indicate thepresence of apatite (P–O bonds) on the surface of sample.These peaks have become sharper when analyzed after 14days. These peaks are the fingerprint peaks for HAp. Theseresults compliment the analysis of the XRD spectra.

� Increase in magnitude of humps at 470 cm�1, 801 and1080 cm�1 (Si–O–Si) with time indicates the re-polymerization of Si–O–Si layer and hence, confirms theanalysis of XRD studies.

3.1.3. FESEM and EDX studiesPowder of the sample has been filtered from SBF and

washed with acetone and DI water four times. In order toremove the moisture, samples have been dried up to 60 1C.Platinum coating has been undertaken to make the samplesconductive. Fig. 3 shows the difference in the morphology ofthe representative samples before and after 14 days during invitro analysis. Change in the morphology can be attributed tothe formation of HAp layer on the surface of samples. FESEMand EDX studies of sample have been undertaken afterconfirmation of presence of apatite layer on the surface ofthe samples from XRD and FTIR studies. FESEM images havebeen taken after 14 days of in vitro analysis when apatite layeris supposed to be fully grown on the surface of the samples.FESEM micrographs confirm the analysis of XRD and FTIRdata. EDX results can provide the information about thecontent of calcium and phosphorus in the samples. Ca/P ratiofrom EDX analysis has been provided for representativesamples in Fig. 3. After 14 days, it has been observed thatCa/P ratio of samples is in the range of 1.62–1.66. Ca/P ratioof human bone is 1.66. In the light of this situation, it can beconcluded that EDX results also confirms the growth of HApon the surface of samples.EDX study has been undertaken for all the samples. Atomic

percentage of constituent elements has been calculated fromnominal composition and compared with atomic percentage ofsame elements as observed from EDX data (Table 2). Trendsof the atomic percentage of elements in nominal and experi-mental compositions of the samples have been observed to besimilar. Moreover, the experimental and nominal values havebeen observed to be close which suggest that the preparedcompositions are similar to the nominal compositions.

3.2. Concentration of ions and degradation behavior of thesystem

Concentration of ions exchanged between the SBF andsample interface has been investigated with the help of AAStechnique. Degradation of samples have been studied in citricbuffer and phosphate buffer saline (PBS) buffer solutions byemploying weight change, pH, XRD and AAS techniques.Both are important parameters to understand the bioactivebehavior and also, to evaluate the samples for clinicalapplications.

3.2.1. Concentration of ionsExchange of ions between SBF solution and sample inter-

face is responsible for the formation of new phases andvariation in the morphology of the samples. AAS of decantSBF has been investigated with 240FS Agilent AtomicAbsorption Spectrometer. Decant of SBF has been filteredwith .22 μm syringe filter. Dilution of SBF was run against thestandard solutions. Concentration of ions has been checkedduring in vitro analysis and results have been presented in Fig.4. With the increase in the content of borate and magnesium insamples, following changes have been observed;

Fig. 3. Representative FESEM micrographs and EDX results (a) Before in vitro and (b) after in vitro.

Table 2Comparison of atomic percentage of elements in nominal composition with atomic percentage of elements as observed from EDX data.

Element MZB-0 (at%) MZB-1 (at%) MZB-2 (at%) MZB-3 (at%) MZB-4 (at%)

Nominalcomposition

EDX Nominalcomposition

EDX Nominalcomposition

EDX Nominalcomposition

EDX Nominalcomposition

EDX

O 55.99 56.12 56.35 56.71 56.71 57.33 57.07 57.89 57.42 58.54Na 14.60 15.78 13.17 14.01 11.74 13.01 10.30 11.31 8.87 9.07Mg 0.72 0.77 1.43 1.62 2.15 2.31 2.86 2.94 3.58 3.89Si 16.49 14.87 16.14 14.61 15.78 13.61 15.42 13.41 15.06 13.25P 1.86 1.78 1.86 1.73 1.86 1.77 1.86 1.76 1.86 1.76Ca 9.62 9.89 9.62 9.86 9.62 9.80 9.62 9.81 9.62 9.89Zn 0.72 0.79 0.72 0.76 0.72 0.77 0.72 0.78 0.72 0.79B 0.00 0.00 0.72 0.70 1.43 1.40 2.15 2.10 2.86 2.81

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� During in vitro analysis, ion concentration of silicon,magnesium, zinc and boron ions in the solution have beenobserved to increase within 50–60 h. It became almoststable up to 250 h with marginal increase in concentration.This slow dissolution of ions after 50–60 h may be due tothe different roles of zinc and magnesium and BO3 units inthe network.

� The ion concentration of phosphorus and calcium has beenobserved to be decreased in the decant within 24 h. duringin vitro analysis. But sudden increase in the concentration

of calcium ions has been observed after 50 h (Fig.4(c))during in vitro analysis. It may be due to the dissociation ofCaCO3 into Caþ and HCO3

� ions(reaction provided in Eq.(1)). After that, Ca and P ions concentration has beenobserved to be regularly decreased which may be related tothe formation of apatite layer on the surface of samples.

� It has been observed that with the increase in the concen-tration of boron and magnesium in the samples, dissolutionrate decreases. It may be correlated with the decrease inpore size of high content borate sample. In order to justify

Fig. 4. Atomic Absorption Spectroscopy result of solution that soaks bioactive samples. Error bar indicates the standard deviation observed for three measurements.

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this statement regarding slower dissolution rate for sampleshaving high content of boron and magnesium, BET studieshave been performed. Details of the experimental set upused for BET analysis has been provided by authorselsewhere [2]. Evaluated pore size from BET study of

prepared samples is provided in Table 1. It can be inferredfrom the table that prepared samples are porous in nature.Without addition of boron, sample (MZB-0) has the highestpore size. With the addition of the boron and magnesium,pore size has been observed to decrease gradually (42 to

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19 nm). These results indicate that slower dissolution ratesat higher contents of boron and magnesium in the samplesmay be related to smaller pore size of the samples.

3.2.2. Degradation studyDegradation behavior of sample is an important parameter

which gives an idea about how fast sample will degrade in thebody. Degradation test of sample has been performed in twodifferent pH media; (i) PBS with pH 7.4 and (ii) Citric bufferwith pH 3.0. PBS has been selected because it is the mostcommon pH in the human body and citric buffer has beenselected because it is released by osteoblast cells during worstconditions in the body [32]. Tests have been performedwithout replacement of buffer solution after 120 h.

Fig. 5. (a) Weight loss trend,(b) pH graph, (c) XRD spectra of samples treated wiformation of ∎ Sodium Calcium Silicate and □ Silicon Phosphate, and (e) Concentrathe standard deviation observed for three measurements.

Weight change, pH, XRD and ion concentration of samplesunder PBS and Citric buffers are the parameters investigatedunder degradation study. When sample come in the contactwith buffer solution there is exchange of ions in betweensample and solution which disturb the stoichiometric weightdistribution of sample. This leads to the change in the netweight of sample. If there is loss of ions from sample (leachingof ions) then this may decrease the weight and loss ofcrystalline phases and vice versa. With the leaching of ions,pH of sample will also increase or decrease depending uponthe nature and concentration of ion leached into the solution.XRD study may provide the information about the new phaseson the sample and AAS study can be used to check the nature(anion or cation) and concentration of leached ions. Therefore,weight loss, pH, XRD and AAS studies can be utilized as

th citric buffer, (d) XRD spectra of sample treated with PBS buffer show thetion of different elements in buffer solutions. Error bars for (a) and (b) indicates

Fig. 6. Swelling ratio percentage of prepared bioactive samples. Error barindicates the standard deviation observed for three measurements.

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complimentary techniques and they are very useful to find outthe exact degradation behavior of sample.

Weight loss percentage of sample has been calculated withthe formula given below;

WL%¼ ðW1�W2ÞW1

� 100 ð2Þ

where WL, W1 and W2 are weight loss, initial and finalweight of the sample. Graphical variation of weight loss data isgiven in Fig. 5(a). It has been observed that MZB-0 has thehighest degradation rate with loss of 0.63 wt% whereas MZB-4 has the lowest degradation rate with loss of 0.40 wt% incitric buffer. Slow leaching may also be due to higher contentof boron and magnesium in the samples which may lead toincrease in the hydrophobic bridging oxygen atoms. TheMgO4

2� units require some modifier cations in the matrix forthe purpose of charge balancing. Presence of borate unitsincrease the hydrophilic bridging oxygens. This may lead toincrease in the network connectivity which may further lead toformation of compact structure with controlled degradationrate. Weight loss in the PBS varies from �0.25(MZB-4) up to-0.43(MZB-0) weight percent. This may be due to slow ionleaching rate of MZB-4 because of its small pore size.Negative weight loss has been reported in the case of PBSbuffer which indicates the gain of weight due to formation ofnew crystalline phases (confirmed in XRD spectra Fig. 5(d)). Ithas been observed by Merolli et.al that these newly formedcrystalline phases of sodium calcium silicate (JCPDF-78-1686)and silicon phosphate (JCPDF-22-1320) are biocompatible andbioactive [33]. XRD spectra of samples immersed in citricbuffer solution have shown amorphous nature of sample after120 h. This may be due to high leaching of ions in the acidicmedium (pH¼3,citric buffer). It has been observed that pH ofcitric buffer solution has increased from 3.00 up to 6.91 forMZB-0 (maximum) and 5.32 for MZB-4 (minimum) as shownin Fig. 5(b). This change in the pH value helps to initiate thehydrolysis process of the samples. But in the case of PBS,there is slight increase in the pH of solution from 7.91 (forMZB-0) up to 8.66 (for MZB-4). Change in the pH may bedue to release of Ca and Mg ions from the whitlockite andcalcite phases. As discussed earlier, whitlockite is a stablephase in the acidic pH (4.5–5),therefore, this phase start todissolve in citric buffer and PBS solution. Concentration ofleached ions in buffer solutions has been investigated withAAS technique for solvent with the filtration from.22 μm filterpaper. Ionic release is an important factor which determine theapatite growth and degradation of bioactive glass or ceramics.Ceramics have different crystalline phases and are supposed totake more time for the dissolution as compared to amorphousglass system. It has been seen from AAS study that concentra-tion of Si, P, Zn, Mg and B ions is higher in the case of citricbuffer solution as compared to PBS buffer(shown in Fig. 5(e)).This observation supports higher change in pH value andconversion of crystalline phases into amorphous in citricbuffer. It has been observed that silicon release is in the rangeof 109.3–95.3 mg/l (Fig.5(e)) for citric buffer and 30.3–23.5 mg/l for PBS buffer. Release of ions from sample is

beneficial to initiate different biological processes like osteo-blast, cell growth and angiogenesis etc. For example, it hasbeen reported [32] that when ion concentration of silica ions isbetween 0.1 and 100 mg/l, it is beneficial for stimulate theosteoblast process. Similarly, calcium in the range of 13.1–90 mg/l helps to initiate the osteoblast proliferation. In thisstudy, silicon and calcium ion concentration lies between 23.5to 30.3 mg/l and 17.5 to 24.5 mg/l respectively. Magnesium isalso present in the extracelluer fluid in the concentration of 17–25.5 mg/l and if its level is increased beyond 25 mg/l, it maylead to muscular paralysis. In our study magnesium ionconcentration has been observed in between 11 and 20 mg/lwhich is the comfortable zone for human tissues.

3.3. Swelling test

Swelling test is an important study to investigate thebehavior of pore size of sample when it comes in contactwith human plasma or PBS buffer. The swelling test ofprepared samples have been performed with conventionalgravimetric procedure as described below. Samples of knownweight are kept in 20 ml of PBS at 37 1C (pH¼7.4). Swollenporous samples were drawn at various time intervals, driedsuperficially by gentle contact with a filter paper and weighedfor the determination of wet weight as a function of theimmersion time. The swelling ratio percentage was calculatedas

%δw ¼ ðWa�WbÞWb

� 100 ð3Þ

Where wa and wb are the sample weights after and beforeswelling, respectively. Each test was repeated three times foreach composition and results were expressed as average valueplus standard deviation (Fig. 6).It is speculated that increase in swelling ratio and pore size

are correlated. Higher value of swelling ratio percentage andpore size of samples for low content borate samples support

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this inference (Fig. 6 and Table 1). Capillaries in the poresavail nutrients from culture media more effectively. Swellingcan enhance the cell adhesion.

3.4. Drug release

Drug release property can be used to explore the possibi-lities of samples as drug carrier agents. Gentamycin as anantibiotic has been tested for drug release study of the samplesbecause this drug has good activity against gram negativemicroorganisms. 1 gm of prepared sample has been immersedin 20 ml of gentamycin solution. After gentamicin wasincorporated into sample, sample has been kept in the solutionup to 24 h. After filtering the powder and drying at 40 1C up to24 h, release of gentamycin from the drug-loaded bioactivesample has been investigated in incubator at 37 1C. One gramof powder has been dipped in the 20 ml of SBF under 37 1C.Gentamycin release was determined by UV analysis. Therelease medium was withdrawn at the predetermined timeintervals and replaced with same amount of fresh SBF solutioneach time. During the drug release mechanism, all the preparedsamples show quick release in first hour and then there isdecrease in the rate of release of drug in SBF (shown in Fig.7). All the samples show similar drug release behavior asreported by mesoporus channel [34]. Reported BET data(Table 1) indicate the mesoporus nature of our samples. Thisstudy shows that prepared samples have good response in drugdelivery phenomena and it is due to their mesoporus behavior.

3.5. Antimicrobial activity

Tendency to kill the microorganisms has been studiedagainst six different gram positive and gram negative micro-organisms.Multiple drug-resistant microorganisms such asMRSA have increased in the world [35]. The developmentof new antimicrobials is the emerging challenge to answer the

Fig. 7. Drug release study of samples. Error bar indicates the standarddeviation observed for three measurements.

problem posed by resistant microorganisms. Keeping theresistance factor in mind and the demand for new antimicrobialagents, we have tested our samples for antimicrobial potential.Antimicrobial results are provided in Fig. 8.

3.5.1. Inoculum preparationA loopful of isolated colonies was inoculated into 5 ml

nutrient broth and incubated at 37 1C for 4 h. The turbidity ofactively growing microbial suspension has been adjusted tomatch the turbidity standard of 0.5 Mc Farland units preparedby mixing 0.5 ml of 1.75% (w/v) barium chloride dihydrate(BaCl2 � 2H2O) to 99.5 ml of 0.18 M (v/v) sulfuric acid duringconstant stirring

3.5.2. Test organismsThe reference strains of bacteria: Staphylococcus aureus

(MTCC-740) Klebsiella pneumonia sub sp. pneumoniae(MTCC-109), Pseudomonas aeruginosa (MTCC-741),)

Fig. 8. (a) Representative figure for microbial activity and (b) bar graphs ofdifferent microbial activity with samples. Error bar indicates the standarddeviation observed for three measurements.

Fig. 9. Cell viability of sample during cytotoxicity test. Error bar indicates thestandard deviation observed for three measurements.

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Salmonella typhimurium (MTCC-1251) and yeast strain:Candida albicans (MTCC-227) have been obtained fromMicrobial Type Culture Collection (MTCC), Institute ofMicrobial Technology (IMTECH), Chandigarh, India and theclinical isolate MRSA has been obtained from Post graduateInstitute of Medical Education and Research, (PGIMER),Chandigarh, India.

3.6. Antimicrobial activity by agar well diffusion assay

The plates containing Muller Hinton agar medium havebeen spread with 0.1 ml of the microbial inoculum. Wells(8 mm diameter) have been cut from agar plates usingsterilized stainless steel cork borer and filled with 0.1 ml ofthe fungal extract. The plates have been incubated at 37 1C for24 h and diameter of resultant zone of each combination ofextract and bacterial strains inhibition has been measured [36].Experiments have been run in triplicate for each combinationof extract and bacterial strains. There are several resources thatcan be tapped for useful products such as antibiotics. Thebacterial cultures used in the present study are responsible forcausing gastrointestinal tract and respiratory infections.

All the samples have been found to be active against almostall the microorganisms tested. S.aureus was found to be themost sensitive organism and the inhibition zone was found tobe in the range of 21–25 mm for boron containing samples.Gram negative bacteria acquire resistance more readily due totheir outer membrane which contains narrow porin channelswhich retard the entry into the cell, of even small hydrophiliccompounds, a lipopolysaccaride moiety which slows down thetrans membrane diffusion of lipopolyphilic antibiotics and theyoften possess a multidrug efflux pump which eliminates manyantibiotics from the cells causing several diseases. All theboron containing samples have shown good antimicrobialpotential against gram negative bacteria viz K.pneumoniae,P.aeruginosa and S.typhimurium with zone of inhibitionranging from 16 to 20 mm, 12 to 20 mm and 04 to 14 mmrespectively. The importance of the reported study becameparamount when resistant strains like MRSA were also foundsensitive to the prepared samples which were not reportedearlier. Prepared boron containing samples have shown notonly activity against bacterial cultures but have also showngood activity against yeast C.albicans with zone of inhibitionranging from 15 to 21 mm. Results have shown that oursamples can be potent antimicrobial agents which can befurther exploited for various pharmaceutical processes.

3.7. Cell cytotoxicity and culture studies

Toxicity and cell attachment (through cell culture investi-gations) have been studied to investigate the friendly beha-vior of sample with cells. Samples have been observed to benon-cytotoxic as per the procedure reported by authorselsewhere [37]. MTT (3-[(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl] tetrazolium bromide) assay has been used forthis study. 10 mL sheep blood has been taken into injectionsyringe containing 3 mL Alsever's solution (anticoagulant)

which was subsequently transferred to sterile centrifugetubes. The blood has been centrifuged at 1600g at roomtemperature for 20 min to separate the plasma from the cells.The supernatant has been discarded and 6 mL PBS was addedwhich was further centrifuged. The red blood cells (RBCs)have been washed thrice with PBS by centrifugation techni-que and the pellet has been re-suspended in 6 mL of PBS.Various dilutions of these cells using PBS have been preparedand counted with the help of a haemocytometer under opticalmicroscope so as to obtain cells equivalent to1� 105 CFU/mL. The following formula has been used todetermine the required number of cells;

Number of cells=mL¼Number of cells counted in 25 squares

� Dilution factor � 104

ð1ÞThe cell suspension thus prepared has been dispensed into

Elisa plates (100 mL/well) and incubated at 37 1C for over-night. The supernatant has been removed carefully and 200 mLof the compound (sample dissolved in DMSO) has been addedand incubated further for 24 h. Supernatant has been removedagain and added to 20 mL MTT solutions (5 mg/mL) to eachwell and incubated further for 3 h at 37 1C on orbital shaker at60 rpm. After incubation, the supernatant has been removedwithout disturbing the cells and 50 mL DMSO has been addedto each well to dissolve the. The wells with untreated cellshave served as control. In the presented study,viable cellpercentage of samples has been calculated by absorbanceintensity. For MZB-0, MZB-1, MZB-2, MZB-3 and MZB-4,the observed cell viabilities are 70.3%, 70.3%, 79.3%, 79.3%,79.3% and 79.3% respectively (Fig. 9). The obtained resultssuggest that all the prepared samples are non-toxic in nature. Itcan also be inferred from the results that increase in the contentof borate and magnesium reduce the toxicity of sample.Only HAp formation ability of material is not enough to

make it an implant material. Before implantation it is alsoimportant to check how it reacts with human osteoblast cells.In order to check the behavior of samples with living cell, thehuman osteosarcoma cell line has been obtained from NationalCenter of Cell Science, Pune,India. DMEM (Dulbecco'sModified Eagle's Medium) has been used with FBS(fetalbovine serum)10%, streptomycin and gentamycin 100 U ml�1

each to maintain cell lines under 37 1C incubation with humidenvironment containing 5% CO2. MTT assay has been used to

Fig. 10. (a) Cell viability bar graph. Error bar indicates the standard deviation observed for three measurements and (b) Representative optical image (at 40�magnification) of MG63 cell line grown on the surface of sample.

V. Anand et al. / Ceramics International 42 (2016) 3638–3651 3649

check the cell integrity. 24 well plates have been used to bindthe MG 63 cell with samples. MG63 cells are seeded on thesterilized plate with concentration 2� 104 cell ml�1. Samplesare kept under 37 1C with 5% CO2 environment for 96 h.Tissue culture treated plastic cover slip (Theromanox) has beenused to grow controlled culture. Glass slices have been kept intriplicate in 24 well plates. Each well plate has been filled with500 μl volume of lymphocyte suspension at the rate of2� 104 cell ml�1. Plates have been incubated for 96 h.500 μl MTT (2 mg ml�1) has been added to the plates before4 h. for termination. After 4 h, blue colored formazan hadappeared in each well plates which was studied with 570 nmUV radiations with the help of Labsystem Multiskan EXELISA reader against a reagent.

Fig. 10 shows the cell viability of samples with respect toMG63 cell. Samples have been compared with commercialavailable culture plate. It has been observed that all theprepared samples successfully provide the positive environ-ment for cell growth (high absorbance indicate good cellgrowth). Leaching of ions have shown impact on the biologicalbehavior of samples. Cell proliferation remains good ifleaching of ions is smooth and regular. Sudden increase inthe concentration of ions may cause the death of cell. Cellviability of all the prepared samples is good and hence, thisobservation compliments the results reported in Fig. 4. It hasbeen already established that presence of zinc ions playimportant role in the growth of cell. It can be seen in Fig.10 that boron and magnesium containing samples enhance thecell proliferation. Observed trends in Fig. 10 may be due to thesmooth dissolution of boron and zinc ions as discussed underthe section concentration of ions and degradation behavior ofthe system.

Many authors [38–42] have investigated the growth ofhydroxylapatite layer on silica based bioactive samples. Ithas been observed that many samples took more than 14 daysto initiate the growth of hydroxylapatite layer. In the presentedwork, authors have reported the faster growth of hydroxyla-patite layer (7th day) during in vitro analysis. Faster is the

growth of hydroxylapatite on the surface of sample, quickerwill be the bond formation between host and implant material.This will lead to recovery of damaged bone in shorter span oftime. Kapoor et.al. and Kansal et al. [43,44] have studied thedegradation behavior of amorphous bioactive samples in twodifferent pH buffer solutions. pH values used were 3 and7.4 which are the same values as used by the authors in thepresented study. Ion leaching for Si, Ca and P observed byKapoor et.al. and Kansal et.al was higher as compared to theion leaching observed by authors for the same ions. Controlledleaching of ions is an important property to improve thetherapeutic efficiency of the treatment. Controlled leaching ofions was observed for the presented system which may beattributed to crystalline nature of the samples. Very fewauthors [45–47] have reported the cell viability greater thancontrol sample. Authors have observed 35% higher viability ascompared to control for MZB-4 sample. It indicates thatprepared sample helps in the proliferation of MG 63 cell lines.Growth of MG 63 cells initiate the osteoblast (bone formation)process. More will be the growth of cells, faster will be therepair of the damaged bone. These results indicate thesignificant contribution by the authors in the presented workin terms of growth of hydroxylapatite layer, degradationbehavior and cell viability properties of the bioactive materials.

4. Conclusions

The prepared samples have shown good bioactivity beha-vior. This feature has been confirmed by the presence ofapatite peaks in XRD and presence of P–O bonds in FTIRspectra at 558 and 601 cm�1 during in vitro studies. Further-more, FESEM and AAS studies compliments the analysis ofXRD and FTIR spectra. During the immersion of samples inSBF, calcium to phosphorus ratio (from EDX data) indicate thegrowth of the apatite phase. It has been inferred from BETstudies that growth of apatite depends upon the porous natureof the samples, High porosity increase the contact area ofsample with SBF which results in the increase in the apatite

V. Anand et al. / Ceramics International 42 (2016) 3638–36513650

growth. Pore size has been found to be lowest in the samplecontaining high content of borate. All the prepared sampleshave been observed to be non-toxic in nature with more than70.3% viable cells. Attachment with MG63 cell line shows thatsamples provide the positive environment for the growth ofcell line. It has been observed that increase in the content ofborate and magnesium leads to enhanced percentage of viablecells. Antimicrobial activity indicate the resistive nature ofsamples towards microorganisms.MZB-4 sample can be con-sidered as the best sample prepared in the laboratory due to thefollowing reasons. This sample has good bioactivity, slowdegradation, �87% drug release, 79.3% cell viability, excel-lent cell proliferation and good tendency to kill microorgan-isms. Results indicate that samples prepared in this study canhave potential clinical applications as osteoconductive carriersfor treating bone infection. Authors recommend the MZB-4composition for further in vivo testing for clinical applications.

Acknowledgments

The authors Vikas Anand and Kulwinder Kaur are gratefulto the financial assistance provided by the UGC, New Delhi(India) through SRF (NET)[F.17-74/2008(SA-I)] and DST,New Delhi (India) through INSPIRE program SRF[IF-120620]respectively.

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