Antibacterial and osteo-stimulatory effects of a borate-based glass series doped with strontium ions

Original Article

Antibacterial and osteo-stimulatoryeffects of a borate-based glass seriesdoped with strontium ions

Yiming Li1,2, Wendy Stone3, Emil H Schemitsch2, Paul Zalzal4,Marcello Papini1, Stephen D Waldman3,5 and Mark R Towler1,3

Abstract

This work considered the effect of both increasing additions of Strontium (Sr2þ) and incubation time on solubility and

both antibacterial and osteo-stimulatory effects of a series of glasses based on the B2O3–P2O5–CaCO3–Na2CO3–TiO2–

SrCO3 series. The amorphous nature of all the glasses was confirmed by X-ray diffraction. Discs of each glass were

immersed in de-ionized water for 1, 7 and 30 days, and the water extracts were used for ion release profiles, pH

measurements and cytotoxicity testing. Atomic absorption spectroscopy was employed to detect the release of Naþ,

Ca2þ and Sr2þ ions from the glasses with respect to maturation, which indicated that the addition of Sr2þ retarded

solubility of the glass series. This effect was also confirmed by weight loss analysis through comparing the initial weight of

glass discs before and after periods of incubation. The incorporation of Sr2þ in the glasses did not influence the pH of the

water extracts when the glasses were stored for up to 30 days. Cytotoxicity testing with an osteoblastic cell line

(MC3T3-E1) indicated that glasses with the higher (20 mol% and 25 mol%) Sr2þ incorporation promoted proliferation

of osteoblast cells, while the glasses with lower Sr2þ contents inhibited cell growth. The glass series, except for Ly-B5

(which contained the highest Sr2þ incorporation; 25 mol%), were bacteriostatic against S. aureus in the short term (1–7

days) as a result of the dissolution products released.

Keywords

Borate-based glass, strontium, solubility, antibacterial effect, biocompatibility

Introduction

In order to improve the osteointegration of Ti6Al4Vtotal hip replacement (THR) devices and promote sta-bility at the implant/bone interface upon implantation,hydroxyapatite (HA) has been applied as a coating,because HA is chemically similar to the mineral phaseof human bone.1 Such implants have been employed inTHR for over 20 years, during which 97.1% survival ata 10-year follow-up clinical study has been recorded.1–3

However, the long-term stability of HA coatings is stillunder debate.3,4 Significant loss of the HA coating onboth immobilised and continuously loaded implantshas been demonstrated in vivo.5 Fractures between thecoating and the Ti6Al4V substrate have been observedafter implantation times as short as 12 weeks and aslong as one year;6,7 the primary reason for the failure atthe interface is the residual stress due to the mismatch

of the coefficients of thermal expansion (CTE) of theceramic and metal components, which can inducemicro-cracking initiating the de-bonding of the coatingfrom the substrate.8–10 The micro-cracking of silicateglass coatings on Ti6Al4V substrates due to mismatchof CTE has also been recorded.11–13 However, unlikeHA and silicate glasses, borate-based glasses can have

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DOI: 10.1177/0885328216672088

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1Department of Mechanical & Industrial Engineering, Ryerson University,

Toronto, ON, Canada2Keenan Research Centre, St. Michael’s Hospital, Toronto, ON, Canada3Chemistry and Biology, Ryerson University, Toronto, ON, Canada4Oakville Memorial Hospital, Oakville, ON, Canada5Chemical Engineering, Ryerson University, Toronto, ON, Canada

Corresponding author:

Mark R Towler, Faculty of Engineering and Architectural Science,

Department of Mechanical & Industrial Engineering, Ryerson University,

350 Victoria Street, Toronto M5B 2K3, ON, Canada.

Email: [email protected]

at RYERSON UNIV on December 5, 2016jba.sagepub.comDownloaded from

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similar CTEs to Ti6Al4V.14 Borate-based glasses arealso capable of forming chemical bonds between boneand the implant onto which they are coated,15 and arereported to offer a favorable substrate for the attach-ment and proliferation of osteogenic cells,16 and cancontribute to the healing of segmental defectsin vivo.17 Borate-based bioactive glasses, then, can beconsidered as coating candidates for THR devices.14

Dissolution products of borate-based glasses havebeen reported to facilitate the formation of newbone.18–20 The rate and extent of ion release will influ-ence new bone formation and borate-based glasses tendto have a high dissolution rate.21 Cell damage can beinduced by high concentrations of dissolution productsand the dramatic change in pH of the environment canoccur as a result of this degradation.22 High concentra-tions of Ca2þ (>32mg/L) can decrease osteoblast viabil-ity,23 and higher than 2.5mM BO3�

3 can inhibit bone cell(MC3TC-E1) proliferation.24 In addition, the resorptionand degradability of the glass coating can result in theloss of the coating–substrate bond strength and subse-quently retard implant fixation.25 It is therefore criticalto tune the solubility of the borate-based glasses to therate of formation of new bone. Addition of Ca2þ hasbeen reported to retard the dissolution rate of the glassesby impeding the migration pathway of Naþ in the glassstructure.26 Sr2þ has a similar ionic structure to Ca2þ,and therefore, Sr2þ incorporation is expected to modifythe dissolution of the glasses. Sr2þ has also been reportedto increase osteoblast proliferation in vivo and stimulatebone formation in vitro.27 Previous studies have investi-gated the influence of different Sr2þ on the solubility andbioactivity of borate-based glasses by incorporatingstrontium by partially substituting it for other modifiers(like MgO or CaO).18,28–32 Varying amounts of glassmodifiers substituting for B2O3 have also been reportedto influence the physical properties of borate-basedglasses.33–35 However, the influence of different amountsof SrCO3 (or SrO) at the expense of B2O3 on the solu-bility and bioactivity of borate-based glasses has notbeen researched.

In addition to introducing chemical bondingbetween the bone and the implant, the bioactive coatingcan be formulated to impart an antibacterial effect tothe surrounding environment as it degrades. Prostheticjoint infection can occur at the time of implantation.Staphylococcus aureus (S. aureus) is often the majorpathogen in metallic implant infections;36,37 a study ofisolates from 242 orthopedic patients confirmed that S.aureus is the most prevalent etiological agent of ortho-pedic infection.38 Previous studies indicated that stron-tium-containing bone cements reduce the cell counts ofS. aureus,39 and boron-containing bioactive glasses(MBG0118 and MBG0123) exert antibacterial effectsagainst S. aureus.40

The aim of this study is to investigate the influence ofa range of Sr2þ contents and incubation time on thesolubility and osteo-stimulatory and antibacterialeffects on S. aureus of borate-based glasses designedfor use as coatings on surgical implants.

Materials and methods

Glass sample preparation

Six glasses (Ly-B0 to Ly-B5) were formulated based onthe B2O3–P2O5–CaCO3–Na2CO3–TiO2–SrCO3 glassseries with increasing amounts of SrCO3 (from 5 to25mol%) at the expense of B2O3. The control glass,Ly-B0, was free of SrCO3. The compositions of theglasses are presented in Table 1. Glasses were preparedby weighing out appropriate amounts of analyticalgrade reagents in powder form, firing the mixtures(1300�C, 1 h) in a silica crucible, and subsequentlyshock quenching them into water. The resulting fritswere dried, ground and sieved to retrieve powderswith a mean particle size of less than 20 mm.

X-ray diffraction

Diffraction patterns were collected using a D2 PHASER(Bruker AXS Inc., WI, USA). Glass powder sampleswere packed into standard stainless steel sampleholders. A generator voltage of 30 kV and a tube currentof 10 mA were employed. Diffractograms were collectedin the range 20�< 2y< 90�, at a scan step size 0.02�and acount time of 0.3 s.

Atomic absorption spectroscopy

Glass powder discs (2.2� 6.4�mm, n¼ 9) fabricatedfor atomic absorption spectroscopy (AAS) were pro-duced by pressing the glass powders into moulds andthen annealing at 50�C above their Tgs, previouslydetermined by a combined differential thermal

Table 1. Compositions of the borate glass series, displayed in

mol%.

LY-B0 LY-B1 LY-B2 LY-B3 LY-B4 LY-B5

B2O3 59 54 49 44 39 34

CaCO3 13 13 13 13 13 13

P2O5 3 3 3 3 3 3

Na2CO3 15 15 15 15 15 15

TiO2 10 10 10 10 10 10

SrCO3 0 5 10 15 20 25

Total 100 100 100 100 100 100

2 Journal of Biomaterials Applications 0(0)



analyser–thermal gravimetric analyser (DTA-TGA,SDT Q600, TA Instruments, New Castle, DE, USA).The discs were then immersed in 15mL de-ionizedwater for 1, 7 and 30 days (three samples of eachglass for each incubation period). Ionic concentrationsof Naþ, Ca2þ and Sr2þ were evaluated from the waterextracts utilising a Perkin Elmer Analyst 800 AtomicAbsorption Spectrometer (Waltham, MA, USA). Thewater extracts were subsequently used for pH analysisand cell culture testing.

pH analysis

Changes in pH of the water extracts were monitored bya Corning 430 pH meter (Corning, NY, USA). Prior totesting, the pH meter was calibrated using pH buffersolution 4.00� 0.02 and 7.00� 0.02 (Fisher Scientific,Pittsburgh, PA, USA). Sterile de-ionized water(pH¼ 7.0) was used as a control and was measured ateach time period for calibration purposes.

Weight loss

Weight loss measurements were carried out afterremoving the glass powder discs from de-ionizedwater after the incubation times of 1, 7 and 30 daysand dried for 24 h at 37�C. The equation used to calcu-late the weight loss (�W) is

�W ¼W0 �W

W0ð1Þ

where W0 is the initial mass of the glass disc, which isapproximately 0.1 g, and W is the mass of the disc aftera certain incubation period.

Agar disk-diffusion test

The antibacterial activity of the borate-based glasseswas evaluated against S. aureus using the agar disk dif-fusion method. Tryptic Soy Broth (TSB; Sigma Aldrich,Oakville, ON, Canada) was used for the culture of S.aureus. All organisms were grown in 100mL TSB to acell concentration of 1� 107cells/mL (20 h, 37�C, aer-obically, 250 r/min). Preparation of the TSA disk-diffu-sion plates involved aseptically spreading 100 mL of theundiluted culture per plate. The pressed glass powderdiscs with heat treatment were also used in the antibac-terial test. Glass powder discs (n¼ 3) were placed on theinoculated plates and the plates were cultured for 1, 7and 30 days at 37�C, sealing the bags to prevent desic-cation. Three glass disks, of different compositions,were assessed per plate. Callipers were used to measurethe diameter of glass powder discs and the halo of

inhibition at three different points for each disk, andthen zone sizes were calculated as follows

Inhibition Zone ðmmÞ ¼Halo��Disc�

2ð2Þ

All glasses were analysed in triplicate and mean zonesizes standard deviations were calculated.

Cytotoxicity testing

Pre-osteoblastic MC3T3-E1 cells (ATCC CRL-2593,ATCC, Manassas, VA, USA) from passages 3–5 wereused for this study and were maintained in aMEMmedia supplemented with 10% FBS and 1% (2mM)L-glutamine (Cambrex, MD, USA) within a cell cultureincubator at 37�C/5% CO2/95% air atmosphere. Cellswere seeded into 24-well plates at a density of 5500cells/cm2 and incubated for 24 h prior to testing.Culture media (1mL) was then further supplementedwith 100mL of liquid extract (from the solubility sam-ples at 30 days for all glasses; n¼ 3 per sample well) andthen incubated for 24 h at 37�/5% CO2. The MTT wasadded in an amount equal to 10% of the culturemedium volume/well. The cultures were then re-incu-bated for a further 2 h (37�C/5% CO2) after which theywere removed from the incubator and the resultant for-mazan crystals dissolved by adding an amount of MTTSolubilisation Solution (10% Triton x-100 in AcidicIsopropanol (0.1 n HCI)) equal to the original culturemedium volume. Once the crystals were fully dissolved,the absorbance was measured at a wavelength of570 nm. All results were expressed relative to the meta-bolic activity of cells seeded (at the same density) ontissue culture plastic (n¼ 3) as controls.

Statistical analysis

One-way analysis of variance (ANOVA) was employedto compare the changes in ion release profiles, pHvalues, weight loss, inhibition zone and MTT assaydata of the experimental materials in relation to (1)different incubation times (e.g. 1, 7 and 30 days), ofeach composition and (2) different glass compositionswith the same incubation time. The comparison of rele-vant means was performed using the post hocBonferroni test. Differences between groups weredeemed significant when p� 0.05.

Results and discussion

X-ray diffraction

X-ray diffraction (XRD) patterns of all the materialsare shown in Figure 1. The broad XRD curves without

Li et al. 3



any detectable sharp peaks confirm the glassy nature ofthe original glasses and annealed discs.41,42 Crystallinephases can inhibit the dissolution of bioactive glasses,influencing glass solubility and biocompatibility.43

Therefore, it is important to retain the amorphousnature of the samples.

AAS

The concentrations of Naþ, Ca2þ and Sr2þ released,with respect to incubation time and glass composition,are shown in Figures 2 to 4, respectively.Unfortunately, the concentration of BO3�

3 cannot beaccurately determined by AAS because of the insensi-tivity of this technique for low atomic number elem-ents.21 For all six glasses, the concentrations of Naþ

and Ca2þ in the water extracts (Figures 2 to 4) werefound to significantly increase with incubation time(p� 0.05, Table 2), while the concentrations of Naþ

and Ca2þ decreased along with increased Sr2þ incorp-oration (p� 0.05, Table 2) in the glasses after 30-dayincubation. For Ly-B0, Ly-B1 and Ly-B2, the concen-trations of Naþ measured after seven-day incubationwere very close to those measured after 30-day incuba-tion, while Naþ concentrations released after seven-dayincubation for the other three glasses were less thanthose after 30-day incubation time. Thus, the releaserate of Naþ in the first week was retarded by morethan 15mol% addition of Sr2þ in the glass series.

The first step in the degradation of a glass in anaqueous environment is the ion exchange44,45 betweenthe glass network modifier cations and Hþ from theimmersing solution. Usually, the Naþ-water reactiondominates the process due to the initial enrichment ofNaþ on the glass surface.26,45 This explains why theconcentrations of Naþ released from each glass arehigher than those of Ca2þ and Sr2þ (Figures 2 to 4).

Additional ions are then transported from the glassbulk to the surface to complete the dissolution process.One of the pathways of ion migration is assisted bycorrelated forward–backward motion of an ion by

20

Ly-B5

Ly-B4

Ly-B3

Ly-B2

Ly-B1

Ly-B0

30 40 50

Degree° (2 Theta)

60 70 80 90

Figure 1. XRD patterns of the original glasses and glass power

discs.

900

Con

cent

ratio

n of

Na+

(mg/

L)

821.2

1 Day7 Days30 Days754.3

743.3 726.6603.3

506.5

750

600

450

300

150

0Ly-B0 Ly-B1 Ly-B2 Ly-B3 Ly-B4 Ly-B5

Na+

Figure 2. Concentration of Naþ from the water extracts of

the glass series versus incubation time.

80

Con

cent

ratio

n of

Sr2+

(mg/

L)

Ly-B0 Ly-B1 Ly-B2 Ly-B3 Ly-B4 Ly-B5

72.963.9

57.9

1 Day

7 Days30 Days

Sr2+

40.35

31.7

60

40

20

0

Figure 4. Concentration of Sr2þ from the water extracts of


35

Con

cent

ratio

n of

Ca2+

(mg/

L)

29.91 Day

7 Days30 Days

18.8

15.714.2

10.6

5.3

30

25

20

15

10

5


Ca2+

Figure 3. Concentration of Ca2þ from the water extracts of





moving to an intermediate position and then returningto its initial site after the passage of the migrating ion.26

The movement of a modifier cation in the matrixinvolves a change in its coordination, where at least afraction of the coordinated oxygen atoms have beenreplaced;26 that is, a number of R–O bonds have tobe broken. It is difficult for Sr2þ to provide such ‘‘tran-sient sites’’26 due to the high strength of the ionic Sr–Obonding;46 thus Sr2þ might block the pathway of othercations. As a result, it is proposed that Sr2þ hinderedthe movement of other dissolution products, reducingsolubility of the glasses.

As expected, the ion release profile of Sr2þ (Figure 4)experienced a significant increase with both incubationtime and Sr2þ incorporation in the glass series (p� 0.05,Table 2). The highest Sr2þ concentrations in the waterextracts ranged from 31.7mg/L to 72.9mg/L (form Ly-B0 to Ly-B5) after 30-day incubation. Previous studieshave reported that Sr2þ concentrations in the rangefrom 8.76mg/L to 87.62mg/L induce stimulatoryeffects on osteoblasts and inhibit bone resorptionin vitro.47,48 It has also been presented that the higherthe Sr2þ concentration, the more pronounced theinhibitory effect on osteoclasts differentiation up toSr2þ concentrations as high as 2102.8mg/L.49

Therefore, addition of Sr2þ to this borate-based glassseries is expected to be beneficial for bone cell prolifer-ation, and this hypothesis will be investigated in thecytotoxicity testing.

Weight loss

As ion release from the glass is accompanied by adecrease in mass, weight loss measurements provide auseful parameter for monitoring the kinetics of glasssolubility. Weight loss of the glass powder discs withrespect to incubation time is shown in Figure 5. Weightloss of the glasses after the 30-day incubation perioddecreased in line with increased Sr2þ incorporationin the glasses (p� 0.05, Table 3), which indicatedthat lower amount of ions released from the glasses

with more Sr2þ incorporation. In addition, for Ly-B0,Ly-B1 and Ly-B2, there was no significant difference(p� 0.05, Table 3) between weight loss recorded after7- and 30-day incubation period, which indicated thatthe amount of ions released from the three glassesreached the limit only after the 7-day incubationperiod. In other words, the dissolution rate of thethree glasses was highest in the first week. However,the weight loss of Ly-B3, Ly-B4 and Ly-B5 increasedsignificantly (p� 0.05, Table 3) from 7-day to 30-dayincubation, which implied that the solubility ofthe glasses in the first seven days was retarded dueto more than 15mol% Sr2þ incorporation in theglasses.

Table 2. Comparison of each ion release profile (n¼ 3) with respect to incubation time, and comparison of each ion concentration

(n¼ 3) after 30-day incubation with respect to different Sr2þ incorporation in the glasses, where p� 0.05 represents significant

difference.

Incubation time (1 day vs. 30 days) Different Sr2þ incorporation

Ions Ly-B0 Ly-B1 Ly-B2 Ly-B3 Ly-B4 Ly-B5 Ly-B0 vs.

Ly-B1

Ly-B0 vs.

Ly-B2

Ly-B0 vs.

Ly-B3

Ly-B0 vs.

Ly-B4

Ly-B0 vs.

Ly-B5

Naþ 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.000

Ca2þ 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Ly-B1 vs.

Ly-B2

Ly-B1 vs.

Ly-B3

Ly-B1 vs.

Ly-B4

Ly-B1 vs.

Ly-B5

Sr2þ 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

60

Wei

ght L

oss

(%)

50

40

30

20

10

0

1 Day 7 Days

Ly-B0Ly-B1Ly-B2Ly-B3Ly-B4Ly-B5

30 Days

Figure 5. Weight Loss of the glass discs versus incubation time.

Table 3. Comparison of weight loss of each glass (n¼ 3) with

respect to incubation time, where p� 0.05 represents significant

difference.

Incubation Time (7 Days vs. 30 Days)


1.000 0.590 0.875 0.001 0.001 0.003

Li et al. 5



pH

pH of the water extracts of the glass series over 1, 7 and30 days are shown in Figure 6. The pH of de-ionizedwater was 7.0. The statistical analysis of pH profilesdemonstrated that the pH of the water extractsincreased significantly with more than 5mol% additionof Sr2þ (p� 0.05, Table 4). However, for each glass, pHvalues did not change with immersion time (p� 0.05,Table 4).

In the reaction between water and glass, Hþ isdonated to NBO and the remaining OH� from thewater molecule is freed. As a consequence, pH of thesolution increases. Based on previous studies, the pH ofsilicate glasses immersed in a neutral aqueous environ-ment for 30 days are in the range of 11–12,40,41 whilethe pH of borate-based glasses are in the range of 9–10.21,50 It is the acidity of B(OH)3 that causes thiseffect.21 However, the pH of the solution still increasesbecause the strong alkaline NaOH overwhelms theweak acidic B(OH)3.

The alkaline pH resulting from the degradation ofthe glasses has a positive influence on bioactivity.51,52 It

has been reported that bone cells respond to pH changeand higher pHs inhibit the activity of osteoclasts redu-cing bone resorption.51 Pro-resorptive agents such asRANKL and parathyroid hormone have little or nostimulatory activity on osteoclasts at pH of 7.4 orabove.52 The results of pH testing also manifest that,for each glass, pH of the water extracts remained in acertain range (p� 0.05, Table 4). Since the mechanismof bone cell formation is very sensitive to change ofacidic balance, precise maintenance of pH value inthe blood and extracellular fluid is required.53

Antibacterial effect

The diameters of inhibition zones of the borate glassesagainst S. aureus with respect to maturation are shownin Figure 7. There is no inhibition zone for Ly-B5against S. aureus. The mean sizes of the inhibitionzones after one-day incubation are 5.6mm for Ly-B0,5.8mm for Ly-B1, 3.5mm for Ly-B2, 4.6mm for Ly-B3and 4.1mm for Ly-B4. Based on statistical analysis,there is no difference (p� 0.05, Table 5) among thesizes of the inhibition zones for these five glasses after

Table 4. Comparison of pH of each glass (n¼ 3) with respect to incubation time, and comparison of pH after 30-day incubation with

respect to different Sr2þ incorporation in the glasses, where p� 0.05 represents significant difference.

Incubation time (1 day vs. 30 days)


0.008 0.293 0.593 0.387 0.105 0.130

Different Sr2þ incorporation

Ly-B0 vs. Ly-B1 Ly-B0 vs. Ly-B2 Ly-B0 vs. Ly-B3 Ly-B0 vs. Ly-B4 Ly-B0 vs. Ly-B5

0.657 0.000 0.000 0.000 0.000

10

Dia

met

er o

f inh

ibiti

on z

one

(mm

)

1-Day

NO

Inhi

bitio

n Z

oon

NO

Inhi

bitio

n Z

oon

NO

Inhi

bitio

n Z

oon

NO

Inhi

bitio

n Z

oon

7-Day30-Day

8

6

4

2


Figure 7. Diameters of inhibition zones of the glasses against S.

aureus with different maturation times, where deviations are

presented.

9.8 1 Day


7 Days

30 Days

pH V

alue

9.6

9.4

9.2

9.0

8.8

8.6

8.818.86

9.02

9.26

9.38

9.57

Figure 6. pH values of the water extracts of the glass series

with different incubation times, where the pH values of 30-day

incubation are tagged on the image.




one-day incubation. In addition, there is no difference(p� 0.05, Table 5) among the sizes of inhibition zonesafter 7-day and 30-day incubation for Ly-B0 and Ly-B1. However, the inhibition zones after the 7-day incu-bation period experienced a significant decrease(p� 0.05, Table 5) for the glasses with 15–25mol%addition of Sr2þ.

Based on the previous studies concerning the anti-bacterial effects of bioactive glasses, some dissolutionproducts such as zinc or silver ions kill bacteria byinhibiting multiple activities in the bacterial cell, suchas glycolysis, trans-membrane proton translocation andacid tolerance.54 Furthermore, the antibacterial effect isproportional to the concentration of these ions.54 Sr2þ

has been reported to exhibit antibacterial activityagainst S. aureus, but at a weak level.55 It is postulatedthat Sr2þ exerts its antibacterial ability by inhibitingbacterial growth and reproduction and impeding per-meability of cytoplasmic membrane, cell wall synthesis,replication of bacterial chromosomes and cell metabol-ism.39 Based on the AAS data, the increased Sr2þ

released from the glasses with higher Sr2þ loadingshas no positive effect on inhibition zone size (Figure7). An inhibition zone also exists for Ly-B0 whichdoes not contain, or subsequently release, Sr2þ. In add-ition, the dissolution mechanism of Sr2þ in TSB cultureof small volume (100mL) might be different from that inde-ionized water. Here, we assume that other dissol-ution products may contribute to the inhibition zone.It has been reported that boron-containing bioactiveglass exerts antibacterial effects against S. aureus dueto BO3�

3 release, but the antibacterial mechanism ofBO3�

3 is still unknown.40 In addition, a boron-basedantibacterial (AN3365) was reported to reveal antibac-terial activity against S. aureus.56 Naþ and Ca2þ canalso inhibit the growth of S. aureus.57,58 The weight lossdata (Figure 5) manifests that lower numbers of dissol-ution products release from the glasses with Sr2þ

incorporation, especially for the glasses with morethan 5mol% addition of Sr2þ, which is in agreement

with the fact that the sizes of the inhibition zonesdecreased or disappeared after seven-day incubationwith more than 5mol% Sr2þ contents in the glasses.Thus, these glass discs are able to inhibit bacterial pro-liferation in one week, but not in the long term. Insummary, the concentrations of all ions may have aninfluence on the sizes of the inhibition zones. In otherwords, a combined or individual effect of some ionsamong BO3�

3 , Sr2þ, Naþ and Ca2þ makes the contribu-tion to the bacteriostatic59 behaviour of the glasses.

Cytotoxicity testing

The cytotoxicity results from glass powder disc extractsafter 30-day incubation are shown in Figure 8. There isno difference (p� 0.05, Table 6) among the cell meta-bolic activity of Ly-B0, Ly-B1, Ly-B2 and Ly-B3glasses, which all experienced significantly reduced pro-liferation compared to control (p� 0.05, Table 6).However, the cell proliferation was significantlyenhanced (p� 0.05, Table 6) in response to the Ly-B4(105%) and Ly-B5 (120%) glass formulations.Compared to the control group, the enhancement ofcell metabolic activity on Ly-B5 was significant(p¼ 0.002).

Based on the results of pH measurement and AASanalysis, concentrations of Sr2þ ions released increasedwith increasing Sr2þ contents in the glass, while theconcentrations of BO3�

3 decreased after 30-day incuba-tion, as would be expected. It has been reported thathigh concentrations of BO3�

3 (>1mg/L) inhibit prolif-eration of osteoblasts (MC3T3-E1),24,60 while concen-trations of Sr2þ in the range from 8.76mg/L to87.62mg/L promote the proliferation of osteoblasticcells (MC3T3-E1) in vitro.43 Therefore, this borate-

125

Nor

mal

ized

cel

l met

abol

ic a

ctiv

ity (

%)

100

75

50

25

0Control Ly-B0 Ly-B1 Ly-B2 Ly-B3 Ly-B4 Ly-B5

Figure 8. Cell metabolic activity normalised by the control

group from sintered glass powder disc extracts after 30-day

incubation.

Table 5. Means comparison of the size of inhibition zones

(n¼ 3) after 1, 7 and 30 days incubation with respect to different

Sr2þ incorporation in the glasses, where p� 0.05 represents

significant difference.


Ly-B0 vs.

Ly-B1

Ly-B0 vs.

Ly-B2

Ly-B0 vs.

Ly-B3

Ly-B0 vs.

Ly-B4

1 Day 1.000 0.205 1.000 0.829

7 Days 1.000 0.000 0.004 0.000

30 Days 0.233

Li et al. 7



based glass series promote the proliferation of osteo-blastic cells with 20mol% and 25mol% Sr2þ incorpo-rated in the glasses.

Conclusion

This study was conducted to investigate the solubilityand antibacterial and osteo-stimulatory effects of anovel borate-based glass series with respect to bothincreasing additions of Sr2þ and incubation time. Theconcentrations of Naþ, Ca2þ and Sr2þ in the waterextracts experienced significant increases with incuba-tion time. However, less Naþ and Ca2þ released fromthe glasses with increasing Sr2þ incorporation after 30-day incubation, indicating that the Sr2þ dopingretarded the dissolution rate of the glasses. Sr2þ incorp-oration also made a contribution to the maintenance ofpH values of the water extracts along with incubationtime. In addition, the glass series promoted prolifer-ation of osteoblastic cells with 20mol% and 25mol%Sr2þ contents, while the other glasses impeded cellgrowth. All members of the glass series, except forLy-B5, exhibited bacteriostatic behaviour against S.aureus in the short term (1–7 days), which might be aresult of a combined or individual effect of some of thedissolution products.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest withrespect to the research, authorship, and/or publication of thisarticle.

Funding

The author(s) disclosed receipt of the following financial sup-port for the research, authorship, and/or publication of thisarticle: The authors gratefully acknowledge the support of

both the Canadian Institute of Health Research (CIHR)and the Natural Sciences and Engineering Research Councilof Canada (NSERC) through the Collaborative HealthResearch Project (CHRP) program (grant no. 315694-DAN).

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Table 6. Comparison of cell metabolic activity (relative to control) (n¼ 3) after 30-day incubation with respect to the control group

and different Sr2þ incorporation in the glasses, where p� 0.05 represents significant difference.


Ly-B0 vs. Ly-B1 Ly-B0 vs. Ly-B2 Ly-B0 vs. Ly-B3 Ly-B0 vs. Ly-B4 Ly-B0 vs. Ly-B5

1.000 1.000 1.000 0.002 0.000

Control vs. Ly-B0 Control vs. Ly-B1 Control vs. Ly-B2 Control vs. Ly-B3 Control vs. Ly-B4 Control vs. Ly-B5

0.003 0.004 0.006 0.019 1.00 0.002




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Antibacterial and osteo-stimulatory effects of a borate-based glass series doped with strontium ions