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

Abstract

This work considered the effect of both increasing additions of Strontium (Sr²⁺) 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 Na⁺, Ca²⁺ and Sr²⁺ ions from the glasses with respect to maturation, which indicated that the addition of Sr²⁺ 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 Sr²⁺ 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%) Sr²⁺ incorporation promoted proliferation of osteoblast cells, while the glasses with lower Sr²⁺ contents inhibited cell growth. The glass series, except for Ly-B5 (which contained the highest Sr²⁺ 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 Ti6Al4V total hip replacement (THR) devices and promote stability at the implant/bone interface upon implantation, hydroxyapatite (HA) has been applied as a coating, because HA is chemically similar to the mineral phase of human bone.¹ Such implants have been employed in THR for over 20 years, during which 97.1% survival at a 10-year follow-up clinical study has been recorded.^1–3 However, the long-term stability of HA coatings is still under debate.^3,4 Significant loss of the HA coating on both immobilised and continuously loaded implants has been demonstrated in vivo.⁵ Fractures between the coating and the Ti6Al4V substrate have been observed after implantation times as short as 12 weeks and as long as one year;^6,7 the primary reason for the failure at the interface is the residual stress due to the mismatch of the coefficients of thermal expansion (CTE) of the ceramic and metal components, which can induce micro-cracking initiating the de-bonding of the coating from the substrate.^8–10 The micro-cracking of silicate glass coatings on Ti6Al4V substrates due to mismatch of CTE has also been recorded.^11–13 However, unlike HA and silicate glasses, borate-based glasses can have similar CTEs to Ti6Al4V.¹⁴ Borate-based glasses are also capable of forming chemical bonds between bone and the implant onto which they are coated,¹⁵ and are reported to offer a favorable substrate for the attachment and proliferation of osteogenic cells,¹⁶ and can contribute to the healing of segmental defects in vivo.¹⁷ Borate-based bioactive glasses, then, can be considered as coating candidates for THR devices.¹⁴

Dissolution products of borate-based glasses have been reported to facilitate the formation of new bone.^18–20 The rate and extent of ion release will influence new bone formation and borate-based glasses tend to have a high dissolution rate.²¹ Cell damage can be induced by high concentrations of dissolution products and the dramatic change in pH of the environment can occur as a result of this degradation.²² High concentrations of Ca²⁺ (>32 mg/L) can decrease osteoblast viability,²³ and higher than 2.5 mM ${BO}_{3}^{3 -}$ can inhibit bone cell (MC3TC-E1) proliferation.²⁴ In addition, the resorption and degradability of the glass coating can result in the loss of the coating–substrate bond strength and subsequently retard implant fixation.²⁵ It is therefore critical to tune the solubility of the borate-based glasses to the rate of formation of new bone. Addition of Ca²⁺ has been reported to retard the dissolution rate of the glasses by impeding the migration pathway of Na⁺ in the glass structure.²⁶ Sr²⁺ has a similar ionic structure to Ca²⁺, and therefore, Sr²⁺ incorporation is expected to modify the dissolution of the glasses. Sr²⁺ has also been reported to increase osteoblast proliferation in vivo and stimulate bone formation in vitro.²⁷ Previous studies have investigated the influence of different Sr²⁺ on the solubility and bioactivity of borate-based glasses by incorporating strontium by partially substituting it for other modifiers (like MgO or CaO).^18,28–32 Varying amounts of glass modifiers substituting for B₂O₃ have also been reported to influence the physical properties of borate-based glasses.^33–35 However, the influence of different amounts of SrCO₃ (or SrO) at the expense of B₂O₃ on the solubility and bioactivity of borate-based glasses has not been researched.

In addition to introducing chemical bonding between the bone and the implant, the bioactive coating can be formulated to impart an antibacterial effect to the surrounding environment as it degrades. Prosthetic joint infection can occur at the time of implantation. Staphylococcus aureus (S. aureus) is often the major pathogen in metallic implant infections;^36,37 a study of isolates from 242 orthopedic patients confirmed that S. aureus is the most prevalent etiological agent of orthopedic infection.³⁸ Previous studies indicated that strontium-containing bone cements reduce the cell counts of S. aureus,³⁹ and boron-containing bioactive glasses (MBG0118 and MBG0123) exert antibacterial effects against S. aureus.⁴⁰

The aim of this study is to investigate the influence of a range of Sr²⁺ contents and incubation time on the solubility and osteo-stimulatory and antibacterial effects on S. aureus of borate-based glasses designed for use as coatings on surgical implants.

Materials and methods

Glass sample preparation

Six glasses (Ly-B0 to Ly-B5) were formulated based on the B₂O₃–P₂O₅–CaCO₃–Na₂CO₃–TiO₂–SrCO₃ glass series with increasing amounts of SrCO₃ (from 5 to 25 mol%) at the expense of B₂O₃. The control glass, Ly-B0, was free of SrCO₃. The compositions of the glasses are presented in Table 1. Glasses were prepared by weighing out appropriate amounts of analytical grade reagents in powder form, firing the mixtures (1300℃, 1 h) in a silica crucible, and subsequently shock quenching them into water. The resulting frits were dried, ground and sieved to retrieve powders with a mean particle size of less than 20 µm.

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

X-ray diffraction

Diffraction patterns were collected using a D2 PHASER (Bruker AXS Inc., WI, USA). Glass powder samples were packed into standard stainless steel sample holders. A generator voltage of 30 kV and a tube current of 10 mA were employed. Diffractograms were collected in the range 20° < 2θ < 90°, at a scan step size 0.02°and a count time of 0.3 s.

Atomic absorption spectroscopy

Glass powder discs (2.2 × 6.4 φmm, n = 9) fabricated for atomic absorption spectroscopy (AAS) were produced by pressing the glass powders into moulds and then annealing at 50℃ above their T_gs, previously determined by a combined differential thermal analyser–thermal gravimetric analyser (DTA-TGA, SDT Q600, TA Instruments, New Castle, DE, USA). The discs were then immersed in 15 mL de-ionized water for 1, 7 and 30 days (three samples of each glass for each incubation period). Ionic concentrations of Na⁺, Ca²⁺ and Sr²⁺ were evaluated from the water extracts utilising a Perkin Elmer Analyst 800 Atomic Absorption Spectrometer (Waltham, MA, USA). The water extracts were subsequently used for pH analysis and cell culture testing.

pH analysis

Changes in pH of the water extracts were monitored by a Corning 430 pH meter (Corning, NY, USA). Prior to testing, the pH meter was calibrated using pH buffer solution 4.00 ± 0.02 and 7.00 ± 0.02 (Fisher Scientific, Pittsburgh, PA, USA). Sterile de-ionized water (pH = 7.0) was used as a control and was measured at each time period for calibration purposes.

Weight loss

Weight loss measurements were carried out after removing the glass powder discs from de-ionized water after the incubation times of 1, 7 and 30 days and dried for 24 h at 37℃. The equation used to calculate the weight loss (ΔW) is

Δ W = \frac{W_{0} - W}{W_{0}}

(1)

where W₀ is the initial mass of the glass disc, which is approximately 0.1 g, and W is the mass of the disc after a certain incubation period.

Agar disk-diffusion test

The antibacterial activity of the borate-based glasses was evaluated against S. aureus using the agar disk diffusion method. Tryptic Soy Broth (TSB; Sigma Aldrich, Oakville, ON, Canada) was used for the culture of S. aureus. All organisms were grown in 100 mL TSB to a cell concentration of 1 × 10⁷cells/mL (20 h, 37℃, aerobically, 250 r/min). Preparation of the TSA disk-diffusion plates involved aseptically spreading 100 µL of the undiluted culture per plate. The pressed glass powder discs with heat treatment were also used in the antibacterial test. Glass powder discs (n = 3) were placed on the inoculated plates and the plates were cultured for 1, 7 and 30 days at 37℃, sealing the bags to prevent desiccation. Three glass disks, of different compositions, were assessed per plate. Callipers were used to measure the diameter of glass powder discs and the halo of inhibition at three different points for each disk, and then zone sizes were calculated as follows

InhibitionZone (mm) = \frac{Halo φ - Disc φ}{2}

(2)

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

Cytotoxicity testing

Pre-osteoblastic MC3T3-E1 cells (ATCC CRL-2593, ATCC, Manassas, VA, USA) from passages 3–5 were used for this study and were maintained in αMEM media supplemented with 10% FBS and 1% (2 mM) l-glutamine (Cambrex, MD, USA) within a cell culture incubator at 37℃/5% CO₂/95% air atmosphere. Cells were seeded into 24-well plates at a density of 5500 cells/cm² and incubated for 24 h prior to testing. Culture media (1 mL) was then further supplemented with 100 µL of liquid extract (from the solubility samples at 30 days for all glasses; n = 3 per sample well) and then incubated for 24 h at 37°/5% CO₂. The MTT was added in an amount equal to 10% of the culture medium volume/well. The cultures were then re-incubated for a further 2 h (37℃/5% CO₂) after which they were removed from the incubator and the resultant formazan crystals dissolved by adding an amount of MTT Solubilisation Solution (10% Triton x-100 in Acidic Isopropanol (0.1 n HCI)) equal to the original culture medium volume. Once the crystals were fully dissolved, the absorbance was measured at a wavelength of 570 nm. All results were expressed relative to the metabolic activity of cells seeded (at the same density) on tissue culture plastic (n = 3) as controls.

Statistical analysis

One-way analysis of variance (ANOVA) was employed to compare the changes in ion release profiles, pH values, weight loss, inhibition zone and MTT assay data of the experimental materials in relation to (1) different incubation times (e.g. 1, 7 and 30 days), of each composition and (2) different glass compositions with the same incubation time. The comparison of relevant means was performed using the post hoc Bonferroni test. Differences between groups were deemed significant when p ≤ 0.05.

Results and discussion

X-ray diffraction

X-ray diffraction (XRD) patterns of all the materials are shown in Figure 1. The broad XRD curves without any detectable sharp peaks confirm the glassy nature of the original glasses and annealed discs.^41,42 Crystalline phases can inhibit the dissolution of bioactive glasses, influencing glass solubility and biocompatibility.⁴³ Therefore, it is important to retain the amorphous nature of the samples.

AAS

The concentrations of Na⁺, Ca²⁺ and Sr²⁺ released, with respect to incubation time and glass composition, are shown in Figures 2 to 4, respectively. Unfortunately, the concentration of ${BO}_{3}^{3 -}$ cannot be accurately determined by AAS because of the insensitivity of this technique for low atomic number elements.²¹ For all six glasses, the concentrations of Na⁺ and Ca²⁺ in the water extracts (Figures 2 to 4) were found to significantly increase with incubation time (p ≤ 0.05, Table 2), while the concentrations of Na⁺ and Ca²⁺ decreased along with increased Sr²⁺ incorporation (p ≤ 0.05, Table 2) in the glasses after 30-day incubation. For Ly-B0, Ly-B1 and Ly-B2, the concentrations of Na⁺ measured after seven-day incubation were very close to those measured after 30-day incubation, while Na⁺ concentrations released after seven-day incubation for the other three glasses were less than those after 30-day incubation time. Thus, the release rate of Na⁺ in the first week was retarded by more than 15 mol% addition of Sr²⁺ in the glass series.

Table 2.

Comparison of each ion release profile (n = 3) with respect to incubation time, and comparison of each ion concentration (n = 3) after 30-day incubation with respect to different Sr²⁺ incorporation in the glasses, where p ≤ 0.05 represents significant difference.

	Incubation time (1 day vs. 30 days)						Different Sr²⁺ 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
Na⁺	0.000	0.000	0.000	0.000	0.000	0.000	0.001	0.000	0.000	0.000	0.000
Ca²⁺	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
Sr²⁺	0.000	0.000	0.000	0.000	0.000	0.000		0.000	0.000	0.000	0.000

The first step in the degradation of a glass in an aqueous environment is the ion exchange^44,45 between the glass network modifier cations and H⁺ from the immersing solution. Usually, the Na⁺-water reaction dominates the process due to the initial enrichment of Na⁺ on the glass surface.^26,45 This explains why the concentrations of Na⁺ released from each glass are higher than those of Ca²⁺ and Sr²⁺ (Figures 2 to 4). Additional ions are then transported from the glass bulk to the surface to complete the dissolution process. One of the pathways of ion migration is assisted by correlated forward–backward motion of an ion by moving to an intermediate position and then returning to its initial site after the passage of the migrating ion.²⁶ The movement of a modifier cation in the matrix involves a change in its coordination, where at least a fraction of the coordinated oxygen atoms have been replaced;²⁶ that is, a number of R–O bonds have to be broken. It is difficult for Sr²⁺ to provide such “transient sites”²⁶ due to the high strength of the ionic Sr–O bonding;⁴⁶ thus Sr²⁺ might block the pathway of other cations. As a result, it is proposed that Sr²⁺ hindered the movement of other dissolution products, reducing solubility of the glasses.

As expected, the ion release profile of Sr²⁺ (Figure 4) experienced a significant increase with both incubation time and Sr²⁺ incorporation in the glass series (p ≤ 0.05, Table 2). The highest Sr²⁺ concentrations in the water extracts ranged from 31.7 mg/L to 72.9 mg/L (form Ly-B0 to Ly-B5) after 30-day incubation. Previous studies have reported that Sr²⁺ concentrations in the range from 8.76 mg/L to 87.62 mg/L induce stimulatory effects on osteoblasts and inhibit bone resorption in vitro.^47,48 It has also been presented that the higher the Sr²⁺ concentration, the more pronounced the inhibitory effect on osteoclasts differentiation up to Sr²⁺ concentrations as high as 2102.8 mg/L.⁴⁹ Therefore, addition of Sr²⁺ to this borate-based glass series is expected to be beneficial for bone cell proliferation, and this hypothesis will be investigated in the cytotoxicity testing.

Weight loss

As ion release from the glass is accompanied by a decrease in mass, weight loss measurements provide a useful parameter for monitoring the kinetics of glass solubility. Weight loss of the glass powder discs with respect to incubation time is shown in Figure 5. Weight loss of the glasses after the 30-day incubation period decreased in line with increased Sr²⁺ incorporation in the glasses (p ≤ 0.05, Table 3), which indicated that lower amount of ions released from the glasses with more Sr²⁺ 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 after 7- and 30-day incubation period, which indicated that the amount of ions released from the three glasses reached the limit only after the 7-day incubation period. In other words, the dissolution rate of the three glasses was highest in the first week. However, the weight loss of Ly-B3, Ly-B4 and Ly-B5 increased significantly (p ≤ 0.05, Table 3) from 7-day to 30-day incubation, which implied that the solubility of the glasses in the first seven days was retarded due to more than 15 mol% Sr²⁺ incorporation in the glasses.

Table 3.

Comparison of weight loss of each glass (n = 3) with respect to incubation time, where p ≤ 0.05 represents significant difference.

Incubation Time (7 Days vs. 30 Days)
Ly-B0	Ly-B1	Ly-B2	Ly-B3	Ly-B4	Ly-B5
1.000	0.590	0.875	0.001	0.001	0.003

pH

pH of the water extracts of the glass series over 1, 7 and 30 days are shown in Figure 6. The pH of de-ionized water was 7.0. The statistical analysis of pH profiles demonstrated that the pH of the water extracts increased significantly with more than 5 mol% addition of Sr²⁺ (p ≤ 0.05, Table 4). However, for each glass, pH values did not change with immersion time (p ≥ 0.05, Table 4).

Table 4.

Comparison of pH of each glass (n = 3) with respect to incubation time, and comparison of pH after 30-day incubation with respect to different Sr²⁺ incorporation in the glasses, where p ≤ 0.05 represents significant difference.

Incubation time (1 day vs. 30 days)
Ly-B0	Ly-B1	Ly-B2	Ly-B3	Ly-B4	Ly-B5
0.008	0.293	0.593	0.387	0.105	0.130
	Different Sr²⁺ 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

In the reaction between water and glass, H⁺ is donated to NBO and the remaining OH⁻ from the water molecule is freed. As a consequence, pH of the solution increases. Based on previous studies, the pH of silicate glasses immersed in a neutral aqueous environment for 30 days are in the range of 11–12,^40,41 while the pH of borate-based glasses are in the range of 9–10.^21,50 It is the acidity of B(OH)₃ that causes this effect.²¹ However, the pH of the solution still increases because the strong alkaline NaOH overwhelms the weak acidic B(OH)₃.

The alkaline pH resulting from the degradation of the glasses has a positive influence on bioactivity.^51,52 It has been reported that bone cells respond to pH change and higher pHs inhibit the activity of osteoclasts reducing bone resorption.⁵¹ Pro-resorptive agents such as RANKL and parathyroid hormone have little or no stimulatory activity on osteoclasts at pH of 7.4 or above.⁵² The results of pH testing also manifest that, for each glass, pH of the water extracts remained in a certain range (p ≥ 0.05, Table 4). Since the mechanism of bone cell formation is very sensitive to change of acidic balance, precise maintenance of pH value in the blood and extracellular fluid is required.⁵³

Antibacterial effect

The diameters of inhibition zones of the borate glasses against S. aureus with respect to maturation are shown in Figure 7. There is no inhibition zone for Ly-B5 against S. aureus. The mean sizes of the inhibition zones after one-day incubation are 5.6 mm for Ly-B0, 5.8 mm for Ly-B1, 3.5 mm for Ly-B2, 4.6 mm for Ly-B3 and 4.1 mm for Ly-B4. Based on statistical analysis, there is no difference (p ≥ 0.05, Table 5) among the sizes of the inhibition zones for these five glasses after one-day incubation. In addition, there is no difference (p ≥ 0.05, Table 5) among the sizes of inhibition zones after 7-day and 30-day incubation for Ly-B0 and Ly-B1. However, the inhibition zones after the 7-day incubation period experienced a significant decrease (p ≤ 0.05, Table 5) for the glasses with 15–25 mol% addition of Sr²⁺.

Table 5.

Means comparison of the size of inhibition zones (n = 3) after 1, 7 and 30 days incubation with respect to different Sr²⁺ incorporation in the glasses, where p ≤ 0.05 represents significant difference.

	Different Sr²⁺ incorporation
	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

Based on the previous studies concerning the antibacterial effects of bioactive glasses, some dissolution products such as zinc or silver ions kill bacteria by inhibiting multiple activities in the bacterial cell, such as glycolysis, trans-membrane proton translocation and acid tolerance.⁵⁴ Furthermore, the antibacterial effect is proportional to the concentration of these ions.⁵⁴ Sr²⁺ has been reported to exhibit antibacterial activity against S. aureus, but at a weak level.⁵⁵ It is postulated that Sr²⁺ exerts its antibacterial ability by inhibiting bacterial growth and reproduction and impeding permeability of cytoplasmic membrane, cell wall synthesis, replication of bacterial chromosomes and cell metabolism.³⁹ Based on the AAS data, the increased Sr²⁺ released from the glasses with higher Sr²⁺ loadings has no positive effect on inhibition zone size (Figure 7). An inhibition zone also exists for Ly-B0 which does not contain, or subsequently release, Sr²⁺. In addition, the dissolution mechanism of Sr²⁺ in TSB culture of small volume (100 µL) might be different from that in de-ionized water. Here, we assume that other dissolution products may contribute to the inhibition zone. It has been reported that boron-containing bioactive glass exerts antibacterial effects against S. aureus due to ${BO}_{3}^{3 -}$ release, but the antibacterial mechanism of ${BO}_{3}^{3 -}$ is still unknown.⁴⁰ In addition, a boron-based antibacterial (AN3365) was reported to reveal antibacterial activity against S. aureus.⁵⁶ Na⁺ and Ca²⁺ can also inhibit the growth of S. aureus.^57,58 The weight loss data (Figure 5) manifests that lower numbers of dissolution products release from the glasses with Sr²⁺ incorporation, especially for the glasses with more than 5 mol% addition of Sr²⁺, which is in agreement with the fact that the sizes of the inhibition zones decreased or disappeared after seven-day incubation with more than 5 mol% Sr²⁺ contents in the glasses. Thus, these glass discs are able to inhibit bacterial proliferation in one week, but not in the long term. In summary, the concentrations of all ions may have an influence on the sizes of the inhibition zones. In other words, a combined or individual effect of some ions among ${BO}_{3}^{3 -}$ , Sr²⁺, Na⁺ and Ca²⁺ makes the contribution to the bacteriostatic⁵⁹ behaviour of the glasses.

Cytotoxicity testing

The cytotoxicity results from glass powder disc extracts after 30-day incubation are shown in Figure 8. There is no difference (p ≥ 0.05, Table 6) among the cell metabolic activity of Ly-B0, Ly-B1, Ly-B2 and Ly-B3 glasses, which all experienced significantly reduced proliferation compared to control (p ≤ 0.05, Table 6). However, the cell proliferation was significantly enhanced (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 of cell metabolic activity on Ly-B5 was significant (p = 0.002).

Table 6.

Comparison of cell metabolic activity (relative to control) (n = 3) after 30-day incubation with respect to the control group and different Sr²⁺ incorporation in the glasses, where p ≤ 0.05 represents significant difference.

	Different Sr²⁺ 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
	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

Based on the results of pH measurement and AAS analysis, concentrations of Sr²⁺ ions released increased with increasing Sr²⁺ contents in the glass, while the concentrations of ${BO}_{3}^{3 -}$ decreased after 30-day incubation, as would be expected. It has been reported that high concentrations of ${BO}_{3}^{3 -}$ (>1 mg/L) inhibit proliferation of osteoblasts (MC3T3-E1),^24,60 while concentrations of Sr²⁺ in the range from 8.76 mg/L to 87.62 mg/L promote the proliferation of osteoblastic cells (MC3T3-E1) in vitro.⁴³ Therefore, this borate-based glass series promote the proliferation of osteoblastic cells with 20 mol% and 25 mol% Sr²⁺ incorporated in the glasses.

Conclusion

This study was conducted to investigate the solubility and antibacterial and osteo-stimulatory effects of a novel borate-based glass series with respect to both increasing additions of Sr²⁺ and incubation time. The concentrations of Na⁺, Ca²⁺ and Sr²⁺ in the water extracts experienced significant increases with incubation time. However, less Na⁺ and Ca²⁺ released from the glasses with increasing Sr²⁺ incorporation after 30-day incubation, indicating that the Sr²⁺ doping retarded the dissolution rate of the glasses. Sr²⁺ incorporation also made a contribution to the maintenance of pH values of the water extracts along with incubation time. In addition, the glass series promoted proliferation of osteoblastic cells with 20 mol% and 25 mol% Sr²⁺ contents, while the other glasses impeded cell growth. All members of the glass series, except for Ly-B5, exhibited bacteriostatic behaviour against S. aureus in the short term (1–7 days), which might be a result of a combined or individual effect of some of the dissolution products.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors gratefully acknowledge the support of both the Canadian Institute of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) through the Collaborative Health Research Project (CHRP) program (grant no. 315694-DAN).

References

Geesink

de Groot

Klein

. Bonding of bone to apatite-coated implants. J Bone Joint Surg Br 1988; 70: 17–22.

Herrera A, Mateo J, Gil-Albarova J, et al. Cementless hydroxyapatite coated hip prostheses. BioMed Res Int 2015; 2015: 1–13.

Mohseni

Zalnezhad

Bushroa

. Comparative investigation on the adhesion of hydroxyapatite coating on Ti–6Al–4V implant: a review paper. Int J Adhes Adhes 2014; 48: 238–257.

Ong

Appleford

. The characterization and development of bioactive hydroxyapatite coatings. JOM 2006; 58: 67–69.

Overgaard

. Calcium phosphate coatings for fixation of bone implants. Evaluated mechanically and histologically by stereological methods. Acta Orthopaed 2001; 71: 1–74.

Ong

Carnes

Bessho

. Evaluation of titanium plasma-sprayed and plasma-sprayed hydroxyapatite implants in vivo. Biomaterials 2004; 25: 4601–4606.

Darimont

Cloots

Heinen

. In vivo behaviour of hydroxyapatite coatings on titanium implants: a quantitative study in the rabbit. Biomaterials 2002; 23: 2569–2575.

Y-P

M-S

S-T

. Plasma-sprayed hydroxyapatite titania composite bond coat for hydroxyapatite coating on titanium substrate. Biomaterials 2004; 25: 4393–4403.

Nakamura

Otsuka

Aoki

. Thermal expansion of hydroxyapatite-β-tricalcium phosphate ceramics. Thermochim Acta 1990; 165: 57–72.

10.

Yang

Y-C

Chang

. Measurements of residual stresses in plasma-sprayed hydroxyapatite coatings on titanium alloy. Surf Coat Technol 2005; 190: 122–131.

11.

Donald

Mallinson

Metcalfe

. Recent developments in the preparation, characterization and applications of glass-and glass–ceramic-to-metal seals and coatings. J Mater Sci 2011; 46: 1975–2000.

12.

Bellucci

Cannillo

Sola

. Coefficient of thermal expansion of bioactive glasses: Available literature data and analytical equation estimates. Ceram Int 2011; 37: 2963–2972.

13.

Pavon

Jimenez-Pique

Anglada

. Stress–corrosion cracking by indentation techniques of a glass coating on Ti6Al4V for biomedical applications. J Eur Ceram Soc 2006; 26: 1159–1169.

14.

Peddi

Brow

Brown

. Bioactive borate glass coatings for titanium alloys. J Mater Sci Mater Med 2008; 19: 3145–3152.

15.

Xiao

Luo

S-H

Wei

X-J

. Evaluation of Ti implants coated with Ag-containing borate bioactive glass for simultaneous eradication of infection and fracture fixation in a rabbit tibial model. J Mater Res 2012; 27: 3147–3156.

16.

Liu

Pan

. Conversion of borate-based glass scaffold to hydroxyapatite in a dilute phosphate solution. Biomed Mater 2010; 5: 015005–015005.

17.

Zobell

Liu

. Healing of critical-size segmental defects in rat femora using strong porous bioactive glass scaffolds. Mater Sci Eng C 2014; 42: 816–824.

18.

Pan H, Zhao X, Zhang X, et al. Strontium borate glass: potential biomaterial for bone regeneration. J R Soc Interface 2009; 7: 1025–1031.

19.

Sheng

MH-C

Taper

Veit

. Dietary boron supplementation enhances the effects of estrogen on bone mineral balance in ovariectomized rats. Biol Trace Elem Res 2001; 81: 29–45.

20.

Uysal

Ustdal

Sonmez

. Stimulation of bone formation by dietary boron in an orthopedically expanded suture in rabbits. Angle Orthod 2009; 79: 984–990.

21.

Huang

Day

Kittiratanapiboon

. Kinetics and mechanisms of the conversion of silicate (45S5), borate, and borosilicate glasses to hydroxyapatite in dilute phosphate solutions. J Mater Sci Mater Med 2006; 17: 583–596.

22.

El-Ghannam

Ducheyne

Shapiro

. Bioactive material template for in vitro, synthesis of bone. J Biomed Mater Res 1995; 29: 359–370.

23.

Maeno

Niki

Matsumoto

. The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture. Biomaterials 2005; 26: 4847–4855.

24.

Brown

Rahaman

Dwilewicz

. Effect of borate glass composition on its conversion to hydroxyapatite and on the proliferation of MC3T3-E1 cells. J Biomed Mater Res A 2009; 88: 392–400.

25.

Sun

Berndt

Gross

. Material fundamentals and clinical performance of plasma-sprayed hydroxyapatite coatings: a review. J Biomed Mater Res 2001; 58: 570–592.

26.

Tilocca

. Sodium migration pathways in multicomponent silicate glasses: Car–Parrinello molecular dynamics simulations. J Chem Phys 2010; 133: 014701–014701.

27.

O’Donnell

Candarlioglu

Miller

. Materials characterisation and cytotoxic assessment of strontium-substituted bioactive glasses for bone regeneration. J Mater Chem 2010; 20: 8934–8941.

28.

Thind

Singh

Kumar

. Compositional dependence of in-vitro bioactivity in sodium calcium borate glasses. J Phys Chem Solid 2009; 70: 1137–1141.

29.

O'Connell

Hanson

O'Shea

. Linear release of strontium ions from high borate glasses via lanthanide/alkali substitutions. J Non-cryst Solid 2015; 430: 1–8.

30.

Jung SB. Borate based bioactive glass scaffolds for hard and soft tissue engineering. Doctoral Dissertations, Paper 2075, 2010.

31.

Shen

Coughlan

Towler

. Degradable borate glass polyalkenoate cements. J Mater Sci Mater Med 2014; 25: 965–973.

32.

Marzouk

ElBatal

. In vitro bioactivity of soda lime borate glasses with substituted SrO in sodium phosphate solution. Process Appl Ceram 2014; 8: 167–177.

33.

Yiannopoulos

Chryssikos

Kamitsos

. Structure and properties of alkaline earth borate glasses. Phys Chem Glasses-Eur J Glass Sci Technol Part B 2001; 42: 164–172.

34.

Pye LD, Fréchette VD and Kreidl NJ. Borate glasses: structure, properties, applications. New York: Springer Science & Business Media, 2012.

35.

Lower

McRae

Feller

. Physical properties of alkaline-earth and alkali borate glasses prepared over an extended range of compositions. J Non-cryst Solid 2001; 293: 669–675.

36.

Murdoch

Roberts

Fowler

. Infection of orthopedic prostheses after Staphylococcus aureus bacteremia. Clin Infect Dis 2001; 32: 647–649.

37.

Friedman

. Concise review of mechanisms of bacterial adhesion to biomaterial surfaces. J Biomed Mater Res 1998; 43: 338–348.

38.

Montanaro

Speziale

Campoccia

. Scenery of Staphylococcus implant infections in orthopedics. Future Microbiol 2011; 6: 1329–1349.

39.

Brauer DS, Karpukhina N, Kedia G, et al. Bactericidal strontium-releasing injectable bone cements based on bioactive glasses. J R Soc Interf 2012; 10: 20120647–20120654.

40.

Munukka

Leppäranta

Korkeamäki

. Bactericidal effects of bioactive glasses on clinically important aerobic bacteria. J Mater Sci Mater Med 2008; 19: 27–32.

41.

Petkov

Ohta

Hou

. Atomic-scale structure of nanocrystals by high-energy X-ray diffraction and atomic pair distribution function analysis: study of FexPd100-x (x= 0, 26, 28, 48) nanoparticles. J Phys Chem C 2007; 111: 714–720.

42.

Warren

Biscce

. The structure of silica glass by X-ray diffraction studies. J Am Ceram Soc 1938; 21: 49–54.

43.

Placek

Coughlan

. Investigating the influence of Na+ and Sr2+ on the structure and solubility of SiO2–TiO2–CaO–Na2O/SrO bioactive glass. J Mater Sci Mater Med 2015; 26: 1–12.

44.

Tilocca

Cormack

. Surface signatures of bioactivity: MD simulations of 45S and 65S silicate glasses. Langmuir 2009; 26: 545–551.

45.

Tilocca

Cormack

. Modeling the water–bioglass interface by ab initio molecular dynamics simulations. ACS Appl Mater Interf 2009; 1: 1324–1333.

46.

Lao

Nedelec

J-M

Jallot

. New strontium-based bioactive glasses: physicochemical reactivity and delivering capability of biologically active dissolution products. J Mater Chem 2009; 19: 2940–2949.

47.

Marie

Ammann

Boivin

. Mechanisms of action and therapeutic potential of strontium in bone. Calcified Tissue Int 2001; 69: 121–129.

48.

Murphy

Boyd

Moane

. The effect of composition on ion release from Ca–Sr–Na–Zn–Si glass bone grafts. J Mater Sci Mater Med 2009; 20: 2207–2214.

49.

Goel

Rajagopal

Ferreira

. Influence of strontium on structure, sintering and biodegradation behaviour of CaO–MgO–SrO–SiO2–P2O5–CaF2 glasses. Acta Biomater 2011; 7: 4071–4080.

50.

Zhao

Huang

Rahaman

. Mechanism for converting Al 2O3-containing borate glass to hydroxyapatite in aqueous phosphate solution. Acta Biomater 2009; 5: 1265–1273.

51.

Arentt

Dempster

. Effect of pH on bone resorption by rat osteoclasts in vitro. Endocrinology 1986; 119: 119–124.

52.

Arnett

. Extracellular pH regulates bone cell function. J Nutr 2008; 138: 415S–418S.

53.

Arnett TR (ed.) Acid–base regulation of bone metabolism. Int Congr Ser 2007; 1297: 255–267.

54.

Boyd

Tanner

. The antibacterial effects of zinc ion migration from zinc-based glass polyalkenoate cements. J Mater Sci Mater Med 2006; 17: 489–494.

55.

Ravi

Balu

Sampath Kumar

. Strontium-substituted calcium deficient hydroxyapatite nanoparticles: synthesis, characterization, and antibacterial properties. J Am Ceram Soc 2012; 95: 2700–2708.

56.

Hernandez

Crépin

Palencia

. Discovery of a novel class of boron-based antibacterials with activity against Gram-negative bacteria. Antimicrob Agent Chemother 2013; 57: 1394–1403.

57.

Zhang

Leppäranta

Munukka

. Antibacterial effects and dissolution behavior of six bioactive glasses. J Biomed Mater Res A 2010; 93: 475–483.

58.

Petersen

Bradford

Weiss

. In vitro and in vivo activities of tigecycline (GAR-936), daptomycin, and comparative antimicrobial agents against glycopeptide-intermediate Staphylococcus aureus and other resistant Gram-positive pathogens. Antimicrob Agent Chemother 2002; 46: 2595–2601.

59.

Vitale-Brovarone

Miola

Balagna

. 3D-glass–ceramic scaffolds with antibacterial properties for bone grafting. Chem Eng J 2008; 137: 129–136.

60.

Hakki

Bozkurt

Hakki

. Boron regulates mineralized tissue-associated proteins in osteoblasts (MC3T3-E1). J Trace Elem Med Biol 2010; 24: 243–250.