Strontium-substituted bioactive glasses in vitro osteogenic and antibacterial effects

Abstract

Objectives

Bioactive glass forms a bone mineral apatite interface and can be engineered to promote optimal bone regeneration. Strontium (Sr 2+ ) stimulates osteoblast and inhibits osteoclast activities in vitro , and is used clinically as a treatment for osteoporosis. Dental bone defect repair requires rapid bone formation for early osseointegration but, can be subject to infection. The aim of this study was to investigate the osteogenic and antibacterial effects of strontium-substituted bioactive glasses in vitro .

Methods

Strontium-substituted bioactive glasses were designed and produced. Then the osteogenic potential and antibacterial effects of bioactive glass particulates were explored.

Results

Alkaline phosphatase activity, cell number, Type I collagen and mineral nodule formation of MC3T3-E1 cells were significantly promoted by the 5% strontium-substituted glass (5Sr). Furthermore, after incubation with 0.001 g and 0.01 g glass particulates, the growth of sub-gingival bacteria, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis was significantly inhibited; the antibacterial activity being dependent on the percentage of strontium in the glasses.

Significance

These results show that strontium-substituted bioactive glasses significantly promote osteogenic responses of MC3T3-E1 osteoblast-like cells and inhibit the growth of A. actinomycetemcomitans and P. gingivalis .

Introduction

When exposed to body fluids, bioactive glass grafts undergo surface reactions to from a strong bond with the living bone with the generation of an hydroxycarbonate apatite (HCA) layer, and therefore they are widely utilized in dental and orthopaedic applications .

Strontium ranelate (SrR), marketed as Protelos ® , is an approved drug for treatment and prevention of osteoporosis, in which, strontium (Sr 2+ ) is reported to be the active component . Numerous studies have demonstrated that Sr 2+ in vitro promotes osteoblastic differentiation and bone nodule formation , stimulates the type I collagen protein levels , increases the expression of dental matrix protein I, and elevates the osteoprotegerin (OPG) secretion . Sr 2+ also inhibits the differentiation and activity of osteoclasts . Therefore, the incorporation of Sr into bioactive glass and used as a bone graft substitute could provide a range of advantages especially if the locally-released Sr is osteogenic.

Strontium has a similar charge-to-size ratio to calcium and because of the properties detailed above, substituting SrO for CaO in bioactive glass has been widely investigated . In a study using simulated body fluid, Fredholm et al. demonstrated that full Sr substitution provides the combined benefits of significantly enhanced early glass dissolution and apatite formation . The mixed Ca-Sr-hydroxyapatite (Sr 5 Ca 5 (PO 4 ) 6 (OH) 2 ) is formed more rapidly . Yet, since Sr is heavier and slightly larger than Ca, expanding the network should increase the ion dissolution rates and therefore Sr has an earlier effect on bone cells compared with Ca .

A bacterial infection can hinder the process of bone healing and sometimes can lead to surgical failure . In recent years, some studies have reported that the incorporation of Sr 2+ into dental and orthopaedic biomaterials will inhibit the bacterial growth, but the antimicrobial mechanism is unclear . Whilst phosphate is essential in biological mineralization, some studies have reported that phosphate ions can also be either bacteriostatic or bactericidal .

We have created a series of bioactive glasses with a fixed 4 mol% P 2 O 5 content, and a range of SrO (substituted for CaO). The osteogenic effects on mouse osteoblast-like cells and antibacterial effects on subgingival bacteria species in vitro were investigated.

Materials and methods

Glass synthesis

Glasses in the system SiO 2 -P 2 O 5 -CaO-Na 2 O-SrO ( Table 1 ) were prepared by the melt-quench route. Briefly, mixtures of analytical grade SiO 2 (Prince Minerals Ltd., Stoke-on-Trent, UK), P 2 O 5 , Na 2 CO 3 , CaCO 3 and SrCO 3 (Sigma–Aldrich Company Ltd., Gillingham, UK) were weighed in the appropriate amounts to give a batch size of 200 g. The batch was mixed thoroughly and placed in a platinum/rhodium crucible, heated and maintained at a temperature of 1460 °C for 90 min in an electrically heated furnace (Lenton EHF 17/3, Hope Valley, UK). After melting, the glass was quenched rapidly into deionized water and the resulting frit was washed with ethanol then dried in a drying cabinet at 37 °C overnight. 100 g of each glass was ground in a Gyro mill (Glen Creston, London, UK) for two periods of 7 min and sieved by a mesh analytical sieve (Endecotts Ltd., London, UK) to obtain fine powder (38 μm diameter). The amorphous structure of the glasses was tested using powder X-ray diffraction (PANalytical, Eindhoven, The Netherlands).

Table 1
Compositions in mol% with Sr substituted Ca.
Glass SiO 2 P 2 O 5 CaO SrO Na 2 O
0Sr 42.00 4.00 39.00 0.00 15.00
5Sr 42.00 4.00 37.05 1.95 15.00
50Sr 42.00 4.00 19.50 19.50 15.00
100Sr 42.00 4.00 0.00 39.00 15.00

Glass powder conditioned culture medium and ion release

Glass particles from each group were immersed in α-Minimum Essential medium (α-MEM, Lonza, London, UK) (1.5 g/L) with 1% antibiotic (penicillin and streptomycin, Invitrogen, London, UK) addition and shaken (60 rpm) at room temperature for 2, 8, 24 and 72 h. At each time point, the samples were centrifuged (800 rpm, 5 min) and filtered with 0.2 μm pore size filters (VWR, Lutterworth, UK) for sterilization.

The filtrate was diluted 1:10 with deionized water and 1% nitric acid and then analyzed in an inductively coupled plasma-optical emission spectroscopy (ICP-OES; Varian Vista-PRO, Varian Ltd., Oxford, UK) to detect silicon, calcium, strontium, and phosphorus concentrations.

Cell culture and cytotoxicity of glass conditioned medium

MC3T3-E1, a mouse osteoblast-like cell line was obtained from the Culture Collections (Public Health England, Porton Down, Salisbury, UK), and maintained in α-MEM under standard conditions (37 °C, 5% CO 2 /95% air, 100% humidity) with 5% foetal bovine serum (FBS, Lonza, London, UK), 1% antibiotic and 1% l -glutamine.

The glass conditioned medium (glass particles immersed for 2, 8, 24 and 72 h) was further supplemented with sterile 1% l -glutamine and 5% FBS, and used to treat MC3T3-E1 for 1 d, 3 d and 5 d. Cytotoxicity of glass conditioned medium on cell growth was visualized by MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay . Briefly, medium was removed and cells were washed twice with PBS, then 30 μL 5 mg/mL tetrazolium salt MTT (Sigma–Aldrich Company Ltd., Gillingham, UK) was added to each well and incubated in 37 °C for 4 h. Formazan crystals generated by mitochondrial enzyme activity were dissolved by dimethyl sulfoxide (DMSO, Sigma–Aldrich Company Ltd., Gillingham, UK) and the intensity of purple coloured reaction product quantified by measuring the absorbance spectra at 570 nm.

Total quantification of cells cultured in glass conditioned medium

According to the cytotoxicity results, 72 h glass conditioned medium was chosen to further explore the effect on cell proliferation. MC3T3-E1 cells were treated for 7 d, 14 d and 21 d. Cell number was ascertained by quantifying the DNA in cultures using the fluorochrome, bisbenzimidazole (Hoechst 33258, Sigma–Aldrich Company Ltd., Gillingham, UK) , in which fluorescence intensity is linearly related to DNA concentration. Briefly, after treatment for the indicated time points, cells were lysed through a freezing and thawing cycle with 100 μL deionized water in each well to rupture cells and release DNA. The resulting lysate was then incubated with fluorochrome bisbenzimidazole (1:50) in TNE buffer with 10 mM Tris, 1 mM EDTA and 2 M NaCl (pH 7.4, all from Sigma–Aldrich Company Ltd., Gillingham, UK) and the resultant fluorescence intensity was measured at 355 nm excitation and 460 nm emission. A calibration curve was prepared using calf thymus DNA (Sigma–Aldrich Company Ltd., Gillingham, UK).

Alkaline phosphatase (ALP) activity in cells cultured in glass conditioned medium

Cells were treated in the same manner as for the cell proliferation experiments. ALP activity was determined by an enzyme histochemical assay , in which ALP converts P-nitrophenyl phosphate to P-nitrophenyl. At each time point, cells were lysed through a freezing and thawing process and reacted with a solution of 2.5 mg/mL 4-nitrophenyl phosphate disodium salt hexahydrate in Tris buffer solution with 1 mM MgCl 2 (pH 9.5, all from Sigma–Aldrich Company Ltd., Gillingham, UK) for 40 min in 37 °C. 0.5 M NaOH was used to stop the reaction and the intensity of yellow reaction product quantified by measuring the absorbance at 405 nm. ALP activity of samples calculated and the enzyme activity was expressed as nmol/mL/min .

Detection and quantification of Type I collagen formation in glass conditioned medium

72 h conditioned medium was further supplemented with 5 mM β-glycerophosphate and 50 μg/mL l -ascorbic acid (Sigma–Aldrich Company Ltd., Gillingham, UK) to make osteogenic medium.

MC3T3-E1 cells were treated for 2, 3 and 4 weeks with glass conditioned osteogenic medium. Type I collagen formation was quantified by measuring Sirius red stain incorporation into the extracellular matrix . After 10 min fixation in 2.5% glutaraldehyde (Sigma–Aldrich Company Ltd., Gillingham, UK) at 4 °C and three times deionized water wash, monolayers were incubated in 0.1% Sirius red F3B in 1.3% saturated aqueous solution of picric acid (all from Sigma–Aldrich Company Ltd., Gillingham, UK) for 1 h at room temperature. Cultures were then washed twice with 0.5% acidified water and three times in deionized water to remove the unincorporated dye. Stained cultures were photographed followed by a dye extraction procedure using a mixture of 0.1 M NaOH and absolute methanol (1:1, all from Sigma–Aldrich Company Ltd., Gillingham, UK) for 30 min at room temperature. 200 μL aliquots of solution and dye were transferred to a 96-well plate and the absorbance measured at 570 nm.

Detection and quantification of mineralization in glass conditioned medium

Cells were treated in the same manner as for the Type I collagen detection experiments. At each time point, medium was removed and cells were washed twice with PBS and fixed in 2.5% glutaraldehyde at 4 °C for 10 min. The cells were then washed twice with excess deionized water prior to the addition of 40 mM Alizarin Red S (pH 4.1, Sigma–Aldrich Company Ltd., Gillingham, UK). The plates were incubated at room temperature for 40 min and then washed three times with deionized water to remove the unincorporated dye.

For quantification of Alizarin Red S staining, an adaptation of the protocol described by Stanford et al. was followed. Alizarin Red S was extracted from the monolayer by incubation in 10% (w/v) cetylpyridinium chloride (CPC, Sigma–Aldrich Company Ltd., Gillingham, UK) in 10 mM sodium phosphate, pH 7.0, for 30 min at room temperature. The dye was removed and 100 μL aliquots were transferred to a 96-well plate and the absorbance reads at 570 nm.

Bacterial studies

Typical subgingival bacteria, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis (kindly gifted by Professor Rob Allaker, Queen Mary University of London), were grown in brain heart infusion agar plates (Fisher Scientific, Loughborough, UK) for 48 h at 37 °C under anaerobic conditions. Log phase cultures were harvested and the bacterial concentrations were measured by absorbance (595 nm).

Glass particulates were sterilized by autoclaving (dry cycle) at 121 °C for 15 min. The glass particulate suspensions in brain heart infusion broth (0.02 g/mL and 0.002 g/mL) were plated in 96-well plates (100 μL/well). The bacteria (10 6 to 10 7 CFU/mL) were added subsequently (100 μL/well). Therefore, the final glass particle working concentrations were 0.01 g/mL and 0.001 g/mL. Strontium chloride (SrCl 2 ·6H 2 O, Sigma–Aldrich Company Ltd., Gillingham, UK) concentrations (4, 2, 1, 0.5 and 0.25 mM) in brain heart infusion broth were used to treat bacteria to investigate the antibacterial effects of Sr 2+ alone. After incubation under anaerobic conditions for 0, 2, 4 and 6 h, 20 μL alamarBlue (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) was added to each well and incubated for 30 min according to the manufacture’s protocol. The plates were next centrifuged for 10 min at 4000 rpm and 100 μL aliquots supernatants were transferred to new black 96-well plates to measure fluorescent intensity at 560 nm excitation and 590 nm emission. Percent inhibition of bacterial growth was then defined as 1 − (mean fluorescence of test wells/mean fluorescence of negative control well) × 100% .

Statistical analyses

The elemental analysis was carried out with three samples per group. Cell assay data with glass conditioned medium are presented as means ± standard deviations and represent data from six replicates per experiment. Four replicates were carried out for the bacterial studies. Comparisons were made using a one-way analysis of variance (ANOVA). Data was adjudged to be significant when P ≤ 0.05.

Materials and methods

Glass synthesis

Glasses in the system SiO 2 -P 2 O 5 -CaO-Na 2 O-SrO ( Table 1 ) were prepared by the melt-quench route. Briefly, mixtures of analytical grade SiO 2 (Prince Minerals Ltd., Stoke-on-Trent, UK), P 2 O 5 , Na 2 CO 3 , CaCO 3 and SrCO 3 (Sigma–Aldrich Company Ltd., Gillingham, UK) were weighed in the appropriate amounts to give a batch size of 200 g. The batch was mixed thoroughly and placed in a platinum/rhodium crucible, heated and maintained at a temperature of 1460 °C for 90 min in an electrically heated furnace (Lenton EHF 17/3, Hope Valley, UK). After melting, the glass was quenched rapidly into deionized water and the resulting frit was washed with ethanol then dried in a drying cabinet at 37 °C overnight. 100 g of each glass was ground in a Gyro mill (Glen Creston, London, UK) for two periods of 7 min and sieved by a mesh analytical sieve (Endecotts Ltd., London, UK) to obtain fine powder (38 μm diameter). The amorphous structure of the glasses was tested using powder X-ray diffraction (PANalytical, Eindhoven, The Netherlands).

Table 1
Compositions in mol% with Sr substituted Ca.
Glass SiO 2 P 2 O 5 CaO SrO Na 2 O
0Sr 42.00 4.00 39.00 0.00 15.00
5Sr 42.00 4.00 37.05 1.95 15.00
50Sr 42.00 4.00 19.50 19.50 15.00
100Sr 42.00 4.00 0.00 39.00 15.00

Glass powder conditioned culture medium and ion release

Glass particles from each group were immersed in α-Minimum Essential medium (α-MEM, Lonza, London, UK) (1.5 g/L) with 1% antibiotic (penicillin and streptomycin, Invitrogen, London, UK) addition and shaken (60 rpm) at room temperature for 2, 8, 24 and 72 h. At each time point, the samples were centrifuged (800 rpm, 5 min) and filtered with 0.2 μm pore size filters (VWR, Lutterworth, UK) for sterilization.

The filtrate was diluted 1:10 with deionized water and 1% nitric acid and then analyzed in an inductively coupled plasma-optical emission spectroscopy (ICP-OES; Varian Vista-PRO, Varian Ltd., Oxford, UK) to detect silicon, calcium, strontium, and phosphorus concentrations.

Cell culture and cytotoxicity of glass conditioned medium

MC3T3-E1, a mouse osteoblast-like cell line was obtained from the Culture Collections (Public Health England, Porton Down, Salisbury, UK), and maintained in α-MEM under standard conditions (37 °C, 5% CO 2 /95% air, 100% humidity) with 5% foetal bovine serum (FBS, Lonza, London, UK), 1% antibiotic and 1% l -glutamine.

The glass conditioned medium (glass particles immersed for 2, 8, 24 and 72 h) was further supplemented with sterile 1% l -glutamine and 5% FBS, and used to treat MC3T3-E1 for 1 d, 3 d and 5 d. Cytotoxicity of glass conditioned medium on cell growth was visualized by MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay . Briefly, medium was removed and cells were washed twice with PBS, then 30 μL 5 mg/mL tetrazolium salt MTT (Sigma–Aldrich Company Ltd., Gillingham, UK) was added to each well and incubated in 37 °C for 4 h. Formazan crystals generated by mitochondrial enzyme activity were dissolved by dimethyl sulfoxide (DMSO, Sigma–Aldrich Company Ltd., Gillingham, UK) and the intensity of purple coloured reaction product quantified by measuring the absorbance spectra at 570 nm.

Total quantification of cells cultured in glass conditioned medium

According to the cytotoxicity results, 72 h glass conditioned medium was chosen to further explore the effect on cell proliferation. MC3T3-E1 cells were treated for 7 d, 14 d and 21 d. Cell number was ascertained by quantifying the DNA in cultures using the fluorochrome, bisbenzimidazole (Hoechst 33258, Sigma–Aldrich Company Ltd., Gillingham, UK) , in which fluorescence intensity is linearly related to DNA concentration. Briefly, after treatment for the indicated time points, cells were lysed through a freezing and thawing cycle with 100 μL deionized water in each well to rupture cells and release DNA. The resulting lysate was then incubated with fluorochrome bisbenzimidazole (1:50) in TNE buffer with 10 mM Tris, 1 mM EDTA and 2 M NaCl (pH 7.4, all from Sigma–Aldrich Company Ltd., Gillingham, UK) and the resultant fluorescence intensity was measured at 355 nm excitation and 460 nm emission. A calibration curve was prepared using calf thymus DNA (Sigma–Aldrich Company Ltd., Gillingham, UK).

Alkaline phosphatase (ALP) activity in cells cultured in glass conditioned medium

Cells were treated in the same manner as for the cell proliferation experiments. ALP activity was determined by an enzyme histochemical assay , in which ALP converts P-nitrophenyl phosphate to P-nitrophenyl. At each time point, cells were lysed through a freezing and thawing process and reacted with a solution of 2.5 mg/mL 4-nitrophenyl phosphate disodium salt hexahydrate in Tris buffer solution with 1 mM MgCl 2 (pH 9.5, all from Sigma–Aldrich Company Ltd., Gillingham, UK) for 40 min in 37 °C. 0.5 M NaOH was used to stop the reaction and the intensity of yellow reaction product quantified by measuring the absorbance at 405 nm. ALP activity of samples calculated and the enzyme activity was expressed as nmol/mL/min .

Detection and quantification of Type I collagen formation in glass conditioned medium

72 h conditioned medium was further supplemented with 5 mM β-glycerophosphate and 50 μg/mL l -ascorbic acid (Sigma–Aldrich Company Ltd., Gillingham, UK) to make osteogenic medium.

MC3T3-E1 cells were treated for 2, 3 and 4 weeks with glass conditioned osteogenic medium. Type I collagen formation was quantified by measuring Sirius red stain incorporation into the extracellular matrix . After 10 min fixation in 2.5% glutaraldehyde (Sigma–Aldrich Company Ltd., Gillingham, UK) at 4 °C and three times deionized water wash, monolayers were incubated in 0.1% Sirius red F3B in 1.3% saturated aqueous solution of picric acid (all from Sigma–Aldrich Company Ltd., Gillingham, UK) for 1 h at room temperature. Cultures were then washed twice with 0.5% acidified water and three times in deionized water to remove the unincorporated dye. Stained cultures were photographed followed by a dye extraction procedure using a mixture of 0.1 M NaOH and absolute methanol (1:1, all from Sigma–Aldrich Company Ltd., Gillingham, UK) for 30 min at room temperature. 200 μL aliquots of solution and dye were transferred to a 96-well plate and the absorbance measured at 570 nm.

Detection and quantification of mineralization in glass conditioned medium

Cells were treated in the same manner as for the Type I collagen detection experiments. At each time point, medium was removed and cells were washed twice with PBS and fixed in 2.5% glutaraldehyde at 4 °C for 10 min. The cells were then washed twice with excess deionized water prior to the addition of 40 mM Alizarin Red S (pH 4.1, Sigma–Aldrich Company Ltd., Gillingham, UK). The plates were incubated at room temperature for 40 min and then washed three times with deionized water to remove the unincorporated dye.

For quantification of Alizarin Red S staining, an adaptation of the protocol described by Stanford et al. was followed. Alizarin Red S was extracted from the monolayer by incubation in 10% (w/v) cetylpyridinium chloride (CPC, Sigma–Aldrich Company Ltd., Gillingham, UK) in 10 mM sodium phosphate, pH 7.0, for 30 min at room temperature. The dye was removed and 100 μL aliquots were transferred to a 96-well plate and the absorbance reads at 570 nm.

Bacterial studies

Typical subgingival bacteria, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis (kindly gifted by Professor Rob Allaker, Queen Mary University of London), were grown in brain heart infusion agar plates (Fisher Scientific, Loughborough, UK) for 48 h at 37 °C under anaerobic conditions. Log phase cultures were harvested and the bacterial concentrations were measured by absorbance (595 nm).

Glass particulates were sterilized by autoclaving (dry cycle) at 121 °C for 15 min. The glass particulate suspensions in brain heart infusion broth (0.02 g/mL and 0.002 g/mL) were plated in 96-well plates (100 μL/well). The bacteria (10 6 to 10 7 CFU/mL) were added subsequently (100 μL/well). Therefore, the final glass particle working concentrations were 0.01 g/mL and 0.001 g/mL. Strontium chloride (SrCl 2 ·6H 2 O, Sigma–Aldrich Company Ltd., Gillingham, UK) concentrations (4, 2, 1, 0.5 and 0.25 mM) in brain heart infusion broth were used to treat bacteria to investigate the antibacterial effects of Sr 2+ alone. After incubation under anaerobic conditions for 0, 2, 4 and 6 h, 20 μL alamarBlue (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) was added to each well and incubated for 30 min according to the manufacture’s protocol. The plates were next centrifuged for 10 min at 4000 rpm and 100 μL aliquots supernatants were transferred to new black 96-well plates to measure fluorescent intensity at 560 nm excitation and 590 nm emission. Percent inhibition of bacterial growth was then defined as 1 − (mean fluorescence of test wells/mean fluorescence of negative control well) × 100% .

Statistical analyses

The elemental analysis was carried out with three samples per group. Cell assay data with glass conditioned medium are presented as means ± standard deviations and represent data from six replicates per experiment. Four replicates were carried out for the bacterial studies. Comparisons were made using a one-way analysis of variance (ANOVA). Data was adjudged to be significant when P ≤ 0.05.

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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Strontium-substituted bioactive glasses in vitro osteogenic and antibacterial effects
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