In the last fifty years the chemical and textural features of melt-derived bioactive glasses, discovered by Hench, have been largely modified to improve their biological performances transforming them into materials with biomedical added value [1
For this reason, from the 1990s the researchers’ attention has been moved to the sol-gel synthesis approach since it provides better textural properties to the glass, such as higher surface area, porosity and homogeneity, involving also lower processing temperatures. These characteristics allow widening the range of compositions showing bioactive behaviour (up to 80 mol % of SiO2
) and increasing the hydroxyapatite (HA) deposition rate [2
]. The HA deposition is in fact the peculiar surface-dependent mechanism characterising bioactive materials and allowing them to form interfacial bonds with hard and soft tissues upon their reaction in physiological fluids [4
From 2000’s mesoporous bioactive glasses (MBGs) have been synthesised combining the sol-gel method with the use of templating agents in order to further improve and control the textural characteristics of sol-gel bioactive glasses. Templating agents are surfactant molecules able to self-assemble in micelles, around which the hydrolysed glass precursors condense forming an ordered mesophase, whose structure depends on several factors (surfactant chemistry and concentration, temperature, pH, etc.) [2
]. After the removal of the surfactant by calcination, an ordered mesoporous structure is obtained. Therefore, the use of these templating agents allows providing MBGs with remarkable structural features: highly ordered and tunable porosity in the range of 2 and 50 nm and higher surface area and pore volume values [7
]. These peculiar characteristics increase MBG reactiveness in body fluids accelerating the process of HA deposition and making these glasses particularly suitable for bone regenerative application [8
In addition to these structural characteristics, the regenerative potential of bioactive glasses is influenced by their ionic dissolution products (Si, Ca, P) [9
]. In fact, it has been demonstrated that the variation of the intracellular ion concentration caused by bioactive glass dissolution leads to the activation of intracellular signalling pathways. This mechanism influences the genic expression of osteoprogenitor cells giving rise to rapid bone regeneration [10
To stimulate a more advanced and specific cell response, therapeutic metallic ions (Cu2+
, etc.) have been recently introduced into the bioactive glass compositions [11
]. These ions are able to stimulate osteogenesis, angiogenesis and to provide antibacterial properties, making them attractive as therapeutic agents in the fields of hard and soft tissue engineering [14
]. Furthermore, the release of therapeutic ions can be synergistically combined with the delivery of pharmaceutical agents or growth factors, which can be loaded into MBG mesopores or adsorbed/grafted on the external surface of the glass particles [7
In addition, MBGs can be exploited as multifunctional carriers in combination with a polymeric vehicle matrix for the development of novel and versatile devices for soft and hard tissue regeneration. With this purpose, the incorporation of Cu-containing MBGs into a thermosensitive hydrogel to obtain a non-invasive injectable formulation for prolonged and localized therapeutic ion release has been recently reported by the authors [16
]. Another intriguing application foresees their incorporation in a Type I collagen matrix to produce bone like, hybrid materials to promote the natural bone remodeling in osteoporotic tissues, as recently reported by Montalbano et al. [17
Among the elements with potential therapeutic properties, strontium has received remarkable interest, due to its well-known role in bone metabolism [18
] by exercising anabolic and anti-catabolic effects. Indeed, due to its chemical analogy to calcium, strontium is able to exploit the calcium sensing receptors present on both osteoblastic and osteoclastic cells to activate their downstream signalling pathways, leading to the promotion of osteoblast replication, differentiation and survival as well as downregulating osteoclast activities [23
]. Furthermore strontium is normally present in the hydroxyapatite crystals of human bones due to its ability of exchange with calcium in the HA reticulum [14
Several studies conducted on bioactive glasses where calcium has been partially substituted by strontium have shown its beneficial effect in promoting bone remodelling. In particular, Moghanian et al. [24
] tested the response of mouse osteoblast-like cells on solid discs composed of Sr-containing 58S glass (60% SiO2
–36% CaO–4% P2
mol %) and evidenced that the presence of strontium was able to increase the metabolic behaviour of osteoblasts and to promote their proliferation and the alkaline phosphatase (ALP) activity. Enhanced osteoblastic activity was also observed by Taherkhani et al. [25
], who exposed human osteosarcoma cells to culture media treated with ionic dissolution products of the same type of glass. More recently, Naruphontjirakul et al. [12
] investigated murine pre-osteoblast cell response to silica nanoparticles obtained by the Stöber method and subsequently enriched with calcium and strontium, as well as the cell response to their ionic dissolution products, and reported an enhanced ALP activity and osteogenic differentiation in presence of nanoparticles containing strontium.
In this work, different molar concentrations of strontium have been incorporated into the framework of a mesoporous glass with SiO2–CaO composition. Sr-containing MBGs have been synthesised by two different approaches, a batch sol-gel procedure and an aerosol-assisted spray-drying method, to produce nano- and micro-sized spheres, respectively. Besides the size of the final particles, the two preparation methods allowed to obtain Sr-substituted samples with different structural features in term of specific surface area, pore volume and pore size and, consequently, to investigate how these aspects may influence the bioactivity and the biological response in terms of cytocompatibility and pro-osteogenic effect.
2. Materials and Methods
2.1. Preparation of Sr-Containing MBGs (Sr-MBGs)
MBGs containing different amount of Sr (2 and 4 mol %) were synthesised through two different synthesis approaches in order to obtain particles with different size and structural parameters (i.e., specific surface area, pore size).
2.1.1. Preparation of Sr-Containing MBG Samples by Aerosol-Assisted Spray Drying Method
Based on a modification of the procedure reported by Pontiroli et al. for a binary SiO2
-CaO composition [26
], MBG micro-particles with different molar percentage of Sr (molar ratio Sr/Ca/Si = 2/13/85 and Sr/Ca/Si = 4/11/85, hereafter named as MBG_Sr2%_SD and MBG_Sr4%_SD respectively) were synthesised by aerosol-assisted spray drying method. Briefly, 2.0 g of the non-ionic block copolymer Pluronic P123 (EO20
, average Mn
~5800, Sigma Aldrich, Milan, Italy) were dissolved in 85.0 g of double distilled H2
O) (solution A). In a separate batch, 10.7 g of TEOS were pre-hydrolysed under acidic conditions using 5.0 g of an aqueous HCl solution at pH = 2 until a transparent solution was obtained (solution B). Solution B was then added drop by drop into solution A and kept stirring for 1 h. Depending on the glass composition, the proper amounts of strontium chloride hexahydrate (SrCl2
O, for analysis EMSURE®
ACS) and calcium nitrate tetrahydrate (Ca(NO3
O, 99%, Sigma Aldrich, Milan, Italy), reported in Table 1
, were added.
The final solution was stirred for 15 min and then sprayed (Büchi, Mini Spray-Dryer B-290, Büchi Labortechnik AG, Flawil, Switzerland) using nitrogen as the atomizing gas with the following parameters: inlet temperature 220 °C, N2 pressure 60 mmHg and feed rate 5 mL/min. The obtained powder was calcined at 600 °C in air for 5 h at a heating rate of 1 °C min−1 using a furnace (Carbolite 1300 CWF 15/5, Carbolite Ltd., Hope Valley, UK), in order to remove the template agent.
2.1.2. Preparation of Sr-Containing MBG Samples by Sol-Gel Synthesis (Base-Catalysed)
MBG nanoparticles containing different amount of Sr (2 mol % of Sr, molar ratio Sr/Ca/Si = 2/13/85, and 4 mol % of Sr, molar ratio Sr/Ca/Si = 4/11/85, named hereafter as MBG_Sr2%_SG and MBG_Sr4%_SG respectively) were synthesised by a base-catalysed sol-gel synthesis, based on a modified procedure reported by the authors [16
]. In particular, 6.6 g cetyltrimethylammonium bromide (CTAB ≥98%, Sigma Aldrich, Milan, Italy) and 12 mL NH4
OH (Ammonium hydroxide solution, Sigma Aldrich, Milan, Italy) were dissolved in 600 mL of ddH2
O under stirring for 30 min. Then, 30 mL of tetraethyl orthosilicate (TEOS, Tetraethyl orthosilicate, Sigma Aldrich, Milan, Italy), calcium nitrate tetrahydrate (Ca(NO3
O, 99%, Sigma Aldrich, Milan, Italy) and strontium chloride hexahydrate (SrCl2
O, for analysis EMSURE®
ACS) as reported in Table 2
, were added and kept under vigorous stirring for 3 h.
The powder was collected by centrifugation (Hermle Labortechnik Z326, Hermle LaborTechnik GmbH, Wehingen, Germany) at 10,000 rpm for 5 min, washed once with distilled water and two times with absolute ethanol. The final precipitate was dried at 70 °C for 12 h and then calcined at 600 °C in air for 5 h at a heating rate of 1 °C min−1 using a furnace (Carbolite 1300 CWF 15/5 Carbolite Ltd., Hope Valley, UK), in order to remove CTAB.
2.2. Characterization of Sr-MBGs
Wide-angle (2ϑ within 15–80°) X-ray diffraction measurements (X’Pert PRO, PANalytical, Almelo, The Netherlands) were performed using CuKα radiation at 40 kV and 40 mA.
MBG_Sr_SD were dispersed on a conductive carbon tape prior to SEM observation (Phenom XL, Phenom-World, Eindhoven, The Netherlands). The MBG_Sr_SG particles morphology was analysed by Field-Emission Scanning Electron Microscopy (FE-SEM) using a ZEISS MERLIN instrument (Oberkochen, Germany). In particular, 10 mg of MBG_Sr_SG powders were dispersed in 10 mL of isopropanol by ultrasonication using an ultrasonic bath (Digitec DT 103H, Bandelin, Berlin, Germany) for 5 min to obtain a stable suspension. A drop of the obtained suspension was deposited on a carbon-coated copper grid (3.05 mm Diam.200 MESH, TAAB, Aldermaston, Berks, UK) and allowed to dry. A Cr layer was deposited on powders before SEM and FE-SEM analyses in order to enhance the sample conductivity.
Nitrogen adsorption/desorption isotherms were measured (ASAP2020, Micromeritics ASAP 2020 Plus Physisorption, Norcross, GA, USA), at the temperature of −196 °C. Before nitrogen adsorption–desorption measurements, each sample was degassed at 150 °C for 3 h. The Brumauer–Emmett–Teller (BET) model was applied to determine the specific surface areas (SSABET) of the samples. The pore size distribution was calculated through the DFT method (Density Functional Theory) using the NLDFT kernel of equilibrium isotherms (desorption branch).
2.3. Sr2+ Ions Release Tests
In order to evaluate the concentration of released Sr2+
ions, the powders were soaked in Tris HCl buffer (Tris(hydroxymethyl)aminomethane (Trizma) (Sigma Aldrich, Milan, Italy) 0.1 M, pH 7.4) at concentration of 250 μg/mL, according to the protocol described by Shi et al. [27
]. In particular, 5 mg of powder were suspended in 20 mL of buffer up to 14 days at 37 °C in an orbital shaker (Excella E24, Eppendorf) with an agitation rate of 150 rpm. At defined time points (3 h, 24 h, 3 days, 7 days and 14 days) half of the supernatant was collected after centrifugation at 10,000 rpm for 5 min (Hermle Labortechnik Z326, Wehingen, Germany) and replaced by the same volume of fresh buffer solution to keep constant the volume of the release medium. The release experiments were carried out in triplicate.
The concentration of Sr ions was measured by Inductively Coupled Plasma Atomic Emission Spectrometry Technique (ICP-AES) (ICP-MS, Thermoscientific, Waltham, MA, USA, ICAP Q), after appropriate dilutions. In order to assess the initial amount of strontium incorporated during the synthesis all samples were dissolved in a mixture of nitric and hydrofluoric acids (0.5 mL of HNO3 and 2 mL of HF for 10 mg of powder) and the resulting solutions were analysed via ICP analysis.
2.4. In Vitro Bioactivity of Sr-Containing MBGs
bioactivity test was performed to evaluate the apatite-forming ability of Sr-MBGs in simulated body fluid (SBF). To this aim, 30 mg of Sr-MBGs were soaked in 30 mL of SBF, according to literature [28
]. The samples were kept immersed at 37 °C up to 14 days in an orbital shaker (Excella E24, Eppendorf, Milan, Italy) with an agitation rate of 150 rpm. At each time point (3 h, 1 day, 3 days, 7 days and 14 days), the suspension was centrifuged at 5000 rpm for 5 min, in order to separate the powder from the solution. The pH of each recovered supernatant was measured, and the powder was washed with distilled water and dried in oven at 70 °C for 12 h prior FE-SEM and XRD analysis to evaluate the apatite layer formation.
2.5. In Vitro Biological Assessment of Sr-Containing MBGs
The biological response to MBG_Sr2%_SD and MBG_Sr2%_SG and to their ionic release products was assessed by following two different experimental approaches. In particular, through a direct contact method, where cells were seeded directly on the MBG particles, and through a not-contact method, according to which the Sr-MBG suspensions were placed in a Transwell® membrane insert (<3 µm pore, SARSTEDT AG & Co., Numbrecht, Germany) to allow the passage of the particle dissolution products.
2.5.1. Inflammatory Response of Sr-Containing MBGs
The inflammatory response test was conducted in direct contact mode, using cells and Sr-MBG particles at concentration of 1 mg/mL. For these tests the murine macrophage cell line J774a.1 (European Collection of Cell Cultures) was used. Before the tests, cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco Invitrogen, Cergy-Pontoise, France) supplemented with 10% fetal bovine serum, penicillin (100 U·mL−1), streptomycin (100 μg·mL−1) and 4 mM l-glutamine. Cells were grown in a 100% humidified incubator at 37 °C with 10% CO2 and passaged 2–3 days before use. Then the J774a.1 cells (2 × 104 mL−1) were seeded onto 24-well tissue culture polystyrene plates (Falcon™), containing the Sr-MBG particles. After 4 h, the RNA from J774.a1 cells was isolated by using the Maxwell® RSC simply RNA Cells Kit (Promega Italia s.r.l, Milan, Italy) and reverse transcribed by the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed through the Applied Biosystems StepOne Plus instrument with 2.2 Step-one software version. Mouse interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor alpha (TNFα) and Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ) were chosen from the collection of the TaqMan Gene Expression Assays as primer sets (Applied Biosystems Assay’s ID: Mm01336189_m1, Mm99999062_m1, Mm00443258_m1, Mm03950126_s1 respectively). Real time PCR was performed in duplicate for all samples in a volume of 20 µL and, after an initial denaturation at 95 °C for 10 min, the PCR amplification was run for 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. The content of cDNA samples was normalized through the comparative threshold cycle (ΔΔCt) method, consisting in the normalization of the number of target gene copies versus the endogenous reference gene YWHAZ.
2.5.2. Biocompatibility Test of Sr-Containing MBGs
Fibroblast cell line L929 was used to assess the biocompatibility of Sr-MBGs. Experimental cell culture medium (BIOCHROM KG, Berlin, Germany), composed by Minimum Eagle’s Medium without L-glutamine, 10% fetal bovine serum, streptomycin (100 g/L), penicillin (100 U/mL), and 2 mmol/L L-glutamine, was placed in 250 mL plastic culture flask (Corning TM, Corning, NY, USA). Cells were cultured at 37 °C in a humidified incubator equilibrated with 5% CO2. Cells were harvested prior to confluence by means of a sterile trypsin-EDTA solution (0.5 g/L trypsin, 0.2 g/L EDTA in normal phosphate buffered saline, pH 7.4), re-suspended in the experimental cell culture medium and diluted to 1 × 105 cells/mL.
A preliminary qualitative assessment was carried out through optical imaging of the cells in direct contact with Sr-MBGs particles. In parallel, cell viability tests were performed in Transwell® permeable inserts. Briefly, fibroblast cells were seeded on polystyrene plate below the Transwell® insert containing 1 mg/mL of Sr-MBG suspension, and after 72 h of incubation cell viability was evaluated through MTT assay. This assay allows assessing the possible toxic effect of particle dissolution products on cells, by evaluating the reduction of the mitochondrial succinate dehydrogenase (SDH) enzyme activity, normally involved in the citric acid cycle. For the execution of the MTT test, cells were incubated with a 1 mg/mL solution of soluble tetrazolium salt (3-(4,5-dimethylthiazol–2yl)-2,5 diphenyl tetrazolium bromide). During the subsequent two hours of incubation at 37 °C, the succinate dehydrogenase enzyme causes the transformation of tetrazolium salts into a yellow soluble substance first and then into a blue water-insoluble product, the formazan precipitate. From the quantification of the precipitate product is possible to evaluate the degree of the enzyme activity and, consequently, the number of metabolically active cells. To perform this evaluation, the formazan precipitate was dissolved with dimethylsulphoxide and was spectrophotometrically measured at a wavelength of 570 nm, providing an optical density (OD) value. Cells grown on polystyrene plate were used as negative control, while cells grown with the addition of 20 µL of a solution of 0.08 mg/mL of Sodium nitroprusside (NPS) were used as the positive one.
2.5.3. Osteogenic Response to Sr-Containing MBGs
Osteoblast-like SAOS-2 cells were cultured at 37 °C in a humidified incubator equilibrated with 5% CO2. Cell suspension was obtained by adding 2 mL of a sterile 0.5% Trypsin-EDTA solution (GIBCO by Life Technology, ref 15400-054, Thermoscientific, Waltham, MA, USA) to a 250 mL cell culture flask (Corning™), re-suspended in the experimental cell culture medium and diluted to 1.45 × 105 cells/mL.
For these experiments, 5 mL of the cell suspension were seeded onto 24-well tissue culture polystyrene plates (Falcon™), provided with the Transwell® insert containing 1 mg/mL of Sr-MBG suspension. After 72 h and 7 days of incubation, the expression of GAPDH, COL1a1, RANKL, SPARC, OPG and ALPL genes as cell differentiation markers was assessed using the real time reverse transcription polymerase chain reaction (qRT-PCR) (Applied Biosystems Assay’s ID: Hs00266705_g1, Hs00164004_m1, Hs00234160_m1, Hs00243519_m1, Hs00900358_m1, Hs01029144_m1, respectively).
The RNA from SAOS-2 cells was isolated by using the Maxwell® RSC simply RNA Cells Kit (Promega), by following the manufacturer’s instructions. RNA was reverse transcribed by the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and RNA quantitation was performed before starting the Rt-PCR using Quantifluor system kit (Promega).
Real-time PCR was performed in the Applied Biosystems StepOne Plus instrument (Applied Biosystems). The content of cDNA samples was normalized by making use of the comparative threshold cycle (ΔΔCt) method.
2.6. Statistical Analysis
Experimental data are reported as mean ± standard deviation. Statistical differences between groups were analysed using two-way ANOVA using Tukey’s post-hoc test and one-way ANOVA using Tukey’s pairwise post-hoc test. Statistical significance was represented as * p < 0.05, ** p < 0.01 and *** p < 0.001.
Mesoporous bioactive glasses incorporating different amounts of strontium were successfully prepared through two different synthesis procedures, a base-catalysed sol-gel and an aerosol-assisted spray-drying method, in the form of nano- and micro-particles, respectively. Besides the size of the final particles, the two synthesis approaches allowed to obtained samples with different specific surface area, pore volume and average pore size.
Release tests carried out in Tris HCl revealed that Sr2+ was almost totally released within 7 days with final released concentrations well correlated to the incorporated amount.
The samples showed an excellent bioactive behaviour, evidenced by the formation of HA deposits after 1 day of soaking in SBF, demonstrating that the partial substitution of calcium with strontium does not significantly affect the surface ion-exchange kinetics.
Both type of Sr-containing MBGs were biocompatible, showed a reduced pro-inflammatory response and were able to stimulate the expression of pro-osteogenic genes (COLL1A1, SPARC and OPG), confirming the potential of Sr2+ as therapeutic element for the stimulation of bone remodelling.
Based on the obtained results, Sr-containing MBGs are very promising candidates for bone regenerative applications as such or in association with drugs as multifunctional carriers in combination with polymeric matrices.