The preparation of the glass samples by melting and casting processes was performed as described previously (16,17). Generally, the glass samples with the composition of Na₂O-K₂O-MgO-CaO-B₂O₃-SiO₂-SrO-P₂O₅ (Table I) were prepared by melting each analytical chemical in a platinum crucible at 1,500°C for 2 h with stirring, and then quenching the melt between cold stainless steel plates into glass flakes. These flakes were used as glass wafers (1-mm thick) in the assay experiments, while some of the glass flakes were crushed into fine particles (105–125 μm in size) and used for the extraction experiments.

Prior to all the experiments, the particles or flakes were soaked in 0.025 M K₂HPO₄ solution for 10 days to allow for the superficial reaction and to avoid the fast borate-releasing stage occurring during the following the experimental processes (18,19). Scanning electron microscope (SEM) observation was performed following the soaking of samples and the partial conversion to hydroxyapatite on the surface of borate glass particles, and prior to commencing the experiments (Fig. 1).

Assessment of the in vitro degradation of strontium in borate glass for each group was conducted by statically immersing the glass particles of each group (1 g) in 0.02 M K₂HPO₄ solution (100 ml; pH 7.0) at 37°C for 28 days. The strontium in the immersion solution at different time points was analyzed by ion chromatography (DX 500 Dionex, USA). The average values were obtained from three parallel experiment samples. The accumulated released irons of strontium at different time points were calculated by the weight of the strontium in the solution divided by that in the original samples.

Canine bone marrow-derived mesenchymal stem cells (MSCs) were used in the present experiment. The animal study was approved by the Committee of Medical Ethics and the Institutional Review Board of Shanghai Sixth People’s Hospital, Shanghai Jiaotong University (China). MSCs were isolated by density gradient centrifugation following iliac bone marrow aspiration. The cells were resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Scientific, HyClone, Logan, UT, USA), 100 IU/ml penicillin (Gibco-BRL) and 100 lg/ml streptomycin (Gibco-BRL), and cultured at 37°C in 5% CO₂ and 95% humidity. After culturing for 7 days in the 75-cm² flask, the adherent cells (passage 0) were washed with phosphate-buffered saline (PBS), and then fresh medium was added every 3–4 days. Following the initiation of cultures for 2 weeks, the cells were washed with PBS, and then lifted by incubation in 0.5 ml of 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid for 2 min at room temperature. Trypsin was neutralized by adding 2 ml complete medium. Each flask of cells was passaged into 75-cm² culture flasks every 3–4 days. After primary culturing for 2 weeks, the second passage MSCs were successfully harvested and used in this study (Fig. 2).

The extraction solutions were prepared by immersing 75 mg partially converted glass particles (average diameter, 105–125 μm) in 10 ml culture medium in a CO₂ incubator at 37°C for 24 h, to ensure the specific surface areas reached 6 cm²/ml (according to DS/EN ISO 10993-12:2009). Subsequently, MSCs (passage 2) were seeded in 96-well plates at a density of 1×10⁴ cells/well in 100 μl FBS culture medium, in a CO₂ incubator at 37°C for 24 h. The medium in the well was then replaced with the prepared extraction solution. After 3 days, the CCK-8 (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) with 10 μl CCK-8 was added to each well using a repeating pipettor, and then mixed on an orbital shaker for 1 min. Following incubation for 2 h, the absorbance of each well was measured spectrophotometrically at 450 nm. Three parallel replicates of each sample at each time point were prepared during the cell viability assay. Statistical analyses were performed using a one-way ANOVA.

The 60-mm petri dishes with affixed partially converted glass particles (105–125 μm; 6 cm²/ml) for each group (A-E, as in Table I) were placed on a small platform rocker in the CO₂ incubator to gently mix in 10 ml culture medium prior to incubation. The rocking cycle consisted of 1 min on and 5 min off. Passage 2 MSCs cells were seeded with culture medium at an initial density of 15,000 cells/cm² and incubated at 37°C in a humidified atmosphere of 5% CO₂. The phase contrast images of MSCs at the interface of each group were observed after 3 and 7 days of culturing.

Similarly, MSCs cells were seeded in the medium containing each group of glass particles (6cm²/ml) at a density of 15,000 cells/cm². After culturing for 7 days, the cells mixed with particles were rinsed with warm PBS twice and incubated for an additional 30 min in serum-free DMEM containing 2 mM calcein acetoxymethyl ester (calcein AM; Biotium, Hayward, CA, USA) and 1 μg/ml propidium iodide (Sigma-Aldrich, USA). The fluorochrome-labeled cultures on the glass wafers were then rinsed with PBS and examined under an epifluorescent microscope fitted with appropriate exciter and emitter filters to detect live (green fluorescent) and dead (red fluorescent) cells.

The global effects of the glass particles with different compositions on the proliferation of MSC cells were assessed by measuring the total cellular DNA content in cultures incubated with the glass particles in place. Triplicate samples of each glass group were added to MSC cultures in 60-mm petri dishes and incubated for 7 days. Following incubation, the cultures were rinsed twice with PBS. The DNA on all dishes was extracted using the TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. The quantification of DNA concentration in each group was measured by a UV spectrophotometer (Eppendorf, Hamburg, Germany).

MSCs were cultured in 60-mm petri dishes affixed with partially converted glass particles for each group (A-E, as in Table I) at an initial seeding density of 15,000 cells/cm². DMEM supplemented with 10% fetal bovine serum and 1% antibiotics mixture (100 μg/ml of penicillin and streptomycin) were used as the culture medium. After being grown to confluence, the culture medium of each group was replaced with osteogenic induction medium, containing 10⁻⁸ M dexamethasone, 0.2 mM ascorbic acid-phosphate and 10 mM β-glycerophosphate (Sigma-Aldrich, Co., St. Louis, MO, USA). Cells were incubated at 37°C, 5% CO₂ and 95% humidity, and the medium was replaced every 2 days.

Total RNA of each group was extracted from cells in the 60-mm petri dishes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA (2 μg) was reverse transcribed into cDNA using the cDNA kit (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. Real-time PCR was then performed to examine the expression of osteogenic genes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in the MSCs. The expression levels of each gene were standardized by the internal control levels of GAPDH. Amplification was denatured at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min; then annealed at 54°C for 30 sec and 72°C for 1 min. The temperature was then returned to 95°C and samples were stored at 4°C overnight. These procedures were performed in a PCR machine (Eppendorf).

The primer sequences used were: Forward: 5′-CCGCACGA CAACCGCACCAT-3′ and reverse: 5′-CGCTCCGGCCCA CAAATCTC-3′ for core binding factor α1 (Cbfa1); forward: 5′-CAGTAGTGACTCATCCGAAG-3′ and reverse: 5′-CTCCTCTTCTTCTTCATCAC-3′ for bone sialoprotein (BSP); forward: 5′-CACCGAGACACCATGAGAGC-3′ and reverse: 5′-TGGTCAGCCAACTCGTCAC-3′ for osteocalcin (OCN); forward: 5′-CTTTTAACTCTGGTAAAGTGG-3′ and reverse: 5′-TTTTGGCTCCCCCCTGCAAAT-3′ for GAPDH.

After 14 and 21 days of culturing, the cells were washed twice with PBS, placed in 500 μl of 1% Triton X-100, and lysed using two freeze-thaw cycles (−80/37°C). Aliquots of the lysate were placed in a 96-well plate for spectrophotometric measurement of ALP activity with p-nitrophenyl phosphate (PNPP) substrate, as previously described (17). ALP activity was normalized with respect to the total protein content obtained from the same cell lysate, and was expressed as nanomoles of PNPP formed per microgram of protein per minute. Total protein content was determined using the Micro-Bicinchoninic acid (BCA) Protein Assay kit (Pierce Biotechnologies, Rockford, IL, USA), according to the manufacturer’s instructions.

Alizarin Red S staining of MSC cultures was conducted after 21 days of culturing. The culture medium was removed from each well and cells were rinsed three times with PBS. After fixing with 4% formaldehyde solution for 30 min at room temperature, the cells were rinsed twice with PBS and fixed cells were stained with 1 ml of 2% Alizarin Red S working solution (Sigma-Aldrich) and incubated for 10–20 min at 37°C. Cells were then rinsed three times and visualized under an optical microscope. The number of mineralized nodules per unit was counted.

During immersion in phosphate solution, the ions of strontium changed over time. As shown in Fig. 3, the concentration reached a stable level after ≥20 days. From the curve, we found that in the solution, the levels of strontium released from the borate glass were relative to its composition in each group, the more strontium the particles contained, the higher the concentration in the corresponding soaking solution.

Results of the CCK-8 assay demonstrated the growth rates of the different groups relative to that of the control group with DMEM+FBS. After culturing for three days, the relative growth rates in the groups containing strontium were between 1.0 and 1.5. The 6Sr group demonstrated a significant difference. However, the 0Sr group demonstrated the lowest relative growth rate (Fig. 4).

Fig. 5 shows the cell morphology (on days 3 and 7) of MSCs incubated with the strontium-containing borate glass particles of different compositions. The optical microscope revealed that all cells proliferated well along the borate glass particles. Additionally, the glass particles and the MSCs were 70% confluent, which was consistent with the results of the CCK-8 assay. On day 7, the 6Sr group showed the greatest amount of proliferation of MSCs among all the groups.

The Live-Dead assay (Fig. 6) provided a direct observation of the proportion of living and dead cells. All the groups showed increased cell proliferation on day 7. The 6Sr group exhibited the least number of dead cells and the greatest number of living cells, on staining. Other groups showed a relatively higher proportion of dead cells than the 6Sr group.

Total DNA concentration, cell growth at the genetic level, indicated that the 6Sr group demonstrated the highest concentration, statistically, than any other group (Fig. 7).

In our experiment, the expression of early, middle and late osteogenic genes in primary canine bone marrow MSCs was examined using real-time RT-PCR on days 7, 14, and 21, respectively, to analyse MSC maturation. It was found that on day 7, compared with the 0Sr group, the Cbfa1 mRNA expression level was significantly higher in the 6Sr, 9Sr and 12Sr groups (P<0.01). On day 14, compared with the 0Sr group, the BSP mRNA level was significantly higher in the 6Sr group (P<0.01). In addition, on day 21, similar to BSP expression, groups 6Sr and 9Sr exhibited higher OCN mRNA expression levels than the 0Sr group (P<0.01) (Fig. 8).

The results of the spectrophotometric measurement of ALP activity of MSCs in all the groups are presented in Fig. 9. On day 14, ALP levels in groups with strontium showed no significant differences compared with the 0Sr group. However, on day 21, the 6Sr group demonstrated a higher ALP activity than the 0Sr group (P<0.01). Moreover, at both time points, the groups containing strontium had a higher ALP level than the 0Sr group. However, the difference was only statistically significant in the 6Sr group on day 21.

Alizarin Red S staining was used to examine the mineralized nodule formation by MSCs cultured with different contents of strontium-incorporated borate medium for 21 days (Fig. 10). Measured by Alizarin Red S staining, the number of mineralized nodules per unit in MSCs was also counted. The result showed that groups 6Sr and 9Sr had the greatest number of mineralized nodules (Fig. 11).