The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro

TLDR: It is shown that ions released from strontium-substituted BG enhance metabolic activity in osteoblasts and inhibit osteoclast activity by both reducing tartrate resistant acid phosphatase activity and inhibiting resorption of calcium phosphate films in a dose-dependent manner.

About: This article is published in Biomaterials.The article was published on 2010-05-01 and is currently open access. It has received 541 citations till now. The article focuses on the topics: Strontium ranelate & Bone resorption.

Figures

Fig. 2. Normalised MTT activity of Saos-2 human osteosarcoma cells treated with dissolution ions from standard BG and BG in which increasing proportions of calcium were substituted for with strontium. Data represent mean þ standard deviation and are normalised to the mean value of controls after 1 day in culture. * indicates significantly higher MTT activity in the indicated group compared to the control group at the same time point. y compared to 0%, z compared to 10%, and x compared to 50% (all at the same time point).

Fig. 1. Elemental concentrations of Ca, P, Si and Sr as determined by ICP-OES in cell culture media over 4 h incubation with BG particles in which 0, 10, 50 or 100% of the calciumwas substituted for with strontium. Dissolution behaviour into (a) RPMI 1640 and (b) DMEM.

Fig. 5. (a) Total number of cells on BG discs after 1, 7 and 14 days in culture as determined by measuring lactate dehydrogenase activity. After 7 days in culture, there were significantly more cells on 100%-strontium-substituted BG discs than on 0%, and after 14 days in culture there were significantly more cells on 50 and 100% strontiumsubstituted BG discs compared to either 0 or 10% (y significantly more cells compared to 0% at the same time point, z compared to 10%). (b) Total alkaline phosphatase (ALP) activity (mean þ standard deviation) in Saos-2 cells on 0% strontium-substituted BG discs and BG inwhich 10, 50 or 100% of the calciumwas substituted for with strontium. After 7 days in culture, cells on 100% strontium-substituted BG discs had significantly higher total ALP activity than those on 0% or 10%-strontium-substituted BG discs. After 2 weeks in culture, cells on discs in which 50 or 100% of the calcium had been substituted for with strontium had significantly higher total ALP activity than those on 0% or 10% strontium-substituted BG discs (y significantly greater total ALP activity compared to 0% at the same time point, z compared to 10%).

Fig. 6. SEM image of osteoclast on 0% strontium-substituted BG disc after 4 days in culture. A large osteoclast with a typical apico-basal polarised resorbing morphology (black arrow) is visible as are many undifferentiated monocytes (white arrows). Scale bar is 100 mm.

Fig. 4. (a) Representative epifluorescense micrograph showing large multinucleated osteoclasts with typical podosome belts (arrows) in cultures of RAW264.7 mouse monocytes after 4 days treatment with RANKL and dissolution ions from 50% strontium-substituted BG. (b) Epifluorescense micrograph of RAW264.7 treated with RANKL and 1 mM pamidronate in which no multinucleated osteoclasts are visible. Red ¼ phalloidin (actin), Blue ¼ DAPI (nuclei). Scale bar in (a) and (b) is 200 mm. (c) Total TRAP activity (mean þ standard deviation) expressed as nmoles p-nitrophenyl liberated per minute in the presence of 20 mM tartrate in RAW264.7 differentiated in BG dissolution ion media or with 1 mM pamidronate (PAM). All cultures were supplemented with 20 ng/mL RANKL. Pamidronate significantly reduced total TRAP activity at all time points compared to all other groups (#). After 4 days, RAW264.7 treated with 100% strontium-substituted BG had significantly reduced TRAP activity compared to all other glass treated groups and controls treated with RANKL alone (CNT) (*compared to CNT at same time point; y compared to 0%; z10%; x50%). After 6 days, cells treated with 10, 50 and 100% strontium-substituted BG had significantly lower TRAP activity when compared to 0% (y). (d) Total number of cells in cultures of RAW264.7 monocytes treated with dissolution ions from BG or pamidronate. There were no significant differences between the number of cells detected in any group.

Frequently asked questions

Q1. What have the authors contributed in "The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro" ?

Here the authors created a BG series ( SiO2–P2O5–Na2O–CaO ) in which 0–100 % of the calcium was substituted by strontium and tested their effects on osteoblasts and osteoclasts in vitro. The authors show that ions released from strontium-substituted BG enhance metabolic activity in osteoblasts. These results suggest that similarly to the osteoporosis drug strontium ranelate, strontium-substituted BG may promote an anabolic effect on osteoblasts and an anti-catabolic effect on osteoclasts. 

Q2. What is the effect of BG on osteoclasts?

Strontium-substituted BG not only stimulated increased osteoblast metabolic activity and inhibited osteoclast differentiation and resorption via their dissolution ions, but also promoted osteoblast proliferation and ALP activity while directly in contact with cells. 

Q3. What is the role of BG in osteoblasts?

As BG are a well-studied biomaterial and excellent system for controlled ion release, strontium-substituted BG may find use in a range of bone regeneration applications including as porous scaffolds, rigid materials and particulate. 

Q4. What is the role of silicon in the cell-stimulating activities of BG?

which is thought to play a role in the cell-stimulating activities of BG dissolution ions due its essential role in bone formation [21], is released into culture media over time due to the breakup of the outer silica layers of the BG network [10]. 

Q5. How thick were the BG rods cut?

Discs 1 mm thick were cut from the rods with a low speed diamond saw (Buehler IsoMet , Dusseldorf, Germany) using isopropanol as a coolant. 

Q6. How long did the BG dissolution ions in culture be incubated?

after 2 weeks in culture, dissolution ions from all formulations of BG enhanced total metabolic activity in Saos-2 cells compared controls. 

Q7. How long did the BG discs undergo a preconditioning?

BG discs were sterilised under UV light for 2 h per side and preconditioned for three days in RPMI 1640 medium (exchanged every day). 

Q8. What is the effect of strontium substitution on osteoblasts?

The effects of strontium substitution in the BG were tested on osteoblast and osteoclast cultures and demonstrate that strontium-substituted BG promote osteoblast proliferation and activity and decrease osteoclast activity and resorption. 

Q9. Why is there concern that long-term treatment with bisphosphonates may freeze bone?

Because of its mode of action, there has been concern that long-term treatment with bisphosphonates may ‘freeze’ bone, prohibiting normal repair processes. 

Q10. What is the mechanism by which strontium ions prevent erosion pit formation in the present study?

The mechanism by which strontium ions prevent erosion pit formation in the present study is not clear, butinhibition of differentiation and disruption of cytoskeletal elements are two possibilities. 

Q11. What was the nih's instructions for analyzing the eroded area?

Micrographs of stained wells were taken on an inverted light microscope and the eroded area was determined using the histogram function in ImageJ (freeware from the NIH). 

Q12. What is the effect of the BG dissolution ions on the saos?

This suggests that dissolution ions released from the strontium-substituted BG further enhance metabolic activity in Saos-2 beyond that caused by the presence of dissolution ions from standard BG. 

Q13. How many BG compositions did the phosphorus concentration change?

The phosphorus concentration in RPMI 1640 changed little over the 4 h release period while in DMEM a general decrease in phosphorus concentration was observed for all BG compositions. 

Q14. What is the correlation coefficient between strontium substitution and erosion pit area?

Mean erosion pit area, an indicator of osteoclast activity, decreased when RAW264.7 monocytes were treated with dissolution ions from BG in which an increasing proportion of calcium was substituted for with strontium (Fig. 3c). 

Q15. How did the TRAP activity of RAW264.7 cells differ from that of other groups?

After 28 days in culture, cells treated with dissolution ions from 100% strontium-substituted BG had significantly higher MTT activity than that in any other group. 

Q16. How does the release of silicon in cell culture media differ from other BGs?

While silicon release into simulated body fluid is reported to happen quickly (30 min–2 h), release into cell culture media proceeds more slowly [20], and although their data show that the siliconconcentration in cell culture media increases with time for all BG compositions and in both media formulations, it does not reach its solubility limit (120 ppm in H20). 

Q17. What is the morphology of the osteoclasts on BG discs?

Osteoclasts on BG discs assumed typical resorption morphologies, which was in contrast to non-resorbing morphologies noted in osteoclasts on tissue culture plastic. 

Q18. How was the statistical significance of the BG assays assessed?

Statistical significance in MTT, TRAP and ALP assays was assessed by ANOVA with post-hoc LSD test to determine statistical significance between individual groups. 

Q19. What did actin staining highlight in mature osteoclasts?

Actin staining highlighted typical podosome belts in mature osteoclasts which appeared as prominent rings encasing multiple nuclei (Fig. 4a). 

Q20. What is the role of RANKL in osteoclast activity?

Recent studies have suggested that modulation of osteoprotegerin and RANKL expression, known regulators of osteoclast activity and resorption, are responsible for this effect [25]. 

Q21. How did the cells with 50 or 100% strontium substitution perform?

After 2 weeks in culture, cells on BG discs with 50 or 100% strontium substitution had significantly higher total ALP activity than those on discs with 0% or 10% strontium substitution.