Strontium Containing Bioactive Glass Coatings for Titanium Implants Stimulate Osteogenesis in vitro
+1,2 Candarlioglu, P L; 1,2Gentleman, E; 3O`Donnell, M D; 1Stevens, M M
+ Department of Materials and 2Institute of Biomedical Engineering, Imperial College London, UK; 3BioCeramic Therapeutics Ltd; London, UK
Senior author [email protected]
1
Introduction:
Titanium is a widely used material for orthopedic implants. However,
titanium alone is bioinert and would benefit from better adherence to
native tissue to stabilize implanted devices. Bioactive glasses are
biocompatible and osteoinductive; however, they possess poor
mechanical properties alone. Therefore, bioactive glasses may provide
an effective coating that increases the bioactivity of inert titanium
implants.
Strontium (Sr) has been found to have osteoblast stimulating and
osteoclast downregulating properties and has been used as a drug to treat
and prevent osteoporosis in form of strontium ranelate. Sr is chemically
similar to Ca and known to stimulate osteoblasts via the calcium –
sensing receptor. It also downregulates osteoclast activity by decreasing
receptor activation of nuclear factor kappa B (RANK) ligand expression,
which is expressed by preosteoclasts and essential during their
maturation [1].
Bioactive glasses dissolve in body fluids and their dissolution ions
promote bone formation by osteoblasts [2]. Based on former research,
we have combined the osteoinductive properties of Sr with the
osteoconductive properties of bioactive glasses and created a more
biocompatible coating material for titanium implants.
Materials and Methods:
SiO2-P2O5-Na2O-CaO-SrO-K2O-MgO-ZnO bioactive glass and glass in
which 10% and 50% of Ca was substituted with Sr were produced by a
melt-quench route. 1.5g/L of glass powder (<38 µm) was added to RPMI
1640 culture medium and incubated on a roller at 37 0C for 4 hours and
then excess glass was filtered. The dissolved ion concentrations were
confirmed via inductively coupled plasma-mass spectrometry (ICP-MS)
(Fig. 1). Bioglass dissolution ions enriched culture media was
supplemented with 10% (w/v) FBS, 2mM L-glutamine, 1% (w/v)
penicillin-streptomycin and bone mineralizing agents (5 mM βglycerophosphate and 50 µg/mL ascorbic acid).SaOs-2 human
osteosarcoma cells were plated at 30,000/cm2 for metabolic activity
(MTT) and alkaline phosphatase (ALP) analyses, whereas for
tetracycline staining a density of 34,400/cm2 was used. 45S5 was used as
control in all experiments. MTT activity based on reduction of a
tetrazolium salt was measured on days 1,7,14 and 21 post - plating. ALP
activity was measured using p-nitrophenyl phosphate as a substrate and
normalized to cell number against lactate dehydrogenase (LDH) enzyme
activity which measures the conversion of tetrazolium salt (INT) to red
formazan.10 µg/mL of tetracycline HCl was added to cultures after 20
days to visualize newly formed mineral and fluorescence images were
taken after 24 hours. A semi-quantitative analysis of images was
performed using Image J.Statistical significance was noted when
ANOVA and post – hoc Tukey test yielded p<0.05.
Results:
Dissolution ions from 50 % Sr glass significantly enhanced metabolic
activity in SaOs-2 compared to those from 0% Sr glass after 21 days in
culture (Fig. 2A). ALP activity after 7 days in culture was enhanced in
all coating glasses compared to 45S5. SaOs-2 exposed to 0, 10 and 50%
Sr – substituted glasses showed greater ALP activity compared to 45S5
after 14 days in culture. Treatment with 10 and 50% Sr – substituted
glasses enhanced ALP activity compared to 0% Sr glass after 21 days in
culture (Fig. 2B). SaOs-2 treated with dissolution ions of 50% Sr glasses
displayed bright staining for tetracycline staining compared to all groups
including 45S5 after 21 days (Fig 3. A – D). A positive correlation
between Sr substitution and tetracycline staining was confirmed by
semi-quantitative analysis of stain area (Fig 3. E). Culture treated with
50 % Sr – substituted glass shows significant increase compared to all
groups.
Discussion:
Sr has a distinct effect on osteoblasts and osteoclasts and has great
potential to enhance bone formation in vivo. Consistent with previous
reports[3], we have shown that incorporation of Sr into bioactive glasses
enhances the bone forming properties of the glasses compared to
controls such as widely used glass formulation, Bioglass® 45S5 (Fig. 2B,
Fig. 3). ALP activity per cell, a marker of actively mineralizing cells was
higher in cultures treated with our standard non - substituted bioactive
glass compared to Bioglass® 45S5. The increasing addition of Sr
resulted in proportional increases in ALP activity. This is in agreement
with our results from tetracycline staining which shows the brightest
staining for bone nodule formation in cultures treated with 50 % Sr –
substituted glasses (Fig. 4D).
These results suggest that Sr incorporation is an improvement of the
original formula in terms of stimulating bone mineralization. Increased
levels of Sr substitution enhance the osteoinductive features of bioactive
glasses which may make them an improved choice for implant coating
purposes.
Figure 1: ICP-MS measurements as parts per million; A Calcium, B
Strontium, C Phosphate, D Silicon. Time was measured in minutes on x
– axis.
Figure 2: A MTT activity after treatment with standard bioactive
glasses or glasses in which some of the Ca had been substituted for with
Sr; B ALP activity measured per cell for SaOs-2 cells treated with
dissolution ions from high phosphate bioactive glasses and 45S5.
Figure 3: Semi – quantitative tetracycline staining A 45S5, B 0%, C
10%, D 50% for Sr substituted glasses Day 21; E Positive stain area.
Scale bar: 1mm (A – D).
References:
1. Suda T et.al. Endocr Rev 1999, 20(3):345
2. Tsigkou O et.al. Biomaterials, 2009, 30(21):3542
3. Verberckmoes SC et.al. Kidney Int, 2003, 64:534
Acknowledgements:
We thank Robert Hill for helpful discussions and suggestions.
Poster No. 1238 • 56th Annual Meeting of the Orthopaedic Research Society