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Osteoporosis is a major worldwide health problem. It is characterized by a low bone mass with microarchitectural deterioration, leading to bone fragility and a consequent increase in fracture risk. In the United States alone, approximately 25% of women over the age of 50 will suffer one or more vertebral compression fractures (VCFs) as a result of osteoporosis.1 In Asian countries, 50% of postmenopausal women have osteoporosis and 30% of elderly women have one or more VCFs.2
Percutaneous vertebroplasty3 is a method of treating VCFs and involves the injection of cement into the fractured vertebral body, thereby stabilizing and reinforcing it. More recently, the technique of kyphoplasty has been described, whereby restoration of vertebral body height is carried out by inflating a pair of balloons inside the vertebral body, before cement is injected.4 The most commonly used cement is polymethyl methacrylate (PMMA), and this has been shown to have good short- and medium-term results.5–8
PMMA bone cements are not designed for vertebroplasty and as such have some potential disadvantages. First, it has a high polymerization temperature,9 can rise up to 120 C, and may represent a danger to the neural elements if leakage occurs.10,11 Second, there is a lack of osseointegration, with formation of a fibrous layer between the bone and cement; there is thus a potential for loosening in the long term.12,13 Third, there is a large mechanical mismatch between PMMA bone cements, which has a compressive strength of 80 MPa (based on ISO 5833) and osteoporotic bone, which has a compressive strength of less than 10 MPa.14,15 This could result in fractures at the adjacent levels after vertebroplasty.
We propose that the ideal bone cement for vertebroplasty should be one that is injectable, has a stiffness close to bone, is bioactive, can osseointegrate, has a low setting temperature, allows immediate load bearing, and is radiopaque. Based on these requirements, a novel bioactive bone cement was developed16 (U.S. Patent No. 6,593,394). It mainly composes of strontium-containing hydroxyapatite (Sr-HA) filler and bisphenol A diglycidylether dimethacrylate (BIS-GMA) resin. Bis-GMA is a bio-glass commonly used in dental practice, and HA is osteoconductive. While strontium belongs to the same group as barium and calcium, is radiopaque, and has been shown to stimulate bone formation17 and inhibit bone resorption in vitro18 as well as in vivo.19–21 Recently, strontium salts have been shown in human clinical trials to be an effective treatment for osteoporosis, and one of a few drugs that acts by promoting bone formation.22–24
The aim of this paper is to review the in vitro, in vivo, and clinical evidence for the effectiveness of this cement.
Materials and Methods
Preparation of Strontium-Containing Hydroxyapatite Bone Cement.
The Sr-HA bone cement contains a filler blend and a resin blend. The filler blend contains Sr-HA (97.0 wt %), fumed silica (2.5 wt %), and benzoyl peroxide (BPO) (0.5 wt %). The resin blend contains BIS-GMA (50 wt %), triethylene glycol dimethacrylate (40 wt %), poly(ethylene glycol) methacrylate (9.75 wt %), and N, N-dimethyl-p-toluidine (DMPT) (0.25 wt %). Sr-HA was synthesized in the author’s laboratory as previously reported.16
Setting Time and Maximum Temperature Determination.
The setting time and maximum temperature were recorded according to ISO 5833 standard.25 In brief, the Sr-HA bone cement was reconstituted in standardized conditions (room temperature of 23 C ± 1 C, humidity of 50% ± 10%), and poured into a mold of 60-mm diameter and 6-mm height. A thermocouple was inserted into the center of the mold, and the temperature measured at 1-minute intervals.
The compressive strength, bending strength, and bending modulus of the Sr-HA bone cement were determined according to ISO 5833 international standard. Six-millimeter-diameter by 12-mm-length cylinders of cement were attested using a MTS materials testing system (MTS 858 Bionix machine, MTS System Inc., Minneapolis, MN). For compressive testing, load was applied axial to the Sr-HA bone cement cylinder under displacement control of 0.1 mm/s. A curve of deformation against load was plotted by operating the MTS until the cylinder fractured or the upper yield point had been passed. The force applied to cause the fracture, or the 2% offset load or the upper yield point load, whichever occurred first, was recorded and the compressive strength (in MPa) defined by dividing the failure load by the original cross-sectional area of the cement cylinder. The final compressive strength was obtained by taking the average value of 6 bone cement cylinder samples.
Four-point bend test was used to determine the bending strength and bending modulus. Rectangular strips of Sr-HA bone cement were prepared as described above, with dimensions of 75 mm length, 10 mm width, and 3.3 mm depth (±0.1 mm). These were placed symmetrically in the 4-point bend test rig and the load applied. The deflection of the strip as a function of the applied force between 15 N and 50 N was recorded. Each experiment was repeated 6 times, and their average values determined.
In Vitro Evaluation of Cell Attachment and Proliferation.
This experiment compares the ability of osteoblasts to attach to conventional hydroxyapatite versus Sr-HA bone cement. Cylindrical samples of both types of bone cement, 13 mm in diameter and 2 mm thick, were prepared, polished, cleaned with Milli-Q water in an ultrasonic bath, dried in an oven at 110 C, and then autoclaved. They were then fixed inside a 24-well cell culture plate using 2% (w/v) agarose, with the polished surface facing up.
Human osteoblasts (SaOS-2 from American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s Modified Eagle Medium containing 10% (v/v) fetal bovine serum at 37 C in a 5% (v/v) CO2 incubator. Cultured cells at about 80% confluence were subcultured with a 1:4 split (i.e., trypsinized cells from a 80 cm2 tissue culture dish were seeded onto 4 × 80 cm2 tissue culture dishes).
For the assessment of attachment to cement, cells were cultured for 2 hours, 4 hours, and 24 hours, respectively (n = 5 for each time point). Cell attachment was expressed as a mean value ± standard deviation (SD) for statistical analysis.
For proliferation evaluation, SaOS-2 cells were plated at a concentration of 3 × 104 cells per well on the Sr-HA bone cement and the HA bone cement. The cells were released by limited digestion with trypsin-EDTA and counted using a hemocytometer at 1 day, 3 days, 5 days, and 7 days, respectively (n = 5 for each day). Cell proliferation was expressed as a mean value ± SD.
Alkaline phosphatase activity was evaluated at days 7 and 14. A purplish blue color develops in the presence of alkaline phosphatase activity, and the reaction was terminated by rinsing in water. The specimens were viewed under a light stereomicroscope equipped with a digital camera.
In Vitro Mineralization.
As strontium has been shown to induce new bone formation, this ability was tested and compared with HA bone cement. SaOS-2 cells were seeded at a concentration of 1 × 105 cells per well on the Sr-HA and HA cement, respectively, and cultured for 14 and 21 days (n = 3 for each time point). The specimens were stained with 2% (w/v) Alizarin red S (pH 4.2). Newly formed bone-like nodules would be stained orange-red. The samples were evaluated using an image analyzer (Image-Pro Plus, Ver 5.0, Media Cybernetics). The amount of bone-like nodules formation was calculated as a percentage of the bone cement disc area. The values were expressed as the mean ± SD. All the above data were statistically analyzed and significant differences were tested by the Student’s t test’, with P value ≤0.05 being significant.
Scanning Electron Microscopy (SEM) and Energy-Dispersive Radiograph (EDX) Analysis.
SEM and EDX analysis examines cell morphology and mineralization. The Sr-HA and HA bone cements were seeded and cultured with SaOS-2 cells as described above. At the appropriate time points, the cells were fixed with 2.5% glutaraldehyde in cacodylate buffer (0.1 mol/L sodium cacodylate-HCl buffer, pH 7.4) for 4 to 24 hours at 4 C. The specimens were serially dehydrated in ethanol, and dried in a critical point dryer using liquid carbon dioxide as transitional fluid. They were then sputter-coated with a 100 Å layer of carbon. The morphology of SaOS-2 cells was observed using a scanning electron microscope (JEOL Stereoscan 360). Minerals were determined by an EDX microanalyzer (Oxford Link model eXL) on 5 random points on each specimen.
In Vivo Evaluation of Biocompatibility, Osteoconduction, and Induction.
This part of the study aims to compare the behavior of the Sr-HA cement with conventional PMMA cements when implanted into the ileum of rabbits. The detailed methodology has been reported previously.26 In brief, the iliac crests of 33 New Zealand white rabbits between 4 to 6 months of age and weighing 2.5 to 3.9 kg were injected with a standard volume of bone cement. 21 rabbits had Sr-HA, while 12 had PMMA (Surgical Simplex P, Howmedica, Ireland) bone cement injection. The rabbits were killed at 1, 3, or 6 months after injection, with each group consisting of 11 rabbits, 7 with Sr-HA, and 4 with PMMA bone cement.
Osseointegration was measured by the affinity index. This is defined as the ratio of the length of the region in which bone is in direct contact with the cement without the presence of intervening tissue divided by the total length of the bone-cement interface, and expressed as a percentage.26 All the measurements were performed using a computer (Leica Qwin Image Processing & Analysis Software, version 2.4) connected to a light microscope (Leica DM-LB) and a digital camera (Leica DC 300). Significance was assessed by the Student’s t test, with P value ≤ 0.05 being significant.
For transmission electron-microscopy (TEM) examination, ultra-thin sections were prepared. The undecalcified samples were embedded in Epon, and TEM foils prepared by sectioning with a diamond knife, followed by carbon coating. The sections were examined with a Tecnai 20 microscope at 200 kV.
Pilot Study in Human Subjects.
After obtaining State Drug Administration’s (China) approval for clinical trials, a pilot study was carried out. Study participants who presented with osteoporotic vertebral compression fractures with persistent pain despite conservative care were offered vertebroplasty. The procedure was performed using a percutaneous transpedicular technique under guidance with image intensification or with CT scan. Pain was assessed using the visual analogue scale, and radiographs of the spine were taken on every outpatient visit. Vertebral body height was measured, presence of adjacent level fractures were noted.
Setting Time and Maximum Temperature
The setting time of Sr-HA bone cement varied between 15 and 18 minutes. The peak curing temperature was 58° C.
The mean compressive strength of the SrHA cement was 40.9 MPa (±8.9). The mean bending strength was 31.3 MPa (±3.2). The bending modulus was 1,408.5 MPa (±142.3). These are less than current PMMA cements (Table 1).
In Vitro Evaluation of Cell Attachment and Proliferation
Both the Sr-HA and the HA bone cements allowed osteoblast attachment (Figure 1). There was a significant increase in the number of osteoblasts adhered to the Sr-HA bone cement compared with the HA bone cement after 4 hours and 24 hours. However, there was no difference in the rate of proliferation of osteoblasts between the two cements (data not shown). Alkaline phosphatase activity was detected at both time points, and in both Sr-HA and HA cements, suggesting that the osteoblastic phenotype was maintained (data not shown).
In Vitro Mineralization
Both the Sr-HA and the HA bone cements have the capacity to promote strong osteoblastic mineralization in vitro (Figure 2). However, on morphometric analysis, significantly more bone was formed on the Sr-HA bone cement than the HA bone cement at both 14 and 21 days (Figure 3).
On evaluation with EDX analyzer, which determines the calcium and phosphorus ratio (Table 2), the matrix was confirmed to be a calcified one with the presence of calcium and phosphorus. The increase in calcium to phosphorus (Ca/P) ratio over time is an indication of progressive mineralization. A significantly higher amount of minerals was detected to be formed on the Sr-HA bone cement as compared with the HA bone cement at 14 and day 21 days (P < 0.05).
In Vivo Evaluation of Biocompatibility, Osteoconduction, and Induction
Significant differences could be seen between PMMA and Sr-HA cements. As expected with PMMA, bone necrosis could be seen with a reduction in the number of trabeculae within the cement implanted area when compared with control. Neither active bone formation nor remodeling was observed (Figure 4).
Sr-HA bone cement showed active bone remodeling at 1 month, with osteoblast laying down an osteoid layer on the cement bone interface, and bone resorption by multinucleated giant cells (osteoclasts), as indicated by the presence of irregular surface pit. New bone growth into the gaps of Sr-HA cement was observed after 2-month implantation (Figure 5).
After 3 months, there was an increase in the thickness of the osteoid layer and osteoblasts and osteoclasts could still be seen. There was good contact between the newly formed bone and the Sr-HA cement, suggesting osseointegration. The affinity index was 73.55% ± 3.50%. This appearance was similar at 6 months (Figure 6), with a further increase in contact between the bone and Sr-HA cement giving an affinity index of 85.15% ± 2.74%. When compared with 3 months, this difference was significant (P < 0.01).
EDX analysis revealed similar composition for the Sr-HA bone cement and bone (Figure 7), indicating bony in-growth into the cement. This is further supported by an analysis of the Ca, Sr, and P ratios in bone, at the bone-cement interface, and within the Sr-HA cement showing no significant differences (Figure 8). All these help confirm that there is intimate contact between the bone and Sr-HA bone cement (Figure 9).
Preliminary Results in Clinical Trial
The main aim was to examine the safety and efficacy of Sr-HA cement in a small group of patients with short- to medium-term follow-up, before a large-scale randomized control study is started.
Twenty-three cases of single-level vertebroplasty were carried out. All patients sustained an osteoporotic spinal fracture in the low thoracic or lumbar spine and were nonresponsive to conservative treatment measures of bracing and analgesics. The mean follow-up was 18 months, with improvement in their pain scores by 85% in 22 of the 23 cases.27,28 Those that were unable to ambulate because of pain were all able to do so afterwards. Follow-up radiographs showed that vertebral body height was maintained (Figure 10), with no adjacent level fractures or recurrence of back symptoms. Three cases were found on postinjection computed tomographic images to have a slight leakage of cement into the spinal canal, but none of the patients had any neurologic sequelae.28
The Sr-HA bone cement has a number advantages over commonly used PMMA cement for vertebroplasty or kyphoplasty. First, osseotegration occurs with Sr-HA and does not with PMMA. The nonbioactive PMMA results in the formation of an intervening soft tissue layer between the bone and cement.29–31 Osseointegration will likely result in better long-term outcome with reduced risk of loosening and also raises the possibility of using the Sr-HA cement for augmenting the vertebral body in young, traumatic fractures.34
Second, PMMA induces local inflammation and bony necrosis; this is likely due to the presence of the toxic methyl methacrylate (MMA) monomer.32–33 In the Sr-HA specimens, no foreign body reaction, no inflammation, and no necrosis was observed by radiographic or histologic analyses. This is because BIS-GMA used as the resin in Sr-HA bone cement is nontoxic to cells.16,34 We demonstrated clearly in the in vivo model that there is no loss of trabecula bone after injection of Sr-HA.
Third, from safety aspects, Sr-HA is superior to PMMA in its lower setting temperature and reduced stiffness to a level closer to bone; it has equivalent properties to PMMA in terms of ease of injection and setting times. It has been postulated by some that the exothermic reaction of PMMA may partly be responsible for the pain relief after vertebroplasty. Our preliminary clinical data would suggest that this is not the case, but rather, the primary reason for pain improvement is increased stability after cement stabilization.
Strontium belongs to the same group as barium and calcium, and itself is radiopaque; therefore, unlike PMMA cement, no additional barium is needed to enhance its radiopacity. Alterations in the ratio of barium to PMMA can significantly affect its mechanical properties.14,15 Our group has previously shown in a porcine model that, when injected into the fractured vertebral body, Sr-HA is effective in fully restoring the stiffness and failure strength to prefracture levels.34
Other bioactive materials for vertebroplasty have been described. Calcium phosphate cements are biocompatible and osteoconductive.35–37 However, its major disadvantage for vertebroplasty is that the material has poor mechanical properties and is not recommended for use in weight-bearing areas. Apatite and wollastonite containing glass ceramic powder and BIS-GMA based resin are also bioactive, but they have a higher mechanical strength than PMMA,38–40 and may therefore aggravate the problem of adjacent level fractures.
The bioactivity and biocompatibility of HA has been well documented both in vitro and in vivo. When added to PMMA, there was a significantly higher cell proliferation and differentiation rate in vitro than PMMA alone.41 This study demonstrated that the addition of strontium to HA can increase the bioactivity of HA, by enhancing osteoblast attachment, and therefore resulting in a quicker rate of bone formation on the cement-bone interface. This would ultimately have the effect of enhancing osseointegration.
Strontium ranelate has been shown to stimulate osteoblast function as well as inhibiting osteoclast bone resorption.42 When given orally, strontium ranelate was shown to be effective in treating osteoporosis.23,43,44 The present study would suggest that strontium administered locally may also have similar effects, although the mechanism of action is not known and is the subject of further investigation.
In principle, depending on clinical needs, the properties of the Sr-HA bone cement can be varied by altering the ratio of the various components within the cement. The polymerization of methacrylate and its derivatives is controlled by BPO and DMPT. Increasing the amount of BPO and DMPT will make the polymerization rate of the bone cement faster, i.e., shorter setting time, but heat release will also be increased. On the other hand, decreasing the amount of BPO and DMPT added to the bone cement can increase the setting time and lower the maximum temperature. However, a prolonged setting time may make the cement difficult to work with, and insufficient amounts of BPO and DMPT can result in incomplete polymerization leading to poor mechanical strength. Thus, in reality, it is difficult to optimize all the properties simultaneously, and a balance should be made on the amount of BPO and DMPT so that its setting time and maximum temperature are suitable to be used in spinal surgery.
While PMMA bone cements are widely used for vertebroplasty and kyphoplasty, the authors believe that there is room for improvement, especially for the development of bioactive cements specifically designed for this indication. The Sr-HA bone cement therefore shows potential for this purpose and is currently undergoing extensive clinical trials to demonstrate its short and long-term effectiveness and safety.
- Currently used PMMA bone cements are not designed for vertebroplasty and kyphoplasty.
- PMMA cements are nonbioactive and have a high stiffness mismatch with osteoporotic bone.
- The new strontium-containing hydroxyapatite cement is superior to PMMA in its handling and mechanical properties, biocompatibility, and osseointegration.
- Strontium delivered locally with hydroxyapatite is able to promote bony ingrowth and attachment.
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