Mehran
Dadkhah
a,
Lucia
Pontiroli
ab,
Sonia
Fiorilli
a,
Antonio
Manca
c,
Francesca
Tallia
ad,
Ion
Tcacencu
e and
Chiara
Vitale-Brovarone
*a
aDepartment of Applied Science and Technology, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy. E-mail: chiara.vitale@polito.it; Tel: +39 011 0904716
bOral Biology, School of Dentistry, University of Leeds, LS9 7TF Leeds, UK
cRadiology Unit, Istituto di Candiolo – Fondazione del Piemonte per l'Oncologia (FPO), IRCCS, Strada Provinciale 142 km 3.95, 10060, Candiolo (Torino), Italy
dDepartment of Materials, Imperial College London, Prince Consort Road, SW7 2BP, London, UK
eDepartment of Dental Medicine, Karolinska Institutet, Huddinge, Sweden
First published on 16th November 2016
In this study, an innovative injectable and bioresorbable composite cement (Spine-Ghost) has been developed by combining a radiopaque glass-ceramic powder (SCNZgc) and spray-dried mesoporous bioactive particles (W-SC) into type III alpha calcium sulphate hemihydrate (α-CSH) (composition α-CSH/SCNZgc/W-SC, 70/20/10 wt%). The Spine-Ghost cement and pure α-CSH (as a reference) were characterised in terms of physical and mechanical properties and compared to a commercial reference (Cerament® – Bonesupport AB, Sweden). The Spine-Ghost cement had a setting time comparable with Cerament® showing a good injectability in the range of 8–20 minutes after the end of mixing. In addition, the Spine-Ghost cement showed a good radiopacity when compared with standard PMMA (BonOs Inject, aap Biomaterials GmbH Germany) and higher compressive strength when compared to healthy cancellous bone. The bioactivity of both Spine-Ghost and Cerament® was evaluated through in vitro soaking in simulated body fluid (SBF). Spine-Ghost samples were highly bioactive, inducing the precipitation of hydroxyapatite crystals in the first week of soaking in vitro. It was also found that the degradation kinetics of the Spine-Ghost cement were faster than those of pure α-CSH and comparable to those of Cerament® after approximately 1 month of soaking in SBF. Moreover, the Spine-Ghost cement was cytocompatible in indirect-contact culture in vitro. Overall results indicate that the Spine-Ghost cement might be a very good candidate for vertebroplasty application and could enhance new bone formation in vivo.
Injectable bone cements (IBCs) play a critical role in determining the effectiveness of VP and KP.
Table 1 reports the list of the most relevant properties for an IBC.10,11
• No toxicity |
• Easy injectability and handling |
• Sufficient radiopacity in order to be able to follow the cement flow under fluoroscopy |
• Working time in the range of 6–10 min |
•Appropriate setting time of about 15 min |
• Appropriate resorption rate (neither too high nor too low) |
• Appropriate mechanical properties for immediate reinforcement of the vertebral body, the value should be comparable to those of the healthy vertebral body (the compressive strength of cancellous bone is about 2–12 MPa) |
• Reasonable cost |
Over the twentieth century, calcium sulphate has been considered as a bone substitute in non-load-bearing bone injuries, due to its superior characteristics such as biocompatibility, biodegradability, osteoconductivity, non-toxicity and mechanical strength12–14 comparable to cancellous bone.15 However, its clinical applications have been limited due to its poor bioactivity,16 lack of osteoinductivity17 and too fast resorption rate in vivo18,19 in comparison with bone regeneration.
Currently, the number of commercial calcium sulphate-based cements is limited: A-GRIX® (AG Digital Technology Corp), Cerament® (BoneSupport AB (Sweden)), OsteoCure® (Futura Biomedical (US)), DentoGen® (Orthogen Corporation), MIIG® X3 and Pro-Dense® (Wright Medical (US)). These cements are composed of one or more solid phases and a liquid phase, which are mixed before operating.20 Among these commercial calcium sulphate-based bone cements, the use of MIIG® in 28 patients was described by Chen and co-workers21 for the open surgical treatment of thoracolumbar burst fractures with posterior instrumentation/decompression combined with vertebroplasty. The safety and efficacy of Cerament® (Bonesupport AB, Sweden) were first described in a prospective non-randomised multicentre study on 15 patients,22 followed by a prospective study on 20 patients and a histological study on a rabbit bone defect documenting bone ingrowth.23
In recent years, several approaches have been proposed for the development of synthetic calcium-based bone cements. For example, Yang and co-workers24 combined calcium sulphate with β-tricalcium phosphate to improve the osteoregenerative capacity. In 2013, Chen and co-workers25 improved the rapid resorption of calcium sulphate by adding dicalcium silicate. In 2015, Tan and co-workers26 reported a composition of calcium sulphate cement/silica-based mesoporous materials (SBA-15) for the controlled release of BMP-2.
Bioactive glasses are a group of surface reactive biomaterials that in certain ranges of compositions are characterised by an excellent osteoconductivity and ability to stimulate bone formation. Mesoporous bioactive glasses (MBGs) are a more recent subcategory of bioactive glasses that have received considerable research interest in the last 5 years.27 This increasing interest is due to their excellent structural features such as a well-ordered mesoporous channel structure, a large specific surface area, a tunable volume and pore size (5 to 20 nm) and a narrow pore diameter distribution.28,29
Calcium phosphate and calcium sulphate cements seem to be applicable as biomaterials for vertebral stabilisation and augmentation.30,31 However these cements have some shortcomings such as poor injectability and handling, low mechanical strength, low viscosity and too fast absorption.30–32 To bypass these limitations, the purpose of our study was to develop an injectable composite cement based on type III alpha calcium sulphate hemihydrate (α-CSH) as a resorbable matrix, enriched with mesoporous glass particles and a glass-ceramic radiopaque phase. Setting time, injectability, compressive strength and in vitro bioactivity and degradability in simulated body fluid (SBF) of the developed injectable cement were assessed. Biological tests using rat bone marrow stromal cells were also carried out in vitro.
In brief, 2.03 g of P123 (EO20PO70EO20, where “EO” is poly(ethylene glycol) and “PO” is poly(propylene glycol)) was dissolved in distilled water (85 mL) until a transparent solution was obtained. In the meantime, 10.73 g of tetraethyl orthosilicate (TEOS) were pre-hydrolysed in an aqueous hydrochloric acid under vigorous stirring to obtain a transparent solution. Subsequently, pre-hydrolysed TEOS was added dropwise to the polymeric solution and stirred for one hour to obtain a transparent solution. Afterwards, 3 g of calcium nitrate tetrahydrate (CaNT) were added to the mixed aqueous solution and stirred for 20 minutes. Spray-drying of the final synthesis solution was carried out using a Büchi Mini Spray-Dryer B-290. The collected sprayed powders were then calcined at 700 °C for 5 hours. The obtained mesoporous powders will be referred to as W-SC hereafter.
The N2 adsorption–desorption isotherm of the W-SC powders was evaluated using a Quantachrome Autosorb-1 instrument. The specific surface area was calculated through the BET (Brunauer–Emmett–Teller) model in the relative pressure range of 0.04–0.1 and pore size distribution was obtained through the DFT (Density Functional Theory) method, using the NLDFT (Nonlocal Density Functional Theory) equilibrium model.
Powders were characterised by X-ray diffraction (XRD, PhilipsX'Pert diffractometer, 2θ within 10–70°) and XRD patterns were analysed using X-Pert high score software. In addition, powders were characterised using a field emission scanning electron microscope (FESEM, Merlin-Zeiss) and an energy dispersive X-ray spectrophotometer (EDS). To prepare the samples for FESEM observations, the mesoporous powders were dispersed in ultrapure isopropanol and sonicated, and then a drop of this suspension was dispensed on a copper grid with holey carbon (200 mesh).
Glass-ceramic particles (SCNZgc) with composition SiO2/CaO/Na2O/ZrO2, 57/30/6/7 mol% were prepared by melting and casting processes. Briefly, silica (SiO2), calcium carbonate (CaCO3), sodium carbonate (Na2CO3) and zirconia (ZrO2) were melted in a platinum crucible at 1600 °C, followed by rapid quenching in water. Thereafter, the glass frit was ceramised at 1150 °C for 3 hours. This process was followed by grinding and sieving to yield powders below 20 μm.34
The phase composition, the microstructural morphology as well as the elemental analysis of Spine-Ghost were characterised by XRD analysis and FESEM coupled with EDS, respectively. Since Spine-Ghost samples were not conductive, they were coated with a metal layer (Cr) to allow for FESEM observation.
The injectability of the Spine-Ghost cement and the CSC was evaluated qualitatively by extruding a certain amount of paste through a 13 gauge vertebroplasty needle (internal diameter 1.803 mm). To optimise the L/P of the Spine-Ghost cement, different L/P ratios were evaluated. For this reason, 5 g of, respectively, Spine-Ghost and calcium sulphate hemihydrate (CSH) powders were mixed with distilled water in a syringe coupled with a vertebroplasty needle (13 gauge), L/P = 0.3, 0.35 and 0.4 mL g−1. The syringe was pressed manually under constant pressure and speed, until the paste could be extruded. The quantity of the cement remaining in the syringe was then visually compared for the different samples.
In order to evaluate the compressive strength of Spine-Ghost, CSC and Cerament®, cylindrical specimens (Φ 13.5 mm, h 10.0 mm) were prepared by using custom made polymeric moulds. The compressive test of the samples was performed under both wet and dry conditions, respectively, after one and seven days from the sample preparation, based on ASTM F2224-09. The test was carried out with a 5 kN load cell, using a crosshead speed of 1 mm min−1 by means of a Zwick/Z100 machine from Zwick/Roell. Three samples per type of cement were tested, the compressive strength was calculated for each group and the results were expressed as M ± SD. Compressive strength (σc) was defined as the maximum pressure load sustained by the sample (Fmax) divided by the cross area of the sample (A), as reported in eqn (1):
σc = Fmax/A | (1) |
Degradation ratio = [(W0 − Wt)/W0] × 100 | (2) |
The morphology and phase composition of the soaked samples were characterised using FESEM/EDS and WAXRD, respectively.
Cytocompatibility was assessed by tetrazolium salt colorimetric assay according to the manufacturer's instructions (Cell Proliferation Kit I (MTT), Roche Diagnostics) after 24 hours of culturing in alpha-minimum essential medium supplemented with 10% fetal bovine serum and antibiotics/antimycotics (all from Life Technologies, Inc.).
Alkaline phosphatase (ALP) activity was used as a marker for the osteogenic differentiation of BMSCs in the presence of the Spine-Ghost discs. For the ALP assay, the normal medium was replaced with commercially available osteogenic medium (Osteogenic Differentiation BulletKit™, Lonza) containing ascorbic acid, beta-glycerophosphate and dexamethasone. After 7 and 14 days of indirect-contact osteogenic culturing, the BMSCs were washed with phosphate buffer, then lysed with 0.2% Triton X-100 (Sigma-Aldrich), and sonicated. ALP activity was quantified by measuring the rate of formation of p-nitrophenol (pNP) produced by hydrolysis of p-nitrophenylphosphate (Sigma-Aldrich) in 1 M diethanolamine solution, buffered to pH 9.8 at 37 °C for 30 min using a spectrophotometer (Labsystems Multiskan MS), measured at 405 nm. The final ALP data were expressed as fold change vs. cells cultured alone.
As previously reported by the authors in ref. 33, W-SC showed a type IV nitrogen adsorption–desorption isotherm, typical of mesoporous materials, with a sharp step at approximately 0.7p/p0. The BET specific surface area is around 190 m2 g−1 and the pore volume 0.25 cm3 g−1. The average pore size, as calculated by the DFT method, was centred at 5 nm.
Fig. 3 shows the FESEM images of the α-CSH powder before and after hydration (CSC) and the Spine-Ghost cement after setting for 24 hours. As it can be observed in Fig. 3(a and b), the α-CSH powders had prismatic crystals with an average length and width of approximately 9 and 3 μm, respectively. The FESEM micrographs of the CSC showed the needle-like crystals of CSD (Fig. 3(c and d)). In addition, the initial composition of the CSC was confirmed by the EDS analysis (Fig. 3e), which detected Ca, O and S. FESEM micrographs of the Spine-Ghost cement are shown in Fig. 3(f and g) at different magnifications. As it can be seen, the Spine-Ghost cement was composed of micron-sized plates, spherical particles (W-SC) and some irregular particles that could be attributed to the three components of the cement calcium sulphate hemihydrate, mesoporous particles and glass-ceramic particles, respectively. The EDS qualitative analysis of the Spine-Ghost sample (Fig. 3h) showed the presence of S, O, Si, Ca, and Zr because of the different cement phases. Fig. 4 shows the EDS mapping analysis of S, O, Si, Ca, Zr and Na for the Spine-Ghost cement, which revealed a homogeneous distribution of the different phases throughout the cement, without segregation.
Fig. 3 FESEM images of (a and b) the α-calcium sulphate hemihydrate powder (α-CSH); (c–e – EDS) the calcium sulphate cement (CSC); (f–h – EDS) the Spine-Ghost cement. |
The setting times of the CSC, Spine-Ghost and Cerament® are reported in Fig. 6a. According to the obtained results, the longest initial and final setting times were observed for the Spine-Ghost cement with (46 ± 0.5) and (57 ± 0.5) minutes, respectively. However, the composite cement hardened within 1 hour, which is a time comparable to Cerament®, taken as a commercial reference. Instead, the CSC exhibited the lowest initial (31 ± 0.5 min) and final (37 ± 0.5 min) setting times.
Wet and dry compressive strengths of the CSC, Spine-Ghost and Cerament® cements were measured after setting of the samples for one and seven days, respectively (Fig. 6b). These results suggested that the CSC had a relatively higher wet compressive strength (approximately 15.8 ± 1.1 MPa) compared to the Spine-Ghost cement (14 ± 0.7 MPa). Conversely, Spine-Ghost exhibited the highest compressive strength after setting for seven days (18.1 ± 0.8 MPa). These results clearly demonstrate that the Spine-Ghost cement has a higher compressive strength under both wet and dry conditions compared to Cerament®.
Fig. 7 FESEM images of W-SC: (a and b) after immersion for 8 hours in SBF; (d and e) after 24 hours of soaking in SBF and related EDS spectra (c and f). |
As previously reported by the authors,33 after soaking in SBF, the diffraction peaks of hydroxyapatite can be identified at approximately 2θ/26°, 29° and 32° after soaking the powders in SBF for 8 hours (Fig. 8(i)). As it can be observed in Fig. 8(ii), by increasing the soaking time up to 24 hours, the intensity of the hydroxyapatite peaks at 26°and 32°/2θ increased and new peaks at 2θ/40°, 47° and 49° appeared.
These results were further supported by the WAXRD analysis of the samples before and after soaking in SBF (Fig. 10). In the CSC, the only diffraction peaks that could be detected after 1 day of soaking were attributed to CSD (Fig. 10a(ii)). After 1 week of soaking (Fig. 10a(iii)), besides the CSD, peaks of HA could be identified at 2θ/25.88°, 31.79° and 34.07°. Conversely, in the WAXRD pattern of the Spine-Ghost sample after 1 day of immersion (Fig. 10b(ii)), the peaks of HA were immediately detected. The diffraction peaks of HA increased in intensity by increasing the soaking time up to 1 week (Fig. 10b(iii)).
Fig. 11 Photographs of (a) calcium sulphate cement (CSC); (b) Spine-Ghost; (c) Cerament® after soaking in SBF for 1, 3, 7, 14, 21 and 28 days. |
The pH values of the SBF solution collected throughout the 28 days of soaking are reported in Fig. 12(b): a sharp increase in the pH value from 7.4 to 7.97 was observed within 72 hours, followed by a gradual decrease to 7.56 after 28 days of soaking. At variance, the immersion of the CSC in SBF led to a slow increase of the pH values from 7.4 to 7.52 within 3 days, followed by a decrease from 7.52 to 7.41 between 3 and 28 days of soaking. For Cerament®, the pH value increased up to 7.55 in the first 3 days and decreased gradually to 7.38 after 28 days of soaking.
Fig. 13 shows FESEM images of the surface of Spine-Ghost and Cerament® cements after 28 days of soaking in SBF. A noticeable layer of bone-like apatite was observed on the surface of the Spine-Ghost cement (Fig. 13(a and b)), as well as on Cerament® (Fig. 13(d and e)).
Fig. 13 FESEM images and EDS of: (a–c) Spine-Ghost cement; (d–f) Cerament®; after soaking in SBF for 28 days. |
For clinical applications it is crucial to evaluate the hardening time at both room temperature (from 20 to 22 °C) and at body temperature. To assess the hardening time of Spine-Ghost, the paste was kept in an oven at 37 °C. At this temperature, the complete hardening of the Spine-Ghost cement was observed after about 40 minutes from the beginning of mixing.
However, the Spine-Ghost cement did not enhance the ALP activity of the BMSCs (Fig. 15). The Spine-Ghost might not be able to support the osteogenic differentiation of the BMSCs in vitro in the present set-up.
Fig. 15 Fold change ALP activity on Spine-Ghost vs. cells cultured alone (=1), indirect-contact culture, 1 μm pore-size inserts, and 12 well-plate. |
The high dissolubility and inorganic ion accumulation of the Spine-Ghost cement in aqueous medium are likely to be the main factor to influence the cell behaviour in vitro.
• It resorbs completely in a relatively short period of time.
• It provides a resorbable scaffold for bone growth.
• It may induce osteoblastic activity by the release of sufficient calcium ions.
• The dissolution rate of calcium sulphate makes it suitable as a delivery vehicle (growth factors, drugs, and antibiotics).42
One of the most popular commercial bone cements is Cerament® (Bonesupport AB, Sweden), which is composed of a mixture of synthetic calcium sulphate hemihydrate, hydroxyapatite and a non-ionic iodinated radiological contrast agent (iohexol).22 After setting, this cement has a compressive strength similar to that of healthy cancellous bone43 and a final setting time in the range of one hour.22 These results show that this injectable material is used as an alternative to PMMA for vertebroplasty.
The self-setting properties of bone cements such as injectability and setting time have crucial effects on their applicability in clinics.44 Injectability is an important property for a bioactive cement, because it grants an easy delivery of the paste through a needle or cannula.45 Injectable bone cements provide the possibility of injecting the paste into skeletal sites of limited accessibility, filling complicated cavities46 and containing defects such as periodontal bony defects, cysts, tumor removal sites and graft substitution gaps.45
Nevertheless, it is not possible to achieve a suitable injectability using a pure calcium sulphate cement due to the lack of inherent viscosity:44 during the injection from the delivery system, the cement paste suffers ‘filter pressing’, so that a separation of the powder and the liquid phase occurs.47 Furthermore, a pure calcium sulphate cement will have a too short final setting time (37 ± 0.5 min), which makes it unsuitable for clinical applications. For these reasons, pure injectable calcium sulphate cements have never been reported, whereas, if other phases are added, it is possible to develop a suitable cement such as Cerament®. In the present study, a novel injectable calcium sulphate-based cement (Spine-Ghost) was developed by incorporating mesoporous powders (W-SC) and glass-ceramic particles (SCNZ) into a calcium sulphate matrix. According to the obtained results, the quantity of the residual Spine-Ghost cement in the syringe, using the optimised L/P ratio (0.4 mL g−1), was less than 1 mL after injection through a 13 gauge vertebroplasty needle. It should be considered that, due to the shear thickening of the paste close to the neck of the syringe, a small amount of cement would always remain in this region even for the cement with full injectability.45 As it can be seen in Fig. 5(c and d) the injectability of the Spine-Ghost cement with the optimum liquid to powder ratio (0.4 mL g−1) is comparable to Cerament®. Moreover it should be mentioned that, during the injection of the Spine-Ghost cement filter pressing or phase separation was not observed (Fig. 5(c)). The absence of any phase separation effect was also confirmed by the EDS mapping analysis in which, in every section of the cement, a homogenous distribution of the different phases was observed.
The setting time of an injectable bone cement is a significant factor for its clinical applications.48 A bone cement with too short or too long setting times is not suitable to be utilised in clinical surgery.49 In fact, in clinical applications, the material needs to be injected before it begins to set and the cement has to reach its final setting time before the treated vertebra is loaded. The setting reaction of the calcium sulphate cement (CSC) can be divided in the following steps: (a) mixing of CSH with a solution leads to the formation of a suspension, (b) saturation of the solution by the dissolution of CSH, (c) precipitation of CSD after the super-saturation of the solution, (d) growth of CSD nuclei to larger crystals and (e) hardening of the cement due to the interlocking of the CSD crystals. Steps (c) and (e) can be identified as the initial and final setting times, respectively.50 Our data showed that the longest initial and final setting times were observed for the Spine-Ghost cement (46 ± 0.5 and 57 ± 0.5 min, respectively) compared to both the CSC (31 ± 0.5 and 37 ± 0.5 min) and Cerament® (40 ± 0.5 and 51 ± 0.5, min). However, the differences were quiet limited and the Spine-Ghost cement could be set within 1 hour, which was comparable with Cerament® from a clinical point of view. In fact, a bone cement with a slightly longer setting time improves the injectability and handling of the cement during the operation before the paste starts to set. Furthermore, the hardening time of the Spine-Ghost cement was evaluated by incubating the cement at a temperature of approximately 37 °C. The obtained results showed that the full hardening time of the Spine-Ghost cement was about 40 min from the beginning of the mixing which was comparable with Cerament® (full hardening within 45 min) supporting the suitability for clinical use.23
Since bone has to withstand high stresses and a challenging body environment, the mechanical properties of a hardened bone cement have been one of the main crucial factors for its clinical use.47
In clinical applications, the compressive strength of a cement material should be analogous or comparable with that of cancellous bone, which is in the range of 2–12 MPa.51,52 It is evident from the results that the hardening process improved the mechanical strength of the cements. Moreover, it is clear that the compressive strength of all the specimens was enhanced by increasing the testing time up to seven days. This increased strength can be explained by the growth of calcium sulphate dihydrate into larger crystals and by the interlocking of these crystals upon hardening of the cement. In this work, the compressive strength of Spine-Ghost under both wet and dry conditions (14 ± 0.7 and 18.1 ± 0.8 MPa, respectively) was higher than the one shown by Cerament® (8.2 ± 0.7 and 7.3 ± 0.6 MPa, respectively) ensuring an enhanced mechanical support compared to the commercial reference. Siemund and co-workers53 described their initial experience with Cerament® in seven patients that underwent vertebroplasty for osteoporotic VCFs, reporting a mean vertebral height loss after treatment of 3.6 mm (range 1.5–5.5) or 21% (range 8–32). These data can be due to incomplete vertebral filling (average injected volume: 1.9 mL, range 0.2–4.0) as a consequence of needle plugging or to a preliminary formulation of Cerament®, but the doubt of cement failure cannot be ruled out. The increase in the mechanical strength of Spine-Ghost can be attributed to the incorporation of the glass-ceramic particles and to the size and flowability of the W-SC particles. The small and spherical W-SC particles can fill the gaps between the hydrating calcium sulphate particles increasing the overall cement density and play a role in the reduction of the crack propagation in the composite. These results are comparable with in-use bone cements which have a compressive strength of around 11 MPa54 and are situated slightly below the upper limit of the compressive strength of cancellous bone (2–12 MPa).51,52 It should also be underlined that Spine-Ghost shows a satisfactory radiopacity (Fig. 14) in comparison with both a standard PMMA similarly opacified with zirconium dioxide (BonOs Inject) and with the calcium sulphate cement Cerament®, chosen as a commercial reference.
The radiopacity of Cerament®, which will fade away over time in the early follow-up, is obtained with an iodinated contrast medium, whereas Spine-Ghost owes its radiopacity to the glass-ceramic phase containing ZrO2 that will keep the bone cement visible until its complete resorption. The long-lasting radiopacity of Spine-Ghost allows for the checking of the integrity of the bone cement during follow-up in the case of pain relapse.
If we compare the safety profile of Cerament® and Spine-Ghost, we should underline that the first contains a liquid iodinated contrast medium (iohexol) that is commonly used intravenously for Computed Tomography. The prevalence of “allergic” reactions to nonionic iodinated contrast medium is generally quite low (reported from 0.3% to 3%)55 but in the cases56 of 2% the reaction can be severe and more difficult to manage during an intervention performed in the prone position. Most of the anaphylactoid reactions are also delayed and can mix up with other common post-procedural symptoms like nausea and pain, thus complicating the patient's management during recovery. In a study on 1000 patients57 who underwent Computed Tomography with intravenous contrast medium, the total adverse reactions that occurred with iohexol were 2.5% that was significantly higher than those that occurred with a different iodinated contrast medium (iodixanol, less than 1%, p < 0.05).
Another important characteristic of biomaterials is their bioactivity, which is defined as the ability of these materials to chemically bond with living bone tissue.58 Hydroxyapatite (HA) is a calcium phosphate based material with a chemical composition and a crystal structure similar to the mineral component of human bone and teeth.59 It plays a significant role in establishing a strong chemical bond with bone tissue. Generally, the ability of a biomaterial to induce hydroxyapatite formation in SBF solution is an accepted way to assess the bioactivity of that material in vitro, and can be used as an index of predictability for its in vivo behaviour. In our study, with the aim of imparting bioactive properties to the bone cement, a spray-dried mesoporous bioactive glass with SiO2/CaO binary composition (W-SC),33 characterised by a spherical morphology, a large exposed surface area and a pore volume, was properly incorporated inside the cement matrix.
The high surface area of W-SC led to an extremely high bioactivity assessed by means of an in vitro bioactivity test showing the formation of a hydroxyapatite layer on the surface of the mesoporous powders after only 8 hours of soaking in SBF. This result is very encouraging and could indicate a potential simulation of bone regeneration in vivo based on the pivotal role of W-SC in the nucleation of HA crystals. In the present study, the very low bioactivity of the calcium sulphate cement was noticeably improved by incorporating the W-SC powders into the CSC. In fact, the in vitro results showed that Spine-Ghost induced the precipitation of a considerable amount of apatite only after 24 hours of immersion, with much faster kinetics if compared to calcium sulphate (Fig. 9). The excellent bioactivity of Spine-Ghost is due to the fast and remarkable release of osteoinductive ions such as Ca and Si from the mesoporous spray-dried powders (W-SC), which play an active biological role in influencing the rate of bone regeneration.60 The release of Si ions in SBF led to a high concentration of surface silanol groups that resulted in a faster nucleation and mineralisation of apatite crystals.29 Besides, as an added value, W-SC powders, because of their well-ordered accessible pores, could potentially provide the ability of loading and releasing drugs and biomolecules in a controlled fashion directly at the intervention site.
Another important feature of a bone cement is its resorbability, which allows new bone tissue to grow into the defect and to replace the cement in a few months.19,25 Over the last few decades, highly resorbable bone substitutes have received considerable interest in bone surgery as they can allow for the reduction of inflammation, stiffness and pain.19,20 In fact, resorption kinetics of degradable materials should match the new bone formation so that these materials are gradually substituted by the new bone tissue, in order to guarantee a continuous support to the vertebra.26 The degradation rate of calcium sulphate-based cements is more rapid than the rate of new bone formation, which often leads to its replacement with fibrous tissue. Therefore, designing calcium sulphate-based bone cements with suitable degradability is a major target. In our study, the degradation rates of calcium sulphate, Spine-Ghost and Cerament® cements were evaluated by their weight loss after soaking in SBF for various time periods. The Spine-Ghost cement showed a weight loss ratio of 83.3% after 28 days of soaking (Fig. 12(a)) and thus faster degradability compared to calcium sulphate (61.5%). The results demonstrated that the Spine-Ghost cement was fully degraded after 32 days of immersion in SBF, hence after a slightly longer time than Cerament®, which showed 100% resorption after 28 days of soaking. It should be underlined that these mass losses were evaluated according to the ISO standards by completely soaking the sample in SBF whereas, once injected, the cement will not be completely surrounded by the physiological fluids and thus the clinical practice generally shows slower resorption rates. Furthermore, the results showed that the pH of the SBF solution increased within 3 days of soaking due to Ca ion release and the formation of phosphate, calcite or apatite.29 It is noteworthy that the immersion of the Spine-Ghost samples in SBF solution led to a slightly alkaline environment, which is an advantage for the nucleation rate of hydroxyapatite crystals.61
These very encouraging results about the mechanical properties, setting times, bioactivity, degradability and biocompatibility of the Spine-Ghost cement, which proved to be equal or superior to the commercial reference, are the groundwork on which more extensive biological tests in vitro and in vivo (in sheep) are ongoing.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6tb02139e |
This journal is © The Royal Society of Chemistry 2017 |