Polymerizable nanoparticulate silica-reinforced calcium phosphate bone cement Saeed Hesaraki,1 Masoud Alizadeh,2 Shokoufeh Borhan,1 Milad Pourbaghi-Masouleh1 1 2
Nanotechnology and Advanced Materials Department, Materials and Energy Research Center, Karaj, Iran Ceramics Department, Materials and Energy Research Center, Karaj, Iran
Received 29 January 2012; accepted 15 April 2012 Published online 16 June 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32731 Abstract: Bone cements based on calcium phosphate powder and different concentrations of colloidal silica suspensions were developed. Setting time and washout behavior of the cements were recorded and compared with those of a control group prepared by the same powder phase and distilled water as liquid. The phase composition, compressive strength, and morphology of the cements were determined after incubation and soaking in simulated body fluid. Proliferation of osteoblasts seeded on samples was also determined as a function of time. The results showed that the long setting time, poor compressive strength, and undesirable washout behavior of the cement made with distilled water were considerably improved by adding colloidal silica in a dose-dependent manner. On the basis of XRD and SEM results, both control group and nanosilica-
added cements composed of nanosized apatite flakes after 7 days soaking, in addition to tetracalcium phosphate residual for the latter. It was found that the rate of hydraulic reactions that are responsible for conversion of the cement reactants to nanostructured apatite was increased by the presence of colloidal silica. Furthermore, the osteoblasts exhibited better proliferation on nanosilica added cements compared to control one. This study suggests better applied properties for nanosilica-added calcium phosphate C 2012 Wiley Periodcement compared to traditional cements. V icals, Inc. J Biomed Mater Res Part B: Appl Biomater 100B: 1627– 1635, 2012.
Key Words: calcium phosphate, hydroxyapatite, bone cement, nanosilica
How to cite this article: Hesaraki S, Alizadeh M, Borhan S, Pourbaghi-Masouleh M. 2012. Polymerizable nanoparticulate silicareinforced calcium phosphate bone cement. J Biomed Mater Res Part B 2012:100B:1627–1635.
INTRODUCTION
The demand for bone repair has increased greatly as a result of musculoskeletal disorders due to incidents, trauma, or diseases like osteoperosis and osteoarthritis and various treatments have been proposed in this regard.1 The successful medical application of calcium phosphate bioceramics in humans has more than 70 years history.2 Among these bioceramics, calcium phosphate cements (CPCs) are the best promising approaches for hard tissue healing because of their similarity to the mineral phase and structure of natural cancellous bone. These materials can be produced by different processing methods and can be fabricated with variable porosity and void volumes. The first such material is comprised of tetracalcium phosphate [TTCP: Ca4(PO4)2O] and dicalcium phosphate [DCPA: CaHPO4]3 and after that, so many other reactants4 including a-tricalcium phosphate (aTCP), dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate (OCP) have also been considered as essential constituents of these cements.5 CPCs exhibit unique properties such as biocompatibility, osteoconductivity, injectability, mouldability, bioresorbability, and low temperature self-setting behavior. These materials
also have potential to act as appropriate drug carriers and form bone resembled apatite in their system which provides an ideal environment for cellular reactions. These properties make CPCs exceptional candidates within other biomaterials. Their relatively convenient handling properties make them suitable to be utilized in head reconstruction surgery, surgery of middle ear and spine and they can be used as bone fillers as well. However, there is few drawbacks such as low mechanical strength and poor washout resistance that restrict CPC applications. Different attempts have been made to improve these deficiencies. Some additives such as resorbable polymers, superplastisizer polymers6 and polymeric fibers have been reported that enhance mechanical strength of conventional CPC. Xu and Quinn7 used 25 vol % restorable polymer with diameter of 322 lm in a cement matrix. It was observed that both work-of-fracture and flexural resistance of the reinforced cement were considerably greater than unreinforced ones. Fabrication of an injectable poly(propylene fumarate)/b-tricalcium phosphate paste composite was another attempt to improve the mechanical properties of cement using cross-linking mechanisms.8 Santos et al.9
Correspondence to: S. Hesaraki; e-mail:
[email protected] Contract grant sponsor: Materials and Energy Research Center
C 2012 WILEY PERIODICALS, INC. V
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studied the effect of adding polyamide fibers on the mechanical properties of calcium phosphate cement based on a-tricalcium phosphate as well as the mechanism involved in increase of mechanical strength. The results demonstrated possibility of the used polymeric fibers for increasing mechanical strength and by the presence of coupling agents for the effective performance of these fibers. In addition to fibers, gelling agents such as sodium alginate, chitosan, carboxymethyl cellulose, and collagen were also used to improve mechanical strength and washout behavior of the cements.10 A fast setting and antiwashout CPCchitosan scaffolds was formulated with tailored micropores.11 Adding chitosan to CPC composition also led to increase in flexural strength, toughness, and strain-tofailure.12 In this study, nanosilica in the form of colloidal system has been employed as a new additive to improve mechanical strength, setting time and undesirable washout behavior of CPCs, contemporaneously. Also, this study demonstrated that the rate of apatite formation and osteoblastic cell proliferation on CPCs increased by using nanosilica in the cement composition. MATERIALS AND METHODS
Starting materials The main starting materials used in this study were: Calcium carbonate (CaCO3, Merck 2069), dicalcium phosohate dihydrate (CaHPO4. 2H2O, Merck 2146) and nanosilica (mean particle size of 60 nm) in a colloidal system, (Shiv Kripa, India). All reagents employed for the preparation of simulated body fluid solution were also purchased from Merck Company (Germany). Tetracalcium phosphate (TTCP) was synthesized through solid state reaction between CaCO3 and CaHPO4. 2H2O according to the method described elsewhere.13 Briefly, an equimolar mixture of calcium carbonate and dicalcium phosphate dihydrate was heated at 1500 C for 6 h and then, the product was quenched to room temperature, crushed, and grounded to a mean particle size of 3 lm using a planetary mill. Cement formulation In this study, the cement consisted of both powder and liquid components. The powder phase (P) comprised of TTCP (50 mol %) and DCPD (50 mol %) and the liquid phase (L) was distilled water or 5 or 10 wt % colloidal silica suspension. In this report, the cements without colloidal silica, with 5 and 10 wt % colloidal silica are called Si-0, Si-5, and Si-10, respectively. Note that the sample prepared with distilled water was used as control group to compare its properties with other experimental groups. To make a consistent paste, liquid and powder were mixed thoroughly at P/L ratio of 3 g/mL and weight of silica phase was included during calculation of P/L ratio. Setting time The initial and final setting time of cements was measured by using a Gilmore needle according to ASTM-C266-89 method. Cement was considered set when the needle loaded
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onto the surface of the sample failed to make an appreciable indent. Total porosity and compressive strength To determine the total porosity of the cements, the prepared pastes of various cements were transferred into Teflon mold to form cylindrical specimens (6 mm in diameter and 12 mm in height). The hardened samples were removed from the mold and incubated at 37 C and 100% relative humidity for 24 h and then dried at 100 C for 24 h. Total porosity (Pt) measurements were performed on dried specimens according to the density method.14 Bulk density (db) was calculated using the following expression: db ¼
M V
(1)
where M is the mass of the specimen and V is its volume. Then, Pt was calculated using Eq. (2): db Pt ¼ 100 1 dp
(2)
where Pt is powder density of the samples obtained by gas picnometer (Micrometrics, Accupy C 1330) using grounded samples passed through a 800 mesh sieve. For the compressive strength (CS) test, the hardened cylindrical samples were incubated at 37 C for 24 h and placed in SBF solution at 37 C for different time intervals up to 7 days. The CS values were recorded using a mechanical testing instrument (Zwick/Roell-HCr 25/400) at crosshead speed of 1 mm/min. It should be noted that the SBF solution was prepared in accordance with the Kokubo’s specification using extra pure reagents.15 Qualitative washout Washout resistance behavior of the samples was qualitatively determined by soaking the as-set cements (the cement sample after initial setting) in SBF solution (37 C) and photographing their structural stability after 24 h using digital camera (Canon sd1100is). In vitro experiments In this part of study, 24 h-incubated disc-shaped cements (1 cm in diameter and 5 mm in thickness) were separately immersed in SBF solution at solid to liquid loading of 2 g/ 100 mL. The samples were kept at 37 C for different periods. The solution was refreshed every 24 h and the ionic concentrations of the extracted solutions were analyzed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The X-ray diffraction (XRD) patterns of the powdered incubated/soaked cements were recorded by X-ray diffraction analysis and compared with the patterns of hardened (set) samples. At the end of each soaking period the samples were washed with distilled water to remove the SBF ingredients and then dried at 70 C. The samples were scanned from 10 to 50 2y (where y is the Bragg angle) using PW 3710 philips diffractometer with a Cu-Ka NANOSILICA-REINFORCED CALCIUM PHOSPHATE BONE CEMENT
ORIGINAL RESEARCH REPORT
radiation and operating voltage of 20 kV. The Fourier transform infrared (FTIR) spectra of the soaked samples were measured between 4000 and 400 cm1 using a FTIR spectrometer (Bruker Vector 33). For this purpose the powdered specimen was mixed with KBr at 1:10 ratio to fabricate a transparent tablet. Surface morphology of the samples after incubation and soaking in SBF solution (for different intervals) was characterized using a scanning electron microscope (SEM, Stereoscan S 360 Cambridge). Due to the poor electrical conductivity of the samples, their surfaces were coated with a thin layer of Gold before testing them. Cell behavior To evaluate biological properties of the cements, osteoblastic cells were derived from newborn rat calvaria and isolated by sequential collagenase digestion from calvaria of newborn (2–5 days) Wistar and cultured in Dulbecco modified Eagle medium (DMEM; Gibco-BRL, Life Technologies, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS; Dainippon Pharmaceutical, Osaka, Japan) and 100 g/mL penicillin–streptomycin (Gibco-BRL, Life Technologies) in 5% CO2 and 95% air atmosphere at 37 C for 1 week. The medium was changed every 2 days. The confluent cells were dissociated with trypsin and subcultured to three passages which were used for tests. The disc-shaped cement specimens (10 mm in diameter and 3 mm in height) were sterilized using 70% ethanol and the osteoblastic cells were seeded onto the tops of the cement discs at 3 104 cells/disc. The specimen/cell constructs were placed into 24-wells culture plates and left undisturbed in an incubator for 4 h to allow the cells to attach to them and then an additional 3 mL of culture medium was added into each well. The cell/specimen constructs were cultured in a humidified incubator at 37 C with 95% air and 5% CO2 for 1, 7, and 14 days. The medium was changed every 3 days. The proliferation of the osteoblastic cells on cement specimens (with or without silica) was determined using the MTT (3-{4,5-dimethylthiazol-2yl}-2,5-diphenyl-2H-tetrazolium bromide) assay. For this purpose, at the end of each evaluating period, the medium was removed and 2 mL of MTT solution was added to each well. Following incubation at 37 C for 4 h in a fully humidified atmosphere at 5% CO2 in air, MTT was taken up by active cells and reduced in the mitochondria to insoluble purple formazan granules. Subsequently, the medium was discarded and the precipitated formazan was dissolved in dimethylsulfoxide, DMSO, (150 mL/well), and optical density of the solution was read using a microplate spectrophotometer (BIO-TEK Elx 800, Highland park, USA) at a wavelength of 570 nm. To observe the morphologies of the cells attached onto the surfaces of the cement specimens, the cells were cultured onto the discs as described above. After 14 days, the culture medium was removed, the cell-cultured specimens were rinsed with phosphate buffered saline (PBS) twice and then the cells were fixed with 500 mL/well of 3% glutaraldehyde solution (diluted from 50% glutaraldehyde solution (Electron Microscopy Science, USA) with PBS). After 30 min, they
were rinsed again and kept in PBS at 4 C. Specimens were then fixed with 1% Osmium tetroxide (Polyscience, Warmington, PA, USA). After cell fixation, the specimens were dehydrated in ethanol solutions of varying concentration (30, 50, 70, 90, and 100%) for about 20 min at each concentration. The specimens were then dried in air, coated with gold and analyzed by SEM (Streoscan S 360, Cambridge). Statistical analysis In this study, data were processed by excel Microsoft 2003 and the results were presented as Mean 6 standard deviation of at least four experiments. The differences between the obtained results of different cements were processed using nonparametric ANOVA (Kruskal–Wallis test) and the p values lower than 0.05 were considered significant. RESULTS
Setting times The initial and final setting times of various calcium phosphate cements are summarized in Table I. According to these results, both initial and final setting times decreased significantly, by adding colloidal silica to the calcium phosphate cement composition and further decrease in setting time values are observed by using more concentrated colloidal silica suspension. Porosity and compressive strength Figure 1(a) shows the total porosity of calcium phosphate cements with different concentration of silica after 24-h incubation at 37 C. A significant decrease in Pt value of the cements is seen by using colloidal silica suspension in the cement composition. Furthermore, the total porosity of Si-10 is significantly higher than that of Si-5 specimens. According to Figure 1(b), the incubated cement without any additive has a low compressive strength value after (9 MPa). The addition of colloidal silica to the cement composition causes to a significant increase in compressive strength so that it reaches to about 20 MPa for Si-5 and 30 MPa for Si-10. The difference between compressive strength of Si-0, Si-5, and Si10 specimens is statistically significant (p < 0.05). A timedependent evolution in compressive strength of all cements is observed after soaking them in SBF solution; however samples with silica additive exhibit higher CS value than control group. The difference between CS values of 7-days soaked Si-5 and Si-10 cements is not statistically significant. Washout Washout resistance of all samples in SBF solution is illustrated in Figure 2. While an approximately complete destruction of Si-0 cement is observed after 24 h, colloidal silica could successfully prevent cement disintegration and retain its structure when confronting with the physiologic solution. Change in SBF ion chemistry Figure 3 shows DCa and DSi (concentration difference between Ca and Si ions in cement-containing SBF and fresh SBF) as a function of time. The DCa
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TABLE I. The Initial and Final Setting Times of Calcium Phosphate Cements Prepared by Different Amounts of Colloidal Silica Compared with Control Group
Initial setting time (min) Final setting time (min)
Si-0
Si-5
Si-10
19 6 4 42 6 4
12 6 2 20 6 3
663 15 6 2
corresponded to Si-5 and Si-10 cements is lower than that of control group [Figure 3(a)]. It should be noted that apatite formation in calcium phosphate cements occurs as a result of dissolution–precipitation mechanism. Since DCa is higher than zero in all evaluating period, it is concluded that dissolution rate is higher than precipitation rate in this evaluating schedule. The increase in dissolution rate fails at 48 h after immersion of nanosilicacontaining samples and then tended to decrease, whereas this time is 120 h for control sample. It reveals that precipitation phenomenon is more active in nanosilica added cements than in control one. Lower level of DCa in the SBF solution of nanosilica added cements can also
FIGURE 2. Visual condition of different cements at 24 h after immersing in SBF solution: (a) Si-0, (b) Si-5, and (c) Si-10.
FIGURE 1. Total porosity of various incubated CPCs (a) and compressive strength of CPCs after incubation and soaking for different times (b).
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predict more apatite deposition rate on surfaces of these cements. Figure 3(b) shows DSi versus time. The Si release increases in the first 24 h of immersion and then a sharp decrease in Si concentration of all SBF samples are observed. It seems that the formation of calcium phosphate layer hinders the release of this ion and the rate of this process decreases after that. It can also be seen that the cement containing higher nanosilica concentration releases more Si ions into the SBF.
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FIGURE 5. The XRD patterns of Si-0 (control group) cement after setting, incubation at 37 for 24 h and soaking in SBF for 1, 3, and 7 days.
FIGURE 3. Concentration difference in Ca (a) and Si (b) ions between cement-containing SBF and fresh SBF during soaking process.
Phase composition Figure 4 shows the XRD pattern of cement powder phase. The individual peaks of tetracalcium phosphate and dicalcium phosphate dihydrate are observed in the powder pat-
tern without any extra impurities. The phase composition of as-set Si-0 cement (cement after setting) as well as progress in formation of apatite product during soaking it in SBF for different periods has been illustrated in Figure 5. The XRD pattern of the as-set cement is closely similar to the cement powder pattern and the presence of apatite phase in this compound is negligible. Detection of diffraction peaks at 2y ¼ 25.8, 31.8, 32.8, and 34 in the XRD patterns of incubated Si-0 cement and increase in their intensities due to the soaking process indicates formation of apatite phase in the cement composition and its dependence on the soaking time. In Si-0, after 7 days soaking, apatite is predominant phase and considerable amount of TTCP is still found. Figure 6 reveals the compositional variations for the colloidal silica-containing samples in the same experimental condition [Figure 6(a): Si-5; Figure 6(b): Si-10]. The XRD patterns of incubated samples are quite similar to the control group and powder phase. However in soaked specimens, the apatite peaks predominate to TTCP after 1 day soaking and the cements just composed of apatite phase after 7 days soaking. The XRD patterns of both Si-5 and Si-10 specimens are extremely similar to each other. The peak broadening is a sign of low crystalinity of the formed apatite phase. Structural groups FTIR spectra of Si-0, Si-5, and Si-10 samples after soaking in SBF solution for 7 days are shown in Figure 7. In all samples, the bands observed at 550, 600, and 1070 cm1 are assigned to the phosphate (PO43) groups in apatite lattice and the bands at 870, 1412, and 1448 cm1 confirms the substitution of carbonate ions in the apatite structure. For Si-5 and Si-10, the absorption bands at 450, 800, and 1250 cm1 are assigned to Si-O-Si modes of vibrations.16 Note that, some absorption bands of PO group come in the same range with Si-O. The formation of apatite phase in all soaked samples can also be determined by the hydroxyl group band of apatite at 3550 cm1.
FIGURE 4. The XRD pattern of powder phase of all CPCs prepared in this study.
Microstructures The SEM images of Si-0, Si-5, and Si-10 samples after 24 h incubation and 1 day and 7 days immersion in SBF are
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other Si-containing cements (p 0.05). At days 7th and 14th, the amount of formazon produced by osteoblasts cultured on the Si-5 and Si-10 is significantly higher than that of Si-0 (p < 0.05), whereas the difference between the number of cells proliferated on Si-5 and Si-10 is not statistically significant. Figure 10 compares the morphology of the osteobalstic cells attached onto the surfaces of Si-0 and Si-10. In both specimens the surface has been covered by polygonal osteoblastic cells with developed cytoplasmic membranes and the cells became confluent on top of the cements after 14 days. Based on this Figure, the confluent cells grown on surface of Si-10 is more compacted and cumulous than Si-0. These results support the MTT data.
DISCUSSION
FIGURE 6. The XRD patterns of silica-containing calcium phosphate cement after setting, incubation at 37 for 24 h and soaking in SBF for 1, 3, and 7 days: (a) Si-5 and (b) Si-10.
shown in Figure 8. In Figure 8(a), the microstructure of control group comprises of cement particles and the exodus of liquid phase from the cement composition produces micropores. Si-5 and Si-10 samples have more compacted structures [Figure 8(b,c)]. It seems that a condensed phase has filled the pores and thus the reactant particles have been covered by a monolithic phase. Figure 8(d–f) shows the microstructure of these three specimens after 1 day immersion in SBF solution. In the control group, aggregated particles with some precipitates on their surfaces are observed. These particles have a network-like connection in Si-5 and are rough and compacted in Si-10. Increasing in soaking time leads to formation of grown plate-like crystals on all cement surfaces with better growth, more tight entanglement and more thickness on Si-5 and Si-10 specimens [Figure 8(g–i)]. In agreement with XRD data, the SEM pictures confirm that the amount of apatite in nanosilica-containing cements is higher than control group. Cell proliferation and morphology Figure 9 shows numbers of viable cells proliferated on various specimens. After cell attachment, the osteoblasts began to proliferate on all specimens due to the significant difference in cell numbers between days 1, 7, and 14th (p < 0.05). At 11th day, no significant difference is observed between the numbers of cells determined on Si-0 and two
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The amount of apatite phase formed through the hydraulic reaction of TTCP and DCPD immediate after setting is the key factor for the antiwashout and mechanical properties of CPCs.17 The problems occur when the content of product phase in asset cement is so little which cannot be even detected by XRD. It leads to poor mechanical strength and cement disintegration upon confronting with physiologic solution. The paste stability or antiwashout behavior which is defined as ability to stay in one piece during setting is a determinant factor for CPC applications in highly blood perfused operations such as vertebroplasty. The cement disintegration results in microparaticles migration to surrounding tissues and leads to inflammatory reactions which decrease the osteoblast viability and differentiation.18 These particles may also act as scaffold for platelet aggregation and enhance coagulation cascade.19 If structural stability of the cement is conserved in the initial moments after implantation, progression of hydraulic reactions will lead to the growth of primary apatite crystals which ensure its strength [see Figure 1(b)] and integrity in physiologic solution and postimplantation. Nanoparticles of SiO2 added as a colloidal system to the CPC composition enhanced poor mechanical strength and washout behavior of the cement. The compressive strength of CPC added with inorganic colloidal silica binder is considerably higher than CPC added with organic superplasticizres.6 It was also found that, in addition to the above mentioned properties, colloidal silica modified the setting time and the rate of conversion of reactants into apatite phase in SBF. Calcium phosphate cement pastes set due to precipitation of apatite nanocrystals in the cement structure from a hydraulic reaction between its starting reactants.17 Mechanical strength of these cements is also governed by entanglement of apatite nanocrystals. The XRD patterns of as-set calcium phosphate cements (Figures 5 and 6) confirm that all of them have the same crystalline phases which are closely similar to the cement powder. In other words, the apatite phase in the as-set cements is not detectable by XRD. Thus, it is suggested that there is another mechanism by which colloidal silica improve cement strength and setting time.
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FIGURE 7. The FTIR spectra of calcium phosphate cements soaked in SBF solution for 7 days.
FIGURE 8. The SEM micrographs of different cements after incubation and soaking in SBF solution: (a) Si-0 incubated for 24 h, (b) Si-5 incubated for 24 h, (c) Si-10 incubated for 24 h, (d) Si-0 soaked in SBF for 1 day, (e) Si-5 soaked in SBF for 1 day, (f) Si-10 soaked in SBF for 1 day, (g) Si-0 Soaked in SBF for 7 days, (e) Si-5 soaked in SBF for 7 days, (i) Si-10 Soaked in SBF for 7 days.
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FIGURE 9. Proliferation of osteoblastic cells on calcium phosphate cements containing different amounts of silica as a function of time (*: p < 0.05).
Colloidal silica is a suspension of fine amorphous, nonporous, and typically spherical silica particles in an aqueous phase in which the particle size ranges from 30 to 100 nm. The subunits of colloidal silica particles are usually unjoined Si(OH)4. Hydrogen ions from the surfaces of colloidal silica particles tend to dissociate in aqueous solution and thus, the ions existed at the surface is OH-, yielding an electrostatically dispersed suspension. When calcium phosphate particles are dispersed in this suspension, the reaction between Ca2þ cations and Si(OH)4 is probable. However, the XRD patterns collected from Si-5 and Si-10 specimens exhibited no further phases such as CaSiO3 (ICDD #2-0689), Ca2SiO4 (ICDD #23-1042) and Ca(OH)2 (ICDD #2-0969). Thus, it is suggested that the cement reactants dispersed among colloidal silica particles reacts to form the nanoapatite crystals through a hydraulic processes (which is discussed in literature17) and simultaneously the colloidal silica particles interact together to polymerize into a 3D network of Si-O-Si groups when the liquid phase of the cement is evaporated or slightly consumed by the reactants. Condensation of the colloidal silica to siloxane occurs through a gelling mechanism20 that improves setting time of the nanocomposite cement. Thus the increased compressive strength and controlled washout of Si-5 and Si-10 cements originate from their reduced porosity and linked reactants. In other words, it is suggested that the condensed silica phase fills spaces between the calcium phosphate particles and reduces microporosity meanwhile simultaneously covers and links reactants together resulting in an increased compressive strength and controlled washout behavior. The reduced porosity [Figure 1(a)] and compacted microstructure [Figure 8(b,c)] of silica-containing cements confirms this suggestion. Another surprising effect of nanosilica on calcium phosphate cement is its effect on the rate of apatite formation in SBF solution. It is suggested that the increased amount of apatite phase in silica-containing cement originates from the presence of condensed SiO2 phase in the cement structure.
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The SiO2 groups which can be converted to Si-OH groups in SBF through hydrolysis reactions are susceptible sites for the apatite nucleation.21 The mechanisms of apatite layer formation on the surfaces of SiO2 based bioactive materials have been well discussed in literatures.22 Difference between Ca2þ concentration of the fresh SBF and specimencontaining SBF can reflect the higher rate of apatite precipitation in nanosilica-containing cements compared with control group. The results of cellular responses showed better cell proliferation and spreading on silica-containing calcium phosphate cement compared with control group. These findings are in agreement with other authors who stated better proliferation of osteoblasts in presence of Si-containing materials.23,24 Several mechanisms involving active or passive have been proposed to explain improved biological behaviors of Si-containing materials. In active mechanisms Si ions released into the medium are seen by the cells and hence, affecting their metabolism. In passive mechanisms Si substitution influences the chemical or topographical features such as grain size, protein conformation at the material surface, and results in change of the cellular behaviors.25
FIGURE 10. Morphology of rat-derived osteoblasts on surfaces of silica-free (control group) and Si-10 specimens after culturing for 14 days.
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In addition to physical, mechanical, and in vitro behavior of CPCs which were improved by using colloidal silica, our experience showed that the rheological and handling properties of CPCs were improved by adding nanosilica which this topic is in our next studying program. CONCLUSIONS
From this study, it is found that colloidal silica is a promising additive to modify undesirable properties of calcium phosphate bone cement. The setting time, compressive strength and structural stability of calcium phosphate cement are thoroughly improved by adding colloidal silica to the cement composition. In addition to enhancement of physical properties, colloidal silica increases the rate of carbonated apatite formation in CPC when soaking them in simulated body fluid solution. Regarding to the beneficial effect of this additive on physical characteristics of CPC and the desirable effect of Si ions incorporated into calcium phosphate ceramics on biological function of osteoblastic cells which is reported by many authors, it is suggested that colloidal silica-added CPCs can be successfully used as bone filler material after passage of the corresponding in vivo tests. ACKNOWLEDGMENTS
The authors would like to thank Dr. Zamanian and Mrs. Khorami for their help in experimental procedures. The assistance of Dr. Nazarian in cellular tests is also acknowledged. REFERENCES 1. Physician’s guide to prevention and treatment of osteoporosis. Maryland: National Osteoperosis Foundation; 1998. 2. LeGeros RZ. 40th Symposium on basic science of ceramics, convention center. Osaka university, 2002. 3. Brown WE, Chow LC. A New Calcium Phosphate Water Setting Cement. Ohio: American Ceramic Society; 1986. 4. Takagi S, Chow LC, Ishikawa K. Formation of hydroxyapatite in new calcium phosphate cements. Biomaterials 1998;19:1593–1599. 5. Bohner M, Gbureck U, Barralet JE. Technological issues for the development of more efficient calcium phosphate bone cements: A critical assessment. Biomaterials 2005;26:6423–6429. 6. Fernandez E, Sarda S, Hamcerencu M, Vlad MD, Gel M, Valls S, Torres R, Lopez J. High-strength apatitic cement by modification with superplasticizers. Biomaterials 2005;26:2289–2296. 7. Xu HH, Quinn JB. Calcium phosphate cements containing resorbable fibers for short-term reinforcement and macroporosity. Biomaterials 2002;23:193–202.
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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | AUG 2012 VOL 100B, ISSUE 6
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