Journal of Dental Sciences (2015) 10, 282e290
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ORIGINAL ARTICLE
Physicochemical properties and biocompatibility of silica doped b-tricalcium phosphate for bone cement Shu-Hsien Huang a, Yi-Jyun Chen a,b,c, Chia-Tze Kao a,b, Chi-Chang Lin d, Tsui-Hsien Huang a,by, Ming-You Shie a*y a
School of Dentistry, Chung Shan Medical University, Taichung, Taiwan Department of Stomatology, Chung Shan Medical University Hospital, Taichung, Taiwan c Dental Department, Taichung Hospital, Ministry of Health and Welfare, Taichung City, Taiwan d Department of Chemical and Materials Engineering, Tunghai University, Taichung City, Taiwan b
Received 7 January 2014; Final revision received 10 June 2014
Available online 17 September 2014
KEYWORDS b-tricalcium phosphate; biocomposites; biodegradation; osteogenic; silica
Abstract Background/purpose: b-Tricalcium phosphate (b-TCP) is an osteoconductive material. Earlier reports revealed that silica plays an important role in bone mineralization, and its incorporation would enhance the biocompatibility of implants. Materials and methods: In this study, silica doping at various concentrations (0 wt.%, 10 wt.%, 20 wt.%, and 30 wt.%) was performed. Si-a ˆ-TCP was obtained upon calcining the as-prepared powders at 1400 C. To check its effectiveness, different Si-a ˆ-TCP samples were prepared to make new bioactive and biodegradable biocomposites for bone repair. Formation of bonelike apatite, the diametral tensile strength, ions released, and weight loss of composites were considered before and after immersion in simulated body fluid. We also examined the behavior of human dental pulp cells (hDPCs) cultured on these materials. Results: The results showed that the apatite deposition ability of the b-TCP was enhanced as the Si content was increased. For composites with >20% Si content, the apatite layer covered the samples. At the end of the immersion point, weight losses of 52%, 45%, 33%, and 26% were observed for the b-TCP containing 0%, 10%, 20%, and 30% Si, respectively. In vitro cell experiments showed that the Si-rich cement promotes hDPC proliferation and differentiation. However, when the Si content in the cement is >20%, the amount of cells and osteogenesis protein of hDPCs were stimulated by Si released from Si-b-TCP. Conclusion: The degradation of b-TCP and osteogenesis of Si give us a strong reason to believe that these Si-based cements may prove to be promising bone repair materials.
* Corresponding author. School of Dentistry, Chung Shan Medical University, Taichung City, Taiwan. E-mail address:
[email protected] (M.-Y. Shie). y
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jds.2014.07.001 1991-7902/Copyright ª 2014, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved.
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Copyright ª 2014, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction The suitable bioactivity and appropriate degradation of materials are required to conform to different clinical requirements for hard tissue repair. Autograft possesses all the characteristics indispensable for new bone formation, osteogenesis, osteoconductivity, and osteoinductivity, and it is currently considered the gold standard in this field. In spite of its strengths, however, there are still various disadvantages when using this material for certain medical clinical applications. This approach regrettably minimizes reconstructive healing and, even worse, sometimes promotes foreign body reaction. Therefore, efforts are ongoing to design better biomaterials that allow good control of material physicochemical properties. Silicate (Si)-based cements have received a considerable amount of positive attention in recent years because these materials have a better bioactivity than calcium phosphatebased materials.1,2 Recently, several studies have indicated that Si-based materials can play an important role in bone formation, at least based on these materials’ Si ion release3 and fast apatite formation ability.4,5 Si-based materials can promote human mesenchymal stem cells, human dental pulp cells (hDPCs), and osteoblast-like cell adhesion, proliferation, and differentiation.6e11 Furthermore, the Sibased cement was able to activate angiogenesis12 and antiosteoclastogenesis.13 These studies demonstrate that Si-based cement has the ability to promote the apatite layer to form in vitro14 and also shows it is better at promoting bone regeneration in vivo.15 However, the slow degradation rate of Si-based materials may result in a decrease in osteoconductivity, which may restrict their application in clinical practice.16 In order to ameliorate its relative disadvantage in regard to material degradation, we used b-tricalcium phosphate (b-TCP) as an additive to see how this would affect its rate of decay. b-TCP is a bioceramic material that is widely used for hard tissue repair. It has a chemical composition similar to apatite present in bone tissue, and has been applied extensively as a bone grafting material.17,18 Previous studies have shown that wollastonite-doped TCP bioceramic has a higher degradation rate and promotes new bone formation better than TCP in vivo.15 Wang et al19 assert that the composite scaffolds containing50% and 80% calcium silicate not only have good osteoconductivity, but also promote rapid bone formation compared with pure b-TCP and calcium silicate scaffolds. Thus, to obtain both osteostimulation and osteoconductivity by taking advantage of the favorable bioactivity of silica and the high degradability of b-TCP, Si-doped b-TCP cements have been produced in the hopes that the right mixture can help to control the degradation rate and improve interactions of the material with human tissue. In this study, Si-doped b-TCP cements with varying ratios were prepared so that we could observe the changes in
physiochemical properties, bioactivity, in vitro degradation behavior, osteogenesis, and antibacterial activity with different ratios of Si doped. It is our hope that this knowledge may help in the design of optimal biomaterials for bone regeneration.
Materials and methods Preparation of Si-doped b-TCP cement The reagent grade SiO2 (Sigma-Aldrich, St. Louis, MO, USA) and b-TCP (Sigma-Aldrich) powders were used as matrix materials. The SiO2 and b-TCP mixtures were sintered at 1400 C for 3 hours using a high-temperature furnace, and then ball-milled in ethyl alcohol using a centrifugal ball mill (S 100; Retsch, Hann, Germany) for 6 hours. The specimen codes “Si10”, “Si20” and “Si30” were used to indicate cement containing 10 SiO2/90 b-TCP, 20 SiO2/80 b-TCP, and 30 SiO2/70 b-TCP (in wt.%), respectively. Si0 is the b-TCP group. The powder was mixed using a liquid/powder ratio of 0.25 mL/g. After mixing with liquids (1M Na2HPO4), the cements were molded in a Teflod mold (diameter, 6 mm; height, 3 mm). The cement quantities were such they fully covered each well of the 24-well plate (GeneDireX, Las Vegas, NV, USA) to a thickness of 2 mm for cell experiments. All samples were stored in an incubator at 100% relative humidity and 37 C for 1 day of hydration.
Setting time and strength After the powder was mixed with liquid, the cement was placed into a cylindrical mold and stored in an incubator at 37 C and 100% relative humidity for hydration. The setting time of the cements was tested according to the standards set by the International Standards Organization 9917-1. The setting time was recorded when the Gilmore needle failed to create a 1-mm-deep indentation in three separate areas. After being taken out of the mold, the composite specimens were incubated at 37 C in 100% humidity for 1 day. Diametral tensile strength (DTS) testing was conducted on an EZ-Test machine (Shimadzu, Kyoto, Japan) at a loading rate of 1 mm/minute. The maximal compression load at failure was obtained from the recorded loadedeflection curves. At least 10 specimens from each group were tested.
In vitro soaking To evaluate the in vitro bioactivity, the cements were immersed in a 10-mL simulated body fluid (SBF) solution at 37 C. The solution in the shaker water bath was not changed daily under a static condition. After soaking for different time durations (from 3 days to 3 months), specimens were removed from the tube and evaluated for several physicochemical properties.
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Morphology The morphology of the cement specimens before and after immersion in SBF were coated with gold and examined under a scanning electron microscope (JSM-6700F, JEOL, Tokyo, Japan) operated in the lower secondary electron image mode at 3 kV accelerating voltage.
Weight loss The degree of degradation was determined by monitoring the weight change of the specimens. After drying at 60 C, the composites were weighed both before and after immersion using a balance. Ten repeated specimens were examined for each of the materials investigated at each time point.
Ion concentration The concentration of Ca, Si, and P ions released from cement on SBF was analyzed using an inductively coupled plasma atomic emission spectrometer (Perkin-Elmer OPT 1 MA 3000DV; Perkin-Elmer, Shelton, CT, USA) after immersion. Three samples were measured for each data point. The results were obtained in triplicate from three separate samples for each test.
Dental pulp cell isolation and culture The hDPCs were freshly derived from caries-free, intact premolars that were extracted for orthodontic treatment purposes, as described previously.12 A sagittal split was performed on each tooth using a chisel, and the pulp tissue was immersed in a phosphate buffered saline (PBS) solution. Pulp tissue was then cut into fragments, distributed into plates, and cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 20% fetal bovine serum (Caisson, North Logan, UT), 1% penicillin (10,000 U/mL)/streptomycin (10,000 mg/mL) (Caisson), and kept in a humidified atmosphere with 5% CO2 at 37 C; the medium was changed every 3 days. The osteogenic differentiation medium was DMEM supplemented with 108M dexamethasone (Sigma-Aldrich), 0.05 g/L L-ascorbic acid (Sigma-Aldrich), and 2.16 g/L glycerol 2-phosphate disodium salt hydrate (Sigma-Aldrich).
Cell viability Cell suspensions at a density of 104 cells/mL were directly seeded over each specimen for different days. Cell cultures were incubated at 37 C in a 5% CO2 atmosphere. After different culturing times, cell viability was evaluated using the PrestoBlue (Invitrogen, Grand Island, NY, USA) assay. Briefly, at the end of the culture period, the medium was discarded and the wells were washed with cold PBS twice. Each well was then filled with the medium with a 1:9 ratio of PrestoBlue in fresh DMEM and incubated at 37 C for 30 minutes, after which the solution in each well was transferred to a new 96-well plate. Plates were read in a multiwell spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. Cells cultured on the
S.-H. Huang et al tissue culture plate without the cement were used as a control (Ctl). The results were obtained in triplicate from three separate experiments in terms of optical density (OD).
Osteogenesis assay The level of alkaline phosphatase (ALP) activity was determined after cell seeding for 3 days and 7 days. The process was as follows: the cells were lysed from disks using 0.2% NP-40, and centrifuged for 10 minutes at 2000 g after washing with PBS. ALP activity was determined using p-nitrophenyl phosphate (Sigma-Aldrich) as the substrate. Each sample was mixed with p-nitrophenyl phosphate in 1M diethanolamine buffer for 15 minutes, after which the reaction was stopped by the addition of 5N NaOH and quantified by absorbance at 405 nm. All experiments were done in triplicate. The osteocalcin (OC) protein released from pulp cells were cultured on different substrates for 7 days and 14 days after cell seeding. An OC enzyme-linked immunosorbent assay kit (Invitrogen) was used to determine OC protein content following the manufacturer’s instruction. The OC protein concentration was measured by correlation with a standard curve. The analyzed blank disks were treated as controls. All experiments were performed in triplicate.
Alizarin Red S stain Accumulated calcium deposition was observed for 14 days using Alizarin Red S staining as described in a previous study.20 To summarize briefly, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and then incubated in 0.5% Alizarin Red S (Sigma-Aldrich) at pH 4.2 for 15 minutes at room temperature in an orbital shaker (25 g). To quantify the stained calcified nodules after staining, samples were immersed with 1.5 mL of 5% sodium dodecyl sulfate (SDS) in 0.5N HCl for 30 minutes at room temperature, following which the tubes were centrifuged at 5000 g for 10 minutes and the supernatant was transferred to the new 96-well plate (GeneDireX). At this time, absorbance was measured at 405 nm (Hitachi).
Antibacterial property To investigate the antibacterial effect of Si-doped b-TCP cement, the hydration composites were mixed with 1 mL Staphylococcus aureus in LB culture medium (4.0 104 bacteria/mL) and cultured for 3 hours and 12 hours. Aliquots of 0.1 mL from each group were mixed with 0.9 mL PrestoBlue for 30 minutes. The solution in each well was transferred to a new 96-well plate. Plates were read in a multiwell spectrophotometer (Hitachi) at 570 nm with a reference wavelength of 600 nm. Cells cultured on the tissue culture plate without the cement were used as a Ctl. The results were obtained in triplicate from three separate experiments in terms of OD.
Statistical analysis A one-way analysis of variance statistical analysis was used to evaluate the significance of the differences between the
Properties of silica-doped b-tricalcium phosphate means in the measured data. Scheffe’s multiple comparison test was used to determine the significance of the deviations in the data for each specimen. In all cases, the results were considered statistically significant if P < 0.05.
Results Setting time and strength After mixing with liquid, the working time of Si0, Si10, Si20, and Si30 was 11 minutes, 11 minutes, 8 minutes, and 7 minutes, respectively (Fig. 1A). As the amount of Si doped increased, the setting time of the cement becomes shorter, going from 70 minutes (Si0 group) all the way down to 31 minutes (Si30 group), which represents a significant difference (P < 0.05). The DTS values of hydration cements ranged from 1.4 MPa to 2.6 MPa, indicating a significant
285 (P < 0.05) increase in the strength as the amount of Si amount is increased (Fig. 1B).
Morphology of immersed cements The results of an examination of the surface microstructure of the specimens before and after immersion in SBF after 3 days and 7 days are shown in Fig. 2. It is readily visible that the Si0 cement has a looser and rougher surface texture, with irregular pores. By contrast, the Si30 cement exhibits a dense and smooth surface containing particle entanglement and micropores. The formation of the bonelike apatite in SBF has proven to be useful in predicting the bone-bonding ability of material in vitro. It can be seen that the apatite layer covers the surfaces of Si20 and Si30 content after immersion for 7 days. However, it is clear that no precipitate has formed on the surfaces of Si0 and Si10.
Weight loss Dissolubility is one of the important factors in biodegradation21 that must be considered when trying to develop a material with a degradation rate most appropriate for hastening and easing the process of bone repair. With this in mind, the degradation rates of the Si-doped b-TCP in SBF solution have been recorded for various time intervals ranging from 3 days to 12 weeks, as shown in Fig. 3. After soaking for 1 week, the Si30 cement shows a relatively modest amount of weight loss (w18.2%), whereas the Si0 cement lost considerably more weight (w26.3%). All samples showed an increased weight loss as the immersion time is increased. The Si30 cement has the lowest dissolution rate and solubility compared with other samples over the whole immersion period, reaching 26.0% after 12 weeks. By contrast, the overall amount of Si0 dissolution reached 52.4% after 12 weeks. At the end of the soaking point, weight losses of approximately 45.3%and 33.4% were observed for Si10 and Si20 cements, respectively, indicating significant differences (P < 0.05).
Ion concentration Variations of Ca, Si, and P ion concentrations in SBF after different soaking periods are shown in Fig. 4. After immersion for 1 day, Ca ion concentration in the Si30 group was decreased to approximately 1.76 mM, which was lower than that in the Si0group (1.92 mM, P < 0.05). As for P ion concentration of SBF, the Si30 group also decreased after 1 day of immersion. Si concentrations increased with increasing incubation time points. Si ion concentration was in the range of 1.2 mM, 1.5 mM, and 1.9 mM at Si10, Si20, and Si30 for 1 week, respectively.
hDPC proliferation
Figure 1 (A) The working, setting time and (B) diametral tensile strength of various Si-doped b-tricalcium phosphate hydrate. Statistical data are presented as mean standard deviation (n Z 6). * Significant difference (P < 0.05) compared to Si0.
Cell behavior and functions associated with the bone cement is closely related to the physicochemical and biological properties of the materials used. To consider the effects of Si doped on osteogenic activity, the biological functions of hDPCs cultured on various cement specimens were evaluated for different time points (Fig. 5). The cell
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Figure 2 Scanning electron micrographs of Si-doped b-tricalcium phosphate cement surfaces before and after immersion in simulated body fluid for 3 days and 7 days.
proliferation gradually increased with the increasing amount of Si doped into b-TCP, indicating a significant difference (P < 0.05) compared with the Si0 cement. For example, on Day 1 the Si30 cement showed an increase of approximately 32% in the OD value in reference to the Si0 cement. The Si0 cement elicited a significant decrease of 25%, 27%, and 20% in comparison with the Si30 cement on Day 1, Day 3, and Day 7 of cell culture, respectively.
Osteogenesis protein secretion
Figure 3 Weight loss of various cements after immersion in simulated body fluid for predetermined time durations.
Osteogenesis differentiation of cells is one of the key steps to determine the level of success in bone formation. The ALP secretion of hDPCs cultured on different cements has also been examined. Fig. 6A shows the analysis of quantitative examination data and the ALP activity of cells cultured on the different composites for 3 days and 7 days. The ALP level increased with increasing of Si content in the cements at all incubation times. A significant 32% and 54% increase (P < 0.05) in the ALP level was measured for Si20 and Si30 in comparison with the Si0 cement for 7 days. Similarly, the OC secretion by cells cultured on the Si30 cement is significantly higher (P < 0.05) than that secreted
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Figure 5 Proliferation of human dental pulp cells treated with various specimens for 7 days. * Significant difference (P < 0.05) compared to Si0.
deposition in the Si0 cement was only lightly stained (Fig. 7). By Week 2, a more intense calcium staining was exhibited. The corresponding quantification of calcium mineral deposition is also shown in Fig. 7B. In the case of the Si-doped cement, significant (1.19-, 1.89-, and 2.13-fold) enhancement (P < 0.05) of calcium content was observed on Si10, Si20, and Si30 compared with Si0 cement on Week 2, respectively.
Antibacterial property Besides the osteostimulation property of the cements, these bone grafts demonstrated that they also had a significant antibacterial effect (Fig. 8). The S. aureus cultures with Ctl and Si0 showed the highest absorbance at the different times observed (P < 0.05). However, the antibacterial activity of Si-doped b-TCP cement was promoted when the Si content was increased. For example, at 24 hours the Si30, Si20, and Si10 cements exhibited a decrease of approximately 46%, 30%, and 9%, respectively, in the OD value referenced to the Si0 cement.
Discussion
Figure 4 (A) Ca, (B) Si, and (C) P ions concentration of simulated body fluid after immersion for different times. Data are presented as mean standard deviation (n Z 6). * Statistically significant difference compared to Si0.
by cells on other samples (Fig. 6B). OC secretion increased 37% and 22% in cells cultured on Si30 and Si20 in comparison with the Si0 cement for 14 days.
Mineralization Bone nodule was clearly seen in cells in the presence of Si20 and Si30 after culture for 1 week, whereas calcium
Calcium phosphate-based cements consist of a powder containing one or several solid compounds of calcium phosphate salts and a liquid phase that can be water or the solution.22 Calcium phosphate cements, especially b-TCP, have a wide application in reconstructive bone surgery. In physiological content, b-TCP quickly degrades by both chemical dissolution and osteoclastic resorption, and affects the bone-remodeling process.23 Thus, this study shows several beneficial effects derived from the simultaneous presence of SiO2 dopants in b-TCP. Furthermore, the effect of SiO2 dopants in b-TCP on hDPC behavior and its activity is demonstrated. The setting time is one of the key factors in clinical requirements. A long setting time could cause problems clinically because of the cement’s inability to maintain
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Figure 6 (A) Alkaline phosphatase activity and (B) osteocalcin (OC) amount of human dental pulp cells cultured on various specimens for different times. * Significant difference (P < 0.05) compared to Si0.
shape and support stresses within this period.24 In this study, when 1M Na2HPO4 was used as a liquid phase, the setting time of the control cement was 70 minutes, consistent with previously reported results.20 In the present study, the setting time of silicate-based cements was proportional to the calcium silicate hydrate amount.25 Calcium silicate hydrate is able to reduce the setting time of the calcium-based cement. Moreover, our results show that a setting time of approximately 30 minutes for injectable bone cements for use in vertebroplasty and kyphoplasty26 is possible. In the bone cement, it is believed that when bonded to natural bone, the apatite layer will precipitate on the surface.14 The formation of the bonelike apatite in SBF has proven to be useful in predicting the bone-bonding ability of materials in vitro. It can be seen that the apatite layer covers the surfaces of specimens with Si20 and Si30
S.-H. Huang et al after immersion. After immersion, the surface of the two samples is completely covered by an apatite layer with tiny ball-like shapes on the surface. However, it is clear that no precipitate has formed on the surfaces of the b-TCP-rich cement (Si0 and Si10). These results are similar to reports by other researchers,15,27 who elucidate the lack of apatite formation observed in b-TCP samples after soaking in SBF for several days.20 The in vitro bioactivity of the silicatebased materials indicates that the presence of PO3 ions 4 in the composition is not an essential requirement for the formation of the apatite layer, which is noteworthy because it is known that PO3 4 depletes calcium and phosphate ions because the PO3 4 ions originate from the in vitro assay solution.25 Most importantly, the SieOH functional group on Si-based has been proven to act as the nucleation center for apatite formation.4 This may explain why the b-TCP cement had no bioactivity. Thus, the Si-doped b-TCP cements were supposed to develop a stronger bond with the surrounding bone tissue compared with the b-TCP-rich cement. As expected, Si-doped b-TCP cements showed an intermediate dissolution behavior similar to that exhibited by pure b-TCP. Moreover, as the Si content is increased, the dissolution rate also decreases steadily. Hence, the degradation rate of the cements may be controlled to a certain extent by varying the Si content in the cement.27 Taking cell functions into account, the appropriate Si released from silicate-based materials may support cell behaviors.28e31 Currently available Si-based materials offer users the ability to control the rate of soluble Si ion, which in turn affects cell adhesion and proliferation.25,32 Regarding biocompatibility, it is certainly evident that no bone grafts showed signs of cytotoxicity. Cell behavior associated with bone cement is closely related to the biological properties of the materials used. To elucidate the effects of Si doping on proliferation and osteogenic activity of pulp, cells cultured on various specimens were evaluated. Cell proliferation was higher when there was a higher Si content in the cement. Si content was likely to be the key factor in cell attachment and proliferation as there was a reciprocal relationship in Ca concentration.3,9,25 There is a genetic control of the cellular response of osteoblasts to the bone cements. Osteoblast differentiation is generally accompanied by ALP and OC secretion. The secretion of ALP from hDPCs was initiated earlier during culture on the higher Si-doped cement surface (Si30) than on the lower Si content cement (Si0), consistent with ALP amounts on the culture medium. It is generally accepted that an increase in the specific activity of ALP in bone cells reflects a shift to a more differentiated state. OC is a late marker of osteogenesis and is the noncollagenous bone-matrix protein characteristic of osteoblast synthetic function. Recent studies also show that Si-based materials promote hDPC proliferation and differentiation.3,9,25 This stimulatory effect may be attributed to the dissolution of Si ions.3,4,33 According to the study, TCP cement always fails to form a chemical bonding with hard tissue at the early stage of the therapy because of its poor bioactivity.27 Taken together, our data indicated that the Si-doped cement might possess a higher potency to interact with hDPCs and to enhance osteogenesis and calcium matrix deposition compared with the pure b-TCP cement. Such a system could combine the benefits of bone substrate properties, such as resorbability and osteoconductivity. Besides
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Figure 7 (A) Alizarin red S staining and (B) quantification of calcium mineral deposits by human dental pulp cells cultured on various cements for 1 week and 2 weeks. * Significant difference (P < 0.05) compared to Si0.
the osteostimulation property of the bone cement, Si-doped b-TCP is also shown to have a significant antibacterial effect. There may be two potential reasons for the antibacterial effect of Si-based cement. One is that Si-based material can release Ca and Si ions, leading to a weak alkaline microenvironment with higher pH value, which may restrain bacterial growth15,34; the other is that the calcium silicate particles themselves could permeate into the bacteria,16,35 which could possibly promote the antibacterial effect. Therefore, Si-doped b-TCP cement could be a promising excellent antibacterial agent.
Conflicts of interest The authors have no conflicts of interest relevant to this article.
Acknowledgments Figure 8 The antibacterial effect of Si-doped b-tricalcium phosphate cement for 3 hours, 12 hours, and 24 hours. * Significant difference (P < 0.05) compared to Si0.
The authors acknowledge receipt of a grant from the National Science Council grant (NSC 101-2314-B-040-011-MY3) of Taiwan.
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