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Modification of dicalcium silicate bone cement biomaterials by using carboxymethyl cellulose
Journal of Non-Crystalline Solids 426 (2015) 164–168
Contents lists available at ScienceDirect
Journal of Non-Crystalline Solids journal homepage: www.elsevier.com/ locate/ jnoncrysol
Modification of dicalcium silicate bone cement biomaterials by using carboxymethyl cellulose Yin Zhang a,b,⁎, Dinggai Wang a, Fei Wang a, Shengxiang Jiang a, Yan Shu a a b
College of Materials Science and Engineering, Nanjing Tech University, Nanjing 210009, China Nanjing Haoqi Advanced Materials Co., Ltd., Nanjing 211300, China
a r t i c l e
i n f o
Article history: Received 11 May 2015 Received in revised form 4 July 2015 Accepted 6 July 2015 Available online xxxx Keywords: Calcium silicate bone cement; Carboxymethylcellulose; Setting time; Compressive strength; Bioactivity
a b s t r a c t To ensure the operability of the clinical, the setting time is one of the most clinically vital factors. Sol–gel technique was used to prepare calcium silicate powders with different molar ratios of CaO/SiO2, and the calcium silicate bone cements (CSCs) were obtained in this study. Functional groups of powder and cements were analyzed by infrared spectroscopy (FT-IR). The doped cement was prepared using carboxymethylcellulose (CMC)-containing calcium silicate powder as solid phase and distilled water as liquid phase. Phase composition, morphology, setting time (St) and compressive strength (Cs) of the doped cement, after mixing with water, were investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), Gillmore needle and electronic universal material testing machine, respectively. In vitro mineralization of doped cement was investigated by SBF immersion test by soaking the samples individually in 10-ml of simulated body fluid (SBF) solution at 37 °C for 0, 1, 3, 7 and 15 days (d), respectively. The results indicated that the doped cement with 0.10% CMC possessed shorter setting time, higher compressive strength, and desirable bioactivity that makes it an attractive choice for use in vertebroplasty and bone filling surgery. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ceramic materials have been widely used in bone repair, bone filling, artificial bone and so on [1], but it had always been difficult to fabricate the complicated structure of dental applications due to limitations, such as brittleness. The biological bone repair materials, having similar mechanical properties to natural bone, were obtained by combining the bioactive ceramic fine particles and organic polymers. Recently, there has been increasing usage of composite materials in bone replacement and bone filling [2–5]. Studies have shown that CaO–SiO2-based ceramic cement has a good biological activity and can be modified by polymer composite materials [6–8]. To ensure the operability of the clinical, the setting time is one of the most clinically relevant factors. Although a standard setting time was not proposed for root canal filling/sealing of the current clinical procedures, an appropriate and sufficient setting time is essential for a successful surgical treatment [9]. After mixing the water and powders, the injected cement could not maintain shape and support stress during this period due to the long setting duration, which could cause clinical problems [10]. However, the setting time was too short to inject into the defection. Some authors [11,12] indicated that an approximate
⁎ Corresponding author at: Nanjing Tech University, NO.5 Xinmofan Road, Nanjing, 210009, China. Tel.: +862583587260. E-mail address: [email protected] (Y. Zhang).
http://dx.doi.org/10.1016/j.jnoncrysol.2015.07.014 0022-3093/© 2015 Elsevier B.V. All rights reserved.
setting time of 15 min for injectable bone cements was applicable to use in dental treatment, vertebroplasty and keratoplasty. Sol–gel method was used to prepare calcium silicate powders with different molar ratios of CaO/SiO2, and CMC was used to modify the calcium silicate bone cements. The setting time, compressive strength and vitro bioactivity were investigated in this study, in addition to other characterizations by XRD, FTIR and SEM. Finally, the hybrid calcium silicate bone cement containing CMC, which can be suitable for clinical application, was prepared in this study. 2. Experimental procedure 2.1. Powder and cement preparation The powder was prepared by sol–gel method described in a previous work [10]. Reagent-grade tetraethyl orthosilicate (Si(OC2H5)4; TEOS, SiO2% ≥ 28.4%, Sinopharm Chemical Reagent Co., Ltd, China) and calcium nitrate (Ca(NO3)2 · 4H2O; 99.0%, XiLong Chemical Co., Ltd, China) were used as precursors for SiO2 and CaO, respectively. 2 mol/L nitric acid (HNO3) and absolute ethanol were used as the catalyst and solvent, respectively. The nominal molar ratios of CaO/SiO2 ranged from 3:7 to 7:3. For simplicity, the sintered powders and the cements derived from such powders were recorded by the same codes throughout this study. As shown in Table 1, for example, the specimen code “C30S70” stands for both sintered powder and cement with CaO/SiO2 = 3:7 (in mol %). The molar ratio of TEOS:(HNO3 + H2O): ethanol was
Y. Zhang et al. / Journal of Non-Crystalline Solids 426 (2015) 164–168 Table 1 Composition (molar ratio), setting time (St) and compressive strength (Cs) of calcium silicate bone cements and doped cements. Composition
Setting time
Compressive strength
Specimen code
CaO/SiO2
Min
MPa
C30S70 C40S60 C50S50 C60S40 C70S30 0.05% CMC/C50S50 0.10% CMC/C50S50 0.20% CMC/C50S50
3:7 4:6 5:5 6:4 7:3 5:5 5:5 5:5
58.0 ± 0.7 37.0 ± 0.8 27.0 ± 0.8 14.0 ± 0.7 11.0 ± 0.9 18.0 ± 0.8 15.0 ± 0.7 13.0 ± 0.9
0.94 ± 0.40 2.05 ± 0.50 15.53 ± 1.80 12.02 ± 1.70 4.87 ± 0.60 13.38 ± 1.40 18.84 ± 2.30 16.35 ± 1.70
1:10:10 in this study. 2 mol/L HNO3, absolute ethanol and the required amount of Ca(NO3)2 · 4H2O were added into TEOS solution successively, with 1 h of stirring after each addition. The prepared sol solution was sealed in a teflon beaker and aged at 60 °C for 2 days, following by evaporation of the solvent in an oven at 120 °C for 2 days to obtain dried gel. This gel was then heated in air to 800 °C for 2 h by using a high-temperature furnace and then cooled to room temperature in the furnace to obtain its powder form. 0.05 wt.%, 0.10 wt.%, 0.20 wt.% carboxymethylcellulose (Shandong Everbright Technology Co., Ltd, China) was added into the sintered powder (C50S50) and coded accordingly (i.e. 0.05% CMC/C50S50). The composite powders were then ball-milled for 12 h in ethanol using a centrifugal ball mill (CQ-QM2L, Nanjing Troon New Instrument Co., Ltd, China) and dried in an oven at 60 °C. For preparing cement, the liquid-to-powder (L/P) ratio of 0.5 ml/g was used for the powders and composite powders. The C50S50 cement without CMC was used as the CSCs control, in regard of adding CMC cements. The mixed cement was poured into a cylindrical stainless steel mold (diameter, 6 mm and high, 12 mm) under a pressure of 0.7 MPa for 1 min using a uniaxial press and incubated at 37 °C and 100% relative humidity, and then allowed to set for 1 day. It is noted that the size (Φ6 mm × 3 mm) of sample was used for soaking test.
165
for predetermined periods of time at 37 °C to evaluate the in vitro bioactivity. After immersing at each time point (0, 1, 3, 7, 15 days), specimens were removed from the centrifuge tube and gently rinsed three times with deionized water and acetone, and then dried at room temperature for XRD, FT-IR and SEM analyses. 3. Results and discussions 3.1. Phase composition and FT-IR spectra of powders Fig. 1a shows the x-ray diffraction analysis of the five CaO–SiO2 powders sintered at 800 °C for 2 h, indicating that the phase evolution is dictated by the Ca–Si ratios of the precursors. The XRD exhibits a distinct and diffuse diffraction peak at around 2θ = 32°–34°, which can be attributed to the β-dicalcium silicate (β-Ca2SiO4, JCPDS 290371) phase. The result suggests that the more content of CaO in precursors, the stronger of the peak intensities of β-Ca2SiO4 and CaO (2θ = 37.5°). It shows that calcium as a promoter played a very important role in the CaO–SiO2 system, additionally, the amount of CaO in the starting materials may affect St [14]. Fig. 1b indicates that the trends in the FT-IR spectra of the prepared powders were similar to those in XRD. For the sample with lowest content of CaO (C30S70), the IR absorption band was assignable to SiO4 asymmetric stretching extends, ranging over a wide wave number from 1300 to 900 cm− 1. The bands at 1550 and 1380 cm−1 were ascribed to the vibration of CO3 groups carbonized from the atmosphere. The band at around 550–530 cm−1 originated from the vibration of the siloxane backbone [15]. When the CaO content was equal to the SiO2 content (C50S50), a broadening and shifting to lower frequency in Si–O–Si asymmetric stretching bands were detected in the FTIR spectra, which indicated that the specimens' structure became
2.2. Test and characterizations Phase analysis of the powder and the cement was carried out using X-ray diffraction (XRD, Geigerflex, Rigaku, Japan) with a monochromated CuKα radiation tube. The surface morphology images were conducted on scanning electron microscope (SEM; S-4800, JEOL, Tokyo, Japan) under an operating current of 20 mA and a voltage of 40 kV. Fourier transform infrared spectroscopy (FT-IR, NEXUS 670, Nicolet, American) was used to analysis the functional groups of samples. According to international standard ISO 9917–1:2007 for water-based cements, a 400-g Gillmore needle with a 1-mm diameter was utilized to measure the setting time of the cement. The final setting time (St) was recorded as the moment when the needle failed to create an indentation of 1 mm in depth in three different areas of the cement cylinder, which were maintained at 37 °C with 100% relative humidity environment. A total of eight specimens were used for testing the St. Compressive strength (Cs) testing was measured on an electronic universal material testing machine (CMT 6203, Shenzhen Shiji Tianyuan Instrument Co., Ltd., Guangdong, China) at a loading rate of 0.5 mm/min. At least 20 specimens were tested for each group. The pH values of SBF solution before and after soaking with the samples for different time point were obtained from the pH meter (PHSJ-3 F, Shanghai Jie Sheng Scientific Instrument Co., Ltd.). The determined values were calculated using mean ± standard deviation. 2.3. In vitro bioactivity test Each 24 h set cylindrical specimen (Φ6 mm × 3 mm) was soaked in SBF solution according to the procedure described by Kokubo et al. [13],
Fig. 1. XRD (a) and FT-IR (b) patterns of calcium silicate powders sintered at 800 °C.
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less ordered as a result of a decrease in the local symmetry triggered by the incorporation of CaO into the silica network [14]. On the contrary, when the Ca/Si ratio was over 1:1, two new bands near 860 and 970 cm−1 emerged, which were associated with the Si–O symmetric stretching mode with one non-bridging silicon-oxygen bond (Si–O– NBO) [14,16]. The calcium amounts largely affected the phase evolution, as indicated by XRD and FTIR results of the powders.
3.2. Phase composition and FT-IR spectra of cements The XRD and FT-IR of the cured cements in powder form after 1 day of hydration are shown in Fig. 2a and b, respectively. It can be seen that the peak at 2θ = 29.4° corresponds to calcium silicate hydrate (CSH, JCPDS 29-378) of hydration products; additionally, the incompletely reacted β-Ca2SiO4 and CaO were also present in these products. The content of CSH increased with the increase of Ca/Si moral ratio in starting materials. It is speculated that the products were related to the hardening mechanism of calcium silicate cement, which could be explained by the effect of β-Ca2SiO4 content on the form of the CSH [14]. It was determined in a previous work that a high CSH content of hydration products leads to shorter St and faster hydration reaction [17]. The diffraction peak near 2θ = 37.5° corresponding to the phase of CaO, the reduce of CaO could significantly reduce the St after mixing with water [18]. As shown in Fig. 2b, the trends in the FT-IR spectra of the cements were similar to those indicated by XRD. The FT-IR spectra of the powder (Fig. 1b) was consistent with that in cement (Fig. 2b) without the emergence of a new band.
3.3. Setting time and compressive strength The setting times (St) and compressive strength (Cs) of the five CSCs and doped cements are shown in Table 1. It can be seen that the St became shorter with increasing CaO/SiO2 ratio by the same L/P = 0.5 mL/g. The St values of the five CSCs were 58 ± 2, 37 ± 2, 27 ± 2, 14 ± 2 and 11 ± 2 min corresponding to C30S70, C40S60, C50S50, C60S40 and C70S30 after mixing with water, respectively. It can be clearly seen that the St of the cement became shorter with increasing CaO content, demonstrating the accelerating role of Ca in the setting reaction [14]. The higher CaO content in the starting materials resulted in more β-Ca2SiO4 and a higher CSH, leading to shorter St and faster hydration reaction. The peak of CaO phase decreased after mixing with water, which resulted in a shorter St. The powder particle size, sintering temperature, liquid phase and composition, and liquid–solid ratio play a very important role in setting time of cement materials [18]. Table 1 shows that the Cs of the five CSCs tends to increase firstly and then reduce. Table 1 illustrates that the Cs values of the five CSCs were 0.94 ± 0.4, 2.05 ± 0.5, 15.53 ± 1.8, 12.02 ± 1.7 and 4.87 ± 0.6 MPa for C30S70, C40S60, C50S50, C60S40 and C70S30, respectively. The Cs of the sample C30S70 with the lowest CaO content is only 0.94 MPa, while the Cs of the sample C70S30 containing the most abundant of CaO is only 4.87 MPa. The maximum Cs of C50S50 reached 15.53 MPa among the five CSCs, which may be due to the differences existed in cement surface structure, such as the role of porosity of hydration products on the mechanical properties of cement [19]. Based on the above, it can be seen that the CaO is a key factor affecting St and Cs. According to the results of the St and Cs values, the C50S50 was regarded as the best powders for compounding CMC. As shown in Table 1, the setting time of the doped cements with 0.05%, 0.10% and 0.20% CMC were significantly decreased to 13 ± 2, 15 ± 2 and 18 ± 2 min, respectively, comparing to the control C50S50 cement (27 ± 2 min). In addition, the Cs of the doped cements changed with increasing the content of CMC. The St of the doped cement containing 0.10% CMC was significantly reduced, while the Cs reached to 18.84 MPa, as compared to the control C50S50 cement (Cs:15.53 MPa). This may be attributed to water absorbing capability of CMC as viscous agent that promoted faster setting. Setting times of the doped cements are inversely proportional to the amount of added CMC. The Cs of the 0.05% CMC/C50S50 was lower than the control C50S50 cement due to the dispersion of a small amount of CMC dispersed that absorbed the water within the sample, resulting in the increase of porosity in the cements. By adding an appropriate amount of CMC (0.10%), which can be combined with the water during the hydration of cement, a network of polymer film can form in the sample, which eventually increases the Cs of the doped cement by improving the compact degree between the β-Ca2SiO4 particles and hydration products. However, when doping with excessive CMC, which would parcel the surface of the β-Ca2SiO4 particle, a weak boundary layer would form to impede the hydration of the β-Ca2SiO4. The hydrated sample, in such a case, would not be able to form a solid crystalline skeleton, which may hinder the development of the Cs [20,21]. 3.4. In vitro bioactivity 3.4.1. pH value Table 2 shows the pH values of the SBF solution after soaking with the 0.10% CMC/C50S50 cement for various periods (0, 1, 3, 7, 15 days). It can be seen that the pH value of the SBF solution quickly reached Table 2 pH values of SBF solution after soaked with the calcium silicate bone cements containing 0.10% CMC for different time point (0, 1, 3, 7, 15 days).
Fig. 2. XRD (a) and FT-IR (b) patterns of calcium silicate bone cements after 1 day of hydration.
Immersion time/day
0
1
3
7
15
pH
7.4
9.6
7.7
7.6
7.6
Y. Zhang et al. / Journal of Non-Crystalline Solids 426 (2015) 164–168
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about 9.6 after immersing in SBF for 1 d. The pH value of the SBF solution tended to decrease after 1 day and became stabilized (pH 7.6–7.7) from 3 days to 15 days. During the soaking process, the H3O+ in solution entered into the surface of the cement, resulting in the increase of the OH− relative concentration of the solution, which enhanced the pH value of SBF solution [18,21].
Fig. 3. XRD patterns of calcium silicate bone cement containing 0.10% CMC before and after immersing in SBF for different time point (0, 1, 3, 7, 15 days).
3.4.2. XRD The XRD pattern of calcium silicate bone cement containing 0.10% CMC before and after immersing in SBF for different time points (0, 1, 3, 7, 15 days) is shown in Fig. 3. The in vitro bioactivity of the 0.10% CMC/C50S50 soaked in SBF solution was tested by observing the hydroxyapatite (Hap) deposited on the surface of the specimens. It shows that the control samples, before soaking in SBF solution, are mostly CSH (JCPDS 29-378) and β-Ca2SiO4 phase. After soaking in SBF for 1, 3, 7 and 15 days, the new visible peaks (2θ = 31.4°–32.6°) attributed to Hydroxyapatite (Hap, JCPDS 24-0033) can be clearly seen. The peaks of CSH became stronger, while the peaks of β-Ca2SiO4 phase gradually disappeared with increasing immersion time. When compared to the control sample, a gradually increased peak intensity
Fig. 4. SEM micrographs of 0.10% CMC/C50S50 cement before and after immersion in SBF for different time point (0, 1, 3, 7, 15 days).
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of the CSH formed in the specimens soaked in SBF can be observed. This phenomenon can be attributed to the adequate reaction of the powders with the water in SBF, which formed more CSH gel and prolonged the soaking time [21,22]. The formation of Hap is due to the reaction of 2+ and OH− during the soaking process [22]. PO3− 4 , Ca
Acknowledgments This work was financially supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References
3.4.3. Morphology Fig. 4 shows the surface morphologies of the cement containing 0.10% CMC before and after soaking in SBF solution. It can be seen that the surface of the 0.10% CMC/C50S50 cement showed significant changes with clusters of smooth precipitated spherulite after immersion, comparing to the control cement (C50S50). By prolonging the soaking time, the Hap spherulites with a greater variety of sizes and content formed apatite layer, which were found on the surfaces of the hybrid cement, especially after immersion in SBF for 7 days. This result revealed their high activity. After soaking 15 days, the spherical apatite with needle surface intertwined together to form apatite layer, indicating that the microspheres gradually grew and changed during immersion process. Previous studies have reported that the examination of apatite formation on the surface of a material in SBF was used to predict the in vivo bioactivity of a biomaterial [23]. In terms of the formation process of Hap on the surface of the dicalcium silicate-based hybrid cement is nature of a process that a new generation grown up and then continue changes [6]. The forming process of Hap major contained the following stages: 1) samples were dissolved and the silicon-rich surface layer was formed, 2) the calcium-phosphate nuclei were formed, followed by the crystal growth and transformation of Hap. The forming process was consistent with 45S5 bioactive glass, which was similar to the formation principle of Hap on the surface of A–W glass-ceramic materials [24]. It is evident from earlier reports that synergistic effect between Si as an effective apatite nucleate and Ca as an apatite precipitation accelerator caused this fast apatite precipitation [6,24].
4. Conclusions A Sol–gel technique was used to prepare calcium silicate powders with different molar ratios of CaO/SiO2. Five cements were obtained by mixing these powders with distilled water. Experimental results indicated that the C50S50 cement exhibited a relatively short setting time and higher compressive strength. Different amounts of CMC were used to modify the C50S50 cement, and the results showed that the incorporation of 0.10 wt.% CMC was most effective at shortening the setting time and enhancing the compressive strength of cements. Moreover, the in vitro bioactivity of 0.10% CMC/C50S50 cement makes it attractive for use in clinical applications of the root canal treatment and vertebroplasty. However, further investigation is necessary to evaluate the effect of CMC on the biocompatibility and the clinical potential of the hybrid CSCs in the further studies.
[1] L.L. Hench, Genetic design of bioactive glass, J. Eur. Ceram. Soc. 29 (7) (2009) 1257–1265. [2] Y. Shirosaki, K. Tsuru, S. Hayakawa, et al., Physical, chemical and in vitro biological profile of chitosan hybrid membrane as a function of organosiloxane concentration, Acta Biomater. 5 (1) (2009) 346–355. [3] S.J. Ding, C.K. Wei, et al., Bio-inspired calcium silicate–gelatin bone grafts for loadbearing applications, J. Mater. Chem. 21 (34) (2011) 12793–12802. [4] C.C. Chen, W.C. Wang, S.J. Ding, In vitro physiochemical properties of a biomimetic gelatin/chitosan oligosaccharide/calcium silicate cement, J. Biomed. Mater. Res. B Appl. Biomater. 95 (2) (2010) 456–465. [5] M.Y. Shie, D.C.H. Chen, C.Y. Wang, et al., Immersion behavior of gelatin-containing calcium phosphate cement, Acta Biomater. 4 (3) (2008) 646–655. [6] C.C. Chen, M.H. Lai, W.C. Wang, et al., Properties of anti-washout-type calcium silicate bone cements containing gelatin, J. Mater. Sci. Mater. Med. 21 (4) (2010) 1057–1068. [7] Q. Lin, X. Lan, Y. Li, et al., Anti-washout carboxymethyl chitosan modified tricalcium silicate bone cement: preparation, mechanical properties and in vitro bioactivity, J. Mater. Sci. Mater. Med. 21 (12) (2010) 3065–3076. [8] T.Y. Chiang, C.C. Ho, D.C.H. Chen, et al., Physicochemical properties and biocompatibility of chitosan oligosaccharide/gelatin/calcium phosphate hybrid cements, Mater. Chem. Phys. 120 (2) (2010) 282–288. [9] E.J. Lee, D.S. Shin, H.E. Kim, et al., Membrane of hybrid chitosan–silica xerogel for guided bone regeneration, Biomaterials 30 (5) (2009) 743–750. [10] J. Camilleri, Evaluation of the physical properties of an endodontic Portland cement incorporating alternative radiopacifiers used as root–end filling material, Int. Endod. J. 43 (3) (2010) 231–240. [11] G. Lewis, Injectable bone cements for use in vertebroplasty and kyphoplasty: Stateof-the-art review, J. Biomed. Mater. Res. B Appl. Biomater. 76 (2) (2006) 456–468. [12] C.C. Chen, C.C. Ho, D.C.H. Chen, et al., Physicochemical properties of calcium silicate cements for endodontic treatment, J. Endod. 35 (9) (2009) 1288–1291. [13] T. Kokubo, Surface chemistry of bioactive glass-ceramics, J. Non-Cryst. Solids 120 (1990) 138–151. [14] J. Serra, P. González, S. Liste, et al., FT-IR and XPS studies of bioactive silica based glasses, J. Non-Cryst. Solids 332 (1) (2003) 20–27. [15] H. Yoshino, K. Kamiya, H. Nasu, IR study on the structural evolution of sol–gel derived SiO2 gels in the early stage of conversion to glasses, J. Non-Cryst. Solids 126 (1) (1990) 68–78. [16] M.M. Pereira, A.E. Clark, L.L. Hench, Homogeneity of bioactive sol–gel-derived glasses in the system CaO–P2O5–SiO2, J. Mater. Synth. Process. 2 (1994) 189–196. [17] F.H. Lin, W.H. Wang, C.P. Lin, Transition element contained partial-stabilized cement (PSC) as a dental retrograde-filling material, Biomaterials 24 (2) (2003) 219–233. [18] M. Bohner, Reactivity of calcium phosphate cements, J. Mater. Chem. 17 (38) (2007) 3980–3986. [19] K. Ishikawa, K. Asaoka, Estimation of ideal mechanical strength and critical porosity of calcium phosphate cement, J. Biomed. Mater. Res. 29 (12) (1995) 1537–1543. [20] Hewen Qian, Chengdong Yin, Changhe Liu, et al., J. Chin. Ceram. Soc. 5 (2) (1996) 69–81 (in Chinese). [21] Fuqin Han, Bo Shao, Qingwen Wang, et al., For. Sci. 45 (7) (2009) 101–105 (in Chinese). [22] M.Y. Shie, H.C. Chang, S.J. Ding, Effects of altering the Si/Ca molar ratio of a calcium silicate cement on in vitro cell attachment, Int. Endod. J. 45 (4) (2012) 337–345. [23] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27 (15) (2006) 2907–2915. [24] S.J. Ding, M.Y. Shie, C.Y. Wang, Novel fast-setting calcium silicate bone cements with high bioactivity and enhanced osteogenesis in vitro, J. Mater. Chem. 19 (8) (2009) 1183–1190.
Contents lists available at ScienceDirect
Journal of Non-Crystalline Solids journal homepage: www.elsevier.com/ locate/ jnoncrysol
Modification of dicalcium silicate bone cement biomaterials by using carboxymethyl cellulose Yin Zhang a,b,⁎, Dinggai Wang a, Fei Wang a, Shengxiang Jiang a, Yan Shu a a b
College of Materials Science and Engineering, Nanjing Tech University, Nanjing 210009, China Nanjing Haoqi Advanced Materials Co., Ltd., Nanjing 211300, China
a r t i c l e
i n f o
Article history: Received 11 May 2015 Received in revised form 4 July 2015 Accepted 6 July 2015 Available online xxxx Keywords: Calcium silicate bone cement; Carboxymethylcellulose; Setting time; Compressive strength; Bioactivity
a b s t r a c t To ensure the operability of the clinical, the setting time is one of the most clinically vital factors. Sol–gel technique was used to prepare calcium silicate powders with different molar ratios of CaO/SiO2, and the calcium silicate bone cements (CSCs) were obtained in this study. Functional groups of powder and cements were analyzed by infrared spectroscopy (FT-IR). The doped cement was prepared using carboxymethylcellulose (CMC)-containing calcium silicate powder as solid phase and distilled water as liquid phase. Phase composition, morphology, setting time (St) and compressive strength (Cs) of the doped cement, after mixing with water, were investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), Gillmore needle and electronic universal material testing machine, respectively. In vitro mineralization of doped cement was investigated by SBF immersion test by soaking the samples individually in 10-ml of simulated body fluid (SBF) solution at 37 °C for 0, 1, 3, 7 and 15 days (d), respectively. The results indicated that the doped cement with 0.10% CMC possessed shorter setting time, higher compressive strength, and desirable bioactivity that makes it an attractive choice for use in vertebroplasty and bone filling surgery. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ceramic materials have been widely used in bone repair, bone filling, artificial bone and so on [1], but it had always been difficult to fabricate the complicated structure of dental applications due to limitations, such as brittleness. The biological bone repair materials, having similar mechanical properties to natural bone, were obtained by combining the bioactive ceramic fine particles and organic polymers. Recently, there has been increasing usage of composite materials in bone replacement and bone filling [2–5]. Studies have shown that CaO–SiO2-based ceramic cement has a good biological activity and can be modified by polymer composite materials [6–8]. To ensure the operability of the clinical, the setting time is one of the most clinically relevant factors. Although a standard setting time was not proposed for root canal filling/sealing of the current clinical procedures, an appropriate and sufficient setting time is essential for a successful surgical treatment [9]. After mixing the water and powders, the injected cement could not maintain shape and support stress during this period due to the long setting duration, which could cause clinical problems [10]. However, the setting time was too short to inject into the defection. Some authors [11,12] indicated that an approximate
⁎ Corresponding author at: Nanjing Tech University, NO.5 Xinmofan Road, Nanjing, 210009, China. Tel.: +862583587260. E-mail address: [email protected] (Y. Zhang).
http://dx.doi.org/10.1016/j.jnoncrysol.2015.07.014 0022-3093/© 2015 Elsevier B.V. All rights reserved.
setting time of 15 min for injectable bone cements was applicable to use in dental treatment, vertebroplasty and keratoplasty. Sol–gel method was used to prepare calcium silicate powders with different molar ratios of CaO/SiO2, and CMC was used to modify the calcium silicate bone cements. The setting time, compressive strength and vitro bioactivity were investigated in this study, in addition to other characterizations by XRD, FTIR and SEM. Finally, the hybrid calcium silicate bone cement containing CMC, which can be suitable for clinical application, was prepared in this study. 2. Experimental procedure 2.1. Powder and cement preparation The powder was prepared by sol–gel method described in a previous work [10]. Reagent-grade tetraethyl orthosilicate (Si(OC2H5)4; TEOS, SiO2% ≥ 28.4%, Sinopharm Chemical Reagent Co., Ltd, China) and calcium nitrate (Ca(NO3)2 · 4H2O; 99.0%, XiLong Chemical Co., Ltd, China) were used as precursors for SiO2 and CaO, respectively. 2 mol/L nitric acid (HNO3) and absolute ethanol were used as the catalyst and solvent, respectively. The nominal molar ratios of CaO/SiO2 ranged from 3:7 to 7:3. For simplicity, the sintered powders and the cements derived from such powders were recorded by the same codes throughout this study. As shown in Table 1, for example, the specimen code “C30S70” stands for both sintered powder and cement with CaO/SiO2 = 3:7 (in mol %). The molar ratio of TEOS:(HNO3 + H2O): ethanol was
Y. Zhang et al. / Journal of Non-Crystalline Solids 426 (2015) 164–168 Table 1 Composition (molar ratio), setting time (St) and compressive strength (Cs) of calcium silicate bone cements and doped cements. Composition
Setting time
Compressive strength
Specimen code
CaO/SiO2
Min
MPa
C30S70 C40S60 C50S50 C60S40 C70S30 0.05% CMC/C50S50 0.10% CMC/C50S50 0.20% CMC/C50S50
3:7 4:6 5:5 6:4 7:3 5:5 5:5 5:5
58.0 ± 0.7 37.0 ± 0.8 27.0 ± 0.8 14.0 ± 0.7 11.0 ± 0.9 18.0 ± 0.8 15.0 ± 0.7 13.0 ± 0.9
0.94 ± 0.40 2.05 ± 0.50 15.53 ± 1.80 12.02 ± 1.70 4.87 ± 0.60 13.38 ± 1.40 18.84 ± 2.30 16.35 ± 1.70
1:10:10 in this study. 2 mol/L HNO3, absolute ethanol and the required amount of Ca(NO3)2 · 4H2O were added into TEOS solution successively, with 1 h of stirring after each addition. The prepared sol solution was sealed in a teflon beaker and aged at 60 °C for 2 days, following by evaporation of the solvent in an oven at 120 °C for 2 days to obtain dried gel. This gel was then heated in air to 800 °C for 2 h by using a high-temperature furnace and then cooled to room temperature in the furnace to obtain its powder form. 0.05 wt.%, 0.10 wt.%, 0.20 wt.% carboxymethylcellulose (Shandong Everbright Technology Co., Ltd, China) was added into the sintered powder (C50S50) and coded accordingly (i.e. 0.05% CMC/C50S50). The composite powders were then ball-milled for 12 h in ethanol using a centrifugal ball mill (CQ-QM2L, Nanjing Troon New Instrument Co., Ltd, China) and dried in an oven at 60 °C. For preparing cement, the liquid-to-powder (L/P) ratio of 0.5 ml/g was used for the powders and composite powders. The C50S50 cement without CMC was used as the CSCs control, in regard of adding CMC cements. The mixed cement was poured into a cylindrical stainless steel mold (diameter, 6 mm and high, 12 mm) under a pressure of 0.7 MPa for 1 min using a uniaxial press and incubated at 37 °C and 100% relative humidity, and then allowed to set for 1 day. It is noted that the size (Φ6 mm × 3 mm) of sample was used for soaking test.
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for predetermined periods of time at 37 °C to evaluate the in vitro bioactivity. After immersing at each time point (0, 1, 3, 7, 15 days), specimens were removed from the centrifuge tube and gently rinsed three times with deionized water and acetone, and then dried at room temperature for XRD, FT-IR and SEM analyses. 3. Results and discussions 3.1. Phase composition and FT-IR spectra of powders Fig. 1a shows the x-ray diffraction analysis of the five CaO–SiO2 powders sintered at 800 °C for 2 h, indicating that the phase evolution is dictated by the Ca–Si ratios of the precursors. The XRD exhibits a distinct and diffuse diffraction peak at around 2θ = 32°–34°, which can be attributed to the β-dicalcium silicate (β-Ca2SiO4, JCPDS 290371) phase. The result suggests that the more content of CaO in precursors, the stronger of the peak intensities of β-Ca2SiO4 and CaO (2θ = 37.5°). It shows that calcium as a promoter played a very important role in the CaO–SiO2 system, additionally, the amount of CaO in the starting materials may affect St [14]. Fig. 1b indicates that the trends in the FT-IR spectra of the prepared powders were similar to those in XRD. For the sample with lowest content of CaO (C30S70), the IR absorption band was assignable to SiO4 asymmetric stretching extends, ranging over a wide wave number from 1300 to 900 cm− 1. The bands at 1550 and 1380 cm−1 were ascribed to the vibration of CO3 groups carbonized from the atmosphere. The band at around 550–530 cm−1 originated from the vibration of the siloxane backbone [15]. When the CaO content was equal to the SiO2 content (C50S50), a broadening and shifting to lower frequency in Si–O–Si asymmetric stretching bands were detected in the FTIR spectra, which indicated that the specimens' structure became
2.2. Test and characterizations Phase analysis of the powder and the cement was carried out using X-ray diffraction (XRD, Geigerflex, Rigaku, Japan) with a monochromated CuKα radiation tube. The surface morphology images were conducted on scanning electron microscope (SEM; S-4800, JEOL, Tokyo, Japan) under an operating current of 20 mA and a voltage of 40 kV. Fourier transform infrared spectroscopy (FT-IR, NEXUS 670, Nicolet, American) was used to analysis the functional groups of samples. According to international standard ISO 9917–1:2007 for water-based cements, a 400-g Gillmore needle with a 1-mm diameter was utilized to measure the setting time of the cement. The final setting time (St) was recorded as the moment when the needle failed to create an indentation of 1 mm in depth in three different areas of the cement cylinder, which were maintained at 37 °C with 100% relative humidity environment. A total of eight specimens were used for testing the St. Compressive strength (Cs) testing was measured on an electronic universal material testing machine (CMT 6203, Shenzhen Shiji Tianyuan Instrument Co., Ltd., Guangdong, China) at a loading rate of 0.5 mm/min. At least 20 specimens were tested for each group. The pH values of SBF solution before and after soaking with the samples for different time point were obtained from the pH meter (PHSJ-3 F, Shanghai Jie Sheng Scientific Instrument Co., Ltd.). The determined values were calculated using mean ± standard deviation. 2.3. In vitro bioactivity test Each 24 h set cylindrical specimen (Φ6 mm × 3 mm) was soaked in SBF solution according to the procedure described by Kokubo et al. [13],
Fig. 1. XRD (a) and FT-IR (b) patterns of calcium silicate powders sintered at 800 °C.
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less ordered as a result of a decrease in the local symmetry triggered by the incorporation of CaO into the silica network [14]. On the contrary, when the Ca/Si ratio was over 1:1, two new bands near 860 and 970 cm−1 emerged, which were associated with the Si–O symmetric stretching mode with one non-bridging silicon-oxygen bond (Si–O– NBO) [14,16]. The calcium amounts largely affected the phase evolution, as indicated by XRD and FTIR results of the powders.
3.2. Phase composition and FT-IR spectra of cements The XRD and FT-IR of the cured cements in powder form after 1 day of hydration are shown in Fig. 2a and b, respectively. It can be seen that the peak at 2θ = 29.4° corresponds to calcium silicate hydrate (CSH, JCPDS 29-378) of hydration products; additionally, the incompletely reacted β-Ca2SiO4 and CaO were also present in these products. The content of CSH increased with the increase of Ca/Si moral ratio in starting materials. It is speculated that the products were related to the hardening mechanism of calcium silicate cement, which could be explained by the effect of β-Ca2SiO4 content on the form of the CSH [14]. It was determined in a previous work that a high CSH content of hydration products leads to shorter St and faster hydration reaction [17]. The diffraction peak near 2θ = 37.5° corresponding to the phase of CaO, the reduce of CaO could significantly reduce the St after mixing with water [18]. As shown in Fig. 2b, the trends in the FT-IR spectra of the cements were similar to those indicated by XRD. The FT-IR spectra of the powder (Fig. 1b) was consistent with that in cement (Fig. 2b) without the emergence of a new band.
3.3. Setting time and compressive strength The setting times (St) and compressive strength (Cs) of the five CSCs and doped cements are shown in Table 1. It can be seen that the St became shorter with increasing CaO/SiO2 ratio by the same L/P = 0.5 mL/g. The St values of the five CSCs were 58 ± 2, 37 ± 2, 27 ± 2, 14 ± 2 and 11 ± 2 min corresponding to C30S70, C40S60, C50S50, C60S40 and C70S30 after mixing with water, respectively. It can be clearly seen that the St of the cement became shorter with increasing CaO content, demonstrating the accelerating role of Ca in the setting reaction [14]. The higher CaO content in the starting materials resulted in more β-Ca2SiO4 and a higher CSH, leading to shorter St and faster hydration reaction. The peak of CaO phase decreased after mixing with water, which resulted in a shorter St. The powder particle size, sintering temperature, liquid phase and composition, and liquid–solid ratio play a very important role in setting time of cement materials [18]. Table 1 shows that the Cs of the five CSCs tends to increase firstly and then reduce. Table 1 illustrates that the Cs values of the five CSCs were 0.94 ± 0.4, 2.05 ± 0.5, 15.53 ± 1.8, 12.02 ± 1.7 and 4.87 ± 0.6 MPa for C30S70, C40S60, C50S50, C60S40 and C70S30, respectively. The Cs of the sample C30S70 with the lowest CaO content is only 0.94 MPa, while the Cs of the sample C70S30 containing the most abundant of CaO is only 4.87 MPa. The maximum Cs of C50S50 reached 15.53 MPa among the five CSCs, which may be due to the differences existed in cement surface structure, such as the role of porosity of hydration products on the mechanical properties of cement [19]. Based on the above, it can be seen that the CaO is a key factor affecting St and Cs. According to the results of the St and Cs values, the C50S50 was regarded as the best powders for compounding CMC. As shown in Table 1, the setting time of the doped cements with 0.05%, 0.10% and 0.20% CMC were significantly decreased to 13 ± 2, 15 ± 2 and 18 ± 2 min, respectively, comparing to the control C50S50 cement (27 ± 2 min). In addition, the Cs of the doped cements changed with increasing the content of CMC. The St of the doped cement containing 0.10% CMC was significantly reduced, while the Cs reached to 18.84 MPa, as compared to the control C50S50 cement (Cs:15.53 MPa). This may be attributed to water absorbing capability of CMC as viscous agent that promoted faster setting. Setting times of the doped cements are inversely proportional to the amount of added CMC. The Cs of the 0.05% CMC/C50S50 was lower than the control C50S50 cement due to the dispersion of a small amount of CMC dispersed that absorbed the water within the sample, resulting in the increase of porosity in the cements. By adding an appropriate amount of CMC (0.10%), which can be combined with the water during the hydration of cement, a network of polymer film can form in the sample, which eventually increases the Cs of the doped cement by improving the compact degree between the β-Ca2SiO4 particles and hydration products. However, when doping with excessive CMC, which would parcel the surface of the β-Ca2SiO4 particle, a weak boundary layer would form to impede the hydration of the β-Ca2SiO4. The hydrated sample, in such a case, would not be able to form a solid crystalline skeleton, which may hinder the development of the Cs [20,21]. 3.4. In vitro bioactivity 3.4.1. pH value Table 2 shows the pH values of the SBF solution after soaking with the 0.10% CMC/C50S50 cement for various periods (0, 1, 3, 7, 15 days). It can be seen that the pH value of the SBF solution quickly reached Table 2 pH values of SBF solution after soaked with the calcium silicate bone cements containing 0.10% CMC for different time point (0, 1, 3, 7, 15 days).
Fig. 2. XRD (a) and FT-IR (b) patterns of calcium silicate bone cements after 1 day of hydration.
Immersion time/day
0
1
3
7
15
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about 9.6 after immersing in SBF for 1 d. The pH value of the SBF solution tended to decrease after 1 day and became stabilized (pH 7.6–7.7) from 3 days to 15 days. During the soaking process, the H3O+ in solution entered into the surface of the cement, resulting in the increase of the OH− relative concentration of the solution, which enhanced the pH value of SBF solution [18,21].
Fig. 3. XRD patterns of calcium silicate bone cement containing 0.10% CMC before and after immersing in SBF for different time point (0, 1, 3, 7, 15 days).
3.4.2. XRD The XRD pattern of calcium silicate bone cement containing 0.10% CMC before and after immersing in SBF for different time points (0, 1, 3, 7, 15 days) is shown in Fig. 3. The in vitro bioactivity of the 0.10% CMC/C50S50 soaked in SBF solution was tested by observing the hydroxyapatite (Hap) deposited on the surface of the specimens. It shows that the control samples, before soaking in SBF solution, are mostly CSH (JCPDS 29-378) and β-Ca2SiO4 phase. After soaking in SBF for 1, 3, 7 and 15 days, the new visible peaks (2θ = 31.4°–32.6°) attributed to Hydroxyapatite (Hap, JCPDS 24-0033) can be clearly seen. The peaks of CSH became stronger, while the peaks of β-Ca2SiO4 phase gradually disappeared with increasing immersion time. When compared to the control sample, a gradually increased peak intensity
Fig. 4. SEM micrographs of 0.10% CMC/C50S50 cement before and after immersion in SBF for different time point (0, 1, 3, 7, 15 days).
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of the CSH formed in the specimens soaked in SBF can be observed. This phenomenon can be attributed to the adequate reaction of the powders with the water in SBF, which formed more CSH gel and prolonged the soaking time [21,22]. The formation of Hap is due to the reaction of 2+ and OH− during the soaking process [22]. PO3− 4 , Ca
Acknowledgments This work was financially supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References
3.4.3. Morphology Fig. 4 shows the surface morphologies of the cement containing 0.10% CMC before and after soaking in SBF solution. It can be seen that the surface of the 0.10% CMC/C50S50 cement showed significant changes with clusters of smooth precipitated spherulite after immersion, comparing to the control cement (C50S50). By prolonging the soaking time, the Hap spherulites with a greater variety of sizes and content formed apatite layer, which were found on the surfaces of the hybrid cement, especially after immersion in SBF for 7 days. This result revealed their high activity. After soaking 15 days, the spherical apatite with needle surface intertwined together to form apatite layer, indicating that the microspheres gradually grew and changed during immersion process. Previous studies have reported that the examination of apatite formation on the surface of a material in SBF was used to predict the in vivo bioactivity of a biomaterial [23]. In terms of the formation process of Hap on the surface of the dicalcium silicate-based hybrid cement is nature of a process that a new generation grown up and then continue changes [6]. The forming process of Hap major contained the following stages: 1) samples were dissolved and the silicon-rich surface layer was formed, 2) the calcium-phosphate nuclei were formed, followed by the crystal growth and transformation of Hap. The forming process was consistent with 45S5 bioactive glass, which was similar to the formation principle of Hap on the surface of A–W glass-ceramic materials [24]. It is evident from earlier reports that synergistic effect between Si as an effective apatite nucleate and Ca as an apatite precipitation accelerator caused this fast apatite precipitation [6,24].
4. Conclusions A Sol–gel technique was used to prepare calcium silicate powders with different molar ratios of CaO/SiO2. Five cements were obtained by mixing these powders with distilled water. Experimental results indicated that the C50S50 cement exhibited a relatively short setting time and higher compressive strength. Different amounts of CMC were used to modify the C50S50 cement, and the results showed that the incorporation of 0.10 wt.% CMC was most effective at shortening the setting time and enhancing the compressive strength of cements. Moreover, the in vitro bioactivity of 0.10% CMC/C50S50 cement makes it attractive for use in clinical applications of the root canal treatment and vertebroplasty. However, further investigation is necessary to evaluate the effect of CMC on the biocompatibility and the clinical potential of the hybrid CSCs in the further studies.
[1] L.L. Hench, Genetic design of bioactive glass, J. Eur. Ceram. Soc. 29 (7) (2009) 1257–1265. [2] Y. Shirosaki, K. Tsuru, S. Hayakawa, et al., Physical, chemical and in vitro biological profile of chitosan hybrid membrane as a function of organosiloxane concentration, Acta Biomater. 5 (1) (2009) 346–355. [3] S.J. Ding, C.K. Wei, et al., Bio-inspired calcium silicate–gelatin bone grafts for loadbearing applications, J. Mater. Chem. 21 (34) (2011) 12793–12802. [4] C.C. Chen, W.C. Wang, S.J. Ding, In vitro physiochemical properties of a biomimetic gelatin/chitosan oligosaccharide/calcium silicate cement, J. Biomed. Mater. Res. B Appl. Biomater. 95 (2) (2010) 456–465. [5] M.Y. Shie, D.C.H. Chen, C.Y. Wang, et al., Immersion behavior of gelatin-containing calcium phosphate cement, Acta Biomater. 4 (3) (2008) 646–655. [6] C.C. Chen, M.H. Lai, W.C. Wang, et al., Properties of anti-washout-type calcium silicate bone cements containing gelatin, J. Mater. Sci. Mater. Med. 21 (4) (2010) 1057–1068. [7] Q. Lin, X. Lan, Y. Li, et al., Anti-washout carboxymethyl chitosan modified tricalcium silicate bone cement: preparation, mechanical properties and in vitro bioactivity, J. Mater. Sci. Mater. Med. 21 (12) (2010) 3065–3076. [8] T.Y. Chiang, C.C. Ho, D.C.H. Chen, et al., Physicochemical properties and biocompatibility of chitosan oligosaccharide/gelatin/calcium phosphate hybrid cements, Mater. Chem. Phys. 120 (2) (2010) 282–288. [9] E.J. Lee, D.S. Shin, H.E. Kim, et al., Membrane of hybrid chitosan–silica xerogel for guided bone regeneration, Biomaterials 30 (5) (2009) 743–750. [10] J. Camilleri, Evaluation of the physical properties of an endodontic Portland cement incorporating alternative radiopacifiers used as root–end filling material, Int. Endod. J. 43 (3) (2010) 231–240. [11] G. Lewis, Injectable bone cements for use in vertebroplasty and kyphoplasty: Stateof-the-art review, J. Biomed. Mater. Res. B Appl. Biomater. 76 (2) (2006) 456–468. [12] C.C. Chen, C.C. Ho, D.C.H. Chen, et al., Physicochemical properties of calcium silicate cements for endodontic treatment, J. Endod. 35 (9) (2009) 1288–1291. [13] T. Kokubo, Surface chemistry of bioactive glass-ceramics, J. Non-Cryst. Solids 120 (1990) 138–151. [14] J. Serra, P. González, S. Liste, et al., FT-IR and XPS studies of bioactive silica based glasses, J. Non-Cryst. Solids 332 (1) (2003) 20–27. [15] H. Yoshino, K. Kamiya, H. Nasu, IR study on the structural evolution of sol–gel derived SiO2 gels in the early stage of conversion to glasses, J. Non-Cryst. Solids 126 (1) (1990) 68–78. [16] M.M. Pereira, A.E. Clark, L.L. Hench, Homogeneity of bioactive sol–gel-derived glasses in the system CaO–P2O5–SiO2, J. Mater. Synth. Process. 2 (1994) 189–196. [17] F.H. Lin, W.H. Wang, C.P. Lin, Transition element contained partial-stabilized cement (PSC) as a dental retrograde-filling material, Biomaterials 24 (2) (2003) 219–233. [18] M. Bohner, Reactivity of calcium phosphate cements, J. Mater. Chem. 17 (38) (2007) 3980–3986. [19] K. Ishikawa, K. Asaoka, Estimation of ideal mechanical strength and critical porosity of calcium phosphate cement, J. Biomed. Mater. Res. 29 (12) (1995) 1537–1543. [20] Hewen Qian, Chengdong Yin, Changhe Liu, et al., J. Chin. Ceram. Soc. 5 (2) (1996) 69–81 (in Chinese). [21] Fuqin Han, Bo Shao, Qingwen Wang, et al., For. Sci. 45 (7) (2009) 101–105 (in Chinese). [22] M.Y. Shie, H.C. Chang, S.J. Ding, Effects of altering the Si/Ca molar ratio of a calcium silicate cement on in vitro cell attachment, Int. Endod. J. 45 (4) (2012) 337–345. [23] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27 (15) (2006) 2907–2915. [24] S.J. Ding, M.Y. Shie, C.Y. Wang, Novel fast-setting calcium silicate bone cements with high bioactivity and enhanced osteogenesis in vitro, J. Mater. Chem. 19 (8) (2009) 1183–1190.