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Insights into structural characteristics and the mechanism of silicate-based calcium phosphates with phosphoric acid modulation
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Insights into structural characteristics and the mechanism of silicate-based calcium phosphates with phosphoric acid modulation Zhengwen Dinga, Qiyi Zhangb, Yanan Wua, Mizhi Jia, Hong Chena, Yonggang Yana,∗ a b
College of Physics, Sichuan University, Chengdu, 610065, China School of Chemical Engineering, Sichuan University, Chengdu, 610065, China
ARTICLE INFO
ABSTRACT
Keywords: Calcium phosphate Tri-calcium silicate Phase transformation Biomaterials
Si-containing calcium phosphate materials recently have attracted attention owning to their biomineral capability and benefit to the proliferation and differentiation of osteoblast-like cells. In this study, tri-calcium silicate (C3S), which can induce apatite layer deposition on its surface, and phosphoric acid are used to prepare silicate-based calcium phosphates via aqueous precipitation method. It's known that calcium phosphate undergoes phase transformation when the pH value changes. So, the different ratios of C3S and phosphoric acid are designed to study the phase transformation of calcium phosphate. The pH, ions release of solution, composition and structure are detected. The results show the phase transformation of calcium phosphate from hydroxyapatite to brushite with the increased content of phosphoric acid. The structure of calcium silicate hydrate (C–S–H) also undergoes a series of changes, including protonation and polymerization. Briefly, this study focuses primarily on the interaction between C–S–H and calcium phosphates. In addition, it may provide a new sight for the sustained delivery of bisphosphonates achieved by depositing on the surface of C3S for the treatment of osteoporosis.
1. Introduction
which are favorable to the biomaterials [12-16]. Some studies have proved that appropriate Si concentration is beneficial to the proliferation and differentiation of osteoblast-like cells. Furthermore, dietary silicon intake can effectively increase bone mineral density to prevent the occurrence of osteoporosis [17]. Tri-calcium silicate (3CaO·SiO2, C3S), a typical Si-containing material, has a self-setting property providing the basic condition for the application as biocement [12,14]. However, there are still many problems unsolved and its application as bone cement is limited. On the one hand, the exist of induction period in the hydration process leads to the long setting time of C3S. This issue has been studied for hundreds of years due to the predominant role in Portland cement as building materials. Though the controversy exists, there is no doubt about the leading function of calcium silicate hydrate (C–S–H), the main product of hydrated C3S, on the solidification of cement [18-20]. On the other hand, higher content of calcium hydroxide (Ca(OH)2, CH) after hydrating may lead to hyperbasicity which goes against the cell growth. Compounding with other materials should be the strategy to reduce these drawbacks of C3S [12,21]. Bioactivity and biocompatibility are two basic properties of the biomaterials for bone repair, which determine the modes of combination between implants and bone tissue as well as inflammation response after implantation, respectively. Certainly, the ability stimulating proliferation and differentiation of osteoblast-like cells is the destination
Hydroxyapatite (Ca10(PO4)6(OH)2, HA), the most stable calcium phosphate in alkaline environment, possesses the similar component with the inorganic part of natural bone tissue that endows it an inherent advantage in the biomaterials for bone repair [1-3]. Plenty of clinical data have demonstrated that the apatite layer is essential for the chemical bonding between bone tissue and the implanted materials [4,5]. With regard to calcium phosphates, inner genius of their chemical constitution makes them possess the abilities of bioactivity, biocompatibility and osteoconduction which lead to a broad application of them as biomaterials [6]. For instance, many studies have been focused on the calcium phosphate cements which are suitable as injectable materials [7]. Certainly, the diversity of calcium phosphate phases which depend on the pH value provides the possibility for the application as bone cement. The choice of the component of calcium phosphate cement also determines its properties, especially the degradation in vivo, which brushite is stable in acidic environment but a higher degradation rate in vivo while HA is stable in vivo [7]. Multifunctionality, various ions, such as Si [2,8], Zn [1,9], Sr [10] and Mg [11], are doped in calcium phosphate to promote bone repair ability. Si-containing materials recently have drawn more attraction owning to their ability to induce the deposition of the apatite on their surfaces ∗
Corresponding author. E-mail address: [email protected] (Y. Yan).
https://doi.org/10.1016/j.ceramint.2019.10.058 Received 16 September 2019; Received in revised form 4 October 2019; Accepted 7 October 2019 0272-8842/ © 2019 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Please cite this article as: Zhengwen Ding, et al., Ceramics International, https://doi.org/10.1016/j.ceramint.2019.10.058
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for the development of biomaterials. Therefore, this study is focused on the preparation of the controllable silicate-based calcium phosphate using phosphoric acid and C3S, which possesses high levels of calcium available for the formation of calcium phosphates, via aqueous precipitation method through which the calcium phosphate deposits on the surface of hydrated C3S with controllable phase by controlling the amount of phosphoric acid without extra by-product. Incorporation of silicate may make calcium phosphate more functional. Many characterization methods were employed to investigate the formation of calcium phosphate on the surface of hydrated C3S and the influence of the amount of phosphoric acid on the phase transition of calcium phosphate. The preparation of silicate-based calcium phosphate could also reveal the mechanism of the bioactivity of C3S that the chemical bonding forms between calcium silicate and calcium phosphate. Another important part in this study is focused on the influence of the amount of phosphoric acid on the structure of C–S–H which may be useful for the research of the hydration of C3S provide effective method to shorten the setting time of C3S-based cement.
Fig. 1. The XRD patterns of the samples with different content of phosphoric acid compared with standard ICDD PDF cards of portlandite (No. 44-1481), C–S–H (No. 33-0306), HA (No. 09-0432) and brushite (No. 09-0077).
2. Experiment and methods 2.1. Preparation of material
centrifuged at 6000 rpm for 10 min. The supernatants were shifted by pipettor for the test of ICP-AES.
The tricalcium silicate was purchased from Kunshan Chinese Technology New Materials Co. Ltd., Kunshan, China. The samples were prepared as follows. Firstly, 5.000 g C3S was added into 1000 ml beaker, 600 ml deionized water and 10% (v/v) ethyl alcohol were used as dispersing agents and then the suspension was stirred using magnetic stirrers for 1 h at 30 °C. Secondly, 0, 1.00, 2.00 and 3.00 ml phosphoric acid (85 wt%, 1.685 g/ml) were added into the suspension dropwise, which were marked as S0, S1, S2 and S3 respectively, following by stirring with magneton for 2 h. Thirdly, the mixtures were aged for 24 h at 30 °C. Lastly, the precipitates were separated from the suspension with a vacuum filtration technique and dried in oven at 80 °C for 10 h. The final obtained materials were ground and sieved through a 200-mesh sift for test. Table 1 showed the designed ratios of raw materials and the molecules, and atoms ratios of Si, Ca and P were also shown. The O atom was not considered as the interference of crystal water.
2.3. Structural characterizations The composition and morphology of the materials were analyzed and characterized by Fourier transforms infrared (FTIR, Nicolet PerkinElmer Co), X-ray diffraction (XRD, X'Pert Pro-MPD), X-ray photoelectron spectroscopy (XPS, XSAM800, Kratos) and scanning electron microscopy (SEM, JEOL JSM 5600LV, Japan). Corresponding to SEM, energy-dispersive X-ray spectroscopy (EDS) was performed to analyze the ratio of the main elements on the surface. 3. Results Fig. 1 showed the XRD patterns of the samples prepared with different contents of phosphoric acid at 30 °C compared with standard ICDD PDF cards. The XRD patterns showed the distinct changes of crystal phase from S0 to S3. For S0, it was obviously that the peaks were attributed to the CH and C–S–H, which were produced by the hydration of C3S. When 1 ml phosphoric acid was introduced, as shown in S1, the peaks of calcium hydroxide at 18°, 28°, 34° and 51° disappeared while some characteristic peaks related to HA appeared at 26°, 32°, 47° and 49°. In S1, the peak of C–S–H at 29° still existed. However, the diffraction peaks of S2, which composed of 2 ml phosphoric, were basically associated with HA and no peak related to the hydration of C3S was shown in S2. With phosphoric acid up to 3 ml, the peaks of S3 were completely attributed to brushite (dicalcium phosphate dihydrate, DCPD). In short, an increasing amount of phosphoric acid was accompanied by the disappearance of C3S-related peaks and the phase transformation from HA to DCPD. The FT-IR spectra of the samples with different content of phosphoric acid were shown in Fig. 2. For S0, the mainly bands were attributed to the vibration of Si–O group. The peak around 960 cm−1 was attributed to asymmetric and symmetric stretching vibrations of Si–O. The group of bands near 500 cm−1 were caused by internal deformation of SiO4 tetrahedra. For S1 and S2, the characteristic peaks were mainly attributed to HA and Si–O groups. The peaks at 603 and 564 cm−1 were the characteristic bending vibration of PO43− and the peaks around 1038 cm−1 were attributed to the vibration of P–O and Si–O group. Owning to carbonization, the bands in the range of 13001500 cm−1 correspond to the asymmetric stretching of CO32− were common shown in S0, S1 and S2. However, the spectrum of S3 showed
2.2. pH value and ions release Each of samples was about 0.3 g and was incubated in plastic bottles filled with 10 ml phosphate buffer solution (PBS; pH value = 7.40) in the shaking water bath with constant temperature of 37 °C and 80 rpm/ min. The solution was refreshed every two days, and the pH value of each bottle was detected by pH meter (PHS-25, LEICI, Shanghai, China) after soaking for different periods (1, 3, 5, 7 and 14 days). The data were handled with mean ± standard deviation (n = 3). The releases of various ions of the samples in deionized water at 37 °C, including Ca, Si and P, were detected by inductively coupled plasma atomic emission spectroscopy (ICP-AES; IRIS Advantage, USA). In brief, the samples weighted 0.1 g were dissolved in 10 ml deionized water and incubated in the shaking water bath for 24 h with constant temperature of 37 °C and 80 rpm/min. The collected suspensions were Table 1 The chemical components of samples.
S0 S1 S2 S3
Raw materials
Molecular ratio
Atom ratio/%
C3S/g
H3PO4/ml
C3S
H3PO4
Si
Ca
P
5 5 5 5
0 1 2 3
3 3 3 3
0 2 4 6
25 21.4 18.8 16.7
75 64.3 56.2 50
0 14.3 25 33.3
2
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Table 2 The release of Ca, Si and P in deionized water at 37 °C and the relative atomic number compared with Ca atoms. Ca
S0 S1 S2 S3
Si
P
ppm
Relative atomic number
ppm
Relative atomic number
100.00 11.70 3.96 23.20
1 1 1 1
7.79 5.98 1.58 6.57
0.11 0.73 0.57 0.40
ppm
Relative atomic number
0.08 0.34 22.00
0.01 0.11 1.22
about S3. Specifically, the content of Ca, Si and P were 5.9, 4.2 and 64.7 times more than that of S2, respectively. Table 2 also showed the relative atomic number compared with Ca atoms. Based on the law of conservation of charge, most Ca ions of S0 were balanced by OH− groups and only a few was balanced by Si-containing groups. With the increased content of phosphoric acid, the Ca ions were gradually balanced by P-containing groups. There was an interesting phenomenon about the relative atomic number of Si that the dissolved calcium ions balanced by Si-containing group of S1 increased sharply compared with S0. Certainly, the relative atomic number of Si decreased with the increased amount of phosphoric acid, which was displayed by S2 and S3. Fig. 4 showed the SEM photographs of samples with different contents of phosphoric acid. From the image of S0, the needle-like crystal deposited on the surface of C–S–H which was attributed to the calcium hydroxide growing during the maturing process. S1 showed the deposition of unregular spherulite which was contributed to the adding of phosphoric acid resulting the formation of HA. With more phosphoric acid up to 2 ml, the plenty of petaloid HA formed on the surface of hydrated C3S. Moreover, there were large lamellae and small plate-like crystal which may be attributed to brushite as shown in S3. Fig. 5 and Table 3 showed the EDS data, especially in Table 3 the exact data were shown that the relative atomic number of Si and P on the surface of samples compared with Ca atoms which was set as 1. It was obviously that the relative content of Si gradually decreased accompanying the increase of P because of the adding of phosphoric acid. Reasonably, the ratio of Ca/P would decrease with the increased content of phosphate and the Ca/P of S2 was 1.73 close to 1.67 of HA. The comparation between the experimental and the theoretical content of Si on the surface among three elements, including Si, Ca and P without O to exclude the interference of crystal water, was shown in Fig. 6. With the adding of phosphoric acid, the contents of Si were lower than the theoretical value. The shielding rates of S2 and S3, which was used to explain the reduction of Si compared with the theoretical value, were higher than 30%. It meant that plenty of calcium phosphate deposited on the surface of hydrated C3S. Fig. 7 showed the XPS spectra of the samples in regard to Ca 2p, Si 2p, O 1s and P 2p with different content of phosphoric acid, and the precise data were displayed in Table 4. The binding energy of Si 2p showed an obvious trend that it moved toward higher site. Especially in the S3 (3 ml phosphoric acid), the binding energy was about 1.2 eV more than that of S2. The binding energy of Ca 2p1/2 increased insignificantly while only slight fluctuations could be seen in Ca 2p3/2. The binding energy of O 1s shifted toward lower sites with the increased amount of phosphate. As for the binding energy of P 2p, there was almost no difference between S1 and S2. With regard to S3, the binding energy of P 2p increased 0.2 eV than that of S1 and S2. In short, the amount of phosphoric acid had a significant and regular influence on the structure of C–S–H.
Fig. 2. FT-IR spectra of the samples with different content of phosphoric acid.
some differences compared with S1 and S2. Due to the acidity of S3, there was no band in the range of 1300-1500 cm−1. The slight shift could be recognized that P–O group at 1056 cm−1 moved toward higher wavenumber compared with S1 and S2. In brief, the obvious P–O group related peaks were shown in FTIR spectra with introduced phosphoric acid and there were some differences among them with different amounts of phosphoric acid. Fig. 3 showed the pH changed with the immersing time in PBS at 37 °C. S0 showed a consistent pH value which was higher than 12 owning to the abundant CH produced by the hydration of C3S. The pH value decreased obviously with the increasing content of phosphoric acid. The pH value of S1 after 14 days immersion was near 10 while the pH value of S2 was 7.6. When the amount of phosphoric acid was up to 3 ml, the pH value of S3 was lower than 7 even immersed for 14 days. Briefly, the amount of phosphoric acid played a distinct influence on the pH value which changed from high alkalinity to weak acidity. Table 2 showed the dissolution of the samples about Ca, Si and P ions in deionized water at 37 °C. S0 performed the highest concentration of Ca and Si compared with the samples with phosphoric acid added. The concentrations of Ca and Si decreased when the amount of phosphoric acid increased up to 2 ml, especially the concentrations of Ca and Si of S2 were pretty low. But, the concentrations of these ions increased rapidly as the amount of phosphoric acid was up to 3 ml
4. Discussion Owning to the dominant function of C3S in Portland cement, which has been widely used for building material, there are plenty of studies
Fig. 3. The changes of pH value vs immersion time in PBS. 3
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Fig. 4. The SEM photographs of S0, S1, S2 and S3 with 10000 magnification.
Fig. 5. The EDS of the main atoms on the surfaces of the samples.
4
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and osteoconduction [3]. It is possible to prepare HA by phosphoric acid and CH with suitable nucleate sites in an alkaline environment [31]. When the phosphoric acid was added drop and drop into the hydrated C3S solution, it can react preferentially with CH owning to the solubility: CH > Ca–OH > Si–O–Ca [18,30]. During the process that 1 ml phosphoric acid is added, the amount of phosphoric acid is insufficient compared with CH, which makes the solution maintain higher pH value than 9 and be suitable for the formation of HA. Therefore, the reaction equation between hydrated C3S and 1 ml phosphoric acid with the hypothesis product of HA, is shown as Eq. (2), and proved by the FTIR, XRD and SEM of S1. Based on the value of x in C–S–H which is around 1.7-1.8, the value of (17–9x) is higher than 0 that means there is little CH existing, which may be a good explanation for higher pH value of S1 in PBS, even no obvious CH peaks are shown in the XRD pattern of S1. With these data, the reaction diagram between C3S and 1 ml phosphate could be described as Fig. 8(b). The Si–O–Ca groups in C–S–H provide nucleate sites for the deposition of phosphate groups via electrostatic interaction and hydrogen bond interaction that can explain the nature of the bioactivity of C3S [13,14]. Because of the insufficient of phosphoric acid, the shielding rate of calcium phosphate on Si is lower which is about 10.9%. As a result, the obvious C–S–H diffraction peaks is shown in S1. There is only minor influence on the Si–O–Ca group in C–S–H, and the binding energy of Si 2p shows just a little shift as shown in Fig. 7. But the effect of 1 ml phosphoric acid on the hydrated C3S is distinctly in some ways. For instance, the pH value in PBS drops to 9.8 from 11.8 of S0. The formation of HA and its shielding function play an inhibition effect on the dissolution of various ions, especially the Ca ions as shown in Table 2.
Table 3 The relative atomic number of Si, P on the surface compared with Ca atom.
S0 S1 S2 S3
Ca
Si
P
Ca/P
1 1 1 1
0.464 0.285 0.235 0.211
0.241 0.578 0.738
4.15 1.73 1.36
Fig. 6. The percentage of Si atoms in the three elements including Si, Ca and P and the shielding rate of Ca and P atoms on Si atom.
9 3CaO SiO2 + 6 H3PO4 + H2 O
xCaO SiO2 yH2 O+ (3
x)Ca(OH)2
9x)Ca(OH)2 + Ca10 (PO4 )6 (OH)2
(2)
focusing on the hydration of C3S though the exact mechanism of hydration is still in doubt [18,19,22,23]. In recent years, increasing attention has been attracted to the application of C3S as biomaterials due to its self-setting property, bioactivity and biological property that the ability to stimulate the proliferation and differentiation of osteoblastlike cells [14,24,25]. It is well-known that the C–S–H and CH are produced when C3S contacts with water [22], which are also proved by XRD and FT-IR in Figs. 1 and 2. The C–S–H, which plays a significant role in the solidification and strength of hardened cement, has been proved that it contains a linear silicate chains of dreierkette form consisting of two paired tetrahedra and a bridging tetrahedron [26]. Previous studies also demonstrate that C–S–H consists of both dimeric and higher polymeric species, such as pentamer and octamer with a 2, 5, 8 … (3n-1) chain length sequence, where n is integer [26-28]. In this study, the linear pentamer is used as the model in the schematic diagram to describe the hydration of C3S which is shown in Fig. 8(a) and the reaction equation can be written as Eq. (1), in which the value of x is variable represented the calcium content and the ratio of Ca/Si in C–S–H with a wide range from 0.5 to 2. Precisely, the value of x is around 1.7-1.8 in C–S–H produced by hydrated C3S [18]. According to previous research, Si–OH and Ca–OH groups have been proved that they do not coexist which means all protons in Si–OH will be replaced by Ca ions to result Si–O–Ca groups [29,30]. In addition, Ca ions in C–S–H are in excess of that required to balance silicate and the additional Ca will be balanced by OH− to form Ca–OH groups. It is possible that the CH precipitates above the Si–O–Ca–OH groups which is displayed in Fig. 8(a). Therefore, it is easy to explain the higher pH value of hydrated C3S: the sustained release of CH on the surface leads to the enrichment of OH− to balance the Ca2+ ions, which is also demonstrated in Table 2 that only small amount of Ca2+ ions are balanced with Si-containing groups.
3CaO SiO2 + H2 O
9[xCaO SiO2 yH2 O] + (17
During the process that 2 ml phosphoric acid is gradually added, the pH value of solution maintains the weakly alkaline environment which is consistently higher than 7.2 and is suitable for the formation of HA. There is no uncertain that CH is consumed completely. Previous studies about the C–S–H structure of hydrated C3S show that around 25% of Ca ions are balanced by OH− and other Ca ions are stored in Si–O–Ca groups [30]. With the calculation, the Ca–OH groups would be replaced thoroughly with 2 ml phosphoric acid. At the meantime, the protonation of Si–O–Ca groups begins to form the Si–OH groups, and finally Ca/ Si value in C–S–H is around 1. Therefore, the schematic diagram could be shown as Fig. 8(c) and the reaction equation with 2 ml phosphoric acid can be shown as Eq. (3) in which the value of x in C–S–H is close to 1. Briefly, the stable silicate-based HA is produced with 2 ml phosphoric acid and the interlayer Ca atoms between C–S–H and HA are shared by them. The XRD and FTIR data about the S2 show the obvious HA-related peaks. The surface morphology shown in Fig. 4 and the EDS data that the Ca/P ratio of S2 is 1.73 also prove the formation of HA. With further analysis of EDS data shown in Fig. 6, the content of Si on the surface of S2 is significantly lower than the theoretical value which is caused by the shielding effect of HA formed on the surface. Moreover, the lowest solubility of Si and Ca ions of S2 shown in Table 2 may be the indirect evidence of the formation of the stable silicate-based HA. Certainly, the influence of 2 ml phosphoric acid on C–S–H becomes relatively obvious which is shown by XPS data that the binding energy of Si 2p shifts toward higher site about 0.298 eV compared with S0, which is attributed to the decalcification of Si–O–Ca resulting in the formation of Si–OH. 9 3CaO SiO2 + 12 H3 PO4 + H2 O
9[xCaO SiO2 yH2 O] + 2Ca10 (PO4)6 (OH)2
(3) With excess level of phosphoric acid up to 3 ml, the final pH value of solution is around 5 in the preparation process, an acidic environment in which dicalcium phosphate is more stable than other calcium phosphates [32]. Certainly, small amounts of other calcium phosphate phases may coexist, such as monocalcium phosphate. The finally
(1)
HA, the most stable phase of calcium phosphate in alkaline environment that the other phases of calcium phosphate will transform toward it eventually, possesses excellent bioactivity, biocompatibility 5
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Fig. 7. The XPS spectra of the samples with the main atoms of Ca 2p, Si 2p, O 1s and P 2p.
of silicate polymerization of C–S–H [33-35]. The possible value of x in Eq. (4) is below 1 which means the ratio of Ca/Si in C–S–H is lower than 1. As a result, the C–S–H with higher degree polymerization covered with brushite is produced in which the ratio of Ca/Si is below 1. In addition, the binding energy of P 2p is 0.2 eV more than S1 and S2 caused by the higher electronegativity of –OH group in brushite.
Table 4 The binding energy of the Si 2p, Ca 2p, O 1s and P 2p. Binding Energy/eV
S0 S1 S2 S3
Si 2p
O 1s
Ca 2p1/2
Ca 2p3/2
P 2p
101.901 102.000 102.199 103.450
531.351 531.100 530.949 530.750
350.351 350.450 350.449 350.550
346.901 346.900 346.999 346.950
132.800 132.799 133.000
3CaO SiO2 + 2H3PO4 + H2 O
xCaO SiO2 yH2 O+ zCaHPO4 ·2H2 O+ else
(4)
Essentially, the bioactivity of C3S is attributed to the interlayer Ca which provides suitable nucleate sites for the deposition of phosphate group via electrostatic interaction and hydrogen bond interaction. Briefly, the preparation of silicate-based calcium phosphates depends on the nucleation and growth of calcium phosphate on the surface of C–S–H and the stability of final product is associated with the proportion between C3S and phosphoric acid. The bioglass with higher content of SiO2 unable to induce the precipitation of apatite layer is perhaps attributed to the short of valuable nucleate sites [36]. With the adding of phosphoric acid, the pH value of C3S is decreased which could reduce the inflammatory response increasing the biocompatibility. The solidification of silicate-based cement is closely related to the polymerization of C–S–H [34,37]. The influence of phosphoric acid on the C–S–H structure may provide a light on shortening the setting time of C3S cement and in our previous study, C3S based cement, which used phosphoric acid as the curing agent, have a significantly reduced setting time [25]. It is versatile that the combination of phosphoric acid and C3S not only shortens the setting time of C3S but also increases the biocompatibility without unwished by-products. A much deeper consideration, bisphosphonates, which are widely used for the treatment of
product of calcium phosphate is primarily brushite which is proved by the XRD data in Fig. 1. The acidity of S3 also suppresses the carbonization so that no CO32− related vibrational absorption peak appears in Fig. 2. With the product of brushite, the reaction equation is shown in Eq. (4), where the maximal content of calcium phosphate is brushite that the value of z is close to 2 and the ‘else’ represents the other calcium phosphate and Fig. 8(d) can be used to describe the deposition of brushite. With excess phosphoric acid, the properties of S3 change a lot by comparation with S1 and S2. Owning to the higher solubility of brushite, the dissolution of Si, Ca and P increases sharply compared with S2 and the shielding of brushite on C–S–H does not prevent the release of Si. With regard to the C–S–H structure, a distinct change can be seen from the XPS data that the binding energy of Si 2p shifts obviously which is up to 1.55 eV compared with S0. During the adding process, the protonation of Si–O–Ca leads to the accumulation of Si–OH groups which provides basic condition for the polymerization. Previous studies have proved that the shift is caused by the increase in the degree 6
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Fig. 8. The schematic diagram of the reaction between C3S and phosphoric acid.
osteoporosis, show a powerful binding force to Ca [38,39]. Similar to the deposition of calcium phosphate, it is possible to achieve the controllable release of bisphosphonates by the carrier of C3S.
[2] W.C. Liu, H.Y. Wang, L.C. Chen, S.W. Huang, C.T. Wu, R.J. Chun, Hydroxyapatite/ tricalcium silicate composites cement derived from novel two-step sol-gel process with good biocompatibility and applications as bone cement and potential coating materials, Ceram. Int. 45 (5) (2019) 5668–5679. [3] J. Venkatesan, S.K. Kim, Nano-hydroxyapatite composite biomaterials for bone tissue engineering–a review, J. Biomed. Nanotechnol. 10 (10) (2014) 3124–3140. [4] H.T. Aiyelabegan, E. Sadroddiny, Fundamentals of protein and cell interactions in biomaterials, Biomed. Pharmacother. 88 (2017) 956–970. [5] C.J. Wilson, R.E. Clegg, D.I. Leavesley, M.J. Pearcy, Mediation of biomaterial-cell interactions by adsorbed proteins: a review, Tissue Eng. 11 (1-2) (2005) 1–18. [6] H.P. Yuan, H. Fernandes, P. Habibovic, J. de Boer, A.M.C. Barradas, A. de Ruiter, W.R. Walsh, C.A. van Blitterswijk, J.D. de Bruijn, Osteoinductive ceramics as a synthetic alternative to autologous bone grafting, P Natl Acad Sci USA 107 (31) (2010) 13614–13619. [7] S. Vahabzadeh, M. Roy, S. Bose, Effects of silicon on osteoclast cell mediated degradation, in vivo osteogenesis and vasculogenesis of brushite cement, J. Mater. Chem. B 3 (46) (2015) 8973–8982. [8] A. Khandan, M. Abdellahi, R.V. Barenji, N. Ozada, E. Karamian, Introducing natural hydroxyapatite-diopside (NHA-Di) nano-bioceramic coating, Ceram. Int. 41 (9) (2015) 12355–12363. [9] A. Grandjean-Laquerrier, P. Laquerriere, E. Jallot, J.M. Nedelec, M. Guenounou, D. Laurent-Maquin, T.M. Phillips, Influence of the zinc concentration of sol-gel derived zinc substituted hydroxyapatite on cytokine production by human monocytes in vitro, Biomaterials 27 (17) (2006) 3195–3200. [10] Y. Huang, M. Hao, X.F. Nian, H.X. Qiao, X.J. Zhang, X.Y. Zhang, G.Q. Song, J.C. Guo, X.F. Pang, H.L. Zhang, Strontium and copper co-substituted hydroxyapatite-based coatings with improved antibacterial activity and cytocompatibility fabricated by electrodeposition, Ceram. Int. 42 (10) (2016) 11876–11888. [11] U. Klammert, T. Reuther, M. Blank, I. Reske, J.E. Barralet, L.M. Grover, A.C. Kubler, U. Gbureck, Phase composition, mechanical performance and in vitro biocompatibility of hydraulic setting calcium magnesium phosphate cement, Acta Biomater. 6 (4) (2010) 1529–1535. [12] W.J. Liu, D. Zhai, Z.G. Huan, C.T. Wu, J. Chang, Novel tricalcium silicate/magnesium phosphate composite bone cement having high compressive strength, in vitro bioactivity and cytocompatibility, Acta Biomater. 21 (2015) 217–227. [13] L.N. Niu, K. Jiao, T.D. Wang, W. Zhang, J. Camilleri, B.E. Bergeron, H.L. Feng, J. Mao, J.H. Chen, D.H. Pashley, F.R. Tay, A review of the bioactivity of hydraulic calcium silicate cements, J. Dent. 42 (5) (2014) 517–533. [14] W.Y. Zhao, J.Y. Wang, W.Y. Zhai, Z. Wang, J. Chang, The self-setting properties and in vitro bioactivity of tricalcium silicate, Biomaterials 26 (31) (2005) 6113–6121. [15] S. Sahmani, A. Khandan, S. Saber-Samandari, M.M. Aghdam, Nonlinear bending and instability analysis of bioceramics composed with magnetite nanoparticles: fabrication, characterization, and simulation, Ceram. Int. 44 (8) (2018) 9540–9549. [16] A. Kordjamshidi, S. Saber-Samandari, M. Ghadiri Nejad, A. Khandan, Preparation of novel porous calcium silicate scaffold loaded by celecoxib drug using freeze drying technique: fabrication, characterization and simulation, Ceram. Int. 45 (11) (2019) 14126–14135. [17] R. Jugdaohsingh, K.L. Tucker, N. Qiao, L.A. Cupples, D.P. Kiel, J.J. Powell, Dietary
5. Conclusion In this study silicate-based calcium phosphates were prepared which phase can be controlled by the proportion between C3S and phosphoric acid via aqueous precipitation method. The phases of calcium phosphate deposited on the surface of hydrated C3S were changing with the increase of phosphoric acid that transformed from HA to brushite. Through a series of methods, the interaction between C–S–H and phosphate was studied that the interlayer Ca was contributed to connection of two phases. The structure of C–S–H underwent a series of changes with increased phosphoric acid, including protonation and polymerization, especially for S3 with 3 ml phosphoric acid. In short, the silicate-based calcium phosphates may be suitable for biomaterial with different modes, such as coating and inorganic active filling matrix. In addition, it provided a new light for the sustained release of bisphosphonates deposited on the surface of hydrated C3S. Declaration of competing interest I would like to declare on behalf of my co-authors that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The research leading to these results has received funding from the National Natural Science Foundation of China (51773123). References [1] D.K. Khajuria, C. Disha, R. Vasireddi, R. Razdan, D.R. Mahapatra, Risedronate/zinchydroxyapatite based nanomedicine for osteoporosis, Mater Sci Eng C Mater Biol Appl 63 (2016) 78–87.
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measured by H-1 NMR relaxometry, J. Phys. Chem. C 117 (1) (2013) 403–412. [30] J.J. Thomas, J.J. Chen, H.M. Jennings, D.A. Neumann, Ca-OH bonding in the C-S-H gel phase of tricalcium silicate and white portland cement pastes measured by inelastic neutron scattering, Chem. Mater. 15 (20) (2003) 3813–3817. [31] A. Osaka, Y. Miura, K. Takeuchi, M. Asada, K. Takahashi, Calcium apatite prepared from calcium hydroxide and orthophosphoric acid, J. Mater. Sci. Mater. Med. 2 (1) (1991) 51–55. [32] J. Zhang, W. Liu, V. Schnitzler, F. Tancret, J.M. Bouler, Calcium phosphate cements for bone substitution: chemistry, handling and mechanical properties, Acta Biomater. 10 (3) (2014) 1035–1049. [33] L. Black, K. Garbev, G. Beuchle, P. Stemmermann, D. Schild, X-ray photoelectron spectroscopic investigation of nanocrystalline calcium silicate hydrates synthesised by reactive milling, Cement Concr. Res. 36 (6) (2006) 1023–1031. [34] K. Kurumisawa, T. Nawa, H. Owada, M. Shibata, Deteriorated hardened cement paste structure analyzed by XPS and Si-29 NMR techniques, Cement Concr. Res. 52 (2013) 190–195. [35] K. Okada, Y. Kameshima, A. Yasumori, Chemical shifts of silicon X-ray photoelectron spectra by polymerization structures of silicates, J. Am. Ceram. Soc. 81 (7) (1998) 1970–1972. [36] R. Li, A.E. Clark, L.L. Hench, An investigation of bioactive glass powders by sol-gel processing, J. Appl. Biomater. 2 (4) (1991) 231–239. [37] S. Papatzani, K. Paine, J. Calabria-Holley, A comprehensive review of the models on the nanostructure of calcium silicate hydrates, Constr. Build. Mater. 74 (2015) 219–234. [38] T.X. Gong, Z.Q. Wang, Y.B. Zhang, C.S. Sun, Q.Z. Yang, T. Troczynski, U.O. Hafeli, Preparation, characterization, release kinetics, and in vitro cytotoxicity of calcium silicate cement as a risedronate delivery system, J. Biomed. Mater. Res. A 102 (7) (2014) 2295–2304. [39] D.L. Kendler, F. Marin, C.A.F. Zerbini, L.A. Russo, S.L. Greenspan, V. Zikan, A. Bagur, J. Malouf-Sierra, P. Lakatos, A. Fahrleitner-Pammer, E. Lespessailles, S. Minisola, J.J. Body, P. Geusens, R. Moricke, P. Lopez-Romero, Effects of teriparatide and risedronate on new fractures in post-menopausal women with severe osteoporosis (VERO): a multicentre, double-blind, double-dummy, randomised controlled trial, Lancet 391 (10117) (2018) 230–240.
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Insights into structural characteristics and the mechanism of silicate-based calcium phosphates with phosphoric acid modulation Zhengwen Dinga, Qiyi Zhangb, Yanan Wua, Mizhi Jia, Hong Chena, Yonggang Yana,∗ a b
College of Physics, Sichuan University, Chengdu, 610065, China School of Chemical Engineering, Sichuan University, Chengdu, 610065, China
ARTICLE INFO
ABSTRACT
Keywords: Calcium phosphate Tri-calcium silicate Phase transformation Biomaterials
Si-containing calcium phosphate materials recently have attracted attention owning to their biomineral capability and benefit to the proliferation and differentiation of osteoblast-like cells. In this study, tri-calcium silicate (C3S), which can induce apatite layer deposition on its surface, and phosphoric acid are used to prepare silicate-based calcium phosphates via aqueous precipitation method. It's known that calcium phosphate undergoes phase transformation when the pH value changes. So, the different ratios of C3S and phosphoric acid are designed to study the phase transformation of calcium phosphate. The pH, ions release of solution, composition and structure are detected. The results show the phase transformation of calcium phosphate from hydroxyapatite to brushite with the increased content of phosphoric acid. The structure of calcium silicate hydrate (C–S–H) also undergoes a series of changes, including protonation and polymerization. Briefly, this study focuses primarily on the interaction between C–S–H and calcium phosphates. In addition, it may provide a new sight for the sustained delivery of bisphosphonates achieved by depositing on the surface of C3S for the treatment of osteoporosis.
1. Introduction
which are favorable to the biomaterials [12-16]. Some studies have proved that appropriate Si concentration is beneficial to the proliferation and differentiation of osteoblast-like cells. Furthermore, dietary silicon intake can effectively increase bone mineral density to prevent the occurrence of osteoporosis [17]. Tri-calcium silicate (3CaO·SiO2, C3S), a typical Si-containing material, has a self-setting property providing the basic condition for the application as biocement [12,14]. However, there are still many problems unsolved and its application as bone cement is limited. On the one hand, the exist of induction period in the hydration process leads to the long setting time of C3S. This issue has been studied for hundreds of years due to the predominant role in Portland cement as building materials. Though the controversy exists, there is no doubt about the leading function of calcium silicate hydrate (C–S–H), the main product of hydrated C3S, on the solidification of cement [18-20]. On the other hand, higher content of calcium hydroxide (Ca(OH)2, CH) after hydrating may lead to hyperbasicity which goes against the cell growth. Compounding with other materials should be the strategy to reduce these drawbacks of C3S [12,21]. Bioactivity and biocompatibility are two basic properties of the biomaterials for bone repair, which determine the modes of combination between implants and bone tissue as well as inflammation response after implantation, respectively. Certainly, the ability stimulating proliferation and differentiation of osteoblast-like cells is the destination
Hydroxyapatite (Ca10(PO4)6(OH)2, HA), the most stable calcium phosphate in alkaline environment, possesses the similar component with the inorganic part of natural bone tissue that endows it an inherent advantage in the biomaterials for bone repair [1-3]. Plenty of clinical data have demonstrated that the apatite layer is essential for the chemical bonding between bone tissue and the implanted materials [4,5]. With regard to calcium phosphates, inner genius of their chemical constitution makes them possess the abilities of bioactivity, biocompatibility and osteoconduction which lead to a broad application of them as biomaterials [6]. For instance, many studies have been focused on the calcium phosphate cements which are suitable as injectable materials [7]. Certainly, the diversity of calcium phosphate phases which depend on the pH value provides the possibility for the application as bone cement. The choice of the component of calcium phosphate cement also determines its properties, especially the degradation in vivo, which brushite is stable in acidic environment but a higher degradation rate in vivo while HA is stable in vivo [7]. Multifunctionality, various ions, such as Si [2,8], Zn [1,9], Sr [10] and Mg [11], are doped in calcium phosphate to promote bone repair ability. Si-containing materials recently have drawn more attraction owning to their ability to induce the deposition of the apatite on their surfaces ∗
Corresponding author. E-mail address: [email protected] (Y. Yan).
https://doi.org/10.1016/j.ceramint.2019.10.058 Received 16 September 2019; Received in revised form 4 October 2019; Accepted 7 October 2019 0272-8842/ © 2019 Elsevier Ltd and Techna Group S.r.l. All rights reserved.
Please cite this article as: Zhengwen Ding, et al., Ceramics International, https://doi.org/10.1016/j.ceramint.2019.10.058
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for the development of biomaterials. Therefore, this study is focused on the preparation of the controllable silicate-based calcium phosphate using phosphoric acid and C3S, which possesses high levels of calcium available for the formation of calcium phosphates, via aqueous precipitation method through which the calcium phosphate deposits on the surface of hydrated C3S with controllable phase by controlling the amount of phosphoric acid without extra by-product. Incorporation of silicate may make calcium phosphate more functional. Many characterization methods were employed to investigate the formation of calcium phosphate on the surface of hydrated C3S and the influence of the amount of phosphoric acid on the phase transition of calcium phosphate. The preparation of silicate-based calcium phosphate could also reveal the mechanism of the bioactivity of C3S that the chemical bonding forms between calcium silicate and calcium phosphate. Another important part in this study is focused on the influence of the amount of phosphoric acid on the structure of C–S–H which may be useful for the research of the hydration of C3S provide effective method to shorten the setting time of C3S-based cement.
Fig. 1. The XRD patterns of the samples with different content of phosphoric acid compared with standard ICDD PDF cards of portlandite (No. 44-1481), C–S–H (No. 33-0306), HA (No. 09-0432) and brushite (No. 09-0077).
2. Experiment and methods 2.1. Preparation of material
centrifuged at 6000 rpm for 10 min. The supernatants were shifted by pipettor for the test of ICP-AES.
The tricalcium silicate was purchased from Kunshan Chinese Technology New Materials Co. Ltd., Kunshan, China. The samples were prepared as follows. Firstly, 5.000 g C3S was added into 1000 ml beaker, 600 ml deionized water and 10% (v/v) ethyl alcohol were used as dispersing agents and then the suspension was stirred using magnetic stirrers for 1 h at 30 °C. Secondly, 0, 1.00, 2.00 and 3.00 ml phosphoric acid (85 wt%, 1.685 g/ml) were added into the suspension dropwise, which were marked as S0, S1, S2 and S3 respectively, following by stirring with magneton for 2 h. Thirdly, the mixtures were aged for 24 h at 30 °C. Lastly, the precipitates were separated from the suspension with a vacuum filtration technique and dried in oven at 80 °C for 10 h. The final obtained materials were ground and sieved through a 200-mesh sift for test. Table 1 showed the designed ratios of raw materials and the molecules, and atoms ratios of Si, Ca and P were also shown. The O atom was not considered as the interference of crystal water.
2.3. Structural characterizations The composition and morphology of the materials were analyzed and characterized by Fourier transforms infrared (FTIR, Nicolet PerkinElmer Co), X-ray diffraction (XRD, X'Pert Pro-MPD), X-ray photoelectron spectroscopy (XPS, XSAM800, Kratos) and scanning electron microscopy (SEM, JEOL JSM 5600LV, Japan). Corresponding to SEM, energy-dispersive X-ray spectroscopy (EDS) was performed to analyze the ratio of the main elements on the surface. 3. Results Fig. 1 showed the XRD patterns of the samples prepared with different contents of phosphoric acid at 30 °C compared with standard ICDD PDF cards. The XRD patterns showed the distinct changes of crystal phase from S0 to S3. For S0, it was obviously that the peaks were attributed to the CH and C–S–H, which were produced by the hydration of C3S. When 1 ml phosphoric acid was introduced, as shown in S1, the peaks of calcium hydroxide at 18°, 28°, 34° and 51° disappeared while some characteristic peaks related to HA appeared at 26°, 32°, 47° and 49°. In S1, the peak of C–S–H at 29° still existed. However, the diffraction peaks of S2, which composed of 2 ml phosphoric, were basically associated with HA and no peak related to the hydration of C3S was shown in S2. With phosphoric acid up to 3 ml, the peaks of S3 were completely attributed to brushite (dicalcium phosphate dihydrate, DCPD). In short, an increasing amount of phosphoric acid was accompanied by the disappearance of C3S-related peaks and the phase transformation from HA to DCPD. The FT-IR spectra of the samples with different content of phosphoric acid were shown in Fig. 2. For S0, the mainly bands were attributed to the vibration of Si–O group. The peak around 960 cm−1 was attributed to asymmetric and symmetric stretching vibrations of Si–O. The group of bands near 500 cm−1 were caused by internal deformation of SiO4 tetrahedra. For S1 and S2, the characteristic peaks were mainly attributed to HA and Si–O groups. The peaks at 603 and 564 cm−1 were the characteristic bending vibration of PO43− and the peaks around 1038 cm−1 were attributed to the vibration of P–O and Si–O group. Owning to carbonization, the bands in the range of 13001500 cm−1 correspond to the asymmetric stretching of CO32− were common shown in S0, S1 and S2. However, the spectrum of S3 showed
2.2. pH value and ions release Each of samples was about 0.3 g and was incubated in plastic bottles filled with 10 ml phosphate buffer solution (PBS; pH value = 7.40) in the shaking water bath with constant temperature of 37 °C and 80 rpm/ min. The solution was refreshed every two days, and the pH value of each bottle was detected by pH meter (PHS-25, LEICI, Shanghai, China) after soaking for different periods (1, 3, 5, 7 and 14 days). The data were handled with mean ± standard deviation (n = 3). The releases of various ions of the samples in deionized water at 37 °C, including Ca, Si and P, were detected by inductively coupled plasma atomic emission spectroscopy (ICP-AES; IRIS Advantage, USA). In brief, the samples weighted 0.1 g were dissolved in 10 ml deionized water and incubated in the shaking water bath for 24 h with constant temperature of 37 °C and 80 rpm/min. The collected suspensions were Table 1 The chemical components of samples.
S0 S1 S2 S3
Raw materials
Molecular ratio
Atom ratio/%
C3S/g
H3PO4/ml
C3S
H3PO4
Si
Ca
P
5 5 5 5
0 1 2 3
3 3 3 3
0 2 4 6
25 21.4 18.8 16.7
75 64.3 56.2 50
0 14.3 25 33.3
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Table 2 The release of Ca, Si and P in deionized water at 37 °C and the relative atomic number compared with Ca atoms. Ca
S0 S1 S2 S3
Si
P
ppm
Relative atomic number
ppm
Relative atomic number
100.00 11.70 3.96 23.20
1 1 1 1
7.79 5.98 1.58 6.57
0.11 0.73 0.57 0.40
ppm
Relative atomic number
0.08 0.34 22.00
0.01 0.11 1.22
about S3. Specifically, the content of Ca, Si and P were 5.9, 4.2 and 64.7 times more than that of S2, respectively. Table 2 also showed the relative atomic number compared with Ca atoms. Based on the law of conservation of charge, most Ca ions of S0 were balanced by OH− groups and only a few was balanced by Si-containing groups. With the increased content of phosphoric acid, the Ca ions were gradually balanced by P-containing groups. There was an interesting phenomenon about the relative atomic number of Si that the dissolved calcium ions balanced by Si-containing group of S1 increased sharply compared with S0. Certainly, the relative atomic number of Si decreased with the increased amount of phosphoric acid, which was displayed by S2 and S3. Fig. 4 showed the SEM photographs of samples with different contents of phosphoric acid. From the image of S0, the needle-like crystal deposited on the surface of C–S–H which was attributed to the calcium hydroxide growing during the maturing process. S1 showed the deposition of unregular spherulite which was contributed to the adding of phosphoric acid resulting the formation of HA. With more phosphoric acid up to 2 ml, the plenty of petaloid HA formed on the surface of hydrated C3S. Moreover, there were large lamellae and small plate-like crystal which may be attributed to brushite as shown in S3. Fig. 5 and Table 3 showed the EDS data, especially in Table 3 the exact data were shown that the relative atomic number of Si and P on the surface of samples compared with Ca atoms which was set as 1. It was obviously that the relative content of Si gradually decreased accompanying the increase of P because of the adding of phosphoric acid. Reasonably, the ratio of Ca/P would decrease with the increased content of phosphate and the Ca/P of S2 was 1.73 close to 1.67 of HA. The comparation between the experimental and the theoretical content of Si on the surface among three elements, including Si, Ca and P without O to exclude the interference of crystal water, was shown in Fig. 6. With the adding of phosphoric acid, the contents of Si were lower than the theoretical value. The shielding rates of S2 and S3, which was used to explain the reduction of Si compared with the theoretical value, were higher than 30%. It meant that plenty of calcium phosphate deposited on the surface of hydrated C3S. Fig. 7 showed the XPS spectra of the samples in regard to Ca 2p, Si 2p, O 1s and P 2p with different content of phosphoric acid, and the precise data were displayed in Table 4. The binding energy of Si 2p showed an obvious trend that it moved toward higher site. Especially in the S3 (3 ml phosphoric acid), the binding energy was about 1.2 eV more than that of S2. The binding energy of Ca 2p1/2 increased insignificantly while only slight fluctuations could be seen in Ca 2p3/2. The binding energy of O 1s shifted toward lower sites with the increased amount of phosphate. As for the binding energy of P 2p, there was almost no difference between S1 and S2. With regard to S3, the binding energy of P 2p increased 0.2 eV than that of S1 and S2. In short, the amount of phosphoric acid had a significant and regular influence on the structure of C–S–H.
Fig. 2. FT-IR spectra of the samples with different content of phosphoric acid.
some differences compared with S1 and S2. Due to the acidity of S3, there was no band in the range of 1300-1500 cm−1. The slight shift could be recognized that P–O group at 1056 cm−1 moved toward higher wavenumber compared with S1 and S2. In brief, the obvious P–O group related peaks were shown in FTIR spectra with introduced phosphoric acid and there were some differences among them with different amounts of phosphoric acid. Fig. 3 showed the pH changed with the immersing time in PBS at 37 °C. S0 showed a consistent pH value which was higher than 12 owning to the abundant CH produced by the hydration of C3S. The pH value decreased obviously with the increasing content of phosphoric acid. The pH value of S1 after 14 days immersion was near 10 while the pH value of S2 was 7.6. When the amount of phosphoric acid was up to 3 ml, the pH value of S3 was lower than 7 even immersed for 14 days. Briefly, the amount of phosphoric acid played a distinct influence on the pH value which changed from high alkalinity to weak acidity. Table 2 showed the dissolution of the samples about Ca, Si and P ions in deionized water at 37 °C. S0 performed the highest concentration of Ca and Si compared with the samples with phosphoric acid added. The concentrations of Ca and Si decreased when the amount of phosphoric acid increased up to 2 ml, especially the concentrations of Ca and Si of S2 were pretty low. But, the concentrations of these ions increased rapidly as the amount of phosphoric acid was up to 3 ml
4. Discussion Owning to the dominant function of C3S in Portland cement, which has been widely used for building material, there are plenty of studies
Fig. 3. The changes of pH value vs immersion time in PBS. 3
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Fig. 4. The SEM photographs of S0, S1, S2 and S3 with 10000 magnification.
Fig. 5. The EDS of the main atoms on the surfaces of the samples.
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and osteoconduction [3]. It is possible to prepare HA by phosphoric acid and CH with suitable nucleate sites in an alkaline environment [31]. When the phosphoric acid was added drop and drop into the hydrated C3S solution, it can react preferentially with CH owning to the solubility: CH > Ca–OH > Si–O–Ca [18,30]. During the process that 1 ml phosphoric acid is added, the amount of phosphoric acid is insufficient compared with CH, which makes the solution maintain higher pH value than 9 and be suitable for the formation of HA. Therefore, the reaction equation between hydrated C3S and 1 ml phosphoric acid with the hypothesis product of HA, is shown as Eq. (2), and proved by the FTIR, XRD and SEM of S1. Based on the value of x in C–S–H which is around 1.7-1.8, the value of (17–9x) is higher than 0 that means there is little CH existing, which may be a good explanation for higher pH value of S1 in PBS, even no obvious CH peaks are shown in the XRD pattern of S1. With these data, the reaction diagram between C3S and 1 ml phosphate could be described as Fig. 8(b). The Si–O–Ca groups in C–S–H provide nucleate sites for the deposition of phosphate groups via electrostatic interaction and hydrogen bond interaction that can explain the nature of the bioactivity of C3S [13,14]. Because of the insufficient of phosphoric acid, the shielding rate of calcium phosphate on Si is lower which is about 10.9%. As a result, the obvious C–S–H diffraction peaks is shown in S1. There is only minor influence on the Si–O–Ca group in C–S–H, and the binding energy of Si 2p shows just a little shift as shown in Fig. 7. But the effect of 1 ml phosphoric acid on the hydrated C3S is distinctly in some ways. For instance, the pH value in PBS drops to 9.8 from 11.8 of S0. The formation of HA and its shielding function play an inhibition effect on the dissolution of various ions, especially the Ca ions as shown in Table 2.
Table 3 The relative atomic number of Si, P on the surface compared with Ca atom.
S0 S1 S2 S3
Ca
Si
P
Ca/P
1 1 1 1
0.464 0.285 0.235 0.211
0.241 0.578 0.738
4.15 1.73 1.36
Fig. 6. The percentage of Si atoms in the three elements including Si, Ca and P and the shielding rate of Ca and P atoms on Si atom.
9 3CaO SiO2 + 6 H3PO4 + H2 O
xCaO SiO2 yH2 O+ (3
x)Ca(OH)2
9x)Ca(OH)2 + Ca10 (PO4 )6 (OH)2
(2)
focusing on the hydration of C3S though the exact mechanism of hydration is still in doubt [18,19,22,23]. In recent years, increasing attention has been attracted to the application of C3S as biomaterials due to its self-setting property, bioactivity and biological property that the ability to stimulate the proliferation and differentiation of osteoblastlike cells [14,24,25]. It is well-known that the C–S–H and CH are produced when C3S contacts with water [22], which are also proved by XRD and FT-IR in Figs. 1 and 2. The C–S–H, which plays a significant role in the solidification and strength of hardened cement, has been proved that it contains a linear silicate chains of dreierkette form consisting of two paired tetrahedra and a bridging tetrahedron [26]. Previous studies also demonstrate that C–S–H consists of both dimeric and higher polymeric species, such as pentamer and octamer with a 2, 5, 8 … (3n-1) chain length sequence, where n is integer [26-28]. In this study, the linear pentamer is used as the model in the schematic diagram to describe the hydration of C3S which is shown in Fig. 8(a) and the reaction equation can be written as Eq. (1), in which the value of x is variable represented the calcium content and the ratio of Ca/Si in C–S–H with a wide range from 0.5 to 2. Precisely, the value of x is around 1.7-1.8 in C–S–H produced by hydrated C3S [18]. According to previous research, Si–OH and Ca–OH groups have been proved that they do not coexist which means all protons in Si–OH will be replaced by Ca ions to result Si–O–Ca groups [29,30]. In addition, Ca ions in C–S–H are in excess of that required to balance silicate and the additional Ca will be balanced by OH− to form Ca–OH groups. It is possible that the CH precipitates above the Si–O–Ca–OH groups which is displayed in Fig. 8(a). Therefore, it is easy to explain the higher pH value of hydrated C3S: the sustained release of CH on the surface leads to the enrichment of OH− to balance the Ca2+ ions, which is also demonstrated in Table 2 that only small amount of Ca2+ ions are balanced with Si-containing groups.
3CaO SiO2 + H2 O
9[xCaO SiO2 yH2 O] + (17
During the process that 2 ml phosphoric acid is gradually added, the pH value of solution maintains the weakly alkaline environment which is consistently higher than 7.2 and is suitable for the formation of HA. There is no uncertain that CH is consumed completely. Previous studies about the C–S–H structure of hydrated C3S show that around 25% of Ca ions are balanced by OH− and other Ca ions are stored in Si–O–Ca groups [30]. With the calculation, the Ca–OH groups would be replaced thoroughly with 2 ml phosphoric acid. At the meantime, the protonation of Si–O–Ca groups begins to form the Si–OH groups, and finally Ca/ Si value in C–S–H is around 1. Therefore, the schematic diagram could be shown as Fig. 8(c) and the reaction equation with 2 ml phosphoric acid can be shown as Eq. (3) in which the value of x in C–S–H is close to 1. Briefly, the stable silicate-based HA is produced with 2 ml phosphoric acid and the interlayer Ca atoms between C–S–H and HA are shared by them. The XRD and FTIR data about the S2 show the obvious HA-related peaks. The surface morphology shown in Fig. 4 and the EDS data that the Ca/P ratio of S2 is 1.73 also prove the formation of HA. With further analysis of EDS data shown in Fig. 6, the content of Si on the surface of S2 is significantly lower than the theoretical value which is caused by the shielding effect of HA formed on the surface. Moreover, the lowest solubility of Si and Ca ions of S2 shown in Table 2 may be the indirect evidence of the formation of the stable silicate-based HA. Certainly, the influence of 2 ml phosphoric acid on C–S–H becomes relatively obvious which is shown by XPS data that the binding energy of Si 2p shifts toward higher site about 0.298 eV compared with S0, which is attributed to the decalcification of Si–O–Ca resulting in the formation of Si–OH. 9 3CaO SiO2 + 12 H3 PO4 + H2 O
9[xCaO SiO2 yH2 O] + 2Ca10 (PO4)6 (OH)2
(3) With excess level of phosphoric acid up to 3 ml, the final pH value of solution is around 5 in the preparation process, an acidic environment in which dicalcium phosphate is more stable than other calcium phosphates [32]. Certainly, small amounts of other calcium phosphate phases may coexist, such as monocalcium phosphate. The finally
(1)
HA, the most stable phase of calcium phosphate in alkaline environment that the other phases of calcium phosphate will transform toward it eventually, possesses excellent bioactivity, biocompatibility 5
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Fig. 7. The XPS spectra of the samples with the main atoms of Ca 2p, Si 2p, O 1s and P 2p.
of silicate polymerization of C–S–H [33-35]. The possible value of x in Eq. (4) is below 1 which means the ratio of Ca/Si in C–S–H is lower than 1. As a result, the C–S–H with higher degree polymerization covered with brushite is produced in which the ratio of Ca/Si is below 1. In addition, the binding energy of P 2p is 0.2 eV more than S1 and S2 caused by the higher electronegativity of –OH group in brushite.
Table 4 The binding energy of the Si 2p, Ca 2p, O 1s and P 2p. Binding Energy/eV
S0 S1 S2 S3
Si 2p
O 1s
Ca 2p1/2
Ca 2p3/2
P 2p
101.901 102.000 102.199 103.450
531.351 531.100 530.949 530.750
350.351 350.450 350.449 350.550
346.901 346.900 346.999 346.950
132.800 132.799 133.000
3CaO SiO2 + 2H3PO4 + H2 O
xCaO SiO2 yH2 O+ zCaHPO4 ·2H2 O+ else
(4)
Essentially, the bioactivity of C3S is attributed to the interlayer Ca which provides suitable nucleate sites for the deposition of phosphate group via electrostatic interaction and hydrogen bond interaction. Briefly, the preparation of silicate-based calcium phosphates depends on the nucleation and growth of calcium phosphate on the surface of C–S–H and the stability of final product is associated with the proportion between C3S and phosphoric acid. The bioglass with higher content of SiO2 unable to induce the precipitation of apatite layer is perhaps attributed to the short of valuable nucleate sites [36]. With the adding of phosphoric acid, the pH value of C3S is decreased which could reduce the inflammatory response increasing the biocompatibility. The solidification of silicate-based cement is closely related to the polymerization of C–S–H [34,37]. The influence of phosphoric acid on the C–S–H structure may provide a light on shortening the setting time of C3S cement and in our previous study, C3S based cement, which used phosphoric acid as the curing agent, have a significantly reduced setting time [25]. It is versatile that the combination of phosphoric acid and C3S not only shortens the setting time of C3S but also increases the biocompatibility without unwished by-products. A much deeper consideration, bisphosphonates, which are widely used for the treatment of
product of calcium phosphate is primarily brushite which is proved by the XRD data in Fig. 1. The acidity of S3 also suppresses the carbonization so that no CO32− related vibrational absorption peak appears in Fig. 2. With the product of brushite, the reaction equation is shown in Eq. (4), where the maximal content of calcium phosphate is brushite that the value of z is close to 2 and the ‘else’ represents the other calcium phosphate and Fig. 8(d) can be used to describe the deposition of brushite. With excess phosphoric acid, the properties of S3 change a lot by comparation with S1 and S2. Owning to the higher solubility of brushite, the dissolution of Si, Ca and P increases sharply compared with S2 and the shielding of brushite on C–S–H does not prevent the release of Si. With regard to the C–S–H structure, a distinct change can be seen from the XPS data that the binding energy of Si 2p shifts obviously which is up to 1.55 eV compared with S0. During the adding process, the protonation of Si–O–Ca leads to the accumulation of Si–OH groups which provides basic condition for the polymerization. Previous studies have proved that the shift is caused by the increase in the degree 6
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Fig. 8. The schematic diagram of the reaction between C3S and phosphoric acid.
osteoporosis, show a powerful binding force to Ca [38,39]. Similar to the deposition of calcium phosphate, it is possible to achieve the controllable release of bisphosphonates by the carrier of C3S.
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5. Conclusion In this study silicate-based calcium phosphates were prepared which phase can be controlled by the proportion between C3S and phosphoric acid via aqueous precipitation method. The phases of calcium phosphate deposited on the surface of hydrated C3S were changing with the increase of phosphoric acid that transformed from HA to brushite. Through a series of methods, the interaction between C–S–H and phosphate was studied that the interlayer Ca was contributed to connection of two phases. The structure of C–S–H underwent a series of changes with increased phosphoric acid, including protonation and polymerization, especially for S3 with 3 ml phosphoric acid. In short, the silicate-based calcium phosphates may be suitable for biomaterial with different modes, such as coating and inorganic active filling matrix. In addition, it provided a new light for the sustained release of bisphosphonates deposited on the surface of hydrated C3S. Declaration of competing interest I would like to declare on behalf of my co-authors that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The research leading to these results has received funding from the National Natural Science Foundation of China (51773123). References [1] D.K. Khajuria, C. Disha, R. Vasireddi, R. Razdan, D.R. Mahapatra, Risedronate/zinchydroxyapatite based nanomedicine for osteoporosis, Mater Sci Eng C Mater Biol Appl 63 (2016) 78–87.
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