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[beta]-Dicalcium Silicate Cement Modified with [beta]-tricalcium Phosphate: In vitro Bioactivity and Mechanical Strength.


Calcium-silicate based cement, are hydraulic materials undergoing a series of physicochemical reactions when hydrated in the presence of moisture. these reactions lead to the formation of hydration product such as hydrated silicate gel (CSH phase) and calcium hydroxide (portlandite), ettringite [1-3].In the last decade, calcium silicate cement has received great consideration in dental and orthopedic surgery since they have the important property to set in a biological medium such as blood, simulated body fluids (SBF) and Saliva being, therefore they can be used for dental and orthopedic surgery [1-4].

Tricalcium phosphates(([Ca.sub.3][(P[O.sub.4]).sub.2]) = [C.sub.3]P) has attracted a great interest for hard tissue repair due to their excellent biocompatibility, good bioactivity, low setting temperature and self-setting characteristic [5]. Tricalcium phosphate ([C.sub.3]P) has three polymorphic forms [alpha], [alpha]' and [beta]. The latter is stable at room temperature and it can be constructively transformed at 1125[degrees]C into [alpha]-[C.sub.3]P [5]. [beta]-[C.sub.3]P is widely used for hard tissue repair. It has a chemical composition similar to apatite present in the bone tissue which favorites its large use as a bone grafting material [6].

The dicalcium silicate [Ca.sub.2]Si[O.sub.4] ([C.sub.2]S) varieties have pulled an extreme consideration because of their broad applications in several fields like cement, ceramics, drugs and bioactive materials [7]. [C.sub.2]S is one of the main components of cement phases which have a good heat-resistant and can spontaneously develop strength, toward water [8]. It exists at range of temperature extended between room temperature and 1500[degrees]C and presents five allotropic forms: [alpha], [[alpha]'.sub.H], [[alpha]'.sub.L], [beta] and [gamma] [9, 10] however their stability ranges on heating and cooling are different [11].

Radiopacity is one of the perfect properties of materials which are susceptible to be utilized in dentistry. This property is likely to be adequate to permit distinction from dentin and adjacent anatomical structures. It's to be noted that calcium silicate cement generally does not have adequate radiopacity to be visualized radiographically [12, 13] hence the necessity to add a radiopacifying agent to cement composition. In the literature, there are many studies which evaluate the radiopacity of many agents as bismuth oxide, zinc oxide, barium sulfate, calcium tungstate, and zirconium oxide [12,13]. However, the conceivable interference of the radiopacifiers with the setting chemistry, biocompatibility, and physical properties of the materials should be further investigated before any clinical recommendation.

In this paper, the effect of the addition amount of [C.sub.3]P and combined ZnO and [Bi.sub.2][O.sub.3] to calcium-silicate cement on the bioactivity and mechanical strength was investigated. Calcium-silicate cement mainly based on dicalcium-silicate ([C.sub.a2]Si[O.sub.4], [C.sub.2]S) was prepared by solid state reaction using mussel shells, Beta-[C.sub.3]P obtained by calcination at 900[degrees]C of [Ca.sub.3][(P[O.sub.4]).sub.2]) = [C.sub.3]P, was added to obtain [C.sub.2]S-[C.sub.3]P, zinc oxide and bismuth oxide was incorporated to prepare radiopaque cement, the obtained material was soaked in a biological medium for different intervals of time ranging from 2 hours to 28 days, the mechanical strength of some elaborated samples was measured at 28 and 72 days.

Materials and Methods

Preparation of Calcium Silicate Cement

In the present study, three basic powders have been used which are dicalcium silicate ([C.sub.2]S), tricalcium phosphate ([C.sub.3]P) and zinc oxide (ZnO). The calcium-silicate cement is mainly based on dicalcium-silicate [C.sub.2]S (75%), Beta-[C.sub.3]P (10%) was added to obtain [C.sub.2]S-[C.sub.3]P. Zinc oxide (15%) was inserted to prepare radiopaque cement. Dicalcium silicate ([C.sub.2]S) was obtained by solid state reaction, starting from an appropriate mixture of mussel shells and silicon powder. The mussel shells are collected from the coast of the Atlantic Ocean of Rabat-Morocco, then shells are dried and finely milled (< 40 [micro]m).The chemical composition of the mussel obtained by the Fluorescent X-ray analysis shows that the fraction of mussel shell contained a significant quantity of calcium oxide (53.95%) (Table. 1). It also contained smaller amounts of impurities, mainly in the form of silicon, sodium, aluminum and magnesium oxides. The loss on ignition (LOI) of shells (43.15%) obtained by calcination of the sample at 1000[degrees]C, correspond to the loss of carbon dioxide due to the partial dissociation of calcium carbonate [14]. Shell and silicon powders are mixed with specific proportions (77% and 23% respectively) then the mixture is treated slowly at different temperatures from 500 to 1050[degrees]C. To reduce the particle size and increase the reactivity of CaC[O.sub.3]; milling in pure acetone or ethanol is made at each change of temperature [15] finally we have obtained a [C.sub.2]S phase at 1050[degrees]C for a heat treatment of 4 hours. Tricalcium phosphate was obtained by calcination of [Ca.sub.3][(P[O.sub.4]).sub.2] at 900 [degrees]C for 3 hours to obtain [beta]-[C.sub.3]P. The obtained material was ground and characterized by X-ray diffraction (XRD) using a Siemens D5000 diffractometer operating with 40 kV and 20 mA, equipped with a copper anticathode and a secondary monochromator ([gamma] = 1.5406 [Angstrom]), it was also characterized by Fourier Transform Infrared (FT-IR) spectroscopy using a JASCO FTIR-4600 in the range of 4004000 cm-1., Also by scanning electron microscopy MEB using FEI FEG 450. The mechanical strength is measured on the cylindrical specimens (1.5 * 0.5 cm) at 28 and 72 days, hydrated on the water with a ratio equal 0.5

In vitro test

The apatite formation ability of the obtained specimens was evaluated by soaking a small pastille (13*1 mm) prepared from synthesized cement then soaking in artificial saliva (AS) for different time periods from 2 hours to 30 days. The AS solution was prepared by dissolving reagent-grade chemicals (Analar Norma Pur) corresponding to SAGF medium [16] in ultra-pure water at a pH of 6.8 using 1 M hydrochloric acid (Pro Analysi). The pH of the solution was fixed at ~6.8 because this value is close to the pH of the mouth saliva. Each sample (pastille) was immersed in a polyethylene bottle containing 10 mL saliva and kept for up to 30 days without shaking in an incubator at 37[degrees]C. After immersion in Artificial Saliva solutions for different time intervals, the pH of solution was measured before the removal of each sample from the solution and put it in acetone solution for at least 24 hours in the aim to stop the hydration reaction then air dried. The samples were further characterized by XRD and FT-IR to control and monitor the present phases in the material during the soaking time.

Results and Discussion

Figure 1 shows the X-ray diffraction patterns of the starting powders. The obvious sharp peaks and low backgrounds suggest that the [C.sub.2]S powder is highly crystalline. The corresponding reflections have been indexed using the The International Centre for Diffraction Data (ICDD), Card No. 01-070-0388 (JCPDS) that are assigned to the [beta]-[C.sub.2]S Phase with small peaks of Wollastonite-2M, according to JCPDS card No. 00-027-0088. Moreover, the polymorphic from obtained [C.sub.3]P was [beta]-[C.sub.3]P according to JCPdS card No. 01-072-7587. Furthermore, the XRD pattern of ZnO is highly crystalline and corresponding to JCPDS card No 01 070-8070. After immersion, pH measuring and air drying, of [C.sub.2]S-[C.sub.3]P sample was analyzed apart by (XRD) Figure 2, show the XRD patterns and identified resulting phases after immersion of the synthetized cement in the artificial saliva, the characteristic peaks of the cement decrease due to a strong hydration and dissolution of the specimens which are favorable to the formation of the hydration products and intermediate phases [14]. We note also the formation of a crystalline phase corresponding to the octa calcium phosphate (OCP = ([Ca.sub.8][H.sub.2][(P[O.sub.4]).sub.6]([H.sub.2]O)), whereas apatite characteristic peaks may appear after 24 h and grow up with time soaking. The presence of tricalcium phosphate enhances and accelerates the formation of HAp (fig.3).

Figure 4 shows the FTIR spectra of the [C.sub.2]S -[C.sub.3]P and for [C.sub.2]S after 24 hours of soaking in artificial saliva. The main bands corresponding to the silicate groups are due to the Si[O.sub.4.sup.4]-tetrashedral. According to Puertas [17] the absorption bands of the different polymorphs of dicalcium silicate are assigned to ([v.sub.1]) (symmetric stretching) located around 800-900 [cm.sup.-1] and ([v.sub.3]) (antisymmetric stretching) are typically located around 800-1000 cm-1 and those of ([v.sub.4]) (the triply degenerated out of plane bending) located around 400-500 [cm.sup.-1]. The spectra of [C.sub.2]S and [C.sub.3]P before soaking presented strong similarities; they exhibited intense bands attributed to P[O.sub.4.sup.3-] of phosphate and Si[O.sub.4.sup.4-] of silicates. The P[O.sub.4.sup.3-] groups are located around: 1010- 1100 [cm.sup.-1] ([v.sub.3]); 962 [cm.sup.-1] ([v.sub.1]) and between 550 and 630 [cm.sup.-1] ([v.sub.4]) [18]. The characteristic bands of HAp presented around 566, 601 and 1019 [cm.sup.-1] could be assigned to the P[O.sub.4.sup.3-] groups [5, 18]. Two marked broad bands at 3300 and 1642 [cm.sup.-1] can be seen which are attributed to the presence of structural OH- groups, these bands were assigned to the bending mode of H-O-H groups and to the O-H stretching mode respectively [18]. Finally, C[O.sub.3.sup.2] bands were detected at 872 [cm.sup.-1] ([v.sub.2]), and between 1410 and 1490 [cm.sup.-1] ([v.sub.3]) [19]. These bands indicated that C[O.sub.3.sup.2-] ions partially substituted phosphate groups in the HAp crystalline structure [18].

After immersion, of specimens in the AS the pH of saliva solution increase (Table 3), accompanied by a decrease of characteristic peaks of the cement resulting in a strong hydration and dissolution of the specimens which are favorable to the formation of the hydration products and intermediate phases. We note also the formation of a crystalline phase corresponding to the octacalcium phosphate (OCP) which is initially formed when the reaction pH is less than 9, whereas apatite characteristic peaks may appear after 24 h and grow up with time soaking at higher pHs. Which proves that OCP phase undergoes a transformation mechanism through nucleation and growth, followed by hydrolysis of the transient OCP phase into the thermodynamically more stable apatite [16]. According to Hench [20], HAp could be formed through a series of reactions including ion exchanges, dissolution, and precipitation, during the immersion of the three powders in the biological medium. The dissolution and fast exchange ions between the AS and the material caused an increase in pH values of the saliva solutions which leads to hydrolysis of the silica groups and creation of silanol groups. The pH values of the medium were measured to be 7.51 and 9.92 respectively, after immersion during 4 and 720 hours (30 days) as summarized in Table 3. This augmentation in the pH of the biological medium breaks Si-O-Si bonds and causes the release of calcium and phosphate. At that point, more Si-OH bonds are formed resulting from the hydrolysis of Si-O-Si bonds caused by the increase of pH then they repolymerize, making a surface poor of Na and Ca cations. This step is followed by dissolution of the cement and migration of [Ca.sup.+2] and P[O.sub.4.sup.-3] to the amorphous Si[O.sub.2] layer creating an amorphous calcium phosphate layer. Further the crystallization of the calcium-phosphate complexes into hydroxycarbonate apatite [16].

The SEM/EDS analysis (fig. 5) shows a fluorescent and compact morphology. There are two types of luminous spots dispersed along the surface of the material; hexagonal leaflets are attributed to zinc oxide (spectre 1, 5 and 6) however large monoclinic spots that are brighter are attributed to bismuth (spectre 3). Spectre 2 was attribuated to [C.sub.2]S with a ratio close to 2.3. Antonio Hungaro Duarte, has summarized Radiopacity of portland cement associated with different radiopacifying agents (Table 4) [12] which prove that zinc oxide and oxide bismuth give some radiopacity of the material. The radiopacity of pure Portland cement and calcium silicate are altogether lower than that of dentin, while all cement/radiopacifier mixtures were significantly more radiopaque than dentin than calcium silicate and Portland cement alone. Whereas substance with bismuth oxide presented the highest radiopacity values. The compact morphology gives a high mechanical strength of the cement. This strength is increased with time of all samples (fig.6). The [C.sub.2]S-[C.sub.3]P-Zn/Bi presents the high mechanical strength value at 28 days while it's the lowest at 72 days this might be explained by the combination of zinc/ bismuth oxide. The [C.sub.2]S-[C.sub.3]P and [C.sub.2]S-[C.sub.3]P-Zn presented the highest strength value at 72 days.


The present paper shed a light on the possibility of elaborating bioactive calcium silicate cement based mainly on dicalcium silicate ([C.sub.2]S) from mussel shell. The bioactivity test was investigated by soaking the cement pastilles for different periods in artificial saliva from 4 hours to 30 days. Whereas the mechanical strength of samples was operated at 28 and 72 days. The result shows that the addition of a small amount of tricalcium phosphate ([C.sub.3]P) (10%) may enhance both the bioactivity and the mechanical strength. The incorporation of zinc oxide (15%) enable obtaining a significante radiopacity of the synthesized material with good mechanical strength, however the sample with bismuth oxide present the highest radiopacity while its strength is less than other samples.

This result demonstrates the possibility of combining small amounts of C3P and ZnO with C2S to obtain a cement with excellent bioactivity, good mechanical strength and significante radiopacity which opens new trend of using this material as a biomaterial in dentistry.


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A. Bouregba (1) *, H. Ez-zaki (1), A. Diouri (1), O. Sassi (2)

(1) Laboratory of Applied Solid State Chemistry, Faculty of Science, Mohammed V University, Rabat, Morocco

(2) Laboratory of physico-chemistry of materials, ENS of Rabat, Mohammed V University, Rabat, Morocco

Received 28 September 2017; Accepted 11 November 2017; Published online 31 December 2017

* Coresponding author: Dr. A. Bouregba;

E-mail: bouregba.fssm@gmail. com

Caption: Figure 1: XRD pattern of starting materials

Caption: Figure 2: XRD pattern of [C.sub.2]S-[C.sub.3]P before and after soaking in artificial saliva

Caption: Figure 3: XRD pattern of [C.sub.2]S and [C.sub.2]S-[C.sub.3]P after 24 hours of soaking in artificial saliva [cm.sup.-1]

Caption: Figure 4: FTIR spectra of [C.sub.2]S-[C.sub.3]P and [C.sub.2]S after 24 hours of soaking in artificial saliva

Caption: Figure 5: SEM/EDS analysis of [C.sub.2]S-[C.sub.3]P-Zn/Bi

Caption: Figure 6: Mechanical strength of synthesized cement at 28 and 72 days
Table 1: Chemical composition of powder mussel shells (wt. %)

Oxides    CaO    Si[O.sub.2]   [Al.sub.2]   [Fe.sub.2]   Na2O
                               [O.sub.3]    [O.sub.3]

Mussel   53.95      0.61          0.23         0.02      0.05

Oxides   MgO    S[O.sub.3]   [K.sub.2]O   [P.sub.2]   LOI *

Mussel   0.67      0.77         0.11         0.3      43.15

Table 2: Composition of the artificial saliva solutions used in the
study (SAGF medium) [16]

Substances             NaCl     KC1     Ca[Cl.sub.2].   K[H.sub.2]
                                         2[H.sub.2]O    P[0.sub.4]

Concentration (g/L)   0.1256   0.9639      0.2278         0.6545

Substances             Urea    N[H.sub.4]Cl   NaHC[O.sub.3]    KSCN

Concentration (g/L)   0.2000      0.178          0.6308       0.1892

Substances             [Na.sub.2]

Concentration (g/L)      0.7632

Table 3: Evolution of pH of the immersion medium
according the soaking periods

Soaking periods (H)    4      24    120     720

pH                    7.51   8.21   8.55   9.9 2

Table 4: Radiopacity of Different Materials

Materials                                Radiopacitv
                                           (mm All

Dicalcium Slicate                          11 [13]
Dicalcium Silicate with Bismuth oxide     7 3 [13]
Portland cement                           1.01 [12]
Portland cement with Zinc oxide           2 64 [12]
Portland cement with Bismuth oxide        5.93 [12]
Dentin                                    1.74 [12]
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Title Annotation:Original Article
Author:Bouregba, A.; Ez-zaki, H.; Diouri, A.; Sassi, O.
Publication:Trends in Biomaterials and Artificial Organs
Date:Jul 1, 2017
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