Accepted Manuscript Novel tricalcium silicate/magnesium phosphate composite bone cement having high compressive strength, in vitro bioactibity and cytocompatibility Wenjuan Liu, Dong Zhai, Zhiguang Huan, Chengtie Wu, Jiang Chang PII: DOI: Reference:

S1742-7061(15)00183-X http://dx.doi.org/10.1016/j.actbio.2015.04.012 ACTBIO 3666

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

20 December 2014 23 March 2015 2 April 2015

Please cite this article as: Liu, W., Zhai, D., Huan, Z., Wu, C., Chang, J., Novel tricalcium silicate/magnesium phosphate composite bone cement having high compressive strength, in vitro bioactibity and cytocompatibility, Acta Biomaterialia (2015), doi: http://dx.doi.org/10.1016/j.actbio.2015.04.012

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Novel tricalcium silicate/magnesium phosphate composite bone cement having high compressive

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strength, in vitro bioactibity and cytocompatibility

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Wenjuan Liua, b, Dong Zhai a, Zhiguang Huan a, Chengtie Wu a, Jiang Changa, *

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a

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Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, People’s

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Republic of China

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai

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b

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of China

University of Chinese Academy of sciences, 319 Yueyang Road, Shanghai 200050, People’s Republic

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* Corresponding author. Tel.:+86 21 52412804; fax: +86 21 52413903.

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Email address: [email protected] (J. Chang).

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1

1

Abstract: Although inorganic bone cements such as calcium phosphate cements have been widely

2

applied in orthopaedic and dental fields because of its self-setting ability, development of high-strength

3

bone cement with bioactivity and biodegradability remains as a major challenge. Therefore, the

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purpose of this study is to prepare a tricalcium silicate/magnesium phosphate (C3S/MPC) composite

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bone cement, which is intended to combine the excellent bioactivity of C3S with remarkable

6

self-setting properties and mechanical strength of MPC. The self-setting and mechanical properties, in

7

vitro induction of apatite formation and degradation behavior, and cytocompatibility of the composite

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cements were investigated. Our results showed that the C3S/MPC composite cement with an optimal

9

composition had compressive strength up to 87 MPa, which was significantly higher than C3S (25 MPa)

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and MPC (64 MPa). The setting time could be adjusted between 3 minutes and 29 minutes with the

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variation of compositions. The hydraulic reaction products of the C3S/MPC composite cement were

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composed of calcium silicate hydrate (CSH) derived from the hydration of C3S and gel-like amorphous

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substance. The C3S/MPC composite cements could induce apatite mineralization on its surface in SBF

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solution and degraded gradually in Tris-HCl solution. Besides, the composite cements showed good

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cytocompatibility and stimulatory effect on the proliferation of MC3T3-E1 osteoblast cells. Our results

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indicated that the C3S/MPC composite bone cement might be a new promising high-strength inorganic

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bioactive material which may hold the potential for bone repair in load-bearing site.

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Keywords: magnesium phosphate, tricalcium silicate, bone cement, orthopaedic, bioactive material

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1

1. Introduction

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Bone cements have gained great interest as their self-setting properties allow for the direct

3

injection and filling to complicated defect sites [1]. Calcium phosphate cements (CPC) are currently the

4

most frequently studied inorganic bone cements due to their good biocompatibility and

5

osteoconductivity [2]. However, CPC still have some drawbacks which hinder their wider application.

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For example, the mechanical strength of CPC is commonly not high enough for bone repair in

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load-bearing defect sites [3]. Another concern over the use of CPC is its relatively low degradation rate

8

which mismatches with the formation of new bone tissue [1, 3, 4].

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In recent years, inorganic cements other than CPC have been developed, aiming at avoiding the

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shortcomings of CPC. Among them, silicate-based cements have received much attention for their

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unique biological properties. As typical silicate bone cement, tricalcium silicate (C3S) is one of the

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main active components in mineral trioxide aggregates (MTA) which has been widely used as

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biocompatible and bioactive root-end filling material [5]. Our previous studies revealed that C3S was

14

bioactive and biodegradable [6-9]. It could induce bone-like apatite mineralization in vitro [6, 7] and its

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ionic extract solution could stimulate proliferation and osteogenic differentiation of bone-related cells

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[6, 10], as well as the odontogenic differentiation of human dental pulp cells [8, 9]. It was proved that

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Si ion released from silicate-based biomaterials plays an important role in stimulating proliferation,

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differentiation, osteogenic gene expression of osteo-related cells [11]. In addition, C3S showed

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moderate in vitro degradation rate [12]. Despite the advantages of bioactivity and degradability, the

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compressive strength of C3S is relatively low and its setting time is too long for clinical application [6].

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Magnesium phosphate cement (MPC) is a kind of inorganic cements developed in recent years.

22

There were several types of MPC with different compositions and reaction products reported in the 3

1

literature. The first MPC reported in the literature composed of magnesium oxide (MgO) and

2

phosphate acid [13]. Later developed compositions of MPC include: (1) MgO and phosphates

3

((NH4)2HPO4, NH4H2PO4, NaH2PO4, Ca(H2PO4)2· H2O et al) [3, 13-16]; (2) magnesium phosphate

4

(Mg3(PO4)2) and ammonium phosphates ((NH4)2 HPO4 or NH4H2PO4) [17-19]; (3) magnesium chloride

5

or magnesium hydroxide (MgCl2, Mg(OH)2) and phosphate acid [20, 21], and so on. The MPCs can

6

form different setting products with the variation of their compositions. For example, MgO and

7

NH4H2 PO4 form NH4MgPO4·6H2O (struvite) and Mg(NH4)2H2(PO4)2·4H2O (schertelite) [13], MgO

8

and NaH2PO4 form amorphous phase [13], Mg3(PO4)2 and (NH4)2 HPO 4 form struvite [17-19], Mg(OH)2

9

and phosphate acid form Mg3(PO4)2 (farringtonite) [21], et al. Among them, the type (1) MPC has been

10

reported to have the characteristics of fast-setting ability and high compressive strength which can

11

reach 58 MPa [3, 13]. The MPC which composes of MgO and NaH2PO4 and formed amorphous

12

products has a lower temperature rise and avoids harmful ammonia release during setting reaction,

13

compared with the MgO and ammonium phosphates composition, therefore it is more suitable to be

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used as a biomedical material. Both in vitro and in vivo evaluations have revealed that MPC had good

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biocompatibility and moderate degradation rate [3, 22-24]. Although it was reported in the previous

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studies that Mg2+ ions could promote proliferation and differentiation of osteoblast cells [25, 26] and

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MPC composed of Mg3(PO4)2 with (NH4)2HPO4 and NH4H2PO4 was bioactive in bone regeneration

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[19], the bioactivity of Mg-containing bone cements with different composition remains to be

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confirmed further.

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It is therefore clear that neither C3S nor MPC alone is suitable to be used as a high-strength

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bioactive bone cement. However, concerning their individual advantages, we assumed that it may be

4

1

possible to obtain a bioactive cement if C3S and MPC are reasonably combined. In fact, our previous

2

studies confirmed that it was feasible to combine the advantages of C3S with CPC or calcium sulfate to

3

obtain improved performance [10]. Therefore, in the present study, we intend to design a C3S/MPC

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composite cement system which combines the bioactive property of C3S and high-strength and

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fast-setting property of MPC. It is anticipated that the composite bone cement may have comparable or

6

higher compressive strength than MPC and shorter setting time than C3S, and in addition, retain the

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bioactivity of C3S. The content of this study includes characterization of the hydration products,

8

mechanical strength and self-setting properties, and the evaluation of in vitro bioactivity, degradation

9

rate and cytocompatibility in order to prove our hypothesis that the combination of C3S and MPC will

10

result in a fast-setting high strength bioactive bone cements.

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2. Materials and methods

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2.1. Preparation of C3S and MPC cement powder

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C3S powder was synthesized by the sol-gel method according to previous publication [27].

15

Briefly, nitric acid (18 mL, 2 mol· L-1) and tetraethyl orthosilicate (TEOS, 0.5 mol) were added into 200

16

mL deionized water and stirred for 0.5 h. Then, Calcium nitrate tetrahydrate (TEOS, 1.5 mol) was

17

added and stirred for 1 h. The mixture was transferred to an oven, kept at 60 °C for 24 h, dried at

18

120 °C for 48 h and finally sintered at 1450 °C in a high-temperature furnace (Nabertherm LHT 08/17,

19

Germany). The obtained powder was ground with a roller type ball mill (170 r·min-1, Shanghai

20

Congming Haidao Qinggong Machinery Company) for 24 h. The mass ratio of zirconia balls (served as

21

abrasive) and C3S powder was 2.5:1. Finally, C3S powder was sieved through a 300-mesh sift. MPC

5

1

powder was prepared by mixing magnesium oxide (MgO), sodium dihydrogen phosphate (NaH2PO4),

2

and sodium borate decahydrate (Na2B4O7·10H2O, borax) as the retardant of the setting reaction of

3

MPC [13]. Less-reactive MgO was synthesized by sintering the light magnesium oxide at 1500 °C for

4

6 hours. Commercial NaH2PO4 and borax were directly used. All source reagents were analytical grade

5

without further purification (Sinopharm chemical reagent Co., Ltd., Shanghai, China). All the three

6

kinds of powders were ground with the roller type ball mill for 8 h, respectively, and the mass ratio of

7

zirconia balls (served as abrasive) and each powder was 2.5:1. After grounding, they were sieved

8

through a 400-mesh sift and then homogeneously mixed (molar ratio of MgO and NaH2PO4: 3.8:1,

9

borax:3 wt.% of MPC) through ball-milling without zirconia balls, which rendered the MPC powders.

10

Then C3S and MPC powders were mixed at different mass ratios as follows: 25% C3S and 75% MPC

11

(C25M75), 50% C3S and 50% MPC (C50M50), 75% C3S and 25% MPC (C75M25). The phase

12

composition of the as-prepared powders was investigated by using the X-ray diffractometer (XRD,

13

Geigerflex, Rigaku Co., Japan) with Cu (Kα) radiation. The particle size distribution and specific

14

surface area (SSA) of C3S, MgO, NaH2PO4 and borax powder (Table 1) were measured by laser

15

diffraction analysis (Mastersizer 3000, Malvern Instruments Ltd., UK)

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2.2 Preparation and characterization of the cement paste

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The cement pastes were prepared by mixing the powders with de-ionized water. Based on our

19

preliminary experiments, the liquid to powder ratios (LPR) were set as 0.5, 0.35, 0.25, 0.18 and 0.13

20

mL· g-1 for C3S, C25M75, C50M50, C75M25 and MPC, respectively, in order to obtain optimal

21

cohesion and workability. The mixtures were stirred for 60 seconds to obtain homogenous pastes, and

6

1

then cast into a cylindrical mold with the size of 6 mm in diameter and 12 mm in height. Cements were

2

de-molded after reaching final setting (the state that the heavy needle of the Vicat apparatus fails to

3

make evident indentations on the surface of the paste, see section 2.3. The storing time in the mold was

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113, 29, 9, 3 and 10 minutes for C3S, C75M25, C50M50, C25M75 and MPC, respectively.) and cured

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in a shaking water bath with constant temperature of 37 °C, 100% humidity and 120 r·min-1 for 7 days,

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and then dehydrated with anhydrous ethanol followed by drying in the air. The phase composition and

7

cross-section of the samples were then investigated by using XRD and scanning electron microscope

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(Hitachi S4800, Hitachi Ltd., Japan) equipped with X-ray energy dispersive spectrum (EDS),

9

respectively.

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2.3 Self-setting properties

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C3S, C25M75, C50M50, C75M25 and MPC samples used for the compressive strength test were

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with the size of 6 mm in diameter and 12 mm in height. They were prepared by the same procedures

14

with those in section 2.2. To investigate the development of compressive strength during the

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self-setting process, the curing time in shaking water bath was set as 1 hour, 2 hours, 1 day, 2 days, 7

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days, 14 days, 21 days and 28 days, respectively. To investigate the effect of LPR on compressive

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strength, another group of C75M25 samples were prepared with LPR of 0.30, 0.32, 0.35, 0.38 and 0.40

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mL· g-1, and cured for 24 hours. Different cement samples with a constant LPR of 0.4 mL· g-1 and

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curing time of 24 hours was prepared for compressive strength test. Then the samples were tested with

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a universal testing machine (AG-1, SHIMADZU Corporation, Japan), at a loading speed of 0.5

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mm· min-1. Three parallel samples were used for each test. In addition, the porosity of the as-prepared

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1

cement cylinders was measured by the Archimede’s principle.

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The setting time of the cements was tested with Vicat apparatus according to ISO-9597-1989E.

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The C3S, C25M75, C50M50, C75M25 and MPC cement pastes were prepared by mixing powders with

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de-ionized water (LPR = 0.5, 0.35, 0.25, 0.18 and 0.13 mL· g-1, respectively). The pastes were filled

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into a cylindrical container, and the setting time was defined as the time from de-ionized water was

6

added into the cement powder to the time when the heavy needle failed to make evident indentations on

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the surface of the paste. Three parallel samples were used for each test. To investigate the effect of LPR

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on setting time, C75M25 samples were prepared with LPR of 0.30, 0.32, 0.35, 0.38 and 0.40 mL· g-1

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and their setting time were further tested. For testing the pH values of the cement paste, 1 g of cement

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powder and de-ionized water (0.5, 0.35, 0.25, 0.18 and 0.13 mL for C3S, C25M75, C50M50, C75M25,

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respectively) were mixed homogeneously for 60 seconds, and then 5 mL of de-ionized water was added.

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Finally, the pH value of the water was measured every 2 minutes until 30 minutes later.

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2.4 Apatite formation on the surface of cement paste

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To evaluate apatite mineralization ability of different cements, disc samples with the size of 6 mm

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in diameter and 2 mm in height were soaked in simulated body fluid (SBF) solution for 7 days in the

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37 °C environment. SBF solution was prepared according to the method reported by Kokubo [28] and

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its composition was presented in Table 2. The disc samples were prepared by filling the cement paste

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described in section 2.2 into a cylindrical mold, followed by de-molding, curing in the shaking water

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bath with constant temperature of 37 °C, 100% humidity and 120 r·min-1 for 7 days and dehydrating.

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Then each of the discs was soaked in SBF solution in polyethylene bottle and the ratio of the surface

8

1

area of the disc to the volume of the solution was 0.1 cm2· mL-1. The bottles with sample and SBF

2

solution inside were put in the shaking water bath. SBF solution was refreshed every 2 days. After 7

3

days’ soaking, the samples were taken out, gently rinsed with de-ionized water and dried in the air for

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24 hours. The composition and morphology of the as-deposited surfaces layer on the discs were

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analyzed by Fourier Transform Infrared Spectroscopy (FTIR; Thermo Nicolet nexus-IR spectrometer;

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Thermo Fisher Scientific, Waltham, MA, USA)，XRD and SEM. The FTIR test was conducted by

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making the powder sample which was shaved from the discs and KBr powder into a tablet with a tablet

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pressing machine, and testing the infrared spectroscopy of the tablet in the range of 4000-400 cm-1, and

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finally, the spectroscopy was subtracted by that of the blank KBr tablet. The cement samples before

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soaking in SBF solution were also characterized by FTIR as comparison.

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2.5 Weight loss

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For the weight loss test, disc samples which were cured in the shaking water bath with constant

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temperature of 37 °C, 100% humidity and 120 r·min-1 for 7 days and with the the size of 6 mm in

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diameter and 2 mm in height were soaked in Tris-HCl buffer solution in polyethylene bottle and the

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ratio of the surface area of the disc to the volume of the solution was 0.1 cm2· mL-1 [29]. The bottles

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with sample and Tris-HCl buffer inside were put in the shaking water bath. After soaking for different

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periods (7, 14, 21, 28 days), and the discs were taken out, dried in the 60 °C oven for 24 hours, and

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weighed on an analytical balance with accuracy of 0.0001 gram. The Tris-HCl solutions were daily

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refreshed. The weight loss ratio was calculated as follows,

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Weight loss (%) = (m0 - mt) / m0 × 100%

9

1

where m0 denoted to the original mass of the discs, and mt the mass after soaking in Tris-HCl solution

2

for different periods. The ion release and pH of each sample in Tris-HCl solution was tested by

3

Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) and pH meter (PHSJ-4A,

4

INESA Instrument, China), respectively. Three parallel samples were used for each test.

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2.6 In vitro cytocompatibility

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In vitro cytocompatibility of the materials was evaluated by the MTT method according to the

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international standard (ISO/EN10993-5) [30], with the MC3T3-E1 osteoblast cell as the experimental

9

cell. The extract solutions were prepared as follows. Firstly, the cement paste was cured in the shaking

10

water bath for 7 days, ground and sieved through a 300-mesh sift. Secondly, 1g of the as-prepared

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powder was mixed with 5 mL cell culture medium (α-MEM) and kept in CO2 incubator at 37 °C for 24

12

hours. Thirdly, the mixture was centrifuged at 4000 rpm for 10 min and the supernatant was collected

13

and filtered through a bacterial filter to obtain the bacteria-free cement extract. Finally, the extract was

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diluted to different concentrations (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mg· mL-1). 100 µl

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MC3T3-E1 cells with the density of 4×104 cells/mL were seeded in each hole of 96-hole plate and

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cured in CO2 incubator for 1 day. Then culture medium was replaced with the cement extracts of

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different concentrations. After culturing for 1, 3, and 7 days, the cement extract was removed and 100

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µl MTT solution (0.5 mg· mL-1) was added into each hole. After 4 hours incubation in the CO2

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incubator, the MTT solution was replaced by 100 µl dimethyl sulfoxide (DMSO). The plate was shaken

20

for 5 minutes and tested for optical density (OD) on plate reader (ELX800; Bio-Tek, Winooski, VT,

21

USA).

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2.7 Statistical analysis

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The data were denoted as mean ± standard deviation (SD). The significant differences between

4

two groups of data were tested by the student’s t-test and if the obtained p < 0.05, the two groups of

5

data were considered significantly different.

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3. Results

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3.1 Characterization of the bone cement powder and paste

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The XRD patterns of the C3S, C25M75, C50M50, C75M25 and MPC powders and paste were

10

shown in Fig. 1. In Fig 1A, the C3S powder was pure crystal phase (ICDD PDF no. 49-0442) (a). In

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MPC powders, there were mainly MgO (ICDD PDF no. 45-0946) and NaH2PO4 (ICDD PDF no.

12

11-0659) phases. For the composite groups of C75M25 (b), C50M50 (c) and C25M75 (d), the crystal

13

phases included C3S, MgO and NaH2PO4. The XRD patterns for cement paste changed compared with

14

the powders (Fig. 1B). For C3S (a), the characteristic peaks in its XRD pattern were attributed to

15

calcium silicate hydrate (CSH, ICDD PDF no. 33-0306) which can be denoted as xCaO· SiO 2·yH2O in

16

which x ranges from 1.2 to 2.3 according to literature report [31-33], calcium carbonate (CaCO3, ICDD

17

PDF no. 05-0586) and unhydrated C3S. In the XRD pattern for MPC, there was only unreacted MgO

18

identified (e) and its hydration product was not discernible as it was an amorphous phase according to

19

previous studies [13]. The hydration products of the composite cements (C25M75, C50M50, C75M25)

20

were composed of CSH and CaCO3 from C3S and the amorphous phases, and no newly formed phase

21

was identified (Fig. 1B, b-d). Moreover, for all the cements, the intensity of diffraction peaks increased

11

1

with the amount of its corresponding component.

2

The cross-sectional surface morphology of cements with various weight ratios of C3S after

3

hydration for 7 days was shown in Fig. 2. It can be seen that C3S presented a microporous structure

4

(Fig. 2a), and SEM image with higher magnification showed that the hydrated C3S was composed of

5

aggregates of needle-like particles (Fig. 2b). In comparison, MPC presented a smoother and more

6

compact structure which was composed of gel-like bulks (Fig. 2i) with notable cracks (Fig. 2j). In the

7

composite samples (Fig. 2c-h), both the particles from C3S and the gel-like bulks from MPC coexisted,

8

and the surface morphologies changed as the weight ratio of C3S varied. It can be seen from the lower

9

magnification images that the structure became smoother and more compact with the increase of MPC

10

content (Fig. 2c, e, g), and C25M75 possessed the most compacted structure with least defects such as

11

pores and cracks (Fig. 2g). From the higher magnification images, it can be seen that the particles were

12

wrapped by the gel-like substance gradually with the increase of MPC content, and in C25M75 it was

13

obvious that the particles was almost completely wrapped in the gel-like matrix.

14

To investigate the distribution of components and possible interaction between C3S and MPC

15

within the composite cements during the hydration, EDS-element mapping was conducted on all the

16

cement samples and the results were presented in Fig. 3. The analyzed area was shown by the

17

back-scattered electron image (Fig. 3a) which showed the composition contrast of the composite

18

cements. For C25 M75, it can be seen that there are mainly two kinds of substance in this image: one is

19

the isolated particle (numbered 1), and the other is the continuous matrix (numbered 2) which wrapped

20

the isolated particles. From the individual element mapping images (Fig. 3b), it can be judged that the

21

isolated particle corresponded to Ca and Si elements, and the continuous matrix corresponded to Na, P

12

1

and Mg elements. The boundaries between the Ca-Si area and the Na-P-Mg area were evident.

2

However, for C75M25 and C50M50, the distribution of Ca, Si elements and Na, P and Mg elements

3

overlapped and phenomenon of element enrichment was not obvious as compared with C25M75 (Fig.

4

S1-S4).

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3.2 Mechanical and self-setting properties

7

The change of compressive strength of different cements with curing time from 1 hour to 7 days

8

was presented in Fig. 4A. The testing point for C3S started from 1 day after hydration because it was

9

too weak to obtain a measurable value before that time point. Among all specimens, C25M75 showed

10

the highest compressive strength among all the cements and increased with prolonged curing time. The

11

compressive strength of C25M75 reached 41 MPa and 87 MPa after curing for 1 hour and 7 days,

12

respectively, which was significantly higher than C3S (beyond measurement and 25MPa after 1 hour

13

and 7 days, respectively) and MPC (9 MPa and 64 MPa after 1 hour and 7 days, respectively). For

14

C50M50 and C75M25, the compressive strength was much lower as compared with C25M75.

15

The effect of LPR on compressive strength of C75M25 samples was presented in Fig. 4B. The

16

compressive strength of C75M25 with LPR of 0.35 mL· g-1 was significantly higher than with LPR of

17

0.30, 0.32, 0.38 and 0.40 mL·g-1.

18

The compressive strength of different cements with a constant LPR of 0.4 mL· g-1 and curing time

19

of 24 hours is presented in Fig. 4C. It can be seen that the compressive strength of MPC and composite

20

cements was significantly lower than C3S. Compared with the compressive strength for 1 day with

21

LPR ratio of 0.5 mL· g-1 (Fig. 4A), the compressive strength of C3S with LPR ratio of 0.4 mL· g-1 (Fig.

13

1

4C) increased, while other formulations decreased. Especially for MPC, the compressive strength with

2

LPR of 0.4 mL· g-1 decreased to 0.7 MPa, as compared with 53 MPa with LPR of 0.13 mL· g-1.

3 4

The porosity of C3S, C75M25, C50M50, C25M75 and MPC cement samples decreased gradually as shown in Fig. 4D.

5

The setting time of C3S, C75M25, C50M50, C25M75 and MPC with different LPR were shown

6

in Fig. 5A. The setting time of the C3S and MPC were 113.3 and 9.7 minutes, respectively. For the

7

composite bone cements, C75M25 had a medium setting time of 29.3 minutes as compared with C3S

8

and MPC. However, it was noted that the C50M50 and C25M75 components had setting times lower

9

than that of MPC, which were 9 and 2.9 minutes, respectively. The C75M25 composite cement was

10

further studied to investigate the influence of LPR on setting time. The results showed that setting time

11

increased with the increase of LPR (Fig. 5B).

12

The pH values in cement pastes during setting reaction were measured to investigate possible

13

influences of pH on the setting reaction. The results (Fig. 6) showed that the pH of C3S paste was

14

around 12. The pH of MPC increased from 7.1 to 8.3 after 30 minutes of setting reaction. The pH of

15

C75M25, C50M50, C25M75 was between that of C3S and MPC and increased gradually with the

16

proceeding of setting reactions.

17 18

3.3 Apatite mineralization

19

Fig. 7 shows the results of FTIR analysis on C3S, C25M75, C50M50, C75M25 and MPC cements

20

before (A) and after (B) soaking in SBF solution for 7 days. The FTIR results showed the formation of

21

crystal hydroxyapatite (HA) on C3S and all the C3S/MPC samples, which was identified by the

14

1

characteristic double peaks (563 and 603 cm-1) of PO43- in the crystalline apatite [34]. In the FTIR

2

spectra of MPC, the absorption peak with the wave number of 603 cm-1 was not found in MPC.

3

The cements after soaking in SBF solution for 7 days were further characterized by XRD (Fig. 8).

4

It can be seen that the diffraction peaks of HA (ICDD PDF no. 09-0432) appeared in the XRD pattern

5

of C3S after soaking in SBF. Although a wide dispersing peak in the similar position appeared in the

6

spectra of MPC, it cannot be assigned to apatite as the typical HA-related PO43- was not presented in

7

FTIR measurement of MPC. For C75M25, C50M50 and C25M75, the diffraction peaks of HA were

8

obvious, which further confirmed their apatite formation ability as has been indicated by the results of

9

FTIR. It was found that the diffraction peaks of MgO became stronger as the content of MPC

10

increased.

11

The SEM images of the cements after soaking in SBF solution for 7 days are shown in Fig. 9. It

12

can be seen that for all the cements a layer of minerals formed on the surface. The microstructure of the

13

minerals layer for C3S was flake-like (Fig. 9b), which, according to the IR and XRD results, should be

14

crystalline HA. For MPC, it was small particles ((Fig. 9j). The surfaces of the composite cements were

15

completely covered with flake like minerals, which were similar to that on C3S.

16 17

3.4 Weight loss

18

The in vitro weight loss behavior of the samples during soaking in Tris-HCl solution was

19

presented in Fig. 10. When soaked in Tris-HCl solution, all the cements dissolved gradually with

20

prolonged soaking time. However, they showed different degradation profiles. The degradation rate of

21

C3S remained nearly constant within 28 days, with the weight loss of 4.42% and 16.16% for 7 days

15

1

and 28 days, respectively. In comparison, MPC degraded faster within the first 7 days (11.74%), and

2

slowed down afterwards with the weight loss of 15.9% after 28 days of soaking. The degradation

3

profiles of C25M75, C50M50 and C75M25 were similar to that of MPC. It can be seen that, the weight

4

loss of the composite cements were between that of C3S and MPC within the first 21 days during

5

soaking. However, after 21 days, they were exceeded by C3S because of the higher degradation rate of

6

the latter. Comparison between the composite cements showed that their weight losses were close to

7

each other, and no significant differences were identified.

8

The concentrations of ions released in Tris-HCl solution during different soaking time were

9

presented in Fig. 11. It can be seen that Ca2+, SiO44- and Mg2+ ions showed sustained release and the

10

release of Mg2+, Na+ and PO43- and B4O72- was much more rapid in the initial 7 days than that in the

11

following 21 days. The pH values of Tris-HCl solution mainly varied from 7.2 to 8.2 during the first 7

12

days of soaking time (Fig. 12).

13 14

3.5 In vitro cytocompatibility

15

The C25M75 composite cement was selected for the evaluation of in vitro cytocompatibility

16

because it showed the highest compressive strength and maintained good apatite mineralization ability.

17

C3S and MPC were selected as control groups. From the cell culture results (Fig. 13), it can be seen

18

that when the concentration of ionic extracts below 12.5 mg· mL-1, MC3T3-E1 cells showed good

19

viability for all the cements, as compared with the blank control group. However, when the ionic

20

extract concentration was above 12.5 mg· mL-1, cell proliferation decreased for all the cements, as

21

compared with the blank control group. In particular, at some concentrations, C25M75 showed

16

1

significantly higher level of cell proliferation as compared with other groups, suggesting the

2

stimulatory effect of C25M75 on the cell proliferation.

3 4

4. Discussion

5

Mechanical property is one important consideration in clinical application of bone cements. As

6

the hydration of C3S is a gradual process, the short-term compressive strength of C3S cements is quite

7

low. In contrast, MPC possesses a high initial compressive strength owing to its fast-setting and

8

relatively compact structure after hydration. Our results confirmed our assumption that the combination

9

of C3S and MPC resulted in cements with high mechanical strength. The most interesting finding is

10

that the C3S/MPC composite cements revealed an even higher compressive strength than MPC

11

cements if the ratio of the C3S and MPC components was optimally selected. In the present study, the

12

compressive strength of C25M75 after curing for 7 days reaches 86 MPa, which is much higher than

13

both components [35-39] and also comparable to the widely used PMMA [40]. An explanation for this

14

enhancement phenomenon is that, in C25M75 the main morphology observed in SEM images is the

15

gel-like bulks generated and CSH particles are wrapped in it, functioning as reinforcement fillers, and

16

the microstructure is compact with less defects. In contrast, with higher content of C3S, the original

17

integrated bulk matrix of MPC becomes discontinuous and disintegrated and thus, the whole structure

18

becomes porous and the porosity increases as shown in Fig. 4D, which leads to decrease of

19

compressive strength. Therefore, it is assumed that the improved mechanical strength is mainly

20

attributed to the compact structure and the enhancement effect of CSH fillers, and the lower

21

compressive strength for C50M50 and C75M25 can be explained by the loose microstructure due to the

17

1

poor match of the two components. In addition, the amorphous phases as the setting products of

2

C75M25, C50M50, C25M75 and MPC may be different, which is another possible explanation for the

3

improved compressive strength of C25M75. Our results indicate that the microstructure of the

4

composite cements is critical for the mechanical strength of the composite cements, and it can be

5

controlled by relative weight ratios of C3S and MPC, and the optimal weight ratio of C3S should be

6

around 25%. Thus, the content of C3S must be controlled within a certain range to obtain high

7

compressive strength.

8

LPR ratio influences the mechanical strength of bone cements by directly influencing the porosity.

9

With the composition constant, compressive strength of C75M25 increased first and then decreased

10

with the increase of LPR, which suggests that an optimal LPR exists for composite cements cement to

11

reach high compressive strength. When the LPR was kept as 0.4 mL·g-1, the compressive strength of

12

MPC and the composite cements decreased significantly. Therefore, both composition and LPR are

13

important factors to consider in optimizing the mechanical property of C3S/MPC composite bone

14

cement.

15

Besides compressive strength, setting time is another important factor that influences the clinical

16

applications of bone cement. The setting process for C3S mainly involves a hydraulic reaction

17

(3CaO·SiO2 + (3-x+y)H2O → xCaO·SiO2·yH2O + (3-x)Ca(OH)2, in which x ranges from 1.2 to 2.3

18

according to literature report) [31-33, 41]. The hydration product CSH is in the form of a solid network

19

which is the main reason for cement setting and strength development. As the hydration of C3S is a

20

gradual process, it takes relatively long time for the cement to set. While for MPC, the setting process

21

is a rapid acid-base reaction between the acid reactant of NaH2PO4 and the basic reactant of MgO [13],

18

1

which is responsible for the fast-setting behavior of MPC. In the C3S/MPC composite cements, the

2

hydration products were the combination of the corresponding ingredients, and the amorphous products

3

of the setting reaction may be different, leading to difference in setting time.

4

inter-reactions between the components in the C3S/MPC composite cements. The hydration of C3S

5

induces formation of Ca(OH)2 which may further react with NaH2PO4 to form amorphous calcium

6

phosphates. The pH values of C3S and MPC cement pastes differ significantly. Thus, the setting

7

reaction of each component in the composite cement may be further affected by the distinct

8

discrepancy of pH between them. The setting time for the composite cements can be modified by

9

adjusting their compositions in the present study. For example, with high amount of MPC (above 50%),

10

the setting time of the composite cements are close to MPC, which may meet the clinical requirement

11

regarding the setting time of a bone cement.

Besides, there may be

12

The formation of a layer of apatite is important for the binding of orthopaedic implant materials to

13

living bone tissue, and this bone binding ability of materials can be evaluated in vitro in SBF solution

14

[28]. The present study shows that C3S and C3S/MPC composite cements induced the formation of

15

apatite layer on their surface in SBF solution. As there was no apatite formed on the surface of MPC

16

after soaking, the apatite layers formed on the surface of composite cements can be attributed to the

17

presence of C3S within the materials. The mechanism of the apatite mineralization is that the Ca2+ ions

18

release firstly from C3S to form a Si-rich layer, which then induces formation of Ca-P nucleation and

19

further apatite crystals [42]. Previous studies on the calcium silicate-containing composite bone

20

cements also revealed similar results, in which C3S contributed to the apatite formation in composite

21

cements [43]. Through this apatite layer, bone and material can form a chemical bond which may lead

19

1

to good bone-material integration [44, 45].

2

For successful bone regeneration, the implant material should be biodegradable so that they can

3

be replaced by regenerated new bone tissue gradually [46]. In the present study, all of the five cements

4

degraded gradually in Tris-HCl buffer solution with prolonged soaking time, and their degradation rates

5

varied with different chemical composition of the cements. In the composite cements, degradation

6

behavior is dominated by MPC as their degradation curves are similar with that of MPC cements.

7

However, the degradation rates of the composites are lower than MPC because of the relatively slower

8

degradation of C3S. Consequently, it is possible to acquire composite cements with different

9

degradation rate by adjusting the composition.

10

Previous studies have shown that C3S and MPC had good cytocompatibility [3, 6, 12]. Our in

11

vitro results showed that the extracts of the composite cements have good cytocompatibility at the

12

concentration lower than 12.5 mg· L-1. Moreover, the extracts of C25M75 composite cement showed

13

stimulatory effect on the proliferation of MC3T3-E1 cells as compared with all other groups. The

14

higher level of cell proliferation of C25M75 may be attributed to synergistic stimulatory effect of Si

15

and Mg ions released from the C25M75 cement on the proliferation of osteoblast-related cells, as the

16

case in akermanite, a Mg and Si-containing bioceramic with osteostimulation property [47]. However,

17

since C25M75 is a multicomponent system, other ions, such as Ca and B may have synergistic effect

18

on cell proliferation. Our results also showed that higher concentration resulted in certain degree of

19

cytotoxicity in all three groups such as pure MPC, C3S and the composite cements. Considering that in

20

the physiological conditions, the actual concentration of the dissolved ions from the implanted cement

21

will not be the same as the ion release obtained by the extraction protocol in current study, this result

20

1

does not mean that the cements are not biocompatible when they are implanted in vivo. Some previous

2

studies with bioactive glasses, calcium silicate bioceramics and MPC cements have demonstrated good

3

in vivo biocompatibility, although cell culture studies also revealed certain degree of cytotoxicity [19,

4

24, 48-50]. An explanation of different cytocompatibility between our results and that from literature

5

for MPC could be the difference in cement composition and cell types, which we know they react

6

differently to different composition of materials. Nevertheless, our results indicate that we do need to

7

consider cytotoxicity effect of the cements if we apply the materials for some applications such as

8

tissue engineering approach, in which a direct contact of cells with the materials scaffolds are required.

9

The MTT assay result suggests that the C25M75 composite cement may have the activity to stimulate

10

bone regeneration as compared with C3S and MPC. However, further investigations are needed to

11

evaluate its bioactivity and biocompatibility both in vitro and in vivo.

12

Although previous studies reported that Mg ions could stimulate proliferation and differentiation

13

of osteoblast cells [25, 26, 51-53], the MPC cements in the present study did not show stimulatory

14

effect on cell proliferation. Therefore, the function of Mg ions may be different in different

15

Mg-containing materials. In addition, it remains to be confirmed whether the bioactive mechanism of

16

the MPC/C3S composite cements are different from that of the pure MPC or C3S.

17 18 19

5. Conclusion

20

In the present study, novel C3S/MPC composite cements with high compressive strength and

21

short setting time were developed. The compressive strength and setting time of the composite cements

21

1

could be modulated by the weight ratio of C3S and MPC. Among the composite cements, C25M75

2

showed the highest compressive strength of 86MPa, which is close to the lower limit of human cortical

3

bone (90-209 MPa) and much higher than C3S and MPC. The composite bone cement has good apatite

4

mineralization ability in SBF solution and degraded gradually with a moderate degradation rate in vitro.

5

Moreover, the composite cement showed good in vitro cytobiocompatibility and stimulated the

6

proliferation of MC3T3-E1 osteoblast cells. These results suggest that, through combination of C3S

7

and MPC, it is possible to obtain composite cements which keep or even surpass the advantages of high

8

compressive strength and bioactivity of its individual components. The bioactive high-strength

9

C3S/MPC composite cement in the present study might have potential for orthopaedic applications.

10 11

Disclosures

12

There is no potential conflict of interest for this study.

13 14

Acknowledgements

15

We acknowledge the financial support from the National Natural Science Foundation of China

16

(Grant No. 81190132), the External Cooperation Program of the Chinese Academy of Sciences (Grant

17

No.GJHZ1211), One-hundred Talent Program of SIC-CAS (Y36ZB1110G), and the Funds of the

18

Clinical Reseach Center for Biomaterials, SIC-CAS (Grant No. BMCRC2010002).

19

22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

References [1] Low KL, Tan SH, Zein SHS, Roether JA, Mourino V, Boccaccini AR. Calcium phosphate-based composites as injectable bone substitute materials. J Biomed Mater Res B 2010;94B:273-86. [2] Vallet-Regi M, Gonzalez-Calbet JM. Calcium phosphates as substitution of bone tissues. Prog Solid State Ch 2004;32:1-31. [3] Wu F, Wei J, Guo H, Chen FP, Hong H, Liu CS. Self-setting bioactive calcium-magnesium phosphate cement with high strength and degradability for bone regeneration. Acta Biomater 2008;4:1873-84. [4] Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Edit 2002;41:3130-46. [5] Gandolfi MG, Taddei P, Tinti A, Prati C. Apatite-forming ability (bioactivity) of ProRoot MTA. Int Endod J 2010;43:917-29. [6] Zhao WY, Wang JY, Zhai WY, Wang Z, Chang J. The self-setting properties and in vitro bioactivity of tricalcium silicate. Biomaterials 2005;26:6113-21. [7] Gou ZG, Chang J, Zhai WY, Wang JY. Study on the self-setting property and the in vitro bioactivity of beta-Ca2SiO4. J Biomed Mater Res B 2005;73B:244-51. [8] Peng WW, Liu WN, Zhai WY, Jiang L, Li LF, Chang J, et al. Effect of tricalcium silicate on the proliferation and odontogenic differentiation of human dental pulp cells. J Endodont 2011;37:1240-6. [9] Du R, Wu T, Liu W, Li L, Jiang L, Peng W, et al. Role of the extracellular signal-regulated kinase 1/2 pathway in driving tricalcium silicate–induced proliferation and biomineralization of human dental pulp cells in vitro. J Endodont 2013;39:1023-9. [10] Huan ZG, Chang J. Novel. bioactive composite bone cements based on the beta-tricalcium phosphate-monocalcium phosphate monohydrate composite cement system. Acta Biomater 2009;5:1253-64. [11] Hoppe A, Güldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011;32:2757-74. [12] Zhao WY, Chang J, Zhai WY. Self-setting properties and in vitro bioactivity of Ca3SiO5/CaSO 4 ·1/2H2O composite cement. Journal of Biomedical Materials Research Part A 2008;85A:336-44. [13] Mestres G, Ginebra MP. Novel magnesium phosphate cements with high early strength and antibacterial properties. Acta Biomater 2011;7:1853-61. [14] Sugama T, Kukacka LE. Magnesium monophosphate cements derived from diammonium phosphate solutions. Cement Concrete Res 1983;13:407-16. [15] Popovics S, Rajendran N, Penko M. Rapid hardening cements for repair of concrete. Aci Mater J 1987;84:64-73. [16] Jia JF, Zhou HJ, Wei J, Jiang X, Hua H, Chen FP, et al. Development of magnesium calcium phosphate biocement for bone regeneration. J R Soc Interface 2010;7:1171-80. [17] Klammert U, Reuther T, Blank M, Reske I, Barralet JE, Grover LM, et al. Phase composition, mechanical performance and in vitro biocompatibility of hydraulic setting calcium magnesium phosphate cement. Acta Biomater 2010;6:1529-35. [18] Moseke C, Saratsis V, Gbureck U. Injectability and mechanical properties of magnesium phosphate cements. J Mater Sci-Mater M 2011;22:2591-8. 23

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[19] Kanter B, Geffers M, Ignatius A, Gbureck U. Control of in vivo mineral bone cement degradation. Acta Biomater 2014;10:3279-87. [20] Tamimi F, Le Nihouannen D, Bassett DC, Ibasco S, Gbureck U, Knowles J, et al. Biocompatibility of magnesium phosphate minerals and their stability under physiological conditions. Acta Biomater 2011;7:2678-85. [21] Lee J, Farag MM, Park EK, Lim J, Yun HS. A simultaneous process of 3D magnesium phosphate scaffold fabrication and bioactive substance loading for hard tissue regeneration. Mater Sci Eng C-Mater Biol Appl 2014;36:252-60. [22] Yu YL, Wang J, Liu CS, Zhang BW, Chen HH, Guo H, et al. Evaluation of inherent toxicology and biocompatibility of magnesium phosphate bone cement. Colloids Surfaces B 2010;76:496-504. [23] Wei J, Jia JF, Wu F, Wei SC, Zhou HJ, Zhang HB, et al. Hierarchically microporous/macroporous scaffold of magnesium-calcium phosphate for bone tissue regeneration. Biomaterials 2010;31:1260-9. [24] Klammert U, Vorndran E, Reuther T, Muller FA, Zorn K, Gbureck U. Low temperature fabrication of magnesium phosphate cement scaffolds by 3D powder printing. J Mater Sci-Mater M 2010;21:2947-53. [25] Wang G, Li J, Zhang W, Xu L, Pan H, Wen J, et al. Magnesium ion implantation on a micro/nanostructured titanium surface promotes its bioactivity and osteogenic differentiation function. Int J Nanomed 2014;9:2387-98. [26] Park JW, Kim YJ, Jang JH, Song H. Osteoblast response to magnesium ion-incorporated nanoporous titanium oxide surfaces. Clin Oral Implan Res 2010;21:1278-87. [27] Zhao WY, Chang J. Sol-gel synthesis and in vitro bioactivity of tricalcium silicate powders. Mater Lett 2004;58:2350-3. [28] Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 2006;27:2907-15. [29] Zhang ML, Lin KL, Chang J. Preparation and characterization of Sr-hardystonite (Sr2ZnSi2O7) for bone repair applications. Mater Sci Eng C-Mater Biol Appl 2012;32:184-8. [30] ISO/EN 10993-5. Biological evaluation of medical devices—Part 5. Tests for cytotoxicity, in vitro methods: 8.2 tests on extract. [31] Kirby DM, Biernacki JJ. The effect of water-to-cement ratio on the hydration kinetics of tricalcium silicate cements: Testing the two-step hydration hypothesis. Cement Concrete Res 2012;42:1147-56. [32] Richardson IG. The nature of C-S-H in hardened cements. Cement Concrete Res 1999;29:1131-47. [33] Richardson IG. The nature of the hydration products in hardened cement pastes. Cement and Concrete Composites 2000;22:97-113. [34] Fowler BO. Infrared studies of apatites .1. vibrational assignments for calcium, strontium, and barium hydroxyapatites utilizing isotopic-substitution. Inorg Chem 1974;13:194-207. [35] Huan ZG, Chang J. Novel tricalcium silicate/monocalcium phosphate monohydrate composite bone cement. J Biomed Mater Res B 2007;82B:352-9. [36] Huan ZG, Chang J. Study on physicochemical properties and in vitro bioactivity of tricalcium silicate-calcium carbonate composite bone cement. J Mater Sci-Mater M 2008;19:2913-8. [37] Liu WN, Chang J, Yue Z. Physicochemical properties and biocompatibility of tricalcium and dicalcium silicate composite cements after hydration. Int J Appl Ceram Technol 2011;8:560-5. 24

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[38] Liu WN, Peng WW, Zhu YQ, Chang J. Physicochemical properties and in vitro biocompatibility of a hydraulic calcium silicate/tricalcium aluminate cement for endodontic use. J Biomed Mater Res B 2012;100B:1257-63. [39] Liu WN, Chang J. Effect of tricalcium aluminate on the properties of tricalcium silicate-tricalcium aluminate mixtures:setting time, mechanical strength, and biocopatity. Int Endod J 2010:10. [40] Kim S, Greenleaf R, Miller MC, Satish L, Kathju S, Ehrlich G, et al. Mechanical effects, antimicrobial efficacy and cytotoxicity of usnic acid as a biofilm prophylaxis in PMMA. J Mater Sci-Mater M 2011;22:2773-80. [41] Peterson VK, Neumann DA, Livingston RA. Hydration of tricalcium and dicalcium silicate mixtures studied using quasielastic neutron scattering. J Phys Chem B 2005;109:14449-53. [42] Niu LN, Jiao K, Wang TD, Zhang W, Camilleri J, Bergeron BE, et al. A review of the bioactivity of hydraulic calcium silicate cements. J Dent 2014;42:517-33. [43] Huan Z, Chang J. Self-setting properties and in vitro bioactivity of calcium sulfate hemihydrate-tricalcium silicate composite bone cements. Acta Biomater 2007;3:952-60. [44] Ohura K, Nakamura T, Yamamuro T, Kokubo T, Ebisawa Y, Kotoura Y, et al. Bone-bonding ability of P2O5-free CaO·SiO2 glasses. J Biomed Mater Res 1991;25:357-65. [45] Xue WC, Liu XY, Zheng XB, Ding CX. In vivo evaluation of plasma-sprayed wollastonite coating. Biomaterials 2005;26:3455-60. [46] Hench LL, Polak JM. Third-generation biomedical materials. Science 2002;295:1014-7. [47] Huang Y, Jin X, Zhang X, Sun H, Tu J, Tang T, et al. In vitro and in vivo evaluation of akermanite bioceramics for bone regeneration. Biomaterials 2009;30:5041-8. [48] Abiraman S, Varma HK, Kumari TV, Umashankar PR, John A. Preliminary in vitro and in vivo characterizations of a sol-gel derived bioactive glass-ceramic system. B Mater Sci 2002;25:419-29. [49] Ni SY, Chang J. In vitro degradation, bioactivity, and cytocompatibility of calcium silicate, dimagnesium silicate, and tricalcium phosphate bioceramics. J Biomater Appl 2009;24:139-58. [50] Xu S, Lin K, Wang Z, Chang J, Wang L, Lu J, et al. Reconstruction of calvarial defect of rabbits using porous calcium silicate bioactive ceramics. Biomaterials 2008;29:2588-96. [51] Zreiqat H, Howlett CR, Zannettino A, Evans P, Schulze-Tanzil G, Knabe C, et al. Mechanisms of magnesium-stimulated adhesion of osteoblastic cells to commonly used orthopaedic implants. J Biomed Mater Res 2002;62:175-84. [52] Yamasaki Y, Yoshida Y, Okazaki M, Shimazu A, Uchida T, Kubo T, et al. Synthesis of functionally graded MgCO3 apatite accelerating osteoblast adhesion. J Biomed Mater Res 2002;62:99-105. [53] Sader MS, LeGeros RZ, Soares GA. Human osteoblasts adhesion and proliferation on magnesium-substituted tricalcium phosphate dense tablets. J Mater Sci-Mater M 2009;20:521-7.

25

1

Figure captions

2

Fig. 1. XRD patterns of (a) C3S, (b) C75M25, (c) C50M50, (d) C25M75 and (e) MPC powders (A),

3

and bone cements after curing under the environment of 37 °C and 100% humidity for 7 days (B). The

4

phases were identified according to the following PDF number: MgO (ICDD PDF no. 45-0946),

5

NaH2PO4 (ICDD PDF no. 11-0659), C3S (ICDD PDF no. 49-0442), CSH (ICDD PDF no. 33-0306),

6

CaCO3 (ICDD PDF no. 05-0586) .

7

Fig. 2. SEM images of (a, b) C3S, (c, d) C75M25, (e, f) C50M50, (g, h) C25M75 and (i, j) MPC bone

8

cements after curing under the environment of 37 °C and 100% humidity for 7 days.

9

Fig. 3. (a) Atomic number contrast image of the C25M75 bone cement by using the back-scattered

10

electron mode of FESEM; (b) Elemental mapping images of the C25M75 bone cement after curing

11

under the environment of 37 °C and 100% humidity for 7 days. The numbers 1 and 2 represent the

12

distribution area of Ca-Si elements and Na-P-Mg elements, respectively.

13

Fig. 4. Compressive strength of the C3S, C75M25, C50M50, C25M75 and MPC bone cements with

14

the LPR of 0.5, 0.35, 0.25, 0.18 and 0.13 mL· g-1, respectively, after curing for different periods (A);

15

compressive strength of C75M25 with the variation of LPR, after curing for 24 hours (B); compressive

16

strength of C3S, C75M25, C50M50, C25M75 and MPC with a constant of LPR (0.4 mL·g-1) , after

17

curing for 24 hours (C), and porosity of C3S, C75M25, C50M50, C25M75 and MPC bone cements,

18

after curing for 7 days. The curve for C3S in (A) starts from 1 day because its compressive strength

19

was too low to be measured before 1 day. The symbol “*” in (A) denotes existence of significant

20

difference between MPC and other groups with the same LPR, and (B) it denotes existence of

21

significant difference between the C75M25 with LPR of 0.35 mL·g-1 and with other LPRs; The symbol

22

“×” in (C) and (D) denotes non-existence of significant difference between two groups. 26

1

Fig. 5. Setting time of the C3S, C75M25, C50M50, C25M75 and MPC bone cements with the LPR of

2

0.5, 0.35, 0.25, 0.18 and 0.13 mL· g-1, respectively (A) and the setting time of C75M25 with the

3

variation of LPR (B). The symbol “×” in (A) and (B) denotes non-existence of significant difference

4

between two groups.

5

Fig. 6. pH values of the C3S, C75M25, C50M50, C25M75 and MPC bone cement pastes with

6

prolonged hydration time.

7

Fig. 7. FTIR spectra of the C3S, C75M25, C50M50, C25M75 and MPC bone cements before (A) and

8

after (B) soaking in SBF solution for 7 days.

9

Fig. 8. XRD patterns of the C3S, C75M25, C50M50, C25M75 and MPC bone cements after soaking in

10

SBF solution for 7 days. The phases were identified according to the following PDF number: HA

11

(ICDD PDF no. 09-0432), CSH (ICDD PDF no. 33-0306), CaCO3 (ICDD PDF no. 05-0586), MgO

12

(ICDD PDF no. 45-0946).

13

Fig. 9. SEM images of (a, b) C3S, (c, d) C75M25, (e, f) C50M50, (g, h) C25M75 and (i, j) MPC bone

14

cements after soaking in SBF solution for 7 days.

15

Fig. 10. Weight loss of the C3S, C75M25, C50M50, C25M75 and MPC bone cements after soaking in

16

Tris-HCl solution for different periods. Significant differences exist between the following groups: (7

17

days) C3S and all the other four cements, and MPC and all the other four groups; (14 days) C3S and

18

C75M25, C3S and MPC, C75M25 and MPC, C25M75 and MPC; (14 days) MPC and C3S, MPC and

19

C75M25, MPC and C25M75; (28 days) C50M50 and MPC;

20

Fig. 11. Ions concentrations in Tris-HCl buffer solutions with different soaking time and cement

21

samples.

27

1

Fig. 12. pH values in Tris-HCl buffer solutions with different soaking time and cement samples.

2

Fig. 13. MTT assay of osteoblast-like (MC3T3-E1) cells cultured in α-MEM medium conditioned with

3

different concentrations of C3S, C25M75 and MPC powder extracts. The symbols *, § and

4

represent significant difference as compared with blank, C3S and MPC, respectively.

5

Fig. S1. (a) Atomic number contrast image of the C3S bone cement by using the back-scattered

6

electron mode of FESEM; (b) Elemental mapping images of the C3S bone cement after curing under

7

the environment of 37 °C and 100% humidity for 7 days.

8

Fig. S2. (a) Atomic number contrast image of the C75M25 bone cement by using the back-scattered

9

electron mode of FESEM; (b) Elemental mapping images of the C75M25 bone cement after curing

□

10

under the environment of 37 °C and 100% humidity for 7 days.

11

Fig. S3. (a) Atomic number contrast image of the C50M50 bone cement by using the back-scattered

12

electron mode of FESEM; (b) Elemental mapping images of the C50M50 bone cement after curing

13

under the environment of 37 °C and 100% humidity for 7 days.

14

Fig. S4. (a) Atomic number contrast image of the MPC bone cement by using the back-scattered

15

electron mode of FESEM; (b) Elemental mapping images of the MPC bone cement after curing under

16

the environment of 37 °C and 100% humidity for 7 days.

17 18

28

1 2 3

Fig. 1

4 29

1 2 3

Fig. 2

4 30

1 2 3

Fig. 3

4 5 6

31

1 2 3

Fig. 4

4 5

32

1 2 3

Fig. 5

4 5

33

1 2 3

Fig. 6

4 5

34

1 2 3 4 5

Fig. 7

6 7

35

1 2 3

Fig. 8

4 5

36

1 2 3

Fig. 9

4 5

37

1 2 3

Fig. 10

4 5

38

1 2 3

Fig. 12

4 5

39

1 2 3

Fig. 13

4 5

40

1 2 3

Table 1. Particle size distribution and specific surface area (SSA) of C3S, MgO, NaH2PO4 and borax powder obtained by laser diffraction analysis. D10, D50 and D90 mean the particle size below which the amount of particles takes up 10%, 50% and 90%, respectively.

C3S

4 5 6 7 8 9 10 11 12 13 14

SSA (m2·g-1)

D10 (µm)

D50 (µm)

D90 (µm)

0.860

2.80

13.10

190.00

MgO

1.425

1.93

5.97

16.10

NaH2PO4

0.360

10.30

25.90

65.20

Borax

0.485

6.28

17.50

36.60

Table 2. Composition of SBF (1000 mL) used in the apatite mineralization study.

Amount Purity (%)

Amount

Purity (%)

NaCl

NaHCO3

KCl

K2HPO4·3H2O

MgCl2·6H2O

8.035g

0.355g

0.255g

0.231g

0.311g

99.5

99.5

99.5

99.0

98.0

1.0M-HCl

CaCl2

NaSO4

Tris

1.0M-HCl

39mL

0.292g

0.072g

6.118g

0-5mL

--

95.0

99.0

99.0

--

15 16 17

41

1 2 3 4

Graphical abstract

5

42