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Novel bioactive composite bone cements based on the β-tricalcium phosphate–monocalcium phosphate monohydrate composite cement system
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Acta Biomaterialia 5 (2009) 1253–1264 www.elsevier.com/locate/actabiomat
Novel bioactive composite bone cements based on the b-tricalcium phosphate–monocalcium phosphate monohydrate composite cement system Zhiguang Huan a,b, Jiang Chang a,* a
Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, People’s Republic of China b Graduate School of the Chinese Academy of Sciences, 319 Yueyang Road, Shanghai 200050, People’s Republic of China Received 26 May 2008; received in revised form 10 September 2008; accepted 8 October 2008 Available online 22 October 2008
Abstract Bioactive composite bone cements were obtained by incorporation of tricalcium silicate (Ca3SiO5, C3S) into a brushite bone cement composed of b-tricalcium phosphate [b-Ca3(PO4)2, b-TCP] and monocalcium phosphate monohydrate [Ca(H2PO4)2H2O, MCPM], and the properties of the new cements were studied and compared with pure brushite cement. The results indicated that the injectability, setting time and short- and long-term mechanical strength of the material are higher than those of pure brushite cement, and the compressive strength of the TCP/MCPM/C3S composite paste increased with increasing aging time. Moreover, the TCP/MCPM/C3S specimens showed significantly improved in vitro bioactivity in simulated body fluid and similar degradability in phosphate-buffered saline as compared with brushite cement. Additionally, the reacted TCP/MCPM/C3S paste possesses the ability to stimulate osteoblast proliferation and promote osteoblastic differentiation of the bone marrow stromal cells. The results indicated that the TCP/MCPM/C3S cements may be used as a bioactive material for bone regeneration, and might have significant clinical advantage over the traditional b-TCP/MCPM brushite cement. Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Calcium phosphate cement; Brushite; In vitro test; Cytotoxicity; Silicate
1. Introduction Materials with self-setting properties have been exploited to augment human bone tissues [1–4], and resorbable calcium phosphate cements based on brushite have raised interests in recent years [5–8]. These kinds of cement are usually presented as a solid phase and a liquid phase. These two components are mixed together in order to form a workable paste that would set into a hard material. The self-setting occurs through an acid–base reaction between a basic calcium phosphate, usually b-tricalcium phosphate (b-TCP), and an acidic phosphate such as orthophosphoric
*
Corresponding author. Tel.: +86 21 52412804; fax: +86 21 52413903. E-mail address: [email protected] (J. Chang).
acid or monocalcium phosphate monohydrate (MCPM) [5– 9]. In clinical applications, the brushite cements can be used in the form of blocks or as a self-setting paste, which could provide scaffolds for bone regeneration and be gradually replaced by tissue [6,7,10]. However, this cement system still has some inherent drawbacks that need to be dealt with. Due to the extremely rapid setting reaction, the brushite cement shows a rather short setting time that makes it difficult to work with, which also results in high porosity and consequently weak mechanical strength [11,12]. In addition, as soluble acidic phosphates are used as sources of phosphate ions, the brushite cement usually will cause a rapid decrease of pH in vivo immediately after implantation. This phenomenon can have an adverse impact on the biocompatibility of the material [13,14]. Other studies also showed that the calcium phosphate cement is less bioactive as compared
1742-7061/$ - see front matter Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2008.10.006
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with some silicate-containing bioactive materials such as AW glass-ceramic and Bioglass [15,16]. Therefore, incorporation of bioactive materials has been investigated to improve the bioactivity of the cement materials [17,18]. Tricalcium silicate (Ca3SiO5) is one of the main components of Portland cement. Once mixed with water, Ca3SiO5 will react with water to create calcium–silicate–hydrate (C–S–H). The polymerization and solidification of the C– S–H network contribute to the self-setting properties and increased mechanical strength of the tricalcium silicate paste after aging. Recent studies have shown that the tricalcium silicate cements are bioactive and can induce bonelike apatite formation in simulated body fluid [19,20], which is partly attributed to the release of silicate ions. The silicate ions are also known to be an effective additive to improve the bioactivity and dissolution rate of hydroxyapatite [21–23]. Furthermore, these materials have the potential to activate bone-related gene expression and stimulate cell proliferation [19,20,24]. It has also been proven that the hydration product of tricalcium silicate cement is degradable in simulated body fluid (SBF) [20]. However, this material shows a rather low self-setting rate, and the setting time of the cement paste is too long (>60 min), which may not be suitable for orthopaedic applications [20]. In this paper, considering the advantages and disadvantages of the brushite and Ca3SiO5 cements, composite cements were designed and prepared by mixing the bTCP/MCPM brushite cement system with bioactive Ca3SiO5. The setting time, workability, mechanical properties, in vitro bioactivity and cytotoxicity of the b-TCP/ MCPM/C3S composite cements were evaluated and compared with those of the brushite cement.
2. Materials and methods 2.1. Material preparation and characterization The brushite cement used in this study was prepared by mixing two phosphate powders in the presence of water. The starting powders were b-TCP (synthesized in-house) and monocalcium phosphate monohydrate (Sinopham Chemical Reagent Co., Ltd). The weight ratio of the two components (b-TCP:MCPM) is 3:2, and they react in the presence of water to form precipitated brushite (dicalcium phosphate dehydrate) according to he reaction presented below [8,25].
mixed with the brushite cement powders. To prepare paste samples, powders were mixed with deionized water using a solid/liquid (S/L) ratio of 2.0 g ml1. The cement paste was prepared in a mortar, thoroughly kneaded with a spatula and poured into a cylindrical polytetrafluoroethylene mold. Air bubbles entrapped in the paste during mixing were allowed to escape by gently shaking the mold (6 mm diameter and 12 mm height). After demolding, the samples were placed in a 100% humidity water bath at 37 °C and kept for 24 h. The phase composition was characterized by X-ray diffraction (XRD; Geigerflex, Rigaku Co., Japan) using monochromated Cu Ka radiation, and the 2h range was from 10° to 60° at a scanning speed of 10° min1. The cross-section of the samples was observed by scanning electron microscopy (SEM) using a scanning electron microscope (JSM-6700F, JEOL, Tokyo, Japan) equipped with an energy dispersive X-ray (EDX) detector (INCA Energy, Oxiford Instruments, UK). 2.2. Setting times and injectability The setting times of the composite pastes with 0%, 10%, 20%, 30%, and 40% content of Ca3SiO5 were measured with the Vicat needle according to ISO9597-1989E. To test the initial setting time of the paste, a needle weighing 280 g and of 1.13 mm diameter is lowered into a specimen of fresh cement paste and the penetration depth is recorded. The cement paste is kept in a standard frustum 40 mm in height. The initial setting time is when the needle penetration is 35 ± 1.0 mm. The final setting time is defined as the time necessary so that the heavy needle (350 g, Ø 2.0 mm) no longer leaves a visible print on the surface of the paste. The measurement was conducted on five specimens from a group of the same composition, and then the average value was calculated. The injectability of the composite paste was evaluated by extruding a certain amount of paste through a disposable syringe by hand according to a modified method described previously [19,26,27]. The syringes have a capacity of 2.5 ml, with an opening nozzle diameter of 2.0 mm. Six grams of as-prepared paste was added into the syringe and, after storage in a water bath at 37 °C for 2 min, the paste was gently extruded from the syringe by hand until it could no longer be continued. Then the weight of the paste expelled from the syringe was measured and the injectability was calculated using Eq. (1) [27]. Each test was repeated at least three times and the average value was calculated.
Ca3 ðPO4 Þ2 þ CaðH2 PO4 Þ2 H2 O þ 7H2 O ! 4CaHPO4 2H2 O Tricalcium silicate (Ca3SiO5) powders were prepared by the sol–gel method, as previously described [19], and they were ground and sieved through a 300-mesh sieve (50 lm). To prepare the brushite/Ca3SiO5 composite cements, Ca3SiO5 powders (0–40 wt.%) were uniformly
Inj% ¼
Paste weight expelled from the syringe Total paste weight before injecting
ð1Þ
2.3. Mechanical test of the cement paste For the compressive mechanical testing, the as-prepared slurries were poured into a cylindrical polytetrafluoroethyl-
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ene mold (6 mm diameter and 12 mm height). Air bubbles entrapped in the paste during mixing were allowed to escape by gently shaking the mold. After aging in a 100% humidity water bath or phosphate-buffered saline (PBS) [28] at 37 °C for 24 h and 4, 7, 14, 21 and 28 days, the reacted paste samples were immediately removed from the molds and the compressive strength was measured at a loading rate of 0.5 mm min1 using a universal testing machine (Instron-1195, USA), according to ASTM D69591. Six replicates were carried out for each group and the results were expressed as mean ± standard deviation (SD). 2.4. pH measurement To test the in vitro pH variation of the cement paste in a simulated body environment, 5 ml fresh cement slurries were directly poured into a 25 ml beaker and then 15 ml of SBF was injected into the container to cover the paste [29,30]. The paste was allowed to set in the SBF without any further shaking, and the test samples were stored in a 37 °C, 100% humidity water bath. After 1 h of immersion, all the SBF used for the immersion was collected by a syringe and its pH value was measured using an electrolyte-type pH meter (pHS-2 C, Jingke Leici Co., Shanghai, China). Fresh SBF, equal in volume to the removed suspension, was then added to the system to renew the immersion medium. The procedure was repeated every hour for a period of 12 h. 2.5. In vitro bioactivity The SBF solution was prepared according to the procedure described by Kokubo [31]. The ion concentrations of the SBF are similar to those in human blood plasma, and the pH value was adjusted to 7.35 [31]. The 24-h-set paste disks (6 mm in diameter and 2 mm in height) were soaked in SBF solution at 37.0 °C in a shaking water bath for 7 days with a surface area-to-volume ratio of 0.1 cm1 [31,32]. After the pre-selected soaking time, the disks were gently rinsed with deionized water to remove SBF solutions followed by drying at room temperature. The samples were characterized by SEM and EDX. 2.6. In vitro degradation of the paste For evaluation of degradation, the 7-day-set paste disks were soaked in PBS at 37 °C in shaking water bath for 4, 7, 10, 14 and 21 days with a surface area-to-volume ratio of 0.1 cm1 [28,31]. The solution was refreshed every day. After the set soaking time, the disks were dried at 60 °C for 24 h and the final weight of each sample was accurately measured. The degradation was calculated by dividing the weight loss by its initial weight. 2.7. Cytotoxicity test for the composite cements The cell proliferation assay was performed by extraction method with Sprague–Dawley rat osteoblast-like cells
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according to the method reported in ISO 10993-5 [33]. The 24-h-set paste with 30% C3S was crushed to powders and sieved through a 300-mesh sieve (50 lm) for further experiments. The dissolution extracts were prepared by adding the cement powders to RPMI 1640 (Gibco, USA) cell culture medium for 1 day at 37 °C in a humidified atmosphere of 5% CO2 and 95% air without agitation. Ratios between the powder weight (mg) and the medium volume (ml) were 6.25 105, 6.25 104, 6.25 103, 6.25 102, 0.625, 6.25, 12.5, 25, 50, 100 and 200 mg ml1. After incubation, the mixture was centrifuged and the supernatant collected. The cell suspension was adjusted to a density of 1 104 cell ml1, and 100 ll of cell suspension was added to each well of a 96-well plate and incubated for 24 h. The culture medium was then removed and replaced by 50 ll of extracts and 50 ll of RPMI 1640 medium supplemented with 20% fetal calf serum (FCS) every second day. The number of viable cells was quantitatively assessed by the MTT test [34]. MTT (Sigma, USA) (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) is a yellow tetrazolium salt that can be enzymatically converted by a living cell to a purple formazan product. The intensity of the color produced is therefore directly proportional to the number of viable cells in culture, and thus to their proliferation in vitro. The absorbance of the color observed can be measured at 590 nm (A590). In brief, after incubating at 37 °C and 5% CO2 for 6 days [35,36], 100 ll of 0.5 mg ml1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was added to extract/cell constructs and cultured for 4 h at 37 °C. Then 100 ll of dimethyl sulfoxide was added to each well, the plate was shaken for 5 min, and the optical density (OD) at 590 nm was measured with an enzyme-linked immunoadsorbent assay late reader (ELX800, Bio-TEK, USA). The medium supplemented with 10% FCS without addition of extracts was used as a control. Six samples per group were tested in the experiment. Results were reported as OD units. 2.8. Alkaline phosphate activity assay Canine bone marrow stromal cells (cBMSC) were isolated and expanded according to the published document with minimal modification [37]. For the assessment of alkaline phosphate (ALP) activity, cBMSC were cultured under the same culture conditions described above for 15 days. ALP activity was quantitatively determined by an assay based on the hydrolysis of p-nitrophenyl phosphate to pnitrophenol according to Lowry et al. [38]. Cells were extracted from the extracts of the 24-h-set paste with 30% C3S and permeablized with the use of 0.1% Triton X-100 solution (Sigma). Cell lysate from each sample was then used for alkaline phosphatase assays. The absorbance was measured at 405 nm with the use of a spectrophotometer (UV–VIS 8500, Shanghai, China), and ALP activity was calculated from a standard curve after normalizing to the total protein content. The results were expressed as
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nanomoles of p-nitrophenol produced per minute per microgram of protein. ALP activity of cells cultured in medium supplemented with 10% FCS without addition of extracts served as a control. Five samples for each material were tested for each incubation time. Each test was performed in triplicate. 2.9. Statistical methods The experimental values were analyzed using Student’s t-test and expressed as mean ± SD. A p-value of <0.05 was considered statistically significant. 3. Results 3.1. Chemical composition and microstructure of the composite cements The results of the XRD, SEM and EDX analyses indicated that both the chemical composition and microstructure of the hydration products of the TCP/MCPM/C3S composite paste samples were significantly different from those of the TCP/MCPM paste sample. The indexed Xray spectrum is presented in Fig. 1. It shows that, for the TCP/MCPM bone cement, the main hydration product was brushite, with a small fraction of non-reacted b-TCP and non-hydrated brushite (monetite) (Fig. 1(A)). However, in the TCP/MCPM/C3S composite samples, peaks for brushite were not observed, and the main hydration products were low-crystallinity calcium-deficient hydroxyapatite (CDHA) and C–S–H, with a larger fraction of non-reacted b-TCP (Fig. 1(B–E)).
Fig. 2 shows the SEM micrographs of the cross-section of the reacted TCP/MCPM and TCP/MCPM/C3S composite paste after setting for 24 h. Fig. 2(A) shows a typical fracture surface for a TCP/MCPM formulation. Here, precipitated brushite and residual b-TCP can be observed as needleshaped crystallites or small particles, respectively. Conversely, for TCP/MCPM/C3S samples with a C3S weight ratio of 10–30%, smooth and fine structures were observed (see, e.g., Fig. 2(B–D)). It is noticed that, as the weight ratio of C3S reached 30% or more, the crystals of hydration products tended to agglomerate within the samples (Fig. 2(E)). The results of EDX analysis indicated that, for the reacted TCP/MCPM brushite paste sample, the Ca:P molar ratio within the material is 1.10, which is between that of brushite (1.00) and b-TCP (1.50). However, a higher Ca:P molar ratio was observed for the TCP/MCPM/C3S paste samples (1.54–1.71) as compared with that of the TCP/MCPM brushite cement, which is close to the theoretical ratio of hydroxyapatite (1.67). It also indicated that an increase in the weight ratio of C3S within the composite cement resulted in an increase in the Ca:P molar ratio within the hydration products, and silicon element was observed for the TCP/MCPM/C3S paste samples. The results of the EDX analysis, in combination with those of the XRD and SEM analyses (Figs. 1 and 2), confirmed that the main hydration product of the TCP/MCPM/C3S was small crystals of calcium-deficient hydroxyapatite. 3.2. Self-setting properties of the composite cement pastes Fig. 3 shows the initial and final setting time of the cement pastes, and the injectability of the pastes is repre-
Fig. 1. XRD patterns of paste samples after aging for 24 h. (A) The TCP/MCPM paste; (B) the TCP/MCPM/C3S composite paste with 10% C3S; (C) the TCP/MCPM/C3S composite paste with 20% C3S; (D) the TCP/MCPM/C3S composite paste with 30% C3S; (E) the TCP/MCPM/C3S composite paste with 40% C3S.
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Fig. 2. SEM micrographs of (A) the TCP/MCPM paste; (B) the TCP/MCPM/C3S composite paste with 10% C3S; (C) the TCP/MCPM/C3S composite paste with 20% C3S; (D) the TCP/MCPM/C3S composite paste with 30% C3S; (E) the TCP/MCPM/C3S composite paste with 40% C3S after aging for 24 h.
Fig. 3. The initial setting time and final setting time of the paste samples with various contents of C3S (S/L = 2.0 g ml1).
Fig. 4. The injectability of the composite pastes with various contents of C3S (S/L = 2.0 g ml1) in 2 min after mixing.
sented in Fig. 4. The TCP/MCPM brushite cement paste set quite rapidly, within 2–3 min, and was consequently unworkable as it failed to be ejected out of the syringe after 2 min. In contrast, it was found that the TCP/ MCPM/C3S composite paste set more slowly (7–25 min for initial setting), and more than 50% of the paste could be extruded through the syringe as the weight ratio of C3S reached 20% or more. The composite paste did not give any demixing or filter pressing during the extrusion. Furthermore, it was observed that both the setting time and the injectability of the TCP/MCPM/C3S composite paste increased with increasing amount of Ca3SiO5 within the composite.
3.3. Mechanical strength of the cement paste Fig. 5(A) shows the compressive strength of the reacted pastes after aging in a 100% humidity water bath at 37 °C for various periods. For the reacted TCP/MCPM cement paste and the TCP/MCPM/C3S composite set formulation with 10% C3S, the compressive strength was low (around 5.0 MPa) and did not change significantly after prolonged aging. In contrast, the compressive strength of the composite paste with more than 10% C3S increased with increasing aging time and amount of C3S. The composite cement with 30% C3S showed the highest compressive strength after prolonged aging (42.5 MPa).
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Fig. 6. Variation in the pH of the suspension after immersion of the paste in SBF.
pastes were higher than that of the TCP/MCPM paste and nearly neutral (6.70–7.35), and after prolonged soaking in SBF, the pH value increased further to 7.59–7.75 after 12 h. 3.5. The bone-like apatite formation on the paste disks after soaking in SBF
Fig. 5. The compressive strength of the paste samples at different aging times in (A) 37 °C and 100% humidity; and (B) PBS.
To further evaluate the in vitro mechanical properties of the material, the materials were soaked in PBS for preset periods and the wet compressive strength was measured after soaking. The results are presented in Fig. 5(B). Comparison between Fig. 5(A) and Fig. 5(B) indicates that a significant decrease in compressive strength was observed for all the samples after aging in PBS as compared with that of the materials aged in the 100% humidity water bath. However, the compressive strength of the composite material with more than 10% C3S still increased with increasing aging time and amount of C3S, and was still significantly higher (23.2 MPa) than that of the reacted TCP/MCPM paste (5.9 MPa) under the same soaking conditions. 3.4. pH measurement Fig. 6 shows the changes in the pH values of the SBF with time in the presence of composite cement pastes. In the case of the TCP/MCPM paste, the pH was initially 4.0 and increased gradually to 5.2 after 12 h. In contrast, the initial pH values of all the TCP/MCPM/C3S composite
To determine the bioactivity, the reacted TCP/MCPM and TCP/MCPM/C3S paste disks were soaked in SBF. The SEM micrographs of the surfaces of the disks after soaking in SBF are presented in Fig. 7. It can be clearly seen that, after soaking in refreshed SBF for 7 days, the TCP/MCPM cement became porous and consisted of spherical b-TCP particles 1–10 lm in diameter, and no bone-like apatite layer was formed on the surface (Fig. 7(A1 and A2)), which corresponded with previous studies on the in vitro aging of the brushite cement [25]. In contrast, newly formed lath-like crystals were observed on the surface of TCP/MCPM/C3S samples after soaking in SBF. When the C3S content within the composite cements increased to 20% or more, many of the crystals formed agglomerates, which further congregated to form a layer that covered the surface of the cement (Fig. 7(D2 and E2)). Such layers have a similar morphology as those formed on the surface of bioactive glass [28] and silicate bioceramics [34] after soaking in SBF, which resulted from the precipitation of bone-like apatite. Furthermore, the result of EDX analysis on the as-observed lath-like crystals suggested that the Ca:P molar ratio is 1.63, which is close to the theoretical ratio of hydroxyapatite (1.67), and such a result, combined with that of SEM analysis (Fig. 7), confirmed the formation of bone-like apatite layer on the surface of reacted TCP/MCPM/C3S composite paste after soaking in SBF. 3.6. In vitro degradation Fig. 8 shows the degradation of the TCP/MCPM and TCP/MCPM/C3S set formulations after soaking in PBS
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Fig. 7. SEM micrographs of the surfaces of the paste samples after soaking in SBF for 7 days. (A1 and A2) the TCP/MCPM paste; (B1 and B2) the TCP/ MCPM/C3S composite paste with 10% C3S; (C1 and C2) the TCP/MCPM/C3S composite paste with 20% C3S; (D1 and D2) the TCP/MCPM/C3S composite paste with 30% C3S; (E1 and E2) the TCP/MCPM/C3S composite paste with 40% C3S.
for various time periods. It was found that early dissolution of set formulations increased upon raising the C3S percentage. After 4 days the C3S percentage had no obvious effect on degradation rate.
3.7. Cytotoxicity test Fig. 9 shows the result of cytotoxicity test on the extracts of both TCP/MCPM and TCP/MCPM/C3S composite set
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Fig. 8. Degradation of the paste samples with various contents of C3S after soaking in PBS for various times.
Fig. 10. The ALP activity of the cBMSC in the presence of the dissolution extracts of the cement pastes. TCP/MCPM/C3S cements significantly enhanced the ALP activity of the cBMSC, which was not observed for TCP/MCPM cement samples. The asterisk (*) indicates that the ALP activity of experimental group was significantly higher than that of the control (p < 0.05).
the extracts was in the range from 6.25 104 to 5 101 mg ml1, while no enhanced proliferation of the cells was observed for the reacted TCP/MCPM paste. 3.8. The effects of cements on ALP activity of cBMSC Cell differentiation was assessed in terms of the ALP activities of the cBMSC after culturing for 15 days (Fig. 10). The results indicated that cells cultured in the extract of TCP/MCPM specimen did not showed higher ALP activity than that of the control, whereas the extract of TCP/MCPM/C3S reacted paste significant enhanced the ALP activity of the cBMSC with the concentration ranging from 6.25 105 to 2.5 101 mg ml1. Fig. 9. Osteoblast cells proliferation in the presence of the dissolution extracts of the powders of the TCP/MCPM and TCP/MCPM/C3S composite pastes with 30% C3S after culturing for 6 days. The asterisk (*) indicates that the cell proliferation of experimental group was significantly different from that of the negative control (p < 0.05).
formulations against osteoblast-like cells after incubation for 6 days. It was observed that the extract of TCP/MCPM paste showed cytotoxicity against osteoblasts when the concentration of the extracts was higher than 12.5 mg ml1 (p < 0.05). In contrast, no significant cytotoxicity against osteoblasts was observed on the samples with 30% C3S and other ratios of C3S (10, 20 and 40 wt.%; data not shown) in the present study, which indicated the superior biocompatibility of the TCP/MCPM/C3S cement as compared with that of the TCP/MCPM cement. Furthermore, it was found that, after incubating for 6 days, the osteoblast-like cell proliferation in the extract of the TCP/ MCPM/C3S reacted paste was significantly higher than in the negative control (p < 0.05) when the concentration of
4. Discussion 4.1. Self-setting properties and mechanical strength of the composite pastes The applicability of injectable self-setting biomaterials is largely dependent on its self-setting characteristics, such as injectability and setting times. Since the original brushite cements showed short setting times and poor injectability, which limit the application of the material, efforts have been taken to overcome the problem, and the use of various additives as retardants has been proven to be an effective way to improve the self-setting properties of the material [12,39,40]. In the present study, the homogeneous TCP/MCPM/C3S composite paste showed a prolonged setting time (7–25 min) and could be mostly injected through the syringe within 2 min after mixing, which is partly attributed to the low hydration rate of the C3S component. According to previous studies on brushite cements,
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a low Ca/P ratio and acidic surrounding pH values (around 4.0) are necessary for the precipitation of brushite crystals [11,41,42], otherwise hydroxyapatite (HA) is formed by preference during the hydration of the cement [41]. The Ca/P ratio in the TCP/MCPM/C3S cement system is higher than in brushite cement, and Ca3SiO5 can react with water to form C–S–H and calcium hydroxide according to the following equation: Ca3 SiO5 þ H2 O ! C–S–H þ CaðOHÞ2 2+
ð2Þ
which results in the release of Ca and OH and an increase in the pH. With the higher Ca/P ratio and the surrounding pH value, CDHA is more likely to be precipitated than brushite crystals during the hydration of TCP/MCPM/C3S composite cement. Since CDHA crystals precipitate much more slowly than brushite crystals [42], the TCP/MCPM/C3S composite paste showed a retarded setting time and consequently improved the injectability compared with the brushite cement paste. Furthermore, the low hydration rate and good injectability of the tricalcium silicate component in the composite cement contributed to the improved self-setting properties of the TCP/ MCPM/C3S composite paste. Taking into account that in clinical applications the cement must be applied before the initial setting time and must be able to be injected during the operation, our results suggest that the TCP/ MCPM/C3S paste is potentially a good injectable material for bone/dental repair. In clinical applications, bone substitute materials are required to provide adequate short- and long-term mechanical support for the defect site, which is important for the bone healing process. Brushite cements are typically weaker than the majority of apatite cements [8], and a main reason for the comparatively low compressive strength is the extremely rapid setting reaction of the brushite cements, which results in high porosity and consequently weak cement. In the present study, the compressive strength of the reacted TCP/MCPM/C3S composite pastes reached 23.2–42.5 MPa, which is higher than that of brushite cements as reported by other researchers [8,11,41]. Such results could be attributed to the lower setting rate and the consequently more compact microstructure of the TCP/ MCPM/C3S composite paste as compared with that of the brushite cement (as seen in Fig. 2). In addition, since Si ions tend to inhibit grain growth of HA crystals [43], the size of the precipitated CDHA crystals within the TCP/MCPM/C3S composite paste were observed to be on the nanoscale, which further promoted the formation of a compact microstructure of the hydrated composite cement [8]. Furthermore, the TCP/MCPM/C3S composite cement showed the ability to self-reinforce during setting. The compressive strength of the composite cements increased with time during the setting both in 100% humidity and in saline such as PBS, which has not been observed for the majority of calcium phosphate cements [5,8,11,28]. Such an ability to self-reinforce is attributed to the fact that the continuous hydration process of Ca3SiO5 resulted in
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progressive polymerization of the calcium silicate hydrate gels and the development of a solid network that enhanced the long-term mechanical strength of the set composite [20,44,45]. This property is important because a self-reinforced cement system can better tolerate the adverse effect of the degradation process on the structure of the cement materials, hence providing more stable long-term mechanical support during the bone regeneration process. 4.2. In vitro bioactivity and degradation As compared with bioactive bone substitution materials such as A-W Bioglass and calcium silicate ceramics, the calcium phosphate cements show a poor ability to induce a bone-like apatite layer on the surface which can form a chemical bond with bone tissue within a short period [18,46]. This was also supported by the finding that no apatite layer deposition was observed for the TCP/MCPM paste after soaking in SBF in our study. In contrast, the combined SEM and EDX analysis suggested that the TCP/MCPM/C3S composite paste induced apatite deposition within 1 week of soaking in SBF, indicating that the composite paste showed excellent bioactivity. Such a result could be attributed to the addition of Ca3SiO5, and the formation of the homogenous apatite layer was dependent on the Ca3SiO5 content. This finding confirmed our assumption that the addition of bioactive Ca3SiO5 could result in bioactive composite cements with controllable bioactivity. As is generally accepted, the increase in the supersaturation of Ca2+ is not enough to promote the formation of new crystals of apatite on the material surface in the simulated body environment [31,47], and this explains the lack of bioactivity of the TCP/MCPM cement. With the addition of Ca3SiO5, HSiO 3 ions are released during the hydration of the composite paste, and this could play an important role in the formation of apatite in the simulated body environment together with Ca2+ ions by providing favorable sites for the nucleation of apatite crystals. It is understandable that when the content of Ca3SiO5 is low, the concentration of HSiO 3 ions that act as the nucleation site for apatite was not adequate to support the formation of an apatite layer on the surface of the composite paste. However, when the content of Ca3SiO5 is increased to a certain extent (20% in the present study), the concentration of HSiO 3 ions is high enough for the ions to be dispersed within the composite paste and provides more nucleation sites for apatite crystals, resulting in the formation of a homogeneous apatite layer throughout the whole composite paste. The TCP/MCPM/C3S composite cement showed a slightly higher degradability than the TCP/MCPM cement. Since the degradability is primarily governed by the chemical composition and the physical characteristics of the material, the higher degradation rate of the TCP/ MCPM/C3S composite cement as compared with that of TCP/MCPM cement may be attributed to the higher degradability of calcium silicate hydrate within the reacted
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composite paste than that of the brushite within the TCP/ MCPM paste [20,28]. As TCP/MCPM brushite cement is generally accepted as a biodegradable material in vivo [6,7,10,39], the results in the present study indicate that the TCP/MCPM/C3S composite cement may also be degradable in clinical applications, with a degradation rate similar to that of TCP/MCPM cement. However, further studies need to be conducted to confirm the in vivo degradation properties of the TCP/MCPM/C3S composite material. 4.3. In vitro cytotoxicity It is generally accepted that the in vitro cell–material interaction is a useful criterion in the evaluation of new biomaterials. It is known that MCPM is weakly acidic, and the implantation of TCP/MCPM paste can result in an acid pH in the environs that may be clinically undesirable [40]. The present study confirmed that hydrated TCP/MCPM paste revealed significant cytotoxicity (p < 0.05) against osteoblasts as the concentration of the paste extracts varied from 25 to 200 mg ml1 (Fig. 9). In contrast, the TCP/MCPM/C3S composite pastes did not cause any significant increase or decrease in the surrounding pH value (Fig. 6), which could be attributed to modification of the MCPM by the OH ions that were released through the hydration of C3S, and therefore the composite cement did not show any cytotoxicity against osteoblasts, as observed in the present study. Moreover, it was observed that the ionic dissolution products of the reacted TCP/ MCPM/C3S composite pastes could provide more than adequate stimulus for cell proliferation. According to previous studies, dissolution extracts of bioactive silicate materials such as bioactive glasses and ceramics could stimulate cell proliferation [21,48]. A recent study also showed that tricalcium silicate paste could stimulate L929 cell proliferation [20]. This stimulatory effect may be due to the dissolution of silicate ions [21]. The present study indicated that the extracts of the reacted TCP/MCPM/C3S composite paste also stimulated proliferation of osteoblasts, suggesting that the composite cement was not only biocompatible and non-cytotoxic, but also possessed excellent bioactivity at the cellular level. 4.4. ALP activity Differentiation of bone marrow stromal cells is one of the key processes for bone regeneration. As a cell surface glycoprotein that is involved in mineralization [49], ALP is the most widely recognized marker of osteoblastic differentiation [50]. In the present study, the ionic extract of the reacted TCP/MCPM/C3S composite paste significantly enhanced the ALP activity of cBMSC, which was not observed for TCP/MCPM set formulation. This result suggested that TCP/MCPM/C3S composite cement possesses a much greater ability to promote osteoblastic differentiation of cBMSC than TCP/MCPM cement. As the previous
studies have shown, Si ions play an important role in bone metabolism and have been shown to raise ALP and osteocalcin expression in human osteoblast-like cells essential in the mineralization process and the bone-bonding mechanism [51,52]. Additionally, Sun et al. found that a silicate ceramic (akermanite) could enhance ALP activity of human bone marrow stromal cells [53]. Therefore, in comparison to TCP/MCPM, the effect of TCP/MCPM/C3S cements on the osteoblastic differentiation of cBMSC may be explained by differences in chemical composition, and the ionic products of the hydrated TCP/MCPM/C3S cement might play an important role in the stimulatory process. More in vitro and vivo studies are necessary to further investigate the mechanism of the stimulatory effects of TCP/MCPM/C3S cements on the osteoblastic differentiation of bone marrow stromal cells. 5. Conclusion In this paper, novel bioactive composite cements were successfully prepared by adding tricalcium silicate into the traditional TCP/MCPM cement system. The TCP/M CPM/C3S composite cements showed a prolonged setting time, significantly improved injectability and mechanical strength compared with the brushite-forming TCP/MCPM cement. Furthermore, the TCP/MCPM/C3S composite cement possessed excellent bioactivity, as indicated by the formation of bone-like apatite in SBF, and showed similar in vitro degradability to that of the TCP/MCPM cement, which is generally considered to be a degradable bone graft material in vivo. The cytotoxicity test showed that the TCP/MCPM/C3S composite cement was not only biocompatible and non-cytotoxic, but also stimulated cell proliferation. Furthermore, the TCP/MCPM/C3S cements possessed the ability to promote osteoblastic differentiation of cBMSC. Our study presents a novel method to design bone cements with improved properties based on traditional calcium phosphate bone cements by adding bioactive self-setting components, and suggests that the addition of bioactive Ca3SiO5 is a possible way to obtain bioactive self-setting composite biomaterials for bone regeneration. Acknowledgements This work was supported by the National Basic Science Research Program of China (973 Program) (Grant No. 2005CB522704), and funds from the Chinese Academy of Sciences for Key Topics in Innovation Engineering (Grant No. KGCX2-YW-207) and the Natural Science Foundation of China (Grant No. 30730034). References [1] Constanz BR, Ison IC, Fulmer M, Poser RD, Smith ST. Skeletal repair by in situ formation of the mineral phase of bone. Science 1995;267:1796–9.
Z. Huan, J. Chang / Acta Biomaterialia 5 (2009) 1253–1264 [2] Schildhauer TA, Bennett AP, Wright TM, Lane JM, O’Leary PF. Intravertebral body reconstruction with an injectable in situ-setting carbonated apatite: biomechanical evaluation of a minimally invasive technique. J Orthop Res 1999;1:67–72. [3] Li YW, Leong JCY, Lu WW, Luk KDK, Cheung KMC, Chiu KY. A novel injectable bioactive bone cement for spinal surgery: a developmental and preclinical study. J Biomed Mater Res 2000;52:164–70. [4] Lidgren L. A composition for an injectable bone mineral substitute material. PCT Int Appl WO 2002005861 A1, 2002. [5] Letmaitre J, Mirtchi A, Mortier A. Calcium phosphate cements for medical use: state of the art and perspectives of development. Silic Ind 1987;10:141–6. [6] Grover LM, Gbureck U, Wright AJ, Tremayne M, Barralet JE. Biologically mediated resorption of brushite cement in vitro. Biomaterials 2006;27:2178–85. [7] Theiss F, Apelt D, Brand B, Kutter A, Zlinszky K, Bohner M, et al. Biocompatibility and resorption of a brushite calcium phosphate cement. Biomaterials 2005;26:4383–94. [8] Charriere E, Terrazzoni S, Pittet C, Mordasini P, Dutoit M, Lemaitre J, et al. Mechanical characterization of brushite and hydroxyapatite cements. Biomaterials 2001;22:2937–45. [9] Gbureck U, Grolms O, Barralet JE, Grover LM, Thull R. Mechanical activation and cement formation of b-tricalcium phosphate. Biomaterials 2003;24:4123–31. [10] Xia Z, Grover LM, Huang Y, Adamopoulos IE, Gbureck U, Triffitt JT, et al. In vitro biodegradation of three brushite calcium phosphate cements by a macrophage cell-line. Biomaterials 2006;27:4557–65. [11] Grover LM, Gbureck U, Young AM, Wright AJ, Barralet JE. Temperature dependent setting kinetics and mechanical properties of b-TCP–pyrophosphoric acid bone cement. J Mater Chem 2005;15: 4955–62. [12] Marino FT, Mastio J, Rueda C, Blanco L, Cabarcos EL. Increase of the final setting time of the brushite cement by using chondroitin 4sulfate and silica gel. J Mater Sci Mater Med 2007;18:1195–201. [13] Ooms EM, Wolke JGC, Heuvel MT, Jeschke B, Jansen JA. Histological evaluation of the bone response to calcium phosphate cement implanted in cortical bone. Biomaterials 2003;24:989–1000. [14] Curran Judith M, Gallagher James A, Hunt John Alan. The inflammatory of biphasic calcium phosphate granules in osteoblast/ macrophage co-culture. Biomaterials 2005;26:5313–20. [15] Kokubo T, Kushiyani M, Ebisawa Y, Kitsugi, Kotani S, Oura K, et al. Apatite formation on bioactive ceramics in body environment. In: Oonishi, Aoki H, Sawai K, editors. Bioceramics. Tokyo: Ishiyaku EuroAmerica; 1988. p. 157–62. [16] Suominen E, Aho AJ, Vedel E, Kangasniemi I, Uusipaikka E, YliUrpo A. Subchondral bone and cartilage repair with bioactive glasses, hydroxyapatite, and hydroxyapatite–glass composite. J Biomed Mater Res 1996;32:543–51. [17] Kobayashi M, Nakamura T, Tamura J, Kokubo T, Kikutani T. Bioactive bone cement: comparison of AW-GC filler with hydroxyapatite and b-TCP fillers on the mechanical and biological properties. J Biomed Mater Res 1997;37:301–13. [18] Kobayashi M, Nakamura T, Okada Y, Fukumoto A, Furukawa T, Kato H, et al. Bioactive bone cement: comparison of apatite and wollastonite containing glass–ceramic, hydrxyapatite and b-tricalcium phosphate fillers on bone-bonding strength. J Biomed Mater Res 1998;42:223–37. [19] Zhao W, Chang J. Sol–gel synthesis and in vitro bioactivity of tricalcium silicate powders. Mater Lett 2004;58:2350–3. [20] 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. [21] Xu JL, Khor KA. Chemical analysis of silica doped hydroxyapatite biomaterials consolidated by a spark plasma sintering method. J Inorg Biochem 2007;101:187–95. [22] Thian ES, Huang J, Best SM, Barber ZH, Brooks RA, Rushton N, et al. The response of osteoblasts to nanocrystalline silicon-substituted hydroxyapatite thin films. Biomaterials 2006;27:2692–8.
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[23] Porter AE. Nanoscale characterization of the interface between bone and hydroxyapatite implants and the effect of silicon on bone apposition. Micron 2006;37:681–8. [24] Xynos ID, Edgar AJ, Buttery LD, Hench LL, Polak JM. Geneexpression profiling of human osteoblasts following treatment with ionic products of biological 45S5 dissolution. J Biomed Mater Res 2001;55:151–7. [25] Mirtchi AA, Lemaitre J, Terao N. Calcium phosphate cements: study of the b-tricalcium phosphate–monocalcium phosphate system. Biomaterials 1989;10:634–8. [26] Bohner M, Baroud G. Injectablity of calcium phosphate pastes. Biomaterials 2005;26:1553–63. [27] Khairoun I, Boltong MG, Driessens FCM, Planell JA. Some factors controlling the injectability of calcium phosphate bone cements. J Mater Sci Mater Med 1998;9:425–8. [28] Grover LM, Knowles JC, Fleming GJP, Barralet. In vitro ageing of brushite calcium phosphate cement. Biomaterials 2003;24:4133–41. [29] Huan ZG, Chang J. Novel tricalcium silicate/monocalcium phosphate monohydrate composite bone cement. J Biomed Mater Res Part B Appl Biomater 2007;82(B):352–9. [30] Nurit J, Margerit J, Terol A, Boudeville P. PH-metric study of the setting reaction of monocalcium phosphate monohydrate/calcium oxide-based cements. J Mater Sci Mater Med 1993;13:1007–14. [31] Kokubo T. Surface chemistry of bioactive glass-ceramics. J NonCrystallogr Solids 1990;120:138–51. [32] Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity. Biomaterials 2006;27:2907–15. [33] ISO/EN 10993-5. Biological evaluation of medical devices – Part 5. Tests for cytotoxicity, in vitro methods: 8.2 tests on extract. [34] Cory AH, Owen TC, Barltrop JA, Cory JG. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 1991;3:207–12. [35] Sogo Y, Ito A, Kamo M, Sakurai T, Onuma K, Ichinose N, et al. Hydrolysis and cytocompatibility of zinc-containing a-tricalcium phosphate powder. Mater Sci Eng C 2004;24:709–15. [36] Xu HHK, Carey LE, Simon CG, Takagi S, Chow LC. Premixed calcium phosphate cements: synthesis, physical properties, and cell cytotoxicity. Dent Mater 2007;23:433–41. [37] Mauney JR, Jaquiery C, Volloch V, Heberer M, Martin I, Kaplan DL. In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering. Biomaterials 2005;26:3173–85. [38] Lowry OH, Roberts NR, Wu M, Hixton WS, Crawford EJ. The quantitative histochemistry of brain II. Enzyme measurements. J Biol Chem 1954;207:19–37. [39] Bohner M, Gbureck U, Barralet JE. Technological issues for the development of more efficient calcium phosphate bone cement: a critical assessment. Biomaterials 2005;26:6423–9. [40] Barralet JE, Grover LM, Gbureck U. Ionic modification of calcium phosphate cement viscosity. Part II. Hypodermic injection and strength improvement of brushite cement. Biomaterials 2004;25: 2197–203. [41] Lilley KJ, Gbureck U, Knowles JC, Farrar DF, Barralet JE. Cement from magnesium substituted hydroxyapatite. J Mater Sci Mater Med 2005;16:455–60. [42] Bohner M. Reactivity of calcium phosphate cements. J Mater Chem 2007:3980–6. [43] Pietak AM, Reid JW, Scott MJ, Sayer M. Silicon substitution in the calcium phosphate bioceramics. Biomaterials 2007;28:4023–32. [44] Wang XP, Ye JD, Wang YJ, Chen L. Self-setting properties of a bdicalcium silicate reinforced calcium phosphate cement. J Biomed Mater Res Part B Appl Biomater 2007;82:93–9. [45] Huan ZG, Chang J. Self-setting properties and in vitro bioactivity of calcium sulfate hemihydrate–tricalcium silicate composite bone cements. Acta Biomater 2007;3:952–60. [46] Kobayashi M, Nakamura T, Shinzato S, Mousa WF, Nishio K, Ohsawa K, et al. Effect of bioactive filler content on mechanical
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Acta Biomaterialia 5 (2009) 1253–1264 www.elsevier.com/locate/actabiomat
Novel bioactive composite bone cements based on the b-tricalcium phosphate–monocalcium phosphate monohydrate composite cement system Zhiguang Huan a,b, Jiang Chang a,* a
Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, People’s Republic of China b Graduate School of the Chinese Academy of Sciences, 319 Yueyang Road, Shanghai 200050, People’s Republic of China Received 26 May 2008; received in revised form 10 September 2008; accepted 8 October 2008 Available online 22 October 2008
Abstract Bioactive composite bone cements were obtained by incorporation of tricalcium silicate (Ca3SiO5, C3S) into a brushite bone cement composed of b-tricalcium phosphate [b-Ca3(PO4)2, b-TCP] and monocalcium phosphate monohydrate [Ca(H2PO4)2H2O, MCPM], and the properties of the new cements were studied and compared with pure brushite cement. The results indicated that the injectability, setting time and short- and long-term mechanical strength of the material are higher than those of pure brushite cement, and the compressive strength of the TCP/MCPM/C3S composite paste increased with increasing aging time. Moreover, the TCP/MCPM/C3S specimens showed significantly improved in vitro bioactivity in simulated body fluid and similar degradability in phosphate-buffered saline as compared with brushite cement. Additionally, the reacted TCP/MCPM/C3S paste possesses the ability to stimulate osteoblast proliferation and promote osteoblastic differentiation of the bone marrow stromal cells. The results indicated that the TCP/MCPM/C3S cements may be used as a bioactive material for bone regeneration, and might have significant clinical advantage over the traditional b-TCP/MCPM brushite cement. Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Calcium phosphate cement; Brushite; In vitro test; Cytotoxicity; Silicate
1. Introduction Materials with self-setting properties have been exploited to augment human bone tissues [1–4], and resorbable calcium phosphate cements based on brushite have raised interests in recent years [5–8]. These kinds of cement are usually presented as a solid phase and a liquid phase. These two components are mixed together in order to form a workable paste that would set into a hard material. The self-setting occurs through an acid–base reaction between a basic calcium phosphate, usually b-tricalcium phosphate (b-TCP), and an acidic phosphate such as orthophosphoric
*
Corresponding author. Tel.: +86 21 52412804; fax: +86 21 52413903. E-mail address: [email protected] (J. Chang).
acid or monocalcium phosphate monohydrate (MCPM) [5– 9]. In clinical applications, the brushite cements can be used in the form of blocks or as a self-setting paste, which could provide scaffolds for bone regeneration and be gradually replaced by tissue [6,7,10]. However, this cement system still has some inherent drawbacks that need to be dealt with. Due to the extremely rapid setting reaction, the brushite cement shows a rather short setting time that makes it difficult to work with, which also results in high porosity and consequently weak mechanical strength [11,12]. In addition, as soluble acidic phosphates are used as sources of phosphate ions, the brushite cement usually will cause a rapid decrease of pH in vivo immediately after implantation. This phenomenon can have an adverse impact on the biocompatibility of the material [13,14]. Other studies also showed that the calcium phosphate cement is less bioactive as compared
1742-7061/$ - see front matter Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2008.10.006
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with some silicate-containing bioactive materials such as AW glass-ceramic and Bioglass [15,16]. Therefore, incorporation of bioactive materials has been investigated to improve the bioactivity of the cement materials [17,18]. Tricalcium silicate (Ca3SiO5) is one of the main components of Portland cement. Once mixed with water, Ca3SiO5 will react with water to create calcium–silicate–hydrate (C–S–H). The polymerization and solidification of the C– S–H network contribute to the self-setting properties and increased mechanical strength of the tricalcium silicate paste after aging. Recent studies have shown that the tricalcium silicate cements are bioactive and can induce bonelike apatite formation in simulated body fluid [19,20], which is partly attributed to the release of silicate ions. The silicate ions are also known to be an effective additive to improve the bioactivity and dissolution rate of hydroxyapatite [21–23]. Furthermore, these materials have the potential to activate bone-related gene expression and stimulate cell proliferation [19,20,24]. It has also been proven that the hydration product of tricalcium silicate cement is degradable in simulated body fluid (SBF) [20]. However, this material shows a rather low self-setting rate, and the setting time of the cement paste is too long (>60 min), which may not be suitable for orthopaedic applications [20]. In this paper, considering the advantages and disadvantages of the brushite and Ca3SiO5 cements, composite cements were designed and prepared by mixing the bTCP/MCPM brushite cement system with bioactive Ca3SiO5. The setting time, workability, mechanical properties, in vitro bioactivity and cytotoxicity of the b-TCP/ MCPM/C3S composite cements were evaluated and compared with those of the brushite cement.
2. Materials and methods 2.1. Material preparation and characterization The brushite cement used in this study was prepared by mixing two phosphate powders in the presence of water. The starting powders were b-TCP (synthesized in-house) and monocalcium phosphate monohydrate (Sinopham Chemical Reagent Co., Ltd). The weight ratio of the two components (b-TCP:MCPM) is 3:2, and they react in the presence of water to form precipitated brushite (dicalcium phosphate dehydrate) according to he reaction presented below [8,25].
mixed with the brushite cement powders. To prepare paste samples, powders were mixed with deionized water using a solid/liquid (S/L) ratio of 2.0 g ml1. The cement paste was prepared in a mortar, thoroughly kneaded with a spatula and poured into a cylindrical polytetrafluoroethylene mold. Air bubbles entrapped in the paste during mixing were allowed to escape by gently shaking the mold (6 mm diameter and 12 mm height). After demolding, the samples were placed in a 100% humidity water bath at 37 °C and kept for 24 h. The phase composition was characterized by X-ray diffraction (XRD; Geigerflex, Rigaku Co., Japan) using monochromated Cu Ka radiation, and the 2h range was from 10° to 60° at a scanning speed of 10° min1. The cross-section of the samples was observed by scanning electron microscopy (SEM) using a scanning electron microscope (JSM-6700F, JEOL, Tokyo, Japan) equipped with an energy dispersive X-ray (EDX) detector (INCA Energy, Oxiford Instruments, UK). 2.2. Setting times and injectability The setting times of the composite pastes with 0%, 10%, 20%, 30%, and 40% content of Ca3SiO5 were measured with the Vicat needle according to ISO9597-1989E. To test the initial setting time of the paste, a needle weighing 280 g and of 1.13 mm diameter is lowered into a specimen of fresh cement paste and the penetration depth is recorded. The cement paste is kept in a standard frustum 40 mm in height. The initial setting time is when the needle penetration is 35 ± 1.0 mm. The final setting time is defined as the time necessary so that the heavy needle (350 g, Ø 2.0 mm) no longer leaves a visible print on the surface of the paste. The measurement was conducted on five specimens from a group of the same composition, and then the average value was calculated. The injectability of the composite paste was evaluated by extruding a certain amount of paste through a disposable syringe by hand according to a modified method described previously [19,26,27]. The syringes have a capacity of 2.5 ml, with an opening nozzle diameter of 2.0 mm. Six grams of as-prepared paste was added into the syringe and, after storage in a water bath at 37 °C for 2 min, the paste was gently extruded from the syringe by hand until it could no longer be continued. Then the weight of the paste expelled from the syringe was measured and the injectability was calculated using Eq. (1) [27]. Each test was repeated at least three times and the average value was calculated.
Ca3 ðPO4 Þ2 þ CaðH2 PO4 Þ2 H2 O þ 7H2 O ! 4CaHPO4 2H2 O Tricalcium silicate (Ca3SiO5) powders were prepared by the sol–gel method, as previously described [19], and they were ground and sieved through a 300-mesh sieve (50 lm). To prepare the brushite/Ca3SiO5 composite cements, Ca3SiO5 powders (0–40 wt.%) were uniformly
Inj% ¼
Paste weight expelled from the syringe Total paste weight before injecting
ð1Þ
2.3. Mechanical test of the cement paste For the compressive mechanical testing, the as-prepared slurries were poured into a cylindrical polytetrafluoroethyl-
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ene mold (6 mm diameter and 12 mm height). Air bubbles entrapped in the paste during mixing were allowed to escape by gently shaking the mold. After aging in a 100% humidity water bath or phosphate-buffered saline (PBS) [28] at 37 °C for 24 h and 4, 7, 14, 21 and 28 days, the reacted paste samples were immediately removed from the molds and the compressive strength was measured at a loading rate of 0.5 mm min1 using a universal testing machine (Instron-1195, USA), according to ASTM D69591. Six replicates were carried out for each group and the results were expressed as mean ± standard deviation (SD). 2.4. pH measurement To test the in vitro pH variation of the cement paste in a simulated body environment, 5 ml fresh cement slurries were directly poured into a 25 ml beaker and then 15 ml of SBF was injected into the container to cover the paste [29,30]. The paste was allowed to set in the SBF without any further shaking, and the test samples were stored in a 37 °C, 100% humidity water bath. After 1 h of immersion, all the SBF used for the immersion was collected by a syringe and its pH value was measured using an electrolyte-type pH meter (pHS-2 C, Jingke Leici Co., Shanghai, China). Fresh SBF, equal in volume to the removed suspension, was then added to the system to renew the immersion medium. The procedure was repeated every hour for a period of 12 h. 2.5. In vitro bioactivity The SBF solution was prepared according to the procedure described by Kokubo [31]. The ion concentrations of the SBF are similar to those in human blood plasma, and the pH value was adjusted to 7.35 [31]. The 24-h-set paste disks (6 mm in diameter and 2 mm in height) were soaked in SBF solution at 37.0 °C in a shaking water bath for 7 days with a surface area-to-volume ratio of 0.1 cm1 [31,32]. After the pre-selected soaking time, the disks were gently rinsed with deionized water to remove SBF solutions followed by drying at room temperature. The samples were characterized by SEM and EDX. 2.6. In vitro degradation of the paste For evaluation of degradation, the 7-day-set paste disks were soaked in PBS at 37 °C in shaking water bath for 4, 7, 10, 14 and 21 days with a surface area-to-volume ratio of 0.1 cm1 [28,31]. The solution was refreshed every day. After the set soaking time, the disks were dried at 60 °C for 24 h and the final weight of each sample was accurately measured. The degradation was calculated by dividing the weight loss by its initial weight. 2.7. Cytotoxicity test for the composite cements The cell proliferation assay was performed by extraction method with Sprague–Dawley rat osteoblast-like cells
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according to the method reported in ISO 10993-5 [33]. The 24-h-set paste with 30% C3S was crushed to powders and sieved through a 300-mesh sieve (50 lm) for further experiments. The dissolution extracts were prepared by adding the cement powders to RPMI 1640 (Gibco, USA) cell culture medium for 1 day at 37 °C in a humidified atmosphere of 5% CO2 and 95% air without agitation. Ratios between the powder weight (mg) and the medium volume (ml) were 6.25 105, 6.25 104, 6.25 103, 6.25 102, 0.625, 6.25, 12.5, 25, 50, 100 and 200 mg ml1. After incubation, the mixture was centrifuged and the supernatant collected. The cell suspension was adjusted to a density of 1 104 cell ml1, and 100 ll of cell suspension was added to each well of a 96-well plate and incubated for 24 h. The culture medium was then removed and replaced by 50 ll of extracts and 50 ll of RPMI 1640 medium supplemented with 20% fetal calf serum (FCS) every second day. The number of viable cells was quantitatively assessed by the MTT test [34]. MTT (Sigma, USA) (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) is a yellow tetrazolium salt that can be enzymatically converted by a living cell to a purple formazan product. The intensity of the color produced is therefore directly proportional to the number of viable cells in culture, and thus to their proliferation in vitro. The absorbance of the color observed can be measured at 590 nm (A590). In brief, after incubating at 37 °C and 5% CO2 for 6 days [35,36], 100 ll of 0.5 mg ml1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was added to extract/cell constructs and cultured for 4 h at 37 °C. Then 100 ll of dimethyl sulfoxide was added to each well, the plate was shaken for 5 min, and the optical density (OD) at 590 nm was measured with an enzyme-linked immunoadsorbent assay late reader (ELX800, Bio-TEK, USA). The medium supplemented with 10% FCS without addition of extracts was used as a control. Six samples per group were tested in the experiment. Results were reported as OD units. 2.8. Alkaline phosphate activity assay Canine bone marrow stromal cells (cBMSC) were isolated and expanded according to the published document with minimal modification [37]. For the assessment of alkaline phosphate (ALP) activity, cBMSC were cultured under the same culture conditions described above for 15 days. ALP activity was quantitatively determined by an assay based on the hydrolysis of p-nitrophenyl phosphate to pnitrophenol according to Lowry et al. [38]. Cells were extracted from the extracts of the 24-h-set paste with 30% C3S and permeablized with the use of 0.1% Triton X-100 solution (Sigma). Cell lysate from each sample was then used for alkaline phosphatase assays. The absorbance was measured at 405 nm with the use of a spectrophotometer (UV–VIS 8500, Shanghai, China), and ALP activity was calculated from a standard curve after normalizing to the total protein content. The results were expressed as
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nanomoles of p-nitrophenol produced per minute per microgram of protein. ALP activity of cells cultured in medium supplemented with 10% FCS without addition of extracts served as a control. Five samples for each material were tested for each incubation time. Each test was performed in triplicate. 2.9. Statistical methods The experimental values were analyzed using Student’s t-test and expressed as mean ± SD. A p-value of <0.05 was considered statistically significant. 3. Results 3.1. Chemical composition and microstructure of the composite cements The results of the XRD, SEM and EDX analyses indicated that both the chemical composition and microstructure of the hydration products of the TCP/MCPM/C3S composite paste samples were significantly different from those of the TCP/MCPM paste sample. The indexed Xray spectrum is presented in Fig. 1. It shows that, for the TCP/MCPM bone cement, the main hydration product was brushite, with a small fraction of non-reacted b-TCP and non-hydrated brushite (monetite) (Fig. 1(A)). However, in the TCP/MCPM/C3S composite samples, peaks for brushite were not observed, and the main hydration products were low-crystallinity calcium-deficient hydroxyapatite (CDHA) and C–S–H, with a larger fraction of non-reacted b-TCP (Fig. 1(B–E)).
Fig. 2 shows the SEM micrographs of the cross-section of the reacted TCP/MCPM and TCP/MCPM/C3S composite paste after setting for 24 h. Fig. 2(A) shows a typical fracture surface for a TCP/MCPM formulation. Here, precipitated brushite and residual b-TCP can be observed as needleshaped crystallites or small particles, respectively. Conversely, for TCP/MCPM/C3S samples with a C3S weight ratio of 10–30%, smooth and fine structures were observed (see, e.g., Fig. 2(B–D)). It is noticed that, as the weight ratio of C3S reached 30% or more, the crystals of hydration products tended to agglomerate within the samples (Fig. 2(E)). The results of EDX analysis indicated that, for the reacted TCP/MCPM brushite paste sample, the Ca:P molar ratio within the material is 1.10, which is between that of brushite (1.00) and b-TCP (1.50). However, a higher Ca:P molar ratio was observed for the TCP/MCPM/C3S paste samples (1.54–1.71) as compared with that of the TCP/MCPM brushite cement, which is close to the theoretical ratio of hydroxyapatite (1.67). It also indicated that an increase in the weight ratio of C3S within the composite cement resulted in an increase in the Ca:P molar ratio within the hydration products, and silicon element was observed for the TCP/MCPM/C3S paste samples. The results of the EDX analysis, in combination with those of the XRD and SEM analyses (Figs. 1 and 2), confirmed that the main hydration product of the TCP/MCPM/C3S was small crystals of calcium-deficient hydroxyapatite. 3.2. Self-setting properties of the composite cement pastes Fig. 3 shows the initial and final setting time of the cement pastes, and the injectability of the pastes is repre-
Fig. 1. XRD patterns of paste samples after aging for 24 h. (A) The TCP/MCPM paste; (B) the TCP/MCPM/C3S composite paste with 10% C3S; (C) the TCP/MCPM/C3S composite paste with 20% C3S; (D) the TCP/MCPM/C3S composite paste with 30% C3S; (E) the TCP/MCPM/C3S composite paste with 40% C3S.
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Fig. 2. SEM micrographs of (A) the TCP/MCPM paste; (B) the TCP/MCPM/C3S composite paste with 10% C3S; (C) the TCP/MCPM/C3S composite paste with 20% C3S; (D) the TCP/MCPM/C3S composite paste with 30% C3S; (E) the TCP/MCPM/C3S composite paste with 40% C3S after aging for 24 h.
Fig. 3. The initial setting time and final setting time of the paste samples with various contents of C3S (S/L = 2.0 g ml1).
Fig. 4. The injectability of the composite pastes with various contents of C3S (S/L = 2.0 g ml1) in 2 min after mixing.
sented in Fig. 4. The TCP/MCPM brushite cement paste set quite rapidly, within 2–3 min, and was consequently unworkable as it failed to be ejected out of the syringe after 2 min. In contrast, it was found that the TCP/ MCPM/C3S composite paste set more slowly (7–25 min for initial setting), and more than 50% of the paste could be extruded through the syringe as the weight ratio of C3S reached 20% or more. The composite paste did not give any demixing or filter pressing during the extrusion. Furthermore, it was observed that both the setting time and the injectability of the TCP/MCPM/C3S composite paste increased with increasing amount of Ca3SiO5 within the composite.
3.3. Mechanical strength of the cement paste Fig. 5(A) shows the compressive strength of the reacted pastes after aging in a 100% humidity water bath at 37 °C for various periods. For the reacted TCP/MCPM cement paste and the TCP/MCPM/C3S composite set formulation with 10% C3S, the compressive strength was low (around 5.0 MPa) and did not change significantly after prolonged aging. In contrast, the compressive strength of the composite paste with more than 10% C3S increased with increasing aging time and amount of C3S. The composite cement with 30% C3S showed the highest compressive strength after prolonged aging (42.5 MPa).
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Fig. 6. Variation in the pH of the suspension after immersion of the paste in SBF.
pastes were higher than that of the TCP/MCPM paste and nearly neutral (6.70–7.35), and after prolonged soaking in SBF, the pH value increased further to 7.59–7.75 after 12 h. 3.5. The bone-like apatite formation on the paste disks after soaking in SBF
Fig. 5. The compressive strength of the paste samples at different aging times in (A) 37 °C and 100% humidity; and (B) PBS.
To further evaluate the in vitro mechanical properties of the material, the materials were soaked in PBS for preset periods and the wet compressive strength was measured after soaking. The results are presented in Fig. 5(B). Comparison between Fig. 5(A) and Fig. 5(B) indicates that a significant decrease in compressive strength was observed for all the samples after aging in PBS as compared with that of the materials aged in the 100% humidity water bath. However, the compressive strength of the composite material with more than 10% C3S still increased with increasing aging time and amount of C3S, and was still significantly higher (23.2 MPa) than that of the reacted TCP/MCPM paste (5.9 MPa) under the same soaking conditions. 3.4. pH measurement Fig. 6 shows the changes in the pH values of the SBF with time in the presence of composite cement pastes. In the case of the TCP/MCPM paste, the pH was initially 4.0 and increased gradually to 5.2 after 12 h. In contrast, the initial pH values of all the TCP/MCPM/C3S composite
To determine the bioactivity, the reacted TCP/MCPM and TCP/MCPM/C3S paste disks were soaked in SBF. The SEM micrographs of the surfaces of the disks after soaking in SBF are presented in Fig. 7. It can be clearly seen that, after soaking in refreshed SBF for 7 days, the TCP/MCPM cement became porous and consisted of spherical b-TCP particles 1–10 lm in diameter, and no bone-like apatite layer was formed on the surface (Fig. 7(A1 and A2)), which corresponded with previous studies on the in vitro aging of the brushite cement [25]. In contrast, newly formed lath-like crystals were observed on the surface of TCP/MCPM/C3S samples after soaking in SBF. When the C3S content within the composite cements increased to 20% or more, many of the crystals formed agglomerates, which further congregated to form a layer that covered the surface of the cement (Fig. 7(D2 and E2)). Such layers have a similar morphology as those formed on the surface of bioactive glass [28] and silicate bioceramics [34] after soaking in SBF, which resulted from the precipitation of bone-like apatite. Furthermore, the result of EDX analysis on the as-observed lath-like crystals suggested that the Ca:P molar ratio is 1.63, which is close to the theoretical ratio of hydroxyapatite (1.67), and such a result, combined with that of SEM analysis (Fig. 7), confirmed the formation of bone-like apatite layer on the surface of reacted TCP/MCPM/C3S composite paste after soaking in SBF. 3.6. In vitro degradation Fig. 8 shows the degradation of the TCP/MCPM and TCP/MCPM/C3S set formulations after soaking in PBS
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Fig. 7. SEM micrographs of the surfaces of the paste samples after soaking in SBF for 7 days. (A1 and A2) the TCP/MCPM paste; (B1 and B2) the TCP/ MCPM/C3S composite paste with 10% C3S; (C1 and C2) the TCP/MCPM/C3S composite paste with 20% C3S; (D1 and D2) the TCP/MCPM/C3S composite paste with 30% C3S; (E1 and E2) the TCP/MCPM/C3S composite paste with 40% C3S.
for various time periods. It was found that early dissolution of set formulations increased upon raising the C3S percentage. After 4 days the C3S percentage had no obvious effect on degradation rate.
3.7. Cytotoxicity test Fig. 9 shows the result of cytotoxicity test on the extracts of both TCP/MCPM and TCP/MCPM/C3S composite set
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Fig. 8. Degradation of the paste samples with various contents of C3S after soaking in PBS for various times.
Fig. 10. The ALP activity of the cBMSC in the presence of the dissolution extracts of the cement pastes. TCP/MCPM/C3S cements significantly enhanced the ALP activity of the cBMSC, which was not observed for TCP/MCPM cement samples. The asterisk (*) indicates that the ALP activity of experimental group was significantly higher than that of the control (p < 0.05).
the extracts was in the range from 6.25 104 to 5 101 mg ml1, while no enhanced proliferation of the cells was observed for the reacted TCP/MCPM paste. 3.8. The effects of cements on ALP activity of cBMSC Cell differentiation was assessed in terms of the ALP activities of the cBMSC after culturing for 15 days (Fig. 10). The results indicated that cells cultured in the extract of TCP/MCPM specimen did not showed higher ALP activity than that of the control, whereas the extract of TCP/MCPM/C3S reacted paste significant enhanced the ALP activity of the cBMSC with the concentration ranging from 6.25 105 to 2.5 101 mg ml1. Fig. 9. Osteoblast cells proliferation in the presence of the dissolution extracts of the powders of the TCP/MCPM and TCP/MCPM/C3S composite pastes with 30% C3S after culturing for 6 days. The asterisk (*) indicates that the cell proliferation of experimental group was significantly different from that of the negative control (p < 0.05).
formulations against osteoblast-like cells after incubation for 6 days. It was observed that the extract of TCP/MCPM paste showed cytotoxicity against osteoblasts when the concentration of the extracts was higher than 12.5 mg ml1 (p < 0.05). In contrast, no significant cytotoxicity against osteoblasts was observed on the samples with 30% C3S and other ratios of C3S (10, 20 and 40 wt.%; data not shown) in the present study, which indicated the superior biocompatibility of the TCP/MCPM/C3S cement as compared with that of the TCP/MCPM cement. Furthermore, it was found that, after incubating for 6 days, the osteoblast-like cell proliferation in the extract of the TCP/ MCPM/C3S reacted paste was significantly higher than in the negative control (p < 0.05) when the concentration of
4. Discussion 4.1. Self-setting properties and mechanical strength of the composite pastes The applicability of injectable self-setting biomaterials is largely dependent on its self-setting characteristics, such as injectability and setting times. Since the original brushite cements showed short setting times and poor injectability, which limit the application of the material, efforts have been taken to overcome the problem, and the use of various additives as retardants has been proven to be an effective way to improve the self-setting properties of the material [12,39,40]. In the present study, the homogeneous TCP/MCPM/C3S composite paste showed a prolonged setting time (7–25 min) and could be mostly injected through the syringe within 2 min after mixing, which is partly attributed to the low hydration rate of the C3S component. According to previous studies on brushite cements,
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a low Ca/P ratio and acidic surrounding pH values (around 4.0) are necessary for the precipitation of brushite crystals [11,41,42], otherwise hydroxyapatite (HA) is formed by preference during the hydration of the cement [41]. The Ca/P ratio in the TCP/MCPM/C3S cement system is higher than in brushite cement, and Ca3SiO5 can react with water to form C–S–H and calcium hydroxide according to the following equation: Ca3 SiO5 þ H2 O ! C–S–H þ CaðOHÞ2 2+
ð2Þ
which results in the release of Ca and OH and an increase in the pH. With the higher Ca/P ratio and the surrounding pH value, CDHA is more likely to be precipitated than brushite crystals during the hydration of TCP/MCPM/C3S composite cement. Since CDHA crystals precipitate much more slowly than brushite crystals [42], the TCP/MCPM/C3S composite paste showed a retarded setting time and consequently improved the injectability compared with the brushite cement paste. Furthermore, the low hydration rate and good injectability of the tricalcium silicate component in the composite cement contributed to the improved self-setting properties of the TCP/ MCPM/C3S composite paste. Taking into account that in clinical applications the cement must be applied before the initial setting time and must be able to be injected during the operation, our results suggest that the TCP/ MCPM/C3S paste is potentially a good injectable material for bone/dental repair. In clinical applications, bone substitute materials are required to provide adequate short- and long-term mechanical support for the defect site, which is important for the bone healing process. Brushite cements are typically weaker than the majority of apatite cements [8], and a main reason for the comparatively low compressive strength is the extremely rapid setting reaction of the brushite cements, which results in high porosity and consequently weak cement. In the present study, the compressive strength of the reacted TCP/MCPM/C3S composite pastes reached 23.2–42.5 MPa, which is higher than that of brushite cements as reported by other researchers [8,11,41]. Such results could be attributed to the lower setting rate and the consequently more compact microstructure of the TCP/ MCPM/C3S composite paste as compared with that of the brushite cement (as seen in Fig. 2). In addition, since Si ions tend to inhibit grain growth of HA crystals [43], the size of the precipitated CDHA crystals within the TCP/MCPM/C3S composite paste were observed to be on the nanoscale, which further promoted the formation of a compact microstructure of the hydrated composite cement [8]. Furthermore, the TCP/MCPM/C3S composite cement showed the ability to self-reinforce during setting. The compressive strength of the composite cements increased with time during the setting both in 100% humidity and in saline such as PBS, which has not been observed for the majority of calcium phosphate cements [5,8,11,28]. Such an ability to self-reinforce is attributed to the fact that the continuous hydration process of Ca3SiO5 resulted in
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progressive polymerization of the calcium silicate hydrate gels and the development of a solid network that enhanced the long-term mechanical strength of the set composite [20,44,45]. This property is important because a self-reinforced cement system can better tolerate the adverse effect of the degradation process on the structure of the cement materials, hence providing more stable long-term mechanical support during the bone regeneration process. 4.2. In vitro bioactivity and degradation As compared with bioactive bone substitution materials such as A-W Bioglass and calcium silicate ceramics, the calcium phosphate cements show a poor ability to induce a bone-like apatite layer on the surface which can form a chemical bond with bone tissue within a short period [18,46]. This was also supported by the finding that no apatite layer deposition was observed for the TCP/MCPM paste after soaking in SBF in our study. In contrast, the combined SEM and EDX analysis suggested that the TCP/MCPM/C3S composite paste induced apatite deposition within 1 week of soaking in SBF, indicating that the composite paste showed excellent bioactivity. Such a result could be attributed to the addition of Ca3SiO5, and the formation of the homogenous apatite layer was dependent on the Ca3SiO5 content. This finding confirmed our assumption that the addition of bioactive Ca3SiO5 could result in bioactive composite cements with controllable bioactivity. As is generally accepted, the increase in the supersaturation of Ca2+ is not enough to promote the formation of new crystals of apatite on the material surface in the simulated body environment [31,47], and this explains the lack of bioactivity of the TCP/MCPM cement. With the addition of Ca3SiO5, HSiO 3 ions are released during the hydration of the composite paste, and this could play an important role in the formation of apatite in the simulated body environment together with Ca2+ ions by providing favorable sites for the nucleation of apatite crystals. It is understandable that when the content of Ca3SiO5 is low, the concentration of HSiO 3 ions that act as the nucleation site for apatite was not adequate to support the formation of an apatite layer on the surface of the composite paste. However, when the content of Ca3SiO5 is increased to a certain extent (20% in the present study), the concentration of HSiO 3 ions is high enough for the ions to be dispersed within the composite paste and provides more nucleation sites for apatite crystals, resulting in the formation of a homogeneous apatite layer throughout the whole composite paste. The TCP/MCPM/C3S composite cement showed a slightly higher degradability than the TCP/MCPM cement. Since the degradability is primarily governed by the chemical composition and the physical characteristics of the material, the higher degradation rate of the TCP/ MCPM/C3S composite cement as compared with that of TCP/MCPM cement may be attributed to the higher degradability of calcium silicate hydrate within the reacted
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composite paste than that of the brushite within the TCP/ MCPM paste [20,28]. As TCP/MCPM brushite cement is generally accepted as a biodegradable material in vivo [6,7,10,39], the results in the present study indicate that the TCP/MCPM/C3S composite cement may also be degradable in clinical applications, with a degradation rate similar to that of TCP/MCPM cement. However, further studies need to be conducted to confirm the in vivo degradation properties of the TCP/MCPM/C3S composite material. 4.3. In vitro cytotoxicity It is generally accepted that the in vitro cell–material interaction is a useful criterion in the evaluation of new biomaterials. It is known that MCPM is weakly acidic, and the implantation of TCP/MCPM paste can result in an acid pH in the environs that may be clinically undesirable [40]. The present study confirmed that hydrated TCP/MCPM paste revealed significant cytotoxicity (p < 0.05) against osteoblasts as the concentration of the paste extracts varied from 25 to 200 mg ml1 (Fig. 9). In contrast, the TCP/MCPM/C3S composite pastes did not cause any significant increase or decrease in the surrounding pH value (Fig. 6), which could be attributed to modification of the MCPM by the OH ions that were released through the hydration of C3S, and therefore the composite cement did not show any cytotoxicity against osteoblasts, as observed in the present study. Moreover, it was observed that the ionic dissolution products of the reacted TCP/ MCPM/C3S composite pastes could provide more than adequate stimulus for cell proliferation. According to previous studies, dissolution extracts of bioactive silicate materials such as bioactive glasses and ceramics could stimulate cell proliferation [21,48]. A recent study also showed that tricalcium silicate paste could stimulate L929 cell proliferation [20]. This stimulatory effect may be due to the dissolution of silicate ions [21]. The present study indicated that the extracts of the reacted TCP/MCPM/C3S composite paste also stimulated proliferation of osteoblasts, suggesting that the composite cement was not only biocompatible and non-cytotoxic, but also possessed excellent bioactivity at the cellular level. 4.4. ALP activity Differentiation of bone marrow stromal cells is one of the key processes for bone regeneration. As a cell surface glycoprotein that is involved in mineralization [49], ALP is the most widely recognized marker of osteoblastic differentiation [50]. In the present study, the ionic extract of the reacted TCP/MCPM/C3S composite paste significantly enhanced the ALP activity of cBMSC, which was not observed for TCP/MCPM set formulation. This result suggested that TCP/MCPM/C3S composite cement possesses a much greater ability to promote osteoblastic differentiation of cBMSC than TCP/MCPM cement. As the previous
studies have shown, Si ions play an important role in bone metabolism and have been shown to raise ALP and osteocalcin expression in human osteoblast-like cells essential in the mineralization process and the bone-bonding mechanism [51,52]. Additionally, Sun et al. found that a silicate ceramic (akermanite) could enhance ALP activity of human bone marrow stromal cells [53]. Therefore, in comparison to TCP/MCPM, the effect of TCP/MCPM/C3S cements on the osteoblastic differentiation of cBMSC may be explained by differences in chemical composition, and the ionic products of the hydrated TCP/MCPM/C3S cement might play an important role in the stimulatory process. More in vitro and vivo studies are necessary to further investigate the mechanism of the stimulatory effects of TCP/MCPM/C3S cements on the osteoblastic differentiation of bone marrow stromal cells. 5. Conclusion In this paper, novel bioactive composite cements were successfully prepared by adding tricalcium silicate into the traditional TCP/MCPM cement system. The TCP/M CPM/C3S composite cements showed a prolonged setting time, significantly improved injectability and mechanical strength compared with the brushite-forming TCP/MCPM cement. Furthermore, the TCP/MCPM/C3S composite cement possessed excellent bioactivity, as indicated by the formation of bone-like apatite in SBF, and showed similar in vitro degradability to that of the TCP/MCPM cement, which is generally considered to be a degradable bone graft material in vivo. The cytotoxicity test showed that the TCP/MCPM/C3S composite cement was not only biocompatible and non-cytotoxic, but also stimulated cell proliferation. Furthermore, the TCP/MCPM/C3S cements possessed the ability to promote osteoblastic differentiation of cBMSC. Our study presents a novel method to design bone cements with improved properties based on traditional calcium phosphate bone cements by adding bioactive self-setting components, and suggests that the addition of bioactive Ca3SiO5 is a possible way to obtain bioactive self-setting composite biomaterials for bone regeneration. Acknowledgements This work was supported by the National Basic Science Research Program of China (973 Program) (Grant No. 2005CB522704), and funds from the Chinese Academy of Sciences for Key Topics in Innovation Engineering (Grant No. KGCX2-YW-207) and the Natural Science Foundation of China (Grant No. 30730034). References [1] Constanz BR, Ison IC, Fulmer M, Poser RD, Smith ST. Skeletal repair by in situ formation of the mineral phase of bone. Science 1995;267:1796–9.
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