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Preparation and in vitro evaluation of strontium-doped calcium silicate/gypsum bioactive bone cement
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Biomed. Mater. 9 045002 (http://iopscience.iop.org/1748-605X/9/4/045002) View the table of contents for this issue, or go to the journal homepage for more
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Biomedical Materials Biomed. Mater. 9 (2014) 045002 (13pp)
doi:10.1088/1748-6041/9/4/045002
Preparation and in vitro evaluation of strontium-doped calcium silicate/gypsum bioactive bone cement Juncheng Wang1, Lei Zhang1, Xiaoliang Sun1, Xiaoyi Chen2, Kailuo Xie1, Mian Lin1, Guojing Yang1, Sanzhong Xu3, Wei Xia4, Zhongru Gou2 1
Department of Orthopedics, Rui’an People’s Hospital & the 3rd Hospital Affiliated to Wenzhou Medical University, Rui’an 325200, People’s Republic of China 2 Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou 310029, People’s Republic of China 3 Department of Orthopedics, The First Affiliated Hospital, School of Medicine of Zhejiang University, Hangzhou 310003, People’s Republic of China 4 Department of Engineering Sciences, The Ångstrom Laboratory, Uppsala University, 75121 Uppsala, Sweden E-mail: [email protected] and [email protected] Received 24 April 2014 Accepted for publication 12 May 2014 Published 12 June 2014 Abstract
The combination of two or more bioactive components with different biodegradability could cooperatively improve the physicochemical and biological performances of the biomaterials. Here we explore the use of α-calcium sulfate hemihydrate (α-CSH) and calcium silicate with and without strontium doping (Sr-CSi, CSi) to fabricate new bioactive cements with appropriate biodegradability as bone implants. The cements were fabricated by adding different amounts (0–35 wt%) of Sr-CSi (or CSi) into the α-CSH-based pastes at a liquid-to-solid ratio of 0.4. The addition of Sr-CSi into α-CSH cements not only led to a pH rise in the immersion medium, but also changed the surface reactivity of cements, making them more bioactive and therefore promoting apatite mineralization in simulated body fluid (SBF). The impact of additives on longterm in vitro degradation was evaluated by soaking the cements in Tris buffer, SBF, and α-minimal essential medium (α-MEM) for a period of five weeks. An addition of 20% Sr-CSi to α-CSH cement retarded the weight loss of the samples to 36% (in Tris buffer), 43% (in SBF) and 54% (in α-MEM) as compared with the pure α-CSH cement. However, the addition of CSi resulted in a slightly faster degradation in comparison with Sr-CSi in these media. Finally, the in vitro cell-ion dissolution products interaction study using human fetal osteoblast cells demonstrated that the addition of Sr-CSi improved cell viability and proliferation. These results indicate that tailorable bioactivity and biodegradation behavior can be achieved in gypsum cement by adding Sr-CSi, and such biocements will be of benefit for enhancing bone defect repair. Keywords: strontium-substituted calcium silicate, gypsum, bioactivity, biodegradability, composite cements (Some figures may appear in colour only in the online journal)
1. Introduction
a common technique in surgical practice. Whereas the limitations in the quantity and donor site complications are associated with autografts, and the concerns related to the immune response and possible transmission of infectious diseases or lack of good osteointegration are the main drawbacks of allografts. Thus the limitations in these treatments promote
In the present ageing societies, a significant increase in pathological or sport bone injuries leads to a growing medical interest in developing new bone augmentation and repair technologies [1–3]. Bone grafting to augment skeletal healing is 1748-6041/14/045002+13$33.00
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the development of synthetic biomaterials with excellent physicochemical and biological performances. In general, an ideal bone implant should possess features of good biocompatibility and excellent bioactivity, as well as an appropriate biodegradation rate to stimulate bone tissue regeneration and remodeling. Calcium sulfate dihydrate (CSD, CaSO4∙2H2O), so-called gypsum, has been used clinically as bone filling material since 1892 by Dreesmann and there were numerous subsequent studies for its use in filling bone defects [4, 5]. Gypsum is used in an hydraulic paste form produced by the hydration of calcium sulfate hemihydrate (CSH, CaSO4∙2H2O), which undergoes in situ setting after filling bone defects, and has good biocompatibility without inducing a significant inflammatory response [6–9]. CSH exists in at least two forms (α and β), and in general, the difference in physicochemical properties of the set paste are usually attributed to their primary particle morphology [5, 10]. Research has shown that the calcium ions released into a solution can stimulate osteogenic differentiation of bone marrow-derived stromal cells [11] and enhance alkaline phosphatase activity as well as gene expression associated with bone regeneration [7]. However, previous studies revealed that the high concentration ion product dissolution from gypsum shows some cytotoxic effects in vitro [12]. In particular, gypsum is usually bioresorbed completely in vivo in four to six weeks [13], which is too fast compared to the new bone tissue formation, and it cannot form a strong bone ingrowth at the early healing stage due to its poor bioactivity. Calcium silicate (CSi; named wollastonite) as one of the bioactive components of apatite-wollastonite (A-W) glass ceramics, has been recently demonstrated to be bioactive [14–16], and can be used to stimulate bone tissue regeneration [17, 18]. However, some drawbacks of CSi are considered because of its poor mechanical property [19, 20] and its fast degradation rate leading to a high Si ion concentration which would have a negative effect on cell growth [21]. Fortunately, studies have demonstrated that the physicochemical properties of bioceramics could be improved by the incorporation of strontium (Sr) which was found to have dual effects of stimulating bone formation and reducing bone resorption [22–24]. Lu’s group developed a strontium-doped hydroxyapatite (SrHA) cement and their widespread studies demonstrated that the Sr-HA cement could significantly stimulate bone formation and osseointegration in normal and osteoporotic bone regeneration [25–29]. Bose et al found that the Sr-doped β-tricalcium phosphate (Sr-TCP) promoted more osteogenesis by excellent early stage bone remodeling as compared to undoped β-TCP, and retarded the degradation rate of TCP [30, 31]. More recently, they found that a tailorable strength and strength degradation behavior can be achieved in β-TCP via compositional modifications using small amount of co-dopants such as Sr and Si [32]. Chang’s group has also investigated the biological response of bone-forming cells to the Sr-containing silicate bioceramics. Their studies demonstrated that the combination of the bioactive elements such as Sr and Si was effective in regulating the osteogenic property of human bone marrow mesenchymal stem cells, and the ionic products from the bioceramics significantly enhanced the ALP
activity and bone-related genes expression [33]. It has been revealed that the incorporation of Sr into CSi (Sr-CSi) can decrease its degradation rate and stimulate the proliferation of osteogenic cells while not change the apatite mineralization ability in simulated body fluid (SBF [34–36]). Moreover, Zhang et al found that partial substitution by Sr may increase HA solubility and inhibit the rapid degradation of borosilicate, but appears to have different mechanisms of physiochemical and biological benefit on different biomaterials [37]. Therefore, the development of Sr-CSi-added, gypsum-based biocement is potentially favorable for enhancing bone tissue regeneration and repair. Therefore, the objective of the present study is to develop a CSH/Sr-CSi biocement which allows setting when in contact with water and enhances bioactivity. The present study summarizes our efforts to prepare the composite biocement, and investigate their surface microstructure, bioactivity, biodegradability and in vitro cell response. 2. Materials and methods 2.1. Powder preparation
The α form of CSH was prepared by the hydrothermal reaction method, in which CSD powder was treated in boiling 15% NaCl solution and stirred for 5 h at 100 °C using 0.1% citric acid as the morphology modifier [38]. After that, the suspension was quickly filtered and immediately washed three times in boiling water and then dried at 120 °C for 6 h. The CSi powder was prepared by the chemical precipitation method as described previously [39]. Firstly, 500 ml Na2SiO3 solution (0.4 mol l−1) was added drop by drop into 500 ml Ca(NO3)2 solution (0.4 mol l−1) whose pH value was kept at 11.4 by rigorous stirring. After titration, the solution was stirred for 24 h, filtered and washed three times with deionized water and three times with ethanol. The precipitates were dried at 80 °C for 24 h and finally calcined at 800 °C for 3 h. Similarly, the Sr-CSi was synthesized by partially replacing the Ca(NO3)2 with 8% Sr(NO3)2 (in molar ratio) and the other conditions remained the same [40]. 2.2. Cement preparation
The α-CSH powders account for 0%, 5%, 10%, 20% and 35% (weight ratio) when mixed with the Sr-CSi. The mixtures were moistened by deionized water (0.4 ml g−1) and vibrated to form an homogeneous paste, and then transferred into a stainless steel mold with an internal diameter (Ø) of 5.0 or 8.0 mm and stored in a 37 °C water bath with 100% humidity for 2 h. After solidifying, the samples were mold released and air-dried at room temperature. The powders and cements were analyzed by x-ray diffraction (XRD; Rigaku), and the crosssectional morphology was observed by scanning electron microscope (SEM; HITACHI, S4800). 2.3. Mechanical test
After air-drying at room temperature for one and seven days, the compressive strength of the cements (Ø 5 × 10 mm) was 2
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10% fetal calf serum (FCS). The medium supplemented with 10% FCS without adding extracts was used as the control. After culturing at 37 °C and 5% CO2 for one, three and five days, 20 μl of methylthiazolyl- diphenyl-tetrazolium bromide (MTT) solution was added to each well and cultured for another 4 h. Then the solution in each well was replaced by 100 μl dimethyl sulfoxide and was shaken for 10 min. The plates were then read in a multifunctional microplate reader SpectraMax (Infinite® M200 Pro, TECAN, CH) at the optical density of 490 nm. Four duplicates were tested in the experiment. The morphology of the cells cultured with the extract solution was observed under SEM.
measured at a loading rate of 1.0 mm min using a universal testing machine (Instron) and four parallel tests were carried out for each group. 2.4. Degradation in vitro
Degradation in vitro was estimated by soaking the cements (W0; Ø 8 × 15 mm) in 0.05 M Tris buffer (pH 7.40), simulated body fluid (SBF) and a serum containing α-minimal essential medium (α-MEM) at 37 °C with a surface area-tovolume ratio of 0.1 cm−1, respectively. The buffer solution was refreshed every 48 h. At the preset time interval, the cements were removed from the liquid, gently rinsed with ethanol, and dried at 80 °C to weight constancy (Wt) before weighing by using an electronic analytical balance (FA2104, Sartorius, Germany). The weight loss (degradation) was expressed as the following equation: weight loss = (W0 − Wt)/W0 × 100%. Meanwhile, the pH value of the media was measured every 48 h before refreshment using an electrolyte type pH meter.
2.8. Statistical analysis
All of the data above was expressed as mean ± standard deviation (SD) and analyzed with the one-way ANOVA. In all cases the results were considered statistically significant with a p value of less than 0.05. 3. Results
2.5. Ion releasing during degradation in vitro
3.1. Phase and morphology of the powders
To investigate the ion release of the composite-cements, the cement samples were soaked in Tris buffer in a water bath at 37 °C for 1–15 days with a surface area-to-volume ratio of 0.1 cm−1. The Ca, Si and Sr ions in the supernatant were measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES; Thermo).
Figure 1 shows the XRD patterns of the powder and sevenday-set cement samples. The major diffraction peaks and the intensity of the pure α-CSH powders are attributed to the standard data (figure 1(a)). As for the CSi and Sr-CSi powders, Sr doping into CSi cannot be found in the XRD pattern compared with pure CSi powder, and there were no other phases such as CaO, SrO, SiO2 or α-CSi (high temperature phase of wollastonite) beside β-CSi (figures 1(b), (c)), indicating it is a single phase product. For the pure CSH-derived cement and the bulk composites with the Sr-CSi additive, the obvious peaks near 20.8°, 11.6° and 29.3°/2θ, corresponding to CSD phases, have been transformed from CSH (figures 1(d), (e)), but the pure Sr-CSi hardly hydrated at all in this time period (figure 1(f)). Figure 2 shows the SEM images of the CSD precursor and the synthetic powders in an aqueous medium, respectively. It is clear that the CSD-to-CSH phase transformation led to significant changes in morphology. The α-CSH powders have stable prismatic shapes with a length-diameter ratio near to 1:1, and an average diameter of about ten microns (figures 2(a), (b)). However, the granular Sr-CSi powders with irregular particle shapes had no significant difference from CSi powder, and meanwhile, these powders exhibit similar particle sizes of several micrometers (figures 2(c), (d)). These observations suggest that the primary individual particle in the composite cement is very tiny, and there are favorable hydration reactions and mechanical strength development.
2.6. Apatite formation in vitro
The SBF with ion concentrations nearly equal to that of human blood plasma was prepared according to the method proposed by Kokubo et al [41]. For characterization of in vitro bioactivity, the 72 h set discs (Ø 5 × 2 mm) were soaked in SBF at 37 °C in a water bath for 72 h with a surface areato-volume ratio of 0.1 cm−1. At the preset time the discs were gently rinsed with ethanol and dried at room temperature for SEM observation. 2.7. Cytocompatibility test
The cytocompatibility of the cements was evaluated by the extraction method, with human fetal osteoblastic cell line hFOB 1.19, according to the international standard [42]. The seven-day-set cement compacts were ground into particles and sieved through a 200 mesh sieve. The extract solutions were prepared by immersing 1.0 g cement particles in 5 ml α-MEM for 24 h at 37 °C in a CO2 incubator. After that, the suspension was centrifuged and the supernatant was filtered through a 0.22 μm Millipore membrane. Then the extract solution was diluted to 1/2, 1/4, 1/8, 1/16, 1/32 and 1/64 of the original concentration with fresh α-MEM. The cell suspension (100 μl) with a density of 1 × 104 cells −1 ml was seeded into each well of the 96-well plate and incubated for 24 h at standard conditions. The culture medium was then removed and replaced by 100 μl diluted extracts with
3.2. Microstructure and mechanical strength of cements
Figure 3 shows the cross-sectional microstructure and biphasic distribution of the CSH/CSix and CSH/Sr-CSix cements. It is clear that the hydration reaction of α-CSH with water resulted in a prismatic-to-rod transformation in the crystal morphology. 3
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Figure 1. XRD patterns of the powders and the 7-day-set cements. (a) α-CSH powder; (b) CSi powder; (c) Sr-CSi powder; (d) pure α-CSH cement; (e) CSH/CSi35 cement; (f) pure Sr-CSi cement.
Figure 2. SEM images of (a) CSD, (b) α-CSH, (c) CSi and (d) Sr-CSi powders.
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Figure 3. SEM images of cross-sectional microstructures of the (a)–(c) CSH/CSix and (d)–(f) CSH/Sr-CSix cements after setting for seven days.
The CSD crystals were tightly packed but the CSi aggregates were distributed unequally in the gypsum substrate. Figure 4 presents the compressive strength of the composite cements with different contents of CSi or Sr-CSi after setting for one and seven days, respectively. The specimens showed slight decreases in strength with the increase of Ca-silicate components. The pure gypsum cement had a compressive strength value of 24.4 MPa on average after one day, significantly (p < 0.05) lower than the cement after setting for seven days (29.8 MPa), indicating an increase of the compressive strength with time proceeding. The addition of CSi up to 10–35% achieved a significantly decreased strength, down to 17.2–9.1 MPa. However, prolongation of setting time positively affected the mechanical strength of the composite cements with an increase of up to 25% of the highest value after setting for 7 days. On the other hand, the Sr-CSi has no significantly (p > 0.05) different effect on the compressive strength of the composite cement compared with pure CSi under the same content condition. These data indicate that the difference is significant but the negative effect of CSi on the compressive strength of the composite cement is still acceptable with the lowest strength ranges from 11.9 to 14.1 MPa, which is superior to human cancellous bone (2.0–10.0 MPa).
Figure 4. Compressive strength of the CSH/CSix and CSH/Sr-CSix composite cements after setting for one and seven days, respectively.
3.3. In vitro bioactivity of cements
As for the surface microstructure and apatite mineralization ability, it can been seen from figure 5(a) that the pure gypsum cement (i.e. CSH/CSi0) showed a much denser surface structure, composed of rod- or plate-like CSD crystals, 5
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Figure 5. SEM images of the surface of the α-CSH cements before (a) and after (b) soaking in SBF for 72 h and EDX spectra (c).
2.89 (15.43/5.33), 2.57 (15.29/5.95) for the CSH/CSi5, CSH/ CSi35, CSH/Sr-CSi5 and CSH/Sr-CSi35, respectively. These values were significantly lower than the Ca/P molar ratio of 4.40 (12.28/2.79, figure 5(c)) for pure gypsum after soaking for same time period, indicating that gypsum cement exhibits a poor apatite deposition ability.
but the surface of the SBF-soaked gypsum was transformed to a porous microstructure (figure 5(b)). The area-scanning EDX analysis confirmed that Ca-phosphate salt was possibly deposited or absorbed onto the soaked cement surface (figure 5(c)), though the Ca/P molar ratio (~4.40) was very high. Figure 6 shows the SEM images of the surface morphology of the composite cements with different ratios (5%, 20%, 35%) of CSi (or Sr-CSi) before and after soaking in SBF for 72 h. The hydrated CSH transforms to rod-like CSD crystals (not shown). When adding CSi, the hybrid composite became a heterogeneous structure with an inhomogeneous distribution of CSi within the gypsum matrix, and there were many rod-like crystals which connected with each other to form the paste. No significant difference between the CSi-added cement and the Sr-CSi-added cement with the same content was observed in structures. With the increase of CSi content, the CSi component occupied more space inside the cement and would finally block the connection of the CSD crystals. In addition, there were irregular aggregates in the CSD crystals, which became more obvious with increasing CSi content. In contrast, it seems that a layer of an apatite-like mineral was deposited onto the surface of the cements after soaking for 72 h, and especially more sphere-like apatite aggregates were deposited on the composites with higher CSi content. The results from the representative EDX analysis (figure 6, inset) of the cement surface layer confirmed that the Ca/P molar ratios were approximately 2.22 (19.20/8.63), 1.87 (12.50/6.67),
3.4. Degradation behavior in vitro
The degradation of the cements was monitored by weight loss and pH variation in three different types of buffer systems. It is seen that the pure gypsum cement (i.e. CSH/CSi0) was degraded rapidly in the Tris buffer and was nearly dissolved completely after 35 days (figure 7(a)). The samples containing 5% CSi or 10% CSi had a slight effect on the degradation of the cements in Tris buffer. While increasing the CSi content up to 20–35%, the degradation rate of the composite cements decreases significantly, with nearly 17–22% of the cement residual in the medium. In fact, the Sr doping in CSi exhibited some degree of inhibition on the degradation rate of the composite cements. For instance, the CSH/CSi35 degraded by 28% after 14 days but only 22% of CSH/Sr-CSi35 was dissolved in the buffer within the same time scale; meanwhile about 31% cement residual remained in the solid phase after 35 days during the same immersion time period (figure 7(b)). In contrast, the cell culture medium (i.e. α-MEM) and SBF adversely affected the degradation behavior of the gypsum-based 6
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Figure 6. SEM images and EDX spectra of the surface of the (a)–(f) CSH/CSix and (g)–(l) CSH/Sr-CSix cements before (a)–(c), (g)–(i) and after (d)–(f), (j)–(l) soaking in SBF. Insets representing the area-scanning EDX spectra.
and especially that enough of the Sr-doped additive (i.e. Sr-CSi) reduces the degradation rate of the composite cements in simulated body environments. Figure 8 presents the changes in pH value of the three types of immersion media during immersion of the cements. It is evident that there is a minimal variation (within 0.2 units) for the medium immersing the pure gypsum cement. As the content of CSi or Sr-CSi increased, the pH value of the solutions increased significantly within the initial seven days and became relatively stable for the remaining soaking procedure. As the CSi turned the solution alkaline when hydrated in water, the more CSi contained in the composites, the more basic the solution would be. However, the Sr doping into CSi weakened the increase of pH value and made the degradation environment moderate. Significant differences (p < 0.05) were identified from the time period of three to nine days between the CSH/CSi and CSH/Sr-CSi composite cements. After that the pH values reduced and the composites containing 20% and 35% Sr-CSi still showed significant differences (p < 0.05) with the CSi
composite cements (figures 7(c)–(f)). The weight loss is seen to slowly increase with immersion time and varies with the CSi content. In particular, CSH/CSi0 showed a fast degradation but CSH/CSi20 and CSH/CSi35 displayed an extremely slow decrease in weight. Since the SBF and α-MEM are both able to induce re-mineralization on the surface of the bioactive materials, hence the apatite coating deposited on the composite cements would retard the biodissolution of the cements in such biomimetic aqueous media. The SEM observation (not shown) also revealed the new apatite-like deposits on the surface of the CSH/CSi35 and CSH/Sr-CSi35 after immersion in SBF and α-MEM for 35 days, respectively. Indeed, the SBF may heavily influence the degradation of the composite cement in comparison with the cell culture medium. However, no significant difference could be seen in the trends among the CSi and Sr-CSi additives and the two types of biomimetic media investigated here, as the weight loss in all cases was the same. These results indicate that the addition of Ca-silicate may retard the degradation rate of the gypsum-based cements, 7
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Figure 7. Weight loss of the CSH/CSix (a), (c), (e) and CSH/Sr-CSix (b), (d), (f) composite cements in Tris buffer (a), (b), SBF (c), (d), and α-MEM (e), (f), respectively.
Figure 8. Changes in pH values of immersion media during immersing the CSH/CSix (a), (c), (e) and CSH/Sr-CSix (b), (d), (f) composite cements.
group. These results suggest that the addition of CSi or Sr-CSi into gypsum cement may cause a pH fluctuation toward high pH values, and perhaps this is of great benefit for osteogenic cell activity and bone regeneration [42, 43]. In addition, the ion concentrations of calcium, silicon and strontium released by the cements with different contents of CSi or Sr-CSi into Tris buffer were measured (not shown). All the ion concentrations increased with the increasing immersion time up to nine days, but after that the calcium concentration became stable. Although the samples with higher CSi or Sr-CSi content released higher amounts of silicon and
strontium into the solution, there was a slight reduction in their concentration, possibly due to the inhibiting effect of higher pH levels on the CSi dissolution as mentioned above. 3.5. In vitro biocompatibility evaluation
As shown in figure 9, the conditioned cell cultures containing ion extract dissolutions from cements show no cytotoxicity against the hFOB 1.19 cells after culturing for one to five days, indicating that the addition of CSi or Sr-CSi did not cause negative effects on the biocompatibility of gypsum-based cements. 8
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Figure 9. MTT test of the cell culture in the absence and presence of the dissolution extracts of the CSD/CSix, and CSD/Sr-CSix cements after culturing for one day (a), (d), three days (b), (e), and five days (c), (f), respectively. (*p < 0.05)
1 and 3 days, respectively. It is clear that no significant difference was observed on the amount or morphology among the three groups on day 1. However, the cells had better aggregation and adhesion in the composite cement extracts compared to the pure gypsum extract on day 3 in terms of SEM observations, implying that the ion extracts dissolution from the composite cements could be regarded as a cell-promoter.
More specifically, the cell proliferation on days 1 through 5 steadily increased in the CSD/CSi cement groups, and the CSD. conditioned medium The cell proliferation on days 1 through 3 steadily increased in the conditioned media with extracts diluted from 1/2 to 1/8, indicating the increasing number of viable cells. As for the cell viability on day 5, the optical density values for the cells in the extracts diluted from 1/8 to 1/64 were significantly higher (p < 0.05) than those in the extracts diluted to 1/2 and 1/4. Interestingly, the CSH/Sr-CSi20 extracts showed lower cell proliferation on day 3 but higher on day 5 than the CSH/Sr-CSi35 extracts, especially the extracts diluted to 1/4, 1/8 and 1/16. This means that the proper concentrations of the extracts may have positive effects to stimulate cell proliferation. Moreover, cell proliferation in most of the extracts with different dilution ratios was significantly higher (p < 0.05) than the control. Figure 10 illustrates the cell morphology in the presence of the ion extracts of the CSH/Sr-CSi0, CSH/Sr-CSi20 and CSH/ Sr-CSi35 cements diluted to 1/4, 1/8, 1/16 after culturing for
4. Discussion Gypsum has enjoyed a long history of clinical use due to its good biodegradability. However, it is generally agreed that the bioresorption of gypsum cement is slightly faster than the formation of new bone, which is harmful to the reconstruction of the bone defect. Our previous primary experiments found that the addition of a high content of CSi (e.g. 28%) into the fiberlike β-CSH powder results in granular materials with poor cohesion ability when in contact with water up to L/P = 0.7, 9
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Figure 10. SEM micrographs of the low- and high-magnified cell morphology in the presence of the dissolution extracts of the CSH/Sr-CSi
cements after culturing for different time periods.
and the β-CSH-based composite powders containing an upper limit of 23% CSi may yield paste at L/P = 0.7. In the present study, a significantly high amount of the incorporation of CSi (i.e. 35%) into the α-CSH powder with prismatic shape particle morphology could produce an homogeneous paste L/P = 0.4. These experimental results were consistent with other reports [5, 44] in that the particle morphology of CSH heavily influences the crystallization and L/P ratio of gypsum cements. On the other hand, our previous experiments have also demonstrated that proper amounts of CSi addition (e.g. 7%) could fill the small pores or voids in the β-CSH-derived cement matrix and readily increase the mechanical strength of the composite cements. However, the composite of Sr-CSi with α-CSH decreased the compressive strength of the composite cements. The reason could be the addition of Sr-CSi which occupies too much space inside the cements and blocks the interactions among the CSD units. The SEM images have clearly shown the bi-component structure of Sr-CSi and CSD in the cements, and the Sr-CSi aggregates are obviously observed to inhomogeneously insert into the gypsum cement. Nevertheless, the compressive strength (11.86–14.11 MPa) of the CSH/Sr-CSi35
cements set for one to seven days was comparable and even superior to that of human cancellous bone. Such favorable mechanical strength will enable the CSH/Sr-CSi cement to develop more promising injectable bone implants. The degradation rate of the gypsum-based cements is a critical design parameter for bone tissue regeneration because appropriate cement degradation provides the space for matrix deposition and tissue growth, which may ultimately lead to improved quantity and quality of regenerated bone [5]. It is worth noting that the CSH/CSi cement showed a significantly lower degradation rate than the pure α-CSH cement. This is because the CSi has a significantly lower dissolution rate than CSD in such weak basic biomimetic media, and thus the adherence of CSi particles on the surface of CSD crystals could reduce the contact of the CSD crystals with the aqueous medium. On the other hand, it is also demonstrated that the CSi-induced apatite remineralization and deposition on the hydraulic composite surface would retard the biodissolution of the cements. Moreover, the doping of Sr into CaSiO3 additionally decreased the degradation rate as well as increasing the pH value of the surrounding environment, which has been proven 10
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by some previous studies [34–36]. The ion release of the CSH/ Sr-CSi cements showed lower Si ion release and increased Sr ion release, which could also be explained by the decreased degradation rate and pH value of the composite cement. On the other hand, the microenvironmental pH is a function of the chemical properties of the biomaterials (e.g. composition, solubility constant, surface chemistry). Thereby, the extremely stable pH is an intrinsic property of pure gypsum cement and cannot be changed, except if its chemical properties are modified (for instance, by adding a new component). Indeed, changing these properties is equivalent to changing the material. However, this new material couldn’t be compared to the original one since besides the chemical properties, the physical properties also change [5, 45]. An extensive number of studies have revealed that the changes in the extracellular fluid pH of the local biological microenvironment can profoundly affect cell metabolism and function [21, 23], and especially that the Ca-silicate-based biomaterials can induce alkalinization of the external medium upon exposure to (simulated) physiological fluids which is favorable for osteogenic cell function and new bone tissue formation and mineralization [24]. Therefore, the CSi-induced pH rise is another factor for improving the biological performances of such gypsum-based cements. In addition, our studies indicate that the pure gypsum cement is poor in inducing apatite mineralization in SBF. Such a situation has been pointed out in documents: pure gypsum always fails to form an effective chemical bond with the newly formed bone tissue at the early stage of therapy due to its poor bioactivity [13]. In contrast, as the CSi (or Sr-CSi) additive increases to a certain amount, the composite cements could induce apatite formation on the surface of the cements within 3 days, which means the composite cements possess good bioactivity. It is confirmed that when CSi ceramics are immersed into (simulated) physiological fluids in vitro, a series of events occur via ion leaching and exchange with the surrounding solution. This process leads to calcium phosphate precipitation and the formation of a bone-like apatite layer onto the ceramic’s surface. The advantage of the formation of such a Ca-Prich layer onto the composite cements in the present study was two-fold. First, the presence of a Ca-P-rich layer at the surface of biomaterials readily promotes adsorption of serum proteins, (especially of fibronectin [46, 47]) followed by attachment, proliferation and differentiation of osteogenic cells [46, 48]. Second, immersion of the composite cement in physiological fluid results in an increase of the pH of the surrounding microenvironment and the rapid increase in the interfacial pH was slowed down by the Ca-P deposition layer, which also retards the biodissolution of the cements. Therefore, the bioactivity of the CSH/CSi cements is highly significant for promoting osteogenic cell proliferation and mineralization and reducing the biodegradation of the material substrate. A cytocompatibility test is a useful criterion in the evaluation of new biomaterials. Although they produced a pH value of around 7.8 during the degradation in the Tris buffer, the composite cements did not induce any cytotoxicity in present study. Neither the proliferation nor the morphology was influenced by the ion extracts. Moreover, the ion release of the composite cements provides more stimulus for cell proliferation than the
pure gypsum cement which may be attributed to the dissolution of silicon [48] and strontium [22–24]. Cell viability and cell number in the strontium-containing conditioned culture medium do not show any statistically significant differences when compared with the medium in the absence of strontium. It is possible that the amount of strontium in the cement was insufficient to stimulate cell proliferation. Several studies demonstrated the high activity of strontium on osteoblastic cells and reported that micromolar concentrations of strontium were effective in improving the cell attachment and the subsequent cell activities and in stimulating bone regeneration [49, 50]. It is known that active osteoblasts are polygonal or spherical in shape, whereas resting osteoblasts are spindle-shaped and flat. SEM observations showed that cells cultured for 24–72 h were viable and proliferated in the conditioned media. The cells were attached, showed a spherical appearance, and were interacting with the surface through cell phyllopodia and extensions. Their spherical shape is the typical morphology of active osteoblasts. These in vitro cell culture results indicate that the ion extract’s dissolution from the Sr-CSi-added composite cements also possibly stimulates cell proliferation, which means such cements are bioactive for the osteogenic cells. 5. Conclusions A novel biphasic bone cement combined with α-CSH and Sr-CSi was successfully prepared and studied in this paper. The mechanical strength of the composite-cement increased with time proceeding after the initial setting but gradually decreased with the increase of the amount of Sr-CSi. The addition of Sr-CSi slowed down the degradation of the composite cement as well as decreased Si and Sr ion release and lowered the pH increase. The cement composites with Sr-CSi showed better bioactivity compared with CSi or pure α-CSH, as indicated by the formation of bone-like apatite in SBF and the cytotoxicity evaluation of the extracts of the cements. Our results demonstrated that controllable bioresorbability and release of biologically active ions within biphasic bone cement provides a rational strategy for combined bone defect repair and antiosteoporotic therapy. Further investigations focusing on large animal models or clinical applications are needed. Acknowledgements This work is supported by the Zhejiang Provincial Natural Science Foundation of China (No. LQ14H060003, LZ14E020001), the National Science Foundation of China (81271956, 51102211, 81301326), and the Fundamental Research Funds for the Central University (2012QN81001). References [1] Ducheyne P, Mauck R L and Smith D H 2012 Biomaterials in the repair of sports injuries Nat. Mater. 24 652–4 [2] Nair M B, Kretlow J D, Mikos A G and Kasper F K 2011 Infection and tissue engineering in segineering bne defects-a mini review Curr. Opin. Biotechnol. 22 721–5 11
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[21] Zhang N, Molenda J A, Fournelle J H, Murphy W L and Sahai N 2010 Effects of pseudowollastonite (CaSiO3) bioceramic on in vitro activity of human mesenchymal stem cells Biomaterials 31 7653–65 [22] Peng S, Liu X S, Huang S, Li Z, Pan H, Zhen W, Luk K D, Guo X E and Lu W W 2011 The cross-talk between osteoclasts and osteoblasts in response to strontium treatment: involvement of osteoprotegerin Bone 49 1290–8 [23] Qiu K, Zhao X J, Wan C X, Zhao C S and Chen Y W 2006 Effect of strontium ions on the growth of ROS17/2.8 cells on porous calcium polyphosphate scaffolds Biomaterials 27 1277–86 [24] Marie P J, Ammann P, Boivin G and Rey C 2001 Mechanisms of action and therapeutic potential of strontium in bone Calcif. Tissue Int. 69 121–9 [25] Wong C T, Chen Q Z, Lu W W, Leong J C, Chan W K, Cheung K M and Luk K D 2004 Ultrastructural study of mineralization of a strontium-containing hydroxyapatite (SrHA) cement in vivo J. Biomed. Mater. Res. A 70A 428–35 [26] Xue W, Moore J L, Hosick H L, Bose S, Bandyopadhyay A, Lu W W, Cheung K M and Luk K D 2006 Osteoprecursor cell response to strontium-containing hydroxyapatite ceramics J. Biomed. Mater. Res. A 79A 804–14 [27] Li Y W, Leong J C, Lu W W, Luk K D, Cheung K M, Chiu K Y and Chow S P 2000 A novel injectable bioactive bone cement for spinal surgery: a developmental and preclinical study J. Biomed. Mater. Res. 52 164–70 [28] Peng S, Liu X S, Zhou G, Li Z, Luk K D, Guo X E, Lu W W 2011 Osteoprotegerin deficiency attenuates strontiummediated inhibition of osteoclastogenesis and bone resorption J. Bone Miner. Res. 26 1272–82 [29] Wong C T, Lu W W, Chan W K, Cheung K M, Luk K D and Lu D S 2004 In vivo cancellous bone remodeling on a strontium-containing hydroxyapatite (Sr-HA) bioactive cement J. Biomed. Mater. Res. 68A 513–21 [30] Banerjee S S, Tarafder S, Davies N M, Bandyopadhyay A and Bose S 2010 Understanding the influence of MgO and SrO binary doping on the mechanical and biological properties of beta-TCP ceramics Acta Biomater. 6 4167–74 [31] Bose S, Tarafder S, Banerjee S S, Davies N M and Bandyopadhyay A 2011 Understanding in vivo response and mechanical property variation in MgO, SrO and SiO2 doped β-TCP Bone 48 1282–90 [32] Bandyopadhyay A, Petersen J, Fielding G, Banerjee S and Bose S 2012 ZnO, SiO2, and SrO doping in resorbable tricalcium phosphates: influence on strength degradation, mechanical properties, and in vitro bone-cell material interactions J. Biomed. Mater. Res. B 100B 2203–12 [33] Zhang M, Wu C, Lin K, Fan W, Chen L, Xiao Y and Chang J 2012 Biological responses of human bone marrow mesenchymal stem cells to Sr-M-Si (M = Zn, Mg) silicate bioceramics J. Biomed. Mater. Res. A 100A 2979–90 [34] Zhang W B, Liu W C, Gu W M, Chen L and Shen Y H 2012 Strontium modification of biomaterial: the effective approach to enhance the bioactivity and biocompatibility of calcium silicate Adv. Mater. Res. 391 195–9 [35] Zhu Y F, Zhu M, He X, Zhang J H and Tao C L 2013 Substitutions of strontium in mesoporous calcium silicate and their physicochemical and biological properties Acta Biomater. 9 6723–31 [36] Wu C, Yogambha R, Danielle K and Hala Z 2007 The effect of strontium incorporation into CaSiO3 ceramics on their physical and biological properties Biomaterials 28 3171–81 [37] Zhang W et al 2011 Effects of strontium in modified biomaterials Acta Biomater. 7 800–8 [38] Hina A and Nancollas G H 2001 Alpha calcium sulfate hemihydrate and a method of making alpha calcium sulfate hemihydrate International Patent WO 0179116
[3] van de Watering F C, Laverman P, Cuijpers V M, Gotthardt M, Bronkhorst EM, Boerman O C, Jansen J A and van den Beuken J J 2013 The biological performance of injectable calcium phosphate/PLGA cement in osteoporotic rats Biomed. Mater. 8 035012 [4] Bahn S L 1966 Plaster: a bone substitute Oral Surg. Oral Med. Oral Pathol. 21 672–81 [5] Thomas M V and Puleo D A 2009 Calcium sulfate: properties and clinical applications J. Biomed. Mater. Res. B 88B 597–610 [6] Stubbs D, Deakin M, Chapman-Sheath P, Bruce W, Debes J, Gillies R M and Walsh W R 2004 In vivo evaluation of resorbable bone graft substitutes in a rabbit tibial defect model Biomaterials 25 5037–44 [7] Lazary A, Balla B, Kosa J P, Bacsi K, Nagy Z, Takacs I, Varga P P, Speer G and Lakatos P 2007 Effect of gypsum on proliferation and differentiation of MC3T3-E1 mouse osteoblastic cells Biomaterials 28 393–9 [8] Kelly C M, Wilkins R M, Gitelis S, Hartjen C, Watson J T and Kim P T 2001 The use of a surgical grade calcium sulfate as a bone graft substitute: results of a multicenter trial Clin. Orthop. Relat. Res. 382 42–50 [9] Scarano A, Orsini G, Pecora G, Iezzi G, Perrotti V and Piattelli A 2007 Peri-implant bone regeneration with calcium sulfate: a light and transmission electron microscopy case report Implant. Dent. 16 195–203 [10] Woo K M, Yu B, Jung H M and Lee Y K 2009 Comparative evaluation of different crystal-structured calcium sulfate as bone- filling materials J. Biomed. Mater. Res. B 91B 545–54 [11] Park E K, Lee Y E, Choi J Y, Oh S H, Shin H I, Kim K H, Kim S Y and Kim S 2004 Cellular biocompatibility and stimulatory effects of calcium metaphosphate on osteoblastic differentiation of human bone marrow-derived stromal cells Biomaterials 25 3403–11 [12] Rauschmann M A, Wichelhaus T A, Stirnal V, Dingeldein E, Zichner L, Schnettler R and Alt V 2005 Nanocrystalline hydroxyapatite composite carrier materials for local delivery of antibiotics in bone infections Biomaterials 26 2677–84 [13] Peltier L F 1959 The use of plaster of Paris to fill large effects in bone: a preliminary report Am. J. Surg. 97 311–15 [14] Siriphannon P, Kameshima Y, Yasumori A, Okada K and Hayashi S 2000 Influence of preparation conditions on the microstructure and bioactivity of α-CaSiO3 ceramics: formation of hydroxyapatite in simulated body fluid J. Biomed. Mater. Res. 52 30–9 [15] Ni S, Chang J, Chou L and Zhai W 2007 Comparison of osteoblast-like cell responses to calcium silicate and tricalcium phosphate ceramics in vitro J. Biomed. Mater. Res. B 80 174–83 [16] Fei L, Wang C, Xue Y, Lin K, Chang J and Sun J 2012 Osteogenic differentiation of osteoblasts induced by calcium silicate and calcium silicate/β-tricalcium phosphate composite bioceramics J. Biomed. Mater. Res. B 100B 1237–44 [17] Xue W, Liu X, Zheng X and Ding C 2005 In vivo evaluation of plasmasprayed wollastonite coating Biomaterials 26 3455–60 [18] Zhu H, Wu B, Feng X and Chen J 2011 Preparation and characterization of bioactive mesoporous calcium silicate– silk fibroin composite films J. Biomed. Mater. Res. B 98B 330–41 [19] Wu C 2009 Methods of improving mechanical and biomedical properties of Ca-Si-based ceramics and scaffolds Expert Rev. Med. Devices 6 237–41 [20] Shirazi F S, Mehrali M, Oshkour A A, Metselaar H S, Kadri N A and Abu Osman N A 2014 Mechanical and physical properties of calcium silicate/alumina composite for biomedical engineering applications J. Mech. Behav. Biomed. Mater. 30 168–75 12
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[46] el-Ghannam A, Ducheyne P and Shapiro I M 1997 Formation of surface reaction products on bioactive glass and their effects on the expression of the osteoblastic phenotype and the deposition of mineralized extracellular matrix Biomaterials 18 295–303 [47] Radin S, Reilly G, Bhargave G, Leboy P S and Ducheyne P 2005 Osteogenic effects of bioactive glass on bone marrow stromal cells J. Biomed. Mater. Res. A 73A 21–9 [48] Miguel S, Kriauciunas R, Tosatti S, Ehrbar M, Ghayor C, Textor M and Weber F E 2010 Enhanced osteoblastic activity and bone regeneration using surface-modified porous bioactive glass scaffolds J. Biomed. Mater. Res. A 94A 1023–33 [49] Yin P, Feng F F, Lei T, Zhong X H and Jian X C 2014 Osteoblastic cell response on biphasic fluorhydroxyapatite/ strontium-substituted hydroxyapatite coatings J. Biomed. Mater. Res. A 102 621–7 [50] Gu Z, Wang H, Li L, Wang Q and Yu X 2012 Cell-mediated degradation of strontium-doped calcium polyphosphate scaffold for bone tissue engineering Biomed. Mater. 7 065007
[39] Lin K, Chang J, Zeng Y and Qian W J 2004 Preparation of macroporous calcium silicate ceramics Mater. Lett. 58 2109–13 [40] Wu C and Hala Z 2007 Preparation and characteristics of strontium containing bioactive CaSiO3 ceramics Key Eng. Mater. 330 499–502 [41] Kokubo T and Takadama H 2006 How useful is SBF in predicting in vivo bone bioactivity Biomaterials 27 2907–15 [42] Shen Y, Liu W, Wen C, Pan H, Wang T and Darvell B W 2012 Bone regeneration: importance of local pH—strontiumdoped borosilicate scaffold J. Mater. Chem. 22 8662–70 [43] Arnett T R 2010 Acidosis, hypoxia and bone Arch. Biochem. Biophys. 503 103–9 [44] Anusavice K J 2003 Phillips’ Science of Dental Materials ed K J Anusavice (St Louis, MO: Saunder) chaper 9 (Gypsum Product) pp 255–81 [45] Barrère F, van Blitterswijk C A and de Groot K 2006 Bone regeneration: molecular and cellular interactions with calcium phosphate ceramics Int. J. Nanomed. 1 317–32
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Preparation and in vitro evaluation of strontium-doped calcium silicate/gypsum bioactive bone cement
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Biomedical Materials Biomed. Mater. 9 (2014) 045002 (13pp)
doi:10.1088/1748-6041/9/4/045002
Preparation and in vitro evaluation of strontium-doped calcium silicate/gypsum bioactive bone cement Juncheng Wang1, Lei Zhang1, Xiaoliang Sun1, Xiaoyi Chen2, Kailuo Xie1, Mian Lin1, Guojing Yang1, Sanzhong Xu3, Wei Xia4, Zhongru Gou2 1
Department of Orthopedics, Rui’an People’s Hospital & the 3rd Hospital Affiliated to Wenzhou Medical University, Rui’an 325200, People’s Republic of China 2 Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou 310029, People’s Republic of China 3 Department of Orthopedics, The First Affiliated Hospital, School of Medicine of Zhejiang University, Hangzhou 310003, People’s Republic of China 4 Department of Engineering Sciences, The Ångstrom Laboratory, Uppsala University, 75121 Uppsala, Sweden E-mail: [email protected] and [email protected] Received 24 April 2014 Accepted for publication 12 May 2014 Published 12 June 2014 Abstract
The combination of two or more bioactive components with different biodegradability could cooperatively improve the physicochemical and biological performances of the biomaterials. Here we explore the use of α-calcium sulfate hemihydrate (α-CSH) and calcium silicate with and without strontium doping (Sr-CSi, CSi) to fabricate new bioactive cements with appropriate biodegradability as bone implants. The cements were fabricated by adding different amounts (0–35 wt%) of Sr-CSi (or CSi) into the α-CSH-based pastes at a liquid-to-solid ratio of 0.4. The addition of Sr-CSi into α-CSH cements not only led to a pH rise in the immersion medium, but also changed the surface reactivity of cements, making them more bioactive and therefore promoting apatite mineralization in simulated body fluid (SBF). The impact of additives on longterm in vitro degradation was evaluated by soaking the cements in Tris buffer, SBF, and α-minimal essential medium (α-MEM) for a period of five weeks. An addition of 20% Sr-CSi to α-CSH cement retarded the weight loss of the samples to 36% (in Tris buffer), 43% (in SBF) and 54% (in α-MEM) as compared with the pure α-CSH cement. However, the addition of CSi resulted in a slightly faster degradation in comparison with Sr-CSi in these media. Finally, the in vitro cell-ion dissolution products interaction study using human fetal osteoblast cells demonstrated that the addition of Sr-CSi improved cell viability and proliferation. These results indicate that tailorable bioactivity and biodegradation behavior can be achieved in gypsum cement by adding Sr-CSi, and such biocements will be of benefit for enhancing bone defect repair. Keywords: strontium-substituted calcium silicate, gypsum, bioactivity, biodegradability, composite cements (Some figures may appear in colour only in the online journal)
1. Introduction
a common technique in surgical practice. Whereas the limitations in the quantity and donor site complications are associated with autografts, and the concerns related to the immune response and possible transmission of infectious diseases or lack of good osteointegration are the main drawbacks of allografts. Thus the limitations in these treatments promote
In the present ageing societies, a significant increase in pathological or sport bone injuries leads to a growing medical interest in developing new bone augmentation and repair technologies [1–3]. Bone grafting to augment skeletal healing is 1748-6041/14/045002+13$33.00
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the development of synthetic biomaterials with excellent physicochemical and biological performances. In general, an ideal bone implant should possess features of good biocompatibility and excellent bioactivity, as well as an appropriate biodegradation rate to stimulate bone tissue regeneration and remodeling. Calcium sulfate dihydrate (CSD, CaSO4∙2H2O), so-called gypsum, has been used clinically as bone filling material since 1892 by Dreesmann and there were numerous subsequent studies for its use in filling bone defects [4, 5]. Gypsum is used in an hydraulic paste form produced by the hydration of calcium sulfate hemihydrate (CSH, CaSO4∙2H2O), which undergoes in situ setting after filling bone defects, and has good biocompatibility without inducing a significant inflammatory response [6–9]. CSH exists in at least two forms (α and β), and in general, the difference in physicochemical properties of the set paste are usually attributed to their primary particle morphology [5, 10]. Research has shown that the calcium ions released into a solution can stimulate osteogenic differentiation of bone marrow-derived stromal cells [11] and enhance alkaline phosphatase activity as well as gene expression associated with bone regeneration [7]. However, previous studies revealed that the high concentration ion product dissolution from gypsum shows some cytotoxic effects in vitro [12]. In particular, gypsum is usually bioresorbed completely in vivo in four to six weeks [13], which is too fast compared to the new bone tissue formation, and it cannot form a strong bone ingrowth at the early healing stage due to its poor bioactivity. Calcium silicate (CSi; named wollastonite) as one of the bioactive components of apatite-wollastonite (A-W) glass ceramics, has been recently demonstrated to be bioactive [14–16], and can be used to stimulate bone tissue regeneration [17, 18]. However, some drawbacks of CSi are considered because of its poor mechanical property [19, 20] and its fast degradation rate leading to a high Si ion concentration which would have a negative effect on cell growth [21]. Fortunately, studies have demonstrated that the physicochemical properties of bioceramics could be improved by the incorporation of strontium (Sr) which was found to have dual effects of stimulating bone formation and reducing bone resorption [22–24]. Lu’s group developed a strontium-doped hydroxyapatite (SrHA) cement and their widespread studies demonstrated that the Sr-HA cement could significantly stimulate bone formation and osseointegration in normal and osteoporotic bone regeneration [25–29]. Bose et al found that the Sr-doped β-tricalcium phosphate (Sr-TCP) promoted more osteogenesis by excellent early stage bone remodeling as compared to undoped β-TCP, and retarded the degradation rate of TCP [30, 31]. More recently, they found that a tailorable strength and strength degradation behavior can be achieved in β-TCP via compositional modifications using small amount of co-dopants such as Sr and Si [32]. Chang’s group has also investigated the biological response of bone-forming cells to the Sr-containing silicate bioceramics. Their studies demonstrated that the combination of the bioactive elements such as Sr and Si was effective in regulating the osteogenic property of human bone marrow mesenchymal stem cells, and the ionic products from the bioceramics significantly enhanced the ALP
activity and bone-related genes expression [33]. It has been revealed that the incorporation of Sr into CSi (Sr-CSi) can decrease its degradation rate and stimulate the proliferation of osteogenic cells while not change the apatite mineralization ability in simulated body fluid (SBF [34–36]). Moreover, Zhang et al found that partial substitution by Sr may increase HA solubility and inhibit the rapid degradation of borosilicate, but appears to have different mechanisms of physiochemical and biological benefit on different biomaterials [37]. Therefore, the development of Sr-CSi-added, gypsum-based biocement is potentially favorable for enhancing bone tissue regeneration and repair. Therefore, the objective of the present study is to develop a CSH/Sr-CSi biocement which allows setting when in contact with water and enhances bioactivity. The present study summarizes our efforts to prepare the composite biocement, and investigate their surface microstructure, bioactivity, biodegradability and in vitro cell response. 2. Materials and methods 2.1. Powder preparation
The α form of CSH was prepared by the hydrothermal reaction method, in which CSD powder was treated in boiling 15% NaCl solution and stirred for 5 h at 100 °C using 0.1% citric acid as the morphology modifier [38]. After that, the suspension was quickly filtered and immediately washed three times in boiling water and then dried at 120 °C for 6 h. The CSi powder was prepared by the chemical precipitation method as described previously [39]. Firstly, 500 ml Na2SiO3 solution (0.4 mol l−1) was added drop by drop into 500 ml Ca(NO3)2 solution (0.4 mol l−1) whose pH value was kept at 11.4 by rigorous stirring. After titration, the solution was stirred for 24 h, filtered and washed three times with deionized water and three times with ethanol. The precipitates were dried at 80 °C for 24 h and finally calcined at 800 °C for 3 h. Similarly, the Sr-CSi was synthesized by partially replacing the Ca(NO3)2 with 8% Sr(NO3)2 (in molar ratio) and the other conditions remained the same [40]. 2.2. Cement preparation
The α-CSH powders account for 0%, 5%, 10%, 20% and 35% (weight ratio) when mixed with the Sr-CSi. The mixtures were moistened by deionized water (0.4 ml g−1) and vibrated to form an homogeneous paste, and then transferred into a stainless steel mold with an internal diameter (Ø) of 5.0 or 8.0 mm and stored in a 37 °C water bath with 100% humidity for 2 h. After solidifying, the samples were mold released and air-dried at room temperature. The powders and cements were analyzed by x-ray diffraction (XRD; Rigaku), and the crosssectional morphology was observed by scanning electron microscope (SEM; HITACHI, S4800). 2.3. Mechanical test
After air-drying at room temperature for one and seven days, the compressive strength of the cements (Ø 5 × 10 mm) was 2
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10% fetal calf serum (FCS). The medium supplemented with 10% FCS without adding extracts was used as the control. After culturing at 37 °C and 5% CO2 for one, three and five days, 20 μl of methylthiazolyl- diphenyl-tetrazolium bromide (MTT) solution was added to each well and cultured for another 4 h. Then the solution in each well was replaced by 100 μl dimethyl sulfoxide and was shaken for 10 min. The plates were then read in a multifunctional microplate reader SpectraMax (Infinite® M200 Pro, TECAN, CH) at the optical density of 490 nm. Four duplicates were tested in the experiment. The morphology of the cells cultured with the extract solution was observed under SEM.
measured at a loading rate of 1.0 mm min using a universal testing machine (Instron) and four parallel tests were carried out for each group. 2.4. Degradation in vitro
Degradation in vitro was estimated by soaking the cements (W0; Ø 8 × 15 mm) in 0.05 M Tris buffer (pH 7.40), simulated body fluid (SBF) and a serum containing α-minimal essential medium (α-MEM) at 37 °C with a surface area-tovolume ratio of 0.1 cm−1, respectively. The buffer solution was refreshed every 48 h. At the preset time interval, the cements were removed from the liquid, gently rinsed with ethanol, and dried at 80 °C to weight constancy (Wt) before weighing by using an electronic analytical balance (FA2104, Sartorius, Germany). The weight loss (degradation) was expressed as the following equation: weight loss = (W0 − Wt)/W0 × 100%. Meanwhile, the pH value of the media was measured every 48 h before refreshment using an electrolyte type pH meter.
2.8. Statistical analysis
All of the data above was expressed as mean ± standard deviation (SD) and analyzed with the one-way ANOVA. In all cases the results were considered statistically significant with a p value of less than 0.05. 3. Results
2.5. Ion releasing during degradation in vitro
3.1. Phase and morphology of the powders
To investigate the ion release of the composite-cements, the cement samples were soaked in Tris buffer in a water bath at 37 °C for 1–15 days with a surface area-to-volume ratio of 0.1 cm−1. The Ca, Si and Sr ions in the supernatant were measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES; Thermo).
Figure 1 shows the XRD patterns of the powder and sevenday-set cement samples. The major diffraction peaks and the intensity of the pure α-CSH powders are attributed to the standard data (figure 1(a)). As for the CSi and Sr-CSi powders, Sr doping into CSi cannot be found in the XRD pattern compared with pure CSi powder, and there were no other phases such as CaO, SrO, SiO2 or α-CSi (high temperature phase of wollastonite) beside β-CSi (figures 1(b), (c)), indicating it is a single phase product. For the pure CSH-derived cement and the bulk composites with the Sr-CSi additive, the obvious peaks near 20.8°, 11.6° and 29.3°/2θ, corresponding to CSD phases, have been transformed from CSH (figures 1(d), (e)), but the pure Sr-CSi hardly hydrated at all in this time period (figure 1(f)). Figure 2 shows the SEM images of the CSD precursor and the synthetic powders in an aqueous medium, respectively. It is clear that the CSD-to-CSH phase transformation led to significant changes in morphology. The α-CSH powders have stable prismatic shapes with a length-diameter ratio near to 1:1, and an average diameter of about ten microns (figures 2(a), (b)). However, the granular Sr-CSi powders with irregular particle shapes had no significant difference from CSi powder, and meanwhile, these powders exhibit similar particle sizes of several micrometers (figures 2(c), (d)). These observations suggest that the primary individual particle in the composite cement is very tiny, and there are favorable hydration reactions and mechanical strength development.
2.6. Apatite formation in vitro
The SBF with ion concentrations nearly equal to that of human blood plasma was prepared according to the method proposed by Kokubo et al [41]. For characterization of in vitro bioactivity, the 72 h set discs (Ø 5 × 2 mm) were soaked in SBF at 37 °C in a water bath for 72 h with a surface areato-volume ratio of 0.1 cm−1. At the preset time the discs were gently rinsed with ethanol and dried at room temperature for SEM observation. 2.7. Cytocompatibility test
The cytocompatibility of the cements was evaluated by the extraction method, with human fetal osteoblastic cell line hFOB 1.19, according to the international standard [42]. The seven-day-set cement compacts were ground into particles and sieved through a 200 mesh sieve. The extract solutions were prepared by immersing 1.0 g cement particles in 5 ml α-MEM for 24 h at 37 °C in a CO2 incubator. After that, the suspension was centrifuged and the supernatant was filtered through a 0.22 μm Millipore membrane. Then the extract solution was diluted to 1/2, 1/4, 1/8, 1/16, 1/32 and 1/64 of the original concentration with fresh α-MEM. The cell suspension (100 μl) with a density of 1 × 104 cells −1 ml was seeded into each well of the 96-well plate and incubated for 24 h at standard conditions. The culture medium was then removed and replaced by 100 μl diluted extracts with
3.2. Microstructure and mechanical strength of cements
Figure 3 shows the cross-sectional microstructure and biphasic distribution of the CSH/CSix and CSH/Sr-CSix cements. It is clear that the hydration reaction of α-CSH with water resulted in a prismatic-to-rod transformation in the crystal morphology. 3
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Figure 1. XRD patterns of the powders and the 7-day-set cements. (a) α-CSH powder; (b) CSi powder; (c) Sr-CSi powder; (d) pure α-CSH cement; (e) CSH/CSi35 cement; (f) pure Sr-CSi cement.
Figure 2. SEM images of (a) CSD, (b) α-CSH, (c) CSi and (d) Sr-CSi powders.
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Figure 3. SEM images of cross-sectional microstructures of the (a)–(c) CSH/CSix and (d)–(f) CSH/Sr-CSix cements after setting for seven days.
The CSD crystals were tightly packed but the CSi aggregates were distributed unequally in the gypsum substrate. Figure 4 presents the compressive strength of the composite cements with different contents of CSi or Sr-CSi after setting for one and seven days, respectively. The specimens showed slight decreases in strength with the increase of Ca-silicate components. The pure gypsum cement had a compressive strength value of 24.4 MPa on average after one day, significantly (p < 0.05) lower than the cement after setting for seven days (29.8 MPa), indicating an increase of the compressive strength with time proceeding. The addition of CSi up to 10–35% achieved a significantly decreased strength, down to 17.2–9.1 MPa. However, prolongation of setting time positively affected the mechanical strength of the composite cements with an increase of up to 25% of the highest value after setting for 7 days. On the other hand, the Sr-CSi has no significantly (p > 0.05) different effect on the compressive strength of the composite cement compared with pure CSi under the same content condition. These data indicate that the difference is significant but the negative effect of CSi on the compressive strength of the composite cement is still acceptable with the lowest strength ranges from 11.9 to 14.1 MPa, which is superior to human cancellous bone (2.0–10.0 MPa).
Figure 4. Compressive strength of the CSH/CSix and CSH/Sr-CSix composite cements after setting for one and seven days, respectively.
3.3. In vitro bioactivity of cements
As for the surface microstructure and apatite mineralization ability, it can been seen from figure 5(a) that the pure gypsum cement (i.e. CSH/CSi0) showed a much denser surface structure, composed of rod- or plate-like CSD crystals, 5
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Figure 5. SEM images of the surface of the α-CSH cements before (a) and after (b) soaking in SBF for 72 h and EDX spectra (c).
2.89 (15.43/5.33), 2.57 (15.29/5.95) for the CSH/CSi5, CSH/ CSi35, CSH/Sr-CSi5 and CSH/Sr-CSi35, respectively. These values were significantly lower than the Ca/P molar ratio of 4.40 (12.28/2.79, figure 5(c)) for pure gypsum after soaking for same time period, indicating that gypsum cement exhibits a poor apatite deposition ability.
but the surface of the SBF-soaked gypsum was transformed to a porous microstructure (figure 5(b)). The area-scanning EDX analysis confirmed that Ca-phosphate salt was possibly deposited or absorbed onto the soaked cement surface (figure 5(c)), though the Ca/P molar ratio (~4.40) was very high. Figure 6 shows the SEM images of the surface morphology of the composite cements with different ratios (5%, 20%, 35%) of CSi (or Sr-CSi) before and after soaking in SBF for 72 h. The hydrated CSH transforms to rod-like CSD crystals (not shown). When adding CSi, the hybrid composite became a heterogeneous structure with an inhomogeneous distribution of CSi within the gypsum matrix, and there were many rod-like crystals which connected with each other to form the paste. No significant difference between the CSi-added cement and the Sr-CSi-added cement with the same content was observed in structures. With the increase of CSi content, the CSi component occupied more space inside the cement and would finally block the connection of the CSD crystals. In addition, there were irregular aggregates in the CSD crystals, which became more obvious with increasing CSi content. In contrast, it seems that a layer of an apatite-like mineral was deposited onto the surface of the cements after soaking for 72 h, and especially more sphere-like apatite aggregates were deposited on the composites with higher CSi content. The results from the representative EDX analysis (figure 6, inset) of the cement surface layer confirmed that the Ca/P molar ratios were approximately 2.22 (19.20/8.63), 1.87 (12.50/6.67),
3.4. Degradation behavior in vitro
The degradation of the cements was monitored by weight loss and pH variation in three different types of buffer systems. It is seen that the pure gypsum cement (i.e. CSH/CSi0) was degraded rapidly in the Tris buffer and was nearly dissolved completely after 35 days (figure 7(a)). The samples containing 5% CSi or 10% CSi had a slight effect on the degradation of the cements in Tris buffer. While increasing the CSi content up to 20–35%, the degradation rate of the composite cements decreases significantly, with nearly 17–22% of the cement residual in the medium. In fact, the Sr doping in CSi exhibited some degree of inhibition on the degradation rate of the composite cements. For instance, the CSH/CSi35 degraded by 28% after 14 days but only 22% of CSH/Sr-CSi35 was dissolved in the buffer within the same time scale; meanwhile about 31% cement residual remained in the solid phase after 35 days during the same immersion time period (figure 7(b)). In contrast, the cell culture medium (i.e. α-MEM) and SBF adversely affected the degradation behavior of the gypsum-based 6
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Figure 6. SEM images and EDX spectra of the surface of the (a)–(f) CSH/CSix and (g)–(l) CSH/Sr-CSix cements before (a)–(c), (g)–(i) and after (d)–(f), (j)–(l) soaking in SBF. Insets representing the area-scanning EDX spectra.
and especially that enough of the Sr-doped additive (i.e. Sr-CSi) reduces the degradation rate of the composite cements in simulated body environments. Figure 8 presents the changes in pH value of the three types of immersion media during immersion of the cements. It is evident that there is a minimal variation (within 0.2 units) for the medium immersing the pure gypsum cement. As the content of CSi or Sr-CSi increased, the pH value of the solutions increased significantly within the initial seven days and became relatively stable for the remaining soaking procedure. As the CSi turned the solution alkaline when hydrated in water, the more CSi contained in the composites, the more basic the solution would be. However, the Sr doping into CSi weakened the increase of pH value and made the degradation environment moderate. Significant differences (p < 0.05) were identified from the time period of three to nine days between the CSH/CSi and CSH/Sr-CSi composite cements. After that the pH values reduced and the composites containing 20% and 35% Sr-CSi still showed significant differences (p < 0.05) with the CSi
composite cements (figures 7(c)–(f)). The weight loss is seen to slowly increase with immersion time and varies with the CSi content. In particular, CSH/CSi0 showed a fast degradation but CSH/CSi20 and CSH/CSi35 displayed an extremely slow decrease in weight. Since the SBF and α-MEM are both able to induce re-mineralization on the surface of the bioactive materials, hence the apatite coating deposited on the composite cements would retard the biodissolution of the cements in such biomimetic aqueous media. The SEM observation (not shown) also revealed the new apatite-like deposits on the surface of the CSH/CSi35 and CSH/Sr-CSi35 after immersion in SBF and α-MEM for 35 days, respectively. Indeed, the SBF may heavily influence the degradation of the composite cement in comparison with the cell culture medium. However, no significant difference could be seen in the trends among the CSi and Sr-CSi additives and the two types of biomimetic media investigated here, as the weight loss in all cases was the same. These results indicate that the addition of Ca-silicate may retard the degradation rate of the gypsum-based cements, 7
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Figure 7. Weight loss of the CSH/CSix (a), (c), (e) and CSH/Sr-CSix (b), (d), (f) composite cements in Tris buffer (a), (b), SBF (c), (d), and α-MEM (e), (f), respectively.
Figure 8. Changes in pH values of immersion media during immersing the CSH/CSix (a), (c), (e) and CSH/Sr-CSix (b), (d), (f) composite cements.
group. These results suggest that the addition of CSi or Sr-CSi into gypsum cement may cause a pH fluctuation toward high pH values, and perhaps this is of great benefit for osteogenic cell activity and bone regeneration [42, 43]. In addition, the ion concentrations of calcium, silicon and strontium released by the cements with different contents of CSi or Sr-CSi into Tris buffer were measured (not shown). All the ion concentrations increased with the increasing immersion time up to nine days, but after that the calcium concentration became stable. Although the samples with higher CSi or Sr-CSi content released higher amounts of silicon and
strontium into the solution, there was a slight reduction in their concentration, possibly due to the inhibiting effect of higher pH levels on the CSi dissolution as mentioned above. 3.5. In vitro biocompatibility evaluation
As shown in figure 9, the conditioned cell cultures containing ion extract dissolutions from cements show no cytotoxicity against the hFOB 1.19 cells after culturing for one to five days, indicating that the addition of CSi or Sr-CSi did not cause negative effects on the biocompatibility of gypsum-based cements. 8
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Figure 9. MTT test of the cell culture in the absence and presence of the dissolution extracts of the CSD/CSix, and CSD/Sr-CSix cements after culturing for one day (a), (d), three days (b), (e), and five days (c), (f), respectively. (*p < 0.05)
1 and 3 days, respectively. It is clear that no significant difference was observed on the amount or morphology among the three groups on day 1. However, the cells had better aggregation and adhesion in the composite cement extracts compared to the pure gypsum extract on day 3 in terms of SEM observations, implying that the ion extracts dissolution from the composite cements could be regarded as a cell-promoter.
More specifically, the cell proliferation on days 1 through 5 steadily increased in the CSD/CSi cement groups, and the CSD. conditioned medium The cell proliferation on days 1 through 3 steadily increased in the conditioned media with extracts diluted from 1/2 to 1/8, indicating the increasing number of viable cells. As for the cell viability on day 5, the optical density values for the cells in the extracts diluted from 1/8 to 1/64 were significantly higher (p < 0.05) than those in the extracts diluted to 1/2 and 1/4. Interestingly, the CSH/Sr-CSi20 extracts showed lower cell proliferation on day 3 but higher on day 5 than the CSH/Sr-CSi35 extracts, especially the extracts diluted to 1/4, 1/8 and 1/16. This means that the proper concentrations of the extracts may have positive effects to stimulate cell proliferation. Moreover, cell proliferation in most of the extracts with different dilution ratios was significantly higher (p < 0.05) than the control. Figure 10 illustrates the cell morphology in the presence of the ion extracts of the CSH/Sr-CSi0, CSH/Sr-CSi20 and CSH/ Sr-CSi35 cements diluted to 1/4, 1/8, 1/16 after culturing for
4. Discussion Gypsum has enjoyed a long history of clinical use due to its good biodegradability. However, it is generally agreed that the bioresorption of gypsum cement is slightly faster than the formation of new bone, which is harmful to the reconstruction of the bone defect. Our previous primary experiments found that the addition of a high content of CSi (e.g. 28%) into the fiberlike β-CSH powder results in granular materials with poor cohesion ability when in contact with water up to L/P = 0.7, 9
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Figure 10. SEM micrographs of the low- and high-magnified cell morphology in the presence of the dissolution extracts of the CSH/Sr-CSi
cements after culturing for different time periods.
and the β-CSH-based composite powders containing an upper limit of 23% CSi may yield paste at L/P = 0.7. In the present study, a significantly high amount of the incorporation of CSi (i.e. 35%) into the α-CSH powder with prismatic shape particle morphology could produce an homogeneous paste L/P = 0.4. These experimental results were consistent with other reports [5, 44] in that the particle morphology of CSH heavily influences the crystallization and L/P ratio of gypsum cements. On the other hand, our previous experiments have also demonstrated that proper amounts of CSi addition (e.g. 7%) could fill the small pores or voids in the β-CSH-derived cement matrix and readily increase the mechanical strength of the composite cements. However, the composite of Sr-CSi with α-CSH decreased the compressive strength of the composite cements. The reason could be the addition of Sr-CSi which occupies too much space inside the cements and blocks the interactions among the CSD units. The SEM images have clearly shown the bi-component structure of Sr-CSi and CSD in the cements, and the Sr-CSi aggregates are obviously observed to inhomogeneously insert into the gypsum cement. Nevertheless, the compressive strength (11.86–14.11 MPa) of the CSH/Sr-CSi35
cements set for one to seven days was comparable and even superior to that of human cancellous bone. Such favorable mechanical strength will enable the CSH/Sr-CSi cement to develop more promising injectable bone implants. The degradation rate of the gypsum-based cements is a critical design parameter for bone tissue regeneration because appropriate cement degradation provides the space for matrix deposition and tissue growth, which may ultimately lead to improved quantity and quality of regenerated bone [5]. It is worth noting that the CSH/CSi cement showed a significantly lower degradation rate than the pure α-CSH cement. This is because the CSi has a significantly lower dissolution rate than CSD in such weak basic biomimetic media, and thus the adherence of CSi particles on the surface of CSD crystals could reduce the contact of the CSD crystals with the aqueous medium. On the other hand, it is also demonstrated that the CSi-induced apatite remineralization and deposition on the hydraulic composite surface would retard the biodissolution of the cements. Moreover, the doping of Sr into CaSiO3 additionally decreased the degradation rate as well as increasing the pH value of the surrounding environment, which has been proven 10
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by some previous studies [34–36]. The ion release of the CSH/ Sr-CSi cements showed lower Si ion release and increased Sr ion release, which could also be explained by the decreased degradation rate and pH value of the composite cement. On the other hand, the microenvironmental pH is a function of the chemical properties of the biomaterials (e.g. composition, solubility constant, surface chemistry). Thereby, the extremely stable pH is an intrinsic property of pure gypsum cement and cannot be changed, except if its chemical properties are modified (for instance, by adding a new component). Indeed, changing these properties is equivalent to changing the material. However, this new material couldn’t be compared to the original one since besides the chemical properties, the physical properties also change [5, 45]. An extensive number of studies have revealed that the changes in the extracellular fluid pH of the local biological microenvironment can profoundly affect cell metabolism and function [21, 23], and especially that the Ca-silicate-based biomaterials can induce alkalinization of the external medium upon exposure to (simulated) physiological fluids which is favorable for osteogenic cell function and new bone tissue formation and mineralization [24]. Therefore, the CSi-induced pH rise is another factor for improving the biological performances of such gypsum-based cements. In addition, our studies indicate that the pure gypsum cement is poor in inducing apatite mineralization in SBF. Such a situation has been pointed out in documents: pure gypsum always fails to form an effective chemical bond with the newly formed bone tissue at the early stage of therapy due to its poor bioactivity [13]. In contrast, as the CSi (or Sr-CSi) additive increases to a certain amount, the composite cements could induce apatite formation on the surface of the cements within 3 days, which means the composite cements possess good bioactivity. It is confirmed that when CSi ceramics are immersed into (simulated) physiological fluids in vitro, a series of events occur via ion leaching and exchange with the surrounding solution. This process leads to calcium phosphate precipitation and the formation of a bone-like apatite layer onto the ceramic’s surface. The advantage of the formation of such a Ca-Prich layer onto the composite cements in the present study was two-fold. First, the presence of a Ca-P-rich layer at the surface of biomaterials readily promotes adsorption of serum proteins, (especially of fibronectin [46, 47]) followed by attachment, proliferation and differentiation of osteogenic cells [46, 48]. Second, immersion of the composite cement in physiological fluid results in an increase of the pH of the surrounding microenvironment and the rapid increase in the interfacial pH was slowed down by the Ca-P deposition layer, which also retards the biodissolution of the cements. Therefore, the bioactivity of the CSH/CSi cements is highly significant for promoting osteogenic cell proliferation and mineralization and reducing the biodegradation of the material substrate. A cytocompatibility test is a useful criterion in the evaluation of new biomaterials. Although they produced a pH value of around 7.8 during the degradation in the Tris buffer, the composite cements did not induce any cytotoxicity in present study. Neither the proliferation nor the morphology was influenced by the ion extracts. Moreover, the ion release of the composite cements provides more stimulus for cell proliferation than the
pure gypsum cement which may be attributed to the dissolution of silicon [48] and strontium [22–24]. Cell viability and cell number in the strontium-containing conditioned culture medium do not show any statistically significant differences when compared with the medium in the absence of strontium. It is possible that the amount of strontium in the cement was insufficient to stimulate cell proliferation. Several studies demonstrated the high activity of strontium on osteoblastic cells and reported that micromolar concentrations of strontium were effective in improving the cell attachment and the subsequent cell activities and in stimulating bone regeneration [49, 50]. It is known that active osteoblasts are polygonal or spherical in shape, whereas resting osteoblasts are spindle-shaped and flat. SEM observations showed that cells cultured for 24–72 h were viable and proliferated in the conditioned media. The cells were attached, showed a spherical appearance, and were interacting with the surface through cell phyllopodia and extensions. Their spherical shape is the typical morphology of active osteoblasts. These in vitro cell culture results indicate that the ion extract’s dissolution from the Sr-CSi-added composite cements also possibly stimulates cell proliferation, which means such cements are bioactive for the osteogenic cells. 5. Conclusions A novel biphasic bone cement combined with α-CSH and Sr-CSi was successfully prepared and studied in this paper. The mechanical strength of the composite-cement increased with time proceeding after the initial setting but gradually decreased with the increase of the amount of Sr-CSi. The addition of Sr-CSi slowed down the degradation of the composite cement as well as decreased Si and Sr ion release and lowered the pH increase. The cement composites with Sr-CSi showed better bioactivity compared with CSi or pure α-CSH, as indicated by the formation of bone-like apatite in SBF and the cytotoxicity evaluation of the extracts of the cements. Our results demonstrated that controllable bioresorbability and release of biologically active ions within biphasic bone cement provides a rational strategy for combined bone defect repair and antiosteoporotic therapy. Further investigations focusing on large animal models or clinical applications are needed. Acknowledgements This work is supported by the Zhejiang Provincial Natural Science Foundation of China (No. LQ14H060003, LZ14E020001), the National Science Foundation of China (81271956, 51102211, 81301326), and the Fundamental Research Funds for the Central University (2012QN81001). References [1] Ducheyne P, Mauck R L and Smith D H 2012 Biomaterials in the repair of sports injuries Nat. Mater. 24 652–4 [2] Nair M B, Kretlow J D, Mikos A G and Kasper F K 2011 Infection and tissue engineering in segineering bne defects-a mini review Curr. Opin. Biotechnol. 22 721–5 11
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