- Email: [email protected]
Preparation of bioactive glass ceramic beads with hierarchical pore structure using polymer self-assembly technique
Materials Chemistry and Physics 115 (2009) 670–676
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Preparation of bioactive glass ceramic beads with hierarchical pore structure using polymer self-assembly technique Hui-suk Yun ∗ , Seung-eon Kim, Young-taek Hyun Center for Future Technology, Korea Institute of Materials Science (KIMS), 531 Changwondero, Changwon, Republic of Korea
a r t i c l e
i n f o
Article history: Received 1 October 2008 Received in revised form 28 January 2009 Accepted 1 February 2009 Keywords: Biomaterials Glasses Porous materials Sol–gel growth
a b s t r a c t Hierarchically mesoporous–macroporous bioactive glass ceramic beads with well interconnected pore structures were fabricated in hydrophobic solvent, chloroform, by the triblock copolymer templating and sol–gel techniques. The beads have oblong pore structure with a size of several hundreds m and these macropore are comprised of several tens of m pores, several of m pores, and several tens of nm pores. The beads show superior apatite-like layer forming activity and good in vitro biodegradability. This simple synthetic method can be adapted for the purpose of preparing various ceramics with hierarchical pore structures, which have excellent potential applications in the field of not only biomaterials such as tissue engineering and drug storage but also catalysis, filters, adsorbents, and optics. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Bioactive glasses (BGs) have been widely studied because they have the ability to chemically bond with living bone tissue and have consequently been used in a variety of medical applications [1–3]. Increasing the specific surface area and pore volume of BGs may greatly accelerate the kinetic deposition process of hydroxycarbonate apatite and allow them to be loaded with the osteogenic agents, thus enhancing their bone-forming bioactivity [4–10]. To this end, the development of mesoporous BGs, which have a large specific surface area and pore volume, has recently accelerated [4–9,11–15]. Highly 2D hexagonal and 3D cubic ordered mesoporous BGs with superior bone-forming bioactivities in vitro compared with normal BGs have therefore been synthesized by templating with a triblock copolymer, Pluronic P123 and F127, respectively. Although all of the reported mesoporous BGs show favorable bioactivity, they are difficult to use as a bone filler or as scaffolds for the regeneration of bone tissue at this stage, because their meso-sized pore are too small to promote cell growth as well as load osteogenic agents such as bone morphogenetic protein [4–9]. Control the pore sizes and structures of BGs is important to maximize their functionality for use as materials in tissue regeneration. Human bone consists of hierarchically 3D interconnected pore structures and cells are sensitive to local environmental features at all length scales from macro down to the molecular [16]. BGs with hierarchical porosity combine the advantages of a high surface area from nanoporous structure with the accessible diffusion pathways associated with a macroporous
structure. BG ceramic scaffolds with hierarchical pore structures were previously reported by our group [12–15]. However, there have been no reports yet concerning the preparation of hierarchically porous BG ceramic beads with pore sizes in the range between 10 nm and 100 m, which can be used as the bone fillers. Hierarchically porous materials have generally been prepared by making use of self-assembled supramolecular structures, the induction of morphological complexity by inducing topological defect structures, and the inorganic replication of organized reaction field [17–24]. These preparation methods required the use of multi-synthetic steps or multi-scale organic templates and the beads produced had hierarchal pore size in the range of several nm to several hundreds of nm, which are still too small to promote cell activity. Herein, we report a novel, simple and reproducible method of preparing hierarchically 3D porous BG beads using the same procedure as that employed for mesoporous BGs, with one additional step: the precursor solution is added to a hydrophobic solvent, chloroform, after appropriate evaporation to induce the self-rearrangement of the triblock copolymer and inorganic framework. Furthermore, we successfully prepared mesoporous BGs with various types of mesopore structure and controlled both the pore size and the material morphology of the beads. This synthetic method allows the well interconnected pore structure, hierarchically porous BG beads in the size range between 10 nm and several hundreds of m with high porosity and good bioactivity. 2. Experimental 2.1. Materials and synthesis
∗ Corresponding author. Tel.: +82 55 280 3351; fax: +82 55 280 3399. E-mail address: [email protected] (H.-s. Yun). 0254-0584/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2009.02.001
In a typical synthesis, 3.46 g of poly (ethylene oxide)132 -poly(prophylene oxide)50 -poly(ethylene oxide)132 ((EO)132 (PO)50 (EO)132 , F108, average Mn = 14,600)
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676 is dissolved in 18.1 ml of ethanol (EtOH). Stock solutions, which were prepared by mixing 1.36 g of calcium nitrate tetrahydrate (CaNT), 0.26 ml of triethyl phosphate (TEP), 6 ml of tetraethyl orthosilicate (TEOS), 0.95 ml of HCl (1 M), 7.62 ml of EtOH and 2.86 ml of H2 O, were added to this solution after stirring them separately for 1 h, and were vigorously stirred together for another 4 h at 40 ◦ C. The molar composition was TEOS:CaNT:TEP:F108 = 1:0.2:0.05:0.008 in this case. The reactant solution was evaporated at 40 ◦ C, 40 RH% for 24 h without stirring to increase of its viscosity. The evaporated sol solutions were dropped into CHCl3 solutions using by a syringe (20–24 G). The obtained gel beads were collected, aged at 40 ◦ C for 24 h and were calcined at 600 ◦ C for 6 h in air to remove the template. In addition, a series of BGs, which were produced using different block copolymers templates, L121 (EO5 PO68 EO5 , Mn = 4400), P123 (EO20 PO70 EO20 , Mn = 5750), and F127 (EO100 PO65 EO100 , Mn = 12,600), were also prepared to study and to compare the formations of the mesoporous BG as well as the hierarchically porous BG. All chemicals were purchased from Aldrich.
2.2. Characterization Structural characterization was carried out by X-ray diffraction (XRD; –2 scanning, Philips-X’pert MPD 3040, using Cu K␣ ( = 1.5406 Å) radiation (40 kV–40 mA), transmission electron microscopy (TEM; JEOL-JEM2100F) with field emission gun at 200 kV, and field emission scanning electron microscopy (FE-SEM; Hitachi-S5500 and JEOL-5800) at an accelerating voltage of 1–5 kV. The specific surface area from meso-sized pore was measured by the N2 -gas adsorption–desorption isotherms using a Brunauer Emmett Teller (BET) apparatus (BelJapan-Belsorp mini II). The pore volume and pore-size distribution were derived from the adsorption branches of the isotherms using the Barrett–Jayner–Halanda (BJH) method. The total pore volume was estimated from the amount adsorbed at a maximum relative pressure. The porosity from macro-sized pore was measured by the Hg intrusion method (AutoPore IV 9510, Micromeritics). The assessment of the in vitro bioactivity of the BGs beads was carried out in simulated body fluid (SBF) at 37 ◦ C. The SBF contained 142.0 mM Na+ , 5 mM K+ ,1.5 mM Mg2+ , 2.5 mM Ca2+ , 147.8 mM HCl− , 4.2 mM HCO3− , 1.0 mM HPO4 2− , and 0.5 mM SO4 2− [13]. Its chemical composition was similar to that of human plasma. The solu-
671
tion had a pH of 7.4 and was kept at 37 ◦ C before use. Concentration of Ca as well as pH levels of the SBF after soaking the BGs beads were determined at different periods by inductive coupled plasma (ICP) atomic emission spectroscopy and pH system (Mettler Toledo). The assessment of the in vitro biodegradability of the BGs beads was carried out in the phosphate buffered saline (PBS) at 37 ◦ C.
3. Results and discussion The formation of BG beads largely depends on the structure of the polymer templates. The precursor solution was added to the hydrophobic solvent, chloroform, drop by drop after adequate evaporation (reduction of 60–65 (v/v%); about 52–55 Brix%) increase the viscosity of the precursor solution to the extent required for producing BG gel beads. 4 types of triblock copolymers were used, L121, P123, F127, and F108, which have different m/n ratio of (EO)m (PO)n (EO)m . The precursor solution containing L121 or P123 as templates formed the gel beads when it was just dropped into chloroform, but the beads gradually lost their shape over time and turned into a gel solution (Fig. 1a). On the other hand, when the precursor solutions containing F127 or F108 were used as the templates, the transparent gel beads were produced when it was just dropped into chloroform, the sizes of the beads gradually shrank, and they turned opaque with time. However, they retained their morphology quite well (Fig. 1b). Fig. 1c and d shows the optical images of the BG beads before and after calcination at 600 ◦ C. The as-synthesized BG beads were well shaped with relatively uniform sizes of 1750 m. The BG beads show a 22% size reduction (1360 m) after calcination, but still have a good morphology without cracks. The size of the beads can be easily controlled from
Fig. 1. Optical images of BG gels templated by (a) P123 and (b) F108 after dropping into CHCl3 . Microscope images of BG beads (c) before and (d) after calcination at 600 ◦ C. Transmission electron microscope (TEM) images of calcined BG synthesized using triblock copolymers; (e and i) L121, (f and j) P123, (g and k) F127, and (h and l) F108 templates. (e–h) Before dropping into CHCl3 and (i–l) after dropping into CHCl3 and standing for 24 h.
672
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
Fig. 2. FE-SEM images (a–f) and TEM images (g–i) of calcined BG beads. (a) Low magnification images of beads and (b and c) high magnification images of surface of beads. FE-SEM images of cross-sectioned BG beads after dropping into CHCl3 and standing for (d) 1 h, (e) 2 h, and (f) 24 h. TEM images of BG beads after dropping into CHCl3 and standing for (g) 1 h, (h) 2 h, and (i–l) 24 h. (j) Inside part of BG beads, (k) outside part of BG beads, and (l) interface between inside and outside part images of BG beads.
Fig. 3. Pore size distribution of BG beads measured by (A) Hg intrusion and (B) N2 adsorption–desorption isotherms.
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
several hundreds of m to several mm by adjusting the syringe size, the viscosity of the precursor and the dropping conditions (Support Information (SI) 1). Similar structural phenomena occurred when a hydrophobic solvent such as hexane, benzene, toluene, diethyl ether, and ethyl acetate was used in place of chloroform, but if the density of the solvent is lower than that of the BG sol beads, the dropped beads immediately sink to the bottom of the beaker and, consequently, the beads can not retain their beads morphology well. If chloroform is not used in this step, a mesoporous structured BG with different pore structures can be obtained, depending on the polymer structure as shown in Fig. 1e–h. L121 induces wormhole-like structure, P123 allows hexagonal structure and both F127 and F108 produce cubic pore structure with high BET surface area. The BET surface area of BG produced using L121, P123, F127, and F108 as templates is 446, 450, 502, and 516 m2 g−1 , respectively. It is worthy to note that all of these pore structures were completely changed by the reaction of gel beads in chloroform as shown in Fig. 1i–l. The BG produced using L121, BG-L121chloro, lost its pore structure resulting in the formation of BG nanoparticles with a relatively homogeneous particle sized of 15 nm. The BG synthesized using P123, BG-P123chloro, also lost its hexagonal pore structure, leading to the formation of BG nanospheres with a heterogeneous size of 35–400 nm. Meanwhile, the pore structures of the BGs obtained using both F127, BG-F127chloro, and F108, BGF108chloro, changed from cubic to cancellous structure with greatly increased pore sizes. More interestingly, the obtained BG beads contain a hierarchical pore structure. Fig. 2 shows the FE-SEM and TEM images of BG-F108chloro (the result obtained for BG-F127 is confirmed by SI 2). The BG-F108chloro beads have well interconnected and hierarchically arranged open pore structure. That is, the large sized pores with size ranging from 7 to 10 m are made up of medium sized pores with a size of several m, and the medium sized pores are built up with the small sized pores with sizes of several tens of nm. The beads also form oblong pore structures with a size of several hundreds m long over the beads. These pore size distributions are relatively sharp and agree well with the results of both the mercury intrusion test and the BJH plot derived from the N2 adsorption isotherm (Fig. 3). That is, in the analysis of the pore size distribution, two relatively sharp peaks centered at 7 and 2 m were observed in the Hg intrusion test and a broad peak centered at 25 nm was detected from the BJH plot derived from the N2 adsorption–desorption test. The pore morphologies gradually changed, starting from the outside of the beads, which is the first part to come into contact with chloroform, and later extending to the inside, depending on the reaction time. Fig. 2d–i shows the FE-SEM and TEM results of BG-F108chloro showing the variability of the pore morphology over time. The pore sizes are increased from outside to inside with increasing the reaction time.
673
After 30 min immersion in chloroform, large pores appear on the outside of the beads (Fig. 2d), their mesopore structure is lost, and large nanopore structures with an average pore size of 40 nm are newly formed (Fig. 2g). The macropores extend from the outside to the inside with increasing pore size over time (Fig. 2e and h) and the hierarchical pore structures are finally obtained after 24 h immersion in chloroform (Fig. 2f and i). However, the pore morphologies are still different on the outside and inside and some meso-sized pores are still remaining in the center of the beads (Fig. 2j–l). The change behavior of the mesopore structure to the hierarchically meso-macropore structure can also be confirmed from the result of the N2 adsorption isotherms (Fig. 4). Prior to its treatment in chloroform, porous BG-F108 has a type IV isotherm with a sharp capillary condensation step at high relative pressures and broad H2 hysteresis loop that is indicative of large uniform cage-type pores. After the reaction in chloroform, drastic changes in both the adsorption–desorption isotherms and pore size distributions can be observed, but the beads still have type IV isotherms with a capillary condensation. The pore size increases with increasing reaction time, with the result that the BET surface area and pore volume of the pores ranging in size from mesopores are decreased. The BET surface area and pore volume of the beads after immersion for 24 h in chloroform are 100 m2 g−1 and 0.16 cm3 g−1 , respectively. On the other hand, BG-F108chloro has a large average porosity of 50% resulting from the macropores, as measured by the Hg intrusion test. These kinds of hierarchical pore structure can obtained only after adequate evaporation and the sol–gel reaction of the precursor solution. If the chloroform is directly added to the precursor solution in the starting step, the separation between the triblock copolymer and inorganic sources occurs, because the triblock copolymers tends to make a reverse micelle due to its self-assembling behavior in chloroform, following which the hydrophobic part comes out of the micelle, and the hydrophilic inorganic sources consequently cannot react with the micelles. If the precursor solution is dropped into chloroform without appreciable evaporation, that is, the precursor solution has low viscosity and hydrolysis and condensation do not sufficiently occur, the dropped beads cannot retain their bead shape, and consequently a heterogeneous pore structure is induced after removing the solvent. Excess evaporation also leads to fail. A possible mechanism for the formation of BG beads with a hierarchical pore structure is illustrated in Scheme 1. Owing to the hydrophobic nature of PO and the hydrophilic nature of EO, PO forms a core and EO exists as a shell around the core in the aqueous condition. According to our synthetic procedure, the inorganic sources, Si-, Ca-, and P-, react with the EO groups and the sol–gel reaction in the inorganic framework proceeds in the first step. These polymer–inorganic composite
Fig. 4. (A) N2 adsorption–desorption isotherms and (B) BJH plots of porous BG beads with different reaction time in chloroform.
674
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
Scheme 1. Suggested mechanism for the formation of the hierarchically porous BG beads.
micelles have a tendency to make reverse micelles in chloroform. That is to say, the EO/inorganic composite part tends to move into the center of the micelle and the PO part attempts to move outside of the cell. The inorganic part exists in the center of the micelle and these reversed micelles try to rearrange each other to produce a hierarchical structure. The ratio of m/n in EOm POn is important when attempting to produce a hierarchical pore structure in this second step. That is, if m is much less than n, the inorganic particles will separate from each other and will exist as nanoparticles after the removal of the polymer template by calcination, without retaining their hierarchical pore structure, because the inorganic particles cannot be interconnected due to their large interparticle distance. We suggest that this is why neither BG-L121chloro (m/n = 0.07) nor BG-P123chloro (m/n = 0.29) are able to form the beads, but rather produce nanoparticles with different particle sizes. On the other hand, if m is sufficiently larger than n, the interparticle distance is short, the particles may interlock with one another after the removal of the polymer template by calcination, and the hierarchical pore structure will be consequently retained. As a result, both BG-F127chloro (m/n = 1.54) and BG-F108chloro (m/n = 2.64), which
have enough longer chain of EO than of PO, can have a hierarchical pore structure. Although this suggested mechanism needs more detailed discussion, the obtained hierarchically porous ceramic beads are of interest, not only for bio-applications but also as potential catalyst, separation, adsorption, or electrode materials, because of their high molecular selectivity ranging from nanopores to macropores as well as their unique and efficient diffusion pathways. The bone-forming bioactivity of BG-F108chloro in vitro was tested in SBF to monitor the formation of hydroxyl apatite (HA) on the surface of the beads [25]. HA provides a suitable substrate for the proliferation and function of osteoblast like cells, which allows for the strong bonding of the materials to the surrounding bone tissue [26]. The FE-SEM images of the beads reveals that the surface of the BG beads undergoes important changes when it reacts with the SBF as shown in Fig. 5a–d. The formation of HA-like nanoparticles is observed on the surface of the BG beads after soaking the sample for 24 h (SI3). This reaction proceed over time and the surface of the BG beads was completely covered with newly formed HA-like nanoparticles having a needle-like morphology along the whole pore structure after being soaked for
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
675
Fig. 5. FE-SEM micrographs of BG beads after they immersed in SBF for (a) 0 h, (b) 24 h, (c) 48 h and (d) 72 h to test in vitro bioactivity of BG beads and cam scope images of BG beads before (e and g) and after (f and h) immersing into PBS for 8 weeks for confirming in vitro biodegradability of BG beads. (g and h) The enlarged image of (e) and (f), respectively.
72 h. Both the Ca2+ concentration and pH level in SBF also reveal these bioactivities of the BG beads (Fig. 6). The Ca2+ levels increase during the 72 h. Thereafter, the Ca2+ concentration in SBF decreases until the end of the assay. The pH evolution follows a profile analogous to that of the Ca2+ content. These results indicate that a Ca2+ –H+ exchange occurs between the BG beads and SBF, and it allows for the deposition of HA on BG beads. In order to study their biodegradability, the dissolution of the BG beads in phosphorous
buffer solution (PBS) was performed as shown in Fig. 5e–h. The BG beads show a weight loss of 10% only after 2 weeks of immersion in PBS, gradually increase their weight loss with time and repeatedly show deformation of BG and formation of HA. These results indicate that the BG beads, which have a hierarchical pore structure, may have good HA-like layer forming activity (bioactivity) in vitro as well as biodegradability. Further studies of the biocompatibility of the BG beads in vitro and in vivo are currently in progress.
676
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
Fig. 6. Variation of (A) calcium content and (B) pH values of SBF as a function of soaking time for the hierarchically porous BG beads.
4. Conclusion Novel hierarchically mesoporous–macroporous BG beads with a well interconnected pore structure, good bioactivity, and biodegradability promising for use as materials for tissue regeneration such as bone fillers with controlled drug delivery functionalities were fabricated by the polymer templating and sol–gel techniques. The self-reassembling reaction of the triblock copolymer in the hydrophobic solution induces the hierarchical pore structure of the BG beads. The ratio of m/n in EOm POn is important to produce a hierarchical pore structure because the interconnecting of the BG nanoparticles after the reaction in chloroform is determined by the interparticle distance. We hope that this simple, reproducible synthetic method can be adapted for the preparation of various hierarchical porous materials, having the potential to be used in applications involving filters, catalysis, biomedical devices, and sensors. Acknowledgment This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-20037-0). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.matchemphys.2009.02.001. References [1] L.L. Hench, R.J. Splinter, W.C. Allen, T.K. Greenlee, Bonding mechanisms at the interface of ceramic prosthetic materials, J. Biomed. Mater. Res. 2 (1971) 117–141. [2] L.L. Hench, Bioceramics, J. Am. Ceram. Soc. 81 (1998) 1705–1727. [3] L.L. Hench, J.M. Dolak, Third generation biomedical materials, Science 295 (2002), 1014 + 1016–1017. [4] X. Yan, C. Yu, X. Zhou, J. Tang, D. Zhao, Highly ordered mesoporous bioactive glasses with superior in vitro bone-forming bioactivities, Angew. Chem. Int. Ed. 43 (2004) 5980–5984. [5] X.X. Yan, H.X. Deng, X.H. Huang, G.Q. Lu, S.Z. Quio, D.Y. Zhao, C.Z. Yu, Mesoporous bioactive glasses. I. Synthesis and structural characterization, J. Non-Cryst. Solid 351 (2005) 3209–3217.
[6] X. Yan, X. Huang, C. Yu, H. Deng, Y. Wang, Z. Zhang, S. Qiao, G. Lu, D. Zhao, The in vitro bioactivity of mesoporous bioactive glasses, Biomaterials 27 (2006) 3396–3403. [7] I. Izquierdo-Barba, L. Ruiz-González, J.C. Doadrio, J.M. González-Calbet, M. Vallet-Regí, Tissue regeneration: a new property of mesoporous materials, Solid State Sci. 7 (2005) 983–989. [8] A. López-Noriega, D. Arcos, I. Izquierdo-Barba, Y. Sakamoto, O. Terasaki, M. Vallet-Regi, Ordered mesoporous bioactive glasses for bone tissue regeneration, Chem. Mater. 18 (2006) 3137–3144. [9] I. Izquierdo-Barba, D. Arcos, Y. Sakamoto, O. Terasaki, A. López-Noriega, M. Vallet-Regí, High-performance mesoporous bioceramics mimicking bone mineralization, Chem. Mater. 20 (2008) 3191–3198. [10] M. Vallet-Regí, C. Victoria Ragel, A.J. Salinas, Glasses with medical applications, Eur. J. Inorg. Chem. (2003) 1029–1042. [11] H.S. Yun, S.E. Kim, Y.T. Hyun, Highly ordered mesoporous bioactive glasses with Im3m symmetry, Mater. Lett. 61 (2007) 4569–4572. [12] H.S. Yun, S.E. Kim, Y.T. Hyun, Design and preparation of bioactive glasses with hierarchical pore networks, Chem. Commun. (2007) 2139–2141. [13] H.S. Yun, S.E. Kim, Y.T. Hyun, S.J. Heo, J.W. Shin, Three-dimensional mesoporousgiantporous inorganic/organic composite scaffolds for tissue engineering, Chem. Mater. 19 (2007) 6363–6366. [14] H.S. Yun, S.E. Kim, Y.T. Hyun, Preparation of 3 dimensional cubic ordered mesoporous bioactive glasses, Solid State Sci. 10 (2008) 1083–1092. [15] H.S. Yun, S.E. Kim, Y.T. Hyun, S.J. Heo, J.W. Shin, Hierarchically mesoporous–macroporous bioactive glasses scaffolds for bone tissue regeneration, J. Biomed. Mater. Res. Part B 87B (2008) 374–380. [16] M.M. Stevens, J.H. George, Exploring and engineering the cell surface interface, Science 310 (2005) 1135–1138. ˝ [17] S. Schacht, Q. Huo, I.G. Voigt-Martin, G.D. Stucky, F. Schuth, Oil–water interface templating of mesoporous macroscale structures, Science 273 (1996) 768–771. [18] S.D. Sims, D. Walsh, S. Mann, Morphosynthesis of macroporous silica frameworks in bicontinuous microemulsions, Adv. Mater. 10 (1998) 151–154. [19] D. Walsh, B. Lebeau, S. Mann, Morphosynthesis of calcium carbonate (vaterite) microsponges, Adv. Mater. 11 (1999) 324–328. [20] T. Sen, G.J.T. Tiddy, J.L. Casci, M.W. Anderson, One-pot synthesis of hierarchically ordered porous-silica materials with three orders of length scale, Angew. Chem. Int. Ed. 42 (2003) 4649–4653. [21] T. Sen, G.J.T. Tiddy, J.L. Casci, M.W. Anderson, Meso-cellular silica foams, macrocellular silica foams and mesoporous solids: a study of emulsion-mediated synthesis, Micropor. Mesopor. Mater. 78 (2005) 255–263. [22] H. Zhang, G.C. Hardy, M.J. Rossinsky, A.I. Cooper, Uniform emulsion-templated silica beads with high pore volume and hierarchical porosity, Adv. Mater. 15 (2003) 78–81. [23] C. Tao, J. Li, Morphosynthesis of microskeletal silica spheres templated by W/O microemulsion, Colloids Surf. A Physicochem. Eng. Aspects 256 (2005) 57–60. [24] J. Wang, Q. Xiao, H. Zhou, P. Sun, Z. yuan, B. Li, D. Ding, A.C. Shi, T. Chen, Budded, mesoporous silica hollow spheres: Hierarchical structure controlled by kinetic self-assembly, Adv. Mater. 18 (2006) 3284–3288. [25] T. Kokubo, Bioactive glass ceramics: properties and applications, Biomaterials 12 (1991) 155–163. [26] S.Y. Ni, J. Chang, L. Chou, A novel bioactive porous CaSiO3 scaffold for bone tissue engineering, J. Biomed. Mater. Res. 76 (2006) 196–205.
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Preparation of bioactive glass ceramic beads with hierarchical pore structure using polymer self-assembly technique Hui-suk Yun ∗ , Seung-eon Kim, Young-taek Hyun Center for Future Technology, Korea Institute of Materials Science (KIMS), 531 Changwondero, Changwon, Republic of Korea
a r t i c l e
i n f o
Article history: Received 1 October 2008 Received in revised form 28 January 2009 Accepted 1 February 2009 Keywords: Biomaterials Glasses Porous materials Sol–gel growth
a b s t r a c t Hierarchically mesoporous–macroporous bioactive glass ceramic beads with well interconnected pore structures were fabricated in hydrophobic solvent, chloroform, by the triblock copolymer templating and sol–gel techniques. The beads have oblong pore structure with a size of several hundreds m and these macropore are comprised of several tens of m pores, several of m pores, and several tens of nm pores. The beads show superior apatite-like layer forming activity and good in vitro biodegradability. This simple synthetic method can be adapted for the purpose of preparing various ceramics with hierarchical pore structures, which have excellent potential applications in the field of not only biomaterials such as tissue engineering and drug storage but also catalysis, filters, adsorbents, and optics. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Bioactive glasses (BGs) have been widely studied because they have the ability to chemically bond with living bone tissue and have consequently been used in a variety of medical applications [1–3]. Increasing the specific surface area and pore volume of BGs may greatly accelerate the kinetic deposition process of hydroxycarbonate apatite and allow them to be loaded with the osteogenic agents, thus enhancing their bone-forming bioactivity [4–10]. To this end, the development of mesoporous BGs, which have a large specific surface area and pore volume, has recently accelerated [4–9,11–15]. Highly 2D hexagonal and 3D cubic ordered mesoporous BGs with superior bone-forming bioactivities in vitro compared with normal BGs have therefore been synthesized by templating with a triblock copolymer, Pluronic P123 and F127, respectively. Although all of the reported mesoporous BGs show favorable bioactivity, they are difficult to use as a bone filler or as scaffolds for the regeneration of bone tissue at this stage, because their meso-sized pore are too small to promote cell growth as well as load osteogenic agents such as bone morphogenetic protein [4–9]. Control the pore sizes and structures of BGs is important to maximize their functionality for use as materials in tissue regeneration. Human bone consists of hierarchically 3D interconnected pore structures and cells are sensitive to local environmental features at all length scales from macro down to the molecular [16]. BGs with hierarchical porosity combine the advantages of a high surface area from nanoporous structure with the accessible diffusion pathways associated with a macroporous
structure. BG ceramic scaffolds with hierarchical pore structures were previously reported by our group [12–15]. However, there have been no reports yet concerning the preparation of hierarchically porous BG ceramic beads with pore sizes in the range between 10 nm and 100 m, which can be used as the bone fillers. Hierarchically porous materials have generally been prepared by making use of self-assembled supramolecular structures, the induction of morphological complexity by inducing topological defect structures, and the inorganic replication of organized reaction field [17–24]. These preparation methods required the use of multi-synthetic steps or multi-scale organic templates and the beads produced had hierarchal pore size in the range of several nm to several hundreds of nm, which are still too small to promote cell activity. Herein, we report a novel, simple and reproducible method of preparing hierarchically 3D porous BG beads using the same procedure as that employed for mesoporous BGs, with one additional step: the precursor solution is added to a hydrophobic solvent, chloroform, after appropriate evaporation to induce the self-rearrangement of the triblock copolymer and inorganic framework. Furthermore, we successfully prepared mesoporous BGs with various types of mesopore structure and controlled both the pore size and the material morphology of the beads. This synthetic method allows the well interconnected pore structure, hierarchically porous BG beads in the size range between 10 nm and several hundreds of m with high porosity and good bioactivity. 2. Experimental 2.1. Materials and synthesis
∗ Corresponding author. Tel.: +82 55 280 3351; fax: +82 55 280 3399. E-mail address: [email protected] (H.-s. Yun). 0254-0584/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2009.02.001
In a typical synthesis, 3.46 g of poly (ethylene oxide)132 -poly(prophylene oxide)50 -poly(ethylene oxide)132 ((EO)132 (PO)50 (EO)132 , F108, average Mn = 14,600)
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676 is dissolved in 18.1 ml of ethanol (EtOH). Stock solutions, which were prepared by mixing 1.36 g of calcium nitrate tetrahydrate (CaNT), 0.26 ml of triethyl phosphate (TEP), 6 ml of tetraethyl orthosilicate (TEOS), 0.95 ml of HCl (1 M), 7.62 ml of EtOH and 2.86 ml of H2 O, were added to this solution after stirring them separately for 1 h, and were vigorously stirred together for another 4 h at 40 ◦ C. The molar composition was TEOS:CaNT:TEP:F108 = 1:0.2:0.05:0.008 in this case. The reactant solution was evaporated at 40 ◦ C, 40 RH% for 24 h without stirring to increase of its viscosity. The evaporated sol solutions were dropped into CHCl3 solutions using by a syringe (20–24 G). The obtained gel beads were collected, aged at 40 ◦ C for 24 h and were calcined at 600 ◦ C for 6 h in air to remove the template. In addition, a series of BGs, which were produced using different block copolymers templates, L121 (EO5 PO68 EO5 , Mn = 4400), P123 (EO20 PO70 EO20 , Mn = 5750), and F127 (EO100 PO65 EO100 , Mn = 12,600), were also prepared to study and to compare the formations of the mesoporous BG as well as the hierarchically porous BG. All chemicals were purchased from Aldrich.
2.2. Characterization Structural characterization was carried out by X-ray diffraction (XRD; –2 scanning, Philips-X’pert MPD 3040, using Cu K␣ ( = 1.5406 Å) radiation (40 kV–40 mA), transmission electron microscopy (TEM; JEOL-JEM2100F) with field emission gun at 200 kV, and field emission scanning electron microscopy (FE-SEM; Hitachi-S5500 and JEOL-5800) at an accelerating voltage of 1–5 kV. The specific surface area from meso-sized pore was measured by the N2 -gas adsorption–desorption isotherms using a Brunauer Emmett Teller (BET) apparatus (BelJapan-Belsorp mini II). The pore volume and pore-size distribution were derived from the adsorption branches of the isotherms using the Barrett–Jayner–Halanda (BJH) method. The total pore volume was estimated from the amount adsorbed at a maximum relative pressure. The porosity from macro-sized pore was measured by the Hg intrusion method (AutoPore IV 9510, Micromeritics). The assessment of the in vitro bioactivity of the BGs beads was carried out in simulated body fluid (SBF) at 37 ◦ C. The SBF contained 142.0 mM Na+ , 5 mM K+ ,1.5 mM Mg2+ , 2.5 mM Ca2+ , 147.8 mM HCl− , 4.2 mM HCO3− , 1.0 mM HPO4 2− , and 0.5 mM SO4 2− [13]. Its chemical composition was similar to that of human plasma. The solu-
671
tion had a pH of 7.4 and was kept at 37 ◦ C before use. Concentration of Ca as well as pH levels of the SBF after soaking the BGs beads were determined at different periods by inductive coupled plasma (ICP) atomic emission spectroscopy and pH system (Mettler Toledo). The assessment of the in vitro biodegradability of the BGs beads was carried out in the phosphate buffered saline (PBS) at 37 ◦ C.
3. Results and discussion The formation of BG beads largely depends on the structure of the polymer templates. The precursor solution was added to the hydrophobic solvent, chloroform, drop by drop after adequate evaporation (reduction of 60–65 (v/v%); about 52–55 Brix%) increase the viscosity of the precursor solution to the extent required for producing BG gel beads. 4 types of triblock copolymers were used, L121, P123, F127, and F108, which have different m/n ratio of (EO)m (PO)n (EO)m . The precursor solution containing L121 or P123 as templates formed the gel beads when it was just dropped into chloroform, but the beads gradually lost their shape over time and turned into a gel solution (Fig. 1a). On the other hand, when the precursor solutions containing F127 or F108 were used as the templates, the transparent gel beads were produced when it was just dropped into chloroform, the sizes of the beads gradually shrank, and they turned opaque with time. However, they retained their morphology quite well (Fig. 1b). Fig. 1c and d shows the optical images of the BG beads before and after calcination at 600 ◦ C. The as-synthesized BG beads were well shaped with relatively uniform sizes of 1750 m. The BG beads show a 22% size reduction (1360 m) after calcination, but still have a good morphology without cracks. The size of the beads can be easily controlled from
Fig. 1. Optical images of BG gels templated by (a) P123 and (b) F108 after dropping into CHCl3 . Microscope images of BG beads (c) before and (d) after calcination at 600 ◦ C. Transmission electron microscope (TEM) images of calcined BG synthesized using triblock copolymers; (e and i) L121, (f and j) P123, (g and k) F127, and (h and l) F108 templates. (e–h) Before dropping into CHCl3 and (i–l) after dropping into CHCl3 and standing for 24 h.
672
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
Fig. 2. FE-SEM images (a–f) and TEM images (g–i) of calcined BG beads. (a) Low magnification images of beads and (b and c) high magnification images of surface of beads. FE-SEM images of cross-sectioned BG beads after dropping into CHCl3 and standing for (d) 1 h, (e) 2 h, and (f) 24 h. TEM images of BG beads after dropping into CHCl3 and standing for (g) 1 h, (h) 2 h, and (i–l) 24 h. (j) Inside part of BG beads, (k) outside part of BG beads, and (l) interface between inside and outside part images of BG beads.
Fig. 3. Pore size distribution of BG beads measured by (A) Hg intrusion and (B) N2 adsorption–desorption isotherms.
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
several hundreds of m to several mm by adjusting the syringe size, the viscosity of the precursor and the dropping conditions (Support Information (SI) 1). Similar structural phenomena occurred when a hydrophobic solvent such as hexane, benzene, toluene, diethyl ether, and ethyl acetate was used in place of chloroform, but if the density of the solvent is lower than that of the BG sol beads, the dropped beads immediately sink to the bottom of the beaker and, consequently, the beads can not retain their beads morphology well. If chloroform is not used in this step, a mesoporous structured BG with different pore structures can be obtained, depending on the polymer structure as shown in Fig. 1e–h. L121 induces wormhole-like structure, P123 allows hexagonal structure and both F127 and F108 produce cubic pore structure with high BET surface area. The BET surface area of BG produced using L121, P123, F127, and F108 as templates is 446, 450, 502, and 516 m2 g−1 , respectively. It is worthy to note that all of these pore structures were completely changed by the reaction of gel beads in chloroform as shown in Fig. 1i–l. The BG produced using L121, BG-L121chloro, lost its pore structure resulting in the formation of BG nanoparticles with a relatively homogeneous particle sized of 15 nm. The BG synthesized using P123, BG-P123chloro, also lost its hexagonal pore structure, leading to the formation of BG nanospheres with a heterogeneous size of 35–400 nm. Meanwhile, the pore structures of the BGs obtained using both F127, BG-F127chloro, and F108, BGF108chloro, changed from cubic to cancellous structure with greatly increased pore sizes. More interestingly, the obtained BG beads contain a hierarchical pore structure. Fig. 2 shows the FE-SEM and TEM images of BG-F108chloro (the result obtained for BG-F127 is confirmed by SI 2). The BG-F108chloro beads have well interconnected and hierarchically arranged open pore structure. That is, the large sized pores with size ranging from 7 to 10 m are made up of medium sized pores with a size of several m, and the medium sized pores are built up with the small sized pores with sizes of several tens of nm. The beads also form oblong pore structures with a size of several hundreds m long over the beads. These pore size distributions are relatively sharp and agree well with the results of both the mercury intrusion test and the BJH plot derived from the N2 adsorption isotherm (Fig. 3). That is, in the analysis of the pore size distribution, two relatively sharp peaks centered at 7 and 2 m were observed in the Hg intrusion test and a broad peak centered at 25 nm was detected from the BJH plot derived from the N2 adsorption–desorption test. The pore morphologies gradually changed, starting from the outside of the beads, which is the first part to come into contact with chloroform, and later extending to the inside, depending on the reaction time. Fig. 2d–i shows the FE-SEM and TEM results of BG-F108chloro showing the variability of the pore morphology over time. The pore sizes are increased from outside to inside with increasing the reaction time.
673
After 30 min immersion in chloroform, large pores appear on the outside of the beads (Fig. 2d), their mesopore structure is lost, and large nanopore structures with an average pore size of 40 nm are newly formed (Fig. 2g). The macropores extend from the outside to the inside with increasing pore size over time (Fig. 2e and h) and the hierarchical pore structures are finally obtained after 24 h immersion in chloroform (Fig. 2f and i). However, the pore morphologies are still different on the outside and inside and some meso-sized pores are still remaining in the center of the beads (Fig. 2j–l). The change behavior of the mesopore structure to the hierarchically meso-macropore structure can also be confirmed from the result of the N2 adsorption isotherms (Fig. 4). Prior to its treatment in chloroform, porous BG-F108 has a type IV isotherm with a sharp capillary condensation step at high relative pressures and broad H2 hysteresis loop that is indicative of large uniform cage-type pores. After the reaction in chloroform, drastic changes in both the adsorption–desorption isotherms and pore size distributions can be observed, but the beads still have type IV isotherms with a capillary condensation. The pore size increases with increasing reaction time, with the result that the BET surface area and pore volume of the pores ranging in size from mesopores are decreased. The BET surface area and pore volume of the beads after immersion for 24 h in chloroform are 100 m2 g−1 and 0.16 cm3 g−1 , respectively. On the other hand, BG-F108chloro has a large average porosity of 50% resulting from the macropores, as measured by the Hg intrusion test. These kinds of hierarchical pore structure can obtained only after adequate evaporation and the sol–gel reaction of the precursor solution. If the chloroform is directly added to the precursor solution in the starting step, the separation between the triblock copolymer and inorganic sources occurs, because the triblock copolymers tends to make a reverse micelle due to its self-assembling behavior in chloroform, following which the hydrophobic part comes out of the micelle, and the hydrophilic inorganic sources consequently cannot react with the micelles. If the precursor solution is dropped into chloroform without appreciable evaporation, that is, the precursor solution has low viscosity and hydrolysis and condensation do not sufficiently occur, the dropped beads cannot retain their bead shape, and consequently a heterogeneous pore structure is induced after removing the solvent. Excess evaporation also leads to fail. A possible mechanism for the formation of BG beads with a hierarchical pore structure is illustrated in Scheme 1. Owing to the hydrophobic nature of PO and the hydrophilic nature of EO, PO forms a core and EO exists as a shell around the core in the aqueous condition. According to our synthetic procedure, the inorganic sources, Si-, Ca-, and P-, react with the EO groups and the sol–gel reaction in the inorganic framework proceeds in the first step. These polymer–inorganic composite
Fig. 4. (A) N2 adsorption–desorption isotherms and (B) BJH plots of porous BG beads with different reaction time in chloroform.
674
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
Scheme 1. Suggested mechanism for the formation of the hierarchically porous BG beads.
micelles have a tendency to make reverse micelles in chloroform. That is to say, the EO/inorganic composite part tends to move into the center of the micelle and the PO part attempts to move outside of the cell. The inorganic part exists in the center of the micelle and these reversed micelles try to rearrange each other to produce a hierarchical structure. The ratio of m/n in EOm POn is important when attempting to produce a hierarchical pore structure in this second step. That is, if m is much less than n, the inorganic particles will separate from each other and will exist as nanoparticles after the removal of the polymer template by calcination, without retaining their hierarchical pore structure, because the inorganic particles cannot be interconnected due to their large interparticle distance. We suggest that this is why neither BG-L121chloro (m/n = 0.07) nor BG-P123chloro (m/n = 0.29) are able to form the beads, but rather produce nanoparticles with different particle sizes. On the other hand, if m is sufficiently larger than n, the interparticle distance is short, the particles may interlock with one another after the removal of the polymer template by calcination, and the hierarchical pore structure will be consequently retained. As a result, both BG-F127chloro (m/n = 1.54) and BG-F108chloro (m/n = 2.64), which
have enough longer chain of EO than of PO, can have a hierarchical pore structure. Although this suggested mechanism needs more detailed discussion, the obtained hierarchically porous ceramic beads are of interest, not only for bio-applications but also as potential catalyst, separation, adsorption, or electrode materials, because of their high molecular selectivity ranging from nanopores to macropores as well as their unique and efficient diffusion pathways. The bone-forming bioactivity of BG-F108chloro in vitro was tested in SBF to monitor the formation of hydroxyl apatite (HA) on the surface of the beads [25]. HA provides a suitable substrate for the proliferation and function of osteoblast like cells, which allows for the strong bonding of the materials to the surrounding bone tissue [26]. The FE-SEM images of the beads reveals that the surface of the BG beads undergoes important changes when it reacts with the SBF as shown in Fig. 5a–d. The formation of HA-like nanoparticles is observed on the surface of the BG beads after soaking the sample for 24 h (SI3). This reaction proceed over time and the surface of the BG beads was completely covered with newly formed HA-like nanoparticles having a needle-like morphology along the whole pore structure after being soaked for
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
675
Fig. 5. FE-SEM micrographs of BG beads after they immersed in SBF for (a) 0 h, (b) 24 h, (c) 48 h and (d) 72 h to test in vitro bioactivity of BG beads and cam scope images of BG beads before (e and g) and after (f and h) immersing into PBS for 8 weeks for confirming in vitro biodegradability of BG beads. (g and h) The enlarged image of (e) and (f), respectively.
72 h. Both the Ca2+ concentration and pH level in SBF also reveal these bioactivities of the BG beads (Fig. 6). The Ca2+ levels increase during the 72 h. Thereafter, the Ca2+ concentration in SBF decreases until the end of the assay. The pH evolution follows a profile analogous to that of the Ca2+ content. These results indicate that a Ca2+ –H+ exchange occurs between the BG beads and SBF, and it allows for the deposition of HA on BG beads. In order to study their biodegradability, the dissolution of the BG beads in phosphorous
buffer solution (PBS) was performed as shown in Fig. 5e–h. The BG beads show a weight loss of 10% only after 2 weeks of immersion in PBS, gradually increase their weight loss with time and repeatedly show deformation of BG and formation of HA. These results indicate that the BG beads, which have a hierarchical pore structure, may have good HA-like layer forming activity (bioactivity) in vitro as well as biodegradability. Further studies of the biocompatibility of the BG beads in vitro and in vivo are currently in progress.
676
H.-s. Yun et al. / Materials Chemistry and Physics 115 (2009) 670–676
Fig. 6. Variation of (A) calcium content and (B) pH values of SBF as a function of soaking time for the hierarchically porous BG beads.
4. Conclusion Novel hierarchically mesoporous–macroporous BG beads with a well interconnected pore structure, good bioactivity, and biodegradability promising for use as materials for tissue regeneration such as bone fillers with controlled drug delivery functionalities were fabricated by the polymer templating and sol–gel techniques. The self-reassembling reaction of the triblock copolymer in the hydrophobic solution induces the hierarchical pore structure of the BG beads. The ratio of m/n in EOm POn is important to produce a hierarchical pore structure because the interconnecting of the BG nanoparticles after the reaction in chloroform is determined by the interparticle distance. We hope that this simple, reproducible synthetic method can be adapted for the preparation of various hierarchical porous materials, having the potential to be used in applications involving filters, catalysis, biomedical devices, and sensors. Acknowledgment This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-20037-0). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.matchemphys.2009.02.001. References [1] L.L. Hench, R.J. Splinter, W.C. Allen, T.K. Greenlee, Bonding mechanisms at the interface of ceramic prosthetic materials, J. Biomed. Mater. Res. 2 (1971) 117–141. [2] L.L. Hench, Bioceramics, J. Am. Ceram. Soc. 81 (1998) 1705–1727. [3] L.L. Hench, J.M. Dolak, Third generation biomedical materials, Science 295 (2002), 1014 + 1016–1017. [4] X. Yan, C. Yu, X. Zhou, J. Tang, D. Zhao, Highly ordered mesoporous bioactive glasses with superior in vitro bone-forming bioactivities, Angew. Chem. Int. Ed. 43 (2004) 5980–5984. [5] X.X. Yan, H.X. Deng, X.H. Huang, G.Q. Lu, S.Z. Quio, D.Y. Zhao, C.Z. Yu, Mesoporous bioactive glasses. I. Synthesis and structural characterization, J. Non-Cryst. Solid 351 (2005) 3209–3217.
[6] X. Yan, X. Huang, C. Yu, H. Deng, Y. Wang, Z. Zhang, S. Qiao, G. Lu, D. Zhao, The in vitro bioactivity of mesoporous bioactive glasses, Biomaterials 27 (2006) 3396–3403. [7] I. Izquierdo-Barba, L. Ruiz-González, J.C. Doadrio, J.M. González-Calbet, M. Vallet-Regí, Tissue regeneration: a new property of mesoporous materials, Solid State Sci. 7 (2005) 983–989. [8] A. López-Noriega, D. Arcos, I. Izquierdo-Barba, Y. Sakamoto, O. Terasaki, M. Vallet-Regi, Ordered mesoporous bioactive glasses for bone tissue regeneration, Chem. Mater. 18 (2006) 3137–3144. [9] I. Izquierdo-Barba, D. Arcos, Y. Sakamoto, O. Terasaki, A. López-Noriega, M. Vallet-Regí, High-performance mesoporous bioceramics mimicking bone mineralization, Chem. Mater. 20 (2008) 3191–3198. [10] M. Vallet-Regí, C. Victoria Ragel, A.J. Salinas, Glasses with medical applications, Eur. J. Inorg. Chem. (2003) 1029–1042. [11] H.S. Yun, S.E. Kim, Y.T. Hyun, Highly ordered mesoporous bioactive glasses with Im3m symmetry, Mater. Lett. 61 (2007) 4569–4572. [12] H.S. Yun, S.E. Kim, Y.T. Hyun, Design and preparation of bioactive glasses with hierarchical pore networks, Chem. Commun. (2007) 2139–2141. [13] H.S. Yun, S.E. Kim, Y.T. Hyun, S.J. Heo, J.W. Shin, Three-dimensional mesoporousgiantporous inorganic/organic composite scaffolds for tissue engineering, Chem. Mater. 19 (2007) 6363–6366. [14] H.S. Yun, S.E. Kim, Y.T. Hyun, Preparation of 3 dimensional cubic ordered mesoporous bioactive glasses, Solid State Sci. 10 (2008) 1083–1092. [15] H.S. Yun, S.E. Kim, Y.T. Hyun, S.J. Heo, J.W. Shin, Hierarchically mesoporous–macroporous bioactive glasses scaffolds for bone tissue regeneration, J. Biomed. Mater. Res. Part B 87B (2008) 374–380. [16] M.M. Stevens, J.H. George, Exploring and engineering the cell surface interface, Science 310 (2005) 1135–1138. ˝ [17] S. Schacht, Q. Huo, I.G. Voigt-Martin, G.D. Stucky, F. Schuth, Oil–water interface templating of mesoporous macroscale structures, Science 273 (1996) 768–771. [18] S.D. Sims, D. Walsh, S. Mann, Morphosynthesis of macroporous silica frameworks in bicontinuous microemulsions, Adv. Mater. 10 (1998) 151–154. [19] D. Walsh, B. Lebeau, S. Mann, Morphosynthesis of calcium carbonate (vaterite) microsponges, Adv. Mater. 11 (1999) 324–328. [20] T. Sen, G.J.T. Tiddy, J.L. Casci, M.W. Anderson, One-pot synthesis of hierarchically ordered porous-silica materials with three orders of length scale, Angew. Chem. Int. Ed. 42 (2003) 4649–4653. [21] T. Sen, G.J.T. Tiddy, J.L. Casci, M.W. Anderson, Meso-cellular silica foams, macrocellular silica foams and mesoporous solids: a study of emulsion-mediated synthesis, Micropor. Mesopor. Mater. 78 (2005) 255–263. [22] H. Zhang, G.C. Hardy, M.J. Rossinsky, A.I. Cooper, Uniform emulsion-templated silica beads with high pore volume and hierarchical porosity, Adv. Mater. 15 (2003) 78–81. [23] C. Tao, J. Li, Morphosynthesis of microskeletal silica spheres templated by W/O microemulsion, Colloids Surf. A Physicochem. Eng. Aspects 256 (2005) 57–60. [24] J. Wang, Q. Xiao, H. Zhou, P. Sun, Z. yuan, B. Li, D. Ding, A.C. Shi, T. Chen, Budded, mesoporous silica hollow spheres: Hierarchical structure controlled by kinetic self-assembly, Adv. Mater. 18 (2006) 3284–3288. [25] T. Kokubo, Bioactive glass ceramics: properties and applications, Biomaterials 12 (1991) 155–163. [26] S.Y. Ni, J. Chang, L. Chou, A novel bioactive porous CaSiO3 scaffold for bone tissue engineering, J. Biomed. Mater. Res. 76 (2006) 196–205.