Revisiting silica based ordered mesoporous materials: medical applications

Abstract

The bioactivity behaviour of SBA-15, MCM-48, MCM-41 mesoporous materials, is revisited in this paper. The influence of their different textural and structural properties on apatite formation is outlined and strategies to modify their kinetics are proven and discussed. On the basis of the experimental data showing the feasibility of control of the bioactivity kinetics on mesoporous materials, together with their controlled drug release abilities, new possibilities for tissue engineering developments are proposed.

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María Vallet-Regí

María Vallet-Regí was born in Las Palmas, Spain, in 1946. She studied chemistry at the Universidad Complutense de Madrid (UCM) and received her PhD at the same University in 1974. She is a full Professor of inorganic chemistry and Head of the Department of Inorganic Chemistry and Bioinorganic at the Faculty of Pharmacy (UCM). Her current research field is solid state chemistry, covering aspects of synthesis, characterisation and reactivity in oxides and bioceramics.

Luisa Ruiz-González

Luisa Ruiz-González studied for her B.S. (1996) and PhD (2002) in chemistry at the Universidad Complutense de Madrid (UCM). She enjoyed a post-doc position at the Oxford University working in the characterization of carbon nanotubes. Her research interest includes synthesis and structural characterization by SAED and HREM techniques of perovskite related, mesoporous and nano materials. She combines research with educational tasks as an assistant at UCM.

Isabel Izquierdo-Barba

I. Izquierdo received her B.S. (1997) and PhD (2003) in pharmacy at the Universidad Complutense de Madrid (UCM). She enjoys a post-doc position at the Stockholm University working in the characterization of mesoporous materials in the Structural Chemistry department.

José M. González-Calbet

José M. González-Calbet was born in Madrid, Spain, in 1952. He studied chemistry at the Universidad Complutense de Madrid (UCM) and received his PhD at the same University in 1979. He is a full Professor of inorganic chemistry and Head of the Department of Inorganic Chemistry at the Faculty of Chemistry (UCM). His current research field is solid state chemistry, covering aspects of reactivity of oxides and structural characterisation by electron microscopy.

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The discovery of ordered silica-based mesoporous materials, by scientists of the Mobil Corporation¹ in 1992, has attracted enormous interest allowing expansion of the potential applications of the host–guest system at the nanometre scale. These applications comprise a broad range of topics, from catalysis to controlled drug release. Actually, mesoporous materials constitute a new generation of materials that show ordered arrangements of channels and cavities of different geometry built up from SiO₂ unities. The pore size is variable (2nm < Φ_p < 50 nm) and can be controlled and modified, in a reasonable range, using in situ² and ex situ³ synthetic strategies. Well-known examples are the 2D-hexagonal (S.G. p6mm) MCM-41⁴ and SBA-15⁵ silica mesoporous materials with pores around 2 and 10 nm, respectively, and the 3D-cubic MCM-48⁶ (S.G. Ia3d ) with pore size between 2 and 4 nm. The feasibility to obtain different pore size and geometries offers a wide range of possibilities for hosting molecules larger than the ones exhibited for classic microporous materials, such as zeolites. Moreover, the absorption properties can be modified, since the pore walls, exhibiting high concentration of silanol groups at the surface, can be functionalized with different chemical species. Taking into account these characteristics, the behaviour of mesoporous materials as drug delivery systems has been developed by this research group since 2001. It is based on the ability of these matrixes to absorb molecules, of pharmacological interest, followed by a potentially controlled release.⁷ This work has opened new directions on the medical research in drug release from a mesoporous matrix.^7–17 Exploring new applications of mesoporous materials is a very attractive goal and, based on their composition and structure, it is possible to consider them as scaffolds in regeneration of hard tissues (bones and tooth). For such a desirable application, the mesoporous materials must exhibit a bioactive behaviour. At this point, it is worth recalling that bioactive glasses,^18–24 that also exhibit silanol groups (Fig. 1), are able to develop an apatite layer. Since Hench et al. discovered Bioglass® in 1971²⁵ many studies, comprising different glasses and calcium silicates compositions, have shown in vitro bioactivity.^22,26–29 Although the mechanism of apatite formation is not completely elucidated, the presence of silanol groups in the surface seems to be crucial. In this sense, several authors propose that silanol groups act as nucleation sites (Fig. 2). However, there are more factors compulsory to the apatite formation;^30,31 among them, it is interesting to point out the textural properties, i.e. those related with the porosity. It has been proved that a direct relation between both pore size and volume and nucleation rate of the apatite layer^32–34 exists.

Fig. 1 Schematic illustration of bioactive glass and ordered mesoporous materials, which shows that both materials exhibit high silanol concentration.

Fig. 2 Hydroxyapatite formation mechanism in bioactive glasses when soaked in physiological fluids.

Taking into account the above considerations, it seems logical to focus on mesoporous silica, because they show high specific surface, high concentration of silanol groups, feasible pore size (2–10 nm) and volume,^35–38 and can also exhibit a bioactive behaviour. In this sense, bioactivity essays were performed in three well known mesoporous materials, SBA-15, MCM-48 and MCM-41. A positive response was found for SBA-15 and MCM-48.³⁹ Although pure MCM-41 is not bioactive, it has been shown that minimal compositional changes can modify this behaviour.^40,41 The aim of this work is to review the potential bioactive behaviour of three matrices in order to analyze the different observed responses, for further applications. In fact, the combination of the recent results obtained for SBA-15 and MCM-48, as well as the possibility not only to promote but also to modify the kinetics of apatite formation in MCM-41, allow us to think about new potential applications in medical science and, more specifically, in biomaterials and tissue engineering. Moreover, development possibilities can be enhanced considering the ability of these materials to accept drugs and osteogenic substances for controlled release. The biocompatibility of these materials makes them appropriate for clinical applications if they are either bioactive or bioinert.

Bioactivity in silica based ordered mesoporous materials

Mesoporous materials with different structural and textural properties were chosen in order to evaluate their influence on the corresponding in vitro response. Bioactivity studies on SBA-15, MCM-48 and MCM-41 materials have been carried out.³⁹ The assessments of in vitro bioactivity were performed in simulated body fluid (SBF),⁴² which has the composition and ionic concentration similar to human plasma, at 37 °C during different immersion periods (Fig. 3). Different characterization techniques (FTIR, SEM-EDS, TEM-ED-EDX) show that, after the in vitro tests, SBA-15 and MCM-48 surfaces are modified, as a consequence of the reaction of the materials with the fluid, giving rise to the appearance of a carbonate hydroxyapatite layer.³⁹ The kinetics of each process is quite different; SBA-15 develops the apatite layer after 30 d while 60 d are required in the case of MCM-48. Nevertheless, after 60 d of immersion the apatite layer did not appear in MCM-41. SEM micrographs and EDS of samples SBA-15 and MCM-48 before and after 15, 30 and 60 d in SBF show that the surface has been covered with needle-type crystal aggregates forming spherical particles. The EDS analysis confirms the presence of Ca and P on the surface. In the case of MCM-41, the surface is not modified and Ca and P have not been detected (Fig. 4).

Fig. 3 Schematic description of the in vitro bioactivity test. Ion concentration in SBF and in human blood plasma are also included.

Fig. 4 SEM micrographs and EDS spectra of SBA-15, MCM-48 and MCM-41 materials at 0, 15, 30 and 60 d.

A TEM study, before and after the treatment in SBF, was also performed (Fig. 5). HREM images before the treatment show the pore size characteristic of the raw material. After the indicated periods of time, the apatite formation is confirmed at SBA-15 and MCM-48 surfaces,³⁹ as shown for SBA-15 in Fig. 5b and d. A careful inspection of the interface between the mesoporous material and the recently formed apatite supplies additional information (see Fig. 1), not previously available in bioactive glasses due to the lack of order. Crystalline particles embedded in an amorphous matrix are found, as clearly observed at the insets of Fig. 5. The amorphous area contains Si while the crystalline one comprises Ca and P. Since only Si is present at the amorphous matrix, it seems logical to relate it with the original mesoporous material that is transformed. This fact could be related to (i) the mesoporous surface modification as consequence of its reaction with the SBF to form the new apatite, the product of this chemical process or (ii) the transformation under the electron beam. A combination of these two factors is probably the more plausible explanation. Minor areas where the mesoporous material has not yet been completely decomposed are also found, as reflected in Fig. 6, corresponding to SBA-15. It is worth mentioning that in such cases the needle like particles are not placed over the mesoporous walls (Fig. 6a). On the left hand of the image, fringes of 8.8 nm periodicity, in agreement to SBA-15, in a clear process of modification, are observed. On the right hand, a small needle like particle is present. This is better seen in the enhanced image (Fig. 6b) where the crystalline nature of the particle imbibed in an amorphous area is also clearly observed. Measured periodicities correspond to the apatite cell. When the FT (Fourier transform) is performed on the amorphous area a broad diffuse scattering appears (Fig. 6c), reflecting, again, the amorphous nature. It is then clear that the mesoporous surface is modified when apatite is formed. This process cannot be studied on bioactive glasses due to their total structural disorder.

Fig. 5 TEM micrographs corresponding to SBA-15: (a) HREM image of the mesoporous material before treatment in SBF; (b) low magnification image after 60 d in SBF, the crystalline (inset A) and amorphous (inset B) nature of the particles and matrix, respectively, are clearly observed; (c) SAED pattern corresponding to the raw SBA-15 mesoporous material; (d) FT corresponding to inset A, characteristic of an apatite cell.

Fig. 6 TEM micrographs showing different features of SBA-15 after treatment: (a) area where the original mesoporous material is not completely transformed; features of both raw material and apatite are evident; (b) enhanced image reflecting the fact that apatite appears over the modified mesoporous material; (c) FT performed at the amorphous area in b.

The different behaviour shown for SBA-15, MCM-48 and MCM-41 can be understood on the basis of the compositional, textural and structural properties of the mesoporous materials, summarized in Table 1. As it has already been mentioned, the silanol groups (Si–OH) can act as nucleation sites of the apatite layer.^22,26–31 In this sense, the silanol group concentration seems to be an important factor to consider. In this case, the Si–OH concentration per surface has been determined by thermogravimetric analyses (TGA) and N₂ adsorption measurements. MCM-41 shows a rather lower concentration of silanol groups (ca. 2.0 mmol SiOH m⁻²) than SBA-15 and MCM-48 (ca. 13 mmol SiOH m⁻²). This could be one reason to explain the absence of bioactivity in MCM-41. Moreover, the textural and structural properties must also be taken into account in order to justify this negative behavior. Both factors are related with the possible ion diffusion inside the mesoporous material. Bigger and more accessible pores will favor the ion diffusion, i.e. the apatite nucleation will be faster.^32–34 Contrarily, the ion diffusion becomes slower and difficult if the pore size decreases, diminishing the nucleation rate. In this sense, SBA-15 shows the largest pore size (8.8 nm) being considerably lower (3.6 nm) in the cases of MCM-48 and MCM-41. This fact could explain the better kinetics observed in SBA-15 compared to MCM-48 and MCM-41. Furthermore, the dimension and shape of the pores is also a very important factor to be considered. In this sense, MCM-41 has pure one dimensional character while MCM-48 and SBA-15 are connected through complementary pores favoring the diffusion paths. However, the identical pore size of MCM-48 and MCM-41 does not justify the great difference on their bioactive behavior, but can be understood taking into account the silanol concentration as well as the structure. MCM-41 wall silanol concentration is lower than in MCM-48 and the 2D-hexagonal lattice without channel connections is less accessible to the ions if compared with the 3D-cubic one exhibited by MCM-48. Moreover, local curvatures for MCM-41 comparing to SBA-15 and MCM-48, being both quite similar, are different and may also play an influence.

Table 1 Bioactive behaviour, textural and compositional parameters in SBA-15, MCM-48, MCM-41, P-MCM-41 and MCM-41/G materials

Matrices	Bioactivity behaviour	Time in SBF/d	S BET/ m^2 g^−1	D p/nm	V p/cm^3 g^−1	V micropore/cm^3 g^−1	[SiOH]/mmol m^−2
SBA-15	Yes	30	787	8.8	1.05	0.061	12.71 × 10^−3
MCM-48	Yes	60	1166	3.6	1.05	—	12.86 × 10^−3
MCM-41	No	60	1157	3.6	0.98	—	2.16 × 10^−3
P-MCM-41	Yes	15	721	3.6	0.91	—	2.93 × 10^−3
MCM-41/G	Yes	1	1131	3.4	0.80	—	1.53 × 10^−3

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The above discussion allows us to understand, on the basis of compositional, textural and structural properties, why apatite formation happens in SBA-15 and MCM-48 in 30 and 60 d, respectively, while MCM-41 does not suffer any alteration, after two months of assay. This does not mean, in principle, that it is impossible to develop the apatite layer, but it is clear that the rate is too slow. Once that the factors playing an important role on the apatite formation have been discussed, the aim is to look for chemical procedures in order to modify the MCM-41 kinetics. On the basis of previous knowledge two different strategies have been applied: (i) doping with phosphorous (ii) addition of small amounts of a second substance that exhibits high bioactivity.

(i) Doping with phosphorous. Taking into account that the bioactivity of silica based ceramic materials has been reported to be strongly dependent upon the phosphorous content of the glass,^43,44 it seems reasonable to think that the doping of MCM-41 with phosphorous could help to obtain a bioactive behaviour from this material. Therefore, phosphorous-doped MCM-41 (P-MCM-41) was prepared as described elsewhere.⁴⁰ The structural and compositional characterization by means of XRD, N₂ adsorption, SEM, TEM and EDS indicates the stabilization of an ordered mesoporous P-containing silica, showing hexagonal symmetry (P6mm), and high specific surface and pore volume (close to 1 cm³ g⁻¹). P MAS-NMR spectroscopy confirms the presence of Si–O–P bonds. A study of the P distribution, by means of EDS, at the surface of the material reveals that P is present in 1% percentage. HREM study, coupled with EDS, reveals that P is mainly located in the outer rim of the particles but not in the inner core. These results evidence that microstructural and textural parameters of MCM-41 are preserved when P is added. The assessment of bioactivity was carried out in SBF, as previously described. In vitro studies, FTIR, SEM, EDS and TEM showed the formation of an apatite-like layer on the mesoporous surface after soaking for 15 d in SBF. These results show that the presence of P, even in amounts as small as 1%, in MCM-41 favours the surface apatite nucleation giving rise to a positive bioactive response for the in vitro test⁴⁰ in a reasonable period of time, 15 d (Table 1)

(ii) Addition of small quantities of bioactive glass. Another strategy used in order to increase the bioactivity process is the preparation of biphasic mixtures, in which one phase acts as apatite nucleation sites, accelerating the process. Bioactivity studies in the hydroxyapatite–bioactive glass biphasic system show an increase of the kinetics against pure hydroxyapatite.^45–49 In the case of MCM-41, a biphasic bioactive mixture was attained using the mesoporous material and a bioactive glass in the system SiO₂–CaO-P₂O₅, against the stability of pure MCM-41.³² Structural and compositional characterization reveals, as in P-MCM-41, that the mesoporous structure is, again, preserved, being the pore volume and size quite similar to the original ones in MCM-41. Following the previously described procedures, bioactivity assessments and corresponding evaluation were performed. In vitro studies indicate that apatite layer formation starts just in 3 h, being completed in 1 d.

The ensemble of results concerning the five materials analysed in this study (Table 1) opens an enormous expectation about their possible applicability. The first hypothesis, on the basis of the evidence of bioactivity, is the possibility to design mesoporous materials in accordance with the desirable response time. In fact, MCM-41 does not develop apatite even after months of treatment, but it is attained after 15 and 1 d by doping with small amounts of P or adding bioactive glass, respectively. If the same procedures were applied to SBA-15 and MCM-48, that exhibit bioactivity after 30 and 60 d of treatment, respectively, it would be possible to adjust the kinetics of the apatite formation process to the required time of a specific problem.

Moreover, these materials can be functionalized with both polar and non polar molecules. This is very important from the biological point of view, since it opens up the possibility to attach different groups with affinity for cells or biological species (such as thiol, acid or amino) to the mesoporous wall. These groups can allow the attachment with specific proteins (osteogenic peptides and growth factors) opening new alternatives in tissue engineering. The loss or failure of an organ or tissue is one of the most frequent and expensive clinical problems. In this sense, regeneration constitutes a target objective of many studies. Tissue engineering is an emerging area directed towards the design of materials than can help an organism to improve its ability of regeneration by recovering both the structure and also its function. There are different strategies but one particularly interesting is the combination of cell harvest with scaffolds by means of in vitro techniques, followed by implantation on the corresponding organ of the host.⁵⁰ For this purpose, biocompatible and bioresorbable scaffolds are desirable to favour the tissue growth and increase the cellular function, i.e., to help the development of cells into a functioning tissue, ready for implantation.

Taking into account these ideas and the mesoporous properties, here reviewed, it is clear that they are promising candidates as scaffolds for tissue regeneration (Fig. 7). For this purpose, it is necessary to find suitable combinations of bioactivity, drug release, porosity and wall functionalization.

Fig. 7 Schematic illustration of tissue engineering technique promoted by bioactive ordered mesoporous materials which can be functionalized with organic groups and release different growth factors for tissue regeneration.

Conclusions

The role that the textural and structural properties of the mesoporous materials play on their bioactive behaviour is established. Alternatively, it is shown how the kinetics of the apatite formation can be modified and improved. The possibility to control the periods of time needed for a positive response, together with the ability to functionalise the surface and the introduction of osteogenic substances inside the pores, open new expectations for designing new mesoporous materials directed to specific medical applications.

References

1. J. S. Beck, C. T.-W. Chu, I. D. Johnson, C. T. Kresge, M. E. Leonowicz, W. J. Roth and J.C. Vartuli, Mobil Oil, US Patent, 5 108 725, 1992.
2. J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D. Schmitt, C. T.-W. Chu, D. H. Olson, E. W. Sheppard, S. B. Higgins and J. L. Schlenker, J. Am. Chem. Soc., 1992, 114, 10834.
3. X. S. Zhao, G. Q. Lu and X. Hu, Chem. Commun., 1999, 1391.
4. A. Firouzi, F. Atef, A. G. Oertly, G. D. Stucky and B. Chmelka, J. Am. Chem. Soc., 1997, 119, 3596.
5. D. Zhao, J. P. Feng, Q. S. Huo, N. Melosh, G. H. Fedrickson, B. F. Chemelka and G. D. Stucky, Science, 1998, 279, 548.
6. M. Kaneda, T. Tsubakiyama, A. Carlsson, Y. Sakamoto, T. Ohsuna, O. Terasaki, S.-H. Joo and R. Ryoo, J. Phys. Chem. B, 2002, 103, 1256.
7. M. Vallet-Regí, A. Rámila, R. P. del Real and J. Pérez-Pariente, Chem. Mater., 2001, 13, 308.
8. B. Muñoz, A. Rámila, I. Díaz, J. Pérez-Pariente and M. Vallet-Regí, Chem. Mater., 2003, 15, 500.
9. A. Ramila, B. Munoz, J. Pérez-Pariente and M. Vallet-Regí, J. Sol–Gel Sci. Technol., 2003, 26, 1199.
10. A. L. Doadrio, E. M. B. Sousa, J. C. Doadrio, J. Perez-Pariente, I. Izquierdo-Barba and M. Vallet-Regí, J. Controlled Release, 2004, 97, 125.
11. M. Vallet-Regí, J. C. Doadrio, A. L. Doadrio, I. Izquierdo-Barba and J. Perez-Pariente, Solid State Ionics, 2004, 172, 435.
12. C. Charnay, S. Bégu, C. Tourné-Péteilh, L. Nicole, D. A. Lerner and J. M. Devoisselle, Eur. J. Pharm. Biopharm., 2004, 57, 533.
13. P. Horcajada, A. Rámila, J. Pérez-Pariente and M. Vallet-Regí, Microporous Mesoporous Mater., 2004, 68, 105.
14. J. Anderson, J. Resenholm, S. Arev and M. Linden, Chem. Mater., 2004, 16, 4160.
15. B. G. Trewyn, C. M. Whitman and V. S.-Y. Lin, Nano Lett., 2004, 4, 2139.
16. C.-Y. Lai, B. G. Trewyn, D. M. Jeftinifa, K. Jeftinifa, S. Xu, S. Jeftinifa and V. Lin, J. Am. Chem. Soc., 2003, 125, 4451.
17. W. Zeng, X. F. Qian, Y. B. Zhang, J. Yin and Z. K. Zhu, Mater. Res. Bull., 2005, 40, 766.
18. L. L. Hench and J. Wilson, Science, 1984, 226, 630.
19. Ö. H. Andersson, K. H. Karlsson, K. Kangasniemi and A. Yli-Urpo, Glastech. Ber., 1988, 61, 300.
20. T. Kokubo, J. Ceram. Soc. Jpn., 1991, 99, 965.
21. R. Li, A. E. Clark and L. L. Hench, J. Appl. Biomat., 1991, 2, 231.
22. M. Vallet-Regí, J. Chem. Soc., Dalton Trans., 2001, 363, 254.
23. H. M. Kim, J. Ceram. Soc. Jpn., 2001, 109, S49.
24. L. L. Hench and J. M. Pollack, Science, 2002, 295, 1014.
25. L. L. Hench, R. J. Splinter, W. C. Allen and T. K. Greenlee, J. Biomed. Mater. Res., 1971, 2, 117.
26. M. Vallet-Regí, C. V. Ragel and J. A. Salinas, Eur. J. Inorg. Chem., 2003, 1029.
27. J. Black and G. Hastig, Handbook of Biomaterial Properties, Chapman and Hall, London, 1998.
28. Handbook of Bioactive Ceramics. Bioactive Glasses and Glass–ceramics, vol. 1, ed. T. Yamamuro, L. L. Hench, J. Wilson, CRC Press, Boca Raton, FL, 1990.
29. M. Brink, T. Turunen, R. P. Happonen and A. Yli-Urpo, J. Biomed. Mater. Res., 1997, 37, 114.
30. P. Li, C. Ohtsuki, T. Kokubo, K. Nakanishi, N. Soga, T. Nakamura and T. Yamamuro, J. Am. Ceram. Soc., 1992, 75, 2097.
31. S. B. Cho, K. Nakanishi, T. Kokubo and N. Soga, J. Am. Ceram. Soc., 1995, 78, 1769.
32. M. M. Pereira, A. E. Clark and L. L. Hench, J. Am. Ceram. Soc., 1995, 78, 2163.
33. M. M. Pereira and L. L. Hench, J. Sol–Gel Sci. Technol., 1996, 7, 59.
34. D. Arcos, D. C. Greenspan and M. Vallet-Regí, Chem. Mater., 2002, 14, 1515.
35. C. T. Kresge, M. E. Leonowicz, W. J. Roth, J. C. Vartulli and J. S. Beck, Nature, 1992, 359, 710.
36. S. Inagaki, Y. Fukushima and K. Kuroda, J. Chem. Soc., Chem. Commun., 1993, 8, 680.
37. Q. Huo, D. I. Margolese, U. Ciesla, P. Y. Feng, T. E. Gier, P. Sieger, R. Leon, P. M. Petroff, F. Schuth and G. D. Stucky, Nature, 1994, 368, 317.
38. Y. Sakamoto, M. Kaneda, O. Terasaki, D. Y. Zhao, J. M. Kim, G. Stucky, H. J. Shin and R. Ryoo, Nature, 2000, 408, 449.
39. I. Izquierdo-Barba, L. Ruiz-González, J. C. Doadrio, J. M. González-Calbet and M. Vallet-Regí, Solid State Sci., 2005, 7, 983.
40. M. Vallet-Regí, I. Izquierdo-Barba, A. Ramila, J. Pérez-Pariente, F. Babonneau and J. M. Gónzalez-Calbet, Solid State Sci., 2005, 7, 233.
41. P. Horcajada, A. Ramila, K. Boulahya, J. M. Gonzalez-Calbet and M. Vallet-Regí, Solid State Sci., 2004, 6, 1295.
42. T. Kokubo, H. Kushitani, S. Sakka, T. Kisugi and T. Yamamuro, J. Biomed. Mater. Res., 1990, 24, 721.
43. M. Vallet-Regí, I. Izquierdo-Barba and A. J.Salinas, J. Biomed. Mater. Res., 1999, 46, 560.
44. M. Vallet-Regí, A. Rámila, S. Padilla and B. Muñoz, J. Biomed. Mater. Res., 2003, 66, 580.
45. I. M. O. Kangasniemi, E. Vedel, J. de Blick-Hogerworst, A. Yli-Urpo and K. de Groot, J. Biomed. Mater. Res., 1993, 27, 1225.
46. J. D. Santos, J. J. Lakhan and F. J. Monteiro, Biomaterials, 1995, 16, 521.
47. M. Vallet-Regí and A. Ramila, Chem. Mater., 2000, 12, 961.
48. M. A. Lopes, J. D. Santos, F. J. Monteiro, C. Othsuki, A. Otaka, S. Kaneko and H. Inove, J. Biomed. Mater. Res., 2001, 54, 463.
49. C. V Ragel, M. Vallet-Regí and L. M. Rodriguez-Lorenzo, Biomaterials, 2002, 23, 1865.
50. K. Y. Lee and D. J. Mooney, Chem. Rev., 2001, 101, 1869.

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