Tissue regeneration: A new property of mesoporous materials

Solid State Sciences 7 (2005) 983–989 www.elsevier.com/locate/ssscie

Tissue regeneration: A new property of mesoporous materials Isabel Izquierdo-Barba a , Luisa Ruiz-González b , Juan C. Doadrio a , José M. González-Calbet b , María Vallet-Regí a,∗ a Dpto. Química Inorgánica y Bioinorgánica, Fac. Farmacia, UCM 28040-Madrid, Spain b Dpto. Química Inorgánica, Fac. Química, UCM 28040-Madrid, Spain

Received 6 April 2005; accepted 25 April 2005 Available online 1 June 2005

Abstract A new application of mesoporous materials as bone regenerators is described. In vitro bioactivity studies by soaking three different mesoporous materials, SBA-15, MCM-48 and MCM-41, in simulated body fluid (SBF) have been carried out. After the in vitro test, the study by Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive spectroscopy, electron diffraction and microscopy shows that an apatite-like layer is formed on the surface of SBA-15 and MCM-48 materials after 30 and 60 days, respectively, allowing their use in biomedical engineering for tissue regeneration.  2005 Elsevier SAS. All rights reserved.

1. Introduction Bioactive materials play an important role in the development of biomedical technology [1] for tissue regeneration. Since the discovery of Bioglass [2], many studies have been carried out [3–5] in silica systems detecting the presence of bioactivity in contact with physiological fluids. This property encompasses the ability of a given material to form interfacial bonds with tissues when in contact with physiological fluid involving, always, the formation of a layer of hydroxycarbonoapatite. Although apatite nucleation and crystallization mechanisms are not yet completely understood, the features of both the substrate and the fluids seem to have important influences. Concerning the solution, parameters such as pH, temperature and ionic concentration determine the type of calcium phosphate formed as well as its precipitation rate [6]. On the other hand, the presence of silanol groups and porosity seems to be crucial in the apatite layer formation. In this sense, Li et al. [7] have reported that this layer is formed on silica gels, but not on dense silica * Corresponding author.

E-mail address: [email protected] (M. Vallet-Regí). 1293-2558/$ – see front matter  2005 Elsevier SAS. All rights reserved. doi:10.1016/j.solidstatesciences.2005.04.003

glasses or quartz. Moreover, the layer formation is enhanced by the presence of pores larger than 2 nm [6–8], and a direct relation between pore size and volume and the nucleation rate has been drawn. Mesoporous silica materials [9,10], having pore sizes in the range of 2–90 nm and inner surface silanol and siloxane reactive groups, are promising candidates as bioactive materials. Furthermore, these pores form ordered arrangements, with high surface area, leading to biomedical applications as systems for drug delivery [11–13]. Such application was first demonstrated using MCM-41 charged with ibuprofen [11] and recently introducing amoxicillin in SBA-15 [14] and ibuprofen in MCM-48 [15]. The possibility to combine both properties, bioactivity and controlled drug release, is a very attractive goal that could be dealt with using mesoporous silica due to their intrinsic textural properties, that can induce bioactivity, together with the option of filling the pores with drugs. Therefore, the aim of this work is to study the bioactivity properties of three mesoporous materials, SBA-15, MCM-48, MCM-41, by means of in vitro assays in simulated body fluids, to evaluate their possible application in biomedical engineering for bone regeneration.

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2. Experimental section The mesoporous materials SBA-15, MCM-48 and MCM41, were synthesized by sol–gel methods in agreement with previously reported procedures [16–18]. To perform the bioactivity assays, the three materials were compacted into disks (13 mm in diameter and 2.5 mm in height) by using uniaxial (2.75 MPa) and isostatic pressure (3 MPa). The assessment of in vitro bioactivity was carried out by soaking the disks in simulated body fluid (SBF) [19], which has a composition and ionic concentration similar to that of human plasma, in periods of 15, 30 and 60 days. After soaking, specimens were removed from the fluid and rinsed with distilled water for 5 minutes. The ionic concentration of solution and the surface of the pieces were studied: (i) calcium ionic concentration in SBF and pH by ionselective electrode technique using Ilyte Na+ , K+ , Ca2+ , pH system, and (ii) silicon and phosphorous for complex formation and UV–Vis spectroscopy in Unicam UV–Visible spectrometer. XRD patterns were acquired in a Philips X’Pert MPD (Cu Kα radiation) diffractometer in the 2θ range of 1–10◦ . TGA measurements were carried out between 303 and 1173 K in air (flow rate 100 ml/min with heating rate of 283 K/min) using a Perkin Elmer instrument. Surface area was determined by N2 adsorption (Barret–Joyner–Halenda method) using a Micromeritics ASAP 2010 porosimeter [20]. The pore size was calculated according to Barret–Joyner–Halenda [21–23] and Kruk–Jaroniec–Sayari approach [23] methods. FTIR spectra were obtained in a Nicolet Nexus spectrometer equipped with a Smart Golden Gate ATR accessory. Elemental analysis was performed in a Macroanalyser Leco CNS-2000-I. TEM was carried out on a JEOL JEM-3000F microscope equipped with an ISIS 300 X-ray microanalysis system (Oxford Instruments) with a LINK “Pentafet” detector for energy dispersive spectroscopy (EDS). SEM-EDS studies were performed in a JEOL 6400 Electron microscope coupled with a LINK AN 10000 system.

3. Results and discussion Small-angle XRD experiments of SBA-15 and MCM41 show well-resolved patterns with (100), (110) and (200) reflections, characteristics of the hexagonal structure of silica SBA-15 and MCM-41 with d(100) spacings of 87 and 37 Å, respectively. The pattern corresponding to MCM-48 can be indexed on the basis of a cubic mesoporous structure (Ia3d symmetry) with d(211) spacing of 38 Å. These data are in good agreement with previously reported patterns [16,18,24]. TEM study confirms the above results. Fig. 1 shows a HREM micrograph and its corresponding

Fast Fourier Transform (FFT) of SBA-15 sample, where the hexagonal symmetry of the pore size distribution can be observed. Low-temperature nitrogen adsorption isotherms (Fig. 2) were performed to calculate the specific surface area, pore volume, and mesopore size distribution, confirming the existence of uniform mesopores with size of 8.8, 3.6 and 3.6 nm, respectively. These and other textural parameters are summarized in Table 1. It is worth mentioning that SBA15, as confirmed by the t-plot analysis, shows micropores in its structure with a volume of 0.061 cm3 /g. Silanol groups concentration (mmol·m−2 ) of the mesoporous materials were determined by TGA (Table 1). Similar results for SBA-15 and MCM-48 materials are found, around 13×10−3 mmol SiOH·m−2 . However, a lower value, 2 × 10−3 mmol SiOH·m−2 , is obtained for MCM-41. The relative variation on the Ca, P and Si concentration in SBF with the soaking time, for each sample, is depicted in Fig. 3. A clear decrease on the Ca and P concentration in SBF of SBA-15 and MCM-48 samples is observed. In the case of MCM-41, the Ca and P content remains constant. Simultaneously, there is an increase on the Si concentration for the three samples, being greater for MCM-41. In the case of SBA-15, Si concentration increases until 30 days after starting the assay and is then maintained. Meanwhile, the pH values (data not shown) increase from initial values of 7.4 up to 7.8 in all the samples. FTIR spectra (Fig. 4) indicate changes at the surface composition of the mesoporous after soaking in SBF for 15, 30 and 60 days. Before treatment, the absorption bands characteristic of silicates, at the wavenumbers 1085, 802, 960 and 472 cm−1 , appear. The most intense band at 1085 cm−1 corresponds to the vibrational mode of asymmetric stretch Si–O–Si; the band at 802 cm−1 to the symmetric stretch Si– O and the strong band at 472 cm−1 to the vibrational mode of bending Si–O–Si. The band at 960 cm−1 can be assigned to a stretching vibration of Si–OH group. Surface modifications of SBA-15 and MCM-48 become more apparent after 30 and 60 days in SBF, respectively, due to the appearance of new vibration bands characteristic of phosphate, at 1050, 955, 604, 562 cm−1 , and carbonate, bands at 1490, 1426, and 872 cm−1 , groups. In addition, the band corresponding to SiOH groups, at 960 cm−1 , disappears. These FTIR spectra are analogous to those of bone hydroxycarbonate apatite [4,25], suggesting the formation of an apatite-like layer at the mesoporous surface. For MCM41, no changes at the surface even after 60 days in SBF are observed. These results fit well with the compositional changes observed in SBF. SEM micrographs and EDS spectra of SBA-15 and MCM-48, before and after different soaking times (15, 30 and 60 days) in SBF, are shown in Fig. 5. Homogeneous surfaces involving only oxygen and silicon (see EDS spectra) can be observed before soaking. Similar results were obtained for MCM-41. After 15 days in SBF, changes on the surface are observed probably due to partial dissolution

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Fig. 1. SBA-15 (a) HREM image and (b) corresponding FFT. Table 1 Textural and compositional parameters and SiOH concentration of SBA-15, MCM-48 and MCM-41 materials

Fig. 2. Low-temperature nitrogen adsorption isotherms corresponding to (a) SBA-15, (b) MCM-48 and (c) MCM-41.

Matrix

SBET (m2 /g)

Dp (nm)

Vp (cm3 /g)

V micropore (cm3 /g)

mmol SiOH·m−2

MCM-41 MCM-48 SBA-15

1157 1166 787

3.6 3.6 8.8

0.98 1.05 1.05

– – 0.061

2.16 × 10−3 12.86 × 10−3 12.71 × 10−3

of the silica network, in accordance to Si variations in SBF (Fig. 3). In both cases, the EDS spectra show the presence of Si and O while a small signal of calcium appears in the SBA-15 spectrum. This phenomenon has not been observed in MCM-48 until 15 days later. After 30 and 60 days, the micrographs evidence that the surface has been covered with needle-type crystal aggregates

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Fig. 3. Variation of the Ca, P, and Si concentration in SBF versus the soaking time.

Fig. 4. FTIR spectra of the mesoporous materials at different soaking times. The presence of phosphate and carbonate groups is evident after 30 and 60 days in SBF for SBA-15 and MCM-48, respectively.

forming spherical particles. The number and size of these particles increase with the soaking time. The EDS analysis confirms the presence of Ca and P on the surface. In the case of MCM-41, Ca and P have not been detected, in agreement with previous analysis and FTIR. Fig. 6(a) shows a low magnification image, corresponding to SBA-15 sample, after 60 days, in which a needle-like particle appears over an amorphous matrix. The crystalline and amorphous nature of the particle and matrix, respectively, are clearly seen in the corresponding higher magnification image (Fig. 6(b)). Moreover, EDS analysis indicates compositional differences. The amorphous area (marked as A in Fig. 6(b)) contains Si while the crystalline one comprises Ca and P. The presence of Si can be understood on the basis of the original SBA-15 sample which decomposes under the electron beam. Distances of 0.8 and 0.54 nm in B area (Fig. 6(c)) are in agreement with the hydroxyapatite ¯ zone axis, as confirmed by the cor[26] structure along [011] responding FFT (Fig. 6(d)). The Ca/P ratio is 1.42, within the limits of Ca deficiency that apatite structure admits. The

combination of these results evidences the formation of an apatite-like structure on the mesoporous surface after soaking in SBF 60 days. HREM study for MCM-48 sample shows, again, the presence of crystalline particles as depicted in Fig. 7(a). Distances between bright contrast dots at the image, as well as the FFT (Fig. 7(b)) are in agreement with the hydroxyapatite unit cell. The Ca/P ratio, as determined, by EDS is 1.47, in accordance with a calcium deficient apatite. The above results show that SBA-15 exhibits in vitro bioactivity because, after 60 days in SBF at 37 ◦ C, its surface was coated with a layer of crystallites with apatite-like structure. Similar results were obtained for MCM-48 after 60 days of soaking in SBF, i.e., an apatite layer is formed as evidenced by the above experimental data. It is then clear that the material is bioactive although it exhibits slower growth kinetics than SBA-15. A different situation has been found for MCM-41 because there was no evidence of apatite formation after soaking for two months. This apparent controversy can be understood on the basis of the textural and

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Fig. 5. SEM micrographs of SBA-15 and MCM-48 at (a) 0, (b) 15, (c) 30 and (d) 60 days in SBF. EDS spectra are shown as inset in each micrograph.

chemical properties of the three materials. The three mesoporous materials studied show differences on the pore size and volume, as summarized in Table 1. It can be observed

that SBA-15 has the largest pore diameter, 8.8 nm against 3.6 nm exhibited by MCM-48 and MCM-41, showing, in addition, micropores that are not present in the other materi-

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Fig. 6. TEM micrographs of a particle formed in SBA-15 after 60 days in SBF. (a) Low magnification image, (b) HREM image showing different compositional and structural areas, marked as A and B, (c) Enlarged image of A area (d) corresponding FFT.

Fig. 7. (a) HREM image of a particle formed in MCM-48 after 60 days in SBF and (b) corresponding FFT.

als. These facts justify the faster apatite formation detected in SBA-15 with respect MCM-48 and MCM-41. Furthermore, since these two substrates have the same pore diameter, a similar ratio would be expected for both. However, there is an enormous difference on the behavior exhibited by MCM-48 and MCM-41 after 60 days of treatment; MCM-48 develops the apatite layer, which does not appear in MCM41. It is clear that to solve this apparent contradiction other parameters must also be taken into account. Indeed, it has been shown that silanol groups behave as nucleation sites for the apatite formation. To evaluate this parameter, the surface silanol concentration was determined (Table 1) by TGA

measurements. The highest value, 13 × 10−3 mmol m−2 , is obtained for SBA-15 and MCM-48 being one order of magnitude higher than the one corresponding to MCM-41. These data can explain the difference on bioactivity for a similar pore size. Although, probably, less significant, it is worth mentioning that despite the similar pore size of both substrates, the slightly larger pore volume exhibited by MCM48, with respect MCM-41 (Table 1) could also contribute to the observed differences. According to the above results it can be concluded that SBA-15 and MCM-48 are bioactive materials, since they develop an apatite layer after being treated in SBF. The

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proven ability of mesoporous materials [11–15] to act as drug-delivery systems, combined with their potential bioactivity, both process exhibiting different kinetics, constitutes an attractive combination of properties that may play an important role on the development and performance of biocomposite materials able to control specific biological applications. These biocompatible and bioactive mesoporous materials will favor the cellular growth and bone regeneration being useful for building macroporous device to be applied in tissue engineering. The material mesoporosity will enhance the tissue oxygenation and also the possibility to introduce different drugs for controlled release. Designing these macroporous scaffolds must have into account that the pore size must be large enough with a high pore interconnection in order to deal with cellular moieties and vascular intergrowth. Nowadays, there are a lot of manufacturing techniques that allow obtaining cellular solids of these characteristics.

Acknowledgements Financial support of CICYT, Spain, through research project MAT02-00025 is acknowledged. We also thank A. Rodríguez (Electron Microscopy Center, Complutense University) and F. Conde (C.A.I. X-Ray Diffraction Centre, Complutense University), for valuable technical and professional assistance.

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