**3.2 Study of in vitro bioactivity**

400 Biomedical Science, Engineering and Technology

SiO2 + 0%PCL + 5%Ketoprofen 21 SiO2 + 6%PCL + 5%Ketoprofen 16 SiO2 + 12%PCL + 5%Ketoprofen 14 SiO2 + 50%PCL + 5%Ketoprofen 8 SiO2 + 0%PCL + 10%Ketoprofen 19 SiO2 + 6%PCL + 10%Ketoprofen 15 SiO2 + 12%PCL + 10%Ketoprofen 12 SiO2 + 50%PCL + 10%Ketoprofen 9 SiO2 + 0%PCL + 15%Ketoprofen 18 SiO2 + 6%PCL + 15%Ketoprofen 14 SiO2 + 12%PCL + 15%Ketoprofen 12 SiO2 + 50%PCL + 15%Ketoprofen 10

Table 4. Variation in the gelification time, controlled by changing the concentration of PCL.

Chromatographic experiments were carried out on a Shimadzu HPLC system, equipped with a Class-VP 5.0 software, an UV spectrophotometric detector SPD-10AVvp and two pumps LC-10ADvp, with low-pressure gradient systems. Samples of solutions were injected by a syringe via a Rheodyne loop injector; the loop volume was 20 μl, the analytical column was a Phenomenex C18 (150 × 4.60 mm; 5 μ); the flow rate of the mobile phase A (water) was set at 0.8 ml/min and that of the mobile phase B (methanol) was set at 0.2 ml/min. The total runtime was 10 minutes. HPLC grade methanol was obtained by Sigma-Aldrich. HPLC grade water was prepared using a Millipore (0.22 μm) system. A standard solution of ketoprofen 3 mM in a simulated body fluid (SBF) was prepared and the samples were taken

The nature of SiO2 gel, poly-ε-caprolactone (PCL) and PCL/SiO2 hybrid materials were ascertained by X-ray diffraction (XRD) analysis using a Philips diffractometer. The presence of hydrogen bonds between organic-inorganic components of the hybrid materials was ascertained by FTIR analysis. Fourier transform infrared (FTIR) transmittance spectra were recorded in the 400-4000 cm-1 region using a Prestige 21 Shimatdzu system, equipped with a DTGS KBr (Deuterated Tryglycine Sulphate with potassium bromide windows) detector, with resolution of 2 cm-1 (45 scans). KBr pelletized

Fig. 8. SiO2/PCL gel after drying.

at the end of the release from the materials.

**Materials prepared Gelation time (20-25°C)** 

In order to study their bioactivity, samples of the studied hybrid materials were soaked in a simulated body fluid (SBF) with ion concentrations nearly equal to those of the human blood plasma, as reported elsewhere and shown in Tab. 5 (Hench & Clark, 1978; Ohtsuki et al., 1992; Paul, 1992). During soaking, the temperature was kept fixed at 37°C. Taking into account that the ratio of the exposed surface to the volume solution influences the reaction, a constant ratio of 50 mm2 ml-1 of solution was respected (Hutmacher et al., 2001).


Table 5. Simulated body fluid (SBF) ionic concentration (mM).

It was shown that SBF reproduces *in vivo* bonelike apatite formation on bioactive glass and ceramic (Hench, 1991). *In vitro* studies using SBF have suggested that bioactive glass and glass-ceramics form bonelike apatite on their surface (see Fig. 9) by providing surface functional groups of silanol (Si—OH), which are effective for apatite nucleation. These groups combine with Ca2+ ions present in the fluid imposing an increase of positive charge on the surface. In addition, Ca2+ ions combine with the negative charge of the phosphate ions to form amorphous phosphate, which spontaneously transforms into hydroxyl-apatite [Ca10(PO4)6(OH)2] where the atomic ratio Ca/P is 1.60 (Ohtsuki et al., 1992). The SBF is already supersaturated with respect to the apatite under normal conditions. Once the apatite nuclei are formed, they can grow spontaneously by consuming the calcium and phosphate ions from the surrounding body fluid. It is known from literature (Hench, 1991; Ohtsuki et al., 1992) that CaO, SiO2-based glasses, CaO, P2O5- based glasses, sodium silicate glasses are more bioactive then ion free glass and ceramic. That is due to the dissolution of appreciable amounts of calcium and phosphate ions, which increase the degree of supersaturation of the surrounding body fluid with respect to the apatite. Moreover, the released ions are exchanged with H3O+ ions in SBF forming silanol groups on their surface; this reaction causes a pH increase of SBF solution and, consequently, Si–OH groups are dissociated into negatively charged units Si–O- that interacts with the positively charged calcium ions to form the calcium silicate.

Fig. 9. Apatite layer on SiO2/PCL surface.

### **3.3 Study of in-vitro release**

For the study of drug release, the discs of the investigated material were soaked in 15 ml of SBF, continuously stirred, at 37°C. The SBF was previously filtered with a Millipore (0.22 µm) system, to avoid any bacterial contamination. Drug release measurements were carried out by means of UV-VIS spectroscopy. Absorbance values were taken at a wavelength λ corresponding to an absorbance maximum value. The calibration curve was determined by taking absorbance versus drug concentration between 0 and 3 mM as parameters. For that interval the calibration curve fits the Lambert and Beers' law (Wang & Pantano, 1992):

$$A = \mathbf{1}, \mathbf{2} \mathbf{6} \cdot \mathbf{C} \tag{8}$$

where A is the absorbance and C is the concentration (mM).

### **4. Characterization**

### **4.1 Sol-gel characterization**

Gelification is the result of hydrolysis and condensation reactions according to the following reactions:

$$\text{Si(OCH}\_3\text{)}\_4 + \text{ nH}\_2\text{O} \quad \Rightarrow \text{ Si(OCH}\_3\text{)}\_{4\text{ }n}\text{(OH)}\_n + \text{ nCH}\_3\text{OH} \tag{9}$$

$$\text{\textbullet -SiOH} \quad \text{\textbullet + CH}\\ \text{\textbullet -Si-Si-Si-} \quad \text{\textbullet -Si-Si-}\\ \text{\textbullet +Si-Si-} \quad \text{\textbullet +Si-Si-}$$

$$\text{\textbullet \text{-}Si-OH} \quad \text{\textbullet \text{ } OH\text{-}Si} \quad \Rightarrow \quad \quad \text{\textbullet \text{-}Si-O-Si} \quad \quad \quad \text{\textbullet \text{ } H\text{-}O} \tag{11}$$

Reaction 12 shows the formation of hydrogen bonds between the carbossilic group of organic polymer and the hydroxyl group of inorganic matrix.

For the study of drug release, the discs of the investigated material were soaked in 15 ml of SBF, continuously stirred, at 37°C. The SBF was previously filtered with a Millipore (0.22 µm) system, to avoid any bacterial contamination. Drug release measurements were carried out by means of UV-VIS spectroscopy. Absorbance values were taken at a wavelength λ corresponding to an absorbance maximum value. The calibration curve was determined by taking absorbance versus drug concentration between 0 and 3 mM as parameters. For that interval the calibration curve fits the Lambert and Beers' law (Wang

Gelification is the result of hydrolysis and condensation reactions according to the following

Si(OCH3)4 + nH2O ⇒ Si(OCH3)4-n (OH)n + nCH3OH (9)

Reaction 12 shows the formation of hydrogen bonds between the carbossilic group of



ܣ ൌ ͳǡʹ ή ܥ) 8 (

Fig. 9. Apatite layer on SiO2/PCL surface.

where A is the absorbance and C is the concentration (mM).

organic polymer and the hydroxyl group of inorganic matrix.

**3.3 Study of in-vitro release** 

& Pantano, 1992):

**4. Characterization** 

reactions:

**4.1 Sol-gel characterization** 

The existence of hydrogen bonds was proved by FTIR measurements. Fig. 10 shows the infrared spectrum of the SiO2 gel (Fig. 10a), the SiO2/PCL (6, 12, 50%wt) gels (Fig. 10b, 10c, 10d) and PCL (Fig. 10e). In Fig. 10a the bands between 3400 and 1600 cm-1 are attributed to water (Sanchez & Ribot, 1994). The bands at 1080 and 470 cm-1 are due to the stretching and bending modes of SiO4 tetrahedra (Sanchez & Ribot, 1994). In the Fig. 10b, 10c, 10d and 10e, the bands at 2928 and 2840 cm-1 are attributed to a symmetric stretching of -CH2- of policaprolattone. The band at 1730 cm-1 is due to the characteristic carboxylic group shifted to low wave numbers. The broad band at 3200 cm-1 is the characteristic O-H group of hydrogen bonds.

Fig. 10. FTIR of (a) SiO2 gel, (b) SiO2+ PCL 6wt% (c) SiO2+ PCL 12wt% (d) SiO2+ PCL 50wt% gels and (e) PCL.

The nature and the microstructure of the hybrid materials have been studied by X-ray diffraction (XRD), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The diffractograms in Fig. 11a show that SiO2 gel exhibits the broad humps which are characteristic for amorphous materials, while the sharp peaks that can be detected on the diffractogram of poly-ε-caprolactone and ketoprofen are typical of crystalline materials (Fig. 11b and 11c). On the other hand the XRD spectrum of hybrid SiO2/PCL exhibits the broad humps characteristic of amorphous materials (Fig. 11d), as well that of SiO2 gel.

Fig. 11. XRD diffractogram of (a) SiO2 gel, (b) PCL, (c) Ketoprofen, (d) SiO2/PCL gel.

SEM micrographs show that no appreciable difference can be observed between the morphology of the four amorphous materials. The samples appear as shown in Fig. 12. The degree of mixing of the organic-inorganic components, i. e. the phase homogeneity, has been ascertained by applying the atomic force microscopy (AFM) in the analysis of the solgel hybrid material.

Fig. 12. SEM micrograph of SiO2/PCL gel.

404 Biomedical Science, Engineering and Technology

diffractogram of poly-ε-caprolactone and ketoprofen are typical of crystalline materials (Fig. 11b and 11c). On the other hand the XRD spectrum of hybrid SiO2/PCL exhibits the broad

humps characteristic of amorphous materials (Fig. 11d), as well that of SiO2 gel.

Fig. 11. XRD diffractogram of (a) SiO2 gel, (b) PCL, (c) Ketoprofen, (d) SiO2/PCL gel.

The AFM contact mode image can be measured in the height mode or in the force mode. Force images (z range in nN) have the advantage that they appear sharper and richer in contrast and that the contours of the nanostructure elements are clearer. In contrast, height images (z range in nm) show a more exact reproduction of the height itself. In this work the height mode has been adopted to evaluate the homogeneity degree of the hybrid materials.

Fig. 13. AFM image showing the microstructure of SiO2 gel .

The AFM topographic images of SiO2 and SiO2/PCL (0, 6, 12, 50 wt%) gel samples are shown in Figs. 13 and 14. It can be observed that the average domain size is less than 130 nm. This result confirms that the synthesized PCL/SiO2 gels can be considered organicinorganic hybrid materials as suggested by literature data (Hench & Clark, 1978).

Fig. 14. AFM image showing the microstructure of SiO2/PCL gel.
