**4.2 Biological characterisation**

The hybrid materials were soaked in SBF, as indicated by Ohtsuki et al. (1992), for in vitro bioactivity tests. The FTIR spectra after several exposures to SBF, 7, 14 and 21 days are shown in Fig. 15b, 15c and 15d. Evidence of the formation of an hydroxyapatite layer is given by the appearance of the 1116 and 1035 cm-1 bands, usually assigned to P-O stretching (Teoli et al., 2006) and of the 580 cm-1 band usually assigned to the P-O bending mode (Teoli et al., 2006). The splitting, already after a 7 day soaking, of the 580 cm-1 band into two others at 610 and 570 cm-1 can be attributed to formation of crystalline hydroxyapatite (Ohtsuki et al.). Finally the band at 800 cm-1 can be assigned to the Si-O-Si band vibration between two adjacent tetrahedral, characteristic of silica gel (Teoli et al., 2006). These considerations support the hypothesis that a surface layer of silica gel forms as supposed in the mechanism proposed in the literature for hydroxyapatite deposition (Allen et al., 2000; Khor et al., 2002). Moreover an evaluation of the morphology of the apatite deposition and a qualitative elemental analysis were also carried out by electron microscopy observations on pelletized discs previously coated with a thin Au film. The EDS reported in Tab. 5 confirm that the surface layer observed in the SEM micrographs (Fig. 16) consists of calcium phosphate and which increases as the PCL.


Table 5. The EDS analyses of hybrid materials 21 days after immersion in SBF.

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 organic-

The hybrid materials were soaked in SBF, as indicated by Ohtsuki et al. (1992), for in vitro bioactivity tests. The FTIR spectra after several exposures to SBF, 7, 14 and 21 days are shown in Fig. 15b, 15c and 15d. Evidence of the formation of an hydroxyapatite layer is given by the appearance of the 1116 and 1035 cm-1 bands, usually assigned to P-O stretching (Teoli et al., 2006) and of the 580 cm-1 band usually assigned to the P-O bending mode (Teoli et al., 2006). The splitting, already after a 7 day soaking, of the 580 cm-1 band into two others at 610 and 570 cm-1 can be attributed to formation of crystalline hydroxyapatite (Ohtsuki et al.). Finally the band at 800 cm-1 can be assigned to the Si-O-Si band vibration between two adjacent tetrahedral, characteristic of silica gel (Teoli et al., 2006). These considerations support the hypothesis that a surface layer of silica gel forms as supposed in the mechanism proposed in the literature for hydroxyapatite deposition (Allen et al., 2000; Khor et al., 2002). Moreover an evaluation of the morphology of the apatite deposition and a qualitative elemental analysis were also carried out by electron microscopy observations on pelletized discs previously coated with a thin Au film. The EDS reported in Tab. 5 confirm that the surface layer observed in the SEM

micrographs (Fig. 16) consists of calcium phosphate and which increases as the PCL.

SiO2 + 0%PCL + Ketoprofen 2.65 1.64 SiO2 + 6%PCL + Ketoprofen 3.13 1.94 SiO2 + 12%PCL + Ketoprofen 5.04 3.23 SiO2 + 50%PCL + Ketoprofen 7.82 4.86

**Atomic %**

**Contents of P Atomic %** 

**Materials soaked in SBF for 21 days Contents of Ca**

Table 5. The EDS analyses of hybrid materials 21 days after immersion in SBF.

inorganic hybrid materials as suggested by literature data (Hench & Clark, 1978).

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

**4.2 Biological characterisation** 

Fig. 15. FTIR spectra of SiO2/PCL gel samples after different times of exposure to SBF: (a) not exposed; (b) 7 days exposed;(c) 14 days exposed; (d) 21 days exposed.

Fig. 16. SEM micrograph of SiO2/PCL gel after being exposed to SBF 21 days.

### **4.3 Release kinetic characterization**

Kinetic measurements of release from the studied materials were carried out in 15 ml of SBF incubated at 37±0.1°C and under continuous magnetic stirring at 150 rpm. Sink conditions were maintained throughout all studies. The discs used were obtained with particle size between 63-125µm compressed at 3 tons and aliquots of 600 µl were withdrawn at 1 h interval and replaced with an equal volume of release medium pre-equilibrated to temperature. Release was essayed by measuring the photometrical absorbance at 259.5 nm. In order to establish the relationship between the UV absorbance of at λ = 259.5 nm and the concentration of the solutions a calibration curve (r2 =0.9907) was drawn for a standard solution with 4 levels of concentration: 0.0 mM, 1.0 mM, 2.0 mM and 3.0 mM (Fig. 17). All the standard solutions were prepared in SBF.

Fig. 18a, 19a, 20a and 18b, 19b, 20b show the drug release rates expressed as a percentage of the drug delivered, related to the drug-loading value, as a function of time. It was observed that from the SiO2+PCL (0, 6, 12, 50 wt%)+ ketoprofen 5wt% gels about 60wt% of the drug was released in a relatively fast manner during the initial 2 hrs and it seems to be completed within 7 hrs without any evident difference in the time of release. For the SiO2+PCL(0, 6, 12, 50 wt%)+ ketoprofen 10 and 15wt % gels about 60wt% of the drug was released during the initial 1 hr and 0,5 hr respectively and it is complete in about 3 hr and 4 hr respectively.

The differences observed in the release behaviour between SiO2+ PCL (0, 6, 12, and 50 wt%) + ketoprofen might be due to the different networks of the four gels that are determined by the different content percentage of PCL. The two stage release observed in all cases suggests that the initial stage of release occurs mainly by dissolution and diffusion of the drug entrapped close to or at the surface of the samples. The second and slower release stage involves the diffusion of the drug entrapped within the inner part of the clusters. An interesting observation is the general presence of an early lag period, which indicates the need for the penetration of the solvent into the structure. Fig. 18b, 19b and 20b show this particular kinetic describing the changes of the release speed during the two stages.

Fig. 17. Calibration curve (259.5 nm) depending on the concentration of Ketoprofen.

Fig. 18. (a) Time-dependent drug release plot for SiO2 + PCL (0, 6, 12, 50%wt) + ketoprofen 5% at 37°C in SBF solution; (b) Time-dependent drug release rate plot for SiO2 + PCL (0, 6, 12, 50%wt) + ketoprofen 5% at 37°C in SBF solution.

Fig. 19. (a) Time-dependent drug release plot for SiO2 + PCL (0, 6, 12, 50%wt) + ketoprofen 10% at 37°C in SBF solution; (b) Time-dependent drug release rate plot for SiO2 + PCL (0, 6, 12, 50%wt) + ketoprofen 10% at 37°C in SBF solution.

Fig. 20. (a) Time-dependent drug release plot for SiO2 + PCL (0, 6, 12, 50%wt) + ketoprofen 15% at 37°C in SBF solution; (b) Time-dependent drug release rate plot for SiO2 + PCL (0, 6, 12, 50%wt) + ketoprofen 15% at 37°C in SBF solution.

## **5. Applications**

408 Biomedical Science, Engineering and Technology

Kinetic measurements of release from the studied materials were carried out in 15 ml of SBF incubated at 37±0.1°C and under continuous magnetic stirring at 150 rpm. Sink conditions were maintained throughout all studies. The discs used were obtained with particle size between 63-125µm compressed at 3 tons and aliquots of 600 µl were withdrawn at 1 h interval and replaced with an equal volume of release medium pre-equilibrated to temperature. Release was essayed by measuring the photometrical absorbance at 259.5 nm. In order to establish the relationship between the UV absorbance of at λ = 259.5 nm and the concentration of the solutions a calibration curve (r2 =0.9907) was drawn for a standard solution with 4 levels of concentration: 0.0 mM, 1.0 mM, 2.0 mM and 3.0 mM (Fig. 17). All

Fig. 18a, 19a, 20a and 18b, 19b, 20b show the drug release rates expressed as a percentage of the drug delivered, related to the drug-loading value, as a function of time. It was observed that from the SiO2+PCL (0, 6, 12, 50 wt%)+ ketoprofen 5wt% gels about 60wt% of the drug was released in a relatively fast manner during the initial 2 hrs and it seems to be completed within 7 hrs without any evident difference in the time of release. For the SiO2+PCL(0, 6, 12, 50 wt%)+ ketoprofen 10 and 15wt % gels about 60wt% of the drug was released during the initial 1 hr and 0,5 hr respectively and it is complete in about 3 hr and 4 hr respectively. The differences observed in the release behaviour between SiO2+ PCL (0, 6, 12, and 50 wt%) + ketoprofen might be due to the different networks of the four gels that are determined by the different content percentage of PCL. The two stage release observed in all cases suggests that the initial stage of release occurs mainly by dissolution and diffusion of the drug entrapped close to or at the surface of the samples. The second and slower release stage involves the diffusion of the drug entrapped within the inner part of the clusters. An interesting observation is the general presence of an early lag period, which indicates the need for the penetration of the solvent into the structure. Fig. 18b, 19b and 20b show this particular kinetic describing the changes of the release speed during

Fig. 17. Calibration curve (259.5 nm) depending on the concentration of Ketoprofen.

**4.3 Release kinetic characterization** 

the standard solutions were prepared in SBF.

the two stages.

Applications for sol-gel processing derive from the various special shapes obtained directly from the gel state (e.g. monoliths, films, fibers, and monisized powders) combined with composition and microstructural control and low processing temperatures. Compared to conventional sources of ceramic raw materials, often minerals dug from the earth, synthetic chemical precursors are a uniform and reproducible source of raw materials than can be made extremely pure through various synthetic means. Low processing temperatures, which result from microstructural control (e.g. high surface areas and small pore sizes), expand glass-forming regions by avoiding crystallization or phase separation, making new materials available to the technologist. The advantages of the sol-gel process (for preparing glass) are shown in Tab. 6 (Brinker & Scherer 1990). The disadvantages of sol-gel processing include the cost of the raw materials, shrinkage that accompanies drying and sintering, and processing time, as it is shown in Tab 7.


Table 6. Some advantages of the Sol-Gel Methods over Conventional Melting for Glass


Table 7. Some disadvantages of the Sol-Gel Methods

There are books (Klein, 1988) and a number of review papers (Dislich, 1986; Johnson, 1985; Klein & Garvey, 1982; Mackenzie, 1988; Uhlmann et al., 1984; Ulrich, 1988a) which discuss this topic in detail and whose primary purpose is to provide a source of references to current technology, and at the same time to analyze critical issues associated with the various classes of applications that must be addressed in order to advance the sol-gel technology; for example, a short outline can be as follows:


In recent years interest in bioactive and biocompatibility of surface-active, biomaterials has grown.

Biomaterials have been used extensively in medical, personal care and food applications, with many similar polymers being used across disciplines. This perspective will emphasize hybrid materials used in medicine and specifically those designed as scaffolds for use in tissue engineering and regenerative medicine. The areas of active research in tissue engineering include: biomaterials design (incorporation of the appropriate chemical, physical, and mechanical/structural properties to guide cell and tissue organization); cell/scaffold integration (inclusion into the biomaterial scaffold of either cells for transplantation or biomolecules to attract cells, including stem cells, from the host to promote integration with the tissue after implantation); and biomolecule delivery (inclusion of growth factors and/or small molecules or peptides that promote cell survival and tissue regeneration). While a significant and growing area of regenerative medicine involves the stimulation of endogenous stem cells, this perspective will emphasize hybrid materials scaffolds used for delivery of cells and biomolecules. The challenges and solutions pursued in designing polymeric biomaterial scaffolds with the appropriate 3-dimensional structure are curently studied.

Ceramics and hybrid dioxide-based materials for the repairing of muscle–skeletal tissues are being increasingly applied over the last half century (Hench, 1991; Li, et al., 1996). Orthopaedic and maxillo–facial prosthesis provide evidence for the enhanced biomechanical performance of titanium and its alloys among metallic prosthetic components (Kitsugi et al., 1996). TiO2-based bioactive ceramic suggests that bone grafting is achieved by supporting the precipitation of calcium (Ca) and phosphorus (P) into a structure similar to the mineral phase of bone. Accordingly, titanium is very promising to develop biomedical materials and devices designed as hard tissue substitutes with improved interface properties (Coreno & Coreno, et al. 2005; Hench, 1991; Li, et al., 1996; Kitsugi et al., 1996).
