**3. Scaffold fabrication**

During the past two decades, biomedical research has advanced extensively to develop potentially applicable scaffolds. Several methods are used decades ago to manufacture these porous structures.

The solvent-casting particulate leaching (SCPL) technique is a standard method to produce polymer-based scaffolds. A polymer is dissolved in an organic solvent that contains mainly salts, with specific dimensions. Then, the mixture is shaped into a three-dimensional mold to produce a scaffold. Thus, when the solvent is removed by simple evaporation, it creates a structure of composite material consisting of the particles together with the polymer. At the end, particles are dissolved in a bath leaving behind a porous structure. In this way, Sola et al. fabricated

innovative 3D porous structures to mimic the bone marrow niche in vitro using polymethyl methacrylate (PMMA) and a flexible polyurethane (PU) and NaCl, as an efficient porogen [16].

The preparation of porous structures from a thermoplastic polymer melt is a convenient route because of the production of scaffolds of many shapes and sizes reproducibly with the use of molds and without involving any solvents. These techniques typically include compression molding, extrusion, and injection molding. Scaffolds of any desired shape could be created by simply changing the mold to use for clinical applications. Moreover, various solid fillers as bioactive molecules could be employed as additives. However, the use of high molding temperatures could degrade and inactivate the biodegradable polymer or the impregnated bioactive molecules [17].

In freeze-gelation method, the porous structure is generated during the freeze of a polymer solution, and then the solvent is extracted by a non-solvent, or the polymer is gelled under the freezing condition. Thus, the porous structure destruction would be avoided during the subsequent drying stage. Porous PLLA, PLGA, chitosan, and alginate scaffolds were successfully fabricated with this method [18].

Various porous biodegradable scaffolds with these polymers have been also fabricated by thermally induced phase separation (TIPS) technique to be used in tissue engineering and drug delivery [19–20]. This technique is based on changes in Gibbs free energy to induce the demixing of a homogeneous polymer solution to obtain a multiphase system [21]. Highly porous scaffolds of biodegradable PCL have been fabricated by this method [22]. Even PLLA scaffolds with hydroxyapatite as filler were successfully fabricated by Ghersi et al. [23].

Three-dimensional printing (3DP) is another method to produce scaffolds for tissue engineering. In 3DP method the solid is created by the reaction of a liquid selectively sprayed onto a powder bed. This is a versatile method to produce scaffolds for tissue engineering [24].

In robocasting process the object is built by printing the required shape layer by layer. For that, a filament of a paste-like material is extruded from a small nozzle while the nozzle is moved across a platform. Many authors have combined this method with sol–gel synthesis, mixing precursors in an aqueous medium. The resulting gels are used to print scaffolds by robocasting. Houmard et al. fabricated in this way highly porous calcium phosphate (CaP) scaffolds for bone-tissue engineering using calcium nitrate tetrahydrate and triethyl phosphite precursors [25].

Besides, several reports on the fabrication of porous scaffolds using sol-gel technique are found in the literature [26–32].

Scaffold could be fabricated using supercritical CO2 as blowing agent, avoiding the use of organic solvent, and thus the presence of solvent residue in the final product due to tradition processes tested until now does not allow the complete removal of the organic solvents involved in the starting solutions, avoiding high temperatures and long processing time (12–48 h) that can imply the stratification of the drug inside the scaffolds due to the separation of the loaded materials from the polymeric solutions. In this way, the efficiency of the generated devices sensibly decreases due to the inhomogeneous distribution of the drug [33].

In supercritical CO2 foaming, the polymer is exposed to carbon dioxide, which plasticizes the polymer by reducing the glass transition temperature. Then in the depressurization step, thermodynamic instability causes supersaturation of the carbon dioxide dissolved in the polymer matrix, and hence, nucleation of cells occurs. Anyway gas foaming could not be used in polymers which have a very low affinity for CO2 because the main requirement of process is that CO2 can be dissolved in a sufficient amount in the polymer. So this technique is more commonly

**199**

*Foaming + Impregnation One-Step Process Using Supercritical CO2*

processing could be used for cartilage tissue engineering [35].

applied to amorphous polymers excluding polymers with high crystallinity or high

In this way, for instance, the PCL scaffolds produced by supercritical fluid processing had a homogeneously interconnected porous structure, and they exhibited a narrow pore size distribution. Consequently, these results indicated that the PCL scaffolds made by supercritical fluid processing offer well-interconnected and nontoxic substrates for cell growth, avoiding problems associated with a solvent residue. This suggests that these elastic PCL scaffolds formed by supercritical fluid

Other authors used supercritical CO2 in the foaming process for the formation of polyvinylidene fluoride copolymerized with hexafluoropropylene loaded scaffolds which is a material that is semicrystalline and biocompatible with a good resistance to acid environments. They concluded that a higher pressure, a lower temperature, and a longer saturation time were more favorable for obtaining uniform foam. Moreover, the average pore cell diameter in low depressurization is larger than that in rapid depressurization. Lower crystallinity and higher melting temperature were

Supercritical CO2 is used sometimes as dryer to prepare scaffolds. In this way polymer solution is prepared, and then this solution is put in contact with scCO2. In

Tang et al. produced porous PCL scaffolds with open and interconnected architectures based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. They achieved the encapsulation growth factor in these porous scaffolds, promoting the osteogenic differentiation and thus having also a significant

A scaffold where a bioactive substance can be incorporated that, for instance, can control proliferation and differentiation of cells is an excellent alternative to be used in tissue engineering. In this way the function of a scaffold is not limited only as a physical support but also as a bioactive element to control cell proliferation and differentiation. Anyway, scaffold impregnation process has been mostly studied for the preparation of long time drug delivery systems, with more or less delay depend-

The conventional impregnation of scaffolds uses organic solvents that dissolve the drug which is going to be incorporated into the scaffolds, but this organic solution should swell and stretch the polymer to allow the diffusion of the drug at adequate

that moment CO2 solubilizes the organic solvent and the scaffold is formed. In this sense a chitosan-based scaffold for tissue engineering applications has been prepared using supercritical CO2 as dryer. The hydrogel fabricated was subsequently processed with supercritical CO2. The highest porosity (87.03%) was achieved at 250 bar, 45°C, and 2 h of processing at 5 g/min CO2 flow rate [37]. However other authors investigated about a new supercritical fluid-assisted technique for the formation of 3D scaffolds to overcome the main difficulty to obtain the coexistence of the macro- and microstructural characteristics necessary for a successful application. The process consists of the formation of a polymeric gel loaded with a solid porogen, then the drying of the gel using supercritical CO2, and the washing with water to eliminate the porogen. In this way Reverchon et al. fabricated (PLLA) scaffolds with elevated porosity (>90%) and interconnectivity and with good mechanical properties [38]. Moreover they produced scaffolds with predetermined shape and size in a relatively short time (<30 h) and without an

*DOI: http://dx.doi.org/10.5772/intechopen.91304*

glass transition temperatures [34].

induced in the formed scaffolds [36].

appreciable solvent residue (<5 ppm).

potential in bone tissue engineering [39].

ing on the final purpose of the delivery.

**4. Impregnation**

### *Foaming + Impregnation One-Step Process Using Supercritical CO2 DOI: http://dx.doi.org/10.5772/intechopen.91304*

*Advanced Supercritical Fluids Technologies*

an efficient porogen [16].

molecules [17].

innovative 3D porous structures to mimic the bone marrow niche in vitro using polymethyl methacrylate (PMMA) and a flexible polyurethane (PU) and NaCl, as

The preparation of porous structures from a thermoplastic polymer melt is a convenient route because of the production of scaffolds of many shapes and sizes reproducibly with the use of molds and without involving any solvents. These techniques typically include compression molding, extrusion, and injection molding. Scaffolds of any desired shape could be created by simply changing the mold to use for clinical applications. Moreover, various solid fillers as bioactive molecules could be employed as additives. However, the use of high molding temperatures could degrade and inactivate the biodegradable polymer or the impregnated bioactive

In freeze-gelation method, the porous structure is generated during the freeze of a polymer solution, and then the solvent is extracted by a non-solvent, or the polymer is gelled under the freezing condition. Thus, the porous structure destruction would be avoided during the subsequent drying stage. Porous PLLA, PLGA, chitosan, and alginate scaffolds were successfully fabricated with this method [18]. Various porous biodegradable scaffolds with these polymers have been also fabricated by thermally induced phase separation (TIPS) technique to be used in tissue engineering and drug delivery [19–20]. This technique is based on changes in Gibbs free energy to induce the demixing of a homogeneous polymer solution to obtain a multiphase system [21]. Highly porous scaffolds of biodegradable PCL have been fabricated by this method [22]. Even PLLA scaffolds with hydroxyapatite as

Three-dimensional printing (3DP) is another method to produce scaffolds for tissue engineering. In 3DP method the solid is created by the reaction of a liquid selectively sprayed onto a powder bed. This is a versatile method to produce scaf-

In robocasting process the object is built by printing the required shape layer by layer. For that, a filament of a paste-like material is extruded from a small nozzle while the nozzle is moved across a platform. Many authors have combined this method with sol–gel synthesis, mixing precursors in an aqueous medium. The resulting gels are used to print scaffolds by robocasting. Houmard et al. fabricated in this way highly porous calcium phosphate (CaP) scaffolds for bone-tissue engineering using calcium nitrate tetrahydrate and triethyl phosphite

Besides, several reports on the fabrication of porous scaffolds using sol-gel

Scaffold could be fabricated using supercritical CO2 as blowing agent, avoiding the use of organic solvent, and thus the presence of solvent residue in the final product due to tradition processes tested until now does not allow the complete removal of the organic solvents involved in the starting solutions, avoiding high temperatures and long processing time (12–48 h) that can imply the stratification of the drug inside the scaffolds due to the separation of the loaded materials from the polymeric solutions. In this way, the efficiency of the generated devices sensibly

In supercritical CO2 foaming, the polymer is exposed to carbon dioxide, which plasticizes the polymer by reducing the glass transition temperature. Then in the depressurization step, thermodynamic instability causes supersaturation of the carbon dioxide dissolved in the polymer matrix, and hence, nucleation of cells occurs. Anyway gas foaming could not be used in polymers which have a very low affinity for CO2 because the main requirement of process is that CO2 can be dissolved in a sufficient amount in the polymer. So this technique is more commonly

decreases due to the inhomogeneous distribution of the drug [33].

filler were successfully fabricated by Ghersi et al. [23].

folds for tissue engineering [24].

technique are found in the literature [26–32].

precursors [25].

**198**

applied to amorphous polymers excluding polymers with high crystallinity or high glass transition temperatures [34].

In this way, for instance, the PCL scaffolds produced by supercritical fluid processing had a homogeneously interconnected porous structure, and they exhibited a narrow pore size distribution. Consequently, these results indicated that the PCL scaffolds made by supercritical fluid processing offer well-interconnected and nontoxic substrates for cell growth, avoiding problems associated with a solvent residue. This suggests that these elastic PCL scaffolds formed by supercritical fluid processing could be used for cartilage tissue engineering [35].

Other authors used supercritical CO2 in the foaming process for the formation of polyvinylidene fluoride copolymerized with hexafluoropropylene loaded scaffolds which is a material that is semicrystalline and biocompatible with a good resistance to acid environments. They concluded that a higher pressure, a lower temperature, and a longer saturation time were more favorable for obtaining uniform foam. Moreover, the average pore cell diameter in low depressurization is larger than that in rapid depressurization. Lower crystallinity and higher melting temperature were induced in the formed scaffolds [36].

Supercritical CO2 is used sometimes as dryer to prepare scaffolds. In this way polymer solution is prepared, and then this solution is put in contact with scCO2. In that moment CO2 solubilizes the organic solvent and the scaffold is formed.

In this sense a chitosan-based scaffold for tissue engineering applications has been prepared using supercritical CO2 as dryer. The hydrogel fabricated was subsequently processed with supercritical CO2. The highest porosity (87.03%) was achieved at 250 bar, 45°C, and 2 h of processing at 5 g/min CO2 flow rate [37].

However other authors investigated about a new supercritical fluid-assisted technique for the formation of 3D scaffolds to overcome the main difficulty to obtain the coexistence of the macro- and microstructural characteristics necessary for a successful application. The process consists of the formation of a polymeric gel loaded with a solid porogen, then the drying of the gel using supercritical CO2, and the washing with water to eliminate the porogen. In this way Reverchon et al. fabricated (PLLA) scaffolds with elevated porosity (>90%) and interconnectivity and with good mechanical properties [38]. Moreover they produced scaffolds with predetermined shape and size in a relatively short time (<30 h) and without an appreciable solvent residue (<5 ppm).

Tang et al. produced porous PCL scaffolds with open and interconnected architectures based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. They achieved the encapsulation growth factor in these porous scaffolds, promoting the osteogenic differentiation and thus having also a significant potential in bone tissue engineering [39].
