**4. Impregnation**

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 depending on the final purpose of the delivery.

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

depressurization rate. Posteriorly the organic solvent should be removed, leaving the impregnated scaffold. This method has several drawbacks, for example, residues of organic solvent in the final product need a last step to dry the scaffold, and the distribution of drug into scaffold is heterogeneous.

SCF impregnation removes all these drawbacks due to its properties as high diffusivity, low surface tension, and the ease of solvent recovery. Nevertheless, this methodology is limited by the solubility of the drug in the SCF, and the polymer can be swollen by the SCF. If these last requirements are fulfilled, a high-quality product free of residual solvents can be obtained, since no organic solvents are involved in process [40, 41]. In this process SCF is put in contact with the active substance that is going to be incorporated into the scaffolds. Then the SCF solubilizes this substance till saturation during the impregnation time. Later, in the depressurization step, the gas rapidly diffuses out of the polymer, deplasticizing it and avoiding the polymers and active substance exposition to high temperatures, which may degrade them.

CO2 is the frequently used SCF because it is not dangerous, not toxic, not flammable, relatively cheap, and classified as a safe solvent. Anyway due to polarity limitations, it is often used as a cosolvent to increase the polarity and selectivity.

Duarte et al. prepared loaded chitosan scaffold able to sustain the release of dexamethasone using supercritical impregnation [42]. Sproule et al. [43] achieved the successful impregnation of a protein in PMMA scaffolds for biomedical applications holding unaltered the protein activity.

Campardelli et al. [9] prepared polycaprolactone/nimesulide patches using supercritical impregnation. In this work the authors achieved the foaming of the polymer in certain conditions at the same time that nimesulide is incorporated into its structure. Thus impregnated scaffolds are prepared in a one-step process for determined conditions. However in other operating conditions, foaming of the polymer is not favored, and scaffolds were not achieved.

Biodegradable PLA/PLGA foams impregnated with indomethacin in scCO2 were studied by Cabezas et al. [44]. Authors observed that drug loading of foams was favored by high values of stirring rate. Moreover little pore sizes were obtained at slow depressurization rates. As it was expected, composition influenced the mechanical resistance, the PLA foams being more fragile.

Fanovich et al. studied the functionalized PCL scaffolds impregnated with natural compounds extracted from Patagonian *Usnea* lichen for tissue engineering [45]. An integrated process at high pressure for extraction/impregnation/foaming of PCL was developed. Authors concluded that the process is successful at 35°C and 15–17 MPa of CO2 by foaming. The same researchers incorporated in a posterior work hydroxyapatite to these scaffolds, concluding that the scaffold obtained from PCL-HA with 20% of the HA shows the highest impregnation yield at 17 MPa and 35°C and subsequently also the best bactericidal effect on the tested *Staphylococcus aureus* strains [46].

The impregnation of chitosan with lactulose using supercritical fluids under various operating conditions, in order to improve the solubility of this natural polymer at neutral or basic pH, was carried out by Diaz et al. The highest impregnation yield was obtained using continuous process, 60-min contact time, 14% (v/v) of cosolvent ethanol/water (95:5), depressurization rate equal to 3.3 bar/min, 100 bar of pressure, and 100°C [47].

The impregnation of 5-fluorouracil, a chemotherapy agent, into a polymer based on D,L-lactide and glycolide was carried out at the same time to the foaming process in a one-step procedure. The possibility of regulating the rate of the scaffold degradation and the kinetics of drug release makes the usage of the copolymer more attractive for a further medical application. Venting rate is revealed to be the

**201**

**case**

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

mechanical strength of the foam preventing pore collapse [48].

*epidermidis*) bacteria and showed good tissue integration [50].

temperature, melting heat, and amount of released quercetin.

of depressurization rate of 0.1–20 MPa min<sup>−</sup><sup>1</sup>

be seen that PCL/quercetin foamed in our facilities [56].

most important factor affecting the probes' pore size and their morphology. Thus, slow venting rates should be used to promote small pores in order to retard the drug release from the polymeric matrix. As it was expected, vigorous stirring rates favor the contact between supercritical CO2 and the swelled polymer, improving the impregnation process. On the other hand, the presence of glycolide enhanced the

In the same way, Salerno et al. prepared porous PCL scaffolds containing three different drugs: 5-fluorouracil, nicotinamide, and triflusal, in order to obtain systems with controlled drug delivery capabilities. ScCO2 saturation and foaming conditions were optimized to create the porosity within the samples and demonstrated that the composition of the starting PCL/drug/solvent mixtures influenced polymer crystallization, scaffold morphology, and pore structure features. Moreover, it was found that drug loading depended on both initial solution composition and drug solubility in scCO2. So, in the case of triflusal that is a highly scCO2-soluble drug, loading efficiency was improved by adding a higher amount of free drug inside of the impregnation vessel. The drug delivery study, as it was expected, indicated that release profiles depended mainly on pore structure and scaffold composition [49]. However, the authors observed that the control on the pore interconnectivity and pore size with this technique still needs to be improved. They proposed the use of natural plasticizers as eugenol to overcome these limitations. Thus, eugenolcontaining PCL scaffolds were prepared by supercritical foaming followed by a one- or a two-step depressurization profile. Moreover these scaffolds presented antimicrobial activity preventing the attachment of Gram-positive (*S. aureus*, *S.* 

A hybrid porous scaffold of PLGA hydroxyapatite and collagen was prepared using a supercritical CO2 saturation technique by Zhang et al. The results showed that the pore size and porosity of the scaffold could be controlled by manipulating these process conditions. The pore size and porosity can be regulated by supercritical CO2 saturation temperature, saturation time, and saturation pressure [51].

**5. One-step supercritical foaming + impregnation process: a particular** 

The experiments were carried out in a plant RESS250 developed by Thar Technologies [56]. PCL and quercetin were mixed physically into an aluminum foil support (ratio 50:1 PCL/Q ), and it was introduced into a stainless steel vessel. Then, CO2 was pumped to the vessel till the desired operating pressure at the same time that the temperature was adjusted is reached. A determined impregnation time was awaited, and once finished, the output valve was opened to vent the CO2 in a range

The generated PCL/quercetin scaffold with higher pore density and smaller pore size was achieved for higher pressure and depressurization rate and lower

. In the SEM image in **Figure 2**, it can

In our facilities PCL scaffolds impregnated with quercetin were prepared using supercritical CO2. PCL is a semicrystalline polyester with a melting point (Tm) of 329–334 K and a glass transition temperature (Tg) of 213 K [52]. Quercetin (Q ) is a flavonoid present in many fruits and vegetables [53]. This flavonoid highlights its antioxidant action, but it has different benefits as antibacterial, cardiovascular health, anti-inflammatory, and anticancer effects [54, 55]. The study was supported by an experimental design to elucidate the influence of pressure (15–30 MPa), temperature (308–333 K), and depressurization rate (0.1–20) on foaming, melting

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

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

*Advanced Supercritical Fluids Technologies*

which may degrade them.

tions holding unaltered the protein activity.

polymer is not favored, and scaffolds were not achieved.

mechanical resistance, the PLA foams being more fragile.

distribution of drug into scaffold is heterogeneous.

depressurization rate. Posteriorly the organic solvent should be removed, leaving the impregnated scaffold. This method has several drawbacks, for example, residues of organic solvent in the final product need a last step to dry the scaffold, and the

SCF impregnation removes all these drawbacks due to its properties as high diffusivity, low surface tension, and the ease of solvent recovery. Nevertheless, this methodology is limited by the solubility of the drug in the SCF, and the polymer can be swollen by the SCF. If these last requirements are fulfilled, a high-quality product free of residual solvents can be obtained, since no organic solvents are involved in process [40, 41]. In this process SCF is put in contact with the active substance that is going to be incorporated into the scaffolds. Then the SCF solubilizes this substance till saturation during the impregnation time. Later, in the depressurization step, the gas rapidly diffuses out of the polymer, deplasticizing it and avoiding the polymers and active substance exposition to high temperatures,

CO2 is the frequently used SCF because it is not dangerous, not toxic, not flammable, relatively cheap, and classified as a safe solvent. Anyway due to polarity limitations, it is often used as a cosolvent to increase the polarity and selectivity. Duarte et al. prepared loaded chitosan scaffold able to sustain the release of dexamethasone using supercritical impregnation [42]. Sproule et al. [43] achieved the successful impregnation of a protein in PMMA scaffolds for biomedical applica-

Campardelli et al. [9] prepared polycaprolactone/nimesulide patches using supercritical impregnation. In this work the authors achieved the foaming of the polymer in certain conditions at the same time that nimesulide is incorporated into its structure. Thus impregnated scaffolds are prepared in a one-step process for determined conditions. However in other operating conditions, foaming of the

Biodegradable PLA/PLGA foams impregnated with indomethacin in scCO2 were studied by Cabezas et al. [44]. Authors observed that drug loading of foams was favored by high values of stirring rate. Moreover little pore sizes were obtained at slow depressurization rates. As it was expected, composition influenced the

Fanovich et al. studied the functionalized PCL scaffolds impregnated with natural compounds extracted from Patagonian *Usnea* lichen for tissue engineering [45]. An integrated process at high pressure for extraction/impregnation/foaming of PCL was developed. Authors concluded that the process is successful at 35°C and 15–17 MPa of CO2 by foaming. The same researchers incorporated in a posterior work hydroxyapatite to these scaffolds, concluding that the scaffold obtained from PCL-HA with 20% of the HA shows the highest impregnation yield at 17 MPa and 35°C and subsequently also the best bactericidal effect on the tested *Staphylococcus* 

The impregnation of chitosan with lactulose using supercritical fluids under various operating conditions, in order to improve the solubility of this natural polymer at neutral or basic pH, was carried out by Diaz et al. The highest impregnation yield was obtained using continuous process, 60-min contact time, 14% (v/v) of cosolvent ethanol/water (95:5), depressurization rate equal to 3.3 bar/min, 100 bar

The impregnation of 5-fluorouracil, a chemotherapy agent, into a polymer based on D,L-lactide and glycolide was carried out at the same time to the foaming process in a one-step procedure. The possibility of regulating the rate of the scaffold degradation and the kinetics of drug release makes the usage of the copolymer more attractive for a further medical application. Venting rate is revealed to be the

**200**

*aureus* strains [46].

of pressure, and 100°C [47].

most important factor affecting the probes' pore size and their morphology. Thus, slow venting rates should be used to promote small pores in order to retard the drug release from the polymeric matrix. As it was expected, vigorous stirring rates favor the contact between supercritical CO2 and the swelled polymer, improving the impregnation process. On the other hand, the presence of glycolide enhanced the mechanical strength of the foam preventing pore collapse [48].

In the same way, Salerno et al. prepared porous PCL scaffolds containing three different drugs: 5-fluorouracil, nicotinamide, and triflusal, in order to obtain systems with controlled drug delivery capabilities. ScCO2 saturation and foaming conditions were optimized to create the porosity within the samples and demonstrated that the composition of the starting PCL/drug/solvent mixtures influenced polymer crystallization, scaffold morphology, and pore structure features. Moreover, it was found that drug loading depended on both initial solution composition and drug solubility in scCO2. So, in the case of triflusal that is a highly scCO2-soluble drug, loading efficiency was improved by adding a higher amount of free drug inside of the impregnation vessel. The drug delivery study, as it was expected, indicated that release profiles depended mainly on pore structure and scaffold composition [49].

However, the authors observed that the control on the pore interconnectivity and pore size with this technique still needs to be improved. They proposed the use of natural plasticizers as eugenol to overcome these limitations. Thus, eugenolcontaining PCL scaffolds were prepared by supercritical foaming followed by a one- or a two-step depressurization profile. Moreover these scaffolds presented antimicrobial activity preventing the attachment of Gram-positive (*S. aureus*, *S. epidermidis*) bacteria and showed good tissue integration [50].

A hybrid porous scaffold of PLGA hydroxyapatite and collagen was prepared using a supercritical CO2 saturation technique by Zhang et al. The results showed that the pore size and porosity of the scaffold could be controlled by manipulating these process conditions. The pore size and porosity can be regulated by supercritical CO2 saturation temperature, saturation time, and saturation pressure [51].
