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Silk fibroin (SF) is a protein that can be used in biomedical field thanks to its biocompatibility, biodegradability and mechanical properties. SF is generally produced in form of aerogel by freeze drying; but, during this process, the delicate nanostructure of the gel can be destroyed, due to the surface tension action on the polymer matrix. The polymer organization at nanoscale (i.e., nanostructure) is preferably required for cells cultivation to ensure their adhesion, differentiation and migration inside the artificial support (i.e., scaffold). In this study, SF aerogels at 10 and 15 % w/w were produced by Supercritical gel drying, potentially useful for nanomedicine applications. SF aerogels were loaded with Ascorbic acid (AA) at 5 % w/w with respect the protein; it was selected since it should favor cell growth and, for example in case of bone tissue engineering applications, for osteoblast differentiation. The obtained aerogels were characterized by scanning electron microscopy and porosity analyses. Moreover, release tests by a spectrophotometer UV/Vis to measure AA release kinetics from SF aerogels and mechanical tests to determine the Young modulus of the produced SF aerogels were also performed. Supercritical assisted process allowed to preserve SF aerogel morphology at nanoscale, thanks to the near zero surface tension of the supercritical mixture (CO2 + organic solvent) at the selected operative conditions (i.e., 200 bar and 35 °C). Moreover, AA release rate can be controlled as well as mechanical properties of the produced aerogels, depending on SF concentration in the starting gel.

In this work an hypothesis of modeling nanofibers network of Poly-L-lactide (PLLA) scaffolds loaded with hydroxyapatite (HA) nanoparticles, suitable for tissue engineering applications, is presented to investigate the mechanical properties by FEM analysis. Scaffolds were produced by Supercritical CO2 drying of polymeric gels. FEM modeling of nanoporous biomaterials involves computational problems such as: the reproduction of the nano-morphology by means of different techniques, such as molecular dynamics simulations that hardly lead to results adherent to the experimental evidence; the reverse engineering to extrapolate geometric information; the identification of a periodic representative volume element (RVE) to reach more coherent results and to reduce the simulation computational effort. Basic modeling assumptions are: a) polymer particles are small enough to exhibit Brownian motion; b) scaffold SEM images show that the porous structure consists of curved fibers that depart from punctiform nuclei realizing a space frame; c) scaffold experimental compressive tests show that the porous material behaves as a soft isotropic material. On the basis of these assumptions, a parametric algorithm that creates a cubic RVE, showing a nanofibers network and having the same porosity of the real material, has been written; RVE size has been optimized on the bases of its material isotropic degree measured by an ad hoc created iterative algorithm for generating a rod spaceframe; RVE mechanical behavior has been optimized by curving appropriately each fiber according to the experimental data and on the basis of SEM imaging diagnostic. Linear FEM simulations on mechanical behavior have given qualitatively and quantitative satisfactory results when compared to the experimental ones.

Supercritical fluids based processes were frequently proposed to realize new kind of supports to deliver poor water-soluble drugs. In some cases, nanostructured aerogels were used as substrates. The matrices constituting the aerogel have to present a high porosity and a high surface area. Different natural and synthetic polymers were used in form of aerogels. Natural polymer-based materials are preferable for their renewability and for environmental reasons; among them, starch is the second most abundant after cellulose and it is attractive for its low cost. In this work, different sources starches (maize, potato and wheat) aerogels were produced using a supercritical carbon dioxide based process. First, a gel is formed by an aqueous solution (hydrogel), then water is replaced by ethanol to form an alcogel; finally, a supercritical gel-drying step is proposed to form the aerogel. The different sources starches were processed and characterized from a macroscopic and a microscopic point of view. The analyses confirmed that the supercritical gel drying process is suitable to form polymeric nanostructured matrices that can be used for delivery systems.

Aerogel-based scaffolds are frequently used for tissue engineering applications, since their nanofibrous structure is suitable for cells adhesion, proliferation and migration. Nevertheless, to achieve proper physico- chemical characteristics and desired mechanical properties, can be necessary to use more polymers together; among them, natural polymers are preferable because they are biocompatible, non-toxic, biodegradable and mucoahesive. In this study, bi-component natural aerogels Alginate-Gelatin (A/G) and Chitosan-Gelatin (CS/G) have been produced by Supercritical gel drying. The results indicate that both A/G and CS/G mixtures formed uniform gels during the preparation step and Supercritical gel drying preserved their delicate nanostructured morphology, thanks to the near zero surface tension of supercritical mixture (CO2 + organic solvent) formed during the drying process, as observed from FESEM pictures. All the aerogels were characterized by porosity values higher than 80%. Moreover, CS/G aerogels crosslinked with Glutaraldehyde (GTA) showed improved thermal behavior increasing gelatin content inside the starting hydrogel.

Microparticles have been utilized as delivery vehicles of soluble factors to modify cellular behavior and therefore enhance tissue engineering regeneration. When incorporated into three-dimensional systems, microparticles can provide geometrical and temporal controlled release of bioactive agents, such as growth factors (GFs) to surrounding cells. This study investigates the effect of GFs release from biopolymer microparticles on osteoblastic differentiation of human mesenchymal stem cells (hMSCs) encapsulated in calcium (Ca)-alginate scaffolds while cultured in a tubular perfusion system bioreactor system. Empirical and deterministic models were used to demonstrate that poly(D,L-lactic-co-glycolic acid)-encapsulated GFs would result in a delayed release profile compared to GFs encapsulated into scaffolds directly. We hypothesized that the dual delivery of human bone-morphogenetic protein 2 (hBMP2) and human vascular endothelial growth factor to cells in dynamic culture would provide molecular and physical cues to promote differentiation. Results indicated that the exposures of hBMP2 and dynamic flow are sufficient in enhancing the osteoblastic differentiation pathway compared to no GF addition and static culture. The GF delivery system in a dynamic flow environment resulted in a synergistic effect on osteoblastic differentiation of hMSCs.

Ethyl Cellulose scaffolds were prepared using a supercritical fluid phase inversion process in which carbon dioxide acts as the non-solvent. Ethyl Cellulose is a derivative of cellulose in which some of the hydroxyl groups on the repeating glucose units are converted into ethyl ether groups; it is a biocompatible but non-biodegradable polymer. It is an extensively studied encapsulating material for the controlled release of pharmaceuticals in tissue engineering applications. Series of experiments were performed at various polymer concentrations, temperatures, pressures, and kind of organic solvent. The structures of the resulting scaffolds were analysed using scanning electron microscopy. We operated with polymer concentrations ranging between 5 and 20 % w/w, using N-methyl pyrrolidone and chloroform as organic solvents and we obtained scaffolds characterized by homogeneous cellular structures with different pore mean size. The mean diameter of the pores ranged from 3 µm to 15 µm, decreasing the polymer concentration from 20 to 5 % w/w. Additional experiments were performed varying the operating pressure from 100 and 200 bar, and varying the operating temperature from 35 to 55 °C. Increasing pressure and/or decreasing temperature, the scaffolds pore size largely decreases; both the effects can be related to carbon dioxide density modification. Ethyl Cellulose scaffolds formation mechanisms were also discussed.

In this paper, different polymers were processed using a Batch Supercritical AntiSolvent precipitation (BSAS) to evaluate the possibility of producing nanoparticles and microparticles of controlled size and distribution. Liquid solutions of a polymer in dimethylsulfoxide (DMSO) or acetone were charged in a vessel; then, supercritical CO2 with the role of antisolvent was pumped up to the desired pressure. The process was performed in two steps: in batch mode for a time t1 and in continuous mode for a time t2, with a constant CO2 flow rate. All the experiments were carried out at a final pressure of 100 bar and a final temperature of 40 °C, whereas the concentration of the liquid solution was varied from 0.1 to 5 mg/mL. SEM images of the processed material were used to study morphology, mean particle size and particle size distribution. Amorphous particles were obtained at all successful BSAS conditions, whereas crystals were sometimes observed when the micronization process failed. Polymers solubilized in acetone, such as cellulose acetate, ethyl cellulose, polymethyl methacrylate (PMMA) and Poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) prevalently precipitated in form of nanoparticles in the range 50-200 nanometers, whereas polymers solubilized in DMSO, such as Poly-vinyl-alcohol (PVA), dextran and inulin precipitated in form of nanoparticles at lower concentrations (in the range 100-200 nanometers) and in form of microparticles at higher concentrations (in the range 0.7-2 µm).

Tissue engineering (TE) is an emerging field aimed at repairing defective tissues and organ, instead of relying on conventional organ transplantation. Among the polymeric materials proposed for TE applications, the water-soluble polymers have been frequently used in form of hydrogels, that are a class of highly hydrated polymer materials. Nevertheless, hydrogel materials have poor mechanical properties; as a result, this scaffolding approach is seldom used for hard tissues applications. With the aim of overcoming this limitation, drying processes have been often applied to eliminate the water from hydrogels and amply the range of applications of the water-soluble polymeric scaffolds, but these techniques present several limitations. For this reason, in this work we proposed a supercritical gel drying method coupled with a water/solvent substitution step. PVA, Alginate and Chitosan hydrogels were processed and opportunely characterized from a macroscopic and microscopic point of view. Supercritical gel drying process confirmed to be effectiveness for the formation of polymeric scaffolds; in particular, we obtained stable 3-D structures with nanometric porous morphologies suitable for TE applications.