Synthesis of Tissue Engineering scaffold

A number of different methods has been described in literature for preparing porous structures to be employed as tissue engineering scaffolds. Each of these techniques presents its own advantages, but none is devoid of drawbacks.

• Nanofiber Self-Assembly: Molecular self-assembly is one of the few methods to create biomaterials with properties similar in scale and chemistry to that of the natural in vivo extracellular matrix (ECM). Moreover, these hydrogel scaffolds have shown superior in vivo toxicology and biocompatibility compared with traditional macroscaffolds and animal-derived materials.

• Textile technologies: these techniques include all the approaches that have been successfully employed for the preparation of non-woven meshes of different polymers. In particular non-woven polyglycolide structures have been tested for tissue engineering applications: such fibrous structures have been found useful to grow different types of cells. The principal drawbacks are related to the difficulties of obtaining high porosity and regular pore size.

• Solvent Casting & Particulate Leaching (SCPL): this approach allows the preparation of porous structures with regular porosity, but with a limited thickness. First the polymer is dissolved into a suitable organic solvent (e.g. polylactic acid could be dissolved into dichloromethane), then the solution is cast into a mold filled with porogen particles. Such porogen can be an inorganic salt like sodium chloride, crystals of saccharose, gelatin spheres or paraffin spheres. The size of the porogen particles will affect the size of the scaffold pores, while the polymer to porogen ratio is directly correlated to the amount of porosity of the final structure. After the polymer solution has been cast the solvent is allowed to fully evaporate, then the composite structure in the mold is immersed in a bath of a liquid suitable for dissolving the porogen: water in case of sodium chloride, saccharose and gelatin or an aliphatic solvent like hexane for paraffin. Once the porogen has been fully dissolved a porous structure is obtained. Other than the small thickness range that can be obtained, another drawback of SCPL lies in its use of organic solvents which must be fully removed to avoid any possible damage to the cells seeded on the scaffold.

• Gas Foaming: to overcome the necessity to use organic solvents and solid porogens a technique using gas as a porogen has been developed. First disc shaped structures made of the desired polymer are prepared by means of compression molding using a heated mold. The discs are then placed in a chamber where are exposed to high pressure CO₂ for several days. The pressure inside the chamber is gradually restored to atmospheric levels. During this procedure the pores are formed by the carbon dioxide molecules that abandon the polymer, resulting in a sponge like structure. The main problems related to such a technique are caused by the excessive heat used during compression molding (which prohibits the incorporation of any temperature labile material into the polymer matrix) and by the fact that the pores do not form an interconnected structure.

• Emulsification/Freeze-drying: this technique does not require the use of a solid porogen like SCPL. First a synthetic polymer is dissolved into a suitable solvent (e.g. polylactic acid in dichloromethane) then water is added to the polymeric solution and the two liquids are mixed in order to obtain an emulsion. Before the two phases can separate, the emulsion is cast into a mold and quickly frozen by means of immersion into liquid nitrogen. The frozen emulsion is subsequently freeze-dried to remove the dispersed water and the solvent, thus leaving a solidified, porous polymeric structure. While emulsification and freeze-drying allows a faster preparation if compared to SCPL, since it does not require a time consuming leaching step, it still requires the use of solvents, moreover pore size is relatively small and porosity is often irregular. Freeze-drying by itself is also a commonly employed technique for the fabrication of scaffolds. In particular it is used to prepare collagen sponges: collagen is dissolved into acidic solutions of acetic acid or hydrochloric acid that are cast into a mold, frozen with liquid nitrogen then lyophilized.

• Thermally Induced Phase Separation (TIPS): similar to the previous technique, this phase separation procedure requires the use of a solvent with a low melting point that is easy to sublime. For example dioxane could be used to dissolve polylactic acid, then phase separation is induced through the addition of a small quantity of water: a polymer-rich and a polymer-poor phase are formed. Following cooling below the solvent melting point and some days of vacuum-drying to sublime the solvent a porous scaffold is obtained. Liquid-liquid phase separation presents the same drawbacks of emulsification/freeze-drying.

• CAD/CAM Technologies: since most of the above described approaches are limited when it comes to the control of porosity and pore size, computer assisted design and manufacturing techniques have been introduced to tissue engineering. First a three-dimensional structure is designed using CAD software, then the scaffold is realized by using ink-jet printing of polymer powders or through Fused Deposition Modeling of a polymer melt.