**5. Processing techniques for scaffold production**

 After the choice of the biomaterial to use for scaffold production, it is quite important to select an adequate processing technique that allows to maintain high levels of control of the macro- and micro-structural properties of the same. The processing methodology must satisfy key requirements such as: process accuracy and repeatability. The scaffolds obtained will present regular shaped pores with consistent pore size and interconnectivity and should not show any physicalchemical variations when produced by the same method. Moreover, the processing conditions must not alter the mechanical properties of the biomaterial, and any toxic solvent used during the process must be totally removed not to limit scaffold clinical use [3, 11]. Among the most spread processing techniques, probably the most known are those that foresee the employment of a porogenous organic or inorganic agent such as sodium chloride, sodium tartrate, sodium citrate, citric acid, or saccharose. However, the use of porogens limits the scaffolds to thin membranes with a thickness of 2 mm to facilitate complete porogen removal [45].

 Mikos *et al.* described solvent casting/particulate leaching for the first time, and it is chosen for the fabrication of porous scaffold used for bone tissue engineering. In this case, the porous agent is dispersed in appropriate solvent and then the dispersion is processed by casting or by freeze-drying. This technique allows obtaining thin membranes with 30–300 micrometer pore size and 20–50% porosity even if the pores have a shifting shape and the interconnectivity is quite low. However, the method presents some disadvantages like time consuming (it is necessary to wait for days or weeks for solvent evaporation) and the use of toxic organic solvents [46].

 In melt molding/particulate leaching, an unrefined thermoplastic polymer is mixed with the porous agent and then the blend is poured in a mold with an appropriate shape. The mold is then heated above the glass transition temperature of the polymer and at last the obtained solid is immersed in a solvent to promote the dissolution of the porogens. The advantage of this methodology is the possibility to monitor the pore size and porosity (generally 80–84%) by varying the amount of porogenous [47]. A good variant of melt molding is extrusion or injection molding proposed by Gomes *et al.,* who replaced the porous agent with a blowing agent based on citric acid. During the heating process, the blowing agent degraded producing carbon dioxide which formed interconnected and well-shaped pores [48].

Gas foaming is an high pressure processing technique described by Mooney *et al* who produced sponges of poly(D,L-lactic-co-glycolic acid) without the use of organic solvents. Solid disks of the polymer are exposed to high pressure CO2

(5.5 MPa) at room temperature followed by a decreasing of gas pressure to reduce its solubility in the polymer bulk. It brings CO2 to abandon the polymer forming well-shaped pores [49].

Phase inversion/particulate leaching is a valid method to obtain polymeric scaffolds. After the polymer solubilization in a suitable solvent, the solution is dissolved in water that provokes the polymer precipitation. Obviously, it is possible to modulate the characteristics of the scaffolds obtained through this method by varying the polymer concentration but also the temperature of the solution. Holy *et al.* used this technique to develop porous PLGA scaffold with architecture similar to osseous trabecular for bone tissue engineering [50].

Another interesting method is the fiber bonding. It allows obtaining scaffolds containing a dense frame of synthetic fibers that form a sufficiently porous threedimensional structure. This technique provides the alignment of the PGA fibers in the desired orientation and subsequently they are covered with a PLLA/methylene chloride solution and heated above the melting temperatures of both polymers. When PLLA is removed through a dissolution process, the PGA fibers remain attached to each other forming a thick net.

In the freeze-drying method, the polymer solution is first frozen rapidly at temperatures below 0°C followed by solvent removal by vacuum sublimation. It can be applied to obtain both natural and synthetic scaffolds [51]. At last, the progress of computer technology led to the development of new techniques like solid freeform fabrication (SSF) whose introduction has signed a new era for manufacturing industry. These techniques allow to produce layer-by-layer 3D objects starting from information generated by CAD system or computer-based medical imaging modalities. Obviously, the use of a computerized production system saves time and modulates with extreme precision parameters related to the micro and macro architecture of the scaffold.

The first SFF technique used for tissue engineering purpose was 3D printing. This technique uses a printer head that places a liquid binder onto thin layers of powder following the object shape generated by a CAD system. Using this technique, Kim *et al.* obtained porous PLGA scaffolds [52], while Zeltinger *et al.* created poly(l-lactic acid) disk shaped scaffolds with two different porosities (75% and 90%) and four different pore size distributions (<38, 38–63, 63–106, and 106–150 μm) [53].

Another interesting SFF methodology is fused deposition modeling (FDM). In this case, a filament of thermoplastic material is fed and melted inside a heated liquifier head and then it is forced out by an extruder and deposited on a platform. Layer by layer, the 3D object is then obtained. By varying the direction of material deposition for each layer, it is possible to change the pore size and interconnectivity of the scaffold. Using this methodology, Hutmacher et al. obtained polycaprolactone scaffolds with honeycomb-like structure and a porosity of 61 +/− 1% and proved their in vitro ability to promote proliferation of primary human fibroblasts and periosteal cells [54].
