Conventional Scaffolds

Several conventional techniques used to produce porous scaffolds, including the salt-leaching and gas-foaming methods, have been used to introduce open pore structures and intercon-nected channels within bioscaffolds. Pores are important to increase the viability of seeded or injected cells within the scaffolds [73–76]. These traditional techniques for preparing 3D scaf-folds are straightforward, cost-effective, and easy to scale up [77, 78].

7.2.1.1 Salt-Leaching Method

Salt-leaching is a simple processing technique to produce 3D biomimetic scaffolds. This method involves making a polymer/organic solvent solution and incorporating porogen particles, which are insoluble in the organic solvent. The solution is then cast into a mold of the desired shape, and the solvent is evaporated away. After the solvent completely evaporates, the final step is to dissolve the porogen particles in an aque-ous solution [49, 50]. The resulting structure has significant porosity as a result of the void spaces left by the dissolved porogen particles. Typi-cally, the porogen is a salt granule or particle.

As with all techniques, there are advantages and disadvantages in using this method. One of the main advantages is that porosity and pore size can be effectively controlled. Materi-als with porosity levels up to 90% and pore-size diameter ranging from 100 to 700 μm have been reported using the salt-leaching technique. The porosity is given by volume fraction occupied by leachable particles. The pore size and pore shape can be modified independently of the porosity by varying the leachable particles’

characteristics (i.e., size and shape) [51, 79, 80].

Another advantage of using the salt-leaching

method is that it is one of the more convenient and straightforward methods for preparing porous 3D scaffolds. The technique involves a minimal number of steps and only requires basic laboratory methods.

One of the major drawbacks to salt leaching is that it only produces thin membranes with a dense surface skin. Moreover, the bioscaffold might contain residual salt particles used during the process. This could negatively impact cell behavior and ultimately tissue growth. In addi-tion to residual salt or other particles remaining on the scaffold, there is often incomplete removal of the solvent during the drying process. Another disadvantage of this method (and other meth-ods employing polymers) is that some of the polymers degrade into acidic by-products.

These acidic degradation products could poten-tially have negative effects on cell adhesion and growth [50]. Finally, another potential drawback to the salt-leaching technique, especially for bioscaffolds needing lower porosity levels, is the lack of interconnectivity between pores. Salt particles that are not in contact with other par-ticles lead to insufficient pore interconnectivity and often become trapped in the polymer scaf-fold [51].

To ameliorate the aforementioned problems, researchers have investigated means to make the process more benign. One method that has been employed is the use of melt polymer solu-tions for the solvent-casting stage, as opposed to using a polymer solution with harsh organic solvents. The melt-molding step consists of mix-ing the polymer powder with salt particles and melting the mixture [81]. The melting step elimi-nates the need for organic solvents, thereby pre-venting the possibility of the scaffold containing residual solvent and harming cells or tissue.

In an attempt to create better interconnectiv-ity between pores and to increase the channel size between pores, a method where by salt par-ticles are partially merged has been proposed.

This method involves merging salt particles

using moisture or using heat to merge sugar particles or paraffin spheres. By merging the porogen material, the probability of isolated particles remaining in the polymer mixture is significantly reduced. Moreover, cellular com-munication and the exchange of nutrients and waste should be improved with better intercon-nectivity between pores [81].

7.2.1.2 Gas-Foaming Method

The gas-foaming method has been demonstrated to produce effective bioscaffolds for tissue engi-neering applications [53, 79, 82, 83]. This method takes advantage of a gas at high pressure to pro-duce a porous structure instead of the harsh sol-vents often used in particle-leaching methods.

The gas-foaming method begins by placing a material, typically polymeric, in a chamber with a gas such as CO2 and increasing the pressure to the point where the gas becomes sufficiently soluble in the polymeric solid phase. This effec-tively saturates the polymer with the gas. The pressure is then lowered to the ambient pres-sure to induce thermodynamic instability of the gaseous phase. The gas begins to phase separate from the polymer, and in an effort to minimize free energy, the gas molecules begin to cluster and cause pore formation. The pores grow by the gas molecules diffusing to the pore nuclei.

The resulting structure is highly porous, but is primarily a closed-pore structure because of the rapid depletion of the gas between pores [52, 79].

The process is limited in its ability to produce consistently repeatable results. Pore formation does not occur in a predictable manner each time the technique is used.

To create better interconnectivity between pores in the gas-foaming method, this technique has been combined with the salt-leaching method. The hybrid technique first creates a composite of the polymer with a porogen. The composite is then placed with a gas in a high-pressure environment to allow the polymer and gas to mix. The pressure is then decreased to the ambient pressure, and the gas molecules create

pores within the structure. Finally, the scaffold is submerged into deionized water (or some other solvent) to dissolve the porogen particle, creating more pores and enhancing pore inter-connectivity [52].

Scaffolds produced using either the gas-foaming method or the hybrid method have been shown to support cellular functions critical for tissue regeneration [52]. These scaffolds have also been used for concomitant release of various biomol-ecules and small molbiomol-ecules in addition to struc-turally supporting tissue growth [52].

7.2.1.3 Textile Fiber Bonding

To produce fibrous 3D scaffolds with good mechanical properties, textile fiber bonding may be employed. Fiber-bonded structures have been shown to perform better than other materi-als in withstanding the in-situ stressors experi-enced during tissue growth [54].

Textile fiber was developed in 1993 by Mikos and colleagues [55]. The process was initially described in a series of steps that include the formation of a composite material of nonbonded fibers embedded in a polymer matrix, subse-quent thermal treatment of the matrix, and finally, selective dissolution of the matrix. The nonbonded fibrous mesh is created by isolating fibers from a thicker multi-lamellar mat. This mat is then either submerged into another polymer solution, thereby ensuring the fibers are immis-cible in the second polymer solution, or the fibers are placed into a mold and the other polymer solution is allowed to fill the remaining mold volume. After the solvent evaporates, the com-posite is heated to a temperature above the melt-ing point of the polymer that comprises the fiber network to form welded points at the crosspoints of the fiber mesh. Finally, the non-fiber polymer is selectively dissolved using a solvent that is immiscible with the fiber network. The resulting fiber matrix is then vacuum-dried to completely remove solvents [55].

Two prominent drawbacks of the fiber-bonding technique include the inability to control pore

7.2 FABRICATION OF 3D BIOSCAFFOLDS 169 size and the technique requires harsh solvents.

As previously mentioned, harsh solvents and inadequate pore size could render the scaffold ineffectual for tissue engineering purposes.

7.2.1.4 Rapid Prototyping/Solid Free-Form Fabrication

Rapid prototyping is another technique to cre-ate porous 3D scaffolds. This method involves using computer-aided design tools to pro-duce a digital representation of a bioscaffold.

A depositor then layers polymeric or other material types to exactly replicate the desired shape. Layer-by-layer assembly, discussed in Chapters 3 and 11, allows for the exact control of morphological characteristics and ultimately the mechanical properties of the bioscaffold. The researcher easily controls pore location and size as well as surface characteristics of the scaffolds to ensure scaf-fold success as a regeneration vehicle. More-over, the predictability of the rapid prototyping method allows for consistent reproduction of various scaffold types with complex shape, size, and other physical requirements [50, 84].

There are several variants of the rapid proto-typing method; however, all variants are catego-rized by whether they use direct or indirect fabrication of the scaffold and whether they employ the melt-dissolution deposition (MDD) technique or the particle-bonding technique [56]. The direct fabrication methods that use the MDD technique are fused deposition method, 3D fiber-deposition technique, precision extru-sion deposition, precise extruextru-sion manufactur-ing, low temperature deposition, multi-nozzle deposition, pressure-assisted microsyringe, rob-ocasting, 3D bioplotter, and rapid prototyping robotic dispensing system [56]. In general, MDD methods involve extruding a strand of material through a small opening in a lateral direction onto previous strands. The melted material eventually solidifies and remains attached to the preceding layer. By controlling the spacing between adjacent strands of polymeric material,

pore size is controlled in the horizontal plane.

For manipulation in the vertical plane, strands are placed at varying angles to achieve specified pore dimensions [56].

A representative MDD method is the fused deposition method (FDM). It begins with feed-ing a strand of material into a liquefier. After melting, the material is extruded as a series of layers. The environment is controlled to main-tain proper contact between layers. The subse-quent scaffold has a honeycomb structure with channel diameters in the hundreds of microm-eters. FDM has also produced scaffolds with polymeric and ceramic components for mechanical strength and scaffolds that support the growth and proliferation of various cell types [56].

A limitation of FDM is that the material being fed into the liquefier has to be of a specific diam-eter and possess certain material properties to physically fit through the rollers and nozzle.

Most natural polymers cannot be used with the FDM because of the high operating tempera-tures required to melt them and produce strands.

The inability to incorporate natural polymers limits the potential biomimicry of the scaffold.

Finally, the ability to achieve sufficient micropo-rosity is inhibited by the deposition of dense filaments.

To remedy these deficiencies, various modifi-cations of the FDM have been employed. Newer methods that eliminate the need for a precursor filament produce scaffolds with sufficient poros-ity and allow for the incorporation of biopoly-mers and biomolecules through lower processing temperatures [56, 85]. Moreover, methods have been developed to significantly reduce the reso-lution of the scaffold, producing filaments that are in the tens of micrometers in diameter [56].

Another development with deposition methods is the ability to create hydrogel materials with well-defined pore structures. These hydrogels could provide the softer scaffolds needed for the regeneration of soft tissues, along with other scaffold properties [57].

The other class of rapid prototyping methods, particle-bonding techniques, imparts advanta-geous features to the resulting bioscaffold. In gen-eral, particle-bonding techniques involve the selective bonding of particles into a thin 2D layer.

These 2D layers are then bonded one by one to produce the desired 3D structure. Various sol-vents have been used to bind the layers, including water with complex sugars [56] and organic sol-vents with synthetic polymers [56]. The technique gives rise to macroporous and microporous struc-tures, but the extent of the micropores is limited by the size of polymer granules in the powder.

The primary advantage of particle-bond methods over melt deposition methods is that the powder causes the scaffold to have a rough exterior, which has been shown to promote cell differentiation, growth, and proliferation [56]. Particle-bonding techniques also do not use heat, which allows for various material types to be used.

The final rapid prototyping methods are indirect methods. They take advantage of a mold to produce a 3D porous scaffold. Indirect methods involve casting material into a mold that will create desired external and internal structures. These methods are useful because they require less raw material and a variety of material blends can be accommodated. The original material properties are maintained throughout processing of the 3D scaffold. Ade-quate microporous and macroporous structures are produced [56, 83, 86].

7.2.1.5 Hydrogels

Hydrogels represent the final class of 3D bioscaf-folds discussed in this survey. Hydrogels are ubiquitous in tissue engineering because they closely approximate the ECM. These bioscaf-folds can be synthesized from a variety of mate-rials, are highly biocompatible owing to their large water content (ranging between 70% and 90%), are processed under mild conditions, and can be augmented to achieve desired mechani-cal properties.

Hydrogel matrices are formed from a variety of synthetic and natural hydrophilic polymers as well as polymeric blends. The most common syn-thetic polymers used include polyvinyl alcohol (PVA), polyethylene glycol (PEG)/polyethylene oxide, and poly-2-hydroxyethyl methacrylate.

Some natural polymers used to create hydrogels are chitosan, alginate, hyaluronic acid, and collagen [87–90].

The chosen material can significantly improve the function of the scaffold. To prevent dissolution of the hydrophilic polymer, crosslinking methods are needed. Some poly-mers used for hydrogels are charged, which serves to create a physical crosslink between oppositely charged macromolecules. Chemical methods involve covalently attaching a linker molecule to polymer chains and then reacting the chain-molecule complex with other macro-molecular chains. Hydrogels can also be formed by polymerizing monomers in the presence of water [58, 87–90].

The mechanical properties of hydrogels are enhanced through the use of ceramics and other blends. To make the scaffold more ame-nable to cell adhesion, given its smooth topog-raphy, a variety of peptide moieties may be attached to the polymer. The RGD (arginine, glutamic acid, aspartic acid) amino-acid sequence is among the oligopeptides used to mimic adhesion proteins that interact with cell surface receptors. Control over hydrogel poros-ity influences diffusion of material in and out of the scaffolds and dictates the ability of cells to infiltrate the scaffold [59]. For better control over hydrogel porosity, 3D printing techniques involving liquid-liquid bonding have been used [91, 92].