**3. Applications of biofabrication to tissue engineering**

#### **3.1. Biofabrication and architectural design of scaffolds**

**Technology Types Materials Advantages Disadvantages**

Good mechanical strength;

materials;

features

Low costs; good mechanical strength;

versatile in lay-down pattern design

no toxic components; water used as binder

Broad range of materials

incorporation of cells and

and conditions;

proteins

Fast processing; low costs;

easy to remove support

easy to achieve small

Good mechanical strength; high accuracy; broad range of materials

wax or wax compounds

ceramics, bulk polymers

thermoplastic polymers/ ceramics

bulk polymers; ceramics

Swollen polymers (hydrogels); thermoplastic polymers; reactive resins; ceramics

SLA Liquid-based Polymers,

320 Advances in Biomaterials Science and Biomedical Applications

SLS Powder-based Metals,

FDM Solid-based Some

3D printing Powder-based powder of

3D plotting Liquid-based or

solid-based

**Table 1.** RP techniques for tissue engineering

**Tissue Engineering Applications**

Bone [39], Heart valves [40].

Bone [41, 42], Cartilage [43].

Bone [44], Adipose [45], Cartilage [46].

Bone [47],

Bone [48], Cartilage [49].

Limited to reactive resins (mostly toxic)

Elevated

Elevated temperatures; small range of bulk

materials

Material must be in powder form; weak bonding between powder particles; rough surface; trapped powder issue; might require postprocessing

Slow processing; no standard condition; time consuming adjustment to new

materials; low mechanical strength

temperatures; local high energy input; uncontrolled porosity The microstructure of scaffolds is increasingly believed to contribute significantly to the diffusion of nutrients and metabolic wastes, spatial organization of cell growth and the development of specific biological functions in tissues. A scaffold with high porosi‐ ty is desirable for the easy diffusion of nutrients and metabolic wastes and is also beneficial for cell migration and neo-vascularization. A high surface area to volume ra‐ tio favors cell attachment and growth. The effect of scaffold pore size on tissue regen‐ eration is also emphasized by experiments demonstrating that (1) an optimum pore size of 5 μm is good for neo-vascularization; (2) 5 - 15 μm pores are beneficial for fi‐ berblast ingrowth; (3) 20 - 125 μm pores can affect the regeneration of adult mammali‐ an skin; and (4) fibrovascular tissues require pores sizes greater than 500 μm [38, 50]. Advanced biofabrication techniques are able to design and precisely control the archi‐ tecture of scaffolds. They can build scaffolds with reproducible morphology and micro‐ structure that varies across the scaffold matrix to resemble natural tissues with complex hierarchical structures.

Conventional lyophilization can only form porous structures with random orientation. An improved technique for fabricating scaffolds with a linearly oriented architecture is called "freeze casting". Freeze casting facilitates directional solidification of solutions or slurries [51]. During freeze casting, the polymer solution is pipetted into a cylindrical mold fitted with a copper bottom plate and secured onto the temperature-controlled copper cold finger of the freeze casting system. The cold finger temperature is low‐ ered at a constant cooling rate to a final temperature, resulting in the directional solid‐ ification of the material dispersion. When the ice is sublimated by freeze drying, the porous microstructure of the resulting scaffold is a negative template of the ice crys‐ tals. Freeze casting has been used to produce a wide range of porous, oriented scaf‐ folds from organic and inorganic materials [51, 52]. This technique is also suitable for the fabrication of nerve conduit scaffolds with a featured porous structure that may guide axon growth.

Different scaffold fabrication techniques can be combined to capitalize on their respective positive features for varying applications. The combination of rapid prototyping with lyo‐ philization, in which the polymer solution is dispensed on substrates with a controllable temperature and the strands formed are frozen and lyophilized to remove the solvent, is called "rapid freeze prototyping" technique [53]. This technique has the advantage of fabri‐ cating scaffolds with both sub-millimeter and micrometer sized pores [54] (Figure 4). The optimized porous scaffolds can accommodate tissue ingrowth at different scales, from cells to tissues. Scaffolds can also be cold processed so that the polymer can be bio-functionalized without compromising their function during manufacturing.

**Figure 4.** Scaffold fabricated by rapid freeze prototyping: (a) camera image of the scaffold, (b) pore size distribution of the scaffold, (c) wall thickness distribution of the scaffold, and (d) 3D reconstructed model of the scaffold using microtomography. (Modified from [54])

#### **3.2. Biofabrication and scaffolds with living cells**

Biofabrication of living structures with desired functionality has become a hot topic in tissue en‐ gineering in past few years. Conventional cell-seeding methods are inadequate for the develop‐ ment of *in vitro* tissue-test systems because they involve random placement of cells and, therefore, lack the precision necessary for spatial control. Conventional cell-seeding methods are also a type of 2D cell culture. In contrast, cell cultures in 3D structures allow for a more natu‐ ral cell attachment and focal adhesion in all directions. The most physiologically relevant cell morphology that can be attained on and in three-dimensional scaffolds will provide the best structural cues to regulate cell function [55, 56]. Different methods of fabricating 3D scaffolds with living cells have been developed. One of the methods is to spray living cells into the scaf‐ folds throughout the electrospinning process to produce nanofibrous 3D tissue scaffolds. In this method, cells are periodically sprayed from a pump-action spray bottle onto the developing scaffold during the electrospinning process [56]. The cells can be layered throughout the thick‐ ness of the scaffolds, but not incorporated into individual polymer nanofibers.

Living cells also can be directly electrospun, as fine composite threads encapsulating living cells, using a coaxial needle configuration and a biocompatible polymer [57, 58]. The poly‐ mer nanofibers accommodate the survival and proliferation of the cells. Advanced rapid prototyping techniques, such as bioprinting, are more capable of incorporating living cells into scaffolds than other techniques. Introducing cells at almost any arbitrary density and precisely into the desired location of a scaffold is possible by means of rapid prototyping. Hydrogel scaffolds, as delivery vehicles for cells, are suitable for bioprinting processes that seed living cells while constructing scaffolds with specific geometries [36]. A pneumatic dis‐ penser system is used to bioprint the cell-associated scaffolds using polymer solution, such as alginate aqueous solutions. The fabrication parameters including pressure and nozzle ve‐ locity can be altered, thus affecting the viability of the cells [59]. Complete biological "scaf‐ fold free" tissue substitutes can also be engineered with specific compositions and shapes, by exploiting cell-cell adhesion and the ability of cultured cells to grow their own ECM; such approaches have the advantage of reducing and mediating inflammatory responses to biomaterials [60]. For this concept, extrusion-based bioprinting is an automated deposition method that can generate a fully biological construct which is structurally and functionally close to native tissues. Spherical or cylindrical multicellular units (the bio-ink) are delivered according to a computer-generated template with the hydrogel (the bio-paper) serving as the support material. The cells neither invade nor rearrange within the hydrogel, which keeps its integrity during post-printing fusion and can be easily removed to free the fused multicellular construct (Figure 5). The authentic tissues can be assembled through cell adhe‐ sion, cell sorting, and tissue fusion processes [37].

**Figure 4.** Scaffold fabricated by rapid freeze prototyping: (a) camera image of the scaffold, (b) pore size distribution of the scaffold, (c) wall thickness distribution of the scaffold, and (d) 3D reconstructed model of the scaffold using micro-

Biofabrication of living structures with desired functionality has become a hot topic in tissue en‐ gineering in past few years. Conventional cell-seeding methods are inadequate for the develop‐ ment of *in vitro* tissue-test systems because they involve random placement of cells and, therefore, lack the precision necessary for spatial control. Conventional cell-seeding methods are also a type of 2D cell culture. In contrast, cell cultures in 3D structures allow for a more natu‐ ral cell attachment and focal adhesion in all directions. The most physiologically relevant cell morphology that can be attained on and in three-dimensional scaffolds will provide the best structural cues to regulate cell function [55, 56]. Different methods of fabricating 3D scaffolds with living cells have been developed. One of the methods is to spray living cells into the scaf‐ folds throughout the electrospinning process to produce nanofibrous 3D tissue scaffolds. In this method, cells are periodically sprayed from a pump-action spray bottle onto the developing scaffold during the electrospinning process [56]. The cells can be layered throughout the thick‐

Living cells also can be directly electrospun, as fine composite threads encapsulating living cells, using a coaxial needle configuration and a biocompatible polymer [57, 58]. The poly‐ mer nanofibers accommodate the survival and proliferation of the cells. Advanced rapid

ness of the scaffolds, but not incorporated into individual polymer nanofibers.

tomography. (Modified from [54])

**3.2. Biofabrication and scaffolds with living cells**

322 Advances in Biomaterials Science and Biomedical Applications

**Figure 5.** Scaffold-free bioprinting technology: (a) the bio-printer: 3D printing is achieved by means of a three-axis positioning system (stage in *y* and printing heads along *x* and *z* (top: Neatco, Carlisle, Canada; bottom: Organovo-In‐ vetech, San Diego)); (b) spheroids with living cells are delivered one by one into the hydrogel bio-paper according to a computer script; (c) layer-by-layer deposition of cylindrical units of bio-paper (shown in blue) and multicellular cylindri‐ cal building blocks. The outcome of printing (spheroids in panel (b), multicellular cylinders in panel (c)) is a set of dis‐ crete units, which post-printing fuse to form a continuous structure. (Modified from [60])

#### **4. Summary**

Engineered scaffolds are playing an increasingly important role in tissue engineering. Scaf‐ folds should not only have porous structures and provide mechanical support to new tissue regrowth but also have a complex mimetic hierarchical structure and biological features. Conventional scaffold fabrication techniques fail to meet these requirements for tissue re‐ generation. Biofabrication technologies have demonstrated potentials in this regard and can be used to create regenerative tissues or organs through the combination of state of the art fabrication techniques, materials science, and cell biology.
