**2. Overview of scaffold-fabrication techniques**

### **2.1. Conventional fabrication techniques**

Many techniques are available to process synthetic and natural biomaterials into various scaffolds. These include conventional techniques, such as solvent-casting and particulateleaching [6], gas foaming [7], phase separation [8], melt molding [9], and freeze drying [10], among others. An overview of these different techniques follows.

**1.** Solvent-casting and particulate-leaching (Figure 1a):

Solvent-casting and particulate-leaching techniques involve using a polymer solution uni‐ formly mixed with salt particles of a specific diameter. The solvent then evaporates leaving behind a polymer matrix with salt particles embedded throughout. The composite is im‐ mersed in water, where the salt leaches out to produce a porous structure [11]. Highly po‐ rous scaffolds with porosity values up to 93% and average pore diameters up to 500 μm can be formed using this technique. A disadvantage of this technique is that it can only be used to produce thin membranes up to 3 mm thick [12].

**2.** Gas foaming (Figure 1b):

During the gas foaming process, molded biodegradable polymers are pressurized at high pressures with gas-foaming agents, such as CO2 and nitrogen [13], water [14], or fluoroform [15], until the polymers are saturated. This results in nucleation and growth of gas bubbles with sizes ranging between 100 and 500 μm in the polymer. This technique has the advant‐ age of being an organic solvent-free process; the major drawback is that the process may yield a structure with largely unconnected pores and a non-porous external surface [16].

**3.** Phase separation (Figure 1c):

During the phase separation process, a polymer solution is quenched and undergoes a liq‐ uid-liquid phase separation to form two phases; a polymer-rich phase and a polymer-poor phase. The polymer-rich phase solidifies and the polymer poor phase is removed, leaving a highly porous polymer network [17]. The micro- and macro-structure of the resulting scaf‐ folds are controlled by varying process parameters such as polymer concentration, quench‐ ing temperature, and quenching rate. The process is conducted at low temperatures, which is beneficial for the incorporation of bioactive molecules in the structure. Using phase sepa‐ ration techniques, nano-scale fibrous structure enables to be formed, which mimics natural extracellular matrix architecture and provides a better environment for cell attachment and function [18].

**4.** Melt molding (Figure 1d):

Melt molding involves filling a mold with polymer powder and a porogen component and then heating to above the glass-transition temperature of the polymer while applying pres‐ sure to the mixture [19]. During the fabrication process, the raw materials will bind together to form a scaffold with designed specified external shape. Once the mold is removed, the porogen is leached out and the porous scaffold is then dried. Melt-molding with porogenleaching is a non-solvent fabrication process that allows independent control of morphology and shape. Drawbacks include the possibility of residual porogen and high processing tem‐ peratures that preclude the ability to incorporate bioactive molecules.

**5.** Freeze drying (Figure 1e):

**2. Overview of scaffold-fabrication techniques**

among others. An overview of these different techniques follows.

**1.** Solvent-casting and particulate-leaching (Figure 1a):

to produce thin membranes up to 3 mm thick [12].

**2.** Gas foaming (Figure 1b):

**3.** Phase separation (Figure 1c):

**4.** Melt molding (Figure 1d):

function [18].

Many techniques are available to process synthetic and natural biomaterials into various scaffolds. These include conventional techniques, such as solvent-casting and particulateleaching [6], gas foaming [7], phase separation [8], melt molding [9], and freeze drying [10],

Solvent-casting and particulate-leaching techniques involve using a polymer solution uni‐ formly mixed with salt particles of a specific diameter. The solvent then evaporates leaving behind a polymer matrix with salt particles embedded throughout. The composite is im‐ mersed in water, where the salt leaches out to produce a porous structure [11]. Highly po‐ rous scaffolds with porosity values up to 93% and average pore diameters up to 500 μm can be formed using this technique. A disadvantage of this technique is that it can only be used

During the gas foaming process, molded biodegradable polymers are pressurized at high pressures with gas-foaming agents, such as CO2 and nitrogen [13], water [14], or fluoroform [15], until the polymers are saturated. This results in nucleation and growth of gas bubbles with sizes ranging between 100 and 500 μm in the polymer. This technique has the advant‐ age of being an organic solvent-free process; the major drawback is that the process may yield a structure with largely unconnected pores and a non-porous external surface [16].

During the phase separation process, a polymer solution is quenched and undergoes a liq‐ uid-liquid phase separation to form two phases; a polymer-rich phase and a polymer-poor phase. The polymer-rich phase solidifies and the polymer poor phase is removed, leaving a highly porous polymer network [17]. The micro- and macro-structure of the resulting scaf‐ folds are controlled by varying process parameters such as polymer concentration, quench‐ ing temperature, and quenching rate. The process is conducted at low temperatures, which is beneficial for the incorporation of bioactive molecules in the structure. Using phase sepa‐ ration techniques, nano-scale fibrous structure enables to be formed, which mimics natural extracellular matrix architecture and provides a better environment for cell attachment and

Melt molding involves filling a mold with polymer powder and a porogen component and then heating to above the glass-transition temperature of the polymer while applying pres‐ sure to the mixture [19]. During the fabrication process, the raw materials will bind together to form a scaffold with designed specified external shape. Once the mold is removed, the

**2.1. Conventional fabrication techniques**

316 Advances in Biomaterials Science and Biomedical Applications

Polymeric porous scaffolds can be prepared by freeze drying. In the freezing stage, the poly‐ mer solution is cooled down to a certain temperature at which all materials are in a frozen state and the solvent forms ice crystals, forcing the polymer molecules to aggregate into the intersti‐ tial spaces. In the second phase, the solvent is removed by applying a pressure lower than the equilibrium vapor pressure of the frozen solvent. When the solvent is completely sublimated, a dry polymer scaffold with an interconnected porous microstructure remains [20, 21]. The po‐ rosity of the scaffolds depends on the concentration of the polymer solution; pore size distribu‐ tion is affected by the freezing temperatures. Apart from fabricating porous scaffolds, this technique is also used to dry biological samples to protect their bioactivities [22].

**Figure 1.** Schematic of conventional scaffold fabrication techniques: (a) solvent-casting and particulate-leaching proc‐ ess: A polymer solution is cast into a mold filled with porogen particles, then the solvent is allowed to evaporate and the porogen is leached out; (b) gas foaming process: Polymer samples are exposed to high pressure allowing satura‐ tion of the gas into the polymer; the subsequent gas pressure reduction causes the nucleation of bubbles; (c) phase separation process: A thermodynamical instability is established in a homogeneous polymer solution that separates into a polymer-rich and a polymer-poor phase; (d) melt molding process. A mold filled with polymer powder and po‐ rogen component is heated to above the polymer glass-transition temperature (Tg) and a pressure (P) is applied to the mixture. The porogen is then leached out, leaving a porous structure; (e) freeze drying process: A polymer solution is cooled down, leading to the formation of solvent ice crystals. Then the solvent is removed by using a pressure lower than the equilibrium vapor pressure of the solvent (P° solution), leaving a porous structure. (Modified from [23])

#### **2.2. Advanced biofabrication techniques**

#### **1.** Electrospinning

Electrospinning is a fabrication technique utilizing electrical charges to draw fine fibers up to the nanometer scale. The technique was invented by Cooley and Morton in 1902. The fi‐ ber electrospinning can also be traced back to the 1930s [24]. In the past decade, significant developments in electrospinning have allowed for creation of scaffolds with different mate‐ rials and, hence, this technique has gained a high popularity in tissue engineering research. Nanofibrous architectures are known to modulate effects on a wide variety of cell behaviors. Nanofibrous architectures can positively affect cell binding and spreading compared to mi‐ cropore and microfibrous architectures (Figure 2). Nanofibrous scaffold architectures have larger surface areas to adsorb proteins than micro-architectures, presenting more binding sites to cell membrane receptors [25]. The exposure of additional cryptic binding sites may also be affected by adsorbed proteins. Furthermore, cells growing in a 3D nanofibrous struc‐ tural environment are able to exchange nutrients and utilize receptors throughout their sur‐ face, while cells in flat culture conditions are limited to nutrient exchange on only one side. Electrospinning techniques have been widely employed to fabricate porous scaffolds with nanofibrous architectures that can mimic the structure and biological functions of the natu‐ ral extracellular matrix [26]. This technique is able to generate fibers with diameters ranging from 2 nm to several micrometers using solutions of both natural and synthetic polymers, with small pore sizes and high surface area to volume ratios. A typical electrospinning setup includes three parts: a syringe pump containing the polymeric materials, a high voltage source to generate high electric field for spinning, and a collector to collect the fibers [27] (Figure 3). During scaffold fabrication, the following electrospinning parameters are very important with respect to the fiber morphology: polymer solution parameters (viscosity, molecular weight of polymer, polymer conductivity, surface tension), processing parameters (applied voltage, distance between tip and collector, flow rate), and environment parame‐ ters (humidity, temperature). Nanofibers with high surface area to volume ratios are most suitable for tissue engineering applications [28].

**Figure 2.** Scaffold architecture affects cell binding and spreading. (Modified from [25])

**Figure 3.** Schematic of electrospinning apparatus. (Modified from [29])

#### **2.** Rapid prototyping

Nanofibrous architectures are known to modulate effects on a wide variety of cell behaviors. Nanofibrous architectures can positively affect cell binding and spreading compared to mi‐ cropore and microfibrous architectures (Figure 2). Nanofibrous scaffold architectures have larger surface areas to adsorb proteins than micro-architectures, presenting more binding sites to cell membrane receptors [25]. The exposure of additional cryptic binding sites may also be affected by adsorbed proteins. Furthermore, cells growing in a 3D nanofibrous struc‐ tural environment are able to exchange nutrients and utilize receptors throughout their sur‐ face, while cells in flat culture conditions are limited to nutrient exchange on only one side. Electrospinning techniques have been widely employed to fabricate porous scaffolds with nanofibrous architectures that can mimic the structure and biological functions of the natu‐ ral extracellular matrix [26]. This technique is able to generate fibers with diameters ranging from 2 nm to several micrometers using solutions of both natural and synthetic polymers, with small pore sizes and high surface area to volume ratios. A typical electrospinning setup includes three parts: a syringe pump containing the polymeric materials, a high voltage source to generate high electric field for spinning, and a collector to collect the fibers [27] (Figure 3). During scaffold fabrication, the following electrospinning parameters are very important with respect to the fiber morphology: polymer solution parameters (viscosity, molecular weight of polymer, polymer conductivity, surface tension), processing parameters (applied voltage, distance between tip and collector, flow rate), and environment parame‐ ters (humidity, temperature). Nanofibers with high surface area to volume ratios are most

suitable for tissue engineering applications [28].

318 Advances in Biomaterials Science and Biomedical Applications

**Figure 2.** Scaffold architecture affects cell binding and spreading. (Modified from [25])

As an alternative to conventional scaffold fabrication methods, a group of techniques based on rapid prototyping (RP) has recently been introduced within the tissue engineering field. RP techniques, based on computer assisted design (CAD) and manufacturing (CAM) techni‐ ques, allow for better control of scaffold internal microstructure and external macroshape compared to conventional fabrication techniques [4, 30]. Three basic RP system types: liq‐ uid-based, solid-based, and powder-based can be selected based on the properties of differ‐ ent scaffold biomaterials. The primary RP processes applied to tissue scaffold fabrication include stereolithography (SLA) [31], selective laser sintering (SLS) [32], fused deposition modeling (FDM) [33], three dimensional (3D) printing [34], and 3D plotting [35]. The choice of materials for the RP techniques includes various polymers, ceramics, and metals. Recent‐ ly, RP techniques have also demonstrated their capacity for embedding living cells [36, 37] and growth factors [38] into scaffolds during the fabrication process and thus their utility for creating biomimetic tissue scaffolds.


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