*2.1. Inkjet Printing*

The principle behind inkjet printing consists of introducing a small volume change upstream of the nozzle, creating a pressure change, which results in a droplet ejection downstream (Figure 1A). This is performed by an inkjet head system which can be either piezoelectric or thermal induced. Thermal induced heads use a resistor as their heating structure. When current passes through the resistor, the fluid in contact is vaporized, creating a bubble that expands in the reservoir. This increases the pressure causing a droplet ejection through the nozzle [8]. Piezoelectric heads cause the volumetric change by applying a voltage pulse to the piezoelectric material [9].

**Figure 1.** Schematic of various 3D printing methodologies. (**A**) Inkjet. A heater or piezo actuator deposits droplets. (**B**) Stereolithography. Layer by layer photopolymerization of a liquid resin by laser. (**C**) Laser induced forward transfer. Droplets of the material induced by a laser source. (**D**) Extrusion. Material exiting a nozzle that is pneumatic, piston, or screw driven. Reproduced with permission from [7].

A wide range of powder materials such as polymers, ceramics, proteins, and cells can be processed using this technique. However, the ink's viscosity is limited to 5–20 Pa.s to avoid high ejection pressures or continuous flow of the material. The main advantages of this technique include high-speed printability, low cost, and the possibility to encapsulate cells in the material [10].

For bone regeneration, bioink patterns using inkjet printing have been studied to control osteoblast differentiation in vitro and bone formation in vivo. A study created patterns of bone morphogenetic protein-2 (BMP-2) within microporous scaffolds (made from DermaMatrix, containing various extracellular molecules such as collagen and fibronectin) and observed cell differentiation in vitro and tissue formation in vivo in the patterned areas [11]. Additionally, another study used inkjet printing to 3D print calcium phosphate scaffolds and incorporated a collagen coating. The implanted scaffolds were osteoconductive while being biodegradable [12].

### *2.2. Laser-Assisted 3D Printing*

Although less commonly used, laser printing technology has emerged from LIFT technology as a promising method for tissue engineering. It has prominent advantages in terms of bioprinting and is also known as laser-assisted bioprinting (LAB). LIFT assisted printers or LAB basically have three main components: (1) a pulsed laser source, (2) a target serving as a support for the printing materials, usually a transparent glass slide or ribbon, and (3) a receiving substrate to collect the materials. During printing, a focused laser pulse stimulates a small area of the target, which comprises an energy-absorbing layer on the surface and bioink solution underneath. Then a portion of the energy-absorbing layer is evaporated, resulting in the formation of a droplet that is collected by the receiving substrate and crosslinked therein [13,14].

Unlike inkjet printers, laser-assisted printers are equipped with no nozzles, obviating direct contact between the dispenser and the bioinks and therefore minimizes the problem of materials/cells clogging. With that, they are compatible with more materials, especially those with high viscosity (1–300 mPa/s), and can maintain cell viability higher than 95% [15]. Those benefits along with products with higher resolution makes laser-assisted printing a promising technology for tissue engineering.

Variations of laser-assisted 3D printing include selective laser sintering (SLS), stereolithography (SLA) (Figure 1B) and LIFT (Figure 1C). In particular, SLS has been extensively used for the regeneration of tissue with complex anatomy like craniofacial bone or cartilage. Developed by Carl Deckard for his Master's thesis at the University of Texas in 1989, SLS uses a high-powered carbon dioxide laser to create structures by fusing the powder layer-by-layer with the underlying powder as support [16]. The laser beam fuses powders selectively on the basis of sectional data from computer-assisted design (CAD). After a layer is created, the powder bed descends and another layer is rolled over. Such process is repeated until the scaffold is completed [17]. SLS can produce tissue engineering scaffolds from a variety of powder materials, including metals, bio-ceramics, and synthetic polymers like polylactic acid (PLA), PCL, poly ethyl ether ketone (PEEK), and poly ether ketone ketone (PEKK) [18,19]. Some researchers also include HA powders in the polymers to increase the osteoinductivity of bone scaffolds [20]. Natural polymers cannot be utilized with this technique due to the high temperatures generated by the laser during printing. However, similar to growth factors, they can be incorporated into the scaffolds post-printing.

PCL is an advantageous material in SLS, mainly because of its low melting (59–64 ◦C) and glass-transition temperatures (−60 ◦C) that facilitate the prototyping process. SLS-printed PCL scaffolds have been used to repair the periodontal [21], craniofacial bone, or osteochondral defects [22–24], and were proved to be biocompatible with adequate strength. Another polymer that has been recently applied in craniofacial regeneration using the SLS technique is PEEK [19], which has more favorable mechanical properties for stress loading than PCL. The manufacturing strategy, however, is not much different from that of PCL scaffolds. Higher performance polymers like PEKK have also been successfully printed by SLS technique [25,26] and introduced in the application of the craniofacial area [27,28].

Nowadays, no polymer processing methods can compete with the SLS regarding the fabrication flexibility and complexity of the 3D shapes obtained. However, the advancement of laser-assisted technologies is obviously restricted by the complicated control of the laser printing system and concerns about the side e ffect of laser exposure.
