*4.3. Cranio-Maxillofacial Tissues*

Craniofacial bones and cartilages comprise the craniofacial skeleton that impart specific appearance and function. It is challenging to reconstruct craniofacial structures due to the complex 3D geometry. With the combination of image-based extraction of craniofacial geometry and the ability to 3D-print shapes with high fidelity, 3D printing technologies are ideally suited for the manufacture of bone and cartilage scaffolds tailored to specific defects. The goal of craniofacial 3D reconstruction is to mimic the external and internal architecture of the host site and to provide essential framework for cell attachment and migration.

Bone is considered the second most transplanted tissue for defects due to trauma, osteoporosis, bone tumors, etc. Many types of biomaterials have been proposed to integrate the desirable properties, such as biocompatibility, printability, osteoconductivity, osteoinductivity, and mechanical properties, attempting to mimic the natural replacement of bone.

Bioceramics are the most commonly selected materials. They are usually composed of calcium and phosphate mineral phases, such as HA, β-tricalcium phosphate (TCP), or bioactive glasses (BGs). They exhibit outstanding biocompatibility and favorable biodegradability. Moreover, 3D printed ceramics can upregulate osteogenesis by creating a bioactive ion-rich cellular micro-environment and promote cell proliferation by close cell-cell interactions [74].  Saijo and colleagues have confirmed this disadvantage by using HA/α -TCP composite scaffolds for maxillomandibular defects, which showed difficulties in composition and fabrication of an ideal scaffold to fulfill strength and dimensional requirements [75]. Still, Shao and colleagues have recently reported that ~10% Mg-substituted wollastonite had much higher flexural strength (31 MPa) than TCP and other calcium-silicate porous bioceramics [76]. By adding a range of metallic ions like Cu2+ and Co2+ into BGs, the angiogenic activity in vivo can be developed, which is beneficial for the healing process [77]. Compared to being used alone, they are more commonly incorporated with other biomaterials such as polymers for the enhancement of osteogenesis and osteoinductivity.

Polymers are another widely-used material that is superior in its printability and efficiency in promoting osteogenesis. The main concerns are its poor cellular interaction and low stiffness. PLA and PGA, for example, are now rarely used for bone scaffolds considering their low compressive strength and osteoconductivity. However, their co-polymer PLGA and another polyester, PCL, have remarkable osteoconductivity and better mechanical properties. By comparison, PCL has a lower rate of degradation and subsequently denser tissues generated [78]. Therefore, it is preferred to be used as the framework of composite scaffolds. As PCL is bioinert, other biological active components such as TCP, HA, decellularized trabecular bone, or growth factors were incorporated into the 3D printing system [21,79,80]. Furthermore, the acidic environment caused by the degradation products of PCL and its hydrophobic nature can be somewhat diminished by the inclusion of hydrophilic polymers like PEG and the surface coating of natural polymers like chitosan [81,82]. PCL scaffolds are well-suited for extrusion-based 3D-printing (Figure 4A), FDM for example, due to the relatively low melting points (62 ◦C). In recent decades, SLS has been developed as a more precise 3D printing technique that can fine-tune the porosity to optimize conditions for cell growth and proliferation. Additionally, a wide range of thermoplastic materials including high performance plastics with specific mechanical properties can be processed by SLS technique. The stiffness of PCL scaffolds manufactured by SLS has been reported to be ~15 to 300 MPa, values that are much higher than conventional 3D

polymers but still lower than human trabecular bone within the condyle (120–450 MPa) or within the mandibular body (112–910 MPa) [22]. Metal-based sca ffolds, on the other hand, are sti ff enough but possess a considerably higher Young's modulus, which would lead to stress shielding issues and therefore failure of the implants. Polyaryletherketones (PEAKs) is a family of high-performance polymers with compatible Young's modulus to natural bone, which would be a suitable property for load bearing orthopedic and craniofacial implants. PEKK is by far a material with the most advantageous performance in the PEAKs family. The PEKK printed by SLS platforms have showed desirable mechanical properties, great biocompatibility, and osteointegration in a craniofacial bone defect model in vivo [18,27,28]. As a promising 3D printing material for bone tissue engineering, more evidence of success is needed for future applications.

The cartilaginous tissues in the craniofacial area primarily include the temporomandibular joint (TMJ) disc, the auricle cartilage, and the nasal cartilage. The bioinks used for cartilage reconstruction should be able to mimic the 3D architecture with mechanical anisotropic, nonlinear, and viscoelastic behavior analogous to native cartilage.

In early approaches, many hydrogels encapsulating the chondrocytes/mesenchymal stem cells (MSCs) with the capability of synthesizing extracellular matrix were fabricated by micro-extrusion technique. The cell-laden hydrogels ranged from natural polymers like alginate and collagen to synthetic polymers like gelatin metacrylamide (GelMA) and polyethylene glycol dimethacrylate (PEGDMA) [83–86]. To improve the mechanical properties, higher polymer concentration and viscosity were preferred. On the other hand, cells proliferate and di fferentiate towards cartilage tissue more readily within lower polymer concentrations. This dilemma increased the challenge of using the hydrogels alone to reconstruct the cartilaginous tissue. The most common solution is to include a sti ffer thermoplastic polymer such as PCL to cell-laden hydrogels by coextrusion or other hybrid strategies (Figure 4B). PCL acts as a frame to reinforce the constructs, and by modulating the polymer percentage, the compressive equilibrium moduli in the range of articular cartilage can be achieved [87–89].

The degradation rate of PCL, which can be up to 2–3 years, is a potential limitation with such multi-material approaches, as residual filaments can act as a barrier to tissue formation. One alternative is applying polymers that have a higher rate of degradation, poly (hydroxymethylglycolide-co-caprolactone) (PHMGCL) or PLGA, for instance [91]. Yet the acidic by-products of PLGA that cause adverse inflammatory response still remains a concern for future applications. Tarafder and colleagues developed a region-variant TMJ disc sca ffold by incorporating the specifically-aligned PCL with PLGA microspheres encapsulating TGFβ3 [92,93]. After seeding with MSCs, multiphase fibrocartilaginous tissues formed and significantly improved the healing process of the perforated disc. The dynamic function was also restored as no arthritis changes were observed on the condyle four weeks post-implantation.

Another way to reduce the residual PCL materials is to increase its porosity by the melt-electrowriting (MEW) technique, which is similar to FDM but using a nozzle tip equipped with voltage. PCL fabricated by MEW can be very thin, with a diameter down to 0.8 μm, and therefore the porosity can be high, up to 93%–98% [94]. In addition, the sti ffness and yielding strains of the resultant sca ffolds were within the range of native cartilage.

**Figure 4.** Craniofacial bone and cartilage reconstruction using PCL as a material for 3D printing. (**A**) Mandibular bone reconstruction. 3D defect model was obtained from the craniofacial CT image data followed by the design of dispensing paths of cells, PCL, and Pluronic F-127 with self-developed software. Multiple cartridges used to deliver and pattern the above ink materials were connected to a microscale nozzle, which dispensed the materials according to the design during 3D printing process. PCL was printed as the framework and the cell-laden hydrogel were dispensed to fill the pores, while Pluronic F-127 were used as sacrificing materials. The osteogenic potential of the scaffold was confirmed by Alizarin Red S staining after being cultured in osteogenic medium for 28 d. (**B**) Auricle cartilage construction. Similarly, a 3D computer-assisted (CAD) model of auricle can be developed from CT or MRI image data and generate a visualized motion program consisting of a command list for XYZ stage movements and air pressure actuation for 3D printing. The concentrations of different ingredients for 3D printing can be optimized by in vitro culture and related tests. Reproduced with permission from [90].