*3.2.1 Pectin-based sponges*

Sponges are comparable to foams with an interconnected network of pores. This type of architecture is beneficial for cell penetration and scaffold colonization, while ensuring adequate diffusion of nutrients to cells within the scaffold. Moreover, a highly porous scaffold with open and connected pores is of critical importance as it allows for the diffusion of nutrients and waste products through the scaffold [6, 7].

Pectin-based sponges are mainly obtained by freeze-drying, also known as lyophilization. This technique consists in freezing a polymer solution followed by the evaporation of the frozen solvent by sublimation. Thus, a solid polymer matrix with numerous and interconnected pores is obtained (**Figure 5**). Before freezing, polymer solutions are generally poured into molds to produce porous scaffolds with the desired shape.

Pectin-based sponges have been principally used to produce scaffolds for wound healing and tissue regeneration. For example, sponges obtained with pectin-based formulations have been used as scaffolds for different types of tissues including cartilage [40, 41], skin [42], and bone [43]. The high hydrophilicity of pectin molecules and the interconnected porosity enables these sponges to entrap a large volume of water creating a 3D hydrogel-based environment that can mimic the natural extracellular matrix [40, 41]. Furthermore, this provides and stabilizes a moist environment for wounds that strongly contributes to accelerating the healing of the wounds [44].

#### **Figure 5.**

*Schematic of the process for obtaining cylindrical porous sponges was obtained by freeze-drying. Magnification of the porous sponges obtained by scanning electron microscopy (image created with BioRender.com).*

### *3.2.2 Complex shaped pectin-based scaffolds*

Producing scaffolds with a customized architecture and by automated and high reproducible approaches is one of the main challenges of TE. The development of pectin-based scaffolds with patient-specific architecture may boost their clinical applications.

Pectin-based scaffolds with complex shapes have been principally obtained by extrusion-based bioprinting so far. Extrusion-based bioprinting is one of the most widely used technology in TE due to its simplicity and versatility in processing a large variety of biomaterials, cells and biomolecules. An extrusion-based bioprinter usually consists of a movable cartridge containing the biomaterial formulation (called '*ink*') and of a movable deposition stage (**Figure 6**). Before bioprinting, the architecture of the scaffolds can be designed by a computer-aided design (CAD) software, or it can be derived from patient medical images acquired, for example, by computed tomography scans or magnetic resonance imaging. The 3D model of the scaffold is subsequently sliced by a computer-aided manufacturing (CAM) software in bioprinting paths and finally converted to a printable code file (called '*G-code*') [45, 46]. During the bioprinting process, the ink is extruded onto the deposition stage following the preprogrammed paths contained in the G-code, in a layer-by-layer process.

The application of pectin-based inks in extrusion-based bioprinting is relatively recent compared to the other fabrication approaches described in the previous sections. Pectin solutions are often not suitable to be processed through extrusion-based bioprinting and structures with poor shape fidelity are often obtained. The first application of pectin as ink for extrusion-based bioprinting dates back to 2017. In this case, pectin was combined with another biopolymer (Pluronic F-127), and complex-shaped scaffolds were bioprinted [47, 48]. Cells were successfully loaded within this formulation and 3D bioprinted to produce living 3D constructs [24]. From that moment, other pectin-based inks have been developed and optimized to produce 3D scaffolds with high shape fidelity [49–51]. For example, pectin-based scaffolds with more complex shapes such as a human ear and nose shape for cartilage tissue regeneration were successfully obtained (**Figure 6**) [41].

**Figure 6.** *Schematic of extrusion-based bioprinting.*
