*4.1. Periodontal Complex*

The concept of using 3D printing for periodontal tissue regeneration is to guide locally available cells to restore periodontal defects, termed guided tissue regeneration (GTR) [59]. These cells can use the support of a 3D printed scaffold, as well as surrounding growth factors, bioactive proteins, etc., to regenerate damaged periodontal tissues [60]. While epithelial tissues regenerate quickly, bone tissues require more time, and therefore a difficulty in periodontal regeneration lies in its tissue complexity (cementum, periodontal ligament, etc.) [59]. To address these challenges, the creation of multiphasic scaffolds has allowed for various properties within a scaffold, which mimics better the composition of the native periodontal complex [59].

PCL scaffolds have been 3D printed and used for periodontal tissue engineering. Improvements to these scaffolds have focused on several key aspects, as stated by Ivanovski and colleagues, relating to periodontal tissue engineering: (1) compartmentalized bone and periodontal attachment tissue formation; (2) cementum formation onto the root surface; (3) correctly oriented periodontal ligament fibers [61]. Lee and colleagues, for instance, 3D printed PCL-HA scaffolds with varying sizes of microchannels to create a compartmentalized multiphasic scaffold [62]. FDM was used to create these scaffolds, which had 100 μm microchannels designed for the cementum/dentin interface, 600 μm for the periodontal ligament (PDL), and 300 μm for the alveolar bone [62]. They found that in vivo implantation of dental pulp stem cells with the scaffold resulted in differentiation of the cell population into putative dentin/cementum, PDL, and alveolar bone [62]. Similarly, Li and colleagues used a freeze-dried PRP coating to improve the biological properties of PCL scaffolds [31]. This coating was applied to the 3D printed PCL scaffold for 5 min at room temperature, then placed at −80 ◦C for 30 min, followed by freeze-drying [31]. The freeze-dried PRP-PCL scaffolds induced significantly greater bone formation compared to traditional PRP-PCL or bare PCL scaffolds [31].

Bioprinting of PDL cells, creating a 3D hydrogel microarray, has been performed to screen for cell-biomaterial interactions [34]. The cells were bioprinted using a pressure-assisted valve-based

bioprinting system placed within a sterile hood and controlled by a computer [34]. The pressure-based system replaces the need for any external stimulation, and thereby minimizes shear forces and high temperatures, allowing for the cells to survive the printing process [34]. This study has found that the viability of the printed periodontal cells was maintained throughout printing, and therefore this methodology can perhaps be used to 3D print periodontal cells directly into future scaffolds [34]. Likewise, Hamlet and colleagues examined alveolar bone regeneration through 3D hyaluronic acid hydrogels containing osteoblasts and found that their hydrogel provided a favorable environment and could stimulate osteogenic gene expression in vitro: they believed that this hydrogel could be optimized as a cell-delivering bioink for future 3D bioprinting applications [63].

The combination of cell sheets with 3D printed scaffolds has also been used for periodontal tissue regeneration. Vaquette and colleagues cultured PDL cell sheets in 24-well plates, which were then combined with an FDM printed PCL scaffold by folding the cell sheet over the scaffold (Figure 2A–C) [64]. They found that the scaffolds incorporating the cell sheet technology had better attachment onto a dentin surface than those without [64]. Farag and colleagues, however, aimed to improve the cell sheet technology by decellularizing the cell sheet after combination with the PCL scaffold (Figure 2D,E) [65]. The decellularization aimed to use the properties of the PDL extracellular matrix to promote periodontal regeneration, while minimizing the immunogenic effects of cellular material. They found that the decellularized cell sheet constructs upregulated the expression of mineralized tissue markers in PDL cells [65]. As an application of scaffold free bioprinting, Bakirci and colleagues developed a novel cell sheet based bioink for 3D bioprinting [66]. Cells were first grown on poly(N-isopropylacrylamide) coated surfaces, harvested, and centrifuged into cell sheet aggregates to be used for bioprinting applications [66]. Although this study developed a cell sheet based bioink using human skin fibroblasts, it illustrates the possibility in developing a PDL cell sheet based bioink, which could then be used for periodontal tissue engineering applications such as scaffolds in treating periodontal-related cases.

Clinically, Rasperini and colleagues reported the use of a SLS printed PCL scaffold to treat a periodontal defect (Figure 2F,G) [21]. A computed tomography scan of the patient's defect was taken to modify the scaffold design and to create a customized scaffold [21]. This scaffold consisted of an internal port for growth factor delivery and pegs perpendicular to the root to facilitate PDL formation [21]. At two weeks, the scaffold was removed and unfortunately the patient showed minimal evidence of bone repair [21]. However, this case highlighted a potential for the use of 3D printed scaffolds in treating periodontal-related cases.
