*4.2. Dental Pulp*

The dental pulp is an unmineralized tissue beneath the mineralized hard exterior of the tooth, which plays a crucial role in tooth vitality; injury, alteration, and/or removal of the pulp may lead to tooth necrosis. Additionally, the pulp may also service to provide immunity, nutrition, and sensation as well [50]. As a result, there is a need to focus on protecting the pulp from trauma, and to regenerate the pulp should it be injured. While there has not been major success in pulp regeneration due to the challenges met in nurturing, revascularizing, and reinnervating the pulp tissue, a promising direction that researchers are exploring is the use of hydrogels to contain and nurture dental pulp cells [68]. By using hydrogels and other biomaterials as cellular scaffolds to mirror the native in vivo environment, researchers can promote cell growth, differentiation, and morphogenesis [50]. However, the major constraint with using hydrogels alone is that spatial manipulation is limited, i.e., researchers cannot fully control multicellular organization and interaction, thus the overall morphogenesis of the artificial gland or tissue. By applying the superior spatial control of 3D cell printing, this issue can be overcome in pulp tissue regeneration [69]. 3D cell printing technology would enable researchers to suspend and position various cells contained in hydrogels as they desire. For example, researchers could print odontoblastoid cells along the dentin walls while having fibroblasts towards the center of the pulp chamber [69]. Furthermore, the enhanced precision gained from 3D bioprinting would

allow researchers to achieve specific cellular interactions, anisotropic mechanical properties, and desired distribution of growth factors [70]. While theoretically the use of 3D cell printing in pulp tissue regeneration sounds feasible, there is a lack of evidence for this to date. Several studies have shown the possibility of successfully 3D printing blood capillaries, however in vivo angiogenesis has not been exhibited in endodontics [37,69].

**Figure 2.** 3D Printed scaffolds for periodontal tissue engineering. (**A**–**C**) Schematic of the scaffold fabrication methodology (**A**). Cross-section showing the fusion of the electrospun fibers with the fused deposition modeling (FDM)-printed compartment of the scaffold (**B**,**C**). (**D**,**E**) Electron spun polycaprolactone (PCL) scaffold (**D**). The PCL scaffold attached to a decellularized sheet (**E**). (**F**,**G**) selective laser sintering (SLS)-printed PCL scaffold to be implanted in patient (**F**). Scaffold placement for implantation (**G**). Reproduced with permission from [21,65,67].

While there is a lack of in vivo studies to date, there are several studies that highlight the potential use of 3D cell printing in dental pulp regeneration. For example, in a study by Athirasala and colleagues, they showed that the mouse odontoblast-like cell line (OD21) could be supported in a novel hydrogel composed of alginate and dentin (Alg-Dent) [71]. While this study did not address more complicated experiments such as the possibility of vasculogenesis and/or angiogenesis, the use of human cells, and cell survivability in root canals and chamber, the study demonstrated the tunability and printability of the scaffold using 3D bioprinting technology. The success of this paper indicated the potential of the scaffold and how 3D bioprinting could further enhance the feasibility of this hydrogel in regenerative

endodontics. Specifically, researchers would be able to localize growth factors and other nutrients precisely to the desired targets such as the peripheral dentin or central pulp to induce cell-specific regeneration as evident in this study.

As previously mentioned, 3D bioprinting methods allow researchers to achieve superior tunability, creating sca ffolds that would not be possible without it. For example, in a recent study by Feng and colleagues, they compared two di fferent techniques in fabricating a PLA sca ffold, either molding via standard extrusion processes or 3D printed, and its influence on dental pulp cells. The results indicated that manufacturing techniques can influence di fferences in cell migration, morphology, and di fferentiation marker expression [72]. Another study by Hu and colleagues used 3D printed molds to create cellularized conduits for peripheral nerve regeneration, which showed comparable results to the use of autografts in repairing peripheral nerve defects (Figure 3) [73]. Thus, further studies should be explored, comparing current sca ffold manufacturing methods and 3D printing and its e ffects on vasculogenesis and angiogenesis in support of pulp regeneration.

**Figure 3.** Cellularized conduits for peripheral nerve regeneration created using 3D printed molds. (**A**) Schematic of the conduit fabrication method. (**B**) Photographs of the rat dorsal side with the biodegradable nerve guidance conduit positioned subcutaneously. The figures are © 2016, Hu Y., Wu Y., et al. (https://doi.org/10.1038/srep32184) used under a Creative Commons Attribution 4.0 International License: http://creativecommons.org/licenses/by/4.0/.

Though there are not many studies to date highlighting the use of 3D printing in pulp regeneration, the current studies that demonstrate the superior tunability and modifications in the mechanical properties of currently viable scaffolds using 3D printing indicates the potential it may have in pulp regeneration. Further studies need to be explored by implementing the results, knowledge, and support gained from current studies in the possibility of inducing vasculogenesis, angiogenesis, and nutritional support in pulp tissue using 3D printing techniques.
