Three-dimensional (3D) printing is an emerging technology in the field of dentistry. It uses a layer-by-layer manufacturing technique to create scaffolds that can be used for dental tissue engineering applications. While several 3D printing methodologies exist, such as selective laser sintering or fused deposition modeling, this paper will review the applications of 3D printing for craniofacial tissue engineering; in particular for the periodontal complex, dental pulp, alveolar bone, and cartilage. For the periodontal complex, a 3D printed scaffold was attempted to treat a periodontal defect; for dental pulp, hydrogels were created that can support an odontoblastic cell line; for bone and cartilage, a polycaprolactone scaffold with microspheres induced the formation of multiphase fibrocartilaginous tissues. While the current research highlights the development and potential of 3D printing, more research is required to fully understand this technology and for its incorporation into the dental field.

For salivary gland (SG) tissue engineering, we cultured acinar NS-SV-AC cell line or primary SG fibroblasts for 14 days in avian egg yolk plasma (EYP). Media or egg white (EW) supplemented the cultures as they grew in 3D-Cryo histology well inserts. In the second half of this manuscript, we measured EYP’s freeze-thaw gelation and freeze-thaw induced gelled EYP (_GEYP), and designed and tested further _GEYP tissue engineering applications. With a 3D-Cryo well insert, we tested _GEYP as a structural support for 3D cell culture or as a bio-ink for 3D-Bioprinting fluorescent cells. In non-printed EYP + EW or _GEYP + EW cultures, sagittal sections of the cultures showed cells remaining above the well’s base. Ki-67 expression was lacking for fibroblasts, contrasting NS-SV-AC’s constant expression. Rheological viscoelastic measurements of _GEYP at 37 °C on seven different freezing periods showed constant increase from 0 in mean storage and loss moduli, to 320 Pa and 120 Pa, respectively, after 30 days. We successfully 3D-printed _GEYP with controlled geometries. We manually extruded _GEYP bio-ink with fluorescence cells into a 3D-Cryo well insert and showed cell positioning. The 3D-Cryo well inserts reveal information on cells in EYP and we demonstrated _GEYP cell culture and 3D-printing applications.

The egg yolk plasma (EYP)—a translucent fraction of the egg yolk (EY) obtained by centrifugation—was tested as a developmentally encouraging, cost-effective, biomaterial for salivary gland (SG) tissue engineering. To find optimal incubating conditions for both the human NS-SV-AC SG acinar cell line and SG fibroblasts, cells were stained with Live/Dead^®. The cellular contents of 96-well plates were analyzed by high content screening image analysis. Characteristically, the EYP biomaterial had lipid and protein content resembling the EY. On its own, the EYP was non-conducive to cell survival. EYP’s pH of 6 mainly contributed to cell death. This was demonstrated by titrating EYP’s pH with different concentrations of either commercial cell culture media, NaOH, or egg white (EW). These additives improved SG mesenchymal and epithelial cell survival. The best combinations were EYP diluted with (1) 70% commercial medium, (2) 0.02 M NaOH, or (3) 50% EW. Importantly, commercial medium-free growth was obtained with EYP + NaOH or EYP + EW. Furthermore, 3D cultures were obtained as a result of EW’s gelatinous properties. Here, the isolation, characterization, and optimization of three EYP-based biomaterial combinations are shown; two were free of commercial medium or supplements and supported both SG cells’ survival.

The Ambystoma maxicanum (axolotl) regenerates strikingly from wounds and amputations. Comparing its healing ability to non-regenerative species such as the mouse should help narrow in on mechanisms to improve human wound healing. Here, the tongue and intermandibular soft tissues of both mice (C57BL/6NCrl) and axolotls were wounded with a 2–2.5 mm punch biopsy. The study aimed to compare the differences between these 2 species following surgical resection with regard to the macroscopic and histological characteristics. These include wound closure times, epithelial wound sealing and thickness as well as acute immune marker myeloperoxidase (MPO) response over 30 days. Post surgery, mice visually showed greater haemorrhage; their wounds immediately collapsed while it took 14 days for the axolotls mandibular void to close. The epithelium sealed the axolotls’ wound margins within 24 h with a maximal mean thickness of 0.42 ± 0.13-fold normalized to unwounded skin. In mice, the epithelium separately sealed the ventral and dorsal sides, respectively at 7 and 7–30 days with mean maximal epithelial thicknesses reaching 13 ± 5.6 and 3.0 ± 0.63-fold. Mean MPO-positive cell values peaked in axolotls at 14 ± 1.5-fold between hours 6–12; while in mice, it peaked at 8.7 ± 0.9-fold between hours 24–96. We conclude that axolotls form smaller blood clots, have a faster and thinner epithelial cell migrating front, and a shorter MPO-positive cell response in comparison to mice. These observations may help refine future oral and facial wound-healing research and treatment.