3D Cell Culture of Human Salivary Glands Using Nature-Inspired Functional Biomaterials: The Egg Yolk Plasma and Egg White.

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.

3D Cultures of Salivary Gland Cells in Native or Gelled Egg Yolk Plasma, Combined with Egg White and 3D-Printing of Gelled Egg Yolk Plasma.

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 Applications of 3D Printing for Craniofacial Tissue Engineering.

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.

3D Culture Histology Cryosectioned Well Insert Technology Preserves the Structural Relationship between Cells and Biomaterials for Time-Lapse Analysis of 3D Cultures.

When performing histology of softer biomaterials, aspiration disrupts the cellular and molecular location information. This study aims to develop a cryosectionable well insert able to preserve the biomaterial and cell's original 3D conformation from the well to histology analysis. The well insert is composed of a paraffin-coated gelatine pill. Within the coated capsule, the human epithelial cell line (NS-SV-AC) is cultured in Matrigel, GrowDex, Myogel, Myogel + GrowDex, or cell culture media for 14 days. At 0 and 14 days, the samples are frozen in liquid nitrogen and cryotome is used to create sections. The slides are stained by Sirius Red and immunohistochemistry using antibodies human collagens I-V and human Ki-67. Sirius Red shows pink shades of biomaterials and the best cellular vertical distribution throughout the sagittal section of the well is achieved with Matrigel, GrowDex, and Myogel + GrowDex; in Myogel and media, the cells sink. For collagen protein expression, only Matrigel induces a notable difference while in the other materials, collagen staining is weak or difficult to distinguish from endogenous collagens. Ki-67 expression is maintained over time. The 3D-cryo well insert provides a new time-lapse histology perspective of analysis for liquid or gel cultures that maintains cells and macromolecules in their unaltered in-well configuration.

Three-dimensionally printed polyetherketoneketone scaffolds with mesenchymal stem cells for the reconstruction of critical-sized mandibular defects.

OBJECTIVE: Additive manufacturing offers a tailored approach to tissue engineering by providing anatomically precise scaffolds onto which stem cells and growth factors can be supplied. Polyetherketoneketone (PEKK), an ideal candidate biomaterial, is limited by a poor implant-bone interface but can be functionalized with adipose-derived stem cells (ADSC) to promote integration. This in vivo study examined the interaction of a three-dimensional printed PEKK/ADSC implant within the critical-sized mandibular defect in a rabbit model. STUDY DESIGN/METHODS: Trapezoidal porous scaffolds with dimensions of 1.5 × 1.0 × 0.5 cm were printed using selective laser sintering. ADSCs were seeded on the scaffolds that were then implanted in marginal defects created in New Zealand rabbits. Rabbits were euthanized at 10- and 20-week intervals. Microcomputed tomography was used to characterize bone ingrowth and was correlated with histological analysis. Stress testing was performed on the scaffolds before and after implantation. RESULTS: All scaffolds were well integrated into adjacent bone. Bone-to-tissue volume increased from 30.34% ( ± 12.46) to 61.27% ( ± 8.24), and trabecular thickness increased from 0.178 mm ( ± 0.069) to 0.331 mm ( ± 0.0306) in the 10- and 20-week groups, respectively, compared to no bone regrowth on the control side (P < 0.05). Histology confirmed integration at the bone-implant interface. Biomechanical testing revealed a compressive resistance 15 times that of bone alone (P < 0.05) CONCLUSION: 3D-printed PEKK scaffolds combined with ADSCs present a promising solution to improve the bone-implant interface and increase the resistance to forces of mastication after mandibular reconstruction. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E392-E398, 2017.

Oral-Facial Tissue Reconstruction in the Regenerative Axolotl.

Absence of large amounts of orofacial tissues caused by cancerous resections, congenital defects, or trauma results in sequelae such as dysphagia and noticeable scars. Oral-neck tissue regeneration was studied in the axolotl (regenerative amphibian) following a 2.5-mm punch biopsy that simultaneously removed skin, connective tissue, muscle, and cartilage in the tongue and intermandibular region. The untreated wound was studied macroscopically and histologically at 17 different time points ranging from 0 to180 days (N = 120 axolotls). At 12 hr, the wound's surface was smoothened and within 1mm, internal lingual muscular modifications occurred; at the same distance, between days 4-7 lingual muscle degradation was complete. Immunofluorescence indicates complete keratinocytes migration by 48 hr. These cells with epidermal Leydig cells, appearing yellow, lead the chin's deep tissue outgrowth until its closure on the 14th day. Regeneration speeds varied and peaked in time for each tissue, (1) deep chin 84.3 µm/hr from 24 to 96 hr, (2) superficial chin 71.1 µm/hr from 7-14 days, and (3) tongue 86.0 µm/hr between 48 hr and 7 days. Immunofluorescence to Col IV showed basement membrane reconnected between days 30-45 coinciding with the chin's dermal tissue's surface area recovery. New muscle appeared at 21 days and was always preceded by the formation of a collagen bed. Both chin tissues regain all surface area and practically all components while the lingual structure lacks some content but is generally similar to the original. The methodology and high-resolution observations described here are the first of its kind for this animal model and could serve as a basis for future studies in oral and facial regenerative research.

Soot aggregate restructuring due to coatings of secondary organic aerosol derived from aromatic precursors.

Restructuring of monodisperse soot aggregates due to coatings of secondary organic aerosol (SOA) derived from hydroxyl radical-initiated oxidation of toluene, p-xylene, ethylbenzene, and benzene was investigated in a series of photo-oxidation (smog) chamber experiments. Soot aggregates were generated by combustion of ethylene using a McKenna burner, treated by denuding, size-selected by a differential mobility analyzer, and injected into a smog chamber, where they were exposed to low vapor pressure products of aromatic hydrocarbon oxidation, which formed SOA coatings. Aggregate restructuring began once a threshold coating mass was reached, and the degree of the subsequent restructuring increased with mass growth factor. Although significantly compacted, fully processed aggregates were not spherical, with a mass-mobility exponent of 2.78, so additional SOA was required to fill indentations between collapsed branches of the restructured aggregates before the dynamic shape factor of coated particles approached 1. Trends in diameter growth factor, effective density, and dynamic shape factor with increasing mass growth factor indicate distinct stages in soot aggregate processing by SOA coatings. The final degree and coating mass dependence of soot restructuring were found to be the same for SOA coatings from all four aromatic precursors, indicating that the surface tensions of the SOA coatings are similar.