The influence of electrospun fibre scaffold orientation and nano-hydroxyapatite content on the development of tooth bud stem cells in vitro.

In stem cell-based dental tissue engineering, the goal is to create tooth-like structures using scaffold materials to guide the dental stem cells. In this study, the effect of fiber alignment and hydroxyapatite content in biodegradable electrospun PLGA scaffolds have been investigated. Fiber orientation of the scaffolds was random or aligned in bundles. For scaffolds with prefabricated orientation, scaffolds were fabricated from PLGA polymer solution containing 0, 10 or 20 % nano-hydroxyapatite. The scaffolds were seeded with porcine cells isolated from tooth buds (dental mesenchymal, dental epithelial, and mixed dental mesenchymal/epithelial cells). Samples were collected at 1, 3 and 6 weeks. Analyses were performed for cell proliferation, ALP activity, and cell morphology. Fiber alignment showed an effect on cell orientation in the first week after cell seeding, but had no long-term effect on cell alignment or organized calcified matrix deposition once the cells reach confluency. Scaffold porosity was sufficient to allow migration of mesenchymal cells. Hydroxyapatite incorporation did not have a positive effect on cell proliferation, especially of epithelial cells, but seemed to promote differentiation. Concluding, scaffold architecture is important to mesenchymal cell morphology, but has no long-term effect on cell alignment or organized ECM deposition. nHA incorporation does have an effect on cell proliferation, differentiation and ECM production, and should be regarded as a bioactive component of dental bioengineered scaffolds.

TyroFill-Titanium Implant Constructs for the Coordinated Repair of Rabbit Mandible and Tooth Defects.

Currently used methods to repair craniomaxillofacial (CMF) bone and tooth defects require a multi-staged surgical approach for bone repair followed by dental implant placement. Our previously published results demonstrated significant bioengineered bone formation using human dental pulp stem cell (hDPSC)-seeded tyrosine-derived polycarbonate scaffolds (E1001(1K)-bTCP). Here, we improved upon this approach using a modified TyroFill (E1001(1K)/dicalcium phosphate dihydrate (DCPD)) scaffold-supported titanium dental implant model for simultaneous bone-dental implant repair. TyroFill scaffolds containing an embedded titanium implant, with (n = 3 each time point) or without (n = 2 each time point) seeded hDPCs and Human Umbilical Vein Endothelial Cells (HUVECs), were cultured in vitro. Each implant was then implanted into a 10 mm full-thickness critical-sized defect prepared on a rabbit mandibulee. After 1 and 3 months, replicate constructs were harvested and analyzed using Micro-CT histological and IHC analyses. Our results showed significant new bone formation surrounding the titanium implants in cell-seeded TyroFill constructs. This study indicates the potential utility of hDPSC/HUVEC-seeded TyroFill scaffolds for coordinated CMF bone-dental implant repair.

Self-Assembled Hydrogel Microparticle-Based Tooth-Germ Organoids.

Here, we describe the characterization of tooth-germ organoids, three-dimensional (3D) constructs cultured in vitro with the potential to develop into living teeth. To date, the methods used to successfully create tooth organoids capable of forming functional teeth have been quite limited. Recently, hydrogel microparticles (HMP) have demonstrated utility in tissue repair and regeneration based on their useful characteristics, including their scaffolding ability, effective cell and drug delivery, their ability to mimic the natural tissue extracellular matrix, and their injectability. These outstanding properties led us to investigate the utility of using HMPs (average diameter: 158 ± 32 µm) derived from methacrylated gelatin (GelMA) (degree of substitution: 100%) to create tooth organoids. The tooth organoids were created by seeding human dental pulp stem cells (hDPSCs) and porcine dental epithelial cells (pDE) onto the HMPs, which provided an extensive surface area for the cells to effectively attach and proliferate. Interestingly, the cell-seeded HMPs cultured on low-attachment tissue culture plates with gentle rocking self-assembled into organoids, within which the cells maintained their viability and morphology throughout the incubation period. The self-assembled organoids reached a volume of ~50 mm3 within two weeks of the in vitro tissue culture. The co-cultured hDPSC-HMP and pDE-HMP structures effectively attached to each other without any externally applied forces. The presence of polarized, differentiated dental cells in these composite tooth-bud organoids demonstrated the potential of self-assembled dental cell HMPs to form tooth-bud organoid-like structures for potential applications in tooth regeneration strategies.

Dental education 2026: A scenario exploration.

PURPOSE: The purpose of the following papers is to explore plausible alternative futures for dental education. The COVID-19 pandemic, challenges emerging from racism in the US, and social unrest were the precipitating factors leading to this consideration of academic dentistry in approximately 5 years. METHODS: In 2020-2021, five teams of six individual followed a seven-step process to develop five different scenarios of dental education in 2026. Four of these scenarios are constructed by considering a range of uncertainties associated with economic sustainability and educational innovation. A fifth scenario describes the optimal case for dental education's role should another pandemic occur. RESULTS: Each scenario is presented as a narrative in three parts: scenario highlights (summary), life in the scenario (fictional case), and scenario details (a description of significant factors within the envisioned future of the scenario). As a strategic tool, these scenarios will assist leaders, institutions, and stakeholders to anticipate and prepare for different futures, identify key indicators that a particular future is emerging, and guide decision-making to create the most desirable future in a changing environment. CONCLUSION: Institutions are encouraged to incorporate these scenarios into their strategic and contingency planning efforts and to use them to generate dialogue during faculty development initiatives. This study provides institutions with a process and model they can follow to create scenarios at the institutional level. The final paper in this collection is a guide that provides ideas and instructions for using the scenarios in curricular and extracurricular activities with faculty members and students.

Identification of adult mineralized tissue zebrafish mutants.

Zebrafish craniofacial, skeletal, and tooth development closely resembles that of higher vertebrates. Our goal is to identify viable adult zebrafish mutants that can be used as models for human mineralized craniofacial, dental, and skeletal system disorders. We used a large-scale forward-genetic chemical N-ethyl-nitroso-urea mutagenesis screen to identify 17 early lethal homozygous recessive mutants with defects in craniofacial cartilage elements, and 7 adult homozygous recessive mutants with mineralized tissue phenotypes including craniofacial shape defects, fused sutures, dysmorphic or missing skeletal elements, scoliosis, and neural arch defects. One mutant displayed both an early lethal homozygous phenotype and an adult heterozygous phenotype. These results extend the utility of the zebrafish model beyond the embryo to study human bone and cartilage disorders.

Tooth Repair and Regeneration: Potential of Dental Stem Cells.

Tooth defects are an extremely common health condition that affects millions of individuals. Currently used dental repair treatments include fillings for caries, endodontic treatment for pulp necrosis, and dental implants to replace missing teeth, all of which rely on the use of synthetic materials. By contrast, the fields of tissue engineering and regenerative medicine and dentistry (TERMD) use biologically based therapeutic strategies for vital tissue regeneration, and thus have the potential to regenerate living tissues. Methods to create bioengineered replacement teeth benefit from a detailed understanding of the molecular signaling networks regulating natural tooth development. We discuss how key signaling pathways regulating natural tooth development are being exploited for applications in TERMD approaches for vital tooth regeneration.

Synergistic roles of Wnt modulators R-spondin2 and R-spondin3 in craniofacial morphogenesis and dental development.

Wnt signaling plays a critical role in craniofacial patterning, as well as tooth and bone development. Rspo2 and Rspo3 are key regulators of Wnt signaling. However, their coordinated function and relative requirement in craniofacial development and odontogensis are poorly understood. We showed that in zebrafish rspo2 and rspo3 are both expressed in osteoprogenitors in the embryonic craniofacial skeleton. This is in contrast to mouse development, where Rspo3 is expressed in osteoprogenitors while Rspo2 expression is not observed. In zebrafish, rspo2 and rspo3 are broadly expressed in the pulp, odontoblasts and epithelial crypts. However, in the developing molars of the mouse, Rspo3 is largely expressed in the dental follicle and alveolar mesenchyme while Rspo2 expression is restricted to the tooth germ. While Rspo3 ablation in the mouse is embryonic lethal, zebrafish rspo3-/- mutants are viable with modest decrease in Meckel's cartilage rostral length. However, compound disruption of rspo3 and rspo2 revealed synergistic roles of these genes in cartilage morphogenesis, fin development, and pharyngeal tooth development. Adult rspo3-/- zebrafish mutants exhibit a dysmorphic cranial skeleton and decreased average tooth number. This study highlights the differential functions of Rspo2 and Rspo3 in dentocranial morphogenesis in zebrafish and in mouse.

Tissue engineered hybrid tooth-bone constructs.

Proper rehabilitation of craniofacial defects is challenging because of the complexity of the anatomy and the component tissue types. The ability to simultaneously coordinate the regeneration of multiple tissues would make reconstruction more efficient and might reduce morbidity and improve outcomes. The craniofacial complex is unique because of the presence of teeth, in addition to skin, bone, cartilage, muscle, vascular, and neural tissues since teeth naturally grow in coordination with the craniofacial skeleton, our group developed an autologous, tooth-bone hybrid model to facilitate repair of mandibular defects in the Yucatan minipig. The hybrid tooth-bone construct was prepared by combining tooth bud cell-seeded scaffolds with autologous iliac crest bone marrow derived stem cell-seeded scaffolds, which were transplanted back into surgically created mandibular defects in the same minipig. The constructs were harvested after 12 and 20 weeks of growth. The resulting bone/tooth constructs were evaluated by X-ray, ultra high-resolution volume computed tomography (VCT), histological, immunohistochemical analyses, and transmission electron microscopy (TEM). The observed formation of small tooth-like structures consisting of organized dentin, enamel, pulp, cementum, periodontal ligament, and surrounded by regenerated alveolar bone, suggests the feasibility for regeneration of teeth and associated alveolar bone, in a single procedure. This model provides an accessible method for future clinical applications in humans.

Calcium phosphate enriched synthetic tyrosine-derived polycarbonate - dicalcium phosphate dihydrate polymer scaffolds for enhanced bone regeneration.

Optimal repair of large craniomaxillofacial (CMF) defects caused by trauma or disease requires the development of new, synthetic osteoconductive materials in combination with cell-based therapies, to overcome the limitations of traditionally used bone graft substitutes. In this study, tyrosine-derived polycarbonate, E1001(1k) scaffolds were fabricated to incorporate the osteoinductive coating, Dicalcium phosphate dihydrate (DCPD). The biocompatibility of E1001(1k)-DCPD, E1001(1k)-ßTCP and E1001(1k) scaffolds was compared using in vitro culture with human dental pulp stem cells (hDPSCs). We found that the DCPD coating was converted to carbonated hydroxyapatite over time in in vitro culture in Osteogenic Media, while the ßTCP did not. hDPSCs exhibited slow initial attachment and proliferation on DCPD E1001(1k) scaffolds, but subsequently improved over time in culture, and promoted osteogenic differentiation. To the best of our knowledge, this study highlights for the first time the effects of Osteogenic Media on phase changes of DCPD, and on DCPD scaffold cytocompatibility with hDPSCs. DCPD showed similar hDPSC biocompatibility and osteoconductivity as compared to ßTCP, and osteogenic differentiation of seeded hDPSCs. These studies suggest that E1001(1k)-DCPD scaffolds are a superior tool for craniofacial bone regeneration and provide the foundation for future in vivo testing.

Corrigendum: Use of Human Dental Pulp and Endothelial Cell Seeded Tyrosine-Derived Polycarbonate Scaffolds for Robust in vivo Alveolar Jaw Bone Regeneration.

[This corrects the article DOI: 10.3389/fbioe.2020.00796.].