Ependymal polarity defects coupled with disorganized ciliary beating drive abnormal cerebrospinal fluid flow and spine curvature in zebrafish.

Idiopathic scoliosis (IS) is the most common spinal deformity diagnosed in childhood or early adolescence, while the underlying pathogenesis of this serious condition remains largely unknown. Here, we report zebrafish ccdc57 mutants exhibiting scoliosis during late development, similar to that observed in human adolescent idiopathic scoliosis (AIS). Zebrafish ccdc57 mutants developed hydrocephalus due to cerebrospinal fluid (CSF) flow defects caused by uncoordinated cilia beating in ependymal cells. Mechanistically, Ccdc57 localizes to ciliary basal bodies and controls the planar polarity of ependymal cells through regulating the organization of microtubule networks and proper positioning of basal bodies. Interestingly, ependymal cell polarity defects were first observed in ccdc57 mutants at approximately 17 days postfertilization, the same time when scoliosis became apparent and prior to multiciliated ependymal cell maturation. We further showed that mutant spinal cord exhibited altered expression pattern of the Urotensin neuropeptides, in consistent with the curvature of the spine. Strikingly, human IS patients also displayed abnormal Urotensin signaling in paraspinal muscles. Altogether, our data suggest that ependymal polarity defects are one of the earliest sign of scoliosis in zebrafish and disclose the essential and conserved roles of Urotensin signaling during scoliosis progression.

Functional comparison of human ACVR1 and zebrafish Acvr1l FOP-associated variants in embryonic zebrafish.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by progressive heterotopic ossification of muscle and connective tissues, is caused by autosomal dominant activating mutations in the type I receptor, ACVR1/ALK2. The classic human FOP variant, ACVR1R206H , shows increased bone morphogenetic protein (BMP) signaling and activation by activins. RESULTS: Here, we performed in vivo functional characterization of human ACVR1R206H and orthologous zebrafish Acvr1lR203H using early embryonic zebrafish dorsoventral patterning as a phenotypic readout for receptor activity. Our results showed that human ACVR1R206H and zebrafish Acvr1lR203H exhibit functional differences in early embryonic zebrafish, and that human ACVR1R206H retained its signaling activity in the absence of a ligand-binding domain (LBD). We also showed, for the first time, that zebrafish Acvr2ba/Acvr2bb receptors are required for human ACVR1R206H signaling in early embryonic zebrafish. CONCLUSIONS: Together, these data provide new insight into ACVR1R206H signaling pathways that may facilitate the design of new and effective therapies for FOP patients.

3D-printed microgels supplemented with dentin matrix molecules as a novel biomaterial for direct pulp capping.

OBJECTIVES: To develop a 3D-printed, microparticulate hydrogel supplemented with dentin matrix molecules (DMM) as a novel regenerative strategy for dental pulp capping. MATERIALS AND METHODS: Gelatin methacryloyl microgels (7% w/v) mixed with varying concentrations of DMM were printed using a digital light projection 3D printer and lyophilized for 2 days. The release profile of the DMM-loaded microgels was measured using a bicinchoninic acid assay. Next, dental pulp exposure defects were created in maxillary first molars of Wistar rats. The exposures were randomly capped with (1) inert material - negative control, (2) microgels, (3) microgels + DMM 500 µg/ml, (4) microgels + DMM 1000 µg/ml, (5) microgels + platelet-derived growth factor (PDGF 10 ng/ml), or (6) MTA (n = 15/group). After 4 weeks, animals were euthanized, and treated molars were harvested and then processed to evaluate hard tissue deposition, pulp tissue organization, and blood vessel density. RESULTS: All the specimens from groups treated with microgel + 500 µg/ml, microgel + 1000 µg/ml, microgel + PDGF, and MTA showed the formation of organized pulp tissue, tertiary dentin, newly formed tubular and atubular dentin, and new blood vessel formation. Dentin bridge formation was greater and pulp necrosis was less in the microgel + DMM groups compared to MTA. CONCLUSIONS: The 3D-printed photocurable microgels doped with DMM exhibited favorable cellular and inflammatory pulp responses, and significantly more tertiary dentin deposition. CLINICAL RELEVANCE: 3D-printed microgel with DMM is a promising biomaterial for dentin and dental pulp regeneration in pulp capping procedures.

Kir2.1 is important for efficient BMP signaling in mammalian face development.

Mutations that disrupt the inwardly rectifying potassium channel Kir2.1 lead to Andersen-Tawil syndrome that includes periodic paralysis, cardiac arrhythmia, cognitive deficits, craniofacial dysmorphologies and limb defects. The molecular mechanism that underlies the developmental consequences of inhibition of these channels has remained a mystery. We show that while loss of Kir2.1 function does not affect expression of several early facial patterning genes, the domain in which Pou3f3 is expressed in the maxillary arch is reduced. Pou3f3 is important for development of the jugal and squamosal bones. The reduced expression domain of Pou3f3 is consistent with the reduction in the size of the squamosal and jugal bones in Kcnj2KO/KO animals, however it does not account for the diverse craniofacial defects observed in Kcnj2KO/KO animals. We show that Kir2.1 function is required in the cranial neural crest for morphogenesis of several craniofacial structures including palate closure. We find that while the palatal shelves of Kir2.1-null embryos elevate properly, they are reduced in size due to decreased proliferation of the palatal mesenchyme. While we find no reduction in expression of BMP ligands, receptors, and associated Smads in this setting, loss of Kir2.1 reduces the efficacy of BMP signaling as shown by the reduction of phosphorylated Smad 1/5/8 and reduced expression of BMP targets Smad6 and Satb2.

Animal models of fibrodysplasia ossificans progressiva.

Fibrodysplasia Ossificans Progressiva is a rare human disease of heterotopic ossification. FOP patients experience progressive development of ectopic bone within fibrous tissues that contributes to a gradual loss of mobility and can lead to early mortality. Due to lack of understanding of the etiology and progression of human FOP, and the fact that surgical interventions often exacerbate FOP disease progression, alternative therapeutic methods are needed, including modeling in animals, to study and improve understanding of human FOP. In this review we provide an overview of the existing animal models of FOP and the key mechanistic findings from each. In addition, we highlight the specific advantages of a new adult zebrafish model, generated by our lab, to study human FOP. Developmental Dynamics 247:279-288, 2018. © 2017 Wiley Periodicals, Inc.

Tissue specific roles for the ribosome biogenesis factor Wdr43 in zebrafish development.

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

Bioelectric signalling via potassium channels: a mechanism for craniofacial dysmorphogenesis in KCNJ2-associated Andersen-Tawil Syndrome.

KEY POINTS: Xenopus laevis craniofacial development is a good system for the study of Andersen-Tawil Syndrome (ATS)-associated craniofacial anomalies (CFAs) because (1) Kcnj2 is expressed in the nascent face; (2) molecular-genetic and biophysical techniques are available for the study of ion-dependent signalling during craniofacial morphogenesis; (3) as in humans, expression of variant Kcnj2 forms in embryos causes a muscle phenotype; and (4) variant forms of Kcnj2 found in human patients, when injected into frog embryos, cause CFAs in the same cell lineages. Forced expression of WT or variant Kcnj2 changes the normal pattern of Vmem (resting potential) regionalization found in the ectoderm of neurulating embryos, and changes the normal pattern of expression of ten different genetic regulators of craniofacial development, including markers of cranial neural crest and of placodes. Expression of other potassium channels and two different light-activated channels, all of which have an effect on Vmem , causes CFAs like those induced by injection of Kcnj2 variants. In contrast, expression of Slc9A (NHE3), an electroneutral ion channel, and of GlyR, an inactive Cl(-) channel, do not cause CFAs, demonstrating that correct craniofacial development depends on a pattern of bioelectric states, not on ion- or channel-specific signalling. Using optogenetics to control both the location and the timing of ion flux in developing embryos, we show that affecting Vmem of the ectoderm and no other cell layers is sufficient to cause CFAs, but only during early neurula stages. Changes in Vmem induced late in neurulation do not affect craniofacial development. We interpret these data as strong evidence, consistent with our hypothesis, that ATS-associated CFAs are caused by the effect of variant Kcnj2 on the Vmem of ectodermal cells of the developing face. We predict that the critical time is early during neurulation, and the critical cells are the ectodermal cranial neural crest and placode lineages. This points to the potential utility of extant, ion flux-modifying drugs as treatments to prevent CFAs associated with channelopathies such as ATS. ABSTRACT: Variants in potassium channel KCNJ2 cause Andersen-Tawil Syndrome (ATS); the induced craniofacial anomalies (CFAs) are entirely unexplained. We show that KCNJ2 is expressed in Xenopus and mouse during the earliest stages of craniofacial development. Misexpression in Xenopus of KCNJ2 carrying ATS-associated mutations causes CFAs in the same structures affected in humans, changes the normal pattern of membrane voltage potential regionalization in the developing face and disrupts expression of important craniofacial patterning genes, revealing the endogenous control of craniofacial patterning by bioelectric cell states. By altering cells' resting potentials using other ion translocators, we show that a change in ectodermal voltage, not tied to a specific protein or ion, is sufficient to cause CFAs. By adapting optogenetics for use in non-neural cells in embryos, we show that developmentally patterned K(+) flux is required for correct regionalization of the resting potentials and for establishment of endogenous early gene expression domains in the anterior ectoderm, and that variants in KCNJ2 disrupt this regionalization, leading to the CFAs seen in ATS patients.

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.

Functional Testing of Bone Morphogenetic Protein (BMP) Pathway Variants Identified on Whole-Exome Sequencing in a Patient with Delayed-Onset Fibrodysplasia Ossificans Progressiva (FOP) Using ACVR1R206H -Specific Human Cellular and Zebrafish Models.

Bone morphogenetic protein (BMP) signaling is critical in skeletal development. Overactivation can trigger heterotopic ossification (HO) as in fibrodysplasia ossificans progressiva (FOP), a rare, progressive disease of massive HO formation. A small subset of FOP patients harboring the causative ACVR1R206H mutation show strikingly mild or delayed-onset HO, suggesting that genetic variants in the BMP pathway could act as disease modifiers. Whole-exome sequencing of one such patient identified BMPR1AR443C and ACVR2AV173I as candidate modifiers. Molecular modeling predicted significant structural perturbations. Neither variant decreased BMP signaling in ACVR1R206H HEK 293T cells at baseline or after stimulation with BMP4 or activin A (AA), ligands that activate ACVR1R206H signaling. Overexpression of BMPR1AR443C in a Tg(ACVR1-R206Ha) embryonic zebrafish model, in which overactive BMP signaling yields ventralized embryos, did not alter ventralization severity, while ACVR2AV173I exacerbated ventralization. Co-expression of both variants did not affect dorsoventral patterning. In contrast, BMPR1A knockdown in ACVR1R206H HEK cells decreased ligand-stimulated BMP signaling but did not affect dorsoventral patterning in Tg(ACVR1-R206Ha) zebrafish. ACVR2A knockdown decreased only AA-stimulated signaling in ACVR1R206H HEK cells and had no effect in Tg(ACVR1-R206Ha) zebrafish. Co-knockdown in ACVR1R206H HEK cells decreased basal and ligand-stimulated signaling, and co-knockdown/knockout (bmpr1aa/ab; acvr2aa/ab) decreased Tg(ACVR1-R206Ha) zebrafish ventralization phenotypes. Our functional studies showed that knockdown of wild-type BMPR1A and ACVR2A could attenuate ACVR1R206H signaling, particularly in response to AA, and that ACVR2AV173I unexpectedly increased ACVR1R206H -mediated signaling in zebrafish. These studies describe a useful strategy and platform for functionally interrogating potential genes and genetic variants that may impact the BMP signaling pathway. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

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.

The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1.

Akt kinases mediate cell growth and survival. Here, we report that a pro-apoptotic kinase, Mst1/STK4, is a physiological Akt1 interaction partner. Mst1 was identified as a component of an Akt1 multiprotein complex isolated from lipid raft-enriched fractions of LNCaP human prostate cancer cells. Endogenous Mst1, along with its paralog, Mst2, acted as inhibitors of endogenous Akt1. Surprisingly, mature Mst1 as well as both of its caspase cleavage products, which localize to distinct subcellular compartments and are not structurally homologous, complexed with and inhibited Akt1. cRNAs encoding full-length Mst1, and N- and C-terminal caspase Mst1 cleavage products, reverted an early lethal phenotype in zebrafish development induced by expression of membrane-targeted Akt1. Mst1 and Akt1 localized to identical subcellular sites in human prostate tumors. Mst1 levels declined with progression from clinically localized to hormone refractory disease, coinciding with an increase in Akt activation with transition from hormone naïve to hormone-resistant metastases. These results position Mst1/2 within a novel branch of the phosphoinositide 3-kinase/Akt pathway and suggest an important role in cancer progression.

Tooth tissue engineering: optimal dental stem cell harvest based on tooth development.

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.

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.

Molecular pedomorphism underlies craniofacial skeletal evolution in Antarctic notothenioid fishes.

BACKGROUND: Pedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon. Despite its importance for evolutionary change, there are few examples of a molecular basis for this phenomenon. Notothenioids represent one of the best described species flocks among marine fishes, but their diversity is currently threatened by the rapidly changing Antarctic climate. Notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy. RESULTS: We compared craniofacial skeletal development in two pelagic notothenioids, Chaenocephalus aceratus and Pleuragramma antarcticum, to that in a benthic species, Notothenia coriiceps, and two outgroups, the threespine stickleback and the zebrafish. Relative to these other species, pelagic notothenioids exhibited a delay in pharyngeal bone development, which was associated with discrete heterochronic shifts in skeletal gene expression that were consistent with persistence of the chondrogenic program and a delay in the osteogenic program during larval development. Morphological analysis also revealed a bias toward the development of anterior and ventral elements of the notothenioid pharyngeal skeleton relative to dorsal and posterior elements. CONCLUSIONS: Our data support the hypothesis that early shifts in the relative timing of craniofacial skeletal gene expression may have had a significant impact on the adaptive radiation of Antarctic notothenioids into pelagic habitats.

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.