Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

Texas A&M College of Dentistry

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization.

Conservation and variation in enamel protein distribution during vertebrate tooth development.

The teeth of many fish, amphibia, and reptiles are attached to the alveolar bone via ankylosis. In contrast, mammalian periodontia are characterized by a gomphosis, an attachment of the tooth root in the alveolar bone socket via periodontal ligament fibers. Among the reptiles, the crocodilians are the only group featuring a gomphosis-type connection between tooth root and alveolar bone, while in other reptiles tooth-root and jawbone are connected via ankylosis. The purpose of the present study was to compare several key features of the crocodilian periodontium with those of the mammalian and noncrocodilian reptile periodontium. As experimental models for our study we chose the periodontium of newborn geckos (Hemidacylus turcicus), juvenile caimans (Caiman crocodilus crocodilus), and 10-day-postnatal Swiss-Webster mice (Mus musculus) as representative models for noncrocodilian reptiles, crocodilian reptiles, and mammals. The caiman periodontium emerged as an intermediary between the mineral-free mouse ligament and the mineralized gecko ankylosis-type attachment. Caiman ligament fibers were less organized than mouse ligament fibers but featured distinct fasciae surrounding ligament fiber bundles. Caiman Hertwig's epithelial root sheath (HERS) was similarly perforated as mouse HERS and distinctly different from the continuous gecko HERS. Both caiman and mouse HERS covered the entire tooth root length, while in the gecko HERS was limited to the coronal portion of the root, allowing for cementoid-mediated ankylosis at the apical tip of the root. We interpret our data to indicate distinct differences in mineral distribution, periodontal ligament fiber organization, and HERS distribution between noncrocodilian reptiles, crocodilian reptiles, and mammals. Mineral deposits in the caiman ligament may reflect an evolutionary position of the caiman periodontium between ankylosis and gomphosis.

Caiman periodontium as an intermediate between basal vertebrate ankylosis-type attachment and mammalian "true" periodontium.

Oral Biology and Chicago.

Our periodontal tissue: a masterpiece of evolution.

Erupting teeth are some of the oldest witnesses of developmental processes in the vertebrate fossil record and provide an important resource for vertebrate cladistics. Here, we have examined a mosasaur jaw fragment from central Texas using ultrathin ground section histology and 3D tomographic imaging to assess features critical for the cladistic placement of mosasaurs among varanoids vs. snakes: (i) the orientation of replacement teeth compared to the major tooth axis, (ii) the occurrence of resorption pits, and (iii) the mode of tooth implantation/attachment to the tooth bearing element (TBE). The replacement tooth studied here developed in an inclined position slightly distal of the deciduous parent tooth, similar to another varanoid squamate, the Gila monster Heloderma suspectum. Ground sections and tomographs also demonstrated that the replacement tooth attachment apparatus was entirely intact and that there was no evidence of mechanical deformation. Sections and tomographs further illustrated that the replacement tooth was located within a bony crypt and the inclination of the crypt matched the inclination of the replacement tooth. These preparations also revealed the presence of a resorption pit within the boundaries of the deciduous tooth that surrounded the developing replacement tooth. This finding suggests that developing mosasaur teeth developed within the walls of resorption pits similar to varanoid tooth germs and unlike developing snake teeth which are surrounded by fibrous connective tissue integuments. Finally, mosasaurs featured pseudo-thecodont tooth implantation with teeth anchored within a socket of mineralized tissue by means of a mineralized periodontal ligament. Together, these data indicate that the moderate inclination of the erupting mosasaur tooth studied here is neither a result of postmortem displacement nor a character representative of snakes, but rather a shared character between Mosasaurs and other varanoids such as Heloderma. In conjunction with the presence of resorption pits and the evidence for pseudothecodont tooth implantation, the tooth eruption and implantation characters described in the present study either place mosasaurs among the varanoids or suggest convergent evolution mechanisms between both clades, with mosasaurs evolving somewhat independently from a common varanoid ancestor. 

Varanoid Tooth Eruption and Implantation Modes in a Late Cretaceous Mosasaur.

The tooth enamel organ (EO) is a complex epithelial cell assembly involved in multiple aspects of tooth development, including amelogenesis. The present study focuses on the role of the nonameloblast layers of the EO, the stratum intermedium, the stellate reticulum, and the outer enamel epithelium (OEE). The secretory stage stratum intermedium was distinguished by p63-positive epithelial stem cell marks, highly specific alkaline phosphatase labeling, as well as multiple desmosomes and gap junctions. At the location of the presecretory stage stellate reticulum, the pre-eruption EO prominently featured the papillary layer (PL) as a keratin immunopositive network of epithelial strands between tooth crowns and oral epithelium. PL cell strands contained numerous p63-positive epithelial stem cells, while BrdU proliferative cells were detected at the outer boundaries of the PL, suggesting that the stellate reticulum/PL epithelial cell sheath proliferated to facilitate an epithelial seal during tooth eruption. Comparative histology studies demonstrated continuity between the OEE and the general lamina of continuous tooth replacement in reptiles, and the outer layer of Hertwig's epithelial root sheath in humans, implicating the OEE as the formative layer for continuous tooth replacement and tooth root extension. Cell fate studies in organ culture verified that the cervical portion of the mouse molar EO gave rise to Malassez rest-like cell islands. Together, these studies indicate that the nonameloblast layers of the EO play multiple roles during odontogenesis, including the maintenance of several p63-positive stem cell reservoirs, a role during tooth root morphogenesis and tooth succession, a stabilizing function for the ameloblast layer, the facilitation of ion transport from the EO capillaries to the enamel layer, as well as safe and seamless tooth eruption.

Daughters of the Enamel Organ: Development, Fate, and Function of the Stratum Intermedium, Stellate Reticulum, and Outer Enamel Epithelium.

The transport of mineral ions from the enamel organ-associated blood vessels to the developing enamel crystals involves complex cargo packaging and carriage mechanisms across several cell layers, including the ameloblast layer and the stratum intermedium. Previous studies have established PHOSPHO1 as a matrix vesicle membrane-associated phosphatase that interacts with matrix vesicles molecules phosphoethanolamine and phosphocholine to initiate apatite crystal formation inside of matrix vesicles in bone. In the present study, we sought to determine the function of Phospho1 during amelogenesis. PHOSPHO1 protein localization during amelogenesis was verified using immunohistochemistry, with positive signals in the enamel layer, ameloblast Tomes' processes, and in the walls of ameloblast secretory vesicles. These ameloblast secretory vesicle walls were also labeled for amelogenin and the exosomal protein marker HSP70 using immunohistochemistry. Furthermore, PHOSPHO1 presence in the enamel organ was confirmed by Western blot. Phospho1(-/-) mice lacked sharp incisal tips, featured a significant 25% increase in total enamel volume, and demonstrated a significant 2-fold reduction in silver grain density of von Kossa stained ground sections indicative of reduced mineralization in the enamel layer when compared to wild-type mice (p < 0.001). Scanning electron micrographs of Phospho1(-/-) mouse enamel revealed a loss of the prominent enamel prism "picket fence" structure, a loss of parallel crystal organization within prisms, and a 1.56-fold increase in enamel prism width (p < 0.0001). Finally, EDS elemental analysis demonstrated a significant decrease in phosphate incorporation in the enamel layer when compared to controls (p < 0.05). Together, these data establish that the matrix vesicle membrane-associated phosphatase PHOSPHO1 is essential for physiological enamel mineralization. Our findings also suggest that intracellular ameloblast secretory vesicles have unexpected compositional similarities with the extracellular matrix vesicles of bone, dentin, and cementum in terms of vesicle membrane composition and intravesicular ion assembly.

Intravesicular Phosphatase PHOSPHO1 Function in Enamel Mineralization and Prism Formation.

MicroRNAs (miRNAs) are critical regulators of the host immune and inflammatory response against bacterial pathogens. In the present review, we discuss target genes, target gene functions, the potential regulatory role of miRNAs in periodontal tissues, and the potential role of miRNAs as biomarkers and therapeutics. In periodontal disease, miRNAs exert control over all aspects of innate and adaptive immunity, including the functions of neutrophils, macrophages, dendritic cells and T and B cells. Previous human studies have highlighted some key miRNAs that are dysregulated in periodontitis patients. In the present study, we mapped the major miRNAs that were altered in our reproducible periodontitis mouse model relative to control animals. The miRNAs that were upregulated as a result of periodontal disease in both human and mouse studies included miR-15a, miR-29b, miR-125a, miR-146a, miR-148/148a and miR-223, whereas miR-92 was downregulated. The association of individual miRNAs with unique aspects of periodontal disease and their stability in gingival crevicular fluid underscores their potential as markers for periodontal disease progression or healthy restitution. Moreover, miRNA therapeutics hold great promise for the future of periodontal therapy because of their ability to modulate the immune response to infection when applied in conjunction with synthetic antagomirs and/or relatively straightforward delivery strategies.

MicroRNAs and immunity in periodontal health and disease.

There is growing concern that titanium particles may play a role in peri-implant breakdown. Ultrasonic scalers are routinely used in the debridement of peri-implant lesions. This in vitro study is designed to evaluate if titanium particles are produced when an ultrasonic scaler is used on an implant.

Titanium particles generated during ultrasonic scaling of implants.

Tooth enamel is a complex mineralized tissue consisting of long and parallel apatite crystals configured into decussating enamel rods. In recent years, multiple approaches have been introduced to generate or regenerate this highly attractive biomaterial characterized by great mechanical strength paired with relative resilience and tissue compatibility. In the present review, we discuss five pathways toward enamel tissue engineering, (i) enamel synthesis using physico-chemical means, (ii) protein matrix-guided enamel crystal growth, (iii) enamel surface remineralization, (iv) cell-based enamel engineering, and (v) biological enamel regeneration based on de novo induction of tooth morphogenesis. So far, physical synthesis approaches using extreme environmental conditions such as pH, heat and pressure have resulted in the formation of enamel-like crystal assemblies. Biochemical methods relying on enamel proteins as templating matrices have aided the growth of elongated calcium phosphate crystals. To illustrate the validity of this biochemical approach we have successfully grown enamel-like apatite crystals organized into decussating enamel rods using an organic enamel protein matrix. Other studies reviewed here have employed amelogenin-derived peptides or self-assembling dendrimers to re-mineralize mineral-depleted white lesions on tooth surfaces. So far, cell-based enamel tissue engineering has been hampered by the limitations of presently existing ameloblast cell lines. Going forward, these limitations may be overcome by new cell culture technologies. Finally, whole-tooth regeneration through reactivation of the signaling pathways triggered during natural enamel development represents a biological avenue toward faithful enamel regeneration. In the present review we have summarized the state of the art in enamel tissue engineering and provided novel insights into future opportunities to regenerate this arguably most fascinating of all dental tissues.

Enamel biomimetics-fiction or future of dentistry.

The function of mammalian periodontal tissues depends on the presence of a nonmineralized periodontal ligament (PDL) juxtaposed in between mineralized tooth anchorage tissues alveolar bone (AB) and root cementum. In the present study we have hypothesized that the Wnt antagonist secreted frizzled related protein 1 (SFRP1) is an essential regulator of periodontal tissue mineral homeostasis. Our immunoreactions and western blot data demonstrated that SFRP1 was substantially expressed higher in PDL fibroblasts than in surrounding AB progenitors and cementoblasts. SFRP1 was also detected at higher levels in PDL fibroblasts than in dental follicle (DF) cells, but the difference was less pronounced. Preferential H3K4me3 active histone mark enrichment on the  promoter and a lack of H3K27me3 repression were most dramatic in PDL progenitors, to a lesser degree in DF cells, and not detected in AB progenitors and cementoblasts. Selective inhibition of SFRP1 using a small molecule inhibitor WAY-316606 demonstrated that SFRP1 block increased PDL cell mineralization and mineralization gene expression such as β-catenin, alkaline phosphatase, osteocalcin, collagen I, and RUNX2. The effect of SFRP1 inhibition on PDL cell mineral homeostasis was confirmed by RNA silencing. These studies also demonstrated that SFRP1 knockdown promotes PDL differentiation through histone H3K4me3-mediated activation of  and . Finally, when SFRP1 inhibition and silencing studies were performed using AB progenitors instead of PDL progenitors, there was little effect on mineralized state control and gene expression, with the exception of osteocalcin, which was dramatically upregulated upon SFRP1 silencing. Together, the results of our study document the highly specific role of the Wnt inhibitor SFRP1 in maintaining the nonmineralized state of PDL progenitors.

The Wnt Antagonist SFRP1: A Key Regulator of Periodontal Mineral Homeostasis.

Periodontal Homeostasis: From Vienna to Texas-A Century of Periodontal Research in the Spirit of Bernhard Gottlieb.

In a new study, LeBlanc and co-workers have discovered an unusually complex dentition in a fossil relative of the modern-day tuatara that features compound occlusal surfaces, thick and prismatic enamel, and a novel enamel-to-bone tooth attachment. This finding suggests that complex dentitions arose independently in several reptilian lineages.

Evolution: Herbivore-Type Teeth in a Cretaceous Tuatara Relative.

The loss of bone following tooth extraction poses a significant clinical problem for maxillofacial esthetics, function, and future implant placement. In the present study, the efficacy of an erythropoietin-impregnated collagen scaffold as an alveolar ridge augmentation material versus a conventional collagen scaffold and a BioOss inorganic bovine bone xenograft was examined. The collagen/Erythropoietin (EPO) scaffold exhibited significantly more rapid and complete osseous regeneration of the alveolar defect when compared to bone xenograft and the collagen membrane alone. The new EPO induced extracellular matrix was rich in Collagen I, Collagen III, Fibronectin (Fn) and E-cadherin, and featured significantly increased levels of the osteogenic transcription factors Runt-related transcription factor 2 (Runx2) and Osterix (Osx). Histomorphometric evaluation revealed a significant two-fold increase in the number of capillaries between the EPO and the BioOss group. Moreover, there was a highly significant 3.5-fold higher level of vascular endothelial growth factor (VEGF) in the collagen/EPO-treated group compared to controls. The significant effect of EPO on VEGF, FN, and RUNX2 upregulation was confirmed in vitro, and VEGF pathway analysis using VEGF inhibitors confirmed that EPO modulated extracellular matrix protein expression through VEGF even in the absence of blood vessels. Together, these data demonstrate the effectiveness of an EPO-impregnated collagen scaffold for bone regeneration as it induces rapid matrix production and osseoinduction adjacent to new capillaries via VEGF.

The Glycoprotein/Cytokine Erythropoietin Promotes Rapid Alveolar Ridge Regeneration In Vivo by Promoting New Bone Extracellular Matrix Deposition in Conjunction with Coupled Angiogenesis/Osteogenesis.

Thomas G. H. Diekwisch

Center for Craniofacial Research and Diagnosis

Texas A&M College of Dentistry

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Thomas G. H. Diekwisch

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Texas A&M College of Dentistry

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Center for Craniofacial Research and Diagnosis