Highly acidic pH facilitates enamel protein self-assembly, apatite crystal growth and enamel protein interactions in the early enamel matrix.

Tooth enamel develops within a pH sensitive amelogenin-rich protein matrix. The purpose of the present study is to shed light on the intimate relationship between enamel matrix pH, enamel protein self-assembly, and enamel crystal growth during early amelogenesis. Universal indicator dye staining revealed highly acidic pH values (pH 3-4) at the exocytosis site of secretory ameloblasts. When increasing the pH of an amelogenin solution from pH 5 to pH 7, there was a gradual increase in subunit compartment size from 2 nm diameter subunits at pH 5 to a stretched configuration at pH6 and to 20 nm subunits at pH 7. HSQC NMR spectra revealed that the formation of the insoluble amelogenin self-assembly structure at pH6 was critically mediated by at least seven of the 11 histidine residues of the amelogenin coil domain (AA 46-117). Comparing calcium crystal growth on polystyrene plates, crystal length was more than 20-fold elevated at pH 4 when compared to crystals grown at pH 6 or pH 7. To illustrate the effect of pH on enamel protein self-assembly at the site of initial enamel formation, molar teeth were immersed in phosphate buffer at pH4 and pH7, resulting in the formation of intricate berry tree-like assemblies surrounding initial enamel crystal assemblies at pH4 that were not evident at pH7 nor in citrate buffer. Amelogenin and ameloblastin enamel proteins interacted at the secretory ameloblast pole and in the initial enamel layer, and co-immunoprecipitation studies revealed that this amelogenin/ameloblastin interaction preferentially takes place at pH 4-pH 4.5. Together, these studies highlight the highly acidic pH of the very early enamel matrix as an essential contributing factor for enamel protein structure and self-assembly, apatite crystal growth, and enamel protein interactions.

Long-acting PFI-2 small molecule release and multilayer scaffold design achieve extensive new formation of complex periodontal tissues with unprecedented fidelity.

The faithful engineering of complex human tissues such as the bone/soft tissue/mineralized tissue interface in periodontal tissues requires innovative molecular cues in conjunction with tailored scaffolds. To address the loss of periodontal bone and connective tissues following periodontal disease, we have generated a polydopamine and collagen coated electrospun PLGA-PCL (PP) scaffold enriched with the small molecule mediator PFI-2 (PP-PFI-pDA-COL-PFI). In vitro 3D studies using PDL progenitors revealed that the PP-PFI-pDA-COL-PFI scaffold substantially enhanced Alizarin Red staining, increased Ca/P ratios 4-fold, and stimulated cell proliferation more than 12-fold compared to PP-controls, suggestive of its potential for mineralized tissue engineering. When applied in our experimental periodontitis model, the PP-PFI-pDA-COL-PFI scaffold resulted in a substantial 34% reduction in alveolar bone defect height, a 25% root-length gain in periodontal attachment, and the formation of highly ordered regenerated acellular cementum twice as thick as in controls. Explaining the mechanism of PFI-2 mineralized tissue regeneration in periodontal tissues, PFI-2 inhibited SETD7-mediated ß-Catenin protein methylation and increased ß-Catenin nuclear localization. Together, dual-level PFI-2 incorporation into a degradable, dopamine/collagen coated PLGA/PCL scaffold backbone resulted in the regeneration of the tripartite periodontal complex with unprecedented fidelity, including periodontal attachment and new formation of mineralized tissues in inflamed periodontal environments.

The Hippo Pathway Effectors YAP/TAZ Are Essential for Mineralized Tissue Homeostasis in the Alveolar Bone/Periodontal Complex.

YAP and TAZ are essential transcriptional co-activators and downstream effectors of the Hippo pathway, regulating cell proliferation, organ growth, and tissue homeostasis. To ask how the Hippo pathway affects mineralized tissue homeostasis in a tissue that is highly reliant on a tight homeostatic control of mineralized deposition and resorption, we determined the effects of YAP/TAZ dysregulation on the periodontal tissues alveolar bone, root cementum, and periodontal ligament. Loss of YAP/TAZ was associated with a reduction of mineralized tissue density in cellular cementum and alveolar bone, a downregulation in collagen I, alkaline phosphatase, and RUNX2 gene expression, an increase in the resorption markers TRAP and cathepsin K, and elevated numbers of TRAP-stained osteoclasts. Cyclic strain applied to periodontal ligament cells resulted in YAP nuclear localization, an effect that was abolished after blocking YAP. The rescue of YAP signaling with the heparan sulfate proteoglycan agrin resulted in a return of the nuclear YAP signal. Illustrating the key role of YAP on mineralization gene expression, the YAP inhibition-related downregulation of mineralization-associated genes was reversed by the extracellular matrix YAP activator agrin. Application of the unopposed mouse molar model to transform the periodontal ligament into an unloaded state and facilitate the distal drift of teeth resulted in an overall increase in mineralization-associated gene expression, an effect that was 10-20% diminished in Wnt1Cre/YAP/TAZ mutant mice. The unloaded state of the unopposed molar model in Wnt1Cre/YAP/TAZ mutant mice also caused a significant three-fold increase in osteoclast numbers, a substantial increase in bone/cementum resorption, pronounced periodontal ligament hyalinization, and thickened periodontal fiber bundles. Together, these data demonstrated that YAP/TAZ signaling is essential for the microarchitectural integrity of the periodontium by regulating mineralization gene expression and preventing excessive resorption during bodily movement of the dentoalveolar complex.

MicroRNAs: Harbingers and shapers of periodontal inflammation.

Periodontal disease is an inflammatory reaction of the periodontal tissues to oral pathogens. In the present review we discuss the intricate effects of a regulatory network of gene expression modulators, microRNAs (miRNAs), as they affect periodontal morphology, function and gene expression during periodontal disease. These miRNAs are small RNAs involved in RNA silencing and post-transcriptional regulation and affect all stages of periodontal disease, from the earliest signs of gingivitis to the regulation of periodontal homeostasis and immunity and to the involvement in periodontal tissue destruction. MiRNAs coordinate periodontal disease progression not only directly but also through long non-coding RNAs (lncRNAs), which have been demonstrated to act as endogenous sponges or decoys that regulate the expression and function of miRNAs, and which in turn suppress the targeting of mRNAs involved in the inflammatory response, cell proliferation, migration and differentiation. While the integrity of miRNA function is essential for periodontal health and immunity, miRNA sequence variations (genetic polymorphisms) contribute toward an enhanced risk for periodontal disease progression and severity. Several polymorphisms in miRNA genes have been linked to an increased risk of periodontitis, and among those, miR-146a, miR-196, and miR-499 polymorphisms have been identified as risk factors for periodontal disease. The role of miRNAs in periodontal disease progression is not limited to the host tissues but also extends to the viruses that reside in periodontal lesions, such as herpesviruses (human herpesvirus, HHV). In advanced periodontal lesions, HHV infections result in the release of cytokines from periodontal tissues and impair antibacterial immune mechanisms that promote bacterial overgrowth. In turn, controlling the exacerbation of periodontal disease by minimizing the effect of periodontal HHV in periodontal lesions may provide novel avenues for therapeutic intervention. In summary, this review highlights multiple levels of miRNA-mediated control of periodontal disease progression, (i) through their role in periodontal inflammation and the dysregulation of homeostasis, (ii) as a regulatory target of lncRNAs, (iii) by contributing toward periodontal disease susceptibility through miRNA polymorphism, and (iv) as periodontal microflora modulators via viral miRNAs.

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel.

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed "shedding", while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel "dahlite" crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.

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) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

Polarized, Amelogenin Expressing Ameloblast-Like Cells from Cervical Loop/Dental Pulp Cocultures in Bioreactors.

The growth of long and polarized ameloblast-like cells has long been heralded as a major prerequisite for enamel tissue engineering. In this study, we have designed three-dimensional bioreactor/scaffold microenvironments to propagate and assess the ability of cervical loop derivatives to become long and polarized ameloblast-like cells. Our studies demonstrated that cervical loop/periodontal progenitor coculture in a growth-factor-enriched medium resulted in the formation of ameloblast-like cells expressing high levels of amelogenin and ameloblastin. Coculture of cervical loop cells with dental pulp cells on tailored collagen scaffolds enriched with leucine-rich amelogenin peptide (LRAP) and early enamel matrix resulted in singular, elongated, and polarized ameloblast-like cells that expressed and secreted ameloblastin and amelogenin enamel proteins. Bioreactor microenvironments enriched with enamel matrix and LRAP also proved advantageous for the propagation of HAT-7 cells, resulting in a ∼20-fold higher expression of amelogenin and ameloblastin enamel proteins compared with controls growing on plain scaffolds. Together, studies presented here highlight the benefits of microgravity culture systems combined with ameloblast-specific microenvironments and tailored scaffolds for the growth of ameloblast-like cells.

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

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.

Histone Methylation Mechanisms Modulate the Inflammatory Response of Periodontal Ligament Progenitors.

Inflammatory conditions affect periodontal ligament (PDL) homeostasis and diminish its regenerative capacity. The complexity of biological activities during an inflammatory response depends on genetic and epigenetic mechanisms. To characterize the epigenetic changes in response to periodontal pathogens we have focused on histone lysine methylation as a relatively stable chromatin modification involved in the epigenetic activation and repression of transcription and a prime candidate mechanism responsible for the exacerbated and prolonged response of periodontal cells and tissues to dental plaque biofilm. To determine the effect of inflammatory conditions on histone methylation profiles, related gene expression and cellular functions of human periodontal ligament (hPDL) progenitor cells, a hPDL cell culture system was subjected to bacterial cell wall toxin exposure [lipopolysaccharide (LPS)]. Chromatin immunoprecipitation-on-chip analysis revealed that healthy PDL cells featured high enrichment levels for the active H3K4me3 mark at COL1A1, COL3, and RUNX2 gene promoters, whereas there were high occupancy levels for the repressive H3K27me3 marks at DEFA4, CCL5, and IL-1ß gene promoters. In response to LPS, H3K27me3 enrichment increased on extracellular matrix and osteogenesis lineage gene promoters, whereas H3K4me3 enrichment increased on the promoters of inflammatory response genes, suggestive of an involvement of epigenetic mechanisms in periodontal lineage differentiation and in the coordination of the periodontal inflammatory response. On a gene expression level, LPS treatment downregulated COL1A1, COL3A1, and RUNX2 expression and upregulated CCL5, DEFA4, and IL-1ß gene expression. LPS also greatly affected PDL progenitor function, including a reduction in proliferation and differentiation potential and an increase in cell migration capacity. Confirming the role of epigenetic mechanisms in periodontal inflammatory conditions, our studies highlight the significant role of histone methylation mechanisms and modification enzymes in the inflammatory response to LPS bacterial cell wall toxins and periodontal stem cell function.

Enamel biomimetics-fiction or future of dentistry.

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.

Microporous Bio-orthogonally Annealed Particle Hydrogels for Tissue Engineering and Regenerative Medicine.

Microporous annealed particle (MAP) hydrogels are an emerging class of biomaterials with the potential to improve outcomes in tissue repair and regeneration. Here, a new MAP hydrogel platform comprising poly(ethylene) glycol (PEG) hydrogel microparticles that are annealed in situ using bio-orthogonal tetrazine click chemistry is reported (i.e., TzMAP hydrogels). Briefly, clickable PEG-peptide hydrogel microparticles with extracellular matrix mimetic peptides to permit cell adhesion and enzymatic degradation were fabricated via submerged electrospraying and stoichiometrically controlled thiol-norbornene click chemistry. Subsequently, unreacted norbornene groups in the microparticles were leveraged for functionalization with bioactive proteins as well as annealing into TzMAP hydrogels via the tetrazine-norbornene click reaction, which is highly selective and proceeds spontaneously without requiring an initiator or catalyst. The results demonstrate that the clickable particles can be easily applied to a tissue-like defect and then annealed into an inherently microporous structure in situ. In addition, the ability to produce TzMAP hydrogels with heterogeneous properties by incorporating multiple types of hydrogel microspheres is demonstrated, first with fluorophore-functionalized hydrogel microparticles and then with protein-functionalized hydrogel microparticles. For the latter, tetrazine-modified alkaline phosphatase was conjugated to PEG hydrogel microparticles, which were mixed with nonfunctionalized microparticles and used to produce TzMAP hydrogels. A biomimetic mineralized/nonmineralized interface was then produced upon incubation in calcium glycerophosphate. Finally, platelet-derived growth factor-BB (PDGF-BB) and human periodontal ligament stem cells (PDLSC) were incorporated into the TzMAP hydrogels during the annealing step to demonstrate their potential for delivering regenerative therapeutics, specifically for periodontal tissue regeneration. In vitro characterization revealed excellent PDGF-BB retention as well as PDLSC growth and spreading. Moreover, PDGF-BB loading increased PDLSC proliferation within hydrogels by 90% and more than doubled the average volume per cell. Overall, these results demonstrate that TzMAP hydrogels are a versatile new platform for the delivery of stem cells and regenerative factors.

Titanium particles generated during ultrasonic scaling of implants.

BACKGROUND: 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. METHODS: New sandblasted, large grit, acid etched (SLA) coated implants were subjected to ultrasonic scaling with stainless steel, titanium, and PEEK plastic tips. The implants were placed in a holding device and the ultrasonic scaler was positioned on the SLA surface under 25 grams of pressure. The implants were subjected to 30 scaling motions. The ultrasonic coolant water was collected and the number of metallic particles were counted under a light microscope. The particles were confirmed to be titanium via elemental analysis. The implants were visually evaluated for damage to the SLA coating. RESULTS: No metallic particles were detected in the water supplied to the ultrasonic scalers (passive control). Metallic particles were detected when implants were subjected to the ultrasonic coolant water only without the scaler tip touching the implant (active control). All implants that were scaled produced metallic particles and showed easily detectable damage to the SLA layer. CONCLUSIONS: All ultrasonic scaling caused the production of titanium particles and caused damage to the SLA coating of the implant. Ultrasonic scalers should be used with great caution in the treatment of peri-implant conditions and care should be taken to not touch the SLA surface of the implant.

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development.

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8-16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.

Intravesicular Phosphatase PHOSPHO1 Function in Enamel Mineralization and Prism Formation.

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.

Integrative Temporo-Spatial, Mineralogic, Spectroscopic, and Proteomic Analysis of Postnatal Enamel Development in Teeth with Limited Growth.

Tooth amelogenesis is a complex process beginning with enamel organ cell differentiation and enamel matrix secretion, transitioning through changes in ameloblast polarity, cytoskeletal, and matrix organization, that affects crucial biomineralization events such as mineral nucleation, enamel crystal growth, and enamel prism organization. Here we have harvested the enamel organ including the pliable enamel matrix of postnatal first mandibular mouse molars during the first 8 days of tooth enamel development to conduct a step-wise cross-sectional analysis of the changes in the mineral and protein phase. Mineral phase diffraction pattern analysis using single-crystal, powder sample X-ray diffraction analysis indicated conversion of calcium phosphate precursors to partially fluoride substituted hydroxyapatite from postnatal day 4 (4 dpn) onwards. Attenuated total reflectance spectra (ATR) revealed a substantial elevation in phosphate and carbonate incorporation as well as structural reconfiguration between postnatal days 6 and 8. Nanoscale liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) demonstrated highest protein counts for ECM/cell surface proteins, stress/heat shock proteins, and alkaline phosphatase on postnatal day 2, high counts for ameloblast cytoskeletal proteins such as tubulin ß5, tropomyosin, ß-actin, and vimentin on postnatal day 4, and elevated levels of cofilin-1, calmodulin, and peptidyl-prolyl cis-trans isomerase on day 6. Western blot analysis of hydrophobic enamel proteins illustrated continuously increasing amelogenin levels from 1 dpn until 8 dpn, while enamelin peaked on days 1 and 2 dpn, and ameloblastin on days 1-5 dpn. In summary, these data document the substantial changes in the enamel matrix protein and mineral phase that take place during postnatal mouse molar amelogenesis from a systems biological perspective, including (i) relatively high levels of matrix protein expression during the early secretory stage on postnatal day 2, (ii) conversion of calcium phosphates to apatite, peak protein folding and stress protein counts, and increased cytoskeletal protein levels such as actin and tubulin on day 4, as well as (iii) secondary structure changes, isomerase activity, highest amelogenin levels, and peak phosphate/carbonate incorporation between postnatal days 6 and 8. Together, this study provides a baseline for a comprehensive understanding of the mineralogic and proteomic events that contribute to the complexity of mammalian tooth enamel development.

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

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.

Depth of cure of contemporary bulk-fill resin-based composites.

This study evaluated the depth of cure (DOC) of packable and flowable bulk-fill resin-based composites (RBCs) including PRG (prereacted glass ionomer) and short-fiber materials. The materials were placed in a black split-mold with a 7 mm deep recess and cured at 700 mW/cm(2) for 20 s using a LED curing light. DOC was assessed using the ISO scraping and Knoops hardness tests. Data (n=5) were computed and analyzed using one-way ANOVA/Scheffe's post hoc test (p<0.05). ISO DOC ranged from 3.66 to 2.54 mm while DOC based on hardness testing ranged from 3 to 1.5 mm. For all materials, a decrease in hardness was observed with increasing depths. The DOC of bulk-fill RBCs was product dependent and greater than standard composites. At 4 mm depth, none of the bulk-fill RBCs had a depth: top hardness ratio of 0.8 and above.

Experimental Validation of Plastic Mandible Models Produced by a "Low-Cost" 3-Dimensional Fused Deposition Modeling Printer.

BACKGROUND: The objective of this study was to investigate the accuracy of 3-dimensional (3D) plastic (ABS) models generated using a low-cost 3D fused deposition modelling printer. MATERIAL/METHODS: Two human dry mandibles were scanned with a cone beam computed tomography (CBCT) Accuitomo device. Preprocessing consisted of 3D reconstruction with Maxilim software and STL file repair with Netfabb software. Then, the data were used to print 2 plastic replicas with a low-cost 3D fused deposition modeling printer (Up plus 2®). Two independent observers performed the identification of 26 anatomic landmarks on the 4 mandibles (2 dry and 2 replicas) with a 3D measuring arm. Each observer repeated the identifications 20 times. The comparison between the dry and plastic mandibles was based on 13 distances: 8 distances less than 12 mm and 5 distances greater than 12 mm. RESULTS: The mean absolute difference (MAD) was 0.37 mm, and the mean dimensional error (MDE) was 3.76%. The MDE decreased to 0.93% for distances greater than 12 mm. CONCLUSIONS: Plastic models generated using the low-cost 3D printer UPplus2® provide dimensional accuracies comparable to other well-established rapid prototyping technologies. Validated low-cost 3D printers could represent a step toward the better accessibility of rapid prototyping technologies in the medical field.

Modulation of Dental Pulp Stem Cell Odontogenesis in a Tunable PEG-Fibrinogen Hydrogel System.

Injectable hydrogels have the great potential for clinical translation of dental pulp regeneration. A recently developed PEG-fibrinogen (PF) hydrogel, which comprises a bioactive fibrinogen backbone conjugated to polyethylene glycol (PEG) side chains, can be cross-linked after injection by photopolymerization. The objective of this study was to investigate the use of this hydrogel, which allows tuning of its mechanical properties, as a scaffold for dental pulp tissue engineering. The cross-linking degree of PF hydrogels could be controlled by varying the amounts of PEG-diacrylate (PEG-DA) cross-linker. PF hydrogels are generally cytocompatible with the encapsulated dental pulp stem cells (DPSCs), yielding >85% cell viability in all hydrogels. It was found that the cell morphology of encapsulated DPSCs, odontogenic gene expression, and mineralization were strongly modulated by the hydrogel cross-linking degree and matrix stiffness. Notably, DPSCs cultured within the highest cross-linked hydrogel remained mostly rounded in aggregates and demonstrated the greatest enhancement in odontogenic gene expression. Consistently, the highest degree of mineralization was observed in the highest cross-linked hydrogel. Collectively, our results indicate that PF hydrogels can be used as a scaffold for DPSCs and offers the possibility of influencing DPSCs in ways that may be beneficial for applications in regenerative endodontics.