Data-driven design of a multiplexed, peptide-sensitized transistor to detect breath VOC markers of COVID-19.

Exhaled human breath contains a rich mixture of volatile organic compounds (VOCs) whose concentration can vary in response to disease or other stressors. Using simulated odorant-binding proteins (OBPs) and machine learning methods, we designed a multiplex of short VOC- and carbon-binding peptide probes that detect a characteristic "VOC fingerprint". Specifically, we target VOCs associated with COVID-19 in a compact, molecular sensor array that directly transduces vapor composition into multi-channel electrical signals. Rapidly synthesizable, chimeric VOC- and solid-binding peptides were derived from selected OBPs using multi-sequence alignment with protein database structures. Selective peptide binding to targeted VOCs and sensor surfaces was validated using surface plasmon resonance spectroscopy and quartz crystal microbalance. VOC sensing was demonstrated by peptide-sensitized, exposed-channel carbon nanotube transistors. The data-to-device pipeline enables the development of novel devices for non-invasive monitoring, diagnostics of diseases, and environmental exposure assessment.

Biomimetic Dentin Repair: Amelogenin-Derived Peptide Guides Occlusion and Peritubular Mineralization of Human Teeth.

Exposure of dentin tubules due to loss of protective enamel (crown) and cementum (root) tissues as a result of erosion, mechanical wear, gingival recession, etc. has been the leading causes of dentin hypersensitivity. Despite being a widespread ailment, no permanent solution exists to address this oral condition. Current treatments are designed to alleviate the pain by either using desensitizers or blocking dentin tubules by deposition of minerals or solid precipitates, which often have short-lived effects. Reproducing an integrated mineral layer that occludes exposed dentin with concomitant peritubular mineralization is essential to reestablish the structural and mechanical integrity of the tooth with long-term durability. Here, we describe a biomimetic treatment that promotes dentin repair using a mineralization-directing peptide, sADP5, derived from amelogenin. The occlusion was achieved through a layer-by-layer peptide-guided remineralization process that forms an infiltrating mineral layer on dentin. The structure, composition, and nanomechanical properties of the remineralized dentin were analyzed by cross-sectional scanning electron microscopy imaging, energy dispersive X-ray spectroscopy, and nanomechanical testing. The elemental analysis provided calcium and phosphate compositions that are similar to those in hydroxyapatite. The measured average hardness and reduced elastic modulus values for the mineral layer were significantly higher than those of the demineralized and sound human dentin. The structural integration of the new mineral and underlying dentin was confirmed by thermal aging demonstrating no physical separation. These results suggest that a structurally robust and mechanically durable interface is formed between the interpenetrating mineral layer and underlying dentin that can withstand long-term mechanical and thermal stresses naturally experienced in the oral environment. The peptide-guided remineralization procedure described herein could provide a foundation for the development of highly effective oral care products leading to novel biomimetic treatments for a wide range of demineralization-related ailments and, in particular, offers a potent long-term solution for dentin hypersensitivity.

Comparison of the sealing ability of various bioceramic materials for endodontic surgery.

OBJECTIVES: Endosequence Bioceramic Root Repair Material (BC-RRM) is used in endodontic microsurgery. It is available as a paste and a putty. However, no studies to date have examined the sealing ability of these forms alone or in combination as root-end filling materials. Hence, this study aimed to compare the sealing properties of these 2 forms of BC-RRM. MATERIALS AND METHODS: Forty-two extracted upper anterior teeth were divided into 3 experimental groups, a positive and negative control. After the root canal treatment, the root ends were resected, retroprepared and retrofilled with either putty, paste + putty or mineral trioxide aggregate (MTA). The teeth were mounted in tubes so the apical 3 mm was submerged in Brain Heart Infusion (BHI) broth. The coronal portions of the canals were inoculated with Enterococcus faecalis and BHI broth and incubated for 30 days. The broth in the tubes was analyzed for colony forming units to check for leakage of bacteria from the canal. The teeth from the groups were sectioned and analyzed using scanning electron microscopy (SEM). The Kruskal-Wallis test and analysis of variance were used to analyze the data with a significance level p < 0.05. RESULTS: The BC-RRM and MTA groups showed similar sealing ability. The positive control showed leakage in all samples. The SEM imaging showed the presence of bacteria in all experimental groups at the material-tooth interface. CONCLUSIONS: No significant differences were noted in the experimental groups, providing sufficient evidence that any combination could be effectively used during endodontic microsurgery.

Airflow dynamics in obese minipigs with obstructive sleep apnea.

OBJECTIVES: Obstructive sleep apnea (OSA) is associated with anatomical restrictions of pharyngeal airway, but the mechanism of airflow dynamics in OSA is largely unknown. This study utilized computational flow dynamics (CFD) to build a 3D model of the pharynx and to test the hypothesis that an increased restriction in the pharynx in OSA/obese minipigs leads to higher resistance, which in turn creates turbulence to induce temporary blockage of pharyngeal airway patency. DESIGN: Of five 9-11-months-old Yucatan minipigs, 3 were non-obese (BMI<35) and two obese (BMI>51). After natural sleep monitoring using BioRadio system, pigs were sedated to collect MRI images and airflow parameters. The MRI images were processed to create 3D configurations of pharynx. These 3D configurations were meshed to create finite element models (FEM) of CFD. The obtained airflow parameters were input into the configurations to identify turbulent airflow and its location. RESULTS: Heavy snoring and multiple >5s hypopnea/apnea episodes (AHI = 32-35) were identified in both obese minipigs during sleep. Compared to the non-obese/non-OSA controls, obese/OSA minipigs showed much lower respiratory tidal volumes and inspiratory airflow speed. FEM simulation found that turbulence was not present in the pharynx in either model. However, a 25% increase of airflow velocity was observed at the narrowest part of the nasal pharynx in the obese/OSA minipig model. CONCLUSIONS: Despite the narrower pharyngeal airway and the higher velocity of airflow, FEM simulation indicated that turbulence was not produced in the obese/OSA minipigs.

Long-term durability of a physician-modified endovascular graft.

We present a unique assessment confirming the long-term durability of a physician-modified endograft deployed as part of an Investigational Device Exemption clinical trial (NCT# 01538056). After receiving an intact postmortem aorta 7 years after the index procedure, we performed microcomputed tomography, necropsy, and metallurgical analysis on the specimen. Microcomputed tomography showed a single strut fracture not noted during previous surveillance. Necropsy revealed no graft fabric compromise, and examination of all three visceral fenestrations showed excellent alignment with no evidence of degradation. Analysis of the strut fracture implicated an initially small, fatigue-induced crack that likely succumbed during postmortem handling.

Biomimetic Tooth Repair: Amelogenin-Derived Peptide Enables in Vitro Remineralization of Human Enamel.

White spot lesions (WSL) and incipient caries on enamel surfaces are the earliest clinical outcomes for demineralization and caries. If left untreated, the caries can progress and may cause complex restorative procedures or even tooth extraction which destroys soft and hard tissue architecture as a consequence of connective tissue and bone loss. Current clinical practices are insufficient in treating dental caries. A long-standing practical challenge associated with demineralization related to dental diseases is incorporating a functional mineral microlayer which is fully integrated into the molecular structure of the tooth in repairing damaged enamel. This study demonstrates that small peptide domains derived from native protein amelogenin can be utilized to construct a mineral layer on damaged human enamel in vitro. Six groups were prepared to carry out remineralization on artificially created lesions on enamel: (1) no treatment, (2) Ca2+ and PO43- only, (3) 1100 ppm fluoride (F), (4) 20 000 ppm F, (5) 1100 ppm F and peptide, and (6) peptide alone. While the 1100 ppm F sample (indicative of common F content of toothpaste for homecare) did not deliver F to the thinly deposited mineral layer, high F test sample (indicative of clinical varnish treatment) formed mainly CaF2 nanoparticles on the surface. Fluoride, however, was deposited in the presence of the peptide, which also formed a thin mineral layer which was partially crystallized as fluorapatite. Among the test groups, only the peptide-alone sample resulted in remineralization of fairly thick (10 µm) dense mineralized layer containing HAp mineral, resembling the structure of the healthy enamel. The newly formed mineralized layer exhibited integration with the underlying enamel as evident by cross-sectional imaging. The peptide-guided remineralization approach sets the foundation for future development of biomimetic products and treatments for dental health care.

Efficacy of different carriers for the triple antibiotic powder during regenerative endodontic procedures.

Chemical debridement during Regenerative Endodontic procedures is important. Previous research studies have evaluated various antibiotics and their concentrations but none have addressed the concern of delivering these materials. Hence, the purpose of this study was to determine what carrier could be used effectively in a clinical setting. Sixty caries-free maxillary incisors were used and inoculated with Enterococcus faecalis and divided into positive and irrigation controls and experimental groups that had triple antibiotic powder (1:1:1 ciprofloxacin:metronidazole:minocycline) delivered using various carriers: saline, cotton, sponge and methylcellulose. Current AAE regenerative protocols were followed. S2 sampling was performed and tested for bacterial presence via culturing and SEM. The results demonstrated that saline was the most effective carrier for the triple antibiotic powder while cotton and sponge were most ineffective. Saline and methylcellulose both reduced bacterial counts to a significant level. Overall, this study demonstrated that saline as a carrier was most effective and should be routinely used.

Analysis of Surface Characteristics of ProTaper Universal and ProTaper Next Instruments by Scanning Electron Microscopy.

BACKGROUND: Many new rotary files systems have been introduced, however, limited research has been conducted related to the surface irregularities of these files and if these have any effects on the files themselves. Hence, the aim of the present study was to analyze surface irregularities of the ProTaper® Universal rotary files (PTU) and the ProTaper Next™ rotary files (PTN) before and after instrumentation in curved canals. The main objective was to investigate the nature of these irregularities and how they might influence the use and fracture of rotary files during root-canal treatments. MATERIAL AND METHODS: The files were examined pre-operatively using a stereomicroscope and scanning electron microscopy(SEM) to analyze surface imperfections and the presence of particles. Mesial roots of forty extracted mandibular molars were selected. Each instrument was used to prepare one of the mesial canals. The files were then rinsed with alcohol, and autoclaved and analyzed again. RESULTS: Of the 80 files used in this study, five files fractured, five files unwound and seven files were curved or bent and they all belonged to the PTU group. Irregularities and debris could be visualized with the SEM on both unused PTU and PTN files. Most of the debris was found associated with deeper milling grooves and defects on the surface of the metal. Surface analysis of the files that were used and sterilized were performed and the SEM images demonstrated organic debris, metal flash, and crack formation and initiation of fractures for both file types. All files showed machining grooves, metal flash, debris, and defects on cutting edges. CONCLUSIONS: These irregularities appear to be critical in the accumulation of debris and initiation of fatigue and crack propagation within the NiTi alloy. The accumulation of debris could be a concern due to the potential exchange of organic debris between patients. Key words:ProTaper® Universal, ProTaper Next™, surface characteristics, SEM.

Assessing the feasibility of yttria-stabilized zirconia in novel designs as mandibular anterior fixed lingual retention after orthodontic treatment.

INTRODUCTION: The purpose of this study is to explore the feasibility of yttria-stabilized zirconia (Y-TZP) in fixed lingual retention as an alternative to stainless steel. METHODS: Exploratory Y-TZP specimens were milled to establish design parameters. Next, the specimens were milled according to ASTM standard C1161-13 and subjected to 4-point flexural tests to determine material properties. Finite element analysis was used to evaluate 9 novel cross-sectional designs, which were compared with stainless steel wire. Each design was analyzed under loading conditions to determine von Mises and bond stresses. The most promising design was fabricated to assess the accuracy and precision of current CAD/CAM milling technology. RESULTS: The superior design had a 1.0 × 0.5 mm semielliptical cross-section and was shown to be fabricated reliably. Overall, the milling indicated a maximum percent standard deviation of 9.3 and maximum percent error of 13.5 with a cost of $30 per specimen. CONCLUSIONS: Y-TZP can be reliably milled to dimensions comparable with currently available metallic retainer wires. Further research is necessary to determine the success of the bonding protocol and the clinical longevity of Y-TZP fixed retainers. Advanced technology is necessary to connect the intraoral scan to an esthetic and patient-specific Y-TZP fixed retainer.

Disinfection Efficacy of Current Regenerative Endodontic Protocols in Simulated Necrotic Immature Permanent Teeth.

INTRODUCTION: The lack of mechanical debridement and reduced concentrations suggested for chemical debridement to maintain stem cell viability call into question the disinfection efficacy of current regenerative protocols. Current protocols vary in the concentration and type of antibiotic medicaments used. The aim of this study was to determine if simulated immature teeth infected with Enterococcus faecalis can be completely disinfected by following current standardized regenerative protocols and to evaluate the probable effects of residual bacteria on stem cell toxicity. METHODS: Sixty-eight caries-free maxillary incisors were used. S1 sampling protocols were validated in both negative and positive control groups via culture, scanning electron microscopy, and confocal laser scanning microscopy. All teeth, except the negative controls, were inoculated with E. faecalis. The teeth were divided into the following groups: group 1, triple antibiotic paste (ciprofloxacin:metronidazole:minocycline) at concentrations of 10, 1, and 0.1 mg/mL; group 2, double antibiotic paste (ciprofloxacin:metronidazole) at concentrations of 10, 1, and 0.1 mg/mL; group 3: Ultracal XS calcium hydroxide (Ultradent, St Louis, MO); and controls, negative and positive controls. Current regenerative protocols recommended by the American Association of Endodontists were followed. S2 sampling was performed after 4 weeks and tested for bacterial presence via culturing, scanning electron microscopy, and confocal laser scanning microscopic analysis. RESULTS: The data showed that calcium hydroxide and the current recommended antibiotic concentrations are not capable of completely eliminating bacteria from simulated necrotic immature permanent teeth. CONCLUSIONS: Overall, this study focuses on the need to re-evaluate the balance between stem cell toxicity and bacterial elimination in order to determine the appropriate concentrations and medicaments for successful regenerative endodontic procedures.

Evaluation of the resistance to cyclic fatigue among ProTaper Next, ProTaper Universal, and Vortex Blue rotary instruments.

INTRODUCTION: The purpose of this study was to compare the fracture resistance to cyclic fatigue of ProTaper Next (PTN; Dentsply Tulsa Dental Specialties, Tulsa, OK), ProTaper Universal (PTU, Dentsply Tulsa Dental Specialties), and Vortex Blue (VB, Dentsply Tulsa Dental Specialties) rotary instruments. METHODS: Twenty instruments each of PTN X1-X5, PTU S1-F5, and VB 20/04-50/04 were rotated until fracture in a simulated canal of 90° and a 5-mm radius using a custom-made testing platform. The number of cycles to fracture (NCF) was calculated. Weibull analysis was used to predict the maximum number of cycles when 99% of the instrument samples survive. RESULTS: VB 20/04-30/04 had significantly higher NCF than PTU S1-F5 and PTN X1-X5. VB 35/04-45/04 had significantly higher NCF than PTU S2-F5 and PTN X2-X5. PTN X1 had higher NCF than PTU S1-F5. PTN X2 had higher NCF than PTU F2-F5. The Weibull distribution predicted the highest number of cycles at which 99% of instruments survive to be 766 cycles for VB 25/04 and the lowest to be 50 cycles for PTU F2. CONCLUSIONS: Under the limitations of this study, VB 20/04-45/04 were more resistant to cyclic fatigue than PTN X2-X5 and PTU S2-F5. PTN X1 and X2 were more resistant to cyclic fatigue than PTU F2-F5. The Weibull distribution appears to be a feasible and potentially clinically relevant model to predict resistance to cyclic fatigue.

Cementomimetics-constructing a cementum-like biomineralized microlayer via amelogenin-derived peptides.

Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides (HABPs) previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 (ADP5) was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein-mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure-function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbidity associated with tooth loss.

Central role of pyrophosphate in acellular cementum formation.

BACKGROUND: Inorganic pyrophosphate (PP(i)) is a physiologic inhibitor of hydroxyapatite mineral precipitation involved in regulating mineralized tissue development and pathologic calcification. Local levels of PP(i) are controlled by antagonistic functions of factors that decrease PP(i) and promote mineralization (tissue-nonspecific alkaline phosphatase, Alpl/TNAP), and those that increase local PP(i) and restrict mineralization (progressive ankylosis protein, ANK; ectonucleotide pyrophosphatase phosphodiesterase-1, NPP1). The cementum enveloping the tooth root is essential for tooth function by providing attachment to the surrounding bone via the nonmineralized periodontal ligament. At present, the developmental regulation of cementum remains poorly understood, hampering efforts for regeneration. To elucidate the role of PP(i) in cementum formation, we analyzed root development in knock-out ((-/-)) mice featuring PP(i) dysregulation. RESULTS: Excess PP(i) in the Alpl(-/-) mouse inhibited cementum formation, causing root detachment consistent with premature tooth loss in the human condition hypophosphatasia, though cementoblast phenotype was unperturbed. Deficient PP(i) in both Ank and Enpp1(-/-) mice significantly increased cementum apposition and overall thickness more than 12-fold vs. controls, while dentin and cellular cementum were unaltered. Though PP(i) regulators are widely expressed, cementoblasts selectively expressed greater ANK and NPP1 along the root surface, and dramatically increased ANK or NPP1 in models of reduced PP(i) output, in compensatory fashion. In vitro mechanistic studies confirmed that under low PP(i) mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PP(i) inhibited mineralization and associated increases in Ank and Enpp1 mRNA. CONCLUSIONS: Results from these studies demonstrate a novel developmental regulation of acellular cementum, wherein cementoblasts tune cementogenesis by modulating local levels of PP(i), directing and regulating mineral apposition. These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration.

Enzyme replacement prevents enamel defects in hypophosphatasia mice.

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity, leading to rickets or osteomalacia and to dental defects. HPP occurs from loss-of-function mutations within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl(-/-), aka Akp2(-/-)) mice closely phenocopy infantile HPP, including the rickets, vitamin B6-responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl(-/-) mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral-targeted TNAP (ENB-0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage-specific expression of TNAP in ameloblasts. In the Alpl(-/-) mice, histological, µCT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter-rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral-targeting, human TNAP at 8.2 mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization and demonstrate the efficacy of mineral-targeted TNAP to prevent enamel defects in HPP.

Engineered Escherichia coli silver-binding periplasmic protein that promotes silver tolerance.

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo.

Self assembled bi-functional peptide hydrogels with biomineralization-directing peptides.

A peptide-based hydrogel has been designed that directs the formation of hydroxyapatite. MDG1, a twenty-seven residue peptide, undergoes triggered folding to form an unsymmetrical beta-hairpin that self-assembles in response to an increase in solution ionic strength to yield a mechanically rigid, self supporting hydrogel. The C-terminal portion of MDG1 contains a heptapeptide (MLPHHGA) capable of directing the mineralization process. Circular dichroism spectroscopy indicates that the peptide folds and assembles to form a hydrogel network rich in beta-sheet secondary structure. Oscillatory rheology indicates that the hydrogel is mechanically rigid (G' 2500Pa) before mineralization. In separate experiments, mineralization was induced both biochemically and with cementoblast cells. Mineralization-domain had little effect on the mechanical rigidity of the gel. SEM and EDXS show that MDG1 gels are capable of directing the formation of hydroxapatite. Control hydrogels, prepared by peptides either lacking the mineral-directing portion or reversing its sequence, indicated that the heptapeptide is necessary and its actions are sequence specific.

The leucine-rich amelogenin peptide alters the amelogenin null enamel phenotype.

INTRODUCTION: The amelogenin proteins secreted by ameloblasts during dental enamel development are required for normal enamel structure. Amelx null (KO) mice have hypoplastic, disorganized enamel similar to that of human patients with mutations in the AMELX gene, and provide a model system for studies of the enamel defect amelogenesis imperfecta. Because many amelogenin proteins are present in developing enamel due to RNA alternative splicing and proteolytic processing, understanding the function of individual amelogenins has been challenging. PURPOSE: Our objective was to better understand the role of LRAP, a 59 amino acid leucine-rich amelogenin peptide, in the development of enamel. APPROACH: Teeth from transgenic mice that express LRAP under control of the Amelx regulatory regions were analyzed for mechanical properties, and transgenic males were mated with female KO mice. Male offspring with a null background that were transgene positive or transgene negative were compared to determine phenotypic differences using microcomputed tomography (microCT) and scanning electron microscopy (SEM). RESULTS: Nanoindentation revealed no differences between LRAP transgenic and wild-type murine enamel. Using microCT, LRAPKO enamel volume and density measurements were similar to those from KO mice. However, in etched samples examined by SEM, the organization of the enamel rod pattern was altered by the presence of the LRAP transgene. CONCLUSIONS: The presence of LRAP leads to changes in enamel appearance compared to enamel from KO mice. Expression of a combination of amelogenin transgenes in KO mice may lead to rescue of the individual characteristics of normal enamel.

Transgenic overexpression of gremlin results in developmental defects in enamel and dentin in mice.

Bone morphogenetic proteins (BMPs) and BMP antagonists play a crucial role in the regulation of tooth development. One of the BMP extracellular antagonists, gremlin, is a highly conserved 20.7-kDa glycoprotein. Previously, researchers reported that transgenic mice overexpressing gremlin under the control of the osteocalcin promoter (gremlin OE) exhibit a skeletal phenotype and tooth fragility. To further define the tooth phenotype, teeth and surrounding supporting tissues, obtained from gremlin OE at ages of 4 weeks, 2 months, and 4 months, were examined. The histological results demonstrate that gremlin OE exhibit an enlarged pulp chamber with ectopic calcification and thinner dentin and enamel compared with wild-type control. In vitro studies using murine pulp cells revealed that gremlin inhibited BMP-4 mediated induction of Dspp. These data provide evidence that balanced interactions between BMP agonists/antagonists are required for proper development of teeth and surrounding tissues. It is clear that these interactions require further investigation to better define the mechanisms controlling tooth root formation (pulp, dentin, cementum, and surrounding tissue) to provide the information needed to successfully regenerate these tissues.

Phosphate: known and potential roles during development and regeneration of teeth and supporting structures.

Inorganic phosphate (P(i)) is abundant in cells and tissues as an important component of nucleic acids and phospholipids, a source of high-energy bonds in nucleoside triphosphates, a substrate for kinases and phosphatases, and a regulator of intracellular signaling. The majority of the body's P(i) exists in the mineralized matrix of bones and teeth. Systemic P(i) metabolism is regulated by a cast of hormones, phosphatonins, and other factors via the bone-kidney-intestine axis. Mineralization in bones and teeth is in turn affected by homeostasis of P(i) and inorganic pyrophosphate (PPi), with further regulation of the P(i)/PP(i) ratio by cellular enzymes and transporters. Much has been learned by analyzing the molecular basis for changes in mineralized tissue development in mutant and knock-out mice with altered P(i) metabolism. This review focuses on factors regulating systemic and local P(i) homeostasis and their known and putative effects on the hard tissues of the oral cavity. By understanding the role of P(i) metabolism in the development and maintenance of the oral mineralized tissues, it will be possible to develop improved regenerative approaches.