Redefining Mucosal Inflammation with Spatial Genomics.

The human oral mucosa contains one of the most complex cellular systems that are essential for normal physiology and defense against a wide variety of local pathogens. Evolving techniques and experimental systems have helped refine our understanding of this complex cellular network. Current single-cell RNA sequencing methods can resolve subtle differences between cell types and states, thus providing a great tool for studying the molecular and cellular repertoire of the oral mucosa in health and disease. However, it requires the dissociation of tissue samples, which means that the interrelationships between cells are lost. Spatial transcriptomic methods bypass tissue dissociation and retain this spatial information, thereby allowing gene expression to be assessed across thousands of cells within the context of tissue structural organization. Here, we discuss the contribution of spatial technologies in shaping our understanding of this complex system. We consider the impact on identifying disease cellular neighborhoods and how space defines cell state. We also discuss the limitations and future directions of spatial sequencing technologies with recent advances in machine learning. Finally, we offer a perspective on open questions about mucosal homeostasis that these technologies are well placed to address.

Cystatin C as a biomarker for cardiorenal syndrome diseases quantitative diagnostics and monitoring via point-of-care.

With heart failure (HF) and renal malfunction becoming global public health issues, there is an urgent need to monitor diseases at home or in the community. Point-of-care testing (POC) would shorten the patients waiting time compared with laboratory molecular analysis. This work evaluates two types of gold nanomaterials, and two assay protocols, to develop a lateral flow (LF) system for Cystatin C (CysC) quantification. Of the protocols, the 'delayed-release' shows the alleviation of the hook effects with 1% BSA running buffer (RB), albeit at increased complexity with three steps of washing. The standard method with sample dilution (1: 150 sample dilution for GNPs, and 1:10 sample dilution for GNRs) can ensure the clinical range detection of CysC as 1 mg/L with partial LF assays. GNPs have stronger optical signal intensity compared with GNRs and developed full LF assays with GNPs require 1:1.5 sample dilution in recombinant Cys C detection. The ideal sample dilution ratio is different for partial and full LF assays. Clinical Relevance- This work is the basis of future work that will use LF devices for human serum/plasma monitoring to assess kidney function related to heart failure during medication. The specificity, sensitivity, and limit of detection will be validated via a clinical trial before potential clinical use.

GSK3 Inhibitor-Induced Dentinogenesis Using a Hydrogel.

Small-molecule drugs targeting glycogen synthase kinase 3 (GSK3) as inhibitors of the protein kinase activity are able to stimulate reparative dentine formation. To develop this approach into a viable clinical treatment for exposed pulp lesions, we synthesized a novel, small-molecule noncompetitive adenosine triphosphate (ATP) drug that can be incorporated into a biodegradable hydrogel for placement by syringe into the tooth. This new drug, named NP928, belongs to the thiadiazolidinone (TDZD) family and has equivalent activity to similar drugs of this family such as tideglusib. However, NP928 is more water soluble than other TDZD drugs, making it more suitable for direct delivery into pulp lesions. We have previously reported that biodegradable marine collagen sponges can successfully deliver TDZD drugs to pulp lesions, but this involves in-theater preparation of the material, which is not ideal in a clinical context. To improve surgical handling and delivery, here we incorporated NP928 into a specifically tailored hydrogel that can be placed by syringe into a damaged tooth. This hydrogel is based on biodegradable hyaluronic acid and can be gelled in situ upon dental blue light exposure, similarly to other common dental materials. NP928 released from hyaluronic acid-based hydrogels upregulated Wnt/ß-catenin activity in pulp stem cells and fostered reparative dentine formation compared to marine collagen sponges delivering equivalent concentrations of NP928. This drug-hydrogel combination has the potential to be rapidly developed into a therapeutic procedure that is amenable to general dental practice.

Scaffold-based developmental tissue engineering strategies for ectodermal organ regeneration.

Tissue engineering (TE) is a multidisciplinary research field aiming at the regeneration, restoration, or replacement of damaged tissues and organs. Classical TE approaches combine scaffolds, cells and soluble factors to fabricate constructs mimicking the native tissue to be regenerated. However, to date, limited success in clinical translations has been achieved by classical TE approaches, because of the lack of satisfactory biomorphological and biofunctional features of the obtained constructs. Developmental TE has emerged as a novel TE paradigm to obtain tissues and organs with correct biomorphology and biofunctionality by mimicking the morphogenetic processes leading to the tissue/organ generation in the embryo. Ectodermal appendages, for instance, develop in vivo by sequential interactions between epithelium and mesenchyme, in a process known as secondary induction. A fine artificial replication of these complex interactions can potentially lead to the fabrication of the tissues/organs to be regenerated. Successful developmental TE applications have been reported, in vitro and in vivo, for ectodermal appendages such as teeth, hair follicles and glands. Developmental TE strategies require an accurate selection of cell sources, scaffolds and cell culture configurations to allow for the correct replication of the in vivo morphogenetic cues. Herein, we describe and discuss the emergence of this TE paradigm by reviewing the achievements obtained so far in developmental TE 3D scaffolds for teeth, hair follicles, and salivary and lacrimal glands, with particular focus on the selection of biomaterials and cell culture configurations.

Translation Approach for Dentine Regeneration Using GSK-3 Antagonists.

The canonical Wnt/ß-catenin signaling pathway is crucial for reparative dentinogenesis following tooth damage, and the modulation of this pathway affects the rate and extent of reparative dentine formation in damaged mice molars by triggering the natural process of dentinogenesis. Pharmacological stimulation of Wnt/ß-catenin signaling activity by small-molecule GSK-3 inhibitor drugs following pulp exposure in mouse molars results in reparative dentinogenesis. The creation of similar but larger lesions in rat molars shows that the adenosine triphosphate (ATP)-competitive GSK-3 inhibitor, CHIR99021 (CHIR), and the ATP noncompetitive inhibitor, Tideglusib (TG), can equally enhance reparative dentine formation to fully repair an area of dentine damage up to 10 times larger, mimicking the size of small lesions in humans. To assess the chemical composition of this newly formed dentine and to compare its structure with surrounding native dentine and alveolar bone, Raman microspectroscopy analysis is used. We show that the newly formed dentine comprises equal carbonate to phosphate ratios and mineral to matrix ratios to that of native dentine, both being significantly different from bone. For an effective dentine repair, the activity of the drugs needs to be restricted to the region of damage. To investigate the range of drug-induced Wnt-activity within the dental pulp, RNA of short-term induced (24-h) molars is extracted from separated roots and crowns, and quantitative Axin2 expression is assayed. We show that the activation of Wnt/ß-catenin signaling is highly restricted to pulp cells in the immediate location of the damage in the coronal pulp tissue with no drug action detected in the root pulp. These results provide further evidence that this simple method of enhancement of natural reparative dentinogenesis has the potential to be translated into a clinical direct capping approach.

Tooth Bioengineering and Regenerative Dentistry.

Over the past 100 y, tremendous progress has been made in the fields of dental tissue engineering and regenerative dental medicine, collectively known as translational dentistry. Translational dentistry has benefited from the more mature field of tissue engineering and regenerative medicine (TERM), established on the belief that biocompatible scaffolds, cells, and growth factors could be used to create functional, living replacement tissues and organs. TERM, created and pioneered by an interdisciplinary group of clinicians, biomedical engineers, and basic research scientists, works to create bioengineered replacement tissues that provide at least enough function for patients to survive until donor organs are available and, at best, fully functional replacement organs. Ultimately, the goal of both TERM and regenerative dentistry is to bring new and more effective therapies to the clinic to treat those in need. Very recently, the National Institutes of Health/National Institute of Dental and Craniofacial Research invested $24 million over a 3-y period to create dental oral and craniofacial translational resource centers to facilitate the development of more effective therapies to treat edentulism and other dental-related diseases over the next decade. This exciting era in regenerative dentistry, particularly for whole-tooth tissue engineering, builds on many key successes over the past 100 y that have contributed toward our current knowledge and understanding of signaling pathways directing natural tooth and dental tissue development-the foundation for current strategies to engineer functional, living replacement dental tissues and whole teeth. Here we use a historical perspective to present key findings and pivotal advances made in the field of translational dentistry over the past 100 y. We will first describe how this process has evolved over the past 100 y and then hypothesize on what to expect over the next century.

Perivascular-Derived Mesenchymal Stem Cells.

Cells have been identified in postnatal tissues that, when isolated from multiple mesenchymal compartments, can be stimulated in vitro to give rise to cells that resemble mature mesenchymal phenotypes, such as odontoblasts, osteoblasts, adipocytes, and myoblasts. This has made these adult cells, collectively called mesenchymal stem cells (MSCs), strong candidates for fields such as tissue engineering and regenerative medicine. Based on evidence from in vivo genetic lineage-tracing studies, pericytes have been identified as a source of MSC precursors in vivo in multiple organs, in response to injury or during homeostasis. Questions of intense debate and interest in the field of tissue engineering and regenerative studies include the following: 1) Are all pericytes, irrespective of tissue of isolation, equal in their differentiation potential? 2) What are the mechanisms that regulate the differentiation of MSCs? To gain a better understanding of the latter, recent work has utilized ChIP-seq (chromatin immunoprecipitation followed by sequencing) to reconstruct histone landscapes. This indicated that for dental pulp pericytes, the odontoblast-specific gene Dspp was found in a transcriptionally permissive state, while in bone marrow pericytes, the osteoblast-specific gene Runx2 was primed for expression. RNA sequencing has also been utilized to further characterize the 2 pericyte populations, and results highlighted that dental pulp pericytes are already precommitted to an odontoblast fate based on enrichment analysis indicating overrepresentation of key odontogenic genes. Furthermore, ChIP-seq analysis of the polycomb repressive complex 1 component RING1B indicated that this complex is likely to be involved in inhibiting inappropriate differentiation, as it localized to a number of loci of key transcription factors that are needed for the induction of adipogenesis, chondrogenesis, or myogenesis. In this review, we highlight recent data elucidating molecular mechanisms that indicate that pericytes can be tissue-specific precommitted MSC precursors in vivo and that this precommitment is a major driving force behind MSC differentiation.

A Mouse Model to Study Reparative Dentinogenesis.

Different animal models have been introduced recently to study the process of reparative dentinogenesis in response to injury-induced pulp exposure. Using a mouse model is advantageous over other animal models since mice can be genetically manipulated to examine specific cellular pathways and lineage trace the progeny of a single cell. However, enabling a standardized molar damage in mice is demanding due to the small size of the teeth compared to the available dental instruments. Here we describe a reproducible and reliable in vivo model that allows us to study dentinogenesis in the first maxillary mouse molar.

Multicentre clinical simulation evaluation of the ISO 80369-6 neuraxial non-Luer connector.

To avoid potentially fatal wrong-route neuraxial drug errors, international standard ISO 80369-6 specifying a non-Luer neuraxial connector design was published in 2016. We describe usability studies used in development of the design. Thirty-eight doctors and 17 nurses performed simulated procedures on manikins, using devices fitted with Luer connectors or draft ISO 80369-6 'non-Luer' connectors. The procedures included spinal anaesthesia; intrathecal chemotherapy; lumbar puncture, cerebrospinal fluid collection and pressure measurement; epidural catheter placement with bolus injection and critical care use. Participants attempted cross connection between neuraxial connectors and a range of other medical device connectors, including those from the ISO 80369 small-bore connector series. Video recording analysis was used for all assessments. Participants subjectively assessed performance of the draft non-Luer connector, including suitability for routine clinical use. Participants performed 198 procedures. The connector achieved easy, leak-free connections. The willingness of participants to use the non-Luer connectors were: spinal anaesthesia 100%; intrathecal chemotherapy 88%; lumbar puncture, cerebrospinal fluid collection and pressure measurement 93%; epidural catheter placement with bolus injection 78%; critical care use 100%. Concerns raised were generally device related, rather than connector related. Most cross-connection attempts failed, even using above clinical forces and, when successful, were judged of low clinical risk potential; the exception was a malaligned connection between the non-Luer slip and female Luer connectors. This led to revision of the dimensional tolerances of the non-Luer connector to reduce this risk, before publication of the final specification in 2016. We conclude that the ISO 80369-6 neuraxial non-Luer connector is suitable for clinical use.

Church leaders' barriers and facilitators before and after implementing a physical activity and nutrition intervention.

Faith-based health promotion programs have been effective in increasing healthy eating (HE) and physical activity (PA). Very few reports exist regarding church leaders' anticipated and experienced barriers and facilitators to program implementation. Pastors (n = 38, 70%) and program coordinators (n = 54, 100%) from churches (N = 54) who attended a program training answered open-ended questions about anticipated barriers and facilitators to implementing the HE and PA parts of the Faith, Activity, and Nutrition (FAN) program. Twelve months later, pastors (n = 49, 92%) and coordinators (n = 53, 98%) answered analogous questions about their experienced barriers and facilitators to implementing the HE and PA parts of the FAN program. Responses were coded using thematic analysis. Similar themes appeared at baseline and follow-up for anticipated and experienced barriers and facilitators. The most common barriers were no anticipated barriers, resistance to change, church characteristics, and lack of participation/motivation. The most common facilitators were internal support, leadership, and communication. Few differences were found between anticipated and experienced barriers and facilitators. Understanding these perspectives, particularly overcoming resistance to change and church characteristics through strong leadership and internal support from church leaders, will improve future program development, resources, and technical assistance in faith-based and non-faith-based communities alike.

WNT10A mutation results in severe tooth agenesis in a family of three sisters.

OBJECTIVES: To identify the genetic basis of severe tooth agenesis in a family of three affected sisters. PATIENTS AND METHODS: A family of three sisters with severe tooth agenesis was recruited for whole-exome sequencing to identify potential genetic variation responsible for this penetrant phenotype. The unaffected father was tested for specific mutations using Sanger sequencing. Gene discovery was supplemented with in situ hybridization to localize gene expression during human tooth development. RESULTS: We report a nonsense heterozygous mutation in exon 2 of WNT10A c.321C>A[p.Cys107*] likely to be responsible for the severe tooth agenesis identified in this family through the creation of a premature stop codon, resulting in truncation of the amino acid sequence and therefore loss of protein function. In situ hybridization showed expression of WNT10A in odontogenic epithelium during the early and late stages of human primary tooth development. CONCLUSIONS: WNT10A has previously been associated with both syndromic and non-syndromic forms of tooth agenesis, and this report further expands our knowledge of genetic variation underlying non-syndromic forms of this condition. We also demonstrate expression of WNT10A in the epithelial compartment of human tooth germs during development.

Regulation of Reactionary Dentine Formation.

During the treatment of dental caries that has not penetrated the tooth pulp, maintenance of as much unaffected dentine as possible is a major goal during the physical removal of decayed mineral. Damage to dentine leads to release of fossilized factors (transforming growth factor-ß [TGF-ß] and bone morphogenic protein [BMP]) in the dentine that are believed to stimulate odontoblasts to secrete new "tertiary" dentine (reactionary dentine). This is formed on the pulpal surface of existing dentine and rethickens the dentine. We have previously shown that activation of Wnt/ß-catenin signaling is pivotal for tooth repair in exposed pulp injury, and the pathway can be activated by small-molecule GSK-3 antagonists, resulting in enhanced reparative dentine formation. Here, we use a nonexposed pulp injury model to investigate the mechanisms of reactionary dentine formation in vivo, using small molecules to modulate the Wnt/ß-catenin, TGF-ß, and BMP pathways. We found that a local increase of Wnt activation at the injury site enhances reactionary dentine secretion. In addition, inhibition of TGF-ß, BMP, or Wnt pathways does not impede reactionary dentine formation, although inhibition of TGF-ß and/or BMP signaling does result in more disorganized, nontubular reactionary dentine. This suggests that Wnt/ß-catenin signaling plays no major role in the formation of reactionary dentine, but in common with reparative dentine formation, exogenous elevation of Wnt/ß-catenin signaling can enhance tertiary dentine formation. Release of latent TGF-ß or BMPs from dentine is not required for the deposition of mineral to form reactionary dentine but does play a role in its organization.

A multicentre audit of HDR/PDR brachytherapy absolute dosimetry in association with the INTERLACE trial (NCT015662405).

A UK multicentre audit to evaluate HDR and PDR brachytherapy has been performed using alanine absolute dosimetry. This is the first national UK audit performing an absolute dose measurement at a clinically relevant distance (20 mm) from the source. It was performed in both INTERLACE (a phase III multicentre trial in cervical cancer) and non-INTERLACE brachytherapy centres treating gynaecological tumours. Forty-seven UK centres (including the National Physical Laboratory) were visited. A simulated line source was generated within each centre's treatment planning system and dwell times calculated to deliver 10 Gy at 20 mm from the midpoint of the central dwell (representative of Point A of the Manchester system). The line source was delivered in a water-equivalent plastic phantom (Barts Solid Water) encased in blocks of PMMA (polymethyl methacrylate) and charge measured with an ion chamber at 3 positions (120° apart, 20 mm from the source). Absorbed dose was then measured with alanine at the same positions and averaged to reduce source positional uncertainties. Charge was also measured at 50 mm from the source (representative of Point B of the Manchester system). Source types included 46 HDR and PDR 192Ir sources, (7 Flexisource, 24 mHDR-v2, 12 GammaMed HDR Plus, 2 GammaMed PDR Plus, 1 VS2000) and 1 HDR 60Co source, (Co0.A86). Alanine measurements when compared to the centres' calculated dose showed a mean difference (±SD) of +1.1% (±1.4%) at 20 mm. Differences were also observed between source types and dose calculation algorithm. Ion chamber measurements demonstrated significant discrepancies between the three holes mainly due to positional variation of the source within the catheter (0.4%-4.9% maximum difference between two holes). This comprehensive audit of absolute dose to water from a simulated line source showed all centres could deliver the prescribed dose to within 5% maximum difference between measurement and calculation.

The CAPS Study: incidence, management and outcomes of cardiac arrest in pregnancy in the UK: a prospective, descriptive study.

OBJECTIVE: To describe the incidence, risks, management and outcomes of cardiac arrest in pregnancy in the UK population, with specific focus on the use of perimortem caesarean section (PMCS). DESIGN: A prospective, descriptive study using the UK Obstetric Surveillance System (UKOSS). SETTING: All UK hospitals with maternity units. POPULATION: All women who received basic life support in pregnancy in the UK between 1 July 2011 and 30 June 2014 (n = 66). METHODS: Prospective case identification through UKOSS monthly mailing. MAIN OUTCOME MEASURES: Cardiac arrest in pregnancy, PMCS, maternal death. RESULTS: There were 66 cardiac arrests in pregnancy, resulting in an incidence of 2.78 per 100 000 maternities (1:36 000; 95% CI 2.2-3.6). In all, 28 women died (case fatality rate 42%); 16 women arrested solely as a consequence of obstetric anaesthesia, 12 of whom were obese. Basic and advanced life support were rapidly delivered. Those who died were more likely to have collapsed at home. Perimortem caesarean section was performed in 49 women, 11 in the emergency department. The time from collapse to PMCS was significantly shorter in women who survived (median interval 3 versus 12 minutes, P = 0.001). Forty-six of 58 babies were born alive; 32 babies to surviving mothers and 14 to women who died. CONCLUSION: Cardiac arrest is rare in the pregnant UK population, however, nearly a quarter of cases are precipitated by obstetric anaesthesia, suggesting an opportunity to reduce the incidence further. Maternal survival rates of 58% were achieved with timely resuscitation, including PMCS, delay in which was associated with maternal death. Inpatient arrests were associated with higher survival rates than arrests that occurred outside the hospital setting. TWEETABLE ABSTRACT: 25% of cardiac arrest in pregnancy is caused by anaesthesia. Rapid perimortem section improves survival.

Non-syndromic severe hypodontia caused by a novel frameshift insertion mutation in the homeobox of the MSX1 gene.

OBJECTIVE: Inherited congenital anomalies in tooth number, particularly hypodontia are relatively common. Although substantial progress has been made that permits a better understanding of the causes of tooth agenesis, overall knowledge of the phenotype:genotype correlations in this anomaly are still lacking. The aim in this study was to identify the causal gene mutation(s) in a family of two sisters with severe hypodontia (oligodontia) including 2nd premolars and 1st and 3rd molars, using whole exome sequencing (WES). METHODS: WES was performed using in-solution hybridization, followed by massively parallel sequencing. RESULTS: A frameshift insertion of 7 basepairs (GCAAGTT) in the homebox of MSX1 gene located in the exon 2 in heterozygous state has been identified in both sisters (NM_002448:exon2:c.572_573ins GCAAGTT: p.F191fs). CONCLUSION: We conclude that this frameshift mutation in the homeodomain (which plays an essential role in DNA binding) of MSX1 gene is responsible for tooth agenesis in this family. This expands the phenotype-genotype correlation associated with MSX1 mutations.

Mesenchymal Cell Community Effect in Whole Tooth Bioengineering.

In vitro expanded cell populations can contribute to bioengineered tooth formation but only as cells that respond to tooth-inductive signals. Since the success of whole tooth bioengineering is predicated on the availability of large numbers of cells, in vitro cell expansion of tooth-inducing cell populations is an essential requirement for further development of this approach. We set out to investigate if the failure of cultured mesenchyme cells to form bioengineered teeth might be rescued by the presence of uncultured cells. To test this, we deployed a cell-mixing approach to evaluate the contributions of cell populations to bioengineered tooth formation. Using genetically labeled cells, we are able to identify the formation of tooth pulp cells and odontoblasts in bioengineered teeth. We show that although cultured embryonic dental mesenchyme cells are unable to induce tooth formation, they can contribute to tooth induction and formation if combined with noncultured cells. Moreover, we show that teeth can form from cell mixtures that include embryonic cells and populations of postnatal dental pulp cells; however, these cells are unable to contribute to the formation of pulp cells or odontoblasts, and at ratios of 1:1, they inhibit tooth formation. These results indicate that although in vitro cell expansion of embryonic tooth mesenchymal cells renders them unable to induce tooth formation, they do not lose their ability to contribute to tooth formation and differentiate into odontoblasts. Postnatal pulp cells, however, lose all tooth-inducing and tooth-forming capacity following in vitro expansion, and at ratios >1:3 postnatal:embryonic cells, they inhibit the ability of embryonic dental mesenchyme cells to induce tooth formation.

Absorbed dose determination in kilovoltage X-ray synchrotron radiation using alanine dosimeters.

Alanine dosimeters from the National Physical Laboratory (NPL) in the UK were irradiated using kilovoltage synchrotron radiation at the imaging and medical beam line (IMBL) at the Australian Synchrotron. A 20 × 20 mm2 area was irradiated by scanning the phantom containing the alanine through the 1 mm × 20 mm beam at a constant velocity. The polychromatic beam had an average energy of 95 keV and nominal absorbed dose to water rate of 250 Gy/s. The absorbed dose to water in the solid water phantom was first determined using a PTW Model 31014 PinPoint ionization chamber traceable to a graphite calorimeter. The alanine was read out at NPL using correction factors determined for 60Co, traceable to NPL standards, and a published energy correction was applied to correct for the effect of the synchrotron beam quality. The ratio of the doses determined by alanine at NPL and those determined at the synchrotron was 0.975 (standard uncertainty 0.042) when alanine energy correction factors published by Waldeland et al. (Waldeland E, Hole E O, Sagstuen E and Malinen E, Med. Phys. 2010, 37, 3569) were used, and 0.996 (standard uncertainty 0.031) when factors by Anton et al. (Anton M, Büermann L., Phys Med Biol. 2015 60 6113-29) were used. The results provide additional verification of the IMBL dosimetry.

Composition of Mineral Produced by Dental Mesenchymal Stem Cells.

Mesenchymal stem cells isolated from different dental tissues have been described to have osteogenic/odontogenic-like differentiation capacity, but little attention has been paid to the biochemical composition of the material that each produces. Here, we used Raman spectroscopy to analyze the mineralized materials produced in vitro by different dental cell populations, and we compared them with the biochemical composition of native dental tissues. We show that different dental stem cell populations produce materials that differ in their mineral and matrix composition and that these differ from those of native dental tissues. In vitro, BCMP (bone chip mass population), SCAP (stem cells from apical papilla), and SHED (stem cells from human-exfoliated deciduous teeth) cells produce a more highly mineralized matrix when compared with that produced by PDL (periodontal ligament), DPA (dental pulp adult), and GF (gingival fibroblast) cells. Principal component analyses of Raman spectra further demonstrated that the crystallinity and carbonate substitution environments in the material produced by each cell type varied, with DPA cells, for example, producing a more carbonate-substituted mineral and with SCAP, SHED, and GF cells creating a less crystalline material when compared with other dental stem cells and native tissues. These variations in mineral composition reveal intrinsic differences in the various cell populations, which may in turn affect their specific clinical applications.