Assessing MTT and sulforhodamine B cell proliferation assays under multiple oxygen environments.

Cell proliferation can be measured directly by counting cells or indirectly using assays that quantitate total protein or metabolic activity. However, for comparing cell proliferation under varying oxygen conditions it is not clear that these assays are appropriate surrogates for cell counting as cell metabolism and protein synthesis may vary under different oxygen environments. We used permeable bottom tissue culture ware to compare proliferation assays as a function of static oxygen concentrations under oxygen partial pressure (pO2) levels ranging from 2 to 139 mmHg. Cell proliferation was measured by cell counting and compared to surrogate methods measuring cell metabolism (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) and total protein (sulforhodamine B) assays under these different environments in Caco-2, MCF-7, MCF-10A and PANC-1 human cell lines. We found that the MTT readings do not correlate with cell number for the Caco-2 and PANC-1 cell lines under different oxygen conditions, whereas the sulforhodamine B protein assays perform well under all conditions. However, within a given oxygen environment, both proliferation assays show a correlation with cell number. Therefore, the MTT assay must be used with caution when comparing cell growth or drug response for cells grown in different oxygen environments. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00584-0.

Current and future directions of drug delivery for the treatment of mental illnesses.

Mental illnesses including anxiety disorders, autism spectrum disorder, post-traumatic stress disorder, schizophrenia, depression, and others exact an immense toll on the healthcare system and society at large. Depression alone impacts 21 million adults and costs over $200 billion annually in the United States. However, pharmaceutical strategies to treat mental illnesses are lagging behind drug development in many other disease areas. Because many of the shortcomings of therapeutics for mental illness relate to delivery problems, drug delivery technologies have the potential to radically improve the effectiveness of therapeutics for these diseases. This review describes the current pharmacotherapeutic approaches to treating mental illnesses as well as drug delivery approaches that have improved existing therapies. Approaches to improve drug bioavailability, provide controlled release of therapeutics, and enable drug targeting to the central nervous system (CNS) will be highlighted. Moreover, next-generation delivery approaches such as environmentally-controlled release and interval/sequential drug release will be addressed. Based on the evolving landscape of the treatment of mental illnesses, the nascent field of drug delivery in mental health has tremendous potential for growth in terms of both economic and patient impact.

Interval delivery of 5HT2A agonists using multilayered polymer films.

There is an urgent unmet medical need to develop therapeutic options for the ~50% of depression patients suffering from treatment-resistant depression, which is difficult to treat with existing psycho- and pharmaco-therapeutic options. Classical psychedelics, such as the 5HT2A agonists, have re-emerged as a treatment paradigm for depression. Recent clinical trials highlight the potential effectiveness of 5HT2A agonists to improve mood and psychotherapeutic growth in treatment-resistant depression patients, even in those who have failed a median of four previous medications in their lifetime. Moreover, microdosing could be a promising way to achieve long-term alleviation of depression symptoms without a hallucinogenic experience. However, there are a gamut of practical barriers that stymie further investigation of microdosing 5HT2A agonists, including: low compliance with the complicated dosing regimen, high risk of diversion of controlled substances, and difficulty and cost administering the long-term treatment regimens in controlled settings. Here, we developed a drug delivery system composed of multilayered cellulose acetate phthalate (CAP)/Pluronic F-127 (P) films for the encapsulation and interval delivery of 5HT2A agonists from a fully biodegradable and biocompatible implant. CAPP film composition, thickness, and layering strategies were optimized, and we demonstrated three distinct pulses from the multilayered CAPP films in vitro. Additionally, the pharmacokinetics and biodistribution of the 5HT2A agonist 2,5-Dimethoxy-4-iodoamphetamine (DOI) were quantified following the subcutaneous implantation of DOI-loaded single and multilayered CAPP films. Our results demonstrate, for the first time, the interval delivery of psychedelics from an implantable drug delivery system and open the door to future studies into the therapeutic potential of psychedelic delivery.

Comparison of calcium-based technologies to remineralise enamel subsurface lesions using microradiography and microhardness.

Assessment of enamel subsurface lesion remineralisation is essential for the evaluation of novel remineralisation technologies. The gold standard to assess subsurface mineral gain of enamel lesions is transverse microradiography (TMR). However, some studies have utilised surface microhardness (SMH) to evaluate efficacy of remineralisation agents. The aim of this study was to assess remineralisation of enamel subsurface lesions using TMR and SMH after in vitro treatment with calcium-containing technologies, and to test correlation between the TMR and SMH measurements. The parameters obtained from the TMR and SMH analyses of enamel subsurface remineralisation were not significantly correlated. Furthermore, the enamel subsurface remineralisation as measured by TMR was significantly correlated with the water-soluble calcium concentration of the remineralisation products. Scanning electron microscopy revealed surface precipitates formed by specific remineralisation treatments obfuscated accurate assessment of remineralisation by SMH. It was concluded that TMR is a more appropriate method for analysis of enamel subsurface remineralisation, and that SMH values of remineralised enamel should be interpreted with caution. Using TMR the level of remineralisation (%R) by the different technologies was CPP-ACP/F (31.3 ± 1.4%); CPP-ACP (24.2 ± 1.4%); CaSO4/K2HPO4/F (21.3 ± 1.4%); f-TCP/F (20.9 ± 1.0%); Nano-HA/F (16.3 ± 0.3%); Nano-HA (15.3 ± 0.6%) and F alone control (15.4 ± 1.3%).

Under-Oil Autonomously Regulated Oxygen Microenvironments: A Goldilocks Principle-Based Approach for Microscale Cell Culture.

Oxygen levels in vivo are autonomously regulated by a supply-demand balance, which can be altered in disease states. However, the oxygen levels of in vitro cell culture systems, particularly microscale cell culture, are typically dominated by either supply or demand. Further, the oxygen microenvironment in these systems is rarely monitored or reported. Here, a method to establish and dynamically monitor autonomously regulated oxygen microenvironments (AROM) using an oil overlay in an open microscale cell culture system is presented. Using this method, the oxygen microenvironment is dynamically regulated via the supply-demand balance of the system. Numerical simulation and experimental validation of oxygen transport within multi-liquid-phase, microscale culture systems involving a variety of cell types, including mammalian, fungal, and bacterial cells are presented. Finally, AROM is applied to establish a coculture between cells with disparate oxygen demands-primary intestinal epithelial cells (oxygen consuming) and Bacteroides uniformis (an anaerobic species prevalent in the human gut).

A multiwell plate-based system for toxicity screening under multiple static or cycling oxygen environments.

Tumor tissue contains a continuous distribution of static and dynamically changing oxygen environments with levels ranging from physiologically normal oxygen down to anoxia. However, in vitro studies are often performed under oxygen levels that are far higher than those found in vivo. A number of devices are available to alter the oxygen environment in cell culture, including designs from our laboratory. However, in our devices and most other designs, changing the media in order to feed or dose cells remains a disruptive factor in maintaining a consistent hypoxic environment. This report presents a novel 96-well plate design that recirculates the local oxygen environment to shield cells during media changes and facilitates toxicity studies of cells cultured under varying oxygen levels. The principle behind the design is presented and the response of human pancreatic cancer PANC-1 cells treated with tirapazamine and doxorubicin under eight different static or cycling oxygen levels was measured. As expected, tirapazamine is progressively more toxic as oxygen levels decrease but retains some toxicity as oxygen is cycled between hypoxic and normoxic levels. Doxorubicin sensitivity is largely unaffected by changing oxygen levels. This technology is ideal for assessing the effects of oxygen as a variable in toxicity screens.

Acceleration of Enamel Subsurface Lesion Remineralisation by Intralesion pH Modulation.

Remineralisation of demineralised enamel subsurface lesions can be enhanced by pretreatment of the lesions with base (NaOH). The aim of this study was to test the effect of intralesion pH modulation on remineralisation of demineralised enamel subsurface lesions by casein phosphopeptide-stabilised amorphous calcium fluoride phosphate (CPP-ACFP) in vitro. Two remineralisation models were utilised, the first involving 60-min cyclic pH modulation for 105 h and the second involved short-term cyclic pH modulation (12-min cycle, 240 min total duration) compared with the equivalent time of continuous treatment (200 min total duration). The intralesion pH modulation was achieved by cyclic exposure to a pH 12.9 NaOH solution and a CPP-ACFP remineralisation solution at pH 5.5. Percent remineralisation was assessed using transverse microradiography with data statistically analysed using a 2-sample Student t test. For the first model, the intralesion pH modulation group had significantly (p < 0.001) higher remineralisation (43.8 ± 6.9%) than the control group (28.2 ± 5.8%) cycled with water. For the second model, the intralesion pH modulation group had significantly (p < 0.001) higher remineralisation (23.1 ± 3.4%) than the group with continuous equivalent CPP-ACFP treatment time (1.9 ± 1.3%). In both models, intralesion pH modulation significantly accelerated remineralisation, and this was attributed to the effect pH modulation had on the diffusion gradients of ions/ion pairs and the degree of saturation with respect to apatite phases within the lesion fluid.

Bioavailable fluoride in calcium-containing dentifrices.

Calcium added to dentifrices can complex with fluoride ions to reduce intra-oral bioavailability and therefore efficacy in preventing dental caries. Six commercially available dentifrices containing different types of calcium and fluoride were analyzed for total and bioavailable fluoride levels by adding 10 g of dentifrice to 30 mL of distilled deionized water and mixing vigorously for 1 min to simulate toothbrushing. One milliliter of the dentifrice/water slurry was immediately centrifuged and the supernatant removed for bioavailable fluoride analysis and the mixed slurry prior to centrifugation used for total fluoride analysis using a modified microdiffusion method. The concentration of fluoride was determined using a fluoride ion-selective electrode calibrated with internal fluoride standards. All the dentifrices had similar total fluoride concentrations to those indicated on their labels (94% to 105%). However, only one dentifrice that contained calcium in the form of casein phosphopeptide amorphous calcium phosphate (CPP-ACP) had almost 100% (97%) of fluoride in bioavailable form. The other dentifrices contained calcium carbonate and they exhibited significantly (p < 0.001) lower bioavailable fluoride levels (27% to 61%), through the generation of poorly soluble fluoride phases. The saliva biomimetic CPP, as CPP-ACP, in a dentifrice stabilised calcium and fluoride ions to maintain fluoride's bioavailability.

Recharge and increase in hardness of GIC with CPP-ACP/F.

OBJECTIVE: To assess the effect of CPP-ACP/F recharging on ion release and hardness of GIC Fuji-Triage (VII) and Fuji-Triage-EP (VII-EP) containing CPP-ACP/F. METHODS: CPP-ACP distribution in Fuji-Triage-EP was determined using immunofluorescence. Thirty blocks of Fuji-Triage and Fuji-Triage-EP with the same surface area were placed individually in 5mL of 50mM lactic acid (pH 5) for three days. Every 12h ten Fuji-Triage and ten Fuji-Triage-EP blocks were treated with 2mL of either MI Paste Plus (CPP-ACP/F) solution (1g paste+4mL water), Placebo MI paste solution (no CPP-ACP/F), or distilled water for 2min. After each 2min treatment the blocks were rinsed with distilled water and placed back into the acid. Calcium, inorganic phosphate and fluoride levels in the acid solution were measured using atomic absorption spectrophotometry, colorimetry and ion specific electrode respectively. Vickers surface hardness of the GIC was also determined. Data were analysed using a two-sample t-test and one-way ANOVA with a Bonferroni-Holm correction for multiple comparisons. RESULTS: CPP-ACP was distributed throughout Fuji-Triage-EP. Significantly (p<0.001) higher calcium, inorganic phosphate and fluoride ion release and greater surface hardness (acid resistance) was observed in both GIC's treated with the CPP-ACP/F paste. Fuji-Triage-EP released higher ion levels and exhibited greater surface hardness (acid resistance) than Fuji-Triage. SIGNIFICANCE: Topical application of CPP-ACP/F paste to GIC Fuji-Triage-EP recharged ion release and increased surface hardness (acid resistance) which may help improve properties and resistance to degradation as well as improve ion release for caries control.

Addition of CPP-ACP to yogurt inhibits enamel subsurface demineralization.

OBJECTIVES: The aim of this study was to investigate the capacity of an approved food additive with anticariogenic properties, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), when added to a processed, sugar-containing yogurt with added lactic-acid bacteria (probiotics), to prevent demineralization of enamel subsurface lesions in vitro. METHODS: Enamel subsurface demineralised lesions were created in thirty extracted human third molars. These were then exposed to artificial saliva (AS) with: 1) Danone yogurt alone; 2) Danone yogurt with 0.2 % w/w CPP-ACP; or 3) Danone yogurt with 0.5 % w/w CPP-ACP at 37 °C for two weeks. The yogurt/AS was replaced with fresh preparations each day. At the completion of each treatment the enamel slabs were embedded, sectioned and analyzed using transverse microradiography to measure changes in enamel lesion depths and subsurface mineral content. Yogurt samples were analysed for soluble calcium (Ca) and inorganic phosphate (Pi) levels and pH. RESULTS: Yogurt alone demineralized enamel subsurface lesions to produce significantly larger lesions. However, the addition of 0.2 % CPP-ACP to the yogurt resulted in significant reduction in demineralization compared with yogurt alone (p < 0.0001). The addition of 0.5 % CPP-ACP to the yogurt produced a net remineralization effect with a significant increase in lesion mineral content (p < 0.0001). The addition of CPP-ACP resulted in a significant (p < 0.0001) dose-related increase in Ca, Pi and pH. CONCLUSIONS: The addition of CPP-ACP to a commercial yogurt exhibited a dose related protective effect with 0.5 % CPP-ACP producing remineralization of existing enamel subsurface lesions under the in vitro experimental conditions. CLINICAL SIGNIfiCANCE: The results of this study suggest that some processed yogurts with added sugar could result in enamel demineralization when frequently consumed by individuals with poor oral hygiene. The addition of CPP-ACP to these yogurts may help prevent demineralization and promote enamel subsurface lesion remineralization, and therefore, make them safer for teeth.

Effects of Bovine Serum Albumin and High pH Pre-Treatment on the Remineralisation of Enamel Subsurface Lesions in vitro.

Accumulated intra-lesion protein such as serum albumin has been speculated to impede remineralisation of carious enamel lesions. The aim of this study was to assess whether intra-lesion bovine serum albumin (BSA) affected subsequent remineralisation of enamel subsurface lesions. Confocal microscopy was used to confirm localisation of BSA in artificial enamel subsurface lesions and its subsequent degradation by a high pH sodium hypochlorite treatment. An in vitro remineralisation experiment tested the effect of intra-lesion BSA, and its degradation by sodium hypochlorite, on remineralisation of subsurface lesions by casein phosphopeptide stabilised amorphous calcium fluoride phosphate. In addition, lesions without BSA were pre-treated with one of 2 high pH solutions (sodium hypochlorite or sodium hydroxide) prior to remineralisation to test whether the high pH pre-treatment influenced remineralisation. Data were obtained on remineralisation using transverse microradiography and were analysed with a one-way ANOVA. Intra-lesion BSA had no significant effect on remineralisation compared with that of control lesions. Pre-treatment of BSA-containing lesions with sodium hypochlorite significantly increased remineralisation. The lesions without BSA that were pre-treated with either sodium hypochlorite or sodium hydroxide also showed the same level of remineralisation as the BSA-containing lesions pre-treated with sodium hypochlorite indicating that the increased remineralisation was pH related. Hence, it was concluded that intra-lesion BSA did not affect remineralisation of artificial enamel subsurface lesions in this model system and that a high pH pre-treatment enhanced remineralisation.

The prebiotic effect of CPP-ACP sugar-free chewing gum.

OBJECTIVES: To determine if chewing gum containing casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) promoted an increase in the abundance of Streptococcus sanguinis and other species associated with dental health in supragingival plaque in a clinical study. MATERIALS AND METHODS: Nineteen participants were recruited for a three-leg cross-over, randomised, controlled clinical trial. Participants chewed a sugar-free gum with or without CPP-ACP six times daily for 20 min over two weeks. The study also involved no gum chewing (no gum) for the same two week period. Participants were randomly assigned to one of the test gums or no gum for each intervention period. Participants abstained from oral hygiene and had washout periods of two weeks between intervention periods. After each intervention period, supragingival plaque was collected and analysed for bacterial composition by sequencing the V4 variable region of the 16S rRNA gene. Data were analysed using a linear mixed model. RESULTS: The CPP-ACP gum intervention produced a significant (p < 0.01) increase in the proportions of S. sanguinis (112%), as well as the commensal species Rothia dentocariosa (127%), Corynebacterium durum (80%) and Streptococcus mitis (55%) when compared with the no gum intervention. All the species that were promoted by the CPP-ACP gum are known to possess one or both of the alkali-producing enzymes arginine deiminase and nitrate reductase. CONCLUSION: This clinical study demonstrated that chewing a sugar-free gum containing CPP-ACP promoted prebiosis by significantly increasing the proportion of S. sanguinis and other health-associated bacterial species in supragingival plaque. CLINICAL SIGNIFICANCE: Regular chewing of CPP-ACP sugar-free gum increases the proportions of health-associated commensal species in supragingival plaque to promote prebiosis and oral homeostasis.

Flow-Encoded Oxygen Control to Track the Time-Dependence of Molecular Changes Induced by Static or Cycling Hypoxia.

Detecting the effects of low oxygen on cell function is often dependent on monitoring the expression of a number of hypoxia markers. The time dependence of the appearance and stability of these markers varies between cell lines. Assessing cellular marker dynamics is also critical to determining how quickly cells respond to transient changes in oxygen levels that occurs with cycling hypoxia. We fabricated a manifold designed to use flow-encoding to produce sequential changes in gas mixtures delivered to a permeable-bottom 96-well plate. We show how this manifold and plate design can be used to expose cells to either static or cycling hypoxic conditions for eight different time periods thereby facilitating the study of the time-response of cells to altered oxygen environments. Using this device, we monitored the time-dependence of molecular changes in human PANC-1 pancreatic carcinoma and Caco-2 colon adenocarcinoma cells exposed to increasing periods of static or cycling hypoxia. Using immunohistochemistry, both cell lines show detectable levels of the marker protein hypoxia-inducible factor-1α (HIF-1α) after 3 h of exposure to static hypoxia. Cycling hypoxia increased the expression level of HIF-1α compared to static hypoxia. Both static and cycling hypoxia also increased glucose uptake and aldehyde dehydrogenase activity. This new device offers a facile screening approach to determine the kinetics of cellular alterations under varying oxygen conditions.

Paper-based passive pumps to generate controllable whole blood flow through microfluidic devices.

Fluid manipulation in microfluidic systems is often controlled by active pumps that are relatively large in size and require external power sources which limit their portability and use in limited-resource settings. In this work, portable, detachable, low-cost, and power-free paper pumps with engineered capillary tubes (referred to as "grooves") that can passively drive viscous fluids based on capillary action are presented. The proposed grooved paper pumps are capable of generating a controllable flow of complex biofluids within microfluidic devices with minimal user intervention and no external power sources. The pumping performance of grooved paper pumps in this study is tested with undiluted, unseparated whole blood samples - demonstrating successful transport of approximately 150 µL of blood within an average time of 5 minutes to 50 minutes, depending on their design parameters. Results for the flow rate of grooved paper pumps indicate that the number of grooves created within the porous paper determines the profile of the generated flow rate. The experimental data also show that as the number of grooves in the pumps is increased, the flow rate approaches a constant value for the entire duration of pumping before the pump becomes saturated. The promising performance of grooved paper pumps with whole blood offers potential applications of these small, disposable pumps in point-of-care diagnostics in which time is crucial and access to external power is limited.

Effects of soy and bovine milk beverages on enamel mineral content in a randomized, double-blind in situ clinical study.

Soy beverages are promoted as healthy alternatives to bovine milk even though they can contain added sugar. OBJECTIVES: To compare enamel mineral content after consumption of bovine milk or a soy beverage in a double-blind, randomized, cross-over in situ clinical study. MATERIALS AND METHODS: Human enamel slabs with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers who consumed 200 ml of either bovine milk or a soy beverage over a 60 s period once a day for 15 days. Enamel lesion depth and mineral content were measured using transverse microradiography. Saliva samples were collected immediately after consuming the beverages and calcium, inorganic phosphate and fluoride levels analysed. Data were statistically analysed using a linear mixed model. RESULTS: Depth of the enamel subsurface lesions increased by 7.1 ±â€¯2.0 µm and mineral content decreased by 47 ±â€¯22 vol% min.µm after consumption of the soy beverage indicating demineralization. However, after consumption of bovine milk the depth of the lesions decreased by 7.6 ±â€¯3.5 µm and mineral content increased by 202 ±â€¯43 vol% min.µm indicating remineralization. The changes were significantly different (p < 0.001) between the two beverages. Fluoride levels were similar in the saliva samples for both beverages, however the calcium and inorganic phosphate levels for the bovine milk group were significantly higher (p < 0.02) than those for the soy beverage group. CONCLUSIONS: In this randomized, double-blind in situ clinical trial consumption of a soy beverage demineralized enamel whereas bovine milk produced remineralization. CLINICAL SIGNIFICANCE: Although soy beverages are promoted as healthy alternatives to bovine milk the added sugar and low calcium bioavailability of the soy drink makes frequent consumption a caries risk. (Trial registration no. ISRCTN19137849).

Self-assembly of dental surface nanofilaments and remineralisation by SnF2 and CPP-ACP nanocomplexes.

Dental caries, erosion and hypersensitivity are major public health problems. SnF2 is used widely in oral care products to help prevent/treat these conditions. Casein phosphopeptide-stabilised amorphous calcium phosphate nanocomplexes (CPP-ACP) are a biomimetic nanotechnology of salivary phosphopeptide-ACP complexes that deliver bioavailable calcium and phosphate ions to promote dental remineralisation (repair). We show here using in vitro studies and a double-blind, randomised controlled, cross-over design in situ clinical trial that SnF2 and CPP-ACP interact to form a nanofilament coating on the tooth surface and that together they are superior in their ability to promote dental remineralisation. Sn(II) by cross-linking the CPP-ACP helps to stabilise the complexes which improves delivery to the tooth surface and enhances binding and ion incorporation into tooth mineral. The combination of SnF2 and CPP-ACP in oral care products may significantly improve their efficacy in prevention/treatment of dental caries/erosion and hypersensitivity.

Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors.

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aß1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aß1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.

Mixing and delivery of multiple controlled oxygen environments to a single multiwell culture plate.

Precise oxygen control is critical to evaluating cell growth, molecular content, and stress response in cultured cells. We have designed, fabricated, and characterized a 96-well plate-based device that is capable of delivering eight static or dynamically changing oxygen environments to different rows on a single plate. The device incorporates a gas-mixing tree that combines two input gases to generate the eight gas mixtures that supply each row of the plate with a different gas atmosphere via a removable manifold. Using air and nitrogen as feed gases, a single 96-well plate can culture cells in applied gas atmospheres with Po2 levels ranging from 1 to 135 mmHg. Human cancer cell lines MCF-7, PANC-1, and Caco-2 were grown on a single plate under this range of oxygen levels. Only cells grown in wells exposed to Po2 ≤37 mmHg express the endogenous hypoxia markers hypoxia-inducible factor-1α and carbonic anhydrase IX. This design is amenable to multiwell plate-based molecular assays or drug dose-response studies in static or cycling hypoxia conditions.

Importance of bioavailable calcium in fluoride dentifrices for enamel remineralization.

OBJECTIVES: To compare remineralization of enamel subsurface lesions by fluoride dentifrices with added calcium in a double-blind, randomized, cross-over, in situ study. METHODS: Human enamel with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers. A slurry (1 g toothpaste/4 ml H2O) was rinsed for 60 s, 4 times per day for 14 days. Seven toothpastes were tested: (i) 1450 ppm F (NaF), (ii) 5000 ppm F (NaF), (iii) 1450 ppm F (MFP) with calcium sodium phosphosilicate (CSP), (iv) 1450 ppm F (MFP) with CaCO3/Arg, (v) 1150 ppm F (SnF2) with amorphous calcium phosphate (ACP), (vi) 1100 ppm F (NaF) with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and (vii) 5000 ppm F (NaF) with functionalized tri-calcium phosphate (TCP). Total (acid soluble) and bioavailable (water soluble) calcium, inorganic phosphate and fluoride levels of the dentifrices were measured using ion chromatography (F/MFP) and spectrophotometry (Ca and inorganic phosphate). Enamel lesion mineral content was measured using transverse microradiography. Data were statistically analysed using a linear mixed model. RESULTS: All calcium and fluoride containing toothpastes released > 90% of bioavailable fluoride and were superior to the respective fluoride alone toothpastes in remineralization of enamel subsurface lesions. The level of remineralization followed the order: CPP-ACP/1l00 ppm F > ACP/1150 ppm F = TCP/5000 ppm F > 5000 ppm F = CaCO3/Arg/1450 ppm F = CSP/1450 ppm F > 1450 ppm F. Bioavailable calcium levels significantly correlated with enhanced remineralization of enamel subsurface lesions. CONCLUSIONS: Bioavailable calcium in fluoride dentifrices enhanced remineralization of enamel subsurface lesions.

Excellent Rectifying Properties of the n-3C-SiC/p-Si Heterojunction Subjected to High Temperature Annealing for Electronics, MEMS, and LED Applications.

This work examines the stability of epitaxial 3C-SiC/Si heterojunctions subjected to heat treatments between 1000 °C and 1300 °C. Because of the potential for silicon carbide in high temperature and harsh environment applications, and the economic advantages of growing the 3C-SiC polytype on large diameter silicon wafers, its stability after high temperature processing is an important consideration. Yet recently, this has been thrown into question by claims that the heterojunction suffers catastrophic degradation at temperatures above 1000 °C. Here we present results showing that the heterojunction maintains excellent diode characteristics following heat treatment up to 1100 °C and while some changes were observed between 1100 °C and 1300 °C, diodes maintained their rectifying characteristics, enabling compatibility with a large range of device fabrication. The parameters of as-grown diodes were J0 = 1 × 10-11 A/mm2, n = 1.02, and +/-2V rectification ratio of 9 × 106. Capacitance and thermal current-voltage analysis was used to characterize the excess current leakage mechanism. The change in diode characteristics depends on diode area, with larger areas (1 mm2) having reduced rectification ratio while smaller areas (0.04 mm2) maintained excellent characteristics of J0 = 2 × 10-10 A/mm2, n = 1.28, and +/-2V ratio of 3 × 106. This points to localized defect regions degrading after heat treatment rather than a fundamental issue of the heterojunction.