Analysis of Neutrophil Responses to Biological Exposures.

Neutrophils are an important part of the innate immune system and among the first cells to respond to infections and inflammation. Responses include chemotaxis towards stimuli, extravasation from the vasculature, and antimicrobial actions such as phagocytosis, granule release, reactive oxygen species (ROS) production, and neutrophil extracellular trap (NET) formation (NETosis). Studying how neutrophils respond to a variety of stimuli, from biomaterial interactions to microbial insults, is therefore an essential undertaking to fully comprehend the immune response. While there are some immortalized cell lines available that recapitulate many neutrophil responses, ex vivo or in vivo studies are required to fully understand the complete range of neutrophil phenotypes. Here we describe two protocols for neutrophil isolation for further ex vivo study: recovery of neutrophils from human peripheral blood, and isolation of neutrophils from the oral cavity. We also discuss an in vivo model of general inflammation with the murine air pouch that can be used to assess numerous parameters of neutrophil and immune activation, including neutrophil recruitment and biological activity. In these protocols, the cells are isolated to allow for a high degree of experimental control. The protocols are relatively straightforward and can be successfully used by labs with no prior primary cell experience. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Neutrophil isolation from human blood Basic Protocol 2: Neutrophil isolation from the oral cavity Basic Protocol 3: Murine air pouch model of general inflammation.

Strontium-loaded hydrogel scaffolds to promote gingival fibroblast function.

Dental implant clinical success is dependent on effective peri-implant tissue attachment to the trans-mucosal portion following placement. Modification of transmucosal implant surfaces can improve cellular adhesion and function leading to formation of an effective soft-tissue seal during healing, of which gingival fibroblasts are prominent cells to migrate to repair wounds and crucial for the development of a collagen rich connective tissue. Biocompatible loaded scaffold materials have been developed to allow local release of molecules with effective biological activity. Our previous studies indicate that strontium can promote gingival fibroblast metabolism, decrease apoptosis and support adhesion to titanium healing abutments. In this study, we developed a strontium-loaded alginate hydrogel scaffold which can be easily personalized to fit over any size and shape of implant transmucosal collar or healing abutment. Results indicate that biologically active strontium ions are effectively released from loaded alginate hydrogel material to promote fibroblast viability and migration to repair in vitro wounds similar to that of strontium citrate solution. Overall, this novel strontium-loaded alginate scaffold device displays good biocompatibility and functionality, demonstrating high potential as a system to provide local delivery of strontium to improve peri-implant mucosal healing following implant placement and clinical success.

Teacher Evaluation of a Self-Directed Career Guidance Intervention for South African Secondary School Learners Amidst Severe COVID-19 Restrictions.

The South African government's COVID-19 pandemic risk mitigation strategies significantly limited social contact, which necessitated a novel approach to existing face-to-face career guidance practices. The Grade 9 Career Guidance Project, originally developed as a group-based career development intervention, required radical adaptation into a self-directed, manualized format to offer career guidance to Grade 9 learners from low-income communities amid a global pandemic. The adaptation and continuation of the project was deemed essential as secondary school learners in low-income communities have limited career guidance support. Furthermore, a close collaboration with the teachers at eight resource-constrained South African secondary schools was vital for successful implementation. To assess the success of the adaptation to a self-directed format, a mixed-methods design was employed, and Life Orientation teachers' evaluative feedback was solicited (n = 11). Favorable quantitative results were obtained; majority of teachers agreed that learners enjoyed the booklet (manualized format) and that it was deemed an adequate substitute to the previous contact-based format of the Career Guidance Project. This was also confirmed by the qualitative findings revealing teachers' satisfaction with the booklet's content, specifically that the booklet is complementary to the Life Orientation curriculum. Qualitative findings identified specific contextual barriers that contributed to some learners struggling to use the booklet optimally. The results suggest that it is feasible and acceptable to implement a self-directed career guidance intervention among secondary school learners amid a global pandemic. Teachers recommended ways to integrate the booklet, resources, and contact sessions as a preferred way forward. These findings have important implications for similar resource-constrained settings that may not have readily access to in-person career guidance and counseling human development.

Mechanical characterization and adhesive properties of a dental adhesive modified with a polymer antibiotic conjugate.

This study is a follow up investigation on recent work by our group demonstrating synthesis, release and strong antibacterial character of resins modified with penicillin V (PV)-based polymer-antibiotic conjugates (PACs). Here, we aimed to evaluate the mechanical, bonding, and other relevant biomedical properties of a commercial adhesive resin modified with PV-PAC. Single Bond Plus (SB+) was modified with PAC containing 1.8 wt% conjugated PV. Adhesive resins were bonded to dentin from extracted human molars and restorative resin added. Beams of cross-sectional area of 0.9 ± 0.1 mm (Kutsch and Young, 2011) (n = 20) were obtained from the molars and tested for micro-tensile bond strength (µTBS) at 24 h and 4 months. For cohesive strength, hourglass beams (10 × 2 × 1 mm; n = 10) were assessed for ultimate tensile strength (UTS), beam-shaped specimens (25x2x2 mm; n = 10) evaluated for flexural strength and modulus (FS/FM) via three-point bending, and cylindrical specimens (3 × 2 mm; n = 10) assessed for ultimate compressive strength (UCS). For surface micro-hardness (MH), cylindrical specimens (3 × 2 mm; n = 6) were assessed before and after an EtOH challenge. The degree of conversion (DC) (5 × 1 mm; n = 6) was determined based on changes in absorbance ratio between peaks at ∼1637 cm-1 and ∼1608 cm-1 before and after curing of adhesive resins using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. To monitor water uptake and diffusion kinetics over a 28-day period, specimens (5 × 1mm) were desiccated, weighed and stored in deionized water. Control and PV-PAC modified adhesive resins demonstrated similar µTBS at 24 h and 4 months; both showing decrease in values after 4 months (p = 0.001 and 0.004). No significant differences between adhesive resins were shown in UTS, FS/FM or UCS (p<0.05). MH of PV-PAC adhesive resin was significantly reduced relative to the control (p<0.001). The DC values of the adhesive resins were not significantly different. While sorption and solubility were no different between materials, the diffusion coefficient of PV-PAC modified adhesive resin was higher than the control (p<0.001). We conclude that incorporation of PV-PAC with 1.8 wt% PV into an adhesive resin does not adversely affect its mechanical, bonding, and physical properties, thus providing a promising option for materials with long-term antibacterial character and on-demand release.

Effect of radio frequency glow-discharge treatment of titanium on human gingival fibroblasts as a function of distance.

The mechanical stability and long-term success of an implant depends on the early healing phase and osseointegration of the bone around it. In addition, a healthy gingival tissue around the implant acts as a barrier that prevents bacteria and pathological byproducts from reaching the implant site. This study investigated the in-vitro attachment and spreading of human gingival fibroblasts (HGF) on bacterial grade polystyrene (PS) at different distances from radio-frequency glow-discharge (RFGD)-treated commercially pure titanium (cpTi) specimens. Controls included sterile cpTi specimens without RFGD treatment. A second set of experiments utilized media transferred to new bacterial grade polystyrene dishes (no cpTi) after the medium was conditioned by exposure to cpTi, either with or without RFGD treatment, for 24 hr. Surface characterization of the dishes was conducted through contact angle measurements and infrared spectroscopy. Cell numbers and surface areas were determined from Image J analysis of multiple microscopic images of fixed, stained cells. The results showed significantly greater numbers and surface areas on bacterial grade PS dishes at distances up to 15 mm from the RFGD-treated cpTi groups than for the controls. Moreover, a significant effect of the conditioned medium from RFGD-treated cpTi versus control cultures was shown on the numbers of fibroblasts attached to bacterial grade polystyrene dishes after 24 hr (p < 0.005) and 48 hr (p = 0.002) incubation. Surface areas of cells exposed to conditioned medium were not significantly different (p ≥ 0.05). Surface characterization of the PS dishes showed a higher value of the critical surface tensions of the treated group when compared to the control group.

Copper@ZIF-8 Core-Shell Nanowires for Reusable Antimicrobial Face Masks.

SARS-CoV-2 and other respiratory viruses spread via aerosols generated by infected people. Face masks can limit transmission. However, widespread use of disposable masks consumes tremendous resources and generates waste. Here, a novel material for treating blown polypropylene filtration media used in medical-grade masks to impart antimicrobial activity is reported. To produce thin copper@ZIF-8 core-shell nanowires (Cu@ZIF-8 NWs), Cu NWs are stabilized using a pluronic F-127 block copolymer, followed by growth of ZIF-8 to obtain uniform core-shell structures. The Cu@ZIF-8 NWs are applied to filtration media by dip coating. Aerosol filtration efficiency decreases upon exposure to ethanol (solvent for dip-coating), but increases with addition of Cu@ZIF-8 NWs. Cu@ZIF-8 NWs shows enhanced antibacterial activity, compared to Cu NWs or ZIF-8 alone, against Streptococcus mutans and Escherichia coli. Antiviral activity against SARS-CoV-2 is assayed using virus-infected Vero E6 cells, demonstrating 55% inhibition of virus replication after 48 h by 1 µg of Cu@ZIF-8 NWs per well. Cu@ZIF-8 NWs' cytotoxicity is tested against four cell lines, and their effect on inflammatory response in A549 cells is examined, demonstrating good biocompatibility. This low-cost, scalable synthesis and straightforward deposition of Cu@ZIF-8 NWs onto filter media has great potential to reduce disease transmission, resource consumption, and environmental impact of waste.

Synthesis and antibacterial activity of polymer-antibiotic conjugates incorporated into a resin-based dental adhesive.

This work reports on polymer-antibiotic conjugates (PACs) as additives to resin-based restorative dental materials as a new strategy to convey sustained antibacterial character to these materials. Such antibacterial performance is expected to improve their longevity in the oral cavity. Using the previously reported ciprofloxacin (Cip)-based PAC as a control, a penicillin V (PV)-based PAC was investigated. The monomer-antibiotic conjugate (MAC) containing a methacrylate monomer group and a PV moiety was prepared via nucleophilic substitution between 2-chloroethyl methacrylate (CEMA) and penicillin V potassium (PVK). The PV-based PAC was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of the MAC with hydroxyethyl methacrylate (HEMA), and further characterized by 1H NMR and gel permeation chromatography (GPC) analysis. Antibiotic resistance was investigated by passaging bacteria in low concentrations of the antibiotic for 19 days, followed by a 48 h challenge at higher concentrations. Our results suggest that the development of antibiotic resistance is unlikely. Zone of inhibition (ZOI) assays revealed no clearing zones around PV-containing resins indicating minimal antibiotic leakage from the material. Similarly, MTT assay demonstrated that the antibiotic-containing specimens did not release cytotoxic byproducts that may inhibit human gingival fibroblast growth. Counting of colony-forming units in an S. mutans biofilm model was used to assess bacterial survival at baseline and after subjecting the antibiotic-containing resin specimens to an enzymatic challenge for 30 days. Significantly reduced bacterial counts were observed as the biofilm aged from 24 to 72 h, and salivary enzymatic exposure did not reduce the antibacterial efficacy of the discs, suggesting that PV-resin will be effective in reducing the re-incidence of dental caries.

Innate Phagocyte Polarization in the Oral Cavity.

The oral cavity is a complex environment constantly exposed to antigens from food and the oral microbiota. Innate immune cells play an essential role in maintaining health and homeostasis in the oral environment. However, these cells also play a significant role in disease progression. This review will focus on two innate phagocytes in the oral cavity: macrophages and neutrophils, and examine their roles during homeostasis and disease development, with a focus on periodontal disease and cancer. Macrophages have a well-known ability to polarize and be activated towards a variety of phenotypes. Several studies have found that macrophages' polarization changes can play an essential role in maintaining health in the oral cavity and contribute to disease. Recent data also finds that neutrophils display phenotypic heterogeneity in the oral cavity. In both cases, we focus on what is known about how these cellular changes alter these immune cells' interactions with the oral microbiota, including how such changes can lead to worsening, rather than improving, disease states.

Biocompatibility, mechanical, and bonding properties of a dental adhesive modified with antibacterial monomer and cross-linker.

OBJECTIVES: This study investigated the antibacterial, cytotoxicity, and mechanical properties of a dental adhesive modified with quaternary ammonium monomer ((2-acryloyloxyethyl)dimethyldodecylammonium bromide) and cross-linker (bis(2-acryloyloxyethyl)methyldodecylammonium bromide). MATERIALS AND METHODS: Monomer (M), cross-linker (C), or a combination of these (M + C) were incorporated into adhesive Adper Single Bond Plus (SB) in 5, 10, or 25% (as wt%). A colony-forming unit and MTT assays were used to evaluate antibacterial properties against Streptococcus mutans and cell viability. Resin-dentin beams (0.9 ± 0.1 mm2) were evaluated for micro-tensile bond strength (µTBS) after 24 h, 6 months, and 3 years. Hourglass specimens were evaluated for ultimate tensile strength (UTS) after 24 h, 1 week, and 6 months. Micro-hardness measurements after softening in ethanol were taken as an indirect assessment of the polymer cross-linking density. Kruskal-Wallis, one-way ANOVA, two-way ANOVA, and Student's t test were used for analysis of the antibacterial, cytotoxicity, µTBS, UTS, and hardness data, all with a significance level of p < 0.05. RESULTS: 10%M and 25%M demonstrated a significant reduction in S. mutans relative to SB (p < 0.001). No differences in cytotoxicity were detected for any of the groups. After 6 months, no changes in µTBS were shown for any of the groups. After 3 years, all groups evidenced a significant decrease in µTBS (p < 0.05) except 5%M, 5%C, and 5%M + 5%C. All groups demonstrated either stable or significantly increased UTS after 6 months. Except for the cross-linker groups, a significant decrease in micro-hardness was shown for all groups after softening in ethanol (p < 0.05). CONCLUSIONS: A 5-10% of monomer may render the resin antibacterial without a compromise to its mechanical and bonding properties. CLINICAL RELEVANCE: Biomodification of a resin adhesive with an antibacterial monomer and cross-linker may help improve the life span of adhesive restorations.

Treponema denticola stimulates Oncostatin M cytokine release and de novo synthesis in neutrophils and macrophages.

Oncostatin M (OSM) is a pleiotropic cytokine elevated in a number of inflammatory conditions including periodontal disease. OSM is produced by a variety of immune cells and has diverse functionality such as regulation of metabolic processes, cell differentiation, and the inflammatory response to bacterial pathogens. The oral cavity is under constant immune surveillance including complementary neutrophil and macrophage populations, due to a persistent symbiotic bacterial presence. Periodontal disease is characterized by a dysbiotic bacterial community, with an abundance of Treponema denticola. Despite strong associations with severe periodontal disease, the source and mechanism of the release of OSM have not been defined in the oral cavity. We show that OSM protein is elevated in the gingival epithelium and immune cell infiltrate during periodontal disease. Furthermore, salivary and oral neutrophil OSM is elevated in correlation with the presence of T. denticola. In an air pouch infection model, T. denticola stimulated higher levels of OSM than the oral pathogen Porphorymonas gingivalis, despite differential recruitment of innate immune cells suggesting T. denticola has distinct properties to elevate OSM levels. OSM release and transcription were increased in isolated human blood, oral neutrophils, or macrophages exposed to T. denticola in vitro as measured by ELISA, qPCR, and microscopy. Using transcription, translation, and actin polymerization inhibition, we found that T. denticola stimulates both OSM release through degranulation and de novo synthesis in neutrophils and also OSM release and synthesis in macrophages. Differential induction of OSM by T. denticola may promote clinical periodontal disease.

Neutrophil Extracellular Traps (NETs): An unexplored territory in renal pathobiology, a pilot computational study.

In the age of modern medicine and artificial intelligence, image analysis and machine learning have revolutionized diagnostic pathology, facilitating the development of computer aided diagnostics (CADs) which circumvent prevalent diagnostic challenges. Although CADs will expedite and improve the precision of clinical workflow, their prognostic potential, when paired with clinical outcome data, remains indeterminate. In high impact renal diseases, such as diabetic nephropathy and lupus nephritis (LN), progression often occurs rapidly and without immediate detection, due to the subtlety of structural changes in transient disease states. In such states, exploration of quantifiable image biomarkers, such as Neutrophil Extracellular Traps (NETs), may reveal alternative progression measures which correlate with clinical data. NETs have been implicated in LN as immunogenic cellular structures, whose occurrence and dysregulation results in excessive tissue damage and lesion manifestation. We propose that renal biopsy NET distribution will function as a discriminate, predictive biomarker in LN, and will supplement existing classification schemes. We have developed a computational pipeline for segmenting NET-like structures in LN biopsies. NET-like structures segmented from our biopsies warrant further study as they appear pathologically distinct, and resemble non-lytic, vital NETs. Examination of corresponding H&E regions predominantly placed NET-like structures in glomeruli, including globally and segmentally sclerosed glomeruli, and tubule lumina. Our work continues to explore NET-like structures in LN biopsies by: 1.) revising detection and analytical methods based on evolving NETs definitions, and 2.) cataloguing NET morphology in order to implement supervised classification of NET-like structures in histopathology images.

Esterase Inhibition and Copper Release from Copper Iodide Dental Adhesives - An In Vitro Study.

PURPOSE: To investigate whether dental adhesives modified with polyacrylic acid copper iodide particles could inhibit esterase activity in vitro and the copper release rate from resin matrices, as well as the correlation between the two variables. MATERIALS AND METHODS: Different concentrations of copper iodide (0.1, 0.5 and 1.0 mg/ml) were incorporated into three commercially available adhesives representative of each type. Disk specimens (n = 3) were fabricated and incubated in cholesterol esterase and pseudo-cholinesterase solutions for 16 days (37°C, pH 7.0). The enzymatic activity and rate of copper release from resin matrices were evaluated at different 4, 8, 12, and 16 days with a UV/visible-light spectrophotometer. RESULTS: Increased copper release and reduced enzymatic activity were observed with higher concentrations of copper iodide (p < 0.001). Greater copper release with reduced enzymatic activity was also demonstrated at the earlier time periods with this relationship reversing over time (p < 0.001). A moderate negative correlation between the variables was evident (-0.441; p = 0.01). CONCLUSIONS: Adhesives containing copper iodide can inhibit esterase activity in a dose- and time-dependent manner. The correlation between the variables suggests that enzymatic activity may depend on the availability of copper.

The Msp Protein of Treponema denticola Interrupts Activity of Phosphoinositide Processing in Neutrophils.

Periodontal disease is a significant health burden, causing tooth loss and poor oral and overall systemic health. Dysbiosis of the oral biofilm and a dysfunctional immune response drive chronic inflammation, causing destruction of soft tissue and alveolar bone supporting the teeth. Treponema denticola, a spirochete abundant in the plaque biofilm of patients with severe periodontal disease, perturbs neutrophil function by modulating appropriate phosphoinositide (PIP) signaling. Through a series of immunoblotting and quantitative PCR (qPCR) experiments, we show that Msp does not alter the gene transcription or protein content of key enzymes responsible for PIP3 signaling: 3' phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase (PI3K), or 5' Src homology 2 domain-containing inositol phosphatase 1 (SHIP1). Instead, using immunoblotting and enzyme-linked immunosorbent assays (ELISAs), we found that Msp activates PTEN through dephosphorylation specifically at the S380 site. Msp in intact organisms or outer membrane vesicles also restricts PIP signaling. SHIP1 phosphatase release was assessed using chemical inhibition and immunoprecipitation to show that Msp moderately decreases SHIP1 activity. Msp also prevents secondary activation of the PTEN/PI3K response. We speculate that this result is due to the redirection of the PIP3 substrate away from SHIP1 to PTEN. Immunofluorescence microscopy revealed a redistribution of PTEN from the cytoplasm to the plasma membrane following exposure to Msp, which may contribute to PTEN activation. Mechanisms of how T. denticola modulates and evades the host immune response are still poorly described, and here we provide further mechanistic evidence of how spirochetes modify PIP signaling to dampen neutrophil function. Understanding how oral bacteria evade the immune response to perpetuate the cycle of inflammation and infection is critical for combating periodontal disease to improve overall health outcomes.

Strontium Effects on Human Gingival Fibroblasts.

Strontium is a naturally occurring alkaline earth metal that has been shown to be useful not only in the treatment and prevention of osteoporosis but also in the treatment of dentinal hypersensitivity in the oral cavity; strontium is also an effective cariostatic, antiplaque, antigingivitis agent. Relatively little is known, however, about the effects of strontium on gingival fibroblasts. The purpose of the present investigation was to conduct in vitro studies on the potential for strontium to positively affect the activity of these cells such that it might be effective in the enhancement of gingival attachment to surfaces, such as healing abutments in implants in the oral cavity. The results indicate that strontium added as strontium citrate (0.5-1.0 mM), both in the absence and presence of a healing abutment, increases human gingival cell activity and decreases apoptosis in these cells. Scanning electron microscopy studies also reveal that the addition of strontium increases attachment of gingival fibroblasts to the surfaces of healing abutments. These studies provide the basis for further investigations on the use of strontium in the prevention and treatment of peri-implantitis by maximizing the formation of a peri-implant soft-tissue barrier.

Danger signals in oral cavity-related diseases.

The oral cavity is a unique environment containing teeth juxtaposed with soft tissues, all of which are constantly bathed in microbial products and host-derived factors. While microbial dysbiosis in the oral cavity clearly leads to oral inflammatory disease, recent advances find that endogenous danger-associated molecular patterns (DAMPs) released from oral and salivary tissue also contribute to the progression of inflammatory and autoimmune disease, respectively. In contrast, DAMPs produced during oral fungal infection actually promote the resolution of infection. Here, we present a review of the literature suggesting a role for signaling by DAMPs, which may intersect with pathogen-associated molecular pattern (PAMP) signaling, in diseases that manifest in the oral cavity, specifically periodontal disease, oropharyngeal candidiasis, and Sjögren's syndrome.

Polymer-antibiotic conjugates as antibacterial additives in dental resins.

Affecting the vast majority of human beings, dental caries is a premier concern of worldwide dental health. As the most commonly used restorative material to treat dental caries, resin-based composites (RBCs) lack antibacterial properties leading to quite limited restoration lifetimes. The objective of this study is to develop a polymer-antibiotic conjugate (PAC) as an effective antibacterial additive for RBCs. A monomer-antibiotic conjugate (MAC) with significant solubility was prepared by an esterification reaction of tert-butyloxycarbonyl (Boc)-protected ciprofloxacin (Cip) and 2-hydroxyethyl methacrylate (HEMA). The Cip-containing PAC with well-controlled molecular weight and composition was synthesized by reversible addition-fragmentation chain transfer (RAFT) copolymerization of the MAC with HEMA (1 : 3 molar ratio), followed by the removal of Boc from the resulting copolymer. The antibacterial dental resin was then prepared by incorporating the PAC into a commercial resin, and their properties and antibacterial performance against Streptococcus mutans were tested. In vitro experiments revealed a very slow release of Cip, which resulted in significant killing effectiveness against Streptococcus mutans nonetheless, as observed through zone of inhibition assessment and SEM imaging. The promising antibacterial properties of these resins indicate that incorporating a PAC as an additive is a valid strategy to generate antibacterial materials for dental applications.

Turning the Spotlight on Lipids in Non-Apoptotic Cell Death.

Although apoptosis has long dominated the spotlight, studies in the past two decades have expanded the repertoire of programmed cell death (PCD). Several forms of non-apoptotic regulated cell death have been identified, with important links to organismal homeostasis and different disease pathologies. Necroptosis, ferroptosis, pyroptosis, and NETosis are the major forms of PCD that have attracted attention. Clear biochemical distinctions differentiate these forms of non-apoptotic PCD at the protein and membrane levels. For instance, pore formation at the plasma membrane is a hallmark of necroptosis and pyroptosis; however, different proteins facilitate pore formation in these processes. Here, we will highlight the role of lipids in different forms of non-apoptotic PCD. In particular, we discuss how lipids can trigger or facilitate the membrane-related changes that result in cell death. We also highlight the use of small molecules in elucidating the mechanisms of non-apoptotic PCD and the potential of lipid biosynthetic pathways to perturb these processes for therapeutic applications as a future avenue of research.

Biocompatibility and bond degradation of poly-acrylic acid coated copper iodide-adhesives.

OBJECTIVE: To investigate the effect of poly-acrylic acid (PAA) copper iodide (CuI) adhesives on bond degradation, tensile strength, and biocompatibility. METHODS: PAA-CuI particles were incorporated into Optibond XTR, Optibond Solo and XP Bond in 0.1 and 0.5mg/ml. Clearfil SE Protect, an MDPB-containing adhesive, was used as control. The adhesives were applied to human dentin, polymerized and restored with composite in 2mm-increments. Resin-dentin beams (0.9±0.1mm2) were evaluated for micro-tensile bond strength after 24h, 6 months and 1year. Hourglass specimens (10×2×1mm) were evaluated for ultimate tensile strength (UTS). Cell metabolic function of human gingival fibroblast cells exposed to adhesive discs (8×1mm) was assessed with MTT assay. Copper release from adhesive discs (5×1mm) was evaluated with UV-vis spectrophotometer after immersion in 0.9% NaCl for 1, 3, 5, 7, 10, 14, 21 and 30 days. SEM, EDX and XRF were conducted for microstructure characterization. RESULTS: XTR and Solo did not show degradation when modified with PAA-CuI regardless of the concentration. The UTS for adhesives containing PAA-CuI remained unaltered relative to the controls. The percent viable cells were reduced for Solo 0.5mg/ml and XP 0.1 or 0.5mg/ml PAA-CuI. XP demonstrated the highest ion release. For all groups, the highest release was observed at days 1 and 14. SIGNIFICANCE: PAA-CuI particles prevented the bond degradation of XTR and Solo after 1year without an effect on the UTS for any adhesive. Cell viability was affected for some adhesives. A similar pattern of copper release was demonstrated for all adhesives.

Sera and salivary matrix metalloproteinases are elevated in patients with vesiculoerosive disease: a pilot study.

BACKGROUND: Oral vesiculoerosive (VE) diseases, such as lichen planus and mucous membrane pemphigoid, are immune-mediated pathoses. Matrix metalloproteinase (MMP)-2 and MMP-9 are elevated in oral lesional biopsy specimens of patients with VE disease. However, the systemic levels and activity of MMP-2 and MMP-9 in this patient population are poorly understood. We performed a pilot study to determine whether the levels and activity of MMP-2 and MMP-9 are elevated in the sera and saliva of patients with VE disease. STUDY DESIGN: We recruited patients with VE disease (n = 10) and healthy controls (n = 19). We collected sera and saliva and performed enzyme-linked immunosorbent assays to measure MMP levels. We used gelatin zymography and Biotrak assays to determine enzyme activity. Data were analyzed using the Mann-Whitney test. RESULTS: There was no difference in the activity of either MMP in the sera or saliva of patients with VE disease compared with controls. Significantly, MMP-2 levels were elevated in the sera of patients with VE disease (P < .0001), whereas MMP-9 was elevated in their saliva (P = .003). CONCLUSIONS: MMP-2 is elevated in the sera of patients with VE disease, and MMP-9 is increased in their saliva. Therefore, these enzymes may be potential markers of disease or therapeutic targets.

Neutrophil transcriptional profile changes during transit from bone marrow to sites of inflammation.

It has recently been established that neutrophils, the most abundant leukocytes, are capable of changes in gene expression during inflammatory responses. However, changes in the transcriptome as the neutrophil leaves the bone marrow have yet to be described. We hypothesized that neutrophils are transcriptionally active cells that alter their gene expression profiles as they migrate into the vasculature and then into inflamed tissues. Our goal was to provide an overview of how the neutrophil's transcriptome changes as they migrate through different compartments using microarray and bio-informatic approaches. Our study demonstrates that neutrophils are highly plastic cells where normal environmental cues result in a site-specific neutrophil transcriptome. We demonstrate that neutrophil genes undergo one of four distinct expression change patterns as they move from bone marrow through the circulation to sites of inflammation: (i) continuously increasing; (ii) continuously decreasing; (iii) a down-up-down; and (iv) an up-down-up pattern. Additionally, we demonstrate that the neutrophil migration signaling network and the balance between anti-apoptotic and pro-apoptotic signaling are two of the main regulatory mechanisms that change as the neutrophil transits through compartments.