A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping.

We recently described cell-projection pumping as a mechanism transferring cytoplasm between cells. The uptake of fibroblast cytoplasm by co-cultured SAOS-2 osteosarcoma cells changes SAOS-2 morphology and increases cell migration and proliferation, as seen by single-cell tracking and in FACS separated SAOS-2 from co-cultures. Morphological changes in SAOS-2 seen by single cell tracking are consistent with previous observations in fixed monolayers of SAOS-2 co-cultures. Notably, earlier studies with fixed co-cultures were limited by the absence of a quantitative method for identifying sub-populations of co-cultured cells, or for quantitating transfer relative to control populations of SAOS-2 or fibroblasts cultured alone. We now overcome that limitation by a novel Cartesian plot analysis that identifies individual co-cultured cells as belonging to one of five distinct cell populations, and also gives numerical measure of similarity to control cell populations. We verified the utility of the method by first confirming the previously established relationship between SAOS-2 morphology and uptake of fibroblast contents, and also demonstrated similar effects in other cancer cell lines including from melanomas, and cancers of the ovary and colon. The method was extended to examine global DNA methylation, and while there was no clear effect on SAOS-2 DNA methylation, co-cultured fibroblasts had greatly reduced DNA methylation, similar to cancer associated fibroblasts.

A Wnt-mediated phenotype switch along the epithelial-mesenchymal axis defines resistance and invasion downstream of ionising radiation in oral squamous cell carcinoma.

BACKGROUND: Dynamic transitions of tumour cells along the epithelial-mesenchymal axis are important in tumorigenesis, metastasis and therapy resistance. METHODS: In this study, we have used cell lines, 3D spheroids and tumour samples in a variety of cell biological and transcriptome analyses to highlight the cellular and molecular dynamics of OSCC response to ionising radiation. RESULTS: Our study demonstrates a prominent hybrid epithelial-mesenchymal state in oral squamous cell carcinoma cells and tumour samples. We have further identified a key role for levels of E-cadherin in stratifying the hybrid cells to compartments with varying levels of radiation response and radiation-induced epithelial-mesenchymal transition. The response to radiation further entailed the generation of a new cell population with low expression levels of E-cadherin, and positive for Vimentin (ECADLow/Neg-VIMPos), a phenotypic signature that showed an enhanced capacity for radiation resistance and invasion. At the molecular level, transcriptome analysis of spheroids in response to radiation showed an initial burst of misregulation within the first 30 min that further declined, although still highlighting key alterations in gene signatures. Among others, pathway analysis showed an over-representation for the Wnt signalling pathway that was further confirmed to be functionally involved in the generation of ECADLow/Neg-VIMPos population, acting upstream of radiation resistance and tumour cell invasion. CONCLUSION: This study highlights the functional significance and complexity of tumour cell remodelling in response to ionising radiation with links to resistance and invasive capacity. An area of less focus in conventional radiotherapy, with the potential to improve treatment outcomes and relapse-free survival.

Career-computer simulation increases perceived importance of learning about rare diseases.

BACKGROUND: Rare diseases may be defined as occurring in less than 1 in 2000 patients. Such conditions are, however, so numerous that up to 5.9% of the population is afflicted by a rare disease. The gambling industry attests that few people have native skill evaluating probabilities. We believe that both students and academics, under-estimate the likelihood of encountering rare diseases. This combines with pressure on curriculum time, to reduce both student interest in studying rare diseases, and academic content preparing students for clinical practice. Underestimation of rare diseases, may also contribute to unhelpful blindness to considering such conditions in the clinic. METHODS: We first developed a computer simulation, modelling the number of cases of increasingly rare conditions encountered by a cohort of clinicians. The simulation captured results for each year of practice, and for each clinician throughout the entirety of their careers. Four hundred sixty-two theoretical conditions were considered, with prevalence ranging from 1 per million people through to 64.1% of the population. We then delivered a class with two in-class on-line surveys evaluating student perception of the importance of learning about rare diseases, one before and the other after an in-class real-time computer simulation. Key simulation variables were drawn from the student group, to help students project themselves into the simulation. RESULTS: The in-class computer simulation revealed that all graduating clinicians from that class would frequently encounter rare conditions. Comparison of results of the in-class survey conducted before and after the computer simulation, revealed a significant increase in the perceived importance of learning about rare diseases (p < 0.005). CONCLUSIONS: The computer career simulation appeared to affect student perception. Because the computer simulation demonstrated clinicians frequently encounter patients with rare diseases, we further suggest this should be considered by academics during curriculum review and design.

Potential Hydrodynamic Cytoplasmic Transfer between Mammalian Cells: Cell-Projection Pumping.

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fibs) and malignant cells (MCs). Others report similar transfer via either tunneling nanotubes (TNTs) or shed membrane vesicles, and this changes the phenotype of recipient cells. Our time-lapse microscopy showed most exchange was from Fibs into MCs, with less in the reverse direction. Although TNTs were seen, we were surprised transfer was not via TNTs but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless by their organellar cargo and the grooves they formed indenting MCs, which was consistent with holotomography. Discrete, rapid, and highly localized transfer events evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell projections normally returns cytoplasm to the cell body. We hypothesize "cell-projection pumping" (CPP), in which cytoplasm in retracting cell projections partially equilibrates into adjacent recipient cells via microfusions that form temporary intercellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modeling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure toward least resistance. Predictions from the model were satisfied when Fibs were cocultured with MCs and fluorescence exchange was related to cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MCs or Fibs was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian intercellular cytoplasmic transfer and communication.

The Role of Thiocyanate in Modulating Myeloperoxidase Activity during Disease.

Thiocyanate (SCN-) is a pseudohalide anion omnipresent across mammals and is particularly concentrated in secretions within the oral cavity, digestive tract and airway. Thiocyanate can outcompete chlorine anions and other halides (F-, Br-, I-) as substrates for myeloperoxidase by undergoing two-electron oxidation with hydrogen peroxide. This forms their respective hypohalous acids (HOX where X- = halides) and in the case of thiocyanate, hypothiocyanous acid (HOSCN), which is also a bactericidal oxidative species involved in the regulation of commensal and pathogenic microflora. Disease may dysregulate redox processes and cause imbalances in the oxidative profile, where typically favoured oxidative species, such as hypochlorous acid (HOCl), result in an overabundance of chlorinated protein residues. As such, the pharmacological capacity of thiocyanate has been recently investigated for its ability to modulate myeloperoxidase activity for HOSCN, a less potent species relative to HOCl, although outcomes vary significantly across different disease models. To date, most studies have focused on therapeutic effects in respiratory and cardiovascular animal models. However, we note other conditions such as rheumatic arthritis where SCN- administration may worsen patient outcomes. Here, we discuss the pathophysiological role of SCN- in diseases where MPO is implicated.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

A novel cell-stiffness-fingerprinting analysis by scanning atomic force microscopy: comparison of fibroblasts and diverse cancer cell lines.

Differing stimuli affect cell stiffness while cancer metastasis is associated with reduced cell stiffness. Cell stiffness determined by atomic force microscopy has been limited by measurement over nuclei to avoid spurious substratum effects in thin cytoplasmic domains, and we sought to develop a more complete approach including cytoplasmic areas. Ninety µm square fields were recorded from ten separate sites of cultured human dermal fibroblasts (HDF) and three sites each for melanoma (MM39, WM175, and MeIRMu), osteosarcoma (SAOS-2 and U2OS), and ovarian carcinoma (COLO316 and PEO4) cell lines, each site providing 1024 measurements as 32 × 32 square grids. Stiffness recorded below 0.8 µm height was occasionally influenced by substratum, so only stiffness recorded above 0.8 µm was analysed, but all sites were included for height and volume analysis. COLO316 had the lowest cell height and volume, followed by HDF (p < 0.0001) and then PEO4, SAOS-2, MeIRMu, WM175, U2OS, and MM39. HDF were more stiff than all other cells (p < 0.0001), while in descending order of stiffness were PEO4, COLO316, WM175, SAOS-2, U2OS, MM39, and MeIRMu (p < 0.02). Stiffness fingerprints comprised scattergrams of stiffness values plotted against the height at which each stiffness value was recorded and appeared unique for each cell type studied, although in most cases the overall form of fingerprints was similar, with maximum stiffness at low height measurements and a second lower peak occurring at high-height levels. We suggest that our stiffness-fingerprint analytical method provides a more nuanced description than previously reported and will facilitate study of the stiffness response to cell stimulation.

Arecoline increases basic fibroblast growth factor but reduces expression of IL-1, IL-6, G-CSF and GM-CSF in human umbilical vein endothelium.

BACKGROUND: Areca nut chewing is associated with oral submucous fibrosis (OSF). Raised vascular basic fibroblast growth factor may induce fibrosis. Arecoline is a muscarinic alkaloid in areca nut, which we earlier reported causes injury and necrosis of human endothelium. MATERIALS AND METHODS: Human umbilical vein endothelial cells were exposed to arecoline with or without tumor necrosis factor-α, and separately to acetylcholine, muscarine, or nicotine. Protein levels of basic fibroblast growth factor, as well as the inflammatory cytokines: granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor, and Interleukins-6, 1-α and 1-ß, were determined by enzyme-linked immunosorbent assay. mRNA levels were established by real-time reverse transcription polymerase chain reaction. RESULTS: Basic fibroblast growth factor was released into the culture medium at arecoline levels causing necrosis (P < 0.05). This contrasted with an opposite effect of arecoline on levels of the inflammatory cytokines (P < 0.05). Tumor necrosis factor-α increased IL-6 and granulocyte-macrophage colony stimulated factor, but arecoline reduced this stimulated expression (P < 0.05). Arecoline had no effect on mRNA for basic fibroblast growth factor, although there was reduced mRNA for the separate inflammatory cytokines studied. The effect of acetylcholine, muscarine, and nicotine was minimal and dissimilar to that of arecoline. CONCLUSIONS: Data raise the possibility that arecoline-induced, vascular basic fibroblast growth factor contributes to OSF, by combining increased growth factor expression with endothelial necrosis, and thus driving fibroblast proliferation.

Arecoline is cytotoxic for human endothelial cells.

BACKGROUND: Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. MATERIALS AND METHODS: Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. RESULTS: Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 µg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 µg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 µg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. CONCLUSIONS: Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis.

Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

BACKGROUND: The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. METHODS: A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. RESULTS: Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. CONCLUSIONS: Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression.

Opposite cytokine synthesis by fibroblasts in contact co-culture with osteosarcoma cells compared with transwell co-cultures.

We recently reported exchange of membrane and cytoplasm during contact co-culture between human Gingival Fibroblasts (h-GF) and SAOS-2 osteosarcoma cells, a process we termed 'cellular sipping' to reflect the manner in which cells become morphologically diverse through uptake of material from the opposing cell type, independent of genetic change. Cellular sipping is increased by Tumor Necrosis Factor-α (TNF-α), and we here show for the first time altered cytokine synthesis in contact co-culture supporting cellular sipping compared with co-culture where h-GF and SAOS-2 were separated in transwells. SAOS-2 had often undetectably low cytokine levels, while Interleukin-6 (IL-6), Granulocyte Colony Stimulating Factor (G-CSF) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) were secreted primarily by TNF-α stimulated h-GF and basic Fibroblast Growth Factor (FGF) was prominent in h-GF lysates (p < 0.001). Contact co-cultures permitting cellular sipping had lower IL-6, G-CSF and GM-CSF levels, as well as higher lysate FGF levels compared with TNF-α treated h-GF alone (p < 0.05). The opposite was the case for co-cultures in transwells, with increased IL-6, G-CSF and GM-CSF levels (p < 0.03) and no clear difference in FGF. We thus demonstrate significant phenotypic change in cultures where cellular sipping occurs, potentially contributing to tumor inflammatory responses.

TNF-α and TGF-ß synergistically stimulate elongation of human endothelial cells without transdifferentiation to smooth muscle cell phenotype.

We earlier reported synergy between tumor necrosis factor-α (TNF-α) and transforming growth factor-ß1 (TGF-ß1) for apoptosis in human umbilical vein endothelium (HUVEC). Here, we study morphological change by circularity measurement of HUVEC surviving this cytokine induced synergistic apoptosis. Contrasting with reports by others studying bovine endothelium, HUVEC did not change morphology in response to TGF-ß1. TNF-α markedly elongated cells (p<0.001) and this further increased with combination of the two cytokines (p<0.001), while elongation was accompanied by increased actin stress fibres. Transdifferentiation of HUVEC to a smooth muscle cell phenotype as reported elsewhere was excluded in the current study.

Membrane and cytoplasmic marker exchange between malignant neoplastic cells and fibroblasts via intermittent contact: increased tumour cell diversity independent of genetic change.

We previously demonstrated that human osteosarcoma cells (SAOS-2) induce contact-dependent apoptosis in endothelium, and expected similar apoptosis in human gingival fibroblasts (h-GF) using SAOS-2 alkaline phosphatase (AP) to identify cells. However, h-GF apoptosis did not occur, despite reduction in AP-negative h-GF number (p < 0.01) and enhancement of this by h-GF TNFα pretreatment (p < 0.01). We suggest that TNFα-enhanced transfer of membrane AP from SAOS-2 to h-GF would explain these data. This idea was investigated using fluorescence prelabelled cells and confocal laser scanning microscopy. Co-cultures of membrane-labelled h-GF (marker-DiO) and SAOS-2 (marker-DiD) generated dual-labelled cells, primarily at the expense of single labelled h-GF (p < 0.001), suggesting predominant membrane transfer from SAOS-2 to h-GF. However, opposite directional transfer predominated when membrane labels were reversed; SAOS-2 further expressed green fluorescent protein (GFP) in cytoplasm and nuclei, and h-GF additionally bore nuclear label (Syto59) (p < 0.001). Cytoplasmic exchange was investigated using h-GF prelabelled with cytoplasmic DDAO-SE and nuclear Syto59, co-cultured with SAOS-2 expressing GFP in cytoplasm and nuclei, and predominant cytoplasmic marker transferred from h-GF to SAOS-2 (p < 0.05). Pretreating h-GF with TNFα increased exchange of membrane markers (p < 0.04) but did not affect either cell surface area profile or circularity. Dual-labelled cells had a morphological phenotype differing from SAOS-2 and h-GF (p < 0.001). Time-lapse microscopy revealed extensive migration of SAOS-2 and cell process contact with h-GF, with the appearance of SAOS-2 indulging in 'cellular sipping' from h-GF. Similar exchange of membrane was seen between h-GF and with other cell lines (melanoma MeIRMu, NM39, WMM175, MM200-B12; osteosarcoma U20S; ovarian carcinoma cells PE01, PE04 and COLO316), while cytoplasmic sharing was also seen in all cell lines other than U20S. We suggest that in some neoplasms, cellular sipping may contribute to phenotypic change and the generation of diverse tumour cell populations independent of genetic change, raising the possibility of a role in tumour progression.

Dental Infection Requiring Hospitalisation Is a Public Health Problem in Australia: A Systematic Review Demonstrating an Urgent Need for Published Data.

Background: The aim of this systematic review was to analyse the published literature on dental infections leading to hospitalisations in Australia. It was hoped that understanding the patterns and trends would form a basis for improved preventive and management policies. Methods: An electronic search was performed using Web of Science, Medline via Ovid and Google Scholar. Inclusion and exclusion criteria were applied. The included studies were analysed for demographics, aetiology, management, length of hospital stay and outcome of dental infections requiring hospitalisation. Results: Nine retrospective studies were eligible for inclusion. A total of 2196 cases of dental infections leading to hospitalisations were reported, with a male predominance (55-67%). Mental health issues, illicit substance abuse and immunosuppression were the main associated comorbidities (up to 58%). Dental caries (59-90%) and pericoronitis (10-19%) were the leading causes of dental infections. Empirical antibiotics were utilised in up to 75% of cases prior to hospital presentation. Six mortalities were reported. Conclusions: The available published data show that dental infection is a significant public health problem. However, only general conclusions were possible due to the variably small sample size and data collection that was inconsistent and incomplete across studies. Improved data collection is required to develop policies for prevention and management.

The Future of Dental Care: The Manipulation of Dental Instruments & Preparation Towards Automated Tooth Cleaning.

Dentistry is an essential practice to maintain the health of the oral cavity. Recent advances in digitization and technology for oral examinations have improved the speed and ease of disease diagnosis and dental treatment. Dental robotics has emerged as a new field of dentistry and offers numerous benefits to dental professionals and society. This paper proposes an innovative design of a dental robot setup with a preliminary study on a head model for the preparation of automated dental exploration in MATLAB and discusses further considerations for automation.

Toxicity of Orthodontic Brackets Examined by Single Cell Tracking.

Subtle toxic effects may be masked in traditional assays that average or summate the response of thousands of cells. We overcome this by using the recent method of single cell tracking in time-lapse recordings. This follows the fate and behavior of individual cells and their progeny and provides unambiguous results for multiple simultaneous biological responses. Further, single cell tracking permits correlation between progeny relationships and cell behavior that is not otherwise possible, including disruption by toxins and toxicants of similarity between paired sister cells. Notably, single cell tracking seems not to have been previously used to study biomaterials toxicity. The culture medium was pre-conditioned by 79 days incubation with orthodontic brackets from seven separate commercial sources. Metal levels were determined by Inductively Coupled Plasma Mass Spectrometry. Metal levels varied amongst conditioned media, with elevated Cr, Mn, Ni, and Cu and often Mo, Pb, Zn, Pd, and Ag were occasionally found. The effect on human dermal fibroblasts was determined by single cell tracking. All bracket-conditioned media reduced cell division (p < 0.05), while some reduced cell migration (p < 0.05). Most bracket-conditioned media increased the rate of asynchronous sister cell division (p < 0.05), a seemingly novel measure for toxicity. No clear effect on cell morphology was seen. We conclude that orthodontic brackets have cytotoxic effects, and that single cell tracking is effective for the study of subtle biomaterials cytotoxicity.

Dental infection and vascular disease.

Periodontitis is a chronic inflammatory response to bacterial plaque in which the anchoring bone and soft tissues supporting teeth are destroyed, resulting in tooth mobility and loss. Dental caries involves the spread of infection from the dentine to the vascular dental pulp and periapical bony tissues, before involvement of adjacent soft tissues and spreading sepsis. Several case-controlled, cross-sectional, and cohort studies report correlation between periodontitis and increased cardiovascular, cerebrovascular, and peripheral artery disease, as determined by clinical disease, angiography, ultrasonography, and reduced flow-mediated dilation. Some studies report a similar relationship of atherosclerosis with periapical infection and potentially also with coronal caries, and this review identifies the need to investigate these associations further. Smoking and cadmium exposure are epidemiologically confounding environmental risk factors shared by atherosclerosis and periodontitis. Further complicating epidemiological studies are the risk factors for both atherosclerosis and periodontitis, with which periodontitis appears to have separate positive feedback relationships. These include diabetes, increased plasma lipid levels, hypertension, and white blood cell count. Animal and human intervention studies provide some direct support of a causal role for periodontitis in atherosclerosis, and possible mechanisms include bacterial invasion of arteries, specific atherogenic properties of oral bacteria, the acute phase response, and cytokine polymorphisms.

Comparison of microsuture, interpositional nerve graft, and laser solder weld repair of the rat inferior alveolar nerve.

PURPOSE: Intraosseous repair of nerves involves difficulty of access and there is concern that bone healing may interfere with repair outcomes. The present report describes the effect of 3 separate repair techniques on recovery from section of the rat intraosseous inferior alveolar nerve, with reference to the mental nerve distal and the trigeminal ganglion proximal to the nerve section. MATERIALS AND METHODS: Unilateral exposure of the inferior alveolar nerves of 28 rats was achieved through bone windows. Nerves were sectioned and rats were assigned to 1 of 4 groups (n = 7): untreated controls, microsuture repair, interpositional nerve grafts from the femoral nerve, or laser solder weld repair. Animals were sacrificed 1 year after surgery for histologic evaluation of the mental nerve, inferior alveolar nerve, and trigeminal ganglion compared with unoperated contralateral nerves. RESULTS: Compared with the unoperated contralateral nerves, nerve section substantially decreased mental nerve fiber number, mental nerve myelination, mental nerve fiber diameter, inferior alveolar nerve vascularity, trigeminal neuron number, and trigeminal neuron horseradish peroxidase tracer uptake and increased trigeminal ganglion degenerate neurons (P < .001). All 3 forms of repair substantially decreased these effects (P < .05). Interpositional nerve graft was least effective (P < .05). Nonetheless, mental nerve fiber diameter was significantly decreased compared with unsectioned nerves after microsuture and laser solder weld repair (P < .05). CONCLUSIONS: Intraosseous repair of the inferior alveolar nerve decreases peripheral and central signs of degeneration. Clinical hyperesthesia after repair may reflect a predominance of small fibers after recovery.

Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, as well as Altered Morphology.

We earlier reported that cell-projection pumping transfers fibroblast contents to cancer cells and this alters the cancer cell phenotype. Here, we report on single-cell tracking of time lapse recordings from co-cultured fluorescent fibroblasts and SAOS-2 osteosarcoma cells, tracking 5201 cells across 7 experiments. The fluorescent lipophilic marker DiD was used to label fibroblast organelles and to trace the transfer of fibroblast cytoplasm into SAOS-2 cells. We related SAOS-2 phenotypic change to levels of fluorescence transfer from fibroblasts to SAOS-2 cells, as well as what we term 'compensated fluorescence', that numerically projects mother cell fluorescence post-mitosis into daughter cells. The comparison of absolute with compensated fluorescence allowed us to deduct if the phenotypic effects in mother SAOS-2 cells were inherited by their daughters. SAOS-2 receipt of fibroblast fluorescence correlated by Kendall's tau with cell-profile area and without evidence of persistence in daughter cells (median tau = 0.51, p < 0.016); negatively and weakly with cell circularity and with evidence of persistence (median tau = -0.19, p < 0.05); and very weakly with cell migration velocity and without evidence of persistence (median tau = 0.01, p < 0.016). In addition, mitotic SAOS-2 cells had higher rates of prior fluorescence uptake (median = 64.9 units/day) than non-dividing cells (median = 35.6 units/day, p < 0.016) and there was no evidence of persistence post-mitosis. We conclude that there was an appreciable impact of cell-projection pumping on cancer cell phenotype relevant to cancer histopathological diagnosis, clinical spread and growth, with most effects being 'reset' by cancer cell mitosis.