Biochemical characterization of the cell-biomaterial interface by quantitative proteomics.

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells. The biochemical basis of the transduction of the physical and mechanical signals to cellular responses is not well understood. The lack of a systematic characterization of cell-biomaterial interaction is the major bottleneck. This study demonstrated the use of a novel subcellular fractionation method combined with quantitative MS-based proteomics to enable the robust and high-throughput analysis of proteins at the adherence interface of Madin-Darby canine kidney cells. This method revealed the enrichment of extracellular matrix proteins and membrane and stress fibers proteins at the adherence surface, whereas it shows depletion of extracellular matrix belonging to the cytoplasmic, nucleus, and lateral and apical membranes. The asymmetric distribution of proteins between apical and adherence sides was also profiled. Apart from classical proteins with clear involvement in cell-material interactions, proteins previously not known to be involved in cell attachment were also discovered.

Metabolic syndrome and three of its components as risk factors for recurrent ischaemic stroke presenting as large-vessel infarction.

BACKGROUND AND PURPOSE: Although a clear protocol for reduction of recurrent ischaemic stroke (RIS) has been established, few studies have compared the stroke subtype distribution and risk factors between RIS and first-ever stroke (FES). METHODS: This one-year hospital-based study enrolled 587 FES and 475 RIS patients. Patients were categorized into four stroke subtypes according to a modified TOAST stroke subtype classification system. Risk factor profiles were compared between the two major stroke groups and between the corresponding four subtypes to discriminate the significant risk factors for RIS. RESULTS: A multivariate regression analysis identified hypertension (OR, 1.87; 95% CI, 1.34-2.62), diabetes mellitus (DM) (OR, 1.57; 95% CI, 1.22-2.02), low high-density lipoprotein (LHDL) (OR, 1.43; 95% CI, 1.08-1.88) and older age as significant RIS risk factors. The significance of the former three RIS factors was further recognized in its large-vessel subtype. Moreover, metabolic syndrome was significantly more common in the recurrent stroke group (P = 0.01), including its large-vessel subtype (P = 0.04). Progressively increasing odds ratios from 1.49 to 2.02, in accordance with increased number of diagnostic components of metabolic syndrome for recurrent large-vessel ischaemic stroke, were noted. CONCLUSIONS: Metabolic syndrome likely plays a crucial role in the development of RIS, including large-vessel infarction in modern-day Taiwan.

Protection offered by root-surface restorative materials against biofilm challenge.

The prevalence of root-surface caries is increasing. We hypothesized that some restorative materials are protective against cariogenic challenge on root surfaces. Our goal was to study the effects of different restorative materials on root surfaces incubated with an oral biofilm generated in an artificial mouth. A biofilm of Streptococcus mutans, Streptococcus sobrinus, Lactobacillus rhamnosus, and Actinomyces naeslundii was co-cultured for 21 days on 24 glass-ionomer cement, resin-modified glass-ionomer cement, or resin-composite-restored root surfaces. These surfaces were then examined with Fourier transform infrared spectroscopy and scanning electron energy-dispersive spectroscopy. Only glass-ionomer restorations showed a significant increase in log calcium-to-phosphorus ratio (P < 0.01), and a significantly lower log amide I-to-hydrogen phosphate ratio on the root surface after incubation in the artificial mouth. Glass-ionomer restoratives conferred a preventive effect on the root surfaces against initial cariogenic challenge with a mixed-species oral biofilm without therapeutic intervention.

Incipient caries lesions on cementum by mono- and co-culture oral bacteria.

OBJECTIVES: There is increasing prevalence of root caries. We hypothesized different biofilms will cause varying demineralization in cementum. This study investigated the extent of demineralization of cementum by oral biofilm formed from three major cariogenic microorganisms: Streptococcus mutans, Lactobacillus acidophilus, and Actinomyces israelii. Sound cementum tooth blocks were incubated with mono-, bi-, and tri-species combinations of the bacteria under investigation. MATERIALS AND METHODS: The matrix (amide I) and phosphate content of the lesions was analyzed by Fourier-transform infrared spectroscopy (FTIR), and calcium and phosphorus levels were analyzed by scanning electron microscopy and energy-dispersive spectroscopy (SEM-EDX). RESULTS: The log[amide I:HPO(4)(2-) absorbance] values showed that A. israelii mono-culture caused significantly more demineralization than the other bacterial cultures. log[Ca:P] showed that all carious lesions were confined to the cementum. CONCLUSIONS: Oral biofilm arising from bacterial species A. israelii alone was the most cariogenic of those tested and produced the most demineralization in incipient carious lesions in cementum.

Knowledge of and attitudes toward severe acute respiratory syndrome among a cohort of dental patients in Hong Kong following a major local outbreak.

OBJECTIVE: To assess the knowledge of and attitudes toward severe acute respiratory syndrome (SARS) among patients attending a teaching dental hospital and private dental practices in Hong Kong during a major local SARS outbreak. METHODS: 250 dental patients were interviewed by questionnaire and 213 were interviewed by phone. RESULTS: Less than one-third (30.0%) of the 463 respondents said they were not afraid of contracting the SARS coronavirus from their dentists and did not avoid dental treatment for that reason. Nearly three-fifths (56.7%) did not worry about contracting SARS from dental treatment. Fewer than 10% of the respondents thought that dentists ran a high risk of contracting SARS. From the patients' experiences, 85.2% and 21.7% of the dentists wore face masks and face shields, respectively, when delivering dental treatment. CONCLUSIONS: The majority of patients interviewed had confidence in their dentists, their treatment environments, and the infection control measures taken, and were not worried about contracting SARS in the dental setting. This perception is an improvement from that described in an earlier study, in which more than half of the patients were concerned about contracting an infection during dental treatment and perceived that the infection control measures undertaken by the dental profession to prevent infectious diseases were not satisfactory. Patients, however, demanded better infection control measures during the SARS outbreak.

Retinal ganglion cells toxicity caused by photosensitising effects of intravitreal indocyanine green with illumination in rat eyes.

AIM: To investigate the effects of indocyanine green (ICG) with or without illumination on rat retinal ganglion cells (RGC) and retinal morphology. METHODS: Intravitreal injections of 1.0 mg/ml ICG solution were performed in rat eyes with or without subsequent illumination for 5 minutes. Eyes in the control group had intravitreal injections of balanced salt solution with illumination. Retrograde labelling of RGC with 6% Fluoro-Gold was performed 1 month later and RGC densities were compared between the three groups. Light microscopy with measurements of outer nuclear layer (ONL) and inner nuclear layer (INL) thicknesses were also performed and compared. RESULTS: Eyes with ICG without illumination showed insignificant reduction in RGC density compared with the control group (p = 0.28), whereas a significant decrease in RGC density was found in eyes that had ICG injection with illumination (p = 0.036). A significant increase in ONL thickness was also observed in the ICG with illumination treated eyes compared with the ICG without illumination and the control groups (p<0.001). No significant difference in INL thickness was observed between the three groups. CONCLUSIONS: Intravitreal injection of 0.1 mg/ml ICG in rat eyes followed by illumination resulted in photosensitising toxicity to RGC. Lower ICG concentration or illumination level should be considered when performing ICG assisted macular surgery.

Sudden cardiac death associates with a large right atrium thrombus in a late survivor of heart transplantation.

A fatal circulatory collapse developed acutely in a patient six years after orthotopic heart transplantation. Autopsy revealed a large, fresh thrombus inside the right atrium which obstructed the orifice of the vena cava completely. The grafted coronary artery and pulmonary vessels were patent. There was no rejection or acute infarct in myocardium. Mildly immature organization was seen on the thrombus-adhered wall. Four chamber enlargement and a rapid decline of right ventricular hypokinesia were detected shortly before death although he did not show active cardiopulmonary symptom. Intracardiac thrombus formation is rather uncommon in late survivor. A synergic effect of atrial turbulence and ventricular hypokinesia in the right heart, superimposed by tissue organization, was proposed for thrombus formation. An empty cardiac output was favored for his mortality. These findings provide a strong evidence to suggest anticoagulation in case of complicated or highly risk of embolization such as turbulence or hypokinesia despite of an absence of active cardiopulmonary symptom.

Retrograde axonal transport in rat ileal mesenteric nerves. Characterization using intravenously administered 125I-nerve growth factor and effect of chemical sympathectomy.

We have previously demonstrated the reproducible occurrence of dystrophic axonopathy and a defect in the retrograde axonal transport of 125I-nerve growth factor (125I-NGF) involving postganglionic sympathetic axons in the alimentary tract of rats with chronic streptozocin (STZ)-induced diabetes. To avoid complexities inherent in monitoring the accumulation of 125I-NGF in the superior mesenteric ganglion as a measure of retrograde transport in the peripheral axons of the extensive alimentary territory, we have examined retrograde axonal transport of 125I-NGF directly in ileal mesenteric nerves. 125I-NGF was injected systemically, and 2-2.5 h later ileal mesenteric nerve pedicles were ligated in vivo for various intervals. Retrogradely transported 125I-NGF in rat mesenteric nerves was measured distal to a ligature placed on the ileal mesenteric pedicle. Transport-unrelated processes, such as mechanical compression or bleeding at the site of ligation, did not contribute significantly to accumulation measured in this fashion. Accumulation of retrogradely transported 125I-NGF at the ligature began 4 h after ligation and remained linear for approximately 12 h. The amount of retrogradely transported 125I-NGF accumulating distal to the ligature reflected the length of the ileal segment served by the pedicle, which allowed the standardization of accumulation based on length of ileum innervated. The results of several experiments showed that 125I-NGF transport originated largely from nerve terminals within the ileal wall with a smaller component from extramural sites, probably terminals within the walls of blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrograde axonal transport of intravenously administered 125I-nerve growth factor in rats with streptozotocin-induced diabetes.

We have previously demonstrated a reproducible axonopathy, involving but not confined to sympathetic neurons, which involves selectively the mesenteric nerves of chronically diabetic rats. We describe a series of experiments aimed at correlating changes in the retrograde transport of 125I-nerve growth factor (125I-NGF) with development of the lesions. 125I-NGF was injected systemically and the time course of accumulation compared in superior cervical ganglion (SCG) and superior mesenteric ganglion (SMG). 125I-NGF accumulated in the SCG with a sharp peak at 12 h, whereas in the SMG, accumulation reached a plateau, remaining at relatively constant amounts for 8 h before decreasing. There was a marked decrease in the accumulation of 125I-NGF (averaging 44% in 11 separate experiments) in the SMG at early times (approximately 12 h). These decreases were seen in animals diabetic for times ranging from 2 days to 10 mo. In contrast, no consistent decrease was observed in the SCG. The time course of 125I-NGF accumulation was unaltered in the SCG of diabetic rats but was clearly retarded in the SMG of diabetic animals. Morphometric examination of the SMG of diabetic animals and controls showed little or no atrophy or neuron loss due to diabetes. We conclude that decreases in the retrograde transport of NGF occur selectively in those ganglia (SMG) serving the alimentary tract in which mesenteric nerves develop distal axonopathy, but not in ganglia whose nerves do not ultimately develop such lesions (SCG). The decreases in retrograde transport precede the development of morphologic lesions in this system. These results are consistent with the suggestion that impairment of axonal transport may play a role in the development of axonopathy.

Retrograde axonal transport of 125I-nerve growth factor in rat ileal mesenteric nerves. Effect of streptozocin diabetes.

The retrograde axonal transport of intravenously (i.v.) administered 125I-nerve growth factor (125I-NGF) was examined in mesenteric nerves innervating the small bowel of rats with streptozocin (STZ) diabetes using methods described in detail in the companion article. The accumulation of 125I-NGF distal to a ligature on the ileal mesenteric nerves of diabetic animals was 30-40% less than in control animals. The inhibition of accumulation of 125I-NGF in diabetic animals was greater at a ligature tied 2 h after i.v. administration than at a ligature tied after 14 h, which suggests that the diabetic animals may have a lag in initiation of NGF transport in the terminal axon or retardation of transport at some site along the axon. The 125I-NGF transport defect was observed as early as 3 days after the induction of diabetes, a time before the development of structural axonal lesions, and did not worsen at later times when dystrophic axonopathy is present. Both the ileal mesenteric nerves, which eventually develop dystrophic axonopathy in experimental diabetes, and the jejunal mesenteric nerves, which never develop comparable structural alterations, showed similar 125I-NGF transport deficits, suggesting that the existence of the transport abnormality does not predict the eventual development of dystrophic axonal lesions. Autoradiographic localization of 125I-NGF in the ileal mesenteric nerves of animals that had been diabetic for 11-13 mo demonstrated decreased amounts of 125I-NGF in transit in unligated paravascular nerve fascicles. There was, however, no evidence for focal retardation of transported 125I-NGF at the sites of dystrophic axonal lesions.

Axonal regeneration of retinal ganglion cells: effect of trophic factors.

A variety of neurotrophic factors can influence the cell functions of the developing, mature and injured retinal ganglion cells. The discovery that retinal ganglion cell loss can be alleviated by neurotrophic factors has generated a great deal of interest in the therapeutic potential of these molecules. Recently, evidence has provided valuable information on the receptors that mediate these events and the intracellular signaling cascades after the binding of these ligands. Signaling by neurotrophic factors does not seem to restrict to retrograde messenger from the target but also includes local interactions with neighbouring cells along the axonal pathways, anterograde signaling from the afferents and autocrine signaling. More insight into the mechanisms of action of neurotrophic factors and the signal transduction pathway leading to the protection and regeneration of retinal ganglion cells may allow the design of new therapeutic strategies.

Developmental expression patterns and localization of DNA-binding protein inhibitor (Id3) in the mouse retina.

Id3 (inhibitor of DNA binding/differentiation), a member of the Id helix-loop-helix protein family, has long been studied as a positive regulator of proliferation and a negative regulator of differentiation. In this study, we examined the expression pattern and cellular phenotypes of Id3 in postnatal and adult mouse retina. Id3 was mainly expressed in the early postnatal inner retina. From the late postnatal development towards adulthood, Id3 expression was confined to the ganglion cell layer and the inner nuclear layer. Colocalization analysis showed that Id3 positive cells were identified as retinal ganglion cells and amacrine cells. The differential expression profiles of Id3 provide the groundwork for the elucidation of its possible role in retinal development.

Coaggregation profiles of the microflora from root surface caries lesions.

Bacterial coaggregation reactions between different species and the auto aggregation of the same species are associated with the initiation and development of dental plaque and biofilms. As no such data is available on isolates from root caries lesions, we evaluated, by a visual, semi-quantitative scoring system and a spectrophotometric, quantitative assay, the coaggregation of 22 different wild-type microbial species comprising ten bacterial genera and a single Candida spp. The quantitative coaggregation assay we used proved to be a more sensitive method than the semi-quantitative, visual evaluation as the results yielded the percent coaggregation. Fusobacterium nucleatum, Lactobacillus acidophilus, Streptococcus bovis II/2 and Gemella morbillorum were observed having higher degrees of autoaggregation than the other examined strains. Significant levels of inter-species coaggregation was seen between: (1) Actinomyces spp. and Veillonella spp.; (2) Actinomyces israelii and Peptostreptococcus prevotii; (3) Campylobacter gracilis and Actinomyces spp.; (4) Prevotella intermedia and nine different species; and (5) Fusobacterium nucleatum and six other species. The single Candida albicans isolate did not coaggregate to a significant extent with any of the 21 bacterial isolates studied. Scanning electron microscopy observation of the coaggregation interactions between bacterial pairs having strong coaggregation reactions revealed varying adhesive patterns. Our findings on coaggregation amongst these isolates imply existence of multiple interactions between the coaggregation-inducing bacterial species in root caries. In particular, Actinomyces spp., Veillonella spp., Prevotella spp. and Fusobacterium spp. appear to play a significant role in this context.

An FTIR study of the effects of artificial saliva on the physical characteristics of the glass ionomer cements used for art.

OBJECTIVE: To investigate the physical and chemical changes of five esthetic restorative materials in an artificial saliva using Fourier transform infrared spectroscopy (FTIR). METHODS: The materials included two glass polyalkenoate (ionomer) cements, one polyacid-modified resin composite, and two resin composites. Five fresh and 20 aged specimens for each material were immersed in 2 ml of artificial saliva at 37 degrees C for 1 week, 2 weeks, 1 month, and 2 months. They were examined by laser surface profilometer and FTIR. RESULTS: Surface roughness average (Ra) value showed significant differences among the physical properties of glass ionomers, polyacid-modified resin composite, and resin composites. SIGNIFICANCE: FTIR spectra showed that a major chemical changes in Fuji IX GP Fast glass ionomer cement in P-NH2 group bonding (900-1000 cm(-1)) that may explain the previously reported increase in microhardness after immersion. There was no direct correlation between surface roughness and spectral changes of the materials tested.

An exploratory study on cultural variations in oral health attitudes, behaviour and values of freshman (first-year) dental students.

OBJECTIVE: To identify similarities and differences in oral health attitudes, behaviour and values among freshman dental students. DESIGN: Cross-cultural survey of dental students. SETTING: 18 cultural areas. PARTICIPANTS AND METHODS: 904 first-year dental students completed the Hiroshima University-Dental Behavioural Inventory (HU-DBI) translated into their own languages. Individual areas were clustered by similarity in responses to the questions. RESULTS: The first group displayed an 'occidental-culture orientation' with the exception of Brazil (Cluster 1 comprised: Australia, United Kingdom, Ireland, Belgium and Brazil, Cluster 2: Germany, Italy, Finland and France). The second group displayed an 'oriental-cultural orientation' with the exception of Greece and Israel (Cluster 3 comprised: China and Indonesia, and Cluster 4: Japan, Korea, Israel, Hong Kong, Malaysia, Thailand and Greece). Australia and United Kingdom were the countries that were most alike. Ireland was the 'neighbour' to these countries. Greece and Malaysia had similar patterns of oral health behaviour although geographic conditions are very different. Although it was considered that in Hong Kong, occidental nations have affected the development of education, it remained in the oriental-culture group. Comparison with the data from the occidentals indicates that a higher percentage of the orientals put off going to the dentist until they have toothache (p < 0.001). Only a small proportion of the occidentals (8%) reported a perception of inevitability in having false teeth, whereas 33% of the orientals held this fatalistic belief (p = 0.001). CONCLUSIONS: Grouping the countries into key cultural orientations and international clusters yielded plausible results, using the HU-DBI.

Role of nerve growth factor in the adult dorsal root ganglia neuron and its response to injury.

The response of dorsal root ganglia (DRG) neurons to NGF deprivation and to axotomy was examined in adult guinea pigs. The success of NGF deprivation by means of an autoimmune approach was monitored by the measurement of serum antibody titer levels against guinea pig NGF with the standard bioassay for NGF activity. That the antibody produced NGF deprivation was confirmed by histologic evidence of neuronal atrophy and apparent cell loss in sections of the superior cervical ganglia (SCG) and by marked decreases (65-80%) of SCG neurotransmitter-synthesizing enzyme activity levels. By using the autoimmune approach a new source of guinea pigs was found which consistently produced high titers of cross-reacting anti-NGF antibodies. Experiments were designed to examine the response of the sensory neuron to injury while chronically deprived of NGF. Total neuronal counts in the sixth lumbar DRG 98 days after sciatic nerve crush showed no difference between NGF-deprived and control ganglia. Measurement of the size spectrum of DRG neurons showed evidence of atrophy of the NGF-deprived neurons in both the uninjured and axotomized side compared to respective controls. The mean volume of uninjured sensory neurons measured in the NGF-deprived guinea pigs was decreased 27.7% (P less than .05) compared with that of control guinea pigs. The degree of regeneration 6 days following a nerve crush was the same in NGF-deprived sensory neurons and in controls when measured by the "pinch test" and by isotope-labeled axonal transport studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor receptors in rat spinal cord: an autoradiographic and immunohistochemical study.

The distribution of nerve growth factor receptors in the lumbar spinal cord of the rat was studied with autoradiographic and immunohistochemical techniques: [125I]nerve growth factor and specific monoclonal antibody (Mab 192) against nerve growth factor receptor were used to localize nerve growth factor binding sites. The distributions of nerve growth factor binding sites with highest density within the superficial layers (laminae I and II) of the dorsal gray matter were virtually identical as demonstrated by these two ligands; this suggests that Mab 192 can be used as a specific probe to identify nerve growth factor receptors in rat nervous system. Nerve growth factor receptor binding sites, as demonstrated by autoradiography, were also found in longitudinal bundles of fibers running dorsolaterally in the lateral funiculus. However, no immunoreactivity was detected in these areas by immunohistochemistry. No specific binding was found in the dorsal horn when [125I]nerve growth factor was co-injected with unlabeled nerve growth factor or after incubation with nonspecific monoclonal antibody. Dorsal root section produced a complete loss of nerve growth factor-specific labeling pattern throughout laminae I-II of the spinal cord. This suggests that nerve growth factor receptors are localized on the nerve terminals of primary afferent fibers which synapse in the region of the spinal cord. The presence of nerve growth factor binding sites in the dorsal horn of the spinal cord is consistent with the possibility that nerve growth factor, or a nerve growth factor-like substance, derived from the central nervous system, may have a role in trophic support of dorsal root ganglion neurons.

Ciliary neurotrophic factor promotes the regrowth capacity but not the survival of intraorbitally axotomized retinal ganglion cells in adult hamsters.

Ciliary neurotrophic factor has recently been shown to promote the axonal regrowth of retinal ganglion cells into a peripheral nerve graft following an intracranial transection of the optic nerve (approximately 7 mm from the optic disc). It is unclear whether the enhancement of axonal regrowth by ciliary neurotrophic factor application correlates with the enhancement of survival of retinal ganglion cells and/or the up-regulation of expression of growth-associated protein-43 messenger RNA in retinas. The present study evaluated the regenerative capacity of retinal ganglion cells following intraorbital transection of the optic nerve (approximately 1.5 mm from the optic disc) and the attachment of a peripheral nerve to the ocular stump of the optic nerve. In addition, we have determined the survival of retinal ganglion cells and the expression of growth-associated protein-43 messenger RNA in ciliary neurotrophic factor-treated retinas following optic nerve transection. The results showed that in the ciliary neurotrophic factor-treated retinas, the number of retinal ganglion cells which had regrown axons into a peripheral nerve is about four times more than the control. In the axotomized retinas, ciliary neurotrophic factor initiated sprouting of axon-like processes at 14 and 28 days post-axotomy and up-regulated the expression level of growth-associated protein-43 messenger RNA at 7, 14 and 28 days post-axotomy. However, ciliary neurotrophic factor did not prevent the death of axotomized retinal ganglion cells. We suggest that one possible mechanism for the axonal regeneration of axotomized retinal ganglion cells by ciliary neurotrophic factor could be mediated by the up-regulation of growth-associated protein-43 gene expression and not by increasing the pool of surviving retinal ganglion cells after axotomy.

Axonal regeneration of retinal ganglion cells depending on the distance of axotomy in adult hamsters.

PURPOSE: To examine the relationship between the distance of axotomy and axonal regeneration of injured retinal ganglion cells (RGCs) systematically and the effect of a predegenerated (pretransected or precrushed) peripheral nerve (PN) graft on axonal regeneration of RGCs axotomized at a definite distance (0.5 mm from the optic disc) in comparison with a normal PN graft. METHODS: The optic nerve (ON) was transected intraorbitally at 0.5, 1, 1.5, 2, or 3 mm or intracranially at 6 to 8 mm from the optic disc, and a PN graft was transplanted onto the ocular ON stump in adult hamsters. Four weeks after grafting, the number of RGCs regenerating their injured axons into the PN graft was investigated in all animals. RESULTS: The number of regenerating RGCs decreased significantly when the distance of axotomy increased from 0.5 to 7 mm. A precrushed PN graft was shown to enhance more injured RGCs to regenerate axons than a normal or pretransected PN graft. CONCLUSIONS: The distance of axotomy on the ON of adult hamsters is critical in determining the number of regenerating RGCs. Thus, experimental strategies to repair the damaged ON by PN transplantation is to attach a precrushed PN graft as close to the optic disc as possible to obtain optimal axonal regeneration of the axotomized RGCs.