The natural compound berberine positively affects macrophage functions involved in atherogenesis.

BACKGROUND AND AIMS: We investigated the effect of berberine (BBR), an alkaloid showing antiatherogenic properties beyond the cholesterol lowering capacity, on macrophage cholesterol handling upon exposure to human serum and on macrophage responses to excess free cholesterol (FC) loading. METHODS AND RESULTS: Mouse and human macrophages were utilized as cellular models. Cholesterol content was measured by a fluorimetric assay; cholesterol efflux, cytotoxicity and membrane FC distribution were evaluated by radioisotopic assays. Monocyte chemotactic protein-1 (MCP-1) secretion was measured by ELISA; membrane ruffling and macropinocytosis were visualized by confocal microscopy. Exposure of cholesterol-enriched MPM to serum in the presence of 1 µM BBR resulted in a reduction of intracellular cholesterol content twice greater than exposure to serum alone (-52%; p < 0.01 and -21%; p < 0.05), an effect not mediated by an increase of cholesterol efflux, but rather by the inhibition of cholesterol uptake from serum. Consistently, BBR inhibited in a dose-dependent manner cholesterol accumulation in human macrophages exposed to hypercholesterolemic serum. Confocal microscope analysis revealed that BBR inhibited macropinocytosis, an independent-receptor process involved in LDL internalization. Macrophage FC-enrichment increased MCP-1 release by 1.5 folds, increased cytotoxicity by 2 fold, and induced membrane ruffling; all these responses were markedly inhibited by BBR. FC-enrichment led to an increase in plasma membrane cholesterol by 4.5 folds, an effect counteracted by BBR. CONCLUSION: We showed novel potentially atheroprotective activities of BBR in macrophages, consisting in the inhibition of serum-induced cholesterol accumulation, occurring at least in part through an impairment of macropinocytosis, and of FC-induced deleterious effects.

Predictors of effectiveness of multidisciplinary rehabilitation treatment on motor dysfunction in multiple sclerosis.

OBJECTIVES: To identify clinical predictors of effectiveness of a motor rehabilitation treatment in a cohort of multiple sclerosis (MS) patients. MATERIALS AND METHODS: We analysed 212 consecutive patients who underwent a short-term (3-7 weeks) intensive (two hours per day, five days per week), individualised, goal-oriented inpatient rehabilitation program. Activity limitation and impairment were measured on admission and discharge of the rehabilitation trial using the motor sub-items of the Functional Independence Measure (mFIM) and the Expanded Disability Status Scale (EDSS) score. Multivariate logistic regression models have been tested to evaluate the role of clinical baseline features on rehabilitation effectiveness. RESULTS: According to pre-defined outcome measures, 75.1% of MS patients improved in either activity limitation (≥5 points delta mFIM) or impairment (≥1.0 delta EDSS score if baseline EDSS was ≤5.5, or ≥0.5 if baseline EDSS was >5.5), and 35.4% of MS patients improved in both outcomes. A relapsing-remitting course of disease, a more severe baseline impairment and activity limitation level, a shorter disease duration and a less severe balance dysfunction were predictive of the effectiveness of rehabilitation. DISCUSSION: These data confirm that an intensive inpatient rehabilitation program is able to produce a short-term relevant improvement on clinical and functional outcome measures and suggest some clinical features which can be considered as potential predictors of the outcome of rehabilitative intervention.

ATM-dependent phosphorylation of nibrin in response to radiation exposure.

Mutations in the gene ATM are responsible for the genetic disorder ataxia-telangiectasia (A-T), which is characterized by cerebellar dysfunction, radiosensitivity, chromosomal instability and cancer predisposition. Both the A-T phenotype and the similarity of the ATM protein to other DNA-damage sensors suggests a role for ATM in biochemical pathways involved in the recognition, signalling and repair of DNA double-strand breaks (DSBs). There are strong parallels between the pattern of radiosensitivity, chromosomal instability and cancer predisposition in A-T patients and that in patients with Nijmegen breakage syndrome (NBS). The protein defective in NBS, nibrin (encoded by NBS1), forms a complex with MRE11 and RAD50 (refs 1,2). This complex localizes to DSBs within 30 minutes after cellular exposure to ionizing radiation (IR) and is observed in brightly staining nuclear foci after a longer period of time. The overlap between clinical and cellular phenotypes in A-T and NBS suggests that ATM and nibrin may function in the same biochemical pathway. Here we demonstrate that nibrin is phosphorylated within one hour of treatment of cells with IR. This response is abrogated in A-T cells that either do not express ATM protein or express near full-length mutant protein. We also show that ATM physically interacts with and phosphorylates nibrin on serine 343 both in vivo and in vitro. Phosphorylation of this site appears to be functionally important because mutated nibrin (S343A) does not completely complement radiosensitivity in NBS cells. ATM phosphorylation of nibrin does not affect nibrin-MRE11-RAD50 association as revealed by radiation-induced foci formation. Our data provide a biochemical explanation for the similarity in phenotype between A-T and NBS.

Thoughts and concerns of patients at hospital discharge after lumbar spine surgery. A qualitative study.

PURPOSE: The increased rate of surgical interventions point to the necessity to investigate the patient's experience from a biopsychosocial perspective. The aim of this study was to investigate the thoughts and concerns of patients undergoing spinal surgery for lumbar degenerative disease at the time of their discharge from hospital. METHODS: Semi-structured interviews were conducted with 28 patients. The questions investigated possible concerns associated to discharging them home. A content analysis was performed by a multidisciplinary group to identify the main themes that emerged from the interviews. RESULTS: The patients were satisfied with the surgeons' preoperative explanations and description of expected prognosis. However, they were disappointed with the lack of information at hospital discharge, in particular regarding practical and behavioral recommendations. The patients expressed clear concerns about being left alone to deal with possible complications or difficulties they may encounter when returning home. CONCLUSION: This study underlined the patients' need for a comprehensive psychological guidance and possibly a person of reference during the post-operative process. Discussing discharge with the patient was emphasized as an important issue to improve patients' compliance to the recovery process itself. Putting these elements into practice should help spine surgeons to manage better hospital discharge.IMPLICATIONS FOR REHABILITATIONA comprehensive discussion with the patient at the time of hospital discharge is clearly stressed as an important issue to improve patients' adherence to the recovery process.The patients complained of a lack of information when returning home, in particular regarding practical and behavioral recommendations.The patients expressed clear concerns about possible complications or difficulties they may encounter when returning home and they expressed their need to know whom they can contact for help if needed.A better understanding of the thoughts and concerns of patients undergoing spinal surgery at the time of their discharge from hospital should help spine surgeons to improve the management of hospital discharge and the post-operative recovery.

IGF-I/IGFBP system: metabolism outline and physical exercise.

The GH/IGF-I system plays a well-known hormonal role and its effects, mainly anabolic and insulin-sensitizing, are mediated through endocrine as well as paracrine/ autocrine mechanisms. This system includes the binding proteins, namely GH binding proteins and IGF-I binding proteins (IGFBP). As expected, this axis plays a key role in organism modification in consequence of a physical exercise. Physical activity, training, and exercise capacity chiefly involve anabolism process modifications of various tissues, in particular muscular adjustments. Numerous investigators found a correlation among the level of exercise tolerance, muscle strength or walking speed and IGF-I/IGFBP-3 concentrations. However, also inverse and absent correlations between circulating IGF-I concentrations and acute or chronic exercise responses have been reported. IGF-I is generally accepted as an important GH mediator with metabolic effects, through both endocrine and paracrine or autocrine mechanisms. GH is the main regulator of the hepatic synthesis of IGF-I and IGFBP-3, which is the most abundant IGF carrier in human plasma. Recently, it has been shown that the physical exercise stimulatory impact on skeletal muscles is mediated through an increased local IGF-I synthesis with an IGFPB involvement. An absent association of exercise performance and circulating IGF-I may indicate that exercise will exert muscle strength by predominately locally derived paracrine or autocrine mediators rather than endocrine circulating IGF-I. The present review considers the general aspects of the IGF/IGFPB system and the role of the IGF/IGFPB system in relation to physical exercise (type, duration, etc.) taking into account the training aspects.

Action Observation Therapy Before Sleep Hours: An EEG Study.

Action Observation Therapy (AOT) is a rehabilitation method which aims at stimulating motor memory by means of the repetitive observation of motor tasks presented through video-clips. Since sleep seems to have a positive effect on learning processes, it is reasonable to hypothesize that the delivery of AOT immediately before sleep hours could enhance the effects of motor training. The objective of the present work was to test the effect of AOT delivered before the sleep hours in terms of improvements in manual dexterity and changes in cortical activity through Electroencephalography (EEG) on healthy subjects. Specifically, EEG traces acquired on a treatment and on a control group before and after three weeks of training during the execution of a Nine Hole Peg Test were analyzed. The spectral analysis of brain signals showed an increased activation of the motor cortex on a subgroup of the treatment subjects. Moreover, a significantly higher involvement of frontal areas was observed in the treatment group.

Selecting a pre-processing pipeline for the analysis of EEG event-related rhythms modulation.

When deciding how to pre-process EEG data, researchers need to make a choice at each single step of the procedure among different possibilities, equally valid. Therefore, in this work, we illustrate how these decisions may affect the quality of the final cleaned data in an Action Observation/Motor Imagery protocol, using quantitative indices. In particular, we showed the effect of segmenting or not the data in epochs around the stimulus presentation time on the independent component analysis (ICA) used for artifact removal. For ICA analysis, we tested two algorithms (SOBI and Extended Infomax). Finally, three re-reference approaches (Common averaged reference-CAR, robust-CAR and reference electrode standardization technique - REST) were also applied and their effects compared. Results showed that the segmenting method has a prominent effect on the cleaning procedure and consequently on final EEG data quality. Extended Infomax is confirmed as the method of choice for the identification of the artifactual components and, finally, CAR and the REST re-referencing techniques led to similar good results.

An update: salivary hormones and physical exercise.

Saliva contains cells and compounds, of local and non-local oral origin, namely inorganic, organic non-protein, protein/polypeptide, and lipid molecules. Moreover, some hormones, commonly assayed in plasma, such as steroids, are detectable in oral fluid and peptide/protein, and non-steroid hormones have been investigated. The sports practice environment and athletes' availability, together with hormone molecule characteristics in saliva and physical exercise behavior effects, confirm this body fluid as an alternative to serum. This review focuses on the relation between salivary steroids and psycho-physiological stress and underlines how the measurement of salivary cortisol provides an approach of self-report psychological indicator and anxiety change in relation to exercise performance. The correlation between salivary and plasma steroid hormone (cortisol, testosterone, and dehydroepiandrosterone (DHEA)) levels, observed during exercise, has been considered, underlining how the type, duration, and intensity of the exercise influence the salivary steroid concentrations in the same way as serum-level variations. Training conditions have been considered in relation to the salivary hormonal response. This review focuses on studies related to salivary hormone measurements, mainly steroids, in physical exercise. Saliva use in physical disciplines, as a real alternative to serum, could be a future perspective.

Comparison of esophagectomy with and without thoracotomy in a low-resource tertiary care center in a developing country.

Esophageal cancer surgery is traditionally performed by a number of open surgical approaches. Open approaches require thoracotomy and laparotomy. Developments in instrumentation and optics have allowed the use of minimally invasive approaches to esophageal cancer, which had been traditionally managed by open operation. Minimally invasive surgery (MIS) avoids thoracotomy and laparotomy and results in quicker return to normal functions and less morbidity. In this prospective study, we compared the immediate surgical and oncologic outcomes of patients who have undergone MIS with those who have had open surgery. From November 1, 2003 to March 30, 2006, 62 cases of carcinoma esophagus were operated in Surgical unit 3 (MIS unit) in the institute. Out of the 62 patients, 34 (54.8%) underwent minimally invasive esophagectomy (MIE), and the remaining 28 patients (45.2%) underwent open surgery. Both operations were done by the same team of surgeons. The groups were compared in terms of perioperative outcomes, morbidity, mortality, and adequacy of oncologic excision. The average duration for MIS was 312.35 min (60-480 min), which was more than that of open group surgery whose average duration was 261.96 min (60-360 min). This difference was found to be not significant (P < 0.110). The average blood loss was 275.74 mL (200-500 mL) in minimally invasive group compared with 312.50 (200-500 mL) in open group (P-value 0.33). Four patients (11.76%) in MIS group had been converted to open surgery. Average duration of hospitalization was 11.9 (4-24) days in MIS group compared with 12.19 (5-24) days in open group (P-value 0.282). Nine (26.47%) patients in MIS group had developed major or minor morbidity. Similarly, eight (28.57%) patients in open group had morbidity. One patient each expired in each group. The morbidity and mortality rates were not statistically significant. There were four leaks (11.76%) in MIS group and three leaks (10.71%) in open group (P-value 0.85). Regarding the extent of nodal clearance, an average number of 9.5 (0-19) nodes were removed in MIS group compared with an average of 7.26 (0-12) nodes in open group (P-value 0.05). Better visibility and magnification enabled more number of lymph nodes to be removed in MIS group. MIE is oncologically safe compared with open surgery. It has almost similar postoperative course, morbidity pattern, and duration of hospital stay as open surgery. Increased duration of procedure compared with open surgery is a disadvantage of MIS, especially in the early part of learning curve.

Human T cell receptor V beta gene polymorphism.

Southern blot hybridizations with human T cell receptor V beta gene probes were used to determine the sizes of the various V beta gene subfamilies. An analysis of DNA samples from 100 unrelated individuals identified a single individual who lacked one V beta gene segment. A second individual had an apparently different repertoire of V beta gene segments in one subfamily, as assayed by hybridization, possibly due to a gene conversion event. An analysis with four restriction enzymes of DNA from 30 consanguineous donors detected restriction fragment length polymorphisms associated with 12 of 14 V beta gene segment subfamilies examined. In an analysis of DNAs from a large panel of unrelated individuals, some alleles at these loci were found to be in linkage disequilibrium, indicating a potentially close physical linkage. The segregation of three polymorphisms, two associated with V beta gene segment loci and one associated with the C beta genes, was compatible with Mendelian inheritance, and demonstrated that highly informative haplotypes could be generated. The high degree of polymorphism observed in the human T cell receptor beta chain complex should allow exploration of possible associations between T cell receptor genes and inherited diseases involving the immune system.

Chronic periaortitis associated with membranous nephropathy: clues to common pathogenetic mechanisms.

Chronic periaortitis (CP) is a rare disease hallmarked by the presence of a periaortic retroperitoneal fibro-inflammatory tissue which can often cause obstructive uropathy. CP is isolated in most cases but it may also be associated with other sclerosing inflammatory and immune-mediated diseases. We here present the case of a patient who was initially diagnosed as having CP and subsequently developed membranous nephropathy and chronic sclerosing sialoadenitis of the right parotid gland. As these conditions were all characterized by either pronounced infiltration of IgG4-positive plasma cells or marked IgG4 tissue deposition, we hypothesize that they are part of the same disease spectrum, and discuss the immune-mediated pathogenetic mechanisms potentially shared by these conditions. In particular, we consider the role of Th2-mediated immune reactions and of immunogenetic factors such as HLA genotype as common determinants of these disorders.

Conservative vs. surgical approach for degenerative meniscal injuries: a systematic review of clinical evidence.

OBJECTIVE: Analyzing the available evidence by comparing the role of arthroscopic surgery and conservative treatment in the management of degenerative meniscopathy. MATERIALS AND METHODS: A literature search was carried out on the PubMed, EMBASE, Scopus, and PEDro databases in May 2019 to identify all the randomized controlled trials (RCTs) comparing arthroscopic surgery to conservative management of painful but stable degenerated menisci. The quality of the RCTs was assessed using the Cochrane Risk of Bias Assessment. RESULTS: A total of 10 studies, including 1525 patients and dealing with conservative treatment vs. arthroscopic surgery were included in this review. In eight studies the effectiveness of exercise therapy was compared to surgery; in one study the effectiveness of intra-articular steroid injection was compared to surgery; in one study the effectiveness of placebo surgery was compared to partial meniscectomy. In all studies, no significant inter-group difference in terms of knee pain and knee function were observed at any follow-up evaluation. CONCLUSIONS: Degenerative meniscal tears, without symptoms of locking and catching, can be successfully managed by a proper regimen of physical therapy as a first line treatment. Surgical approach might be considered in case of poor response after conservative treatment.

Functional and computational assessment of missense variants in the ataxia-telangiectasia mutated (ATM) gene: mutations with increased cancer risk.

The functional consequences of missense variants are often difficult to predict. This becomes especially relevant when DNA sequence changes are used to determine a diagnosis or prognosis. To analyze the consequences of 12 missense variants in patients with mild forms of ataxia-telangiectasia (A-T), we employed site-directed mutagenesis of ataxia-telangiectasia mutated (ATM) cDNA followed by stable transfections into a single A-T cell line to isolate the effects of each allele on the cellular phenotype. After induction of the transfected cells with CdCl2, we monitored for successful ATM transcription and subsequently assessed: 1) intracellular ATM protein levels; 2) ionizing radiation (IR)-induced ATM kinase activity; and 3) cellular radiosensitivity. We then calculated SIFT and PolyPhen scores for the missense changes. Nine variants produced little or no correction of the A-T cellular phenotype and were interpreted to be ATM mutations; SIFT/PolyPhen scores supported this. Three variants corrected the cellular phenotype, suggesting that they represented benign variants or polymorphisms. SIFT and PolyPhen scores supported the functional analyses for one of these variants (c.1709T>C); the other two were predicted to be "not tolerated" (c.6188G>A and c.6325T>G) and were classified as "operationally neutral." Genotype/phenotype relationships were compared: three deleterious missense variants were associated with an increased risk of cancer (c.6679C>T, c.7271T>G, and c.8494C>T). In situ mutagenesis represents an effective experimental approach for distinguishing deleterious missense mutations from benign or operationally neutral missense variants.

A single ataxia telangiectasia gene with a product similar to PI-3 kinase.

A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer.

Salivary free insulin-like growth factor-i levels: effects of an acute physical exercise in athletes.

BACKGROUND/AIMS: The offer of human saliva IGF-I (sIGF-I) measurement in athletes investigation is a new proposal. The aim was to investigate the physical exercise effect on sIGF-I and explore plasma free IGF-I relation. MATERIALS AND METHODS: Saliva and blood were collected from well-trained athletes, investigated immediately before and at the end of a physical exercise test. RESULTS: sIGF-I was significantly increased at the end of the physical exercise. The plasma free IGF-I concentrations did not demonstrate any difference. The saliva total protein level (sTP) was also significantly increased. A positive correlation between sTP and sIGF-I, was observed, both before and after physical exercise, and between salivary and plasma free IGF-I only after physical exercise. The salivary free IGF-I level significantly increased after physical exercise, moreover a correlation with the plasma levels exists in post-exercise condition. CONCLUSION: The physical exercise affects sIGF-I as well as the sTP. The correlation between plasma and salivary free IGF-I levels only in post-exercise condition suggests further studies to investigate the effects of different type and duration of physical exercise. The comparison with other salivary biochemical parameter investigation would also further increase comprehension on the role of salivary IGF-I.

A fast computational method for determining equilibrium concentration profiles in intermixed nanoislands.

An efficient computational method for finding the equilibrium concentration profiles which minimize the free energy of intermixed heteroepitaxial islands of assigned shape and average composition is described. A combination of a Monte Carlo method and continuum elasticity theory solved by a finite element method is shown to provide the desired profiles allowing for a significant computational gain with respect to atomistic approaches. The role played by dimensionality (ridges versus islands) and by entropy is discussed.

Cough sensors. II. Transient receptor potential membrane receptors on cough sensors.

The transient receptor potential (TRP) family of channels is represented by at least six members in primary sensory neurons. These include the TRP vanilloid subtypes 1 (TRPV1), 2, 3, and 4, the cold and menthol receptor TRPM8, and TRPA1. Much interest has been directed to the study of the TRPV1, because capsaicin has been instrumental in discovering the unique role of a subset of primary sensory neurons in causing nociceptive responses, in activating reflex pathways including cough, and in producing neurogenic inflammation. TRPV1 is now regarded as an integrator of diverse sensory modalities because it undergoes marked plasticity and sensitization through a variety of mechanisms, including activation of G-protein-coupled or tyrosine kinase receptors. Evidence in experimental animals and in patients with airway diseases indicates a marked hypersensitivity to cough induced by TRPV1 agonists. Recent studies with newly developed high-affinity and selective TRPV1 antagonists have revealed that TRPV1 inhibition reduces cough induced by citric acid or antigen challenge.

Effects of different classroom temperatures on cardiac autonomic control and cognitive performances in undergraduate students.

OBJECTIVE: Indoor microclimate may affect students' wellbeing, cardiac autonomic control and cognitive performance with potential impact on learning capabilities. To assess the effects of classroom temperature variations on the autonomic profile and students' cognitive capabilities. APPROACH: Twenty students attending Humanitas University School, (14M, age 21 ± 3 years) underwent a single-lead ECG continuous recording by a portable device during a 2 h lecture when classroom temperature was set 'neutral' (20 °C-22 °C, Day 1) and when classroom temperature was set to 24 °C-26 °C (Day 2). ECGs were sent by telemetry to a server for off-line analysis. Spectral analysis of RR variability provided indices of cardiac sympathetic (LFnu), vagal (HF, HFnu) and cardiac sympatho-vagal modulation (LF/HF). Symbolic analysis of RR variability provided the percentage of sequences of three heart periods with no significant change in RR interval (0V%) and with two significant variations (2V%) reflecting cardiac sympathetic and vagal modulation, respectively. Students' cognitive performance (memory, verbal comprehension and reasoning) was assessed at the end of each lecture using the Cambridge Brain Sciences cognitive evaluation tool. MAIN RESULTS: Classroom temperature and CO2 were assessed every 5 min. Classroom temperatures were 22.4 °C ± 0.1 °C (Day 1) and 26.2 °C ± 0.1 °C (Day 2). Student's thermal comfort was lower during Day 2 compared to Day 1. HR, LF/HF and 0V% were greater during Day 2 (79.5 ± 12.1 bpm, 6.9 ± 7.1 and 32.8% ± 10.3%) than during Day 1 (72.6 ± 10.8 bpm, 3.4 ± 3.7, 21.4% ± 9.2%). Conversely, 2V% was lower during Day 2 (23.1% ± 8.1%) than during Day 1 (32.3% ± 11.4%). Short-term memory, verbal ability and the overall cognitive C-score scores were lower during Day 2 (10.3 ± 0.3; 8.1 ± 1.2 and 10.9 ± 2.0) compared to Day 1 (11.7 ± 2.1; 10.7 ± 1.7 and 12.6 ± 1.8). SIGNIFICANCE: During Day 2, a shift of the cardiac autonomic control towards a sympathetic predominance was observed compared to Day 1, in the presence of greater thermal discomfort. Furthermore, during Day 2 reduced cognitive performances were found.

Ethanol causes neurogenic vasodilation by TRPV1 activation and CGRP release in the trigeminovascular system of the guinea pig.

Ethanol stimulating transient receptor potential vanilloid 1 (TRPV1) on primary sensory neurons promotes neurogenic inflammation, including calcitonin gene-related peptide (CGRP)-mediated coronary dilation. Alcoholic beverages trigger migraine attacks and activation of trigeminal neurons plays a role in migraine. We have investigated in guinea pigs whether ethanol by TRPV1 stimulation causes neurogenic inflammation in the trigeminovascular system. Ethanol-evoked release of neuropeptides from slices of dura mater was abolished by Ca(2+) removal, capsaicin pretreatment and the TRPV1 antagonist, capsazepine. Intragastric ethanol increased plasma extravasation in dura mater, an effect abolished by capsazepine and the NK1 receptor antagonist, SR140333, and caused vasodilation around the middle meningeal artery, an effect abolished by capsazepine and the CGRP receptor antagonist, BIBN4096BS. Vasodilation of meningeal vessels by TRPV1 activation and CGRP release may be relevant to the mechanism by which alcohol ingestion triggers migraine attacks.

Analysis of living cells grown on different titanium surfaces by time-lapse confocal microscopy.

In this study we have combined fluorescence- and reflection-confocal laser scanning microscopy for the simultaneous visualization of living cells and surface topography beneath them. To this purpose we have designed a specific flow chamber and we have tested it with osteoblasts grown on an opaque, thick support, made of smooth or sandblasted titanium. Cells were loaded with Calcein-AM or tetramethylrhodamine methyl ester (TMRM), two probes employed as indicators of cell viability/morphology and mitochondrial membrane potential, respectively. Besides the acquisition of stacks of confocal sections, the system allowed also vertical views and faithful three-dimensional reconstruction of the samples. Confocal microscope implemented with our flow chamber proved to be a promising tool for time-lapse investigation of cell-biomaterial interactions.