Pore size regulates cell and tissue interactions with PLGA-CaP scaffolds used for bone engineering

A common subject in bone tissue engineering is the need for porous scaffolds to support cell and tissue interactions aiming at repairing bone tissue. As poly(lactide-co-glycolide)calcium phosphate (PLGACaP) scaffolds can be manufactured with different pore sizes, the aim of this study was to evaluate the effect of pore diameter on osteoblastic cell responses and bone tissue formation. Scaffolds were prepared with 85% porosity, with pore diameters in the ranges 470590, 590850 and 8501200 mu m. Rat bone marrow stem cells differentiated into osteoblasts were cultured on the scaffolds for up to 10 days to evaluate cell growth, alkaline phosphatase (ALP) activity and the gene expression of the osteoblast markers RUNX2, OSX, COL, MSX2, ALP, OC and BSP by real-time PCR. Scaffolds were implanted in critical size rat calvarial defects for 2, 4, and 8 weeks for histomorphometric analysis. Cell growth and ALP activity were not affected by the pore size; however, there was an increase in the gene expression of osteoblastic markers with the increase in the pore sizes. At 2 weeks all scaffolds displayed a similar amount of bone and blood vessels formation. At 4 and 8 weeks much more bone formation and an increased number of blood vessels were observed in scaffolds with pores of 470590 mu m. These results show that PLGACaP is a promising biomaterial for bone engineering. However, ideally, combinations of larger (similar to 1000 mu m) and smaller (similar to 500 mu m) pores in a single scaffold would optimize cellular and tissue responses during bone healing. Copyright (C) 2011 John Wiley & Sons, Ltd.

Does the neonatal clinical risk for illness severity influence pain reactivity and recovery in preterm infants?

Background: The biobehavioural pain reactivity and recovery of preterm infants in the neonatal period may reflect the capacity of the central nervous system to regulate neurobiological development. Objective: The aim of the present study was to analyse the influence of the neonatal clinical risk for illness severity on biobehavioural pain reactivity in preterm infants. Methods: Fifty-two preterm infants were allocated into two groups according to neonatal severity of illness, as measured by the Clinical Risk Index for Babies (CRIB). The low clinical risk (LCr) group included 30 neonates with CRIB scores <4, and the high clinical risk (HCr) group included 22 neonates with CRIB scores >= 4. Pain reactivity was assessed during a blood collection, which was divided into five phases (baseline, antisepsis, puncture, recovery-dressing and recovery-resting). Behavioral pain reactivity was measured using the scores, and magnitude of responses in Neonatal Facial Coding System (NFCS) and Sleep-Wake States Scale (SWS). The heart rate was continuously recorded. Results: The HCr demonstrated a higher magnitude of response on the SWS score from the baseline to the puncture phase than the LCr. Also, the HCr exhibited a higher mean heart rate and minimum heart rate than the LCr in the recovery-resting phase. In addition, the HCr exhibited a higher minimum heart rate from the baseline to the recovery-resting phase than the LCr. Conclusion: The infants exhibiting a high neonatal clinical risk showed high arousal during the puncture procedure and higher physiological reactivity in the recovery phase.

On the Deactivation Mechanisms of Adenine-Thymine Base Pair

In this contribution, the multiconfigurational second-order perturbation theory method based on a complete active space reference wave function (CASSCF/CASPT2) is applied to study all possible single and double proton/hydrogen transfers between the nucleobases in the adenine-thymine (AT) base pair, analyzing the role of excited states with different nature [localized (LE) and charge transfer (CT)] and considering concerted as well as step-wise mechanisms. According to the findings, once the lowest excited states, localized in adenine, are populated during UV irradiation of the Watson-Crick base pair, the proton transfer in the N-O bridge does not require high energy in order to populate a CT state. The latter state will immediately relax toward a crossing with the ground state, which will funnel the system to either the canonical structure or the imino-enol tautomer. The base pair is also capable of repairing itself easily since the imino-enol species is unstable to thermal conversion.

A clinical experience of the supraclavicular flap used to reconstruct head and neck defects in late-stage cancer patients

The supraclavicular island flap has been widely used in head and neck reconstruction, providing an alternative to the traditional techniques like regional or free flaps, mainly because of its thin skin island tissue and reliable vascularity. Head and neck patients who require large reconstructions usually present poor clinical and healing conditions. An early experience using this flap for late-stage head and neck tumour treatment is reported. Forty-seven supraclavicular artery flaps were used to treat head and neck oncologic defects after cutaneous, intraoral and pharyngeal tumour resections. Dissection time, complications, donor and reconstructed area outcomes were assessed. The mean time for harvesting the flaps was 50 min by the senior author. All donor sites were closed primarily. Three cases of laryngopharyngectomy reconstruction developed a small controlled (salivary) leak that was resolved with conservative measures. Small or no strictures were detected on radiologic swallowing examinations and all patients regained normal swallowing function. Five patients developed donor site dehiscence. These wounds were treated with regular dressing until healing was complete. There were four distal flap necroses in this series. These necroses were debrided and closed primarily. The supraclavicular flap is pliable for head and neck oncologic reconstruction in late-stage patients. High-risk patients and modified radical neck dissection are not contraindications for its use. The absence of the need to isolate the pedicle offers quick and reliable harvesting. The arc of rotation on the base of the neck provides adequate length for pharyngeal, oral lining and to reconstruct the middle and superior third of the face. (C) 2012 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Effect of Ultrasonic Activation on pH and Calcium Released by Calcium Hydroxide Pastes in Simulated External Root Resorption

Introduction: The purpose of this study was to analyze the influence of ultrasonic activation of calcium hydroxide (CH) pastes on pH and calcium release in simulated external root resorptions. Methods: Forty-six bovine incisors had their canals cleaned and instrumented, and defects were created in the external middle third of the roots, which were then used for the study. The teeth were externally made impermeable, except for the defected area, and divided into the following 4 groups containing 10 samples each according to the CH paste and the use or not of the ultrasonic activation: group 1: propylene glycol without ultrasonic activation, group 2: distilled water without ultrasonic activation, group 3: propylene glycol with ultrasonic activation, and group 4: distilled water with ultrasonic activation. After filling the canals with the paste, the teeth were restored and individually immersed into flasks with ultrapure water. The samples were placed into other flasks after 7, 15, and 30 days so that the water pH level could be measured by means of a pH meter. Calcium release was measured by means of an atomic absorption spectrophotometer. Six teeth were used as controls. The results were statistically compared using the Kruskal-Wallis and Mann-Whitney U tests (P < .05). Results: For all periods analyzed, the pH level was found to be higher when the CH paste was activated with ultrasound. Calcium release was significantly greater (P < .05) using ultrasonic activation after 7 and 30 days. Conclusions: The ultrasonic activation of CH pastes favored a higher pH level and calcium release in simulated external root resorptions. (J Endod 2012;38:834-837)

Avaliação clínica e radiográfica do extravasamento da pasta de hidróxido de cálcio em dentes com lesões periapicais

It has been evaluated clinically and radiographically the effect of the extrusion of calcium hydroxide paste in teeth with periapical lesions. Twenty-five patients with teeth showing pulp necrosis and periapical lesions were submitted to endodontic treatment. Dressing calcium hydroxide paste, iodoform and propylene glycol were used. Clinically, the symptoms were evaluated after treatment and, radiographically, the reabsorption of extravasated paste and repair process of the periapical lesion. Only three patients had symptoms severe in the first 24 hours, the paste was completely reabsorbed within 15 days and no interference in the repair process.

Anorexia: a failure in the work of melancholia

The self-reproach against their own bodies seen in patients with eating disorders has led us to posit the existence of failures in the work of melancholia. Defined by Freud in 1915, this process of melancholia is aimed at repairing a loss felt as unbearable by the ego and that triggers off a violent struggle with ambivalent feelings toward the lost object. The resulting hatred is aimed at the shadow of the object that falls on the ego. Especially in anorexia nervosa, there seems to be a regressive movement that goes beyond this.

On the cellular stress response to Staphylococcus aureus alpha-toxin

Staphylococcus aureus alpha-hemolysin was the first bacterial toxin recognized to form pores in the plasma membrane of eukaryotic cells. It is secreted as a water-soluble monomer that upon contact with target membranes forms an amphiphatic heptameric beta-barrel which perforates the bilayer. As a consequence, red cells undergo colloidosmotic lyses, while some nucleated cells may succumb to necrosis or programmed cell death. However, most cells are capable of repairing a limited number of membrane lesions, and then respond with productive transcriptional activation of NF-kB. In the present study, by using microarray and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), data from a previously performed serial analysis of gene expression (SAGE) were extended and verified, revealing that immediate early genes (IEGs) such as c-fos, c-jun and egr-1 are strongly induced at 2-8 h after transient toxin treatment. Activating protein 1 (AP-1: c-Fos, c-Jun) binding activity was increased accordingly. As IEGs are activated by growth factors, these findings led to the discovery that -toxin promotes cell cycle progression of perforated cells in an EGFR-dependent fashion. Although the amount of c-fos mRNA rose rapidly after toxin treatment, c-Fos protein expression was observed only after a lag of about 3 h. Since translation consumes much ATP, which transiently drops after transient membrane perforation, the suspicion arised that membrane-perforation caused global, but temporary downregulation of translation. In fact, eIF2α became heavily phosphorylated minutes after cells had been confronted with the toxin, resulting in shutdown of protein synthesis before cellular ATP levels reached the nadir. GCN2 emerged as a candidate eIF2α kinase, since its expression rapidly increased in toxin-treated cells. Two hours after toxin treatment, GADD34 transcripts, encoding a protein that targets the catalytic subunit of protein phosphatase 1 (PP1) to the endoplasmic reticulum, were overexpressed. This was followed by dephosphorylation of eIF2α and resumption of protein synthesis. Addition of tautomycetin, a specific inhibitor of PP1, led to marked hyperphosphorylation of eIF2α and significantly reduced the drop of ATP-levels in toxin-treated cells. A novel link between two major stress-induced signalling pathways emerged when it was found that both translational arrest and restart were under the control of stress-activated protein kinase (SAPK) p38. The data provide an explanation for the indispensible role of p38 for defence against the archetypal threat of membrane perforation by agents that produce small transmembrane-pores.

Effects of pesticides on honey bees (Apis mellifera L.): study of a specific route of exposure and evaluation of biochemical-physiological changes in the assessment of the pesticides toxicity

In this study, some important aspects of the relationship between honey bees (Apis mellifera L.) and pesticides have been investigated. In the first part of the research, the effects of the exposure of honey bees to neonicotinoids and fipronil contaminated dusts were analyzed. In fact, considerable amounts of these pesticides, employed for maize seed dressing treatments, may be dispersed during the sowing operations, thus representing a way of intoxication for honey bees. In particular, a specific way of exposure to this pesticides formulation, the indirect contact, was taken into account. To this aim, we conducted different experimentations, in laboratory, in semi-field and in open field conditions in order to assess the effects on mortality, foraging behaviour, colony development and capacity of orientation. The real dispersal of contaminated dusts was previously assessed in specific filed trials. In the second part, the impact of various pesticides (chemical and biological) on honey bee biochemical-physiological changes, was evaluated. Different ways and durations of exposure to the tested products were also employed. Three experimentations were performed, combining Bt spores and deltamethrin, Bt spores and fipronil, difenoconazole and deltamethrin. Several important enzymes (GST, ALP, SOD, CAT, G6PDH, GAPDH) were selected in order to test the pesticides induced variations in their activity. In particular, these enzymes are involved in different pathways of detoxification, oxidative stress defence and energetic metabolism. The results showed a significant effect on mortality of neonicotinoids and fipronil contaminated dusts, both in laboratory and in semi-field trials. However, no effects were evidenced in honey bees orientation capacity. The analysis of different biochemical indicators highlighted some interesting physiological variations that can be linked to the pesticide exposure. We therefore stress the attention on the possibility of using such a methodology as a novel toxicity endpoint in environmental risk assessment.

Suche nach Zielgenen des T-Box-Transkriptionsfaktors Optomotor-blind aus Drosophila melanogaster

Zusammenfassung:rnrnDas Ziel der Arbeit bestand darin mehr über die Funktion des T-Box Transkriptionsfaktors Omb zu erfahren. Dm omb ist der nächste Verwandte zu Hs Tbx2/3, die wegen ihrer Rolle bei verschiedenen Krebsarten für die Entwicklung neuer Therapien bedeutsam sind. rnIn drei, von Herrn Pflugfelder hergestellten, omb Allelen l(1)omb282, l(1)omb12, l(1)omb15 wurden neue Mutationen kartiert. Dabei handelt es sich um zwei missense-Mutationen und eine Stopmutation. Sie betreffen Aminosäurereste, die in allen T-Box Proteinen konserviert sind und daher vermutlich lebenswichtige Proteinabschnitte betreffen. In EMSA Versuchen konnte gezeigt werden, dass die missense-Mutationen die DNA-Bindung des Omb-T Proteins verhindern.rnFür die Suche nach Omb Zielgenen wurden Gene und phylogenetisch konservierte TBE-Genabschnitte auf ihre Regulation durch Omb getestet. Dabei wurde das Expressionsmuster von Genen mitels in situ und das Muster von enhancer getriebener β-Gal Expression histochemisch oder durch Immunfärbung von wildtypischen und l(1)omb15 Larven des dritten Stadiums verglichen. rnUpstream der mirr Transkriptionseinheit wurde ein cis-regulatorisches TBE-Fragment identifiziert, das ein Aktivitätsmuster in Flügelimaginalscheiben zeigte, welches dem von Mirr nahe kommt. Sowohl ein Omb Verlust als auch die Mutation der TBE Sequenz führten zu einer ähnlichen ektopischen Aktivierung des Fragments, was auf eine Abhängigkeit von Omb hinweist. rnIn der intronischen Sequenz von inv wurde ebenfalls ein TBE-Fragment entdeckt, das eine β-Gal-Aktivität in Flügelscheiben des späten L3 Stadiums anterior der A/P Grenze zeigte. Diese Expression könnte sich mit der späten für en/inv beschriebenen Expression (Blair, 1992) decken. Immunfärbungen bestätigten, dass der Verlust dieser Aktivität in omb0 tatsächlich durch den Verlust von Omb hervorgerufen wird und nicht durch eine Entwicklungsverzögerung der Larven verursacht wird.rnSchließlich wurde durch die Reparatur von TBX Expressionsvektoren eine Konstruktreihe (Legler, 2010) fertiggestellt, mit deren Hilfe die Auswirkungen einer Überexpression auf die Zellmotilität in Drosophila untersucht werden kann. Das soll helfen den Einfluss von TBX Proteinen auf die Invasivität von Krebszellen zu verstehen.rn

Valutazione degli effetti dei parametri operativi nel processo di laser ablation su materiali compositi CFRP a matrice epossidica

L’attività sperimentale presentata in questo elaborato riguarda lo studio di una particolare applicazione che impiega la tecnologia laser per la lavorazione di materiali compositi ed è stata interamente svolta, in particolar modo nella sua parte operativa, presso i laboratori della Facoltà di Ingegneria a Bologna. Il lavoro di tesi ha come obiettivo fondamentale la valutazione degli effetti che i parametri di processo possono avere sulla qualità risultante nel procedimento di ablazione per i materiali compositi. Per questa indagine sono stati utilizzati campioni piani (tutti identici tra loro) con rinforzo in fibra di carbonio e matrice in resina epossidica, i quali sono stati lavorati con un laser Nd:YAG (λ = 1064 nm) funzionante in regime continuo. L’idea alla base dell’intera attività sperimentale è stata quella di realizzare una ablazione ottimale, rimuovendo dai campioni esclusivamente la resina (in maniera locale) e tentando, allo stesso tempo, di ottenere il minimo danneggiamento possibile per le fibre. Le prove effettuate non costituiscono naturalmente un punto di arrivo, bensì rappresentano piuttosto un punto di partenza per acquisire informazioni preliminari che potranno consentire, nel prossimo futuro, di proseguire con il perfezionamento del processo e la messa a punto dei parametri, al fine di conseguire una lavorazione che dia risultati effettivamente ottimali ed interessanti per l’eventuale applicazione industriale.

Biomedical and industrial applications of atmospheric pressure non-equilibrium plasmas

This dissertation will be focused on the characterization of an atmospheric pressure plasma jet source with an application oriented diagnostic approach and the description of processes supported by this plasma source. The plasma source investigated is a single electrode plasma jet. Schlieren images, optical emission spectra, temperature and heat flux profiles are analyzed to deeply investigate the fluid dynamic, the chemical composition and the thermal output of the plasma generated with a nanosecond-pulsed high voltage generator. The maximum temperature measured is about 45 °C and values close to the room temperature are reached 10 mm down the source outlet, ensuring the possibility to use the plasma jet for the treatment of thermosensitive materials, such as, for example, biological substrate or polymers. Electrospinning of polymeric solution allows the production of nanofibrous non-woven mats and the plasma pre-treatment of the solutions leads to the realization of defect free nanofibers. The use of the plasma jet allows the electrospinnability of a non-spinnable poly(L-lactic acid) (PLLA) solution, suitable for the production of biological scaffold for the wound dressing.

Cost-Effectiveness of Alternative Retrofit Options to Support Decision Making

How to evaluate the cost-effectiveness of repair/retrofit intervention vs. demolition/replacement and what level of shaking intensity can the chosen repairing/retrofit technique sustain are open questions affecting either the pre-earthquake prevention, the post-earthquake emergency and the reconstruction phases. The (mis)conception that the cost of retrofit interventions would increase linearly with the achieved seismic performance (%NBS) often discourages stakeholders to consider repair/retrofit options in a post-earthquake damage situation. Similarly, in a pre-earthquake phase, the minimum (by-law) level of %NBS might be targeted, leading in some cases to no-action. Furthermore, the performance measure enforcing owners to take action, the %NBS, is generally evaluated deterministically. Not directly reflecting epistemic and aleatory uncertainties, the assessment can result in misleading confidence on the expected performance. The present study aims at contributing to the delicate decision-making process of repair/retrofit vs. demolition/replacement, by developing a framework to assist stakeholders with the evaluation of the effects in terms of long-term losses and benefits of an increment in their initial investment (targeted retrofit level) and highlighting the uncertainties hidden behind a deterministic approach. For a pre-1970 case study building, different retrofit solutions are considered, targeting different levels of %NBS, and the actual probability of reaching Collapse when considering a suite of ground-motions is evaluated, providing a correlation between %NBS and Risk. Both a simplified and a probabilistic loss modelling are then undertaken to study the relationship between %NBS and expected direct and indirect losses.

Comparison of low-strength compression stockings with bandages for the treatment of recalcitrant venous ulcers

OBJECTIVE: To compare the proportion and rate of healing, pain, and quality of life of low-strength medical compression stockings (MCS) with traditional bandages applied for the treatment of recalcitrant venous leg ulcers. METHODS: A single-center, randomized, open-label study was performed with consecutive patients. Sigvaris prototype MCS providing 15 mm Hg-25 mm Hg at the ankle were compared with multi-layer short-stretch bandages. In both groups, pads were placed above incompetent perforating veins in the ulcer area. The initial static pressure between the dressing-covered ulcer and the pad was 29 mm Hg and 49 mm Hg with MCS and bandages, respectively. Dynamic pressure measurements showed no difference. Compression was maintained day and night and changed every week. The primary endpoint was healing within 90 days. Secondary endpoints were healing within 180 days, time to healing, pain (weekly Likert scales), and monthly quality of life (ChronIc Venous Insufficiency Quality of Life [CIVIQ] questionnaire). RESULTS: Of 74 patients screened, 60 fulfilled the selection criteria and 55 completed the study; 28 in the MCS and 27 in the bandage group. Ulcers were recurrent (48%), long lasting (mean, 27 months), and large (mean, 13 cm2). All but one patient had deep venous reflux and/or incompetent perforating veins in addition to trunk varices. Characteristics of patients and ulcers were evenly distributed (exception: more edema in the MCS group; P = .019). Healing within 90 days was observed in 36% with MCS and in 48% with bandages (P = .350). Healing within 180 days was documented in 50% with MCS and in 67% with bandages (P = .210). Time to healing was identical. Pain scored 44 and 46 initially (on a scale in which 100 referred to maximum and 0 to no pain) and decreased within the first week to 20 and 28 in the MCS and bandage groups, respectively (P < .001 vs .010). Quality of life showed no difference between the treatment groups. In both groups, pain at 90 days had decreased by half, independent of completion of healing. Physical, social, and psychic impairment improved significantly in patients with healed ulcers only. CONCLUSION: Our study illustrates the difficulty of bringing large and long-standing venous ulcers to heal. The effect of compression with MCS was not different from that of compression with bandages. Both treatments alleviated pain promptly. Quality of life was improved only in patients whose ulcers had healed.

Intensity-modulated radiotherapy for a rendu-osler-weber disease patient with recurrent severe epistaxis: a case report

We present a case of a Rendu-Osler-Weber disease patient with recurrent life threatening epistaxis demanding multiple blood transfusions despite of repetitive endoscopic laser and electrocoagulations, endovascular embolisation, septodermoplasty, and long-term intranasal dressings. As alternative treatment modalities repeatedly failed and the patient became almost permanently dependent on nasal dressing, we performed a highly conformal intensity-modulated radiotherapy of the nasal cavity; a total dose of 50 Gy in 2 Gy single fractions was applied. The therapy was very well tolerated, no acute toxicities occurred. Two weeks after the last radiation dose had been applied, the nasal dressing could be removed without problems. Endoscopical control revealed an almost avascular white mucosa without any trace of bleeding spots; previously existing hemangiomas and crusts had disappeared. After a 1-year-follow up, the patient had no significant recurrent epistaxis.