Bioactive Membranes and Nanocoatings for Guided Tissue Regeneration in Periodontal Diseases

Periodontal diseases are highly prevalent in population of all ages. Initiated by bacterial accumulation at the interface of bone and soft tissue, they lead to the loss of gingival tissue adherent to the root surface and, eventually, to tooth loss. Regenerative approaches to treat periodontitis offer exciting possibilities; guided tissue/bone regeneration (GTR/GBR) approaches are promising because, through the insertion of a physical barrier, they can exclude unwanted epithelial and gingival connective tissue cells from the healing area and allow bone tissue cells to repopulate the bony defect. Different resorbable and non-resorbable membranes have been developed. Expanded polytetrafluoroethylene (ePTFE) membranes are the “gold standard” for GTR/GBR applications but they are non-resorbable and they need a second surgical operation to repair dehiscence. Biodegradable synthetic membranes avoid a second surgical operation but they show drawbacks concerning the capacity of space maintenance, early/late absorption, mechanical properties and bacterial infection during degradation. Collagen membranes have advantages related to collagen biological properties but are characterized by low mechanical strength. The “ideal” membrane for use in periodontal regenerative therapy has yet to be developed. The main purpose of this thesis was the design of biologically active products, with improved osteoconductive and antimicrobial properties, for GTR/GBR applications in periodontal diseases. In a more traditional approach, a commercially available membrane (based on PTFE) was surface modified by environmentally friendly technique to allow rapid bone re-growth and exert antimicrobial action. Binding ability of 3,4-dihydroxy-DL-phenylalanine (DOPA) to samples of any type, size and shape was exploited to improve PTFE surface properties. In particular, a hydroxyapatite nanoparticles (HAp) coating was applied by DOPA self-polymerization on PTFE surface in the presence of HAp nanoparticles, to promote the bone re-growth properties of PTFE films. Chemical composition analysis demonstrated the successful deposition of polyDOPA and HAp on coated films. Morphological and topographical characterizations further confirmed the total surface coverage causing an increase in surface roughness (39.8±5.2 nm for PTFE films vs 236.5±12.0 nm for polyDOPA/HAp coated films) and wettability (110.8±2.8° for PTFE films vs 46.1±12.4° for polyDOPA/HAp coated samples). A discontinuous HAp coating was still present after 14 days of incubation of coated PTFE films in phosphate buffered saline. Pre-osteoblastic MC3T3-E1 cells cultured on polyDOPA/HAp coated films showed a pronounced increase of cell proliferation and adhesion. Regarding the antimicrobial action, silver nanoparticles (AgNPs) have been selected due to their good antimicrobial efficacy against bacteria, viruses and other eukaryotic micro-organisms. The successful deposition of AgNPs on PTFE surface, through the functional groups of DOPA, has been demonstrated by physico-chemical and morphological analyses. Nanoparticles exhibited a diameter around 68 nm and were homogeneously distributed on the surface. In vitro cell tests with fibroblast NIH 3T3 cells showed an inhibition of cells proliferation on AgNPs functionalized films after 3 days of culture, while good cell adhesion was observed with cells randomly distributed on sample surface and extensively spread. The antimicrobial efficiency was demonstrated against S. aureus and Ag release was sustained for at least 14 days. The mussel-inspired coated PTFE membrane could find potential application as GTR/GBR strategy for the treatment of periodontal diseases. In a highly innovative approach, a bi-layered bioabsorbable membrane was developed, by the assembly of a compact and a porous layer. GTR/GBR membranes can be considered an interface-implant between gingival connective tissue/epithelium and alveolar bone tissue. Developing a multi-component structure membrane with compositional and structural gradients that meet the local functional requirements could represent a challenge. Binary blends of poly(DL-lactide-co-ε-caprolactone) (PLCL) and poly(DL-lactide-co-glycolide) (PLGA) with various compositions (100/0, 75/25, 50/50, 25/75, 0/100 wt/wt) were prepared by solvent casting technique as compact layer of the bi-layered membrane. Morphological analysis did not evidence phase separation between PLCL and PLGA and the behavior of blend glass transition temperatures as a function of composition suggested some degree of blend compatibility. However, blends elastic modulus showed a negative deviation from the additive law of mixture. In vitro cell tests with fibroblast NIH 3T3 cells showed improved cell adhesion and growth on PLCL/PLGA 25/75 blend. Due to its biocompatibility, its superior mechanical properties (E=10.2±0.6 MPa, σmax=0.8±0.0 MPa, and εmax=548.8±57.9%) and compatibility between the components, PLCL/PLGA 25/75 blend was selected for this application. Compact films were then surface modified via layer-by-layer (LbL) technique to enhance fibroblast cell response and confer antibacterial efficacy. A surface priming treatment (aminolysis) was optimized before depositing LbL coating. The following parameters were used: C=0.08 g/mL, t=8 min and T=37 °C. Then, multilayered chondroitin sulfate/chitosan (CHS/CH) coatings were deposited on the aminolysed films. The feasibility of multilayer coating was confirmed by QCM-D analysis. Further confirmations derived from water contact angle measurements (contact angle jumped alternatively between 45° and 65° depending on the outmost layer component) and FTIR-ATR analysis (appearance of absorbance peaks characteristics of CHS and CH). FTIC-labelled CH was also employed to follow LbL built up by fluorescence microscopy analysis. In vitro cell tests demonstrated the ability of coated samples to improve NIH 3T3 fibroblast adhesion and proliferation. Biocompatibility properties increased with increasing the layer number and were superior in the case of CH-terminating layers but no antibacterial activity was observed for films coated with 16 layers. Three dimensional sponge-like composite membranes fabricated by freeze-drying, with a composition similar to natural bone, and based on β-tricalcium phosphate (TCP) dispersed in a chitosan/gelatin (CH/G) network cross-linked with genipin (GP) and disodium phosphate salt (DSP) were developed as porous layer of the bi-layered device. Three membranes were developed (CH/G, CH/G+GP-DSP and CH/G/TCP+GP-DSP) and characterized. Successful double cross-linking of CH/G network was confirmed by Kaiser test, chemical and thermal analysis. All membranes showed a typical foam-like morphology with interconnected pores having an average diameter of 100-200 μm. Both cross-linking and TCP presence caused a marked increase of membrane stability in water solution, as well as of tensile modulus and maximum tensile strength (respectively, 14.9±5.1 MPa and 0.6±0.0 MPa for CH/G, and 29.4±2.7 MPa and 0.8±0.1 MPa for CH/G/TCP+GP-DSP.). Compared to CH/G samples, CH/G+GP-DSP and CH/G/TCP+GP-DSP membranes showed improved MG-63 human osteoblast-like cells response, in terms of cell viability and morphology. The assembly process of the compact and porous layer was developed based on the insertion of an intermediate adhesive layer composed by a polyvinylpyrrolidone/polyethylene glycol 70/30 wt/wt blend. Preliminary characterizations were carried out. Morphological analysis did not show changes in compact and porous layer structure due to the presence of the adhesive. The final device showed an elastic modulus of about 61 MPa in dry condition that markedly decreased in wet state (to about 5 MPa). Qualitative analysis of membrane manageability revealed its ability to adapt to mandible conformation after immersion in physiological solution. Despite the need for additional tests, the bi-layered membrane appeared promising for GTR/GBR applications

Smoking cessation interventions in nurses and other health care workers

Introduction: Tobacco smoking can be considered an old and a new challenge for public health. The aim of this review was to analyse different smoking cessation interventions aiming at health care workers.Methods: A literature search of electronic journal databases for studies on smoking cessation interventions among health care workers was performed according to PRISMA criteria, using the MEDLINE and Scopus databases.Results: Smoking restriction policies shouldn’t be considered as actual interventions, being ineffective, unpopular and reducing willingness to quit smoking in many subjects. Even though pharmacological therapies based on bupropion SR and transdermal nicotine patches grant significant results on the short-term (weeks and months), smoking recurrence rates are high and individualised interventions should be preferred or integrated since they seem to grant better results on the longterm (years).Conclusions: There is evidence that smoking cessation interventions among health care workers can be effective. This is of particular interest both for reducing tobacco smoking prevalence among this type of workers and for helping them to be useful model for the general population

Molecular basis for endocrine disruption by pesticides targeting aromatase and estrogen receptor

The intensive use of pesticides has led to their increasing presence in water, soil, and agricultural products. Mounting evidence indicates that some pesticides may be endocrine disrupting chemicals (EDCs), being therefore harmful for the human health and the environment. In this study, three pesticides, glyphosate, thiacloprid, and imidacloprid, were tested for their ability to interfere with estrogen biosynthesis and/or signaling, to evaluate their potential action as EDCs. Among the tested compounds, only glyphosate inhibited aromatase activity (up to 30%) via a non-competitive inhibition or a mixed inhibition mechanism depending on the concentration applied. Then, the ability of the three pesticides to induce an estrogenic activity was tested in MELN cells. When compared to 17\u3b2-estradiol, thiacloprid and imidacloprid induced an estrogenic activity at the highest concentrations tested with a relative potency of 5.4 7 10 1210 and 3.7 7 10 129, respectively. Molecular dynamics and docking simulations predicted the potential binding sites and the binding mode of the three pesticides on the structure of the two key targets, providing a rational for their mechanism as EDCs. The results demonstrate that the three pesticides are potential EDCs as glyphosate acts as an aromatase inhibitor, whereas imidacloprid and thiacloprid can interfere with estrogen induced signaling

Integration of Biomechanical and Biological Characterization in the Development of Porous Poly(Caprolactone)-Based Membranes for Abdominal Wall Hernia Treatment.

AIMS: Synthetic meshes are the long-standing choice for the clinical treatment of abdominal wall hernias: the associated long-term complications have stimulated the development of a new-generation of bio-resorbable prostheses. In this work, polycaprolactone (PCL) porous membranes prepared by solvent casting/porogen leaching of PCL/poly(ethylene glycol) (PEG) blends with different compositions (different PCL/PEG weight ratio and PEG molecular weight) were investigated to be applied in the field. An optimal porous membrane structure was selected based on the evaluation of physicochemical, biomechanical and in-vitro biological properties, compared to a reference commercially available hernia mesh (CMC). FINDINGS: Selected PCL7-2i membranes (derived from PCL/PEG 70/30, PCL: Mw 70,000-90,000 Da; PEG: 35,000 Da) showed suitable pore size for the application, intermediate surface hydrophilicity and biomimetic mechanical properties. In-vitro cell tests performed on PCL7-2i membranes showed their cytocompatibility, high cell growth during 21 days, a reduced production of pro-inflammatory IL-6 respect to CMC and a significant secretion of Collagen Type I. CONCLUSIONS: PCL7-2i membranes showed biomimetic biomechanical properties and in-vitro biological properties similar to or even better than - in the case of anti-inflammatory behavior and collagen production - CMC, a commercially available product, suggesting potentially improved integration in the host tissue

Functional and clinical relevance of novel mutations in a large cohort of patients with Cockayne syndrome

BACKGROUND: Cockayne syndrome (CS) is a rare, autosomal recessive multisystem disorder characterised by prenatal or postnatal growth failure, progressive neurological dysfunction, ocular and skeletal abnormalities and premature ageing. About half of the patients with symptoms diagnostic for CS show cutaneous photosensitivity and an abnormal cellular response to UV light due to mutations in either the ERCC8/CSA or ERCC6/CSB gene. Studies performed thus far have failed to delineate clear genotype-phenotype relationships. We have carried out a four-centre clinical, molecular and cellular analysis of 124 patients with CS

The LiberAction Project: Implementation of a Pediatric Liberation Bundle to Screen Delirium, Reduce Benzodiazepine Sedation, and Provide Early Mobilization in a Human Resource-Limited Pediatric Intensive Care Unit

Background: Delirium, bed immobilization, and heavy sedation are among the major contributors of pediatric post-intensive care syndrome. Recently, the Society of Critical Care Medicine has proposed the implementation of daily interventions to minimize the incidence of these morbidities and optimize children functional outcomes and quality of life. Unfortunately, these interventions require important clinical and economical efforts which prevent their use in many pediatric intensive care units (PICU). Aim: First, to evaluate the feasibility and safety of a PICU bundle implementation prioritizing delirium screening and treatment, early mobilization (<72 h from PICU admission) and benzodiazepine-limited sedation in a human resource-limited PICU. Second, to evaluate the incidence of delirium and describe the early mobilization practices and sedative drugs used during the pre- and post-implementation periods. Third, to describe the barriers and adverse events encountered during early mobilization. Methods: This observational study was structured in a pre- (15th November 2019-30th June 2020) and post-implementation period (1st July 2020-31st December 2020). All patients admitted in PICU for more than 72 h during the pre and post-implementation period were included in the study. Patients were excluded if early mobilization was contraindicated. During the pre-implementation period, a rehabilitation program including delirium screening and treatment, early mobilization and benzodiazepine-sparing sedation guidelines was developed and all PICU staff trained. During the post-implementation period, delirium screening with the Connell Assessment of Pediatric Delirium scale was implemented at bedside. Early mobilization was performed using a structured tiered protocol and a new sedation protocol, limiting the use of benzodiazepine, was adopted. Results: Two hundred and twenty-five children were enrolled in the study, 137 in the pre-implementation period and 88 in the post-implementation period. Adherence to delirium screening, benzodiazepine-limited sedation and early mobilization was 90.9, 81.1, and 70.4%, respectively. Incidence of delirium was 23% in the post-implementation period. The median cumulative dose of benzodiazepines corrected for the total number of sedation days (mg/kg/sedation days) was significantly lower in the post-implementation period compared with the pre-implementation period: [0.83 (IQR: 0.53-1.31) vs. 0.74 (IQR: 0.55-1.16), p = 0.0001]. The median cumulative doses of fentanyl, remifentanil, and morphine corrected for the total number of sedation days were lower in the post-implementation period, but these differences were not significant. The median number of mobilizations per patient and the duration of each mobilization significantly increased in the post-implementation period [3.00 (IQR: 2.0-4.0) vs. 7.00 (IQR: 3.0-12.0); p = 0.004 and 4 min (IQR: 3.50-4.50) vs. 5.50 min (IQR: 5.25-6.5); p < 0.0001, respectively]. Barriers to early mobilization were: disease severity and bed rest orders (55%), lack of physicians' order (20%), lack of human resources (20%), and lack of adequate devices for patient mobilization (5%). No adverse events related to early mobilization were reported in both periods. Duration of mechanical ventilation and PICU length of stay was significantly lower in the post-implementation period as well as the occurrence of iatrogenic withdrawal syndrome. Conclusion: This study showed that the implementation of a PICU liberation bundle prioritizing delirium screening and treatment, benzodiazepine-limited sedation and early mobilization was feasible and safe even in a human resource-limited PICU. Further pediatric studies are needed to evaluate the clinical impact of delirium, benzodiazepine-limited sedation and early mobilization protocols on patients' long-term functional outcomes and on hospital finances

PIK3R1W624R Is an Actionable Mutation in High Grade Serous Ovarian Carcinoma.

Identifying cancer drivers and actionable mutations is critical for precision oncology. In epithelial ovarian cancer (EOC) the majority of mutations lack biological or clinical validation. We fully characterized 43 lines of Patient-Derived Xenografts (PDXs) and performed copy number analysis and whole exome sequencing of 12 lines derived from naïve, high grade EOCs. Pyrosequencing allowed quantifying mutations in the source tumours. Drug response was assayed on PDX Derived Tumour Cells (PDTCs) and in vivo on PDXs. We identified a PIK3R1W624R variant in PDXs from a high grade serous EOC. Allele frequencies of PIK3R1W624R in all the passaged PDXs and in samples of the source tumour suggested that it was truncal and thus possibly a driver mutation. After inconclusive results in silico analyses, PDTCs and PDXs allowed the showing actionability of PIK3R1W624R and addiction of PIK3R1W624R carrying cells to inhibitors of the PI3K/AKT/mTOR pathway. It is noteworthy that PIK3R1 encodes the p85α regulatory subunit of PI3K, that is very rarely mutated in EOC. The PIK3R1W624R mutation is located in the cSH2 domain of the p85α that has never been involved in oncogenesis. These data show that patient-derived models are irreplaceable in their role of unveiling unpredicted driver and actionable variants in advanced ovarian cancer

Faulty cardiac repolarization reserve in alternating hemiplegia of childhood broadens the phenotype

Alternating hemiplegia of childhood is a rare disorder caused by de novo mutations in the ATP1A3 gene, expressed in neurons and cardiomyocytes. As affected individuals may survive into adulthood, we use the term 'alternating hemiplegia'. The disorder is characterized by early-onset, recurrent, often alternating, hemiplegic episodes; seizures and non-paroxysmal neurological features also occur. Dysautonomia may occur during hemiplegia or in isolation. Premature mortality can occur in this patient group and is not fully explained. Preventable cardiorespiratory arrest from underlying cardiac dysrhythmia may be a cause. We analysed ECG recordings of 52 patients with alternating hemiplegia from nine countries: all had whole-exome, whole-genome, or direct Sanger sequencing of ATP1A3. Data on autonomic dysfunction, cardiac symptoms, medication, and family history of cardiac disease or sudden death were collected. All had 12-lead electrocardiogram recordings available for cardiac axis, cardiac interval, repolarization pattern, and J-point analysis. Where available, historical and prolonged single-lead electrocardiogram recordings during electrocardiogram-videotelemetry were analysed. Half the cohort (26/52) had resting 12-lead electrocardiogram abnormalities: 25/26 had repolarization (T wave) abnormalities. These abnormalities were significantly more common in people with alternating hemiplegia than in an age-matched disease control group of 52 people with epilepsy. The average corrected QT interval was significantly shorter in people with alternating hemiplegia than in the disease control group. J wave or J-point changes were seen in six people with alternating hemiplegia. Over half the affected cohort (28/52) had intraventricular conduction delay, or incomplete right bundle branch block, a much higher proportion than in the normal population or disease control cohort (P = 0.0164). Abnormalities in alternating hemiplegia were more common in those ≥16 years old, compared with those <16 (P = 0.0095), even with a specific mutation (p.D801N; P = 0.045). Dynamic, beat-to-beat or electrocardiogram-to-electrocardiogram, changes were noted, suggesting the prevalence of abnormalities was underestimated. Electrocardiogram changes occurred independently of seizures or plegic episodes. Electrocardiogram abnormalities are common in alternating hemiplegia, have characteristics reflecting those of inherited cardiac channelopathies and most likely amount to impaired repolarization reserve. The dynamic electrocardiogram and neurological features point to periodic systemic decompensation in ATP1A3-expressing organs. Cardiac dysfunction may account for some of the unexplained premature mortality of alternating hemiplegia. Systematic cardiac investigation is warranted in alternating hemiplegia of childhood, as cardiac arrhythmic morbidity and mortality are potentially preventable

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

UV-sensitive syndrome (UVSS) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma1, 2, 3, 4. Despite mild clinical features, cells from individuals with UVSS, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER)2, 4, 5, which removes DNA damage in actively transcribed genes6. Three of the seven known UVSS cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively)7, 8. The remaining four individuals with UVSS, one of whom is described for the first time here, formed a separate UVSS-A complementation group1, 9, 10; however, the responsible gene was unknown. Using exome sequencing11, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UVSS-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders