Three-Dimensional Bioprinting of Organoid-Based Scaffolds (OBST) for Long-Term Nanoparticle Toxicology Investigation.

The toxicity of nanoparticles absorbed through contact or inhalation is one of the major concerns for public health. It is mandatory to continually evaluate the toxicity of nanomaterials. In vitro nanotoxicological studies are conventionally limited by the two dimensions. Although 3D bioprinting has been recently adopted for three-dimensional culture in the context of drug release and tissue regeneration, little is known regarding its use for nanotoxicology investigation. Therefore, aiming to simulate the exposure of lung cells to nanoparticles, we developed organoid-based scaffolds for long-term studies in immortalized cell lines. We printed the viscous cell-laden material via a customized 3D bioprinter and subsequently exposed the scaffold to either 40 nm latex-fluorescent or 11-14 nm silver nanoparticles. The number of cells significantly increased on the 14th day in the 3D environment, from 5 × 105 to 1.27 × 106, showing a 91% lipid peroxidation reduction over time and minimal cell death observed throughout 21 days. Administered fluorescent nanoparticles can diffuse throughout the 3D-printed scaffolds while this was not the case for the unprinted ones. A significant increment in cell viability from 3D vs. 2D cultures exposed to silver nanoparticles has been demonstrated. This shows toxicology responses that recapitulate in vivo experiments, such as inhaled silver nanoparticles. The results open a new perspective in 3D protocols for nanotoxicology investigation supporting 3Rs.

GSK-3 inhibition reverts mesenchymal transition in primary human corneal endothelial cells.

Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK-3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of α-SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na+/K+ ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed that CHIR99021 induced downregulation of EnMT markers (α-SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the ß-catenin and TGFß pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT, providing a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy.

Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium.

Nanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle-mediated RNA interference therapy aimed at enhancing its proliferative capacity, necessary for tissue regeneration. This work develops a strategy for siRNA nanoninjection to the human corneal endothelium. Nanoneedles can deliver p16-targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is nontoxic ex vivo, and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNA interference therapy for the treatment of endothelial corneal dysfunction.

Fluctuations in Corneal Endothelial LAP2 Expression Levels Correlate with Passage Dependent Declines in Their Cell Proliferative Activity.

The corneal endothelium is the inner corneal mono-layered epithelium, fundamental for preserving corneal hydration and transparency. However, molecular mechanisms that regulate corneal endothelial cells (CEnCs), in particular regarding their proliferative capacity, have been only partially elucidated. CEnCs are quiescent in vivo and they easily undergo endothelial to mesenchymal transition (EnMT) in vitro. This study aims to analyze CEnCs behavior and expression in vitro, either in sub-confluent growing (S) or confluent (C) CEnCs cultures. Primary rabbit and human CEnCs were cultured and used for RT-PCR, immunofluorescence or western blot analysis. These methods allowed identifying a novel molecular marker, LAP2, that is upregulated in S while downregulated in C human or rabbit CEnCs. Those results were observed for several subsequent passages in culture and this, together with the correlation between ki67 and LAP2 expression, suggested LAP2 as a novel possible indicator for culture ageing. Finally, treatment with FGF and TGFß in rCEnCs highlighted how LAP2 can vary as the cells regulate their proliferative state. In conclusion, we have identified a novel marker for CEnCs, LAP2, that regulates its expression depending on the cells sub/confluent state and that correlates with CEnCs proliferation.

Short-term lung function changes predict mortality in patients with fibrotic hypersensitivity pneumonitis.

BACKGROUND AND OBJECTIVE: A proportion of patients with fibrotic hypersensitivity pneumonitis (fHP) follow a progressive disease course despite immunosuppressive treatment. Little is known about predictors of mortality in fHP. We aimed to investigate the impact of short-term lung function changes in fHP on mortality. METHODS: Baseline demographics for 145 consecutive patients with a multi-disciplinary team diagnosis of fHP, as well as baseline and 1-year follow-up of lung function, baseline echocardiographic findings, bronchoalveolar lavage (BAL) cellularity and all-cause mortality were recorded. Changes in forced vital capacity (FVC) ≥ 5% and ≥10%, and diffusion capacity of the lung for carbon monoxide (DLCO) ≥ 10% and ≥15% at 1 year were calculated. Cox proportional hazards analysis was performed to test for associations with mortality. RESULTS: Baseline lung function severity, age, presence of honeycombing on computed tomography (CT) and echocardiographic pulmonary arterial systolic pressure (PASP) ≥ 40 mm Hg were associated with early mortality, while BAL lymphocytosis was associated with improved survival. A decline in FVC ≥ 5% (hazard ratio [HR]: 3.10, 95% CI: 2.00-4.81, p < 0.001), FVC ≥ 10% (HR: 3.11, 95% CI: 1.94-4.99, p < 0.001), DLCO ≥ 10% (HR: 2.80, 95% CI: 1.78-4.42, p < 0.001) and DLCO ≥ 15% (HR: 2.92, 95% CI: 1.18-4.72, p < 0.001) at 1 year was associated with markedly reduced survival on univariable and multivariable analyses after correcting for demographic variables, disease severity, honeycombing on CT and treatment, as well as BAL lymphocytosis and PASP ≥ 40 mm Hg on echocardiography, in separate models. CONCLUSION: Worsening in FVC and DLCO at 1 year, including a marginal decline in FVC ≥ 5% and DLCO ≥ 10%, is predictive of markedly reduced survival in fHP.

Challenges and resources in adult life with Joubert syndrome: issues from an international classification of functioning (ICF) perspective.

BACKGROUND: Joubert Syndrome (JS) is a rare inherited neurodevelopmental disorder defined by a characteristic cerebellar and brainstem malformation (i.e. the molar tooth sign) and variable organ involvement. The aim of the present study was to describe functional limitations and disabilities in a large sample of adult patients with a diagnosis of JS. METHODS: We administered the International Classification of Functioning (ICF) checklist to thirty-six adult Italian patients with JS or their caregivers through telephone calls. RESULTS: None-to-mild impairment was documented for basic cognitive and mental functions, whereas severe deficit emerged for higher-order skills and language. A mismatch between individuals' capacity for daily activity and social participation and the actual performance in these fields emerged, suggesting that adults with JS may greatly benefit from external support from the caring environment. Indeed, specific facilitators were highlighted, including communication technologies as well as family members, healthcare professionals and peers support. Mild-to-severe barriers have been identified by adult patients with JS in the domains of services, systems and policies. CONCLUSIONS: These findings highlight challenges and barriers for adults with JS in areas of daily functioning that may be improved by investing in rehabilitation care models that embed social support programs and policies into clinical interventions.IMPLICATIONS FOR REHABILITATIONChildren with Joubert Syndrome, a child-onset rare inherited neurodevelopmental condition, are growing up and becoming adults; a life course approach in rehabilitation is needed;There is a substantial lack of information on the long-term adaptive daily functioning of children with a diagnosis of Joubert Syndrome;In this paper, the International Classification of Functioning (ICF) was applied to assess the daily functioning in people with JS;Severe deficits emerged for high-order skills and language, whereas the use of communication technologies and the engagement of family members were highlighted as key facilitators;These findings highlight the need for a change of paradigm in the care model of subjects with JS, with the embedding of social support in rehabilitation programs.

3D Printed Masks for Powders and Viruses Safety Protection Using Food Grade Polymers: Empirical Tests.

The production of 3D printed safety protection devices (SPD) requires particular attention to the material selection and to the evaluation of mechanical resistance, biological safety and surface roughness related to the accumulation of bacteria and viruses. We explored the possibility to adopt additive manufacturing technologies for the production of respirator masks, responding to the sudden demand of SPDs caused by the emergency scenario of the pandemic spread of SARS-COV-2. In this study, we developed different prototypes of masks, exclusively applying basic additive manufacturing technologies like fused deposition modeling (FDM) and droplet-based precision extrusion deposition (db-PED) to common food packaging materials. We analyzed the resulting mechanical characteristics, biological safety (cell adhesion and viability), surface roughness and resistance to dissolution, before and after the cleaning and disinfection phases. We showed that masks 3D printed with home-grade printing equipment have similar performances compared to the industrial-grade ones, and furthermore we obtained a perfect face fit by customizing their shape. Finally, we developed novel approaches to the additive manufacturing post-processing phases essential to assure human safety in the production of 3D printed custom medical devices.

Bionic for Training: Smart Framework Design for Multisensor Mechatronic Platform Validation.

Home monitoring supports the continuous improvement of the therapy by sharing data with healthcare professionals. It is required when life-threatening events can still occur after hospital discharge such as neonatal apnea. However, multiple sources of external noise could affect data quality and/or increase the misdetection rate. In this study, we developed a mechatronic platform for sensor characterizations and a framework to manage data in the context of neonatal apnea. The platform can simulate the movement of the abdomen in different plausible newborn positions by merging data acquired simultaneously from three-axis accelerometers and infrared sensors. We simulated nine apnea conditions combining three different linear displacements and body postures in the presence of self-generated external noise, showing how it is possible to reduce errors near to zero in phenomena detection. Finally, the development of a smart 8Ws-based software and a customizable mobile application were proposed to facilitate data management and interpretation, classifying the alerts to guarantee the correct information sharing without specialized skills.

Follow-up of Acute Respiratory Disorders in Cyclists Competing in the 100th Giro d'Italia.

Acute respiratory disorder is a common sub-clinical condition affecting elite cyclists. Monitoring the perturbations of the immunological cells in the respiratory tract, indicative of a likely proinflammatory state, during an International Cycling Union world tour is a challenging task. The aim of this study was to follow up on the sign and symptoms of upper way respiratory infections with or without asthma, using non-invasive methods, during a 21-day race (100° Giro d'Italia, 2017). Nine male elite cyclists of the Bahrain Merida Team were evaluated before the training season and daily during the race. Clinical history, skin prick and spirometric test, acute respiratory symptoms were measured using validated questionnaires, and values of fraction of exhaled nitric oxide were collected longitudinally. Four of the 9 athletes had allergies with/or consistent abnormal spirometric curves before the race. During the race, 5 athletes had a fraction of exhaled nitric oxide values >20 ppb which correlated with respiratory symptoms collected through questionnaires. These were related to the environmental characteristics of the places travelled through in the race. The athletes with a predisposition to chronic respiratory inflammation in the pre-competitive season were more likely to develop acute respiratory symptoms during the race.

Author Correction: In-vivo vascular application via ultra-fast bioprinting for future 5D personalised nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A fine-tuned ß-catenin regulation during proliferation of corneal endothelial cells revealed using proteomics analysis.

Corneal endothelial (CE) dysfunction is the main indication for corneal transplantation, an invasive procedure with several limitations. Developing novel strategies to re-activate CE regenerative capacity is, therefore, of fundamental importance. This goal has proved to be challenging as corneal endothelial cells (CEnC) are blocked in the G0/G1 phase of the cell cycle in vivo and, albeit retaining proliferative capacity in vitro, this is further hindered by endothelial-to-mesenchymal transition. Herein we investigated the mechanisms regulating CEnC proliferation in vitro. Comparing the proteome of non-proliferating (in vivo-G0/G1) and proliferating (in vitro-G2/M) rabbit CEnC (rCEnC), 77 proteins, out of 3,328 identified, were differentially expressed in the two groups (p < 0.005). Literature and Gene Ontology analysis revealed ß-catenin and transforming growth factor (TGF-ß) pathways to be correlated with the identified proteins. Treatment of rCEnC with a ß-catenin activator and inhibitor showed that ß-catenin activation was necessary during rCEnC proliferation, but not sufficient for its induction. Furthermore, both pro-proliferative activity of basic fibroblast growth factor and anti-proliferative effects of TGF-ß were regulated through ß-catenin. Overall, these results provide novel insights into the molecular basis underlying the proliferation process that CEnC re-activate in vitro, consolidating the role of ß-catenin and TGF-ß.

In-vivo vascular application via ultra-fast bioprinting for future 5D personalised nanomedicine.

The design of 3D complex structures enables new correlation studies between the engineering parameters and the biological activity. Moreover, additive manufacturing technology could revolutionise the personalised medical pre-operative management due to its possibility to interplay with computer tomography. Here we present a method based on rapid freeze prototyping (RFP) 3D printer, reconstruction cutting, nano dry formulation, fast freeze gelation, disinfection and partial processes for the 5D digital models functionalisation. We elaborated the high-resolution computer tomography scan derived from a complex human peripheral artery and we reconstructed the 3D model of the vessel in order to obtain and verify the additive manufacturing processes. Then, based on the drug-eluting balloon selected for the percutaneous intervention, we reconstructed the biocompatible eluting-freeform coating containing 40 nm fluorescent nanoparticles (NPs) by means of RFP printer and we tested the in-vivo feasibility. We introduced the NPs-loaded 5D device in a rat's vena cava. The coating dissolved in a few minutes releasing NPs which were rapidly absorbed in vascular smooth muscle cell (VSMC) and human umbilical vein endothelial cell (HUVEC) in-vitro. We developed 5D high-resolution self-dissolving devices incorporating NPs with the perspective to apply this method to the personalised medicine.

Functional associations of pleuroparenchymal fibroelastosis and emphysema with hypersensitivity pneumonitis.

BACKGROUND: Pleuroparenchymal fibroelastosis (PPFE) has been described in hypersensitivity pneumonitis (HP) yet its functional implications are unclear. Combined pulmonary fibrosis and emphysema (CPFE) has occasionally been described in never-smokers with HP, but epidemiological data regarding its prevalence is sparse. CTs in a large HP cohort were therefore examined to identify the prevalence and effects of PPFE and emphysema. METHODS: 233 HP patients had CT extents of interstitial lung disease (ILD) and emphysema quantified to the nearest 5%. Lobar percentage pleural involvement of PPFE was quantified on a 4-point categorical scale: 0 = absent, 1 = affecting <10%, 2 = affecting 10-33%, 3 = affecting >33%. Marked PPFE reflected a total lung score of ≥3/18. Results were evaluated against FVC, DLco and mortality. RESULTS: Marked PPFE prevalence was 23% whilst 23% of never-smokers had emphysema. Following adjustment for patient age, gender, smoking status, and ILD and emphysema extents, marked PPFE independently linked to reduced baseline FVC (p = 0.0002) and DLco (p = 0.002) and when examined alongside the same covariates, independently linked to worsened survival (p = 0.01). CPFE in HP demonstrated a characteristic functional profile of artificial lung volume preservation and disproportionate DLco reduction. CPFE did not demonstrate a worsened outcome when compared to HP patients without emphysema beyond that explained by CT extents of ILD and emphysema. CONCLUSIONS: PPFE is not uncommon in HP, and is independently associated with impaired lung function and increased mortality. Emphysema was identified in 23% of HP never-smokers. CPFE appears not to link to a malignant microvascular phenotype as outcome is explained by ILD and emphysema extents.

Pleuroparenchymal Fibroelastosis: A Review of Histopathologic Features and the Relationship Between Histologic Parameters and Survival.

Pleuroparenchymal fibroelastosis (PPFE) is now a defined clinicopathologic entity in the updated 2013 ATS/ERS classification of idiopathic interstitial pneumonias (IIPs), which has led to a significant increase in cases being diagnosed at our institution. We have therefore reviewed 43 PPFE cases (58 biopsies in total) to assess whether any clinical or histopathologic features provide prognostic information. A semiquantatitive grading system was used to assess extent of fibroblastic foci, intra-alveolar fibroelastosis, visceral pleural fibrosis, chronic inflammation in areas of fibrosis, vascular fibrointimal thickening, and presence of granulomas. Other patterns of interstitial lung disease were also noted, if present. All biopsies showed intra-alveolar fibroelastosis, fibroblastic foci at the leading edge of fibrosis and chronic inflammation within areas of fibrosis, 91% showed vascular fibrointimal thickening of vessels, 73% showed pleural fibrosis, and 35% showed granulomas. Ten cases showed a coexistent IIP (5 showed usual interstitial pneumonia, 5 showed features of hypersensitivity pneumonitis). There was no significant correlation with mortality and severity of histologic parameters, other than a significant decrease in mortality in PPFE with coexistent granulomas, after adjusting for age and gender (hazard ratio, 0.27; P=0.049). Male gender was also associated with an increased risk of mortality, after adjusting for age (hazard ratio, 4.8; P=0.045). PPFE is more common than previously thought, not infrequently showing coexistent pathology, specifically usual interstitial pneumonia and granulomatous lung disease, our data suggesting the latter may have prognostic significance.

Mydriatics release from solid and semi-solid ophthalmic formulations using different in vitro methods.

The aim of the present paper was the development of semi-solid (hydrogels) and solid (film) ophthalmic formulations for the controlled release of two mydriatics: phenylephrine and tropicamide. The formulations - based on polyvinylalcohol and hyaluronic acid - were characterized, and release studies were performed with three different in vitro set-ups, i.e. Franz-type diffusion cell, vial method and inclined plane; for comparison, a solution and a commercial insert, both clinically used to induce mydriasis, were evaluated. Both gels and film allowed for a controlled release of drugs, appearing a useful alternative for mydriatics administration. However, the release kinetic was significantly influenced by the method used, highlighting the need for optimization and standardization of in vitro models for the evaluation of drug release from ophthalmic dosage forms.

Oxygen therapy in COPD patients with isolated nocturnal hypoxemia; comparison of quality of life and sleep between bronchitis and emphysema phenotype: A prospective observational study.

BACKGROUND: COPD is a heterogeneous disease composed by two main phenotypes: bronchitis (COPDb) and emphysema (COPDe) with different clinical presentation, physiology, imaging, response to therapy and decline in lung function. The aim of this study is to evaluate whether nocturnal hypoxemic COPDb and COPDe have a different behaviour during sleep and the effect of nocturnal oxygen supplementation (nO2LT). MATERIALS AND METHODS: 75 COPDb and 120 COPDe were enrolled. All patients performed polysomnography, Pittsburgh and Maugeri Foundation Respiratory Failure questionnaire, and pulmonary function before and after six months of nO2LT. RESULTS: At baseline, compared to COPDb, COPDe have decreased sleep efficiency (SE) (67.5±6% vs. 76.9±3% p<0.05) and higher arousals (A/I) (18.1±3 event/h vs. 8.7±1 event/h p<0.05). Oxygen desaturation index (ODI) was increased during REM (7.1±1 event/h vs. 2.3±0.5 event/h p<0.05). nO2LT in COPDe improves SE (77±4% vs. 67.5±6% p<0.05) and decreases A/I (9±5 event/h vs. 18.1±3 event/h p<0.05). ODI during REM (3.5±2 event/h vs. 7.1±1 p<0.05) decreases and quality of life (QoL) improves (MFR-28 total 56±22 vs 45±20 p<0.05), due to an improvement in cognitive abilities (45±30 vs 33±31 p<0.05) and daily activities (61±29 vs 53±21 p>0.05). In COPDb nO2LT reduces ST90 (15±6% vs. 43±8% p<0.05) less than in COPDe (15±6% vs. 8±4% p<0.05); improves A/I (10±2 event/h vs. 8.7±1 p<0.05) and there is no evidence of an improvement in QoL. CONCLUSIONS: Six months of nO2LT improve quality of life in COPDe, not in COPDb. We found a difference in sleep quality between COPDe and COPDb.

A new approach to detect early lung functional impairment in very light smokers.

PURPOSE: The aim of our study is to investigate if lung carbon monoxide diffusing capacity (DLCO) measured during effort is able to detect early respiratory functional impairment. METHODS: We enrolled 25 very light smokers and 20 healthy non smokers. Subjects underwent plethysmography, DLCO (single breath technique) and calculated effective pulmonary blood flow (Qc) by rebreathing method. During exercise by cycle ergometer (duration 10±2min; recovery 11±3min) DLCO and Qc were calculated at 25% and 50% of theoretical maximum workload. RESULTS: At baseline lung function and Qc did not differ between groups. DLCO and DLCO/Qc measured during exercise were significantly greater in non smokers (p<0.001); Qc was not statistically different. In very light smokers, DLCO, DLCO/Qc measured during exercise significantly correlated with the number of pack years (r=-0.60 p<0.001; r=-0.58 p<0.05; r=-0.55 p<0.05, respectively). CONCLUSIONS: In very light smokers there is lung function impairment and our data show that DLCO during exercise may reveal this underlying early damage.

Impaired lung transfer factor in fibromyalgia syndrome.

OBJECTIVES: The aim of this study was to evaluate whether pulmonary diffusing capacity is impaired in patients with fibromyalgia (FM) as it is in those with other diseases characterised by autonomic nerve system (ANS) dysfunction such as type 1 diabetes. METHODS: Forty-five consecutive anti-nuclear antibody (ANA)-negative female Caucasian patients aged 50.1± 5.6 years with FM and compared with 45 healthy female control volunteers matched in terms of age and body mass index (BMI). The autonomic function has been evaluated by means of standard electrocardiography (ECG), finger blood pressure respiration, and muscle sympathetic nerve activity (MSNA) at rest and during a stepwise tilt test up to 75°. Their autonomic profiles were drawn up on the basis of MSNA, plasma catecholamine levels, and spectral indices of cardiac sympathetic and vagal modulation, and sympathetic vasomotor control computed by means of the spectrum analysis of RR and systolic arterial pressure (SAP) variability. Lung volumes and dynamic spirometry parameters were assessed by means of plethysmography. All of the patients were clinically evaluated and completed the FQI and COMPASS questionnaire. RESULTS: There was no difference in lung volumes between the FM patients and healthy controls, but DLCO (83±4 vs. 96±5; p<0.001), Kco (84±5 vs 98±5; p<0.001), DM (12.7±2.4 vs 13.6±1.8; p<0.05) and Vc (48±3.9 vs 65±7; p<0.001) were significantly reduced in the patients. The COMPASS-31, RCS and pain VAS scores significantly correlated with DLCO, Kco and Vc with the correlation being particularly close in the case of Vc. Furthermore, univariate Cox proportional hazard analysis showed that the three scores were all significantly associated with an increased risk of impaired DLCO (respectively, χ(2) 16.21, p<0.0005; χ(2) 7.09, p<0.005; χ(2) 6.37, p<0.01). CONCLUSIONS: FM impairs DLCO mainly as a result of a reduction in Vc, and that this defect is inversely proportional to the severity of the dysfunction suggesting a relationship between impaired DLCO and autonomic nerve dysfunction.