64 research outputs found
Endotelio corneale umano: dagli studi in vitro all’applicazione dell’ ingegneria tissutale
No full textL'endotelio corneale regola lo stato di idratazione stromale necessario per la trasparenza corneale. In età adulta, la densità cellulare endoteliale (ECD) diminuisce annualmente dello 0,6%. Poiché le cellule endoteliali corneali umane hanno ridotta capacità proliferativa in vivo, la loro perdita è compensata dalla migrazione e allargamento delle cellule vicine. Quando l' ECD scende al di sotto del valore soglia di 500 cellule/mm2, in seguito a invecchiamento o trauma o una condizione patologica, l'endotelio non è in grado di garantire una corretta idratazione corneale, causando edema, opacità corneale e disturbi visivi. Il trapianto corneale, con le relative limitazioni, ad oggi è l'unico trattamento efficiente per le malattie endoteliali corneali. Tuttavia, la carenza mondiale di cornee donatrici sta diventando sempre più un problema non trascurabile, con solo 1 cornea disponibile ogni 70 cornee richieste. Questo ha indotto a sviluppare strategie alternative per il trattamento di malattie endoteliali corneali, tra cui gli approcci di ingegneria tissutale.
L'ingegneria tissutale è un approccio terapeutico emergente che combina l'utilizzo di cellule endoteliali corneali con l’utilizzo di un appropriato biomateriale per la coltura ed il trapianto di queste cellule. Il nostro gruppo di ricerca ha precedentemente dimostrato che il legame di un decapeptide contenente il motivo peptidico RGD (Arg-Gly-Asp) allo scaffold di chitina garantisce il mantenimento del comportamento delle cellule epiteliali corneali umane. Le caratteristiche dello scaffold sono state ottimizzate per produrre un substrato con proprietà biomeccaniche simili allo stroma corneale umano per trasparenza, architettura, rigidità e resistenza meccanica.
In questo progetto di ricerca, il nostro obiettivo è quello di studiare l'utilizzo della chitina funzionalizzata con l’ RGD come potenziale substrato anche per l'adesione e l'espansione delle cellule corneali endoteliali umane. Gli esperimenti sono stati condotti al fine di ottenere un tessuto endoteliale ingegnerizzato e, in una prospettiva futura, una cornea umana tridimensionale con tutti i suoi strati (epitelio + stroma + endotelio).
Tuttavia, l'ingegneria tissutale dell’ endotelio corneale è una sfida complessa per diversi motivi: a) le cellule corneali endoteliali hanno una bassa capacità proliferativa che deve essere stimolata finemente in vitro con terreni di coltura appropriati; b) durante la coltura in vitro, le cellule corneali endoteliali vanno incontro a senescenza prematura (in particolare nelle colture cellulari derivate da donatori più anziani) e a una trasformazione fenotipica assumendo un fenotipo mesenchimale, la cosiddetta transizione endoteliale-mesenchimale; e) pochi marcatori molecolari specifici definiscono la qualità delle cellule corneali endoteliali, necessari per controllare le loro funzioni fisiologiche cellulari; d) infine, per l’approccio di ingegneria tissutale dell’ endotelio corneale, non è stato ancora sviluppato un biomateriale in grado di creare un microambiente favorevole all'attività delle cellule corneali endoteliali.
Per questo motivo, in questo progetto di ricerca abbiamo affrontato alcune sfide che rendono difficile l’utilizzo delle cellule corneali endoteliali, in termini di (I) ottimizzazione della tecnica di coltura delle cellule corneali endoteliali umane (Capitolo I), (II) identificazione di marcatori funzionali specifici delle cellule corneali endoteliali (Capitolo II), (III) prevenzione della transizione endotelio-mesenchimale che induce ad un trans-differenziamento cellulare verso un fenotipo mio-fibroblastico che causa una perdita della funzione cellulare (Capitolo III) e (IV) analisi dello scaffold selezionato per coltivare le cellule corneali endoteliali (Capitolo IV).The corneal endothelium (CE) is the innermost layer of the cornea that regulates the stromal hydration state required to maintain corneal transparency. During adulthood, the endothelial cell density (ECD) decreases by 0.6% each year. As human corneal endothelial cells (hCECs) do not proliferate, the loss of aging-induced hCECs is compensated by migration and enlargement of neighbouring cells. When ECD falls below a threshold of 500 cells/mm2, by aging or trauma/disease, the endothelium does not have enough pumping power to guarantee a correct corneal hydration, leading to oedema, corneal opacity, and visual impairment. Corneal transplantation, with related problems, is the only efficient treatment for corneal endothelial diseases up to date. However, the worldwide donor corneas shortage is increasingly becoming a non-negligible issue, with only 1 cornea available for 70 needed. This has led to investigate alternative strategies for treating corneal endothelial diseases, such as tissue engineering approaches.
Corneal endothelial tissue engineering is an emerging therapeutic approach that involves the use of hCECs combined with a biomaterial to create tissue engineered grafts for transplantation. Our research group has previously demonstrated that proper binding of an RGD (Arg-Gly-Asp) peptide to the chitin scaffold guaranteed maintenance of human corneal epithelial cells behaviour. Scaffold characteristics were optimised to produce a substrate with biomechanical properties resembling the human corneal stroma for transparency, architecture, stiffness, and mechanical strength.
In this research project, our aim is to investigate the use of this functionalized biological scaffold as a potential substrate also for hCECs adhesion and expansion. The experiments were carried out in order to obtain a functional tissue engineered endothelial graft and, from a future perspective, a three-dimensional human cornea with all its layers (epithelium + stroma + endothelium). If successful, this elegant approach has the potential to increase access to corneal therapy by treating multiple patients.
However, CE tissue engineering is a major challenge for several reasons: a) the hCECs have a low natural proliferative capacity that must be finely stimulated in vitro with an appropriate mitogen-rich medium; b) during in vitro expansion, hCECs undergo premature senescence (particularly in cultures derived from older donors) and phenotypic transformation to a mesenchymal phenotype, so-called Endothelial-Mesenchymal Transition (EnMT), which must be prevented c) few specific molecular markers define the quality of cultured hCECs, which are needed to control their physiological cell functions; d) finally, to develop a tailored engineered corneal endothelium, a substrate material that is able to create a favourable microenvironment for hCECs activity has not been yet developed.
Thus, in this research project we analysed some challenges faced with hCECs in terms of (I) optimization of hCECs culture techniques (Chapter I), (II) identification of specific hCECs functional markers (Chapter II), (III) prevention of EnMT which leads to a cellular trans-differentiation towards a myofibroblastic phenotype causing a cellular loss of function (Chapter III), and (IV) analysis of the identified scaffold to make bioengineered corneal endothelial grafts (Chapter IV)
Impact of culture media on primary human corneal endothelial cells derived from old donors
No full text: Corneal endothelial dysfunction is a major indication for corneal transplantation. However, a global shortage of donor corneal tissues and risks associated with corneal surgeries have prompted exploration of alternative options, including tissue-engineered grafts or cell injection therapy. Nonetheless, these approaches require a controlled culture of primary human corneal endothelial cells (HCEnCs). Although HCEnCs established from young donors are generally more proliferative and maintain a better phenotype, corneas from old donors are more frequently accessible from eye banks due to a lower corneal endothelial cell count than the necessary threshold required for transplantation. In this study, we investigated various culture media to evaluate which one is the most appropriate for stimulating the proliferation while maintaining cell morphology and function of HCEnCs derived from old donors (age >65 years). All experiments were performed on paired research-grade donor corneas, divided for the conditions under investigation in order to minimize the inter-donor variability. Cell morphology as well as expression of specific markers were assessed at both mRNA (CD166, SLC4A11, ATP1A1, COL8A1, α-SMA, CD44, COL1A1, CDKN2A, LAP2A and LAP2B) and protein (ZO-1, α-SMA, Ki67 and LAP2) levels. Results obtained showed how the Dual Media formulation maintained the hexagonal phenotype more efficiently than Single Medium, but cell size gradually increased with passages. In contrast, the Single Medium provided a higher proliferation rate and a prolonged in vitro expansion but acquired an elongated morphology. To summarize, Single medium and Dual media preserve morphology and functional phenotype of HCEnCs from old donor corneas at low passages while maintenance of the same cell features at high passages remains an active area of research. The new insights revealed within this work become particularly relevant considering that the elderly population a) is the main target of corneal endothelial therapy, b) represents the majority of corneal donors. Therefore, the proper expansion of HCEnCs from old donors is essential to develop novel personalised therapeutic strategies and reduce requirement of human corneal tissues globally.Corneal endothelial dysfunction is a major indication for corneal transplantation. However, a global shortage of donor corneal tissues and risks associated with corneal surgeries have prompted exploration of alternative options, including tissue-engineered grafts or cell injection therapy. Nonetheless, these approaches require a controlled culture of primary human corneal endothelial cells (HCEnCs). Although HCEnCs established from young donors are generally more proliferative and maintain a better phenotype, corneas from old donors are more frequently accessible from eye banks due to a lower corneal endothelial cell count than the necessary threshold required for transplantation. In this study, we investigated various culture media to evaluate which one is the most appropriate for stimulating the proliferation while maintaining cell morphology and function of HCEnCs derived from old donors (age >65 years). All experiments were performed on paired research-grade donor corneas, divided for the conditions under investigation in order to minimize the inter-donor variability. Cell morphology as well as expression of specific markers were assessed at both mRNA (CD166, SLC4A11, ATP1A1, COL8A1, α-SMA, CD44, COL1A1, CDKN2A, LAP2A and LAP2B) and protein (ZO-1, α-SMA, Ki67 and LAP2) levels. Results obtained showed how the Dual Media formulation maintained the hexagonal phenotype more efficiently than Single Medium, but cell size gradually increased with passages. In contrast, the Single Medium provided a higher proliferation rate and a prolonged in vitro expansion but acquired an elongated morphology. To summarize, Single medium and Dual media preserve morphology and functional phenotype of HCEnCs from old donor corneas at low passages while maintenance of the same cell features at high passages remains an active area of research. The new insights revealed within this work become particularly relevant considering that the elderly population a) is the main target of corneal endothelial therapy, b) represents the majority of corneal donors. Therefore, the proper expansion of HCEnCs from old donors is essential to develop novel personalised therapeutic strategies and reduce requirement of human corneal tissues globally
Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium
No full textNanoneedles 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
No full textThe 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 TGF beta 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
Comparison between MERRA-2 and CWEEDS for use in pavement mechanistic-empirical design in Canada
No full textTo improve the climate resiliency of existing and new pavements, it is important to carry out pavement designs using continuous climate records at high temporal frequencies. Over the years, significant research efforts have been dedicated to obtain high-quality climatic data for pavement design including the latest adoption of the Modern-Era Retrospective analysis for Research and Applications, version 2 (MERRA-2). The purpose of this study is to assess how MERRA-2 performs when compared to the Canadian Weather Energy and Engineering Datasets (CWEEDS), which provides hourly meteorological data for many parts of the country from various periods. In a first part, climate parameters at nine locations were directly compared to determine the correlation between two datasets. In a second part, long-term performances were simulated for typical flexible pavement to assess the relative impact of each climate scenario. As detailed in this paper, observed differences between MERRA-2 and CWEEDS indicate the need for further improvement of climate data quality and availability for designing resilient pavements in Canada.The presentation of the authors' names and (or) special characters in the title of the pdf file of the accepted manuscript may differ slightly from what is displayed on the item page. The information in the pdf file of the accepted manuscript reflects the original submission by the author
Regenerative Medicine of Epithelia: Lessons From the Past and Future Goals
No full textThis article explores examples of successful and unsuccessful regenerative medicine on human epithelia. To evaluate the applications of the first regenerated tissues, the analysis of the past successes and failures addresses some pending issues and lay the groundwork for developing new therapies. Research should still be encouraged to fill the gap between pathologies, clinical applications and what regenerative medicine can attain with current knowledge
13C-breath tests in the study of microsomial liver function
No full textConventional liver tests can be used to estimate a mixture of injury and function but none of these may be regarded as a reliable marker either to quantify functional hepatic reserve or to reflect life-threatening complications of acute and chronic liver diseases. To overcome this limit, many dynamic tests have been developed in order to evaluate the "hepatic functional mass". Among these tests we can include breath tests with 13C-labeled substrates undergoing different metabolic pathways. As concerning the evaluation of microsomal function, two main categories of breath tests have been developed based on the limiting step in the different substrates metabolism. The first group include aminopyrine, caffeine and diazepam, all substrates with a metabolism independent from hepatic blood flow and dependent almost exclusively from the enzymatic activity of different cytochromes P450. The other group is composed of substrates with flow dependent metabolism like methacetin, phenacetin, erythromycin. The aim of this review is to describe the clinical applications of microsomal liver breath tests in different hepatic diseases
High dosage rifaximin for the treatment of small intestinal bacterial overgrowth.
No full textBACKGROUND: Rifaximin is a broad spectrum non-absorbable antibiotic used for treatment of small intestinal bacterial overgrowth. Doses of 1200 mg/day showed a decontamination rate of 60% with low side-effects incidence. AIMS: To assess efficacy, safety and tolerability of rifaximin 1600 mg with respect to 1200 mg/day for small intestinal bacterial overgrowth treatment. METHODS: Eighty consecutive small intestinal bacterial overgrowth patients were enrolled. Diagnosis of small intestinal bacterial overgrowth based the clinical history and positivity to H(2)/CH(4) glucose breath test. Patients were randomized in two 7-day treatment groups: rifaximin 1600 mg (group 1); rifaximin 1200 mg (group 2). Glucose breath test was reassessed 1 month after. Compliance and side-effect incidence were also evaluated. RESULTS: One drop-out was observed in group 1 and two in group 2. Glucose breath test normalization rate was significantly higher in group 1 with respect to group 2 both in intention-to-treat (80% vs. 58%; P < 0.05) and per protocol analysis (82% vs. 61%; P < 0.05). No significant differences in patient compliance and incidence of side effects were found between groups. CONCLUSIONS: Rifaximin 1600 mg/day showed a significantly higher efficacy for small intestinal bacterial overgrowth treatment with respect to 1200 mg with similar compliance and side-effect profile
A high-quality hourly, daily and monthly solar irradiance dataset in China during 1981-2014 based on MERRA-2 Reanalysis products
No full textA high-quality hourly, daily and monthly solar irradiance dataset in China during 1981-2014 based on MERRA-2 Reanalysis products
Wenmin Qin1, Lunche Wang1, Ming Zhang1, Lan Feng1, Yulong Zhong1, Qiqi Zhu1，Hejin Fang1, Hong Cai1, Chao Yang2
1Hubei Key Laboratory of Critical Zone Evolution, School of Geography and Information Engineering, China University of Geosciences, Wuhan 430074, China;
2 Institute of Geodesy and Geophysics, Chinese Academy of Sciences, Wuhan 430077, China;
Corresponding author: Wenmin Qin, Hubei Key Laboratory of Critical Zone Evolution, School of Geography and Information Engineering, China University of Geosciences, Lumo road 388, Hongshan District, Wuhan 430074, China; Tel.: +86 18163315797; E-mail: [email protected].
Abstract: Solar irradiance (SI) is the main driving factor contributing to climate change and energy balance between the land and atmosphere. High-quality records of global solar irradiance (GHI), direct normal irradiance (DNI) and diffuse solar irradiance (DIF) are of vital importance for solar applications, but the solar radiation observations are sparse around the world. As an alternative, numerous SI reanalysis data in grid format have been developed in regional and global scales. Among them, the MERRA-2 (Modern-Era Retrospective Analysis for Research and Applications, version 2) products could provide high quality SI records with acceptable accuracy and long temporal ranges. This study attempted to improve the accuracy of GHI records derived from MERRA-2 products, and to generate grid DNI and DIF datasets for all-sky conditions over mainland China during 1981-2014, based on the REST2 model and cloud transmittance estimates combining sunshine observations. The results indicate that the estimated GHI values (GHInew) show higher agreements with GHI measurement at 17 CMA (China meteorological administrations) stations than that for the GHI records derived from MERRA-2 products (MERRA-2 GHI). Then, grid GHI, DNI and DIF datasets (0.50° (lat) *0.625° (lon)) throughout China were constructed. The results indicated that the MERRA-2 GHI records may overestimate the GHI values over mainland China. The grid GHI, DNI and DIF dataset generated in this study can assist in numerous solar studies and applications
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