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Realizzazione di un patch miocardico bioingegnerizzato mediante impianto di cellule staminali mesenchimali su matrici polimeriche tridimensionali
No full textIntroduzione
Dopo un danno il tessuto miocardico non è in grado di replicarsi e viene
sostituito da tessuto fibroso che comporta un successivo rimodellamento e dilatazione
del ventricolo stesso che porterà verso l’ insufficienza cardiaca.
Negli ultimi anni, particolare attenzione è stata rivolta alla terapia cellulare per
il trattamento del miocardio ischemico e dell’insufficienza cardiaca postinfartuale.
Inizialmente questa terapia prevedeva l’iniezione di sospensioni cellulari
direttamente a livello miocardico o direttamente nel circolo coronarico nel tentativo
di evidenziare un miglioramento della funzione contrattile della zona lesa.
Da differenti ricerche si è evidenziato come tra i differenti elementi cellulari
staminali, le cellule staminali mesenchimali siano quelle più indicate per la terapia
cardiaca.
Si tratta di elementi pluripotenti in grado di differenziarsi in cellule endoteliali
e cardiomiociti.
Tuttavia se impiantate direttamente sul tessuto miocardico leso non sono in
grado di sopravvivere per la mancanza di ossigeno e di substrati.
Una strategia promettente per risolvere questo problema sembrerebbe poter venire
dall’ingegneria tissutale.
Essa infatti utilizza biomateriali realizzati con polimeri naturali o di sintesi in grado
di consentire il corretto sviluppo e la corretta differenziazione degli elementi cellulari
inizialmente in vitro ed il loro successivo impianto in vivo.
Obiettivi
Muovendo dalla conoscenza dei patch bio-ingegnerizzati e dall’utilizzo di
cellule staminali, l’obiettivo di questo studio è quello di realizzare un patch
miocardico bio-ingegnerizzato mediante impianto di cellule staminali mesenchimali
su matrici polimeriche tridimensionali realizzate mediante tecnica di elettrofilatura.
Materiali e metodi
Durante la prima fase del protocollo di lavoro, è stata realizzata una matrice
polimerica di supporto che consentisse la crescita e la differenziazione degli elementi
cellulari staminali.
A questo scopo è stato utilizzato come polimero il Polycaprolactone con un
peso molecolare medio di 80kDa (Sigma Aldrich) disciolto in soluzione 3:1 con
difenilformammide (DMF; Sigma Aldrich).
La soluzione così realizzata è stata posta in una pompa siringa (Harvard
Apparatus, Hollison, MA) ed iniettata attraverso un ago con diametro medio 0,84mm
collegato con un generatore di corrente ad alto voltaggio (Gamma High Voltage
Research, Ormond Beach, FL)
Ad un voltaggio di 24 kV la soluzione fluida iniettata è stata polimerizzata per
formare una rete manometrica i cui prodotti di degradazione sono stati valutati sia in
vivo che in vitro.
Si è successivamente provveduto, all’isolamento di cellule staminali
mesenchimali da midollo osseo di ratto, alla loro caratterizzazione, amplificazione in
coltura e valutazione delle unità formanti colonie (CFU).
La loro successiva differenziazione in cardiomiociti è stata caratterizzata
attraverso reazioni di immunoistochimica.
Le colture cellulari ottenute sono state quindi inoculate sulle matrici polimeriche
precedentemente realizzate.
Infine sono state monitorate le caratteristiche morfologiche, conseguenti a tale
ancoraggio mediante microscopia elettronica a scansione (SEM).
Risultati
Lo scaffold ottenuto ha un diametro medio delle fibre di circa 300nm, appare di
buona qualità e non presenta alcun “bead”.
La caratterizzazione fisica ha mostrato come lo spessore delle fibre influenzi la
sua degradazione in vivo ed in vitro.
Le cellule mesenchimali mostrano all’analisi immunocitochimica l’espressione
di alcuni markers di cardiomiociti.
L’analisi al SEM ha evidenziato una buona adesione ed espansione degli
elementi cellulari allo scaffold.Background
Following injury, the myocardium cannot replicate and is replaced with fibrous tissue,
eventually inducing ventricular remodeling and dilation, and ultimately leading to heart failure.
Therefore, how to restore the injured myocardium and ameliorate cardiac dysfunction is a
major issue.
Mesenchymal stem cells (MSC) are pluripotent progenitor cells able to differentiate into
vascular endothelial cells and cardiomyocytes.
Many researchers have demonstrated that MSC implantation induces myocardial
regeneration and improves cardiac function through myogenesis and angiogenesis.
Even though implanted cells can survive and differentiate into cardiomyocytes in the injured
myocardium, because of the lack of oxygen and adequate nutrients, the infarcted myocardium is not
an environment conducive to cell survival.
To solve these problems, cardiac tissue engineering has become a promising strategy.
It is a relatively new discipine that intends to overcome the obstacles to prolonging patients’ life
after myocardial infarction, is continuously improving.
It comprises a biomaterial based ‘vehicle’, either a porous scaffold or dense patch, made of either
natural or synthetic polymeric materials, to aid transportation of cells into the diseased region in the
heart.
Biomaterials suggested for this specific tissue-engineering application need to have particular
mechanical properties matching those of native myocardium, so that the delivered donor cells
integrate and remain intact in vivo.
Objective
Our objective was to realize an in vitro bioengineered graft developed by culturing
mesenchymal stem cells (MSC) from neonatal rats on electrospun, nanofibrous and biodegradable
scaffold.
Electrostatic fiber spinning, or electrospinning, is a process that produces ultrafine fibers in
the form of a non-woven mesh through the action of a high electric field.
The resulting fiber diameters are in the submicron range, and are unattainable by other fiber
spinning techniques.
Due to the small fiber diameters, the meshes have a high specific surface area conjectured to be
beneficial for cell attachment and proliferation.
Materials and methods
Polycaprolactone (PCL) with an average molecular weight of 80 kDa (Sigma Aldrich) was
dissolved in a 3:1 mixture of chloroform and dipheniformammide (DMF; Sigma Aldrich) to obtain
a 8 wt% solution.
The polymer solution was delivered with a syringe pump (Harvard Apparatus, Holliston,
MA) to a stainless steel capillary (inner diameter=0.84 mm; Aisi 304) connected to a high voltage
power supply (Gamma High Voltage Research, Ormond Beach, FL).
At a voltage of 24 kV, a fluid jet was ejected from the capillary, and a non-woven fibrous
mesh was created. Degradation rate of the scaffolds was also evaluated in vitro (with Ringer
solution) and in vivo (in rats).
Bone marrow cells were obtained by flushing femurs and tibias with phosphate-buffered
saline (PBS). The obtained bone marrow cells were cultured in Dulbecco’s Modified Eagle’s
Medium (Gibco BRL, USA) supplemented with 10% foetal bovine serum (FBS) and 50 IU/mL
penicillin–streptomycin (Gibco BRL) and was kept at 37°C in humidified air with 5% CO2.
The non-adherent cells were discarded with the media changes, which were performed every
3 days. When the cultures became nearly confluent, adherent cells were detached with trypsin–
EDTA and subsequently passaged. After four to five passages, cells were harvested and then used
for cell implantation.
Then, immunocitochemical analysis of some specific cardiac differentiative markers was
assessed. After that, the cell-polymer constructs were cultured with cardiac supplements for up to 4
weeks.
Finally Scanning electron microscopy (SEM) examinations were performed at 1, 3 and 7
days after the seeding.
Results
The obtained PCL scaffold had an average diameter of 300nm with good quality and did not
show any “bead”.
The fiber diameter also significantly influenced both in vitro degradation and in vivo
biodegradation rates.
Mesenchymal stem cells shown at the immunocitochemical analysis de novo expression of
some cardiomyocite markers (alpha sarcomeric Actin, tropomyosin).
Penetration of cells and abundant extracellular matrix were observed in the cell-polymer
constructs after 1 week.
SEM showed that the surfaces of the cell-polymer constructs were partially covered with
cells after one week.
Conclusion
We have established a versatile in vitro system for bioengineered graft combining a
nanofibrous electrospun biodegradable scaffold and MSC derived cardiomyocites .
In the future this graft may be used on an animal model to investigate the effect of
mesenchymal stem cells on myocardial infarction
Hemodynamic evaluation of ICU patients undergoing partial ventilatory assistance: role of asynchrony
Full text linkEditorial comment to intraoperative haemodynamic monitoring and management of adults having non-cardiac surgery: guidelines of the German society of Anaesthesiology and Intensive care medicine in collaboration with the German Association of the Scientific medical societies
No full textIn this issue, the German Society of Anaesthesiology and
Intensive Care Medicine in collaboration with the German
Association of the Scientific Medical Societies provide
guidelines on intraoperative haemodynamic monitoring and
management of adults having non-cardiac surgery.
The document brings together a broad variety of data
regarding perioperative haemodynamic management.
Although a wide range of studies have been covered, no
systematic literature search was conducted, and the methodology
for appraising the evidence and formulating the recommendations
is not described. Therefore, these guidelines
should be viewed as a consensus of expert opinions, with
questions identified by anaesthesiologists from three German
university hospitals
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Evolution of a hillslope by rock avalanches: insights from analog models
No full textRock avalanches are among the most hazardous processes on hillslopes because of high velocity, great dimensions, and long run-out distance. For this reason, understanding the dynamics and factors of rock avalanches and their role in hillslope evolution is crucial. Studies evidenced that occurrence and evolution of these phenomena are influenced by lithological, structural, and climatic factors. Statistical analysis on natural cases demonstrated correlations between slope geometry and rock avalanche volume. Most of the studies referred to experimental tests which represent powerful tools to understand these landslides. Many models focused on the mechanism leading to high velocity and long run-out, but few studies discuss the role of rock avalanches in the evolution of a bedrock hillslope. The influence of slope geometry and physical properties of the substratum on the dynamics of rock avalanches is poorly constrained. We present results from analog models of a hillslope evolving by base level lowering. We tested several slope widths and two analog materials. The experimental apparatus allowed for checking the mass of mobilized material at each step and for taking a 3D scan of the whole surface. Our results, coupled with a statistical analysis, indicated that hillslope evolution is influenced by the material internal friction and by the friction with box walls (i.e., valley walls) when the slope is narrow. Widening the slope, the influence of lateral friction disappears, confirming observations in other models and nature. These results represent a new contribution to understand the dynamics of rock avalanches on bedrock hillslopes
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