Poly(ethylenglycol) mimics adhesive capability of the ECM treatment on 3D polylactide-based scaffolds to study in vitro human hepatocarcinoma processes

EVALUATION OF STABILITY AND ENZYMATIC ACTIVITIES OF PROTEOLYTIC ENZYMES USED IN PANCREATIC ISLET TRANSPLANTATION

In pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (Coll-G and Coll-H). In a well defined composition Coll-G/Coll-H together enzymes working on hydrophobic amminoacid, the neutral protease (Dispase) or the thermolysin (Thermostable Neutral Protease), are used in Langerhans islets purification. By electrophoresis and gelatin zymography approaches, in combination to densitometry quantitative valuation we have compared in composition, stability and autodigestion processes C. hystoliticum collagenases, Neutral protease and Thermolysin from two different producers, Roche and Serva.…

Analysis of intracellular FGF-2 trafficking in Sk-Hep1 cells.

Bi-layer PCL/PLA scaffold prepared by melt for interface tissue engineering

The development of porous multilayer devices allow controlling chemical, physical and mechanical properties by tuning the properties of each single layer. For instance, this feature is of main concern for the production of porous devices designed to regenerate diseased zones at the interface of tissue presenting intrinsic anisotropic structures that gradually change from one tissue to another. In this context, synthetic biodegradable polymers commonly used biomedical applications include polylactic acid (PLA) and polycaprolactone (PCL). In this work, a novel bi-layered multiphasic scaffold (BLS) is presented. It is composed by a PLA-layer presenting pore size in the range of 90-110 μm while…

Preparation of three-layered porous PLA/PEG scaffold: relationship between morphology, mechanical behavior and cell permeability.

Interface tissue engineering (ITE) is used to repair or regenerate interface living tissue such as for instance bone and cartilage. This kind of tissues present natural different properties from a biological and mechanical point of view. With the aim to imitating the natural gradient occurring in the bone-cartilage tissue, several technologies and methods have been proposed over recent years in order to develop polymeric functionally graded scaffolds (FGS). In this study three-layered scaffolds with a pore size gradient were developed by melt mixing polylactic acid (PLA) and two water-soluble porogen agents: sodium chloride (NaCl) and polyethylene glycol (PEG). Pore dimensions were controll…

A poly-L-lactic acid/ collagen/glycosaminoglycan matrix for tissue engineering applications

Adhesion of tissue cells to biomaterials is a prerequisite of paramount importance for the effectiveness of a tissue engineering construct (cell and scaffolds). Functionalization of polymeric scaffolds with organic polymers, such as collagen or proteoglycans, is a promising approach in order to improve the cytocompatibility. As a matter of fact, organic polymers, isolated directly from the extracellular matrix, contain a multitude of surface ligand (fibronectin, laminin, vitronectin) and arginine–glycine–aspartic acid-containing peptides that promote cell adhesion. In tissue engineering, the combination of organic and synthetic polymers gives rise to scaffolds characterized simultaneously …

Analysis of FGF-2 traffiking in Sk-Hep1 cells

The role of poly(ethylenglycol) molecular weight on viability and cell adhesion in polylactide based scaffolds

A new method to valued efficiency of enzyme blend for pancreas tissue digestion.

One of the best successful example of cell therapy is represented by islet transplantation since the '90. However islet isolation methods are not completely standardized yet. More than half of isolation procedures failed to isolate adequate islets for transplantation, due to variable pancreas condition, and to unpredictable enzymatic blend efficiency. Enzymes used for pancreas digestion are purified from Clostridium histolyticum; these enzymes has a broad substrate specificity and potent collagenolytic activity compared to vertebrate collagenases. However, a major obstacle in human islet isolation successful is due to the variability in composition and concentration of the collagenases used…

The effect of processing and molecular weight of poly(ethylenglycol) of polylactide-based scaffolds on viability and cell adhesion of SkHep1 cells

Large-scale manufacturing of radiation sculptured therapeutic nanogels

Nanogels (NGs), or small particles formed by physically or chemically crosslinked polymer networks, represent a niche in the development of “smart” nanoparticles for drug delivery and diagnostics. Yet, they offerunique advantages over other systems, including a large and flexible surface for multivalent bio-conjugation; an internal 3D aqueous environment for incorporation and protection of (bio)molecular drugs; the possibility to entrap light-activemolecules, metal or mineral nanoparticles for imaging or phototherapeutic purposes; stimuli-responsiveness to achieve temporal and/or site control of the release function and biocompatibility. The availability of inexpensive, robust and versatile…

Bio-affinity of MC3T3-E1 osteoblastic cells with polymeric scaffold in Poly L-Lactid Acid (PLLA) for bone substitution.

Damage to an organ or tissue remains a problem despite advances in medical technology. Available treatments include organs transplantation, surgical reconstruction such as mechanical devices. However, all these methodologies have several contraindications. In recent years, tissue engineering methods is considered as means to replace diseased or damaged organs. For these kind of application, the choice of scaffolding material is crucial to the success of the technique. In the function of the application, the synthetic scaffolds should meet several criteria, including: good biocompatibility, sufficient mechanical properties, and adequate biodegradability. The aim of our study was to evaluate …

Integration of PCL and PLA in a monolithic porous scaffold for interface tissue engineering.

A novel bi-layered multiphasic scaffold (BLS) have been fabricated for the first time by combining melt mixing, compression molding and particulate leaching. One layer has been composed by polylactic acid (PLA) presenting pore size in the range of 90-110µm while the other layer has been made of polycaprolactone (PCL) with pores ranging from 5 to 40µm. The different chemo-physical properties of the two biopolymers combined with the tunable pore architecture permitted to realize monolithic functionally graded scaffolds engineered to be potentially used for interface tissues regenerations. BLS have been characterized from a morphological and a mechanical point of view. In particular, mechanica…

Localization of neutral ceramidase and sphingosine kinase1 in hepatoma and breast carcinoma cells and their shed vesicles

Vesicles mediated cell-cell and cell-matrix interactions

Fed-batch pre-industrial production and purification of a consensus tetratricopeptide repeat (CTPR) scaffold as a container for Fluorescent Proteins (FPs)

White light-emitting diodes (WLEDs) are big news in the field of lighting, however, current production processes are still very expensive or based on unsustainable inorganic metals such as inorganic phosphorus. The EU-funded ARTIBLED project aims to produce low-cost and high-efficiency Bio-hybrid light-emitting diodes (Bio-HLEDs). This can be achieved using artificially synthesized fluorescent proteins linked in biological scaffolds like the packaging to obtain LED for lighting applications containing a biogenic phosphor. This study aims to optimize the protein scaffold CTPR10 production process to obtain a high number of scaffolds with a good purity level for Bio-HLEDs construction. Differ…

Viabilità e adesione cellulare di cellule di epatocarcinoma umano su supporti 3D a base di poli(acido D,L lattico)

Mathematical and numerical modeling of an airlift perfusion bioreactor for tissue engineering applications

The Tissue Engineering (TE) strategy is widely focused on the development of perfusion bioreactors to promote the production of three-dimensional (3D) functional tissues. To optimize tissue production, it is worth investigating the engineering parameters of a bioreactor system for identifying a beneficial range of operation variables. Mathematical and numerical modeling of a perfusion bioreactor is capable to provide relevant insights into the fluid flow and nutrients transport while predicting experimental data and exploring the impact of changing operating parameters, such as fluid velocities. In this work, the hydrodynamic parameters and oxygen transport were investigated using mathemati…

Radiation engineered functionalized nanogels as platform for biomedical nanocarriers

Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone …

Dexamethasone dipropionate loaded nanoparticles of α-elastin-g-PLGA for potential treatment of restenosis.

A graft copolymer of α-elastin with poly(lactic-co-glycolic) acid (PLGA) has been synthesized and successfully employed to produce nanoparticles. Exploiting the known biological activity of α-elastin to promote the maintenance of smooth muscle cells (SMCs) contractile phenotype and the antiproliferative effect of glucocorticoids, the aim of this research was to produce drug-loaded nanoparticles suitable for potential treatment of restenosis. In particular, nanoparticles of α-elastin-g-PLGA with a mean size of 200 nm have been produced and loaded with dexamethasone dipropionate (10% w/w), chosen as a model drug that inhibits proliferation of vascular SMCs. These nanoparticles are able to pro…

Comparative study on enzymatic activity and molecules stability of some commercial proteolytic enzymes used in pancreatic islet isolation.

In pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (COLL G and COLL H). They are used in a defined tissue dissociation enzyme (TDE) mixtures together neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). The TDE mixtures were in part responsible for the success of the Edmonton protocol; however, just to now, people working in islets purification found discrepancy in an application to another one. This variability in application see in the enzymatic blend composition the higher accused, such as the contamination from endotoxines due to extractive production…

SCAFFOLDS POLILATTIDICI PER APPLICAZIONI BIOMEDICHE

Melt Processed PCL/PEG Scaffold with Discrete Pore Size Gradient for Selective Cellular Infiltration

In order to develop scaffold able to mimic the natural gradient properties of tissues, biphasic and triphasic approaches were adopted. In this work, polycaprolactone/polyethylene glycol (PCL/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching without harmful solvents. The method permitted to develop three-layer scaffolds with high control of porosity and pore size. The mechanical properties were evaluated under physiological condition in order to simulate the real conditions of work. Co-culture of osteoblastic and fibroblastic mice cells were carried out in order to study the differential cellular permeation through the different pore size layers.

Minimalism in radiation synthesis of biomedical functional nanogels.

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly(N-vinyl pyrrolidone)-grafted-(aminopropyl)methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to …

ENGINEERED NANOGELS FOR BIOLOGICAL DRUGS TRANSPORT

Nowadays the nanocarrier strategy, aimed to rebuild the tumor therapy approach in a winning way, is growing very fast. This is in the direction to engender nanocarriers having synergistic action by the “biological drugs” transported, the capability to recognize specific targets and the molecular architecture that allows them to perform at the best their therapeutic functions. We are building engineered nanocarriers having different functions (control cell cycle, induce apoptosis, etc.) as well as the capability to recognize specific targets (tumor cells, angiogenic and/or sprouting endothelial cells); moreover, they must have also the ability to escape the host immunosystem, such that not t…

An active form of sphingosine kinase-1 is released in the extracellular medium as component of membrane vesicles shed by two human tumor cell line.

Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form i…

Scaffold con gradiente della dimensione dei pori per la migrazione selettiva di cellule eterotipiche

A Fibrillar Biodegradable Scaffold for Blood Vessels Tissue Engineering

In recent years there has been a growing interest for the development of tubular scaffolds employed to assist the replacement of small blood vessels. Materials designed for this purpose need to be biodegradable, have good mechanical properties and improve cell adhesion, proliferation and differentiation. To obtain biomaterials with these properties, electrospinning seems to be one of the most useful technique. Several biodegradable synthetic polymers or constituents of the extracellular matrix (ECM) have been electrospun showing optimal mechanical properties and biodegradability. However, such polymers are lacking in versatile chemical structure affordable to immobilize growth factors or ch…

Heparin functionalized polyaspartamide/polyester scaffold for potential blood vessel regeneration

An interesting issue in tissue engineering is the development of a biodegradable vascular graft able to substitute a blood vessel and to allow its complete regeneration. Here, we report a new scaffold potentially useful as a synthetic vascular graft, produced through the electrospinning of α,β-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-D,L-aspartamide-graft-polylactic acid (PHEA-EDA-g-PLA) in the presence of polycaprolactone (PCL). The scaffold degradation profile has been evaluated as well as the possibility to bind heparin to electrospun fibers, being it a known anticoagulant molecule able to bind growth factors. In vitro cell compatibility has been investigated using human vascular e…

Pre-vascularized PLLA scaffolds: A new approcah to develop deep tissue regeneration

Shedding of Membrane Vesicles Mediates Fibroblast Growth Factor-2 Release from Cells

Fibroblast growth factor-2 (FGF-2), a polypeptide with regulatory activity on cell growth and differentiation, lacks a conventional secretory signal sequence, and its mechanism of release from cells remains unclear. We characterized the role of extracellular vesicle shedding in FGF-2 release. Viable cells released membrane vesicles in the presence of serum. However, in serum-free medium vesicle shedding was dramatically down-regulated, and the cells did not release FGF-2 activity into their conditioned medium. Addition of serum to serum-starved cells rapidly induced intracellular FGF-2 clustering under the plasma membrane and into granules that colocalized with patches of the cell membrane …

Structure and biological evaluation of amino-functionalized PVP nanogels for fast cellular internalization

Abstract Aminopropyl methacrylamide chloride-graft-poly(N-vinyl pyrrolidone) nanogels (NGs) were designed to exploit the favorable properties of poly(N-vinyl pyrrolidone) (PVP), such as its high affinity to water and complexation ability of ions, molecules and macromolecules, with the availability of primary amino groups for bioconjugation reactions. A thorough structural characterization of the nanoscalar networks was performed via 1 H NMR and solid state 13 C NMR spectroscopies, while solid state NMR relaxation time measurements completed the NGs description in terms of polymer network density. Information on the hydrodynamic size and surface charge densities were sought via dynamic light…

Heparin functionalized polyaspartamide/polyester scaffold for potential blood vessel regeneration

Radiation sculpured nanogels as platform for novel therapeutic systems

Water-borne polymeric nanoparticles for glutathione-mediated intracellular delivery of anticancer drugs

A new family of water-borne, biocompatible and carboxyl- functionalized nanogels was developed for glutathione- mediated delivery of anticancer drugs. Poly(N-vinyl- pyrrolidone)-co-acrylic acid nanogels were generated by e- beam irradiation of aqueous solutions of a crosslinkable polymer, using industrial-type linear accelerators and set- ups. Nanogels physico-chemical properties and colloidal stability, in a wide pH range, were investigated. In vitro cell studies proved that the nanogels are fully biocompatible and able to quantitatively bypass cellular membrane. An anticancer drug, doxorubicin (DOX), was linked to the carboxyl groups of NGs through a spacer containing a disulphide cleavab…

Production of Injectable Marine Collagen-Based Hydrogel for the Maintenance of Differentiated Chondrocytes in Tissue Engineering Applications

Cartilage is an avascular tissue with limited ability of self-repair. The use of autologous chondrocyte transplants represent an effective strategy for cell regeneration

Chemical hydrogels based on a hyaluronic acid-graft-α-elastin derivative as potential scaffolds for tissue engineering

In this work hyaluronic acid (HA) functionalized with ethylenediamine (EDA) has been employed to graft α-elastin. In particular a HA-EDA derivative bearing 50 mol% of pendant amino groups has been successfully employed to produce the copolymer HA-EDA-g-α-elastin containing 32% w/w of protein. After grafting with α-elastin, remaining free amino groups reacted with ethylene glycol diglycidyl ether (EGDGE) for producing chemical hydrogels, proposed as scaffolds for tissue engineering. Swelling degree, resistance to chemical and enzymatic hydrolysis, as well as preliminary biological properties of HA-EDA-g-α-elastin/EGDGE scaffold have been evaluated and compared with a HA-EDA/EGDGE scaffold. T…

Dexamethasone Dipropionate Loaded Nanoparticles Of -Elastin-G-Plga For Potential Treatment Of Restenosis

Macromolecular engineering of hydrogels: from macro to nano scalar materials for different levels of biointeraction

Radiation engineered polymer nanocarriers for a new generation of nanoparticles based tumor specific radiopharmaceuticals

Electrospun PCL/GO-g-PEG structures: Processing-morphology-properties relationships

Abstract Polycaprolactone (PCL) biocomposite nanofiber scaffolds with different concentrations of graphene oxide (GO) and GO surface grafted with poly(ethylene glycol) (GO-g-PEG) were prepared by electrospinning. Morphological, mechanical as well as wettability characterizations of electrospun nanofibers were carried out. Results showed that the average diameter of PLA/GO electrospun nanofibers decreased upon increasing the filler content. Differently, the diameter increased while using GO-g-PEG. Both nanofillers enhanced the electrospun PCL hydrophilicity even if PCL/GO-g-PEG samples exhibited improved wettability. The Young moduli of the composite nanofiber mats were improved by adding GO…

3D polylactide-based scaffolds for studying human hepatocarcinoma processes in vitro

We evaluated the combination of leaching techniques and melt blending of polymers and particles for the preparation of highly interconnected three-dimensional polymeric porous scaffolds for in vitro studies of human hepatocarcinoma processes. More specifically, sodium chloride and poly(ethylene glycol) (PEG) were used as water-soluble porogens to form porous and solvent-free poly(L,D-lactide) (PLA)-based scaffolds. Several characterization techniques, including porosimetry, image analysis and thermogravimetry, were combined to improve the reliability of measurements and mapping of the size, distribution and microarchitecture of pores. We also investigated the effect of processing, in PLA-ba…

Large-scale radiation manufacturing of hierarchically assembled nanogels

Nanogels (NGs), or small particles formed by physically or chemically crosslinked polymer networks, represent a niche in the development of "smart" nanoparticles for drug delivery and diagnostics. They offer unique advantages over other systems, including a large and flexible surface for multivalent bio-conjugation; an internal 3D aqueous environment for incorporation and protection of (bio)molecular drugs; the possibility to entrap active metal or mineral cores for imaging or phototherapeutic purposes; stimuli-responsiveness to achieve temporal and/or site control of the release function and biocompatibility. The availability of inexpensive and robust preparation methodologies is at the ba…

Use of Modified 3D Scaffolds to Improve Cell Adhesion and Drive Desired Cell Responses.

In the most common approach of tissue engineering, a polymeric scaffold with a well-defined architecture has emerged as a promising platform for cells adhesion and guide their proliferation and differentiation into the desired tissue or organ. An ideal model for the regeneration should mimic clinical conditions of tissue injury, create a permissive microenvironment for diffusion of nutrients, gases and growth factors and permit angiogenesis. In this work, we used a 3D support made of synthetic resorbable polylactic acid (PLLA), which has considerable potential because of its well-known biocompatibility and biodegradability. One of the factors that influence cell adhesion to the scaffold is …

Oligonucleotide-decorated carboxyl-modified PVP nanogels: a new platform for diagnosis and gene-therapy.

Nanogels, or nanoscalar chemically crosslinked polymer networks, were generated by simultaneous crosslinking of Poly(N-vinyl pyrrolidone) (PVP) and grafting of acrylic acid (AA) monomer through e-beam irradiation of their aqueous solutions. The yield of the process is very high and no separation or complex purification procedures are required since no recourse to surfactants or organic solvents is made. The administered irradiation dose (40 kGy) imparts also sterility to the obtained nanogels in the form of aqueous dispersions. Control of particle size and size-distribution can be achieved by proper selection of the irradiation conditions. In particular, we obtained nanogel variants with av…

Recovery of Bioactive Compounds from Marine Organisms: Focus on the Future Perspectives for Pharmacological, Biomedical and Regenerative Medicine Applications of Marine Collagen

Marine environments cover more than 70% of the Earth’s surface and are among the richest and most complex ecosystems. In terms of biodiversity, the ocean represents an important source, still not widely exploited, of bioactive products derived from species of bacteria, plants, and animals. However, global warming, in combination with multiple anthropogenic practices, represents a serious environmental problem that has led to an increase in gelatinous zooplankton, a phenomenon referred to as jellyfish bloom. In recent years, the idea of “sustainable development” has emerged as one of the essential elements of green-economy initiatives; therefore, the marine environment has been re-evaluated …

Vesicle mediated cell-cell and cell-matrix interactions

A new method to value efficiency of enzyme blends for pancreatic tissue digestion.

Islet transplantation, since the 90’s, has been resulting to be one of the best successful example of human cell therapy. Nevertheless, islet isolation procedure is not completely standardized; in fact, more than fifty percent of islets procedures don’t arrive to their transplantation. This is due both to the variability of donor’s pancreas and to an unpredictable enzymatic blend efficiency. Enzymes used in pancreas digestion are extracted from Clostridium histolyticum bacteria and digest several substrates. In particular they have strong collagenolytic activity compared to vertebrate collagenases. However, several impediments persist in human islet isolation success probably due to the var…

Poly lactic acid based scaffolds as graft for small-diameter arterial replacement.

Vascular Tissue engineering (VTE) has emerged as a promising approach to develop blood vessel substitutes. Investigators have explored the use of arterial tissue cells combined with various types of natural and synthetic scaffolds to make tubular constructs in order to develop a functional small-diameter arterial replacement graft. The grafts must mimic the unique viscoelastic nature of an artery and be non-disruptive to blood ?ow. Moreover, after implantation, the scaffold must be gradually populated by cells and replaced by extra cellular matrix; with this respect, it is crucial that this replacement takes place with a well-defined timescale. In this work tubular scaffolds for VTE were pr…

PLLA/Fibrin Tubular Scaffold: A New Way for Reliable Endothelial Cell Seeding

In the present work a simple and quick technique for cell seeding into tubular-shaped scaffolds, which allows a homogeneous cell distribution, was tested. The poly-L-lactide (PLLA) scaffolds, prepared via diffusion induced phase separation (DIPS), were filled with fibrin gel in order to obtain a hybrid scaffold for Vascular Tissue Engineering applications. The formation of immobilized fibrin networks on the inner surface of the tubular scaffolds was observed using confocal microscopy and SEM. Morphological analysis of the so-obtained scaffold revealed that the fibrin gel is uniformly distributed on the internal surface of the scaffold, leading to an organized structure. Moreover a penetrati…

Preparation and characterization of 3D PLA porous scaffolds

Intracellular trafficking of endogenous fibroblast growth factor‐2

We have previously reported how the release of fibroblast growth factor-2 (FGF-2) is mediated by shed vesicles. In the present study, we address the question of how newly synthesized FGF-2 is targeted to the budding vesicles. Considering that in vitro cultured Sk-Hep1 hepatocarcinoma cells release FGF-2 and shed membrane vesicles only when cultured in the presence of serum, we added serum to starved cells and monitored intracellular movements of the growth factor. FGF-2 was targeted both to the cell periphery and to the nucleus and nucleolus. Movements toward the cell periphery were not influenced by drugs affecting microtubules, but were inhibited by cytocalasin B. Involvement of actin in …

Large-scale Radiation Manufacturing of Hierarchically Assembled Nanogels

Nanogels (NGs), or small particles formed by physically or chemically crosslinked polymer networks, represent a niche in the development of “smart” nanoparticles for drug delivery and diagnostics. They offer unique advantages over other systems, including a large and flexible surface for multivalent bio-conjugation; an internal 3D aqueous environment for incorporation and protection of (bio)molecular drugs; the possibility to entrap active metal or mineral cores for imaging or phototherapeutic purposes; stimuli-responsiveness to achieve temporal and/or site control of the release function and biocompatibility. The availability of inexpensive and robust preparation methodologies is at the ba…

Biocompatibility evaluation of PLLA scaffolds for vascular tissue engineering

Poly-L-lactic acid (PLLA), a hemicrystalline material, has been extensively studied in applications of engineered tissues, because it is biodegradable, absorbable and it supports cell attachment and growth. The purpose of this study is to evaluate tissue/ material interactions, neovascularization and the biocompatibility of PLLA by optical and scanning electron microscopy in a model of animal implant. PLLA porous disks were implanted into the dorsal subcutis of BALB/C mice for 1, 2, 3, and 8 weeks. The bioptic samples of excised PLLA and the surrounding tissue were evaluated for inflammatory response and tissue ingrowth. The samples were divided in two halves: one was fixed in neutral buffe…

Localisation of neutral ceramidase and sphingosine kinase 1 in hepatoma and breastr carcinoma cells and their shed vesicles

Meccanismi non convenzionali di secrezione dell’FGF-2

PLLA-fibrin scaffolds for Vascular Tissue Engineering

Polymeric Nanogels: Nanocarriers For Drug Delivery Application.

The application of nanotechnology to medicine has enabled the development of functionalised nanoparticles that, acting as carriers, can be loaded with drugs or genetic material to be released with a controlled mechanism in specific districts of the organism. Even though nanomedicine is a relative new branch of science, many type of nanocarriers for drug delivery have been developed over the past 30 years, such as liposomes, dendrimers, quantum dots, solid lipid nanoparticles, viruses and virus-like nanoparticles as well as a wide var iety of polymeric nanoparticles. Among these last, in our opinion, nanogels deserve a special attention. Nanogels are nanoscalar polymer networks, with a tende…

3d collagen hydrogel promotes in vitro langerhans islets vascularization through ad-mvfs angiogenic activity

Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33–955 μm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-…

Oligonucleotides-decorated-poly(N-vinyl pyrrolidone) nanogels for gene delivery

Pulsed electron-beam irradiation of a semi-dilute poly(N-vinyl pyrrolidone) (PVP) aqueous solution in the presence of acrylic acid has led to a carboxyl functionalized nanogel system. Nanoparticles hydrodynamic size and surface charge density, in water and as a function of pH, were investigated by dynamic light scattering and laser doppler velocimetry, respectively. Nanogels (NGs) were proved not to be cytotoxic at the cellular level. Indeed, they rapidly bypass the cellular membrane to accumulate in specific cell portions of the cytoplasm, in the perinuclear area. The availability of pendant carboxyl groups on the crosslinked PVP NGs core prompted us to attempt their decoration with a sing…

Pore size graded scaffold for selective cellular permeation

A new method to value efficiency of enzyme blends for pancreas tissue digestion

In pancreatic islets isolation for cell therapy the major enzymes used are obtained from Clostridium hystoliticum: class I and class II collagenases. They are used in a defined tissue dissociation enzyme mixture together with neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). However, just to now, people working in islets production found variable outcomes in isolation procedures mainly due to large variability in enzymatic blend composition and efficacy. Using electrophoresis and gelatin zymography approaches together with densitometry evaluation assays we compared the composition, stability and auto-digestion processes of C. hystoliticum collagenases, Neutral prote…

Tissue engineered vascular grafts based on poly-lactic acid blends

A great deal of research has been pursued in the last decade with the goal of developing blood vessel substitutes. Tissue engineering has emerged as a promising approach to address the shortcomings of current options. One of the major tasks in this research field is the possibility to tune the biodegradability of the implantable devices (scaffolds). After implantation, the scaffold has to be replaced by extra cellular matrix; with this respect, it is crucial that this replacement takes place with appropriate dynamics and a well-defined timescale. In this work tissue-engineered vascular graft were produced, utilizing several PLLA/PLA blends (100/0, 90/10, 75/25 wt/wt) in order to tune their …

Nanocarriers to targeted terapy: a goal for biomedical applications.

The reasons for the growing interest in biomaterials for application in the biomedical field are related to need to find alternative and more effective than conventional procedures used in drug treatment of several diseases. The traditional methods of drug delivery has limitations due to the drug’s toxicity in the body. The development of nanocarriers, which can be functionalized and loaded with drugs or genetic material and can be released into the body with a controlled mechanism, was a real goal in the field of biomedical applications. Synthetic hydrogels have found many applications especially in tissue-engineering scaffolds, as carriers for implantable and drug delivery devices. Hydrog…

CLUSTERING OF SPECIFIC MOLECULES IN SHED VESICLES.

In several tumor cell lines serum addition causes release of vesicles that bud from the cell surface and can be purified from cell conditionated media. These vesicles are known to be involved in cell migration and tumor progression. We recently demonstrated that FGF-2, a growth factor devoid of the classical signaling sequence, is secreted as a component of these vesicles. In order to analyze how molecules are clustered in shed vesicles we followed their intracellular movements by immunofluorescence techniques. The role of cytoskeletal components was analyzed using molecules such as paclitaxol, nocodazole, colchicin and cytochalasin which destabilize their organization. In the absence of se…