Novel inulin-based mucoadhesive micelles loaded with corticosteroids as potential transcorneal permeation enhancers

In this work a new copolymer of inulin (INU) derivatized with ethylendiamine (EDA) and retinoic acid (RA), named INU-EDA-RA, was synthetized, characterized and employed to produce micelles as carriers for topical administration of corticosteroids for the potential treatment of diseases of posterior eye segment. Spectroscopic analysis confirmed a molar derivatization degree of 11.30 and 4.30% in EDA and RA, respectively. INU-EDA-RA micelles are capable of strong mucoadhesive interactions which result time-independent and stable over time but concentration depending. Moreover micelles are able to encapsulate efficiently from 3 to 13% (w/w) of lipophilic drugs, as dexamethasone, triamcinolone …

The protease complex consisting of dipeptidyl peptidase IV and seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices

Abstract Dipeptidyl peptidase IV (DPP4/CD26) and seprase/fibroblast activation protein α are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH2 terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existen…

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.…

A 3D‑scaffold of PLLA induces the morphological differentiation and migration of primary astrocytes and promotes the production of extracellular vesicles

The present study analyzed the ability of primary rat astrocytes to colonize a porous scaffold, mimicking the reticular structure of the brain parenchyma extracellular matrix, as well as their ability to grow, survive and differentiate on the scaffold. Scaffolds were prepared using poly-L-lactic acid (PLLA) via thermally-induced phase separation. Firstly, the present study studied the effects of scaffold morphology on the growth of astrocytes, evaluating their capability to colonize. Specifically, two different morphologies were tested, which were obtained by changing the polymer concentration in the starting solution. The structures were characterized by scanning electron microscopy, and a…

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…

Computational modeling and experimental characterization of fluid dynamics in micro-CT scanned scaffolds within a multiple-sample airlift perfusion bioreactor

The perfusion of flow during cell culture induces cell proliferation and enhances cellular activity. Perfusion bioreactors offer a controlled dynamic environment for reliable in vitro applications in the tissue engineering field. In this work, to evaluate the effects of the operating parameters of a custom-made bioreactor, numerical simulations were performed to solve the fluid velocity profile inside the bioreactor containing multi-grid support that allows allocating of multiple seeded scaffolds at the same time. The perfusion system exhibited a uniform distribution of liquid velocities within the regions, suitable for cell growth on seeded scaffolds. The effects of the porous microstructu…

Multi-Functional Nanogels for Tumor Targeting and Redox-Sensitive Drug and siRNA Delivery

(1) Background: A new family of nanosystems able to discern between normal and tumor cells and to release a therapeutic agent in controlled way were synthetized by e-beam irradiation. This technique permits to obtain biocompatible, sterile, carboxyl-functionalized polyvinylpyrrolidone (PVP-co-acrylic acid) nanogels (NGs); (2) Methods: Here, we performed a targeting strategy based on the recognition of over-expressed proteins on tumor cells, like the folate receptor. The selective targeting was demonstrated by co-culture studies and flow cytometry analysis, using folate conjugated NGs. Moreover, nanoparticles were conjugated to a chemotherapeutic drug or to a pro-apoptotic siRNA through a gl…

Could beta-catenin regulate the expression of proteolitic enzymes during angiogenesis?

During angiogenesis several modifications occurs at endothelial cell plasma membrane level as: MT-MMPs and serine integral membrane proteases (SIMPs) over-expression. Moreover, when endothelial cells during angiogenesis acquired a mesenchymal phenotype the cell-cell interactions mediate by cadherins are lost and β-catenin, a stabilizer of interaction occurring between cadherins and cytoscheleton, can translocate to the nucleus where acts as transcription factor in association to TCF/LEF. Our experiments were focalized to the expression/activation of proteolytic enzymes when cell-cell contacts are perturbed, by mechanical or site specific perturbations. We have analyzed the mRNA, proteins an…

Co-Deposition and Characterization of Hydroxyapatite-Chitosan and Hydroxyapatite-Polyvinylacetate Coatings on 304 SS for Biomedical Devices

During the last decades, biomaterials have been deeply studied to perform and improve coatings for biomedical devices. Metallic materials, especially in the orthopedic field, represent the most common material used for different type of devices thanks to their good mechanical properties. Nevertheless, low/medium resistance to corrosion and low osteointegration ability characterizes these materials. To overcome these problems, the use of biocoatings on metals substrate is largely diffused. In fact, biocoatings have a key role to confer biocompatibility properties, to inhibit corrosion and thus improve the lifetime of implanted devices. In this work, the attention was focused on Hydroxyapatit…

PLLA biodegradable scaffolds for angiogenesis via Diffusion Induced Phase Separation (DIPS)

A critical obstacle in tissue engineering is the inability to maintain large masses of living cells upon transfer from the in vitro culture conditions into the host in vivo. Capillaries, and the vascular system, are required to supply essential nutrients, including oxygen, remove waste products and provide a biochemical communication “highway”. For this reason it is mandatory to manufacture an implantable structure where the process of vessel formation – the angiogenesis – can take place. In this work PLLA scaffolds for vascular tissue engineering were produced by dip-coating via Diffusion Induced Phase Separation (DIPS) technique. The scaffolds, with a vessel-like shape, were obtained by p…

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 COMPOSITE PLLA SCAFFOLD FOR REGENERATION OF COMPLEX TISSUES

A composite biodegradable scaffold incorporating an integrated network of synthetic blood vessels was designed and prepared, in line with the requirements of a scaffold effectively supporting the regeneration of highly vascularized tissues. In other words, this composite scaffold should allow the regeneration of complex injured tissue (e.g. dermis) and, at the same time, favour the development of a vascular network on its inner, i.e. a 3D polymeric scaffolds embedding synthetic blood vessel-like structures for nutrient supply and metabolite removal. PLLA assures a high degree of biocompatibility and a low level of inflammation response upon implantation, while the embedded tubular vessel-li…

Additive Manufacturing of Multi‐Scale Porous Soft Tissue Implants That Encourage Vascularization and Tissue Ingrowth

Medical devices, such as silicone-based prostheses designed for soft tissue implantation, often induce a suboptimal foreign-body response which results in a hardened avascular fibrotic capsule around the device, often leading to patient discomfort or implant failure. Here, it is proposed that additive manufacturing techniques can be used to deposit durable coatings with multiscale porosity on soft tissue implant surfaces to promote optimal tissue integration. Specifically, the “liquid rope coil effect”, is exploited via direct ink writing, to create a controlled macro open-pore architecture, including over highly curved surfaces, while adapting atomizing spray deposition of a silicone ink t…

Polyactide Biodegradable Scaffolds for Tissue Engineering Applications: Phase Separation-Based Techniques

One of the most reliable techniques for the preparation of biodegradable scaffolds suitable for tissue engineering applications (e.g. regeneration of wounded/damaged tissues) is based on liquid/liquid phase separation of ternary solvent/antisolvent/polymer solutions. In particular, two phase separation protocols are examined here: Thermally Induced Phase Separation (TIPS) and Diffusion Induces Phase Separation (DIPS). According to the former protocol, a thermodynamically stable polymeric ternary solution is brought below its metastability/instability point (spinodal/binodal curve) by quench in a cooling medium: under opportune conditions, a foam-like structure is formed by nucleation and 3-…

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 …

Continuous Microfiber Wire Mandrel‐Less Biofabrication for Soft Tissue Engineering Applications

Suture materials are the most common bioimplants in surgical and clinical practice, playing a crucial role in wound healing and tendon and ligament repair. Despite the assortment available on the market, sutures are still affected by significant disadvantages, including failure in mimicking the mechanical properties of the tissue, excessive fibrosis, and inflammation. This study introduces a mandrel-less electrodeposition apparatus to fabricate continuous microfiber wires of indefinite length. The mandrel-less biofabrication produces wires, potentially used as medical fibers, with different microfiber bundles, that imitate the hierarchical organization of native tissues, and tailored mechan…

Titolo: Novel proteases from marine organisms with potential interest in restoration procedure

Poly-left-lactic acid tubular scaffolds via diffusion induced phase separation: Control of morphology

n this work, tubular poly-left-lactic acid scaffolds for vascular tissue engineering applications were produced by an innovative two-step method. The scaffolds were obtained by performing a dip-coating around a nylon fiber, followed by a diffusion induced phase separation process. Morphological analysis revealed that the internal lumen of the as-obtained scaffold is equal to the diameter of the fiber utilized; the internal surface is homogeneous with micropores 1–2 μm large. Moreover, a porous open structure was detected across the thickness of the walls of the scaffold. An accurate analysis of the preparation process revealed that it is possible to tune up the morphology of the scaffold (w…

PLLA scaffolds with controlled architecture as potential microenvironment for in vitro tumor model

Abstract The "microenvironment" where a tumor develops plays a fundamental role in determining its progression, the onset of metastasis and, eventually, its resistance to therapies. Tumor cells can be considered more or less invasive depending both on the nature of the cells and on the site where they are located. Commonly adopted laboratory culture protocols for the investigation of tumor cells take usually place on standard two-dimensional supports. However, such cultures do not allow for reproduction of the biophysical properties of the tumor’s microenvironment, thus causing the cells to lose most of their relevant characteristics. In this work MDA-MB 231 breast cancer cells were cultiva…

Differential expression of proteolytic enzymes during epithelial-mesenchymal transaction of endothelial cells.

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

Seprase-DPPIV Association and Prolyl Peptidase and Gelatinase Activities of the Protease Complex

Effect of E. coli biofilm formation and removal on passive films on AISI 316L during fermentation processes

Abstract 316L coupons were sanitized in hot water vapour inducing iron enrichment in passive films. Coupons were then immersed in a pilot fed-batch fermenter in presence of E. coli. Sanitization causes iron enrichment in passive films. Fermentation causes the growth of biofilm on the SS, constituted by bacteria embedded in an extracellular polymeric substance. During fermentation SS open circuit potential is very negative due to low oxygen concentration on its surface, while the chelating action of siderophores induces chromium enrichment in the passive film. Disinfection in NaClO for 30 min allows removal of biofilm and formation of a protective passive film.

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…

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesi…

Proteolytic enzymes expression during epithelial/mesenchymal transaction of endothelial cells

Shed membrane vesicles and selective localization of gelatinases and MMP-9/TIMP-1 complexes.

Galvanic Deposition of Hydroxyapatite/Chitosan/Collagen Coatings on 304 Stainless Steel

The galvanic deposition method was used to deposit Hydroxyapatite/Chitosan/Collagen coatings on 304 stainless steel. Galvanic deposition is an alternative and valid way to fabricate bio-coatings with high biocompatibility and good anticorrosion properties. Physical-chemical characterizations were carried out to investigate chemical composition and morphology of the samples. Coatings consist of a mixture of calcium phosphate (Brushite and Hydroxyapatite) with chitosan and collagen. Corrosion tests were performed in the simulated body fluid (SBF) after different aging times. Results show that, in comparison with bare 304 stainless steel, coating shifts corrosion potential to anodic values and…

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…

Spatial distribution of two maternal messengers encoding two sea urchin cell surface proteins

We have localized, byin situ hybridization, two messengers, called bepl and bep4 (butanol extracted proteins), coding for cell surface proteins, involved in cell interaction, in sections of eggs of the sea urchinP. lividus. These maternal RNAs are spatially distributed in a gradient with a maximum at one pole of the egg. These results represent the first demonstration of the existence of a gradient of informational molecules in the sea urchin egg,i.e., the biological material for which the gradient theory was first proposed.

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…

Physical and biological properties of electrospun poly( d , l ‐lactide)/nanoclay and poly( d , l ‐lactide)/nanosilica nanofibrous scaffold for bone tissue engineering

Electrospun scaffolds exhibiting high physical performances with the ability to support cell attachment and proliferation are attracting more and more scientific interest for tissue engineering applications. The inclusion of inorganic nanoparticles such as nanosilica and nanoclay into electrospun biopolymeric matrices can meet these challenging requirements. The silica and clay incorporation into polymeric nanofibers has been reported to enhance and improve the mechanical properties as well as the osteogenic properties of the scaffolds. In this work, for the first time, the physical and biological properties of polylactic acid (PLA) electrospun mats filled with different concentrations of n…

Physico-chemical and mechanical characterization of in-situ forming xyloglucan gels incorporating a growth factor to promote cartilage reconstruction

Abstract The development of growth factors is very promising in the field of tissue regeneration but specifically designed formulations have to be developed in order to enable such new biological entities (NBEs). In particular, the range of therapeutic concentrations is usually very low compared to other active proteins and the confinement in the target site can be of crucial importance. In-situ forming scaffolds are very promising solutions for minimally invasive intervention in cartilage reconstruction and targeting of NBEs. In this work injectable, in-situ forming gels of a temperature responsive partially degalactosylated xyloglucan (Deg-XG) incorporating the growth factor FGF-18 are fo…

Vesicles mediated cell-cell and cell-matrix interactions

Sterilization of macroscopic poly(l-lactic acid) porous scaffolds withdense carbon dioxide: Investigation of the spatial penetration of thetreatment and of its effect on the properties of the matrix

Abstract In this work the sterilization with dense carbon dioxide of poly( l -lactic acid) (PLLA) porous scaffolds intended for tissue engineering applications was investigated with the main objective of confirming the three-dimensional efficacy of the treatment and of analysing the scaffold properties after CO2 treatment. For this purpose the scaffold was contaminated with a conventional bacterium (Escherichia coli) and with spores (Streptomyces coelicolor), a species more fascinating and difficult to inactivate. Contamination was performed in such a way to soak the whole matrix with bacteria and spores. The effect of pressure and treatment time on the efficacy of the sterilization was eva…

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)

An acid extract from dissociation medium of sea urchin embryos, induces mesenchyme differentiation

Abstract When material extracted by 1 M acetic acid from the dissociation medium of sea urchin embryos is added at low concentrations to isolated primary mesenchyme cells, it induces skeletogenesis. The same material added to dissociated blastula cells, or to embryos at the blastula stage, stimulates skeleton formation and pigment cell differentiation. On dissociated cells, it also increases cell reaggregation, thymidine incorporation and survival. On embryos, it induces exogastrulation and appearence of extraembryonic pigment cells. The activity of the extract is resistant to raised temperatures and partially to tryptic digestion but is abolished by trypsin treatment followed by heating. T…

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…

Clustering of type-II transmembrane serine proteases (TTSPs) and matrix metalloproteases (MMPs) in the cell surface and shed membrane vesicles of active endothelial cells

Comparative analyses of endothelial and tumoral cells cultured in 2D and 3D type-1 collagen fibril gels

Use of recombinant collagenases class I and II in optimization of cell purification for tissue engineering applications

Sterilization of three-dimensional tissue engineering scaffolds by supercritical carbon dioxide

Medical devices, implants or patient-care equipment that will come into intimate contact with a patient must be effectively decontaminated to prevent infection or disease transmission. Non-sterile devices have significant ramifications for patient morbidity and mortality and two processes must be undertaken in order to make an item acceptable for use or implantation: cleaning and disinfection (or sterilization) [1]. Sterilization is the destruction of living organisms, and must be done without damaging the material surface and without compromising the bulk material strength or biocompatibility of implantable device. Common sterilization processes include steam autoclaving, gamma irradiation…

Radiation-tailored xyloglucan-doxorubicin nanoparticles for cancer therapy

Polysaccharides are long chains of monosaccharides linked by glycosidic bonds. They are widely utilized in biomedical applications, for tissue engineering and wound management, and as excipients of pharmaceutical formulations. In cancer therapy, the development of nanoscale drug delivery systems aims at addressing issues related to the low efficacy of chemotherapeutics and protein drugs due to poor solubility and stability, and to off-target effects that severely affect patients’ body conditions. Biocompatibility, availability of functional groups, amenability of chemical derivatisation and multifunctional conjugation with drugs and targeting ligands make polysaccharide nanoparticles intere…

3D Collagen matrix improves islets of Langerhans differentiation and function

The importance of preserve cell-cell contact and cell-matrix interaction are both fundamental for maintain the differentiation state. We use both standardized method of isolation and culture in 3D matrix which improves islet cell survival and function for more than one week in colture.

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

Roles of molecules involved in epithelial/mesenchymal transition during angiogenesis

Formation of vessels requires "epithelial-mesenchymal" transition of endothelial cells, with several modifications at the level of endothelial cell plasma membranes. These processes are associated with redistribution of cell-cell and cell-substrate adhesion molecules, cross talk between external ECM and internal cytoskeleton through focal adhesion molecules and the expression of several proteolytic enzymes, including matrix metalloproteases and serine proteases. These enzymes with their degradative action on ECM components, generate molecules acting as activators and/or inhibitors of angiogenesis. The purpose of this review is to provide an overview of the molecules involved in epithelial-m…

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.

Identification and characterization of a constitutive HSP75 in sea urchin embryos.

Abstract An antiserum against a hsp of the 70-kDa family was prepared, by means of a fusion protein, which was able to detect a constitutive 75-kDa hsc in the sea urchinP. lividus.This hsc was present both during oogenesis and at all developmental stages. A two-dimensional electrophoresis has revealed four isolectric forms of this 75-kDa hsc. The amino acid sequence of the fragment used to prepare the anti-hsp70 antibodies revealed a 43% identity with the corresponding part of sea urchin sperm receptor, and in mature eggs a brighter immunofluorescence was seen all around the cell cortex where the receptor for sea urchin sperm is localized. In oocytes the hsp75 was localized in the cytoplasm…

Role of MT1-MMP in cell-cell contact formation of endothelial cells

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 …

Engineering approaches in siRNA delivery.

siRNAs are very potent drug molecules, able to silence genes involved in pathologies development. siRNAs have virtually an unlimited therapeutic potential, particularly for the treatment of inflammatory diseases. However, their use in clinical practice is limited because of their unfavorable properties to interact and not to degrade in physiological environments. In particular they are large macromolecules, negatively charged, which undergo rapid degradation by plasmatic enzymes, are subject to fast renal clearance/hepatic sequestration, and can hardly cross cellular membranes. These aspects seriously impair siRNAs as therapeutics. As in all the other fields of science, siRNAs management ca…

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…

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

PHEA-Dox nanoparticles as pH-sensitive model for drug delivery in tumour treatment.

PHEA-Dox nanoparticles as pH-sensitive model for drug delivery in tumour treatment. S. Camporaa, G. Adamoa, N. Maurob, C. Scialabbab, M. Licciardib, G. Giammonab and G. Ghersia. aDipartmento di “Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche” (STEBICEF), Università di Palermo, Viale delle Scienze Ed. 16, 90128 Palermo, Italy. bLaboratory of Biocompatible Polymers, Dipartmento di “Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche” (STEBICEF), Università di Palermo, Via Archirafi, 32 90123 Palermo, Italy. Classical chemotherapeutic applications, using molecules such as doxorubicin (Dox), have side effects due to an unspecific action. In order to obtain a specific release of…

COLLAGENASI RICOMBINANTI DI C. HISTOLYTICUM E METODO PER LA LORO PRODUZIONE.

La presente descrizione si riferisce alla produzione di collagenasi ricombinanti, in particolare è qui descritto un metodo per la produzione di collagenasi Col ricombinante di Clostridium histolyticum caratterizzato da una resa maggiore a circa 140mg/l di coltura di detta collagenasi in forma biologicamente attiva, collagenasi prodotte mediante detto metodo, composizioni che le comprendono e loro uso.

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…

Functionalization of nanoparticles in specific targeting and mechanism release

The development of various nanotechnologies have provided a new field of research, which allows the manipulation of molecular components of matter and covers a vast array of nanodevices. The “smart” multifunctional nanostructures should work as customizable, targeted drug-delivery vehicles capable of carrying large doses of therapeutic agents into malignant cells. Some nanomedical approaches are based on the use of functionalized nanoparticles (NPs), not only to reduce toxicity and side effects of drugs but, also in potential the biological barriers crossing on, such as: the blood–brain barrier, different cellular compartments, including the nucleus. Currently, many materials are used for n…

Expression of proteolitic enzymes in induced angiogenesis

Angiogenesis (growth of new blood vessels) plays a key role in tissue repair, such as in cancer progression (Folkman 1995. New Engl. J. Med. 333, 1757-63). At the beginning of the process is observed the matrix degradation and endothelial cells (ECs) migration inside to the connective tissue in proximity of vessel walls. During angiogenesis several modifications occurs at plasma membrane level; a redistribution of cell-cell and cell-matrix adhesion molecules (Bazzoni et al 1999), such as an increased expression of several proteolytic enzymes, including MMPs and serine proteases, as plasminogen activators and SIMPs, were observed (Ghersi et al. 2006. Cancer Res. 66, 4652-61; Ghersi 2008 Fron…

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…

Polyaminoacid–doxorubicin prodrug micelles as highly selective therapeutics for targeted cancer therapy

An amphiphilic copolymer carrying high-dose doxorubicin (21% on a weight basis), PHEA–EDA–P,C–Doxo, was prepared by coupling doxorubicin with a biocompatible polyaminoacid through a pH-sensitive spacer. Additional derivatization with 4-pentynoic acid endows it with self-assembling properties by means of π–π stacking. These micelles can be triggered to promptly release drug in lysosomes (∼40% in 12 h) through pH-dependent micelle hydrolysis after uptake. In vitro tests on co-cultures of cancer (MDA-MB 231) and normal (HB-2) breast cells proved that the conjugate was selectively internalized into the former rather than normal cells, exploiting the caveolae-dependent endocytosis pathway, expla…

Vibrio Proteases for Biomedical Applications: Modulating the Proteolytic Secretome of V. alginolyticus and V. parahaemolyticus for Improved Enzymes Production

Proteolytic enzymes are of great interest for biotechnological purposes, and their large-scale production, as well as the discovery of strains producing new molecules, is a relevant issue. Collagenases are employed for biomedical and pharmaceutical purposes. The high specificity of collagenase-based preparations toward the substrate strongly relies on the enzyme purity. However, the overall activity may depend on the cooperation with other proteases, the presence of which may be essential for the overall enzymatic activity, but potentially harmful for cells and tissues. Vibrios produce some of the most promising bacterial proteases (including collagenases), and their exo-proteome includes s…

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

Self-organized environment-sensitive inulin–doxorubicin conjugate with a selective cytotoxic effect towards cancer cells

An inulin-based random copolymer bearing high dose doxorubicin (18.45% on a weight basis), INU-EDA-P, C-DOXO, was prepared by coupling doxorubicin with inulin though a citraconylamide bridge used as a pH sensitive spacer. A further conjugation with pentynoic acid via an amidic bond led to the hydrophobization of the copolymer which allows the acquisition of a self-assembling ability at low concentration (0.33 mg mL(-1)) combining both Pi-Pi stacking and London interactions. Drug release studies were carried out at different pH demonstrating a remarkable pH dependency, where the maximum release rate was observed at pH mimicking cancer tissue and lysosomal environments. Besides, by measuring …

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 …

Tissue Dissociation and Primary Cells Isolation Using Recombinant Collagenases Class I and II

Collagenases class I (Col G) and class II (Col H) currently available for tissue dissociation are produced from Clostridium histolyticum (human pathogen) strains. In the processes of extraction of the cells from the tissue, combined activity of both classes of enzymes is required. CI and CII are complementary in degrading collagen. ABIEL recently produced the collagenase class I and II using the recombinant DNA technologies (PCT WO 2011/073925 A9). The enzymes were produced in E. coli and purified by affinity chromatography. The method of production adopted allows absolute control of the final composition of these enzymes, as well as their stability, purity, activity, absence of toxicity an…

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…

PLLA biodegradable scaffolds for Vascular Tissue Engineering (VTE) applications via dip drawing and Diffusion Induced Phase Separation (DIPS)

C. HYSTOLITICUM RECOMBINANT COLLAGENASES AND METHOD FOR THE MANUFACTURE THEREOF

The present invention relates to the production of recombinant collagenases, and in particular describes a method for the production of recombinant clostridium histolyticum collagenases Col characterized by a yield higher than approximately 140 mg/l of culture of said collagenases in soluble, biologically active form, collagenases produced by this method, compositions comprising these collagenases and the use thereof.

CLUSTERING OF TYPE-II TRANSMEMBRANE SERINE PROTEASES (TTSPS) AND MATRIX METALLOPROTEINASES (MMPS) IN THE CELL SURFACE AND SHED MEMBRANE VESICLES OF ACTIVE ENDOTHELIAL CELLS.

Heparin functionalized polyaspartamide/polyester scaffold for potential blood vessel regeneration

Core-shell PLA/Kef hybrid scaffolds for skin tissue engineering applications prepared by direct kefiran coating on PLA electrospun fibers optimized via air-plasma treatment

Abstract Over the recent years, there is a growing interest in electrospun hybrid scaffolds composed of synthetic and natural polymers that can support cell attachment and proliferation. In this work, the physical and biological properties of polylactic acid (PLA) electrospun mats coated with kefiran (Kef) were evaluated. Gravimetric, spectroscopic (FTIR-ATR) and morphological investigations via scanning electron microscopy confirmed the effective formation of a thin kefiran layer wrapped on the PLA fibers with an easy-tunable thickness. Air plasma pre-treatment carried out on PLA (P-PLA) affected both the morphology and the crystallinity of Kef coating as confirmed by differential scanning…

Radiation sculpured nanogels as platform for novel therapeutic systems

GENERATION OF PREVASCULARIZED PLLA BIODEGRADABLE SCAFFOLDS BY DIP DRAWING AND DIFFUSION INDUCED PHASE SEPARATION (DIPS).

A critical obstacle in tissue engineering is to develop a massive structure of living cells upon transfer from the in vitro culture conditions into the host in vivo. A vascular network is required to supply essential nutrients, including oxygen, remove metabolic waste products and provide a biochemical communication “highway”. For these reasons to build an implantable structure in which vessel formation (angiogenesis) take place is mandatory. PLLA scaffolds usable in vascular tissue engineering were generated by dip-coating via Diffusion Induced Phase Separation (DIPS) technique. The scaffolds, with a vessel-like shape, were obtained by performing a DIPS process around a nylon fibre whose d…

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…

Water-borne e-beam crosslinked nanogels as nanomaterials platform for drug delivery

Comprehensive analysis of a Vibrio parahaemolyticus strain extracellular serine protease VpSP37

Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain be…

Procedimento di preparazione di scaffolds polimerici preangiogenizzati.

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…

Proteolytic enzymes clustered in specialized plasma membrane domains generate permissive substrates for endothelial cell migration

FOLATE-DECORATED E-BEAM CROSSLINKED NANOGELS TO ENHANCE UPTAKE BY TUMOR CELLS

E-Beam Crosslinked Polymeric and Bio-Hybrid Nanocarriers for Drug Delivery and Bioimaging

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

Muscle degeneration in neuramindase 1 deficient mice results from infiltration of the muscle fibers by expanded connective tissue

AbstractNeuraminidase 1 (NEU1) regulates the catabolism of sialoglycoconjugates in lysosomes. Congenital NEU1 deficiency in children is the basis of sialidosis, a severe neurosomatic disorder in which patients experience a broad spectrum of clinical manifestations varying in the age of onset and severity. Osteoskeletal deformities and muscle hypotonia have been described in patients with sialidosis. Here we present the first comprehensive analysis of the skeletal muscle pathology associated with loss of Neu1 function in mice. In this animal model, skeletal muscles showed an expansion of the epimysial and perimysial spaces, associated with proliferation of fibroblast-like cells and abnormal …

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

Mucoadhesive micelles based on inulin derivative for ocular release of corticosteroids

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…

Supercritical carbon dioxide induces sterilization of PLLA scaffolds contaminated by E. coli.

The common sterilization techniques are based on physical processes that involve, for example, the use of autoclaves or systems to radiation such as γ-rays that can cause a structural change of the polymer treated. Therefore, the use of supercritical carbon dioxide (scCO2) is an excellent alternative, as it does not induce any variation of biomaterials treated (Perrut M., 2012). It's a good candidate because is readily available at low cost, non-toxic and non-flammable, it has an easily accessible critical point (7.38 MPa and 304.2 K) and excellent transport properties and wettability (White A. et al., 2005). We report the development of a supercritical CO2 based process capable of steriliz…

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…

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 …

Proteolytic enzymes in membrane domains of endothelial cells cultured in type-I collagen 3D gels

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 …

Double Flow Bioreactor for In Vitro Test of Drug Delivery.

In this work, double-structured polymeric scaffolds were produced, and a double flow bioreactor was designed and set up in order to create a novel system to carry out advanced in vitro drug delivery tests. The scaffolds, consisting of a cylindrical porous matrix, are able to host cells, thus mimicking a three-dimensional tumor mass: moreover, a “pseudo-vascular” structure was embedded into the matrix, with the aim of allowing a flow circulation. The structure that emulates a blood vessel is a porous tubular-shaped scaffold prepared by Diffusion Induced Phase Separation (DIPS), with an internal lumen of 2 mm and a wall thickness of 200 micrometers. The as-prepared vessel was incorporated…

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

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…

Tailoring PLLA scaffolds for tissue engineering applications: Morphologies for 2D and 3D cell cultures

PLLA scaffold suitable for dermis regeneration were realized by Thermally Induced Phase Separation (TIPS) starting from a ternary solution PLLA/dioxane/water. The reconstruction of a complex tissues as the dermis implies the use of different cellular types (coculture), with different growth behaviour (2D vs. 3D). The scaffolds present an homogeneous porous surface to allow the keratinocytes 2D growth and a porous internal structure for the fibroblasts 3D growth. Our results show that the porosity of the surface can be tuned by changing the chemical nature of the sample holder (aluminium, teflon, polypropylene). A large variety of morphologies, in terms of average pore size and interconnecti…

Isolation of Adult Rat Cardiomyocytes Using Recombinant Collagenases

Direct isolation of primary cells from tissues and organs allows for the maintenance of important cell characteristics and properties for in vitro studies and a plethora of biomedical applications. Dissociation of cells from the organ of interest is possible due to the enzymatic activity of collagenases. The choice and the dose of these enzymes is the critical step to obtain the maximum number of cells with intact structure and function. In this contest, Abiel collagenases class I (Col G) and class II (Col H) were synthesised using recombinant DNA technologies and their ability to degrade collagen in cell isolation from different tissues was tested. Examples of cells isolated with these enz…

Meccanismi non convenzionali di secrezione dell’FGF-2

Espressione ed associazione di enzimi proteolitici durante la transizione epitelio/mesenchima in cellule endoteliali.

Tuning of the Mg Alloy AZ31 Anodizing Process for Biodegradable Implants

Coatings were grown on the AZ31 Mg alloy by a hard anodizing process in the hot glycerol phosphate-containing electrolyte. Anodizing conditions were optimized, maximizing corrosion resistance estimated by impedance measurements carried out in Hank's solution at 37 °C. A post anodizing annealing treatment (350 °C for 24 h) allowed us to further enhance the corrosion resistance of the coatings mainly containing magnesium phosphate according to energy-dispersive X-ray spectroscopy and Raman analyses. Gravimetric measurements revealed a hydrogen evolution rate within the limits acceptable for application of AZ31 in biomedical devices. In vitro tests demonstrated that the coatings are biocompati…

PLLA-fibrin scaffolds for Vascular Tissue Engineering

Activation of EDTA-resistant Gelatinases in Malignant Human Tumors

Abstract Among the many proteases associated with human cancer, seprase or fibroblast activation protein α, a type II transmembrane glycoprotein, has two types of EDTA-resistant protease activities: dipeptidyl peptidase and a 170-kDa gelatinase activity. To test if activation of gelatinases associated with seprase could be involved in malignant tumors, we used a mammalian expression system to generate a soluble recombinant seprase (r-seprase). In the presence of putative EDTA-sensitive activators, r-seprase was converted into 70- to 50-kDa shortened forms of seprase (s-seprase), which exhibited a 7-fold increase in gelatinase activity, whereas levels of dipeptidyl peptidase activity remaine…

The Foreign Body Response to an Implantable Therapeutic Reservoir in a Diabetic Rodent Model

Advancements in type 1 diabetes mellitus treatments have vastly improved in recent years. The move toward a bioartificial pancreas and other fully implantable systems could help restore patient's glycemic control. However, the long-term success of implantable medical devices is often hindered by the foreign body response. Fibrous encapsulation "walls off" the implant to the surrounding tissue, impairing its functionality. In this study we aim to examine how streptozotocin-induced diabetes affects fibrous capsule formation and composition surrounding implantable drug delivery devices following subcutaneous implantation in a rodent model. After 2 weeks of implantation, the fibrous capsule sur…

Patent number RM2009A000073: Procedimento di preparazione di scaffold polimerici preangiogenizzati

Classe proposta A61F - Filtri da inserire nei vasi sanguigni; protesi; apparecchi ortopedici e curativi per contraccezione; fomentazione; trattamento o protezione degli occhi e delle orecchie; bendaggi, garze per medicazioni e assorbenti; necessario per il pronto soccorso

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…

PROCEDIMENTO DI PREPARAZIONE DI SCAFFOLD POLIMERICI PREANGIOGENIZZATI

MDA-MB-231 and 8701BC breast cancer lines promote the migration and invasiveness of ECV304 cells on 2D and 3D type-I collagen matrix

Tumor angiogenesis is a multiphasic process, having the extracellular matrix remodeling as critical step. Different classes of proteolytic enzymes in matrix digestion/remodeling are involved. The role of lytic enzymes and their activation mode have not been completely elucidated. Herein, the crosstalk between endothelia and tumor cells, by realization of bi- and three-dimensional endothelial and breast cancer cells co-cultures, were studied in vitro. Particularly, the effects of two tumor conditioned media (TCM) were assessed about endothelial proliferation, migration, and invasiveness. An increase in expression of pro-MMP9 was detected when endothelial cells were cultured in the presence o…

Preparation, characterization and in vitro test of composites poly-lactic acid/hydroxyapatite scaffolds for bone tissue engineering.

Abstract In this work, the possibility to produce composite Poly-L-lactic acid (PLLA)/Hydroxyapatite (HA) porous scaffolds via Thermally Induced Phase Separation (TIPS) for bone tissue engineering applications was investigated. Several PLLA/HA wt/wt ratios (95/5, 90/10, 70/30, 50/50, 34/66) were tested and the as-obtained scaffolds were characterized via Scanning Electron Microscopy, Wide Angle X-Ray Diffraction, Thermogravimetric analysis, Gas Pycnometry, Differential Scanning Calorimetry and mechanical compression test. Morphological analysis revealed an open structure with interconnected pores and HA particles embedded in the polymer matrix. Finally, cell cultures were carried out into t…

Clustering and Proteolytic enzymes complexes formation in specialized plasma membra domains of endothelial cells. Role in angiogenesis.

Identification of proteolytic enzymes from Eriphia verrucosa and Palinurus elephas capable to degrade gliadin

In small intestinal disease, coeliac sprue, proline-rich gluten peptides from wheat, rye and barley are relatively resistant to gastrointestinal digestion, and therefore remain in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. Since most serine endopeptidases are unable to hydrolyse proline residues, proline specific proteases may be therapeutic keys in digestive diseases. Partial hydrolysis reduces the risk of allergenic sensitization while total hydrolysis ensures the elimination of the allergenicity of whey protein (Villad´oniga and others 2007). Kimoto and others (1998) reported that 18-, 31-, 37- and 58-kDa wheat allergens were recognized by the …

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…

Microscopic evidence of the primary astrocytes' morphological differentiation and migration inside porous Poly-L-lactic acid 3D‑scaffolds

Tissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro and/or in vivo tissues with morphological and functional features similar to the biological tissue of the human body [1]. In the attempt to construct suitable tissue models, a critical step is the setting of 3D scaffolds that mimic the supportive structures of a natural extracellular matrix microenvironment into which cells are normally embedded. In this context, the generation of 3D cultures of brain cells is of particular interest. For instance, the poly L‐lactic acid (PLLA) polymer is wildly used because of its biocompatible and biodegradable potential; the PLLA scaffold topography simulates the n…

Overespression of MT1-MMP modified the integrity of endothelial cell contact

Assessing the Effects of VEGF Releasing Microspheres on the Angiogenic and Foreign Body Response to a 3D Printed Silicone-Based Macroencapsulation Device.

Macroencapsulation systems have been developed to improve islet cell transplantation but can induce a foreign body response (FBR). The development of neovascularization adjacent to the device is vital for the survival of encapsulated islets and is a limitation for long-term device success. Previously we developed additive manufactured multi-scale porosity implants, which demonstrated a 2.5-fold increase in tissue vascularity and integration surrounding the implant when compared to a non-textured implant. In parallel to this, we have developed poly(ε-caprolactone-PEG-ε-caprolactone)-b-poly(L-lactide) multiblock copolymer microspheres containing VEGF, which exhibited continued release of bioa…

Pore size graded scaffold for selective cellular permeation

E-beam crosslinked nanogels conjugated with monoclonal antibodies in targeting strategies

Abstract Poly(N-vinyl pyrrolidone)-based-nanogels (NGs), produced by e-beam irradiation, are conjugated with monoclonal antibodies (mAb) for active targeting purposes. The uptake of immuno-functionalized nanogels is tested in an endothelial cell line, ECV304, using confocal and epifluorescence microscopy. Intracellular localization studies reveal a faster uptake of the immuno-nanogel conjugate with respect to the ‘bare’ nanogel. The specific internalization pathway of these immuno-nanogels is clarified by selective endocytosis inhibition experiments, flow cytometry and confocal microscopy. Active targeting ability is also verified by conjugating a monoclonal antibody which recognizes the αv…

The use of Diffusion Induced Phase Separation (DIPS) technique for the preparation of biodegradable scaffolds for angiogenesis

Matrix metalloproteases, serine proteases and their inhibitors in angiogenesis

Functionalized Poly(N-isopropylacrylamide)-Based Microgels in Tumor Targeting and Drug Delivery

Over the past several decades, the development of engineered small particles as targeted and drug delivery systems (TDDS) has received great attention thanks to the possibility to overcome the limitations of classical cancer chemotherapy, including targeting incapability, nonspecific action and, consequently, systemic toxicity. Thus, this research aims at using a novel design of Poly(N-isopropylacrylamide) p(NIPAM)-based microgels to specifically target cancer cells and avoid the healthy ones, which is expected to decrease or eliminate the side effects of chemotherapeutic drugs. Smart NIPAM-based microgels were functionalized with acrylic acid and coupled to folic acid (FA), targeting the f…

Recent developments and applications of smart nanoparticles in biomedicine

Abstract Over the last decades, nanotechnology applied in medicine (nanomedicine) has sparked great interest from the scientific community, thanks to the possibility to engineer nanostructured materials, including nanoparticles (NPs), for a specific application. Their small size confers them unique properties because they are subject to physical laws in the middle between classical and quantum physics. This review is proposed to explain better how to design a specific NP and clarify the relationship between the type, size, and shape of NPs and the specific medical applications. NPs are classified into inorganic (metallic NPs, quantum dots, carbon-based nanostructures, mesoporous silica NPs)…

Removal of old protein layers from surfaces of works of art by new enzymes

The aim of the present project is to set up bio-cleaning protocols in order to easily remove altered protein layers (e.g. animal glues) by enzymatic proteins (proteases). Protein molecules were isolated from marine invertebrate organisms, characterized by size exclusion highpressure liquid chromatography (Waters: SEC-HPLC, BioSuite 250 to 10㎛ SEC 7.5 x 300 mm) and their gelatinase activity analyzed by zymography on polyacrilamide gel. The remarkably proteolytic activity and the reaction temperature range of these enzymes, 4° to 37℃, made it possible to hypothesize their use in bioremediation projects. Tests were performed on protein coating found on some polychrome wooden artifacts exposed …

CLUSTERING OF PROTEOLYTIC ENZYMES ON SPECIALIZED PLASMA MEMBRANE DOMAINS OF ENDOTHELIAL CELLS. ROLE IN ANGIOGENESIS

Vascular Endothelial Growth Factor-Releasing Microspheres Based on Poly(ε-Caprolactone-PEG-ε-Caprolactone)-b-Poly(L-Lactide) Multiblock Copolymers Incorporated in a Three-Dimensional Printed Poly(Dimethylsiloxane) Cell Macroencapsulation Device

Pancreatic islet transplantation is a promising advanced therapy that has been used to treat patients suffering from diabetes type 1. Traditionally, pancreatic islets are infused via the portal vein, which is subsequently intended to engraft in the liver. Severe immunosuppressive treatments are necessary, however, to prevent rejection of the transplanted islets. Novel approaches therefore have focused on encapsulation of the islets in biomaterial implants which can protect the islets and offer an organ-like environment. Vascularization of the device’s surface is a prerequisite for the survival and proper func- tioning of transplanted pancreatic islets. We are pursuing a prevascularization s…

Deposition and characterization of Hydroxyapatite-Chitosan coatings on 304 SS for biomedical devices

During the last years biomaterials have been largely investigated in order to perform and improve biomedical devices. As regards orthopedic field, the most common equipment used (such as implants, bone grafts or screws) are constituted by metallic materials (steel and titanium alloys), characterized by low/medium resistance to corrosion and low osteointegration ability. Furthermore, these factors could produce local inflammations of the tissues surrounding the implants, increasing kinetics of corrosion phenomena. Scientific community has focused the attention on biocoatings interposed between metal and aggressive environment in order to inhibit corrosion. Furthermore, these coatings are abl…

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…