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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 mathematical equations and Computational Fluid Dynamics (CFD) analysis modeling on a novel perfusion bioreactor working as an airlift external loop. Mathematical models and numerical simulation were associated with experimental results at different configurations to evaluate the effect of the reactor parameters on its hydrodynamics. All predictions of gas holdup and liquid circulation velocity from the modeling were in good agreement with the experimental data. A Poly-L-lactic acid (PLLA) scaffold was produced by Thermally Induced Phase Separation (TIPS) and used as 3D support for biological tests in static and dynamic conditions to assess cell viability and proliferation inside the bioreactor. The whole set of fluid-dynamic and biological results showed that our bioreactor environment offers the benefits of an in vitro potential system to produce functional engineered tissues.

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-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF–islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation.

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 is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.

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 is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.

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, and it was further enhanced when GO was surface grafted with PEG, in particular at low concentrations. This suggests the improved dispersion of GO-g-PEG due to the surface grafting with PEG. Furthermore, in order to assess the potential applications of the electrospun nanofibers in tissue engineering, MC3T3-E1 cells were cultured on PCL and PCL/GO-g-PEG scaffolds, thus providing preliminary and comparative proliferation rate assays.

Cartilage is an avascular tissue with limited ability of self-repair. The use of autologous chondrocyte transplants represent an effective strategy for cell regeneration, however, preserving the differentiated state, which ensures the ability to regenerate damaged cartilage, represents the main challenge during in vitro culturing. For this purpose, we produced an injectable marine collagen-based hydrogel, by mixing native collagen from the jellyfish Rhizostoma pulmo with hydroxy-phenyl-propionic acid (HPA)-functionalized marine gelatin. This biocompatible hydrogel formulation, due to the ability of enzymatically reticulate using horseradish peroxidase (HPR) and H2O2, gives the possibility of trap cells inside, in the absence of cytotoxic effects, during the cross-linking process. Moreover, it enables the modulation of the hydrogel stiffness merely varying the concentration of H2O2 without changes in the concentration of polymer precursors. The maintenance of differentiated chondrocytes in culture was then evaluated via morphological analysis of cell phenotype, GAG production and cytoskeleton organization. Additionally, gene expression profiling of differentiation/dedifferentiation markers provided evidence for the promotion of the chondrogenic gene expression program. This, combined with the biochemical properties of marine collagen, represents a promising strategy for maintaining in vitro the cellular phenotype in the aim of the use of autologous chondrocytes in regenerative medicine practices.

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.

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 by the mechanical strength of synthetic materials and the biocompatibility of natural materials. In this work, porous poly-L-lactide acid scaffolds were functionalized with a synthetic collagen–glycosaminoglycans matrix in order to improve cell adhesion. For this purpose, a protocol for collagen–glycosaminoglycans conjugation into the pores of the scaffolds was set up. Moreover, an innovative protocol for the quantification of the conjugated glycosaminoglycans inside the scaffolds was created and adopted. The results have confirmed the effectiveness of the developed protocol: a collagen–glycosaminoglycans conjugation, with an efficiency of about 21% was obtained inside the scaffold. Moreover, SEM analysis highlighted the presence of the homogeneous synthetic matrix into the bulk of porous scaffolds. Finally, cell culture assays carried out by utilizing mouse embryonic fibroblasts showed that cell proliferation on poly-L-lactide acid-collagen–glycosaminoglycans scaffold is higher than on poly-L-lactide acid collagen scaffold (utilized as control). Therefore, it can be stated that the presence of glycosaminoglycans not only increases the mechanical strength of the matrix, thanks to their cross-linking effect, but also it seems to lead to a more significant cell growth. Overall, it is reasonable to state that the concerned protocol may be proposed as a reliable route to increase the rate of proliferation and in some cases to stimulate the cell differentiation in tissue engineering devices.

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 penetration of the gel into the porous wall of the scaffold was observed. The in vitro endothelial cell cultures carried out in the scaffolds highlighted a faster cell proliferation inside the hybrid scaffold with respect to simple PLLA scaffold. Results show that the fibrin/PLLA hybrid scaffold may be favourably used for Vascular Tissue Engineering applications.

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 endothelial cells (ECV 304) and the ability of heparinized PHEA-EDA-g-PLA/PCL scaffold to retain basic fibroblast growth factor has been evaluated in comparison with not heparinized sample.

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, mechanical tests have been carried out both in air and immersing the specimens in phosphate buffered saline (PBS) solution at 37°C, in order to evaluate the elastic modulus and the interlayer adhesion strength. Fibroblasts and osteoblasts have been cultured and co-cultured in order to investigate the cells permeation trough the different layers. The results indicate that the presented method is appropriate for the preparation of multiphasic porous scaffolds with tunable morphological and mechanical characteristics. Furthermore, the cells seeded were found to grow with a different trend trough the different layers thus demonstrating that the presented device has good potential to be used in interface tissue regeneration applications.

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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 FGF-2 trafficking toward the cell periphery was supported by coimmunoprecipitation and immune localization experiments. Colocalization of FGF-2 granules moving to the cell periphery and FM4-64-labelled intracellular lipids were not observed. Ouabain and methylamine, two inhibitors of FGF-2 release, were analyzed for their effects on FGF-2 intracellular localization and on vesicle shedding. Ouabain inhibited FGF-2 movements toward the cell periphery. The FGF-2 content of shed vesicles was therefore reduced. Methylamine inhibited vesicle shedding; in its presence, FGF-2 clustered at the cell periphery, but the rate of its release decreased. FGF-2 targeting to the nucleus and nucleolus was not affected by cytocalasin B, whereas it was inhibited by drugs that modify microtubule dynamics. Neither ouabain, nor methylamine interfered with FGF-2 translocation to the nucleus and nucleolus. FGF-2 targeting to the budding vesicles and to the nucleus and nucleolus is therefore mediated by fundamentally different mechanisms.

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 controlled by NaCl granulometry while PEG solvation created a micropores network within the devices. Scaffolds were characterized from a morphological and mechanical point of view in order to find a correlation between the preparation method, the pore architecture and compressive mechanical behavior. Biological tests were also performed in order to study the effect of pore size gradient on the permeation of different cell lines in co-culture. To imitate the physiological work condition, compressive tests were also performed in phosphate buffered saline (PBS) solution at 37 °C. The presented preparation method permitted to prepare three-layered scaffolds with high control of porosity and pore size distribution. Furthermore mechanical behaviors were found to be strongly affected by pore architecture of tested devices as well as the permeation of osteoblast and fibroblast in-vitro.

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 with typical features of shed vesicle membranes. Shed vesicles carried three FGF-2 isoforms (18, 22, 24 kDa). Addition of vesicles to endothelial cells stimulated chemotaxis and urokinase plasminogen activator production, which were blocked by anti-FGF-2 antibodies. Treatment of intact vesicles with 2.0 m NaCl or heparinase, which release FGF-2 from membrane-bound proteoglycans, did not abolish their stimulatory effect on endothelial cells, indicating that FGF-2 is carried inside vesicles. The comparison of the stimulatory effects of shed vesicles and vesicle-free conditioned medium showed that vesicles represent a major reservoir of FGF-2. Thus, FGF-2 can be released from cells through vesicle shedding.

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 single strand oligonucleotide, which holds a terminal amino group. The recognition ability of the attached single helix of its complementary strand was investigated. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 39774.

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 buffered formaldehyde for 24 hours, embedded in parraffin blocks, and cut into 8-μm-thick sections. The other half was fixed with 4% glutaraldehyde, dehydrated, gold coated, and was then observed with an scanning electron microscope (FEGESEM QUANTA 200 FEI). The anatomopathological examination of the implant site revealed no significant inflammatory response. The histological analysis revealed a thin fibrous capsule and a minimal inflammatory reaction with polymorphonuclear leukocytes, lymphocytes, monocytes and plasma cells around the implant, which died out during the second week. The presence of neutrophils and lymphocytes was detected until the second week after the implantation and a trama of connective tissue was found on the pores of the scaffold during the first week. The presence of giant cells was detected in the final stage of the inflammatory response and healing, which mainly occurred during the second week after the implantation. In addition, a significant angiogenic activity on the external implant surface was observed during the third week. 8 weeks after the implant, the inflammatory cell response was absolutely minimal, with an intense angiogenic activity occurring within the scaffold. This study shows the following results: PLLA has a high degree of biocompatibility and angiogenic potential; moreover, scaffolds are biocompatible in terms of cell survival and proliferation, as they stimulate a minimal inflammatory response while supporting cell infiltration, the matrix apposition and neo vascularization., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

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 prolong the drug release and show a pronounced sensibility to elastase. Drug unloaded nanoparticles stimulate the differentiation of human umbilical artery smooth muscle cells (HUASMCs) toward the contractile phenotype as demonstrated by immunofluorescence, flow cytofluorimetric, and western blotting analyses. Finally, drug-loaded nanoparticles efficiently reduce viability of HUASMCs as evidenced by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay.

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 endothelial cells (ECV 304) and the ability of heparinized PHEA-EDA-g-PLA/PCL scaffold to retain basic fibroblast growth factor has been evaluated in comparison with not heparinized sample.

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 scattering (DLS) and ζ-potential measurements. These measurements, carried out both in water and in buffer and at the variance of pH, temperature and time, support the excellent colloidal stability of the amino-graft PVP nanogels and their redispersability from the dry physical form. The total absence of in vitro toxicity and immunogenicity was ascertained first, and then cell localization studies in cell cultures of fluorescent variants were performed. Furthermore, the possibility of decorating the nanogels with bovine serum albumin (BSA) to obtain a stable bio-hybrid colloidal nanoconstruct was established. All the gathered evidences qualify these materials as potential candidates to be used as nanocarriers for intra-cellular delivery of protein and other biomolecular drugs.

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 both quantify the available functional groups for bioconjugation and follow nanogels localization in cell cultures. Finally, a model protein, bovine serum albumin, was conjugated to the nanogels to demonstrate the attachment of biologically relevant molecules for targeting purposes in drug delivery. The described approach provides a novel strategy to fabricate biohybrid nanogels with a very promising potential in nanomedicine.

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-based blends, on the simultaneous bulk/surface modifications and pore architectures in the scaffolds, and assessed the effects on human hepatocarcinoma viability and cell adhesion. The influence of PEG molecular weight on the scaffold morphology and cell viability and adhesion were also investigated. Morphological studies indicated that it was possible to obtain scaffolds with well-interconnected pores of assorted sizes. The analysis confirmed that SK-Hep1 cells adhered well to the polymeric support and emitted surface protrusions necessary to grow and differentiate three-dimensional systems. PEGs with higher molecular weight showed the best results in terms of cell adhesion and viability.

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 average hydrodynamic diameters in the range 30 – 100 nm with PDI lower than 0.3 and negative surface charge density. Confirmation of the molecular architecture and detection of the surface composition have been sought through complementary spectroscopic techniques, such as FTIR and XPS. Preliminarily to any biological evaluation or bio-conjugation, absence of cytotoxicity was tested through MTT assay. Studies on nanogel viability thought cell compartments, carried out with fluorescent PVP nanogels variants incubated for different times in cell cultures and observed by confocal microscopy, showed cytoplasmic distribution of nanogels and preferential localization of around the nuclei during the first 7 hours and progressive disappearance during the following 17 hours. Nanogels secretion as waste products could be hypothesized. The availability of accessible carboxyl groups for subsequent facile bio-conjugation reactions makes this family of functional nanogels very promising for decoration with oligonucleotides. As a proof of concept, a fluorescent oligonucleotide semi-helix was conjugated through an amino group attached to the terminal 3’. The recognition ability of the conjugated forward semi-helix toward the reverse semi-helix was proved by spectrofluorimetric readings using a reverse oligo carrying a fluorescence quencher.

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 variability in composition and concentration of collagenases used during the digestion phase‏. In islets isolation’s process neutral protease is also used and plays an important role. However, it should be considered as double-edged sword: it contributes to accelerate the tissue dissociation but, sometimes, its action could result decreased in islet yield, through their fragmentation, breakdown, inactivation‏. Proteases’ activities cannot be preciously adjusted in a narrow range since it doesn’t exist any approach showing the best possible dosage and composition of enzymes that can be used in a successful process of human islets pancreas extraction. At the moment, the available data on commercial enzymatic activity are not sufficient to predict their efficiency in the process of pancreas digestion and consequently it is very difficult to select which enzyme batches can be successfully used. For these reasons we apply to generate an innovative evaluation assay to select enzymes useful in islet pancreas isolation procedure.

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 in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis.

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 polymeric scaffolds having specific characteristics about biocompatibility and biodegradability.

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. Different fed-batch fermentation procedures were investigated and it was possible to produce more than two times of biomass and intracellular proteins compared to a conventional fed-batch strategy. The improvement in production leads both to the reduction of costs related to the amount of IPTG used and the isolation of a consistent amount of CTPR10 through rapid and highly efficient purification techniques. The realization of this project represents a significant milestone for Europe which will be at the forefront of innovation in the lighting sector.

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 methods to obtain them. Using electrophoresis and gelatin zymography approaches; together densitometry evaluation assays we compared in composition, stability and auto-digestion processes C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have investigated about the stability of analyzed enzymes when they are in solution in relation to their storage processes at different temperatures (-20°C; 0°C and r.t.); such as, concerning their digestive activity when applied at different temperatures (working temperatures: 25°C; 30°C; 37°C and 42 °C). Our results shown an heterogeneous composition of the different blend of collagenases from Roches and Serva, furthermore, heterogeneity is observed by lot to lot; on the other hand, we have found several more proteins and/or fragments respect the HPLC profiles publish by vendors. In gelatin zymographyes several digestive bands were catalytic active showing very high complex degradative patterns. Additionally, in neutral protease from Serva contaminants with gelatinolytic activities were detected; not in thermolysin. In stability experiments we investigated an inactivation/auto-digestive processes of analyzed enzymes; nevertheless, at different working temperatures, a more stable activity of analyzed molecules at 25°C were observed; activity that can be compared to that detectable at 37°C at the beginning of digestive process, at this temperature in the times inactivation enzymes were observed. These data take together strongly imply a not controlled digestive processes due to several contaminants present in the blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/degradative activities mean the impossibility to control the islets digestion process due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes. 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 protease and Thermolysin from Roches and Serva. Moreover, we have assessed: –the stability of enzymes when they are in solution at different temperatures (-20 C; 0 C and room temperature); –their digestive activity when applied at different working temperatures (25 C; 30 C; 37 C and 42 C). Our results shown a heterogeneous composition of the different enzymatic blend enzymes analyzed. Furthermore, heterogeneity is observed among different batch enzymes; we found several more proteins and/or fragments compare to HPLC profiles published by vendors. In gelatin zymographyes several digestive bands were catalytic, showing very high complex degradative patterns, in part active even in condition of calcium deprivation. Additionally, in neutral protease from Serva (and not in Thermolysin) contaminants with gelatinolytic activities were detected. An auto-digestive/inactivation processes of enzymes occurred at different working temperatures, reduced by lowering temperature up to 25 C. These data taken together strongly imply a not controlled digestive processes due to several contaminants in enzyme blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/degradative activities obstacles the possibility to control islet digestion due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes.

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 the other layer was made of PCL with pores ranging from 5 to 40 μm. The sponges were prepared by combining melt mixing and particulate leaching. In brief, PLA or PCL with PEG and NaCl were fed to a batch mixer and processed until a constant value of torque was achieved. The blends were then compression molded in a laboratory press in appropriate cylindrical molds. Finally, the porogen parts of the blends (NaCl and PEG) were removed by selective leaching in demineralized water. BLS have been characterized from a morphological and a mechanical point of view. Fibroblasts and osteoblasts were cultured and co-cultured in order to investigate the cells permeation trough the different layers.

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. The presence of α-elastin grafted to HA-EDA improves attachment, viability and proliferation of primary rat dermal fibroblasts and human umbilical artery smooth muscle cells. Biological performance of HA-EDA-g-α-elastin/EGDGE scaffold resulted comparable to that of a commercial collagen type I sponge (Antema®), chosen as a positive control.

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. On analyzed enzymes we have, also, explored about their stability when in solution in relation to storage processes at different temperatures (-20°C; 0°C and r.t.) and on their type-I collagen degradation activity when used at different temperatures (working temperatures: 25°C; 30°C; 37°C and 42 °C). Our results revealed an heterogeneous composition of the different blend of collagenases respect the HPLC profiles publish by vendors and an heterogeneity by lot to lot; moreover, we observed contamination from other enzymes in analysed samples. In fact, in gelatin zymographyes several digestive bands were catalytic active showing very high complex degradative patterns. Additionally, neutral protease from Serva contains gelatinolytic activities; not detected in thermolysin Roche. These data strongly suggest that a not controlled digestive processes can be associate to contaminants present in the blends and also due to auto-catalytic processes. In particular, the presence of low molecular weight gelatine/degradative activities denote the islets digestion process can not be control. Generation of recombinant collagenases probably could be the solution to overcome the variability in the extractive processes.