The extracellular matrix (ECM) represents the quintessential material for tissue engineering (TE)... more The extracellular matrix (ECM) represents the quintessential material for tissue engineering (TE) applications, because it provides a structural support and regulates tissue development. Therefore, the main challenge in TE is to recreate ECM analogues that recapitulate the structural and molecular microenvironment to promote and guide tissue growth. Apart from composition, the distribution and presentation of molecular cues within the matrix
Journal of Materials Science: Materials in Medicine, 2012
PEGylated silica nanoparticles, giving very stable aqueous sols, were successfully functionalised... more PEGylated silica nanoparticles, giving very stable aqueous sols, were successfully functionalised with rhodamine, one of the more stable fluorophore; they were also decorated with the targeting agent folic acid (FA) and charged with the well known drug doxorubicin. Rhodamine functionalization required a modification of the synthesis route of the nanoparticles (NP). Functionalization with FA required activation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Folate decorated NP were easily charged with doxorubicin. The experimental results proved the successfulness of the functionalization. The bond to the NP does not reduce the therapeutic efficacy of the drug. The calculated encapsulation efficiency (32 %) was only a little lower than the value (47 %) reported for the very popular PEGylated PLGA NP.
Peptide or protein ligands can be used for molecular decoration to enhance the functionality of s... more Peptide or protein ligands can be used for molecular decoration to enhance the functionality of synthetic materials. However, some skepticism has arisen about the efficacy of such strategy in practical contexts since serum proteins largely adsorb. To address this issue, it is crucial to ascertain whether a chemically conjugated integrin-binding peptide is fully recognized by a cell even if partially covered by a physisorbed layer of serum protein; in more general terms, if competitive protein fragments physisorbed onto the surface are distinguishable from those chemically anchored to it. Here, we engraft an RGD peptide on poly-ε-caprolactone (PCL) surfaces and follow the dynamics of focal adhesion (FA) and cytoskeleton assembly at different times and culture conditions using a variety of analytical tools. Although the presence of serum protein covers the bioconjugated RGD significantly, after the first adhesion phase cells dig into the physisorbed layer and reach the submerged signal to establish a more stable adhesion structure (mature FAs). Although the spreading area index is not substantially affected by the presence of the RGD peptide, cells attached to chemically bound signals develop a stronger adhesive interaction with the materials and assemble a mechanically stable cytoskeleton. This demonstrates that cells are able to discriminate, via mechanosensoring, between adhesive motives belonging to physisorbed proteins and those firmly anchored on the material surface.
The design of porous scaffolds able to promote and guide cell proliferation, colonization, and bi... more The design of porous scaffolds able to promote and guide cell proliferation, colonization, and biosynthesis in three dimensions is key determinant in bone tissue engineering (bTE). The aim of this study was to assess the role of the micro-architecture of poly(epsilon-caprolactone) scaffolds in affecting human mesenchymal stem cells' (hMSCs) spatial organization, proliferation, and osteogenic differentiation in vitro. Poly(epsilon-caprolactone) scaffolds for bTE and characterized by mono-modal and bi-modal pore size distributions were prepared by the combination of gas foaming and selective polymer extraction from co-continuous blends. The topological properties of the pore structure of the scaffolds were analyzed and the results correlated with the ability of hMSCs to proliferate, infiltrate, and differentiate in vitro in three dimensions. Results showed that the micro-architecture of the pore structure of the scaffolds plays a crucial role in defining cell seeding efficiency as well as hMSCs' three-dimensional colonization, proliferation, and osteogenic differentiation. Taken all together, our results indicated that process technologies able to allow a fine-tune of the topological properties of biodegradable porous scaffolds are essential for bTE strategies.
A big challenge in tumor targeting by nanoparticles (NPs), taking advantage of the enhanced perme... more A big challenge in tumor targeting by nanoparticles (NPs), taking advantage of the enhanced permeability and retention effect, is the fabrication of small size devices for enhanced tumor penetration, which is considered fundamental to improve chemotherapy efficacy. The purposes of this study are (i) to engineer the formulation of doxorubicin-loaded poly(D,L-lactic-co-glycolic acid) (PLGA)-block-poly(ethylene glycol) (PEG) NPs to obtain <100 nm devices and (ii) to translate standard 2D cytotoxicity studies to 3D collagen systems in which an initial step gradient of the NPs is present. Doxorubicin release can be prolonged for days to weeks depending on the NP formulation and the pH of the release medium. Sub-100 nm NPs are effectively internalized by HeLa cells in 2D and are less cytotoxic than free doxorubicin. In 3D, <100 nm NPs are significantly more toxic than larger ones towards HeLa cells, and the cell death rate is affected by the contributions of drug release and device transport through collagen. Thus, the reduction of NP size is a fundamental feature from both a technological and a biological point of view and must be properly engineered to optimize the tumor response to the NPs.
Thrombospondin-2 (TSP2)-null mice have increased endocortical bone and increased marrow stromal c... more Thrombospondin-2 (TSP2)-null mice have increased endocortical bone and increased marrow stromal cell (MSC) numbers. Similarly, in vitro, TSP2-null MSC show increased proliferation, and this is blocked by exogenous TSP2. TSP2-null MSC also exhibit delayed osteogenesis and enhanced adipogenesis compared to wild-type (WT) cells. Our goal is to determine whether TSP2 influences MSC differentiation indirectly, through its effect on cell proliferation, or whether TSP2 directly modulates MSC phenotype. Preliminary observations suggest that, in both WT and TSP2-null MSC, there is an inverse relationship between cell number and osteogenic potential. Furthermore, culture of TSP2-null MSC with a low serum concentration (1%) reduces cell number to a level comparable with that of WT cells cultured with 10% FBS. In fact, on a per cell basis, these two groups display a similar degree of mineralization. In parallel experiments, we are using siRNA to inhibit TSP2 gene expression in high-density MC3T3-E1 preosteoblasts. Transient transfection of siRNA reduced TSP2 mRNA and protein expression for seven days without affecting actin mRNA or cell number. At day 21, mineralization was reduced in cells transfected with TSP2 siRNA but not in cells transfected with a scrambled RNAi or in non-transfected cells. Cell number did not vary. In contrast to the experiments comparing WT and TSP2-null MSC, this experiment suggests that the effect of TSP2 on osteogenic differentiation is not linked to proliferation. We will use RNAi in cells cultured with various concentrations of serum to evaluate further the relationship between TSP2, differentiation and cell number.
The cell recognition of bioactive ligands immobilized on polymeric surfaces is strongly dependent... more The cell recognition of bioactive ligands immobilized on polymeric surfaces is strongly dependent on ligand presentation at the cell/material interface. While small peptide sequences such as Arg-Gly-Asp (RGD) are being widely used to obtain biomimetic interfaces, surface characteristics after immobilization as well as presentation of such ligands to cell receptors deserve more detailed investigation. Here, we immobilized an RGD-based sequence on poly(ε-caprolactone) (PCL), a largely widespread polymeric material used in biomedical applications, after polymer aminolysis. The surface characteristics along with the efficacy of the functionalization was monitored by surface analysis (FTIR-ATR, contact angle measurements, surface free energy determination) and spectrophotometric assays specially adapted for the analytical quantification of functional groups and/or peptides at the interface. Particular attention was paid to the evaluation of a number, morphology, and penetration depth of immobilized functional groups and/or peptides engrafted on polymeric substrates. In particular, a typical morphology in peptide distribution was evidenced on the surface raised from polymer crystallites, while a significant penetration depth of the engrafted molecules was revealed. NIH3T3 fibroblast adhesion studies verified the correct presentation of the ligand with enhanced cell attachment after peptide conjugation. Such work proposes a morphological and analytical approach in surface characterization to study the surface treatment and the distribution of ligands immobilized on polymeric substrates.
The physico-chemical properties of nanoparticles (NPs), such as small dimensions, surface charge ... more The physico-chemical properties of nanoparticles (NPs), such as small dimensions, surface charge and surface functionalization, control their capability to interact with cells and, in particular, with sub-cellular components. This interaction can be also influenced by the adsorption of molecules present in biological fluids, like blood, on NP surface. Here, we analysed the effect of serum proteins on 49 and 100 nm red fluorescent polystyrene NP uptake in porcine aortic endothelial (PAE) cells, as a model for vascular transport. To this aim, NP uptake kinetic, endocytic pathway and intracellular trafficking were studied by monitoring NPs inside cells through confocal microscopy and multiple particle tracking (MPT). We demonstrated that NPs are rapidly internalized by cells in serum-free (SF) medium, according to a saturation kinetic. Conversely, in 10% foetal bovine serum-enriched (SE) medium, NP uptake rate results drastically reduced. Moreover, NP internalization depends on an active endocytic mechanism that does not involve clathrin-and caveolae-mediated vesicular transport, in both SE and SF media. Furthermore, MPT data indicate that NP intracellular trafficking is unaffected by protein presence. Indeed, approximately 50-60% of internalized NPs is characterized by a sub-diffusive behaviour, whereas the remaining fraction shows an active motion. These findings demonstrate that the unspecific protein adsorption on NP surface can affect cellular uptake in terms of internalization kinetics, but it is not effective in controlling active and cellular-mediated uptake mechanisms of NPs and their intracellular routes.
Journal of Materials Science: Materials in Medicine, 2010
The ability to genetically modify cells seeded inside synthetic hydrogel scaffolds offers a suita... more The ability to genetically modify cells seeded inside synthetic hydrogel scaffolds offers a suitable approach to induce and control tissue repair and regeneration guiding cell fate. In fact the transfected cells can act as local in vivo bioreactor, secreting plasmid encoded proteins that augment tissue regeneration processes. We have realized a DNA bioactivated high porous poly(ethylene glycol) (PEG) matrix by polyethyleneimine (PEI)/DNA complexes adsorption. As the design of the microarchitectural features of a scaffold also contributes to promote and influence cell fate, we appropriately designed the inner structure of gene activated PEG hydrogels by gelatine microparticles templating. Microarchitectural properties of the scaffold were analysed by scanning electron microscopy. 3D cell migration and transfection were monitored through time-lapse videomicroscopy and confocal laser scanning microscopy.
Journal of Materials Science: Materials in Medicine, 2007
Recent studies, on cells cultured in 3D collagen gels, have shown that, beside from their well kn... more Recent studies, on cells cultured in 3D collagen gels, have shown that, beside from their well known biochemical role, fibronectin (FN) and laminin (LM) affect cell functions via a modification of mechanical and structural properties of matrix due to interaction with collagen molecules. Though biochemical properties of FN and LM have been widely studied, little is known about their role in collagen matrix assembly. The aim of this work was to characterize FN-and LM-based collagen semi-interpenetrating polymer networks (semi-IPNs), in order to understand how these biomacromolecular species can affect collagen network assembly and properties. Morphology, viscoelasticity and diffusivity of collagen gels and FN-and LM-based collagen semi-IPNs were analysed by Confocal Laser Scanning microscopy (CLSM), Environmental Scanning Electron microscopy (ESEM), Transmission Electron microscopy (TEM), Rheometry and Fluorescence Recovery After Photobleaching (FRAP) techniques. It was found that FN and LM were organized in aggregates, interspersed in collagen gel, and in thin fibrils, distributed along collagen fibres. In addition, high FN and LM concentrations affected collagen fibre assembly and structure and induced drastic effects on rheological and transport properties.
The use of scaffold-aided strategies for the regeneration of biological tissues requires the fulf... more The use of scaffold-aided strategies for the regeneration of biological tissues requires the fulfilment of an accurate architectural design, that is, micro and macrostructure, with the final goal of realizing architectures to adopt as guidance for those cell activities specific to the formation of novel tissues. Here, highly porous scaffolds made up of biodegradable poly(ε-caprolactone) (PCL) have been realized by thermally induced phase separation (TIPS). Two different polymer/solvent systems, derived by the dissolution of PCL in dioxane and DMSO respectively, were investigated. The aim was to demonstrate the high potential of TIPS technique, in imprinting specific pore features to the polymer matrices, by a conscious selection of polymer/solvent systems. The investigation of pore architecture by SEM/mercury intrusion porosimetry/image analyses, firstly allow to detect remarkable variations in porosity (from 92% to 78%,) and pore sizes, ranging from micro-scale (ca 10 µm) to macro-scale (greater than 100 µm) as a function of the used polymer/solvent systems. Moreover, experimental and theoretical evidences referred to scaffold shaped in custom-made molds--a thin Teflon ring between two copper plates--allow exploring how the sensitivity of polymer solution features (i.e., crystallinity, thermal inertia) to the cooling temperature can affect the alignment of polymer phases and, ultimately, scaffold pore anisotropy. Analytical results supported by preliminary biological studies demonstrate the higher ability of PCL/dioxane solution to promote the formation of aligned pores which provide a morphological guidance to cell advance during the preliminary stage of culture. These findings, taken as a whole, put the basis for a better informed regeneration of structurally complex tissues based on the modeling of scaffold micro and macro-architecture by thermodynamic forces.
Realization of systems able to both recruit cells and influence their fate (affecting their proce... more Realization of systems able to both recruit cells and influence their fate (affecting their processes) represents a new approach for tissue regeneration. We investigated the potency of gene activated matrix (GAM) and implemented the GAM strategy in order to achieve a control of gene expression, as well as a specific cell recruitment. To this aim we developed a 3D DNA bio-activated collagen matrix by Poly (ethylenimine) (PEI)/DNA complex immobilization in the matrix through biotin/avidin bond. Moreover, we realised a serum based chemotactic gradient within the matrix in order to directionally attract NIH3T3 cells. In this system, cells are recruited and forced to migrate through the matrix where they find the bound PEI/DNA complexes and are transfected. The transfected cells can act as local in vivo bioreactors, secreting plasmid encoded proteins that augment tissue repair and regeneration. 3D cell migration and cell transfection were monitored through time-lapse video microscopy and fluorescence microscopy. Cell transfection was also quantified through FACS analysis. Results show that our engineered matrix is able to recruit external cells and transfect them once internalized, therefore it could help in tissue repairing strategy.
Essential to the design of genetic bioreactors used in the human body is a consideration of how t... more Essential to the design of genetic bioreactors used in the human body is a consideration of how the properties of biomaterials can combine to envelope, spatially guide, reprogramme by gene transfer, and then release cells. In order to approach this goal, poly(ethylene glycol) (PEG) matrices with modulated structural features and defined spatial patterns of bioactive signals have been designed and produced. In particular, within such PEG matrices, both an adhesive RGD peptide gradient, to directionally attract NIH3T3 cells, and a designed spatial distribution of immobilized poly(ethylenimine) (PEI)/DNA complexes, to obtain a localized transfection, have been realized. These bioactive biomaterials have been designed bearing in mind that cells following an RGD gradient migrate through the matrix, in which they find the bound DNA and become transfected. Both cell migration and transfection have been monitored by fluorescence microscopy. Results show that this system is able to envelope cells, spatially guide them towards the immobilized gene complexes and locally transfect them. Therefore, the system, acting as a genetic bioreactor potentially useful for the regulation of biology at a distance, could be used to directly control cell trafficking and activation in the human body, and has many potential biomedical applications.
The use of scaffold-based strategies in the regeneration of biological tissues requires that the ... more The use of scaffold-based strategies in the regeneration of biological tissues requires that the design of the microarchitecture of the scaffold satisfy key microstructural and biological requirements. Here, we examined the ability of a porous poly(epsilon-caprolactone) (PCL) scaffold with novel bimodal-micron scale (mu-bimodal) porous architecture to promote and guide the in vitro adhesion, proliferation and three-dimensional (3-D) colonization of human mesenchymal stem cells (hMSCs). The mu-bimodal PCL scaffold was prepared by a combination of gas foaming (GF) and selective polymer extraction (PE) from co-continuous blends. The microarchitectural properties of the scaffold, in particular its morphology, porosity distribution and mechanical compression properties, were analyzed and correlated with the results of the in vitro cell-scaffold interaction study, carried out for 21days under static conditions. Alamar Blue assay, scanning electron microscopy, confocal laser scanning microscopy and histological analyses were performed to assess hMSC adhesion, proliferation and 3-D colonization. The results showed that the combined GF-PE technique allowed the preparation of PCL scaffold with a unique multiscaled and highly interconnected microarchitecture that was characterized by mechanical properties suitable for load-bearing applications. Study of the cell-scaffold interaction also demonstrated the ability of the scaffold to support hMSC adhesion and proliferation, as well as the possibility to promote and guide 3-D cell colonization by appropriately designing the microarchitectural features of the scaffold.
Understanding the influence of a controlled spatial distribution of biological cues on cell activ... more Understanding the influence of a controlled spatial distribution of biological cues on cell activities can be useful to design &amp;amp;amp;amp;amp;amp;quot;cell instructive&amp;amp;amp;amp;amp;amp;quot; materials, able to control and guide the formation of engineered tissues in vivo and in vitro. To this purpose, biochemical and mechanical properties of the resulting biomaterial must be carefully designed and controlled. In this work, the effect of covalently immobilized RGD peptide gradients on poly(ethylene glycol) diacrylate hydrogels on cell behaviour was studied. We set up a mechanical device generating gradients based on a fluidic chamber. Cell response to RGD gradients with different slope (0.7, 1 and 2 mM cm(-1)) was qualitatively and quantitatively assessed by evaluating cell adhesion and, in particular, cell migration, compared to cells seeded on hydrogels with uniform distribution of RGD peptides. To evaluate the influence of RGD gradient and to exclude any concentration effect on cell response, all analyses were carried out in a specific region of the gradients which displayed the same average concentration of RGD (1.5 mM). Results suggest that cells recognize the RGD gradient and adhere onto it assuming a stretched shape. Moreover, cells tend to migrate in the direction of the gradient, as their speed is higher than that of cells migrating on hydrogels with a uniform distribution of RGD and increases by increasing RGD gradient steepness. This increment is due to an augmentation of bias speed component of the mean squared speed, that is, the drift of the cell population migrating on the anisotropic surface provided by the RGD gradient.
The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum ... more The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum dots, polymeric nanoparticles) within and across cells in a very efficient manner. However, a clear understanding of the detailed uptake mechanism remains elusive. In this work, we investigate the journey of gH625-functionalized polystyrene nanoparticles in mouse-brain endothelial cells from their interaction with the cell membrane to their intracellular final destination. The aim is to elucidate how gH625 affects the behavior of the nanoparticles and their cytotoxic effect. The results indicate that the mechanism of translocation of gH625 dictates the fate of the nanoparticles, with a relevant impact on the nanotoxicological profile of positively charged nanoparticles.
Journal of Vacuum Science & Technology B: Microelectronics and Nanometer Structures, 1990
Abstract The purpose of this paper is to present experimental results on optical properties of x‐... more Abstract The purpose of this paper is to present experimental results on optical properties of x‐ray mask substrates relevant to x‐ray lithography systems utilizing optical alignment between mask and wafer. Data on mask substrates of several materials including B‐ ...
A genetically modified recombinant gH625-c-prune was prepared through conjugation of c-prune with... more A genetically modified recombinant gH625-c-prune was prepared through conjugation of c-prune with gH625, a peptide encompassing 625-644 residues of the glycoprotein H of herpes simplex virus 1, which has been proved to possess the ability to carry cargo molecules across cell membranes. C-prune is the C-terminal domain of h-prune, overexpressed in breast, colorectal, and gastric cancers, interacting with multiple partners, and representing an ideal target for inhibition of cancer development. Its C-terminal domain results in an intrinsically disordered domain (IDD), and the peculiar properties of gH625 render it an optimal candidate to act as a carrier for this net negatively charged molecule by comparison with the positively charged TAT. A characterization of the recombinant gH625-c-prune fusion protein was conducted by biochemical, cellular biology and confocal microscopy means in comparison with TAT-c-prune. The results showed that the gH625-c-prune exhibited the ability to cross biomembranes, opening a new scenario on the use of gH625 as a novel multifunctional carrier.
The extracellular matrix (ECM) represents the quintessential material for tissue engineering (TE)... more The extracellular matrix (ECM) represents the quintessential material for tissue engineering (TE) applications, because it provides a structural support and regulates tissue development. Therefore, the main challenge in TE is to recreate ECM analogues that recapitulate the structural and molecular microenvironment to promote and guide tissue growth. Apart from composition, the distribution and presentation of molecular cues within the matrix
Journal of Materials Science: Materials in Medicine, 2012
PEGylated silica nanoparticles, giving very stable aqueous sols, were successfully functionalised... more PEGylated silica nanoparticles, giving very stable aqueous sols, were successfully functionalised with rhodamine, one of the more stable fluorophore; they were also decorated with the targeting agent folic acid (FA) and charged with the well known drug doxorubicin. Rhodamine functionalization required a modification of the synthesis route of the nanoparticles (NP). Functionalization with FA required activation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Folate decorated NP were easily charged with doxorubicin. The experimental results proved the successfulness of the functionalization. The bond to the NP does not reduce the therapeutic efficacy of the drug. The calculated encapsulation efficiency (32 %) was only a little lower than the value (47 %) reported for the very popular PEGylated PLGA NP.
Peptide or protein ligands can be used for molecular decoration to enhance the functionality of s... more Peptide or protein ligands can be used for molecular decoration to enhance the functionality of synthetic materials. However, some skepticism has arisen about the efficacy of such strategy in practical contexts since serum proteins largely adsorb. To address this issue, it is crucial to ascertain whether a chemically conjugated integrin-binding peptide is fully recognized by a cell even if partially covered by a physisorbed layer of serum protein; in more general terms, if competitive protein fragments physisorbed onto the surface are distinguishable from those chemically anchored to it. Here, we engraft an RGD peptide on poly-ε-caprolactone (PCL) surfaces and follow the dynamics of focal adhesion (FA) and cytoskeleton assembly at different times and culture conditions using a variety of analytical tools. Although the presence of serum protein covers the bioconjugated RGD significantly, after the first adhesion phase cells dig into the physisorbed layer and reach the submerged signal to establish a more stable adhesion structure (mature FAs). Although the spreading area index is not substantially affected by the presence of the RGD peptide, cells attached to chemically bound signals develop a stronger adhesive interaction with the materials and assemble a mechanically stable cytoskeleton. This demonstrates that cells are able to discriminate, via mechanosensoring, between adhesive motives belonging to physisorbed proteins and those firmly anchored on the material surface.
The design of porous scaffolds able to promote and guide cell proliferation, colonization, and bi... more The design of porous scaffolds able to promote and guide cell proliferation, colonization, and biosynthesis in three dimensions is key determinant in bone tissue engineering (bTE). The aim of this study was to assess the role of the micro-architecture of poly(epsilon-caprolactone) scaffolds in affecting human mesenchymal stem cells&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; (hMSCs) spatial organization, proliferation, and osteogenic differentiation in vitro. Poly(epsilon-caprolactone) scaffolds for bTE and characterized by mono-modal and bi-modal pore size distributions were prepared by the combination of gas foaming and selective polymer extraction from co-continuous blends. The topological properties of the pore structure of the scaffolds were analyzed and the results correlated with the ability of hMSCs to proliferate, infiltrate, and differentiate in vitro in three dimensions. Results showed that the micro-architecture of the pore structure of the scaffolds plays a crucial role in defining cell seeding efficiency as well as hMSCs&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; three-dimensional colonization, proliferation, and osteogenic differentiation. Taken all together, our results indicated that process technologies able to allow a fine-tune of the topological properties of biodegradable porous scaffolds are essential for bTE strategies.
A big challenge in tumor targeting by nanoparticles (NPs), taking advantage of the enhanced perme... more A big challenge in tumor targeting by nanoparticles (NPs), taking advantage of the enhanced permeability and retention effect, is the fabrication of small size devices for enhanced tumor penetration, which is considered fundamental to improve chemotherapy efficacy. The purposes of this study are (i) to engineer the formulation of doxorubicin-loaded poly(D,L-lactic-co-glycolic acid) (PLGA)-block-poly(ethylene glycol) (PEG) NPs to obtain <100 nm devices and (ii) to translate standard 2D cytotoxicity studies to 3D collagen systems in which an initial step gradient of the NPs is present. Doxorubicin release can be prolonged for days to weeks depending on the NP formulation and the pH of the release medium. Sub-100 nm NPs are effectively internalized by HeLa cells in 2D and are less cytotoxic than free doxorubicin. In 3D, <100 nm NPs are significantly more toxic than larger ones towards HeLa cells, and the cell death rate is affected by the contributions of drug release and device transport through collagen. Thus, the reduction of NP size is a fundamental feature from both a technological and a biological point of view and must be properly engineered to optimize the tumor response to the NPs.
Thrombospondin-2 (TSP2)-null mice have increased endocortical bone and increased marrow stromal c... more Thrombospondin-2 (TSP2)-null mice have increased endocortical bone and increased marrow stromal cell (MSC) numbers. Similarly, in vitro, TSP2-null MSC show increased proliferation, and this is blocked by exogenous TSP2. TSP2-null MSC also exhibit delayed osteogenesis and enhanced adipogenesis compared to wild-type (WT) cells. Our goal is to determine whether TSP2 influences MSC differentiation indirectly, through its effect on cell proliferation, or whether TSP2 directly modulates MSC phenotype. Preliminary observations suggest that, in both WT and TSP2-null MSC, there is an inverse relationship between cell number and osteogenic potential. Furthermore, culture of TSP2-null MSC with a low serum concentration (1%) reduces cell number to a level comparable with that of WT cells cultured with 10% FBS. In fact, on a per cell basis, these two groups display a similar degree of mineralization. In parallel experiments, we are using siRNA to inhibit TSP2 gene expression in high-density MC3T3-E1 preosteoblasts. Transient transfection of siRNA reduced TSP2 mRNA and protein expression for seven days without affecting actin mRNA or cell number. At day 21, mineralization was reduced in cells transfected with TSP2 siRNA but not in cells transfected with a scrambled RNAi or in non-transfected cells. Cell number did not vary. In contrast to the experiments comparing WT and TSP2-null MSC, this experiment suggests that the effect of TSP2 on osteogenic differentiation is not linked to proliferation. We will use RNAi in cells cultured with various concentrations of serum to evaluate further the relationship between TSP2, differentiation and cell number.
The cell recognition of bioactive ligands immobilized on polymeric surfaces is strongly dependent... more The cell recognition of bioactive ligands immobilized on polymeric surfaces is strongly dependent on ligand presentation at the cell/material interface. While small peptide sequences such as Arg-Gly-Asp (RGD) are being widely used to obtain biomimetic interfaces, surface characteristics after immobilization as well as presentation of such ligands to cell receptors deserve more detailed investigation. Here, we immobilized an RGD-based sequence on poly(ε-caprolactone) (PCL), a largely widespread polymeric material used in biomedical applications, after polymer aminolysis. The surface characteristics along with the efficacy of the functionalization was monitored by surface analysis (FTIR-ATR, contact angle measurements, surface free energy determination) and spectrophotometric assays specially adapted for the analytical quantification of functional groups and/or peptides at the interface. Particular attention was paid to the evaluation of a number, morphology, and penetration depth of immobilized functional groups and/or peptides engrafted on polymeric substrates. In particular, a typical morphology in peptide distribution was evidenced on the surface raised from polymer crystallites, while a significant penetration depth of the engrafted molecules was revealed. NIH3T3 fibroblast adhesion studies verified the correct presentation of the ligand with enhanced cell attachment after peptide conjugation. Such work proposes a morphological and analytical approach in surface characterization to study the surface treatment and the distribution of ligands immobilized on polymeric substrates.
The physico-chemical properties of nanoparticles (NPs), such as small dimensions, surface charge ... more The physico-chemical properties of nanoparticles (NPs), such as small dimensions, surface charge and surface functionalization, control their capability to interact with cells and, in particular, with sub-cellular components. This interaction can be also influenced by the adsorption of molecules present in biological fluids, like blood, on NP surface. Here, we analysed the effect of serum proteins on 49 and 100 nm red fluorescent polystyrene NP uptake in porcine aortic endothelial (PAE) cells, as a model for vascular transport. To this aim, NP uptake kinetic, endocytic pathway and intracellular trafficking were studied by monitoring NPs inside cells through confocal microscopy and multiple particle tracking (MPT). We demonstrated that NPs are rapidly internalized by cells in serum-free (SF) medium, according to a saturation kinetic. Conversely, in 10% foetal bovine serum-enriched (SE) medium, NP uptake rate results drastically reduced. Moreover, NP internalization depends on an active endocytic mechanism that does not involve clathrin-and caveolae-mediated vesicular transport, in both SE and SF media. Furthermore, MPT data indicate that NP intracellular trafficking is unaffected by protein presence. Indeed, approximately 50-60% of internalized NPs is characterized by a sub-diffusive behaviour, whereas the remaining fraction shows an active motion. These findings demonstrate that the unspecific protein adsorption on NP surface can affect cellular uptake in terms of internalization kinetics, but it is not effective in controlling active and cellular-mediated uptake mechanisms of NPs and their intracellular routes.
Journal of Materials Science: Materials in Medicine, 2010
The ability to genetically modify cells seeded inside synthetic hydrogel scaffolds offers a suita... more The ability to genetically modify cells seeded inside synthetic hydrogel scaffolds offers a suitable approach to induce and control tissue repair and regeneration guiding cell fate. In fact the transfected cells can act as local in vivo bioreactor, secreting plasmid encoded proteins that augment tissue regeneration processes. We have realized a DNA bioactivated high porous poly(ethylene glycol) (PEG) matrix by polyethyleneimine (PEI)/DNA complexes adsorption. As the design of the microarchitectural features of a scaffold also contributes to promote and influence cell fate, we appropriately designed the inner structure of gene activated PEG hydrogels by gelatine microparticles templating. Microarchitectural properties of the scaffold were analysed by scanning electron microscopy. 3D cell migration and transfection were monitored through time-lapse videomicroscopy and confocal laser scanning microscopy.
Journal of Materials Science: Materials in Medicine, 2007
Recent studies, on cells cultured in 3D collagen gels, have shown that, beside from their well kn... more Recent studies, on cells cultured in 3D collagen gels, have shown that, beside from their well known biochemical role, fibronectin (FN) and laminin (LM) affect cell functions via a modification of mechanical and structural properties of matrix due to interaction with collagen molecules. Though biochemical properties of FN and LM have been widely studied, little is known about their role in collagen matrix assembly. The aim of this work was to characterize FN-and LM-based collagen semi-interpenetrating polymer networks (semi-IPNs), in order to understand how these biomacromolecular species can affect collagen network assembly and properties. Morphology, viscoelasticity and diffusivity of collagen gels and FN-and LM-based collagen semi-IPNs were analysed by Confocal Laser Scanning microscopy (CLSM), Environmental Scanning Electron microscopy (ESEM), Transmission Electron microscopy (TEM), Rheometry and Fluorescence Recovery After Photobleaching (FRAP) techniques. It was found that FN and LM were organized in aggregates, interspersed in collagen gel, and in thin fibrils, distributed along collagen fibres. In addition, high FN and LM concentrations affected collagen fibre assembly and structure and induced drastic effects on rheological and transport properties.
The use of scaffold-aided strategies for the regeneration of biological tissues requires the fulf... more The use of scaffold-aided strategies for the regeneration of biological tissues requires the fulfilment of an accurate architectural design, that is, micro and macrostructure, with the final goal of realizing architectures to adopt as guidance for those cell activities specific to the formation of novel tissues. Here, highly porous scaffolds made up of biodegradable poly(ε-caprolactone) (PCL) have been realized by thermally induced phase separation (TIPS). Two different polymer/solvent systems, derived by the dissolution of PCL in dioxane and DMSO respectively, were investigated. The aim was to demonstrate the high potential of TIPS technique, in imprinting specific pore features to the polymer matrices, by a conscious selection of polymer/solvent systems. The investigation of pore architecture by SEM/mercury intrusion porosimetry/image analyses, firstly allow to detect remarkable variations in porosity (from 92% to 78%,) and pore sizes, ranging from micro-scale (ca 10 µm) to macro-scale (greater than 100 µm) as a function of the used polymer/solvent systems. Moreover, experimental and theoretical evidences referred to scaffold shaped in custom-made molds--a thin Teflon ring between two copper plates--allow exploring how the sensitivity of polymer solution features (i.e., crystallinity, thermal inertia) to the cooling temperature can affect the alignment of polymer phases and, ultimately, scaffold pore anisotropy. Analytical results supported by preliminary biological studies demonstrate the higher ability of PCL/dioxane solution to promote the formation of aligned pores which provide a morphological guidance to cell advance during the preliminary stage of culture. These findings, taken as a whole, put the basis for a better informed regeneration of structurally complex tissues based on the modeling of scaffold micro and macro-architecture by thermodynamic forces.
Realization of systems able to both recruit cells and influence their fate (affecting their proce... more Realization of systems able to both recruit cells and influence their fate (affecting their processes) represents a new approach for tissue regeneration. We investigated the potency of gene activated matrix (GAM) and implemented the GAM strategy in order to achieve a control of gene expression, as well as a specific cell recruitment. To this aim we developed a 3D DNA bio-activated collagen matrix by Poly (ethylenimine) (PEI)/DNA complex immobilization in the matrix through biotin/avidin bond. Moreover, we realised a serum based chemotactic gradient within the matrix in order to directionally attract NIH3T3 cells. In this system, cells are recruited and forced to migrate through the matrix where they find the bound PEI/DNA complexes and are transfected. The transfected cells can act as local in vivo bioreactors, secreting plasmid encoded proteins that augment tissue repair and regeneration. 3D cell migration and cell transfection were monitored through time-lapse video microscopy and fluorescence microscopy. Cell transfection was also quantified through FACS analysis. Results show that our engineered matrix is able to recruit external cells and transfect them once internalized, therefore it could help in tissue repairing strategy.
Essential to the design of genetic bioreactors used in the human body is a consideration of how t... more Essential to the design of genetic bioreactors used in the human body is a consideration of how the properties of biomaterials can combine to envelope, spatially guide, reprogramme by gene transfer, and then release cells. In order to approach this goal, poly(ethylene glycol) (PEG) matrices with modulated structural features and defined spatial patterns of bioactive signals have been designed and produced. In particular, within such PEG matrices, both an adhesive RGD peptide gradient, to directionally attract NIH3T3 cells, and a designed spatial distribution of immobilized poly(ethylenimine) (PEI)/DNA complexes, to obtain a localized transfection, have been realized. These bioactive biomaterials have been designed bearing in mind that cells following an RGD gradient migrate through the matrix, in which they find the bound DNA and become transfected. Both cell migration and transfection have been monitored by fluorescence microscopy. Results show that this system is able to envelope cells, spatially guide them towards the immobilized gene complexes and locally transfect them. Therefore, the system, acting as a genetic bioreactor potentially useful for the regulation of biology at a distance, could be used to directly control cell trafficking and activation in the human body, and has many potential biomedical applications.
The use of scaffold-based strategies in the regeneration of biological tissues requires that the ... more The use of scaffold-based strategies in the regeneration of biological tissues requires that the design of the microarchitecture of the scaffold satisfy key microstructural and biological requirements. Here, we examined the ability of a porous poly(epsilon-caprolactone) (PCL) scaffold with novel bimodal-micron scale (mu-bimodal) porous architecture to promote and guide the in vitro adhesion, proliferation and three-dimensional (3-D) colonization of human mesenchymal stem cells (hMSCs). The mu-bimodal PCL scaffold was prepared by a combination of gas foaming (GF) and selective polymer extraction (PE) from co-continuous blends. The microarchitectural properties of the scaffold, in particular its morphology, porosity distribution and mechanical compression properties, were analyzed and correlated with the results of the in vitro cell-scaffold interaction study, carried out for 21days under static conditions. Alamar Blue assay, scanning electron microscopy, confocal laser scanning microscopy and histological analyses were performed to assess hMSC adhesion, proliferation and 3-D colonization. The results showed that the combined GF-PE technique allowed the preparation of PCL scaffold with a unique multiscaled and highly interconnected microarchitecture that was characterized by mechanical properties suitable for load-bearing applications. Study of the cell-scaffold interaction also demonstrated the ability of the scaffold to support hMSC adhesion and proliferation, as well as the possibility to promote and guide 3-D cell colonization by appropriately designing the microarchitectural features of the scaffold.
Understanding the influence of a controlled spatial distribution of biological cues on cell activ... more Understanding the influence of a controlled spatial distribution of biological cues on cell activities can be useful to design &amp;amp;amp;amp;amp;amp;quot;cell instructive&amp;amp;amp;amp;amp;amp;quot; materials, able to control and guide the formation of engineered tissues in vivo and in vitro. To this purpose, biochemical and mechanical properties of the resulting biomaterial must be carefully designed and controlled. In this work, the effect of covalently immobilized RGD peptide gradients on poly(ethylene glycol) diacrylate hydrogels on cell behaviour was studied. We set up a mechanical device generating gradients based on a fluidic chamber. Cell response to RGD gradients with different slope (0.7, 1 and 2 mM cm(-1)) was qualitatively and quantitatively assessed by evaluating cell adhesion and, in particular, cell migration, compared to cells seeded on hydrogels with uniform distribution of RGD peptides. To evaluate the influence of RGD gradient and to exclude any concentration effect on cell response, all analyses were carried out in a specific region of the gradients which displayed the same average concentration of RGD (1.5 mM). Results suggest that cells recognize the RGD gradient and adhere onto it assuming a stretched shape. Moreover, cells tend to migrate in the direction of the gradient, as their speed is higher than that of cells migrating on hydrogels with a uniform distribution of RGD and increases by increasing RGD gradient steepness. This increment is due to an augmentation of bias speed component of the mean squared speed, that is, the drift of the cell population migrating on the anisotropic surface provided by the RGD gradient.
The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum ... more The membranotropic peptide gH625 is able to transport different cargos (i.e., liposomes, quantum dots, polymeric nanoparticles) within and across cells in a very efficient manner. However, a clear understanding of the detailed uptake mechanism remains elusive. In this work, we investigate the journey of gH625-functionalized polystyrene nanoparticles in mouse-brain endothelial cells from their interaction with the cell membrane to their intracellular final destination. The aim is to elucidate how gH625 affects the behavior of the nanoparticles and their cytotoxic effect. The results indicate that the mechanism of translocation of gH625 dictates the fate of the nanoparticles, with a relevant impact on the nanotoxicological profile of positively charged nanoparticles.
Journal of Vacuum Science & Technology B: Microelectronics and Nanometer Structures, 1990
Abstract The purpose of this paper is to present experimental results on optical properties of x‐... more Abstract The purpose of this paper is to present experimental results on optical properties of x‐ray mask substrates relevant to x‐ray lithography systems utilizing optical alignment between mask and wafer. Data on mask substrates of several materials including B‐ ...
A genetically modified recombinant gH625-c-prune was prepared through conjugation of c-prune with... more A genetically modified recombinant gH625-c-prune was prepared through conjugation of c-prune with gH625, a peptide encompassing 625-644 residues of the glycoprotein H of herpes simplex virus 1, which has been proved to possess the ability to carry cargo molecules across cell membranes. C-prune is the C-terminal domain of h-prune, overexpressed in breast, colorectal, and gastric cancers, interacting with multiple partners, and representing an ideal target for inhibition of cancer development. Its C-terminal domain results in an intrinsically disordered domain (IDD), and the peculiar properties of gH625 render it an optimal candidate to act as a carrier for this net negatively charged molecule by comparison with the positively charged TAT. A characterization of the recombinant gH625-c-prune fusion protein was conducted by biochemical, cellular biology and confocal microscopy means in comparison with TAT-c-prune. The results showed that the gH625-c-prune exhibited the ability to cross biomembranes, opening a new scenario on the use of gH625 as a novel multifunctional carrier.
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Papers by D. Guarnieri