Shedding light on azopolymer brush dynamics by fluorescence correlation spectroscopy.

Understanding the response to illumination at a molecular level as well as characterising polymer brush dynamics are key features that guide the engineering of new light-stimuli responsive materials. Here, we report on the use of a confocal microscopy technique that was exploited to discern how a single molecular event such as the photoinduced isomerisation of azobenzene can affect an entire polymeric material at a macroscopic level leading to photodriven mass-migration. For this reason, a set of polymer brushes, containing azobenzene (Disperse Red 1, DR) on the side chains of poly(methacrylic acid), was synthesised and the influence of DR on the polymer brush dynamics was investigated for the first time by Fluorescence Correlation Spectroscopy (FCS). Briefly, two dynamics were observed, a short one coming from the isomerisation of DR and a long one related to the brush main chain. Interestingly, photoinduced polymer aggregation in the confocal volume was observed.

High sensitive and direct fluorescence detection of single viral DNA sequences by integration of double strand probes onto microgels particles.

A novel class of probes for fluorescence detection was developed and combined to microgel particles for a high sensitive fluorescence detection of nucleic acids. A double strand probe with an optimized fluorescent-quencher couple was designed for the detection of different lengths of nucleic acids (39 nt and 100 nt). Such probe proved efficient in target detection in different contests and specific even in presence of serum proteins. The conjugation of double strand probes onto polymeric microgels allows for a sensitive detection of DNA sequences from HIV, HCV and SARS corona viruses with a LOD of 1.4 fM, 3.7 fM and 1.4 fM, respectively, and with a dynamic range of 10(-9)-10(-15) M. Such combination enhances the sensitivity of the detection of almost five orders of magnitude when compared to the only probe. The proposed platform based on the integration of innovative double strand probe into microgels particles represents an attractive alternative to conventional sensitive DNA detection technologies that rely on amplifications methods.

Vasculogenic potential evaluation of bottom-up, PCL scaffolds guiding early angiogenesis in tissue regeneration.

Vascularization is a key factor in the successful integration of tissue engineered (TE) grafts inside the host body. Biological functions of the newly formed tissue depend, in fact, on a reliable and fast spread of the vascular network inside the scaffold. In this study, we propose a technique for evaluating vascularization in TE constructs assembled by a bottom-up approach. The rational, ordered assembly of building blocks (BBs) into a 3D scaffold can improve vessel penetration, and-unlike most current technologies-is compatible with the insertion of different elements that can be designed independently (e.g. structural units, growth factor depots etc.). Poly(ε-caprolactone) scaffolds composed of orderly and randomly assembled sintered microspheres were used to assess the degree of vascularization in a pilot in vivo study. Scaffolds were implanted in a rat subcutaneous pocket model, and retrieved after 7 days. We introduce three quantitative factors as a measure of vascularization: the total percentage of vascularization, the vessels diameter distribution and the vascular penetration depth. These parameters were derived by image analysis of microcomputed tomographic scans of biological specimens perfused with a radiopaque polymer. The outcome of this study suggests that the rational assembly of BBs helps the onset and organization of a fully functional vascular network.

Surface decoration with gH625-membranotropic peptides as a method to escape the endo-lysosomal compartment and reduce nanoparticle toxicity.

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.

Three-dimensional cellular distribution in polymeric scaffolds for bone regeneration: a microCT analysis compared to SEM, CLSM and DNA content.

In orthopaedic surgery the tissues damaged by injury or disease could be replaced using constructs based on biocompatible materials, cells and growth factors. Scaffold design, porosity and early colonization are key components for the implant success. From biological point of view, attention may be also given to the number, type and size of seeded cells, as well as the seeding technique and cell morphological and volumetric alterations. This paper describes the use of the microCT approach (to date used principally for mineralized matrix quantification) to observe construct colonization in terms of cell localization, and make a direct comparison of the microtomographic sections with scanning electron microscopy images and confocal laser scanning microscope analysis. Briefly, polycaprolactone scaffolds were seeded at different cell densities with MG63 osteoblastic-like cells. Two different endpoints, 1 and 2 weeks, were selected for the three-dimensional colonization and proliferation analysis of the cells. By observing all images obtained, in addition to a more extensive distribution of cells on scaffolds surfaces than in the deeper layers, cell volume increased at 2 weeks compared to 1 week after seeding. Combining the cell number quantification by deoxyribonucleic acid analysis and the single cell volume changes by confocal laser scanning microscope, we validated the microCT segmentation method by finding no statistical differences in the evaluation of the cell volume fraction of the scaffold. Furthermore, the morphological results of this study suggest that an effective scaffold colonization requires a precise balance between different factors, such as number, type and size of seeded cells in addition to scaffold porosity.

Multiplex single particle analysis in microfluidics.

A straightforward way to measure separated micrometric sized particles in microfluidic flow is reported. The light scattering profile (LSP) of each single particle is fully characterized by using a CMOS-camera based small angle light scattering (SALS) apparatus, ranging from 2° up to 30°. To ensure controlled particle passage through the incident laser, a viscoelastic 3D alignment effect by viscoelastic induced particle migration has been implemented in a simple and cost-effective microfluidic device. Different polystyrene particle sizes are measured in microfluidic flows and the obtained scattering signatures are matched with the Lorenz-Mie based scattering theory. The results confirm the possibility of using this apparatus for real multiplex particle analyses in microfluidic particle flows.

In vitro strategies for mimicking dynamic cell-ECM reciprocity in 3D culture models.

The extracellular microenvironment regulates cell decisions through the accurate presentation at the cell surface of a complex array of biochemical and biophysical signals that are mediated by the structure and composition of the extracellular matrix (ECM). On the one hand, the cells actively remodel the ECM, which on the other hand affects cell functions. This cell-ECM dynamic reciprocity is central in regulating and controlling morphogenetic and histogenetic processes. Misregulation within the extracellular space can cause aberrant bidirectional interactions between cells and ECM, resulting in dysfunctional tissues and pathological states. Therefore, tissue engineering approaches, aiming at reproducing organs and tissues in vitro, should realistically recapitulate the native cell-microenvironment crosstalk that is central for the correct functionality of tissue-engineered constructs. In this review, we will describe the most updated bioengineering approaches to recapitulate the native cell microenvironment and reproduce functional tissues and organs in vitro. We have highlighted the limitations of the use of exogenous scaffolds in recapitulating the regulatory/instructive and signal repository role of the native cell microenvironment. By contrast, strategies to reproduce human tissues and organs by inducing cells to synthetize their own ECM acting as a provisional scaffold to control and guide further tissue development and maturation hold the potential to allow the engineering of fully functional histologically competent three-dimensional (3D) tissues.

Three-dimensional poly(ε-caprolactone) bioactive scaffolds with controlled structural and surface properties.

The requirement of a multifunctional scaffold for tissue engineering capable to offer at the same time tunable structural properties and bioactive interface is still unpaired. Here we present three-dimensional (3D) biodegradable polymeric (PCL) scaffolds with controlled morphology, macro-, micro-, and nano-mechanical performances endowed with bioactive moieties (RGD peptides) at the surface. Such result was obtained by a combination of rapid prototyping (e.g., 3D fiber deposition) and surface treatment approach (aminolysis followed by peptide coupling). By properly designing process conditions, a control over the mechanical and biological performances of the structure was achieved with a capability to tune the value of compressive modulus (in the range of 60-90 MPa, depending on the specific lay-down pattern). The macromechanical behavior of the proposed scaffolds was not affected by surface treatment preserving bulk properties, while a reduction of hardness from 0.50-0.27 GPa to 0.1-0.03 GPa was obtained. The penetration depth of the chemical treatment was determined by nanoindentation measurements and confocal microscopy. The efficacy of both functionalization and the following bioactivation was monitored by analytically quantifying functional groups and/or peptides at the interface. NIH3T3 fibroblast adhesion studies evidenced that cell attachment was improved, suggesting a correct presentation of the peptide. Accordingly, the present work mainly focuses on the effect of the surface modification on the mechanical and functional performances of the scaffolds, also showing a morphological and analytical approach to study the functionalization/bioactivation treatment, the distribution of immobilized ligands, and the biological features.

Fluorescent (rhodamine), folate decorated and doxorubicin charged, PEGylated nanoparticles synthesis.

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.

Processing/structure/property relationship of multi-scaled PCL and PCL-HA composite scaffolds prepared via gas foaming and NaCl reverse templating.

In this study, we investigated the processing/structure/property relationship of multi-scaled porous biodegradable scaffolds prepared by combining the gas foaming and NaCl reverse templating techniques. Poly(ε-caprolactone) (PCL), hydroxyapatite (HA) nano-particles and NaCl micro-particles were melt-mixed by selecting different compositions and subsequently gas foamed by a pressure-quench method. The NaCl micro-particles were finally removed from the foamed systems in order to allow for the achievement of the multi-scaled scaffold pore structure. The control of the micro-structural properties of the scaffolds was obtained by the optimal combination of the NaCl templating concentration and the composition of the CO2-N2 mixture as the blowing agent. In particular, these parameters were accurately selected to allow for the fabrication of PCL and PCL-HA composite scaffolds with multi-scaled open pore structures. Finally, the biocompatibility of the scaffolds has been assessed by cultivating pre-osteoblast MG63 cells in vitro, thus demonstrating their potential applications for bone regeneration.

Solvent and melting induced microspheres sintering techniques: a comparative study of morphology and mechanical properties.

In this work we propose a bottom up approach founded on the assembly of building blocks by solvent induced microparticle sintering to realize multifunctional polymer scaffolds with predefined pore dimension and fully percolative pathway, able to include interspersing microdepot for the release of bioactive molecules. The aim of this study was to develop a versatile method of microspheres sintering based on the partial dissolution of the surface of adjacent microparticles and to compare it with melting induced microspheres sintering, just developed in a previous work. The two techniques were compared in terms of morphology, porosity and mechanical properties. The high potential of customizing the sintering process by the proper selection of the sintering techniques as well as microparticles with different features (e.g., material, size, shape, inner porosity) allows obtaining a wide pattern of micro/nanostructures with bio-inspired mechanical response so satisfying all basic requirements of a "smart" scaffold for bone tissue engineering.

Active targeting of cancer cells by CD44 binding peptide-functionalized oil core-based nanocapsules.

Selectivity in tumor targeting is one of the major issues in cancer treatment. Therefore, surface functionalization of drug delivery systems with active moieties, able to selectively target tumors, has become a worldwide-recognized strategy. The CD44 receptor is largely used as a biomarker, being overexpressed in several tumors, and consequently as a target thanks to the identification of the CD44 binding peptide. Here we implemented the CD44 binding peptide logic onto an oil core-polymer multilayer shell, taking into account and optimizing all relevant features of drug delivery systems, such as small size (down to 100 nm), narrow size distribution, drug loading capability, antifouling and biodegradability. Besides promoting active targeting, the oil core-based system enables the delivery of natural and synthetic therapeutic compounds. Biological tests, using curcumin as a bioactive compound and fluorescent tag, demonstrated that CD44 binding peptide-functionalized nanocapsules selectively accumulate and internalize in cancer cells, compared to the control, thanks to ligand-receptor binding.

Effects of pulsating heat source on interstitial fluid transport in tumour tissues.

Macromolecules and drug delivery to solid tumours is strongly influenced by fluid flow through interstitium, and pressure-induced tissue deformations can have a role in this. Recently, it has been shown that temperature-induced tissue deformation can influence interstitial fluid velocity and pressure fields, too. In this paper, the effect of modulating-heat strategies to influence interstitial fluid transport in tissues is analysed. The whole tumour tissue is modelled as a deformable porous material, where the solid phase is made up of the extracellular matrix and cells, while the fluid phase is the interstitial fluid that moves through the solid matrix driven by the fluid pressure gradient and vascular capillaries that are modelled as a uniformly interspersed fluid point-source. Pulsating-heat generation is modelled with a time-variable cosine function starting from a direct current approach to solve the voltage equation, for different pulsations. From the steady-state solution, a step-variation of vascular pressure included in the model equation as a mass source term via the Starling equation is simulated. Dimensionless 1D radial equations are numerically solved with a finite-element scheme. Results are presented in terms of temperature, volumetric strain, pressure and velocity profiles under different conditions. It is shown that a modulating-heat procedure influences velocity fields, that might have a consequence in terms of mass transport for macromolecules or drug delivery.

Design of porous polymeric scaffolds by gas foaming of heterogeneous blends.

One of the challenges in tissue engineering scaffold design is the realization of structures with a pre-defined multi-scaled porous network. Along this line, this study aimed at the design of porous scaffolds with controlled porosity and pore size distribution from blends of poly(epsilon-caprolactone) (PCL) and thermoplastic gelatin (TG), a thermoplastic natural material obtained by de novo thermoplasticization of gelatin. PCL/TG blends with composition in the range from 40/60 to 60/40 (w/w) were prepared by melt mixing process. The multi-phase microstructures of these blends were analyzed by scanning electron microscopy and dynamic mechanical analysis. Furthermore, in order to prepare open porous scaffolds for cell culture and tissue replacement, the TG and PCL were selectively extracted from the blends by the appropriate combination of solvent and extraction parameters. Finally, with the proposed combination of gas foaming and selective polymer extraction technologies, PCL and TG porous materials with multi-scaled and highly interconnected porosities were designed as novel scaffolds for new-tissue regeneration.

Effect of peristaltic-like movement on bioengineered intestinal tube.

The intestine is a highly heterogeneous hollow organ with biological, mechanical and chemical differences between lumen and wall. A functional human intestine model able to recreate the in vivo dynamic nature as well as the native tissue morphology is demanded for disease research and â€‹drug discovery. Here, we present a system, which combines an engineered three-dimensional (3D) tubular-shaped intestine model (3D In-tube) with a custom-made microbioreactor to impart the key aspects of the in vivo microenvironment of the human intestine, mimicking the rhythmic peristaltic movement. We adapted a previously established bottom-up tissue engineering approach, to produce the 3D tubular-shaped lamina propria and designed a glass microbioreactor to induce the air-liquid interface â€‹condition and peristaltic-like motion. Our results demonstrate the production of a villi-like protrusion and a correct spatial differentiation of the intestinal epithelial cells in enterocyte-like as well as mucus-producing-like cells on the lumen side of the 3D In-tube. This dynamic platform offers a proof-of-concept model of the human intestine.

A BIOPHYSICAL ANALYSIS TO ASSESS X-RAY SENSITIVITY OF HEALTHY AND TUMOUR CELLS.

The mechanobiology is providing novel perspectives in the study of cancer and is contributing to evaluate the cancer responses, from a biophysical point of view, to classical therapeutic approaches- radiotherapy and chemotherapy. Here we have explored the effects of two doses (4 and 8 Gy) of 6 MeV photons on spreading, focal adhesions, migration and mechanical properties of BALB/c 3T3 and their SV40 transformed equivalent, SVT2. Cell biophysical responses to 4 and 8 Gy were analysed and compared with those reported in previous published work when lower doses (1 and 2 Gy) were administered Panzetta et al. (Effects of high energy X-rays on cell morphology and functions. Proc. Book 2017;16:116). We observed that the range of sensitivity to ionising radiations profoundly changes depending on the patho-physiological state of cells. In particular, we found that X-rays induce morphological and functional variations in both cell lines (decreased motility, increased adhesion and increased cytoskeleton stiffness). These changes were slightly dependent on doses in the case of SVT2 cells and may indicate a possible mechanical normalisation in their phenotype. Nevertheless, the responses of BALB/c 3T3 were negligible only for the low dose of 1 Gy and increased significantly in a dose-dependent manner with higher doses. We believe that the characterisation of X-rays effects on the cell mechanobiology could shed new light in the design and customisation of radiotherapy treatments.

Silk-ELR co-recombinamer covered stents obtained by electrospinning.

In the field of tissue engineering the choice of materials is of great importance given the possibility to use biocompatible polymers produced by means of biotechnology. A large number of synthetic and natural materials have been used to this purpose and processed into scaffolds using Electrospinning technique. Among materials that could be used for the fabrication of scaffold and degradable membranes, natural polymers such as collagen, elastin or fibroin offer the possibility to design structures strictly similar to the extracellular matrix (ECM). Biotechnology and genetic engineering made possible the advent of a new class of biopolymers called protein-based polymers. One example is represented by the silk-elastin-proteins that combine the elasticity and resilience of elastin with the high tensile strength of silk-fibroin and display engineered bioactive sequences. In this work, we use electrospinning technique to produce a fibrous scaffold made of the co-recombinamer Silk-ELR. Obtained fibres have been characterized from the morphological point of view. Homogeneity and morphology have been explored using Scanning Electron Microscopy. A thorough study regarding the influence of Voltage, flow rate and distance have been carried out to determine the appropriate parameters to obtain the fibrous mats without defects and with a good distribution of diameters. Cytocompatibility has also been in vitro tested. For the first time we use the co-recombinamer Silk-ELR for the fabrication of a 2.5 angioplasty balloon coating. This structure could be useful as a coated scaffold for the regeneration of intima layer of vessels.

Gene delivery systems for gene therapy in tissue engineering and central nervous system applications.

The present review aims to describe the potential applications of gene delivery systems to tissue engineering and central nervous system diseases. Some key experimental work has been done with interesting results, but the subject is far from being fully explored. The combined approach of gene therapy and material science has a huge potential to improve the therapeutic approaches now available for a wide range of medical applications. Focus is given to this multidisciplinary strategy in neurodegenerative pathologies, where the use of polymeric matrices as gene carriers might make a crucial difference.

Azobenzene-based polymers: emerging applications as cell culture platforms.

The fabrication of biomaterials whose properties are activated or inhibited on demand via light is appealing for fundamental biological studies as well as for the development of new applications in tissue engineering and regenerative medicine. One of the most widely used molecules in light-controlled systems is azobenzene for its ability to isomerise in response to light. In this minireview, the fundamental landmarks towards the application of azobenzene-containing materials as cell culture substrates will be highlighted, foreseeing their massive use as next-generation cell-instructive materials.

The performance of poly-epsilon-caprolactone scaffolds in a rabbit femur model with and without autologous stromal cells and BMP4.

The ability of a cellular construct to guide and promote tissue repair strongly relies on three components, namely, cell, scaffold and growth factors. We aimed to investigate the osteopromotive properties of cellular constructs composed of poly-epsilon-caprolactone (PCL) and rabbit bone marrow stromal cells (BMSCs), or BMSCs engineered to express bone morphogenetic protein 4 (BMP4). Highly porous biodegradable PCL scaffolds were obtained via phase inversion/salt leaching technique. BMSCs and transfected BMSCs were seeded within the scaffolds by using an alternate flow perfusion system and implanted into non-critical size defects in New Zealand rabbit femurs. In vivo biocompatibility, osteogenic and angiogenic effects induced by the presence of scaffolds were assessed by histology and histomorphometry of the femurs, retrieved 4 and 8 weeks after surgery. PCL without cells showed scarce bone formation at the scaffold-bone interface (29% bone/implant contact and 62% fibrous tissue/implant contact) and scarce PCL resorption (16%). Conversely, PCL seeded with autologous BMSCs stimulated new tissue formation into the macropores of the implant (20%) and neo-tissue vascularization. Finally, the BMP4-expressing BMSCs strongly favoured osteoinductivity of cellular constructs, as demonstrated by a more extensive bone/scaffold contact.