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At present just about 30% of the waste plastic collected is efficiently recycled, while the rest is incinerated, disposed in landfills, or can end up in compost and be released in the environment, inducing a very negative effect on safety and health of flora and fauna. Sustainable management of hardly recyclable plastic waste generated by light weight single use packaging and agricultural films can be improved by applying biotechnological approaches, combining microorganisms, new enzymes, earthworms, and insects to work collaboratively, not only to promote the degradation of these plastics but also to obtain, by-products of the biodegradation process to be valorized as fertilizers, functional polysaccharides, etc. In order to develop a feasible process, mapping and characterization of the most diffused agri-food waste plastic were conducted isolating the main types of plastic involved. Plastic waste in agriculture is mainly constituted by polyethylene (PE) both linear low density (LLDPE) and high density (HDPE), polypropylene (PP) and polystyrene (PS), whereas in food packaging polyethylene is still present together with a large presence of polypropylene, polystyrene and polyethylene terephthalate (PET). Combining plastic presence and availability of organisms for their degradability, representative samples of plastics (PE, PET, PS) were selected for analysis of deterioration and potential subsequent biodegradation by enzymes and organisms. To monitor the plastic degradability by enzymes, and larvae, methods for the plastic analysis were set, outlining some differences in virgin and post consumer plastic in particular after use in agriculture, assessing the possibility to monitor the degradability of plastic with time and different treatments, in particular, some evidence of polyethylene degradability from larvae of Tenebrio molitor was observed.

Background: In situ cardiac tissue engineering aims to heal the infarcted myocardium by guiding tissue regeneration within the patient body. A key step in this approach is the design of a bioactive scaffold, able to stimulate tissue repair at the site of damage. In the development of bioactive scaffolds, molecular imprinting nanotechnology has been recently proposed as a new functionalization strategy. Objectives: In this work, Molecularly Imprinted Particles (MIP) with recognition properties towards the stromal-derived factor-1 (SDF-1) were synthesized, characterized and used for the functionalization of a biomimetic scaffold. MIP are expected to favor the enrichment of the SDF-1 bioactive molecule within the scaffold, thereby promoting myocardial regeneration. Methods: MIP were obtained by precipitation polymerization, using the SDF-1 molecule as a template. Alginate/gelatin/elastin sponges were fabricated by freeze-drying and functionalized by MIP deposition. Morphological, physicochemical and functional analyses were performed both on MIP and on MIP-modified scaffolds. A preliminary biological in vitro investigation was also carried out using rat cardiac progenitor cells (rCPCs). Results: Imprinted nanoparticles with an average diameter between 0.6 and 0.9 µm were obtained. Infrared analysis of MIP confirmed the expected chemical structure. Recognition and selectivity tests showed that MIP were able to selectively recognize and rebind the template, even after their deposition on the scaffold. In vitro biological tests showed that cell adhesion to the scaffold was promoted by MIP functionalization. Conclusion: Results obtained in the present study suggest that biomimetic alginate/gelatin/elastin sponges, functionalized by MIP with recognition properties towards SDF-1, could be successfully used for tissue engineering approaches to repair the infarcted heart.

In the present work, a new combination of synthetic and natural biomaterials is proposed for bone tissue engineering (BTE) applications. In order to mimic the inorganic and organic phases of bone extracellular matrix (ECM), a bioactive glass-ceramic deriving from a SiO2–P2O5–CaO–MgO–Na2O–K2O parent glass, acting as a substrate in form of a slice, was surface-functionalised with a type I collagen-based coating. In particular, the collagen was blended with a water soluble polyurethane (PUR), synthesised from poly(ethylene glycol), 1,6-hexamethylene diisocyanate and N-BOC-serinol. The PUR was designed to expose amino groups on the polymeric chain, which can be exploited for the blend stabilisation through crosslinking. The newly synthesised PUR demonstrated to be non-cytotoxic, as assessed by a biological test with MG-63 osteoblast-like cells. The collagen/PUR blend showed good biocompatibility as well. The polymeric coating on the glass-ceramic samples was produced by surface-silanisation, followed by further chemical grafting of the blend, using genipin as a crosslinker. The glass-ceramic surface was characterised at each functionalisation step, demonstrating that the procedure allowed obtaining a covalent link between the blend and the substrate. Finally, biological tests performed using human periosteal derived precursor cells demonstrated that the proposed polymer-coated material was a good substrate for bone cell adhesion and growth, and a good candidate to mimic the composite nature of the bone ECM.

Chemotherapeutics represent the standard treatment for a wide range of cancers. However, these agents also affect healthy cells, thus leading to severe off-target effects. Given the non-selectivity of the commonly used drugs, any increase in the selective tumor tissue uptake would represent a significant improvement in cancer therapy. Recently, the use of gene therapy to completely remove the lesion and avoid the toxicity of chemotherapeutics has become a tendency in oncotherapy. Ideally, the genetic material must be safely transferred from the site of administration to the target cells, without involving healthy tissues. This can be achieved by encapsulating genes into non-viral carriers and modifying their surface with ligands with high selectivity and affinity for a relevant receptor on the target cells. Hence, in this work we evaluate the use of terpolymer-based nanocapsules for the targeted delivery of DNA toward cancer cells. The surface of the nanocapsules is decorated with folic acid to actively target the folate receptors overexpressed on a variety of cancer cells. The nanocapsules demonstrate a good ability of encapsulating and releasing DNA. Moreover, the presence of the targeting moieties on the surface of the nanocapsules favors cell uptake, opening up the possibility of more effective therapies.

The use of injectable scaffolds to repair the infarcted heart is receiving great interest. Thermosensitive polymers, in situ polymerization, in situ cross-linking, and self-assembling peptides are the most investigated approaches to obtain injectability.Aim of the present work was the preparation and characterization of a novel bioactive scaffold, in form of injectable microspheres, for cardiac repair. Gellan/gelatin microspheres were prepared by a water-in-oil emulsion and loaded by adsorption with Insulin-like growth factor 1 to promote tissue regeneration. Obtained microspheres underwent morphological, physicochemical and biological characterization, including cell culture tests in static and dynamic conditions and in vivo tests. Morphological analysis of the microspheres showed a spherical shape, a microporous surface and an average diameter of 66 ± 17µm (under dry conditions) and 123 ± 24 µm (under wet conditions). Chemical Imaging analysis pointed out a homogeneous distribution of gellan, gelatin and Insulin-like growth factor-1 within the microsphere matrix. In vitro cell culture tests showed that the microspheres promoted rat cardiac progenitor cells adhesion, and cluster formation. After dynamic suspension culture within an impeller-free bioreactor, cells still adhered to microspheres, spreading their cytoplasm over microsphere surface. Intramyocardial administration of microspheres in a cryoinjury rat model attenuated chamber dilatation, myocardial damage and fibrosis and improved cell homing.Overall, the findings of this study confirm that the produced microspheres display morphological, physicochemical, functional and biological properties potentially adequate for future applications as injectable scaffold for cardiac tissue engineering.

In recent years, there has been an increasing interest toward the covalent binding of bioactive peptides from extracellular matrix proteins on scaffolds as a promising functionalization strategy in the development of biomimetic matrices for tissue engineering. A totally new approach for scaffold functionalization with peptides is based on Molecular Imprinting technology. In this work, imprinted particles with recognition properties toward laminin and fibronectin bioactive moieties were synthetized and used for the functionalization of biomimetic sponges, which were based on a blend of alginate, gelatin, and elastin. Functionalized sponges underwent a complete morphological, physicochemical, mechanical, functional, and biological characterization. Micrographs of functionalized sponges showed a highly porous structure and a quite homogeneous distribution of imprinted particles on their surface. Infrared and thermal analyses pointed out the presence of interactions between blend components. Biodegradation and mechanical properties appeared adequate for the aimed application. The results of recognition tests showed that the deposition on sponges did not alter the specific recognition and binding behavior of imprinted particles. In vitro biological characterization with cardiac progenitor cells showed that early cell adherence was promoted. In vivo analysis showed that developed scaffolds improved cardiac progenitor cell adhesion and differentiation toward myocardial phenotypes.

Bioactive materials should maintain their properties during implantation and for long time in contact with physiological fluids and tissues. In the present research, five different bioactive materials (a bioactive glass and four different chemically treated bioactive titanium surfaces) have been studied and compared in terms of mechanical stability of the surface bioactive layer-substrate interface, their long term bioactivity, the type of hydroxyapatite matured and the stability of the hydroxyapatite-surface bioactive layer interface. Numerous physical and chemical analyses (such as Raman spectroscopy, macro and micro scratch tests, soaking in SBF, Field Emission Scanning Electron Microscopy equipped with Energy Dispersive Spectroscopy (SEM-EDS), zeta potential measurements and Fourier Transformed Infra-Red spectroscopy (FTIR) with chemical imaging) were used. Scratch measurements evidenced differences among the metallic surfaces concerning the mechanical stability of the surface bioactive layer-substrate interface. All the surfaces, despite of different kinetics of bioactivity, are covered by a bone like carbonate-hydroxyapatite with B-type substitution after 28 days of soaking in SBF. However, the stability of the apatite layer is not the same for all the materials: dissolution occurs at pH around 4 (close to inflammation condition) in a more pronounced way for the surfaces with faster bioactivity together with detachment of the surface bioactive layer. A protocol of characterization is here suggested to predict the implant-bone interface stability.

Introduction: Injectable scaffolds are emerging as a promising strategy in the field of myocardial tissue engineering. Among injectable scaffolds, microparticles have been poorly investigated. The goal of this study was the development of novel gelatin/gellan microparticles that could be used as an injectable scaffold to repair the infarcted myocardium. In particular, the effect of particle size on cardiac progenitor cell response was investigated. Methods: Particles were produced by a water-in-oil emulsion method. Phosphatidylcholine was used as a surfactant. Particles with different diameter ranges (125–300 µm and 350–450 µm) were fabricated using two different surfactant concentrations. Morphological, physicochemical, and functional characterizations were carried out. Cardiac progenitor cell adhesion and growth on microparticles were tested both in static and dynamic suspension culture conditions. Results: Morphological analysis of the produced particles showed a spherical shape and porous surface. The hydrophilicity of particle matrix and the presence of intermolecular interactions between gellan and gelatin were pointed out by the physicochemical characterization. A weight loss of 75 ± 5 % after 90 days of hydrolytic degradation was observed. Injectability through a narrow needle (26 G) and persistence of the microparticles at the injection site were preliminarily verified by ex vivo test. In vitro cell culture tests showed a preservation of rat cardiac progenitor biologic properties and indicated a preferential cell adherence to microparticles with a smaller size. Conclusion: Overall, the obtained results indicate that the produced gelatin/gellan microparticles could be potentially employed as injectable scaffolds for myocardial regeneration.

Tissue engineering has emerged as a viable approach to treat disease or repair damage in tissues and organs. One of the key elements for the success of tissue engineering is the use of a scaffold serving as artificial extracellular matrix (ECM). The ECM hosts the cells and improves their survival, proliferation, and differentiation, enabling the formation of new tissue. Here, we propose the development of a class of protein/polysaccharide-based porous scaffolds for use as ECM substitutes in cardiac tissue engineering. Scaffolds based on blends of a protein component, collagen or gelatin, with a polysaccharide component, alginate, were produced by freeze-drying and subsequent ionic and chemical crosslinking. Their morphological, physicochemical, and mechanical properties were determined and compared with those of natural porcine myocardium. We demonstrated that our scaffolds possessed highly porous and interconnected structures, and the chemical homogeneity of the natural ECM was well reproduced in both types of scaffolds. Furthermore, the alginate/gelatin (AG) scaffolds better mimicked the native tissue in terms of interactions between components and protein secondary structure, and in terms of swelling behavior. The AG scaffolds also showed superior mechanical properties for the desired application and supported better adhesion, growth, and differentiation of myoblasts under static conditions. The AG scaffolds were subsequently used for culturing neonatal rat cardiomyocytes, where high viability of the resulting cardiac constructs was observed under dynamic flow culture in a microfluidic bioreactor. We therefore propose our protein/polysaccharide scaffolds as a viable ECM substitute for applications in cardiac tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 769-781, 2018.

A large number of pathologies require the resection of the bowel and anastomoses to rejoin the two remaining stumps to regain lumen patency. Various materials have been used to rejoin one bowel end to the other such as catgut, stainless steel, and absorbable sutures. The present method for anastomosis surgery uses an entero-entero anastomosis (EEA) circular stapler with only a staple line. This method can have some drawbacks, such as intracellular fluid leakage and local inflammations. The aim of this study is to design and develop a novel bioartificial polymer with a ring shape made of polyvinyl alcohol (PVA) and gelatin (80/20 ratio (w/w)) loaded both directly with acetylsalicylic acid and with nanoparticles incorporating the same drug to reduce local inflammation even for a prolonged period of time. A physical method (8 cycles freezing/thawing) was used to obtain a crosslinked bioartificial shape memory ring. Mechanical analysis showed a storage modulus having a comparable value with that of the human colon. HPLC analysis pointed out a sustained and prolonged release of the anti-inflammatory drug both immediately after anastomosis surgery and during healing period. Cell culture tests indicated the cytocompatibility of the bioartificial device. A shape memory of the hydrogel prepared in ring form was observed at 37 °C after immersion in water. These bioartificial devices can represent a new approach to serve as a multifunctional anastomotic ring.

The purpose of this work was the preparation and characterization of polymeric membranes for the selective recognition of saccharides using molecular imprinting technology associated to phase inversion. A system able to bind saccharides with high selectivity is particularly important in the pharmaceutical sector, since some of these compounds are constituents of molecules which can exert serious toxic effects even at very low concentrations. Two polymeric matrices were prepared using poly(ethylene-co-vinyl alcohol) copolymers, with an ethylene molar content of 32% and 44%, and imprinted with two different saccharide molecules: maltose and 2-keto 3-deoxy-D-manno-octulosonate (KDO). Matrices imprinted against maltose and KDO showed an easy template extraction, high binding capability and satisfactory selectivity, particularly for matrix with an ethylene molar content of 44%.

A safe and effective use of nanoparticles in biology and medicine requires a thorough understanding, down to the molecular level, of how nanoparticles interact with cells in the physiological environment. This study evaluated the two-way interaction between inorganic nanomaterials (INMs) and cells from A549 human lung carcinoma cell line. The interaction between silica and zinc oxide INMs and cells was investigated using both standard methods and advanced characterization techniques. The effect of INMs on cell properties was evaluated in terms of cell viability, chemical modifications, and volume changes. The effect of cells and culture medium on INMs was evaluated using dynamic light scattering (DLS), scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDS), high performance liquid chromatography (HPLC), gas chromatography-mass spectroscopy (GC-MS), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). No cytotoxic effect was detected in the case of silicon oxide INMs, while for high doses of zinc oxide INMs a reduction of cell survival was observed. Also, increased cell volume was recorded after 24 h incubation of cells with zinc oxide INMs. A better dimensional homogeneity and colloidal stability was observed by DLS for silicon oxide INMs than for zinc oxide INMs. SEM-EDS analysis showed the effectiveness of the adopted dispersion procedure and confirmed in the case of zinc oxide INMs the presence of residual substances derived from organosilane coating. HPLC and GC-MS performed on INMs aqueous dispersions after 24 h incubation showed an additional peak related to the presence of an organic contaminant only in the case of zinc oxide INMs. FTIR Chemical Imaging carried out directly on the cells showed, in case of incubation with zinc oxide INMs, a modification of the spectra in correspondence of phospholipids, nucleic acids and proteins characteristic absorption bands when compared with untreated cells. Overall, our results confirm the importance of developing new experimental methods and techniques for improving the knowledge about the biosafety of nanomaterials.

Adequate mechanical properties to withstand the surgical procedure and decoration with bioactive molecules promoting tissue regeneration are crucial aspects in the development of successful matrice...

Bioactive materials, able to induce hydroxyapatite precipitation in contact with body fluids, are of great interest for their bone bonding capacity. . The aim of this paper is to compare bioactive materials with different surface features to verify the mechanisms of action and the relationship with kinetics and type of precipitated hydroxyapatite over time. Four different surface treatments for Ti/Ti6Al4V alloy and a bioactive glass were selected and a different mechanism of bioactivity is supposed for each of them. Apart from the conventional techniques (FESEM, XPS and EDX), less common characterizations (zeta potential measurements on solid surfaces and FTIR chemical imaging) were applied. The results suggest that the OH groups on the surface have several effects: the total number of the OH groups mainly affects hydrophilicity of surfaces, while the isoelectric points, surface charge and ions attraction mainly depend on OH acidic/basic strength. Kinetics of hydroxyapatite precipitation is faster when it involves a mechanism of ion exchange while it is slower when it is due to electrostatic effects . The electrostatic effect cooperates with ion exchange and it speeds up kinetics of hydroxyapatite precipitation. Different bioactive surfaces are able to differently induce precipitation of type A and B of hydroxyapatite, as well as different degrees of crystallinity and carbonation. STATEMENT OF SIGNIFICANCE: The bone is made of a ceramic phase (a specific type of hydroxyapatite), a network of collagen fibers and the biological tissue. A strong bond of an orthopedic or dental implant with the bone is achieved by bioactive materials where precipitation and growth of hydroxyapatite occurs on the implant surface starting from the ions in the physiological fluids. Several bioactive materials are already known and used, but their mechanism of action is not completely known and the type of precipitated hydroxyapatite not fully investigated. In this work, bioactive titanium and bioglass surfaces are compared through conventional and innovative methodologies. Different mechanisms of bioactivity are identified, with different kinetics and the materials are able to induce precipitation of different types of hydroxyapatite, with different degree of crystallinity and carbonation.

Cystic fibrosis (CF) is a progressive genetic disease caused by mutations in the gene that produces the CF transmembrane conductance regulator (CFTR) protein. The malfunction of the CFTR protein causes a thick buildup of mucus in the lungs that clogs the airways and traps bacteria, thus leading to infections, extensive lung damage and respiratory failure. Micro-delivery systems are currently being investigated as an efficient way to cross the viscous and complex architecture of the CF mucus. In this study, we produced synthetic and natural microparticles (MPs) based on poly(dl‑lactide‑co‑glycolide) (PLGA) or gellan gum through tailored water/oil emulsion procedures. Morphological and physico-chemical characterizations were carried out on both classes of MPs showing particles having diameters within suitable ranges to reach the CF airways. In vitro biocompatibility tests were also performed on both MPs using a human lung cancer cell line (A549) demonstrating that treatment with MPs induces no cytotoxic effects. Both classes of MPs were loaded with a mucolytic agent (N‑acetyl cysteine, NAC) and their release kinetics evaluated using high performance liquid chromatography (HPLC). The analysis pointed out that the amount of NAC released from MPs resulted in a dose-dependent increment, with a rapid release kinetic to satisfy the requirement for inducing an early mucus degradation. Finally, mucolytic action of NAC-loaded MPs was evaluated in an artificial sputum model through its rheological analysis obtaining the lowest viscosity profile after the addition of drug-loaded MPs. Taken together, gained results allowed us to select suitable MPs as potential drug targeting platforms having a mucolytic action for CF treatment.

3D scaffolds used to repair damaged tissues should be able to mimic both composition and functions of natural extracellular matrix, which is mainly composed of polysaccharides and proteins. In our previous research new biomimetic sponges, based on blends of alginate with gelatin, were produced and characterized for myocardial tissue engineering applications. It was observed that these scaffolds can potentially function as a promising cardiac extracellular matrix substitute, but a reinforcement is required to improve their suturing properties. Aim of the present work was the development of a suturable biomimetic patch by the inclusion of a synthetic mesh within an alginate/gelatin scaffold. The mesh, produced by dry spinning, was made of eight superimposed layers of polycaprolactone microfibers, each one rotated of 45° with respect to the adjacent one. Reinforced scaffolds were obtained through the use of a mold, specially designed to place the fibrous mesh exactly in the center of the sponge. Both the reinforcement mesh and the reinforced scaffold were characterized. A perfect integration between the mesh and the sponge was observed. The fibrous mesh reduced the capacity of the sponge to absorb water, but the degree of hydrophilicity of the material was still comparable with that of natural cardiac tissue. The reinforced system showed a suitable stability in aqueous environment and it resulted much more resistant to suturing than not reinforced scaffold and even than human arteries. Polycaprolactone mesh was not cytotoxic and the reinforced scaffold was able to support cardiomyocytes adhesion and proliferation. Overall, the obtained results confirmed that the choice to modify the alginate/gelatin sponges through the insertion of an appropriate reinforcement system turned out to be correct in view of their potential use in myocardial tissue engineering.

The protection from ischaemia‐reperfusion‐associated myocardial infarction worsening remains a big challenge. We produced a bioartificial 3D cardiac patch with cardioinductive properties on stem cells. Its multilayer structure was functionalised with clinically relevant doses of adenosine. We report here the first study on the potential of these cardiac patches in the controlled delivery of adenosine into the in vivo ischaemic‐reperfused pig heart. A Fourier transform infrared chemical imaging approach allowed us to perform a characterisation, complementary to the histological and biochemical analyses on myocardial samples after in vivo patch implantation, increasing the number of investigations and results on the restricted number of pigs (n = 4) employed in this feasibility step. In vitro tests suggested that adenosine was completely released by a functionalised patch, a data that was confirmed in vivo after 24 hr from patch implantation. Moreover, the adenosine‐loaded patch enabled a targeted delivery of the drug to the ischaemic‐reperfused area of the heart, as highlighted by the activation of the pro‐survival signalling reperfusion injury salvage kinases pathway. At 3 months, though limited to one animal, the used methods provided a picture of a tissue in dynamic conditions, associated to the biosynthesis of new collagen and to a non‐fibrotic outcome of the healing process underway. The synergistic effect between the functionalised 3D cardiac patch and adenosine cardioprotection might represent a promising innovation in the treatment of reperfusion injury. As this is a feasibility study, the clinical implications of our findings will require further in vivo investigation on larger numbers of ischaemic‐reperfused pig hearts.

Supplemental material, Supplement_Material_(Fig_1_and_2) for Influence of injectable microparticle size on cardiac progenitor cell response by Elisabetta Rosellini, Niccoletta Barbani, Caterina Frati, Denise Madeddu, Diana Massai, Umberto Morbiducci, Luigi Lazzeri, Angela Falco, Costanza Lagrasta, Alberto Audenino, Maria Grazia Cascone and Federico Quaini in Journal of Applied Biomaterials & Functional Materials

Evolution of biomaterials for implants progressively shifted the focus from adequate mechanical strength to improved biocompatibility and absence of toxicity and, finally, to fast tissue integration. Recently, new frontiers and challenges of titanium implants have been addressed with the focus on bioactivity and fighting bacterial infection and biofilm formation. This is closely related to the clinical demand of multifunctional implants able to simultaneously have a number of specific responses with respect to body fluids, cells and pathogenic agents. Nanotechnologies can have a substantial and effective impact in regulating the tissue-implant interface through controlled topography and surface functionalization at the nanoscale and some of them are here explored. Titanium and titanium alloys surface treated in order to be bioactive (induced precipitation of hydroxyapatite in contact with physiologic fluids) and antibacterial are here characterized and compared. They have in common surface topography at the nanoscale and functionalization with antibacterial metal ions or nanoparticles, but differ in surface reactivity in contact with the bodyfluids (simulated through SBF-Simulated Body Fluid, albumin and/or hydrogen peroxide complex solutions). The focus is on comparison of their mechanisms of action, kinetics of surface activity and biological response. A bioactive glass belonging to the system SiO2-Na2O-CaO-P2O5-B2O3-Al2O3 is used as reference for comparison with a classic bioactive material; the same glass doped with silver ions by ion exchange in aqueous solutions of silver nitrate was used as reference of antibacterial action through Ag ions release. Protocols of analysis suitable for evaluating and comparing the mechanisms of bioactivity and antibacterial action (FESEM, Raman, XPS, electrokinetic and electrophoretic zeta potential measurements), kinetics of surface reactivity (release of antibacterial agents, FTIR, cross section observation after soaking in SBF), biocompatibility (corrosion resistance and ion release in complex solutions) and biological response (biofilm formation, osteoblast adhesion and differentiation) of a wide range of inorganic biomaterials have been assessed and are here described and discussed.

Tissue engineering has emerged as a viable approach to treat disease or repair damage in tissues and organs. One of the key elements for the success of tissue engineering is the use of a scaffold serving as artificial extracellular matrix (ECM). The ECM hosts the cells and improves their survival, proliferation, and differentiation, enabling the formation of new tissue. Here, we propose the development of a class of protein/polysaccharide-based porous scaffolds for use as ECM substitutes in cardiac tissue engineering. Scaffolds based on blends of a protein component, collagen or gelatin, with a polysaccharide component, alginate, were produced by freeze-drying and subsequent ionic and chemical crosslinking. Their morphological, physicochemical, and mechanical properties were determined and compared with those of natural porcine myocardium. We demonstrated that our scaffolds possessed highly porous and interconnected structures, and the chemical homogeneity of the natural ECM was well reproduced in both types of scaffolds. Furthermore, the alginate/gelatin (AG) scaffolds better mimicked the native tissue in terms of interactions between components and protein secondary structure, and in terms of swelling behavior. The AG scaffolds also showed superior mechanical properties for the desired application and supported better adhesion, growth, and differentiation of myoblasts under static conditions. The AG scaffolds were subsequently used for culturing neonatal rat cardiomyocytes, where high viability of the resulting cardiac constructs was observed under dynamic flow culture in a microfluidic bioreactor. We therefore propose our protein/polysaccharide scaffolds as a viable ECM substitute for applications in cardiac tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 769-781, 2018.

Cardiovascular diseases, especially myocardial infarction, are the leading cause of morbidity and mortality in the world, also resulting in huge economic burdens on national economies. A cardiac patch strategy aims at regenerating an infarcted heart by providing healthy functional cells to the injured region via a carrier substrate, and providing mechanical support, thereby preventing deleterious ventricular remodeling. In the present work, polyaniline (PANI) was doped with camphorsulfonic acid and blended with poly(glycerol-sebacate) at ratios of 10, 20 and 30 vol.% PANI content to produce electrically conductive composite cardiac patches via the solvent casting method. The composites were characterized in terms of their electrical, mechanical and physicochemical properties. The in vitro biodegradability of the composites was also evaluated. Electrical conductivity increased from 0 S cm−1 for pure PGS to 0.018 S cm−1 for 30 vol.% PANI–PGS samples. Moreover, the conductivities were preserved for at least 100 h post fabrication. Tensile tests revealed an improvement in the elastic modulus, tensile strength and elasticity with increasing PANI content. The degradation products caused a local drop in pH, which was higher in all composite samples compared with pure PGS, hinting at a buffering effect due to the presence of PANI. Finally, the cytocompatibility of the composites was confirmed when C2C12 cells attached and proliferated on samples with varying PANI content. Furthermore, leaching of acid dopants from the developed composites did not have any deleterious effect on the viability of C2C12 cells. Taken together, these results confirm the potential of PANI–PGS composites for use as substrates to modulate cellular behavior via electrical stimulation, and as biocompatible scaffolds for cardiac tissue engineering applications.

The objective of this study was the preparation and physico-chemical, mechanical, biological, and functional characterization of a multifunctional coating for an innovative, fully implantable device. The multifunctional coating was designed to have three fundamental properties: adhesion to device, close mechanical resemblance to human soft tissues, and control of the inflammatory response and tissue repair process. This aim was fulfilled by preparing a multilayered coating based on three components: a hydrophilic primer to allow device adhesion, a poly(vinyl alcohol) hydrogel layer to provide good mechanical compliance with the human tissue, and a layer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fibers. The use of biopolymer fibers offered the potential for a long-term interface able to modulate the release of an anti-inflammatory drug (dexamethasone), thus contrasting acute and chronic inflammation response following device implantation. Two copolymers, poly(vinyl acetate-acrylic acid) and poly(vinyl alcohol-acrylic acid), were synthetized and characterized using thermal analysis (DSC, TGA), Fourier transform infrared spectroscopy (FT-IR chemical imaging), in vitro cell viability, and an adhesion test. The resulting hydrogels were biocompatible, biostable, mechanically compatible with soft tissues, and able to incorporate and release the drug. Finally, the multifunctional coating showed a good adhesion to titanium substrate, no in vitro cytotoxicity, and a prolonged and controlled drug release.

In this study biomimetic poly(glycerol sebacate) PGS matrix was developed for cardiac patch application. The rationale was that such matrices would provide conducive environment for the seeded cells at the interphase with PGS. From the microstructural standpoint, PGS was fabricated into dense films and porous PGS scaffolds. From the biological aspect, biomimetic PGS membranes were developed via covalently binding peptides Tyr-Ile-Gly-Ser-Arg (YIGSR) and Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), corresponding to the epitope sequences of laminin and fibronectin, respectively onto the surface. To improve and enhance homogenous binding of peptides onto the PGS surface, chemical modification of its surface was carried out. A sequential regime of alkaline hydrolysis with 0.01 M NaOH for 5 min and acidification with 0.01 M HCl for 25 s was optimal. More COOH chemical group was exposed without causing deleterious effect on the bulk properties of the polymer as revealed by the physicochemical analysis carried out. HPLC analysis, chemical imaging and ToF-SIMS were able to establish the successful homogenous functionalization of PGS membranes with the peptides. Finally, the developed biomimetic membranes supported the adhesion and growth of rat and human cardiac progenitor cells.

The influence of conventional sterilization methods such as gamma irradiation, ethylene oxide treatment and steam exposure on the physical properties of poly(glycerol sebacate) (PGS) was investigated. Scanning electron microscope (SEM) observations, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and Raman spectra demonstrated that the applied sterilization methods do not induce adverse surface modifications. The sterilized samples also maintained their thermal and mechanical properties post-sterilization. In addition, gamma sterilized PGS films did not induce any toxicity when films were in contact with murine fibroblast 3T3 cells. PGS is compatible for all the sterilization methods used in this study, which is of relevance for the wider application of PGS in medical devices.

This study covers the preparation of microspheres for the controlled and targeted release of paclitaxel, using novel degradable polymers as carrier materials. Paclitaxel-loaded microspheres were prepared by oil-in-water single-emulsion solvent extraction/evaporation technique by using a series of polyurethanes and a block copolymer; the physicochemical properties of these polymers were modulated by changing nature and composition of their structural units. The obtained microparticles showed a regular morphology and properties (diameter: 1–100 µm; resuspension index: 18.8–100%; encapsulation efficiency: 26.6–97.2%) depending on polymer hydrophilicity and emulsifier used. In vitro release curves showed in all cases almost zero-order kinetics after an initial low burst effect (from 1 to 8.4%), which is required to minimize the drug side effects. This work also proposes a novel strategy to combine a controlled and a targeted release through the functionalization of the polymer matrix with peptide sequences. An RGD-functionalized polyurethane was used to successfully prepare paclitaxel-loaded microparticles. Studies on the preparation of polymer microspheres are reported. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

The biomaterial scaffold plays a key role in most tissue engineering strategies. Its surface properties, micropatterning, degradation, and mechanical features affect not only the generation of the tissue construct in vitro, but also its in vivo functionality. The area of myocardial tissue engineering still faces significant difficulties and challenges in the design of bioactive scaffolds, which allow composition variation to accommodate divergence in the evolving myocardial structure. Here we aimed at verifying if a microstructured bioartificial scaffold alone can provoke an effect on stem cell behavior. To this purpose, we fabricated microstructured bioartificial polymeric constructs made of PLGA/gelatin mimicking anisotropic structure and mechanical properties of the myocardium. We found that PLGA/gelatin scaffolds promoted adhesion, elongation, ordered disposition, and early myocardial commitment of human mesenchymal stem cells suggesting that these constructs are able to crosstalk with stem cells in a precise and controlled manner. At the same time, the biomaterial degradation kinetics renders the PLGA/gelatin constructs very attractive for myocardial regeneration approaches.

Background: The accumulation of amyloid beta protein in the brain causes the cognitive impairment observed in neurodegenerative pathologies such as Alzheimer's disease. The present study aimed to test the hypothesis that a rapid removal of amyloid beta protein peptides from the blood by an extracorporeal purification system could represent an alternative solution for the treatment of patients suffering from this neurodegenerative disease. Methods: In this regard, we investigated the specific recognition properties of a molecularly imprinted membrane based on poly(ethylene-co-vinyl alcohol) toward the amyloid beta protein fragment 25-35 (AbP), the more neurotoxic domain of amyloid beta protein. A chemical modification of the copolymer backbone using succinic anhydride was also performed to favor the formation of carboxylic groups and thus improve imprinting performance. Results: The physico-chemical, morphological, mechanical and functional characterisations gave interesting results confirming the ability of imprinted membranes to in vitro rebind AbP. Conclusions: This work represents a proof of concept regarding the development of a biocompatible polymer membrane capable of selectively removing amyloid beta peptide from the blood and consequently from the cerebrospinal fluid.

Over the past decade, a large number of strategies and technologies have been developed to reduce heart failure progression. Among these, cardiac tissue engineering is one of the most promising. Aim of this study is to develop a 3D scaffold to treat cardiac failure. A new three-block copolymer, obtained from δ-valerolactone and polyoxyethylene, was synthesised under high vacuum without catalyst. Copolymer/gelatine blends were microfabricated to obtain a ECM-like geometry. Structures were studied under morphological, mechanical, degradation and biological aspects. To prevent left ventricular remodelling, constructs were biofunctionalises with molecularly imprinted nanoparticles towards the matrix metalloproteinase MMP-9. Results showed that materials are able to reproduce the ECM structure with high resolution, mechanical properties were in the order of MPa similar to those of the native myocardium and cell viability was verified. Nanoparticles showed the capability to rebind MMP-9 (specific rebinding 18.67) and to be permanently immobilised on the scaffold surface.

The bioartificial chitosan–poly(vinyl alcohol) blends were toughened by means of a dehydrothermal treatment (DHT), to facilitate the formation of hydrogen bonds between the macromolecules. The materials were characterized by stress–strain test, contact angle measurement, spotlight Fourier transform infrared spectroscopy and chemical imaging, weight loss in water, swelling in water vapor saturated atmosphere, Alamar blue test to evaluate the indirect cytotoxicity, and the diffusive permeation, through membranes made with the blends, of D(+)glucose, vitamin B12, and bovine serum albumin. The results were compared with those of the blends crosslinked by glutaraldehyde (GTA). The Young's modulus ranges between 10.56 and 16.12 MPa, and it is higher for the blends subjected to DHT than for those crosslinked by GTA, a fact explainable by the elasticity of the latter, due to the flexible bridges connecting the different chains. The contact angles indicate a scarce wettability, and then a scarce hydrophilicity, which is confirmed by the chemical imaging of the surfaces, made in the total reflection (microATR) mode, of the films toughened by DHT: the ν(OH) band in the 4000–3000 cm-1 is nearly absent in all the regions of the maps. Moreover, the correlation maps indicate a homogeneous distribution of the two components within the blends. The weight loss in water is generally less than 15%, and increases with the content of the ionizable macromolecule chitosan in the blends, a trend shown also by the swelling after exposure to water vapors. Alamar blue test shows that none of the eluates, after being in contact with the materials up to seven days, appears cytotoxic toward murine fibroblasts. As concerning the diffusive permeation, it appears good for D(+)glucose, quite good for vitamin B12, and scarce for bovine serum albumin. In conclusion, the chitosan–poly(vinyl alcohol) blends studied appear to be suitable for their use as biomaterials.

In recent years, research in the field of myocardial tissue engineering has advanced thanks to the development of new biomaterials and a more clear understanding of processes that are at the basis of cardiac tissue growth. However, classical porous scaffolds developed during these years to try to reconstruct and mimic heart function have proven to be inadequate because they are not able to reproduce the typical myocardial environment. One approach to increase functionality of tissue-engineered constructs relies on attempts to mimic the microarchitecture of natural tissues, since it is well known that topology is one of the principal stimuli that cells need to activate their functions. The aim of this work was the realization of three-dimensional microfabricated scaffolds, with cardiac extracellular matrix (ECM)-like architecture. For this purpose, samples of pig myocardium were decellularized, embedded in paraffin wax and analyzed under an optical microscope, in order to evaluate the geometrical features of the cardiac ECM. On the basis of these data, a simplified model of the cardiac ECM microarchitecture was designed. Microfabricated scaffolds were realized with Soft Lithography technique, using a bioartificial blend, based on alginate, gelatin and a novel poly(N-isopropylacrylamide)-based copolymer, which we synthesized. The scaffolds were characterized in terms of topological and mechanical properties. Moreover, cell adhesion, proliferation, and differentiation tests were performed. The microfabricated scaffolds showed they matched the anisotropic mechanical properties of adult human left ventricular myocardium, while at the same time being able to promote myoblast alignment in the absence of external stimuli.

The aim of this work was the preparation of blends based on alginate and gelatin, with different weight ratio, to combine the advantages of these two natural polymers for application in cardiac tissue engineering. The physicochemical characterization, performed by Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analysis, revealed a good miscibility and the presence of interactions among the functional groups of pure biopolymers. Concerning the swelling and degradation tests, performed in different solutions simulating body fluids, both swelling degree and weight losses were higher in phosphate buffer saline (PBS) and for the blends with a higher content of gelatin. These results indicated a better stability of the blends in cell culture medium than in PBS and suggested a mainly hydrolytic degradation process. Cell culture tests, carried out using C2C12 myoblasts, showed a good cell proliferation for all the blends containing more than 60% of gelatin, with the alginate/gelatin 20:80 showing the best response. The same blend was the only one on which cell differentiation was observed. The results obtained in the biological characterization allow to select the alginate/gelatin 20:80 blend as a suitable material to prepare scaffolds for myocardial tissue engineering.

In this work, bioartificial binary blends between poly(e-caprolactone) (PCL) and a polysaccharide (chitosan (CS) or starch (S)) with different contents of the natural polymer (5–30 wt.%) were produced. Melt-mixing and double-precipitation were the methods used for the obtainment of PCL/S and PCL/CS blends, respectively. Tubular scaffolds were produced from bioartificial blends by melt-extrusion. Physico-chemical characterisation was performed by differential scanning calorimetry analysis (DSC), thermogravimetry (TGA), scanning electron microscopy (SEM), infrared analysis (FTIR-ATR and micro-ATR mapping), atomic force microscopy (AFM) and stress–strain tests. Blends were not miscible, phase-separated systems, showing a homogeneous composition and morphology only at low polysaccharide content (≤ 10 wt.%). The biocompatibility of bioartificial guides was investigated by culturing NIH-3T3 mouse fibroblasts. Cells response showed the following order: PCL/S > PCL > PCL/CS. For each blend type, biocompatibility increased with decreasing the polysaccharide content. In vitro cell tests using S5Y5 neuroblastoma cells, carried out on the most biocompatible blends, assessed their absence of cytotoxicity towards these model cells of the nervous tissue. Results showed that blends with a low chitosan or starch content (≤ 10 wt.%) are promising for the regeneration of tissues requiring tubular scaffolds, such as the peripheral nerves.

In the present work the synthesis and the chemical and thermal characterization of poly(methyl methacrylate-co-butyl methacrylate) copolymer, in three different macromolecular compositions, are reported. The aim of the present work was the identification of a standard method to obtain copolymers with controlled macromolecular composition, molecular weights and particle size distribution, together with the identification of the effect of the macromolecular composition on the material properties. A monomer-starved seeded semi-batch emulsion reaction was carried out and optimized, monitoring the kinetic of the copolymerization through the evaluation of residual monomer amounts. Then, an evaluation of the macromolecular composition was performed by Fourier transform infrared spectroscopy analysis. Molecular weight, molecular weight distribution, latex characteristics and thermal behaviour were also investigated.

Bioartificial biodegradable materials were prepared mixing chitosan (CHI) and poly(vinyl alcohol) (PVA), then manufactured as films, and finally cross-linked with glutaraldehyde (GTA), both in the absence and in the presence of the edible hexa-alcohol sorbitol (SOR), as a plasticizer. The release of the components into water was tested by high performance liquid chromatography (HPLC); no release of CHI and scarce release of PVA were found. The water uptake was tested by measuring the swelling of the materials, after incubating them for 20 h in an atmosphere saturated with water vapor at 37°C. The swelling percentage increases with increasing CHI content in the blends, although it is the less hydrophilic polymer. This behavior was attributed to the difficulty of water to diffuse through the crystalline PVA structure, which is partially altered in the blends. The addition of SOR enhances the water sorption, as expected. The biodegradability of the materials was tested using the specific enzyme chitosanase, and was found to depend on the blend composition, as well as to be enhanced by the addition of SOR. The initial degradation rates were calculated; the maximum rates were found when the CHI to PVA ratio was 80:20 for all systems. The results of the enzymatic degradation generally agree with those of the swelling. The cross-linked blends were also tested as drug-delivery systems. The drugs chosen were the vitamin L-ascorbic acid (AsA) and the anti-cancer drug paclitaxel (PTX). The effective diffusion coefficients, D eff , were evaluated for the release of both the drugs from each material. Those of AsA are greater, of many powers of ten, than those of PTX, owing mainly to the hydrophilic nature of the first drug and to the hydrophobic of the second one. In conclusion, these materials seem available for biomedical use.

Molecular imprinting is a technique for the synthesis of polymers capable to bind target molecules selectively. The imprinting of large proteins, such as cell adhesion proteins or cell receptors, opens the way to important and innovative biomedical applications. However, such molecules can incur into important conformational changes during the preparation of the imprinted polymer impairing the specificity of the recognition cavities. The “epitope approach” can overcome this limit by adopting, as template, a short peptide sequence representative of an accessible fragment of a larger protein. The resulting imprinted polymer can recognize both the template and the whole molecule thanks to the specific cavities for the epitope. In this work two molecularly imprinted polymer formulations (a macroporous monolith and nanospheres) were obtained using the protected peptide Z-Thr-Ala-Ala-OMe, as template, and Z-Thr-Ile-Leu-OMe, as analogue for the selectivity evaluation, methacrylic acid, as functional monomer, and trimethylolpropane trimethacrylate and pentaerythritol triacrylate (PETRA), as cross-linkers. Polymers were synthesized by precipitation polymerization and characterized by standard techniques. Polymerization and rebinding solutions were analyzed by high performance liquid chromatography. The highly cross-linked polymers retained about 70% of the total template amount, against (20% for the less cross-linked ones). The extracted template amount and the rebinding capacity decreased with the cross-linking degree, while the selectivity showed the opposite behaviour. The PETRA cross-linked polymers showed the best recognition (MIP 2−, α= 1.71) and selectivity (MIP 2+, α′= 5.58) capabilities. The cytotoxicity tests showed normal adhesion and proliferation of fibroblasts cultured in the medium that was put in contact with the imprinted polymers.

The preparation of novel polymeric systems through Molecular Imprinting Technology (MIT) for potential application in extracorporeal blood purification is described. Membranes based on poly(ethylene‐co‐vinyl alcohol) material, produced through a phase inversion method, were modified introducing in their structure specific binding sites for lipid and/or protein molecules. Membranes prepared are intended to selectively remove low density lipoproteins and cholesterol (LDL) from the plasma, by using interactions at a molecular level, between the molecularly imprinted membrane and specific target molecules, created during the preparation procedure. The binding performances of membranes and their potentiality as adsorbents for two different model target compounds, a phospholipid (phosphotidylcholine, PC) and a protein (α‐amylase enzyme) were investigated, showing improved adsorption capacity with respect to unmodified control membranes. In addition, molecularly imprinted poly(ethylene‐co‐vinyl alcohol)...

Scaffolds for cardiac patch application must meet stringent requirements such as biocompatibility, biodegradability, and facilitate vascularization in the engineered tissue. Here, a bioactive, biocompatible, and biodegradable electrospun scaffold of poly(glycerol sebacate)-poly(e-caprolactone) (PGS-PCL) is proposed as a potential scaffold for cardiac patch application. The fibers are smooth bead free with average diameter = 0.8 ± 0.3 μm, mean pore size = 2.2 ± 1.2 μm, porosity = 62 ± 4%, and permeability higher than that of control biological tissue. For the first time, bioactive PGS-PCL fibers functionalized with vascular endothelial growth factor (VEGF) are developed, the approach used being chemical modification of the PGS-PCL fibers followed by subsequent binding of VEGF via amide bonding. The approach results in uniform immobilization of VEGF on the fibers; the concentrations are 1.0 μg cm(-2) for the PGS-PCL (H) and 0.60 μg cm(-2) for the PGS-PCL (L) samples. The bioactive scaffold supports the attachment and growth of seeded myogenic and vasculogenic cell lines. In fact, rat aortic endothelial cells also display angiogenic features indicating potential for the formation of vascular tree in the scaffold. These results therefore demonstrate the prospects of VEGF-functionalized PGS-PCL fibrous scaffold as promising matrix for cardiac patch application.

Purpose Nano-drug delivery systems based on polymeric biomaterials have received considerable interest as drug delivery vehicles. In this work, radical polymerization was carried out in order to obtain nanoparticles based on a new acrylate terpolymer (PBMA-(PEG)MEMA-PDMAEMA). Methods Nanoparticles were developed in the form both of nanospheres and nanocapsules, an innovative kind of hollow nanoparticles with a great potential because of their low effective density and high specific surface area. The ability of the nanoparticles to load and then release a nucleic acid (DNA) to be used in cancer treatment was also investigated. Results Scanning electron microscopy analysis showed a spherical shape, nanometric dimensions, and a homogeneous distribution of the nanoparticles, also confirmed by dynamic light scattering measurements. Fourier-transform infrared spectroscopy chemical imaging analysis carried out on the nanocapsules before and after removal of the core demonstrated the presence of the cavity. High-performance liquid chromatography analysis confirmed good encapsulation efficiency of DNA both for nanospheres and nanocapsules. Drug release tests showed controlled release kinetics for both the systems with a high release of DNA in the first hours. In vitro MTT assay showed that the particles do not have cytotoxic effects on the cells. Conclusions The preliminary investigation showed that the terpolymer-based nanoparticles developed in this study could be good candidates to be used as innovative and versatile gene delivery systems.