Elisabetta Rosellini

Preparation and characterization of alginate/gelatin blend films for cardiac tissue engineering

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.

Strategies for the chemical and biological functionalization of scaffolds for cardiac tissue engineering: a review

The development of biomaterials for cardiac tissue engineering (CTE) is challenging, primarily owing to the requirement of achieving a surface with favourable characteristics that enhances cell attachment and maturation. The biomaterial surface plays a crucial role as it forms the interface between the scaffold (or cardiac patch) and the cells. In the field of CTE, synthetic polymers (polyglycerol sebacate, polyethylene glycol, polyglycolic acid, poly-l-lactide, polyvinyl alcohol, polycaprolactone, polyurethanes and poly(N-isopropylacrylamide)) have been proven to exhibit suitable biodegradable and mechanical properties. Despite the fact that they show the required biocompatible behaviour, most synthetic polymers exhibit poor cell attachment capability. These synthetic polymers are mostly hydrophobic and lack cell recognition sites, limiting their application. Therefore, biofunctionalization of these biomaterials to enhance cell attachment and cell material interaction is being widely investigated. There are numerous approaches for functionalizing a material, which can be classified as mechanical, physical, chemical and biological. In this review, recent studies reported in the literature to functionalize scaffolds in the context of CTE, are discussed. Surface, morphological, chemical and biological modifications are introduced and the results of novel promising strategies and techniques are discussed.

Development and characterization of novel electrically conductive PANI-PGS composites for cardiac tissue engineering applications.

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 30vol.% 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 0Scm(-1) for pure PGS to 0.018Scm(-1) for 30vol.% PANI-PGS samples. Moreover, the conductivities were preserved for at least 100h 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.

Biomimetic poly(glycerol sebacate) (PGS) membranes for cardiac patch application.

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 25s 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.

Hydroxyapatite/gelatin/gellan sponges as nanocomposite scaffolds for bone reconstruction

The aim of this work was the morphological, physicochemical, mechanical and biological characterization of a new composite system, based on gelatin, gellan and hydroxyapatite, and mimicking the composition of natural bone. Porous scaffolds were prepared by freeze–drying technique, under three different conditions of freezing. The morphological analysis showed a homogeneous porosity, with well interconnected pores, for the sample which underwent a more rapid freezing. The elastic modulus of the same sample was close to that of the natural bone. The presence of interactions among the components was demonstrated through the physicochemical investigation. In addition, the infrared chemical imaging analysis pointed out the similarity among the composite scaffold and the natural bone, in terms of chemical composition, homogeneity, molecular interactions and structural conformation. Preliminary biological characterization showed a good adhesion and proliferation of human mesenchymal stem cells.

Bioactive Electrospun Fibers of Poly(glycerol sebacate) and Poly(ε‐caprolactone) for Cardiac Patch Application

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(ε-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.

Three-dimensional microfabricated scaffolds with cardiac extracellular matrix-like architecture.

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.

Molecularly imprinted nanoparticles with recognition properties towards a laminin H–Tyr–Ile–Gly–Ser–Arg–OH sequence for tissue engineering applications

Nanotechnology is an emerging field that promises to revolutionize medicine and is increasingly used in tissue engineering applications. Our research group proposed for the first time molecular imprinting as a new nanotechnology for the creation of advanced synthetic support structures for cell adhesion and proliferation. The aim of this work was the synthesis and characterization of molecularly imprinted polymers with recognition properties towards a laminin peptide sequence and their application as functionalization structures in the development of bioactive materials. Nanoparticles with an average diameter of 200 nm were synthesized by precipitation polymerization of methacrylic acid in the presence of the template molecule and trimethylpropane trimethacrylate as the cross-linking agent. The imprinted nanoparticles showed good performance in terms of recognition capacity and selectivity. The cytotoxicity tests showed normal vitality of C2C12 myoblasts cultured in the medium that was put in contact with the imprinted polymers. After the deposition on the polymeric film surface, the imprinted particles maintained their specific recognition and rebinding behaviour, showing an even higher quantitative binding than free nanoparticles. Preliminary in vitro cell culture tests demonstrated the ability of functionalized materials to promote cell adhesion, proliferation and differentiation, suggesting that molecular imprinting can be used as an innovative functionalization technique.

Chitosan-Based Macromolecular Biomaterials for the Regeneration of Chondroskeletal and Nerve Tissue

The use of materials, containing the biocompatible and bioresorbable biopolymer poly()-2-amino-2-deoxy--D-glucan, containing some N-acetyl-glucosamine units (chitosan, CHI) and/or its derivatives, to fabricate devices for the regeneration of bone, cartilage and nerve tissue, was reviewed. The CHI-containing devices, to be used for bone and cartilage regeneration and healing, were tested mainly for in vitro cell adhesion and proliferation and for insertion into animals; only the use of CHI in dental surgery has reached the clinical application. Regarding the nerve tissue, only a surgical repair of a 35 mm-long nerve defect in the median nerve of the right arm at elbow level with an artificial nerve graft, comprising an outer microporous conduit of CHI and internal oriented filaments of poly(glycolic acid), was reported. As a consequence, although many positive results have been obtained, much work must still be made, especially for the passage from the experimentation of the CHI-based devices, in vitro and in animals, to their clinical application.

Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering.

The aim of this work was the development of new synthetic polymeric systems, functionalized by surface chemical modification with bioactive peptides, for myocardial tissue engineering. Polycaprolactone and a poly(ester-ether-ester) block copolymer synthesized in our lab, polycaprolactone–poly(ethylene oxide)–polycaprolactone (PCL–PEO–PCL), were used as the substrates to be modified. Two pentapeptides, H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) from fibronectin and H-Tyr-Ile-Gly-Ser-Arg-OH (YIGSR) from laminin, were used for the functionalization. Polymeric membranes were obtained by casting from solutions and then functionalized by means of alkaline hydrolysis and subsequent coupling of the bioactive molecules through 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride/N-hydroxysuccinimide chemistry. The hydrolysis conditions, in terms of hydrolysis time, temperature, and sodium hydroxide concentration, were optimized for the two materials. The occurrence of the coupling reaction was demonstrated by infrared spectroscopy, as the presence on the functionalized materials of the absorption peaks typical of the two peptides. The peptide surface density was determined by chromatographic analysis and the distribution was studied by infrared chemical imaging. The results showed a nearly homogeneous peptide distribution, with a density above the minimum value necessary to promote cell adhesion. Preliminary in vitro cell culture studies demonstrated that the introduction of the bioactive molecules had a positive effect on improving C2C12 myoblasts growth on the synthetic materials.