Biomimetic Polymers for Cardiac Tissue Engineering.

Heart failure is a morbid disorder characterized by progressive cardiomyocyte (CM) dysfunction and death. Interest in cell-based therapies is growing, but sustainability of injected CMs remains a challenge. To mitigate this, we developed an injectable biomimetic Reverse Thermal Gel (RTG) specifically engineered to support long-term CM survival. This RTG biopolymer provided a solution-based delivery vehicle of CMs, which transitioned to a gel-based matrix shortly after reaching body temperature. In this study we tested the suitability of this biopolymer to sustain CM viability. The RTG was biomolecule-functionalized with poly-l-lysine or laminin. Neonatal rat ventricular myocytes (NRVM) and adult rat ventricular myocytes (ARVM) were cultured in plain-RTG and biomolecule-functionalized-RTG both under 3-dimensional (3D) conditions. Traditional 2D biomolecule-coated dishes were used as controls. We found that the RTG-lysine stimulated NRVM to spread and form heart-like functional syncytia. Regarding cell contraction, in both RTG and RTG-lysine, beating cells were recorded after 21 days. Additionally, more than 50% (p value < 0.05; n = 5) viable ARVMs, characterized by a well-defined cardiac phenotype represented by sarcomeric cross-striations, were found in the RTG-laminin after 8 days. These results exhibit the tremendous potential of a minimally invasive CM transplantation through our designed RTG-cell therapy platform.

Physical characterization of alginate-Pluronic F127 gel for endoluminal NABDs delivery.

Here we focus the attention on the physical characteristics of a highly biocompatible hydrogel made up of crosslinked alginate and Pluronic F127 (PF127). This is a composite polymeric blend we propose for artery endoluminal delivery of an emerging class of molecules named nucleic acid based drugs (NABDs). The physical characterization of our composite gel, i.e. mesh size distribution and PF127-alginate mutual organization after crosslinking, can significantly determine the NABDs release kinetics. Thus, to explore these aspects, different technical approaches, i.e. rheology, low/high field NMR and TEM, were used. While rheology provided information at the macroscopic and nano-level, the other three approaches gave details at the nano-level. We observe that Pluronic micelles, organizing in cubic ordered domains, generate, upon alginate crosslinking, the formation of meshes (≈ 150 nm) larger than those occurring in a Pluronic-free alginate network (≈ 25 nm). Nevertheless, smaller alginate meshes are still on and can just host un-structured Pluronic micelles and water. Accordingly, the gel structure is quite inhomogeneous, where big meshes (filled by crystalline Pluronic) co-exist with smaller meshes (hosting water and un-structured PF127 micelles). While big meshes offer a considerable hindering action on a diffusing solute, smaller ones represent a sort of free space where solute diffusion is faster. The presence of big and small meshes indicates that drug release may follow a double kinetics characterized by a fast and slow release. Notably, this behavior is considered appropriate for endoluminal drug release to the arterial wall.

EMID2 is a novel biotherapeutic for aggressive cancers identified by in vivo screening.

BACKGROUND: New drugs to tackle the next pathway or mutation fueling cancer are constantly proposed, but 97% of them are doomed to fail in clinical trials, largely because they are identified by cellular or in silico screens that cannot predict their in vivo effect. METHODS: We screened an Adeno-Associated Vector secretome library (> 1000 clones) directly in vivo in a mouse model of cancer and validated the therapeutic effect of the first hit, EMID2, in both orthotopic and genetic models of lung and pancreatic cancer. RESULTS: EMID2 overexpression inhibited both tumor growth and metastatic dissemination, consistent with prolonged survival of patients with high levels of EMID2 expression in the most aggressive human cancers. Mechanistically, EMID2 inhibited TGFß maturation and activation of cancer-associated fibroblasts, resulting in more elastic ECM and reduced levels of YAP in the nuclei of cancer cells. CONCLUSION: This is the first in vivo screening, precisely designed to identify proteins able to interfere with cancer cell invasiveness. EMID2 was selected as the most potent protein, in line with the emerging relevance of the tumor extracellular matrix in controlling cancer cell invasiveness and dissemination, which kills most of cancer patients.

Recovery from Food Waste-Biscuit Doughs Enriched with Pomegranate Peel Powder as a Model of Fortified Aliment.

The aim of the present work is the characterization of biscuit doughs enriched with pomegranate peel powder (PPP) at 3 (PPP3) and 5 (PPP5) wt% in the prospect of developing a fortified aliment as a support of the therapy of chronic inflammatory diseases of the intestinal tract. The total phenolic content of the powder was preliminarily evaluated. Then, the main compounds present in the PPP were identified by HPLC-ESI-TOF-MS analysis, being mainly hydrolysable tannins. The PPP was then treated at 180 °C for 20 min to mimic the baking treatment, and its water-soluble fraction (PPPwsf) was then added in the Caco-2 cell culture as a model of the intestinal epithelial barrier to verify its dose-dependent toxicity, ability in counteracting the oxidative stress, and anti-inflammatory action. Rheological experiments were performed to predict the macroscopic behavior of the PPP-added doughs during lamination and biscuit baking. SEM investigations gave their contribution to the microscopic comprehension of the dough structure. Finally, a consumer panel composed by thirty volunteers was enrolled to express its opinion on the sensory agreeableness of the biscuits prepared with two different concentrations of PPP compared with the reference dough. The discussion is focused on the biological effects of the main components found in the PPP.

Mechanical spectroscopy and relaxometry on alginate hydrogels: a comparative analysis for structural characterization and network mesh size determination.

The structure of calcium-saturated alginate hydrogels has been studied by combining rheological determinations and relaxometry measurements. The mechanical spectroscopy analyses performed on alginate gel cylinders at different polysaccharide concentration allowed estimating their main structural features such as the average mesh size. The calculation was based on the introduction of a front factor in the classical rubber elasticity approach which was correlated to the average length of the Guluronic acid blocks along the polysaccharide chain. Transverse relaxation time (T(2)) determinations performed on the cylinders revealed the presence of two relaxation rates of the water entrapped within the hydrogel network. The cross-correlation of the latter data with the rheological measurements allowed estimating the mesh size distribution of the hydrogel network. The results obtained for the hydrogel cylinders were found to be consistent with the relaxometric analysis performed on the alginate microbeads where, however, only one type of water bound into the network structure was detected. A good correlation was found in the average mesh size determined by means of relaxometric measurements on alginate microbeads and by a statistical analysis performed on TEM micrographs. Finally, the addition of a solution containing calcium ions allowed investigating further the different water relaxation modes within alginate hydrogels.

Polysaccharide-based polyanion--polycation--polyanion ternary systems. A preliminary analysis of interpolyelectrolyte interactions in dilute solutions.

The present contribution deals with the preparation and characterization of ternary mixtures of polysaccharides with potential applications in the field of tissue engineering. Two natural polyanions, i.e., alginate and hyaluronic acid, and a polycation, a lactose-modified chitosan (chitlac), were mixed in dilute conditions. The miscibility between the three components was explored in the presence of different amounts of supporting simple salt. These analyses allowed to identify the experimental conditions avoiding polymer phase separation and leading to either solution of independent polymers or soluble nonstoichiometric interpolyelectrolyte complexes. The characterization of the interpolyelectrolyte complexes was tackled by means of viscometry, light scattering, fluorescence quenching, and energy transfer. The electrostatic interactions taking place among the different polyelectrolytes led to synergistic effects on the viscosity of the polymer mixtures which strongly depend on the ionic strength. It has been found that, starting from binary soluble complexes of alginate and chitlac, the addition of hyaluronan led to the dissolution of the complexes. At variance, the addition of alginate to a phase-separated binary mixture of hyaluronan and chitlac led to the formation of soluble complexes composed of all three polysaccharides and, eventually, to their dissolution. In addition, the results showed that at low ionic strength the overall properties of the ternary mixtures depend on their order of mixing.

Viscoelastic behavior of cardiomyocytes carrying LMNA mutations.

BACKGROUND: Laminopathies are genetic diseases caused by mutations in the nuclear lamina. OBJECTIVE: Given the clinical impact of laminopathies, understanding mechanical properties of cells bearing lamin mutations will lead to advancement in the treatment of heart failure. METHODS: Atomic force microscopy (AFM) was used to analyze the viscoelastic behavior of neonatal rat ventricular myocyte cells expressing three human lamin A/C gene (LMNA) mutations. RESULTS: Cell storage modulus was characterized, by two plateaus, one in the low frequency range, a second one at higher frequencies. The loss modulus instead showed a "bell" shape with a relaxation toward fluid properties at lower frequencies. Mutations shifted the relaxation to higher frequencies, rendering the networks more solid-like. This increase of stiffness with mutations (solid like behavior) was at frequencies around 1 Hz, close to the human heart rate. CONCLUSIONS: These features resulted from a combination of the properties of cytoskeleton filaments and their temporary cross-linker. Our results substantiate that cross-linked filaments contribute, for the most part, to the mechanical strength of the cytoskeleton of the cell studied and the relaxation time is determined by the dissociation dynamics of the cross-linking proteins. The severity of biomechanical defects due to these LMNA mutations correlated with the severity of the clinical phenotype.

The degree of compactness of the incipient High Methoxyl Pectin networks. A rheological insight at the sol-gel transition.

Fractal analysis can be properly applied to complex structures, like physical and chemical networks formed by particles or polymers, when they exhibit self-similarity over an extended range of length scales and, hence, can be profitably used not only for their morphological characterization but also for individuating possible relationships between morphology and mechanisms of aggregation and crosslinking, as well as between morphology and physical properties. Several experimental methods are available to determine the fractal dimension of gel networks, including various scattering techniques and microscopies, permeability measurements and rheology. The present study regards the self-assembly kinetics of High Methoxyl Pectin (HMP) solutions with different pectin and sucrose concentrations investigated by rheological measurements to highlight the effects of pectin and sucrose concentrations on the gel point and to evaluate the degree of compactness of the incipient gel networks through an interpretation of the viscoelastic response at the sol-gel transition.

Agarose/κ-carrageenan-based hydrogel film enriched with natural plant extracts for the treatment of cutaneous wounds.

Hydrogels for complex and chronic wound dressings must be conformable, absorb and retain wound exudates and maintain hydration. They can incorporate and release bioactive molecules that can accelerate the healing process. Wound dressings have to be in contact with the wound and epidermis, even for long periods, without causing adverse effects. Hydrogel dressing formulations based on biopolymers derived from terrestrial or marine flora can be relatively inexpensive and well tolerated. In the present article hydrogel films composed by agarose (1.0 wt%), κ-carrageenan at three different concentrations (0.5, 1.0 and 1.5 wt%) and glycerol (3.0 wt%) were prepared without recourse to crosslinking agents, and characterized for their mechanical properties, morphology, swelling and erosion behavior. The films resulted highly elastic and able to absorb and retain large amounts of fluids without losing their integrity. One of the films was loaded with the aqueous extract from Cryphaea heteromalla (Hedw.) D. Mohr for its antioxidant properties. Absence of cytotoxicity and ability to reduce the oxidative stress were demonstrated on NIH-3T3 fibroblast cell cultures. These results encourage further biological evaluations to assess their impact on the healing process.

Structural characterization of calcium alginate matrices by means of mechanical and release tests.

In this paper we have concentrated on the characterization of calcium alginate hydrogels loaded with a model drug (myoglobin) by means of a mechanical approach; in addition, release tests of myoglobin from alginate hydrogels were performed. At a fixed temperature, relaxation tests (mechanical study) were carried out on matrices constituted by different polymer concentrations. The interpretation of the relaxation behavior of the different matrices was conducted using the generalized Maxwell model; as a result of this investigation it was possible to conclude that for polymer concentrations greater than 0.5 g/ 100 mL the matrices behaved as solid materials. In addition, it was observed that the mechanical properties of the matrices increased with polymer concentration. With regard to the release tests, the diffusion coefficient of myoglobin in the matrix in relation to polymer concentrations was determined. The mechanical and release data where then analyzed by Flory's theory and by a modified free-volume theory, respectively, to estimate the network mesh size xi. The comparison between the mesh sizes obtained by the two approaches showed a satisfactory agreement for polymer concentrations greater than 0.5 g/100 mL. It should be noted that the approach proposed here to determine the polymeric network meshes is absolutely general and can be advantageously applied to the characterization of other similar polymeric systems.