Development of hyaluronan-based membranes for the healing of intestinal surgical wounds: a preliminary study.

Implantable membranes based on alginate and hyaluronic acid (HA) were manufactured to obtain a rapidly resorbing pliable mesh for the in situ administration of HA to intestinal tissue. Morphological analyses of this interpenetrated matrix pointed out a homogeneous polymeric texture while degradation studies demonstrated that the material is able to dissolve in physiological solutions within few days. Biological studies in vitro showed that the membrane is biocompatible towards human dermal fibroblasts and that liquid extracts from the HA-containing membrane can stimulate wound healing. A preliminary in vivo biocompatibility study on rats showed that the membranes in direct contact with the intestine did not elicit any acute adverse reaction or immune response, while only a mild inflammatory reaction was noticed at the mesenteric or serosal region. Overall, these results appear to support the application of these polysaccharide-based materials for intestinal wound healing.

Boric Acid Induced Transient Cross-Links in Lactose-Modified Chitosan (Chitlac).

The present paper explores the effect of boric acid on Chitlac, a lactose-modified chitosan which had previously shown interesting biological and physical-chemical features. The herewith-reported experimental evidences demonstrated that boric acid binds to Chitlac, producing conformational and association effects on the chitosan derivative. The thermodynamics of boric acid binding to Chitlac was explored by means of 11B NMR, circular dichroism (CD), and UV-vis spectroscopy, while macromolecular effects were investigated by means of viscometry and dynamic light scattering (DLS). The experimental results revealed a chain-chain association when limited amounts of boric acid were added to Chitlac. However, upon exceeding a critical boric acid limit dependent on the polysaccharide concentration, the soluble aggregates disentangle. The rheological behavior of Chitlac upon treatment with boric acid was explored showing a dilatant behavior in conditions of steady flow. An uncommonly high dependence in the scaling law between the zero-shear viscosity and the concentration of Chitlac was found, i.e., η0 ∝ CCTL5.8, pointing to interesting potential implications of the present system in biomaterials development.

Silver-containing antimicrobial membrane based on chitosan-TPP hydrogel for the treatment of wounds.

Treatment of non-healing wounds represents hitherto a severe dilemma because of their failure to heal caused by repeated tissue insults, bacteria contamination and altered physiological condition. This leads to face huge costs for the healthcare worldwide. To this end, the development of innovative biomaterials capable of preventing bacterial infection, of draining exudates and of favoring wound healing is very challenging. In this study, we exploit a novel technique based on the slow diffusion of tripolyphosphate for the preparation of macroscopic chitosan hydrogels to obtain soft pliable membranes which include antimicrobial silver nanoparticles (AgNPs) stabilized by a lactose-modified chitosan (Chitlac). UV-Vis and TEM analyses demonstrated the time stability and the uniform distribution of AgNPs in the gelling mixture, while swelling studies indicated the hydrophilic behavior of membrane. A thorough investigation on bactericidal properties of the material pointed out the synergistic activity of chitosan and AgNPs to reduce the growth of S. aureus, E. coli, S. epidermidis, P. aeruginosa strains and to break apart mature biofilms. Finally, biocompatibility assays on keratinocytes and fibroblasts did not prove any harmful effects on the viability of cells. This novel technique enables the production of bioactive membranes with great potential for the treatment of non-healing wounds.

Polysaccharide-based networks from homogeneous chitosan-tripolyphosphate hydrogels: synthesis and characterization.

Polysaccharide networks, in the form of hydrogels and dried membranes based on chitosan and on the cross-linker tripolyphosphate (TPP), were developed using a novel approach. TPP was incorporated into chitosan by slow diffusion to favor a controlled gelation. By varying chitosan, TPP, and NaCl concentration, transition from inhomogeneous to homogeneous systems was achieved. Rheology and uniaxial compression tests enabled to identify the best performing hydrogel composition with respect to mechanical properties. FTIR, (31)P NMR, and spectrophotometric methods were used to investigate the interaction chitosan-TPP, the kinetics of phosphates diffusion during the dialysis and the amount of TPP in the hydrogel. A freeze-drying procedure enabled the preparation of soft pliable membranes. The lactate dehydrogenase assay demonstrated the biocompatibility of the membranes toward fibroblasts. Overall, we devised a novel approach to prepare homogeneous macroscopic chitosan/TPP-based biomaterials with tunable mechanical properties and good biocompatibility that show good potential as novel polysaccharide derivatives.

Biophysical functionality in polysaccharides: from Lego-blocks to nano-particles.

The objective of the paper is to show the very important biophysical concepts that have been developed with polysaccharides. In particular, an attempt will be made to relate "a posteriori" the fundamental aspects, both experimental and theoretical, with some industrial applications of polysaccharide-based materials. The overview of chain conformational aspects includes relationships between topological features and local dynamics, exemplified for some naturally occurring carbohydrate polymers. Thus, by using simulation techniques and computational studies, the physicochemical properties of aqueous solutions of polysaccharides are interpreted. The relevance of conformational disorder-order transitions, chain aggregation, and phase separation to the underlying role of the ionic contribution to these processes is discussed. We stress the importance of combining information from analysis of experimental data with that from statistical-thermodynamic models for understanding the conformation, size, and functional stability of industrially important polysaccharides. The peculiar properties of polysaccharides in industrial applications are summarized for the particularly important example of nanoparticles production, a field of growing relevance and scientific interest.

Marine polysaccharides in microencapsulation and application to aquaculture: "from sea to sea".

This review's main objective is to discuss some physico-chemical features of polysaccharides as intrinsic determinants for the supramolecular structures that can efficiently provide encapsulation of drugs and other biological entities. Thus, the general characteristics of some basic polysaccharides are outlined in terms of their conformational, dynamic and thermodynamic properties. The analysis of some polysaccharide gelling properties is also provided, including the peculiarity of the charged polysaccharides. Then, the way the basic physical chemistry of polymer self-assembly is made in practice through the laboratory methods is highlighted. A description of the several literature procedures used to influence molecular interactions into the macroscopic goal of the encapsulation is given with an attempt at classification. Finally, a practical case study of specific interest, the use of marine polysaccharide matrices for encapsulation of vaccines in aquaculture, is reported.

Development of biodegradable membranes for the delivery of a bioactive chitosan-derivative on cartilage defects: A preliminary investigation.

Biodegradable membranes for cartilage applications were manufactured starting from polymeric networks of a lactose-modified chitosan (CTL), previously proposed for chondrocytes stimulation. This implantable biomaterial was conceived as a reservoir of a bioactive polymer that could promote the activity of chondrocytes and the healing of cartilage defects. Freeze-drying of reticulated hydrogels enabled to obtain pliable membranes with a homogeneous polymeric texture, as pointed out by scanning electron microscopy analyses. Swelling tests and dimensional evaluations showed that the material is able to absorb physiological fluids and expand gradually upon rehydration. This feature was evaluated on a simulated cartilage defect on pig's humerus (ex vivo), which revealed the capability of the membranes to progressively fit the tissue voids on the damaged cartilage. The rheological properties of the rehydrated membranes pointed out their peculiar strain-stiffening behavior, which represents a promising feature for the regeneration of tissues subjected to variable mechanical loads and deformations. Biological in vitro studies demonstrated the biocompatibility of the membranes in contact with primary chondrocytes and osteoblasts. Taken together, these results represent a starting point for the development of a novel generation of implantable biomaterials for cartilage treatment based on CTL.

The effects of alginate encapsulation on NIT-1 insulinoma cells: viability, growth and insulin secretion.

Transplantation of microencapsulated insulin-secreting cells is proposed as a promising therapy for the treatment of type I diabetes mellitus. In recent years, important advances have been made in the field of immunoisolation and many studies have shown that alginate provides some major advantages for encapsulation over other systems. Since it is known that the extracellular matrix influences the behaviour of encapsulated cells, the aim of the present work has been to study the consequences of encapsulation on some cell functions. For this purpose, cell growth and dynamics of insulin release of NIT-1 cells entrapped in alginate capsules compared with those exhibited by free NIT-1 cells were investigated by means of growth curves, assays, Trypan blue staining and ELISA test. All investigations performed allowed us to conclude that alginate-entrapped NIT-1 cells maintain their growth features and secretory functions although with some important differences. In particular, alginate encapsulation affects the cellular growth profile and causes the lost of time dependence of insulin secretion profile.

Nucleation, reorganization and disassembly of an active network from lactose-modified chitosan mimicking biological matrices.

Developing synthetic materials able to mimic micro- and macrorheological properties of natural networks opens up to novel applications and concepts in materials science. The present contribution describes an active network based on a semi-synthetic polymer, a lactitol-bearing chitosan derivative (Chitlac), and a transient inorganic cross-linker, boric acid. Due to the many and diverse anchoring points for boric acid on the flanking groups of Chitlac, the cross-links constantly break and reform in a highly dynamic fashion. The consequence is a network with unusual non-equilibrium and mechanical properties closely resembling the rheological behavior of natural three-dimensional arrangements and of cytoskeleton. Concepts like network nucleation, reorganization and disassembly are declined in terms of amount of the cross-linker, which acts as a putative motor for remodeling of the network upon application of energy. The out-of-equilibrium and non-linear behavior render the semi-synthetic system of great interest for tissue engineering and for developing in-vitro mimics of natural active matrices.

Alginate/lactose-modified chitosan hydrogels: a bioactive biomaterial for chondrocyte encapsulation.

A new bioactive scaffold was prepared from a binary polysaccharide mixture composed of a polyanion (alginate) and a polycation (a lactose-modified chitosan, chitlac). Its potential use for articular chondrocytes encapsulation and cartilage reconstructive surgery applications has been studied. The hydrogel combines the ability of alginate to act as a 3D supporting structure with the capability of the second component (chitlac) to provide interactions with porcine articular chondrocytes. Physico-chemical characterization of the scaffold was accomplished by gel kinetics and compression measurements and demonstrated that alginate-chitlac mixture (AC-mixture) hydrogels exhibit better mechanical properties when compared with sole alginate hydrogels. Furthermore, biochemical and biological studies showed that these 3D scaffolds are able to maintain chondrocyte phenotype and particularly to significantly stimulate and promote chondrocyte growth and proliferation. In conclusion, the present study can be considered as a first step towards an engineered, biologically active scaffold for chondrocyte in vitro cultivation, expansion, and cell delivery.

Mimicking mechanical response of natural tissues. Strain hardening induced by transient reticulation in lactose-modified chitosan (chitlac).

The effect of transient cross-links has been explored on a lactose-modified chitosan, which previously had shown interesting biological features. The presence of galactose side chains and of the polyol spacer resulted particularly appealing for the reticulation by borate ions. The interaction between chitlac and borax was investigated by means of 11B NMR while rheology pointed to a marked non-linear behavior depending on the amount of borax added to the system. The presence of limited amount of cross-linking ion led to dilatant behavior when the steady flow curve was measured. In addition, strain stiffening was noticed on elastic response upon exceeding a critical stress, indicating a transient nature in the formation of the cross-links. The non-linear response of chitlac in the presence of borax compared surprisingly well with the one showed by proteins composing the natural ECM pointing at a possible role of mechanotransduction in the biological significance of the modified chitosan.

Antibacterial-nanocomposite bone filler based on silver nanoparticles and polysaccharides.

Injectable bone fillers represent an attractive strategy for the treatment of bone defects. These injectable materials should be biocompatible, capable of supporting cell growth and possibly able to exert antibacterial effects. In this work, nanocomposite microbeads based on alginate, chitlac, hydroxyapatite and silver nanoparticles were prepared and characterized. The dried microbeads displayed a rapid swelling in contact with simulated body fluid and maintained their integrity for more than 30 days. The evaluation of silver leakage from the microbeads showed that the antibacterial metal is slowly released in saline solution, with less than 6% of silver released after 1 week. Antibacterial tests proved that the microbeads displayed bactericidal effects toward Staphylococcus aureus, Pseudomonas aeruginosa and Staphylococcus epidermidis, and were also able to damage pre-formed bacterial biofilms. On the other hand, the microbeads did not exert any cytotoxic effect towards osteoblast-like cells. After characterization of the microbeads bioactivity, a possible means to embed them in a fluid medium was explored in order to obtain an injectable paste. Upon suspension of the particles in alginate solution or alginate/hyaluronic acid mixtures, a homogenous and time-stable paste was obtained. Mechanical tests enabled to quantify the extrusion forces from surgical syringes, pointing out the proper injectability of the material. This novel antibacterial bone filler appears as a promising material for the treatment of bone defects, in particular when possible infections could compromise the bone-healing process. Copyright © 2016 John Wiley & Sons, Ltd.

Exploiting natural polysaccharides to enhance in vitro bio-constructs of primary neurons and progenitor cells.

Current strategies in Central Nervous System (CNS) repair focus on the engineering of artificial scaffolds for guiding and promoting neuronal tissue regrowth. Ideally, one should combine such synthetic structures with stem cell therapies, encapsulating progenitor cells and instructing their differentiation and growth. We used developments in the design, synthesis, and characterization of polysaccharide-based bioactive polymeric materials for testing the ideal composite supporting neuronal network growth, synapse formation and stem cell differentiation into neurons and motor neurons. Moreover, we investigated the feasibility of combining these approaches with engineered mesenchymal stem cells able to release neurotrophic factors. We show here that composite bio-constructs made of Chitlac, a Chitosan derivative, favor hippocampal neuronal growth, synapse formation and the differentiation of progenitors into the proper neuronal lineage, that can be improved by local and continuous delivery of neurotrophins. STATEMENT OF SIGNIFICANCE: In our work, we characterized polysaccharide-based bioactive platforms as biocompatible materials for nerve tissue engineering. We show that Chitlac-thick substrates are able to promote neuronal growth, differentiation, maturation and formation of active synapses. These observations support this new material as a promising candidate for the development of complex bio-constructs promoting central nervous system regeneration. Our novel findings sustain the exploitation of polysaccharide-based scaffolds able to favour neuronal network reconstruction. Our study shows that Chitlac-thick may be an ideal candidate for the design of biomaterial scaffolds enriched with stem cell therapies as an innovative approach for central nervous system repair.

On the demixing of hyaluronan and alginate in the gel state.

The manuscript focuses on the demixing of hyaluronan and alginate in the hydrogel state. Binary solutions of the two polysaccharides have been treated with Ca2+ as the alginate cross-linking ion and the radial distribution of the two components in the hydrogels was measured by means of 1H NMR. These results revealed the presence of alginate-enriched and hyaluronan-enriched domains stemming from a polysaccharide demixing. The hydrogels were characterized by means of uniaxial compression and creep-compliance measurements which showed that the demixing increased the overall resistance of the hydrogel to stress. In addition, due to the viscoelastic properties of hyaluronan, a marked increase of the Newtonian viscosity of the constructs was noticed. The peculiarity of the effect of hyaluronan was demonstrated by the use of an alginate unable to form gel by binding non-calcium binding alginate, i.e. mannuronan, ruling out the effect of viscosity over the time-dependent behavior of the mixed hyaluronan-alginate hydrogels.

Adhesive coatings based on melanin-like nanoparticles for surgical membranes.

Adhesive coatings for implantable biomaterials can be designed to prevent material displacement from the site of implant. In this paper, a strategy based on the use of melanin-like nanoparticles (MNPs) for the development of adhesive coatings for polysaccharidic membranes was devised. MNPs were synthesized in vitro and characterized in terms of dimensions and surface potential, as a function of pH and ionic strength. The in vitro biocompatibility of MNPs was investigated on fibroblast cells, while the antimicrobial properties of MNPs in suspension were evaluated on E. coli and S. aureus cultures. The manufacturing of the adhesive coatings was carried out by spreading MNPs over the surface of polysaccharidic membranes; the adhesive properties of the nano-engineered coating to the target tissue (intestinal serosa) were studied in simulated physiological conditions. Overall, this study opens for novel approaches in the design of naturally inspired nanostructured adhesive systems.

Butyrate-Loaded Chitosan/Hyaluronan Nanoparticles: A Suitable Tool for Sustained Inhibition of ROS Release by Activated Neutrophils.

Tissue damage caused by excessive amounts of neutrophil-derived reactive oxygen species (ROS) occurs in many inflammatory diseases. Butyrate is a short-chain fatty acid (SCFA) with known anti-inflammatory properties, able to modulate several neutrophil functions. Evidence is provided here that butyrate inhibits neutrophil ROS release in a dose and time-dependent fashion. Given the short half-life of butyrate, chitosan/hyaluronan nanoparticles are next designed and developed as controlled release carriers able to provide cells with a long-lasting supply of this SCFA. Notably, while the inhibition of neutrophil ROS production by free butyrate declines over time, that of butyrate-loaded chitosan/hyaluronan nanoparticles (B-NPs) is sustained. Additional valuable features of these nanoparticles are inherent ROS scavenger activity, resistance to cell internalization, and mucoadhesiveness. B-NPs appear as promising tools to limit ROS-dependent tissue injury during inflammation. Particularly, by virtue of their mucoadhesiveness, B-NPs administered by enema can be effective in the treatment of inflammatory bowel diseases.

Cell-compatible covalently reinforced beads obtained from a chemoenzymatically engineered alginate.

A chemoenzymatic strategy has been exploited to make covalently linked alginate beads with high stability. This was achieved by grafting mannuronan (alginate with 100% mannuronic acid (M)) with methacrylate moieties and then performing two enzymatic steps converting M to guluronic acid (G) in alternating sequences (MG-blocks) and in G-blocks. In this way a methacrylate grafted alginate with better gel-forming ability was achieved. Covalent bindings were introduced into the beads by using a photoinitiating system that initiated polymerization of the methacrylate moieties. The covalent links were demonstrated by beads remaining intact after treatment with EDTA. The new chemoenzymatic photocrosslinked (CEPC) beads were compatible with cells with low post-encapsulation ability like C2C12 myoblasts and human pancreatic islets. The islets continued secreting insulin after encapsulation. On contrary, cells with a high post-encapsulation proliferative ability like 293-endo cells died within 2-week post-encapsulation. The exceptional stability and the cell compatibility of the new CEPC beads make them interesting as bioreactors for delivering therapeutic proteins in future applications.

Hyaluronan delivery by polymer demixing in polysaccharide-based hydrogels and membranes for biomedical applications.

Alginate-based membranes containing hyaluronic acid (HA) were manufactured by freeze-drying calcium-reticulated hydrogels. The study of the distribution of the two macromolecules within the hydrogel enabled to highlight a polymer demixing mechanism that tends to segregate HA in the external parts of the constructs. Resistance and pliability of the membranes were tuned, while release and degradation studies enabled to quantify the diffusion of both polysaccharides in physiological solution and to measure the viable lifetime of the membranes. Biological studies in vitro proved that the liquid extracts from the HA-containing membranes stimulate wound healing and that fibroblasts are able to colonize the membranes. Overall, such novel alginate-HA membranes represent a promising solution for several medical needs, in particular for wound treatment, giving the possibility to provide an in situ administration of HA from a resorbable device.

Adhesive and sealant interfaces for general surgery applications.

The main functions of biological adhesives and sealants are to repair injured tissues, reinforce surgical wounds, or even replace common suturing techniques. In general surgery, adhesives must match several requirements taking into account clinical needs, biological effects, and material features; these requirements can be fulfilled by specific polymers. Natural or synthetic polymeric materials can be employed to generate three-dimensional networks that physically or chemically bind to the target tissues and act as hemostats, sealants, or adhesives. Among them, fibrin, gelatin, dextran, chitosan, cyanoacrylates, polyethylene glycol, and polyurethanes are the most important components of these interfaces; various aspects regarding their adhesion mechanisms, mechanical performance, and resistance to body fluids should be taken into account to choose the most suitable formulation for the target application. This review aims to describe the main adhesives and sealant materials for general surgery applications developed in the past decades and to highlight the most important aspects for the development of future formulations.