Strain Hardening in Highly Acetylated Chitosan Gels.

Strain hardening has recently emerged as a near-universal response of biological tissues to mechanical stimulation as well as a powerful regulator of cell fate. Understanding the mechanistic basis for this nonlinear elasticity is crucial for developing bioinspired materials that mimic extracellular matrix mechanics. Here, we show that covalent networks built from highly acetylated chitosans exhibit strain hardening at physiological pH and osmolarity. While varying the chitosan physical-chemical composition and network connectivity, we provide evidence that temporary nodes arising from the entangling of chains between stable cross-links are at the root of nonlinear elasticity. The contour length (Lc) of the said chains revealed that the larger the chain length between the cross-links, the greater is the entanglement over disentanglement upon network stretching. To this end, we calculated that the minimum number of Khun's segments in Lc that contributes to the onset of strain hardening is 15. Furthermore, we identified a relationship between critical strain marking nonlinear elasticity and the network connectivity, being similar to that found for the cytoskeletal collagen matrix, indicating the potential use of semiflexible (neutral pH-soluble) chitosans in assembling extracellular matrix mimics.

Long lasting mucoadhesive membrane based on alginate and chitosan for intravaginal drug delivery.

The intravaginal route of administration can be exploited to treat local diseases and for systemic delivery. In this work, we developed an alginate/chitosan membrane sufficiently stable in a simulated vaginal fluid and able to dissolve over time at a very slow and linear rate. The membrane demonstrated good mechanical properties both in its swollen and dry form. As a study case, we evaluated the viability of this potential drug delivery system for the treatment of bacterial vaginosis, a common disease affecting women in their reproductive age. Metronidazole was effectively included in the alginate/chitosan membrane and its bactericide effect was demonstrated against Staphylococcus aureus and Gardnerella vaginalis, simultaneously showing good biocompatibility with a cervix epithelial cell line. Since this alginate/chitosan membrane is stable in a simulated vaginal environment, is easy to fabricate and can be used for the controlled release of a model drug, it represents a promising drug delivery system for local intravaginal applications.

On the Mechanism of Genipin Binding to Primary Amines in Lactose-Modified Chitosan at Neutral pH.

The present manuscript deals with the elucidation of the mechanism of genipin binding by primary amines at neutral pH. UV-VIS and CD measurements both in the presence of oxygen and in oxygen-depleted conditions, combined with computational analyses, led to propose a novel mechanism for the formation of genipin derivatives. The indications collected with chiral and achiral primary amines allowed interpreting the genipin binding to a lactose-modified chitosan (CTL or Chitlac), which is soluble at all pH values. Two types of reaction and their kinetics were found in the presence of oxygen: (i) an interchain reticulation, which involves two genipin molecules and two polysaccharide chains, and (ii) a binding of one genipin molecule to the polymer chain without chain-chain reticulation. The latter evolves in additional interchain cross-links, leading to the formation of the well-known blue iridoid-derivatives.

Glycosylated-Chitosan Derivatives: A Systematic Review.

Chitosan derivatives, and more specifically, glycosylated derivatives, are nowadays attracting much attention within the scientific community due to the fact that this set of engineered polysaccharides finds application in different sectors, spanning from food to the biomedical field. Overcoming chitosan (physical) limitations or grafting biological relevant molecules, to mention a few, represent two cardinal strategies to modify parent biopolymer; thereby, synthetizing high added value polysaccharides. The present review is focused on the introduction of oligosaccharide side chains on the backbone of chitosan. The synthetic aspects and the effect on physical-chemical properties of such modifications are discussed. Finally, examples of potential applications in biomaterials design and drug delivery of these novel modified chitosans are disclosed.

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.

Lactose-Modified Chitosan Gold(III)-PEGylated Complex-Bioconjugates: From Synthesis to Interaction with Targeted Galectin-1 Protein.

Galectins (Gal) are a family of glycan-binding proteins characterized by their affinity for ß-galactosides. Galectin-1 (Gal-1), a dimeric lectin with two galactoside-binding sites, regulates cancer progression and immune responses. Coordination chemistry has been engaged to develop versatile multivalent neoglycoconjugates for binding Gal-1. In this study we report a fast and original method to synthesize hybrid gold nanoparticles in which a hydrochloride lactose-modified chitosan, named CTL, is mixed with dicarboxylic acid-terminated polyethylene glycol (PEG), leading to shell-like hybrid polymer-sugar-metal nanoparticles (CTL-PEG-AuNPs). The aim of this paper is to preliminarily study the interaction of the CTL-PEG-AuNPs with a target protein, namely, Gal-1, under specific conditions. The molecular interaction has been measured by Transmission Electron Microscopy (TEM), UV-vis, and Raman Spectroscopy on a large range of Gal-1 concentrations (from 0 to 10-12 M). We observed that the interaction was strongly dependent on the Gal-1 concentration at the surface of the gold nanoparticles.

Complex Coacervates between a Lactose-Modified Chitosan and Hyaluronic Acid as Radical-Scavenging Drug Carriers.

Complex coacervation of two oppositely charged polysaccharides, namely a lactose-modified chitosan (CTL) and hyaluronan (HA), was investigated in this study. Coacervates of the two polysaccharides were prepared by drop-by-drop injection of HA into CTL. Transmittance and dynamic light scattering (DLS) measurements in combination with TEM analyses demonstrated the formation of spheroidal colloids in the nano-/microsize range showing good homogeneity. Strikingly, the presence of 150 mM supporting NaCl did not hamper the colloid formation. Stability studies on selected formulations demonstrated that HA/CTL coacervates were stable up to 3 weeks at 37 °C and behaved as pH-responsive colloids since transition from entangled to disentangled chains was attained for a proper pH range. The possibility of freeze-drying the coacervates for storage purposes and the ability of encapsulating selected payloads were investigated as well, for two values of the fraction of the lactitol side-chain substitution (FL). Finally, biological tests using human neutrophils were undertaken at acidic pH value (pH = 6.0): under such experimental conditions, akin to those frequently occurring in the inflammatory microenvironment, coacervates scavenged reactive oxygen species (ROS) generated by these cells in basal conditions. Given the well documented bioactivity of CTL with respect to chitosan toward cartilage regeneration, these findings point to a possible application of HA/CTL-based colloids as scavenging and bioactive carriers for the delivery of therapeutic molecules at confined inflamed sites such as knee joints.

Alginate membranes loaded with hyaluronic acid and silver nanoparticles to foster tissue healing and to control bacterial contamination of non-healing wounds.

Chronic non-healing wounds are a clinically important problem in terms of number of patients and costs. Wound dressings such as hydrogels, hydrocolloids, polyurethane films and foams are commonly used to manage these wounds since they tend to maintain a moist environment which is shown to accelerate re-epithelialization. The use of antibacterial compounds is important in the management of wound infections. A novel wound-dressing material based on a blended matrix of the polysaccharides alginate, hyaluronic acid and Chitlac-silver nanoparticles is here proposed and its application for wound healing is examined. The manufacturing approach to obtain membranes is based on gelling, foaming and freeze-casting of alginate, hyaluronic acid and Chitlac-silver nanoparticles mixtures using calcium ions as the cross-linking agent. Comprehensive evaluations of the morphology, swelling kinetics, permeability, mechanical characteristics, cytotoxicity, capability to inhibit metalloproteinases and of antibacterial property were conducted. Biological in vitro studies demonstrated that hyaluronic acid released by the membrane is able to stimulate the wound healing meanwhile the metal silver exploits an efficient antibacterial activity against both planktonic bacteria and biofilms. Overall, the experimental data evidence that the studied material could be used as antibacterial wound dressing for wound healing promotion.

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.

Adhesion of human gingival fibroblasts/Streptococcus mitis co-culture on the nanocomposite system Chitlac-nAg.

Composite materials are increasingly used as dental restoration. In the field of biomaterials, infections remain the main reason of dental devices failure. Silver, in the form of nanoparticles (AgNPs), ions and salt, well known for its antimicrobial properties, is used in several medical applications in order to avoid bacterial infection. To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized AgNPs with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a coculture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release raised up to 20 %. Moreover the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces with the co-culture system even more when saliva is added. Integrin ß1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKCα translocated into nuclei. These data confirm that Chitlac-nAg have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.

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.

Silk Fibroin-Enriched Bioink Promotes Cell Proliferation in 3D-Bioprinted Constructs.

Three-dimensional (3D) bioprinting technology enables the controlled deposition of cells and biomaterials (i.e., bioink) to easily create complex 3D biological microenvironments. Silk fibroin (SF) has recently emerged as a compelling bioink component due to its advantageous mechanical and biological properties. This study reports on the development and optimization of a novel bioink for extrusion-based 3D bioprinting and compares different bioink formulations based on mixtures of alginate methacrylate (ALMA), gelatin and SF. The rheological parameters of the bioink were investigated to predict printability and stability, and the optimal concentration of SF was selected. The bioink containing a low amount of SF (0.002% w/V) was found to be the best formulation. Light-assisted gelation of ALMA was exploited to obtain the final hydrogel matrix. Rheological analyses showed that SF-enriched hydrogels exhibited greater elasticity than SF-free hydrogels and were more tolerant to temperature fluctuations. Finally, MG-63 cells were successfully bioprinted and their viability and proliferation over time were analyzed. The SF-enriched bioink represents an excellent biomaterial in terms of printability and allows high cell proliferation over a period of up to 3 weeks. These data confirm the possibility of using the selected formulation for the successful bioprinting of cells into extracellular matrix-like microenvironments.

Ionic Strength Impacts the Physical Properties of Agarose Hydrogels.

Agarose is a natural polysaccharide known for its ability to form thermoreversible hydrogels. While the effects of curing temperature and polysaccharide concentration on mechanical properties have been discussed in the literature, the role of ionic strength has been less studied. In the present manuscript, we investigate the effects of supporting salt concentration and the role of cation (i.e. Na+ or Li+, neighbors in the Hofmeister series), on the setting and performance of agarose hydrogels. Compressive and rheological measurements show that the supporting salts reduce the immediate elastic response of agarose hydrogels, with Li+ showing a stronger effect than Na+ at high ionic strength, while they significantly increase the extent of linear stress-strain response (i.e., linear elasticity). The presence of increasing amounts of added supporting salt also leads to a reduction in hysteresis during mechanical deformation due to loading and unloading cycles, which is more pronounced with Li+ than with Na+. The combination of rheological measurements and NMR relaxometry shows a mesh size in agarose hydrogels in the order of 6-17 nm, with a thickness of the water layer bound to the biopolymer of about 3 nm. Of note, the different structuring of the water within the hydrogel network due to the different alkali seems to play a role for the final performance of the hydrogels.

Nano-composite scaffolds for bone tissue engineering containing silver nanoparticles: preparation, characterization and biological properties.

In this study nano-composite scaffolds to be used as bone grafts have been endowed with antibacterial properties owing to the presence of silver nanoparticles. The alginate/hydroxyapatite composite scaffolds were prepared by internal gelation followed by a freeze-drying procedure to obtain a porous structure. The nanoparticles were prepared in presence of a lactose modified-chitosan and this colloidal solution was adsorbed on the scaffolds by exploiting electrostatic interactions. The adsorption and release of the silver from the composite scaffold was measured by ICP-AES and spectrofluorimetry measurements. Micro-computed tomography analysis of the scaffolds showed a homogeneous porous structure with average pore sizes of 341.5 µm and porosity of 80 %. In vitro biological tests (MTS and killing kinetics assays) demonstrated that silver does not affect the ability of the scaffolds to promote osteoblasts proliferation and that at the same time it exerts a strong bactericidal effect against both Gram+ and Gram- bacterial strains. Overall, the combined results indicate that these biocompatible antimicrobial scaffolds possess ideal characteristics for tissue engineering applications.

In vitro antimicrobial properties of silver-polysaccharide coatings on porous fiber-reinforced composites for bone implants.

Biostable fiber-reinforced composite (FRC) implants prepared from bisphenol-A-dimethacrylate and triethyleneglycoldimethacrylate resin reinforced with E-glass fibers have been successfully used in cranial reconstructions in 15 patients. Recently, porous FRC structures were suggested as potential implant materials. Compared with smooth surface, porous surface allows implant incorporation via bone ingrowth, but is also a subject to bacterial attachment. Non-cytotoxic silver-polysaccharide nanocomposite coatings may provide a way to decrease the risk of bacterial contamination of porous FRC structures. This study is focused on the in vitro characterization of the effect porosity on the antimicrobial efficiency of the coatings against Staphylococcus aureus and Pseudomonas aeruginosa by a series of microbiological tests (initial adhesion, antimicrobial efficacy, and biofilm formation). Characterization included confocal laser scanning microscopy and scanning electron microscopy. The effect of porosity on the initial attachment of S. aureus was pronounced, but in the case of P. aeruginosa the effect was negligible. There were no significant effects of the coatings on the initial bacterial attachment. In the antimicrobial efficacy test, the coatings were potent against both strains regardless of the sample morphology. In the biofilm tests, there were no clear effects either of morphology or of the coating. Further coating development is foreseen to achieve a longer-term antimicrobial effect to inhibiting bacterial implant colonization.

The effect of a silver nanoparticle polysaccharide system on streptococcal and saliva-derived biofilms.

In this work, we studied the antimicrobial properties of a nanocomposite system based on a lactose-substituted chitosan and silver nanoparticles: Chitlac-nAg. Twofold serial dilutions of the colloidal Chitlac-nAg solution were both tested on Streptococcus mitis, Streptococcus mutans, and Streptococcus oralis planktonic phase and biofilm growth mode as well as on saliva samples. The minimum inhibitory and bactericidal concentrations of Chitlac-nAg were evaluated together with its effect on sessile cell viability, as well as both on biofilm formation and on preformed biofilm. In respect to the planktonic bacteria, Chitlac-nAg showed an inhibitory/bactericidal effect against all streptococcal strains at 0.1% (v/v), except for S. mitis ATCC 6249 that was inhibited at one step less. On preformed biofilm, Chitlac-nAg at a value of 0.2%, was able to inhibit the bacterial growth on the supernatant phase as well as on the mature biofilm. For S. mitis ATCC 6249, the biofilm inhibitory concentration of Chitlac-nAg was 0.1%. At sub-inhibitory concentrations, the Streptococcal strains adhesion capability on a polystyrene surface showed a general reduction following a concentration-dependent-way; a similar effect was obtained for the metabolic biofilm activity. From these results, Chitlac-nAg seems to be a promising antibacterial and antibiofilm agent able to hinder plaque formation.

Antibacterial and bioactive multilayer electrospun wound dressings based on hyaluronic acid and lactose-modified chitosan.

Antibacterial multilayer electrospun matrices based on hyaluronic acid (HA) and a lactose-modified chitosan (CTL) were synthetized (i) by combining electrospun polycaprolactone (PCL) and polysaccharidic matrices in a bilayer device and (ii) by sequentially coating the PCL mat with CTL and HA. In both cases, the antibacterial activity was provided by loading rifampicin within the PCL support. All matrices disclosed suitable morphology and physicochemical properties to be employed as wound dressings. Indeed, both the bilayer and coated fibers showed an optimal swelling capacity (3426 ± 492 % and 1435 ± 251 % after 7 days, respectively) and water vapor permeability (160 ± 0.78 g/m2h and 170 ± 12 g/m2h at 7 days, respectively). On the other hand, the polysaccharidic dressings were completely wettable in the presence of various types of fluids. Depending on the preparation method, a different release of both polysaccharides and rifampicin was detected, and the immediate polysaccharide dissolution from the bilayer structure impacted the antibiotic release (42 ± 4 % from the bilayer structure against 25 ± 2 % from the coated fibers in 4 h). All the multilayer matrices, regardless of their production strategy and composition, revealed optimal biocompatibility and bioactivity with human dermal fibroblasts, as the released bioactive polysaccharides induced a faster wound closure in the cell monolayer (100 % in 24 h) compared to the controls (78 ± 8 % for untreated cells and 89 ± 5 % for cells treated with PCL alone, after 24 h). The inhibitory and bactericidal effects of the rifampicin loaded matrices were assessed on S. aureus, S. epidermidis, E. coli, and P. aeruginosa. The antibacterial matrices were found to be highly effective except for E. coli, which was more resistant even at higher amounts of rifampicin, with a bacterial concentration of 6.4 ± 0.4 log CFU/mL and 6.8 ± 0.3 log CFU/mL after 4 h in the presence of the rifampicin-loaded bilayer and coated matrices, respectively.

Immediate stress dissipation in dual cross-link hydrogels controls osteogenic commitment of mesenchymal stem cells.

In vitro studies of mesenchymal stem cells (MSCs) differentiation have been predominantly performed with non-physiologically elastic materials. Here we report the effect of different viscoplastic ECM mimics on the osteogenic engagement of MSCs in 2D. We have developed soft hydrogels, composed of a lactose-modified chitosan, using a combination of permanent and temporary cross-links. The presence of temporary cross-links has a minor effect on the shear modulus of the hydrogels, but causes an immediate relaxation (dissipation) of the applied stress. This material property leads to early osteogenic commitment of MSCs, as evidenced by gene expression of runt-related transcription factor 2 (RUNX2), type 1 collagen (COL1A1), osteocalcin (OCN), alkaline phosphatase enzyme activity (ALP) and calcium deposit formation. In contrast, cells cultured on purely elastic hydrogels with only permanent cross-link begin to differentiate only after a longer period of time, indicating a dissipation-mediated mechano-sensing in the osteogenic commitment of MSCs.