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.

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.

Controlled Quenching of Agarose Defines Hydrogels with Tunable Structural, Bulk Mechanical, Surface Nanomechanical, and Cell Response in 2D Cultures.

The scaffolding of agarose hydrogel networks depends critically on the rate of cooling (quenching) after heating. Efforts are made to understand the kinetics and evolution of biopolymer self-assembly upon cooling, but information is lacking on whether quenching might affect the final hydrogel structure and performance. Here, a material strategy for the fine modulation of quenching that involves temperature-curing steps of agarose is reported. Combining microscopy techniques, standard and advanced macro/nanomechanical tools, it is revealed that agarose accumulates on the surface when the curing temperature is set at 121 °C. The inhomogeneity can be mostly recovered when it is reduced to 42 °C. This has a drastic effect on the stiffness of the surface, but not on the viscoelasticity, roughness, and wettability. When hydrogels are strained at small/large deformations, the curing temperature has no effect on the viscoelastic response of the hydrogel bulk but does play a role in the onset of the non-linear region. Cells cultured on these hydrogels exhibit surface stiffness-sensing that affects cell adhesion, spreading, F-actin fiber tension, and assembly of vinculin-rich focal adhesions. Collectively, the results indicate that the temperature curing of agarose is an efficient strategy to produce networks with tunable mechanics and is suitable for mechanobiology studies.

Hydrogels based on methylated-alginates as a platform to investigate the effect of material properties on cell activity. The role of material compliance.

Alginate-based hydrogels with tunable mechanical properties are developed by chemical methylation of the polysaccharide backbone, which was performed either in homogeneous phase (in solution) or in heterogeneous phase (on hydrogels). Nuclear Magnetic Resonance (NMR) and Size Exclusion Chromatography (SEC-MALS) analyses of methylated alginates allow to identify the presence and location of methyl groups on the polysaccharide, and to investigate the influence of methylation on the stiffness of the polymer chains. The methylated polysaccharides are employed for the manufacturing of calcium-reticulated hydrogels for cell growth in 3D. The rheological characterization shows that the shear modulus of hydrogels is dependent on the amount of cross-linker used. Methylated alginates represent a platform to explore the effect of mechanical properties on cell activity. As an example, the effect of compliance is investigated using hydrogels displaying similar shear modulus. An osteosarcoma cell line (MG-63) was encapsulated in the alginate hydrogels and the effect of material compliance on cell proliferation and localization of YAP/TAZ protein complex is investigated by flow cytometry and immunohistochemistry, respectively. The results point out that an increase of material compliance leads to an increase of the proliferative rate of cells and correlates with the translocation of YAP/TAZ inside the cell nucleus.

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.

Correction: CTL-doxorubicin (DOX)-gold complex nanoparticles (DOX-AuGCs): from synthesis to enhancement of therapeutic effect on liver cancer model.

[This corrects the article DOI: 10.1039/D0NA00758G.].

DOTAREM (DOTA)-Gold-Nanoparticles: Design, Spectroscopic Evaluation to Build Hybrid Contrast Agents to Applications in Nanomedecine.

Introduction: The realization of MRI contrast agents through chemical protocols of functionalization is a strong domain of research. In this work, we developed and formulated a novel hybrid gold nanoparticle system in which a gold salt (HAuCl4) is combined with dotarem, an MRI contrast agent (DOTA) by chelation (Method IN) and stabilized by a lactose-modified chitosan polymer (CTL; Chitlac) to form DOTA IN-CTL AuNPs. Result and Discussion: The authors demonstrate the biological efficiency of these nanoparticles in the case of three cell lines: Mia PaCa-2 (human pancreatic cancer cell line), TIB-75 (murine liver cell line) and KKU-M213 (cholangiocarcinoma cell line). DOTA IN-CTL AuNPs are stable under physiological conditions, are nontoxic, and are very efficient as PTT agents. The highlights, such as high stability and preliminary MRI in vitro and in vivo models, may be suitable for diagnosis and therapy. Conclusion: We proved that DOTA IN-CTL AuNPs have several advantages: i) Biological efficacy on three cell lines: MIA PaCa-2 (human pancreatic cancer cell line), TIB-75 (murine liver cell line) and KKU-M213 (cholangiocarcinoma cell line); ii) high stability, and no-toxicity; iii) high efficiency as a PPT agent. The study conducted on MRI in vitro and in vivo models will be suitable for diagnosis and therapy.

Hyaluronic acid/lactose-modified chitosan electrospun wound dressings - Crosslinking and stability criticalities.

Polysaccharide electrospun wound dressings should be an effective strategy in the field of wound care, as they combine an extracellular matrix-like structure with excellent biomimicry. However, their high hydrophilicity and large surface area cause a rapid dissolution in aqueous environments, compromising their clinical employment. In the present paper, electrospun membranes prepared using hyaluronic acid, a bioactive lactose-modified chitosan (CTL), and polyethylene oxide have been crosslinked using glutaraldehyde, genipin, EDC/NHS or thermal treatments, obtaining very poor results in terms of membrane stability. Therefore, carbonyldiimidazole (CDI) and methacrylic anhydride were investigated in an innovative way, where CDI proved to be the best compromise between nanofiber water resistance, architecture maintenance and degradability. Indeed, the swelling and degradation behavior as well as the water vapor permeability of these matrices were tested, revealing the effectiveness of the electrospun products in absorbing large amount of liquid while maintaining the balance between water retention and gas permeability.

Sulfated lactose-modified chitosan. A novel synthetic glycosaminoglycan-like polysaccharide inducing chondrocyte aggregation.

Lactose-modified chitosan (CTL) is sulfated using SO3·py or SO3·DMF as sulfating agents. The two products are characterized by elemental analysis, FT-IR, 1H,13C-DEPT-HSQC and 1H,13C-HSQC-TOCSY experiments which allow the extent and selectivity of chemical sulfation to be determined. Dynamic Light Scattering shows a pH-dependent association of the sulfated polysaccharides which are described as flexible by the Smidsrød's B parameter and the intrinsic viscosity at infinite ionic strength. Shear viscosity and intrinsic viscosity show that sulfation protocols lead to chain scission which is more pronounced when SO3·DMF is used. The sulfated samples are able to induce aggregation of human bone marrow mesenchymal stem cells, resulting in the formation of smaller nodules compared to the unmodified CTL sample. Over time, the sample with the higher degree of sulfation allows further aggregation between cell clusters while the sample with the lower degree of sulfation shows dissolution of the aggregates.

Alginate bone scaffolds coated with a bioactive lactose modified chitosan for human dental pulp stem cells proliferation and differentiation.

Bioactive and biodegradable porous scaffolds can hasten the healing of bone defects; moreover, patient stem cells seeded onto scaffolds can enhance the osteoinductive and osteoconductive properties of these biomaterials. In this work, porous alginate/hydroxyapatite scaffolds were functionalized with a bioactive coating of a lactose-modified chitosan (CTL). The highly interconnected porous structure of the scaffold was homogeneously coated with CTL. The scaffolds showed remarkable stability up to 60 days of aging. Human Dental Pulp Stem Cells (hDPSCs) cultured in the presence of CTL diluted in culture medium, showed a slight and negligible increase in terms of proliferation rate; on the contrary, an effect on osteogenic differentiation of the cells was observed as a significant increase in alkaline phosphatase activity. hDPSCs showed higher cell adhesion on CTL-coated scaffolds than on uncoated ones. CTL coating did not affect cell proliferation, but stimulated cell differentiation as shown by alkaline phosphatase activity analysis.

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.

Development of di-methacrylate quaternary ammonium monomers with antibacterial activity.

Nine antibacterial di-methacrylate monomers based on bis-quaternary ammonium salts (bis-QAMs) were synthesized and structurally characterized. The biological activity of the bis-QAMs was tested in terms of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) on different bacterial strains achieving promising results and, in most cases, a complete bactericidal effect using a bis-QAM concentration lower than 1 mg/mL. Two of the structures showed comparable and superior activity against S. mutans than the commercial monomer 12-methacryloyloxydodecyl pyridinium bromide (MDBP). All the bis-QAMs here described were able to inhibit S. mutans biofilm formation at a concentration equal to the MIC value. From the analysis of the obtained data, some correlation regarding the structure and the antibacterial activity of the bis-QAMs could be drawn: a flexible alkyl C12 spacer between the two quaternary ammonium moieties increased the monomer antibacterial effect in comparison to the aromatic ones; the equilibrium between hydrophobic and hydrophilic moieties was directly correlated to the bactericidal range of action; the increase of the steric hindrance of the ammonium side groups might be both advantageous or disadvantageous to the antibacterial efficacy depending on the whole monomer chemical structure. Even though the possible correlation between the monomer structures and their bacteriostatic or bactericidal effect is under investigation, the monomers exhibited low cytotoxicity on human dental pulp stem cells, confirming their promising potential in the dental materials' field. STATEMENT OF SIGNIFICANCE: The use of dental resins with antibacterial monomers might prevent the formation of secondary caries at the restoration margins. For this purpose, a series of di-methacrylate bis-quaternary ammonium monomers (QAMs) was developed. Unlike antibacterial mono-methacrylate monomers already described in the literature, the synthesized di-methacrylate monomers have the potential of acting as cross-linkers stabilizing the polymeric network and bear two quaternary ammonium groups that increase their antibacterial ability. The QAMs exert bactericidal activity on both Gram(+) and Gram(-) bacterial strains maintaining at the same time good biocompatibility with the oral environment. Some structural elements of the monomers were clearly related to high antibacterial properties, and this can help design new active structures and better understand their mechanism of action.

Insights into Mechanical Behavior and Biological Properties of Chia Seed Mucilage Hydrogels.

In this contribution we report insights on the rheological properties of chia (Salvia hispanica) seed mucilage hydrogels. Creep experiments performed in steady state conditions allowed calculation of Newtonian viscosities for chia hydrogels with different polymer concentration, pointing at inter-chain interactions as the main responsible for the different behavior toward network slipping under constant stress. A combination of oscillatory frequency and stress sweep tests highlighted a moderate effect of temperature in influencing hydrogel mechanics. The latter results prompted us to investigate potential biological functions for this set of biomaterials. Lactate Dehydrogenase assay proved the lack of cytotoxicity of chia suspensions toward Human Mesenchymal Stem Cells from adipose tissue used here as a cell model. Differentiation experiments were finally undertaken to verify the influence of chia samples on osteo-induction triggered by chemical differentiation factors. Alkaline Phosphatase enzyme activity assay and Alizarin red staining demonstrated that chia mucilage did not alter in vitro stem cell differentiation. Collectively, this set of experiments revealed an almost inert role associated with chia suspensions, indicating a possible application of chia-based networks as scaffold models to study osteogenesis in vitro.

Temporary/Permanent Dual Cross-Link Gels Formed of a Bioactive Lactose-Modified Chitosan.

Mounting evidences have recognized that dual cross-link and double-network gels can promisingly recapitulate the complex living tissue architecture and overcome mechanical limitations of conventional scaffolds used hitherto in regenerative medicine. Here, dual cross-link gels formed of a bioactive lactose-modified chitosan reticulated via both temporary (boric acid-based) and permanent (genipin-based) cross-linkers are reported. While boric acid rapidly binds to lactitol flanking diols increasing the overall viscosity, a slow temperature-driven genipin binding process takes place allowing for network strengthening. Combination of frequency and stress sweep experiments in the linear stress-strain region shows that ultimate gel strength, toughness, and viscoelasticity depend on polymer-to-genipin molar ratio. Notably, herewith it is demonstrated that linear stretching correlates with strain energy dissipation through boric acid binding/unbinding dynamics. Strain-hardening effect in the nonlinear regime, along with good biocompatibility in vitro, points at an interesting role of present system as biological extracellular matrix substitute.

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.

Binary Solutions of Hyaluronan and Lactose-Modified Chitosan: The Influence of Experimental Variables in Assembling Complex Coacervates.

A miscibility study between oppositely charged polyelectrolytes, namely hyaluronic acid and a lactose-modified chitosan, is here reported. Experimental variables such as polymers' weight ratios, pH values, ionic strengths and hyaluronic acid molecular weights were considered. Transmittance analyses demonstrated the mutual solubility of the two biopolymers at a neutral pH. The onset of the liquid-liquid phase separation due to electrostatic interactions between the two polymers was detected at pH 4.5, and it was found to be affected by the overall ionic strength, the modality of mixing and the polymers' weight ratio. Thorough Dynamic Light Scattering (DLS) measurements were performed to check the quality of the formed coacervates by investigating their dimensions, homogeneity and surface charge. The whole DLS results highlighted the influence of the hyaluronic acid molecular weight in affecting coacervates' dispersity and size.

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.

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.