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.

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.

N-isopropyl chitosan. A pH- and thermo-responsive polysaccharide for gel formation.

The present contribution deals with the synthesis and characterization of N-isopropyl chitosan in which the introduction of hydrophobic groups leads to an increased flexibility of the polysaccharide backbone. The isopropyl groups extend the solubility of the modified-chitosan samples and render the modified chitosan a pH- and thermo-sensitive system for hydrogel formation. Indeed, upon varying the pH of the system and/or its temperature within a range compatible with biological applications, a non-reversible sol-gel transition occurs, as determined through extended rheological analyses. The modified chitosan samples show a very good biocompatibility as determined through preliminary viability and cell growth experiments.

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.

Biomimetic, Multiresponsive, and Self-Healing Lactose-Modified Chitosan (CTL)-Based Gels Formed via Competitor-Assisted Mechanism.

The present paper describes an original method to form under physiological conditions homogeneous lactose-modified chitosan (CTL) gels avoiding syneresis. Specifically, combination of boric acid-i.e., the cross-linker-and mannitol-i.e., a polyol competitor for boron binding-were exploited to reduce the very fast kinetics of CTL/boron self-assembly. Resulting gels were homogeneous as proved by scattering analyses. An in-depth rheological characterization was undertaken to identify the correct mannitol-to-boron ratio at which gels showed homogeneity without weakening. Stress sweep and frequency sweep tests were performed to investigate the viscoelastic properties of these dynamic networks, highlighting a marked strain-hardening behavior, which is pivotal in native tissues. Notably, herein we report for the first time that CTL-boric acid gels are multiresponsive systems, whose mechanics can be tailored by different stimuli such as the presence of small molecules like glucose. Moreover, we demonstrate that these networks spontaneously self-heal after breakage. The biocompatibility of such gels was studied under 2D and 3D conditions toward three different cell models, namely, pig primary chondrocytes, human Dental Pulp Stem Cells (hDPSCs), and mouse fibroblasts. Giving the peculiar mechanical performance of selected systems and considering the well-known bioactivity of the chitosan derivative, CTL-boric acid networks are promising candidates as multiresponsive gels to be used in the field of tissue engineering, especially for articular cartilage regeneration.

The role played by the molecular weight and acetylation degree in modulating the stiffness and elasticity of chitosan gels.

A broad library of chitosans was produced varying the molecular weight and the fraction of acetylated units, FA. The produced chitosans were used for the formation of wall-to-wall cylindrical gels through a controlled external gelation using tripolyphosphate (TPP) as cross-linker. The resulting gels were analyzed by rheometry. Viscosity average degree of polymerization (DPv¯) > 152 was shown to be required for the formation of stable gels. Both gel stiffness and gel rupture strength were proportional to the molecular weight regardless of the applied deformation. Increasing acetylation produced a marked reduction of the shear modulus, but, in parallel, switched the networks from rigid and brittle to weak and elastic. Intriguingly, gels made of chitosan with FA = 0.37 displayed notable elasticity, i.e. up to 90% of applied strain falls into linear regime. These findings suggest that the frequency of glucosamine (D unit) and N-acetyl-glucosamine (A unit) contribute to a subtle structure-property relationship in chitosan-TPP gels.

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.

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.

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.

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.

Myoblast adhesion, proliferation and differentiation on human elastin-like polypeptide (HELP) hydrogels.

BACKGROUND: The biochemical, mechanical and topographic properties of extracellular matrix are crucially involved in determining skeletal muscle cell morphogenesis, proliferation and differentiation. Human elastin-like polypeptides (HELPs) are recombinant biomimetic proteins designed to mimic some properties of the native matrix protein; when employed as myoblast adhesion substrates, they stimulate in vitro myogenesis. Given the influence that the biophysical properties of extracellular matrix have on skeletal muscle cells, the aim of this work was to investigate the effects of HELP hydrogels on myoblasts' viability and functions. METHODS: We recently synthesized a novel polypeptide, HELPc, by fusing the elastin-like backbone to a 41aa sequence present in the α2 chain of type IV collagen, containing two arginyl-glycyl-aspartic acid (RGD) motifs. To obtain hydrogels, the enzymatic cross-linking of the HELPc was accomplished by transglutaminase. Here, we employed both non-cross-linked HELPc glass coatings and cross-linked HELPc hydrogels at different monomer densities, as adhesion substrates for C2C12 cells, used as a myoblast model. RESULTS: By comparing cell adhesion, proliferation and differentiation, we revealed several striking differences. Depending on support rigidity, adhesion to HELPc substrates dictated cell morphology, spreading, focal adhesion formation and cytoskeletal organization. Hydrogels greatly stimulated cell proliferation, particularly in low-serum medium, and partially inhibited myogenic differentiation. CONCLUSIONS: On the whole, the results underline the potential of these genetically engineered polypeptides as a tool for dissecting crucial steps in myogenesis.

Insight into the ionotropic gelation of chitosan using tripolyphosphate and pyrophosphate as cross-linkers.

Ionotropic gelation of chitosan by means of opposite charged ions represents an efficient alternative to covalent reticulation because of milder condition of use and, in general, higher biocompatibility of the resulting systems. In this work 90° light scattering (turbidimetry), circular dichroism (CD) and 1H NMR measurements have been performed to study the interactions between the biopolymer and ionic cross-linkers tripolyphosphate (TPP) and pyrophosphate (PPi) in dilute solutions. Thereafter, a dialysis-based technique was exploited to fabricate tridimensional chitosan hydrogels based on both polyanions. Resulting matrices showed a different mechanical behavior because of their peculiar mesh-texture at micro/nano-scale: in the present contribution we demonstrate that TPP and PPi favor the formation of homogeneous and inhomogeneous systems, respectively. The different texture of networks could be exploited in future for the preparation of systems for the controlled release of molecules.