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Final Report Summary - EMP-ECM (Towards Engineered Multicomponent Polysaccharide Hydrogels for Surrogate Extracellular Matrices)

The challenge in development of surrogate extracellular matrices, ECMs is to design and prepare synthetic materials capable of influencing cell differentiation, proliferation, survival and migration through both biochemical interactions and mechanical cues. Current effort in the engineering of synthetic ECM has focused on installing molecular features (peptides, proteins and bio-interactive polymers) within insoluble scaffolds, either by self-assembly or through covalent modifications of polymer or biopolymer networks. Apart from their direct role in cell interaction, bioactive molecules or peptide sequences are found to affect the hierarchical structural organization, surface properties and mechanical properties of the resulting material, thus affecting indirectly the cellular response.
Particularly appealing materials explored as synthetic ECMs are hydrogels-3D cross-linked insoluble, hydrophilic networks of polymers whose physical characteristics resemble those of native ECMs. These polymers can absorb large amounts of water or biological fluids (up to 99 wt%) due to the interaction of water molecules with the polymer backbone. One of the advantages of using hydrogels as scaffolds for tissue engineering is that one can easily adjust both their physicochemical and mechanical properties to meet the demands for tissue scaffolds and cell encapsulation. Biocompatible hydrogel scaffolds can be obtained by selecting biocompatible synthetic or natural polymers and cross-linkers, for example polysaccharides. Although polysaccharides being- non-toxic, hemocompatible, and relatively cheap – possess many of the favorable properties required for biomaterials, some of them (such as alginate and pectin) do not facilitate cell attachment. The means most commonly used to overcome this obstacle is to graft to the polysaccharide chain an integrin-binding peptide, Arg-Gly-Asp (RGD). The effect of RGD incorporation on the bioactivity of a polysaccharide hydrogel, has been extensively explored. However less is known about its effect on the hydrogel hierarchical structure and its structure-properties relations.
The overall aim of the proposed research is to develop a fundamental understanding of the structure-mechanical properties-function relations of multicomponent polysaccharide hydrogels used in tissue engineering applications and to apply this understanding in the development of engineering principles that can serve as a generic guide for the design of polysaccharide-based materials for biological applications. To this end, the three following specific aims will be addressed:
Aim 1 – Composition–structure relationships: Characterize the interrelations between the chemical compositions of the building blocks (both polymers and peptides) on the resulting structure of the bioactive gels. In particular, determine the effect of different RGD peptides and heparin on the nanostructure of the polysaccharides, starting from molecular conformations in dilute solutions all the way up to self-assembled gels.
Aim 2 – Structure–function relationships: Systematically investigate the effect of the different structures characterized in Aim 1 on the physical properties of the hydrogel constructs fabricated from the components. described above.
Aim 3 – Biofunctionality: Evaluate the cellular response of the hydrogels synthesized and characterized in Aims 1 and 2. Determine whether there is a correlation between physical properties of polysaccharide hydrogels and the cellular response.

Over the research period of the project we studied the effect of conjugated G4RGDS on the structural features of HA-, Alginate- and Chitosan in aqueous solutions by SAXS and rheology. We showed that the fraction of the bounded peptide , determines the behavior of a polysaccharide-peptide conjugates in solution, regardless of the specific nature of the polysaccharide. We also explored the effect of self-assembling RGD-containing peptides on the spatial organization of polysaccharidee network. Three peptide sequences designed to self assemble into an "unstructured" manner (G6KRGDY), into spherical micelles (A6KRGDY), and V6KRGDY into cylindrical micelles (V6KRGDY) were covalently attached to alginate that was then geled by addition of calcium ions. Rheology and small-angle X-ray scattering (SAXS) analysis showed that the peptides' ability to self-assemble in aqueous solution affects the spatial organization of the alginate and the mechanical properties of the alginate/peptide hybrid hydrogel, both when the peptide is covalently attached to the alginate backbone and when peptide and alginate solutions are simply mixed together. Therefore we concluded that possible intermolecular interactions between the peptides and the polymer should be taken into consideration in the design of hybrid biomaterials. In addition, a detailed structural analysis of the conjugated architecture in solution can be used a tool to tailor the properties of polysaccharide/peptide hybrid hydrogels.
The findings of the project resulted in two manuscripts and were presented in more than ten international conferences.

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