Polysaccharide-based hydrogels are remarkable materials for the development of tissue engineering strategies as they meet several critical requirements for such applications and they may partly mimic the extracellular matrix. Chitosan is widely envisioned as hydrogel in biomedical fields for its bioresorbability, biocompatibility, and fungistatic and bacteriostatic properties. In this study, we report that the modulation of the polymer concentration, the degree of acetylation, the gelation processes [or neutralization routes (NR)] in the preparation of different chitosan-based hydrogels lead to substantially and significantly different biological responses. We show that it is possible to tune the physicochemical characteristics, mechanical properties, and biological responses of such matrices. Physical hydrogels prepared from highly acetylated chitosan were softer, degraded quickly in vivo, and were not suitable for in vitro culture of human mesenchymal stem and progenitor derived endothelial cells. In contrast, for a same chitosan concentration and obtained by the same processing route, a low degree of acetylation chitosan hydrogel provided a more elastic material, better cell adhesion on its surface and tissue regeneration, and restored tissue neo-vascularization as well. This work offers promising and innovative perspectives for the design of hydrogel materials with tunable properties for tissue engineering and regenerative medicine.

SummaryMature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of “soft” tissue from which cells can easily be isolated and studied, the “hard” mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation under physiological conditions. We obtained mature osteoblasts and characterized them on the basis of the following parameters: the strong expression of osteoblastic markers, such as Runx2 and Col-I; the achievement of specific dimensions (the cell volume increases 26-fold compared to the osteoblast precursors); and the production of an abundant extracellular matrix also called osteoid. We demonstrated that the differentiation of osteoblast precursors into mature osteoblasts requires the continuous activation of Bone Morphogenetic Protein (BMP) receptors, which we established with the immobilization of a BMP-2mimetic peptide on a synthetic matrix mimicking in vivo microenvironment. Importantly, we demonstrated that the organization of the F-actin network and acetylated microtubules of the cells were modified during the differentiation process. We showed that the perturbation of the F-actin cytoskeleton organization abolished the differentiation process. In addition, we demonstrated that expression of the Runx2 gene is required for this differentiation. These findings demonstrate the retro-regulation of cytoplasmic and genic components due to the continuous induction of BMP-2 and also provide more detailed insights into the correct signaling of BMPs for cell differentiation in bone tissue.

How endothelial cells (ECs) express the particular filopodial or lamellipodial form of the actin machinery is critical to understanding EC functions such as angiogenesis and sprouting. It is not known how these mechanisms coordinately promote lumen formation of ECs. Here, adhesion molecules (RGD peptides) and inductor molecules (BMP-2 mimetic peptides) are micropatterned onto polymer surfaces by a photolithographic technique to induce filopodial and lamellipodial migration modes. Firstly, the effects of peptide microgeometrical distribution on EC adhesion, orientation and morphogenesis are evaluated. Large micropatterns (100 μm) promote EC orientation without lumen formation, whereas small micropatterns (10-50 μm) elicit a collective cell organization and induce EC lumen formation, in the case of RGD peptides. Secondly, the correlation between EC actin machinery expression and EC self-assembly into lumen formation is addressed. Only the filopodial migration mode (mimicked by RGD) but not lamellipodial migration mode (mimicked by BMP-2) promotes EC lumen formation. This work gives a new concept for the design of biomaterials for tissue engineering and may provide new insight for angiogenesis inhibition on tumors.