Search

Physicochemical features of a cell nanoenvironment exert important influence on stem cell behavior and include the influence of matrix elasticity and topography on differentiation processes. The presence of growth factors such as TGF-β and BMPs on these matrices provides chemical cues and thus plays vital role in directing eventual stem cell fate. Engineering of functional biomimetic scaffolds that present programmed spatio-temporal physical and chemical signals to stem cells holds great promise in stem cell therapy. Progress in this field requires tacit understanding of the mechanistic aspects of cell-environment nanointeractions, so that they can be manipulated and exploited for the design of sophisticated next generation biomaterials. In this review, we report and discuss the evolution of these processes and pathways in the context of matrix adhesion as they might relate to stemness and stem cell differentiation. Super-resolution microscopy and single-molecule methods for in vitro nano-manipulation are helping to identify and characterize the molecules and mechanics of structural transitions within stem cells and matrices. All these advances facilitate research toward understanding of stem cell niche and consequently to developing new class of biomaterials helping the “used biomaterials” for applications in tissue engineering and regenerative medicine.

Different studies have established a link between adult mesenchymal stem cells (MSCs) and perivascular cells, such as pericytes, which envelop the vascular tubes. This link was validated mainly through shared marker signatures and raised the hypothesis that all MSCs are pericytes. A controversy remains, however, as to whether there are functions specifi c to only one cell type. A specifi c role of pericytes in vascular tube stabilization that is not shared with MSCs is reported. An experimental model is introduced for the induction of endothelial cell (EC) tubulogenesis after 24 h of incubation on micropatterned polymer surfaces. Pericytes or MSCs are added separately to this system to evaluate their effect on tubular stabilization. In the absence of additional cells or in the presence only of MSCs the tubular structures are lost after 36 h. Addition of only pericytes, however, stabilizes the EC vasculogenic tubes. Furthermore, only pericytes, but not MSCs, are able to migrate through a mimetic basement membrane and interact with ECs to stabilize lumen structures. It is suggested that pericytes have specifi c cell functions of potential major importance in vascular and bone-tissue regeneration. The use of pericytes in tissue engineering will thus become critical. Blood vessels are composed of two interacting cell types

We present a method of preparing and characterizing nanostructured bioactive motifs using a combination of nanoimprint lithography and surface functionalization. Nanodots were fabricated on silicon surfaces and modified with a cell-adhesive RGD peptide for studies in human mesenchymal stem cell adhesion and differentiation. We report that bioactive nanostructures induce mature focal adhesions on human mesenchymal stem cells with an impact on their behavior and dynamics specifically in terms of cell spreading, cell-material contact, and cell differentiation.

Summary Mature 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.

H ow endothelial cells (ECs) express the particular fi lopodial 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 fi lopodial and lamellipodial migration modes. Firstly, the effects of peptide microgeometrical distribution on EC adhesion, orientation and morphogenesis are evaluated. Large micropatterns (100 μ m) promote EC orientation without lumen formation, whereas small micropatterns (10‐50 μ m) 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 fi lopodial 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.

Stem cells cultured on extracellular matrix (ECM) with different stiffnesses have been shown to engage into different lineage commitments. However, in vivo, the components of the ECM are known to bind and strongly interact with growth factors. The effect, on the stem cell fate, of the cooperation between the mechanical properties and the growth factor in the same microenvironment has not yet been investigated. Here, we propose a protocol for mimicking this stem cell microenvironment with an in vitro system. This system consists in grafting (without using a spacer) biomolecules that contain N-termini groups onto hydrogel (poly(acrylamide-co-acrylic acid)) surfaces of various stiffnesses ranging from 0.5 to 70 kPa. First, we demonstrate that the commitment of mesenchymal stem cell populations changes in response to the substrate's rigidity, with myogenic differentiation occurring at 13-17 kPa and osteogenic differentiation at 45-49 kPa. Chemical grafting of soft and stiff matrices with an osteogenic factor (BMP-2(mimetic peptide)) results only in osteogenic differentiation. Also, when grafted on even softer gels (0.5-3.5 kPa), the BMP-2(mimetic peptide) had no effect on the stem cell differentiation. We prove that correct organization of F-actin cytoskeleton due to the mechanical properties of the microenvironment is necessary for BMP-induced smad1/5/8 phosphorylation and nuclear translocation. These results suggest that stem cell differentiation is dictated mechanically, but in the presence of a biochemical factor, the effect of the mechanical factor on stem cell commitment is modified. This can explain the diversity of stem cell behaviors in vivo where different growth factors are sequestrated on the ECM.

International audience; The differentiation of stem cells can be modulated by physical factors such as the micro- and nano-topography of the extracellular matrix. One important goal in stem cell research is to understand the concept that directs differentiation into a specific cell lineage in the nanoscale environment. Here, we demonstrate that such paths exist by controlling only the micro- and nano-topography of polymer surfaces. Altering the depth (on a nanometric scale) of micro-patterned surface structures allowed increased adhesion of human mesenchymal stem cells (hMSCs) with specific differentiation into osteoblasts, in the absence of osteogenic medium. Small (10 nm) depth patterns promoted cell adhesion without noticeable differentiation, whereas larger depth patterns (100 nm) elicited a collective cell organization, which induced selective differentiation into osteoblast-like cells. This latter response was dictated by stress through focal-adhesion-induced reorganization of F-actin filaments. The results have significant implications for understanding the architectural effects of the in vivo microenvironment and also for the therapeutic use of stem cells.

Angiogenesis, the formation of new blood vessels by sprouting from pre-existing ones, is critical for the establishment and maintenance of complex tissues. Angiogenesis is usually triggered by soluble growth factors such as VEGF. However, geometrical cues also play an important role in this process. Here we report the induction of angiogenesis solely by SVVYGLR peptide micropatterning on polymer surfaces. SVVYGLR peptide stripes were micropatterned onto polymer surfaces by photolithography to study their effects on endothelial cell (EC) behavior. Our results showed that the EC behaviors (cell spreading, orientation and migration) were significantly more guided and regulated on narrower SVVYGLR micropatterns (10 and 50 µm) than on larger stripes (100 µm). Also, EC morphogenesis into tube formation was switched on onto the smaller patterns. We illustrated that the central lumen of tubular structures can be formed by only one-to-four cells due to geometrical constraints on the micropatterns which mediated cell-substrate adhesion and generated a correct maturation of adherens junctions. In addition, sprouting of ECs and vascular networks were also induced by geometrical cues on surfaces micropatterned with SVVYGLR peptides. These micropatterned surfaces provide opportunities for mimicking angiogenesis by peptide modification instead of exogenous growth factors. The organization of ECs into tubular structures and the induction of sprouting angiogenesis are important towards the fabrication of vascularized tissues, and this work has great potential applications in tissue engineering and tissue regeneration.