Julien E. Gautrot

Extracellular-matrix tethering regulates stem-cell fate

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

Surface-initiated polymer brushes in the biomedical field : applications in membrane science, biosensing, cell culture, regenerative medicine and antibacterial coatings

Surface-initiated polymer brushes in the biomedical field : applications in membrane science, biosensing, cell culture, regenerative medicine and antibacterial coatings

Polymer Brushes Showing Non-Fouling in Blood Plasma Challenge the Currently Accepted Design of Protein Resistant Surfaces

Ultra-low-fouling poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)) brushes have been synthesized for the first time. Similar to the so far only ultra-low-fouling surface, poly(carboxybetaine acrylamide), the level of blood plasma fouling was below the detection limit of surface plasmon resonance (SPR, 0.03 ng·cm(-2)) despite being a hydrogen bond donor and displaying a moderate wettability, thus challenging the currently accepted views for the design of antifouling properties. The antifouling properties were preserved even after two years of storage. To demonstrate the potential of poly(HPMA) brushes for the preparation of bioactive ultra-low fouling surfaces a label-free SPR immunosensor for detection of G Streptococcus was prepared.

Biofunctionalized Protein Resistant Oligo(ethylene glycol)-Derived Polymer Brushes as Selective Immobilization and Sensing Platforms

Poly(oligo(ethylene glycol) methacrylate) (POEGMA) brushes are extremely protein resistant polymer coatings that can reduce nonspecific adsorption of proteins from complex mixtures such as blood, sera and plasma. These coatings can be prepared via atom transfer radical polymerization with excellent control of their thickness and grafting density. We studied their direct functionalization with streptavidin and developed an assay for determining which coupling conditions afford the highest streptavidin loading efficiency. Disuccinimidyl carbonate was found to be the most efficient activating agent for covalent capture of the receptor. Using infrared and X-ray photoelectron spectroscopy, fluorescence microscopy, surface plasmon resonance, and ellipsometry, we examined how structural parameters such as the length of the oligo(ethylene glycol) side chain affect streptavidin functionalization, but also immobilization of biotinylated antibodies, subsequent selective secondary recognition and nonspecific binding of proteins. We found evidence that large macromolecules cannot infiltrate dense polymer brushes and that bulky antibody recognition occurs in the upper part of these coatings.

Exploiting the superior protein resistance of polymer brushes to control single cell adhesion and polarisation at the micron scale.

The control of the cell microenvironment on model patterned substrates allows the systematic study of cell biology in well defined conditions, potentially using automated systems. The extreme protein resistance of poly(oligo(ethylene glycol methacrylate)) (POEGMA) brushes is exploited to achieve high fidelity patterning of single cells. These coatings can be patterned by soft lithography on large areas (a microscope slide) and scale (substrates were typically prepared in batches of 200). The present protocol relies on the adsorption of extra-cellular matrix (ECM) proteins on unprotected areas using simple incubation and washing steps. The stability of POEGMA brushes, as examined via ellipsometry and SPR, is found to be excellent, both during storage and cell culture. The impact of substrate treatment, brush thickness and incubation protocol on ECM deposition, both for ultra-thin gold and glass substrates, is investigated via fluorescence microscopy and AFM. Optimised conditions result in high quality ECM patterns at the micron scale, even on glass substrates, that are suitable for controlling cell spreading and polarisation. These patterns are compatible with state-of-the-art technologies (fluorescence microscopy, FRET) used for live cell imaging. This technology, combined with single cell analysis methods, provides a platform for exploring the mechanisms that regulate cell behaviour.

The nanoscale geometrical maturation of focal adhesions controls stem cell differentiation and mechanotransduction.

We show that the nanoscale adhesion geometry controls the spreading and differentiation of epidermal stem cells. We find that cells respond to such hard nanopatterns similarly to their behavior on soft hydrogels. Cellular responses were seen to stem from local changes in diffusion dynamics of the adapter protein vinculin and associated impaired mechanotransduction rather than impaired recruitment of proteins involved in focal adhesion formation.

Cell sensing of physical properties at the nanoscale: Mechanisms and control of cell adhesion and phenotype.

UNLABELLED The chemistry, geometry, topography and mechanical properties of biomaterials modulate biochemical signals (in particular ligand-receptor binding events) that control cells-matrix interactions. In turn, the regulation of cell adhesion by the biochemical and physical properties of the matrix controls cell phenotypes such as proliferation, motility and differentiation. In particular, nanoscale geometrical, topographical and mechanical properties of biomaterials are essential to achieve control of the cell-biomaterials interface. The design of such nanoscale architectures and platforms requires understanding the molecular mechanisms underlying adhesion formation and the assembly of the actin cytoskeleton. This review presents some of the important molecular mechanisms underlying cell adhesion to biomaterials mediated by integrins and discusses the nanoscale engineered platforms used to control these processes. Such nanoscale understanding of the cell-biomaterials interface offers exciting opportunities for the design of biomaterials and their application to the field of tissue engineering. STATEMENT OF SIGNIFICANCE Biomaterials design is important in the fields of regenerative medicine and tissue engineering, in particular to allow the long term expansion of stem cells and the engineering of scaffolds for tissue regeneration. Cell adhesion to biomaterials often plays a central role in regulating cell phenotype. It is emerging that physical properties of biomaterials, and more generally the microenvironment, regulate such behaviour. In particular, cells respond to nanoscale physical properties of their matrix. Understanding how such nanoscale physical properties control cell adhesion is therefore essential for biomaterials design. To this aim, a deeper understanding of molecular processes controlling cell adhesion, but also a greater control of matrix engineering is required. Such multidisciplinary approaches shed light on some of the fundamental mechanisms via which cell adhesions sense their nanoscale physical environment.

Recent advances in entropy-driven ring-opening polymerizations

Entropy-driven ring-opening polymerization (ED-ROP) of unstrained macrocyclic monomers and/or oligomers employs the ring-chain equilibria between macrocycles and their corresponding polymers and the associated increase of conformational freedom to achieve high molecular weight materials. The principles of building macrocyclic compounds, their use in ED-ROP, and the practical considerations of polymerizations are described, and recent progress in this area is discussed through selected examples. The various polymerization techniques used for ED-ROP are discussed, including anionic, radical, coordination/insertion, ring-opening metathesis, and enzymatic polymerization methods. Emphasis is placed on the potential of ED-ROP in the synthesis of biomaterials and the development of enzyme-catalyzed green systems.

Formation of Pickering Emulsions Using Ion-Specific Responsive Colloids

The ability to control the dispersion, aggregation, and assembly of colloidal systems is important for a number of applications, for instance, Pickering emulsions, drug and gene delivery, control of fluid rheology, and the formation of colloidal crystal arrays. We generated a responsive colloidal system based on polymer-brush-grafted silica nanoparticles and demonstrated that such a colloidal system can be used to produce stable oil-in-water Pickering emulsions. Cationic poly(2-(methacryloyloxy)-ethyl-trimethyl-ammonium chloride) (PMETAC) brushes were grown from silica nanoparticles (diameter ∼320 nm) through surface-initiated atom-transfer radical polymerization (ATRP). PMETAC brushes are attractive coatings for controlling the behavior of colloidal systems, owing to their ion-specific collapse resulting in the switching of surface hydrophilicity. Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta-potential measurements indicated the successful grafting of PMETAC brushes on nanoparticles. The resulting colloidal dispersion was shown to be responsive to perchlorate ions (ClO(4)(-)), which triggered particle aggregation and enabled the generation of Pickering emulsions. The onset of aggregation depended on the polymer chain length. Aggregation was not affected by the initiator density and brush conformational changes. Further studies suggested that particle aggregation and the formation of stable Pickering emulsions were not simply due to brush collapse but also were due to a gradual shielding of electrostatic repulsion. Finally, the stability and homogeneity of the resulting Pickering emulsions were studied.

Shape-Induced Terminal Differentiation of Human Epidermal Stem Cells Requires p38 and Is Regulated by Histone Acetylation

Engineered model substrates are powerful tools for examining interactions between stem cells and their microenvironment. Using this approach, we have previously shown that restricted cell adhesion promotes terminal differentiation of human epidermal stem cells via activation of serum response factor (SRF) and transcription of AP-1 genes. Here we investigate the roles of p38 MAPK and histone acetylation. Inhibition of p38 activity impaired SRF transcriptional activity and shape-induced terminal differentiation of human keratinocytes. In addition, inhibiting p38 reduced histone H3 acetylation at the promoters of SRF target genes, FOS and JUNB. Although histone acetylation correlated with SRF transcriptional activity and target gene expression, treatment with the histone de-acetylase inhibitor, trichostatin A (TSA) blocked terminal differentiation on micro-patterned substrates and in suspension. TSA treatment simultaneously maintained expression of LRIG1, TP63, and ITGB1. Therefore, global histone de-acetylation represses stem cell maintenance genes independent of SRF. Our studies establish a novel role for extrinsic physical cues in the regulation of chromatin remodeling, transcription, and differentiation of human epidermal stem cells.