Dr. Aleeza Farrukh

Hydrogels mimicking the mechanical and biochemical features of the cellular microenvironment allow cell encapsulation and facilitate in vitro 3D culture. In addition to biocompatibility and reactivity in physiological conditions, a key criterion for crosslinking chemistry is appropriate gelation kinetics to allow mixing and homogeneous distribution of cells with the hydrogel precursors. We have previously presented aryl methylsulfone/thiol (MS/SH) reaction as a thiol-reactive cross-linking system for cell encapsulation in star polyethylene glycol (PEG4) hydrogels with a gelation kinetics in minutes time scale. Remaining experimental challenges for this system are a finer modulation of gelation kinetics and streamlining the synthesis of the prepolymer. Here we present the possibility to tune the gelation kinetics by introducing an electron-withdrawing substituent at p-position of the aryl MS ring. This variant also presents synthetic advantages. We study the influence of the p-substituent on the physicochemical properties of MS/SH crosslinked hydrogels, and their performance for cell encapsulation. We compare these properties with the PEG-MS variant containing an electron-donating linker. The new star poly(ethylene glycol)-4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)benzamide (PEG4-CONH-TzMS) shows superior properties as cell encapsulating hydrogel in terms of ease of mixing polymer precursors, faster gelation, homogenous cell distribution and high enzymatic stability.

Abstract Neuro-regeneration after trauma requires growth and reconnection of neurons to reestablish information flow in particular directions across the damaged tissue. To support this process, biomaterials for nerve tissue regeneration need to provide spatial information to adhesion receptors on the cell membrane and to provide directionality to growing neurites. Here, photoactivatable adhesive peptides based on the CASIKVAVSADR laminin peptidomimetic are presented and applied to spatiotemporal control of neuronal growth to biomaterials in vitro. The introduction of a photoremovable group [6-nitroveratryl (NVOC), 3-(4,5-dimethoxy-2-nitrophenyl)butan-2-yl (DMNPB), or 2,2′-((3′-(1-hydroxypropan-2-yl)-4′-nitro-[1,1′-biphenyl]-4-yl)azanediyl)bis(ethan-1-ol) (HANBP)] at the amino terminal group of the K residue temporally inhibited the activity of the peptide. The bioactivity was regained through controlled light exposure. When used in neuronal culture substrates, the peptides allowed light-based control of the attachment and differentiation of neuronal cells. Site-selective irradiation activated adhesion and differentiation cues and guided seeded neurons to grow in predefined patterns. This is the first demonstration of ligand-based light-controlled interaction between neuronal cells and biomaterials.

Strategies for neural tissue repair heavily depend on our ability to temporally reconstruct the natural cellular microenvironment of neural cells. Biomaterials play a fundamental role in this context, as they provide the mechanical support for cells to attach and migrate to the injury site, as well as fundamental signals for differentiation. This review describes how different cellular processes (attachment, proliferation, and (directional) migration and differentiation) have been supported by different material parameters, in vitro and in vivo. Although incipient guidelines for biomaterial design become visible, literature in the field remains rather phenomenological. As in other fields of tissue regeneration, progress will depend on more systematic studies on cell-materials response, better understanding on how cells behave and understand signals in their natural milieu from neurobiology studies, and the translation of this knowledge into engineered microenvironments for clinical use.

The ability to guide the growth of neurites is relevant for reconstructing neural networks and for nerve tissue regeneration. Here, a biofunctional hydrogel that allows light-based directional control of axon growth in situ is presented. The gel is covalently modified with a photoactivatable derivative of the short laminin peptidomimetic IKVAV. This adhesive peptide contains the photoremovable group 2-(4′-amino-4-nitro-[1,1′-biphenyl]-3-yl)propan-1-ol (HANBP) on the Lys rest that inhibits its activity. The modified peptide is highly soluble in water and can be simply conjugated to −COOH containing hydrogels via its terminal −NH2 group. Light exposure allows presentation of the IKVAV adhesive motif on a soft hydrogel at desired concentration and at defined position and time point. The photoactivated gel supports neurite outgrowth in embryonic neural progenitor cells culture and allows site-selective guidance of neurites extension. In situ exposure of cell cultures using a scanning laser allows outgrowth of neurites in desired pathways.

Abstract The integrin α5β1 is overexpressed in colon, breast, ovarian, lung and brain tumours, and has been identified as key component in mechanosensing. In order to study how dynamic changes in α5β1 engagement affect cellular behaviour, photoactivatable derivatives of α5β1-specific ligands are presented in this article. A photoremovable protecting group (PRPG) was introduced into the ligand structure at a relevant position for integrin recognition. The presence of the chromophore temporarily inhibited ligand bioactivity. Light exposure at a cell-compatible dose efficiently cleaved the protecting group and restored functionality. The photoactive ligand had an azide end-functional group for covalent immobilisation onto biomaterials by click chemistry. Selective cell responses (attachment, spreading, migration) to the activated ligand on the surface are achieved by controlled exposure to light, at similar levels to the native ligand. Spatial and temporal control of the cellular response is demonstrated, including the possibility of in situ activation. Photoactivatable integrin-selective ligands in model microenvironments will allow the study of cellular behaviour in response to changes in the activation of individual integrins as consequence of dynamic variations in matrix composition.

Optoregulated biointerfaces offer the possibility to manipulate the interactions between cell membrane receptors and the extracellular space. This Invited Feature Article summarizes recent efforts by our group and others during the past decade to develop light-responsive biointerfaces to stimulate cells and elicit cellular responses using photocleavable protecting groups (PPG) as our working tool. This article begins by providing a brief introduction to available PPGs, with a special focus on the widely used o-nitrobenzyl family, followed by an overview of molecular design principles for the control of bioactivity in the context of cell–material interactions and the characterization methods to use in following the photoreaction at surfaces. We present various light-guided cellular processes using PPGs, including cell adhesion, release, migration, proliferation, and differentiation, both in vitro and in vivo. Finally, this Invited Feature Article closes with our perspective on the current status and future challenges of this topic.

Summary Engineering of biomaterials with specific biological properties has gained momentum as a means to control stem cell behavior. Here, we address the effect of bifunctionalized hydrogels comprising polylysine (PL) and a 19-mer peptide containing the laminin motif IKVAV (IKVAV) on embryonic and adult neuronal progenitor cells under different stiffness regimes. Neuronal differentiation of embryonic and adult neural progenitors was accelerated by adjusting the gel stiffness to 2 kPa and 20 kPa, respectively. While gels containing IKVAV or PL alone failed to support long-term cell adhesion, in bifunctional gels, IKVAV synergized with PL to promote differentiation and formation of focal adhesions containing β1-integrin in embryonic cortical neurons. Furthermore, in adult neural stem cell culture, bifunctionalized gels promoted neurogenesis via the expansion of neurogenic clones. These data highlight the potential of synthetic matrices to steer stem and progenitor cell behavior via defined mechano-adhesive properties.

Biomaterials for cell culture allowing simple and quantitative presentation of instructive cues enable rationalization of the interplay between cells and their surrounding microenvironment. Poly(acrylamide) (PAAm) hydrogels are popular 2D-model substrates for this purpose. However, quantitative and reproducible biofunctionalization of PAAm hydrogels with multiple ligands in a trustable, controlled, and independent fashion is not trivial. Here, we describe a method for bifunctional modification of PAAm hydrogels with thiol- and amine- containing biomolecules with controlled densities in an independent, orthogonal manner. We developed copolymer networks of AAm with 9% acrylic acid and 2% N-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acrylamide. The covalent binding of thiol- and amine-containing chromophores at tunable concentrations was demonstrated and quantified by UV spectroscopy. The morphology, mechanical properties, and homogeneity of the copolymerized hydrogels were characterized by scanning electron microscopy, dynamic mechanical analysis, and confocal microscopy studies. Our copolymer hydrogels were bifunctionalized with polylysine and a laminin-mimetic peptide using the specific chemistries. We analyzed the effect of binding protocol of the two components in the maturation of cultured postmitotic cortical neurons. Our substrates supported neuronal attachment, proliferation, and neuronal differentiation. We found that neurons cultured on our hydrogels bifunctionalized with ligand-specific chemistries in a sequential fashion exhibited higher maturation at comparable culture times than using a simultaneous bifunctionalization strategy, displaying a higher number of neurites, branches, and dendritic filopodia. These results demonstrate the relevance of quantitative and optimized coupling chemistries for the performance of simple biomaterials and with sensitive cell types.

Poly(acrylamide) P(AAm) gels have become relevant model substrates to study cell response to the mechanical and biochemical properties of the cellular microenvironment. However, current bioconjugation strategies to functionalize P(AAm) gels, mainly using photoinduced arylazide coupling, show poor specificity and hinder conclusive studies of material properties and cellular responses. We describe methylsulfonyl-containing P(AAm) hydrogels for cell culture. These gels allow easy, specific and functional covalent coupling of thiol containing bioligands in tunable concentrations under physiological conditions, while retaining the same swelling, porosity, cytocompatibility, and low protein adsorption of P(AAm) gels. These materials allow quantitative and standardized studies of cell-materials interactions with P(AAm) gels.