Open Access

Hydrogels are useful temporal matrices for cell culture technologies. The successful mixing and encapsulation of cells within the gel requires the selection of efficient and cytocompatible gelation reactions occurring in the minute timescale under physiological conditions. The thiol-methylsulfonyl (MS) chemical reaction is introduced here as a novel chemistry to encapsulate cells in polymeric matrices. Thiol-MS crosslinking does not require a light activation step and can occur within the seconds-to-minutes timescale by adjusting the pH in the physiological range 8.0-6.6. This reaction is cytocompatible and the reaction product is hydrolytically stable in cell culture media up to 4 weeks. Cell encapsulation protocols enabling comfortable handling and yielding homogenous distribution of the embedded cells are described. All these features are relevant for the application of this crosslinking reaction to biomedical scenarios. Finally, this manuscript also compares the performance of thiol-MS hydrogels with the established thiol-maleimide and thiol-vinylsulfone hydrogels. The benefit of thiol-MS crosslinking in terms of control over hydrogelation kinetics is demonstrated.

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.

The restoration of neuronal activity after injury or during aging requires neuron repopulation at the site of injury, directional regeneration of new nerves and oriented generation of new synapses. The ECM protein Laminin is abundant in neuronal microenvironment and is known to be involved in directing neuronal migration, differentiation and neurite development. In this thesis, a strategy for in vitro directional neurite growth in soft hydrogels is presented. It is based on the spatiotemporal control of the availability of Laminin adhesive motifs within synthetic hydrogels using light as an external guiding trigger. Different variants of Laminin mimetic peptides containing the IKVAV were selected as ligands to mediate control over axonal growth on biomaterials. The photo- cleavable groups 3-(4,5-dimethoxy-2-nitrophenyl)-2-butanol (DMNPB), 6-nitroveratryl alcohol (NVOC) and 2,2'-((3'-(1-hydroxypropan-2-yl)-4'-nitro-[1,1'-biphenyl] 4-yl)azanediyl)bis(ethan-1-ol) (HANBP) were inserted at the K rest of the peptide to temporally block IKVAV bioactivity. Poly(acrylamide) (PAAm) hydrogel films with varied stiffness from 0.2-70 kPa were used as 2D substrates to study IKVAV-guided directional growth of axons. Two novel acryl monomers carrying methylsulfone (MS) side chains were developed to tune specific coupling of thiol terminated IKVAV to the PAAm gel at physiological conditions. The ability of the photoactivatable IKVAV-containing peptides to trigger and support neurite outgrowth was studied and compared in different cell biology experiments using neural progenitor cells from mouse embryo. The ability of the photoactivatable IKVAV-containing peptides to trigger and support spatial organization of neurons was demonstrated by using masked irradiation. The in-situ light exposure of IK(HANBP)VAV by scanning lasers allowed spatially directed neurite development in 2D cell cultures. In the last part of the Thesis, an attempt to extend the photoactivation strategy to 3D environments was made by using two-photon activatable chromophores. The p-methoxynitrobiphenyl (PMNB) photoremovable group was introduced at aspartic acid residue of RGD sequence, a common adhesive motif used for cell attachment to biomaterials. Degradable hydrogels modified with RGD(PMNB)fC peptide were developed and 3D resolved spatial photoactivation inside the gel using two-photon laser guided migration of fibroblasts L929 within the 3D network was established. These results demonstrate that photoactivatable adhesive peptides can be used for spatiotemporal activation of attachment, migration and directional growth of cells in 2D and 3D cultures and provide a tool to control and pattern cell processes in relevant biomedical applications

Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1β and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1β and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses.