RESUMEN
In fiber suspensions with low optical contrast, the in situ characterization of structural properties with conventional microscopy methods fails. However, overlaying subsequent images of multiple particle tracking (MPT) videos including short trajectories usually discarded in MPT analysis allowed for direct visualization of individual fibers and the network structure of lyophilized collagen I (Coll) distributed in hydrochloric acid solutions. MPT yielded a broad distribution of mean square displacements (MSDs). Freely diffusing tracer particles yielded viscosities indicating that, irrespective of concentration, a constant amount of Coll is dissolved in the aqueous phase. Particles found elastically trapped within fibrous Coll structures exhibited a broad range of time-independent MSDs and we propose a structure comprising multiple fiber bundles with dense regions inaccessible to tracers and elastic regions of different stiffness in between. Bulky aggregates inaccessible to the 0.2 µm tracers exist even at low Coll concentrations, a network of slender fibers evolves above the sol-gel transition and these fibers densify with increasing Coll concentration. This novel MPT-based imaging technique possesses great potential to characterize the fiber distribution in and structural properties of a broad range of biological and technical suspensions showing low contrast when imaged with conventional techniques. Thus, MPT imaging and microrheology will help to better understand the effect of fiber distribution and network structure on the viscoelastic properties of complex suspensions.
RESUMEN
For over half a century, hematopoietic stem cells (HSCs) have been used for transplantation therapy to treat severe hematologic diseases. Successful outcomes depend on collecting sufficient donor HSCs as well as ensuring efficient engraftment. These processes are influenced by dynamic interactions of HSCs with the bone marrow niche, which can be revealed by artificial niche models. Here, a multifunctional nanostructured hydrogel is presented as a 2D platform to investigate how the interdependencies of cytokine binding and nanopatterned adhesive ligands influence the behavior of human hematopoietic stem and progenitor cells (HSPCs). The results indicate that the degree of HSPC polarization and motility, observed when cultured on gels presenting the chemokine SDF-1α and a nanoscale-defined density of a cellular (IDSP) or extracellular matrix (LDV) α4ß1 integrin binding motif, are differently influenced on hydrogels functionalized with the different ligand types. Further, SDF-1α promotes cell polarization but not motility. Strikingly, the degree of differentiation correlates negatively with the nanoparticle spacing, which determines ligand density, but only for the cellular-derived IDSP motif. This mechanism potentially offers a means of predictably regulating early HSC fate decisions. Consequently, the innovative multifunctional hydrogel holds promise for deciphering dynamic HSPC-niche interactions and refining transplantation therapy protocols.
RESUMEN
Multiple particle tracking (MPT) microrheology was employed for monitoring the development of extracellular matrix (ECM) mechanical properties in the direct microenvironment of living cells. A customized setup enabled us to overcome current limitations: (i) Continuous measurements were enabled using a cell culture chamber, with this, matrix remodeling by fibroblasts in the heterogeneous environment of macroporous scaffolds was monitored continuously. (ii) Employing tracer laden porous scaffolds for seeding human mesenchymal stem cells (hMSCs), we followed conventional differentiation protocols. Thus, we were, for the first time able to study the massive alterations in ECM elasticity during hMSC differentiation. (iii) MPT measurements in 2D cell cultures were enabled using a long distance objective. Exemplarily, local mechanical properties of the ECM in human umbilical vein endothelial cell (HUVEC) cultures, that naturally form 2D layers, were investigated scaffold-free. Using our advanced setup, we measured local, apparent elastic moduli G0,app in a range between 0.08 and 60 Pa. For fibroblasts grown in collagen-based scaffolds, a continuous decrease of local matrix elasticity resulted during the first 10 hours after seeding. The osteogenic differentiation of hMSC cells cultivated in similar scaffolds, led to an increase of G0,app by 100 %, whereas after adipogenic differentiation it was reduced by 80 %. The local elasticity of ECM that was newly secreted by HUVECs increased significantly upon addition of protease inhibitor and in high glucose conditions even a twofold increase in G0,app was observed. The combination of these advanced methods opens up new avenues for a broad range of investigations regarding cell-matrix interactions and the propagation of ECM mechanical properties in complex biological systems. STATEMENT OF SIGNIFICANCE: Cells sense the elasticity of their environment on a micrometer length scale. For studying the local elasticity of extracellular matrix (ECM) in the direct environment of living cells, we employed an advanced multipleparticle tracking microrheology setup. MPT is based on monitoring the Brownian motion oftracer particles, which is restricted by the surrounding network. Network elasticity can thusbe quantified. Overcoming current limitations, we realized continuous investigations of ECM elasticityduring fibroblast growth. Furthermore, MPT measurements of stem cell ECM showed ECMstiffening during osteogenic differentiation and softening during adipogenic differentiation.Finally, we characterized small amounts of delicate ECM newly secreted in scaffold-freecultures of endothelial cells, that naturally form 2D layers.