A method for preparing porous hydrogel scaffolds suitable for bioengineering applications based on colloidal‐crystal templating is demonstrated. Templated scaffolds are shown to have highly interconnected porosities at moderate void fractions due to their ordered arrangement of voids, and thus combine abundant pathways for cell migration (see Figure) and mass transport with compressive moduli comparable to soft tissues.

Small-angle neutron scattering and dynamic and static light scattering measurements were used to probe the structures of aqueous and organic-solvent-based magnetic fluids comprising dispersed magnetite nanoparticles (∼10 nm in diameter) stabilized against flocculation by adsorbed alkanoic acid layers. A core−shell model fitted to a set of neutron scattering spectra obtained from contrast variation experiments allowed the determination of the iron oxide core size and size distribution, the thicknesses of the surfactant shells, and the spatial arrangement of the individual particles. The magnetic colloidal particles appear to form compact fractal clusters with a fractal dimension of 2.52 and a correlation length of ∼350 Å in aqueous magnetic fluids, consistent with the structures of clusters observed directly using cryo-TEM (transmission electron microscopy), whereas chainlike clusters with a fractal dimension of 1.22 and a correlation length of ∼400 Å were found for organic-solvent-based magnetic fluids. The differences in cluster structure were attributed to the relative strengths of the particle−particle interaction energies. Weak interactions in the organic-solvent-based systems dictate the formation of small structures for which the apparent fractal dimensions are naturally small, whereas significantly stronger interparticle interactions in aqueous magnetic fluids result in larger, more compact clusters with higher fractal dimensions. The growth of the aqueous clusters beyond a certain size was inhibited by an increasingly high energy barrier (balance between repulsive electrostatic and attractive van der Waals interactions) with increasing cluster size. The aqueous clusters were stable against further growth when diluted with a surfactant solution but grew in time when diluted with pure water. In the latter case, the loss of part of the stabilizing secondary surfactant layer to the aqueous phase to satisfy thermodynamic partitioning constraints led to a destabilization in its dispersion. Light scattering studies indicated a change in the fractal dimension from 2.52 to about 1.20 as the clusters grew.

Immunotherapies harness the inherent potential of the body to destroy foreign or infected cells, and are currently being investigated as treatments for cancer. One way to boost native immune responses might be to engineer ectopic lymphoid tissue, providing a supportive microenvironment for immune cell priming, and/or bringing together immune cells at a desired location (e.g., solid tumor sites). Here we describe the development and in vitro testing of composite macroporous poly(ethylene glycol) (PEG) hydrogel scaffolds infused with collagen as a tissue engineering platform for immunotherapy. The PEG hydrogel with ordered, interconnected pores provided mechanical stability and the potential to depot supporting cytokines/chemokines, while an infused collagen matrix supported intra-scaffold migration of loaded T cells and dendritic cells. Rapid, nearly unconstrained T cell migration through scaffolds was achieved by using inverse opal supporting structures with 80 microm macropores. In addition, we demonstrated that the lymphoid tissue chemokine CCL21 could be bound to the inverse opal gel walls of these scaffolds, to provide motility-inducing cues for T cells within these structures. This hybrid scaffold approach combines the strengths of the synthetic and biopolymer hydrogels used in a highly synergistic fashion, allowing each material to compensate for limiting properties of its partner.

Homeostatic chemokines such as CCL19, CCL21, and CXCL13 are known to elicit chemotaxis from naive T and B cells and play a critical role in lymphocyte homing to appropriate zones within secondary lymphoid organs (SLO). Here we tested whether CCL21 and CXCL13 modulate murine lymphocyte motility in the absence of concentration gradients, using videomicroscopy to directly observe the migration of single cells. CCL21 treatment of T cells induced rapid polarization and sustained random migration with average speeds of 5.16 ± 2.08 μm/min; B cell migration (average velocity 4.10 ± 1.58 μm/min) was similarly induced by CXCL13. Migration required the presence of both chemokine and adhesion ligands and was sustained for >24 h. Furthermore, in in vitro assays modeling the relative infrequency of Ag-specific T cell-dendritic cell (DC) encounters during primary immune responses, we found that CCL21 addition to T-DC cocultures accelerated the kinetics of CD69 up-regulation and enhanced by 2-fold the proliferation of Ag-specific T cells in a manner dependent on G-protein-coupled receptor signaling in T cells. These results suggest that homeostatic chemokines could substantially impact the dynamics and priming of lymphocytes within SLO even in the absence of significant concentration gradients.

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