(A-5) Electrospun, photoresponsive hydrogels, layered to establish complex cellular microenvironments with high-resolution features

Introduction:: When modeling tissue in vitro, the biomaterial system used must be able to sufficiently mimic the complex functional properties of the tissue. Hydrogels are often used because they are soft materials with many physiologically relevant properties, and they can be designed to include specific biochemical and functional cues that can influence cell fate within an engineered tissue construct. Research continues to look to address challenges in replicating physiological environments through the development of processes that enable specification of construct properties at increasing resolution.
  Multi-layered hydrogels and their fabrication processes have been of interest in biomaterials research because of their potential to enable heterogeneity to be established across the z-space a construct occupies. Control of biochemical and mechanical properties within a single layer allows a layered construct to mimic anisotropic physiological properties of tissue across all three dimensions. Fibrous hydrogels offer additional opportunities to introduce topographical features that can direct cell behaviors and might be used in a layered construct.
  In this work, we aim to leverage the strengths of both multi-layered hydrogels and photosensitive, fibrous hydrogels to fabricate a construct that contains both cells and high-resolution cell-instructive cues. Here, we demonstrate the potential to use electrospun hydrogel fibers to align, with accuracy on the order of ~100 μm, specific photopatterned features in cell-containing constructs that are two-layers thick. Current work is focused on understanding cell responses to the differing cues.

Materials and Methods:: A combination of norbornene-modified polyethylene glycol and thiol-modified polyethylene glycol were combined to create an electrospinning solution. By design, when nanofibers were photo-crosslinked, all thiols would be consumed through reaction with norbornene, but excess norbornenes would remain for further reaction with thiolated molecules, so this material is referred to simply as NorPEG. This solution included 10 wt% 20 kDa NorPEG, 7 wt% 10 kDa PEG-thiol, I2959 photoinitiator, and non-reactive polyethylene oxide to facilitate electrospinning.
  Two-layered fibrous hydrogels were constructed as follows. Glass coverslips were surfacemodified to covalently crosslink to fibers deposited on them. NorPEG was electrospun onto the modified coverslips and UV-crosslinked. For directional fiber alignment, coverslips were placed on a spinning mandril.
  Fibers were patterned with a thiolated peptide at 1 mM (containing rhodamine B for fluorescent imaging alone, or RGD for cell culture) via photomasks. Each photomask was a member of a pair that, when aligned, create a complete pattern. To ensure accurate pattern placement, a 3D-printed device was designed and used to place the photomask on the hydrogel.
  Hydrogels were layered by stacking two coverslips with corresponding patterns, inverting one to “sandwich” both hydrogels between glass. During patterning, one photomask from each pair was inverted, producing an inverted pattern to allow for this coverslip inversion. A 3D-printed device was designed to align the coverslips during stacking.
  Cell-culture was performed with myoblasts (C2C12s) in standard growth media and according to typical cell culture protocols. Cells were fixed and stained with 488-phalloidin and DAPI.

Results, Conclusions, and Discussions:: NorPEG hydrogel fibers were determined to be the well suited for electrospinning and layering, based on qualitative observation of electrospinning performance and fiber stability during physical manipulation.
  Upon imaging of hydrogel fibers upon which fluorescently labeled peptides were patterned using photomasks, patterns were observed with high fidelity to photomask design. These patterns were maintained throughout the layering process. The patterns on each layer were considered well-aligned, as any misalignment was on the order of ~100 µm. This is on the order of the resolution limits of biofabrication technologies such as bioprinting. Epifluorescence images display the successful construction of two-layered hydrogels in which each layer has unique biochemical properties, specified by photopatterned molecules, and mechanical properties, specified by aligned fiber direction in that layer (Figure 1). Ongoing work to improve alignment accuracy is investigating the design of 3D printed alignment devices, minimizing coverslip sliding after stacking, and factors related to material characteristics (e.g. swelling) and immobilization.
  Imaging RGD-functionalized hydrogels displayed cells responding to the layer-specific presence of RGD and direction of fiber alignment (Figure 2). On RGD patterns, there is increased cell adhesion. The mean confluency for RGD-patterned regions after seeding was 65+/-22%, and for non-RGD-patterned regions, it was 12+/-3% (p-value = ~0.0004). Additionally, the cells respond to the differing topographical cues, as observed in epifluorescence images of the layered hydrogel nanofibers. On each layer, cell morphologies become extended and uniaxial with alignment parallel to the direction of that layer’s fibers.
  Altogether, we have demonstrated the ability to construct a multi-layered fibrous hydrogel, and specify different physical properties and biochemical signals within each layer to control the behavior of cells seeded in the hydrogel construct. Electrospun, fibrous hydrogels have excellent properties to mimic body tissues for in vitro study of cell behavior and ongoing work looks to improve the accuracy of alignment as well as cellular complexity within the constructs. Applications in understanding multicellular behaviors, for example in stem cell niches, are currently under consideration.

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