Nanofibrous hydrogels with spatially patterned biochemical signals to control cell behavior

Abstract

The ability to spatially pattern biochemical signals into nanofibrous materials using thiol-ene reactions of thiolated molecules to presented norbornene groups is demonstrated. This approach is used to pattern three molecules independently within one scaffold, to pattern molecules through the depth of a scaffold, and to spatially control cell adhesion and morphology.

Keywords: alignment; electrospinning; nanofibers; patterning; tissue engineering.

Figure 1

Electrospun nanofibrous hydrogel formation, crosslinking, and biochemical ligand patterning. (A) Synthesis of norbornene-hyaluronic acid (NorHA) with norbornene group shown in red. (B) Electrospinning process and (C) morphology of nanofibers post electrospinning (Scale bar: 5μm). (D) Schematic illustrating gross fiber appearance (top row) and the corresponding molecular reactions (bottom row) associated with the steps to crosslink and pattern biochemical ligands in nanofibrous hydrogels. Crosslinking occurs in the dry state via UV light initiated thiol-ene reactions of a di-thiol and norbornene groups on NorHA (to stabilize the nanofibrous structure upon hydration). Subsequent patterning is achieved by exposing scaffolds to UV light through a photomask in the presence of a UV initiator and thiolated biomolecules to react with remaining norbornene groups.

Figure 2

Spatial resolution of biomolecules within a nanofibrous scaffold. (A–B) Photomask and corresponding patterns after UV light mediated covalent attachment of a thiolated peptide fluorophore (GCEEE-FITC) to an electrospun NorHA hydrogel. (A) and (B-right) are each one focal plane in a confocal image. (B-left) Intensity profile of a horizontal line across the image. Scale bars: (A,B-right) 25 μm, (B-left) 100 μm. (C) Demonstration of patterning multiple ligands onto the same electrospun NorHA hydrogel. Patterning included 200 μm lines of thiolated peptide fluorophore (GCDD-Rho), 200 μm lines of a second thiolated peptide fluorophore (GCEEE-FITC), and 100 μm circles of a third thiolated peptide fluorophore (GCEE-Methoxycoumarin). The overlaid image demonstrates patterning of three different biomolecules onto the same nanofibrous scaffold. Scale bars: 100 μm. (D) Patterns with depth in a thick scaffold. 200 μm lines were patterned through the top of the scaffold and x-y and z-projections are shown for the top (right) and bottom (left) of the scaffold indicating patterning to a depth of 1.5 mm. Scale bars: 50 μm.

Figure 3

Patterning of biochemical ligands alters cell attachment and morphology. (A) Photomask (white areas indicate areas permitting transmittance of UV light) used to covalently attach thiolated RGD to the nanofibrous scaffold (PBL – Polymeric Biomaterials Lab). (B–D) 3T3 fibroblasts adhere to the RGD pattern with high fidelity. Scale bars: (B,D) 100 μm, (C) 500 μm. Quantification of the number of cells per area (E), aspect ratio (F), and cell area (G) shows differences in cell adhesion, elongation, and morphology between nanofiber regions patterned with RGD and nanofiber regions without RGD. (p<0.001)

Figure 4

Altered cell behavior in the presence of topographical cues and spatial patterning of RGD. (A) Cells orient and elongate with aligned nanofibrous topography on NorHA scaffolds with uniform RGD, (B) whereas cell orientation and elongation is abrogated in the absence of aligned nanofibers. Scale bars: 100 μm. (C–D) Cells orient and elongate with aligned nanofibrous topography on 100 μm wide cell adhesive lines of RGD parallel to nanofiber orientation. Scale bars: (C) 200 μm, (D) 100 μm. (E) Counts of nuclei (DAPI) as a function of horizontal position, and (F) angle of cells from image (C). (G–H) Cells elongate and orient with aligned nanofibrous topography (horizontal) but are spatially restricted (vertically) when RGD is patterned in 100 μm lines perpendicular to nanofiber orientation. Scale bars: (G) 200 μm, (H) 100 μm. (I) Counts of nuclei (DAPI) as a function of horizontal position, and (J) angle of cells from image (G). Staining in all images: Green: F-Actin (FITC-Phalloidin), Blue: Nuclei (DAPI), Red: RGD (GCDD-Rho).