Assistant Professor University of Virginia, United States
Introduction:: Recapitulating the cell microenvironment to enhance tissue regeneration is an ongoing challenge in biomaterial development because it often requires expensive and exogenous growth factor delivery. To overcome this obstacle, we exploit the properties of heparin within Microporous Annealed Particle (MAP)1 hydrogel scaffolds to capture endogenous growth factors for enhanced chemotaxis mimicking the cell environment. Via affinity interactions, heparin, a strongly negatively charged proteoglycan, regulates localization and retention of cell-secreted growth factors which comprise an essential component of the microenvironment in uninjured skin tissue and direct cell behavior in wounds. MAP hydrogel is an injectable biomaterial composed of spherical particles that uses a secondary photo-crosslinking technique to create a stable structure with cell-scale porosity. To encourage chemotaxis, a heterogeneous scaffold with heparin particle “microislands” is created by ratiometrically mixing heparin particle and non-heparin particle populations. Previous studies found improved cell migration in a 2D MAP scaffold model with 10% density of heparin-MAP (hep-MAP) microislands matching physiological skin concentration (2.6mg/ml) compared to non-heparin MAP (No Hep) and homogenous hep-MAP2. To build on these positive results, we tested a range of heparin concentrations and density distributions within a more physiologically relevant, 3D cell migration assay. Assessing cell spheroid behavior in 3D allows us to gain further and more accurate insight into the influence of heparin on cell spreading. Here we present results of cell migration of human dermal fibroblasts with variable ratios and concentrations of heparin in a novel 3D scaffold.
Materials and Methods:: Heparin was thiolated with PDPH (3-(2-pyridyldithio) propionyl hydrazide) and thiolation was determined with a deprotection assay. MAP microgels were synthesized with a high-throughput microfluidic technique with a PEG-maleimide backbone and MMP-2 degradable crosslinker. Heparin was incorporated into the particles at concentrations of either 0, 2.6, 6.2, or 8.2mg/ml (1X, 2X, and 3X physiological skin concentration). Each hep-MAP population was mixed as a fraction (0, 10, 20, or 30% density) of microislands with the No Hep microgels. Human dermal fibroblasts (HDFs) were prepared for a novel 3D scaffold spheroid sprouting migration assay. A primary layer of 15ul of the desired gel type was placed into a well of a 96 well plate, centrifuged, and photo-crosslinked with UV light. The cell spheroid was placed in the center of the scaffold and incubated for 30 minutes. The secondary layer of gel was then placed on top of the spheroid, and assembled in the same manner as the first. Using z-stack imaging with confocal microscopy, spheroid volume was analyzed over 48 hours within each heparin concentration and for each distribution of hep-MAP.
Results, Conclusions, and Discussions:: The 3D migration assay was first developed with the robust fibroblast cell line, NIH/3T3s, and optimized for primary HDF spheroids. This novel 3D assay minimizes stresses on the cell spheroids, reduces required microgel usage, and maximizes reproducibility. These scaffolds allowed us to probe directionality of migration and cell behavior in response to the different hep-MAP microgel fractions in a 3D manner as opposed to previous methods. Four MAP gel formulations (No heparin, 1X, 2X, 3X Hep) were mechanically matched at 20kPa and size-matched to 75um particle diameters. With these 3D in vitro assays, we observed a longer timepoint was needed than in 2D to distinguish differences between volumes for heparin fractions in all concentrations, likely due to the increased dimensionality. Our study found that at 48 hours, cell spheroid volume increased in 30% hep-MAP for all heparin concentrations compared to the No Hep scaffolds. Comparing the different concentration conditions (1X, 2X and 3X hep-MAP) to the No Hep control scaffolds, we found that 3X hep-MAP at 30% of the scaffold led to the highest spheroid volume, while the other heparin concentrations trended towards higher spheroid volumes than No Hep but not significantly. These data confirm improved cell migration with heterogeneous hep-MAP distributions in a 3D model. They also suggest that higher concentrations of heparin may be sequestering more growth factors to act as chemotactic cues and accelerating cell migration.
Here we present a high throughput 3D MAP gel scaffold preparation that maintains cell viability for migration assays. We took advantage of this by expanding our testing parameters of heparin concentration and microgel fraction and optimized gel formulations which will be further explored in an in vivo diabetic murine skin wound healing model. We anticipate iterations of the presented MAP scaffold and heterogenous microisland populations will be effective for tissue regeneration applications beyond wound healing due to its modularity, high throughput capabilities, and physiological relevance.
Acknowledgements (Optional): : JT supported by NSF GRFP, Biorender was used to construct the figure
