Cassandra L. Petrou,ab Tyler J. D’Ovidio, b Deniz A. Bolukbas,cd Sinem Tas,cd R. Dale Brown,e Ayed Allawzi,e Sandra Lindstedt,dfg Eva Nozik-Graycke Kurt R. Stenmark,e Darcy E. Wagner cd and Chelsea M. Magin *ab Fibrotic disorders account for over one third of mortalities worldwide. Despite great efforts to study the cellular and molecular processes underlying fibrosis, there are currently few effective therapies. Dual-stage polymerization reactions are an innovative tool for recreating heterogeneous increases in extracellular matrix (ECM) modulus, a hallmark of fibrotic diseases in vivo. Here, we present a clickable decellularized ECM (dECM) crosslinker incorporated into a dynamically responsive poly(ethylene glycol)-a-methacrylate (PEGaMA) hybrid-hydrogel to recreate ECM remodeling in vitro. An off-stoichiometry thiol–ene Michael addition between PEGaMA (8-arm, 10 kg mol1) and the clickable dECM resulted in hydrogels with an elastic modulus of E = 3.6 0.24 kPa, approximating healthy lung tissue (1–5 kPa). Next, residual aMA groups were reacted via a photo-initiated homopolymerization to increase modulus values to fibrotic levels (E = 13.4 0.82 kPa) in situ. Hydrogels with increased elastic moduli, mimicking fibrotic ECM, induced a significant increase in the expression of myofibroblast transgenes. The proportion of primary fibroblasts from dual-reporter mouse lungs expressing collagen 1a1 and alphasmooth muscle actin increased by approximately 60% when cultured on stiff and dynamically stiffened hybrid-hydrogels compared to soft. Likewise, fibroblasts expressed significantly increased levels of the collagen 1a1 transgene on stiff regions of spatially patterned hybrid-hydrogels compared to the soft areas. Collectively, these results indicate that hybrid-hydrogels are a new tool that can be implemented to spatiotemporally induce a phenotypic transition in primary murine fibroblasts in vitro.
Kolene E. Bailey, Christopher Pino, Mallory L. Lennon, Anne Lyons, Jeffrey G. Jacot, Steven R. Lammers, Melanie Koenigshoff and Chelsea M. Magin. American Journal of Respiratory Cell and Molecular Biology 2019. Maintaining the three-dimensional (3D) architecture and cellular complexity of lung tissue ex vivo can enable elucidation of the cellular and molecular pathways underlying chronic pulmonary diseases. Precision-cut lung slices (PCLS) are one human-lung model with the potential to support critical mechanistic studies and early drug discovery. However, many studies report a short culture period of seven to ten days. Here, we systematically evaluated poly(ethylene glycol)-based hydrogel platforms for the encapsulation of PCLS. We demonstrated the ability to support ex vivo culture of embedded PCLS over 21 days compared to control PCLS floating in media. Our customized hydrogels maintained PCLS architecture (no change in porosity), viability (4.7-fold increase, p<0.0001), and cellular phenotype as measured by surfactant protein C (1.8-fold increase, p<0.0001) and vimentin expression (no change) compared to non-encapsulated controls. Collectively, these results demonstrate that hydrogel biomaterials support the extended culture times required to study chronic pulmonary diseases ex vivo using PCLS technology.
Tyler J. D’Ovidio, Aidan R.W. Friederich, Nic de Herrera, Duncan Davis-Hall, Ethan E. Mann and Chelsea M. Magin. Biomedical Physics & Engineering Express 2019, 5, 065027. Hypergranulation, bacterial infection, and device dislodgment are common complications associated with percutaneous gastronomy (PG) tube placement for enteral feeding largely attributable to delayed stoma tract maturation around the device. Stoma tract maturation is a wound-healing process that requires collective and complete migration of an advancing epithelial layer. While it is widely accepted that micropatterned surfaces enhance cell migration when cells are cultured directly on the substrate, few studies have investigated the influence of apical contact guidance from micropatterned surfaces on cell migration, as occurs during stoma tract formation. Here, we developed 2D and 3D in vitro epithelial cell migration assays to test the effect of various Sharklet micropatterns on apically-guided cell migration. The 2D modified scratch wound assay identified a Sharklet micropattern (+10SK50×50) that enhanced apical cell migration by 4-fold (p = 0.0105) compared to smooth controls over the course of seven days. The best-performing micropattern was then applied to cylindrical prototypes with the same outer diameter as a pediatric PG tube. These prototypes were evaluated in the novel 3D migration assay where magnetic levitation aggregated cells around prototypes to create an artificial stoma. Results indicated a 50% increase (p < 0.0001) in cell migration after seven days along Sharklet-micropatterned prototypes compared to smooth controls. The Sharklet micropattern enhanced apically-guided epithelial cell migration in both 2D and 3D in vitro assays. These data suggest that the incorporation of a Sharklet micropattern onto the surface of a PG tube may accelerate cell migration via apical contact, improve stoma tract maturation, and reduce skin-associated complications.
Duncan Davis-Hall, Vy Nguyen, Tyler J. D’Ovidio, Ganna Bilousova and Chelsea M. Magin. Advanced Biosystems 2019, 3, 1900022. The extracellular matrix (ECM) controls keratinocyte proliferation, migration, and differentiation through β‐integrin signaling. Wound‐healing research requires expanding cells in vitro while maintaining replicative capacity; however, early terminal differentiation under traditional culture conditions limits expansion. Here, a design of experiments approach identifies poly(ethylene glycol)‐based hydrogel formulations with mechanical properties (elastic modulus, E = 20.9 ± 0.56 kPa) and bioactive peptide sequences that mimic the epidermal ECM. These hydrogels enable systematic investigation of the influence of cell‐binding domains from fibronectin (RGDS), laminin (YIGSR), and collagen IV (HepIII) on keratinocyte stemness and β1 integrin expression. Quantification of 14‐day keratin protein expression shows four hydrogels improve stemness compared to standard techniques. Three hydrogels increase β1 integrin expression, demonstrating a positive linear relationship between stemness and β1 integrin expression. Multifactorial statistical analysis predicts an optimal peptide combination ([RGDS] = 0.67 mm, [YIGSR] = 0.13 mm, and [HepIII] = 0.02 mm) for maintaining stemness in vitro. Best‐performing hydrogels exhibit no decrease in Ki‐67‐positive cells compared to standards (15% decrease, day 7 to 14; p < 0.05, Tukey Test). These data demonstrate that precisely designed hydrogel biomaterials direct integrin expression and promote proliferation, improving the regenerative capability of cultured keratinocytes for basic science and translational work.
Kolene E. Bailey, Michael L. Floren, Tyler J. D’Ovidio, Steven R. Lammers, Kurt R. Stenmark, and Chelsea M. Magin. American Journal of Physiology-Lung Cellular and Molecular Physiology 2018, 316, L303. This review highlights how advances in lung tissue characterization reveal dynamic changes in the structure, mechanics and composition of the extracellular matrix in chronic pulmonary diseases, and how this information paves the way for tissue-informed engineering of more organotypic models of human pathology. Current translational challenges are discussed as well as opportunities to overcome these barriers with precision biomaterial design and advanced biomanufacturing techniques that embody the principles of personalized medicine to facilitate the rapid development of novel therapeutics for this devastating group of chronic diseases.