Alveolar epithelial cells and microenvironmental stiffness synergistically drive fibroblast activation in three-dimensional hydrogel lung models
Thomas Caracena, Rachel Blomberg, Rukshika S. Hewawasam, Zoe E. Fry, David W.H. Riches, and Chelsea M. Magin. Biomaterials Science (2022), 10:7133-7148 (link)
Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease that progressively and irreversibly alters the lung parenchyma, eventually leading to respiratory failure. The study of this disease has been historically challenging due to the myriad of complex processes that contribute to fibrogenesis and the inherent difficulty in accurately recreating the human pulmonary environment in vitro. Here, we describe a poly(ethylene glycol) PEG hydrogel-based three-dimensional model for the co-culture of primary murine pulmonary fibroblasts and alveolar epithelial cells that reproduces the micro-architecture, cell placement, and mechanical properties of healthy and fibrotic lung tissue. Co-cultured cells retained normal levels of viability up to at least three weeks and displayed differentiation patterns observed in vivo during IPF progression. Interrogation of protein and gene expression within this model showed that myofibroblast activation required both extracellular mechanical cues and the presence of alveolar epithelial cells. Differences in gene expression indicated that cellular co-culture induced TGF-β signaling and proliferative gene expression, while microenvironmental stiffness upregulated the expression of genes related to cell–ECM interactions. This biomaterial-based cell culture system serves as a significant step forward in the accurate recapitulation of human lung tissue in vitro and highlights the need to incorporate multiple factors that work together synergistically in vivo into models of lung biology of health and disease.
Engineering Hybrid-Hydrogels Comprised of Healthy or Diseased Decellularized Extracellular Matrix to Study Pulmonary Fibrosis
Kamiel S. Saleh, Rukshika Hewawasam, Predrag Serbedzija, Rachel Blomberg, Saif E. Noreldeen, Benjamin Edelman, Bradford J. Smith, David W.H. Riches, and Chelsea M. Magin. 2022 CMBE Young Innovators. Cellular and Molecular Bioengineering (2022). (link)
Idiopathic pulmonary fibrosis is a chronic disease characterized by progressive lung scarring that inhibits gas exchange. Evidence suggests fibroblast-matrix interactions are a prominent driver of disease. However, available preclinical models limit our ability to study these interactions. We present a technique for synthesizing phototunable poly(ethylene glycol) (PEG)-based hybrid-hydrogels comprising healthy or fibrotic decellularized extracellular matrix (dECM) to decouple mechanical properties from composition and elucidate their roles in fibroblast activation. Here, we engineered and characterized phototunable hybrid-hydrogels using molecular techniques such as ninhydrin and Ellman’s assays to assess dECM functionalization, and parallel-plate rheology to measure hydrogel mechanical properties. These biomaterials were employed to investigate the activation of fibroblasts from dual-transgenic Col1a1-GFP and aSMA-RFP reporter mice in response to changes in composition and mechanical properties. We show that reacting functionalized dECM from healthy or bleomycin-injured mouse lungs with PEG alpha-methacrylate (aMA) in an off-stoichiometry Michael-addition reaction created soft hydrogels mimicking a healthy lung elastic modulus (4.99 ± 0.98 kPa). Photoinitiated stiffening increased the material modulus to fibrotic values (11.48 ± 1.80 kPa). Percent activation of primary murine fibroblasts expressing Col1a1 and aSMA increased by approximately 40% following dynamic stiffening of both healthy and bleomycin hybrid-hydrogels. There were no significant differences between fibroblast activation on stiffened healthy versus stiffened bleomycin-injured hybrid-hydrogels. Phototunable hybrid-hydrogels provide an important platform for probing cell-matrix interactions and developing a deeper understanding of fibrotic activation in pulmonary fibrosis. Our results suggest that mechanical properties are a more significant contributor to fibroblast activation than biochemical composition within the scope of the hybrid-hydrogel platform evaluated in this study.
Engineering Tissue-Informed Biomaterials to Advance Pulmonary Regenerative Medicine
Donald R. Campbell Jr, Christiana N. Senger, Amy L. Ryan, and Chelsea M. Magin. Fronteirs in Medicine | Pulmonary Medicine (2021). (link)
Biomaterials intentionally designed to support the expansion, differentiation, and three-dimensional (3D) culture of induced-pluripotent stem cells (iPSCs) may pave the way to cell-based therapies for chronic respiratory diseases. These conditions are endured by millions of people worldwide and represent a significant cause of morbidity and mortality. Currently, there are no effective treatments for the majority of advanced lung diseases and lung transplantation remains the only hope for many chronically ill patients. Key opinion leaders speculate that the novel coronavirus, COVID-19, may lead to long-term lung damage, further exacerbating the need for regenerative therapies. New strategies for regenerative cell-based therapies harness the differentiation capability of human iPSCs for studying pulmonary disease pathogenesis and treatment. Excitingly, biomaterials are a cell culture platform that can be precisely designed to direct stem cell differentiation. Here, we present a closer look at the state-of-the-art of iPSC differentiation for pulmonary engineering, offer evidence supporting the power of biomaterials to improve stem cell differentiation, and discuss our perspective on the potential for tissue-informed biomaterials to transform pulmonary regenerative medicine.
Clickable decellularized extracellular matrix as a new tool for building hybrid-hydrogels to model chronic fibrotic diseases in vitro
Cassandra L. Petrou, Tyler J. D’Ovidio, Deniz A. Bolukbas, Sinem Tas, R. Dale Brown. Ayed Allawzi, Sandra Lindstedt, Eva Nozik-Grayck, Kurt R. Stenmark, Darcy E. Wagner, and Chelsea M. Magin. Journal of Materials Chemistry B (2020). (link)
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)-α-methacrylate (PEGαMA) hybrid-hydrogel to recreate ECM remodeling in vitro. An off-stoichiometry thiol–ene Michael addition between PEGαMA (8-arm, 10 kg mol−1) 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 αMA 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 alpha-smooth 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.
Embedding of Precision-Cut Lung Slices in Engineering Hydrogel Biomaterials Supports Extended ex vivo Culture
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. (link)
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.
Micropattern-mediated apical guidance accelerates epithelial cell migration to improve healing around percutaneous gastrostomy tubes
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. (link)
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.
Peptide-Functionalized Hydrogels Modulate Integrin Expression and Stemness in Adult Human Epidermal Keratinocytes
Duncan Davis-Hall, Vy Nguyen, Tyler J. D’Ovidio, Ganna Bilousova and Chelsea M. Magin. Advanced Biosystems 2019, 3, 1900022. (link)
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.
Tissue-Informed Engineering Strategies for Modeling Human Pulmonary Diseases
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. (link)
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.
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