Pulmonary Fibrosis Stakeholder Summit: A Joint NHLBI, Three Lakes Foundation, and Pulmonary Fibrosis Foundation Workshop Report.

Despite progress in elucidation of disease mechanisms, identification of risk factors, biomarker discovery, and the approval of two medications to slow lung function decline in idiopathic pulmonary fibrosis and one medication to slow lung function decline in progressive pulmonary fibrosis, pulmonary fibrosis remains a disease with a high morbidity and mortality. In recognition of the need to catalyze ongoing advances and collaboration in the field of pulmonary fibrosis, the NHLBI, the Three Lakes Foundation, and the Pulmonary Fibrosis Foundation hosted the Pulmonary Fibrosis Stakeholder Summit on November 8-9, 2022. This workshop was held virtually and was organized into three topic areas: 1) novel models and research tools to better study pulmonary fibrosis and uncover new therapies, 2) early disease risk factors and methods to improve diagnosis, and 3) innovative approaches toward clinical trial design for pulmonary fibrosis. In this workshop report, we summarize the content of the presentations and discussions, enumerating research opportunities for advancing our understanding of the pathogenesis, treatment, and outcomes of pulmonary fibrosis.

Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade increasing evidence from preclinical models suggests that cells, which are not normally resident in the lung can be utilized to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathologic remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "-omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

An Introduction to Engineering and Modeling the Lung.

Over the last decade, the field of lung biology has evolved considerably due to many advancements, including the advent of single-cell RNA (scRNA) sequencing, induced pluripotent stem cell (iPSC) reprogramming, and 3D cell and tissue culture. Despite rigorous research and tireless efforts, chronic pulmonary diseases remain the third leading cause of death globally, with transplantation being the only option for treating end-stage disease. This chapter will introduce the broader impacts of understanding lung biology in health and disease, provide an overview of lung physiology and pathophysiology, and summarize the key takeaways from each chapter describing engineering translational models of lung homeostasis and disease. This book is divided into broad topic areas containing chapters covering basic biology, engineering approaches, and clinical perspectives related to (1) the developing lung, (2) the large airways, (3) the mesenchyme and parenchyma, (4) the pulmonary vasculature, and (5) the interface between lungs and medical devices. Each section highlights the underlying premise that engineering strategies, when applied in collaboration with cell biologists and pulmonary physicians, will address critical challenges in pulmonary health care.

Engineering Dynamic 3D Models of Lung.

The lung parenchyma-consisting of gas-filled alveoli, vasculature, and connective tissue-is the site for gas exchange in the lung and plays a critical role in a number of chronic lung diseases. In vitro models of lung parenchyma can, therefore, provide valuable platforms for the study of lung biology in health and disease. Yet modeling such a complex tissue requires integrating multiple components, including biochemical cues from the extracellular environment, geometrically defined multicellular interactions, and dynamic mechanical inputs such as the cyclic stretch of breathing. In this chapter, we provide an overview of the broad spectrum of model systems that have been developed to recapitulate one or more features of lung parenchyma, and some of the scientific advances generated by those models. We discuss the use of both synthetic and naturally derived hydrogel materials, precision-cut lung slices, organoids, and lung-on-a-chip devices, with perspectives on the strengths, weaknesses, and potential future directions of these engineered systems.

Embedding of Precision-Cut Lung Slices in Engineered Hydrogel Biomaterials Supports Extended Ex Vivo Culture.

Maintaining the three-dimensional 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 short culture times of 7-10 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 for at least 21 days compared with control PCLS floating in media. These customized hydrogels maintained PCLS architecture (no difference), viability (4.7-fold increase, P < 0.0001), and cellular phenotype as measured by SFTPC (1.8-fold increase, P < 0.0001) and vimentin expression (no change) compared with nonencapsulated controls. Collectively, these results demonstrate that hydrogel biomaterials support the extended culture times required to study chronic pulmonary diseases ex vivo using PCLS technology.

Tissue-informed engineering strategies for modeling human pulmonary diseases.

Chronic pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH), and chronic obstructive pulmonary disease (COPD), account for staggering morbidity and mortality worldwide but have limited clinical management options available. Although great progress has been made to elucidate the cellular and molecular pathways underlying these diseases, there remains a significant disparity between basic research endeavors and clinical outcomes. This discrepancy is due in part to the failure of many current disease models to recapitulate the dynamic changes that occur during pathogenesis in vivo. As a result, pulmonary medicine has recently experienced a rapid expansion in the application of engineering principles to characterize changes in human tissues in vivo and model the resulting pathogenic alterations in vitro. We envision that engineering strategies using precision biomaterials and advanced biomanufacturing will revolutionize current approaches to disease modeling and accelerate the development and validation of personalized therapies. 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.

Role of mucins in lung homeostasis: regulated expression and biosynthesis in health and disease.

In humans and mice, the first line of innate defense against inhaled pathogens and particles in the respiratory tract is airway mucus. The primary solid components of the mucus layer are the mucins MUC5AC and MUC5B, polymeric glycoproteins whose changes in abundance and structure can dramatically affect airway defense. Accordingly, MUC5AC/Muc5ac and MUC5B/Muc5b are tightly regulated at a transcriptional level by tissue-specific transcription factors in homeostasis and in response to injurious and inflammatory triggers. In addition to modulated levels of mucin gene transcription, translational and post-translational biosynthetic processes also exert significant influence upon mucin function. Mucins are massive macromolecules with numerous functional domains that contribute to their structural composition and biophysical properties. Single MUC5AC and MUC5B apoproteins have molecular masses of >400 kDa, and von Willebrand factor D-like as well as other cysteine-rich domain segments contribute to mucin polymerization and flexibility, thus increasing apoprotein length and complexity. Additional domains serve as sites for O-glycosylation, which increase further mucin mass several-fold. Glycosylation is a defining process for mucins that is specific with respect to additions of glycans to mucin apoprotein backbones, and glycan additions influence the physical properties of the mucins via structural modifications as well as charge interactions. Ultimately, through their tight regulation and complex assembly, airway mucins follow the biological rule of 'form fits function' in that their structural organization influences their role in lung homeostatic mechanisms.

Hydrogel-embedded precision-cut lung slices support ex vivo culture of in vivo -induced premalignant lung lesions.

Lung cancer is the leading cause of global cancer death and prevention strategies are key to reducing mortality. Medical prevention may have a larger impact than treatment on mortality by targeting high-risk populations and reducing their lung cancer risk. Premalignant lesions (PMLs) that can be intercepted by prevention agents are difficult to study in humans but easily accessible in murine preclinical carcinogenesis studies. Precision-cut lung slices (PCLS) are underutilized as an ex vivo model for lung cancer studies due to limited culture time. Embedding PCLS within bioengineered hydrogels extends PCLS viability and functionality for up to six weeks. Here, we embedded PCLS generated from urethane-induced murine PMLs in cell-degradable and non-degradable hydrogels to study viability and activity of the tissues over six weeks. PMLs in hydrogel-embedded PCLS maintained viability, gene expression, and proliferation. Treatment of hydrogel-embedded PCLS containing urethane-induced PMLs with iloprost, a known lung cancer prevention agent, recapitulated in vivo gene expression and activity. These studies also showed that iloprost reduced proliferation and PML size in hydrogel-embedded PCLS, with some differences based on hydrogel formulation and suggested that hydrogel-embedded PCLS models may support long-term culture of in vivo generated PMLs to improve preclinical studies of lung cancer and prevention agents.

Dynamically stiffening biomaterials reveal age- and sex-specific differences in pulmonary arterial adventitial fibroblast activation.

Respiratory diseases like pulmonary arterial hypertension (PAH) frequently exhibit sexual dimorphism. Female PAH patients are more susceptible to the disease but have increased survival rates. This phenomenon is known as the estrogen paradox, and the underlying mechanisms are not fully understood. During PAH progression in vivo , human pulmonary arterial adventitial fibroblasts (hPAAFs) differentiate into an activated phenotype. These cells produce excess, aberrant extracellular matrix proteins that stiffen the surrounding pulmonary arterial tissues. Here, we employed dynamic poly(ethylene glycol)-alpha methacrylate (PEGαMA)-based biomaterials to study how the age and sex of human serum influenced hPAAF activation in response to microenvironmental stiffening in vitro . Results showed female and male cells responded differently to increases in microenvironmental stiffness and serum composition. Male hPAAFs were less activated than female cells on soft hydrogels and more responsive to increases in microenvironmental stiffness regardless of serum composition. Female hPAAF activation followed this pattern only when cultured in younger (age < 50) female serum or when older (age ≥ 50) female serum was supplemented with estradiol. Otherwise, female hPAAF activation was relatively high on both soft and stiffened hydrogels, with little difference in activation between the two conditions. Collectively, these results suggest that it may be possible to model the estrogen paradox observed in PAH in vitro and that it is critical for researchers to report cell sex and serum source when conducting in vitro experimentation.

Dynamically stiffening biomaterials reveal age- and sex-specific differences in pulmonary arterial adventitial fibroblast activation.

Respiratory diseases like pulmonary arterial hypertension (PAH) frequently exhibit sexual dimorphism. Female PAH patients are more susceptible to the disease but have increased survival rates. This phenomenon is known as the estrogen paradox, and the underlying mechanisms are not fully understood. During PAH progression in vivo, human pulmonary arterial adventitial fibroblasts (hPAAFs) differentiate into an activated phenotype. These cells produce excess, aberrant extracellular matrix proteins that stiffen the surrounding pulmonary arterial tissues. Here, we employed dynamic poly(ethylene glycol)-alpha methacrylate (PEGαMA)-based biomaterials to study how the age and sex of human serum influenced hPAAF activation in response to microenvironmental stiffening in vitro. Results showed female and male cells responded differently to increases in microenvironmental stiffness and serum composition. Male hPAAFs were less activated than female cells on soft hydrogels and more responsive to increases in microenvironmental stiffness regardless of serum composition. Female hPAAF activation followed this pattern only when cultured in younger (age < 50) female serum or when older (age ≥ 50) female serum was supplemented with estradiol. Otherwise, female hPAAF activation was relatively high on both soft and stiffened hydrogels, with little difference in activation between the two conditions. Collectively, these results suggest that it may be possible to model the estrogen paradox observed in PAH in vitro and that it is critical for researchers to report cell sex and serum source when conducting in vitro experimentation.

Hydrogel-Embedded Precision-Cut Lung Slices Model Lung Cancer Premalignancy Ex Vivo.

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best prevention, 50% of lung cancer diagnoses occur in people who have quit smoking. Research into treatment options for high-risk patients is constrained to rodent models, which are time-consuming, expensive, and require large cohorts. Embedding precision-cut lung slices (PCLS) within an engineered hydrogel and exposing this tissue to vinyl carbamate, a carcinogen from cigarette smoke, creates an in vitro model of lung cancer premalignancy. Hydrogel formulations are selected to promote early lung cancer cellular phenotypes and extend PCLS viability to six weeks. Hydrogel-embedded PCLS are exposed to vinyl carbamate, which induces adenocarcinoma in mice. Analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content after six weeks reveals that vinyl carbamate induces premalignant lesions with a mixed adenoma/squamous phenotype. Putative chemoprevention agents diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue are validated with hydrogel-embedded human PCLS and results show increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the foundation for more sophisticated ex vivo models that enable the study of carcinogenesis and chemoprevention strategies.

Challenges and Opportunities for Commercializing Technologies in the Pulmonary Arena: An Official American Thoracic Society Report.

"Translational medicine" has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new "bench-to-bedside" approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.

Engineered antifouling microtopographies: an energetic model that predicts cell attachment.

We have developed a model for the prediction of cell attachment to engineered microtopographies based on two previous models: the attachment point theory and the engineered roughness index (ERI) model. The new surface energetic attachment (SEA) model is based on both the properties of the cell-material interface and the size and configuration of the topography relative to the organism. We have used Monte Carlo simulation to examine the SEA model's ability to predict relative attachment of the green alga Ulva linza to different locations within a unit cell. We have also compared the predicted relative attachment for Ulva linza, the diatom Navicula incerta, the marine bacterium Cobetia marina, and the barnacle cyprid Balanus amphitrite to a wide variety of microtopographies. We demonstrate good correlation between the experimental results and the model results for all tested experimental data and thus show the SEA model may be used as a powerful indicator of the efficacy for antifouling topographies.

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEGαMA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Chemical Modification of Human Decellularized Extracellular Matrix for Incorporation into Phototunable Hybrid-Hydrogel Models of Tissue Fibrosis.

Tissue fibrosis remains a serious health condition with high morbidity and mortality rates. There is a critical need to engineer model systems that better recapitulate the spatial and temporal changes in the fibrotic extracellular microenvironment and enable study of the cellular and molecular alterations that occur during pathogenesis. Here, we present a process for chemically modifying human decellularized extracellular matrix (dECM) and incorporating it into a dynamically tunable hybrid-hydrogel system containing a poly(ethylene glycol)-α methacrylate (PEGαMA) backbone. Following modification and characterization, an off-stoichiometry thiol-ene Michael addition reaction resulted in hybrid-hydrogels with mechanical properties that could be tuned to recapitulate many healthy tissue types. Next, photoinitiated, free-radical homopolymerization of excess α-methacrylates increased crosslinking density and hybrid-hydrogel elastic modulus to mimic a fibrotic microenvironment. The incorporation of dECM into the PEGαMA hydrogel decreased the elastic modulus and, relative to fully synthetic hydrogels, increased the swelling ratio, the average molecular weight between crosslinks, and the mesh size of hybrid-hydrogel networks. These changes were proportional to the amount of dECM incorporated into the network. Dynamic stiffening increased the elastic modulus and decreased the swelling ratio, average molecular weight between crosslinks, and the mesh size of hybrid-hydrogels, as expected. Stiffening also activated human fibroblasts, as measured by increases in average cellular aspect ratio (1.59 ± 0.02 to 2.98 ± 0.20) and expression of α-smooth muscle actin (αSMA). Fibroblasts expressing αSMA increased from 25.8 to 49.1% upon dynamic stiffening, demonstrating that hybrid-hydrogels containing human dECM support investigation of dynamic mechanosensing. These results improve our understanding of the biomolecular networks formed within hybrid-hydrogels: this fully human phototunable hybrid-hydrogel system will enable researchers to control and decouple the biochemical changes that occur during fibrotic pathogenesis from the resulting increases in stiffness to study the dynamic cell-matrix interactions that perpetuate fibrotic diseases.

Tissue-engineered models of lung cancer premalignancy.

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best preventive action, nearly 50% of all lung cancer diagnoses occur in people who have already quit smoking. Research into treatment options for these high-risk patients has been constrained to rodent models of chemical carcinogenesis, which are time-consuming, expensive, and require large numbers of animals. Here we show that embedding precision-cut lung slices within an engineered hydrogel and exposing this tissue to a carcinogen from cigarette smoke creates an in vitro model of lung cancer premalignancy. Hydrogel formulations were selected to promote early lung cancer cellular phenotypes and extend PCLS viability up to six weeks. In this study, hydrogel-embedded lung slices were exposed to the cigarette smoke derived carcinogen vinyl carbamate, which induces adenocarcinoma in mice. At six weeks, analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content revealed that vinyl carbamate induced the formation of premalignant lesions with a mixed adenoma/squamous phenotype. Two putative chemoprevention agents were able to freely diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue were validated with hydrogel-embedded human PCLS and results showed increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the starting point for more sophisticated ex vivo models and a foundation for the study of carcinogenesis and chemoprevention strategies.

3D-bioprinted, phototunable hydrogel models for studying adventitial fibroblast activation in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease of the lung vasculature, characterized by elevated pulmonary blood pressure, remodeling of the pulmonary arteries, and ultimately right ventricular failure. Therapeutic interventions for PAH are limited in part by the lack ofin vitroscreening platforms that accurately reproduce dynamic arterial wall mechanical properties. Here we present a 3D-bioprinted model of the pulmonary arterial adventitia comprised of a phototunable poly(ethylene glycol) alpha methacrylate (PEG-αMA)-based hydrogel and primary human pulmonary artery adventitia fibroblasts (HPAAFs). This unique biomaterial emulates PAH pathogenesisin vitrothrough a two-step polymerization reaction. First, PEG-αMA macromer was crosslinked off-stoichiometry by 3D bioprinting an acidic bioink solution into a basic gelatin support bath initiating a base-catalyzed thiol-ene reaction with synthetic and biodegradable crosslinkers. Then, matrix stiffening was induced by photoinitiated homopolymerization of unreacted αMA end groups. A design of experiments approach produced a hydrogel platform that exhibited an initial elastic modulus (E) within the range of healthy pulmonary arterial tissue (E= 4.7 ± 0.09 kPa) that was stiffened to the pathologic range of hypertensive tissue (E= 12.8 ± 0.47 kPa) and supported cellular proliferation over time. A higher percentage of HPAAFs cultured in stiffened hydrogels expressed the fibrotic marker alpha-smooth muscle actin than cells in soft hydrogels (88 ± 2% versus 65 ± 4%). Likewise, a greater percentage of HPAAFs were positive for the proliferation marker 5-ethynyl-2'-deoxyuridine (EdU) in stiffened models (66 ± 6%) compared to soft (39 ± 6%). These results demonstrate that 3D-bioprinted, phototunable models of pulmonary artery adventitia are a tool that enable investigation of fibrotic pathogenesisin vitro.

Alveolar epithelial cells and microenvironmental stiffness synergistically drive fibroblast activation in three-dimensional hydrogel lung models.

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-ß 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.

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 αSMA-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 (αMA) 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 αSMA 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. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00726-y.

Antifouling performance of cross-linked hydrogels: refinement of an attachment model.

Poly(ethylene glycol) dimethacrylate (PEGDMA), PEGDMA-co-glycidyl methacrylate (PEGDMA-co-GMA), and PEGDMA-co-hydroxyethyl methacrylate (PEGDMA-co-HEMA) hydrogels were polymerized using ammonium persulfate and ascorbic acid as radical initiators. Surface energies of the hydrogels and a standard, poly(dimethylsiloxane) elastomer (PDMSe), were characterized using captive bubble and sessile drop measurements, respectively (γ = 52 mN/m, γ(0) = 19 mN/m). The chemical composition of the hydrogels was characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. All three hydrogel compositions reduced significantly (p = 0.05) initial attachment of zoospores of the green alga Ulva linza (up to 97%), cells of the diatom Navicula incerta (up to 58%) and the bacterium Cobetia marina (up to 62%), compared to a smooth PDMSe standard. A shear stress (45 Pa), generated in a water channel, eliminated up to 95% of the initially attached cells of Navicula from the smooth hydrogel surfaces relative to smooth PDMSe surfaces. Compared to the PDMSe standard, 79% of the cells of C. marina were removed from all smooth hydrogel compositions when exposed to a 50 Pa wall shear stress. Attachment of spores of the green alga Ulva to microtopographies replicated in PEGDMA-co-HEMA was also evaluated. The Sharklet AF microtopography patterned, PEGDMA-co-HEMA surfaces reduced attachment of spores of Ulva by 97% compared to a smooth PDMSe standard. The attachment densities of spores to engineered microtopographies in PDMSe and PEGDMA-co-HEMA were shown to correlate with a modified attachment model through the inclusion of a surface energy term. Attachment densities of spores of Ulva to engineered topographies replicated in a material other than PDMSe are now correlated with the attachment model (R(2) = 0.80).