X-Ray Dark-Field Imaging of Lung Cancer in Mice

Lung cancer accounts for 1.6 million deaths per year worldwide. The majority of patients are diagnosed at advanced stages of the disease and often present with metastasis. Thus, the 5-year survival rate of lung cancer remains around 15%. Early diagnosis of lung cancer allows for better control of the disease with 5-year survival rates up to around 70%. Chest radiography is the most common technique for visualizing lungs. However, small lesions in the lung are often missed by conventional X-ray radiography. New technological advances, such as grating-based imaging, allow for better contrast in soft tissue. Grating-based imaging depends on the interactions between the specimen and the X-rays while they pass through, resulting in interference and refraction of the beam. Contrast acquisition from these interactions are categorized as interferometric methods. X-ray dark-field imaging relies on quantification of small-angle scattering of the X-rays during this traverse and has shown success in obtaining enhanced contrast from soft tissues such as the lung. In in vivo models, dark-field imaging has been shown to be superior to conventional radiography for visualization of pulmonary diseases including lung cancer. In this chapter, we summarize applications of this technology for imaging of lung cancer in small animals and discuss its future perspectives and potential challenges in translation

Current and Future Engineering Strategies for ECMO Therapy

Extracorporeal membrane oxygenation (ECMO) is a last resort therapy for patients with respiratory failure where the gas exchange capacity of the lung is compromised. Venous blood is pumped through an oxygenation unit outside of the body where oxygen diffusion into the blood takes place in parallel to carbon dioxide removal. ECMO is an expensive therapy which requires special expertise to perform. Since its inception, ECMO technologies have been evolving to improve its success and minimize the complications associated with it. These approaches aim for a more compatible circuit design capable of maximum gas exchange with minimal need for anticoagulants. This chapter summarizes the basic principles of ECMO therapy with the latest advancements and experimental strategies aiming for more efficient future designs

3D printing of decellularised porcine lung ECM

Statement of Purpose: Chronic lung diseases are one of the major health problems that cause death and disability. Approximately 65 million people suffer from chronic lung diseases, and the number of patients is predicted to increase worldwide 1 . Lung transplantation is the only available treatment option for patients at end-stage disease. However, there is a chronic shortage of donor organs, resulting in a large unmet clinical need. To tackle this issue, the concept of transplantable bioengineered lungs has been proposed as a solution which might help to meet current transplantation needs 2 One of the ways to potentially build complex lung structure is 3D bioprinting, but bioinks which are compatible with 3D printers, support cell growth, and can maintain appropriate mechanical stability are unknown. In this regard, decellularized extracellular matrix (dECM) based materials are considered as a potential novel source of material for bioinks because they have been shown in other contexts to provide a suitable microenvironment for regeneration. However, there has been no investigation of their use in 3D printing of complex shapes. In this study, we evaluated the rheological properties of porcine lungderived dECM solutions and hydrogels to assess their suitability for 3D printing and to determine parameters which can be used to produce stable structures

How to build a lung : latest advances and emerging themes in lung bioengineering

Chronic respiratory diseases remain a major cause of morbidity and mortality worldwide. The only option at end-stage disease is lung transplantation, but there are not enough donor lungs to meet clinical demand. Alternative options to increase tissue availability for lung transplantation are urgently required to close the gap on this unmet clinical need. A growing number of tissue engineering approaches are exploring the potential to generate lung tissue ex vivo for transplantation. Both biologically derived and manufactured scaffolds seeded with cells and grown ex vivo have been explored in pre-clinical studies, with the eventual goal of generating functional pulmonary tissue for transplantation. Recently, there have been significant efforts to scale-up cell culture methods to generate adequate cell numbers for human-scale bioengineering approaches. Concomitantly, there have been exciting efforts in designing bioreactors that allow for appropriate cell seeding and development of functional lung tissue over time. This review aims to present the current state-of-the-art progress for each of these areas and to discuss promising new ideas within the field of lung bioengineering

Clickable, hybrid hydrogels as tissue culture platforms for modeling chronic pulmonary diseases in vitro

Statement of Purpose: Many chronic pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and chronic obstructive pulmonary disease (COPD), are complex and poorly understood. While great progress has been made to elucidate the cellular and molecular pathways underlying these diseases, treatment options remain limited. The dynamic alterations in mechanical properties and composition of the ECM that occur during pathologic tissue remodeling have been extensively studied as a major driver of cellular activation and disease progression. However, current in vitro models of pulmonary tissues rely almost exclusively on naturally derived materials, such as Matrigel, collagen or decellularized ECM (dECM), which provide biological activity but cannot be easily tuned to emulate the time-dependent changes in mechanical properties that occur during disease progression. We aim to develop a new class of clickable, dynamically tunable hybrid hydrogels that will allow for the manipulation of microenvironmental mechanical properties through a two-stage polymerization process while also maintaining the complex biological composition of the lung ECM to provide a new tool for studying cell behavior in vitro. Using PH as a model, this hydrogel system will contain dECM from healthy and pathologic lung tissue in order to study the influence of both composition and dynamic mechanical properties on the initiation and progression of PH. Here, we determined the primary amine content in Rat-Tail Collagen Type I (Col I) and three decellularized porcine lung samples. We converted free amines to thiol groups using Traut’s reagent. These thiol groups will ultimately be used to crosslink polyethylene glycol alpha methacrylate (PEGαMA) off-stoichiometry in a Michael addition reaction to form the hybrid hydrogel that can later be stiffened through a secondary, light-initiated homopolymerization of MA moieties to emulate disease progression in vitro (Fig 1A)

A Semi-quantitative Scoring System for Green Histopathological Evaluation of Large Animal Models of Acute Lung Injury

Acute respiratory distress syndrome (ARDS) is a life-threatening, high mortality pulmonary condition characterized by acute lung injury (ALI) resulting in diffuse alveolar damage. Despite progress regarding the understanding of ARDS pathophysiology, there are presently no effective pharmacotherapies. Due to the complexity and multiorgan involvement typically associated with ARDS, animal models remain the most commonly used research tool for investigating potential new therapies. Experimental models of ALI/ARDS use different methods of injury to acutely induce lung damage in both small and large animals. These models have historically played an important role in the development of new clinical interventions, such as fluid therapy and the use of supportive mechanical ventilation (MV). However, failures in recent clinical trials have highlighted the potential inadequacy of small animal models due to major anatomical and physiological differences, as well as technical challenges associated with the use of clinical co-interventions [e.g., MV and extracorporeal membrane oxygenation (ECMO)]. Thus, there is a need for larger animal models of ALI/ARDS, to allow the incorporation of clinically relevant measurements and co-interventions, hopefully leading to improved rates of clinical translation. However, one of the main challenges in using large animal models of preclinical research is that fewer species-specific experimental tools and metrics are available for evaluating the extent of lung injury, as compared to rodent models. One of the most relevant indicators of ALI in all animal models is evidence of histological tissue damage, and while histological scoring systems exist for small animal models, these cannot frequently be readily applied to large animal models. Histological injury in these models differs due to the type and severity of the injury being modeled. Additionally, the incorporation of other clinical support devices such as MV and ECMO in large animal models can lead to further lung damage and appearance of features absent in the small animal models. Therefore, semi-quantitative histological scoring systems designed to evaluate tissue-level injury in large animal models of ALI/ARDS are needed. Here we describe a semi-quantitative scoring system to evaluate histological injury using a previously established porcine model of ALI via intratracheal and intravascular lipopolysaccharide (LPS) administration. Additionally, and owing to the higher number of samples generated from large animal models, we worked to implement a more sustainable and greener histopathological workflow throughout the entire process

ECM-alginate microcarriers for alveolar regeneration in COPD

With 65 million patients globally, chronic obstructive pulmonary disease (COPD) is the third-leading cause of death. It is characterized by progressive and irreversible airflow limitation in the lung resulting from chronic airway inflammation and/or loss of alveolar tissue. Current treatments for COPD can only stop or slow down disease progression with no cure. The only option for end-stage disease is lung transplantation with restricted efficacy due to scarcity of donor lungs and high rejection rates. Hence, novel therapeutic strategies are urgently needed.To address this, we aimed to develop alveolar type 2 (AT2) cell-bearing microcarriers derived from decellularized extracellular matrix (dECM) for alveolar regeneration in COPD lungs. Both AT2 cells and dECM have demonstrated regenerative effects in lung diseases in previous studies but were never reported for use in COPD therapy.dECM was obtained with perfusion-based decellularization of murine, porcine, or human lungs followed by enzymatic digestion. Size-tunable monodispersed dECM-alginate microcarriers were formed in a custom-made microfluidic device.The size of the microcarriers was directly dependent on the flow rate of alginate solution and dECM properties. Cell-bearing microcarriers with various alginate to dECM ratios could be generated ranging from sizes of 30 to 100 µm, which is in the range of individual mouse and human alveoli.With our custom-built microfluidic chip, we are able to generate monodispersed dECM-alginate microcarriers which can be loaded with stem or progenitor cells. Microcarriers are versatile platforms that can support transplanted cells while providing healthy ECM in vivo, and thus has potential in COPD treatment

Toxicological effects of zinc oxide nanoparticle exposure: an in vitro comparison between dry aerosol air-liquid interface and submerged exposure systems

Engineered nanomaterials (ENMs) are increasingly produced and used today, but health risks due to their occupational airborne exposure are incompletely understood. Traditionally, nanoparticle (NP) toxicity is tested by introducing NPs to cells through suspension in the growth media, but this does not mimic respiratory exposures. Different methods to introduce aerosolized NPs to cells cultured at the air-liquid-interface (ALI) have been developed, but require specialized equipment and are associated with higher cost and time. Therefore, it is important to determine whether aerosolized setups induce different cellular responses to NPs than traditional ones, which could provide new insights into toxicological responses of NP exposure. This study evaluates the response of human alveolar epithelial cells (A549) to zinc oxide (ZnO) NPs after dry aerosol exposure in the Nano Aerosol Chamber for In Vitro Toxicity (NACIVT) system as compared to conventional, suspension-based exposure: cells at ALI or submerged. Similar to other studies using nebulization of ZnO NPs, we found that dry aerosol exposure of ZnO NPs via the NACIVT system induced different cellular responses as compared to conventional methods. ZnO NPs delivered at 1.0 mg/cm2 in the NACIVT system, mimicking occupational exposure, induced significant increases in metabolic activity and release of the cytokines IL-8 and MCP-1, but nodifferences were observed using traditional exposures. While factors associated with the method of exposure, such as differing NP aggregation, may contribute toward the different cellular responses observed, our results further encourage the use of more physiologically realistic exposure systems for evaluating airborne ENM toxicity

Increased particle flow rate from airways precedes clinical signs of ARDS in a porcine model of LPS-induced acute lung injury

Acute respiratory distress syndrome (ARDS) is a common cause of death in the intensive care unit, with mortality rates of ~30-40%. To reduce invasive diagnostics such as bronchoalveolar lavage and time-consuming in-hospital transports for imaging diagnostics, we hypothesized that particle flow rate (PFR) pattern from the airways could be an early detection method and contribute to improving diagnostics and optimizing personalized therapies. Porcine models were ventilated mechanically. Lipopolysaccharide (LPS) was administered endotracheally and in the pulmonary artery to induce ARDS. PFR was measured using a customized particles in exhaled air (PExA 2.0) device. In contrast to control animals undergoing mechanical ventilation and receiving saline administration, animals who received LPS developed ARDS according to clinical guidelines and histologic assessment. Plasma levels of TNF-α and IL-6 increased significantly compared with baseline after 120 and 180 min, respectively. On the other hand, the PFR significantly increased and peaked 60 min after LPS administration, i.e., ~30 min before any ARDS stage was observed with other well-established outcome measurements such as hypoxemia, increased inspiratory pressure, and lower tidal volumes or plasma cytokine levels. The present results imply that PFR could be used to detect early biomarkers or as a clinical indicator for the onset of ARDS

Monitoring lung injury with particle flow rate in LPS- and COVID-19-induced ARDS

In severe acute respiratory distress syndrome (ARDS), extracorporeal membrane oxygenation (ECMO) is a life-prolonging treatment, especially among COVID-19 patients. Evaluation of lung injury progression is challenging with current techniques. Diagnostic imaging or invasive diagnostics are risky given the difficulties of intra-hospital transportation, contraindication of biopsies, and the potential for the spread of infections, such as in COVID-19 patients. We have recently shown that particle flow rate (PFR) from exhaled breath could be a noninvasive, early detection method for ARDS during mechanical ventilation. We hypothesized that PFR could also measure the progress of lung injury during ECMO treatment. Lipopolysaccharide (LPS) was thus used to induce ARDS in pigs under mechanical ventilation. Eight were connected to ECMO, whereas seven animals were not. In addition, six animals received sham treatment with saline. Four human patients with ECMO and ARDS were also monitored. In the pigs, as lung injury ensued, the PFR dramatically increased and a particular spike followed the establishment of ECMO in the LPS-treated animals. PFR remained elevated in all animals with no signs of lung recovery. In the human patients, in the two that recovered, PFR decreased. In the two whose lung function deteriorated while on ECMO, there was increased PFR with no sign of recovery in lung function. The present results indicate that real-time monitoring of PFR may be a new, complementary approach in the clinic for measurement of the extent of lung injury and recovery over time in ECMO patients with ARDS