Sound-guided assessment and localization of pulmonary air leak.

Pulmonary air leak is the most common complication of lung surgery, with air leaks that persist longer than 5 days representing a major source of post-surgery morbidity. Clinical management of air leaks is challenging due to limited methods to precisely locate and assess leaks. Here, we present a sound-guided methodology that enables rapid quantitative assessment and precise localization of air leaks by analyzing the distinct sounds generated as the air escapes through defective lung tissue. Air leaks often present after lung surgery due to loss of tissue integrity at or near a staple line. Accordingly, we investigated air leak sounds from a focal pleural defect in a rat model and from a staple line failure in a clinically relevant swine model to demonstrate the high sensitivity and translational potential of this approach. In rat and swine models of free-flowing air leak under positive pressure ventilation with intrapleural microphone 1 cm from the lung surface, we identified that: (a) pulmonary air leaks generate sounds that contain distinct harmonic series, (b) acoustic characteristics of air leak sounds can be used to classify leak severity, and (c) precise location of the air leak can be determined with high resolution (within 1 cm) by mapping the sound loudness level across the lung surface. Our findings suggest that sound-guided assessment and localization of pulmonary air leaks could serve as a diagnostic tool to inform air leak detection and treatment strategies during video-assisted thoracoscopic surgery (VATS) or thoracotomy procedures.

Opto-electromechanical quantification of epithelial barrier function in injured and healthy airway tissues.

The airway epithelium lining the luminal surface of the respiratory tract creates a protective barrier that ensures maintenance of tissue homeostasis and prevention of respiratory diseases. The airway epithelium, unfortunately, is frequently injured by inhaled toxic materials, trauma, or medical procedures. Substantial or repeated airway epithelial injury can lead to dysregulated intrinsic repair pathways and aberrant tissue remodeling that can lead to dysfunctional airway epithelium. While disruption in the epithelial integrity is directly linked to degraded epithelial barrier function, the correlation between the structure and function of the airway epithelium remains elusive. In this study, we quantified the impact of acutely induced airway epithelium injury on disruption of the epithelial barrier functions. By monitoring alternation of the flow motions and tissue bioimpedance at local injury site, degradation of the epithelial functions, including mucociliary clearance and tight/adherens junction formation, were accurately determined with a high spatiotemporal resolution. Computational models that can simulate and predict the disruption of the mucociliary flow and airway tissue bioimpedance have been generated to assist interpretation of the experimental results. Collectively, findings of this study advance our knowledge of the structure-function relationships of the airway epithelium that can promote development of efficient and accurate diagnosis of airway tissue injury.

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue.

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

Imaging-guided bioreactor for de-epithelialization and long-term cultivation of ex vivo rat trachea.

Recent synergistic advances in organ-on-chip and tissue engineering technologies offer opportunities to create in vitro-grown tissue or organ constructs that can faithfully recapitulate their in vivo counterparts. Such in vitro tissue or organ constructs can be utilized in multiple applications, including rapid drug screening, high-fidelity disease modeling, and precision medicine. Here, we report an imaging-guided bioreactor that allows in situ monitoring of the lumen of ex vivo airway tissues during controlled in vitro tissue manipulation and cultivation of isolated rat trachea. Using this platform, we demonstrated partial removal of the rat tracheal epithelium (i.e., de-epithelialization) without disrupting the underlying subepithelial cells and extracellular matrix. Through different tissue evaluation assays, such as immunofluorescent staining, DNA/protein quantification, and electron beam microscopy, we showed that the epithelium of the tracheal lumen can be effectively removed with negligible disruption in the underlying tissue layers, such as cartilage and blood vessel. Notably, using a custom-built micro-optical imaging device integrated with the bioreactor, the trachea lumen was visualized at the cellular level, and removal of the endogenous epithelium and distribution of locally delivered exogenous cells were demonstrated in situ. Moreover, the de-epithelialized trachea supported on the bioreactor allowed attachment and growth of exogenous cells seeded topically on its denuded tissue surface. Collectively, the results suggest that our imaging-enabled rat trachea bioreactor and localized cell replacement method can facilitate creation of bioengineered in vitro airway tissue that can be used in different biomedical applications.

Homogeneous Distribution of Exogenous Cells onto De-epithelialized Rat Trachea via Instillation of Cell-Loaded Hydrogel.

Injured or diseased airway epithelium due to repeated environmental insults or genetic mutations can lead to a functional decline of the lung and incurable lung diseases. Bioengineered airway tissue constructs can facilitate in vitro investigation of human lung diseases and accelerate the development of effective therapeutics. Here, we report robust tissue manipulation modalities that allow: (i) selective removal of the endogenous epithelium of in vitro cultured airway tissues and (ii) spatially uniform distribution and prolonged cultivation of exogenous cells that are implanted topically onto the denuded airway lumen. Results obtained highlight that our approach to airway tissue manipulation can facilitate controlled removal of the airway epithelium and subsequent homogeneous distribution of newly implanted cells. This study can contribute to the creation of innovative tissue engineering methodologies that can facilitate the treatment of lung diseases, such as cystic fibrosis, primary ciliary dyskinesia, and chronic obstructive pulmonary disease.

Non-destructive vacuum-assisted measurement of lung elastic modulus.

In living tissues, mechanical stiffness and biological function are intrinsically linked. Alterations in the stiffness of tissues can induce pathological interactions that affect cellular activity and tissue function. Underlying connections between tissue stiffness and disease highlights the importance of accurate quantitative characterizations of soft tissue mechanics, which can improve our understanding of disease and inform therapeutic development. In particular, accurate measurement of lung mechanical properties has been especially challenging due to the anatomical and mechanobiological complexities of the lung. Discrepancies between measured mechanical properties of dissected lung tissue samples and intact lung tissues in vivo has limited the ability to accurately characterize integral lung mechanics. Here, we report a non-destructive vacuum-assisted method to evaluate mechanical properties of soft biomaterials, including intact tissues and hydrogels. Using this approach, we measured elastic moduli of rat lung tissue that varied depending on stress-strain distribution throughout the lung. We also observed that the elastic moduli of enzymatically disrupted lung parenchyma increased by at least 64%. The reported methodology enables assessment of the nonlinear viscoelastic characteristics of intact lungs under normal and abnormal (i.e., injured, diseased) conditions and allows measurement of mechanical properties of tissue-mimetic biomaterials for use in therapeutics or in vitro models. STATEMENT OF SIGNIFICANCE: Accurate quantification of tissue stiffness is critical for understanding mechanisms of disease and developing effective therapeutics. Current modalities to measure tissue stiffness are destructive and preclude accurate assessment of lung mechanical properties, as lung mechanics are determined by complex features of the intact lung. To address the need for alternative methods to assess lung mechanics, we report a non-destructive vacuum-based approach to quantify tissue stiffness. We applied this method to correlate lung tissue mechanics with tissue disruption, and to assess the stiffness of biomaterials. This method can be used to inform the development of tissue-mimetic materials for use in therapeutics and disease models, and could potentially be applied for in-situ evaluation of tissue stiffness as a diagnostic or prognostic tool.

Load-bearing biodegradable PCL-PGA-beta TCP scaffolds for bone tissue regeneration.

A biocompatible and biodegradable scaffold with load-bearing ability is required to enhance the repair of bone defects by facilitating the attachment, and proliferation of cells, and vascularization during new bone formation. However, it is challenging to maintain the porosity and biodegradability, as well as mechanical properties (especially compressive strength), at the same time. Therefore, in the present work, a biodegradable composite structure of poly(caprolactone) (PCL) was designed using compression molding with varying amounts of poly(glycolic acid) (PGA) (25, 50, 75 wt%) and fixed amount (20 wt%) of beta tricalcium phosphate (beta TCP). It was hypothesized that the fabricated composite structure will develop porosity during the degradation of the PGA and that the corresponding decrease in mechanical properties will be compensated by new bone formation and ingrowth, in vivo. Accordingly, we have systematically studied the effects of sample composition on time-dependent dissolution and mechanical properties of the PGA/beta TCP scaffolds. The compressive strength increased up to ~92 MPa at 50% compression of the designed PCL-PGA samples. Furthermore, the dissolution rate, as well as weight loss, was observed to increase with an increase in the PGA amount in PCL. Based on the mechanical properties and dissolution data, it is concluded that the PCL-PGA scaffolds with beta TCP can be suitable candidates for bone tissue engineering applications, specifically for the reconstruction of bone defects, where strength and biodegradation are both important characteristics.