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BACKGROUND Airway release (AR) maneuvers performed during airway resection to reduce anastomotic tension have not been thoroughly studied. METHODS This study retrospectively analyzed consecutive resections for postintubation stenosis (PITS) and primary tracheal neoplasms (PTNs) at Massachusetts General Hospital (Boston, MA). Anastomotic complications were defined as stenosis, separation, necrosis, granulation tissue, and air leak. Logistic regression modeling was used to identify factors associated with AR and adverse outcome. RESULTS From 1993 to 2019, 545 patients with PITS (375; 68.8%) and PTNs (170; 31.2%) underwent laryngotracheal, tracheal, or carinal (resections and reconstructions; 5.7% (31 of 545) were reoperations. AR was performed in 11% (60 of 545): in 3.8% of laryngotracheal resections (6 of 157; all laryngeal), in 9.8% of tracheal resections (34 of 347; laryngeal, 12, and hilar, 22), and in 49% of carinal resections (20 of 41; laryngeal, 1, and hilar, 19). Mean resected length was 3.5 cm (range, 1to- 6.3 cm) with AR and 3.0 cm (range, 0.8 to 6.5 cm) without AR (P < .01). Operative mortality was 0.7% (4 of 545); all 4 anastomoses were intact until death. Anastomotic complications were present in 5% of patients who underwent AR (3 of 60) and in 9.3% (45 of 485) of patients who did not. AR was associated with resection length of 4 cm or longer (odds ratio [OR], 6.15; 95% confidence interval [CI], 1.37 to 27.65), PTNs (OR, 7.81; 95% CI, 3.31 to 18.40), younger age (OR, 0.96; 95% CI, 0.94 to 0.98), and lung resection (OR, 6.09; 95% CI, 1.33 to 27.90). Anastomotic complications in patients with tracheal anastomoses were associated with preexisting tracheostomy (OR, 2.68; 95% CI, 1.50 to 4.80), but not release. CONCLUSIONS Tracheal reconstruction succeeds, even when anastomotic tension requires AR. Because intraoperative assessment may underestimate tension, lowering the threshold for AR seems prudent, particularly in patients with diabetes.

Lung regeneration is dependent on the availability of progenitor lung cells. Large numbers of self-renewing, patient-specific induced pluripotent stem cell-derived alveolar epithelial cells (iPSC-AECs) are needed to adequately recellularize whole-organ constructs. Prior methods to generate functional iPSC-AECs are not feasible for large-scale cell production. We present a novel protocol to produce iPSC-AECs, which is scalable for whole-organ regeneration. Differentiation of iPSCs was performed with genetically modified iPSCs with fluorescent reporters, which underwent differentiation in a stepwise protocol mimicking lung development. Cells were purified, sorted, and embedded in a liquid Matrigel precursor either to form adherent droplets or to form cell-laden Matrigel spheroids, which were subsequently transferred to spinner flasks with media as floating droplets. After culture, monolayer spheres of iPSC-AECs were isolated to form single cell suspensions. Equal numbers of iPSC-AECs from the two culture conditions were seeded into decellularized lung scaffolds. IPSC-AECs cultured in floating droplets were significantly more proliferative than those in adherent droplets, with significantly higher total cell counts and Ki67 expression. Equivalent expression of the distal lung markers was observed for both culture conditions. Lungs recellularized from both culture groups had similar histological appearance. Media changes took significantly less time with the floating droplet method and was more cost effective. The floating droplet culture method demonstrated enhanced proliferative capacity, stable distal lung epithelial phenotype, and reduced resources compared with prior culture methods. In this study, we provide a means for iPSC-AEC production for regeneration of whole-lung constructs. IMPACT STATEMENT: We describe a novel culture method for induced pluripotent stem cell-derived alveolar epithelial cell (AEC) expansion with enhanced proliferative capacity and reduced resource requirements compared with previously described methods. This method is scalable for human whole-lung regeneration bioengineering or could be automated for commercial cell production. This culture method may have implications for the differentiation of type I AECs from type II AECs.

Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro., The authors form pre-epicardial cells (PECs) from hiPSC-derived lateral plate mesoderm on treating with BMP4, RA and VEGF, and co-culture these PECs with cardiomyocytes, inducing cardiomyocyte aggregation, proliferation and network formation with more mature structures and improved beating/contractility.

Lung transplantation remains the only curative treatment for end-stage pulmonary disease. Lung ischemia-reperfusion injury (IRI) is a major contributor to primary allograft dysfunction and donor organ nonutilization. The alveolar macrophage is a key inflammatory mediator in IRI.

BACKGROUND: Vaping, including the use of electronic cigarettes (e-cigarettes), has become increasingly prevalent, yet the associated long-term health risks are largely unknown. Given the prevalence of use, particularly among adolescents early in their lifespan, it is vital to understand the potential chronic pathologic sequelae of vaping. METHODS: We present the cases of four patients with chronic lung disease associated with e-cigarette use characterized by clinical evaluation, with pulmonary function tests (PFTs), chest high-resolution computed tomography (HRCT), endobronchial optical coherence tomography (EB-OCT) imaging, and histopathologic assessment. RESULTS: Each patient presented with shortness of breath and chest pain in association with a 3- to 8-year history of e-cigarette use, with mild progressive airway obstruction on PFTs and/or chest HRCT findings demonstrating evidence of air trapping and bronchial wall thickening. EB-OCT imaging performed in two patients showed small airway–centered fibrosis with bronchiolar narrowing and lumen irregularities. The predominant histopathologic feature on surgical lung biopsy was small airway–centered fibrosis, including constrictive bronchiolitis and MUC5AC overexpression in all patients. Patients who ceased vaping had a partial, but not complete, reversal of disease over 1 to 4 years. CONCLUSIONS: After thorough evaluation for other potential etiologies, vaping was considered to be the most likely common causal etiology for all patients due to the temporal association of symptomatic chronic lung disease with e-cigarette use and partial improvement in symptoms after e-cigarette cessation. In this series, we associate the histopathologic pattern of small airway–centered fibrosis, including constrictive bronchiolitis, with vaping, potentially defining a clinical and pathologic entity associated with e-cigarette use. (Funded in part by the National Institutes of Health.)

OBJECTIVES Emphysema affects millions of patients worldwide. Cell transplantation and tissue engineering are promising approaches for the regeneration of gas exchange tissue in vivo. A reproducible and resource-efficient animal model with relevant pathological and physiological features is critical to assess efficacy of novel therapies. Here, we share a method for rapid development of emphysema in an adaptive immune-deficient rat with METHODS Porcine pancreatic elastase (PPE) was intratracheally administered to male RNU rats. Rats were monitored for 21 days after which subjects underwent lung computed tomography (CT) scans. Rats were then weighed, intubated and mechanically ventilated to measure dynamic compliance. After sacrifice, lungs were fixed, and histological sections were quantitatively assessed for emphysematous changes. RESULTS A single instillation of elastase was enough to produce anatomic and physiological evidence of emphysema. Weight change for doses of 16 and 32 units PPE/100 g were significantly lower than controls (P = 0.028 and P = 0.043, respectively). Compliance values for doses of 16 and 32 units PPE/100 g were significantly higher than controls (P = 0.037 and P = 0.006, respectively). Lung hyperlucency was confirmed by CT with mean Hounsfield units for a dose of 32 units PPE/100 g being significantly lower than controls (P CONCLUSIONS We present an efficient method for emphysema development in immune-deficient rats as a tool to evaluate human biological therapeutics. Changes in dynamic compliance, histology and cross-sectional imaging recapitulate human emphysema.

PURPOSE: To assess the technical success and complication rates of CT-guided fiducial marker placement for the localization of pulmonary nodules and to assess the surgical localization failure rate. MATERIALS AND METHODS: This was a single-center, retrospective analysis of consecutive patients who underwent CT-guided fiducial marker placement procedures between 2014 and 2020. End points included the technical success of the fiducial marker placement, procedural complications, and the surgical localization failure rate. A two-sample t test and a Fisher exact test were used to compare continuous and categorical variables, respectively. Multivariate logistic regression was used to identify independent risk factors for complications. RESULTS: A total of 198 preoperative CT-guided fiducial marker placement procedures were performed in 190 patients (mean age, 64 years ± 12 [standard deviation]; 121 women) to localize 205 nodules (mean size, 10 mm ± 4; mean distance to the pleura, 10 mm ± 9). The technical success rate was 98.5% (195 of 198). There were no major complications. A total of 202 nodules were resected during 193 procedures performed 5 days ± 13 after the fiducial marker placement (range, 0–123 days). Surgical localization failure occurred in one patient (0.5%). Of the resected nodules, 146 were lung cancers, 26 nodules were metastases, two were carcinoid tumors, and 28 were benign. CONCLUSION: The CT-guided fiducial marker placement of pulmonary nodules was safe, effective, and resulted in a low surgical localization failure rate. Keywords: CT, Percutaneous, Thorax, Lung

Lung transplantation is currently the only curative treatment for patients with end-stage lung disease; however, donor organ shortage and the need for intense immunosuppression limit its broad clinical application. Bioartificial lungs created by combining native matrix scaffolds with patient-derived cells might overcome these problems. Decellularization involves stripping away cells while leaving behind the extracellular matrix scaffold. Cadaveric lungs are decellularized by detergent perfusion, and histologic examination confirms the absence of cellular components but the preservation of matrix proteins. The resulting lung scaffolds are recellularized in a bioreactor that provides biomimetic conditions, including vascular perfusion and liquid ventilation. Cell seeding, engraftment, and tissue maturation are achieved in whole-organ culture. Bioartificial lungs are transplantable, similarly to donor lungs, because the scaffolds preserve the vascular and airway architecture. In rat and porcine transplantation models, successful anastomoses of the vasculature and the airway were achieved, and gas exchange was evident after reperfusion. However, long-term function has not been achieved because of the immaturity of the vascular bed and distal lung epithelia. The goal of this strategy is to create patient-specific transplantable lungs using induced pluripotent stem cell (iPSC)-derived cells. The repopulation of decellularized scaffolds to create transplantable organs is one of possible future clinical applications of iPSCs.

Current reconstruction methods of the laryngotracheal segment fail to replace the complex functions of the human larynx. Bioengineering approaches to reconstruction have been limited by the complex tissue compartmentation of the larynx. We attempted to overcome this limitation by bioengineering laryngeal grafts from decellularized canine laryngeal scaffolds recellularized with human primary cells under one uniform culture medium condition. First, we developed laryngeal scaffolds which were generated by detergent perfusion-decellularization over 9 days and preserved their glycosaminoglycan content and biomechanical properties of a native larynx. After subcutaneous implantations in rats for 14 days, the scaffolds did not elicit a CD3 lymphocyte response. We then developed a uniform culture medium that strengthened the endothelial barrier over 5 days after an initial growth phase. Simultaneously, this culture medium supported airway epithelial cell and skeletal myoblast growth while maintaining their full differentiation and maturation potential. We then applied the uniform culture medium composition to whole laryngeal scaffolds seeded with endothelial cells from both carotid arteries and external jugular veins and generated reendothelialized arterial and venous vascular beds. Under the same culture medium, we bioengineered epithelial monolayers onto laryngeal mucosa and repopulated intrinsic laryngeal muscle. We were then able to demonstrate early muscle formation in an intramuscular transplantation model in immunodeficient mice. We supported formation of three humanized laryngeal tissue compartments under one uniform culture condition, possibly a key factor in developing complex, multicellular, ready-to-transplant tissue grafts. Impact Statement For patients undergoing laryngectomy, no reconstruction methods are available to restore the complex functions of the human larynx. The first promising preclinical results have been achieved with the use of biological scaffolds fabricated from decellularized tissue. However, the complexity of laryngeal tissue composition remains a hurdle to create functional viable grafts, since previously each cell type requires tailored culture conditions. In this study, we report the de novo formation of three humanized laryngeal tissue compartments under one uniform culture condition, a possible keystone in creating vital composite tissue grafts for laryngeal regeneration.

Objectives The study objectives were to evaluate the immediate, short-, and medium-term efficacy and safety of pulmonary artery branch sealing using an ultrasonic vessel-sealing device in minimally invasive anatomic lung resection. Methods This study consists of a prospective, phase 2, multicenter, international clinical trial (clinicaltrials.gov: NCT02719717) that enrolled patients planned for video-assisted thoracoscopic surgery/robotic anatomic lung resection in 7 centers (United States, Canada, United Kingdom). Pulmonary artery branches of 7 mm or less were sealed and divided with an ultrasonic energy vessel-sealing device. The remainder of the lobectomy was performed according to surgeon preference. Intraoperative, in-hospital, and 30-day postoperative bleeding and complications were prospectively recorded. Results A total of 150 patients with a minimum of 1 pulmonary artery branch sealed with an ultrasonic vessel-sealing device were prospectively enrolled in the trial. Resections included 139 lobectomies and 11 segmentectomies. A total of 424 pulmonary artery branches were divided: 239 with the ultrasonic vessel-sealing device, 181 with endostaplers, and 4 with endoscopic clips. The mean and median pulmonary artery diameters were 4.7 mm/5.0 mm, 10.3 mm/10.0 mm, and 6.5 mm/6.5 mm for each method, respectively. Three of the pulmonary artery branches divided with the ultrasonic vessel-sealing device (1.3%) and 4 pulmonary artery branches divided with endostaplers (2.2%) bled intraoperatively. Among the patients with seal failures, 1 patient required conversion to thoracotomy. There was no postoperative bleeding from divided pulmonary artery branches with either sealing method. There was no mortality at 30 days. Conclusions Pulmonary artery branch sealing with ultrasonic energy during video-assisted thoracoscopic surgery lobectomy is safe for vessels 7 mm or less. The use of an ultrasonic device is a reasonable sealing method for pulmonary artery branches 7 mm or less.

During heart maturation, epicardial-derived cells emanating from the pro-epicardial organ envelope the developing heart, then integrate within the myocardium and undergo epithelial-mesenchymal transition (EMT) to become fibroblasts, smooth muscle cells, and endothelial cells that enable several processes of healthy heart morphogenesis (i.e., ventricular thickening, compaction, and angiogenesis). Directed cardiac differentiation of human induced-pluripotent stem cells (hiPSCs) holds great potential for generating several cardiovascular cell types, which can theoretically be combined to model cardiac development more completely and engineer sophisticated myocardial grafts. Here, we show that differentiating hiPSC-derived lateral plate mesoderm (LPM) with BMP4, RA and VEGF (BVR) for 96 hours can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) with high efficiency (86.8 ± 4.1% WT1 + by FACS), that also show significant increases in WT1, TBX18, SEMA3D , TBX5, BNC, and SCX transcript expression within 7 days. This protocol was successfully reproduced in three commercial lines of hiPSCs. BVR stimulation after Wnt inhibition of LPM demonstrated efficient co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidated CMs into dense aggregates, which then formed a connected beating syncytium with significantly enhanced contractility (0.029 ± 0.002 mN/mm 2 ; pUPK1B, ITGA4, and ALDH1A2 transcript expressions. Our study also demonstrated that PECs secrete IGF2 and stimulate CM proliferation in co-culture (37.2 ± 2.1% vs. 28.1 ± 1%, p=0.016). These characteristics suggest PECs could play a key role in enhancing cell-cell signaling and tissue organization, which may help to recapitulate important aspects of cardiac morphogenesis in vitro and advance the maturation of bioengineered cardiac constructs.

The catastrophic global effects of the SARS-CoV-2 pandemic highlight the need to develop novel therapeutics strategies to prevent and treat viral infections of the respiratory tract. To enable this work, we need scalable, affordable, and physiologically relevant models of the human lung, the primary organ involved in the pathogenesis of COVID-19. To date, most COVID-19 in vitro models rely on platforms such as cell lines and organoids. While 2D and 3D models have provided important insights, human distal lung models that can model epithelial viral uptake have yet to be established. We hypothesized that by leveraging techniques of whole organ engineering and directed differentiation of induced pluripotent stem cells (iPSC) we could model human distal lung epithelium, examine viral infection at the tissue level in real time, and establish a platform for COVID-19 related research ex vivo. In the present study, we used type 2 alveolar epithelial cells (AT2) derived from human iPSCs to repopulate whole rat lung acellular scaffolds and maintained them in extended biomimetic organ culture for 30 days to induce the maturation of distal lung epithelium. We observed emergence of a mixed type 1 and type 2 alveolar epithelial phenotype during tissue formation. When exposing our system to a pseudotyped lentivirus containing the spike of wildtype SARS-CoV-2 and the more virulent D614G, we observed progression of the infection in real time. We then found that the protease inhibitor Camostat Mesyalte significantly reduced viral transfection in distal lung epithelium. In summary, our data show that a mature human distal lung epithelium can serve as a novel moderate throughput research platform to examine viral infection and to evaluate novel therapeutics ex vivo.

Chad E Eckert,1 Jason L Harris,1 Jordan B Wong,1 Suzanne Thompson,2 Edmund S Kassis,3 Masahiro Tsuboi,4 Harald C Ott,5 Seth Force6 1Ethicon Inc., Research and Development, Cincinnati, OH 45242, USA; 2Ethicon Inc., Preclinical Center of Excellence, Cincinnati, OH 45242, USA; 3Ethicon Inc., Medical Affairs, Cincinnati, OH 45242, USA; 4Division of Thoracic Surgery and Oncology, National Cancer Center Hospital East, Kashiwanoha, Kashiwa, Chiba 277-8577, Japan; 5Department of Surgery, Division of Thoracic Surgery, Massachusetts General Hospital, Boston, MA 02114, USA; 6Department of Surgery, Division of Cardiothoracic Surgery, Emory University Hospital, Atlanta, GA 30322, USA Purpose: Thoracic air leaks are a common complication following pulmonary resections. Limitations in clinical studies and preclinical models have hindered efforts to understand the pathophysiology of air leaks. With an emphasis on staple-line specific air leaks, we hypothesize that ventilation modality – intraoperative positive pressure vs postoperative negative pressure – and stapler design may play a role in air leaks. Methods: Using a novel physiologic lung model, air leaks associated with graduated and uniform staple designs were evaluated under positive and negative pressure ventilation, simulating perioperative breathing in porcine lungs. Air leak incidence, air leak volume, and air leak rate were captured along with ventilation pressure and tidal volume. Results: In all cases, negative pressure ventilation was associated with a higher occurrence of leaks when compared to positive pressure ventilation. Lungs leaked more air and at a faster rate under negative pressure ventilation compared to positive pressure ventilation. Graduated staple designs were associated with higher occurrence of leaks as well as larger leak rates when compared to uniform staples. Tissue thickness was not associated with differences in air leaks when tested with appropriate staple heights. Conclusion: Using a novel lung model to investigate the pathophysiology of air leaks, we have identified breathing modality and staple design as two important variables that may impact air leaks. This work will help guide device design and drive future studies in human tissue, and it may help inform clinical practice to ultimately improve patient outcomes. Keywords: prolonged air leak, lung physiology, preclinical model, surgical stapler, ventilation mechanics

Objective: A novel ex vivo model is described to advance the understanding of prolonged air leaks, one of the most common postoperative complications following thoracic resection procedures. Methods: As an alternative to in vivo testing, an ex vivo model simulating the various physiologic environments experienced by an isolated lung during the perioperative period was designed and built. Isolated porcine lungs were perfused and ventilated during open chest and closed chest simulations, mimicking intra and postoperative ventilation conditions. To assess and validate system capabilities, nine porcine lungs were tested by creating a standardized injury to create an approximately 250 cc/min air leak. Air leak rates, physiologic ventilation, and perfusion parameters were continuously monitored, while gas transfer analysis was performed on selected lungs. Segmental ventilation was monitored using electrical impedance tomography. Results: The evaluated lungs produced flow–volume and pressure–volume loops that approximated standard clinical representations under positive (mechanical) and negative (physiological) pressure ventilation modalities. Leak rate was averaged across the ventilation phases, and sharp increases in leak rate were observed between positive and negative pressure phases, suggesting that differences or changes in ventilation mechanics may strongly influence leak development. Conclusion: The successful design and validation of a novel ex vivo lung model was achieved. Model output paralleled clinical observations. Pressure modality may also play a significant role in air leak severity. Significance: This work provides a foundation for future studies aimed at increasing the understanding of air leaks to better inform means of mitigating the risk of air leaks under clinically relevant conditions.

Context.—Lesion localization during intraoperative frozen section of lung resection specimens can be challenging. Imaging could aid lesion localization while enabling 3-dimensional specimen analysis.Objective.—To assess the feasibility of integrating micro–computed tomography (micro-CT) into the perioperative evaluation of fresh surgical lung resection specimens.Design.—Fresh lung specimens from patients with a presumptive diagnosis of lung cancer were imaged with micro-CT prior to routine histopathologic and molecular analysis. Micro-CT images were assessed to determine image quality, lesion size, and distance from lesion to the nearest surgical margin. Micro-CT measurements were compared to pathologic measurements using Bland-Altman analysis.Results.—A total of 22 specimens from 21 patients were analyzed (mean image acquisition time, 13 ± 6 minutes). Histologic quality of imaged specimens was indistinguishable from a control group of nonimaged lung specimens. Artifacts, most commonly from specimen deflation (n = 8), obscured fine detail on micro-CT images of 10 specimens. Micro-CT could successfully localize the target lesion in the other 12 specimens. Distance to the nearest surgical margin was determined in 10 specimens. Agreement of micro-CT with final pathology was good, with a mean difference of −2.8% (limits of agreement −14.5% to 20.0%) for lesion size and −0.5 mm (limits of agreement −4.4 to 3.4 mm) for distance to nearest surgical margin.Conclusions.—Micro-CT of fresh surgical lung specimens is feasible and has the potential to evaluate the size and location of lesions within resection specimens, as well as distance to the nearest surgical margin, all without compromising specimen integrity.

Decellularized native extracellular matrix (ECM) biomaterials are widely used in tissue engineering and have reached clinical application as biomesh implants. To enhance their regenerative properties and postimplantation performance, ECM biomaterials could be functionalized via immobilization of bioactive molecules. To facilitate ECM functionalization, we developed a metabolic glycan labeling approach using physiologic pathways to covalently incorporate click-reactive azide ligands into the native ECM of a wide variety of rodent tissues and organs in vivo, and into the ECM of isolated rodent and porcine lungs cultured ex vivo. The incorporated azides within the ECM were preserved after decellularization and served as chemoselective ligands for subsequent bioconjugation via click chemistry. As proof of principle, we generated alkyne-modified heparin, immobilized it onto azide-incorporated acellular lungs, and demonstrated its bioactivity by Antithrombin III immobilization and Factor Xa inhibition. The herein reported metabolic glycan labeling approach represents a novel platform technology for manufacturing click-reactive native ECM biomaterials, thereby enabling efficient and chemoselective functionalization of these materials to facilitate tissue regeneration and repair.

Rationale: Early, accurate diagnosis of interstitial lung disease (ILD) informs prognosis and therapy, especially in idiopathic pulmonary fibrosis (IPF). Current diagnostic methods are imperfect. High-resolution computed tomography has limited resolution, and surgical lung biopsy (SLB) carries risks of morbidity and mortality. Endobronchial optical coherence tomography (EB-OCT) is a low-risk, bronchoscope-compatible modality that images large lung volumes in vivo with microscopic resolution, including subpleural lung, and has the potential to improve the diagnostic accuracy of bronchoscopy for ILD diagnosis. Objectives: We performed a prospective diagnostic accuracy study of EB-OCT in patients with ILD with a low-confidence diagnosis undergoing SLB. The primary endpoints were EB-OCT sensitivity/specificity for diagnosis of the histopathologic pattern of usual interstitial pneumonia (UIP) and clinical IPF. The secondary endpoint was agreement between EB-OCT and SLB for diagnosis of the ILD fibrosis pattern. Methods: EB-OCT was performed immediately before SLB. The resulting EB-OCT images and histopathology were interpreted by blinded, independent pathologists. Clinical diagnosis was obtained from the treating pulmonologists after SLB, blinded to EB-OCT. Measurements and Main Results: We enrolled 31 patients, and 4 were excluded because of inconclusive histopathology or lack of EB-OCT data. Twenty-seven patients were included in the analysis (16 men, average age: 65.0 yr): 12 were diagnosed with UIP and 15 with non-UIP ILD. Average FVC and Dl(CO) were 75.3% (SD, 18.5) and 53.5% (SD, 16.4), respectively. Sensitivity and specificity of EB-OCT was 100% (95% confidence interval, 75.8–100.0%) and 100% (79.6–100%), respectively, for both histopathologic UIP and clinical diagnosis of IPF. There was high agreement between EB-OCT and histopathology for diagnosis of ILD fibrosis pattern (weighted κ: 0.87 [0.72–1.0]). Conclusions: EB-OCT is a safe, accurate method for microscopic ILD diagnosis, as a complement to high-resolution computed tomography and an alternative to SLB.

Summary Background Esophageal perforation is a morbid condition and remains a therapeutic challenge. We report the outcomes of a large institutional experience with esophageal perforation and identify risk factors for morbidity and mortality. Methods A retrospective analysis was conducted on 142 patients who presented with a thoracic or gastroesophageal junction esophageal perforation from 1995 to 2020. Baseline characteristics, operative or interventional strategies, and outcomes were analyzed by etiology of the perforation and management approach. Multivariable cox and logistic regression models were constructed to identify predictors of mortality and morbidity. Results Overall, 109 (77%) patients underwent operative intervention, including 80 primary reinforced repairs and 21 esophagectomies and 33 (23%) underwent esophageal stenting. Stenting was more common in iatrogenic (27%) and malignant (64%) perforations. Patients who presented with a postemetic or iatrogenic perforation had similar 90-day mortality (16% and 16%) and composite morbidity (51% and 45%), whereas patients who presented with a malignant perforation had a 45% 90-day mortality and 45% composite morbidity. Risk factors for mortality included age >65 years (hazard ratio [HR] 1.89 [1.02–3.26], P = 0.044) and a malignant perforation (HR 4.80 [1.31–17.48], P = 0.017). Risk factors for composite morbidity included pleural contamination (odds ratio [OR] 2.06 [1.39–4.43], P = 0.046) and sepsis (OR 3.26 [1.44–7.36], P = 0.005). Of the 33 patients who underwent stent placement, 67% were successfully managed with stenting alone and 30% required stent repositioning. Conclusions Risk factors for morbidity and mortality after esophageal perforation include advanced age, pleural contamination, septic physiology, and malignant perforation. Primary reinforced repair remains a reasonable strategy for patients with an esophageal perforation from a benign etiology.

Early, accurate diagnosis of interstitial lung disease (ILD) is critical for clinical management and therapeutic decision-making, especially distinguishing idiopathic pulmonary fibrosis (IPF) from non-IPF ILD. Unfortunately, current diagnostic imaging methods are limited in resolution and surgical biopsy methods are invasive. In this study, we evaluate the accuracy of endobronchial optical coherence tomography (EB-OCT) as a low-risk method for in vivo ILD diagnosis in patients undergoing surgical biopsy. EB-OCT was able to distinguish diagnostic microanatomical features of IPF from non-IPF ILDs, independently compared against surgical biopsy. These findings support the potential of OCT as a minimally-invasive method for IPF diagnosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal form of fibrotic interstitial lung disease (ILD). Early diagnosis of IPF is essential, but often requires invasive surgery. We conduct a prospective study evaluating the diagnostic accuracy of endobronchial optical coherence tomography (EB-OCT) for IPF diagnosis as compared to concurrent surgical lung biopsy (SLB) and clinical follow-up diagnosis. EB-OCT was performed immediately prior to SLB in 27 ILD patients. EB-OCT was 100% sensitive and 100% specific for both histologic and clinical follow up diagnosis of IPF. The results demonstrate the potential of EB-OCT as minimally-invasive, low-risk in vivo method for microscopic IPF diagnosis.

Lung transplantation is the only treatment for end-stage lung disease; however, donor organ shortage and intense immunosuppression limit its broad clinical impact. Bioengineering of lungs with patient-derived cells could overcome these problems. We created bioartificial lungs by seeding human-derived cells onto porcine lung matrices and performed orthotopic transplantation to assess feasibility and in vivo function. Porcine decellularized lung scaffolds were seeded with human airway epithelial cells and human umbilical vein endothelial cells. Following in vitro culture, the bioartificial lungs were orthotopically transplanted into porcine recipients with planned 1-day survival (n = 3). Lungs were assessed with histology and in vivo function. Orthotopic transplantation of cadaveric lungs was performed as control. Engraftment of endothelial and epithelial cells in the grafts were histologically demonstrated. Technically successful orthotopic anastomoses of the vasculatures and airway were achieved in all animals. Perfusion and ventilation of the lung grafts were confirmed intraoperatively. The gas exchange function was evident immediately after transplantation; PO2 gradient between pulmonary artery and vein were 178 ± 153 mm Hg in the bioartificial lung group and 183 ± 117 mm Hg in the control group. At time of evaluation 24 hours after reperfusion, the pulmonary arteries were found to be occluded with thrombus in all bioartificial lungs. Engineering and orthotopic transplantation of bioartificial lungs with human cells were technically feasible in a porcine model. Early gas exchange function was evident. Further progress in optimizing recellularization and maturation of the grafts will be necessary for sustained perfusability and function.

Approximately 10% to 15% of patients with solitary fibrous tumors of the pleura (SFTP) have recurrence after resection. Many are not candidates for reresection and lack effective treatments. We explored the expression of programmed death ligand 1 (PD-L1) as a biomarker for candidacy for treatment with immune checkpoint inhibitors.We reviewed the medical records of 52 patients with primary SFTP and 5 with recurrent SFTP. We performed immunohistochemistry on tumor tissue to determine the expression of PD-L1 and infiltration by cluster of differentiation 8 (CD8)-positive immune cells.Any PD-L1 expression was observed in 11 primary SFTP (21.2%). Overall, PD-L1 expression level was less than 1% in 10 patients (19.2%) and greater than 1% in 1 (1.9%). Tumor infiltration by CD8-positive immune cells was absent or rare in 13 patients (25%), less than 5% in 31 (59.6%), and 5% to 25% in 8 (15.4%). There were no associations between PD-L1 expression or immune cell infiltrates and known risk factors for recurrence or a prognostic risk score classification. Time to recurrence was strongly associated with the risk score classification (P.001), but it was not associated with PD-L1 expression (P = .296) or immune cell infiltrates (P = .619). In recurrent SFTP, PD-L1 was expressed in 4 of 10 tumors (40%; all1% expression). There was no correlation in PD-L1 expression between primary and recurrent SFTP samples.A small subset of SFTP express PD-L1 at low levels (1%) but exhibit colocalization of CD8-positive immune cells suggesting an inducible expression mechanism. The role of PD-L1 merits exploration in the clinical setting in patients with advanced SFTP when alternative treatments or clinical trials are considered.

The human trachea is a vital structure with complex architecture and well-defined mechanical properties. Trauma, neoplasm, congenital defects and iatrogenic injury can lead to loss of patency and necessitate surgical intervention to reestablish its function as a viable and disease resistant airway. Due to the trachea’s structural redundancy, and longitudinal elasticity, circumferential tracheal resection and primary anastomosis is safe in the majority of patients. For those who require removal of more than half of the trachea, options are limited. This unmet need, and the apparent simplicity of the trachea as a hollow tube, have inspired surgeons and scientists to engineer tracheal substitutes over nearly a century. None of these implants have achieved long term patency. As a result, patients have suffered from complications of premature clinical translation. While experimental research in this area has to continue, engineered tissues are not a viable form of treatment at this point in time.

In reconstructive surgery, transfer of patients' tissue (autologous flaps) is routinely used to repair large soft tissue defects caused by surgery, trauma, chronic diseases, or malformations; unfortunately, this strategy is not always possible and often creates a secondary defect in the donor site of the tissue. Tissue-engineered synthetic flaps are currently unable to repair clinically-relevant, large-volume defects; allogenic flaps from cadaveric donors could provide a ready-to-use biological alternative if treated with methods to avoid the immune-rejection of the donor's cells. Here, we describe the successful decellularization of a large (> 800 cc) human-derived adipose flap through a perfusion apparatus; we demonstrate the complete removal of the immunogenic cellular components of the flap with the retention of its structural components and vascular network. Our aim is to obtain a universally compatible, off-the-shelf acellular allogenic flap that could be recellularized with cells from recipient patients to provide a tissue-engineered allogenic/autologous alternative for reconstruction of large-volume soft-tissue defects.

Patients with short bowel syndrome lack sufficient functional intestine to sustain themselves with enteral intake alone. Transplantable vascularized bioengineered intestine could restore nutrient absorption. Here we report the engineering of humanized intestinal grafts by repopulating decellularized rat intestinal matrix with human induced pluripotent stem cell-derived intestinal epithelium and human endothelium. After 28 days of in vitro culture, hiPSC-derived progenitor cells differentiate into a monolayer of polarized intestinal epithelium. Human endothelial cells seeded via native vasculature restore perfusability. Ex vivo isolated perfusion testing confirms transfer of glucose and medium-chain fatty acids from lumen to venous effluent. Four weeks after transplantation to RNU rats, grafts show survival and maturation of regenerated epithelium. Systemic venous sampling and positron emission tomography confirm uptake of glucose and fatty acids in vivo. Bioengineering intestine on vascularized native scaffolds could bridge the gap between cell/tissue-scale regeneration and whole organ-scale technology needed to treat intestinal failure patients., There is a need for humanised grafts to treat patients with intestinal failure. Here, the authors generate intestinal grafts by recellularizing native intestinal matrix with human induced pluripotent stem cell-derived epithelium and human endothelium, and show nutrient absorption after transplantation in rats.

The purposes of this study were to review current progress being made in engineering bioartificial lungs for transplantation and identify tangible benefits to the implementation of this research into development of 4D lung models for disease modeling, drug screening, and personalized medicine that could be realized in the next 5 years. We focused primarily on the generation and analysis of decellularized lung scaffolds, the repopulation of acellular lung scaffolds by primary cells and pluripotent stem cells-derived cells, and current disease modeling approaches. Bioengineered lung has significant potential in transplantation and facilitates future biomedical research by establishing of 4D cell culture model on the scaffold; it will benefit the implementation of the development of disease modeling, drug screening, and personalized medicine.

Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5+TP63+ basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair.

Lung tissue engineering on native extracellular matrix involves combining essential cell populations with corresponding scaffolds. Functional regeneration will require both airway and alveolar epithelial repopulation, along a proximal-to-distal axis. Basal epithelial cells (BECs) can be isolated from donor lung tissue and used for airway recellularization. Yet, a robust source of distal alveolar epithelial cells remains a challenge. Inhibiting the Notch pathway by targeting γ-secretase can direct patient-derived BECs toward a distal Type-2 pneumocyte (AT2)-like fate in vitro, as demonstrated by increased surfactant protein-C (SP-C, +22.06-fold), ABCA3 (+2.79-fold), and LAMP1 (+11.48-fold) gene expression. Notch inhibition was confirmed by decreased HES1 (-2.69-fold) and by a Notch-responsive luciferase reporter. Notch inhibition following delivery of BECs to the airways of lung scaffolds and ex vivo biomimetic culture enabled greater induction of distal AT2-like fate (+27.61-fold SP-C, -3.73-fold HES1 vs in vitro). Regenerated lungs displayed alveolar recellularization with organized architecture and morphology. Revascularization of the scaffold with pulmonary artery endothelial cells (PAECs) further aided BEC distalization during ex vivo regeneration (+2.38-fold SP-C, -6.17-fold HES1, vs no PAECs). Addition of air ventilation during ex vivo lung culture further enhanced this response (+2.54 SP-C vs no ventilation). These results demonstrate an exciting potential of BECs for lung tissue regeneration and endogenous repair. The microenvironment and reparative niche are also important drivers of epithelial cell fate during regeneration.

Background: Lung transplantation remains the only treatment for many end-stage lung diseases. However, donor organ shortage and the need for immunosuppression limit clinical impact. Cadaveric lungs can be decellularized to yield scaffolds that provide a suitable platform for tissue regeneration. Directed differentiation of human iPSCs toward a lung epithelial fate is a promising approach to facilitate the goal of whole organ regeneration with patient-specific cells. Objective: To investigate the capacity of iPSC-derived alveolar cells to form distal pulmonary epithelium on acellular lung scaffolds. Methods: Human iPSCs carrying Nkx2.1-GFP and SPC-TdTomato reporters were differentiated towards a lung alveolar fate by established protocols. SP-C+ alveolar cells were delivered to decellularized rat lungs (n=6) to repopulate the distal niche. Epithelial tissue formation was induced through extended ex vivo biomimetic culture. Regenerated lungs were analyzed for tissue architecture and cell fate. Acellular human lung slices were also repopulated with iPSC-derived cells in vitro. Seeded lung slices (n=12) were cultured, then tissue structure and cell fate were also analyzed. Results: Formation of early distal lung epithelial tissue with mature type II alveolar cell phenotype, expressing Nkx2.1, E-Cadherin, SP-C, SP-B was observed. We also identified AQP5 positive cells, which may identify a type 1 alveolar fate. Similar findings were seen on human lung slices. Conclusions: Human iPSC-derived alveolar epithelial cells can form distal lung epithelium on both rodent and human lung scaffolds, demonstrating the future potential for patient-specific lung regeneration at a transplantable scale.

Background: Recellularization of acellular lung scaffolds is a promising approach for lung bioengineering. High level tissue maturation requires cues driving cell proliferation, migration, and fate. The Hippo pathway can mediate these processes in proximal and distal lung epithelium. We examined effects of Hippo modulation on proximal and distal epithelium using pharmacological inhibition and biomechanical stimulation. Methods: Proximal epithelium was studied using airway basal cells (BEAS2Bs). Distal epithelium was studied using induced pluripotent stem cell (iPSC)-derived alveolar type II progenitor cells (AT2s). In vitro, we used soft (4kPa) and stiff (50kPa) hydrogels and the small molecule XMU-MP-1 (inhibits Hippo, activating Yap). In ex vivo scaffold recellularization culture, we tested air/liquid ventilation and Hippo inhibition by XMU-MP-1. Results: In BEAS2Bs, Yap downstream gene CTGF decreased expression on soft hydrogels (2.03-fold) while increasing with XMU-MP-1 treatment (30.73-fold). Air ventilation of BEAS2B-recellularized lung scaffolds enhanced CTGF expression (3.04-fold), tissue morphology, and cell coverage. XMU-MP-1 treatment of AT2s in vitro increased CTGF expression (37.66-fold) and surfactant protein C (SP-C) expression (14.63-fold). Liquid ventilation of AT2-recellularized lung scaffolds enhanced SP-C expression (11.16-fold) and XMU-MP-1 treatment increased CTGF expression (16.55-fold). Conclusions: Hippo pathway modulation can be used to enhance ex vivo lung epithelial regeneration. Hippo inhibition may cause proliferation and migration of airway cells while increasing distal surfactant production in AT2s, enhancing gas exchange function.

Video-assisted thoracic surgery (VATS) and robotically assisted surgery are used increasingly for minimally invasive diagnostic and therapeutic resection of pulmonary nodules. Unsuccessful localization of small, impalpable, or deep pulmonary nodules can necessitate conversion from VATS to open thoracotomy. Preoperative localization techniques performed by radiologists have improved the success rates of VATS resection for small and subsolid nodules. Any center at which VATS diagnostic resection of indeterminate pulmonary nodules is performed should be supported by radiologists who offer preoperative nodule localization. Many techniques have been described, including image-guided injection of radioisotopes and radiopaque liquids and placement of metallic wires, coils, and fiducial markers. These markers enable the surgeon to visualize the position of an impalpable nodule intraoperatively. This article provides details on how to perform each percutaneous localization technique, and a group of national experts with established nodule localization programs describe their preferred approaches. Special reference is made to equipment required, optimization of marker placement, prevention of technique-specific complications, and postprocedural treatment. This comprehensive unbiased review provides valuable information for those who are considering implementation or optimization of a nodule localization program according to workflow patterns, surgeon preference, and institutional resources in a particular center. ©RSNA, 2019.

Background To evaluate the management, complications of treatment, and outcomes of postintubation tracheal stenosis. Methods A retrospective review was performed of records from a prospective database of all patients undergoing tracheal or laryngotracheal resection from 1993 to 2017 for postintubation tracheal stenosis. Redo operations after failure of initial resection and reconstruction for postintubation tracheal stenosis were included. Results There were 392 patients whose ages ranged from 3 months to more than 84 years. A tracheostomy was performed in 275 as part of their care before surgery (present at time of resection in 123), dilations in 201, laser treatment in 82, T tubes in 66, and stents in 44 patients. Median length of resection was 3 cm. Laryngeal release was required in 15 of 392 (3.8%). Operative mortality was 0.8% (3 of 392); T tubes, tracheostomy present at resection, requirement for postoperative tracheostomy, and laryngeal involvement adversely impacted outcomes. Patients having tracheal resection and reconstruction had good or satisfactory outcomes in 96% (289 of 301) compared with 85% (77 of 91) having laryngotracheal resection. Complications within 30 days and at more than 30 days occurred in 116 patients and 14 patients, respectively. There were 96 anastomotic complications—68% minor (65 of 96), and 32% major (31 of 96). Necrosis of cartilage occurred in 12 patients and dehiscence in 14 patients. Conclusions Despite advances in care postintubation tracheal stenosis remains a challenging problem. Laryngotracheal resection and tracheostomy lead to worse outcomes. Excellent surgical results can be obtained for postintubation tracheal stenosis. Good results require careful evaluation, management of comorbid conditions, meticulous technique, minimizing tension, and preservation of blood supply.

The lung's primary function is to exchange gases to provide oxygen to the circulating blood and remove carbon dioxide from it. Maintenance of this function relies on a highly adaptive vascular system and specialized epithelial cells lining the respiratory tract along a proximodistal axis. Coordinated cell differentiation along this axis during lung development provides an important road map for regenerative medicine. Throughout adult life, endogenous stem and progenitor cell populations maintain an inherent regenerative potential to secure homeostasis of the respiratory tract after injury. Novel methods have been developed for airway and lung tissue engineering, at least conceptually enabling in vivo and ex vivo repair. Among those, the use of biological scaffolds to support recellularization and regeneration has further advanced the goal of personalized, patient-specific therapeutics for the lung.

Islet transplantation is superior to extrinsic insulin supplementation in the treating severe Type 1 diabetes. However, its efficiency and longevity are limited by substantial islet loss post-transplantation due to lack of engraftment and vascular supply. To overcome these limitations, we developed a novel approach to bio-fabricate functional, vascularized islet organs (VIOs) ex vivo. We endothelialized acellular lung matrixes to provide a biocompatible multicompartment scaffold with an intact hierarchical vascular tree as a backbone for islet engraftment. Over seven days of culture, islets anatomically and functionally integrated into the surrounding bio-engineered vasculature, generating a functional perfusable endocrine organ. When exposed to supra-physiologic arterial glucose levels in vivo and ex vivo, mature VIOs responded with a physiologic insulin release from the vein and provided more efficient reduction of hyperglycemia compared to intraportally transplanted fresh islets. In long-term transplants in diabetic mice, subcutaneously implanted VIOs achieved normoglycemia significantly faster and more efficiently compared to islets that were transplanted in deviceless fashion. We conclude that ex vivo bio-fabrication of VIOs enables islet engraftment and vascularization before transplantation, and thereby helps to overcome limited islet survival and function observed in conventional islet transplantation. Given recent progress in stem cells, this technology may enable assembly of functional personalized endocrine organs.

The careful study of cell-based lung repair and regeneration ex vivo may one day provide us with the necessary tools to create a patient-derived alternative to cadaveric donor lungs for transplantation. Many parameters must be monitored and optimized to advance this aim. The use of rat lungs as a small-scale model for lung regeneration is an efficient way to develop the key improvements required for optimal tissue repair and regeneration. In this study, we report the use of a novel high-throughput, automated, multichannel lung bioreactor system, which allows for culture and analysis of rodent scale isolated lungs. In this model, five decellularized rat lungs can be repopulated with human primary endothelial and epithelial cells, cultured under varying perfusion and ventilation parameters in parallel, and analyzed for standardized endpoints. As a proof of principle, we report a multiphase organ culture protocol, which achieves consistent tissue regeneration across lungs at multiple points during culture, and further promotes overall tissue maturation through the application of lung ventilation. IMPACT STATEMENT: This work presents methods for ex vivo lung recellularization and biomimetic culture in a high-throughput and consistent manner. These methods allow for the testing of multiple variables, all of which are simultaneously controlled and monitored on a single fully automated pump system, and subsequent assessment of both epithelial and endothelial repair and tissue regeneration. This system provides a controlled environment for tissue repair, wherein key variables can be modified, monitored, reproduced, and optimized to advance the goal of ex vivo tissue regeneration based on native organ scaffolds.

Whole lung extracellular matrix scaffolds can be created by perfusion of cadaveric organs with decellularizing detergents, providing a platform for organ regeneration. Lung epithelial engineering must address both the proximal airway cells that function to metabolize toxins and aid mucociliary clearance and the distal pneumocytes that facilitate gas exchange. Engineered pulmonary vasculature must support in vivo blood perfusion with low resistance and intact barrier function and be antithrombotic. Repopulating the native lung matrix with sufficient cell numbers in appropriate anatomic locations is required to enable organ function.

Rationale: More than 25 million individuals have heart failure worldwide, with ≈4000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only ≈2500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. Objective: The objective of this study is to translate previous work to human scale and clinically relevant cells for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human induced pluripotent stem cell–derived cardiomyocytes. Methods and Results: To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiomyocytes derived from nontransgenic human induced pluripotent stem cells and generated tissues of increasing 3-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole-heart scaffolds with human induced pluripotent stem cell–derived cardiomyocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue and showed electrical conductivity, left ventricular pressure development, and metabolic function. Conclusions: Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human induced pluripotent stem cell–derived cardiomyocytes and enable the bioengineering of functional human myocardial-like tissue of multiple complexities.

A key challenge to the clinical implementation of decellularized scaffold-based tissue engineering lies in understanding the process of removing cells and immunogenic material from a donor tissue/organ while maintaining the biochemical and biophysical properties of the scaffold that will promote growth of newly seeded cells. Current criteria for evaluating whole organ decellularization are primarily based on nucleic acids, as they are easy to quantify and have been directly correlated to adverse host responses. However, numerous proteins cause immunogenic responses and thus should be measured directly to further understand and quantify the efficacy of decellularization. In addition, there has been increasing appreciation for the role of the various protein components of the extracellular matrix (ECM) in directing cell growth and regulating organ function. We performed in-depth proteomic analysis on four types of biological scaffolds and identified a large number of both remnant cellular and ECM proteins. Measurements of individual protein abundances during the decellularization process revealed significant removal of numerous cellular proteins, but preservation of most structural matrix proteins. The observation that decellularized scaffolds still contain many cellular proteins, although at decreased abundance, indicates that elimination of DNA does not assure adequate removal of all cellular material. Thus, proteomic analysis provides crucial characterization of the decellularization process to create biological scaffolds for future tissue/organ replacement therapies.