Spontaneous reversal of stenosis in tissue-engineered vascular grafts.

We developed a tissue-engineered vascular graft (TEVG) for use in children and present results of a U.S. Food and Drug Administration (FDA)-approved clinical trial evaluating this graft in patients with single-ventricle cardiac anomalies. The TEVG was used as a Fontan conduit to connect the inferior vena cava and pulmonary artery, but a high incidence of graft narrowing manifested within the first 6 months, which was treated successfully with angioplasty. To elucidate mechanisms underlying this early stenosis, we used a data-informed, computational model to perform in silico parametric studies of TEVG development. The simulations predicted early stenosis as observed in our clinical trial but suggested further that such narrowing could reverse spontaneously through an inflammation-driven, mechano-mediated mechanism. We tested this unexpected, model-generated hypothesis by implanting TEVGs in an ovine inferior vena cava interposition graft model, which confirmed the prediction that TEVG stenosis resolved spontaneously and was typically well tolerated. These findings have important implications for our translational research because they suggest that angioplasty may be safely avoided in patients with asymptomatic early stenosis, although there will remain a need for appropriate medical monitoring. The simulations further predicted that the degree of reversible narrowing can be mitigated by altering the scaffold design to attenuate early inflammation and increase mechano-sensing by the synthetic cells, thus suggesting a new paradigm for optimizing next-generation TEVGs. We submit that there is considerable translational advantage to combined computational-experimental studies when designing cutting-edge technologies and their clinical management.

Factors Influencing Poor Outcomes in Synthetic Tissue-Engineered Tracheal Replacement.

OBJECTIVES: Humans receiving tissue-engineered tracheal grafts have experienced poor outcomes ultimately resulting in death or the need for graft explantation. We assessed the performance of the synthetic scaffolds used in humans with an ovine model of orthotopic tracheal replacement, applying standard postsurgical surveillance and interventions to define the factors that contributed to the complications seen at the bedside. STUDY DESIGN: Large animal model. SETTING: Pediatric academic research institute. SUBJECTS AND METHODS: Human scaffolds were manufactured with an electrospun blend of polyethylene terephthalate and polyurethane reinforced with polycarbonate rings. They were seeded with autologous bone marrow-derived mononuclear cells and implanted in sheep. Animals were evaluated with routine bronchoscopy and fluoroscopy. Endoscopic dilation and stenting were performed to manage graft stenosis for up to a 4-month time point. Grafts and adjacent native airway were sectioned and evaluated with histology and immunohistochemistry. RESULTS: All animals had signs of graft stenosis. Three of 5 animals (60%) designated for long-term surveillance survived until the 4-month time point. Graft dilation and stent placement resolved respiratory symptoms and prolonged survival. Necropsy demonstrated evidence of infection and graft encapsulation. Granulation tissue with signs of neovascularization was seen at the anastomoses, but epithelialization was never observed. Acute and chronic inflammation of the native airway epithelium was observed at all time points. Architectural changes of the scaffold included posterior wall infolding and scaffold delamination. CONCLUSIONS: In our ovine model, clinically applied synthetic tissue-engineered tracheas demonstrated infectious, inflammatory, and mechanical failures with a lack of epithelialization and neovascularization.

Mouse Model of Tracheal Replacement With Electrospun Nanofiber Scaffolds.

OBJECTIVES: The clinical experience with tissue-engineered tracheal grafts (TETGs) has been fraught with graft stenosis and delayed epithelialization. A mouse model of orthotopic replacement that recapitulates the clinical findings would facilitate the study of the cellular and molecular mechanisms underlying graft stenosis. METHODS: Electrospun nanofiber tracheal scaffolds were created using nonresorbable (polyethylene terephthalate + polyurethane) and co-electrospun resorbable (polylactide-co-caprolactone/polyglycolic acid) polymers (n = 10/group). Biomechanical testing was performed to compare load displacement of nanofiber scaffolds to native mouse tracheas. Mice underwent orthotopic tracheal replacement with syngeneic grafts (n = 5) and nonresorbable (n = 10) and resorbable (n = 10) scaffolds. Tissue at the anastomosis was evaluated using hematoxylin and eosin (H&E), K5+ basal cells were evaluated with the help of immunofluorescence testing, and cellular infiltration of the scaffold was quantified. Micro computed tomography was performed to assess graft patency and correlate radiographic and histologic findings with respiratory symptoms. RESULTS: Synthetic scaffolds were supraphysiologic in compression tests compared to native mouse trachea ( P < .0001). Nonresorbable scaffolds were stiffer than resorbable scaffolds ( P = .0004). Eighty percent of syngeneic recipients survived to the study endpoint of 60 days postoperatively. Mean survival with nonresorbable scaffolds was 11.40 ± 7.31 days and 6.70 ± 3.95 days with resorbable scaffolds ( P = .095). Stenosis manifested with tissue overgrowth in nonresorbable scaffolds and malacia in resorbable scaffolds. Quantification of scaffold cellular infiltration correlated with length of survival in resorbable scaffolds (R2 = 0.95, P = .0051). Micro computed tomography demonstrated the development of graft stenosis at the distal anastomosis on day 5 and progressed until euthanasia was performed on day 11. CONCLUSION: Graft stenosis seen in orthotopic tracheal replacement with synthetic tracheal scaffolds can be modeled in mice. The wide array of lineage tracing and transgenic mouse models available will permit future investigation of the cellular and molecular mechanisms underlying TETG stenosis.

Quantification of tissue-engineered trachea performance with computational fluid dynamics.

OBJECTIVES/HYPOTHESIS: Current techniques for airway characterization include endoscopic or radiographic measurements that produce static, two-dimensional descriptions. As pathology can be multilevel, irregularly shaped, and dynamic, minimal luminal area (MLA) may not provide the most comprehensive description or diagnostic metric. Our aim was to examine the utilization of computational fluid dynamics (CFD) for the purpose of defining airway stenosis using an ovine model of tissue-engineered tracheal graft (TETG) implantation. STUDY DESIGN: Animal research model. METHODS: TETGs were implanted into sheep, and MLA was quantified with imaging and endoscopic measurements. Graft stenosis was managed with endoscopic dilation and stenting when indicated. Geometries of the TETG were reconstructed from three-dimensional fluoroscopic images. CFD simulations were used to calculate peak flow velocity (PFV) and peak wall shear stress (PWSS). These metrics were compared to values derived from a quantitative respiratory symptom score. RESULTS: Elevated PFV and PWSS derived from CFD modeling correlated with increased respiratory symptoms. Immediate pre- and postimplantation CFD metrics were similar, and implanted sheep were asymptomatic. Respiratory symptoms improved with stenting, which maintained graft architecture similar to dilation procedures. With stenting, baseline PFV (0.33 m/s) and PWSS (0.006 Pa) were sustained for the remainder of the study. MLA measurements collected via bronchoscopy were also correlated with respiratory symptoms. PFV and PWSS found via CFD were correlated (R2 = 0.92 and 0.99, respectively) with respiratory symptoms compared to MLA (R2 = 0.61). CONCLUSIONS: CFD is valid for informed interventions based on multilevel, complex airflow and airway characteristics. Furthermore, CFD may be used to evaluate TETG functionality. LEVEL OF EVIDENCE: NA. Laryngoscope, E272-E279, 2018.

The Heart and Great Vessels.

Cardiovascular disease is the leading cause of mortality worldwide. We have made large strides over the past few decades in management, but definitive therapeutic options to address this health-care burden are still limited. Given the ever-increasing need, much effort has been spent creating engineered tissue to replaced diseased tissue. This article gives a general overview of this work as it pertains to the development of great vessels, myocardium, and heart valves. In each area, we focus on currently studied methods, limitations, and areas for future study.

Designing a tissue-engineered tracheal scaffold for preclinical evaluation.

OBJECTIVE: Recent efforts to tissue engineer long-segment tracheal grafts have been complicated by stenosis and malacia. It has been proposed that both the mechanical characteristics and cell seeding capacity of TETG scaffolds are integral to graft performance. Our aim was to design a tracheal construct that approximates the biomechanical properties of native sheep trachea and optimizes seeding with bone marrow derived mononuclear cells prior to preclinical evaluation in an ovine model. METHODS: A solution of 8% polyethylene terephthalate (PET) and 3% polyurethane (PU) was prepared at a ratio of either 8:2 or 2:8 and electrospun onto a custom stainless steel mandrel designed to match the dimensional measurements of the juvenile sheep trachea. 3D-printed porous or solid polycarbonate C-shaped rings were embedded within the scaffolds during electrospinning. The scaffolds underwent compression testing in the anterior-posterior and lateral-medial axes and the biomechanical profiles compared to that of a juvenile ovine trachea. The most biomimetic constructs then underwent vacuum seeding with ovine bone marrow derived mononuclear cells. Fluorometric DNA assay was used to quantify scaffold seeding. RESULTS: Both porous and solid rings approximated the biomechanics of the native ovine trachea, but the porous rings were most biomimetic. The load-displacement curve of scaffolds fabricated from a ratio of 2:8 PET:PU most closely mimicked that of native trachea in the anterior-posterior and medial-lateral axes. Solid C-ringed scaffolds had a greater cell seeding efficiency when compared to porous ringed scaffolds (Solid: 19 × 104 vs. Porous: 9.6 × 104 cells/mm3, p = 0.0098). CONCLUSION: A long segment tracheal graft composed of 2:8 PET:PU with solid C-rings approximates the biomechanics of the native ovine trachea and demonstrates superior cell seeding capacity of the two prototypes tested. Further preclinical studies using this graft design in vivo would inform the rational design of an optimal TETG scaffold.

Near Complete Obliteration of the Left Hemithorax by Congenital Lobar Emphysema in an Adult.

Congenital lobar emphysema is a rare pulmonary malformation typically diagnosed during infancy and is characterized by bullous disease. A 28-year-old woman, who presented with 1 week of progressive dyspnea and chest pain, was found to have left hemithoracic lung hyperinflation with perfusion deficit upon radiographic evaluation. Bullous disease was found intraoperatively to originate from 1 lower lobe segment. Normal parenchymal lung expansion occurred following resection of the affected segment. Therefore, we present a rare case of congenital lobar emphysema resulting in near-complete occupancy of the left hemithorax and diagnosed in a previously asymptomatic adult without history of predisposing factors.

Endoscopic management of tissue-engineered tracheal graft stenosis in an ovine model.

OBJECTIVE: To evaluate the safety and efficacy of bronchoscopic interventions in the management of tissue-engineered tracheal graft (TETG) stenosis. STUDY DESIGN: Animal research study. METHODS: TETGs were constructed with seeded autologous bone marrow-derived mononuclear cells on a bioartificial graft. Eight sheep underwent tracheal resection and orthotopic implantation of this construct. Animals were monitored by bronchoscopy and fluoroscopy at 3 weeks, 6 weeks, 3 months, and 4 months. Bronchoscopic interventions, including dilation and stenting, were performed to manage graft stenosis. Postdilation measurements were obtained endoscopically and fluoroscopically. RESULTS: Seven dilations were performed in six animals. At the point of maximal stenosis, the lumen measured 44.6 ± 8.4 mm2 predilation and 50.7 ± 14.1 postdilation by bronchoscopy (P = 0.3517). By fluoroscopic imaging, the airway was 55.9 ± 12.9 mm2 predilation and 65.9 ± 22.4 mm2 postdilation (P = 0.1303). Stents were placed 17 times in six animals. Pre- and poststenting lumen sizes were 62.8 ± 38.8 mm2 and 80.1 ± 54.5 mm2 by bronchoscopy (P = 0.6169) and 77.1 ± 38.9 mm2 and 104 ± 60.7 mm2 by fluoroscopy (P = 0.0825). Mortality after intervention was 67% with dilation and 0% with stenting (P = 0.0004). The average days between bronchoscopy were 8 ± 2 for the dilation group and 26 ± 17 in the stenting group (P = 0.05). One hundred percent of dilations and 29% of stent placements required urgent follow-up bronchoscopy (P = 0.05). CONCLUSION: Dilation has limited efficacy for managing TETG stenosis, whereas stenting has a more lasting clinical effect. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:2219-2224, 2017.

Objective characterization of airway dimensions using image processing.

OBJECTIVES: With the evolution of medical and surgical management for pediatric airway disorders, the development of easily translated techniques of measuring airway dimensions can improve the quantification of outcomes of these interventions. We have developed a technique that improves the ability to characterize endoscopic airway dimensions using common bronchoscopic equipment and an open-source image-processing platform. METHODS: We validated our technique of Endoscopic Airway Measurement (EAM) using optical instruments in simulation tracheas. We then evaluated EAM in a large animal model (Ovis aries, n = 5), comparing tracheal dimensions obtained with EAM to measurements obtained via 3-D fluoroscopic reconstruction. The animal then underwent resection of the measured segment, and direct measurement of this segment was performed and compared to radiographic measurements and those obtained using EAM. RESULTS: The simulation tracheas had a direct measurement of 13.6, 18.5, and 24.2 mm in diameter. The mean difference of diameter in simulation tracheas between direct measurements and measurements obtained using EAM was 0.70 ± 0.57 mm. The excised ovine tracheas had an average diameter of 18.54 ± 0.68 mm. The percent difference in diameter obtained from EAM and from 3-D fluoroscopic reconstruction when compared to measurement of the excised tracheal segment was 4.98 ± 2.43% and 10.74 ± 4.07% respectively. Comparison of these three measurements (EAM, measurement of resected trachea, 3-D fluoroscopic reconstruction) with repeated measures ANOVA demonstrated no statistical significance. CONCLUSIONS: Endoscopic airway measurement (EAM) provides equivalent measurements of the airway with the improved versatility of measuring non-circular and multi-level dimensions. Using optical bronchoscopic instruments and open-source image-processing software, our data supports preclinical and clinical translation of an accessible technique to provide objective quantification of airway diameter.

Hyperglycemic Ins2AkitaLdlr⁻/⁻ mice show severely elevated lipid levels and increased atherosclerosis: a model of type 1 diabetic macrovascular disease.

Accelerated atherosclerosis is the leading cause of death in type 1 diabetes, but the mechanism of type 1 diabetes-accelerated atherosclerosis is not well understood, in part due to the lack of a good animal model for the long-term studies required. In an attempt to create a model for studying diabetic macrovascular disease, we have generated type 1 diabetic Akita mice lacking the low density lipoprotein receptor (Ins2(Akita)Ldlr⁻/⁻). Ins2(Akita)Ldlr⁻/⁻ mice were severely hyperglycemic with impaired glucose tolerance. Compared with Ldlr⁻/⁻ mice, 20-week-old Ins2(Akita)Ldlr⁻/⁻ mice fed a 0.02% cholesterol AIN76a diet showed increased plasma triglyceride and cholesterol levels, and increased aortic root cross-sectional atherosclerotic lesion area [224% (P < 0.001) in males and 30% (P < 0.05) in females]. Microarray and quantitative PCR analyses of livers from Ins2(Akita)Ldlr⁻/⁻ mice revealed altered expression of lipid homeostatic genes, including sterol-regulatory element binding protein (Srebp)1, liver X receptor (Lxr)α, Abca1, Cyp7b1, Cyp27a1, and Lpl, along with increased expression of pro-inflammatory cytokine genes, including interleukin (Il)1α, Il1ß, Il2, tumor necrosis factor (Tnf)α, and Mcp1. Immunofluorescence staining showed that the expression levels of Mcp1, Tnfα, and Il1ß were also increased in the atherosclerotic lesions and artery walls of Ins2(Akita)Ldlr⁻/⁻ mice. Thus, the Ins2(Akita)Ldlr⁻/⁻ mouse appears to be a promising model for mechanistic studies of type 1 diabetes-accelerated atherosclerosis.

Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.

The nuclear hormone receptor pregnane X receptor (PXR; also called SXR) functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Although many clinically relevant PXR ligands have been shown to affect cholesterol levels, the role of PXR in cholesterol homeostasis and atherosclerosis has not been thoroughly investigated. Here, we report that activation of PXR by feeding the PXR agonist pregnenolone 16alpha-carbonitrile (0.02%) for 2 weeks to wild-type (WT) mice significantly increased total cholesterol levels and atherogenic lipoproteins VLDL and LDL levels, but had no effect in PXR knockout (PXR(-/-)) mice. Chronic PXR activation in atherosclerosis prone apolipoprotein E deficient (ApoE(-/-)) mice was found to decrease HDL levels and increase atherosclerotic cross-sectional lesion area at both the aortic root and in the brachiocephalic artery by 54% (P < 0.001) and 116% (P < 0.01), respectively. PXR activation significantly regulated genes in the liver involved in lipoprotein transportation and cholesterol metabolism, including CD36, ApoA-IV, and CYP39A1, in both WT and ApoE(-/-) mice. Furthermore, PXR activation can increase CD36 expression and lipid accumulation in peritoneal macrophages of ApoE(-/-) mice. In summary, PXR activation in WT mice increases levels of the atherogenic lipoproteins VLDL and LDL, whereas in ApoE(-/-) mice, PXR increases atherosclerosis, perhaps by diminishing levels of the antiatherogenic ApoA-IV and increasing lipid accumulation in macrophages.