Fusobacterium nucleatum subsp. polymorphum recovered from malignant and potentially malignant oral disease exhibit heterogeneity in adhesion phenotypes and adhesin gene copy number, shaped by inter-subspecies horizontal gene transfer and recombination-derived mosaicism.

Fusobacterium nucleatum is an anaerobic commensal of the oral cavity associated with periodontitis and extra-oral diseases, including colorectal cancer. Previous studies have shown an increased relative abundance of this bacterium associated with oral dysplasia or within oral tumours. Using direct culture, we found that 75 % of Fusobacterium species isolated from malignant or potentially malignant oral mucosa were F. nucleatum subsp. polymorphum. Whole genome sequencing and pangenome analysis with Panaroo was carried out on 76 F. nucleatum subsp. polymorphum genomes. F. nucleatum subsp. polymorphum was shown to possesses a relatively small core genome of 1604 genes in a pangenome of 7363 genes. Phylogenetic analysis based on the core genome shows the isolates can be separated into three main clades with no obvious genotypic associations with disease. Isolates recovered from healthy and diseased sites in the same patient are generally highly related. A large repertoire of adhesins belonging to the type V secretion system (TVSS) could be identified with major variation in repertoire and copy number between strains. Analysis of intergenic recombination using fastGEAR showed that adhesin complement is shaped by horizontal gene transfer and recombination. Recombination events at TVSS adhesin genes were not only common between lineages of subspecies polymorphum, but also between different subspecies of F. nucleatum. Strains of subspecies polymorphum with low copy numbers of TVSS adhesin encoding genes tended to have the weakest adhesion to oral keratinocytes. This study highlights the genetic heterogeneity of F. nucleatum subsp. polymorphum and provides a new framework for defining virulence in this organism.

Radial Scars on Screening Digital Breast Tomosynthesis: Upstaging Rates and Management Strategies.

BACKGROUND. Radial scars are more commonly identified on digital breast tomosynthesis (DBT) than on digital mammography (DM). Nonetheless, universal guidelines for radial scar management in the current era of DBT are lacking. OBJECTIVE. The purpose of this study was to determine the upstaging rates of screening DBT-detected radial scars with and without atypia and to identify features related to upstaging risk. METHODS. This retrospective study included patients who underwent core needle biopsy (CNB) showing a radial scar after screening DBT and DM from January 1, 2013, to December 31, 2020. Patients without surgical excision or at least 2 years of imaging follow-up after CNB were excluded. Rates of upstaging to breast cancer (ductal carcinoma in situ [DCIS] or invasive disease) were compared between radial scars with and without atypia at CNB. Associations of upstaging with patient, imaging, and pathologic variables were explored using standard statistical tests. RESULTS. Of 165 women with 171 radial scars, the final study sample included 153 women (mean age, 56 years; range, 33-83 years) with 159 radial scars that underwent surgical excision (80.5%, 128/159) or at least 2 years of imaging follow-up (19.5%, 31/159). Seven radial scars were upstaged to DCIS and one to invasive disease. Therefore, the up-staging rate of radial scars to cancer was 5.0% (8/159). The upstaging rate of radial scars without atypia at CNB was 1.6% (2/129) and that of radial scars with atypia was 20.0% (6/30) (p < .001). On multivariable analysis, features associated with higher upstaging risk included a prior breast cancer diagnosis (62.5% vs 4.8%; p = .01) and the presence of atypia at CNB (75.0% vs 15.9%; p = .02). The upstaging rate according to mammographic finding type was 7.1% (1/14) for asymmetries, 12.5% (2/16) for masses, 5.3% (5/95) for architectural distortion, and 0.0% (0/34) for calcifications. CONCLUSION. Screening-detected radial scars without atypia at CNB have a low upstaging rate to breast cancer of 1.6%. CLINICAL IMPACT. Imaging surveillance rather than surgery is a reasonable approach for radial scars without atypia, particularly for those presenting as calcifications.

Analytic Morphomics in Myositis-Related Interstitial Lung Disease.

PURPOSE: Interstitial lung disease (ILD) is the most common non-musculoskeletal manifestation of idiopathic inflammatory myopathies (IIM). Identification of body composition change may enable early intervention to improve prognosis. We investigated muscle quantity and quality derived from cross-sectional imaging in IIM, and its relationship to ILD severity. METHODS: A retrospective cohort study assessing IIM of ILD patients (n = 31) was conducted. Two datasets separated in time were collected, containing demographics, biochemical data, pulmonary function testing and thoracic CT data. Morphomic analysis of muscle quantity (cross-sectional area) and quality (density in Hounsfield Units) on thoracic CT were analysed utilising a web-based tool allowing segmentation of muscle and fat. Bilateral erector spinae and pectoralis muscle (ESM&PM) were measured at defined vertebral levels. RESULTS: FVC and DLCO decreased but within acceptable limits of treatment response (FVC: 83.7-78.7%, p < 0.05, DLCO 63.4-60.6%, p < 0.05). The cross-sectional area of the PM and ESM increased (PM: 39.8 to 40.7 cm2, p = 0.491; ESM: 35.2 to 39.5 cm2, p = 0.098). Density significantly fell for both the PM and ESM (PM: 35.3-31 HU, p < 0.05; ESM: 38-33.7, p < 0.05). Subcutaneous fat area increased from 103.9 to 136.1 cm2 (p < 0.05), while the visceral fat area increased but not reaching statistical significance. The change in PM density between time points demonstrated an inverse correlation with DLCO (p < 0.05, R = - 0.49). CONCLUSION: Patients with IIM ILD demonstrated significant body composition changes on CT imaging unlikely to be detected by traditional measurement tools. An increase in muscle area with an inverse decrease in density suggests poor muscle quality.

Replacing Plain Radiograph with ultra-low dose CT thorax in cystic fibrosis (CF) in the era of CFTR modulation and its impact on cumulative effective dose.

BACKGROUND: Medical radiation exposure is of increasing concern in patients with cystic fibrosis (PWCF) due to improving life expectancy. We aimed to assess and quantify the cumulative effective dose (CED) in PWCF in the context of CFTR-modulator therapy and the advancement of dose reduction techniques. METHODS: We performed a retrospective observational study in a single University CF centre over a 11-year period. We included PWCF, aged over 18 years who exclusively attended our institution. Relevant clinical data (demographics, transplantation history and modulator status) and radiological data (modality, quantity, and radiation exposure measured as CED) were collected. For those on modulator therapy the quantified imaging and radiation data was dichotomised into pre-and-post therapy periods. RESULTS: The study included 181 patients: 139 on CFTR modulator therapy, 15 transplant recipients and 27 with neither exposure. 82% of patients received <25 mSv over the study period. Mean study duration was 6.9 ± 2.6 years pre-modulation and 4.2 ± 2.6 years post-modulation. Pre-modulation CT contributed 9.6% of total chest imaging (n = 139/1453) and 70.9% of the total CED. Post-modulation CT use increased contributing 42.7% of chest imaging (n = 444/1039) and comprised 75.8% of CED. Annual CED was 1.55 mSv pre and 1.36 mSv post modulation (p = 0.41). Transplant recipients had an annual CED of 64 ± 36.1mSv. CONCLUSION: Chest CT utilisation for PWCF is rising in our institution, replacing chest radiography amidst CFTR-modulation. Despite the increasing use of CT, no significant radiation dose penalty was observed with a reduction in mean annual CED, primarily due to the influence of CT dose reduction strategies.

Salinity and temperature affect the symbiont profile and host condition of Florida USA blue crabs Callinectes sapidus.

Subtropical Florida blue crabs, Callinectes sapidus, exhibit differing life history traits compared to their temperate counterparts, likely influencing symbiont infection dynamics. Little information exists for Florida C. sapidus symbiont profiles, their distribution among various habitats, and influence on crab condition. Using histopathology, genomics, and transmission electron microscopy, we describe the first symbiont profiles for Florida C. sapidus occupying freshwater to marine habitats. Twelve symbiont groups were identified from 409 crabs including ciliophorans, digenean, microsporidian, Haplosporidia, Hematodinium sp., Nematoda, filamentous bacteria, gregarine, Callinectes sapidus nudivirus, Octolasmis sp., Cambarincola sp., and putative microcell. Overall, 78% of C. sapidus were documented with one or more symbiont groups demonstrating high infection rates in wild populations. Environmental variables water temperature and salinity explained 48% of the variation in symbiont groups among Florida habitats, and salinity was positively correlated with C. sapidus symbiont diversity. This suggests freshwater C. sapidus possess fewer symbionts and represent healthier individuals compared to saltwater populations. Crab condition was examined using the reflex action mortality predictor (RAMP) to determine if reflex impairment could be linked to symbiont prevalence. Symbionts were found positively correlated with crab condition, and impaired crabs were more likely to host symbionts, demonstrating symbiont inclusion may boost predictive ability of the RAMP application. The microsporidian symbiont group had a particularly strong effect on C. sapidus reflex response, and impairment was on average 1.57 times higher compared to all other symbiont groups. Our findings demonstrate the importance of considering full symbiont profiles and their associations with a spatially and temporally variable environment to fully assess C. sapidus population health.

Introduction of a daily peer learning process with added value for faculty and trainees.

OBJECTIVE: To implement a new daily peer learning (PL) conference which incorporates faculty and trainees within the abdominal imaging division of a large academic medical center, and to determine participants' level of satisfaction and preference over the pre-existing peer review (PR) model. METHODS: We replaced our pre-existing PR-based tool with a daily hour-long case-based PL teaching conference over a 3-month pilot period. Faculty and trainees were surveyed about their experience at the end of the pilot period. RESULTS: A total of 711 cases were logged during the pilot period (median 9 cases per day). We received 30 survey responses from a total of 48 eligible participants. Survey responses from both faculty and trainees on the new PL conference were overwhelmingly favorable, including unanimous support for permanently replacing the existing PR tool with the new PL conference. CONCLUSION: Our successful pilot of a daily PL conference replacing an existing PR tool adds to the growing body of evidence of radiologists strongly supporting PL based processes over PR. Our paradigm of actively involving trainees within the process can serve as a model for other institutions.

An evaluation of information online on artificial intelligence in medical imaging.

BACKGROUND: Opinions seem somewhat divided when considering the effect of artificial intelligence (AI) on medical imaging. The aim of this study was to characterise viewpoints presented online relating to the impact of AI on the field of radiology and to assess who is engaging in this discourse. METHODS: Two search methods were used to identify online information relating to AI and radiology. Firstly, 34 terms were searched using Google and the first two pages of results for each term were evaluated. Secondly, a Rich Search Site (RSS) feed evaluated incidental information over 3 weeks. Webpages were evaluated and categorized as having a positive, negative, balanced, or neutral viewpoint based on study criteria. RESULTS: Of the 680 webpages identified using the Google search engine, 248 were deemed relevant and accessible. 43.2% had a positive viewpoint, 38.3% a balanced viewpoint, 15.3% a neutral viewpoint, and 3.2% a negative viewpoint. Peer-reviewed journals represented the most common webpage source (48%), followed by media (29%), commercial sources (12%), and educational sources (8%). Commercial webpages had the highest proportion of positive viewpoints (66%). Radiologists were identified as the most common author group (38.9%). The RSS feed identified 177 posts of which were relevant and accessible. 86% of posts were of media origin expressing positive viewpoints (64%). CONCLUSION: The overall opinion of the impact of AI on radiology presented online is a positive one. Consistency across a range of sources and author groups exists. Radiologists were significant contributors to this online discussion and the results may impact future recruitment.

Mindfulness, happiness, and anxiety in a sample of college students before and after taking a meditation course.

OBJECTIVE: To explore differences in mindfulness, happiness, and perceived anxiety in a sample of college students before and after taking a meditation course. PARTICIPANTS: Participants were college students at a primarily undergraduate institution enrolled in an experiential meditation class (n = 74) and a non-meditation class comparison group (n = 73). METHODS: The study design was a before-after observational study with two groups and three dependent variables: the Mindfulness Attention Awareness Scale, the State Trait Anxiety Inventory and the Subjective Happiness Scale. RESULTS: Students in the meditation course increased average subjective happiness and mindfulness attention awareness. Mindfulness scores increased and anxiety decreased more for students in the meditation class compared to students in the psychosocial class. CONCLUSIONS: This research provides evidence that taking a semester long meditation course is associated with improvements in college student well-being.

Extending Johnson's Typology: Additional Manifestations of Dating Violence and Coercive Control.

In this study, we explored patterns of violence and coercive control in young adult dating relationships by testing and extending Johnson's typology of intimate partner violence. Young adults (N = 398) between 18 and 27 years old completed an online survey about experiences of violence and coercive control in current and past dating relationships. Using cluster analysis, we classified relationships as no/low coercive control and high coercive control. We then categorized relationship types according to Johnson's typology using the coercive control clusters and the absence/presence of violence. In total, 35% of relationships were abusive (i.e., violent and/or high coercive control), with 24% of all reported relationships including violence with and without high coercive control, and 11% including nonviolent coercive control. Among violent relationships, situational couple violence was more common than other types of dating violence, and two additional types of violence were found: (a) violence toward a nonviolent coercive controlling partner and (b) nonviolent coercive control toward an intimate terrorist, both of which are potentially types of resistance distinct from Johnson's concept of violent resistance. Additionally, victims of intimate terrorism and victims of nonviolent coercive control were significantly more fearful of their partners than victims of situational couple violence, and victims of situational couple violence did not differ in their fear of partners compared to respondents in nonabusive relationships. These findings identify additional abusive relationship types and elucidate the importance of extending Johnson's typology to more comprehensively capture the complex dynamics of coercive control and/or violence in young adult dating relationships.

The evolving role of radiological imaging in cystic fibrosis.

PURPOSE OF REVIEW: Radiological imaging has a crucial role in pulmonary evaluation in cystic fibrosis (CF), having been shown to be more sensitive than pulmonary function testing at detecting structural lung changes. The present review summarizes the latest published information on established and evolving pulmonary imaging techniques for assessing people with this potentially life-limiting disorder. RECENT FINDINGS: Chest computed tomography (CT) has taken over the predominant role of chest radiography in many centres for the initial assessment and surveillance of CF lung disease. However, several emerging techniques offer a promising means of pulmonary imaging using less ionizing radiation. This is of particular importance given these patients tend to require repeated imaging throughout their lives from a young age. Such techniques include ultra-low-dose CT, tomosynthesis, dynamic radiography and magnetic resonance imaging. In addition, deep-learning algorithms are anticipated to improve diagnostic accuracy. SUMMARY: The recent introduction of triple-combination CF transmembrane regulator therapy has put further emphasis on the need for sensitive methods of monitoring treatment response to allow for early adaptation of treatment regimens in order to limit irreversible lung damage. Further research is needed to establish how emerging imaging techniques can contribute to this safely and effectively.

Paradoxical scintigraphy bone scan findings with malignancy-associated extreme hypercalcemia.

We report the first case of extreme hypercalcemia (Ca 2+ >6.0 mmol/L) as the initial presentation of de novo metastatic breast cancer. Following treatment and stabilization of the patient, imaging revealed a large breast mass and widespread osseous metastases. Whole body bone scintigraphy demonstrated significant extra osseous uptake of radiotracer in the lungs, liver, and kidneys-a rare phenomenon secondary to profound hypercalcemia. Biopsy revealed estrogen receptor (ER) positive breast carcinoma, for which the patient was treated.

Extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture.

Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically.

Decellularized Human Gut as a Natural 3D Platform for Research in Intestinal Fibrosis.

BACKGROUND: The current methodologies for the identification of therapeutic targets for inflammatory bowel disease (IBD) are limited to conventional 2-dimensional (2D) cell cultures and animal models. The use of 3D decellularized human intestinal scaffolds obtained from surgically resected intestine and engineered with human intestinal cells may provide a major advancement in the development of innovative intestinal disease models. The aim of the present study was to design and validate a decellularization protocol for the production of acellular 3D extracellular matrix (ECM) scaffolds from the human duodenum. METHODS: Scaffolds were characterized by verifying the preservation of the ECM protein composition and 3D architecture of the native intestine and were employed for tissue engineering with primary human intestinal myofibroblasts for up to 14 days. RESULTS: Engrafted cells showed the ability to grow and remodel the surrounding ECM. mRNA expression of key genes involved in ECM turnover was significantly different when comparing primary human intestinal myofibroblasts cultured in 3D scaffolds with those cultured in standard 2D cultures on plastic dishes. Moreover, incubation with key profibrogenic growth factors such as TGFß1 and PDGF-BB resulted in markedly different effects in standard 2D vs 3D cultures, further emphasizing the importance of using 3D cell cultures. CONCLUSIONS: These results confirm the feasibility of 3D culture of human intestinal myofibroblasts in intestinal ECM scaffolds as an innovative platform for disease modeling, biomarker discovery, and drug testing in intestinal fibrosis.

Non-Invasive Longitudinal Bioluminescence Imaging of Human Mesoangioblasts in Bioengineered Esophagi.

IMPACT STATEMENT: Methodologies for incorporation of cells into tissue-engineered grafts, particularly at the later preclinical stages, are suboptimal and non-validated, and monitoring cell fate within scaffolds cultured in bioreactors and in vivo is challenging. In this study, we demonstrate how bioluminescence imaging (BLI) can overcome these difficulties and allow quantitative cell tracking at multiple stages of the bioengineering preclinical pipeline. Our robust bioluminescence-based approach allowed reproducible longitudinal monitoring of mesoangioblast localization and survival in 2D/3D tissue culture, in organ-scale bioreactors, and in vivo. Our findings will encourage the use of BLI in tissue engineering studies, improving the overall quality of cell-scaffold interaction research.

Cirrhotic Human Liver Extracellular Matrix 3D Scaffolds Promote Smad-Dependent TGF-ß1 Epithelial Mesenchymal Transition.

An altered liver microenvironment characterized by a dysregulated extracellular matrix (ECM) supports the development and progression of hepatocellular carcinoma (HCC). The development of experimental platforms able to reproduce these physio-pathological conditions is essential in order to identify and validate new therapeutic targets for HCC. The aim of this work was to validate a new in vitro model based on engineering three-dimensional (3D) healthy and cirrhotic human liver scaffolds with HCC cells recreating the micro-environmental features favoring HCC. Healthy and cirrhotic human livers ECM scaffolds were developed using a high shear stress oscillation-decellularization procedure. The scaffolds bio-physical/bio-chemical properties were analyzed by qualitative and quantitative approaches. Cirrhotic 3D scaffolds were characterized by biomechanical properties and microarchitecture typical of the native cirrhotic tissue. Proteomic analysis was employed on decellularized 3D scaffolds and showed specific enriched proteins in cirrhotic ECM in comparison to healthy ECM proteins. Cell repopulation of cirrhotic scaffolds highlighted a unique up-regulation in genes related to epithelial to mesenchymal transition (EMT) and TGFß signaling. This was also supported by the presence and release of higher concentration of endogenous TGFß1 in cirrhotic scaffolds in comparison to healthy scaffolds. Fibronectin secretion was significantly upregulated in cells grown in cirrhotic scaffolds in comparison to cells engrafted in healthy scaffolds. TGFß1 induced the phosphorylation of canonical proteins Smad2/3, which was ECM scaffold-dependent. Important, TGFß1-induced phosphorylation of Smad2/3 was significantly reduced and ECM scaffold-independent when pre/simultaneously treated with the TGFß-R1 kinase inhibitor Galunisertib. In conclusion, the inherent features of cirrhotic human liver ECM micro-environment were dissected and characterized for the first time as key pro-carcinogenic components in HCC development.

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors.

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.

Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells.

Liver transplantation is the definitive treatment of liver failure but donor organ shortage limits its availability. Stem cells are highly expandable and have the potential to differentiate into any specialist cell. Use of patient-derived induced Pluripotent Stem Cells (hiPSCs) has the additional advantage for organ regeneration therapies by removing the need for immunosuppression. We compared hepatocyte differentiation of human embryonic stem cells (hESCs) and hiPSCs in a mouse decellularised liver scaffold (3D) with standard in vitro protocol (2D). Mouse livers were decellularised preserving micro-architecture, blood vessel network and extracellular matrix. hESCs and hiPSCs were primed towards the definitive endoderm. Cells were then seeded either in 3D or 2D cultures and the hepatocyte differentiation was continued. Both hESCs and hiPSCs differentiated more efficiently in 3D than in 2D, with higher and earlier expression of mature hepatocyte marker albumin, lipid and glycogen synthesis associated with a decrease in expression of fetal hepatocyte marker alpha-fetoprotein. Thus we conclude that stem cell hepatocyte differentiation in 3D culture promotes faster cell maturation. This finding suggests that optimised 3D protocols could allow generation of mature liver cells not achieved so far in standard 2D conditions and lead to improvement in cell models of liver disease and regenerative medicine applications.

A comparison of tracheal scaffold strategies for pediatric transplantation in a rabbit model.

OBJECTIVES/HYPOTHESIS: Despite surgical advances, childhood tracheal stenosis is associated with high morbidity and mortality. Various tracheal scaffold strategies have been developed as the basis for bioengineered substitutes, but there is no consensus on which may be superior in vivo. We hypothesized that there would be no difference in morbidity and mortality between three competing scaffold strategies in rabbits. STUDY DESIGN: Pilot preclinical study. METHODS: Tracheal scaffolds were prepared by three methods that have been applied clinically and reported: preserved cadaveric ("Herberhold") allografts, detergent-enzymatically decellularized allografts, and synthetic scaffolds (nanocomposite polymer [polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU)]). Scaffolds were implanted into cervical trachea of New Zealand White rabbits (n = 4 per group) without cell seeding. Control animals (n = 4) received autotransplanted tracheal segments using the same technique. Animals underwent bronchoscopic monitoring of the grafts for 30 days. Macroscopic evaluation of tissue integration, graft stenosis, and collapsibility and histological examinations were performed on explants at termination. RESULTS: All surgical controls survived to termination without airway compromise. Mild to moderate anastomotic stenosis from granulation tissue was detected, but there was evidence suggestive of vascular reconnection with minimal fibrous encapsulation. In contrast, three of the four animals in the Herberhold and POSS-PCU groups, and all animals receiving decellularized allografts, required early termination due to respiratory distress. Herberhold grafts showed intense inflammatory reactions, anastomotic stenoses, and mucus plugging. Synthetic graft integration and vascularization were poor, whereas decellularized grafts demonstrated malacia and collapse but had features suggestive of vascular connection or revascularization. CONCLUSIONS: There are mirror-image benefits and drawbacks to nonrecellularized, decellularized, and synthetic grafts, such that none emerged as the preferred option. Results from prevascularized and/or cell-seeded grafts (as applied clinically) may elucidate clearer advantages of one scaffold type over another. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E449-E457, 2017.

Tracheal Replacement Therapy with a Stem Cell-Seeded Graft: Lessons from Compassionate Use Application of a GMP-Compliant Tissue-Engineered Medicine.

Tracheal replacement for the treatment of end-stage airway disease remains an elusive goal. The use of tissue-engineered tracheae in compassionate use cases suggests that such an approach is a viable option. Here, a stem cell-seeded, decellularized tissue-engineered tracheal graft was used on a compassionate basis for a girl with critical tracheal stenosis after conventional reconstructive techniques failed. The graft represents the first cell-seeded tracheal graft manufactured to full good manufacturing practice (GMP) standards. We report important preclinical and clinical data from the case, which ended in the death of the recipient. Early results were encouraging, but an acute event, hypothesized to be an intrathoracic bleed, caused sudden airway obstruction 3 weeks post-transplantation, resulting in her death. We detail the clinical events and identify areas of priority to improve future grafts. In particular, we advocate the use of stents during the first few months post-implantation. The negative outcome of this case highlights the inherent difficulties in clinical translation where preclinical in vivo models cannot replicate complex clinical scenarios that are encountered. The practical difficulties in delivering GMP grafts underscore the need to refine protocols for phase I clinical trials. Stem Cells Translational Medicine 2017;6:1458-1464.

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds.

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3-8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.