Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.

Tobacco smoking and somatic mutations in human bronchial epithelium.

Tobacco smoking causes lung cancer1-3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6-10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.

Higher throughput drug screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia.

BACKGROUND: Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies. METHODS: We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. RESULTS: Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro. CONCLUSION: Our study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.

Bioengineered airway epithelial grafts with mucociliary function based on collagen IV- and laminin-containing extracellular matrix scaffolds.

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air-liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering.

RATIONALE: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. OBJECTIVES: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. METHODS: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. MEASUREMENTS AND MAIN RESULTS: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. CONCLUSIONS: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

Health-related quality of life following aortopexy for tracheomalacia: a cross-sectional study.

OBJECTIVES: The objective was to measure health-related quality of life (HRQoL) of children following treatment of all-cause tracheomalacia with aortopexy. METHODS: Children ≥5 years and parents of children <18 years who had undergone aortopexy completed the Paediatric Quality of Life Inventory (PedsQL4.0). Scores were compared to published norms. RESULTS: Completed questionnaires were received from 35 parents (65%) and 10 children (38%). Median age at aortopexy was 9.8 months (1 month-12.7 years) and median years of follow-up was 2.6 (4 months-6.9 years). Children who completed questionnaires had a median age of 8.4 (5.7-13.4) years. Parent and child-reported total PedsQL scores were 69.61 (SD : 19.74), and 63.15 (SD : 20.40) respectively. Half of parents and 80% of children reported scores suggesting poor HRQoL outcomes. Parent-reported total, physical and psycho-social scores were lower than those of healthy children and those with acute illness but comparable to children with chronic health conditions and cardiovascular disease. Similarly, children themselves reported comparable total scores to children with chronic illness but child-reported psycho-social scores were lower in the aortopexy group than any other group. There was no association between PedsQL scores and cause of malacia, age or time since aortopexy. The presence of complex congenital comorbidities had a significant (p < 0.05) impact on HRQoL scores. CONCLUSIONS: Following aortopexy children remain at risk of poor HRQoL, especially those with complex comorbidities. HRQoL reported by both parent and child provides important insight into the lives of children following this procedure. Further longitudinal and qualitative study are required to better understand this complex group.

Age-specific nasal epithelial responses to SARS-CoV-2 infection.

Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection.

Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study.

BACKGROUND: Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up. METHODS: A 12-year-old boy was born with long-segment congenital tracheal stenosis and pulmonary sling. His airway had been maintained by metal stents, but, after failure, a cadaveric donor tracheal scaffold was decellularised. After a short course of granulocyte colony stimulating factor, bone marrow mesenchymal stem cells were retrieved preoperatively and seeded onto the scaffold, with patches of autologous epithelium. Topical human recombinant erythropoietin was applied to encourage angiogenesis, and transforming growth factor ß to support chondrogenesis. Intravenous human recombinant erythropoietin was continued postoperatively. Outcomes were survival, morbidity, endoscopic appearance, cytology and proteomics of brushings, and peripheral blood counts. FINDINGS: The graft revascularised within 1 week after surgery. A strong neutrophil response was noted locally for the first 8 weeks after surgery, which generated luminal DNA neutrophil extracellular traps. Cytological evidence of restoration of the epithelium was not evident until 1 year. The graft did not have biomechanical strength focally until 18 months, but the patient has not needed any medical intervention since then. 18 months after surgery, he had a normal chest CT scan and ventilation-perfusion scan and had grown 11 cm in height since the operation. At 2 years follow-up, he had a functional airway and had returned to school. INTERPRETATION: Follow-up of the first paediatric, stem-cell-based, tissue-engineered transplant shows potential for this technology but also highlights the need for further research. FUNDING: Great Ormond Street Hospital NHS Trust, The Royal Free Hampstead NHS Trust, University College Hospital NHS Foundation Trust, and Region of Tuscany.

ß-Catenin determines upper airway progenitor cell fate and preinvasive squamous lung cancer progression by modulating epithelial-mesenchymal transition.

Human lung cancers, including squamous cell carcinoma (SCC) are a leading cause of death and, whilst evidence suggests that basal stem cells drive SCC initiation and progression, the mechanisms regulating these processes remain unknown. In this study we show that ß-catenin signalling regulates basal progenitor cell fate and subsequent SCC progression. In a cohort of preinvasive SCCs we established that elevated basal cell ß-catenin signalling is positively associated with increased disease severity, epithelial proliferation and reduced intercellular adhesiveness. We demonstrate that transgene-mediated ß-catenin inhibition within keratin 14-expressing basal cells delayed normal airway repair while basal cell-specific ß-catenin activation increased cell proliferation, directed differentiation and promoted elements of early epithelial-mesenchymal transition (EMT), including increased Snail transcription and reduced E-cadherin expression. These observations are recapitulated in normal human bronchial epithelial cells in vitro following both pharmacological ß-catenin activation and E-cadherin inhibition, and mirrored our findings in preinvasive SCCs. Overall, the data show that airway basal cell ß-catenin determines cell fate and its mis-expression is associated with the development of human lung cancer.

Use of Simulation to Visualize Healthcare Worker Exposure to Aerosol in the Operating Room.

SUMMARY STATEMENT: Simulation resources offer an opportunity to highlight aerosol dispersion within the operating room environment. We demonstrate our methodology with a supporting video that can offer operating room teams support in their practical understanding of aerosol exposure and the importance of personal protective equipment.

Lessons learned from pre-clinical testing of xenogeneic decellularized esophagi in a rabbit model.

Decellularization of esophagi from several species for tissue engineering is well described, but successful implantation in animal models of esophageal replacement has been challenging. The purpose of this study was to assess feasibility and applicability of esophageal replacement using decellularized porcine esophageal scaffolds in a new pre-clinical model. Following surgical replacement in rabbits with a vascularizing muscle flap, we observed successful anastomoses of decellularized scaffolds, cues of early neovascularization, and prevention of luminal collapse by the use of biodegradable stents. However, despite the success of the surgical procedure, the long-term survival was limited by the fragility of the animal model. Our results indicate that transplantation of a decellularized porcine scaffold is possible and vascular flaps may be useful to provide a vascular supply, but long-term outcomes require further pre-clinical testing in a different large animal model.

Cell-intrinsic differences between human airway epithelial cells from children and adults.

The airway epithelium is a protective barrier that is maintained by the self-renewal and differentiation of basal stem cells. Increasing age is a principle risk factor for chronic lung diseases, but few studies have explored age-related molecular or functional changes in the airway epithelium. We retrieved epithelial biopsies from histologically normal tracheobronchial sites from pediatric and adult donors and compared their cellular composition and gene expression profile (in laser capture-microdissected whole epithelium, fluorescence-activated cell-sorted basal cells, and basal cells in cell culture). Histologically, pediatric and adult tracheobronchial epithelium was similar in composition. We observed age-associated changes in RNA sequencing studies, including higher interferon-associated gene expression in pediatric epithelium. In cell culture, pediatric cells had higher colony formation ability, sustained in vitro growth, and outcompeted adult cells in a direct competitive proliferation assay. Our results demonstrate cell-intrinsic differences between airway epithelial cells from children and adults in both homeostatic and proliferative states.

Subglottic ectopic thymus mimicking airway haemangioma.

Subglottic haemangioma presents as progressive obstruction in the neonatal and infantile airway, with a soft lesion seen during endoscopy. Diagnosis is based on macroscopic findings, biopsy is not usually performed and propranolol is first-line treatment. In contrast, ectopic thymus is a rare differential diagnosis for subglottic mass made by histopathological examination after excision or autopsy. In this article, we present a case of an infant with a subglottic lesion with endoscopic features consistent with haemangioma. After initial clinical response to propranolol, the patient represented with progressive stridor no longer responding to therapy. Open excision of the lesion was performed, and histopathology revealed ectopic thymus tissue. In this case, ectopic thymus tissue mimicked the presentation of subglottic haemangioma, and confirmation bias persisted due to an apparent initial clinical response to treatment with propranolol. In cases of subglottic mass refractory to medical treatment, excision of the lesion should be considered.

Micro-CT Imaging of Pediatric Thyroglossal Duct Cysts: A Prospective Case Series.

Objectives: To determine the feasibility of micro-CT as a high-resolution 3D imaging tool for thyroglossal duct cysts and to evaluate its role augmenting traditional histopathological examination of resected specimens. Methods: A single centre, prospective case series of consecutive children undergoing excision of a thyroglossal duct cyst was performed at a quaternary paediatric referral hospital in the United Kingdom. Consecutive children listed for excision of a thyroglossal duct cyst whose parents agreed to participate were included and there were no exclusion criteria. Results: Surgically excised thyroglossal duct cyst or remnant specimens from five patients (two males, three females) were examined using micro-CT alongside traditional histopathological examination. In all cases, micro-CT imaging was able to demonstrate 3D imaging datasets of the specimens successfully and direct radio-pathological comparisons were made (Figures 1-5, Supplementary Video 1). Conclusions: The study has shown the feasibility and utility of post-operative micro-CT imaging of thyroglossal duct cysts specimens as a visual aid to traditional histopathological examination. It better informs the pathological specimen sectioning using multi-planar reconstruction and volume rendering tools without tissue destruction. In the complex, often arborised relationship between a thyroglossal duct cyst and the hyoid, micro-CT provides valuable image plane orientation and indicates proximity of the duct to the surgical margins. This is the first case series to explore the use of micro-CT imaging for pediatric thyroglossal duct specimens and it informs future work investigating the generalizability of micro-CT imaging methods for other lesions, particularly those from the head and neck region where precisely defining margins of excision may be challenging.

A 5-year retrospective study assessing the association between seasonal and meteorological change and incidence of aneurysmal subarachnoid haemorrhage.

OBJECTIVE: Seasonal variation in incidence of spontaneous subarachnoid haemorrhage (SAH) is well recognised. This retrospective single-centre study aimed to characterise seasonality of SAH in the temperate climate of London, UK and to determine associations of incidence with meteorological variables. We further investigated whether associations vary according to location of aneurysm. METHODS: Admission data on 647 patients admitted with spontaneous SAH from December 2003 to August 2008 was analysed using our neurosurgical referrals database. Average monthly incidence of SAH was correlated with local temperature, atmospheric pressure and humidity data. In a subset of 467 patients, impact of aneurysm location on seasonal variation was evaluated. RESULTS: A non-significant bi-annual peak incidence was observed in Spring and Autumn with a trough in Summer (chi(2) = 1.5, p = 0.47). This trend was particularly marked with middle cerebral and posterior communicating artery aneurysms. However, anterior communicating artery aneurysmal SAH peaked in Summer only. SAH incidence correlated significantly with average humidity (coefficient 0.213, CI (0.02-0.404), p = 0.035) and peak humidity (coefficient 0.128, CI (0.008-0.248), p = 0.041). Temperature and atmospheric pressure did not correlate with incidence. CONCLUSIONS: This study illustrates a pattern of variation in SAH incidence similar to that seen in other populations and climates. However, our data suggests that this pattern differs according to aneurysm location. Unusually, we also find that humidity, and not temperature or atmospheric pressure, correlates with SAH incidence. Seasonal variability in aneurysm rupture is likely to be multifactorial, but meteorological factors may play an important role.

Late Recovery of Parathyroid Function after Total Thyroidectomy in Children and Adults: Is There a Difference?

BACKGROUND: Parathyroid failure after total thyroidectomy is the commonest adverse event amongst both children and adults. The phenomenon of late recovery of parathyroid function, especially in young patients with persistent hypoparathyroidism, is not well understood. This study investigated differences in rates of parathyroid recovery in children and adults and factors influencing this. METHODS: A joint dual-centre database of patients who underwent a total thyroidectomy between 1998 and 2018 was searched for patients with persistent hypoparathyroidism, defined as dependence on oral calcium and vitamin D supplementation at 6 months. Demographic, surgical, pathological, and biochemical data were collected and analysed. Parathyroid Glands Remaining in Situ (PGRIS) score was calculated. RESULTS: Out of 960 patients who had total thyroidectomy, 94 (9.8%) had persistent hypoparathyroidism at 6 months, 23 (24.5%) children with a median [range] age 10 [0-17], and 71 (75.5%) adults aged 55 [25-82] years, respectively. Both groups were comparable regarding sex, indication, extent of surgery, and PGRIS score. After a median follow-up of 20 months, the parathyroid recovery rate was identical for children and adults (11 [47.8%] vs. 34 [47.9%]; p = 0.92). Sex, extent, and indication for surgery had no effect on recovery (all p > 0.05). PGRIS score = 4 (HR = 0.48) and serum calcium >2.25 mmol/L (HR = 0.24) at 1 month were associated with a decreased risk of persistent hypoparathyroidism on multivariate analysis (p < 0.05). CONCLUSION: Almost half of patients recovered from persistent hypoparathyroidism after 6 months; therefore, the term persistent instead of permanent hypoparathyroidism should be used. Recovery rates of parathyroid function in children and adults were similar. Regardless of age, predictive factors for recovery were PGRIS score = 4 and a serum calcium >2.25 mmol/L at 1 month.

Quality of life can be good after slide tracheoplasty for long-segment tracheal stenosis.

OBJECTIVES: The objectives of this study were to measure 'health-related quality of life' (HRQoL) in children following slide tracheoplasty for long-segment tracheal stenosis (LSTS) and to explore the relationship of comorbidities and parental mental health with HRQoL outcomes. METHODS: A cross-sectional study was undertaken with children who had undergone slide tracheoplasty. Participants included parents and children (age 5-15 years) recruited over a 13-month period, who were asked to complete validated measures of HRQoL, development and behaviour. Scores were compared to published norms. RESULTS: Forty-two children (male 69%; n = 29) were included; mean age was 5.3 (standard deviation 3.5) years and mean follow-up was 45 (range 4-179) months. Mean total HRQoL scores for children with repaired LSTS did not differ from those of healthy norms other than for children aged 13-23 months, but 10 children (24%) had scores >2 SD below the mean for healthy children. HRQoL was poorer in children with non-cardiac congenital comorbidities than in those with isolated LSTS (mean scores 60.34 ± 17.19 and 85.52 ± 12.19, respectively, P = 0.01). There was good agreement between children's and parents' scores, although children rated their HRQoL as better than their parents did. Anxious parents rated their children's HRQoL as significantly worse than non-anxious parents (P<0.001). CONCLUSIONS: Older children with isolated LSTS can have excellent HRQoL after surgery. Younger children, at an earlier time point postoperatively, and those with non-cardiac congenital comorbidities have poorer HRQoL. Further longitudinal evaluation is required to identify psycho-social (including parental) predictors of outcome which may inform, or be amenable to, intervention.

Expansion of Human Airway Basal Stem Cells and Their Differentiation as 3D Tracheospheres.

Although basal cells function as human airway epithelial stem cells, analysis of these cells is limited by in vitro culture techniques that permit only minimal cell growth and differentiation. Here, we report a protocol that dramatically increases the long-term expansion of primary human airway basal cells while maintaining their genomic stability using 3T3-J2 fibroblast coculture and ROCK inhibition. We also describe techniques for the differentiation and imaging of these expanded airway stem cells as three-dimensional tracheospheres containing basal, ciliated, and mucosecretory cells. These procedures allow investigation of the airway epithelium under more physiologically relevant conditions than those found in undifferentiated monolayer cultures. Together these methods represent a novel platform for improved airway stem cell growth and differentiation that is compatible with high-throughput, high-content translational lung research as well as human airway tissue engineering and clinical cellular therapy.

Monitoring neovascularization and integration of decellularized human scaffolds using photoacoustic imaging.

Tissue engineering is a branch of regenerative medicine that aims to manipulate cells and scaffolds to create bioartificial tissues and organs for patients. A major challenge lies in monitoring the blood supply to the new tissue following transplantation: the integration and neovascularization of scaffolds in vivo is critical to their functionality. Photoacoustic imaging (PAI) is a laser-generated ultrasound-based technique that is particularly well suited to visualising microvasculature due to the high optical absorption of haemoglobin. Here, we describe an early proof-of-concept study in which PAI in widefield tomography mode is used to image biological, decellularized human tracheal scaffolds. We found that PAI allowed the longitudinal tracking of scaffold integration into subcutaneous murine tissue with high spatial resolution at depth over an extended period of time. The results of the study were consistent with post-imaging histological analyses, demonstrating that PAI can be used to non-invasively monitor the extent of vascularization in biological tissue-engineered scaffolds. We propose that this technique may be a valuable tool for studies designed to test interventions aimed at improving the speed and extent of scaffold neovascularization in tissue engineering. With technological refinement, it could also permit in vivo monitoring of revascularization in patients, for example to determine timing of heterotopic graft transfer.